WO2004010848A2 - Oncogenes useful for the identification of urinary bladder cancer - Google Patents

Oncogenes useful for the identification of urinary bladder cancer Download PDF

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WO2004010848A2
WO2004010848A2 PCT/US2003/023555 US0323555W WO2004010848A2 WO 2004010848 A2 WO2004010848 A2 WO 2004010848A2 US 0323555 W US0323555 W US 0323555W WO 2004010848 A2 WO2004010848 A2 WO 2004010848A2
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seq
sequence
nucleic acid
expression
amplification
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WO2004010848A8 (en
WO2004010848A3 (en
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Peter Schraml
Sanja Tomovska
Guido Sauter
Ronald Simon
Juha Kononen
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Diomeda Life Sciences, Inc.
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Priority to AU2003259269A priority Critical patent/AU2003259269A1/en
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Publication of WO2004010848A8 publication Critical patent/WO2004010848A8/en
Publication of WO2004010848A3 publication Critical patent/WO2004010848A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds

Definitions

  • the present invention relates to the detection and identification of nucleic acids and proteins that serve as cancer markers in biological samples. More specifically the present invention relates to the diagnosis and prognosis of bladder cancer by specifically detecting the presence of nucleic acids and/or their products which, when present individually or together in a biological sample are indicative of the presence of cancer in the organism. In particular, these markers are useful in detecting or modulating particularly aggressive forms of cancer. The invention further relates to methods for identifying and using candidate agents and/or targets which modulate certain cancers.
  • Occupational exposure may account for 21-2S* ⁇ ibladder cancer in white males in the United States. Almost all cases of bladder cancer are of the (95%), and among those, approximately 80% appear initially as more or less wo ⁇ sitffe ⁇ entiated, superficial papillary neoplasms with a tendency for multifocal or diffuse involvement of the urothelial surface and/or recurrent tumor episodes, but with limited potential- or' invasive growth. Most tumors are detected as non-invasive papillary tumors (stage pTa).
  • CIS carcinoma- in-situ
  • morphologic definition is arbitrary and generally defined as a total replacement of the urothelial surface by cells which bear morphologic features of the carcinoma, but which lack architectural alterations other than an increase in the number of cell layers, i.e., a flat lesion.
  • CIS constitute for another 5-10% of bladder neoplasias.
  • CIS are thought to represent the precursor lesions of most if not all solid invasive bladder carcinomas.
  • the stages pT1 and pT2-4 must be distinguished.
  • invasion is limited to the lamina propria.
  • These tumors have a good prognosis. They can be cured by transurethral resection in about 80%. Prognosis is much worse in pT2-4 carcinomas. Despite aggressive surgery (cystectomy), only about 50% of these patients survive.
  • Oncogenes that are known to play a role in bladder sancer include erbB-2 (Coombs, L.M., et al., BrJ Cancer, 63: 601-608 (1991)), Epr ⁇ l ⁇ ssial Growth Factor Receptor (EGFR) (Neal, D., et al., Lancet, 1: 366-368 (1985); Nfe ⁇ t .E., et al., Cancer, 65: 1619-1625 (1990), c-myc (Masters, J.R., et al., Urol Res, 16: 3 ⁇ ft344 (1988)), Cyclin D1 (Lee, O, et al., Cancer, 79: 780-789
  • Tumor suppressor genes that Ka g R&heen found inactivated in bladder carcinomas include p53 (Kamb, A., et al., Science; 2gSi.4' ⁇ 6-4W (1994)); Kamb, A., et al., Science, 264: 436-440 (1994); Stadler, W., Cance*@3s&.5 : 2060-2063 (1994), rb (Cordon-Cardo, C, et al., J Natl Cancer Inst, 84: 1251-125Q*f992)) and MTS1 (Kamb, A., et al., Science, 265: 416-417 (1994)); Kamb, A., et at , Sfcfe ⁇ ee, 264: 436-440 (1994); Stadler, W., Cancer Res,
  • Cytogenetic changes that can be found in bladder cancer are thought to provide cfues.where yet unidentified oncogenes and tumor suppressor genes may be located. Chromosomal deletions, which are frequently contributing to tumor suppressor gene inactivation, may pinpoint the location of tumor suppressor genes. Typical deletion sites in bladder cancer include 2q, 3p, 4p, 5q, 8p, 9q, 10p, 11p, 11q, 12q and 16q.
  • An increase in the copy number of genes (gene amplification) is one of the possible causes for oncogene overexpression.
  • the term "overexpression' is an abnormal or over-representation of a gene product expressed by a gene In bladder cancer, 22 different amplification sites have previously been published (Table 1).
  • CGH is based on the simultaneous hybridization of tumor and normal DNA to normal metaphase spreads (Kallioniemi, A., et al., Science, 258: 818-821 (1992) and disclosed in U.S. Patents 6,335,167 which is incorporated herein by reference).
  • CGH allows one to identify all c etions and amplifications of a tumor in one examination, even from formalin fixed tumof ⁇ r. Over the last few years, CGH has proved to be an excellent screening tool for ampli ⁇ &stions. CGH has lead to the identification of more than 30 yet unknown amplification sfes.in various tumors.
  • Bladder cancer tends to recur often, thus, consistent follow-up is imperative for thesss iagnosed patients.
  • Follow-up usually consists of cystoscopy, biopsy and x-ray exa fcafe .. Some of those procedures, though necessary, are considered invasive methods; ws#E .are often uncomfortable for the patient.
  • Bladder washings obtained during cystose ⁇ sjoan be then analyzed histologically and for DNA content. However, that approach is not &fe£9 s..successful, particularly when a limited number of cells is present in the sample.
  • FISFi fluorescense in situ hybridization
  • amplified and/or deleted genes are important to the management of cancer, for example, bladder cancer, for several reasons: (1 ) to improve prognostication; (2) to detect amplification and/or deletion events that are associated with the development of drug resistance; and (3) to improve therapy.
  • the present invention provides methods for the diagnosis and prognosis of bladder cancer.
  • the method comprises identifying a gene or set of genes that exist within an amplicon, specifically the region designated as 6p22, and determining the expression of the gene(s) thereof in a first tissue type of a first individual, and comparing this to the expression of the gene from a second unaffected individual. A difference in the expression indicates that the first individual has an increased risk of cancer.
  • this invention provides cDNA sequences from a number of genes wH i map to this region. Also provided is a contig (a series of clones that contigea ⁇ sly spans this amplicon) which can be used to prepare probes specific for the amplicom The probes can be used to detect chromosomal abnormalities at chromosome 6.
  • the probes disd& «M ⁇ ere can be used in kits for the detection of a chromosomal abnormality at ates&position FLpter 0.11 - 0.19 on human chromosome 6.
  • the kits include a which contains a labeled nucleic acid probe which binds selectively to a targeif fCjlypucleotide sequence at about FLpter 0.11 - 0.19 on human chromosome 6.
  • the p afes ⁇ preferably includes at least one nucleic acid that specifically hybridizes to a nucleic acid selected from the nucleic acids disclosed here. Even more p ⁇ a ly, the probes comprise one or more nucleic acids selected from the nucleic acKte; .disclosed here.
  • the probes are labeled with digoxigenin or jetirr.
  • the kit may further include a reference probe specific to a sequence in the centromere of chromosome 6.
  • the present invention includes preferred nucleic acid sequences useful for hybridizing to amplified nucleic acids that are indicative of bladder cancer.
  • Another aspect of the invention makes use of fluorescense in situ hybridization (FISH) on tissue microarray sections by hybridizing YAC/PAC or BAG probes comprising SEQ ID NO:1 to SEQ ID NO: 4 or fragments for use therewith.
  • FISH fluorescense in situ hybridization
  • Another aspect of the invention is an oligonucleotide comprising a target- binding sequence consisting of the sequence of any one of SEQ ID NO:1 to SEQ ID NO: 4, and optionally a contiguous sequence required for an amplification reaction.
  • Another aspect of the invention is a combination of oligonucleotides used in a detection assay specific for a bladder target nucleic acid sequence.
  • the combination comprises a first oligonucleotide that serves as a first amplification primer that hybridizes specifically to a first -specific sequence contained in the 6p22 region of an expressed gene sequence, or that is contained in the E2F3 expressed gene sequence; a second oligonucleotide that serves as a second amplification primer that specifically hybridizes to a different, non-overlapping second sequence sr ⁇ feias BIG1 contained in the 6p22 region.
  • oligonucleotides are a first oligonucleotide comprising a sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4; the second
  • ⁇ jf ycleotide comprising a sequence selected from the group consisting of SEQ ID NO:1 , S iQ NO:2, SEQ ID NO:3, and SEQ ID NO.4, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:S;T?5;7slS£Q ID NO:4; and the third oligonucleotide comprises a sequence selected ttei ⁇ the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.
  • Another embodiment makes"y®?iOt an aptamer probe to detect either the transcript or protein related to the genes of ⁇ :te:: ⁇ resent invention.
  • the method comprises asss ig.for the expressed gene sequence encoding the E2F3, BIG1, and/or BIG2 product.
  • the nucleic acid sample is RNA, more preferably mRNA, isolated fr ⁇ rrrhuman bladder.
  • the detecting step detects a signal in an assay, such as a homogeneous detection assay or an antibody based assay, such as:, IHC, sandwich assay or antibody biochips. These assays are well known by those skilled in the art. Furthermore an assay could be devised wherein detection occurs indirectly, such as a first fragment unlabled fragment is bound to the target and a secondary labeled fragment is introduced for detection of the first.
  • a method of screening drug candidates comprises providing a cell that expresses an expression profile gene or fragments thereof.
  • Preferred embodiments of the expression profile gene as described herein include the sequence comprising E2F3, BIG1 , BIG2 or a fragment thereof.
  • the method further includes adding a drug candidate to the cell and determining the effect of the drug candidate on the expression of the expression profile gene.
  • the method of screening drug candidates includes comparing the level of expression in the absence of the drug candidate to the level of expression in the presence of the drug candidate, wherein the concentration of the drug candidate can vary when present, and wherein the comparison can occur after addition or removal of the drug candidate.
  • the cell expresses at least one expression profile genes.
  • the profile genes may show an increase or decrease.
  • Also provided herein is a method of screening for a bioactive agent' ⁇ spable of binding to E2F3, BIG1, BIG2 or a fragment thereof, the method comprising combining E2F3, BIG1 , BIG2 or fragment thereof and a candidate bioactive agenf, and determining the binding of the candidate agent to the E2F3, BIG1 , BIG2 or fragment thereof.
  • iftar ⁇ ethod comprises combining E2F3, BIG1 , BIG2 or fragment thereof and a and determining the effect of the candidate agent on the bioactivity of E2F3; li ⁇ s BIG2 or the fragment thereof.
  • E2F3, BIG1 , BIG2 has the U ⁇ r*sfetty. of a bladder cancer modulating protein.
  • Also provided herein is a method of evaluating the eifest.of a candidate cancer drug comprising administering the drug to a transgenic animal, expressing or over-expressing E2F3, BIG1 , BIG2 or a fragment thereof, or an animal lacking E2F3, BIG1, BIG2 for example as a result of a gene knockout. Additionally, provided herein is a method of evaluating the effect of a candidate cancer drug comprising administering the drug to a patient and removing a cell sample from the patient. The expression profile of the cell is then determined. This method may further comprise comparing the expression profile to an expression profile of a healthy individual.
  • a method for inhibiting the activity of a bladder cancer modulating protein comprises binding an inhibitor to the protein expressed by E2F3, BIG1, and/or BIG2.
  • the invention provides a method for neutralizing the effect of a bladder cancer modulating protein.
  • the method comprises contacting an agent specific for the protein with the protein in an amount sufficient to effect neutralization.
  • the protein is expressed by one or a combination of E2F3, BIG1, and/or BIG2.
  • methods of inhibiting bladder cancer can be performed by administering any inhibitor of E2F3, BIG1 , and/or BIG2 activity to a cell or individual.
  • a E2F3, BIG1 , and/or BIG2 inhibitor is an antisense molecule to E2F3, BIG1 , and/or BIG2, respectively.
  • a biochip comprising a nucleic aei&segment comprising SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 3, SEQ ID NO: 4, oi» ⁇ s fragment thereof, wherein the biochip comprises fewer than 1000 nucleic acid ' j ⁇ S ⁇ t ⁇ es. Preferably at least two nucleic acid segments are included.
  • the biochip could also * contain capture molecules other than nucleic acids, for example antibodies, antibody ffr ⁇ ants, aptamers, protein scaffolds etc.
  • A* r sG «fy s» ⁇ ded herein are methods of eliciting an immune response in an individual.
  • a method provided herein comprises administering to an individual a composite* comprising E2F3, BIG1 , BIG2 or a fragment thereof.
  • compositionsr-eapsfela of eliciting an immune response in an individual comprising E2F3, BIG1 , BIG2 or a fragment thereof and a pharmaceutically acceptable carrier.
  • said composition comprises a nucleic acid comprising a sequence encoding E2F3, BIG1 , BIG2 or a fragment thereof and a pharmaceutically acceptable carrier.
  • chromosome sample refers to a tissue or cell sample prepared for standard in situ hybridization methods described below. The sample is prepared such that individual chromosomes remain substantially intact and typically comprises metaphase spreads or interphase nuclei prepared according to standard techniques.
  • Nucleic acid refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, would encompass known analogs of natural nucleotides that can function in a similar manner as naturally occurring nucleotides.
  • an "isolated" polynucleotide is a polynucleotide which is substantially separated from other contaminants that naturally accompany it, e.g., protein, other polynucleotide sequences.
  • the term embraces polynucleotide sequences ⁇ , -i' zfa have been removed or purified from their naturally-occurring environment or clone libr ry . and include recombinant or cloned DNA isolates and chemically synthesized analogues ⁇ F's"- ⁇ !3;S)gues biologically synthesized by heterologous systems.
  • “Su-s&sqwence or fragment” refers to a sequence of nucleic acids that comprise a part of longer sequence of nucleic acids.
  • a “probe” or a “nucfefesisstd probe,” as used herein, is defined to be a collection of one or more nucleic aorcifpagments whose hybridization to a target can be detected.
  • the probe is labeled as dese foed: below so that its binding to the target can be detected.
  • the probe is produced from a: source of nucleic acids from one or more particular (preselected) portions of the genomevfor example one or more clones, an isolated whole chromosome or chromosome fragment, or a collection of polymerase chain reaction (PCR) amplification products.
  • the probes of the present invention are produced from nucleic acids found in the 6p22 amplicon as described herein.
  • the probe may be processed in some manner, for example, by blocking or removal of repetitive nucleic acids or enrichment with unique nucleic acids.
  • probe may be used herein to refer not only to the detectable nucleic acids, but to the detectable nucleic acids in the form in which they are applied to the target, for example, with the blocking nucleic acids, etc.
  • the blocking nucleic acid may also be referred to separately. What "probe” refers to specifically is clear from the context in which the word is used.
  • “Hybridizing” refers the binding of two single stranded nucleic acids via complementary base pairing.
  • Bind(s) substantially or “binds specifically” or “binds selectively” or “hybridizes specifically” refer to complementary hybridization between an oligonucleotide and a target sequence and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target polynucleotide sequence. These terms also refer to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.
  • a complex mixture e.g., total cellular
  • stringent conditions refers to conditions under which a probe will hybridize to its target subseq&face, but to no other sequences. Stringent conditions are sequence-dependent anc .' ⁇ l be different in different circumstances. Longer sequences hybridize specifically at rug ⁇ r,, temperatures. Generally, stringent conditions are selected to be about 5° C io ⁇ . than the thermal melting point (Tm) for the specific sequence at a defined ionic pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic acfcffeancentration) at which 50% of the probes complementary to the target sequence hybridizKitolhe target sequence at equilibrium.
  • Tm thermal melting point
  • stringent conditions will be those : ,y «hich the salt concentration is at least about 0.02 Na ion concentration (or other salts) ' a£y? ..7.0 to 8.3 and the temperature is at least about 60° C for short probes. Stringent also be achieved with the addition of destabilizing agents such as formamide.
  • polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 85% sequence identity, more preferably at least 90%, and most preferably at least 95% compared to a reference sequence using the methods described below using standard parameters.
  • nucleic acid sequences are said to be “identical” if the sequence of nucleotides in the two sequences is the same when aligned for maximum correspondence as described below.
  • complementary to is used herein to mean that the complementary sequence is identical to all or a portion of a reference polynucleotide sequence.
  • Sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two sequences over a “comparison window” to identify and compare local regions of sequence similarity.
  • a “comparison window”, as used herein, refers to a segment of at least about 20 contiguous positions, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are' optimally aligned.
  • Percentage of sequence identity is determined by comparing two optimally aBfg&sel.sequences over a comparison window, wherein the portion of the in the comparison window may comprise additions or deletions (i.e., ga ⁇ as compared to the reference sequence (which does not comprise additions or cSt ⁇ f ⁇ &o ⁇ s) for optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino tsc ' uiresidue occurs in both sequences to yield the number of matched positions, dividing-fee. number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • nucleotide sequences are substantially identical if two molecules hybridize to the same sequence under stringent conditions.
  • Stringent conditions are sequence dependent and will be different in different circumstances.
  • stringent conditions are selected to be about 5° C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • stringent conditions will be those in which the salt concentration is about 0.02 molar or lower at pH 7 and the temperature is at least about 60° C.
  • Figure 1 A model of bladder cancer development and progression.
  • Figure 2 provides CGH profiles of tumors and a tumor cell line with 6p amplification. CGH profiles are shown next to idf ⁇ gfams of chromosomes 6. In all tumors circumscribed peaks are visible at 6p21.3-22 f ⁇ c ⁇ ating high level amplification of this region.
  • Figure 3A, 3B, 3C and 3D represent a combined physic&ki&anscript and FISH map of the amplified region at 6p22. Clones marked in red constitute ⁇ s.Eore of the amplicon (1.7 Mb in size). Average copy number for each clone in the map «c « counted from 10-30 tumors placed into a tissue microarray and assayed with a labeled DNA-probe.
  • Clones marked in red define the minimal commonly amplified rs s,at 6p22. Genes and transcripts mapping to the region are also indicated.
  • a photograph of a Western blot demonstrating a high amount of E2F3 protein in >1s; ds?v:cancer cell lines with 6p22 amplification (CRL1472, HTB5, HTB9) as compared to b ⁇ fefeccancer cell lines with normal 6p22 copy number (CRL 7882, RT1 2) or other cancer fe:C.HL60.
  • Figure 5 is a photograph of a cell nucleus with multiple copies of the E2F3 gene, demonstrated with hybridizing tissue microarray sections with green FISH probe.
  • Figure 6 is a photograph of a cell nucleus from a tumor that has equal numbers of E2F3 and centromere 6 FISH probe signals.
  • Figure 7A is a Northern blot indicating that no E2F3 transcript was seen in cell line CRL-1479, whereas cell lines with E2F3 amplification CHTB-5 CRL1427 and ACC5637 show high level of E2F3 expression.
  • Figure 7B is a Western blot indicating that the protein expression level in the third amplified cell line (ACC 5637) was comparable to the expression in non- amplified cell lines.
  • Figure 8 is a photograph of an amplified tumor stained with E2F3 antibody.
  • Figure 9 is a photograph of a non-amplified tumor stained with E2F3 antibody.
  • Figure 10 graphically represents E2F3 alterations and prognosis of patient survival.
  • Figure 11 graphically represents E2F3 alterations and prognosis of patient survival.
  • Figure 12 is the nucleic acieteequence of the gene identified as BIG1 located in the 6p22 region.
  • Figure 13 is the nucleic acid sequence ofih gene identified as BIG2 located in the 6p22 region.
  • Figure 14 is the nucleic acid sequence of the gene:J entified as E2F3 located in the 6p22 region.
  • Figure 15 is a schematic of BIG-2: (A) assembling of a 3.144ii?? ; !fdnigene" from cDNA sequences ENSESTT00002382100, NM_175879, dJ434O11 , ar ⁇ fci Q8N9R5;; (B) predicted intron-exon structure; and (C) predicted protein domains. . significant similarity was found for the MBOAT domain.
  • R ⁇ £,yse;16 is a schematic of BIG-1 : (A) intron-exon structure; (B) predicted protein domai Y ix. Significant matches were found for the UPF0004, Radical_SAM, and TRAM domains:
  • Figure 17 is a Northern -analysis of 9 bladder tumor cell lines.
  • the 2.6 kb BIG-1 mRNA is abundantly expressedttrr 6p22.3 amplified cell lines CRL-1472, 5637, and 5-HTB.
  • Northern blot staining with met ylene blue showed presence of RNA in all cell lines (control in the lower panel).
  • the target genes are unknown for the majority of loci harboring gene amplifications such as 1q21-31 , 2q13, 3p22-24, 6p22, 8p11 , 8q21, 9p21, 10p13-14, 13q13; " ⁇ q31-33, 18p11, 20q, 21p11, 22q11-13, Xp11- 13, and Xq21-22.2 (Kallioniemi, A., et al:, U nes Chrom Cancer, 12: 213-219 (1995); Kallioniemi, A., et al, Science, 258: 818-821 (tJ ) . Mitelman, F., Catalog of chromosome aberrations in cancer, 5th edn.
  • the present invention exploits the genome-era information resources and tissue a ⁇ ta jptetforms to quickly identify and examine loci harboring gene amplifications.
  • CGH comparative genomic hybridization
  • 6p22 is the most frexpsfttsite of high level amplification in bladder cancer at which no oncogene is cys «j ⁇ tly known (Table 2);
  • 6p+ is the genomic alteration detectable by CGH which is &sf ⁇ 'sfe ⁇ ngly linked to a high tumor grade (Table 3); and
  • 6p+ is strongly (and independef FarmStumor grade) associated with rapid tumor cell proliferation (Table 4).
  • the final order of the clones within the physical map contig differed markedly from the order of the clones indicated by the publicly available databases.
  • the final, verified genomic contig contained the 6p22 amplicon, but included also by-stander sequences on both sides of the core amplification unit.
  • FISH studies were performed on bladder cancer tissue microarray constructed from o specimens showing 6p alterations. Individual contig clones were used as probes in
  • GS-BIG FISH assay revealed amplification in 86 out of 1 ,029 -ansiyzed tumors pointing to an overall amplification frequency of about 8%.
  • the breakdowrrc? BiG1 amplification frequency according to histological tumor type is shown below in TaMe 6.
  • GS-BIG amplification associates strongly with high tumor grade and advanced sl . ⁇ 0.0001 each, Chi-square statistical test of significance). Furthermore, GS- 3l& ⁇ mplification is very rare in early-stage tumors (0.7 % of pTaG1/G2 tumors) but re ⁇ ftfe Sy common in invasively growing cancers (15 % of pT1 -4 tumors).
  • DNA probes derived from the GS-BIG region can be used to detect bladder cancer, for example on voided urine cells, and GS-BIG amplification can be used to differentiate between tumors of low and high malignancy potential.
  • the positive predictive value of GS-BIG6 amplification for invasive tumor growth or high grade would be about 93%.
  • the present invention describes a genomic sequence originating from chromosomal region 6p22 (Genomic Sequence for Bladder Invasive Growth 6, GS-BIG) that undergoes amplification in bladder cancer, and teaches the use of this sequence in detection of bladder cancer.
  • E2F3 belongs to a family of cell cycle regulatory transcription factors that are controlled by the retinoblastoma tumor suppressor (Humbert, P.O., et al., Genes Dev, 14: 690-703 (2000); Lees, J.A., et al., 13: 7813-7825 (1993); Leone, G., et al., Genes Dev, 12: 2120-30 (1998); Leone, G., et al., Genes Dev, 12: 2120-30 (1998); Wv, : L, et al., Nature, 414: 457-62 (2001).
  • E2F1 , E2F2, or E2F3 with DP1 serve as transcriptional activators of genes that promote cell cycling, whereas compl xes of E2F/DP with pRb repress transcription and inhibit cell growth (Leone, G., et al., ( S& s.Dev, 12: 2120-30 (1998); Nevins, J.R. Cell Growth Differ, 9: 585-93 (1998); Trimawte f . J.M. & Lees, J.A. Nat Rev Mol Cell Biol, 3: 11-20 (2002).
  • mice models two isoformacHiE2F3 have been identified that are expressed via an alternative translation site'
  • E2F3A, i$ a truncated variant
  • E2F3B a truncated variant
  • E2F3B lacks 101 N- terminal amino acids relative to the full-length pr ⁇ l s,: including a moderately conserved sequence of unknown function, which is pres sir.Only in the growth- promoting E2F family members.
  • E2F3A (like E2Ff s ⁇ :E2F2) promotes cell proliferation
  • E2F3B is believed to inhibit cell cycle by E2F4- and E2F; ⁇ ' -mediated transcriptional activation of p16 (Gaubatz, S., et al. Mol Cell, 6: 729-35 (2000); Wu, L, et al., Nature, 414: 457-62 (2001).
  • p16 Gaubatz, S., et al. Mol Cell, 6: 729-35 (2000)
  • Wu, L, et al., Nature, 414: 457-62 (2001) it is not known whether these two functional isoforms exist in humans, or play similar roles as in mice.
  • Recent reports have indicated that different members of the E2F gene family could play specific and diverse roles in tumorigenesis of various human malignancies.
  • E2F1 increased copy numbers and overexpression of E2F1 were found in an erythroleukemia cell line (Saito, M., et al., Genomics, 25: 130-8 (1995), and E2F5 was amplified and upregulated in 4.2% of breast cancers (Polanowska, J., et al., Genes Chromosomes Cancer, 28: 126-30 (2000). Decreased expression of E2F1 was recently found to be associated with an increased risk of progression to metastases in bladder cancer (Rabbani, F., et al., J Natl Cancer Inst, 91: 874-81 (1999).
  • E2F3 E2F3 in human cancers.
  • overexpression of E2F3 could facilitate cell cycle progression and an increased cell proliferation rate.
  • overexpression of E2F3 could provide a growth advantage to cells exhibiting this alteration, resulting in clonal selection explaining the presence of 6p22 amplifications in advanced-stage tumors.
  • TMA Tissue microarray
  • CGH Comparative Genome Hybridization
  • E2F3 expression would correlate with tumor proliferation.
  • the Ki67 labeling index (LI) was analyzed for each tumor. There was a strong association between a high Ki67 LI and E2F3 expression (p ⁇ 0.0001 ), which was independent of grade and stage. Thus leading to the conclusion that E2F3 is frequently amplified and overexpressed in invasively growing bladder cancer (stage pT1-4). E2F3 overexpression appears to provide a growth advantaege to tumor cells by activating cell proliferation in a subset of bladder tumors.
  • E2F3 gene amplification FISH analyses were performed in two steps: prescreening and establishing optimal hybridization conditions was done on a mini-TMA containing specimens with 6p22 amplification (as identified by CGH), followed by a large-scale TMA analysis of 2317 clinical specimens.
  • the 6p22-amplification specific mini-TMA revealed E2F3 amplification in all 3 cell lines and 7 primary tumors that had shown 6p22 amplification by CGH.
  • the average E2F3 copy numbers in these tumors ranged between 7 and 31.
  • FISH FISH was successful in 1272 of 2317 (55%) samples of the large TMA. FISH related problems (weak hybridization, background, tissue damage) were responsible for about two thirds, TMA linked problems (too few or absence'sCtumor cells on the TMA spot) were causing about one third of the non-informative cas ⁇ , E2F3 amplification was detected in 145 of 1037 (14.0%) interpretable tumorsr ⁇ nly the first biopsy of each patient included). Examples of amplified and non-amplified i&? ⁇ ;s are shown in Figures 5 and 6. The associations with tumor phenotype are summarized in Table 7.
  • E2F3 amplification was singly associated with high tumor grade and advanced stage (p ⁇ 0.0001 each). Most si> ⁇ l ingly, E2F3 amplification was rare in pTaG1/G2 tumors (4 of 398; 1%) while 22% (TtQr. ⁇ f 489) of the invasively growing TCC (pT1-4) had E2F3 amplification. E2F3 amplification was most frequent in muscle invasive cancers of several histological subtypes including TCC (29.2%), small cell carcinomas (35.7%), sarcomatoid cancers (22.2%), and probably adenocarcinomas (1 of 4 samples amplified).
  • E2F3 positive and negative tumors are shown in Figures tfssi 9.
  • E2F3 positivity was found in 57 of 106 amplified TCCs (53.8%), but in only m&f 640 (14.22%) non- amplified tumors. This association held also true within the pT1-4 TCCs that could be analyzed by both FISH and IHC (p ⁇ 0.0001).
  • E2F3 positive tumors increase ⁇ from 10% in pTaG1/G2 tumors (49 of 492) to 20.9% in invasively growing pT1-4 TCCs (1'25Tof 608).
  • Table 8 E2F3 Alterations and Tumor Phenotype in Urinary Bladder Cancer
  • Non-amplified cases include gains, a ⁇ number of analyzable samples, b) Mean Ki-67 LI. c) 95% Cl of Ki- 67 LI. d) Student'aT-Test
  • IHC negative neg/weak.
  • IHC positive mod/strong E2F3 alterations and prognosis
  • E2F3 amplification and E2F3 expression were associated with poor tumor specific survival if all patients were included in the analysis ( Figures 10 and 11). There was no association between E2F3 alterations and tumor specific survival within the subgroup of pT2-4 TCCs. E2F3 amplification and E2F3 overexpression provided no prognostic significance among pTa/ pT1 tumors, neither for recurrence nor for progression.
  • a PAC probe containing the E2F3-specific STS marker stSG15990 was ordered from the Sanger Centre, Cambridge UK, to determine whether E2F3 is present within the 6p22 amplicon.
  • the application of a mini-TMA composed of CGH amplified tumors allowed us t ⁇ .directly assess the amplification status of E2F3 not only in primary bladder tumors, in bladder cancer cell lines, in a single hybridization experiment.
  • E2F3 overexpression by IHC was found in 53:&?-3.i ) of E2F3 amplified, but in only 14.2% of non-amplified tumors.
  • these data suggest that E2F3 overexpression is not present in all amplified tumor samples. This finding could be in par xiue to technical reasons, as none of the currently commercially available antibodies- targeting E2F3 (including that one used in our study) is specifically recommended for immunohistochemistry on formalin fixed tissues. It is possible, therefore, that the use of a non-optimized antibody has resulted in an unusual high fraction of IHC false negative samples.
  • Amplification may be a mechanism that is particularly effective to simultaneously overexpress multiple adjacent genes which may jointly provide a growth advantage to amplified tumor cells.
  • neighboring oncogenes that sometimes undergo coamplification have recently been identified at various genomic regions such as MDM2, GLI, CDK4, and SAS at 12q13-q15 (Reifenberger, Gêt et al., Cancer Res, 54: 4299-303 (1994) or CCNDI , EMS1 , and INT2 at 11q13 (Hui, R., et al., Oncogme 15: 1617-23 (1997).
  • CDK5AL1 a gene of unknown function that shares protein sequence similafB* « ,,with CDK5RAP1 (which is involved in the regulation of neuronal differentiation) maps ⁇ Lte&egion adjacent to E2F3.
  • SOX4 a transcription factor that is a member of the high mobfii ⁇ sup (HMG)-box family of DNA binding protein (Farr, C.J., et al., Mamm Genome, 4: 57 ⁇ 1993), and PRL (the gene encoding
  • Prolactin map to region between 1 and'-i iScwetromeric to E2F3.
  • Prolactin (PRL) is a protein hormone closely related to growth hor T a ⁇ scl, mainly secreted in the anterior pituitary lactotrope and the decidualized stromai ' ⁇ tt' ⁇ e. human endometrium (DiMattia, G.E., et al., J Biol Chem, 265: 16412-21 ⁇ i
  • Northern analysis demonstrated no correlation between amplification and'' overexpression of these genes in 8 cell lines (Bruch, J., et al., Cancer Res, 60: 4SK «% 30 (2000).
  • Transcripts termed BIG1 and BIG2 represent genes that map to the core GS-BIG region and are overexpressed at least at the mRNA level. Thus, these genes may either alone or in combination with each other form an oncogene cluster that provides a growth advantage to bladder cancer cells.
  • the occurrence of a subgroup of tumors having unequivocal E2F3 protein overexpression in the absence of amplification suggests that other mechanisms than amplification may also cause E2F3 activation in bladder cancer.
  • E2F3 expression supports an oncogenic role of E2F3 alterations through cell cycle activation. This is consistent with the known role of E2F3 as a gene activator essentially required for S-phase induction (Leone, G., et al., Genes Dev, 12: 2120-30 (1998).
  • E2F3 as a gene activator essentially required for S-phase induction
  • accelerated cell proliferation is an important prerequisite for tumor growth, but it does not necessarily determine aggressiveness.
  • Other factors like invasive growth and, most importantly, the metastatic potential of a tumor are key determinants for the clinical outcome of a cancer patient.
  • E2F3 amplification was i&'s .in only 4 of 398 pTaG1/G2 tumors (1%).
  • 6p22 amplification detection eo ⁇ i- ⁇ have clinical utility in the difficult cytological distinction of low and high malignant bladdes ⁇ «?pla.sm's on voided urine cells.
  • the positive predictive value of E2F3 amplifi ⁇ ai ⁇ a'f ⁇ r. invasive tumor growth or high grade would be about 93%.
  • E2F3 is included in a bladder cancer amplicon at 6p22. Taking together the known cell cycle activating role of E2F3, its overexpression in amplified tumors and the association with cell proliferation in vivo, it appears that E2F3 could be one of probably multiple target genes inside the 6p22 amplification whose overexpression gives growth advantage to tumor cells.
  • the present invention demonstrates how a combination of genomic technologies, microarray discovery platforms and bioinformatics resources can be used to rapidly identify, validate and characterize target genes for genetic alterations and associate these changes to specific medical conditions.
  • clones and sequence information provided herein can be used to detect amplifications, or other chromosomal abnormalities, at 6p22 in a biological sample.
  • the methods involve hybridization of probes that specifically bind one or more nucleic acid sequences of the target amplicon with nucleic acids present in a biological sample or derived from ⁇ biological sample.
  • a biological sample is a sample of biological tissue or fluid containi «-
  • the biological sample is a cell or tissue suspeot&i*&f being cancerous (transformed).
  • sample also includes- si-tpsernatant (containing cells) or the cells themselves from cell cultures, cells from tissue other media in which it may be desirable to detect chromosomal abnormalities.
  • a biological sample is prepared by cells, either as single cell suspensions or as tissue preparation, on solid supp ⁇ rta such as glass slides and fixed by choosing a fixative which provides the best spalist resolution of the cells and the optimal hybridization efficiency. Selecting Probes
  • any of the SEQ ID NOs, or the cDNAs disclosed herein are suitable for use in detecting the 6p22 amplicon.
  • Methods of preparing probes are well known to those of skill in the art (see, e.g. Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989) or Current Protocols in Molecular Biology, F. Ausubel et al., ed. Greene Publishing and Wiley-lnterscience, New York (1987)).
  • the probes are most easily prepared by combining and labeling one or more of the SEQ ID NOs.
  • the constructs Prior to use, the constructs are fragmented to provide smaller nucleic acid fragments that easily penetrate the cell and hybridize to the target nucleic acid. Fragmentation can be by any of a number of methods well known to hose of skill in the art. Preferred methods include treatment with a restriction enzyme to selectively cleave the molecules, or alternatively to briefly heat the nucleic acids in the presence of Mg 2+ .
  • Probes are preferably fragmented to an average fragment length ranging from about 50 bp to about 2000 bp, more preferably from about 100 bp to about 1000 bp and most preferably from about 150 bp to about 500 bp.
  • probes can be produced by amplifying (e.g. via PCR) selected subsequences from the 6p22 amplicon disclosed herein.
  • amplifying e.g. via PCR
  • sequences provided herein permit one of skill to select primers that amplify sequences from one or more exons located within the 6p22 amplicon.
  • Particularly preferred probes include nucleic acids from probes RP1-46C2, RP1-2??tvfi.i, RP-177P22, RP-44C7 and RP3-348I23, which corresponds to genomic region betwefe ⁇ rSTS markers G717699 and G26264.
  • the cDNAs are particularly useful T ⁇ s ⁇ sntifying cells that have increased expression of the corresponding genes, instance, Northern blot analysis.
  • nucleic acids are well known to those of sk»ft the art.
  • Preferred labels are those that are suitable for use in in situ hybridization.
  • Tfte,. nucleic acid probes may be detectably labeled prior to the hybridization reaction:
  • a detectable label which binds to the hybridization product may be used.
  • detectable labels include any material having a detectable physical or chemical property and have been well-developed in the field of immunoassays.
  • a "label” is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
  • Useful labels in the present invention include radioactive labels (e.g. 32 P, 125 l, 14 C, 3 H, and 35 S), fluorescent dyes (e.g. fluorescein, rhodamine, Texas Red, etc.), electron-dense reagents (e.g. gold), enzymes (as commonly used in an ELISA), colorimetric labels (e.g. colloidal gold), magnetic labels (e.g. DynabeadsTM), and the like.
  • radioactive labels e.g. 32 P, 125 l, 14 C, 3 H, and 35 S
  • fluorescent dyes e.g. fluorescein, rhodamine, Texas Red, etc.
  • electron-dense reagents e.g. gold
  • enzymes as commonly used in an ELISA
  • colorimetric labels e.g. colloidal gold
  • magnetic labels
  • stains directly labeled with fluorescent labels are preferred for chromosome hybridization.
  • a direct labeled probe is a probe to which a detectable label is attached. Because the direct label is already attached to the probe, i subsequent steps are required to associate the probe with the detectable label.
  • an indirect labeled probe is one which bears a moiety to which a detectable label is subsequently bound, typically after the probe is hybridized with the target nucleic acf ⁇ t ⁇ .
  • the label must be detectible in as low copy number as possible thereby/ ximizing the sensitivity of the assay and yet be detectible above any backgrounti ; y» 8i.
  • a label must be chosen that provides a highly localized signal thereby provt5 j;:a,high degree of spatial resolution when physically mapping the stain against the chrorvm s' Particularly preferred fluorescent labels include fluorescein-12-dUTP and Texas W& fcs&dUTP.
  • the labels may be coupled to the pr fets. a variety of means known to those of skill in the art.
  • tJf ⁇ nucleic acid probes will be labeled using nick translation or random primer extension (Rsghy, et al., J. Mol. Biol., 113: 237 (1977) or Sambrook, et al., Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1985)).
  • nick translation or random primer extension Rsghy, et al., J. Mol. Biol., 113: 237 (1977) or Sambrook, et al., Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1985)
  • the probes of this invention need not be absolutely specific for the targeted 6p22 region of the genome.
  • probes are intended to produce “staining contrast.”
  • “Contrast” is quantified by the ratio of the probe intensity of the target region of the genome to that of the other portions of the genome.
  • a DNA library produced by cloning a particular chromosome e.g. chromosome 7
  • the library contains both sequences found only on that chromosome, and sequences shared with other chromosomes. Roughly half the chromosomal DNA falls into each class.
  • the target chromosome would be twice as bright (contrast ratio of 2) as the other chromosomes since it would contain signal from the both the specific and the shared sequences in the stain, whereas the other chromosomes would only be stained by the shared sequences.
  • the other chromosomes would only be stained by the shared sequences.
  • Detecting the 6p22 Amplicon As explained above, detection of amplification in the 6p22 amplicon r indicative of the presence and/or prognosis of a bladder cancers.
  • a 6p22 amplification is detected through the hybridization of a probe of this invention to a target nucleic acid (e.g. a chromosomal ya «:(i» n which it is desired to screen for the amplification.
  • a target nucleic acid e.g. a chromosomal ya «:(i» n which it is desired to screen for the amplification.
  • Suitable hybridization formats - ⁇ r ⁇ A known to those of skill in the art and include, but are not limited to, variations of Sou't ' fesrBlots, in situ hybridization and quantitative amplification methods such as quantit fe «RGR (see, e.g. Sambrook, supra, Kallioniemi, et al., Proc.
  • the 6p22 amplicon is fdeotified using in situ hybridization.
  • in situ hybridization comprises the following major steps: (1) fixation of tissue or biological structure to analyzed; (2) prehybridization treatment of the biological structure to increase accessibility of target DNA, and to reduce nonspecific binding; (3) hybridization of the mixture of nucleic acids to the nucleic acid in the biological structure or tissue; (4) posthybridization washes to remove nucleic acid fragments not bound in the hybridization and (5) detection of the hybridized nucleic acid fragments.
  • the reagent used in each of these steps and their conditions for use vary depending on the particular application.
  • human genomic DNA is used as an agent to block such hybridization.
  • the preferred size range is from about 200 bp to about 1000 bases, more preferably between about 400 to about 800 bp for double stranded, nick translated nucleic acids.
  • Hybridization protocols for the particular applications disclosed here are described in Pinkel, et al., Proc. Natl. Acad. Sci. USA, 85: 9138-9142 (1988) and in EPO Pub. No. 430,402. Suitable hybridization protocols can also be found in Methods o/in Molecular Biology Vol. 33: In Situ Hybridization Protocols, K. H. A. Choo, ed., Humana Press, Totowa, N.J., (1994). In a particularly preferred embodiment, the hybridization protocol of Kallioniemi, et al., Proc. Natl Acad Sci USA, 89: 5321-5325 (1992) is used.
  • Paraffin embedded tumor sections can to-f ⁇ , «ad. r as can fresh or frozen material. Because FISH can be applied to the limited material'? Huch. preparations prepared from uncultured primary tumors can also be used (see, e.g., ss oniemi, A., et al., Cytogenet. Cell Genet, 60: 190-193 (1992)). For instance, small biopsy tissue, samples from tumors can be used for touch preparations (see, e.g., Kallioniemi;' A., et al., Cytogenet. Cell Genet, 60: 190-193 (1992)). Small numbers of cells obtained from aspiration biopsy or cells in bodily fluids (e.g., blood, urine, sputum and the like) can also be analyzed. For prenatal diagnosis, appropriate samples will include amniotic fluid and the like.
  • bodily fluids e.g., blood, urine, sputum and the like
  • a genomic or cDNA (typically fragmented and separated on an electrophoretic gel) is hybridized to a probe specific for the target region.
  • a probe specific for the target region e.g., 6p22
  • a control non amplified
  • the cDNA sequences disclosed here can also be used for detecting mutations (e.g., substitutions, insertions, and deletions) within the corresponding endogenous genes.
  • mutations e.g., substitutions, insertions, and deletions
  • nucleic acid hybridization techniques generally described above can be adapted to detect such much mutations.
  • oligonucleotide probes that distinguish between mutant and wild-type forms of the target gene can be used in standard hybridization assays.
  • amplification e.g., using PCR
  • kits Containing 6p22 Amplicon Probes This invention also provides diagnostic kits for the of chromosomal abnormalities at 6p22.
  • the kits include vim, or more probes to the 6p22 amplicon described herein.
  • the kits can additionaliy'i ⁇ e blocking probes, instructional materials describing how to use the kit contents in;'* detecting 6p22 amplicons.
  • kits may also include one or more of the following: vasr ⁇ salabels or labeling agents to facilitate the detection of the probes, reagents for the hybri Bjr ⁇ fen including buffers, a metaphase spread, bovine serum albumin (BSA) and otherl ⁇ se ing agents, sampling devices including fine needles, swabs, aspirators and the like, stle, and negative hybridization controls and so forth.
  • vasr ⁇ salabels or labeling agents to facilitate the detection of the probes
  • reagents for the hybri Bjr ⁇ fen including buffers, a metaphase spread, bovine serum albumin (BSA) and otherl ⁇ se ing agents
  • sampling devices including fine needles, swabs, aspirators and the like, stle, and negative hybridization controls and so forth.
  • cDNA clones One may express the desired ⁇ >tides encoded by the cDNA clones disclosed here in a recombinantly engineered -Y l such as bacteria, yeast, insect (especially employing baculoviral vectors), and mammalian cells. It is expected that those of skill in the art are knowledgeable in the numerous. expression systems available for expression of the cDNAs. No attempt to describe in detail the various methods known for the expression of proteins in prokaryotes or eukaryotes will be made.
  • the expression of natural or synthetic nucleic acids 5 encoding polypetides of the invention will typically be achieved by operably linking the DNA or cDNA to a promoter (which is either constitutive or inducible), followed by incorporation into an expression vector.
  • the vectors can be suitable for replication and integration in either prokaryotes or eukaryotes.
  • Typical expression vectors contain transcription and translation terminators, initiation sequences, and promoters 0 useful for regulation of the expression of the DNA encoding the polypeptides.
  • expression plasmids which contain, at the minimum, a strong promoter to direct transcription, a ribosome binding site for translational initiation, and a transcription/translation terminator.
  • E. coli Examples of regulatory regions suitable for this purpose in E. coli are the promoter and operator region of the E. coli tryptophan biosynthetic pathway as described by Yanofsky, O, J. Bacteriol., 158:1018-1024 (1984) and the leftward promoter of phage lambda (P L ) as described by Herskowitz, I. and Hagen, D., Ann. Rev. Genet, 14:399-445 (1980). The inclusion of selection markers in DNA vectors
  • E. coli 20 transformed in E. coli is also useful.
  • markers include genes specifying resistance to ampicillin, tetracycline, or chlor ⁇ mphenicol.
  • Expression systems are available using E. coli, Bacillus sp. Palva, I., & (*L, Gene, 22:229-235 (1983); Mosbach, K., et al., Nature, 302:543-545 and Salmoneii$sR.,coli systems are preferred.
  • the polypeptides produced by prokaryote cells may not necessarily 1 properly.
  • the expressed polypeptides may first be dei ⁇ fteif.and then renatured. This can be accomplished by solibilizing the bacterialiy ⁇ tf ⁇ ced proteins in a chaotropic agent such as guanidine HCI and reducing all the cyst ac&residues with a reducing agent such as beta-
  • yeast expression plasmids like YEp6, YEp13, YEp4 can be used as vectors.
  • a gene of interest can be fused to any of the promoters in various yeast vectors.
  • the above-mentioned plasmids have been fully described in the literature (Botstein, et al., Gene, 8:17-24 (1979); Broach, et al., Gene, 8:121-133 (1979)).
  • Illustrative of cell cultures useful for the production of the polypeptides are cells of insect or mammalian origin. Mammalian cell systems often will be in the form of monolayers of cells although mammalian cell suspensions may also be used. Illustrative examples of mammalian cell lines include VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, W138, BHK, Cos-7 or MDCK cell lines.
  • the vector e. g., a plasmid, which is used to transform the host cell, preferably contains DH/ sequences to initiate transcription and sequences to control the translation of gene sequence. These sequences are referred to as expression
  • ca ⁇ rol sequences are often obtained from the SV-40 promoter (Science, 222:524-52?;?/? 983), the CMV I.E. Promoter (Proc. Natl. Acad. Sci., 81:659-663 (1984)) or the meWsv hionein promoter (Nature, 296:39-42 (1982).
  • the cloning vector containing the expressfes control sequences is cleaved using restriction enzymes and adjusted in size as neos ssy or desirable and ligated with the desired DNA by means well known in the art.
  • ion terminator sequences from known mammalian genes need to be incorporated iiif ⁇ ihe vector.
  • An example of a terminator sequence is the polyadenlyation sequescelrom the bovine growth hormone gene. Sequences for accurate splicing of the transcript may also be included.
  • An example of a splicing sequence is the VP1 intron from SV40 (Sprague, J. et al., J. Virol., 45: 773-781 (1983)).
  • gene sequences to control replication in the host cell may be incorporated into the vector such as those found in bovine papilloma virus type- vectors.
  • nucleic acids and encoded polypeptides of the invention can be used directedly to inhibit the endogenous genes or their gene products.
  • inhibitory nucleic acids may be used to specifically bind to a complementary nucleic acid sequence. By binding to the appropriate target sequence, an RNA-RNA, a DNA-DNA, or RNA-DNA duplex is formed. These nucleic acids are often termed
  • inhibitors because they are usually complementary to the sense or coding strand of the gene, although approaches for use of “sense” nucleic acids have also been developed.
  • inhibitory nucleic acids refers to both “sense” and “antisense” nucleic acids. Inhibitory nucleic acid methods encompass a number of different approaches to altering expression of specific genes that operate by different mechanisms.
  • inhibitory nucleic acid therapy approaches can be classified into those that target DNA sequences, those that target RNA sequences (including pre- mRNA and mRNA), those that target proteins (sense strand approaches), and those that cause cleavage or chemical modification of the target nucleic acids (ribozymes).
  • These different types of inhibitory nucleic acid ; ';?chnology are described, for instance, in Helene, C. and Toulme, J., Biochim. Biophys. ⁇ - ⁇ .1049: 99-125 (1990).
  • Inhibitory nucleic acid complementary to regions of c-my ⁇ vraRNA has been shown to inhibit c-myc protein expression in a human promyelocytic leufe&ir ⁇ .
  • the encoded polypeptides of the invention can also be used to design molecu! ⁇ , « ⁇ a ⁇ tidic or nonpeptidic) that inhibit the endogenous proteins by, for instance, between the protein and a second molecule specifically recognized toy the protein.
  • Methods for designing such molecules are well known to those skilled in.the art.
  • polypeptides can be designed which have sequence identity with the encoded proteins or may comprise modifications (conservative or non- conservative) of the sequences. The modifications can be selected, for example, to alter their in vivo stability. For instance, inclusion of one or more D-amino acids in the peptide typically increases stability, particularly if the D-amino acid residues are substituted at one or both termini of the peptide sequence.
  • polypeptides can also be modified by linkage to other molecules.
  • different N- or C-terminal groups may be introduced to alter the molecule's physical and/or chemical properties. Such alterations may be utilized to affect, for example, adhesion, stability, bio-availability, localization or detection of the molecules.
  • a wide variety of labels may be linked to the terminus, which may provide, directly or indirectly, a detectable signal.
  • the polypeptides may be modified in a variety of ways for a variety of end purposes while still retaining biological activity.
  • bioactive agent or “drug candidate” or grammatical equivalents as used herein describes any molecule, e.g., protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, etc., to be tested for bioactive agents that are capabf ⁇ " ⁇ sf directly or indirectly altering the cancer phenotype or the expression of an expressed' rer ⁇ leic acid sequence, positioned with the 6p22 region, identified herein, including both r - c acid sequences and protein sequences.
  • the bioactive ge ts modulate the expression profiles, or expression profile nucleic acids or proteins ovided herein.
  • the candidate agent su i&sw&es a cancer phenotype, for example to a normal tissue fingerprint.
  • the ctsfalisiate agent preferably suppresses a severe cancer phenotype.
  • a plurality /tessay mixtures are run in parallel with different agent concentrations to obtain a differen ife'iresponse to the various concentrations.
  • one of these concentrations serves a&S t negative control, i.e., at zero concentration or below the level of detection.
  • a candidate agent will neutralize the effect of E2F3, BIG-1 or BIG- .
  • neutralize is meant that activity of a protein is either inhibited or counter acte ⁇ g ⁇ lnst so as to have substantially no effect on a cell.
  • Candidate agents encompass numerous chemical classes, though typically they are organic molecules; preferably small organic compounds having a molecular weight of more than 100 and less than about 2,500 daltons. Preferred small molecules are less than 2000, or less than 1500 or less than 1000 or less than 500 D.
  • Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups.
  • the candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Particularly preferred are peptides.
  • Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and ⁇ biochemical means. Known pharmacological agents may be subjected to directed or rartAom chemical modifications, such as acylation, alkylation, esterification, amidification to roduce structural analogs.
  • lifecandidate bioactive agents are proteins.
  • protein herein is meant at least two c ⁇ ws"l»& .attached amino acids, which includes proteins, polypeptides, oligopeptides and peptidfe;>j-The protein may be made up of naturally occurring amino acids and peptide bonds, structures.
  • amino acid or “peptide residue”, as both naturally occurring and synthetic amino acids.
  • homo-p e ⁇ 's ⁇ i'ne, citrulline and noreleucine are considered amino acids for the purposes of the invention.
  • Amino acid also includes imino acid residues such as proline and The side chains may be in either the (R) or the (S) configuration.
  • the amino acids are in the (S) or L-configuration. If non- naturally occurring side chains are used, non-amino acid substituents may be used, for example to or retard in vivo degradations.
  • the candidate bioactive agents are naturally occurring proteins or fragments of naturally occurring proteins. Thus, for example, cellular extracts containing proteins, or random or directed digests of proteinaceous cellular extracts, may be used. In this way libraries of procaryotic and eucaryotic proteins may be made for screening in the methods of the invention. Particularly preferred in this embodiment are libraries of bacterial, fungal, viral, and mammalian proteins, with the latter being preferred, and human proteins being especially preferred.
  • the candidate bioactive agents are peptides of from about 5 to about 30 amino acids, with from about 5 to about 20 amino acids being preferred, and from about 7 to about 15 being particularly preferred.
  • the peptides may be digests of naturally occurring proteins as is outlined above, random peptides, or "biased” random peptides.
  • randomized or grammatical equivalents herein is meant that each nucleic acid and peptide consists of essentially random nucleotides and amino acids, respectively. Since generally these random peptides (or nucleic acids, discussed below) are chemically synthesized, they may incorporate any nucleotide or amino acid at any position.
  • the synthetic process can be designed to generate randomized proteins or nucleic acids, to allow the formation of all or most of the possible combinations over the length of the sequence, thus forming a library of randomized candidate bioactive proteinaceous agents.
  • the library is fully randomized, with no sequence preferences or constants at an ⁇ position.
  • the library is biased. That is, some positions wif s-the sequence are either held constant, or are selected from a limited number of possi&iifes.
  • the nucleotides or amino acid res Sr ⁇ are randomized within a defined class, for example, of hydrophobic amino acids, hydr ⁇ p?iS ⁇ s;asidues, sterically biased (either small or large) residues, towards the creation of domains, the creation of cysteines, for cross-linking, prolines for SH-3 domains, &s x@s.,.
  • the candidate bioactive agents are nucleic acids, a&xfepianed above.
  • nucleic acid candidate bioactive agents may be naturally occurring nucleic acids, random nucleic acids, or "biased" random nucleic acids.
  • digests of procaryotic or eucaryotic genomes may be used as is outlined above for proteins.
  • the candidate bioactive agents are organic chemical moieties, a wide variety of which are available in the literature.
  • the sample containing the target sequences to be analyzed is added to the biochip.
  • the target sequence is prepared using known techniques.
  • the sample may be treated to lyse the cells, using known lysis buffers, electroporation, etc., with purification and/or amplification such as PCR occurring as needed, as will be appreciated by those in the art.
  • an in vitro transcription with labels covalently attached to the nucleosides is done.
  • the nucleic acids are labeled with biotin-FITC or PE, or with cy3 or cy5.
  • the target sequence is labeled with, for example, a fluorescent, a chemiluminescent, a chemical, or a radioactive signal, to provide a means of detecting the target sequence's specific binding to a probe.
  • the label also can be an enzyme, such as, alkaline phosphatase or horseradish peroxidase, which wten provided with an appropriate substrate produces a product that can be detected.
  • the label can be a labeled compound or small molecule, such as an enzyme inh ⁇ ESfe, that binds but is not catalyzed or altered by the enzyme.
  • the label also can be a mofefe or compound, such as, an epitope tag or biotin which specifically binds to streptavidin.
  • a mofefe or compound such as, an epitope tag or biotin which specifically binds to streptavidin.
  • the streptavidin is labeled as described above, thereby, arting a detectable signal for the bound target sequence. As known in the art, unbound is removed prior to analysis.
  • these assays can be direct hybridization assays or can comprise which include the use of multiple probes, as is generally outlined in U.S. Pat. N6s?.5i.6&1 ,702; 5,597,909;
  • nucleic acid is prepared as outlined above, and then added to the biochip comprising?; a plurality of nucleic acid probes, under conditions that allow the formation of a hybridization complex.
  • a variety of hybridization conditions may be used in the present invention, including high, moderate and low stringency conditions as outlined above.
  • the assays are generally run under stringency conditions which allows formation of the label probe hybridization complex only in the presence of target. Stringency can be controlled by altering a step parameter that is a thermodynamic variable, including, but not limited to, temperature, formamide concentration, salt concentration, chaotropic salt concentration pH, organic solvent concentration, etc.
  • reaction may include a variety of other reagents may be included in the assays. These include reagents like salts, buffers, neutral proteins, e.g. albumin, detergents, etc which may be used to facilitate optimal hybridization and detection, and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease r ⁇ hibitors, anti-microbial agents, etc., may be used, depending on the sample preparation methods and purity of the target.
  • the screens are don& ⁇ i ⁇ anitfy drugs or bioactive agents that modulate the cancer phenotype.
  • a preferred embodiment is in the screenuig- feandidate agents that can induce or suppress a particular expression profile, thus pre1fer:fe ; generating the associated phenotype. That is, candidate agents that can mimic or poiss ®. an expression profile in, for example, bladder cancer similar to the expression ⁇ r ⁇ ffe:. of normal bladder tissue is expected to result in a suppression of the bladder cantsjsiptienotype.
  • mimicking an expression profile, or changing one prc ⁇ teio another is the goal.
  • screens can be run to alter the expression of the genes individually. That is, screening for modulation of regulation of expression of a single gene can be done; that is, rather than try to mimic all or part of an expression profile, screening for regulation of individual genes can be done. Thus, for example, particularly in the case of target genes whose presence or absence is unique between two states, screening is done for modulators of the target gene expression.
  • screening is done to alter the biological function of the expression product of the expressed gene. Again, having identified the importance of a gene in a particular state, screening for agents that bind and/or modulate the biological activity of the gene product can be run as is more fully outlined below.
  • screening of candidate agents that modulate the cancer phenotype either at the gene expression level or the protein level can be done.
  • screens can be done for novel genes that are induced in response to a candidate agent.
  • a screen as described above can be performed to ; ientify genes that are specifically modulated in response to the agent. Comparing expansion profiles between normal tissue and agent treated cancer tissue reveals genes frigee not expressed in normal tissue or cancer tissue, but are expressed in agent treate ⁇ ?sue.
  • agent specific sequences can be identified and used by any of the method& «fcts.cribed herein for expressed genes or proteins. In particular these sequences and they encode find use in marking or identifying agent treated cells.
  • antibeeS . can be raised against the agent induced proteins and used to target novel therapeutics fee.treated cancer tissue sample.
  • a candidate a- .vis.administered to a population of bladder cancer cells that thus has an associated biads';3P, ⁇ cancer expression profile.
  • administration or “contacting” herein is meant that the canari. agent is added to the cells in such a manner as to allow the agent to act upon the ceil; ⁇ shether by uptake and intracellular action, or by action at the cell surface.
  • nucleic acid encoding a proteinaceous candidate agent i.e. a pepfeie
  • the cells can be washed if desired and are allowed to incubate under preferably physiological conditions for some period of time.
  • the cells are then harvested and a new gene expression profile is generated, as outlined herein.
  • bladder cancer tissue may be screened for agents that reduce or suppress the bladder cancer phenotype.
  • a change in at least one gene of the expression profile indicates that the agent has an effect on bladder cancer activity.
  • screens may be done on individual genes and gene products (proteins). That is, having identified a particular expressed gene as important in a particular state, screening of modulators of either the expression of the gene or the gene product itself can be done.
  • the gene products of expressed genes are sometimes referred to herein as " expressed proteins” or “cancer modulating proteins.” Additionally, “modulator” and “modulating” proteins are sometimes used interchangeably herein.
  • the t ⁇ s ressed protein is termed E2F3, BIG-1 or BIG-2. These sequences can be iderti ⁇ s described herein for expressed sequences (SEQs).
  • the expressed protein may fSfta.fragment, or alternatively, be the full length protein to the fragment shown herein.
  • the expressed protein is a fragment.
  • the amino a : ⁇ &squence which is used to determine sequence identity or similarity is that identified as S ⁇ CF ⁇ O: 2.
  • the sequences are naturally occurring alleli ' C'vsx3 s&of a protein having the sequence identified as SEQ ID NO: 2.
  • the expressed protein is a fragment of approxiYftft y 14 to 24 amino acids long. More preferably the fragment is a soluble fragment.
  • the fragment includes a non-transmembrane region.
  • the fragment has an N-terminal Cys to aid in solubility.
  • the C- terminus of the fragment is kept as a free acid and the n-terminus is a free amine to aid in coupling, i.e., to cysteine.
  • the fragment of approximately 14 to 24 amino acids long. More preferably the fragment is a soluble fragment.
  • a E2F3, BIG-1 or BIG-2 fragment has at least one E2F3, BIG-1 or BIG- 2 bioactivity as defined below.
  • expressed proteins are conjugated to an immunogenic agent as discussed herein. In one embodiment the expressed protein is conjugated to BSA.
  • screening for modulators of expression of specific genes can be done. This will be done as outlined above, but in general the expression of only one or a few genes are evaluated.
  • screens are designed to first find candidate agents that can bind to expressed proteins, and then these agents may be used in assays that evaluate the ability of the candidate agent to modulate expressed activity.
  • assays there are a number of different assays which may be run; binding assays and activity assays.
  • binding assays are done.
  • purified or isolated gene product is used; that is, the gene products of one or more expssssed nucleic acids are made. In general, this is done as is known in the art.
  • antibodies are generated to the protein gene products, and standard immunoassays are run to determine the amount of protein present.
  • cells comprising ti'tesxpressed proteins can be used in the assays.
  • the methods comprise combining a expressed prolfet ⁇ r ⁇ EKlla candidate bioactive agent, and determining the binding of the candidate agent to th : passed protein.
  • Preferred embodiments utilize the human expressed protein, although proteins may also be used, for example for the development of animal modete ⁇ >P'hsman disease.
  • variant or derivative expressc :,r ⁇ &teins may be used.
  • the expressed protein or the candidate agent is non-diffusably bound to an insoluble support having isolated sample receiving areas (e.g. a microtiter plate, an array, eit .
  • isolated sample receiving areas e.g. a microtiter plate, an array, eit .
  • soluble assays known in the art may be performed.
  • the insoluble supports may be made of any composition to which the compositions can be bound, is readily separated from soluble material, and is otherwise compatible with the overall method of screening.
  • the surface of such supports may be solid or porous and of any convenient shape. Examples of suitable insoluble supports include microtiter plates, arrays, membranes and beads.
  • Microtiter plates and arrays are especially convenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples.
  • the particular manner of binding of the composition is not crucial so long as it is compatible with the reagents and overall methods of the invention, maintains the activity of the composition and is nondiffusable.
  • Preferred methods of binding include the use of antibodies (which do not sterically block either the ligand binding site or activation sequence when the protein is bound to the support), direct binding to "sticky" or ionic supports, chemical cross-linking, the synthesis of the protein or agent on the surface, etc. Following binding of the protein or agent, excess unbound material is removed by washing. The sample receiving areas may then be blocked through incubation with bovine serum albumin (BSA), casein or other innocuous protein or other moiety.
  • BSA bovine serum albumin
  • the expressed protein is bound to the support, and a candidate bioactive agent is added to the assay.
  • the. candidate agent is bound to the support and the expressed protein is added.
  • Novel biding agents include specific antibodies, non-natural binding agents identified in screens of chemical libraries, peptide analogs, etc.
  • screening assays feagents that have a low toxicity for human cells.
  • assays may be usedffethis purpose, including labeled in vitro protein-protein binding assays, shift assays, immunoassays for protein binding, functional assays assays, etc.) and the like.
  • the determinatio ⁇ &f «l'?'® -binding of the candidate bioactive agent to the expressed protein may be done in p. ,rr ⁇ m.ber of ways.
  • the candidate bioactive agent is labelled; a-rt ⁇ fr ⁇ ding determined directly. For example, this may be done by attaching all or a pa#fm.of the expressed protein to a solid support, adding a labelled candidate agent (for exam le, a fluorescent label), washing off excess reagent, and determining whether the la ⁇ &Ha present on the solid support.
  • a labelled candidate agent for exam le, a fluorescent label
  • washing off excess reagent for exam le, a fluorescent label
  • Various blocking and washing steps may be utilized as ⁇ s known in the art.
  • label herein is meant that the compound is either directly or indirectly labeled with a label which provides a detectable signal, e.g. radioisotope, fluorescers, enzyme, antibodies, particles such as magnetic particles, chemiluminescers, or specific binding molecules, etc.
  • Specific binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin, etc.
  • the complementary member would normally be labeled with a molecule which provides for detection, in accordance with known procedures, as outlined above.
  • the label can directly or indirectly provide a detectable signal.
  • the proteins may be labeled at tyrosine positions using 125 1, or with fluorophores.
  • more than one component may be labeled with different labels; using 125 1 for the proteins, for example, and a fluorophor for the candidate agents.
  • the binding of the candidate bioactive agent is determined through the use of competitive binding assays.
  • the competitor is a binding moiety known to bind to the target molecule (i.e. bladder cancer), such as an antibody, peptide, binding partner, ligand, etc. Under certain circumstances, there may be competitive binding as between the bioactive agent and the binding moiety, with the binding moiety displacing the bioactive agent.
  • the candidate bioactive agent is labeled. Either the? candidate bioactive agent, or the competitor, or both, is added first to the protein fosr ⁇ i time sufficient to allow binding, if present. Incubations may be performed at any tt €W ⁇ jeirature which facilitates optimal activity, typically between 4 and 40° C.
  • Incubai ⁇ s ⁇ riods are selected for optimum activity, but may also be optimized to facilitate put screening. Typically between 0.1 and 1 hour will be sufficient. Excess reagsefe is. generally removed or washed away. The second component is then added, an&ii.fe ⁇ resence or absence of the labeled component is followed, to indicate binding.
  • the compefe ⁇ is added first, followed by the candidate bioactive agent.
  • either component can be labeled.
  • the presence of label in the wash solution indicates displacement by the agent.
  • the candidate bioactive agent is labeled
  • the presence of the label on the support indicates displacement.
  • the candidate bioactive agent is added first, with incubation and washing, followed by the competitor.
  • the absence of binding by the competitor may indicate that the bioactive agent is bound to the expressed protein with a higher affinity.
  • the methods comprise differential screening to identity bioactive agents that are capable of modulating the activity of the expressed proteins.
  • the methods comprise combining a expressed protein and a competitor in a first sample.
  • a second sample comprises a candidate bioactive agent, a expressed protein and a competitor.
  • the binding of the competitor is determined for both samples, and a change, or difference in binding between the two samples indicates the presence of an agent capable of binding to the expressed protein and potentially modulating its activity. That is, if the binding of the competitor is different in the second sample relative to the first sample, the agent is capable of binding to the expressed protein.
  • a preferred embodiment utilizes differential screening- ( ⁇ identify drug candidates that bind to the native expressed protein, but cannot bind to rrtc ⁇ fied expressed proteins.
  • the structure of the expressed protein may be modeled, and used in rational drug design to synthesize agents that interact with that site.
  • Drug c t' ⁇ rte es that affect bladder cancer bioactivity are also identified by screening drugs for ⁇ ?f ⁇ ility to either enhance or reduce the activity of the protein.
  • Positive negative controls may be used in the assays.
  • Preferably all control and ifefK-amples are performed in at least triplicate to obtain statistically significant results. of all samples is for a time sufficient for the binding of the agent to the protein.
  • Folf ⁇ Nivkss ieubation all samples are washed free of non-specifically bound material and the anvs ⁇ t.of bound, generally labeled agent determined. For example, where a radiolabel is employed, the samples may be counted in a scintillation counter to determine the amou ⁇ i ⁇ f bound compound.
  • a variety of other reagents may be included in the screening assays. These include reagents like salts, neutral proteins, e.g.
  • albumin may be used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions.
  • reagents that otherwise improve the efficiency of the assay such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may be used.
  • the mixture of components may be added in any order that provides for the requisite binding.
  • methods for screening for a bioactive agent capable of modulating the activity of expressed proteins comprise the steps of adding a candidate bioactive agent to a sample of expressed proteins, as above, and determining an alteration in the biological activity of expressed proteins.
  • “Modulating the activity” of bladder cancer includes an increase in activity, a decrease in activity, or a change in the type or kind of activity present.
  • the candidate agent should both bind to cancer proteins (although this may not be necessary), and alter its biological or biochemical activity as defined herein.
  • the methods include both in vitro screening methods, as are generally outlined above, and in vivo screening of cells for alterations in the presence, distribution, activity or amount of expressed proteins.
  • the methods comprise combining a bladder - smcer sample and a candidate bioactive agent, and evaluating the effect on bladder carfesa activity, respectively.
  • cancer activity or grammatical equivalents herein is meant at least one of cancer's biological activities, including, but not limited to, cell division, ppvferably in bladder tissue, cell proliferation, tumor growth, and transformation of cells.
  • cancer activity includes activation of E2F3, BIG-1 , BIG-2 or a by E2F3, BIG-1 or BIG-2.
  • An inhibitor of cancer activity is an agent which inhibits &w f- -i .-.or more cancer activities.
  • the activity of the e' jr ⁇ assed protein is increased; in another preferred embodiment, the activity of the expressecl. rotein is decreased.
  • bioactive agents that are antagonists are preferred in some embodiments, and bioactive agents that are agonists may be preferred in other embodiments.
  • the invention provides methods for screening for bioactive agents capable of modulating the activity of a expressed protein.
  • the methods comprise adding a candidate bioactive agent, as defined above, to a cell comprising expressed proteins.
  • Preferred cell types include almost any cell.
  • the cells contain a recombinant nucleic acid that encodes a expressed protein.
  • a library of candidate agents are tested on a plurality of cells.
  • the assays are evaluated in the presence or absence or previous or subsequent exposure of physiological signals, for example hormones, antibodies, peptides, antigens, cytokines, growth factors, action potentials, pharmacological agents including chemotherapeutics, radiation, carcinogenics, or other cells (i.e. cell-cell contacts).
  • physiological signals for example hormones, antibodies, peptides, antigens, cytokines, growth factors, action potentials, pharmacological agents including chemotherapeutics, radiation, carcinogenics, or other cells (i.e. cell-cell contacts).
  • the determinations are determined at different stages of the cell cycle process.
  • GS-BIG protein activity includes at least one of the following: cancer activity, binding to GS-BIG protein, activation of GS-BIG protein or activation of substrates of GS-BIG protein by GS-BIG protein.
  • An inhibitor of GS- BIG protein inhibits at least one of GS-BIG protein's bioactivities.
  • a method of inhibiting bladder cancer cell division comprises administration of a bladder cancer inhibitor.
  • a method of inhibiting tumor growth comprises administration of a bladder cancer inhibitor.
  • the inhibitor is an inhibitor of one or more of the GS-BIG protein.
  • a ⁇ 'i r . embodiment methods of treating cells or individuals with cancer are provided.
  • th ⁇ ii ⁇ o is an inhibitor of GS-BIG6.
  • a expresst ⁇ ⁇ jiwfein inhibitor is an antibody as discussed above.
  • the inhibitor ⁇ y"& ⁇ vpjaiis,ense molecule.
  • Antisense molecules as used herein include antisense or sense comprising a singe-stranded nucleic acid sequence (either RNA or DNA) ca ⁇ ⁇ of binding to target mRNA (sense) or DNA (antisense) sequences for expressed m ⁇ tesules.
  • a preferred antisense molecule is for one or more of the GS-BIG proteins o f ⁇ sa: ligand or activator thereof.
  • Antisense or sense oligonucleotides comprise a fragment generally at least about 14 nucleotides, preferably from about 14 to 30 nucleotides.
  • the ability to derive an antisense or a sense oligonucleotide, based upon a cDNA sequence encoding a given protein is described in, for example, Stein and Cohen, Cancer Res., 48: 2659 (1988) and van der Krol, et al., BioTechniques, 6: 958 (1988).
  • Antisense molecules may be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, as described in WO 91/04753.
  • Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors.
  • conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell.
  • a sense or an antisense oligonucleotide may be introduced into a cell containing the target nucleic acid sequence by formation of an oligonucleotide-lipid complex, as described in WO 90/10448. It is understood that the use of antisense molecules or knock out and knock in models may also be used in screening assays as discussed above, in addition to methods of treatment.
  • the compounds having the desired pharmacological activity may be administered in a physiologically acceptable carrier to a host, as previously described.
  • the agents may be administered in a variety of ways, orally, parenterally e.g., cf ⁇ cutaneously, intraperitoneally, intravascularly, etc.
  • the compounds may be formulated in a variety of ways.
  • concentration? Sfeerapeutically active compound in the formulation may vary from about 0.1-100 wt. 7c/: !: T ⁇ agents may be administered alone or in combination with other treatments, i.e., ratif ⁇ t ⁇ ifrrav
  • the pharmaceutical composiiK'jvs.f(T ; ??;fha prepared in various forms, such as granules, tablets, pills, suppositories, capsules; ⁇ w ⁇ ssions, salves, lotions and the like.
  • Pharmaceutical grade organic or inorganic carriei & &4fm diluents suitable for oral and topical use can be used to make up compositions c ⁇ i'itM- «M ⁇ g..the therapeutically-active compounds.
  • Diluents known to the art include as ssus media, vegetable and animal oils and fats. Stabilizing agents, wetting and emulsify ⁇ ,;.
  • the invention provides methods for identifying cells containing variant cancer genes comprising determining all or part of the sequence of at least one endogeneous cancer gene in a cell. As will be appreciated by those in the art, this may be done using any number of sequencing techniques.
  • the invention provides methods of identifying the cancer genotype of an individual comprising determining all or part of the sequence of at least one cancer gene of the individual. This is generally done in at least one tissue of the individual, and may include the evaluation of a number of tissues or different samples of the same tissue. The method may include comparing the sequence of the sequenced gene to a known gene, i.e. a wild- type gene.
  • the sequence of all or part of the expressed gene can then be compared to the sequence of a known expressed gene to determine if any differences exist. This can be done using any number of known homology programs, such as Bestfit, etc.
  • the presence of a difference in the sequence between the expressed gene of the patient and the known expressed gene is indicative of a d ⁇ ss se state or a propensity for a disease state, as outlined herein.
  • the expressed genes are used as probes to determine the*: umber of copies of the expressed gene in the genome.
  • expressed genes are used as probed to determine the chromosome? tr ⁇ Jization of the expressed genes.
  • Information such as chromosomal localization finds - uGiin.providing a diagnosis or prognosis in particular when chromosomal abnormaH ' tfes:* ⁇ h, as translocations, and the like are identified in expressed gene loci.
  • methods of modulating in cells or organisms are provided.
  • the methods to a cell an antibody that reduces or eliminates the biological activity of an expressed protein.
  • the methods comprise administering to a cell or organism a recombinant nucleic acid encoding a expressed protein.
  • this may be accomplished in any number of ways.
  • the activity of the expressed gene is increased by increasing the amount in the cell, for example by overexpressing the endogenous protein or by administering a gene encoding the sequence, using known gene-therapy techniques, for example.
  • the gene therapy techniques include the 5 incorporation of the exogenous gene using enhanced homologous recombination (EHR).
  • EHR enhanced homologous recombination
  • the activity of the endogeneous gene is decreased, for example by the administration of an inhibitor of cancer, such as an antisense nucleic acid.
  • the expressed proteins of the present invention may be 0 used to generate polyclonal and monoclonal antibodies to expressed proteins, which are useful as described herein.
  • the expressed proteins can be coupled, using standard technology, to affinity chromatography columns. These columns may then be used to purify expressed antibodies.
  • the antibodies are generated to epitopes unique to a expressed protein; that is, the 5 antibodies show little or no cross-reactivity to other proteins. These antibodies find use in a number of applications.
  • the expressed antibodies may be coupled to standard affinity chromatography columns and used to purify expressed proteins.
  • the antibodies may also be used as blocking polypeptides, as outlined above, since th ⁇ v s will specifically bind to the expressed protein.
  • a therapeutically effective dose of a expressed or modulator thereof is admuvlatered to a patient.
  • therapeutically effective dose herein is meant a dose that profe s. the effects for which it is administered. The exact dose will depend on the purposc ⁇ the treatment, and will be ascertainable by one skilled in the art using known techniques?. h , ⁇ s known in the art, adjustments for 5 degradation, systemic versus localized delivery, art S ⁇ feof new protease synthesis, as well as the age, body weight, general health, sex, diet;' ⁇ IttwKiaf. administration, drug interaction and the severity of the condition may be necessary, ar ⁇ te:4il:be ascertainable with routine experimentation by those skilled in the art.
  • a "patient” for the purposes of the present invention includes both humari&x 3.0:. and other animals, particularly mammals, and organisms. Thus the methods are amicable to both human therapy and veterinary applications.
  • the patient is a mammal, and in the most preferred embodiment the patient is hurr ⁇ n.
  • the administration of the expressed proteins and modulators of the present invention can be done in a variety of ways as discussed above, including, but not limited to, orally, subcutaneously, intravenously, intranasally, transdermally, intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally, or intraocularly.
  • the expressed proteins and modulators may be directly applied as a solution or spray.
  • compositions of the present invention comprise a expressed protein in a form suitable for administration to a patient.
  • the pharmaceutical compositions are in a water soluble form, such as being present as pharmaceutically acceptable salts, which is meant to include both acid and base addition salts.
  • “Pharmaceutically acceptable acid addition salt” refers to those salts that retain the biological effectiveness of the free bases and that are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like
  • organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid,
  • “PharmaceufBa acceptable base addition salts” include those derived from inorganic bases suti?..as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, t?j>per, manganese, aluminum salts and the like. Particularly preferred are the ammoniuh' potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceut ⁇ c ⁇ jiPcceptable organic non-toxic bases include salts of primary, secondary, and tertiary amifrw,..
  • substituted amines including naturally occurring substituted amines, cyclic amines a'tv ⁇ i- ⁇ ic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethy »? « ⁇ , ⁇ tripropylamine, and ethanolamine.
  • compositions may also include one or mo c the following: carrier proteins such as serum albumin; buffers; fillers such as ,,; microcrystalline cellulose, lactose, corn and other starches; binding agents; sweeteners and other flavoring agents; coloring agents; and polyethylene glycol.
  • carrier proteins such as serum albumin
  • buffers such as ,,; microcrystalline cellulose, lactose, corn and other starches
  • binding agents such as ,,; microcrystalline cellulose, lactose, corn and other starches
  • binding agents such as ,,; microcrystalline cellulose, lactose, corn and other starches
  • binding agents such as ,,; microcrystalline cellulose, lactose, corn and other starches
  • sweeteners and other flavoring agents such as , lactose, corn and other starches
  • binding agents such as ,, and microcrystalline cellulose, lactose, corn and other starches
  • sweeteners and other flavoring agents such as , lactose, corn and other starches
  • expressed proteins and modulators are administered as therapeutic agents, and can be formulated as outlined above.
  • expressed genes (including both the full-length sequence, partial sequences, or regulatory sequences of the expressed coding regions) can be administered in gene therapy applications, as is known in the art. These expressed genes can include antisense applications, either as gene therapy (i.e. for incorporation into the genome) or as antisense compositions, as will be appreciated by those in the art.
  • expressed genes are administered as DNA vaccines, either single genes or combinations of expressed genes. Naked DNA vaccines are generally known in the art. Brower, Nature Biotechnology, 16:1304- 1305 (1998).
  • expressed genes of the present invention are used as DNA vaccines.
  • Methods for the use of genes as DNA vaccines are well known to one of ordinary skill in the art, and include placing a expressed gene or portion of a expressed gene under the control of a promoter for expression in a patient with bladder cancer.
  • the expressed gene used for DNA vaccines can encode full-length expressed proteins, but more preferably encodes portions of the expressed proteins including peptides derived from the expressed protein.
  • a patient is. immunized with a DNA vaccine comprising a plurality of nucleotide from a expressed gene.
  • the [D?& ⁇ «accines include a gene encoding an adjuvant molecule with the DNA vaccine.
  • SucH"fr j.wyant molecules include cytokines that increase the immunogenic response to the encoded by the DNA vaccine. Additional or alternative adjuvants are knoes'teibose of ordinary skill in the art and find use in the invention.
  • expressed genes find use in generatit ⁇ t;$ animal models of cancer.
  • gene.vtfo-srapy technology wherein antisense RNA directed to the cancer gene will also diminish' or repress expression of the gene.
  • An animal generated as such serves as an animal model of cancer that finds use in screening bioactive drug candidates.
  • gene knockout technology for example as a result of homologous recombination with an appropriate gene targeting vector, will result in the absence of the cancer protein.
  • tissue-specific expression or knockout of the cancer protein may be necessary.
  • transgenic animals can be generated that overexpress the expressed protein.
  • promoters of various strengths can be employed to express the transgene.
  • the number of copies of the integrated transgene can be determined and compared for a determination of the expression level of the transgene. Animals generated by such methods find use as animal models of expressed and are additionally useful in screening for bioactive molecules to treat disorders related to the expressed protein.
  • & -ks? ⁇ les herein concern human cells and the language is primarily directed to human aw' ⁇ sicns, the concept of this invention is applicable to genomes from any plant or anima ⁇ . ' rV ⁇ g nomes compared need only be related closely enough to have sufficient substa ⁇ ft& ⁇ 't ⁇ atical sequences for a meaningful analysis. For example, a human genome and primate could be compared according to the methods of this invention.
  • Stage pT1 was defined by presence of both unequivocal tumor invasion of the suburothelial stroma and tumor-free fragments of the muscular bladder wall. Carcinomas with stroma invasion but absence of muscular bladder wall in the biopsy were classified as at least pT1 (pT1-). Clinical data were available from 1123 patients. The medium follow-up period was 42 months (range 1 - 236 months). Time to recurrence and time to progression (to stage pT2 or higher) were selected as study endpoints for patients with pTa and pT1 tumors.
  • tissue microarray sections- ⁇ r ated according to the Paraffin Pretreatment Reagent Kit protocol (purchase'ii*tos*:Vysis, Downers Grove, IL) before hybridization.
  • FISH was performed with a digoxigei ⁇ & ⁇ tAC probe (PAC dJ177P22, Sanger Centre, UK) containing the E2F3 gene and a Spect ⁇ as'R rlabeled chromosome 6 centromeric probe (CEP6) as a reference (purchaset - reVysis).
  • Hybridization and posthybridization washes were according to the 'LSI procft £f '. (Vysis).
  • Probe visualization using fluorescent isothiocyanate (FITC)-conjugated sheep anti-digoxigenin was as described (Wagner, U., et al., Am J Pathol, 151 : 753-759 (1997). Slides were counterstained with 125 ng/ml 4', 6-diamino-2-phenylindole in an antifade solution. Amplification was defined as presence (in 5% of tumor cells) of at least 3 times as many E2F3 gene signals than centromere 6 signals.
  • the IHC staining intensity (scored in a four step scale including 0, 1+, 2+, and 3+) and the fraction of positive tumor cells was recorded for each tissue spot. Based on these values, a final IHC result was calculated according to the following criteria: Negative: no staining at all, or 1 + staining intensity in no more than 10% of tumor cells; positive: at least 2+ staining intensity in more than 10% of tumor cells.
  • the rabbit monoclonal antibody MIB1 (1 :800, Dianova, Hamburg, Germany) ims employed to detect Ki67 protein (expressed in all cells in G1 , S, G2 and M pha ⁇ s>).,as previously described (Moch, H., et al., Urol Res, 25: S25-30 (1997). Nuclei ws ⁇ s'. ' .considered Ki67 positive if any nuclear staining was seen. The Ki67 labeling inde ⁇ ), (percentage of Ki67 positive cells) was determined on each arrayed tumor sam ⁇ J ? ⁇ , scoring at least 300 cells each. Tumors with Ki67 negative mitoses were excluded Western analysis
  • Protein was extracted from ab ⁇ c ⁇ R2 ⁇ 06 cells from each of the 8 bladder tumor cell lines as described (Leone, G., et al., C mm ⁇ Dev, 12: 2120-30 (1998). Ten ⁇ g protein of each sample was subjected to SDS-PAGE ⁇ '.10% polyacrylamide gels. Proteins were transferred onto PVDF membrane (Bio-Rad, Glatitesgg, Switzerland). The membrane was blocked in TBS (25 mM Tris at pH 7.4, 137 m ' ⁇ it ⁇ J 2.7 mM KCl) containing 10% skim milk at RT for 2 hr.
  • TBS 25 mM Tris at pH 7.4, 137 m ' ⁇ it ⁇ J 2.7 mM KCl
  • Blots were then incubated wit ⁇ rnQUse monoclonal E2F3 Ab-4 antibody (5 ⁇ g/ml) (Lab Vision, CA, U.S.A.) in TBS containing'. 5% skim milk overnight at 4°C, and washed subsequently in TBS containing 0.1% Tween 20 for 30 minutes. Blots were incubated with a second antibody (1 :2000) (goat anti-mouse IgG, Fc, AP127P; Juro Supply AG, Lucerne, Switzerland) for 1 hour at room temperature, and washed for 30 minutes. Blots were processed with the ECL system (Amersham Pharmacia Biotech, Dubendorf, Switzerland).
  • the combined physical, transcript and FISH map of the amplified region at 6p22 is a!$ ia if Figures 3A, 3B, 3C and 3D.
  • Clones marked in red constitute the core of the a ⁇ vp r ⁇ .n (1.7 Mb in size). Average copy number for each clone in the map was counted tumors placed into a tissue microarray and assayed with a labeled DNA-probe.
  • Rhe ar marked in red define the minimal commonly amplified region at 6p22. Genes and to the region are also indicated.
  • Bioinformatic tools were used to identify and ' ctesp-sterize novel genes within GS-BIG.
  • S& ⁇ n software tool
  • cDNA sequences were mapped to th' ⁇ corresponding genomic sequences using NCBI's pairwise BLAST tool (http://www.ncbi.nlm.nih.gov/blast/bl2seq/bl2.html).
  • NM_175879, dJ434O11 , and Q8N9R5 showed overlapping sequences, thus allowing for assembling of a "unigene” which we termed "BIG-2".
  • the unigene spans about 110 kb of genomic sequence, including 13 exons with a total cDNA size of 3.14 kb.
  • GenScan-analysis of the cDNA sequence revealed a 1488 bp (495 aa) coding sequence.
  • the predicted protein contains an MBOAT domain motif ( Figure 15).
  • the MBOAT (membrane bound O-acyl transferase) family of membrane proteins contains a variety of acyltransferase enzymes and has been suggested to play a role in WNT signaling. This supports potential growth-promoting role for BIG-2.
  • TRAM RNA-binding domain, common to tK ⁇ W ⁇ acil methylation and adenine thiolation enzymes.
  • the TRAM domain is present in - ⁇ w ⁇ al other proteins associated with the translation machinery and in a family of small, unci E ⁇ gs ⁇ terized archaeal proteins that are predicted to play a role in the regulation of tRNA mo f&stion or translation (Anantharaman, V., et al., FEMS Microbiol Lett, 197: 215-221 (20 ⁇
  • FISH Fluorescence in situ hybridization
  • the tissue microarray sections were treated according to the Paraffin Pretreatment Reagent Kit protocol (purchased from Vysis, Downers Grove, IL) before hybridization.
  • FISH was performed with a digoxigenated PAC or BAC clones (Sanger Centre, UK) and a Spectrum Red-labeled chromosome 6 centromeric probe (CEP6) as a reference (purchased from Vysis).
  • CEP6 Spectrum Red-labeled chromosome 6 centromeric probe
  • Hybridization and posthybridization washes were according to the 'LSI procedure' (Vysis).
  • Contingency table tests were applied to study the relationship between histologic tumor iy ⁇ '& ⁇ e, stage and GS-BIG6 amplification. Student t tests were employed to examine the a& ⁇ a ⁇ ions of the Ki67 LI with GS- BIG6 amplification. Analysis of Variance (ANOVA) waa i ⁇ sd to determine the parameters with greatest influence on tumor cell proliferation. ⁇ wival curves were plotted according to the Kaplan-Meier method and analyzed for statiSfe-al differences using a log rank test. Patients with pTa/pT1 tumors were censored at the time of their last clinical control showing no evidence of disease or at the date when cystectomy was performed. Patients with pT2-4 carcinomas were censored at the time of their last clinical control or at the time of death if they died from causes not related to their tumor.

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Abstract

Studies by Comparative Genome Hybridization (CGH) have shown that 6p22 is frequently amplified in invasive urinary bladder cancer. The E2F3 gene maps to 6p22 and encodes a transcription factor important for cell cycle regulation and DNA replication. To determine whether E2F3 resides in the 6p22 amplicon, 7 primary tumors and 3 cell lines known to have 6p22 amplification by CGH were examined by fluorescence in situ hybridization (FISH) using an E2F3-specific probe. All ten 6p22 amplified tumors showed E2F3 amplification. To further investigate the role of E2F3 in bladder cancer, a tissue microarray containing samples from 2317 bladder tumors was used for FISH and immunohistochemical expression analysis. E2F3 amplification was strongly associated with invasive tumor phenotype and high tumor grade (p<0.0001 each). Only 4 of 398 pTaG1/G2 tumors (1%) but 110 of 489 pT1-4 carcinomas (22%) had E2F3 amplification. A high E2F3 expression level was associated with high grade, advanced stage, and E2F3 gene amplification (p<0.0001 each). To evaluate whether E2F3 expression would correlate with tumor proliferation, the Ki67 labeling index (LI) was analyzed for each tumor. There was a strong association between a high Ki67 LI and E2F3 expression (p<0.0001), which was independent of grade and stage. We conclude that E2F3 is frequently amplified and overexpressed in invasively growing bladder cancer (stage pT1-4). E2F3 expression appears to provide a growth advantage to tumor cells by activating cell proliferation in a subset of bladder tumors.

Description

ONCOGENES USEFUL FOR THE IDENTIFICATION OF URINARY BLADDER CANCER
BACKGROUND OF THE INVENTION
1. Field of the Invention The present invention relates to the detection and identification of nucleic acids and proteins that serve as cancer markers in biological samples. More specifically the present invention relates to the diagnosis and prognosis of bladder cancer by specifically detecting the presence of nucleic acids and/or their products which, when present individually or together in a biological sample are indicative of the presence of cancer in the organism. In particular, these markers are useful in detecting or modulating particularly aggressive forms of cancer. The invention further relates to methods for identifying and using candidate agents and/or targets which modulate certain cancers.
2. Description of the State of Art Cancer of the bladder is the fifth most common cancer in the United States with an annual incidence of about 18 cases per 100,000 or over 50,000 new cases per year, leatifnfΛα. more than 10,000 deaths annually. A model of bladder carcinoma developrrr® ,is shown in Figure 1. The incidence (80% of the cases) is highest in the 50-79 year <ϊ,e group; the disease prevalence peaks in the seventh decade of life with a strong m8fe redominance, that is, a male:female ratio of 5:1. Bladder cancer accounts for 7% crS t ew cases of cancer among men and 3% among women, as well as 2% of cancer tføeths among men and 1% among women. Occupational exposure may account for 21-2S*^øibladder cancer in white males in the United States. Almost all cases of bladder cancer are of the
Figure imgf000002_0001
(95%), and among those, approximately 80% appear initially as more or less woϋ sitffeεentiated, superficial papillary neoplasms with a tendency for multifocal or diffuse involvement of the urothelial surface and/or recurrent tumor episodes, but with limited potential- or' invasive growth. Most tumors are detected as non-invasive papillary tumors (stage pTa).
These tumors recur in 50-60% of cases, but they rarely progress into a life- threatening muscle-invasive carcinoma (stage pT2-4). It is currently debated whether these invasive recurrences of patients with previous pTa tumors represent true tumor progression or de novo carcinomas. Of special interest is flat carcinoma- in-situ (CIS) of the bladder, a lesion presenting problems in diagnosis and of unpredictable behavior (e.g. recurrence and progression) and where morphologic definition is arbitrary and generally defined as a total replacement of the urothelial surface by cells which bear morphologic features of the carcinoma, but which lack architectural alterations other than an increase in the number of cell layers, i.e., a flat lesion. CIS constitute for another 5-10% of bladder neoplasias. CIS are thought to represent the precursor lesions of most if not all solid invasive bladder carcinomas. Among the invasive carcinomas the stages pT1 and pT2-4 must be distinguished. In pT1 carcinomas invasion is limited to the lamina propria. These tumors have a good prognosis. They can be cured by transurethral resection in about 80%. Prognosis is much worse in pT2-4 carcinomas. Despite aggressive surgery (cystectomy), only about 50% of these patients survive.
It is likely that development and progression of bladder cancer is driven by a malfunction of specific genes (i.e. overexpression of oncogenes or inactivation of tumor suppressor genes). Oncogenes that are known to play a role in bladder sancer include erbB-2 (Coombs, L.M., et al., BrJ Cancer, 63: 601-608 (1991)), Eprάlϊssial Growth Factor Receptor (EGFR) (Neal, D., et al., Lancet, 1: 366-368 (1985); Nfeβt .E., et al., Cancer, 65: 1619-1625 (1990), c-myc (Masters, J.R., et al., Urol Res, 16: 3Φft344 (1988)), Cyclin D1 (Lee, O, et al., Cancer, 79: 780-789
(1997)), and h-RAS (tSwwles, M.A., et al., Cancer-Res, 53: 133-139 (1993)). Tumor suppressor genes that KagR&heen found inactivated in bladder carcinomas include p53 (Kamb, A., et al., Science; 2gSi.4'\6-4W (1994)); Kamb, A., et al., Science, 264: 436-440 (1994); Stadler, W., Cance*@3s&.5 : 2060-2063 (1994), rb (Cordon-Cardo, C, et al., J Natl Cancer Inst, 84: 1251-125Q*f992)) and MTS1 (Kamb, A., et al., Science, 265: 416-417 (1994)); Kamb, A., et at , Sfcfeπee, 264: 436-440 (1994); Stadler, W., Cancer Res, 54: 2060-2063 (1994); Cairnsr.-P^ βf a/., Cancer Res, 54: 1422-1424 (1994); Cairns, P., et al., Science, 265: 415-416 (1§£$ . These genes have been intensively studied in bladder carcinomas but their malfuncffoπ does not appear to sufficiently explain bladder cancer progression or to provide clinicalf useful prognostic information. It is likely that most oncogenes and tumor suppressor ' genes involved in bladder cancer biology remain to be identified.
Cytogenetic changes that can be found in bladder cancer are thought to provide cfues.where yet unidentified oncogenes and tumor suppressor genes may be located. Chromosomal deletions, which are frequently contributing to tumor suppressor gene inactivation, may pinpoint the location of tumor suppressor genes. Typical deletion sites in bladder cancer include 2q, 3p, 4p, 5q, 8p, 9q, 10p, 11p, 11q, 12q and 16q. An increase in the copy number of genes (gene amplification) is one of the possible causes for oncogene overexpression. The term "overexpression' is an abnormal or over-representation of a gene product expressed by a gene In bladder cancer, 22 different amplification sites have previously been published (Table 1).
Table 1
Figure imgf000004_0001
1 1 , 20-22: Coombs, L.M., et. al., BrJ Cancer, 63:601-608 (1991); Sen, S., et al., Oncogene, 14:2195-2200 (1997); Monni, O., et al., Blood, 90:1168-1174 (1997); Houldsworth, J., et al., Blood, 87:25-29 (1996).
2 23, 24: Visakorpi, T., et al., Cancer Res, 55:342-347 (9195); Anzick, S., Science, 217:965-968 (1997).
3 2i«ϊdanyi, M., et al., J Pathol, 175:211-7 (1995).
4 14, 26-2&Sauter, G., et al., IntJ Cancer, 57:508-514 (1994); Bischoff, J.R., et al., Embo J, 17SSS2-3065 (1988); Guan, X.Y., et al., Cancer Res, 56:3446-50 (1996); Tanner, M.M., ed ., Cancer Res, 56:3441-5 (1996)).
Most of these^sf^røere initially detected either by comparative genomic hybridization (CGH) or by eoRS ftlαnal cytogenetics (HSR's). Only a few of these amplification sites correspond to kπe&isReacogenes. Importantly, all currently known oncogenes have been found overexpresses i -fefestder cancer much more frequently than amplified. For example, erbB-2, EGFR, and c lsr ϊ tare all amplified in 10% but overexpressed in 50% of bladder carcinomas (Sauter, & , ,β&alr Cancer Res, 53: 2199-2203 (1993); Sauter, G., et al., Int J Cancer, 57: 508-514 (19S Bringuier, et al., Oncogene, 12: 1747-1753 (1996); Wagner, U., et al., J Pathol \n press)-, Thi suggests that mechanisms other than amplification can lead to overexpression of these oncogenes in a considerable fraction of tumors. The same may apply to unknown oncogenes located at other amplification sites. At least some of these unknown oncogenes may therefore be much more important than expected from their amplification frequency. importantly, there is increasing evidence for a simultaneous overexpression of multiple genes with potential oncogenic function in amplified tumors. This has for example been shown for CDK4 and MDM2 at 12q14 (Anzick, S., et al., Science, 277: 965-968 (1997)), cyclin D1 and EMS1 at 11q13 (Bringuier, et al., Oncogene, 12: 1747-1753 (1996)), and AIB1 , AIB3/4, or AURORA2/BTAK at 20 (Anzick, S., et al., Science, 277: 965-968 (1997); Bischoff, J.R., et al., Embo J, 17: 3052-3065 (1988); Guan, X.Y., et al., Cancer Res, 56: 3446-50 (1996); Tanner, M.M., et al., Cancer Res, 56: 3441-5 (1996). These observations raise the possibility that amplification can lead to a growth advantage of tumor cells because a set of multiple genes rather than only one gene is overexpressed. It could be speculated that these oncogenes for which amplification is uncommon may not strongly benefit from this activation mechanism because of a lack of neighbor genes of which a co-oyerexpression could lead to an additional growth advantage.
Eleven of the 22 known amplification units (disclosed above in Table 1) in urinary bladder cancer have been initially detected by CGH. CGH is based on the simultaneous hybridization of tumor and normal DNA to normal metaphase spreads (Kallioniemi, A., et al., Science, 258: 818-821 (1992) and disclosed in U.S. Patents 6,335,167 which is incorporated herein by reference). CGH allows one to identify all c etions and amplifications of a tumor in one examination, even from formalin fixed tumofδϊr. Over the last few years, CGH has proved to be an excellent screening tool for ampliϊ&stions. CGH has lead to the identification of more than 30 yet unknown amplification sfes.in various tumors. Strategies to identify the genes that give amplified tumor csife selective growth advantage include the search for genes which are consistently μas tøSthe amplicon and also overexpressed in amplified tumors. Several genes that arαtecrøsistently overexpressed in amplification units detected by CGH have recently beew feswibed. Provided that functional analyses reveal clues for an oncogenic function, sucii'& aes may be considered as potential oncogenes. Examples for potential oncogenic targcfti øs found in amplicons that were initially described by CGH include the androgen recepteπgene.,. BCL-2, REL, PAX7-FKHR fusion gene, telomerase at 3q, AIB1 and BTAK at 20-% ^ fe&ar-Hall, S., et al., Genes Chromosomes Cancer, 17: 7-13 (1996); Soder, A., et al., Ohcs^gsne, 14: 1013-1021 (1997); Sen, S., et al., Oncogene, 14: 2195-2200 (1997); Monni, O..r et al., Blood, 90: 1168-1174 (1997); Houldsworth, J., et al., Blood, 87: 25-29 (1996); Visakorpi, T., et al., Cancer Res, 55: 342-347 (1995); Anzick, S., et al., Science, 277: 965-968 (1997)). However, only very few of these "CGH" genes have been identified through traditional positional cloning methods. Based on the human genome sequence data it has become much more fruitful to investigate positional candidate genes, i.e. genes that are known to be located within a region of interest perhaps even having a putative function consistant with a "cancer gene."
To date, large sets of formalin fixed tumors for gene amplification (erbB-2, EGFR, c-myc) and chromosomal deletions (p53, 9p, 9q, 8p, 8q) have been analizyed by FISH (Sauter, G., et al., Cancer Res, 53: 2199-2203 (1993); Sauter, G., et al., Int J Cancer, 57: 508-514 (1994); Sauter, G., et al., Am J Pathol, 144: 756-766 (1994); Wagner, U., et al., Am J Pathol, 151: 753-759 (1997); Sauter, G., et al., Int J Cancer, 64: 99-103 (1995); Sauter, G., Am J Pathol, 146: 1131-1139 (1995); Sauter, G., Cytometry, 21 : 40-46 (1995)) which allowed for the the identification of significant heterogeneity of both amplifications and deletions in these tumors. In several FISH studies, on large series of well characterized carcinomas, it was shown that major genetic differences existed between pTa and pT1 carcinomas while the differences were much less evident between pT1 carcinomas and the more advanced muscle invasive cancers (Sauter, G., et al., Am J Pathol, 144: 756-766 (1994); Wagner^ U., et al., Am J Pathol, 151: 753-759 (1997); Sauter, G., Am J Pathol, 146: 1131-113B' (1995); Sauter, G., et al., Urol Res, 25: S37-44 (1997); Sauter, G., J Pathol in press). Bladder cancer tends to recur often, thus, consistent follow-up is imperative for thesss iagnosed patients. Follow-up usually consists of cystoscopy, biopsy and x-ray exa fcafe .. Some of those procedures, though necessary, are considered invasive methods; ws#E .are often uncomfortable for the patient. Bladder washings obtained during cystoseα sjoan be then analyzed histologically and for DNA content. However, that approach is not &fe£9 s..successful, particularly when a limited number of cells is present in the sample. Uπf&fvjregtely, cytogenetic results on bladder cancer are often difficult to obtain due to the- -p v; growth of tumor cells in culture. In these cases, fluorescense in situ hybridization (FISFi hasibeen used as an alternative technique in detecting numerical chromosome cbs^as. To date, FISH has been performed on cultured cells and paraffin embedded tissue:sections of bladder tumors with good results.
Thus, additional methods for the diagnosis and prognosis of bladder cancer would be desirable. While acade ia and industry has made an effort to identify novel sequences, there has not been an equal effort exerted to identify the function of the novel sequences that lie in amplified regions. Accordingly, provided herein are methods that can be used in diagnosis and prognosis of bladder cancer. Further provided are methods that can be used to screen candidate bioactive agents for the ability to modulate bladder cancer. Additionally, provided herein are molecular targets for therapeutic intervention in bladder cancers.
The identification of amplified and/or deleted genes is important to the management of cancer, for example, bladder cancer, for several reasons: (1 ) to improve prognostication; (2) to detect amplification and/or deletion events that are associated with the development of drug resistance; and (3) to improve therapy. SUMMARY OF THE INVENTION
The present invention provides methods for the diagnosis and prognosis of bladder cancer. The method comprises identifying a gene or set of genes that exist within an amplicon, specifically the region designated as 6p22, and determining the expression of the gene(s) thereof in a first tissue type of a first individual, and comparing this to the expression of the gene from a second unaffected individual. A difference in the expression indicates that the first individual has an increased risk of cancer.
In addition this invention provides cDNA sequences from a number of genes wH i map to this region. Also provided is a contig (a series of clones that contigeaαsly spans this amplicon) which can be used to prepare probes specific for the amplicom The probes can be used to detect chromosomal abnormalities at chromosome 6.
The probes disd& «Mιere can be used in kits for the detection of a chromosomal abnormality at ates&position FLpter 0.11 - 0.19 on human chromosome 6. The kits include a
Figure imgf000007_0001
which contains a labeled nucleic acid probe which binds selectively to a targeif fCjlypucleotide sequence at about FLpter 0.11 - 0.19 on human chromosome 6. The p afesϊpreferably includes at least one nucleic acid that specifically hybridizes
Figure imgf000007_0002
to a nucleic acid selected from the nucleic acids disclosed here. Even more p ^a ly, the probes comprise one or more nucleic acids selected from the nucleic acKte; .disclosed here. In a preferred embodiment, the probes are labeled with digoxigenin or jetirr. The kit may further include a reference probe specific to a sequence in the centromere of chromosome 6. In particular, the present invention includes preferred nucleic acid sequences useful for hybridizing to amplified nucleic acids that are indicative of bladder cancer.
According to one aspect of the invention, there is provided a nucleic acid sequence identified herein as SEQ ID NO:1 to SEQ ID NO: 4. Another aspect of the invention makes use of fluorescense in situ hybridization (FISH) on tissue microarray sections by hybridizing YAC/PAC or BAG probes comprising SEQ ID NO:1 to SEQ ID NO: 4 or fragments for use therewith.
Another aspect of the invention is an oligonucleotide comprising a target- binding sequence consisting of the sequence of any one of SEQ ID NO:1 to SEQ ID NO: 4, and optionally a contiguous sequence required for an amplification reaction.
Another aspect of the invention is a combination of oligonucleotides used in a detection assay specific for a bladder target nucleic acid sequence. The combination comprises a first oligonucleotide that serves as a first amplification primer that hybridizes specifically to a first -specific sequence contained in the 6p22 region of an expressed gene sequence, or that is contained in the E2F3 expressed gene sequence; a second oligonucleotide that serves as a second amplification primer that specifically hybridizes to a different, non-overlapping second sequence srøfeias BIG1 contained in the 6p22 region. Particularly preferred combinations of oligonucleotides are a first oligonucleotide comprising a sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4; the second
< jf ycleotide comprising a sequence selected from the group consisting of SEQ ID NO:1 , S iQ NO:2, SEQ ID NO:3, and SEQ ID NO.4, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:S;T?5;7slS£Q ID NO:4; and the third oligonucleotide comprises a sequence selected tteiϊthe group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.
Another embodiment makes"y®?iOt an aptamer probe to detect either the transcript or protein related to the genes of β:te::ρresent invention.
One embodiment of the method comprises asss ig.for the expressed gene sequence encoding the E2F3, BIG1, and/or BIG2 product. to:pτeferred embodiments, the nucleic acid sample is RNA, more preferably mRNA, isolated frørrrhuman bladder. In preferred embodiments, the detecting step detects a signal in an assay, such as a homogeneous detection assay or an antibody based assay, such as:, IHC, sandwich assay or antibody biochips. These assays are well known by those skilled in the art. Furthermore an assay could be devised wherein detection occurs indirectly, such as a first fragment unlabled fragment is bound to the target and a secondary labeled fragment is introduced for detection of the first.
The present invention provides methods for screening for compositions which modulate bladder cancer. In one aspect, a method of screening drug candidates comprises providing a cell that expresses an expression profile gene or fragments thereof. Preferred embodiments of the expression profile gene as described herein include the sequence comprising E2F3, BIG1 , BIG2 or a fragment thereof. The method further includes adding a drug candidate to the cell and determining the effect of the drug candidate on the expression of the expression profile gene.
In one embodiment, the method of screening drug candidates includes comparing the level of expression in the absence of the drug candidate to the level of expression in the presence of the drug candidate, wherein the concentration of the drug candidate can vary when present, and wherein the comparison can occur after addition or removal of the drug candidate. In a preferred embodiment, the cell expresses at least one expression profile genes. The profile genes may show an increase or decrease.
Also provided herein is a method of screening for a bioactive agent' ©spable of binding to E2F3, BIG1, BIG2 or a fragment thereof, the method comprising combining E2F3, BIG1 , BIG2 or fragment thereof and a candidate bioactive agenf, and determining the binding of the candidate agent to the E2F3, BIG1 , BIG2 or fragment thereof.
Further provided herein is a method for screening for a bioactive agent capab e-QiΦ AaXlng the bioactivity of E2F3, BIG1 , BIG2 or a fragment thereof. In one embodiment? iftarøethod comprises combining E2F3, BIG1 , BIG2 or fragment thereof and a
Figure imgf000009_0001
and determining the effect of the candidate agent on the bioactivity of E2F3; li^ s BIG2 or the fragment thereof. In one embodiment, E2F3, BIG1 , BIG2 has the Uκr*sfetty. of a bladder cancer modulating protein. Also provided herein is a method of evaluating the eifest.of a candidate cancer drug comprising administering the drug to a transgenic animal, expressing or over-expressing E2F3, BIG1 , BIG2 or a fragment thereof, or an animal lacking E2F3, BIG1, BIG2 for example as a result of a gene knockout. Additionally, provided herein is a method of evaluating the effect of a candidate cancer drug comprising administering the drug to a patient and removing a cell sample from the patient. The expression profile of the cell is then determined. This method may further comprise comparing the expression profile to an expression profile of a healthy individual.
In one aspect of the invention, a method for inhibiting the activity of a bladder cancer modulating protein are provided. The method comprises binding an inhibitor to the protein expressed by E2F3, BIG1, and/or BIG2.
In another aspect, the invention provides a method for neutralizing the effect of a bladder cancer modulating protein. The method comprises contacting an agent specific for the protein with the protein in an amount sufficient to effect neutralization. In a preferred embodiment, the protein is expressed by one or a combination of E2F3, BIG1, and/or BIG2.
As described herein, methods of inhibiting bladder cancer can be performed by administering any inhibitor of E2F3, BIG1 , and/or BIG2 activity to a cell or individual. In one embodiment, a E2F3, BIG1 , and/or BIG2 inhibitor is an antisense molecule to E2F3, BIG1 , and/or BIG2, respectively.
Moreover, provided herein is a biochip comprising a nucleic aei&segment comprising SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 3, SEQ ID NO: 4, oi»<s fragment thereof, wherein the biochip comprises fewer than 1000 nucleic acid'jϊSΦtøes. Preferably at least two nucleic acid segments are included. The biochip could also* contain capture molecules other than nucleic acids, for example antibodies, antibody ffrø ants, aptamers, protein scaffolds etc.
A*rsG«fy s»λάded herein are methods of eliciting an immune response in an individual. In or-c^E^sbo iment a method provided herein comprises administering to an individual a composite* comprising E2F3, BIG1 , BIG2 or a fragment thereof. In another aspect, said compositi&'js'rørøprises a nucleic acid comprising a sequence encoding E2F3, BIG1 , BIG2 or a fragϊrøs heεeof.
Further provided herein are compositionsr-eapsfela of eliciting an immune response in an individual. In one embodiment, a com position- provided herein comprises E2F3, BIG1 , BIG2 or a fragment thereof and a pharmaceutically acceptable carrier. In another embodiment, said composition comprises a nucleic acid comprising a sequence encoding E2F3, BIG1 , BIG2 or a fragment thereof and a pharmaceutically acceptable carrier.
Additional advantages, and novel features of this invention shall be set forth in part in the description and examples that follow, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by the practice of the invention. The advantages of the invention may be realized and attained by means of the instrumentalities and in combinations particularly pointed out in the appended claims.
DEFINITIONS A "chromosome sample" as used herein refers to a tissue or cell sample prepared for standard in situ hybridization methods described below. The sample is prepared such that individual chromosomes remain substantially intact and typically comprises metaphase spreads or interphase nuclei prepared according to standard techniques. "Nucleic acid" refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, would encompass known analogs of natural nucleotides that can function in a similar manner as naturally occurring nucleotides.
An "isolated" polynucleotide is a polynucleotide which is substantially separated from other contaminants that naturally accompany it, e.g., protein,
Figure imgf000011_0001
other polynucleotide sequences. The term embraces polynucleotide sequences^, -i' zfa have been removed or purified from their naturally-occurring environment or clone libr ry . and include recombinant or cloned DNA isolates and chemically synthesized analogues ϋF's"-ϊ!3;S)gues biologically synthesized by heterologous systems. "Su-s&sqwence or fragment" refers to a sequence of nucleic acids that comprise a part of longer sequence of nucleic acids.
A "probe" or a "nucfefesisstd probe," as used herein, is defined to be a collection of one or more nucleic aorcifpagments whose hybridization to a target can be detected. The probe is labeled as dese foed: below so that its binding to the target can be detected. The probe is produced from a: source of nucleic acids from one or more particular (preselected) portions of the genomevfor example one or more clones, an isolated whole chromosome or chromosome fragment, or a collection of polymerase chain reaction (PCR) amplification products. The probes of the present invention are produced from nucleic acids found in the 6p22 amplicon as described herein. The probe may be processed in some manner, for example, by blocking or removal of repetitive nucleic acids or enrichment with unique nucleic acids. Thus the word "probe" may be used herein to refer not only to the detectable nucleic acids, but to the detectable nucleic acids in the form in which they are applied to the target, for example, with the blocking nucleic acids, etc. The blocking nucleic acid may also be referred to separately. What "probe" refers to specifically is clear from the context in which the word is used. "Hybridizing" refers the binding of two single stranded nucleic acids via complementary base pairing.
"Bind(s) substantially" or "binds specifically" or "binds selectively" or "hybridizes specifically" refer to complementary hybridization between an oligonucleotide and a target sequence and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target polynucleotide sequence. These terms also refer to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA. The term "stringerrfeconditions" refers to conditions under which a probe will hybridize to its target subseq&face, but to no other sequences. Stringent conditions are sequence-dependent anc .'^l be different in different circumstances. Longer sequences hybridize specifically at rug^r,, temperatures. Generally, stringent conditions are selected to be about 5° C io^ . than the thermal melting point (Tm) for the specific sequence at a defined ionic
Figure imgf000012_0001
pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic acfcffeancentration) at which 50% of the probes complementary to the target sequence hybridizKitolhe target sequence at equilibrium. Typically, stringent conditions will be those: ,y«hich the salt concentration is at least about 0.02 Na ion concentration (or other salts) 'a£y? ..7.0 to 8.3 and the temperature is at least about 60° C for short probes. Stringent
Figure imgf000012_0002
also be achieved with the addition of destabilizing agents such as formamide.
One of skill will recognize that the precise sequence of the particular probes described herein can be modified to a certain degree to produce probes that are "substantially identical" to the disclosed probes, but retain the ability to bind substantially to the target sequences. Such modifications are specifically covered by reference to the individual probes herein. The term "substantial identity" of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 85% sequence identity, more preferably at least 90%, and most preferably at least 95% compared to a reference sequence using the methods described below using standard parameters.
Two nucleic acid sequences are said to be "identical" if the sequence of nucleotides in the two sequences is the same when aligned for maximum correspondence as described below. The term "complementary to" is used herein to mean that the complementary sequence is identical to all or a portion of a reference polynucleotide sequence.
Sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two sequences over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window", as used herein, refers to a segment of at least about 20 contiguous positions, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are' optimally aligned.
Optimal alignment of sequences for comparison rrey.be conducted by the local homology algorithm of Smith and Waterman, Adv. Apph Math., 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, Φfø Biol., 48:443 (1970), by the search for similarity method of Pearson and Lipman, Pϊϋ-/;;'Matl. Acad. Sci. (U.S.A.), 85: 2444 (1988), by computerized implementations of these ' t& s.
"Percentage of sequence identity" is determined by comparing two optimally aBfg&sel.sequences over a comparison window, wherein the portion of the
Figure imgf000013_0001
in the comparison window may comprise additions or deletions (i.e., ga^as compared to the reference sequence (which does not comprise additions or cStøfø&oαs) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino tsc'uiresidue occurs in both sequences to yield the number of matched positions, dividing-fee. number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to the same sequence under stringent conditions. Stringent conditions are sequence dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 5° C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Typically, stringent conditions will be those in which the salt concentration is about 0.02 molar or lower at pH 7 and the temperature is at least about 60° C. BRIEF DESCRIPTION OF THE DRAWINGS
The accompanying drawings, which are incorporated in and form a part of the specification, illustrate the preferred embodiment of the present invention, and together with the description serve to explain the principles of the invention.
In the Drawings: Figure 1 : A model of bladder cancer development and progression.
Figure 2 provides CGH profiles of tumors and a tumor cell line with 6p amplification. CGH profiles are shown next to idføgfams of chromosomes 6. In all tumors circumscribed peaks are visible at 6p21.3-22 fπc^ating high level amplification of this region. Figure 3A, 3B, 3C and 3D represent a combined physic&ki&anscript and FISH map of the amplified region at 6p22. Clones marked in red constitute©s.Eore of the amplicon (1.7 Mb in size). Average copy number for each clone in the map «c« counted from 10-30 tumors placed into a tissue microarray and assayed with a labeled DNA-probe. Clones marked in red define the minimal commonly amplified rs s,at 6p22. Genes and transcripts mapping to the region are also indicated.
Figure imgf000014_0001
a photograph of a Western blot demonstrating a high amount of E2F3 protein in >1s; ds?v:cancer cell lines with 6p22 amplification (CRL1472, HTB5, HTB9) as compared to bϊέ fefeccancer cell lines with normal 6p22 copy number (CRL 7882, RT1 2) or other cancer fe:C.HL60. Figure 5 is a photograph of a cell nucleus with multiple copies of the E2F3 gene, demonstrated with hybridizing tissue microarray sections with green FISH probe. Figure 6 is a photograph of a cell nucleus from a tumor that has equal numbers of E2F3 and centromere 6 FISH probe signals.
Figure 7A is a Northern blot indicating that no E2F3 transcript was seen in cell line CRL-1479, whereas cell lines with E2F3 amplification CHTB-5 CRL1427 and ACC5637 show high level of E2F3 expression.
Figure 7B is a Western blot indicating that the protein expression level in the third amplified cell line (ACC 5637) was comparable to the expression in non- amplified cell lines.
Figure 8 is a photograph of an amplified tumor stained with E2F3 antibody. Figure 9 is a photograph of a non-amplified tumor stained with E2F3 antibody.
Figure 10 graphically represents E2F3 alterations and prognosis of patient survival.
Figure 11 graphically represents E2F3 alterations and prognosis of patient survival. Figure 12 is the nucleic acieteequence of the gene identified as BIG1 located in the 6p22 region.
Figure 13 is the nucleic acid sequence ofih gene identified as BIG2 located in the 6p22 region.
Figure 14 is the nucleic acid sequence of the gene:J entified as E2F3 located in the 6p22 region.
Figure 15 is a schematic of BIG-2: (A) assembling of a 3.144ii??;!fdnigene" from cDNA sequences ENSESTT00002382100, NM_175879, dJ434O11 , arøfci Q8N9R5;; (B) predicted intron-exon structure; and (C) predicted protein domains. . significant similarity was found for the MBOAT domain. Rξ£,yse;16 is a schematic of BIG-1 : (A) intron-exon structure; (B) predicted protein domai Y ix. Significant matches were found for the UPF0004, Radical_SAM, and TRAM domains:
Figure 17 is a Northern -analysis of 9 bladder tumor cell lines. The 2.6 kb BIG-1 mRNA is abundantly expressedttrr 6p22.3 amplified cell lines CRL-1472, 5637, and 5-HTB. Northern blot staining with met ylene blue showed presence of RNA in all cell lines (control in the lower panel). Figure 18 graphically displays quantitative mRNA expression analysis of BIG- 1 relative to bladder cell line 7882 showing the weakest BIG-1 mRNA expression (=1 fold expression).
DETAILED DESCRIPTION OF INVENTION Gene amplification plays an important role in the initiation and progression of bladder cancer where more than 30 different genomic loci have been identified that recurrently harbor DNA amplifications (Kallioniemi, A., et al., Genes Chrom Cancer, 12: 213-219 (1995); Mitelman, F., Catalog of chromosome aberrations in cancer, 5th edn. Wiley-Liss: New York; Richter, J., et al., Am J Pathol, 153: 1615-1621 (1998); Richter, J., et al., Cancer Res, 57: 2860-2864 (1997); Simon, R., et al., J Pathol, 185: 345-351 (1998); Voorter, O, et al., Am J Pathol, 146: 1341-1354 (1995). Several of these amplifications contain known oncogenes such as HER2 at 17q21 , CCND1 at 11q13, MYC at 8q24, EGFR at 7p13, or MDM2 and CDK4 at 12q13-15 (Dalla-Favera, et al., Proc Natl Acad Sci U SA, 79: 7824-7 (1982); Kondo, I. & Shimizu, N. Cytogenet Cell Genet, 35: 9-14 (1983); Popescu, N.C., et al., Genomics, 4: 362-366 (1989); Reifenberger, G., et al., Cancer Res, 54: 4299-303 (1994); Xiong, Y., et al., Genomics, 13: 575-584 (1992). However, the target genes are unknown for the majority of loci harboring gene amplifications such as 1q21-31 , 2q13, 3p22-24, 6p22, 8p11 , 8q21, 9p21, 10p13-14, 13q13; " ^q31-33, 18p11, 20q, 21p11, 22q11-13, Xp11- 13, and Xq21-22.2 (Kallioniemi, A., et al:, U nes Chrom Cancer, 12: 213-219 (1995); Kallioniemi, A., et al, Science, 258: 818-821 (tJ ) . Mitelman, F., Catalog of chromosome aberrations in cancer, 5th edn. Wiley-i5.fe New York; Richter, J., et al., Am J Pathol, 153: 1615-1621 (1998); Richter, J., et al., Caram-Res, 57: 2860-2864 (1997); Richter, J., et al., Cancer Res, 59: 5687-5691 (1999); Sae&føs . et al., Path Res Pract, 190: 281-282 (1994); Simon, R., et al., J Pathol, 185: 345-3S "Bf f iξ );
Terracciano, L., et al., J Pathol, 189: 230-235 (1999); Voorter, O, et al., Am J ^S§ ϊ 146: 1341-1354 (1995); Zhao, J., et al., Cancer Res, 59: 4658-4661 (1999)).
The present invention exploits the genome-era information resources and tissue aι ta jptetforms to quickly identify and examine loci harboring gene amplifications.
Based on comparative genomic hybridization (CGH) (CGH is disclosed in U.S. Patent No. 6,335,167, which isincorporated herein in its entirety) data of more than 300 bladder carcinomas, was analyzed. This is dramatically more than the sum of all other published CGH analyses of bladder carcinomas (33+14+46=93) (Richter, J., et al., Am J Pathol, 153: 1615-1621 (1998); Richter, J., et al., Cancer Res, 57: 2860- 2864 (1997); Richter, J., et al., Cancer Res, 59: 5687-5691 (1999); Simon, R., et al., J Pathol, 185: 345-351 (1998); Simon, R., et al., Int J Oncol, 17: 1025-1029 (2000); Terracciano, L, et al., J Pathol, 189: 230-235 (1999); Zhao, J., et al., Cancer Res, 59: 4658-4661 (1999). One of the most common sites of high-level amplification is 6p22, which was present in 10 of 172 advanced stage tumors analyzed. Importantly, these analyses lead to the identification of many tumors and cancer cell lines with high level amplifications at various sites (Table 2, Figure 2).
Figure imgf000017_0001
Among these amplification sites the re§κ¥£.6p22 attracted our main interest because of three reasons: (1) 6p22 is the most frexpsfttsite of high level amplification in bladder cancer at which no oncogene is cys«jϊtly known (Table 2); (2) 6p+ is the genomic alteration detectable by CGH which is &sfδ'sfeøngly linked to a high tumor grade (Table 3); and (3) 6p+ is strongly (and independef ».Stumor grade) associated with rapid tumor cell proliferation (Table 4).
Table 3
Genomic alterations and histologic grade in 94 invasive bladder carcinomas (pT1-4)
Figure imgf000018_0001
Table 4
Genomic alterations and tumor cell proliferation (Ki67 labeling index) in 94 invasive bladder carcinomas (pT1-4)
Figure imgf000019_0001
amean ± standard deviation b statistical significance was independent of histolό ft pςie c statistical significance was independent of tumor stage-
One candidate gene on 6p22 was cyclin D3. However, immesKsbis pchemical analyses revealed no association between cyclin D3 expression and 6p2 -1 amplification suggesting that an other gene (or genes) must be the target at the Sfsl 'i ID) While about 25% of 300 bladder carcinomas expressed cyclin D3, 6 of 6 tumors with higtiievel 6p22 amplification were cyclin D3 negative.
To identify the gene(s) amplified at 6p22 we first constructed a physical map and DNA clone contig of the amplified region at 6p22, covering over 5Mb of DNA, shown in Figure 3A, 3B, 3C and 3D. Using the latest version of the publicly available human genome map, large-insert BAG DNA clones and STS (sequence-tagged- sites) markers spanning the amplified region were selected. The contig spanning 6p22 amplicon was constructed by sequence alignment of the overlapping clones, and in the case of BAG clones that were not fully sequenced, by PCR analysis of STS sites within the BACs. The final order of the clones within the physical map contig differed markedly from the order of the clones indicated by the publicly available databases. The final, verified genomic contig contained the 6p22 amplicon, but included also by-stander sequences on both sides of the core amplification unit. To narrow down to the minimal commonly amplified region within the 6p22 contig, FISH studies were performed on bladder cancer tissue microarray constructed from o specimens showing 6p alterations. Individual contig clones were used as probes in
FISH analyses, and the avarage copy number was counted from 10-30 tumor specimens for each contig clone. The clones that showed the most frequent amplification pattern on cell lines and clinical specimens define the core amplicon unit, designated as the GS-BIG region (Figure 3A, 3B, 3C and 3D). This core region of the amplification spans over 1.7 Mb of genomic DNA (SEQ ID NO: 1), see sequence listing on CDROM submitted herewith, which is incorporated herein in its entirety by this reference, and these genes of interest were identified: BIG-1 (SEQ ID NO:2); BRSWMSEQ ID NO:3) and E2F3 (SEQ ID. NO: 4). Figures 12, 13 and 14, respectively. E353 will be discussed in further detail below.
As the next stc .the clinical relevance of the BIG1 amplification in bladder cancer was validated by paralyzing more than 2,000 primary bladder tumor specimens using tissue micro&s?? ?fs (Table 5).
Table 5
Figure imgf000021_0001
GS-BIG FISH assay revealed amplification in 86 out of 1 ,029 -ansiyzed tumors pointing to an overall amplification frequency of about 8%. The breakdowrrc? BiG1 amplification frequency according to histological tumor type is shown below in TaMe 6. Table 6 GS-BIG Amplification and Tumor Phenotype
Figure imgf000022_0002
a for definition see text b contingency table analysis ° only first biopsies of patients with TCC included d only first biopsies of patients with muscle invasive carcinomas (pT2-4) included
Within transitional cell carcinoma, the most common histological subtype of bladdei ": fl.ncer, GS-BIG amplification associates strongly with high tumor grade and advanced sl . <0.0001 each, Chi-square statistical test of significance). Furthermore, GS- 3l&βmplification is very rare in early-stage tumors (0.7 % of pTaG1/G2 tumors) but reϊftfe Sy common in invasively growing cancers (15 % of pT1 -4 tumors).
Next, which molecular features '3 $'%;ή i.for tumors with GS-BIG amplification were analyzed. First, immunohistϋ ;fes is ry results with a marker for cellular proliferation (Ki67) were analyzed. This
Figure imgf000022_0001
and grade independent association of GS-BIG (p<0.0001 , ANOVA) ampHtto^ with high cell proliferation rate. Second, the pattern of GS-BIG amplification was corπpsFedto other gene amplifications known to occur in bladder cancer. This showed that GS-BIG, is the most common gene amplification in invasive bladder cancer (15%), followed by Her2 (13.5%) and Cyclin D1 (11.1%) amplifications. Finally, association of GS-BIG amplification to patient outcome was analyzed. When all tumor subgroups are analyzed it appears that GS-BIG amplification confers poor prognosis (p<0.05, Mann-Whitney).
These results show that DNA probes derived from the GS-BIG region can be used to detect bladder cancer, for example on voided urine cells, and GS-BIG amplification can be used to differentiate between tumors of low and high malignancy potential. Based on an expected fraction of 65% pTaG1/G2 tumors in consecutive series of bladder tumors, the positive predictive value of GS-BIG6 amplification for invasive tumor growth or high grade would be about 93%. To summarize, the present invention describes a genomic sequence originating from chromosomal region 6p22 (Genomic Sequence for Bladder Invasive Growth 6, GS-BIG) that undergoes amplification in bladder cancer, and teaches the use of this sequence in detection of bladder cancer.
Identification of E2F3 in the p622 Region E2F3 belongs to a family of cell cycle regulatory transcription factors that are controlled by the retinoblastoma tumor suppressor (Humbert, P.O., et al., Genes Dev, 14: 690-703 (2000); Lees, J.A., et al., 13: 7813-7825 (1993); Leone, G., et al., Genes Dev, 12: 2120-30 (1998); Leone, G., et al., Genes Dev, 12: 2120-30 (1998); Wv,:L, et al., Nature, 414: 457-62 (2001). Heterodimers of E2F1 , E2F2, or E2F3 with DP1 serve as transcriptional activators of genes that promote cell cycling, whereas compl xes of E2F/DP with pRb repress transcription and inhibit cell growth (Leone, G., et al., (S& s.Dev, 12: 2120-30 (1998); Nevins, J.R. Cell Growth Differ, 9: 585-93 (1998); Trimawtef. J.M. & Lees, J.A. Nat Rev Mol Cell Biol, 3: 11-20 (2002). In mice models, two isoformacHiE2F3 have been identified that are expressed via an alternative translation site' |'©^.Y., et al., Oncogene, 19: 3422-33 (2000). In mice the full-length form, named E2F3A, i$:<o»φressed only at the G1/S boundary. In contrast, a truncated variant (E2F3B) is present iHmι%hout the cell cycle. E2F3B lacks 101 N- terminal amino acids relative to the full-length prαl s,: including a moderately conserved sequence of unknown function, which is pres sir.Only in the growth- promoting E2F family members. Whereas E2F3A (like E2Ff sαά:E2F2) promotes cell proliferation, E2F3B is believed to inhibit cell cycle by E2F4- and E2F;δ'-mediated transcriptional activation of p16 (Gaubatz, S., et al. Mol Cell, 6: 729-35 (2000); Wu, L, et al., Nature, 414: 457-62 (2001). However, it is not known whether these two functional isoforms exist in humans, or play similar roles as in mice. Recent reports have indicated that different members of the E2F gene family could play specific and diverse roles in tumorigenesis of various human malignancies. For example, increased copy numbers and overexpression of E2F1 were found in an erythroleukemia cell line (Saito, M., et al., Genomics, 25: 130-8 (1995), and E2F5 was amplified and upregulated in 4.2% of breast cancers (Polanowska, J., et al., Genes Chromosomes Cancer, 28: 126-30 (2000). Decreased expression of E2F1 was recently found to be associated with an increased risk of progression to metastases in bladder cancer (Rabbani, F., et al., J Natl Cancer Inst, 91: 874-81 (1999). Whereas almost all research on E2F3 has been performed in cell line or mice models so far, little is known about the potential role of E2F3 in human cancers. Based on the rate limiting role of E2F3 for cell proliferation (Humbert, P.O., et al., Genes Dev, 14: 690-703 (2000) it is possible that overexpression of E2F3 could facilitate cell cycle progression and an increased cell proliferation rate. In turn, overexpression of E2F3 could provide a growth advantage to cells exhibiting this alteration, resulting in clonal selection explaining the presence of 6p22 amplifications in advanced-stage tumors. Tissue microarray (TMA) technology (Kononen, J., etal., Nat Med, 4: 844-847 (1998)), which is incorporated herein by reference, was applied to study E2F3 alterations in urinary bladder cancer. A TMA containing 2317 bladder cancers was used to examine the impact of E2F3 gene copy number changes oirthe protein expression level, tumor phenotype, and clinical outcome.
Studies by Comparative Genome Hybridization (CGH) have shown that 6p22 is fre «flt!y amplified in invasive urinary bladder cancer. The E2F3 gene maps to the center
Figure imgf000024_0001
GS-BIG region at 6p22. To validate that E2F3 is part of the amplicon more th'&wΛ'fc.primary tumors and 3 cell lines known to have 6p22 amplification by CGH wer«c«:amined by fluorescence in situ hybridization (FISH) using an probe containing
Figure imgf000024_0002
All ten 6p22 amplified specimens (3 cell lines + 7 primary tumors) showed Ki' 'smplification. This prometed an evaluation of the clinical frquency and prognos ffesigruficance of E2F3 amplification event in a large bladder cancer specimen cohort. Tissuwriicroarray containing a total of 2,317 bladder tumors were used for FISH and immuna istochemical expression analysis. E2F3 amplification was strongly associated rii r invasive tumor phenotype and high tumor grade (p<0.0001 each). Only 4 of 398 pTaG1/G2 tumors (1%) but 110 of 489 pT1-4 carcinomas (22%) had E2F3 amplification. A higrv'E2F3 expression level was associated with high grade, advanced stage, and E2F3 gene amplification (p<0.0001 each). To evaluate whether E2F3 expression would correlate with tumor proliferation, the Ki67 labeling index (LI) was analyzed for each tumor. There was a strong association between a high Ki67 LI and E2F3 expression (p<0.0001 ), which was independent of grade and stage. Thus leading to the conclusion that E2F3 is frequently amplified and overexpressed in invasively growing bladder cancer (stage pT1-4). E2F3 overexpression appears to provide a growth advantaege to tumor cells by activating cell proliferation in a subset of bladder tumors.
E2F3 gene amplification FISH analyses were performed in two steps: prescreening and establishing optimal hybridization conditions was done on a mini-TMA containing specimens with 6p22 amplification (as identified by CGH), followed by a large-scale TMA analysis of 2317 clinical specimens. The 6p22-amplification specific mini-TMA revealed E2F3 amplification in all 3 cell lines and 7 primary tumors that had shown 6p22 amplification by CGH. The average E2F3 copy numbers in these tumors ranged between 7 and 31.
FISH was successful in 1272 of 2317 (55%) samples of the large TMA. FISH related problems (weak hybridization, background, tissue damage) were responsible for about two thirds, TMA linked problems (too few or absence'sCtumor cells on the TMA spot) were causing about one third of the non-informative cas ^, E2F3 amplification was detected in 145 of 1037 (14.0%) interpretable tumorsr^nly the first biopsy of each patient included). Examples of amplified and non-amplified i&?κ§ι;s are shown in Figures 5 and 6. The associations with tumor phenotype are summarized in Table 7.
Table 7 Histological and Clinical Parameters of 2317 Arrayed Bladder Cancer Samples
Figure imgf000026_0001
Within transitional cell csϊόΦ .,. which is by far the most common bladder cancer subtype, E2F3 amplification was singly associated with high tumor grade and advanced stage (p<0.0001 each). Most si>τl ingly, E2F3 amplification was rare in pTaG1/G2 tumors (4 of 398; 1%) while 22% (TtQr.αf 489) of the invasively growing TCC (pT1-4) had E2F3 amplification. E2F3 amplification was most frequent in muscle invasive cancers of several histological subtypes including TCC (29.2%), small cell carcinomas (35.7%), sarcomatoid cancers (22.2%), and probably adenocarcinomas (1 of 4 samples amplified).
E2F3 expression in bladder tumor cell lines
To determine whether E2F3 amplification leads to elevated E2F3 expression, Northern and Western blot analyses were performed using 3 amplified and 5 non- amplified bladder tumor cell lines. Two E2F3-specific transcripts of approximately 4 kb and 6 kb were detected in 4 cell lines. Three cell lines showed only one transcript. No E2F3 transcript was seen in cell line CRL-1479 (Figure 7A). All three amplified cell lines (5-HTB, ACC 5637, and CRL-1472), showed increased RNA expression, although the upper E2F3 transcript was missing in 5-HTB and CRL-1472. Only the cell lines with the highest RNA expression (5-HTB, HTB-9 and CRL-1472) had also distinct protein overexpression. The protein expression level in the third amplified cell line (ACC 5637) was comparable to the expression in non-amplified cell lines (Figures 4 and 7B). E2F3 immunohistochemistry (IHC) on tumor microarrays
Differential staining intensity was analyzed within 1334 first biopsy tumor tissue spots on the bladder cancer TMA. Nearly one fifth of the tumors exhibited varying degrees of positive staining, as shown fetaw in Table 8. Examples of E2F3 positive and negative tumors are shown in Figures tfssi 9. E2F3 expression by IHC was significantly linked to E2F3 copy number changes (p«c\.0001). E2F3 positivity was found in 57 of 106 amplified TCCs (53.8%), but in only m&f 640 (14.22%) non- amplified tumors. This association held also true within the
Figure imgf000027_0001
pT1-4 TCCs that could be analyzed by both FISH and IHC (p <0.0001). Among thes^'tømors there were 253 (63.6%) tumors that were negative for both IHC and FISH, 4f Y&ϋ . E2F3 amplification only (10.3%), 50 with strong IHC positivity only (12.56%), and 54 ' vs, ϊ, both strong expression and amplification (13.57%). E2F3 positivity was sigπ ifea i ;more frequent in small cell carcinomas (55.6% positive) than in other histology suti'^^Table 8). Within transitional cell carcinomas, E2F3 expression was linked to advanced :&Jege, and high grade (p<0.0001 each). The frequency of E2F3 positive tumors increaseάfrom 10% in pTaG1/G2 tumors (49 of 492) to 20.9% in invasively growing pT1-4 TCCs (1'25Tof 608). Table 8 E2F3 Alterations and Tumor Phenotype in Urinary Bladder Cancer
Figure imgf000028_0001
oe
Figure imgf000029_0001
a) number of tumors with interpretable result^ only first biopsies are considered) b) only pT2-4 c) only TCC d) amplified versus non-amplified tumog e) pT1 -excluded amp: RATIO >= 3 gain: RATIO >=1.5
E2F3 expression and tumor cell proliferation (Ki67 LI)
Both gene amplification and protein overexpression were significantly associated with rapid tumor cell proliferation (p<0.0001 each). ANOVA analysis including E2F3 expression/amplification and either tumor stage or grade showed that both E2F3 expression and amplification were independent predictors of rapid tumor cell proliferation (p<0.0001 each). Accordingly, the separate analyses of tumors of identical grades and stages lead to either significant differences in the proliferation between E2F3 negative and positive tumors or at least to a clear tendency towards a higher Ki67 LI in E2F3 positive tumors (Table 9).
Table 9 E2F3 Amplification/Overexpression in Relation to Ki-67 LI
Figure imgf000031_0001
Only first i rø fsies of patients with TCC (n=1953) included. Non-amplified cases include gains, a^number of analyzable samples, b) Mean Ki-67 LI. c) 95% Cl of Ki- 67 LI. d) Student'aT-Test
IHC negative: neg/weak. IHC positive: mod/strong E2F3 alterations and prognosis
Both E2F3 amplification and E2F3 expression were associated with poor tumor specific survival if all patients were included in the analysis (Figures 10 and 11). There was no association between E2F3 alterations and tumor specific survival within the subgroup of pT2-4 TCCs. E2F3 amplification and E2F3 overexpression provided no prognostic significance among pTa/ pT1 tumors, neither for recurrence nor for progression.
Previous CGH studies have repeatedly highlighted 6p22 as an amplification site in bladder cancer (Richter, J., et al., Am J Pathol, 153: 1615-1621 (1998); Richter, J., et al., Cancer Res, 59: 5687-5691 (1999). No known oncogene has previously been linked to this chromosomal alteration. A rapid search of the publicly available genome databases revealed that E2F3, a key gene for G1/S transition (Leone, G., et al., Genes Dev, 12: 2120-30 (1998), has been mapped to 6p22. A PAC probe containing the E2F3-specific STS marker stSG15990 was ordered from the Sanger Centre, Cambridge UK, to determine whether E2F3 is present within the 6p22 amplicon. The application of a mini-TMA composed of CGH amplified tumors allowed us tα.directly assess the amplification status of E2F3 not only in primary bladder tumors,
Figure imgf000032_0001
in bladder cancer cell lines, in a single hybridization experiment.
The presence of E2F3,.in the 6p22 amplicons of all tumors (preselected according to the CGH data) prove , strong evidence for an involvement of E2F3 in bladder cancer and prompted us to I fe. analyze its role in urinary bladder tumors. Because Western blot analysis suggested P&&R.2F3 amplification can lead to E2F3 overexpression, we proceeded to analyze the pres?@& a©e.and significance of E2F3 amplification/ expression in clinical tumor samples on ourφfea cter cancer TMA containing tissue from 2317 different tumors (Richter, J., et al., mλPathol, 157: 787-94 (2000).
Using the TMA approach, E2F3 overexpression by IHC was found in 53:&?-3.i ) of E2F3 amplified, but in only 14.2% of non-amplified tumors. Despite the strong association between E2F3 amplification and overexpression, these data suggest that E2F3 overexpression is not present in all amplified tumor samples. This finding could be in par xiue to technical reasons, as none of the currently commercially available antibodies- targeting E2F3 (including that one used in our study) is specifically recommended for immunohistochemistry on formalin fixed tissues. It is possible, therefore, that the use of a non-optimized antibody has resulted in an unusual high fraction of IHC false negative samples. However, the lack of detectable protein expression also in 1/4 E2F3 amplified bladder cancer cell lines analyzed by Western blotting supports the IHC findings, raising the possibility that E2F3 protein expression may be down-regulated in some 6p22 amplified tumors, e.g. by post- transcriptional or post-translational regulatory mechanisms that are involved in fine- tuning functionally active E2F3 levels. Alternatively, our data could indicate that E2F3 is not the (only) amplification target at 6p22, but that overexpression of one or several other genes in the same amplicon is required to drive the growth advantage of amplified cells in 6p22 amplified tumors without E2F3 protein overexpression. There is a growing amount of evidence indicating that the molecular mechanisms of gene amplification does not follow the simple one gene-one amplicon concept. Amplification may be a mechanism that is particularly effective to simultaneously overexpress multiple adjacent genes which may jointly provide a growth advantage to amplified tumor cells. For example, neighboring oncogenes that sometimes undergo coamplification have recently been identified at various genomic regions such as MDM2, GLI, CDK4, and SAS at 12q13-q15 (Reifenberger, G„ et al., Cancer Res, 54: 4299-303 (1994) or CCNDI , EMS1 , and INT2 at 11q13 (Hui, R., et al., Oncogme 15: 1617-23 (1997). Only few known genes are located in direct genomic neighborhooϊsfciQf E2F3. CDK5AL1 , a gene of unknown function that shares protein sequence similafB*«,,with CDK5RAP1 (which is involved in the regulation of neuronal differentiation) maps^Lte&egion adjacent to E2F3. SOX4, a transcription factor that is a member of the high mobfiiϋ^^sup (HMG)-box family of DNA binding protein (Farr, C.J., et al., Mamm Genome, 4: 57ϊ^ 1993), and PRL (the gene encoding
Prolactin) map to region between 1 and'-i iScwetromeric to E2F3. Prolactin (PRL) is a protein hormone closely related to growth hor T a^scl, mainly secreted in the anterior pituitary lactotrope and the decidualized stromai '^tt' ^e. human endometrium (DiMattia, G.E., et al., J Biol Chem, 265: 16412-21 φi However, Northern analysis demonstrated no correlation between amplification and'' overexpression of these genes in 8 cell lines (Bruch, J., et al., Cancer Res, 60: 4SK«% 30 (2000). Transcripts termed BIG1 and BIG2 represent genes that map to the core GS-BIG region and are overexpressed at least at the mRNA level. Thus, these genes may either alone or in combination with each other form an oncogene cluster that provides a growth advantage to bladder cancer cells. The occurrence of a subgroup of tumors having unequivocal E2F3 protein overexpression in the absence of amplification suggests that other mechanisms than amplification may also cause E2F3 activation in bladder cancer.
The strong and independent association of E2F3 expression with tumor cell proliferation, and the central role of E2Fs in cell cycle control, supports an oncogenic role of E2F3 alterations through cell cycle activation. This is consistent with the known role of E2F3 as a gene activator essentially required for S-phase induction (Leone, G., et al., Genes Dev, 12: 2120-30 (1998). However, accelerated cell proliferation is an important prerequisite for tumor growth, but it does not necessarily determine aggressiveness. Other factors, like invasive growth and, most importantly, the metastatic potential of a tumor are key determinants for the clinical outcome of a cancer patient.
Using a bladder cancer TMA resource allowed the rapid and successful analysis of more than 1000 carcinomas on both DNA and protein level demonstrating the advantages of the microarray format for large-scale molecular analysis on clinical tissue material. The findings suggest an important role of E2F3 amplification/overexpression in invasive bladder cancer. A total of 29.2% of muscte. invasive bladder cancers showed E2F3 amplification. To our knowledge, this makes E2F3 the most frequently amplified gene in invasive bladder cancer. Using the same methodological criteria including FISH protocols, copy number cutoff levels and TMA resources? ,we have observed a lower frequency fur amplifications of HER2 (14%) and CCND1 (1R%) in pT2-4 bladder cancer (unpublished data). Remarkably, E2F3 amplification was i&'s .in only 4 of 398 pTaG1/G2 tumors (1%). Thus, 6p22 amplification detection eoϊi-ϊ have clinical utility in the difficult cytological distinction of low and high malignant bladdes Λ «?pla.sm's on voided urine cells. Based on an expected fraction of 65% pTaG1/G2'tάϊ f^ .in consecutive series of bladder tumors, the positive predictive value of E2F3 amplifiόai^a'før. invasive tumor growth or high grade would be about 93%. The marked discrepano^Urt e-frequency of E2F3 amplification between pTaG1/G2 and pT1-4 carcinomas is Ge&si&tetrt with models suggesting that pTaG1/G2 tumors are genetically stable tumors wiUrøm∑ich lower likelihood to acquire chromosomal rearrangements than invasively growing tørøars that are characterized by a much higher number of genomic alterations (Richter, JJ, et al., Am J Pathol, 153: 1615-1621 (1998); Richter, J., et al., Cancer Res, 57: 2860- 2864 (1997); Richter, J., et al., Cancer Res, 59: 5687-5691 (1999); Simon, R., et al., J Pathol, 185: 345-351 (1998); Simon, R., et al., Cancer Res, 61 : 4514-4519 (2001); Simon, R., et al., Oncogene, 21: 2476-83 (2002); Zhao, J., et al., Cancer Res, 59: 4658-4661 (1999).
In summary, E2F3 is included in a bladder cancer amplicon at 6p22. Taking together the known cell cycle activating role of E2F3, its overexpression in amplified tumors and the association with cell proliferation in vivo, it appears that E2F3 could be one of probably multiple target genes inside the 6p22 amplification whose overexpression gives growth advantage to tumor cells. The present invention demonstrates how a combination of genomic technologies, microarray discovery platforms and bioinformatics resources can be used to rapidly identify, validate and characterize target genes for genetic alterations and associate these changes to specific medical conditions.
Detection of 6p22 Abnormalities
One of skill in the art will appreciate that the clones and sequence information provided herein can be used to detect amplifications, or other chromosomal abnormalities, at 6p22 in a biological sample. Generally the methods involve hybridization of probes that specifically bind one or more nucleic acid sequences of the target amplicon with nucleic acids present in a biological sample or derived from ^biological sample. As used herein, a biological sample is a sample of biological tissue or fluid containi«-|,;τ-?Jls desired to be screened for chromosomal abnormalities (e.g. amplifications undeletions). In a preferred embodiment, the biological sample is a cell or tissue suspeot&i*&f being cancerous (transformed). Methods of isolating biological samples are
Figure imgf000035_0001
to those of skill in the art and include, but are not limited to, aspirations, tissue
Figure imgf000035_0002
biopsies, and the like. Frequently the sample will be a "clinical sample"
Figure imgf000035_0003
derived from a patient. It will be recognized that the term "sample" also includes- si-tpsernatant (containing cells) or the cells themselves from cell cultures, cells from tissue
Figure imgf000035_0004
other media in which it may be desirable to detect chromosomal abnormalities. In a preferred embodiment, a biological sample is prepared by
Figure imgf000035_0005
cells, either as single cell suspensions or as tissue preparation, on solid suppαrta such as glass slides and fixed by choosing a fixative which provides the best spalist resolution of the cells and the optimal hybridization efficiency. Selecting Probes
Any of the SEQ ID NOs, or the cDNAs disclosed herein are suitable for use in detecting the 6p22 amplicon. Methods of preparing probes are well known to those of skill in the art (see, e.g. Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989) or Current Protocols in Molecular Biology, F. Ausubel et al., ed. Greene Publishing and Wiley-lnterscience, New York (1987)).
The probes are most easily prepared by combining and labeling one or more of the SEQ ID NOs. Prior to use, the constructs are fragmented to provide smaller nucleic acid fragments that easily penetrate the cell and hybridize to the target nucleic acid. Fragmentation can be by any of a number of methods well known to hose of skill in the art. Preferred methods include treatment with a restriction enzyme to selectively cleave the molecules, or alternatively to briefly heat the nucleic acids in the presence of Mg2+. Probes are preferably fragmented to an average fragment length ranging from about 50 bp to about 2000 bp, more preferably from about 100 bp to about 1000 bp and most preferably from about 150 bp to about 500 bp.
Alternatively, probes can be produced by amplifying (e.g. via PCR) selected subsequences from the 6p22 amplicon disclosed herein. The sequences provided herein permit one of skill to select primers that amplify sequences from one or more exons located within the 6p22 amplicon.
Particularly preferred probes include nucleic acids from probes RP1-46C2, RP1-2??tvfi.i, RP-177P22, RP-44C7 and RP3-348I23, which corresponds to genomic region betwefeϋ<rSTS markers G717699 and G26264. In addition, the cDNAs are particularly useful Tώs^sntifying cells that have increased expression of the corresponding genes,
Figure imgf000036_0001
instance, Northern blot analysis.
One of skill will appreciate t? &:®i.ng the sequence information and clones provided herein, one of skill in the art carriώ&feis,the same or similar probes from other human genomic libraries using routine mei oc i^g. Southern or Northern Blots). Labeling Probes
Methods of labeling nucleic acids are well known to those of sk»ft the art. Preferred labels are those that are suitable for use in in situ hybridization. Tfte,. nucleic acid probes may be detectably labeled prior to the hybridization reaction: Alternatively, a detectable label which binds to the hybridization product may be used. Such detectable labels include any material having a detectable physical or chemical property and have been well-developed in the field of immunoassays.
As used herein, a "label" is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Useful labels in the present invention include radioactive labels (e.g. 32 P, 125l, 14C, 3H, and 35S), fluorescent dyes (e.g. fluorescein, rhodamine, Texas Red, etc.), electron-dense reagents (e.g. gold), enzymes (as commonly used in an ELISA), colorimetric labels (e.g. colloidal gold), magnetic labels (e.g. Dynabeads™), and the like. Examples of labels which are not directly detected but are detected through the use of directly detectable label include biotin and dioxigenin as well as haptens and proteins for which labeled antisera or monoclonal antibodies are available.
The particular label used is not critical to the present invention, so long as it does not interfere with the in situ hybridization of the stain. However, stains directly labeled with fluorescent labels (e.g. fluorescein-12-dUTP, Texas Red-5-dUTP, etc.) are preferred for chromosome hybridization.
A direct labeled probe, as used herein, is a probe to which a detectable label is attached. Because the direct label is already attached to the probe, i subsequent steps are required to associate the probe with the detectable label. In contrss%,an indirect labeled probe is one which bears a moiety to which a detectable label is subsequently bound, typically after the probe is hybridized with the target nucleic acfέt±.
In addition the label must be detectible in as low copy number as possible thereby/ ximizing the sensitivity of the assay and yet be detectible above any backgrounti;y» 8i. Finally, a label must be chosen that provides a highly localized signal thereby provt5 j;:a,high degree of spatial resolution when physically mapping the stain against the chrorvm s' Particularly preferred fluorescent labels include fluorescein-12-dUTP and Texas W& fcs&dUTP.
The labels may be coupled to the pr fets. a variety of means known to those of skill in the art. In a preferred embodimenttJf^nucleic acid probes will be labeled using nick translation or random primer extension (Rsghy, et al., J. Mol. Biol., 113: 237 (1977) or Sambrook, et al., Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1985)). One of skill in the art will appreciate that the probes of this invention need not be absolutely specific for the targeted 6p22 region of the genome. Rather, the probes are intended to produce "staining contrast." "Contrast" is quantified by the ratio of the probe intensity of the target region of the genome to that of the other portions of the genome. For example, a DNA library produced by cloning a particular chromosome (e.g. chromosome 7) can be used as a stain capable of staining the entire chromosome. The library contains both sequences found only on that chromosome, and sequences shared with other chromosomes. Roughly half the chromosomal DNA falls into each class. If hybridization of the whole library were capable of saturating all of the binding sites on the target chromosome, the target chromosome would be twice as bright (contrast ratio of 2) as the other chromosomes since it would contain signal from the both the specific and the shared sequences in the stain, whereas the other chromosomes would only be stained by the shared sequences. Thus, only a modest decrease in hybridization of the shared sequences in the stain would substantially enhance the contrast. Thus contaminating sequences which only hybridize to non-targeted sequences, for example, impurities in a library, can be tolerated in the stain to the extent that the sequences do not reduce the staining contrast below useful levels.
Detecting the 6p22 Amplicon As explained above, detection of amplification in the 6p22 amplicon r indicative of the presence and/or prognosis of a bladder cancers.
In a preferred embodiment, a 6p22 amplification is detected through the hybridization of a probe of this invention to a target nucleic acid (e.g. a chromosomal ya «:(i» n which it is desired to screen for the amplification. Suitable hybridization formats -ώϋrøϊAknown to those of skill in the art and include, but are not limited to, variations of Sou't'fesrBlots, in situ hybridization and quantitative amplification methods such as quantit fe«RGR (see, e.g. Sambrook, supra, Kallioniemi, et al., Proc. Natl Acad Sci USA, 89: 5321^^5.(1992), and PCR Protocols, A Guide to Methods and Applications, Innis et al., N^s^ Ac. Press, Inc. N.Y., (1990)). In situ Hybridization
In a preferred embodiment, the 6p22 amplicon is fdeotified using in situ hybridization. Generally, in situ hybridization comprises the following major steps: (1) fixation of tissue or biological structure to analyzed; (2) prehybridization treatment of the biological structure to increase accessibility of target DNA, and to reduce nonspecific binding; (3) hybridization of the mixture of nucleic acids to the nucleic acid in the biological structure or tissue; (4) posthybridization washes to remove nucleic acid fragments not bound in the hybridization and (5) detection of the hybridized nucleic acid fragments. The reagent used in each of these steps and their conditions for use vary depending on the particular application.
In some applications it is necessary to block the hybridization capacity of repetitive sequences. In this case, human genomic DNA is used as an agent to block such hybridization. The preferred size range is from about 200 bp to about 1000 bases, more preferably between about 400 to about 800 bp for double stranded, nick translated nucleic acids.
Hybridization protocols for the particular applications disclosed here are described in Pinkel, et al., Proc. Natl. Acad. Sci. USA, 85: 9138-9142 (1988) and in EPO Pub. No. 430,402. Suitable hybridization protocols can also be found in Methods o/in Molecular Biology Vol. 33: In Situ Hybridization Protocols, K. H. A. Choo, ed., Humana Press, Totowa, N.J., (1994). In a particularly preferred embodiment, the hybridization protocol of Kallioniemi, et al., Proc. Natl Acad Sci USA, 89: 5321-5325 (1992) is used.
Typically, it is desirable to use dual color FISH, in wifeb two probes are utilized, each labeled by a different fluorescent dye. A test probu't' t hybridizes to the region of interest is labeled with one dye, and a control probe tharø ridizes to a different region is labeled with a second dye. A nucleic acid that hybridizes^a stable portion of the chromosome of interest, such as the centromere region, ia- fer). most useful as the control probe. In this way, differences between efficiency of hybridization from sample to sample can be accounted for. T ιa.FISH methods for detecting chromosomal abnormalities can be performed Orrrwrøgram quantities of the subject nucleic acids. Paraffin embedded tumor sections can to-f ^,«ad.r as can fresh or frozen material. Because FISH can be applied to the limited material'? Huch. preparations prepared from uncultured primary tumors can also be used (see, e.g., ss oniemi, A., et al., Cytogenet. Cell Genet, 60: 190-193 (1992)). For instance, small biopsy tissue, samples from tumors can be used for touch preparations (see, e.g., Kallioniemi;' A., et al., Cytogenet. Cell Genet, 60: 190-193 (1992)). Small numbers of cells obtained from aspiration biopsy or cells in bodily fluids (e.g., blood, urine, sputum and the like) can also be analyzed. For prenatal diagnosis, appropriate samples will include amniotic fluid and the like.
Southern Blots
In a Southern Blot, a genomic or cDNA (typically fragmented and separated on an electrophoretic gel) is hybridized to a probe specific for the target region. Comparison of the intensity of the hybridization signal from the probe for the target region (e.g., 6p22) with the signal from a probe directed to a control (non amplified) such as centromeric DNA, provides an estimate of the relative copy number of the target nucleic acid. Detecting Mutations in Genes from the 6p22 Amplicon
The cDNA sequences disclosed here can also be used for detecting mutations (e.g., substitutions, insertions, and deletions) within the corresponding endogenous genes. One of skill will recognize that the nucleic acid hybridization techniques generally described above can be adapted to detect such much mutations. For instance, oligonucleotide probes that distinguish between mutant and wild-type forms of the target gene can be used in standard hybridization assays. In some embodiments, amplification (e.g., using PCR) can be used to increase copy number of the target sequence prior to hybridization.
Kits Containing 6p22 Amplicon Probes This invention also provides diagnostic kits for the
Figure imgf000040_0001
of chromosomal abnormalities at 6p22. In a preferred embodiment, the kits include vim, or more probes to the 6p22 amplicon described herein. The kits can additionaliy'i ϊ e blocking probes, instructional materials describing how to use the kit contents in;'* detecting 6p22 amplicons. The kits may also include one or more of the following: vasrøsalabels or labeling agents to facilitate the detection of the probes, reagents for the hybri Bjrøfen including buffers, a metaphase spread, bovine serum albumin (BSA) and otherl^se ing agents, sampling devices including fine needles, swabs, aspirators and the like, stle, and negative hybridization controls and so forth.
Expression of cDNA clones One may express the desired ρε >tides encoded by the cDNA clones disclosed here in a recombinantly engineered -Y l such as bacteria, yeast, insect (especially employing baculoviral vectors), and mammalian cells. It is expected that those of skill in the art are knowledgeable in the numerous. expression systems available for expression of the cDNAs. No attempt to describe in detail the various methods known for the expression of proteins in prokaryotes or eukaryotes will be made.
In brief summary, the expression of natural or synthetic nucleic acids 5 encoding polypetides of the invention will typically be achieved by operably linking the DNA or cDNA to a promoter (which is either constitutive or inducible), followed by incorporation into an expression vector. The vectors can be suitable for replication and integration in either prokaryotes or eukaryotes. Typical expression vectors contain transcription and translation terminators, initiation sequences, and promoters 0 useful for regulation of the expression of the DNA encoding the polypeptides. To obtain high level expression of a cloned gene, it is desirable to construct expression plasmids which contain, at the minimum, a strong promoter to direct transcription, a ribosome binding site for translational initiation, and a transcription/translation terminator.
15 Examples of regulatory regions suitable for this purpose in E. coli are the promoter and operator region of the E. coli tryptophan biosynthetic pathway as described by Yanofsky, O, J. Bacteriol., 158:1018-1024 (1984) and the leftward promoter of phage lambda (PL) as described by Herskowitz, I. and Hagen, D., Ann. Rev. Genet, 14:399-445 (1980). The inclusion of selection markers in DNA vectors
20 transformed in E. coli is also useful. Examples of such markers include genes specifying resistance to ampicillin, tetracycline, or chlorømphenicol. Expression systems are available using E. coli, Bacillus sp. Palva, I., & (*L, Gene, 22:229-235 (1983); Mosbach, K., et al., Nature, 302:543-545 and Salmoneii$sR.,coli systems are preferred.
25. The polypeptides produced by prokaryote cells may not necessarily 1 properly. During purification from E. coli, the expressed polypeptides may first be deiϊδfteif.and then renatured. This can be accomplished by solibilizing the bacterialiy μ tføced proteins in a chaotropic agent such as guanidine HCI and reducing all the cyst ac&residues with a reducing agent such as beta-
30 mercaptoethanol. The
Figure imgf000041_0001
are then renatured, either by slow dialysis or by gel filtration. U.S. Pat. No. 4,5Ti?5ft3;, which is hereby incorporated by reference. A variety of eukaryotic expression systems such as yeast, insect cell lines and mammalian cells, are known to those of skill in the art. As explained briefly below, the polypeptides may also be expressed in these eukaryotic systems.
Synthesis of heterologous proteins in yeast is well known and described. Methods in Yeast Genetics, Sherman, F., et al., Cold Spring Harbor Laboratory
(1982) is a well recognized work describing the various methods available to produce the polypeptides in yeast. A number of yeast expression plasmids like YEp6, YEp13, YEp4 can be used as vectors. A gene of interest can be fused to any of the promoters in various yeast vectors. The above-mentioned plasmids have been fully described in the literature (Botstein, et al., Gene, 8:17-24 (1979); Broach, et al., Gene, 8:121-133 (1979)).
Illustrative of cell cultures useful for the production of the polypeptides are cells of insect or mammalian origin. Mammalian cell systems often will be in the form of monolayers of cells although mammalian cell suspensions may also be used. Illustrative examples of mammalian cell lines include VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, W138, BHK, Cos-7 or MDCK cell lines.
As indicated above, the vector, e. g., a plasmid, which is used to transform the host cell, preferably contains DH/ sequences to initiate transcription and sequences to control the translation of
Figure imgf000042_0001
gene sequence. These sequences are referred to as expression
Figure imgf000042_0002
When the host cell is of insect or mammalian origin illustrative expression ca^rol sequences are often obtained from the SV-40 promoter (Science, 222:524-52?;?/? 983), the CMV I.E. Promoter (Proc. Natl. Acad. Sci., 81:659-663 (1984)) or the meWsv hionein promoter (Nature, 296:39-42 (1982). The cloning vector containing the expressfes control sequences is cleaved using restriction enzymes and adjusted in size as neos ssy or desirable and ligated with the desired DNA by means well known in the art.
As with yeast, when higher animal host cells are employed, polyadenlyation or trarrs«? ion terminator sequences from known mammalian genes need to be incorporated iiifάihe vector. An example of a terminator sequence is the polyadenlyation sequescelrom the bovine growth hormone gene. Sequences for accurate splicing of the transcript may also be included. An example of a splicing sequence is the VP1 intron from SV40 (Sprague, J. et al., J. Virol., 45: 773-781 (1983)). Additionally, gene sequences to control replication in the host cell may be incorporated into the vector such as those found in bovine papilloma virus type- vectors. Saveria-Campo, M., 1985, "Bovine Papilloma virus DNA a Eukaryotic Cloning Vector" in DNA Cloning Vol. 11 a Practical Approach Ed. D. M. Glover, IRL Press, Arlington, Va. pp. 213-238.
The nucleic acids and encoded polypeptides of the invention can be used directedly to inhibit the endogenous genes or their gene products. For instance, inhibitory nucleic acids may be used to specifically bind to a complementary nucleic acid sequence. By binding to the appropriate target sequence, an RNA-RNA, a DNA-DNA, or RNA-DNA duplex is formed. These nucleic acids are often termed
"antisense" because they are usually complementary to the sense or coding strand of the gene, although approaches for use of "sense" nucleic acids have also been developed. The term "inhibitory nucleic acids" as used herein, refers to both "sense" and "antisense" nucleic acids. Inhibitory nucleic acid methods encompass a number of different approaches to altering expression of specific genes that operate by different mechanisms.
In brief, inhibitory nucleic acid therapy approaches can be classified into those that target DNA sequences, those that target RNA sequences (including pre- mRNA and mRNA), those that target proteins (sense strand approaches), and those that cause cleavage or chemical modification of the target nucleic acids (ribozymes). These different types of inhibitory nucleic acid;';?chnology are described, for instance, in Helene, C. and Toulme, J., Biochim. Biophys. Λ -^.1049: 99-125 (1990). Inhibitory nucleic acid complementary to regions of c-my^vraRNA has been shown to inhibit c-myc protein expression in a human promyelocytic leufe&irø. cell line, HL60, which overexpresses the c-myc protoncogene. See Wickstrom E. L., s &L, PNAS (USA), 85: 1028-1032 (1988) and Harel-Bellan, A., et al., Exp. Med., 168T-2SS0b2318 (1988).
The encoded polypeptides of the invention can also be used to design molecu!© ,«{ aρtidic or nonpeptidic) that inhibit the endogenous proteins by, for instance,
Figure imgf000043_0001
between the protein and a second molecule specifically recognized toy the protein. Methods for designing such molecules are well known to those skilled in.the art. For instance, polypeptides can be designed which have sequence identity with the encoded proteins or may comprise modifications (conservative or non- conservative) of the sequences. The modifications can be selected, for example, to alter their in vivo stability. For instance, inclusion of one or more D-amino acids in the peptide typically increases stability, particularly if the D-amino acid residues are substituted at one or both termini of the peptide sequence.
The polypeptides can also be modified by linkage to other molecules. For example, different N- or C-terminal groups may be introduced to alter the molecule's physical and/or chemical properties. Such alterations may be utilized to affect, for example, adhesion, stability, bio-availability, localization or detection of the molecules. For diagnostic purposes, a wide variety of labels may be linked to the terminus, which may provide, directly or indirectly, a detectable signal. Thus, the polypeptides may be modified in a variety of ways for a variety of end purposes while still retaining biological activity. The term "candidate bioactive agent" or "drug candidate" or grammatical equivalents as used herein describes any molecule, e.g., protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, etc., to be tested for bioactive agents that are capabfø "<sf directly or indirectly altering the cancer phenotype or the expression of an expressed' rerøleic acid sequence, positioned with the 6p22 region, identified herein, including both r - c acid sequences and protein sequences. In preferred embodiments, the bioactive ge ts modulate the expression profiles, or expression profile nucleic acids or proteins ovided herein. In a particularly preferred embodiment, the candidate agent su i&sw&es a cancer phenotype, for example to a normal tissue fingerprint. Similarly, the ctsfalisiate agent preferably suppresses a severe cancer phenotype. Generally a plurality /tessay mixtures are run in parallel with different agent concentrations to obtain a differen ife'iresponse to the various concentrations. Typically, one of these concentrations serves a&S t negative control, i.e., at zero concentration or below the level of detection.
In one aspect, a candidate agent will neutralize the effect of E2F3, BIG-1 or BIG- .φ * By "neutralize" is meant that activity of a protein is either inhibited or counter acte ^gølnst so as to have substantially no effect on a cell.
Candidate agents encompass numerous chemical classes, though typically they are organic molecules; preferably small organic compounds having a molecular weight of more than 100 and less than about 2,500 daltons. Preferred small molecules are less than 2000, or less than 1500 or less than 1000 or less than 500 D. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Particularly preferred are peptides.
Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical andϊbiochemical means. Known pharmacological agents may be subjected to directed or rartAom chemical modifications, such as acylation, alkylation, esterification, amidification to roduce structural analogs.
In a preferred embodiment, lifecandidate bioactive agents are proteins. By "protein" herein is meant at least two cσws"l»& .attached amino acids, which includes proteins, polypeptides, oligopeptides and peptidfe;>j-The protein may be made up of naturally occurring amino acids and peptide bonds,
Figure imgf000045_0001
structures. Thus "amino acid", or "peptide residue", as
Figure imgf000045_0002
both naturally occurring and synthetic amino acids. For example, homo-p e ^'s^i'ne, citrulline and noreleucine are considered amino acids for the purposes of the invention. "Amino acid" also includes imino acid residues such as proline and
Figure imgf000045_0003
The side chains may be in either the (R) or the (S) configuration. In the prefe re embodiment, the amino acids are in the (S) or L-configuration. If non- naturally occurring side chains are used, non-amino acid substituents may be used, for example to
Figure imgf000045_0004
or retard in vivo degradations. In a preferred embodiment, the candidate bioactive agents are naturally occurring proteins or fragments of naturally occurring proteins. Thus, for example, cellular extracts containing proteins, or random or directed digests of proteinaceous cellular extracts, may be used. In this way libraries of procaryotic and eucaryotic proteins may be made for screening in the methods of the invention. Particularly preferred in this embodiment are libraries of bacterial, fungal, viral, and mammalian proteins, with the latter being preferred, and human proteins being especially preferred.
In a preferred embodiment, the candidate bioactive agents are peptides of from about 5 to about 30 amino acids, with from about 5 to about 20 amino acids being preferred, and from about 7 to about 15 being particularly preferred. The peptides may be digests of naturally occurring proteins as is outlined above, random peptides, or "biased" random peptides. By "randomized" or grammatical equivalents herein is meant that each nucleic acid and peptide consists of essentially random nucleotides and amino acids, respectively. Since generally these random peptides (or nucleic acids, discussed below) are chemically synthesized, they may incorporate any nucleotide or amino acid at any position. The synthetic process can be designed to generate randomized proteins or nucleic acids, to allow the formation of all or most of the possible combinations over the length of the sequence, thus forming a library of randomized candidate bioactive proteinaceous agents.
In one embodimer^Lthe library is fully randomized, with no sequence preferences or constants at an^position. In a preferred embodiment, the library is biased. That is, some positions wif s-the sequence are either held constant, or are selected from a limited number of possi&iifes. For example, in a preferred embodiment, the nucleotides or amino acid res Srø are randomized within a defined class, for example, of hydrophobic amino acids, hydrϋp?iSκs;asidues, sterically biased (either small or large) residues, towards the creation of
Figure imgf000046_0001
domains, the creation of cysteines, for cross-linking, prolines for SH-3 domains, &s x@s.,. threonines, tyrosines or histidines for phosphorylation sites, etc., or to purinesv.ete- In a preferred embodiment, the candidate bioactive agents are nucleic acids, a&xfepianed above.
As de-scribed above generally for proteins, nucleic acid candidate bioactive agents may be naturally occurring nucleic acids, random nucleic acids, or "biased" random nucleic acids. For example, digests of procaryotic or eucaryotic genomes may be used as is outlined above for proteins.
In a preferred embodiment, the candidate bioactive agents are organic chemical moieties, a wide variety of which are available in the literature. After the candidate agent has been added and the cells allowed to incubate for some period of time, the sample containing the target sequences to be analyzed is added to the biochip. If required, the target sequence is prepared using known techniques. For example, the sample may be treated to lyse the cells, using known lysis buffers, electroporation, etc., with purification and/or amplification such as PCR occurring as needed, as will be appreciated by those in the art. For example, an in vitro transcription with labels covalently attached to the nucleosides is done. Generally, the nucleic acids are labeled with biotin-FITC or PE, or with cy3 or cy5.
In a preferred embodiment, the target sequence is labeled with, for example, a fluorescent, a chemiluminescent, a chemical, or a radioactive signal, to provide a means of detecting the target sequence's specific binding to a probe. The label also can be an enzyme, such as, alkaline phosphatase or horseradish peroxidase, which wten provided with an appropriate substrate produces a product that can be detected. Alternatively, the label can be a labeled compound or small molecule, such as an enzyme inhϊESfe, that binds but is not catalyzed or altered by the enzyme. The label also can be a mofefe or compound, such as, an epitope tag or biotin which specifically binds to streptavidin. Fs&the example of biotin, the streptavidin is labeled as described above, thereby, arting a detectable signal for the bound target sequence. As known in the art, unbound
Figure imgf000047_0001
is removed prior to analysis.
As will be appreciated by those in life rt,, these assays can be direct hybridization assays or can comprise
Figure imgf000047_0002
which include the use of multiple probes, as is generally outlined in U.S. Pat. N6s?.5i.6&1 ,702; 5,597,909;
5,545,730; 5,594,117; 5,591 ,584; 5,571,670; 5,580,731 ; 5, 57 f '45, 591,584;
5,624,802; 5,635,352; 5,594,118; 5,359,100; 5,124,246; and 5,681 , 693f;.aft,af which are hereby incorporated by reference. In this embodiment, in general, the tart fe:; nucleic acid is prepared as outlined above, and then added to the biochip comprising?; a plurality of nucleic acid probes, under conditions that allow the formation of a hybridization complex. A variety of hybridization conditions may be used in the present invention, including high, moderate and low stringency conditions as outlined above. The assays are generally run under stringency conditions which allows formation of the label probe hybridization complex only in the presence of target. Stringency can be controlled by altering a step parameter that is a thermodynamic variable, including, but not limited to, temperature, formamide concentration, salt concentration, chaotropic salt concentration pH, organic solvent concentration, etc.
These parameters may also be used to control non-specific binding, as is generally outlined in U.S. Pat. No. 5,681 ,697. Thus it may be desirable to perform certain steps at higher stringency conditions to reduce non-specific binding.
The reactions outlined herein may be accomplished in a variety of ways, as will be appreciated by those in the art. Components of the reaction may be added simultaneously, or sequentially, in any order, with preferred embodiments outlined below. In addition, the reaction may include a variety of other reagents may be included in the assays. These include reagents like salts, buffers, neutral proteins, e.g. albumin, detergents, etc which may be used to facilitate optimal hybridization and detection, and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease røhibitors, anti-microbial agents, etc., may be used, depending on the sample preparation methods and purity of the target. Once the assay is run, the data is analyzed to detøfsnine the expression levels, and changes in expression levels as between states, of indP ual genes, forming a gene expression profile.
The screens are don&Φiύanitfy drugs or bioactive agents that modulate the cancer phenotype. Specifically,
Figure imgf000048_0001
types of screens that can be run. A preferred embodiment is in the screenuig- feandidate agents that can induce or suppress a particular expression profile, thus pre1fer:fe ;generating the associated phenotype. That is, candidate agents that can mimic or poiss ®. an expression profile in, for example, bladder cancer similar to the expression μrøffe:. of normal bladder tissue is expected to result in a suppression of the bladder cantsjsiptienotype. Thus, in this embodiment, mimicking an expression profile, or changing one prcϊδteio another, is the goal.
In a preferred embodiment, as for the diagnosis and prognosis applications, having;identified the expressed genes important in any one state, screens can be run to alter the expression of the genes individually. That is, screening for modulation of regulation of expression of a single gene can be done; that is, rather than try to mimic all or part of an expression profile, screening for regulation of individual genes can be done. Thus, for example, particularly in the case of target genes whose presence or absence is unique between two states, screening is done for modulators of the target gene expression.
In a preferred embodiment, screening is done to alter the biological function of the expression product of the expressed gene. Again, having identified the importance of a gene in a particular state, screening for agents that bind and/or modulate the biological activity of the gene product can be run as is more fully outlined below.
Thus, screening of candidate agents that modulate the cancer phenotype either at the gene expression level or the protein level can be done.
In addition screens can be done for novel genes that are induced in response to a candidate agent. After identifying a candidate agent based upon its ability to suppress a bladder cancer expression pattern leading to a normal expression pattern, or modulate a single expressed gene expression profile so as to mimic the expression of the gene from normal tissue, a screen as described above can be performed to ;ientify genes that are specifically modulated in response to the agent. Comparing expansion profiles between normal tissue and agent treated cancer tissue reveals genes fr iare not expressed in normal tissue or cancer tissue, but are expressed in agent treate^ ?sue. These agent specific sequences can be identified and used by any of the method&«fcts.cribed herein for expressed genes or proteins. In particular these sequences and
Figure imgf000049_0001
they encode find use in marking or identifying agent treated cells. In addition, antibeeS .can be raised against the agent induced proteins and used to target novel therapeutics fee.treated cancer tissue sample.
Thus, in one embodiment, a candidate a- .vis.administered to a population of bladder cancer cells, that thus has an associated biads';3P,τcancer expression profile. By "administration" or "contacting" herein is meant that the can fete. agent is added to the cells in such a manner as to allow the agent to act upon the ceil;^shether by uptake and intracellular action, or by action at the cell surface. In some embodiments, nucleic acid encoding a proteinaceous candidate agent (i.e. a pepfeie) may be put into a viral construct such as a retroviral construct and added to the cell, such that expression of the peptide agent is accomplished; see PCT US97/01019, hereby expressly incorporated by reference.
Once the candidate agent has been administered to the cells, the cells can be washed if desired and are allowed to incubate under preferably physiological conditions for some period of time. The cells are then harvested and a new gene expression profile is generated, as outlined herein.
Thus, for example, bladder cancer tissue may be screened for agents that reduce or suppress the bladder cancer phenotype. A change in at least one gene of the expression profile indicates that the agent has an effect on bladder cancer activity. By defining such a signature for the particular phenotype, screens for new drugs that alter the phenotype can be devised. With this approach, the drug target need not be known and need not be represented in the original expression screening platform, nor does the level of transcript for the target protein need to change.
In a preferred embodiment, as outlined above, screens may be done on individual genes and gene products (proteins). That is, having identified a particular expressed gene as important in a particular state, screening of modulators of either the expression of the gene or the gene product itself can be done. The gene products of expressed genes are sometimes referred to herein as " expressed proteins" or "cancer modulating proteins." Additionally, "modulator" and "modulating" proteins are sometimes used interchangeably herein. In one embodiment, the t≤s ressed protein is termed E2F3, BIG-1 or BIG-2. These sequences can be iderti^ s described herein for expressed sequences (SEQs). The expressed protein may fSfta.fragment, or alternatively, be the full length protein to the fragment shown herein. fcRfe My, the expressed protein is a fragment. In a preferred embodiment, the amino a :ϊ &squence which is used to determine sequence identity or similarity is that identified as S^CF^ O: 2. In another embodiment, the sequences are naturally occurring alleli'C'vsx3 s&of a protein having the sequence identified as SEQ ID NO: 2. In another embodime?Λi !he.,sequences are sequence variants as further described herein. Preferably, the expressed protein is a fragment of approxiYftft y 14 to 24 amino acids long. More preferably the fragment is a soluble fragment. Preferably, the fragment includes a non-transmembrane region. In a preferred embodiment, the fragment has an N-terminal Cys to aid in solubility. In one embodiment, the C- terminus of the fragment is kept as a free acid and the n-terminus is a free amine to aid in coupling, i.e., to cysteine. Preferably, the fragment of approximately 14 to 24 amino acids long. More preferably the fragment is a soluble fragment. In another embodiment, a E2F3, BIG-1 or BIG-2 fragment has at least one E2F3, BIG-1 or BIG- 2 bioactivity as defined below.
In one embodiment the expressed proteins are conjugated to an immunogenic agent as discussed herein. In one embodiment the expressed protein is conjugated to BSA.
Thus, in a preferred embodiment, screening for modulators of expression of specific genes can be done. This will be done as outlined above, but in general the expression of only one or a few genes are evaluated.
In a preferred embodiment, screens are designed to first find candidate agents that can bind to expressed proteins, and then these agents may be used in assays that evaluate the ability of the candidate agent to modulate expressed activity. Thus, as will be appreciated by those in the art, there are a number of different assays which may be run; binding assays and activity assays.
In a preferred embodiment, binding assays are done. In general, purified or isolated gene product is used; that is, the gene products of one or more expssssed nucleic acids are made. In general, this is done as is known in the art. For exampfe, antibodies are generated to the protein gene products, and standard immunoassays are run to determine the amount of protein present. Alternatively, cells comprising ti'tesxpressed proteins can be used in the assays.
'Hteyπ a preferred embodiment, the methods comprise combining a expressed prolfetørøEKlla candidate bioactive agent, and determining the binding of the candidate agent to th : passed protein. Preferred embodiments utilize the human expressed protein, although
Figure imgf000051_0001
proteins may also be used, for example for the development of animal modeteτ>P'hsman disease. In some embodiments, as outlined herein, variant or derivative expressc :,r^&teins may be used.
Generally, in a preferred embodiment of the metrto^s.herein, the expressed protein or the candidate agent is non-diffusably bound to an insoluble support having isolated sample receiving areas (e.g. a microtiter plate, an array, eit . It is understood that alternatively, soluble assays known in the art may be performed. The insoluble supports may be made of any composition to which the compositions can be bound, is readily separated from soluble material, and is otherwise compatible with the overall method of screening. The surface of such supports may be solid or porous and of any convenient shape. Examples of suitable insoluble supports include microtiter plates, arrays, membranes and beads. These are typically made of glass, plastic (e.g., polystyrene), polysaccharides, nylon or nitrocellulose, teflon™, etc. Microtiter plates and arrays are especially convenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples. The particular manner of binding of the composition is not crucial so long as it is compatible with the reagents and overall methods of the invention, maintains the activity of the composition and is nondiffusable. Preferred methods of binding include the use of antibodies (which do not sterically block either the ligand binding site or activation sequence when the protein is bound to the support), direct binding to "sticky" or ionic supports, chemical cross-linking, the synthesis of the protein or agent on the surface, etc. Following binding of the protein or agent, excess unbound material is removed by washing. The sample receiving areas may then be blocked through incubation with bovine serum albumin (BSA), casein or other innocuous protein or other moiety.
In a preferred embodiment, the expressed protein is bound to the support, and a candidate bioactive agent is added to the assay. Alternatively, the. candidate agent is bound to the support and the expressed protein is added. Novel biding agents include specific antibodies, non-natural binding agents identified in screens of chemical libraries, peptide analogs, etc. Of particular interest are screening assays" feagents that have a low toxicity for human cells. A wide variety of assays may be usedffethis purpose, including labeled in vitro protein-protein binding assays,
Figure imgf000052_0001
shift assays, immunoassays for protein binding, functional assays
Figure imgf000052_0002
assays, etc.) and the like.
The determinatioιτ&f«l'?'® -binding of the candidate bioactive agent to the expressed protein may be done in p. ,rrøm.ber of ways. In a preferred embodiment, the candidate bioactive agent is labelled; a-rtΦfrøding determined directly. For example, this may be done by attaching all or a pa#fm.of the expressed protein to a solid support, adding a labelled candidate agent (for exam le, a fluorescent label), washing off excess reagent, and determining whether the laβ&Ha present on the solid support. Various blocking and washing steps may be utilized as ϊs known in the art. By "labeled" herein is meant that the compound is either directly or indirectly labeled with a label which provides a detectable signal, e.g. radioisotope, fluorescers, enzyme, antibodies, particles such as magnetic particles, chemiluminescers, or specific binding molecules, etc. Specific binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin, etc. For the specific binding members, the complementary member would normally be labeled with a molecule which provides for detection, in accordance with known procedures, as outlined above. The label can directly or indirectly provide a detectable signal.
In some embodiments, only one of the components is labeled. For example, the proteins (or proteinaceous candidate agents) may be labeled at tyrosine positions using 125 1, or with fluorophores. Alternatively, more than one component may be labeled with different labels; using 125 1 for the proteins, for example, and a fluorophor for the candidate agents.
In a preferred embodiment, the binding of the candidate bioactive agent is determined through the use of competitive binding assays. In this embodiment, the competitor is a binding moiety known to bind to the target molecule (i.e. bladder cancer), such as an antibody, peptide, binding partner, ligand, etc. Under certain circumstances, there may be competitive binding as between the bioactive agent and the binding moiety, with the binding moiety displacing the bioactive agent. In one embodiment, the candidate bioactive agent is labeled. Either the? candidate bioactive agent, or the competitor, or both, is added first to the protein fosrøi time sufficient to allow binding, if present. Incubations may be performed at any tt€Wϊjeirature which facilitates optimal activity, typically between 4 and 40° C. Incubaiϊ s^ riods are selected for optimum activity, but may also be optimized to facilitate
Figure imgf000053_0001
put screening. Typically between 0.1 and 1 hour will be sufficient. Excess reagsefe is. generally removed or washed away. The second component is then added, an&ii.feφresence or absence of the labeled component is followed, to indicate binding.
In a preferred embodiment, the compefeϊis added first, followed by the candidate bioactive agent. Displacement of the comρsfl?«®ϊs an indication that the candidate bioactive agent is binding to the expressed proteiirsnd thus is capable of binding to, and potentially modulating, the activity of the expressecfcprotein. In this embodiment, either component can be labeled. Thus, for example, if trie; competitor is labeled, the presence of label in the wash solution indicates displacement by the agent. Alternatively, if the candidate bioactive agent is labeled, the presence of the label on the support indicates displacement.
In an alternative embodiment, the candidate bioactive agent is added first, with incubation and washing, followed by the competitor. The absence of binding by the competitor may indicate that the bioactive agent is bound to the expressed protein with a higher affinity. Thus, if the candidate bioactive agent is labeled, the presence of the label on the support, coupled with a lack of competitor binding, may indicate that the candidate agent is capable of binding to the expressed protein. In a preferred embodiment, the methods comprise differential screening to identity bioactive agents that are capable of modulating the activity of the expressed proteins. In this embodiment, the methods comprise combining a expressed protein and a competitor in a first sample. A second sample comprises a candidate bioactive agent, a expressed protein and a competitor. The binding of the competitor is determined for both samples, and a change, or difference in binding between the two samples indicates the presence of an agent capable of binding to the expressed protein and potentially modulating its activity. That is, if the binding of the competitor is different in the second sample relative to the first sample, the agent is capable of binding to the expressed protein. Alternatively, a preferred embodiment utilizes differential screening- (^identify drug candidates that bind to the native expressed protein, but cannot bind to rrtc^fied expressed proteins. The structure of the expressed protein may be modeled, and used in rational drug design to synthesize agents that interact with that site. Drug c t' άrte es that affect bladder cancer bioactivity are also identified by screening drugs for κ?føility to either enhance or reduce the activity of the protein.
Positive
Figure imgf000054_0001
negative controls may be used in the assays. Preferably all control and ifefK-amples are performed in at least triplicate to obtain statistically significant results.
Figure imgf000054_0002
of all samples is for a time sufficient for the binding of the agent to the protein. FolfόNivkss ieubation, all samples are washed free of non-specifically bound material and the anvsϋ∞t.of bound, generally labeled agent determined. For example, where a radiolabel is employed, the samples may be counted in a scintillation counter to determine the amouπ iαf bound compound. A variety of other reagents may be included in the screening assays. These include reagents like salts, neutral proteins, e.g. albumin, detergents, etc which may be used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may be used. The mixture of components may be added in any order that provides for the requisite binding.
Screening for agents that modulate the activity of expressed proteins may also be done. In a preferred embodiment, methods for screening for a bioactive agent capable of modulating the activity of expressed proteins comprise the steps of adding a candidate bioactive agent to a sample of expressed proteins, as above, and determining an alteration in the biological activity of expressed proteins. "Modulating the activity" of bladder cancer includes an increase in activity, a decrease in activity, or a change in the type or kind of activity present. Thus, in this embodiment, the candidate agent should both bind to cancer proteins (although this may not be necessary), and alter its biological or biochemical activity as defined herein. The methods include both in vitro screening methods, as are generally outlined above, and in vivo screening of cells for alterations in the presence, distribution, activity or amount of expressed proteins. Thus, in this embodiment, the methods comprise combining a bladder - smcer sample and a candidate bioactive agent, and evaluating the effect on bladder carfesa activity, respectively. By "cancer activity" or grammatical equivalents herein is meant at least one of cancer's biological activities, including, but not limited to, cell division, ppvferably in bladder tissue, cell proliferation, tumor growth, and transformation of cells.' ft^'tae, embodiment, cancer activity includes activation of E2F3, BIG-1 , BIG-2 or a
Figure imgf000055_0001
by E2F3, BIG-1 or BIG-2. An inhibitor of cancer activity is an agent which inhibits &w f- -i .-.or more cancer activities. At least 3 main protein targets exist within the GS-Brø©:*B rα: E F3, B1G1 and BIG2. These targets, separately and in combination with orø?ftϊs&$aer will be referred to herein as "GS-BIG proteins".
In a preferred embodiment, the activity of the e' jrøassed protein is increased; in another preferred embodiment, the activity of the expressecl. rotein is decreased. Thus, bioactive agents that are antagonists are preferred in some embodiments, and bioactive agents that are agonists may be preferred in other embodiments. In a preferred embodiment, the invention provides methods for screening for bioactive agents capable of modulating the activity of a expressed protein. The methods comprise adding a candidate bioactive agent, as defined above, to a cell comprising expressed proteins. Preferred cell types include almost any cell. The cells contain a recombinant nucleic acid that encodes a expressed protein. In a preferred embodiment, a library of candidate agents are tested on a plurality of cells.
In one aspect, the assays are evaluated in the presence or absence or previous or subsequent exposure of physiological signals, for example hormones, antibodies, peptides, antigens, cytokines, growth factors, action potentials, pharmacological agents including chemotherapeutics, radiation, carcinogenics, or other cells (i.e. cell-cell contacts). In another example, the determinations are determined at different stages of the cell cycle process.
In this way, bioactive agents are identified. Compounds with pharmacological activity are able to enhance or interfere with the activity of the expressed protein. In one embodiment, " GS-BIG protein activity" as used herein includes at least one of the following: cancer activity, binding to GS-BIG protein, activation of GS-BIG protein or activation of substrates of GS-BIG protein by GS-BIG protein. An inhibitor of GS- BIG protein inhibits at least one of GS-BIG protein's bioactivities.
In one embodiment, a method of inhibiting bladder cancer cell division is provided. The method comprises administration of a bladder cancer inhibitor.
In another embodiment, a method of inhibiting tumor growth is provided. The reethod comprises administration of a bladder cancer inhibitor. In a preferred em feciiiKient, the inhibitor is an inhibitor of one or more of the GS-BIG protein.
In a Φ'i r . embodiment, methods of treating cells or individuals with cancer are provided. The u.?s'ώ? 1,.comprises administration of a bladder cancer inhibitor. In a preferred embodiment, th©iiΛ o is an inhibitor of GS-BIG6.
In one embodiment, a expresst^ ϊ jiwfein inhibitor is an antibody as discussed above. In another embodiment, the inhibitor ιy"&εvpjaiis,ense molecule. Antisense molecules as used herein include antisense or sense
Figure imgf000056_0001
comprising a singe-stranded nucleic acid sequence (either RNA or DNA) ca ^ ^of binding to target mRNA (sense) or DNA (antisense) sequences for expressed møtesules. A preferred antisense molecule is for one or more of the GS-BIG proteins o fόsa: ligand or activator thereof. Antisense or sense oligonucleotides, according to the: present invention, comprise a fragment generally at least about 14 nucleotides, preferably from about 14 to 30 nucleotides. The ability to derive an antisense or a sense oligonucleotide, based upon a cDNA sequence encoding a given protein is described in, for example, Stein and Cohen, Cancer Res., 48: 2659 (1988) and van der Krol, et al., BioTechniques, 6: 958 (1988).
Antisense molecules may be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, as described in WO 91/04753. Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell. Alternatively, a sense or an antisense oligonucleotide may be introduced into a cell containing the target nucleic acid sequence by formation of an oligonucleotide-lipid complex, as described in WO 90/10448. It is understood that the use of antisense molecules or knock out and knock in models may also be used in screening assays as discussed above, in addition to methods of treatment.
The compounds having the desired pharmacological activity may be administered in a physiologically acceptable carrier to a host, as previously described. The agents may be administered in a variety of ways, orally, parenterally e.g., cfλ cutaneously, intraperitoneally, intravascularly, etc. Depending upon the manner of intro ^flαn, the compounds may be formulated in a variety of ways. The concentration? Sfeerapeutically active compound in the formulation may vary from about 0.1-100 wt. 7c/:!: T^agents may be administered alone or in combination with other treatments, i.e., ratiføtøifrrav
The pharmaceutical composiiK'jvs.f(T; ??;fha prepared in various forms, such as granules, tablets, pills, suppositories, capsules; ^w^ssions, salves, lotions and the like. Pharmaceutical grade organic or inorganic carriei & &4fm diluents suitable for oral and topical use can be used to make up compositions cϋi'itM-«Mδg..the therapeutically-active compounds. Diluents known to the art include as ssus media, vegetable and animal oils and fats. Stabilizing agents, wetting and emulsify^,;. agents, salts for varying the osmotic pressure or buffers for securing an adequate pH value, and skin penetration enhancers can be used as auxiliary agents. Without being bound by theory, it appears that the various expressed sequences are important in bladder cancer. Accordingly, disorders based on mutant or variant cancer genes may be determined. In one embodiment, the invention provides methods for identifying cells containing variant cancer genes comprising determining all or part of the sequence of at least one endogeneous cancer gene in a cell. As will be appreciated by those in the art, this may be done using any number of sequencing techniques. In a preferred embodiment, the invention provides methods of identifying the cancer genotype of an individual comprising determining all or part of the sequence of at least one cancer gene of the individual. This is generally done in at least one tissue of the individual, and may include the evaluation of a number of tissues or different samples of the same tissue. The method may include comparing the sequence of the sequenced gene to a known gene, i.e. a wild- type gene.
The sequence of all or part of the expressed gene can then be compared to the sequence of a known expressed gene to determine if any differences exist. This can be done using any number of known homology programs, such as Bestfit, etc. In a preferred embodiment, the presence of a difference in the sequence between the expressed gene of the patient and the known expressed gene is indicative of a dϊss se state or a propensity for a disease state, as outlined herein. Irrøpreferred embodiment, the expressed genes are used as probes to determine the*: umber of copies of the expressed gene in the genome.
In another pr^rød embodiment expressed genes are used as probed to determine the chromosome? trøJization of the expressed genes. Information such as chromosomal localization finds - uGiin.providing a diagnosis or prognosis in particular when chromosomal abnormaH'tfes:*^ h, as translocations, and the like are identified in expressed gene loci.
Thus, in one embodiment, methods of modulating
Figure imgf000058_0001
in cells or organisms are provided. In one embodiment, the methods
Figure imgf000058_0002
to a cell an antibody that reduces or eliminates the biological activity of an
Figure imgf000058_0003
expressed protein. Alternatively, the methods comprise administering to a cell or organism a recombinant nucleic acid encoding a expressed protein. As will be appreciated by those in the art, this may be accomplished in any number of ways. In a preferred embodiment, for example when the expressed sequence is down- regulated in cancer, the activity of the expressed gene is increased by increasing the amount in the cell, for example by overexpressing the endogenous protein or by administering a gene encoding the sequence, using known gene-therapy techniques, for example. In a preferred embodiment, the gene therapy techniques include the 5 incorporation of the exogenous gene using enhanced homologous recombination (EHR). Alternatively, for example when the expressed sequence is up-regulated in cancer, the activity of the endogeneous gene is decreased, for example by the administration of an inhibitor of cancer, such as an antisense nucleic acid.
In one embodiment, the expressed proteins of the present invention may be 0 used to generate polyclonal and monoclonal antibodies to expressed proteins, which are useful as described herein. Similarly, the expressed proteins can be coupled, using standard technology, to affinity chromatography columns. These columns may then be used to purify expressed antibodies. In a preferred embodiment, the antibodies are generated to epitopes unique to a expressed protein; that is, the 5 antibodies show little or no cross-reactivity to other proteins. These antibodies find use in a number of applications. For example, the expressed antibodies may be coupled to standard affinity chromatography columns and used to purify expressed proteins. The antibodies may also be used as blocking polypeptides, as outlined above, since th©vswill specifically bind to the expressed protein. 0 In one embodiffwnt, a therapeutically effective dose of a expressed or modulator thereof is admuvlatered to a patient. By "therapeutically effective dose" herein is meant a dose that profe s. the effects for which it is administered. The exact dose will depend on the purposc^the treatment, and will be ascertainable by one skilled in the art using known techniques?. h ,\s known in the art, adjustments for 5 degradation, systemic versus localized delivery, art S ^feof new protease synthesis, as well as the age, body weight, general health, sex, diet;' ■IttwKiaf. administration, drug interaction and the severity of the condition may be necessary, arøte:4il:be ascertainable with routine experimentation by those skilled in the art.
A "patient" for the purposes of the present invention includes both humari&x 3.0:. and other animals, particularly mammals, and organisms. Thus the methods are amicable to both human therapy and veterinary applications. In the preferred embodtrftent the patient is a mammal, and in the most preferred embodiment the patient is hurrøn. The administration of the expressed proteins and modulators of the present invention can be done in a variety of ways as discussed above, including, but not limited to, orally, subcutaneously, intravenously, intranasally, transdermally, intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally, or intraocularly. In some instances, for example, in the treatment of wounds and inflammation, the expressed proteins and modulators may be directly applied as a solution or spray.
The pharmaceutical compositions of the present invention comprise a expressed protein in a form suitable for administration to a patient. In the preferred embodiment, the pharmaceutical compositions are in a water soluble form, such as being present as pharmaceutically acceptable salts, which is meant to include both acid and base addition salts. "Pharmaceutically acceptable acid addition salt" refers to those salts that retain the biological effectiveness of the free bases and that are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like. "PharmaceufBa acceptable base addition salts" include those derived from inorganic bases suti?..as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, t?j>per, manganese, aluminum salts and the like. Particularly preferred are the ammoniuh' potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutϊc≥ϊψjiPcceptable organic non-toxic bases include salts of primary, secondary, and tertiary amifrw,.. substituted amines including naturally occurring substituted amines, cyclic amines a'tv^i-^ ic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethy »?«©,■ tripropylamine, and ethanolamine.
The pharmaceutical compositions may also include one or mo c the following: carrier proteins such as serum albumin; buffers; fillers such as ,,; microcrystalline cellulose, lactose, corn and other starches; binding agents; sweeteners and other flavoring agents; coloring agents; and polyethylene glycol. Adϋi ή';?5..are well known in the art, and are used in a variety of formulations.
In a preferred embodiment, expressed proteins and modulators are administered as therapeutic agents, and can be formulated as outlined above. Similarly, expressed genes (including both the full-length sequence, partial sequences, or regulatory sequences of the expressed coding regions) can be administered in gene therapy applications, as is known in the art. These expressed genes can include antisense applications, either as gene therapy (i.e. for incorporation into the genome) or as antisense compositions, as will be appreciated by those in the art.
In a preferred embodiment, expressed genes are administered as DNA vaccines, either single genes or combinations of expressed genes. Naked DNA vaccines are generally known in the art. Brower, Nature Biotechnology, 16:1304- 1305 (1998).
In one embodiment, expressed genes of the present invention are used as DNA vaccines. Methods for the use of genes as DNA vaccines are well known to one of ordinary skill in the art, and include placing a expressed gene or portion of a expressed gene under the control of a promoter for expression in a patient with bladder cancer. The expressed gene used for DNA vaccines can encode full-length expressed proteins, but more preferably encodes portions of the expressed proteins including peptides derived from the expressed protein. In a preferred embodiment a patient is. immunized with a DNA vaccine comprising a plurality of nucleotide
Figure imgf000061_0001
from a expressed gene. Similarly, it is possible to immunize a patient with a
Figure imgf000061_0002
of expressed genes or portions thereof as defined herein. Without being bound LH!,theory, expression of the polypeptide encoded by the DNA vaccine, cytotoxic T-cells, ϊ ^iper T-cells and antibodies are induced which recognize and destroy or eliminate cells e^ssssing expressed proteins.
In a preferred embodiment, the [D?&Λ«accines include a gene encoding an adjuvant molecule with the DNA vaccine. SucH"fr j.wyant molecules include cytokines that increase the immunogenic response to the
Figure imgf000061_0003
encoded by the DNA vaccine. Additional or alternative adjuvants are knoes'teibose of ordinary skill in the art and find use in the invention.
In another preferred embodiment expressed genes find use in generatitøt;$ animal models of cancer. For example, as is appreciated by one of ordinary skill in the art, when the cancer gene identified is repressed or diminished in cancer tissue, gene.vtfo-srapy technology wherein antisense RNA directed to the cancer gene will also diminish' or repress expression of the gene. An animal generated as such serves as an animal model of cancer that finds use in screening bioactive drug candidates. Similarly, gene knockout technology, for example as a result of homologous recombination with an appropriate gene targeting vector, will result in the absence of the cancer protein. When desired, tissue-specific expression or knockout of the cancer protein may be necessary.
It is also possible that the expressed protein is overexpressed in bladder cancer. As such, transgenic animals can be generated that overexpress the expressed protein. Depending on the desired expression level, promoters of various strengths can be employed to express the transgene. Also, the number of copies of the integrated transgene can be determined and compared for a determination of the expression level of the transgene. Animals generated by such methods find use as animal models of expressed and are additionally useful in screening for bioactive molecules to treat disorders related to the expressed protein.
The invention is further illustrated by the following non-limited examples. All scientific and technical terms have the meanings as understood by one with ordinary skill in the art. The specific examples which follow illustrate the methods in which the compositions of the present invention may be prepared and are not to be construed as limiting the invention in sphere or scope. The methods may be adapted to variation in order to produce compositions embraced by this invention but not specifically αfetosed. Further, variations of the methods to produce the same compositions in sβaewhat different fashion will be evident to one skilled in the art.
Although the & -ks?φles herein concern human cells and the language is primarily directed to human aw'ϊsicns, the concept of this invention is applicable to genomes from any plant or animaϊ.' rV^g nomes compared need only be related closely enough to have sufficient substaπft&^'tøatical sequences for a meaningful analysis. For example, a human genome and
Figure imgf000062_0001
primate could be compared according to the methods of this invention.
EXAMPLES MATERIALS AND METHODS Comparative genomic hybridization (CGH)
A review of the CGH profiles of 278 primary bladder carcinomas and 20 cell lines previously examined in our laboratory revealed 10 tumors and 3 cell lines with distϊπefφeaks around 6p22. Examples of CGH profiles are shown in Figure 10. Bladder cancer tissue microarray (TMA)
The composition of our bladder cancer TMA containing 2317 formalin-fixed paraffin embedded tissues from 1853 bladder cancer patients was previously described (Richter, J., et al., Am J Pathol, 157: 787-94 (2000); Simon, R., et al., Oncogene, 21 : 2476-83 (2002). Some of the clinical data were updated for this study. All slides of all tumors were reviewed by one pathologist (GS). Tumor stage and grade were defined according to UICC and WHO (Mostofi, F. Histological typing of urinary bladder tumors. World Health Organization: Geneva; UICC. TNM Classification of malignant tumours, 4 edn. Springer: Berlin 1992). Stage pT1 was defined by presence of both unequivocal tumor invasion of the suburothelial stroma and tumor-free fragments of the muscular bladder wall. Carcinomas with stroma invasion but absence of muscular bladder wall in the biopsy were classified as at least pT1 (pT1-). Clinical data were available from 1123 patients. The medium follow-up period was 42 months (range 1 - 236 months). Time to recurrence and time to progression (to stage pT2 or higher) were selected as study endpoints for patients with pTa and pT1 tumors. Follow-up information was considered complete enough to include a pTa/pT1 cancer patient in the study if cystoscopies had been performed at least at 3, 9 and 15 months, then annually until the endpoint of this study (recurrence, last control). To include a patient for analyses of time to progression, longer intervals between controls were accepted if the last follow up control rated out progression. Recurrences were defined as cystoscopically visible tumors. Tum&a progression was defined as the presence of muscle invasion (stage pT2 or higher) in
Figure imgf000063_0001
biopsy. An overview of the histological and clinical data is given above in t'Hfes. 5 and 7 Fluorescence in situ hybridikasϊ& (FISH)
The tissue microarray sections- ^ r ated according to the Paraffin Pretreatment Reagent Kit protocol (purchase'ii*tos*:Vysis, Downers Grove, IL) before hybridization. FISH was performed with a digoxigeiϊ&ϊ tAC probe (PAC dJ177P22, Sanger Centre, UK) containing the E2F3 gene and a Spectϊαas'R rlabeled chromosome 6 centromeric probe (CEP6) as a reference (purchaset - reVysis). Hybridization and posthybridization washes were according to the 'LSI procft £f '. (Vysis). Probe visualization using fluorescent isothiocyanate (FITC)-conjugated sheep anti-digoxigenin (Roche Diagnostics, Rotkreuz, Switzerland) was as described (Wagner, U., et al., Am J Pathol, 151 : 753-759 (1997). Slides were counterstained with 125 ng/ml 4', 6-diamino-2-phenylindole in an antifade solution. Amplification was defined as presence (in 5% of tumor cells) of at least 3 times as many E2F3 gene signals than centromere 6 signals.
Immunohistochemistry. Standard indirect immunoperoxidase procedures were used for immunohistochemistry (ABC-Elite, Vector Laboratories, Burlingame, CA). The monoclonal antibody E2F3 Ab-4 (Lab Vision Corporation, CA) was tested on array sections containing formalin-fixed paraffin embedded, E2F3 amplified and non- amplified bladder tumors. Optimal staining of the cell nuclei (1 :100 dilution of primary antibody) could best be achieved after pretreatment in 1 mM EDTA at 99 °C for 40 minutes for antigen retrieval. The primary antibody was omitted for negative controls. Diaminobenzidine was used as a chromogen. The IHC staining intensity (scored in a four step scale including 0, 1+, 2+, and 3+) and the fraction of positive tumor cells was recorded for each tissue spot. Based on these values, a final IHC result was calculated according to the following criteria: Negative: no staining at all, or 1 + staining intensity in no more than 10% of tumor cells; positive: at least 2+ staining intensity in more than 10% of tumor cells.
The rabbit monoclonal antibody MIB1 (1 :800, Dianova, Hamburg, Germany) ims employed to detect Ki67 protein (expressed in all cells in G1 , S, G2 and M pha^s>).,as previously described (Moch, H., et al., Urol Res, 25: S25-30 (1997). Nuclei wsεs'.'.considered Ki67 positive if any nuclear staining was seen. The Ki67 labeling inde ^ ), (percentage of Ki67 positive cells) was determined on each arrayed tumor samμJ ?γ, scoring at least 300 cells each. Tumors with Ki67 negative mitoses were excluded
Figure imgf000064_0001
Western analysis
Protein was extracted from abϋcώR2κΛ06 cells from each of the 8 bladder tumor cell lines as described (Leone, G., et al., C mm^Dev, 12: 2120-30 (1998). Ten μg protein of each sample was subjected to SDS-PAGE ^'.10% polyacrylamide gels. Proteins were transferred onto PVDF membrane (Bio-Rad, Glatitesgg, Switzerland). The membrane was blocked in TBS (25 mM Tris at pH 7.4, 137 m 'ϊ it§J 2.7 mM KCl) containing 10% skim milk at RT for 2 hr. Blots were then incubated witϊϊ rnQUse monoclonal E2F3 Ab-4 antibody (5 μg/ml) (Lab Vision, CA, U.S.A.) in TBS containing'. 5% skim milk overnight at 4°C, and washed subsequently in TBS containing 0.1% Tween 20 for 30 minutes. Blots were incubated with a second antibody (1 :2000) (goat anti-mouse IgG, Fc, AP127P; Juro Supply AG, Lucerne, Switzerland) for 1 hour at room temperature, and washed for 30 minutes. Blots were processed with the ECL system (Amersham Pharmacia Biotech, Dubendorf, Switzerland).
Statistics All tissue samples on the TMA were utilized for comparisons of amplification and overexpression of E2F3. Only the first biopsy was used for further statistical analyses in patients having more than one tumor on the TMA. Contingency table analysis and Chi-square tests were applied to study the relationship between histology tumor type, grade, stage and E2F3 expression/amplification. Student t tests were employed to examine the associations of the Ki67 LI with E2F3 expression/amplification. Analysis of Variance (ANOVA) was utilized to determine the parameters with greatest influence on tumor cell proliferation. Survival curves were plotted according to the Kaplan-Meier method and analyzed for statistical differences using a log rank test. Patients with pTa/pT1 tumors were censored at the time of their last clinical control showing no evidence of disease or at the date when cystectomy was performed. Patients with pT2-4 carcinomas were censored at the time of their last clinical control or at the time of death if they died from causes not related to their tumor.
Example II Analysis of GS-BIG
The combined physical, transcript and FISH map of the amplified region at 6p22 is a!$ ia if Figures 3A, 3B, 3C and 3D. Clones marked in red constitute the core of the aπvp rø.n (1.7 Mb in size). Average copy number for each clone in the map was counted
Figure imgf000065_0001
tumors placed into a tissue microarray and assayed with a labeled DNA-probe. doite ar marked in red define the minimal commonly amplified region at 6p22. Genes and
Figure imgf000065_0002
to the region are also indicated.
Characterization of novel genes within G f f *
Bioinformatic tools were used to identify and'ctesp-sterize novel genes within GS-BIG. For a first rough prediction of locations of
Figure imgf000065_0003
in the genomic sequences, and identification of coding sequences, the S&^ n" software tool (Stanford University, Dept of Mathematics; http://genes.mit.edu/GENSi'S&N,html) was utilized. For further detailed analysis, cDNA sequences were mapped to th'© corresponding genomic sequences using NCBI's pairwise BLAST tool (http://www.ncbi.nlm.nih.gov/blast/bl2seq/bl2.html). Gene Code's "Sequencher 4.1.2" software package was utilized to assemble contigs from BAG or cDNA sequences prior to the BLAST analysis. For prediction of protein motifs, the ScanSite motif search tool (http://scansite.mit.edu/) was employed.
BIG-2 Closer examination of the cDNA sequences ENSESTT00002382100,
NM_175879, dJ434O11 , and Q8N9R5 showed overlapping sequences, thus allowing for assembling of a "unigene" which we termed "BIG-2". The unigene spans about 110 kb of genomic sequence, including 13 exons with a total cDNA size of 3.14 kb. GenScan-analysis of the cDNA sequence revealed a 1488 bp (495 aa) coding sequence. The predicted protein contains an MBOAT domain motif (Figure 15). The MBOAT (membrane bound O-acyl transferase) family of membrane proteins contains a variety of acyltransferase enzymes and has been suggested to play a role in WNT signaling. This supports potential growth-promoting role for BIG-2.
BIG-1 BIG-1 spans 684.69 kb of genomic DNA, including 14 exons. The 1740 bp mRNA translates into a protein of 579 amino acids, see Figure 16. Three functional domains have been identified using ScanSite's MotifScan software. The function of the UPF0004 domain is unknown, but it is almost always found in conjunction with Radical_SAM and TRAM. Radical_SAM is believed to be involved in radical-based catalysis in a number of previously well-studied but unresolved biochemical pathways, deluding unusual methylations, isomerization, sulfur insertion, ring formation, artcisrohic oxidation and protein radical formation (Sofia, H. J., et al., Nucleic Acids Res, 2 ':fJW-1106 (2001). TRAM presumably represents a RNA-binding domain, common to tKϊW τacil methylation and adenine thiolation enzymes. The TRAM domain is present in -^w^al other proteins associated with the translation machinery and in a family of small, unci Eβgsϊterized archaeal proteins that are predicted to play a role in the regulation of tRNA mo f&stion or translation (Anantharaman, V., et al., FEMS Microbiol Lett, 197: 215-221 (20ϋ
Northern blot analysis of 9 bladder tumor cell M As shown in Figure 17 the 2.6 kb BIG-1 mRNA is aiiferø&antly expressed in
6p22.3 amplified cell lines CRL-1472, 5637, and 5-HTB. Northern Mot staining with methylene blue showed presence of RNA in all cell lines (control in th&.lower panel). Quantitative mRNA expression analysis of BIG-1 relative to bladder cell Iiπer7882 showing the weakest BIG-1 mRNA expression (=1 fold expression) is shown in Figure 18.
Fluorescence in situ hybridization (FISH)
The tissue microarray sections were treated according to the Paraffin Pretreatment Reagent Kit protocol (purchased from Vysis, Downers Grove, IL) before hybridization. FISH was performed with a digoxigenated PAC or BAC clones (Sanger Centre, UK) and a Spectrum Red-labeled chromosome 6 centromeric probe (CEP6) as a reference (purchased from Vysis). Hybridization and posthybridization washes were according to the 'LSI procedure' (Vysis). Probe visualization using fluorescein isothiocyanate (FITC)-conjugated sheep anti-digoxigenin (Roche Diagnostics,
Rotkreuz, Switzerland). Slides were counterstained with 125 ng/ml 4',6-diamino-2- phenylindole in an antifade solution. Amplification was defined as presence (in ≥ 5% of tumor cells) of either more than 10 gene signals or tight clusters of at least 5 gene signals or more than 3 times as many E2F3 than centromere 6 signals. Immunohistochemistry
Standard indirect immunoperoxidase procedures were used for immunohistochemistry (ABC-Elite, Vector Laboratories, Burlingame, CA). The primary antibody was omitted for negative controls. Diaminobenzidine was used as a chromogen. The rabbit monoclonal antibody MIB1 (1 :800, Dianova, Hamburg, Germany) was employed to detect Ki67 protein (expressed in all cells in G1, S, G2 and M phase) as previously described (Houldsworth, J, et al., Blood, 87:25-29 (1996). Nuclei were considered Ki67 positive if any nuclear staining was seen. The Ki67 ii Jag index (LI) (percentage of Ki67 positive cells) was determined on each
Figure imgf000067_0001
sample by scoring at least 300 cells each. Tumors with Ki67 negative mitoses wei
Figure imgf000067_0002
from analyses.
Statistics
Contingency table
Figure imgf000067_0003
tests were applied to study the relationship between histologic tumor iyμ'& ^ξ e, stage and GS-BIG6 amplification. Student t tests were employed to examine the a&^a^ ions of the Ki67 LI with GS- BIG6 amplification. Analysis of Variance (ANOVA) waa i^sd to determine the parameters with greatest influence on tumor cell proliferation. θ wival curves were plotted according to the Kaplan-Meier method and analyzed for statiSfe-al differences using a log rank test. Patients with pTa/pT1 tumors were censored at the time of their last clinical control showing no evidence of disease or at the date when cystectomy was performed. Patients with pT2-4 carcinomas were censored at the time of their last clinical control or at the time of death if they died from causes not related to their tumor.
The foregoing description is considered as illustrative only of the principles of the invention. Further, since numerous modifications and changes will readily occur to those skilled in the art, it is not desired to limit the invention to the exact construction and process shown as described above. Accordingly, all suitable modifications and equivalents may be resorted to falling within the scope of the invention as defined by the claims that follow. The words "comprise," "comprising," "include," "including," and "includes" when used in this specification and in the following claims are intended to specify the presence of stated features, integers, components, or steps, but they do not preclude the presence or addition of one or more other features, integers, components, steps, or groups thereof.

Claims

CLAIMSThe embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:
1. An isolated nucleic acid molecule comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.
2. The isolated nucleic acid of claim 1, wherein the sequence is SEQ ID NO:1.
3. The isolated nucleic acid of claim 1 , wherein the sequence is SEQ ID NO:2.
4. The isolated nucleic acid of claim 1 , wherein the sequence is SEQ ID NO:3.
5. The isolated nucleic acid of claim 1, wherein the sequence is SEQ ID NO:4.
6. A method of detecting bladder cancer comprising, obtaining a biological sample from a subject suspected of having bladder cancer; ar#l, detecting abnormal expression of the GS-BIG region.
7. The method of claim 6, wherein said biological sample is tissue from said subject.
f?I The method of claim 7, wherein said biological sample is a biomolecule
Figure imgf000069_0001
said tissue sample.
9 The methαα'-GføfeϊiCTi 8, wherein said biomolecule is a nucleic acid.
10. The method of claim 8,' v i'?.ft?asaid biomolecule is a protein.
11. The method of claim 6, wherein satøiwjfcject is a mammal.
12. The method of claim 11 , wherein said mammal; .is a human.
13. The method of claim 6, wherein a labeled fragment isolated from SEQ ID NO:1 is hybridized to said biological sample.
14. The method of claim 6, wherein a labeled fragment isolated from SEQ ID NO:2 is hybridized to said biological sample.
15. The method of claim 6, wherein a labeled fragment isolated from SEQ
ID NO:3 is hybridized to said biological sample.
17. The method of claim 6, wherein a labeled fragment isolated from SEQ ID NO: 4 is hybridized to said biological sample.
18. The method of claim 6, wherein detecting abnormal expression of the GS-BIG region is accomplished by PCR-based detection.
19. The method of claim 6, wherein detecting abnormal expression of the GS-BIG region is accomplished by an antibody based assay.
20. The method of claim 6, wherein detecting abnormal expression of the GS-BIG region is accomplished by means of specific affinity wherein molecules having specific affinity to fragments from the GS-BIG region are detected.
21. The method of claim 6, wherein detection occurs using an assay in which the first fragment is unlabeled, and a secondary step for detection is introduced.
'S A method of treating bladder cancer comprising administering to a patient having 1 :fe: er cancer a compound that reduces the level of expression in the 6p22 region.
23. The method of
Figure imgf000070_0001
said compound comprises an antisense oligonucleotide that complemeπis^SKJ lP. NO:2, SEC ID NO:3 or SEC ID NO:4.
24. The method of claim 23, further comprising admirtteiir ir!g.additional antisense oligonucleotides so that the expression of SEC ID NOS:2-4 aie ©".: . suppressed.
25. The method of claim 22, wherein said compound is ligand binding molecule that inactivates the protein expressed by SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.
26. The method of claim 22, wherein said compound comprises a plurality of ligand binding molecules that will inactivate at least two genes expressed within the 6p22 region.
27. An isolated nucleic acid molecule comprising a polynucleotide sequence sharing at least 75 percent homology to a sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.
28. The isolated nucleic acid of claim 27, wherein said polynucleotide is at least 75 percent homologous to sequence is SEQ ID NO:1.
29. The isolated nucleic acid of claim 27, wherein said polynucleotide is at least 75 percent homologous to sequence is SEQ ID NO:2.
30. The isolated nucleic acid of claim 27, wherein said polynucleotide is at least 75 percent homologous to sequence is SEQ ID NO:3.
31. The isolated nucleic acid of claim 27, wherein said polynucleotide is at least 75 percent homologous to sequence is SEQ ID NO:4.
32. The isolated nucleic acid of claim 27, wherein said polynucleotide is at least 85 percent homologous to sequence is SEQ ID NO:1.
33. The isolated nucleic acid of claim 27, wherein said polynucleotide is at least 85 percent homologous to sequence is SEQ ID NO:2.
34. The isolated nucleic acid of claim 27, wherein said polynucleotide is at least 85 percent homologous to sequence is SEQ ID NO:3.
35. The isolated nucleic acid of claim 27, wherein said polynucleotide is at least 85 percent homologous to sequence is SEQ ID NO:4.
36. The isolated nucleic acid of claim 27, wherein said polynucleotide is at least 95 percent homologous to sequence is SEQ ID NO:1.
37. The isolated nucleic acid of claim 27, wherein said polynucleotide is at least 95 percent homologous to sequence is SEQ ID NO:2.
38. The isolated nucleic acid of claim 27, wherein said polynucleotide is at least 95 percent homologous to sequence is SEQ ID NO:3.
39. The isolated nucleic acid of claim 27, wherein said polynucleotide is at least 95 percent homologous to sequence is SEQ ID NO:4.
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WO2006060742A2 (en) * 2004-12-02 2006-06-08 Oncotech, Inc. Reagents and methods for predicting drug resistance
WO2009001315A3 (en) * 2007-06-26 2009-11-12 Staahl Ulf Use of a class of genes encoding lysophospholipid acyl transferases for application in agriculture, biotechnology, and medicine
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Title
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SIMON R. ET AL.: 'E2F3 amplification and overexpression is associated with invasive tumor growth and rapid tumor cell proliferation in urinary bladder cancer' PATHOLOGY RESEARCH AND PRACTICE vol. 199, no. 4, 14 June 2003, pages 198 - 199, XP002977514 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006060742A2 (en) * 2004-12-02 2006-06-08 Oncotech, Inc. Reagents and methods for predicting drug resistance
WO2006060742A3 (en) * 2004-12-02 2007-01-25 Oncotech Inc Reagents and methods for predicting drug resistance
WO2009001315A3 (en) * 2007-06-26 2009-11-12 Staahl Ulf Use of a class of genes encoding lysophospholipid acyl transferases for application in agriculture, biotechnology, and medicine
WO2015073949A1 (en) * 2013-11-15 2015-05-21 The University Of North Carolina At Chapel Hill Method of subtyping high-grade bladder cancer and uses thereof

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