WO2001022864A2 - Sequences caracteristiques du cancer de la vessie - Google Patents

Sequences caracteristiques du cancer de la vessie Download PDF

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Publication number
WO2001022864A2
WO2001022864A2 PCT/US2000/041005 US0041005W WO0122864A2 WO 2001022864 A2 WO2001022864 A2 WO 2001022864A2 US 0041005 W US0041005 W US 0041005W WO 0122864 A2 WO0122864 A2 WO 0122864A2
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Prior art keywords
bladder cancer
sequences
gene
patient
tcc
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PCT/US2000/041005
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English (en)
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WO2001022864A3 (fr
WO2001022864A9 (fr
Inventor
Elena Feinstein
Orna Mor
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Quark Biotech, Inc.
Kohn, Kenneth, I.
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Priority to IL14879800A priority Critical patent/IL148798A0/xx
Priority to EP00977267A priority patent/EP1248855A4/fr
Priority to CA002385893A priority patent/CA2385893A1/fr
Priority to AU14926/01A priority patent/AU1492601A/en
Priority to US09/825,682 priority patent/US6998232B1/en
Publication of WO2001022864A2 publication Critical patent/WO2001022864A2/fr
Publication of WO2001022864A3 publication Critical patent/WO2001022864A3/fr
Publication of WO2001022864A9 publication Critical patent/WO2001022864A9/fr

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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
    • 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
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to the identification of polynucleotide sequences that are differentially expressed in bladder cancer. More specifically, the present invention relates to the use of the sequences and gene products for diagnosis and as probes.
  • Bladder cancer is the second most-common genitourinary cancer in the United States, with only prostate cancer being more frequently diagnosed. Bladder cancer accounts for approximately two percent of all malignant tumors and approximately seven percent of all urinary tract malignancies in U.S. men. Over 54,000 new cases were estimated to be diagnosed in the United States in 1998, with approximately 12,500 deaths predicted [American Cancer Society, 1998]. The prevalence of bladder cancer is higher in industrialized nations, perhaps reflecting increased exposure to environmental carcinogens. Men are three times more frequently affected than women. The disease usually occurs between 60-70 years of age and the age-adjusted bladder cancer rate in white men is almost twice that of black men.
  • bladder cancers are carcinomas of the transitional epithelium of the bladder's mucosal lining (transitional cell carcinoma (TCC)). Although 90 percent of the cases are localized at diagnosis, up to 80 percent recur. A number of etiological factors are associated with the development of bladder cancer, but in industrialized countries, cigarette smoking is the most significant. Specific chemicals have also been identified as causing bladder cancer, as have a number of occupational exposures to less well-defined specific agents. Treatment with cytostatic drugs, especially cyclophosphamide, is associated with increased risk of bladder cancer, as is treatment with radiotherapy for uterine cancer.
  • Bladder cancer is a potentially preventable disease, with a significant morbidity and mortality in many parts of the world.
  • Tumors are graded according to the degree of cellular abnormality, with the most atypical cells being designated as high-grade (i.e., G3 grade) tumors.
  • the major prognostic factors in carcinoma of the bladder are the depth of invasion into the bladder wall and the degree of differentiation of the tumor. The higher the grade of the tumor at the diagnosis, the higher the incidence of death from the disease within two years.
  • non-invasive tumors being papillary tumors which do not invade the lamina propria, and are classified as non-invasive TCC, i.e., "Ta" tumors, may recur, but nearly 70% will not progress further.
  • a tumor which does not invade the muscle but does enter the lamina propria presents in many cases a worse prognosis.
  • Such tumors are classified as non-invasive TCC, i.e., T1 tumors.
  • G1 grade tumors Most superficial tumors are well differentiated and classified as G1 grade tumors. Patients in whom superficial tumors are less differentiated, large, multiple, or associated with carcinoma in situ in other areas of the bladder mucosa, (classified as G2-G3 tumors) are at greatest risk for recurrence and the development of invasive cancer. Invasive bladder tumors tend to spread rapidly to the regional lymph nodes and then into adjacent structures. Overall, the five-year survival rate of TCC is 76 percent for whites and 55 percent for blacks.
  • carcinoma of the bladder is frequently multifocal.
  • the entire bladder epithelium and the lining of the entire urothelial cell tract can undergo malignant change. After apparently successful treatment of a bladder lesion, new tumors may occur at the same site (recurrence) or in other urothelial cells in the bladder. Approximately 30 percent of bladder carcinomas present as multiple lesions at the time of initial diagnosis.
  • bladder cancer The early diagnosis of bladder cancer is central to the effective treatment of TCC.
  • the detection of bladder tumors relies on intravenous pyelogram or other contrast studies to rule out urothelial involvement in the kidneys or ureters, and invariably cystoscopy which remains the accepted standard for diagnosis of mucosal abnormalities.
  • cystoscopy There are no presently reliable methods available to easily and specifically identify the presence of bladder cancer cells.
  • a variety of new technologies and potential tumor markers are being studied in bladder cancer and some are being translated into clinical use.
  • Adverse prognostic features associated with a greater risk of disease progression include the presence of multiple aneuploid cell lines, nuclear p53 overexpression, and expression of the Lewis-x blood group antigen [Hudson and Herr, 1995; Lacombe et al., 1996]. It has been postulated that p53 may be useful for predicting the level of aggression of the tumor and to identify patients who will not benefit from chemotherapy. However, only a very small, select group of patients with invasive disease may benefit from this approach [Ozen, 1998].
  • a method of diagnosing the presence of bladder cancer in a patient by analyzing a tissue sample from the patient for the presence of a least one expresses gene wherein the presence of the expressed gene is indicative of bladder cancer.
  • a polynucleotide sequence whose expression is indicative of bladder cancer.
  • a marker for bladder cancer is also provided.
  • methods of diagnosising bladder cancer by screening for the presenceof at least one expressed gene wherein the presence of the expressed gene is indicative of bladder cancer.
  • Methods of treating and regulating bladder cancer-associated pathologies by administering to a patient a therapeutically effective amount of a ribozyme, antisense olignucleofide, or agonist against the nucleic acids sequences of the present invention are also provided.
  • nucleic acid sequences associated with bladder cancer are provided. More specifically, the sequences of the present invention are set forth in Table I or II or have a complementary or allelic variation sequence thereto. The nucleic acid sequences of the complementary or allelic variation sequences are provided in Tables III and IV, respectively.
  • Bladder cancers can also be referred to as transitional cell carcinomas or "TCC”.
  • the present invention further provides a method of diagnosing the presence of bladder cancer in a patient, including the steps of analyzing a tissue sample from the patient for the presence of at least one expressed gene (up-regulated) wherein the mRNA from the expressed gene hybridizes to at least one of the sequences in Tables I or II, with hybridization occurring under conditions sufficiently stringent to require at least 95% base pairing.
  • the present invention provides antibodies directed against the gene products of the sequences of the present invention.
  • the antibodies can be either monoclonal, polyclonal or recombinant and be used in immunoassays as described in the Methods herein below.
  • regulate or modulate or control is meant that the process is either induced or inhibited to the degree necessary to effect a change in the process and the associated disease state in the patient. Whether induction or inhibition is being contemplated is apparent from the process and disease being treated and is known to those skilled in the medical arts.
  • the present invention identifies genes for gene therapy, diagnostics and therapeutics that have direct causal relationships between a disease and its related pathologies and up- or down- regulator (responder) genes. That is, the present invention is initiated by a physiological relationship between cause and effect.
  • the present invention identifies polynucleotide sequences named in Tables I and II, and set forth in Tables III and IV, respectively, that can be utilized diagnostically in bladder cancer. Sequences named in Table I were found to match sequences in data banks and were newly found in the present application to be upregulated in TCC. The sequences named in Table II are either genes with unknown protein product or of unknown genes. All the sequences named in both Tables I and II were found to be associated with TCC relative to normal bladder samples.
  • sequences are markers or probes for genes that are regulated in bladder carcinoma.
  • regulated it is meant that the genes can be either upregulated or downregulated, depending upon the specific gene.
  • these partial sequences are designated “Expressed Sequence Tags” (ESTs) and are markers for the genes actually expressed in vivo and are ascertained as described herein.
  • ESTs comprise DNA sequences corresponding to a portion of nuclear encoded mRNA. The EST has a length that allows for PCR (polymerase chain reaction), use as a hybridization probe and is a unique designation for the gene with which it hybridizes (generally under conditions sufficiently stringent to require at least 95% base pairing).
  • WO 93/00353 For a detailed description and review of ESTs and their functional utility see WO 93/00353 which is incorporated in its entirety by reference. WO 93/00353 further describes how the EST sequences can be used to identify the transcribed genes. The Example herein also describes a method of identification.
  • the present invention also provides a method of diagnosing the presence of bladder cancer in a patient, by the expression of at least one expressed gene (upregulated) identified by the sequences of the present invention set forth in Tables I and II.
  • Methods of identification of hybridization results can include, but are not limited to, immunohistochemical staining of the tissue samples. Further for identification of the gene, in situ hybridization, Southern blotting, single strand conformational polymorphism, restriction endonuclease fingerprinting (REF), PCR amplification and DNA-chip analysis using nucleic acid sequence of the present invention as probes/primers can be used.
  • purified, isolated and cloned bladder cancer associated genes identified by the probes and/or sequences hybridizing under stringent conditions with 95% homology and set forth herein, or a complementary or allelic variation sequence and human homologs, as relevant, thereto, are disclosed.
  • the present invention further provides proteins encoded by the identified genes.
  • the present invention further provides antibodies directed against these proteins.
  • the present invention further provides transgenic animals and cell lines carrying at least one expressible gene identified by the present invention.
  • the present invention further provides knock-out eukaryotic organisms in which at least one nucleic acid sequences as identified by the probes of the present invention and prepared as described in the Methods.
  • the present invention provides a method of identifying bladder cancer, and particularly early stage associated pathologies in a patient.
  • the present invention provides a treatment by administering to a patient a therapeutically effective amount of an antagonist of at least one protein as encoded by the nucleic acid sequences or sequences identified herein or by the probes of the present invention.
  • the present invention provides a method of regulating bladder cancer-associated pathologies in a patient in need of such treatment by administering to a patient a therapeutically effective amount of at least one antisense oligonucleofide against the nucleic acid sequences or dominant negative peptide directed against the sequences or their proteins.
  • the present invention provides a method of preparation of a library using proprietary methods of library preparation as follows: the SDGI method, as described in US Patent Application USSN 09/538,709 of same under the assignee, filed 30 March, 2000, and incorporated herein by reference in its entirety, the Antisense method as described in US Provisional Patent Application SN 60/157,843 of same assignee, filed 6 October, 1999, and incorporated herein by reference in its entirety, and suppressive subtraction hybridization (SSH) (Diatchenko et al., 1996).
  • SSH suppressive subtraction hybridization
  • Negative dominant peptide refers to a partial cDNA sequence that encodes for a part of a protein, i.e. a peptide (see Herskowitz, 1987). This peptide can have a different function from the protein from which it was derived. It can interact with the full protein and inhibit its activity or it can interact with other proteins and inhibit their activity in response to the full protein. Negative dominant means that the peptide is able to overcome the natural proteins and fully inhibit their activity to give the cell a different characteristics like resistance or sensitization to killing. For therapeutic intervention either the peptide itself is delivered as the active ingredient of a pharmaceutical composition or the cDNA can be delivered to the cell utilizing the same methods as for antisense delivery.
  • the antagonist or regulating agent or active ingredient is dosed and delivered in a pharmaceutically acceptable carrier as described herein below.
  • the term antagonist or antagonizing is used in its broadest sense.
  • Antagonism can include any mechanism or treatment which results in inhibition, inactivation, blocking or reduction in gene activity or gene product and for example preventing progression from non-invasive to invasive. It should be noted that the inhibition of a gene or gene product provides for an increase in a corresponding function that the gene or gene product was regulating.
  • the antagonizing step can include blocking cellular receptors for the gene products and can include antisense treatment as discussed herein.
  • AS oligonucleotide sequences can be short sequences of DNA, typically 15-30 mer but can be as small as 7 mer (Wagner et al, 1996), designed to complement a target mRNA of interest and form an RNA.AS duplex. This duplex formation can prevent processing, splicing, transport or translation of the relevant mRNA.
  • AS nucleotide sequences can elicit cellular RNase H activity when hybridized with their target mRNA, resulting in mRNA degradation (Calabretta et al, 1996). In that case, RNase H cleaves the RNA component of the duplex and can release the AS to further hybridize with additional molecules of the target RNA.
  • An additional mode of action results from the interaction of AS with genomic DNA to form a triple helix which can be transcriptionally inactive.
  • the sequence target segment for the antisense oligonucleotide is selected such that the sequence exhibits suitable energy related characteristics important for oligonucleotide duplex formation with their complementary templates, and shows a low potential for self-dimerization or self-complementation [Anazodo et al., 1996].
  • the computer program OLIGO Primary Analysis Software, Version 3.4
  • the program allows the determination of a qualitative estimation of these two parameters (potential self-dimer formation and self- complimentary) and provides an indication of "no potential” or "some potential” or "essentially complete potential”.
  • segments are generally selected that have estimates of no potential in these parameters. However, segments can be used that have "some potential" in one of the categories. A balance of the parameters is used in the selection as is known to those of skill in the art. Further, the oligonucleotides are also selected as needed so that analogue substitution do not substantially affect function, as is known to those of skill in the art.
  • Phosphorothioate antisense oligonucleotides do not normally show significant toxicity at concentrations that are effective, exhibit sufficient pharmacodynamic half-lives in animals (Agarwal et al., 1996) and are nuclease resistant. Antisense induced loss-of-function phenotypes related with cellular development were shown for the glial fibrillary acidic protein (GFAP), for the establishment of tectal plate formation in chick (Galileo et al., 1991) and for the N- myc protein which is responsible for the maintenance of cellular heterogeneity in neuroectodermal cultures (ephithelial vs.
  • GFAP glial fibrillary acidic protein
  • N- myc protein which is responsible for the maintenance of cellular heterogeneity in neuroectodermal cultures
  • bFgF basic fibroblast growth factor
  • Ribozymes can be utilized. This is particularly necessary in cases where antisense therapy is limited by stoichiometric considerations (Sarver et al., 1990, Gene Regulation and Aids, pp. 305-325). Ribozymes can then be used that target the same sequence. Ribozymes are RNA molecules that possess RNA catalytic ability (see Cech for review) that cleave a specific site in a target RNA. The number of RNA molecules that are cleaved by a ribozyme is greater than the number predicted by stochiochemistry. (Hampel and Tritz, 1989; Uhlenbeck, 1987).
  • Ribozymes catalyze the phosphodiester bond cleavage of RNA.
  • ribozyme structural families include Group I introns, RNase P, the hepatitis delta virus ribozyme, hammerhead ribozymes and the hairpin ribozyme originally derived from the negative strand of the tobacco ringspot virus satellite RNA (sTRSV) (Sullivan, 1994; U.S. Patent No. 5,225,347, columns 4-5).
  • the latter two families are derived from viroids and virusoids, in which the ribozyme is believed to separate monomers from oligomers created during rolling circle replication (Symons, 1989 and 1992).
  • ribozyme motifs are most commonly adapted for trans-cleavage of mRNAs for gene therapy (Sullivan, 1994).
  • the ribozyme type utilized in the present invention is selected as is known by those of skill in the art. Hairpin ribozymes are now in clinical trial and are the preferred type. In general, the ribozyme is from 30-100 nucleotides in length.
  • Modifications or analogues of nucleotides can be introduced to improve the therapeutic properties of the nucleotides. Improved properties include increased nuclease resistance and/or increased ability to permeate cell membranes.
  • Nuclease resistance is provided by any method known in the art that does not interfere with biological activity of the antisense oligodeoxy- nucleotides, cDNA and/or ribozymes as needed for the method of use and delivery (Iyer et al., 1990; Eckstein, 1985; Spitzer and Eckstein, 1988; Woolf et al., 1990; Shaw et al., 1991).
  • Modifications that can be made to oligonucleotides in order to enhance nuclease resistance include, but are not limited to, modifying the phophorous or oxygen heteroatom in the phosphate backbone. These modifications also include preparing methyl phosphonates, phosphorothioates, phosphorodithioates and morpholino oligomers.
  • the modification is provided by having phosphorothioate bonds linking between the four to six 3'-terminus nucleotide bases.
  • phosphorothioate bonds can link all the nucleotide bases.
  • Other modifications known in the art can be used where the biological activity is retained, but the stability to nucleases is substantially increased.
  • the present invention also includes all analogues of, or modifications to, an oligonucleotide of the invention that does not substantially affect the function of the oligonucleotide.
  • the nucleotides can be selected from naturally occurring or synthetic modified bases. Naturally occurring bases include adenine, guanine, cytosine, thymine and uracil.
  • Modified bases of the oligonucleotides include xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza cytosine and 6-aza thymine, psuedo uracil, 4-thiuracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8- thiolalkyl adenines, 8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanines, 8-amino guanine, 8-thiol guanine, 8-thioalkyl guanines, 8-hydroxyl guanine and other substituted guanines, other aza and deaza adenines, other aza and deaza guanines, 5-trifluoromethyl uracil and 5-
  • analogues of nucleotides and/or polynucleotides can be prepared wherein the structure of the nucleotide and/or polynucleotide is fundamentally altered and that are better suited as therapeutic or experimental reagents.
  • An example of a nucleotide analogue is a peptide nucleic acid (PNA) wherein the deoxyribose (or ribose) phosphate backbone in DNA (or RNA) is replaced with a polyamide backbone which is similar to that found in peptides.
  • PNA analogues have been shown to be resistant to degradation by enzymes and to have extended lives in vivo and in vitro.
  • PNAs have been shown to bind stronger to a complementary DNA sequence than a DNA molecule. This observation is attributed to the lack of charge repulsion between the PNA strand and the DNA strand.
  • Other modifications that can be made to oligonucleotides include polymer backbones, cyclic backbones, or acyclic backbones.
  • the active ingredients of pharmaceutical compositions can include oligonucleotides that are nuclease resistant as are needed for the practice of the invention or a fragment thereof shown to have the same effect when targeted against the appropriate sequence(s) and/or ribozymes.
  • Combinations of active ingredients as disclosed in the present invention can be used, including combinations of antisense sequences.
  • the antisense oligonucleotides (and/or ribozymes) and cDNA of the present invention can be synthesized by any method known in the art for ribonucleic or deoxyribonucleic nucleotides.
  • an Applied Biosystems 380B DNA synthesizer can be used.
  • fragments are used, two or more such sequences can be synthesized and linked together for use in the present invention.
  • nucleotide sequences of the present invention can be delivered either directly or with viral or non-viral vectors. When delivered directly the sequences are generally rendered nuclease resistant. Alternatively the sequences can be incorporated into expression cassettes or constructs such that the sequence is expressed in the cell as discussed herein below. Generally the construct contains the proper regulatory sequence or promotor to allow the sequence to be expressed in the targeted cell.
  • the proteins of the present invention can be produced recombinantly (see generally Marshak et al, 1996 "Strategies for Protein Purification and Characterization. A laboratory course manual.”, CSHL Press) and analogues can be due to post-translational processing.
  • analogue as used herein is defined as a nucleic acid sequence or protein which has some differences in their amino acid/nucleotide sequences as compared to the native sequence of the sequences disclosed herein. Ordinarily, the analogue is generally at least 70% homologous over any portion that is functionally relevant. In more preferred embodiments the homology is at least 80% and can approach 95% homology to the protein/nucleotide sequence.
  • the amino acid or nucleotide sequence of an analog can differ from that of the primary sequence when at least one residue is deleted, inserted or substituted, but the protein or nucleic acid molecule remains functional. Differences in glycosylation can provide protein analogues.
  • “Functionally relevant” refers to the biological property of the molecule and in this context means an in vivo effector or antigenic function or activity that is directly or indirectly performed by a naturally occurring protein or nucleic acid molecule.
  • Effector functions include, but are not limited to include, receptor binding, any enzymatic activity or enzyme modulatory activity, any carrier binding activity, any hormonal activity, any activity in promoting or inhibiting adhesion of cells to extracellular matrix or cell surface molecules, or any structural role as well as having the nucleic acid sequence encode functional protein and can be expressible.
  • the antigenic functions essentially mean the possession of an epitope or antigenic site that is capable of cross-reacting with antibodies raised against a naturally occurring protein.
  • Biologically active analogues share an effector function of the native which can, but do not necessarily, additionally possess an antigenic function.
  • the method includes preparing cell fractionations; extracting intact total RNA from membrane bound polysomes and free polysomes; preparing cDNA probes from template RNA derived from the extracted polysomes; performing microarray-based comparison of the relative abundance of the different RNA species; analyzing the results; and identifying genes or clones encoding membranal or secreted proteins.
  • membranal and secreted proteins are both accessible and critical for transduction of numerous intra- and intercellular signals, they are generally viewed as preferred targets for pharmacological use and intervention. Therefore, the a priori classification of arrayed unknown gene sequences into those that potentially code for secreted and membranal proteins is of great value for the optimization of a high-throughput process of identifying potential drug targets. Furthermore, the identification of genes which express membranal or secreted proteins that are differentially expressed in different cellular situations is of the utmost importance in designing therapeutic or diagnostic tools for TCC.
  • a method of identifying clones which encode membranal and secreted proteins was employed by preparing bladder cancer cell fractionations, preparing cDNA probes from template RNA derived from membrane-bound polysomes and free-polysomes, performing a microarray-based comparison of the relative abundance of different RNA species, analyzing the results and thereby identifying genes encoding for membranal and secreted proteins. Since membranal and secreted proteins are generally viewed as preferred targets for pharmacological intervention, the present invention thus provides a method of identifying likely targets for TCC diagnosis and therapy.
  • the probes were prepared from normal healthy bladder samples and from TCC tumors. Only intact RNA with a proper histological report indicating the existence of TCC was used. All normal and tumor material was collected from two separate clinical centers. Such approach minimizes the influence of local specific surgical bias or subjectivity of the pathological report.
  • Probe 1 was common to all hybridizations (common control probe). RNA from TCC samples was mixed with RNA from normal bladder samples. An equal amount of the RNA mixture was labeled with Cy3 and used in all hybridizations; and Probe 2. In each of the hybridizations, a different RNA sample from a single donor was used (test probe).
  • a common control for all the hybridizations enables comparison of the results between the different hybridizations. If the common control (probe 1) hybridization results are similar in pattern in different hybridizations, comparison can be made between the results of probe 2 hybridizations and all hybridizations.
  • the hybridizations were carried out in three separate sets, but the same common control was used in all sets.
  • Set 1 includes hybridizations 2-11 (TC2A- TC11A)
  • set 2 includes hybridizations 16-25 (TC16A-TC25A)
  • set 3 includes hybridizations 28-41 (TC28A-TC41A).
  • TCC cell line -T24- (from ATCC) was used for cellular fractionation.
  • Membrane-bound polysomes were separated from free polysomes using a sucrose step gradient.
  • RNA coding for potentially secreted proteins was isolated from this microsomal-membranal fraction and separated from RNA coding for intracellular proteins. Hybridization was performed as described hereunder.
  • Probes used were as follows: Probe 1. Free polysomal RNA fraction labeled with Cy3; and Probe 2. Membrane-bound RNA fraction with Cy5.
  • microarray was made up of cDNA clones derived from 3 different libraries:
  • SDGI library (Described in US Patent Application USSN 09/538,709 of same applicant company, filed 30 March, 2000, and incorporated herein by reference in its entirety): A pool of non-invasive TCC, invasive TCC and normal bladder was used for library preparation. 4550 clones from the SDGI library were included in the TCC chip.
  • Antisense library (Described in US Provisional Patent Application SN 60/157,843 of same applicant company, filed 6 October, 1999, and incorporated herein by reference in its entirety): The same cDNA pool used for the SDGI library was used for the preparation of a library enriched for antisense sequences. 450 clones from this library were included in the TCC chip.
  • SSH library (Diatchenko et al., 1996). A subtraction library was made as follows. A normal bladder RNA pool was used for subtraction from non-invasive TCC RNA pool. The subtracted cDNA was used for the microarray printing. 5000 clones from the SSH library were used for printing.
  • PCR Polymerase chain reaction
  • ELISAs where appropriate are one of the immunoassays employed to assess a specimen.
  • ELISA assays are well known to those skilled in the art. Both polyclonal and monoclonal antibodies can be used in the assays. Where appropriate other immunoassays, such as radioimmunoassays (RIA) can be used as are known to those in the art. Available immunoassays are extensively described in the patent and scientific literature.
  • Antibodies can be either monoclonal, polyclonal or recombinant. Conveniently, the antibodies can be prepared against the immunogen or portion thereof for example a synthetic peptide based on the sequence, or prepared recombinantly by cloning techniques or the natural gene product and/or portions thereof can be isolated and used as the immunogen. Immunogens can be used to produce antibodies by standard antibody production technology well known to those skilled in the art as described generally in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988 and Borrebaeck, Antibody Engineering - A Practical Guide, W.H. Freeman and Co., 1992. Antibody fragments can also be prepared from the antibodies and include Fab, F(ab') 2 , and Fv by methods known to those skilled in the art.
  • polyclonal antibodies For producing polyclonal antibodies a host, such as a rabbit or goat, is immunized with the immunogen or immunogen fragment, generally with an adjuvant and, if necessary, coupled to a carrier; antibodies to the immunogen are collected from the sera. Further, the polyclonal antibody can be absorbed such that it is monospecific. That is, the sera can be absorbed against related immunogens so that no cross-reactive antibodies remain in the sera rendering it monospecific.
  • the technique involves hyperimmunization of an appropriate donor with the immunogen, generally a mouse, and isolation of splenic antibody producing cells. These cells are fused to a cell having immortality, such as a myeloma cell, to provide a fused cell hybrid which has immortality and secretes the required antibody. The cells are then cultured, in bulk, and the monoclonal antibodies harvested from the culture media for use.
  • an appropriate donor with the immunogen
  • the immunogen generally a mouse
  • splenic antibody producing cells are fused to a cell having immortality, such as a myeloma cell, to provide a fused cell hybrid which has immortality and secretes the required antibody.
  • the cells are then cultured, in bulk, and the monoclonal antibodies harvested from the culture media for use.
  • RNAs from antibody producing B-lymphocytes of animals, or hybridoma are reverse- transcribed to obtain complimentary DNAs (CDNAs).
  • CDNAs complimentary DNAs
  • Antibody cDNA which can be full or partial length, is amplified and cloned into a phage or a plasmid.
  • the cDNA can be a partial length of heavy and light chain cDNA, separated or connected by a linker.
  • the antibody, or antibody fragment is expressed using a suitable expression system to obtain recombinant antibody.
  • Antibody cDNA can also be obtained by screening pertinent expression libraries.
  • the antibody can be bound to a solid support substrate or conjugated with a detectable moiety or be both bound and conjugated as is well known in the art.
  • a solid support substrate for a general discussion of conjugation of fluorescent or enzymatic moieties see Johnstone & Thorpe, Immunochemistry in Practice, Blackwell Scientific Publications, Oxford, 1982.
  • the binding of antibodies to a solid support substrate is also well known in the art. (see for a general discussion Harlow & Lane Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Publications, New York, 1988 and Borrebaeck, Antibody Engineering - A Practical Guide, W.H.
  • the detectable moieties contemplated with the present invention can include, but are not limited to, fluorescent, metallic, enzymatic and radioactive markers such as biotin, gold, ferritin, alkaline phosphatase, ⁇ - galactosidase, peroxidase, urease, fluorescein, rhodamine, tritium, 14 C and iodination.
  • fluorescent, metallic, enzymatic and radioactive markers such as biotin, gold, ferritin, alkaline phosphatase, ⁇ - galactosidase, peroxidase, urease, fluorescein, rhodamine, tritium, 14 C and iodination.
  • the present invention provides for transgenic gene and polymorphic gene animal and cellular (cell lines) models as well as for knockout models. These models are constructed using standard methods known in the art and as set forth in United States Patents 5,487,992, 5,464,764, 5,387,742, 5,360,735, 5,347,075, 5,298,422, 5,288,846, 5,221 ,778, 5,175,385, 5,175,384,5,175,383, 4,736,866 as well as Burke and Olson (1991), Capecchi (1989), Davies et al. (1992), Dickinson et al. (1993), Duff and Lincoln (1995), Huxley et al. (1991), Jakobovits et al. (1993), Lamb et al.
  • one parent strain instead of carrying a direct human transgene can have the homologous endogenous gene modified by gene targeting such that it approximates the transgene. That is, the endogenous gene has been "humanized” and/or mutated (Reaume et al, 1996). It should be noted that if the animal and human sequence are essentially homologous a "humanized” gene is not required.
  • the transgenic parent can also carry an overexpressed sequence, either the nonmutant or a mutant sequence and humanized or not as required. The term transgene is therefore used to refer to all these possibilities.
  • cells can be isolated from the offspring which carry a transgene from each transgenic parent and that are used to establish primary cell cultures or cell lines as is known in the art.
  • a parent strain is homozygous for the transgene.
  • the endogenous nontransgene in the genome that is homologous to the transgene is nonexpressive.
  • nonexpressive is meant that the endogenous gene is not expressed and that this nonexpression is heritable in the offspring.
  • the endogenous homologous gene could be "knocked- out” by methods known in the art.
  • the parental strain that receives one of the transgenes could carry a mutation at the endogenous homologous gene rendering it nonexpressed.
  • gene therapy refers to the transfer of genetic material (e.g DNA or RNA) of interest into a host to treat or prevent a genetic or acquired disease or condition phenotype.
  • the genetic material of interest encodes a product (e.g. a protein, polypeptide, peptide, functional RNA, antisense) whose production in vivo is desired.
  • the genetic material of interest can encode a hormone, receptor, enzyme, polypeptide or peptide of therapeutic value.
  • the genetic material of interest encodes a suicide gene.
  • ex vivo and (2) in vivo gene therapy Two basic approaches to gene therapy have evolved: (1) ex vivo and (2) in vivo gene therapy.
  • ex vivo gene therapy cells are removed from a patient, and while being cultured are treated in vitro.
  • a functional replacement gene is introduced into the cell via an appropriate gene delivery vehicle/method (transfection, transduction, homologous recombination, etc.) and an expression system as needed and then the modified cells are expanded in culture and returned to the host/patient.
  • These genetically reimplanted cells have been shown to express the transfected genetic material in situ.
  • target cells are not removed from the subject rather the genetic material to be transferred is introduced into the cells of the recipient organism in situ, that is within the recipient.
  • the host gene if the host gene is defective, the gene is repaired in situ [Culver, 1998]. These genetically altered cells have been shown to express the transfected genetic material in situ.
  • the gene expression vehicle is capable of delivery/ transfer of heterologous nucleic acid into a host cell.
  • the expression vehicle can include elements to control targeting, expression and transcription of the nucleic acid in a cell selective manner as is known in the art. It should be noted that often the 5'UTR and/or 3'UTR of the gene can be replaced by the 5'UTR and/or 3'UTR of the expression vehicle. Therefore as used herein the expression vehicle can, as needed, not include the 5'UTR and/or 3'UTR of the actual gene to be transferred and only include the specific amino acid coding region.
  • the expression vehicle can include a promotor for controlling transcription of the heterologous material and can be either a constitutive or inducible promotor to allow selective transcription. Enhancers that can be required to obtain necessary transcription levels can optionally be included. Enhancers are generally any non- translated DNA sequence which works contiguously with the coding sequence (in cis) to change the basal transcription level dictated by the promoter.
  • the expression vehicle can also include a selection gene as described herein below. Vectors can be introduced into cells or tissues by any one of a variety of known methods within the art.
  • nucleic acids by infection offers several advantages over the other listed methods. Higher efficiency can be obtained due to their infectious nature. Moreover, viruses are very specialized and typically infect and propagate in specific cell types. Thus, their natural specificity can be used to target the vectors to specific cell types in vivo or within a tissue or mixed culture of cells. Viral vectors can also be modified with specific receptors or ligands to alter target specificity through receptor mediated events.
  • DNA viral vector for introducing and expressing recombinant sequences is the adenovirus derived vector Adenop53TK.
  • This vector expresses a herpes virus thymidine kinase (TK) gene for either positive or negative selection and an expression cassette for desired recombinant sequences.
  • TK herpes virus thymidine kinase
  • This vector can be used to infect cells that have an adenovirus receptor which includes most cancers of epithelial origin as well as others.
  • This vector as well as others that exhibit similar desired functions can be used to treat a mixed population of cells and can include, for example, an in vitro or ex vivo culture of cells, a tissue or a human subject. Additional features can be added to the vector to ensure its safety and/or enhance its therapeutic efficacy.
  • Such features include, for example, markers that can be used to negatively select against cells infected with the recombinant virus.
  • An example of such a negative selection marker is the TK gene described above that confers sensitivity to the antibiotic gancyclovir. Negative selection is therefore a means by which infection can be controlled because it provides inducible suicide through the addition of antibiotic. Such protection ensures that if, for example, mutations arise that produce altered forms of the viral vector or recombinant sequence, cellular transformation can not occur.
  • features that limit expression to particular cell types can also be included. Such features include, for example, promoter and regulatory elements that are specific for the desired cell type.
  • recombinant viral vectors are useful for in vivo expression of a desired nucleic acid because they offer advantages such as lateral infection and targeting specificity.
  • Lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. The result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. This is in contrast to vertical-type of infection in which the infectious agent spreads only through daughter progeny.
  • Viral vectors can also be produced that are unable to spread laterally. This characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.
  • viruses are very specialized infectious agents that have evolved, in many cases, to elude host defense mechanisms.
  • viruses infect and propagate in specific cell types.
  • the targeting specificity of viral vectors utilizes its natural specificity to specifically target predetermined cell types and thereby introduce a recombinant gene into the infected cell.
  • the vector to be used in the methods of the invention depends on desired cell type to be targeted and is known to those skilled in the art. For example, if breast cancer is to be treated then a vector specific for such epithelial cells are used. Likewise, if diseases or pathological conditions of the hematopoietic system are to be treated, then a viral vector that is specific for blood cells and their precursors, preferably for the specific type of hematopoietic cell, is used.
  • Retroviral vectors can be constructed to function either as infectious particles or to undergo only a single initial round of infection.
  • the genome of the virus is modified so that it maintains all the necessary genes, regulatory sequences and packaging signals to synthesize new viral proteins and RNA. Once these molecules are synthesized, the host cell packages the RNA into new viral particles which are capable of undergoing further rounds of infection.
  • the vector's genome is also engineered to encode and express the desired recombinant gene.
  • the vector genome is usually mutated to destroy the viral packaging signal that is required to encapsulate the RNA into viral particles. Without such a signal, any particles that are formed do not contain a genome and therefore cannot proceed through subsequent rounds of infection.
  • the specific type of vector depends upon the intended application.
  • the actual vectors are also known and readily available within the art or can be constructed by one skilled in the art using well-known methodology.
  • the recombinant vector can be administered in several ways. If viral vectors are used, for example, the procedure can take advantage of their target specificity and consequently, do not have to be administered locally at the diseased site. However, local administration can provide a quicker and more effective treatment, administration can also be performed by, for example, intravenous or subcutaneous injection into the subject. Injection of the viral vectors into a spinal fluid can also be used as a mode of administration, especially in the case of neuro-degenerative diseases. Following injection, the viral vectors circulate until they recognize host cells with the appropriate target specificity for infection. An alternate mode of administration can be by direct inoculation locally at the site of the disease or pathological condition or by inoculation into the vascular system supplying the site with nutrients or into the spinal fluid.
  • Local administration is advantageous because there is no dilution effect and, therefore, a smaller dose is required to achieve expression in a majority of the targeted cells. Additionally, local inoculation can alleviate the targeting requirement required with other forms of administration since a vector can be used that infects all cells in the inoculated area. If expression is desired in only a specific subset of cells within the inoculated area, then promoter and regulatory elements that are specific for the desired subset can be used to accomplish this goal.
  • Such non-targeting vectors can be, for example, viral vectors, viral genome, plasmids, phagemids and the like.
  • Transfection vehicles such as liposomes can also be used to introduce the non- viral vectors described above into recipient cells within the inoculated area. Such transfection vehicles are known by one skilled within the art.
  • the compound of the present invention is administered and dosed in accordance with good medical practice, taking into account the clinical condition of the individual patient, the site and method of administration, scheduling of administration, patient age, sex, body weight and other factors known to medical practitioners.
  • the pharmaceutically "effective amount" for purposes herein is thus determined by such considerations as are known in the art. The amount must be effective to achieve improvement including but not limited to improved survival rate or more rapid recovery, or improvement or elimination of symptoms and other indicators as are selected as appropriate measures by those skilled in the art.
  • the compound of the present invention can be administered in various ways. It should be noted that it can be administered as the compound or as pharmaceutically acceptable salt and can be administered alone or as an active ingredient in combination with pharmaceutically acceptable carriers, diluents, adjuvants and vehicles.
  • the compounds can be administered orally, subcutaneously or parenterally including intravenous, intraarterial, intramuscular, intraperitoneally, and intranasal administration as well as intrathecal and infusion techniques. Implants of the compounds are also useful.
  • the patient being treated is a warm-blooded animal and, in particular, mammals including man.
  • the pharmaceutically acceptable carriers, diluents, adjuvants and vehicles as well as implant carriers generally refer to inert, non-toxic solid or liquid fillers, diluents or encapsulating material not reacting with the active ingredients of the invention.
  • the doses can be single doses or multiple doses over a period of several days, but single doses are preferred.
  • the doses can be single doses or multiple doses over a period of several days.
  • the treatment generally has a length proportional to the length of the disease process and drug effectiveness and the patient species being treated.
  • the pharmaceutical formulations suitable for injection include sterile aqueous solutions or dispersions and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • the carrier can be a solvent or dispersing medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Nonaqueous vehicles such as cottonseed oil, sesame oil, olive oil, soybean oil, corn oil, sunflower oil, or peanut oil and esters, such as isopropyl myristate, can also be used as solvent systems for compound compositions.
  • various additives which enhance the stability, sterility, and isotonicity of the compositions including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added.
  • antimicrobial preservatives including antimicrobial preservatives, antioxidants, chelating agents, and buffers
  • Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like.
  • Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. According to the present invention, however, any vehicle, diluent, or additive used have to be compatible with the compounds.
  • Sterile injectable solutions can be prepared by incorporating the compounds utilized in practicing the present invention in the required amount of the appropriate solvent with various of the other ingredients, as desired.
  • a pharmacological formulation of the present invention can be administered to the patient in an injectable formulation containing any compatible carrier, such as various vehicle, adjuvants, additives, and diluents; or the compounds utilized in the present invention can be administered parenterally to the patient in the form of slow-release subcutaneous implants or targeted delivery systems such as monoclonal antibodies, vectored delivery, iontophoretic, polymer matrices, liposomes, and microspheres.
  • any compatible carrier such as various vehicle, adjuvants, additives, and diluents
  • the compounds utilized in the present invention can be administered parenterally to the patient in the form of slow-release subcutaneous implants or targeted delivery systems such as monoclonal antibodies, vectored delivery, iontophoretic, polymer matrices, liposomes, and microspheres.
  • Examples of delivery systems useful in the present invention include: 5,225,182; 5,169,383; 5,167,616; 4,959,217; 4,925,678; 4,487,603; 4,486,194; 4,447,233; 4,447,224; 4,439,196; and 4,475,196.
  • Many other such implants, delivery systems, and modules are well known to those skilled in the art.
  • a pharmacological formulation of the compound utilized in the present invention can be administered orally to the patient. Conventional methods such as administering the compounds in tablets, suspensions, solutions, emulsions, capsules, powders, syrups and the like are usable. Known techniques which deliver it orally or intravenously and retain the biological activity are preferred.
  • the compound of the present invention can be administered initially by intravenous injection to bring blood levels to a suitable level.
  • the patient's levels are then maintained by an oral dosage form, although other forms of administration, dependent upon the patient's condition and as indicated above, can be used.
  • the quantity to be administered vary for the patient being treated and vary from about 100 ng/kg of body weight to 100 mg/kg of body weight per day and preferably are from 10 ⁇ g/kg to 10 mg/kg per day.
  • RNA is prepared from the cytoplasmic extracts and from the nuclear fractions.
  • fluorescent cDNA probes are prepared. Each condition is labeled with a different fluorescent dye.
  • a probe can be composed of a mixture of Cy3 -dCTP cDNA prepared from RNA extracted from stressed cells and with Cy5-dCTP cDNA prepared from RNA extracted from nonstressed cells. The probes are used for hybridization to micro-array containing individually spotted cDNA clones derived from cells that were exposed to stress.
  • Oligonucleotides were used for Differential display.
  • the oligonucleotides are generally those described in the Delta RNA Fingerprinting kit (Clonetech Labs. Inc.).
  • Amplification reactions Each reaction is done in 20 ⁇ l and contains 50 ⁇ M dNTP mix, 1 ⁇ M from each primer, 1x polymerase buffer, 1 unit expand Polymerase (Beohringer Mannheim), 2 ⁇ Ci [ ⁇ - 32 P]dATP and 1 ⁇ l cDNA template. Cycling conditions are generally: three minutes at 95°C, then three cycles of two minutes at 94°C, five minutes at 40°C, five minutes at 68°C. This is followed by 27 cycles of one minute at 94°C, two minutes at 60°C, two minutes at 68°C. Reactions were terminated by a seven minute incubation at 68°C and addition of 20 ⁇ l sequencing stop solution (95% formamide, 10mM NaOH, 0.025% bromophenol blue, 0.025% xylene cyanol).
  • Gel analysis Generally 3-4 ⁇ l are loaded onto a 5% sequencing polyacrylamide gel and samples are electrophoresed at 2000 volts/40 milliamperes until the slow dye (xylene cyanol) is about 2 cm from the bottom. The gel is transferred to a filter paper, dryed under vacuum and exposed to x-ray film.
  • Reverse transcription as above but with 2 ⁇ g polyA+ selected mRNA.
  • cDNA from the previous step is treated with alkali to remove the mRNA, precipitated and dissolved in 20 ⁇ l H 2 0. 5 ⁇ I buffer, 2 ⁇ l 10mM dATP, H 2 O to 48 ⁇ l and 2 ⁇ l terminal deoxynucleotide transferase (TdT) are added. The reaction is incubated two to four hours at 37°C. 5 ⁇ l oligo dT (1 ⁇ g/ ⁇ l) was added and incubated at 60°C for five minutes.
  • cDNA with Dpnll is digested by adding 3 ⁇ l Dpnll reaction buffer 20 V and Dpnll to 25 ⁇ l cDNA and incubated five hours at 37° C. 50 ⁇ l TE is added and extracted with phenol/chloroform. cDNA is precipitated and dissolved to a concentration of 10ng/ ⁇ l.
  • Dpnll digested cDNA 4 ⁇ l from each oligo and 5 ⁇ l ligation buffer x10 and annealed at 60°C for ten minutes. 2 ⁇ l ligase is added and incubated overnight at 16°C. The ligation mixture is diluted by adding 140 ⁇ l TE. Amplification is carried out in a volume of 200 ⁇ l using appropriate primer and 2 ⁇ l ligation product and repeated in twenty tubes for each sample. Before adding Taq DNA polymerase, the tubes are heated to 72°C for three minutes. PCR conditions are as follows: five minutes at 72°C, twenty cycles of one minute at 95°C and three minutes at 72°C, followed by ten minutes at 72°C.
  • Amplification Amplification of subtracted DNA in a final volume of 200 ⁇ l as follows: Buffer, nucleotides and 20 ⁇ l of the diluted DNA are added, heated to 72° C, and Taq DNA polymerase added. Incubate at 72°C for five minutes and add apprpriate oligo. Ten cycles of one minute at 95°C, three minutes at 70°C are performed. Incubate ten minutes at 72°C. The amplification is repeated in four separate tubes.
  • the amplified DNA is extracted with phenol/chloroform, precipitated and all four tubes combined in 40 ⁇ l 0.2xTE, and digested with Mung Bean Nuclease as follows: To 20 ⁇ 1 DNA 4 ⁇ l buffer, 14 ⁇ l H 2 O and 2 ⁇ l Mung Bean Nuclease (10 units/ ⁇ l) added. Incubate at 30°C for thirty-five minutes + First Differential Product (DPI). Repeat subtraction hybridization and PCR amplification at driver: differential ratio of 1 :400 (DPII) and 1 :40,000 (DPMI) using appropriate oligonucleotides. Differential products are then cloned into a Bluescript vector at the BAM HI site for analysis of the individual clones.
  • DPII Differential Product
  • RNA isolated as described herein above is labeled with fluorescent dNTP's using a reverse transcription reaction to generate a labeled cDNA probe.
  • mRNA is extracted from cells cultured in non-stress conditions and labeled with Cy3-dCTP (Amersham) and mRNA extracted from cells cultured under stress conditions is labeled with Cy5-dCTP (Amersham).
  • the two labeled cDNA probes are then mixed and hybridized onto a microarray (Schena et al, 1996) composed of for example 2000 cDNA clones derived from a cDNA library prepared from appropriate cells cultured under the stress conditions.
  • the microarray is scanned using a laser scanner and amount of fluorescence of each of the fluorescence dyes is measured for each cDNA clone on the micro-array giving an indication of the level of mRNA in each of the original mRNA populations being tested. Comparison of the fluorescence on each cDNA clone on the micro-array between the two different fluorescent dyes is a measure for the differential expression of the indicated genes between the two experimental conditions.
  • C6 Hela and Jurkat cell lines (ATCC) were grown either in DMEM (C6 and HeLa) or in RPMI (Jurkat) supplemented with 10% FCS.
  • C6 cells were grown either under normal oxygen conditions or under hypoxia for 4 or 16 hours (0.5%O 2 and 5%CO 2 ).
  • Jurkat cells were grown either at 37°C or exposed to 43°C for one or four hours.
  • HeLa cells were kept under normal tissue culture conditions. One to three hours prior to harvesting (for non-treated cells) or prior to treatment, culture medium was replaced with a fresh one. Cells were then washed with ice cold PBS-/-, harvested on ice and either directly processed to extraction of total RNA or collected by centrifugation (400g, 5 minutes). The cell pellet was immediately frozen in liquid nitrogen and kept at -70° until used for fractionation to isolate nuclei, polysomes or microsomes.
  • Nuclear and polysomal subcellular fractions were obtained in a single fractionation procedure as previously described
  • RNA sample After addition of 1mg/ml of heparin it was layered on top of a sucrose gradient (0.5M - 1.5M) and centrifuged (Beckman, SW41 , 36K, 4°C, 110'). Upon centrifugation, fractions were collected into tubes containing 0.5% SDS. The polysomal fraction was further used for extraction of RNA
  • Hela or Jurkat cell pellets were thawed on ice and lysed with 0.25M sucrose lysis buffer (250mM sucrose, 50mM TEA (triethanolamine), 50mM KOAc pH 7.5, 6mM Mg(OAc)2, 1mM EDTA, 1 mM DTT, 0.5mM PMSF, 200 units/ml RNasine (Promega), 0.1 mg/ml heparin) - 1 ml per 10 8 cells.
  • Cells were homogenized on ice using a Teflon homogenizer (Heidolph, 1000 rpm). Following centrifugation (600g, 10 minutes, 4°C), the supernatant was collected and kept on ice.
  • the nuclei pellet was washed with 1ml of lysis buffer and re-centrifuged (600g, 10 minutes, 4°C) to release the contaminating microsomes. The resultant supernatant was combined with the previous one. Total supernatant material was further centrifuged to precipitate mitochondria (10,000g, 10 minutes, 4°C) and heparin (1mg/ml) was added to the collected supernatant. The latter (5 ml) was layered over the two-step sucrose gradient (2.5 ml of 2.05M sucrose in lysis buffer/2.5 ml of 1.5M sucrose in lysis buffer).
  • Middle phase 2.5 ml containing membrane bound polysomes (MBP) and pellet containing free polysomes (FP) were further used for RNA extraction.
  • RNA was purified from both fractions with phenolxhloroform (1 :1) and precipitated with 0.3M NaOAc, 20 ⁇ g/ml glycogen (Roche) and 50% isopropanol. Following centrifugation (10,000g, 20 minutes, 4°C), RNA pellets were washed with 70% ethanol, dried and dissolved in H 2 0.
  • a cDNA microarray containing 1847 cDNA fragments was constructed from clones of subtracted cDNA libraries derived from C6 glioma cells enriched for hypoxia-responsive mRNAs. Specifically, mRNA prepared from C6 cells cultured under hypoxic conditions for 4 and 16 hours, respectively, was subjected to bidirectional subtraction (PCR-Select cDNA subtraction kit, Clontech) followed by cloning into pBluescript. Three sublibraries were prepared. Sublibrary A was enriched for cDNA species up-regulated after four hours of hypoxia: sublibrary B, for cDNA species up-regulated after 16 hours of hypoxia; and sublibrary C, for cDNA species down-regulated after 16 hours of hypoxia.
  • cDNA clones were selected from sublibrary A, 884 from sublibrary B, and 373 from sublibrary C.
  • the array also contained a set of control genes whose response to hypoxia in C6 glioma (and other cells) is well documented including vascular- endothelial growth factor (VEGF) 8 '9-25 glucose transporter 1(Glut1)9.26 and lactate dehydrogenase (LDH)27.
  • VEGF vascular- endothelial growth factor
  • Glut19.26 glucose transporter 1(Glut1)9.26
  • LDH lactate dehydrogenase
  • This microarray contained 1418 human cDNA clones collected from a library derived from the human F2054 fibroblast cell line.
  • the library was prepared in pBluescript, the average insert size being about 500 bp.
  • cDNA probes were synthesized from 50 ⁇ g of RNA using reverse transcriptase (Superscript, Gibco-BRL) and 18-mer oligo-dT primer.
  • the hybridization probe was composed of two cDNA populations, derived from two different RNA sources: one labelled with Cy3-dCTP and the other with Cy5-dCTP (Amersham), as previously described28.
  • the following types of probes were used for hybridizations to different DNA microarrays:
  • C ⁇ -specific microarray This microarray was utilized for hybridizations of C6-derived probes: (1) total RNA normoxia (Cy3)/total RNA four hours hypoxia (Cy 5); (2) total RNA normoxia (Cy 3)/ total RNA 16 hours hypoxia (Cy 5); (3) nuclear RNA normoxia (Cy3)/nuclear RNA 4 hours hypoxia (Cy5); (4) nuclear RNA normoxia (Cy3)/nuclear RNA 16 hrs hypoxia (Cy5).
  • F2054-specific microarray This microarray was utilized for hybridization of probes derived from heat shock experiments with Jurkat cells: (1)total RNA 37°C(Cy3)/total RNA 1 hour 43°C(Cy 5); (2) total RNA 37°C (Cy 3)/ total RNA four hours 43°C (Cy 5); (3) nuclear RNA 37°C (Cy3)/nuclear RNA 1hr 43°C (Cy5); (4) polysomal RNA 37°C (Cy3)/polysomal RNA 1 hour 43°C (Cy5); (5) polysomal RNA 37°C (Cy3)/polysomal RNA four hours 43°C (Cy5)
  • the a priori probability was first estimated for a selected clone to be MBP-resident ( ⁇ 1 in equations (1 ,2)). This estimation is required since the relative portions of the two clone populations both on the array and in the expressed RNA population are probably not equal (more cytosolic/nuclear clones are expected), thereby biasing in advance their probability of detection.
  • Bioinformatics sequence analysis of selected EST clones The EST sequences were extended using QBI's propriety clustering software (manuscript in preparation). Putative coding regions for the obtained contig sequences were defined by two criteria: (1) open reading frame (ORF) longer than 50 amino acids, flanked by untranslated region(s) containing multiple stop codons in all three frames; and (2) when a potential 5' UTR was observed, the presence of an initiation methionine was required; (it was sometimes possible to define it relative to the poly-A tail).
  • ORF open reading frame
  • the contigs were characterized in two steps. First, homology searches (BLAST) were performed to identify homologous sequences in the nucleotide and protein non-redundant (nr) databases of Genbank. Then the contig sequences were characterized by prediction of motifs and domains using
  • Table I The sequences named in Table I are identified by clone number and accession number. This list includes sequences of known genes whose function in bladder cancer was heretofore unknown and which were now found to upregulated in bladder cancer. Nucleic acid sequences are provided in Table III.
  • Table II The sequences named in Table II are identified by clone number. This list includes sequences of novel genes which have no identity to known proteins or genes in the gene databases. Nucleic acid sequences are provided in Table IV. In both Tables I and II, the differential expression pattern of the different hybridization probes is provided. In both Table I and II, the genes listed were found to be upregulated in at least 60% of TCC samples and unchanged in at least 75% of the normal samples.
  • Table I and II show the genes as described in biological NCBI databases, with the Genebank number of each gene as presented in the NCBI database.
  • the location of the clone in the TCC microarray of the present invention is set forth in the tables, with their clone ID and the location of the clone in the TCC microarray.
  • hybridization set 1 (Columns 5-14) which includes hybridizations TC2A-TC11A, show all the results as compared to the TC7A (normal) hybridization result.
  • the results are the calculated differential expression (see above for explanation of the calculations) of normal urothel samples (samples designated TC7A-TC11A) and of TCC samples (samples designated TC2A- TC6A).
  • hybridization set 2 which includes hybridizations TC16A-TC25A, all the results were calculated in comparison to the TC22A (normal) hybridization result.
  • Set 2 is shown in columns 15-24 wherein there is shown the calculated differential expression (see above for explanation of the calculations) of normal urothel samples (samples designated TC21A-TC24A) and of TCC samples (samples designated TC16A-TC20A and TC25A).
  • set 3 which includes hybridizations TC28A-TC41A, all the results were calculated compared to the reference normal probe from TC47A.
  • the results are the calculated differential expression (see above for explanation of the calculations) of normal urothel samples (samples designated TC35A-TC38A and samples TC46A- TC48A) and of TCC samples (samples designated TC28A-TC34A and TC39- TC45A).
  • the first set of raw data show the description of the grade of the TCC tumor i.e. G1-G3 or high / low grade (when available).
  • the second set of raw data are for tumor material, this shows the description of the stage of the tumor i.e. Ta, T1 , TIS (tumor in situ) or INV (invasive TCC).
  • the type of sample e.g. normal.
  • yeast artificial chromosome Nature, Vol. 362, pp. 255-261 (1993).
  • TNCTTTNC >29_TCC_17A5_M13F_E04_034.abl.fa TIME: Wed Aug 9 12:48:30 2000 trimming information: raw_sequence : 861 (high quality : 83-477) sequence: 99-187 [length:89]

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Abstract

L'invention porte: sur un procédé de diagnostic du cancer de la vessie chez un patient par analyse d'un prélèvement de tissu pour y rechercher au moins un gène exprimé dont la présence est révélatrice du cancer de la vessie; sur une séquence de polynucléotide dont la présence est révélatrice du cancer de la vessie; sur un marqueur de cancer de la vessie; sur des procédés de diagnostic du cancer de la vessie par criblage pour détecter la présence d'au moins un gène exprimé dont la présence est révélatrice du cancer de la vessie; et sur des procédés de traitement et régulation des pathologies associées au cancer de la vessie par administration à un patient de doses à efficacité thérapeutique d'un ribozyme, d'un oligonucléotide antisens, ou d'un agoniste, agissant contre les séquences d'acides nucléiques de la présente invention.
PCT/US2000/041005 1999-09-27 2000-09-27 Sequences caracteristiques du cancer de la vessie WO2001022864A2 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
IL14879800A IL148798A0 (en) 1999-09-27 2000-09-27 Sequences characteristic of bladder cancer
EP00977267A EP1248855A4 (fr) 1999-09-27 2000-09-27 Sequences caracteristiques du cancer de la vessie
CA002385893A CA2385893A1 (fr) 1999-09-27 2000-09-27 Sequences caracteristiques du cancer de la vessie
AU14926/01A AU1492601A (en) 1999-09-27 2000-09-27 Sequences characteristic of bladder cancer
US09/825,682 US6998232B1 (en) 1999-09-27 2001-04-04 Methods of diagnosing bladder cancer

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US15615399P 1999-09-27 1999-09-27
US60/156,153 1999-09-27

Related Child Applications (1)

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US09/825,682 Continuation-In-Part US6998232B1 (en) 1999-09-27 2001-04-04 Methods of diagnosing bladder cancer

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Cited By (4)

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Publication number Priority date Publication date Assignee Title
WO2002086084A2 (fr) * 2001-04-04 2002-10-31 Quark Biotech, Inc. Caracteristiques sequentielles d'un cancer de la vessie
US6998232B1 (en) 1999-09-27 2006-02-14 Quark Biotech, Inc. Methods of diagnosing bladder cancer
EP2292796A1 (fr) * 2005-02-10 2011-03-09 Oncotherapy Science, Inc. Procédé de diagnostic de cancer de la vessie
US11130789B2 (en) 2004-07-23 2021-09-28 Pacific Edge Limited Urine markers for detection of bladder cancer

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US5856136A (en) * 1996-07-03 1999-01-05 Incyte Pharmaceuticals, Inc. Human stem cell antigens

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DE19818619A1 (de) * 1998-04-21 1999-10-28 Metagen Gesellschaft Fuer Genomforschung Mbh Menschliche Nukleinsäuresequenzen aus Blase-Tumor

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US5856136A (en) * 1996-07-03 1999-01-05 Incyte Pharmaceuticals, Inc. Human stem cell antigens

Non-Patent Citations (1)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6998232B1 (en) 1999-09-27 2006-02-14 Quark Biotech, Inc. Methods of diagnosing bladder cancer
WO2002086084A2 (fr) * 2001-04-04 2002-10-31 Quark Biotech, Inc. Caracteristiques sequentielles d'un cancer de la vessie
WO2002086084A3 (fr) * 2001-04-04 2003-03-27 Quark Biotech Inc Caracteristiques sequentielles d'un cancer de la vessie
US11130789B2 (en) 2004-07-23 2021-09-28 Pacific Edge Limited Urine markers for detection of bladder cancer
EP2292796A1 (fr) * 2005-02-10 2011-03-09 Oncotherapy Science, Inc. Procédé de diagnostic de cancer de la vessie
EP2295601A1 (fr) * 2005-02-10 2011-03-16 Oncotherapy Science, Inc. Procédé de diagnostic de cancer de la vessie
US7998695B2 (en) 2005-02-10 2011-08-16 Oncotherapy Science, Inc. Method of diagnosing bladder cancer
US8685641B2 (en) 2005-02-10 2014-04-01 Oncotherapy Science, Inc. Method of screening compounds for treating bladder cancer

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WO2001022864A3 (fr) 2001-11-01
EP1248855A2 (fr) 2002-10-16
EP1248855A4 (fr) 2004-12-22
AU1492601A (en) 2001-04-30
WO2001022864A9 (fr) 2002-12-12
IL148798A0 (en) 2002-09-12
CA2385893A1 (fr) 2001-04-05

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