WO2000012526A1 - NOVEL TARGETS OF p53 REGULATORY ACTIVITY - Google Patents

Abstract

Novel nucleic acid molecules and encoded proteins are provided, which are targets of the activity of the p53 tumor suppressor protein. Several p53-induced target nucleic acid molecules, and one p53-repressed target, are described. The proteins encoded by these p53-induced or -repressed target genes are expected to be useful for modulation of cellular proliferation.

Description

NOVEL TARGETS OF p53 REGULATORY ACTIVITY

This application claims priority to U.S. Provisional Application No. 60/098,251, filed August 28, 1998, the entirety of which is incorporated by reference herein. Pursuant to 35 U.S.C. §202 (c) , it is acknowledged that the U.S. Government has certain rights in the invention described herein, which was made in part with funds from the National Institutes of Health.

FIELD OF THE INVENTION

This invention relates to diagnosis and treatment of neoplastic diseases. More specifically, this invention provides novel nucleic acid molecules involved in p53 -mediated regulation of cell cycle progression or apoptosis.

BACKGROUND OF THE INVENTION

Several publications are referenced in this application to more fully describe the state of the art to which this invention pertains. The disclosure of each such publication is incorporated by reference herein.

The p53 tumor suppressor protein is a nuclear phosphoprotein that functions in cell -cycle arrest, programmed cell death (apoptosis) , inhibition of tumor growth, and preservation of genetic stability. It performs these functions through involvement in several biochemical pathways, including transcriptional activation, transcriptional suppression and inhibition of DNA replication. Loss of proper p53 function in cells is one step in the progression toward a neoplastic phenotype ; more than 50% of human cancers have mutations in the p53 gene (see V.E. Velculescu & .S. El-Deiry, Clin. Chem., 42:858-868, 1996; A.J. Levine, Cell 88: 321- 331, 1997) .

DNA binding and transcriptional activation are among the best-understood functions of p53. Nearly every tumor-derived p53 mutant has lost its ability to bind DNA and transcriptionally activate target genes. This observation suggests that these properties of p53 are critical to its role in the control of cell proliferation. The diverse nature of the genes that p53 transcriptionally regulates suggests that p53 may be involved in pathways of cell -cycle control, angiogenesis, DNA repair, differentiation, growth factor signaling and apoptosis . Cell cycle arrest is one of the major roles of p53 regulatory function, operating through the cdk4-Rb pathway to control the Gl-to-S phase transition. The retinoblastoma protein (Rb) regulates E2F-DP transcription factor complexes, which themselves regulate a number of genes required to initiate or propagate the S phase of the cell cycle (for review, see Levine, 1997, supra) . p53 regulates the cdk4 -Rb pathway by turning on transcription of _p2i^{WAF1 CIP1} (El-Deiry et al . , Cell 75: 817- 825, 1993), which in turn acts on cyclin-CDK complexes (including Cyclin D/CDK4) and proliferating cell nuclear antigen (PCNA) to stop DNA replication.

There is also evidence to indicate that p53 can regulate the Gl/S phase transition by at least one p21- independent mechanism, e.g., through another of its downstream targets, GADD45. Moreover, p53 has also been implicated in a G2/M phase checkpoint by a mechanism not fully understood, as well as a third checkpoint, GO arrest, perhaps through the p53 target Gasl .

The other major function of p53 is to regulate apoptosis. Experimental evidence indicates that p53 activates apoptosis after DNA damage, such as that which results from treatment with chemo- or radiotherapy. p53 regulates a number of genes involved in apoptosis, including bax, bcl -2, insulin-like growth-factor binding protein-3 ( IGF-BP3) , fas, and the genes encoding TRAIL receptors DR4 and Killer/DR5. The genes for Bax, IGF-BP3 and Killer/DR5 have been shown to contain p53 -dependent , cis-acting, DNA-responsive elements. Inactivation or mutation of p53 has been reported to enhance cell sensitivity to cytotoxic agents which induce DNA damage. However, the status (wild type or mutant) of p53 in tumor cells does not always correlate with chemosensitivity . It is clear from the foregoing discussion that the regulation of molecules involved in cell growth, proliferation and apoptosis by p53 is not sufficiently understood for the purpose of designing rational therapies to modulate these processes. Accordingly, there is a need to identify additional downstream targets of p53 function. Once identified, such p53 targets may be utilized or regulated in cell growth arrest- or apoptosis-based therapeutic approaches to the elimination of tumor cells.

SUMMARY OF THE INVENTION

The present invention provides novel nucleic acid molecules that are targets of p53 activity and whose encoded proteins may be involved in modulation of cellular proliferation. These molecules are referred to herein collectively as "p53 targets" .

According to one aspect of the invention, a p53-inducible isolated nucleic acid molecule is provided, which includes an open reading frame encoding a human homolog of Drosophila melanogaster peroxidasin. In a preferred embodiment, the nucleic acid molecule encodes a polypeptide comprising part or all of amino acid SEQ ID NO: 15. Preferably, the nucleic acid molecule comprises part or all of SEQ ID NO: 3 or SEQ ID NO: 11. In another preferred embodiment, the nucleic acid molecule comprises a site for p53 interaction, which comprises the consensus sequence of SEQ ID NO: 24, or which comprises SEQ ID NO: 23. According to another aspect of the invention, a p53-inducible isolated nucleic acid molecule is provided, which includes an open reading frame encoding a human oxidase enzyme that comprises a motif conserved with bacterial flavin-oxidases . In a preferred embodiment, the nucleic acid molecule encodes a polypeptide comprising part or all of SEQ ID NO: 16. Preferably, the nucleic acid molecule comprises part or all of SEQ ID NO:4, SEQ ID NO: 5 or SEQ ID NO: 12. In another preferred embodiment, the nucleic acid molecule comprises a site for p53 interaction, which comprises the consensus sequence of SEQ ID NO: 25, or which comprises SEQ ID NOS:26, 27, 28 or 29.

According to another aspect of the invention, a p53-inducible isolated nucleic acid molecule is provided, which includes an open reading frame encoding a secreted, cysteine-rich polypeptide with proteinase inhibitor activity. In a preferred embodiment, the nucleic acid molecule encodes a polypeptide comprising part or all of SEQ ID NO: 17. Preferably, the nucleic acid molecule comprises part or all of SEQ ID NO : 8 or SEQ ID NO: 13.

According to another aspect of the invention, an isolated nucleic acid is provided, which is downregulated in response to p53 induction in a cultured cell, and which includes an open reading frame encoding a human transcription factor comprising a POU-domain like sequence and two homeo-domain like sequences. In a preferred embodiment, the nucleic acid molecule encodes a polypeptide comprising part or all of SEQ ID NO: 18. Preferably, the nucleic acid molecule comprises part or all of SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 14.

According to another aspect of the invention, isolated nucleic acid molecules are provided, which are up-regulated in response to p53 induction in a cultured cell, and which comprise part or all of one or more sequences selected from the group consisting of SEQ ID NOS. 1, 2, 6 and 7.

According to another aspect of the invention, a method is provided for isolating protein coding sequences corresponding to any one or more of the aforementioned isolated nucleic acid molecules. The method comprises assembling a hybridization reaction mixture in which is dispensed on or more of the isolated nucleic acid molecules in a single stranded form, and a test sample that comprises the corresponding protein coding sequence, in a single-stranded form, under conditions enabling hybridization of the isolated nucleic acid molecule and the protein coding sequence, and thereafter separating and cloning the protein coding sequence. According to another aspect of the invention, isolated proteins produced by expression of the aforementioned protein coding sequences are provided. In a preferred embodiment, the proteins comprise amino acid sequences substantially the same as any one of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18.

According to another aspect of the invention, recombinant DNA molecules are provided, which comprise the aforementioned nucleic acid molecules or protein coding sequences, operably linked to vectors for transforming cells. Cells transformed with those recombinant DNA molecules are also provided, as well as cellular assay systems utilizing those recombinant molecules . According to another aspect of the invention, oligonucleotides between about 10 and about 100 nucleotides in length are provided, which specifically hybridize with portions of the nucleic acid molecules of the invention. According to another aspect of the invention, antibodies are provided which are immunologically specific for part or all of any of the proteins of the invention.

According to another aspect of the invention, kits are provided, which comprise containers containing one or more of the nucleic acid molecules, oligonucleotides, polypeptides or antibodies of the invention. Additional reagents and instructions for using the kit components are provided in a preferred embodiment.

The nucleic acids, proteins and antibodies of the present invention are useful as diagnostic and therapeutic agents for the detection and treatment of cancer and other proliferative diseases. They should also find utility as research tools and will facilitate the elucidation of the mechanistic action of the novel genetic and protein interactions involved in the control of p53 -mediated cell cycle control and apoptosis.

Additional features and advantages of the present invention will be better understood by reference to the drawings, detailed description and examples that follow. BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1. Schematic diagram showing the functional domain structure of the human peroxidasin molecule. The locations of DNA probes used in Northern blotting analysis are labeled with the letters A through D.

Fig. 2. Schematic diagram of region of PRG3 exhibiting a shared motif with bacterial flavin-oxidases . Sequences of the shared motifs of PRG3 (residues 103-139 of SEQ ID NO: 16), sulfhydryl dehydrogenase (SEQ ID

N0:19), NADH oxidase (SEQ ID NO:20) and flavocytochrome c (SEQ ID NO: 21) are shown below.

Fig. 3. Schematic diagram of predicted motif structure of PRG5 protein. PRG5 has a predicted signal peptide sequence at the amino terminus, and consists of 123 amino acids with the signal peptide and 107 amino acids without the signal peptide. PRG5 is a cysteine rich protein (16 out of 107 amino acids) as indicated by vertical lines. The sequences shown below are PRG5 (SEQ ID NO: 17) compared with antileukoproteinase (SEQ ID

NO: 22), another cysteine rich protein.

Fig. 4. Fig. 4A is a histogram showing the results of transcription activation experiments using a reporter gene containing the putative p53 interacting site from PRG2. Reporter plasmids containing the site were exposed to wildtype p53 or a transactivation- negative mutant p53 (additional control comprised the reporter gene not exposed to any p53) . Fold-activation is shown on the Y axis. Fig. 4B shows a region of the exon of the PRG2 gene where the p53 interacting site is located. The 16 bp site is shown (SEQ ID NO:23, along with a consensus sequence of the site (SEQ ID NO: 24) .

Fig. 5. Fig. 5A is a schematic diagram showing the location of five potential p53 -responsive elements in the PRG5 gene. A p53 binding consensus sequence (SEQ ID NO: 25) is shown, which was constructed from the five potential p53 -responsive elements (A is SEQ ID NO: 26; B is SEQ ID NO: 27; C is SEQ ID NO: 28; D combines B and C; E is SEQ ID NO: 29) . Fig. 5B is a histogram showing activation of a reporter gene containing one of fragments A-E. The Y axis is luciferase activity expressed as fluorescence units) .

Fig. 6. Histogram showing the inhibition of cell growth by PRG2 and PRG5. 293 cells were transfected with expression plasmids, β-gal (Neo^s) , vector (pcDNA3.1, Neo^R) , PRG2 (Neo^R) , or PRG5 (Neo^R) . After two days of transfection, cells were passaged to three dishes and continued to culture an additional two weeks in the presence of 600 μg/ml of G418. Drug resistant colonies were quantified by crystal violet staining followed by elution with methanol and measurement of OD at 595 nm.

Fig. 7. Nucleotide and deduced amino acid sequence of PRG3 cDNA. Fig. 8. Nucleotide and deduced amino acid sequence of PRG5 cDNA.

Fig. 9. Nucleotide and deduced amino acid sequence of PRG6 cDNA.

Fig. 10. Alignment of amino acid sequences of PRG2 gene product and Drosophila peroxidasin (SEQ ID

NO: 30) . The amino acid sequence homology was calculated using the Smith-Waterman algorithm. *=identical amino acids, .=similar amino acids.

DETAILED DESCRIPTION OF THE INVENTION

I . Definitions ;

Various terms relating to the biological molecules of the present invention are used hereinabove and also throughout the specifications and claims. The terms "substantially the same, " "percent similarity" and "percent identity" are defined in detail below. With reference to nucleic acids of the invention, the term "isolated nucleic acid" is sometimes used. This term, when applied to DNA, refers to a DNA molecule that is separated from sequences with which it is immediately contiguous (in the 5¹ and 3' directions) in the naturally occurring genome of the organism from which it was derived. For example, the "isolated nucleic acid" may comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a procaryote or eucaryote. An "isolated nucleic acid molecule" may also comprise a cDNA molecule. With respect to RNA molecules of the invention, the term "isolated nucleic acid" primarily refers to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term may refer to an RNA molecule that has been sufficiently separated from RNA molecules with which it would be associated in its natural state (i.e., in cells or tissues), such that it exists in a "substantially pure" form (the term "substantially pure" is defined below) .

With respect to protein, the term "isolated protein" or "isolated and purified protein" is sometimes used herein. This term refers primarily to a protein produced by expression of an isolated nucleic acid molecule of the invention. Alternatively, this term may refer to a protein which has been sufficiently separated from other proteins with which it would naturally be associated, so as to exist in "substantially pure" form.

The term "substantially pure" refers to a preparation comprising at least 50-60% by weight the compound of interest (e.g., nucleic acid, oligonucleotide, protein, etc.). More preferably, the preparation comprises at least 75% by weight, and most preferably 90-99% by weight, the compound of interest. Purity is measured by methods appropriate for the compound of interest (e.g. chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like) .

Nucleic acid sequences and amino acid sequences can be compared using computer programs that align the similar sequences of the nucleic or amino acids thus define the differences. As one example, the CLUSTLW program and parameters employed therein may be utilized (Thompson et al . , 1994, supra) . However, equivalent alignments and similarity/identity assessments can be obtained through the use of any standard alignment software. For instance, the BLAST programs used to query sequence similarity in GenBank and other public databases may be used. The GCG Wisconsin Package version 9.1, available from the Genetics Computer Group in Madison, Wisconsin, and the default parameters used (gap creation penalty=12, gap extension penalty=4) by that program may also be used to compare sequence identity and similarity.

The term "substantially the same" refers to nucleic acid or amino acid sequences having sequence variation that do not materially affect the nature of the protein (i.e. the structure, stability characteristics, substrate specificity and/or biological activity of the protein) . With particular reference to nucleic acid sequences, the term "substantially the same" is intended to refer to the coding region and to conserved sequences governing expression, and refers primarily to degenerate codons encoding the same amino acid, or alternate codons encoding conservative substitute amino acids in the encoded polypeptide. With reference to amino acid sequences, the term "substantially the same" refers generally to conservative substitutions and/or variations in regions of the polypeptide not involved in determination of structure or function.

The terms "percent identical" and "percent similar" are also used herein in comparisons among amino acid and nucleic acid sequences. When referring to amino acid sequences, "percent identical" refers to the percent of the amino acids of the subject amino acid sequence that have been matched to identical amino acids in the compared amino acid sequence by a sequence analysis program. "Percent similar" refers to the percent of the amino acids of the subject amino acid sequence that have been matched to identical or conserved amino acids.

Conserved amino acids are those which differ in structure but are similar in physical properties such that the exchange of one for another would not appreciably change the tertiary structure of the resulting protein. Conservative substitutions are defined in Taylor (1986,

J. Theor. Biol. 119:205). When referring to nucleic acid molecules, "percent identical" refers to the percent of the nucleotides of the subject nucleic acid sequence that have been matched to identical nucleotides by a sequence analysis program. In an exemplary embodiment of the invention, the Smith-Waterman algorithm is used to determine percent similarity and percent identity among protein sequences.

With respect to antibodies, the term "immunologically specific" refers to antibodies that bind to one or more epitopes of a protein of interest, but which do not substantially recognize and bind other molecules in a sample containing a mixed population of antigenic biological molecules.

With respect to oligonucleotides or other single-stranded nucleic acid molecules, the term "specifically hybridizing" refers to the association between two single-stranded nucleic acid molecules of sufficiently complementary sequence to permit such hybridization under pre-determined conditions generally used in the art (sometimes termed "substantially complementary") . In particular, the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA or RNA molecule, to the substantial exclusion of hybridization of the oligonucleotide with single-stranded nucleic acids of non-complementary sequence.

A "coding sequence" or "coding region" refers to a nucleic acid molecule having sequence information necessary to produce a gene product, when the sequence is expressed. The term "operably linked" or "operably inserted" means that the regulatory sequences necessary for expression of the coding sequence are placed in a nucleic acid molecule in the appropriate positions relative to the coding sequence so as to enable expression of the coding sequence. This same definition is sometimes applied to the arrangement other transcription control elements (e.g. enhancers) in an expression vector.

Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell. The terms "promoter" , "promoter region" or "promoter sequence" refer generally to transcriptional regulatory regions of a gene, which may be found at the 5 ' or 3 ' side of the coding region, or within the coding region, or within introns. Typically, a promoter is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3 ' direction) coding sequence. The typical 5' promoter sequence is bounded at its 3 ' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence is a transcription initiation site (conveniently defined by mapping with nuclease SI) , as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase .

A "vector" is a replicon, such as plasmid, phage, cosmid, or virus to which another nucleic acid segment may be operably inserted so as to bring about the replication or expression of the segment.

The term "nucleic acid construct" or "DNA construct" is sometimes used to refer to a coding sequence or sequences operably linked to appropriate regulatory sequences and inserted into a vector for transforming a cell . This term may be used interchangeably with the term "transforming DNA" . Such a nucleic acid construct may contain a coding sequence for a gene product of interest, along with a selectable marker gene and/or a reporter gene.

The term "selectable marker gene" refers to a gene encoding a product that, when expressed, confers a selectable phenotype such as antibiotic resistance on a transformed cell. The term "reporter gene" refers to a gene that encodes a product which is easily detectable by standard methods, either directly or indirectly.

A "heterologous" region of a nucleic acid construct is an identifiable segment (or segments) of the nucleic acid molecule within a larger molecule that is not found in association with the larger molecule in nature. Thus, when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism. In another example, coding sequence is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene) . Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.

A cell has been "transformed" or "transfected" by exogenous or heterologous DNA when such DNA has been introduced inside the cell . The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA. A "clone" is a population of cells derived from a single cell or common ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of stable growth in vi tro for many generations.

II. Description As described above, p53 plays a pivotal and complex role in preventing malignant transformation. In some instances, p53 has been found to activate apoptosis after treatment of cells with chemotherapeutic agents or radiation. However, the molecular mechanism of p53- dependent apoptosis remains unclear, making it difficult to design or implement therapeutic strategies based on p53. P53 is also involved in regulation of cell cycle progression, and this function also has yet to be fully elucidated. Identification of downstream targets of p53 function in apoptotic or cell cycle signaling will facilitate rational development of diagnostic and therapeutic agents based on interaction with p53.

In accordance with the present invention, differential display analysis has been utilized to identify six cellular cDNA segments corresponding to genes whose RNA levels change in response to induction of p53 activity in human EB1 cells. The differential display analysis that led to the identification of these p53 target nucleic acid molecules is described in detail in Example 1.

The EB1 cells in which the changes in mRNA levels was observed comprise an inducible p53, the induction of which leads to apoptosis of the cells. The steady state levels of five of the mRNAs (referred to herein as PRG1 , PRG2 , PRG3 , PRG4 and PRG5 , respectively) were elevated, while the level of one (referred to herein as PRG6) decreased in response to p53 induction. Nucleotide sequences for each of the p53 targets initially obtained are set forth below. PRG1: Two nucleotide sequences found in PRG1 are set forth as SEQ ID NOS: 1 and 2, respectively. SEQ ID NO:l:

GTCGTGTGTC ACATTCAGAA GGCCTTTAAT GCCTGTTTGT CAAATTCTTG GCTAATTAAC AACTGCTGTG TGAGGTAAGC CATGGAGGAG GCCGTGTGAA AND SEQ ID NO : 2 :

GCTTGGCAGA TGAAGAGATT GGAAGTGAAC CACTGAGATG ACTGATGAGC GTCCTCAGCA GGCTGTGTTG CAGGGGGAAG AGTGCAGGTT GTTTATAAGG CCTGGGTCGT Neither of SEQ ID NOS: 1 or 2 correspond to a

Genbank or EST entry. PRG1 was up-regulated following p53 induction in EB1 cells.

PRG2 : A nucleotide sequence found in PRG2 is set forth below as SEQ ID NO: 3.

ACTGCTGTGA AGACTGTAGG ACCAGGGGGC AGTTCAATGC CTTTTCCTAT CATTTCCGAG GCAGACGGTC TCTTGAGTTC AGCTACCAGG AGGACAAGCC GACCAAGAAA ACAAGACCAC GGAAAATACC CAGTGTTGGG AGACAGGGGG AACATCTCAG CAACAGCACC TCAGCCTTCA GCACACGCTC AGATGCATCT GGGACAAATG ACTTCAGAGA GTTTGTTCTG GAAATGCAGA AGACCATCAC

AGACCTCAGA ACACAGATAA AGAAACTTGA ATCACGGCTC AGTACCACAG AGTGCGTGGA TGCCGGGGGC GAATCTCACG CCAACAACAC CAAGTGGAAA AAAGATGCAT GCACCATTTG TGAATGCAAA GACGGGCAGG TCACCTGCTT CGTGGAAGCT TGCCCC PRG2 was further characterized, as described below and in detail in Examples 1 and 2.

PRG3 : Two nucleotide sequences found in PRG3 are set forth as SEQ ID NOS: 4 and 5, respectively. SEQ ID NO:4:

TTATTAAACC CTCACTAAAT GCTGGTAGCC AGTTCATGTT GAGAAGCTGA GGACTAGGTC TACACTGGCG CCCTCTGCAT TCAGGTCAAG GGCCAAATGG GAATGTGCAG AAGGCAGGGG A AND SEQ ID NO : 5 : CCATTGATGC CTCTTCTCTA GATTCCGCCA TTCGTTAGGA GTTGCAAAGC TGTGGTATTC TCATTCTAGC ATTGCTCCTT TCCTTCTTGG GAATGCTTCT GTAGAGAGAA ACTACCTCTC TCCGGGATGC TTTTTTT

PRG3 was further characterized, as described below and in greater detail in Example 2.

PRG4: Two nucleotide sequences found in PRG4 are set forth as SEQ ID NOS: 6 and 7, respectively.

SEQ ID NO: 6: TGCTTGCTCT TCTTTAATAA GAGCAGTGGC TTTCAAAACA CAATGTCATT

TGTTCATCTT GGAACTGCTA TCCACAAGCC AAACAGCTCC TGTTAGATAG

GCACTC

AND SEQ ID NO : 7 :

CCCAAATACT TTTGATATAG CATCTTGCTA CCTGAGGAAC GCAAGGAGAA TAATACTCAT AAGCAGAGCT CTGTTTTCCC ATAGGACTGA CTCTAACTAT

GTGAAGAACT GCTACATATC TACAGTGCTT AATAGGCGGT GC

Neither of SEQ ID NOS : 4 or 5 correspond to a

Genbank or EST entry. PRG4 was up-regulated following p53 induction in EB1 cells.

PRG5: A sequence found in PRG5 is set forth below as SEQ ID NO: 8.

ATTAACCCTC ACTAAATGCT GGGGACCAAC GGAAAGAGTT CACGATGGGA

GGCCTGGGGC CCTGCCCGCT GGACAGCACT ATCTCTACCA GCGGTGGTTC CGGCCTTCTG ATAATCACTG GCCTGCTGAC ACTTCCCTGC AACCCATCCA

CCCCTGGTTT CTCCTCCTGG GAGTCAAAGT CCATAGCCTG AGCTCGGAGG AAGGCCTCTG TATCACCCCA GTACTCTGCA CCACTGCCAT ACGAGCTTCC CACCCTTCCT AACGCTTTCA CACCAATCCG TACATGCTGC TTCCTCCACC AAAAATGCCC AATTCAGGCA GACCCTGACC TCTCCCTCAG GCAGCCCAAC CATCCAGAAT GAATATTCTT GCAGAGTTTT CCAAACATCA GTCATTCACC

TCTTTCATGA TTTTCACCAT ACCTACAAAA TAGCACCATG ATAGGTTGCA CGCTGCCTGT ACCACCGTTT ACTTAATGTT TTCTTTAAAT GGCTCACTTT TGTATATAAA TAAATTCATT TCAAAAAAAA AGATATCACT CAG

PRG5 was further characterized, as described below and in detail in Example 2.

PRG6 : Two sequences found in PRG6 are set forth below as SEQ ID NOS: 9 and 10, respectively. SEQ ID NO: 9:

TCAGGGGTAC TGCCACCTAG CAGGCTTTAT TGGGAAGGGA CAAAGCCTCA GGAGCTGGGT GCCCCAGAGG CTGCTGGGTC TTGAGCCACA GCTGCAGCCA AT

AND SEQ ID NO: 10: ACGCGAATGG TACCCGAGCC TGGCGACCAT GCAGGAGTCG CTGCGGGTGA AGCAGCTGGC CGAAGAGCAG AAGCGTCGGG AGAGGGAGCA GCACATCGCA

PRG6 has been further characterized, as described below and in detail in Example 2.

The predicted amino acid sequence of SEQ ID

NO: 3 above (PRG2) was 37% identical and 46% similar to Drosophila peroxidasin (amino acids 1448-1498) , an extracellular matrix-associated peroxidase (dPxn) . This sequence also completely matched the sequence of GenBank clone KIAA0230, which was originally isolated in a a random screen from a human monocyte cancer cell line. Full length cDNA and deduced amino acid sequences of the KIAA0230 clone are set forth herein as SEQ ID NO: 11 and SEQ ID NO: 15, respectively, and are referred to hereinafter as the complete sequences of PRG2.

As described in Example 1, the predicted full- length protein sequence of PRG2 showed significant homology to the Drosophila peroxidasin (Fig. 10) (percentages of identity and similarity were determined using the Smith-Waterman algorithm (Smith, T. F. and Waterman, M. (1981). J. Mol. Biol., 147:195-197). Thus, PRG2 appears to be the human homolog (hPxn) of the Drosophila gene (Nelson et al . , 1994) . In Drosophila, dPxn is expressed exclusively in hemocytes derived from head mesoderm at a very early stage of differentiation. dPxn exists as a homotrimer with a unique hybrid structure that combines an enzymatically functional peroxidase domain with motifs that are typically found in extracellular matrix-associated proteins (Bornstein, 1992) . It is a secreted protein that contains a secretory recognition sequence (a signal peptide sequence) at its amino terminus. dPxn catalyzes H₂0₂- driven radioiodination, oxidations and the formation of dityrosine in vitro. It is also thought to function in extracellular matrix consolidation, phagocytosis and defense. Activation of hPxn suggests that reactive oxygen species (ROS) production by peroxidases may be a trigger for p53 -dependent apoptosis. It is important to point out that the potentially multifunctional hPxn protein may participate in a number of biological processes in human cells, as dPxn does in Drosophila . These processes are likely to involve removal and destruction of cells by induction of programmed cell death by p53, consolidation of extracellular matrix by covalent cross-link formation and protection of the organism against non-self.

To identify the predicted amino acid sequences for PRG3 , PRG5 and PRG6, we made cDNA libraries from the mRNA isolated from EBl cells treated with 0.1 mM ZnCl₂ for 9 hours. One half million bacteria colonies were screened by hybridization using the original cDNA fragment for PRG3 , 5, or 6 as a probe. Positive clones were analyzed by sequencing. Full length cDNA and deduced amino acid sequences of PRG3 are set forth herein as SEQ ID NOS : 12 and 16, respectively. The 3474 bp cDNA contains a 1042 bp 5'-noncoding sequence, a 1008 bp coding sequence followed by 1422 bp 3'-noncoding sequence (Fig. 7). The predicted amino acid sequence revealed that PRG3 consists of 336 amino acids with an approximate molecular weight 37 kDa. PRG3 is a basic protein (pi = 9.27) and does not show significant homology to any mammalian protein in the GenBank database. The predicted localization of the protein is in the cytoplasm (PSORT II Prediction) . Interestingly, PRG3 shares a motif with bacterial flavin- oxidases (Fig. 2) , suggesting that PRG3 may have oxidase activity. Full length cDNA and deduced amino acid sequences of PRG5 are set forth herein as SEQ ID NOS: 13 and 17, respectively. The 1411 bp cDNA contains a 64 bp 5'-noncoding sequence, a 369 bp coding sequence followed by 978 bp 3'-noncoding sequence (Fig. 8). The predicted amino acid sequence of PRG5 suggests that it is a small secreted protein. PRG5 has a predicted signal peptide sequence at its NH₂-terminus and consists of 123 amino acids (MW = 13.3 kDa) with the signal peptide and 107 amino acids (MW = 11.7 kDa) without the signal peptide (Fig. 3) . The striking feature of the PRG5 protein is its high content of cysteine residues (16 out of 107 amino acids) . PRG5 shares a similar cysteine motif with antileukoproteinase (ALP) or secretory leukocyte proteinase inhibitor (SLPI) , an elastase and cathepsin G proinflammatory serine proteinase inhibitor secreted from epithelial cells (Fig. 3) (Eisenberg et al . , 1990; Thompson and Ohlsson, 1986; Vogelmeier et al . , 1991). Thus, it is reasonable to assume that PRG5 product has a proteinase inhibitor activity. Full length cDNA and deduced amino acid sequences of PRG6 are set forth herein as SEQ ID NOS: 14 and 18, respectively. The 696 bp cDNA contains a 636 bp coding sequence followed by 60 bp 3'-noncoding sequence in the cDNA (Fig. 9) . The predicted amino acid sequence of PRG6 revealed that PRG6 consists of 212 amino acids with an approximate molecular weight 24.3 kDa. PRG6 is a basic protein (pi = 9.83) and does not show significant homology to any protein in the GenBank database. The predicted localization of the protein is in the nucleus (PSORT II Prediction) . Since PRG6 protein contains a POU-domain like sequence and two homeo-domain like sequences, it might function as a transcription factor. The chromosomal locations of each of PRG 1-6 have been determined. These locations are set forth in Example 2.

In addition, p53 -mediated transcriptional regulation of PRG3 and PRG5 has been further elucidated. As described in Example 2, potential p53 -responsive sequences have been identified in both PRG3 and PRG5. PRG2, PRG3 and PRG5 also have been found to exhibit a growth inhibitory effect upon overexpression in several cultured cell lines. This suggests that PRG2 , PRG3 and PRG5 are involved in p53 -mediated growth suppression pathways.

Reactive oxygen species (ROS) are powerful inducers of apoptosis. PRG2 and PRG3 may play a role in redox regulation. The predicted proteins encoded by PRG2 , and 3 are potentially a heme-peroxidase and a flavin-oxidase, respectively. The proteins encoded by these genes may collectively increase the intracellular content of ROS, which in turn may damage mitochondria. The enzymes or enzyme homologs that have the potential to produce ROS in cells have been identified by others in a different human colorectal cancer cell line undergoing p53-dependent apoptosis (Polyak et al . , 1997). The observations made in accordance with this invention and by others are consistent with the view that p53 -mediated apoptosis involves the intracellular production of ROS. Indeed, p53-induced apoptosis in smooth muscle cells seems to be ROS-dependent (Johnson et al . , 1996) . Since one of the previously identified potentially ROS- generating enzymes, PIG3, failed to induce apoptosis when overexpressed in cells (Polyak et al . , 1997), PRG2 and

PRG3 might be unique ROS-generating enzymes that directly initiate apoptosis. If this is the case, cell growth might be controlled by inhibiting or activating these enzyme activities. This would be particularly important for the treatment of cancer.

It has been shown that certain proteinase inhibitors inhibit cell growth in cultured cells and, in particular, sometimes prevent angiogenesis or metastasis of cancer. PRG5 shares a similar cysteine motif with antileukoproteinase (ALP) or secretory leukocyte proteinase inhibitor (SLPI) . ALP or SLPI is a serine proteinase inhibitor that inhibits proinflammatory serine proteincase, such as elastase, cathepsin G (Eisenberg et al . , 1990; Thompson and Ohlsson, 1986; Vogelmeier et al . , 1991), chymase (Walter et al . , 1996), and tryptase

(Hochstrasser et al . , 1993; Pemberton et al . , 1998). Thus, PRG5 might be proved useful as a therapeutic in degenerative and inflammatory diseases that lead to proteolytic damage to these and other tissues. Recent study showed that chymase and tryptase produced by inflammatory mast cells induced angiogenesis during skin carcinogenesis (Coussens et al . , 1999) . It might be speculated that PRG5 inhibits angiogenesis in tumor by inhibiting chymase and tryptase. As discussed in greater detail below, several utilities for the p53 target nucleic acids and proteins of the invention can be envisioned. First, the identification of three novel proteins differentially expressed by p53 expression (PRG3, 5, and 6) provides the opportunity to further explore the p53 response pathway with the possibility that this exploration, aided by the insights provided by these induced proteins, will lead to the identification of new targets for drug development. Further analysis of the one repressed mRNA (PRG6) might reveal that inhibition of its encoded protein ameliorates the consequences of the loss of p53 function. If this is the case, then it could prove to be a target for drug screening. Second, the proteins identified by us might prove to be useful as therapeutic genes for gene therapy applications. Furthermore, since proteins encoded by PRG2 and 5 are secreted protein, and also since the proteins showed growth suppression activities on human cancer cells, they might be useful as cancer therapeutic reagents themselves. Alternatively, any interacting molecules, directly or indirectly, to the proteins encoded by PRG2 , 3 , and 5 might be proved to be new useful therapeutic targets for controlling cell growth. As a potential, the protein product from PRG5 might be proved useful as a therapeutic in degenerative and inflammatory diseases that lead to proteolytic damage to these and other tissues. There might be a possibility that PRG5 can control angiogenesis of tumor cells as well (Coussens et al . , 1999). Although specific clones are described and exemplified herein, this invention is intended to encompass nucleic acid sequences and proteins from humans and other species that are sufficiently similar to be used interchangeably with the exemplified nucleic acids and proteins for the purposes described below. It will be appreciated by those skilled in the art that nucleic acids from diverse species, and particularly mammalian species, should possess a sufficient degree of homology with the human clones so as to be interchangeably useful in various applications. The present invention, therefore, is drawn to nucleic acids and encoded proteins from any species in which they are found, preferably of mammalian origin, and most preferably of human origin. Additionally, in the same manner that structural homologs of human p53 targets are considered to be within the scope of this invention, functional homologs are also considered to be within the scope of this invention. Allelic variants and natural mutants of the genes in which SEQ ID NOS: 1-14 are found are likely to exist within the human genome and within the genomes of other species. Because such variants are expected to possess certain differences in nucleotide and amino acid sequence, this invention provides isolated nucleic acid molecules having at least about 65% (preferably over 75%, more preferably over 85%) sequence homology in the coding region with the nucleotide sequences set forth as SEQ ID NOS: 1-14 (and, most preferably, specifically comprising coding regions found in any of SEQ ID NOS: 1-14), and isolated proteins having at least about 65% sequence homology (preferably at least 75%, more preferably at least 85%) with the amino acid sequences set forth as SEQ ID NOS: 15-18 (and most preferably, comprising one of SEQ ID NOS: 15-18) . Nucleic acid sequences and amino acid sequences can be compared using computer programs that align the similar sequences of the nucleic or amino acids and thus define the differences. Examples are set forth in the definitions above.

This invention also provides full length cDNAs or exons of genomic DNAs obtained by hybridization with one or more of SEQ ID NOS 1-14, and isolated proteins produced by expression of those DNAs. Because of the natural sequence variation likely to exist among the proteins and nucleic acids encoding them, one skilled in the art would expect to find up to about 25-35% nucleotide sequence variation, while still maintaining the unique properties of the proteins of the present invention. Such an expectation is due in part to the degeneracy of the genetic code, as well as to the known evolutionary success of conservative amino acid sequence variations, which do not appreciably alter the nature of the protein. Accordingly, such variants are considered substantially the same as one another and are included within the scope of the present invention.

The following description sets forth the general procedures involved in practicing the present invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. Unless otherwise specified, general cloning procedures, such as those set forth in Sambrook et al . , Molecular Cloning, Cold Spring Harbor Laboratory (1989) (hereinafter "Sambrook et al . " ) or Ausubel et al . (eds) Current Protocols in Molecular Biology, John Wiley & Sons (1999) (hereinafter "Ausubel et al . " ) are used.

III. Preparation of p53 Target Nucleic Acid Molecules, Encoded Proteins and Iromunoloqicallv Specific Antibodies

A. Nucleic Acid Molecules

Nucleic acid molecules encoding the p53 targets of the invention may be prepared by two general methods: (1) they may be synthesized from appropriate nucleotide triphosphates, or (2) they may be isolated from biological sources. Both methods utilize protocols well known in the art . The availability of nucleotide sequence information, such as the cDNAs having any of SEQ ID NOS: 1-14, enables preparation of an isolated nucleic acid molecule of the invention by oligonucleotide synthesis. Synthetic oligonucleotides may be prepared by the phosphoramadite method employed in the Applied Biosystems 38A DNA Synthesizer or similar devices. The resultant construct may be purified according to methods known in the art, such as high performance liquid chromatography (HPLC) . Long, double-stranded polynucleotides must be synthesized in stages, due to the size limitations inherent in current oligonucleotide synthetic methods.

Thus, a long, double-stranded molecule may be synthesized as several smaller segments of appropriate complementarity. Complementary segments may be annealed such that each segment possesses appropriate cohesive termini for attachment of an adjacent segment. Adjacent segments may be ligated by annealing cohesive termini in the presence of DNA ligase to construct an entire double- stranded molecule. A synthetic DNA molecule so constructed may then be cloned and amplified in an appropriate vector.

In a preferred embodiment, any of SEQ ID NOS: 1-14 is used to isolate longer or full-length corresponding p53 target nucleic acid sequences from appropriate biological sources using methods known in the art. In a preferred embodiment, cDNA clones are isolated from libraries of human origin. This has been done for PRG3 , 5 and 6. In an alternative embodiment, genomic clones may be isolated. Such clones have the advantage of possessing 5¹ and 3' untranslated regions that may be involved in regulation of expression of the p53 target gene. Alternatively, cDNA or genomic clones from other species, preferably mammalian species, may be obtained.

In accordance with the present invention, nucleic acids having the appropriate level sequence homology with any of SEQ ID NOS: 1-14 may be identified by using hybridization and washing conditions of appropriate stringency. For example, hybridizations may be performed, according to the method of Sambrook et al . , using a hybridization solution comprising: 5X SSC, 5X Denhardt's reagent, 1.0% SDS, 100 μg/ml denatured, fragmented salmon sperm DNA, 0.05% sodium pyrophosphate and up to 50% formamide. Hybridization is carried out at 37_42oc for at least six hours. Following hybridization, filters are washed as follows: (1) 5 minutes at room temperature in 2X SSC and 1% SDS; (2) 15 minutes at room temperature in 2X SSC and 0.1% SDS; (3) 30 minutes-1 hour at 37^QC in IX SSC and 1% SDS; (4) 2 hours at 42- 65oin IX SSC and 1% SDS, changing the solution every 30 minutes .

One common formula for calculating the stringency conditions required to achieve hybridization between nucleic acid molecules of a specified sequence homology (Sambrook et al . , 1989):

T_m = 81.5°C + 16.6Log [Na+] + 0.41(% G+C) - 0.63 (% formamide) - 600/#bp in duplex

As an illustration of the above formula, using [N+] = [0.368] and 50% formamide, with GC content of 42% and an average probe size of 200 bases, the T_m is 57°C. The T_ra of a DNA duplex decreases by 1 - 1.5°C with every 1% decrease in homology. Thus, targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42 °C. Such sequences would be considered substantially complementary to the probe. In a preferred embodiment, the hybridization is at 37°C and the final wash is at 42 °C, in a more preferred embodiment the hybridization is at 42° and the final wash is at 50°, and in a most preferred embodiment the hybridization is at 42°C and final wash is at 65°C, with the above hybridization and wash solutions. Conditions of high stringency include hybridization at 42 °C in the above hybridization solution and a final wash at 65°C in 0.1X SSC and 0.1% SDS for 10 minutes.

It should be understood that the hybridization methods described above are used to isolate full-length cDNA or genomic clones that encode the polypeptides associated with the p53 target cDNA segments having any of SEQ ID NOS: 1-14. These cDNA and genomic clones, their encoded proteins, and antibodies immunologically specific for those proteins, are considered to be part of the present invention, inasmuch as the isolation of SEQ ID NOS: 1-14 enables those molecules to be isolated and/or produced.

Nucleic acids of the present invention may be maintained as DNA in any convenient cloning vector. In a preferred embodiment, clones are maintained in plasmid cloning/expression vector, such as pBluescript (Stratagene, La Jolla, CA) , which is propagated in a suitable E. coli host cell. Optionally, the vectors may contain inducible promoter sequences, such as metallothionine, tetracycline or dexamethasone responsive promoters . Nucleic acid molecules of the invention include cDNA, genomic DNA, RNA, and fragments thereof which may be single- or double-stranded. Thus, this invention provides oligonucleotides (sense or antisense strands of DNA or RNA) having sequences capable of hybridizing with at least one sequence of a nucleic acid molecule of the present invention, such as selected segments of the cDNAs having SEQ ID NOS: 1-14. Such oligonucleotides are useful as probes for detecting corresponding genes in test samples of potentially malignant cells or tissues, e.g. by PCR amplification, or for the isolation of homologous sequences from other species.

In addition, 5' and 3' regulatory sequences of the p53 targets can be used to advantage. For instance, a promoter sequence can be cloned upstream of a reporter gene, so that drug screening can be performed to isolate compounds that induce expression of the gene. Reporters may include LacZ, Luciferase, GFP, Hygromycin, or other negative or positive selection markers.

B. Proteins and Antibodies

Proteins encoded by the p53 targets of the present invention may be prepared in a variety of ways, according to known methods. In a preferred embodiment, full-length cDNAs or coding regions of genomic DNAs obtained using any of SEQ ID NOS: 1-14 are themselves employed for production of the encoded proteins using in vi tro expression methods known in the art. For example, a cDNA or gene may be cloned into an appropriate in vi tro transcription vector, such a pSP64 or pSP65 for in vi tro transcription, followed by cell-free translation in a suitable cell-free translation system, such as wheat germ or rabbit reticulocytes . In vi tro transcription and translation systems are commercially available, e.g., from Promega Biotech, Madison, Wisconsin or BRL, Rockville, Maryland.

According to a particularly preferred embodiment, larger quantities of p53 target -encoded proteins may be produced by expression in a suitable procaryotic or eucaryotic system. For example, part or all of a selected DNA molecule may be inserted into a plasmid vector adapted for expression in a bacterial cell (such as E. coli) or a yeast cell (such as Saccharomyces cerevisiae) , or into a baculovirus vector for expression in an insect cell. Such vectors comprise the regulatory elements necessary for expression of the DNA in the host cell, positioned in such a manner as to permit expression of the DNA in the host cell. Such regulatory elements required for expression include promoter sequences, transcription initiation sequences and, optionally, enhancer sequences .

The proteins produced by gene expression in a recombinant procaryotic or eucyarotic system may be purified according to methods known in the art. In a preferred embodiment, a commercially available expression/secretion system can be used, whereby the recombinant protein is expressed and thereafter secreted from the host cell, to be easily purified from the surrounding medium. If expression/secretion vectors are not used, an alternative approach involves purifying the recombinant protein by affinity separation, such as by immunological interaction with antibodies that bind specifically to the recombinant protein. Such methods are commonly used by skilled practitioners. The proteins of the invention, prepared by the aforementioned methods, may be analyzed according to standard procedures.

The present invention also provides antibodies capable of immunospecifically binding to the proteins of the invention. Polyclonal or monoclonal antibodies directed toward any of these proteins may be prepared according to standard methods. Monoclonal antibodies may be prepared according to general methods of Kόhler and Milstein, following standard protocols. In a preferred embodiment, antibodies have been prepared, which react immunospecifically with various epitopes of the proteins.

Polyclonal or monoclonal antibodies that immunospecifically interact with the proteins can be utilized for identifying and purifying such proteins. For example, antibodies may be utilized for affinity separation of proteins with which they immunospecifically interact. Antibodies may also be used to immunoprecipitate proteins from a sample containing a mixture of proteins and other biological molecules. Other uses of antibodies are described below.

IV. Uses of p53 Target Nucleic Acids, Proteins and Antibodies

Cellular signaling molecules, including proteins involved in apoptosis pathways, have received a great deal of attention as potential mediators of selective killing of tumor cells or arrest of tumor cell growth, for the elimination of cancer from the body. As potential signaling molecules involved in p53 -mediated regulation of cell growth or apoptosis, the novel p53 targets described herein are expected to be particularly useful as diagnostic and therapeutic agents. Such molecules will also provide valuable research tools.

The novel p53 target nucleic acids described herein may be used for a variety of purposes in accordance with the present invention. DNA, RNA, or fragments thereof may be used as probes to detect the presence of and/or expression of the corresponding genes, as well as to identify alterations of those genes that result in loss of function, as they may arise in various cancers. Methods in which nucleic acids of the invention may be utilized as probes for such assays include, but are not limited to: (1) in si tu hybridization; (2)

Southern hybridization (3) northern hybridization; (4) assorted amplification reactions such as polymerase chain reactions (PCR) ; and (5) DNA arrays, which are sometimes referred to as "gene chips" . In addition, as discussed above, the nucleic acids of the invention may also be utilized as probes to identify and isolate full length corresponding genes, or related genes either from humans or from other species. As is well known in the art, hybridization stringencies may be adjusted to allow hybridization of nucleic acid probes with complementary sequences of varying degrees of homology. Thus, the p53 target nucleic acids may be used to advantage to identify and characterize other genes of varying degrees of relation to the sequences, thereby enabling further characterization of the signaling cascade involved in the control of cell proliferation in different cell types. p53 target nucleic acid molecules of the invention, or fragments thereof, may also be utilized to control expression of the genes, thereby regulating the amount of protein available to participate in p53- mediated signaling pathways. Alterations in the physiological amount of such proteins may act synergistically with chemotherapeutic agents used to treat cancer.

The proteins encoded by the p53 target nucleic acid molecules of the invention may be used as targets to screen for agents that affect their expression or activity, according to methods known in the art. Additionally, they may be used to identify genes encoding other proteins with which they interact (e.g., by the "interaction trap" technique) , which should further accelerate elucidation of the cellular signaling mechanisms in which they may be involved (Golemis, et al . , Interaction trap/two-hybrid systems to identify interacting proteins, Unit 20.1.1-20.1.28 in Current Protocols in Molecular Biology, eds. F.M. Ausubel, et al . John Wiley & Sons, NY (1996) .

Genes or cDNAs of the invention that are induced by p53 may be used for gene therapy applications. For example, the genes or their cDNAs cloned into expression cassettes can be delivered as naked DNA or within a viral vector to tumor cells lacking functional p53. This would restore a gene function downstream of p53 that cannot be activated in the p53 -deficient tumor cell. As described above, these downstream targets of p53 are expected to function in blocking cell proliferation, inducing apoptosis, or both, thereby reducing or eliminating the tumor.

As mentioned above, the p53 target gene that is repressed by p53 is expected to be particularly useful as a target in a screen for drugs that antagonize or block its expression, or the activity of the encoded gene product. Such drugs would inhibit a function that is normally inhibited by p53, and therefore could be used to block cell proliferation or induce apoptosis in p53- deficient cells.

Purified proteins encoded by the p53 target nucleic acids, or fragments thereof, may be used to produce polyclonal or monoclonal antibodies which also may serve as sensitive detection reagents for the presence and accumulation of the proteins in cultured cells or tissues from living patients (the term "patients" refers to both humans and animals) .

Recombinant techniques enable expression of fusion proteins containing part or all of the proteins of interest. The full length proteins or fragments of the protein may be used to advantage to generate an array of monoclonal antibodies specific for various epitopes of the proteins, thereby providing even greater sensitivity for detection of the protein in cells or tissue.

Polyclonal or monoclonal antibodies immunologically specific for the p53 target proteins may be used in a variety of assays designed to detect and quantitate proteins, which may serve as a prognostic indicator for malignant disease. Such assays include, but are not limited to: (1) flow cytometric analysis; (2) immunochemical localization in cultured cells or biopsy tissue; and (3) immunoblot analysis (e.g., dot blot, Western blot) of extracts from various cells and tissues. Additionally, as described above, antibodies can be used for purification of the proteins (e.g., affinity column purification, immunoprecipitation) . Antibodies may also be utilized as therapeutic agents to block the normal functionality of the proteins in a target cell population.

Alternatively, the possibility exists that antibodies to one or more of these proteins may in fact activate receptors associated with the protein ligands and have a therapeutic value in inducing apoptosis or cell cycle arrest. Precedent exists for such a function, for instance, with anti-Fas antibodies. An antibody for such a purpose may prove superior to the true ligand, and it may be deliverable intravenously, depending on its specificity for cancer cells, and absence of toxicity. The antibody also may be injected directly into a tumor. From the foregoing discussion, it can be seen that the p53 target nucleic acids of the invention, and antibodies immunologically specific for their encoded proteins, can be used to detect gene expression and protein accumulation for purposes of assessing the genetic and protein interactions involved in the regulation of apoptosis, cell cycle arrest or other p53- mediated function in both wild-type p53 positive and negative cells. Aberrant signal transduction in cells is often correlated with cellular transformation and cancer of various tissue types. It is expected that these tools will be particularly useful for diagnosis and prognosis of human neoplastic disease as described above.

Although the compositions of the invention have been described with respect to human diagnostics and therapeutics, it will be apparent to one skilled in the art that these tools will also be useful in animal and cultured cell experimentation with respect to various malignancies and/or other conditions manifested by altered patterns of apoptosis or lack of apoptosis in response to DNA damaging agents or ionizing radiation. They can also be used to generate animal model systems, e.g. "knockout" animals that do not express the gene, as a model of cancer susceptibility. Such animals are useful as models for human disease and treatment thereof, inasmuch as they may exhibit phenotypes that may mimic a developmental abnormality and may have a predisposition to cancer, especially following exposure to carcinogens. In connection with this embodiment, it is preferable to clone mouse homologs of the human p53 targets of the invention, according to standard methods as described above .

According to another aspect of the invention, kits are provided to facilitate the use of the nucleic acids, proteins and other biological molecules of the invention. In one embodiment, the kits comprise various nucleic acid molecules of the invention, for use as probes in the variety of assays described above . In another embodiment, the kits comprise one or more of the PRG proteins described above. In another embodiment, the kits comprise DNA constructs encoding the proteins, fragments and mutants thereof, and appropriate substrates, along with instructions on how to use the constructs. In another embodiment, the kits comprise aliquots of transgenic cells and instructions for their use. In another embodiment, the kits may comprise antibodies and other reagents for performing immunological assays. The kits may also comprise, optionally, various additional reagents, such as growth media, enzyme substrates and standard solutions for calibrating product formation.

The following examples are set forth to illustrate embodiments of the invention. They are not intended to limit the scope of the invention in any way.

EXAMPLE 1 Isolation of Differentially Expressed cDNAs from p53 -Dependent Apoptotic Cells: Activation of the Human Homolog of the Drosophila Peroxidasin Gene

Using a modified differential display technique (Liang and Pardee, Science 257: 967-971, 1992), we have identified six differentially expressed genes in human colon cancer EBl cells undergoing p53 -dependent apoptosis (Shaw et al . , PNAS 89: 4495-4499, 1992). This example describes the identification of those genes and the further characterization of one of the genes, a human homolog of the Drosophila peroxidasin gene.

MATERIALS AND METHODS

Cell lines and cul ture

The human colon cancer cell lines EB, EBl, and EBl-DH were maintained in McCoy's 5A medium supplemented with 10% fetal bovine serum. EBl was cultured in the presence of 200 μg/ml of G418, and EBl-DH was cultured in the presence of 200 μg/ml of G418 and hygromycin. A wild-type human p53 cDNA under the control of the rat metallothionein MT-1 promoter was stably transfected into p53-null EB cells to establish the EBl cell line (Shaw et al . , 1987) . EBl cells show massive apoptosis upon induction of wild-type p53 by exposure to 0.1 mM ZnCl₂ for 2 to 3 days. EBl-DH cells are an apoptosis-resistant derivative of EBl cells.

Differential display- To identify genes regulated by p53 in EBl cells at an early stage of p53 -dependent apoptosis, total RNA samples were isolated from EBl cells before and after treatment with 0.1 mM ZnCl₂ for 9 hours . RNA samples were prepared by the guanidine isothiocyanate/CsCl ultracentrifugation method (Sambrook et al . , 1989) . Differential display was performed using a Delta RNA fingerprint kit from Clontech (Palo Alto, CA) using the protocol suggested by the company with minor modifications. Briefly, total RNA samples were first treated with RNAse-free DNAse and then used as a template for first-strand DNA synthesis by Superscript II reverse transcriptase (Gibco BRL, Gaithersburg, MD) . A "cDNA synthesis primer" was used in this reaction. Differential display PCR fingerprint reactions were performed with the combination of a "P-primer" (PI to P10) and a "T-primer" (Tl to T9) , giving a total of 90 combinations. All reactions contained [ -³³P] cLATP, and all were carried out with two different amounts of first - strand cDNA to eliminate false signals. All PCR amplifications were performed using a Perkin-Elmer GeneAmp 9600 with the following program: 94°C 5 min, 40°C 5 min, 68°C 5 min (1 cycle) / 94°C 30 sec, 40°C 30 sec, 68°C 5 min (2 cycles) / 94°C 20 sec, 60°C 30 sec, 68°C 2 min (23 cycles) / 68°C 7 min. PCR products were separated on a denaturing 5% polyacrylamide/8 M urea gel in 0.5X TBE buffer. Gels were dried under vacuum, and exposed to X-ray film. DNA fragments were identified, cut out from the gels, and eluted in water. Each eluted DNA fragment was then reamplified with the same primer set as that used in the original reaction. The reamplified fragments were subcloned into the pGEM5 vector (Promega, Madison, Wl) . DNA inserts purified from plasmids were used to make ³²P-labeled probes for Northern blotting to confirm that the corresponding mRNAs were expressed at different levels under the two sets of conditions. The DNA sequence of each differentially expressed mRNA clone was determined by automated sequencing (ABI370: Applied Biosystems, Foster City, CA) . DNA sequences were analyzed by homology search (BLAST) using Genbank database. One of the cDNA clone, KIAA0230, which is identical to PRG2 was obtained from Kazusa DNA research Institute, Japan. The GenBank accession numbers of PRGs described in this manuscript are: AF147071, AF147072 (PRG1) , AF147073 (PRG2), AF147074, AF147075 (PRG3), AF147076, AF147077 (PRG4), AF147078 (PRG5) , AF147079, AF147080 (PRG6) .

Northern blot analysis

Total RNA isolated from EB, EBl, or EBl-DH cells was separated on MOPS agarose gels containing 1% folmaldehyde . RNA was transferred to Nytran membranes (Schleicher & Schuell, Keene, NH) and fixed using a Stratalinker UV-irradiation system (Stratagene, La Jolla, CA) . DNA probes were ³²PdCTP-labeled with the Prime- It II kit (Stratagene, La Jolla, CA) . Hybridizations were carried out in Qickhybri solution (Stratagene, La Jolla, CA) as recommended by the manufacturer. Membranes were washed with 0.2 x SSPE and 0.1% SDS at 60°C, unless otherwise indicated. To analyze the 4.5-kb Pxn mRNA, we used four cDNA probes corresponding to different fragments within the coding region of the gene: probe A, amino acids 1-161; probe B, amino acids 501-636; probe C, amino acids 587-751; and probe D, amino acids 1315-1452. Human multiple-tissue RNA blots (Clontech, Palo Alto, CA) were probed with PRG2 cDNA fragment.

RESULTS AND DISCUSSION

Identification of differentially expressed transcripts in p53 -induced apoptosis .

The human EB colon cancer cell line lacks p53. EBl cells were established from EB cells by stable transfection of a wild-type p53 -expressing gene under the control of the rat metallothionein MT-1 promoter. EBl cells show massive apoptosis upon induction of wild-type p53 by exposure to 0.1 mM ZnCl₂ for 2 to 3 days. To identify potential target genes of p53 in a p53 -dependent apoptotic pathway, we analyzed mRNAs from a very early stage of p53-induced apoptosis. We compared the mRNA prepared from EBl cells treated with 0.1 mM ZnCl₂ for 9 hr to that prepared from untreated cells using a modified differential display technique (Liang et al . , 1992) . We identified six differentially expressed genes: five were up-regulated (PRG1-5) and one was down-regulated (PRG6) as shown in Table 1. We found two matched sequences in the GenBank database. PRG2 has an identical sequence to KIAA0230 (D86983) , which shows a high homology to Drosophila peroxidasin, an extracellular matrix- associated peroxidase. PRG5 has an identical sequence within the human chromosome DNA 20ql2-13.2 (Z93016) and PRG6 has an identical sequence within the human chromosome DNA 19pl3.2 (AD000092). In both cases, the coding sequences have not been identified. The other three clones did not correspond to sequences found in GenBank database .

Expression analysis of the genes in EBl cells undergoing p53 -dependent apoptosis . The expression levels of newly identified transcripts upon p53 induction were characterized by Northern blot using each cloned fragment as a probe. Total RNA samples were prepared from cells treated with 0.1 mM ZnCl₂ for 0, 4, 9, 13 hr. Steady-state levels of PRG1 to 5 mRNA are highest (approximately 10 -fold induction) at 9 hr after p53 induction. After 9 hr, these mRNA levels decrease. The mRNA level of PRG6 decreased to the lowest level at the 9 hr post p53 induction. The parental EB cell line did not show any changes in the steady-state levels of these mRNAs . The apoptosis-resistant variant of EBl, termed EBl-DH, showed only a slight difference in PRG mRNA expression, although p21 (Wafl/Cipl) mRNA, a well characterized p53 target gene, was strongly induced in this cell line. These results indicate that the p53 -dependent transcriptional activation machinery is intact even in the apoptosis- resistant EBl-DH cell line. We observed an increased expression of PRG6 mRNA in EBl-DH cells at the 13 hr post p53 induction. This suggests that the increased amount of PRG6 might be required for preventing p53-depndent apoptosis. We conclude that differential expression of the PRG mRNAs is specific to the active process of apoptosis.

PRG2 is the human homolog of Drosophila peroxidasin, an extracellular matrix-associated peroxidase .

The PRG2 sequence showed a 37% identity and a 46% similarity in amino acid level with Drosophila peroxidasin (amino acids 1448-1498) , an extracellular matrix-associated peroxidase) . The entire PRG2 sequence (416 nt) showed a complete match to the sequence of a KIAA0230 clone in the GenBank database. The KIAA0230 clone was originally isolated in a random screen from a human monocyte cancer cell line. The predicted amino acid sequence of KIAA0230 (1480 amino acids) and the sequence of the Drosophila peroxidasin polypeptide (1535 amino acids) were highly related (between amino acids 36 and 1315 the identity is 45.4%, and similarity is 67.8%). From these results we conclude that PRG2 is a human homolog of Drosophila peroxidasin. Peroxidasin has a unique hybrid structure that combines an enzymatically functioning peroxidase domain with motifs that are typically found in extracellular matrix-associated proteins (Nelson et al . , 1994; Bornstein, 1992). It is a secreted protein that contains a secretory recognition sequence (a signal peptide sequence) at its amino terminus (amino acids 1-23) . We found a signal sequence candidate in the human homolog (amino acids 1-29) . This suggests that the human homolog can be secreted from the cells. Furthermore, hPxn contains four lg C2-like repeats (Fig. 1) that share characteristic residues with the lg loops of Drosophila and vertebrate neural/cell adhesion molecules, such as fascilins, neuroglian, NCAM (neural cell adhesion molecule) , LI (neural adhesion molecule LI) , and DCC (deleted in colorectal carcinoma) (Pulido et al . , 1992; Walsh et al . , 1997). hPxn contains also leucine-rich repeats at the N-terminus and a cystein-rich motif found in type-I and II thrombospondin and procollagen (Bornstein, 1992) .

Central to human oxidative defense are the heme proteins found in leukocytes : myeloperoxidase (MPG) and eosinophil peroxidase (EPO) of leukocytes, and the lactoperoxidase (LPO) in various secretions. Together with thyroid peroxidase (TPO) , they form a gene family. MPO and EPO are made and stored in macrophages . They usually act within the cell on phagocytosed material, but can also be released from cells upon stimulation (Abrams et al., 1993; Abrams et al . 1992).

The amino acid sequence of the active enzyme domain of peroxidasin is very similar to that of the other human peroxidases MPO, EPO, and TPO, as well as LPO. This result strongly supports the idea that PRG2 , a human peroxidasin, functions as a peroxidase enzyme.

Expression profile of PRG2 in human organs .

The PRG2 mRNA expression profile in human tissues was examined. It was found that PRG2 mRNA is expressed in most of the tissues, with the exception of the brain and leukocytes. The expression is highest in the heart, placenta, spleen, ovary, and intestines. The predicted size of PRG2 mRNA is 7.5 kb, but we detected also a 4.5-kb mRNA in testes . The shorter mRNA is detected in EBl cells undergoing p53 -dependent apoptosis. It is important to point out that the p53 protein is always overexpressed in testes relative to other tissues, and that the reason for its high level expression in this tissue has not yet been elucidated. It is possible that transcription of the 4.5-kb mRNA in testes is regulated by p53 as found in EBl cells.

Al ternative form of hPxn contains the complete peroxidase enzyme domain . To characterize the 4.5-kb hPxn mRNA, we performed Northern blot analyses using various portions of the hPxn cDNA as probes. The smaller 4.5-kb mRNA induced in EBl cells undergoing p53 -dependent apoptosis hybridized with probes C and D. This indicates that the 4.5-kb mRNA shares a common sequence with the cDNA at its 3' -terminal portion, including the peroxidase domain. Importantly, as determined by blotting with the cDNA probes A and B, the N-terminal portion of hPxn includes a signal sequence for secretion that is not present in the smaller 4.5-kb mRNA. These results indicate that the N- terminus of the hPxn protein induced by p53 in apoptotic EBl cells or in testes is different from the hPxn protein expressed in a variety of other organs and tissues. They suggest also that the smaller protein may lack a signal peptide and localize in the cytoplasm. The 4.5-kb transcript appears to encode a complete peroxidase enzyme, and it is possible that induction of this mRNA increases cellular ROS production.

EXAMPLE 2

Chromosomal Localization of PRG genes,

Regulation of PRG Gene Expression and Functions of Encoded Proteins

Chromosomal localization of PRG genes .

To identify chromosomal localization of the PRG genes, genomic DNA fragments containing PRGl, 3, 4, 5, or 6 were newly isolated from BAC libraries. The purified genomic DNA fragments were labeled with digoxygen-dUTP and hybridized to synchronized human lymphocyte metaphase spreads (FISH) . These results, performed on both DA-DAPI and PI banded metaphase chromosomes, clearly showed localization of the PRGs . PRGl is localized to 19ql3.2- 13.3, PRG3 is localized to 10q21.3-22.1 , PRG4 is localized to 17pll.2, PRG5 is localized to 20ql2-13, PRG6 is localized to 19pl3.2-13.1. The chromosomal localizations of the genomic DNA fragments that contains either PRG5 or 6 are available in GenBank (accession numbers: Z93016 for PRG5 and AD000092 for PRG6) and our results confirmed these localizations.

We performed a positional cloning in human YAC genomic library to isolate genomic DNA fragment for PRG2. We obtained independent RH (Radiation Hybrid) score vectors from EBl (European Bioinformatics Institute, UK) and GENETHON (France) for the PRG2 sequence and calculated maps for both vectors to the top of chromosome 2 (between contigs WI-1412 and WI-17765) using the Whitehead Institute/MIT RH mapping service. Based on these results, we identified 11 YAC clones that cover this entire region and overlap each other through a YAC database at the Genome Database. Eleven yeast clones were obtained from Genome Systems Inc. and total DNA samples were prepared from them. PCR analysis using 2 sets of primers (a 5' -primer set and a 3' -primer set) revealed that one YAC clone (clone E, the address name is 940gl0) contained PRG2 gene. Since 2 sets of primers successfully amplified DNA fragments, we assume that this clone (estimated insert size 1 Mbp) contains the PRG2 genomic DNA, at least from its 3' -end to the 5' -end of the common exon between 7.5 kb and 4.5 kb mRNA. Based on these results, we mapped the PRG2 gene to chromosome 2p24.3.

Transcriptional regulation of PRG2 and 5 by p53 . p53 functions as a transcription factor with interacting a specific DNA sequence in the gene (p53- responsive element) and activates transcription from the promoter. We analyzed PRG2 and PRG5 genomic DNA and found potential p53 -responsive sequences.

PRG2

We have identified a potential p53 -responsive sequence in a coding sequence located between 1698 to 1713. We cloned this 16 bp fragment into pTILuc, a luciferase reporter plasmid with minimum promoter function (Horikoshi et al . , 1995). The luciferase reporter plasmid with one copy or two copies of this 16 pb fragment was co-transfecterd into H1299 cells (p53 -/- ) with wild-type or 175RH mutant p53 , a transactivation negative mutant p53, expression plasmid. As shown in Fig. 4a, these reporter plasmids clearly responded to wild-type p53, but not to mutant p53, and the activities were induced 11-fold (a single copy) or 15-fold (two copies) by wild-type p53. Next, we analyzed the genomic structure of the exons that contain this 16 bp . The sequence of the genomic DNA revealed that this 16 bp fragment is localized in a single exon (Fig. 4b) , therefore, we conclude that this 16 bp fragment is a potential p53 -responsive element in PRG2 gene.

PRG5

Based on the genomic DNA sequence of PRG5 gene in Genbank (accession number: Z93016) , we have identified genomic structure of PRG5 gene. We have identified 5 potential p53 -responsive elements (Fig. 5a) . We have constructed luciferase reporter plasmids as we described above for each candidate fragment. As shown in Fig. 5b, we found that the luciferase activities from the reporter plasmid containing fragment D or E were strongly induced by p53, about 13-fold or 97-fold, respectively. From these results, we concluded that fragment D (11848-12098) and fragment E (18201-18288) are potential p53 -responsive sequences in PRG5 gene.

Transcriptional regulation of PRG3 by p53 .

The expression profile of PRG3 by p53 was studied by Northern blotting. Poly (A) ⁺ RNA samples were prepared from EBl cells after treatment with 0.1 mM ZnCl₂ for 0, 4, 9, and 14 hr. Membranes were probed with DNA fragment from PRG3 coding region (nucleotide position 1096-2106) under a high-stringency condition for hybridization. We have identified additional 1.8 kb mRNA addition to previously identified 4.0 kb transcript. Only 4.0 kb mRNA was detectable when probed with a DNA fragment from 3' end of untranslated region (UTR) of the PRG3. These results indicated that these two PRG3 mRNA transcripts differ in their 3 'UTR.

We analyzed transcriptional regulation of PRG3 by p53 in other cell line. Human osteosarcoma cells (Saos2, p53 -/-) were infected by recombinant retrovirus carrying p53 gene. Poly (A) ⁺ RNA sample was prepared after 24 hr of infection and PRG3 mRNA level was identified by Northern blotting with using a coding sequence as a probe . PRG3 mRNA was induced by the induction of p53 expression also in Saos2 cells suggesting that PRG3 is an immediate p53 target gene.

Expression profiles of PRG3 and 6 in human tissues .

The expression profile of PRG3 and 6 mRNA in human tissues was examined. A human multiple-tissue Northern blot (Clontech) was probed with a cloned PRG3 or 6 cDNA fragment.

The relative amount of PRG3 mRNA expression appeared to vary across the different tissues. The highest level of PRG3 mRNA was observed in heart, moderate level was present in liver, while low level of expression was detected in brain, placenta, lung, kidney, pancreas, and skeletal muscle. The same membrane was reprobed with glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) gene to normalize the expression. The difference of expression may indicate a tissue specific regulation of PRG3 gene expression and the possible involvement of PRG3 function in cellular differentiation.

The relative amount of PRG6 mRNA expression appeared to be constant across the different tissues, except in liver. These results may suggest that the repression of PRG6 is conducted by a liver specific factor (s) .

Growth inhibi tory function of PRG2 , 3 , and 5. Since PRGs are potential candidates for downstream effector molecules of p53 in an apoptosis pathway, we tested whether PRGs have any effect on cell growth. 293 cells were transfected with β-Gal expression plasmid (negative control) , expression vector (positive control), and PRG2 or 3 , or 5 expression plasmid. After two days of transfection, cells were passaged into three dishes and continued in culture for an additional two weeks in the presence of 600 μg/ml of G418. As shown in Fig. 6, PRG2 and PRG5 inhibited colony formation about by 50%. Interestingly, PRG3 did not inhibit colony formation in 293 cells, but we found that PRG3 inhibited colony formation in Saos2 cells and in Hs68 cells. It is reasonable to assume that the growth inhibitory effects by the overexpression of PRG2 , 3 , or 5 are caused by the induction of apoptosis since the expression of PRG2 , 3, and 5 are induced by p53 in an apoptotic cell line but not in an apoptosis resistant cell line.

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The present invention is not limited to the embodiments described and exemplified above, but is capable of variation and modification within the scope of the appended claims .

Claims

We claim:

1. A p53-inducible isolated nucleic acid molecule that include an open reading frame encoding a human homolog of Drosophila melanogaster peroxidasin.

2. The nucleic acid molecule of claim 1, which comprises part or all of SEQ ID NO : 3.

3. A human peroxidasin encoded by the isolated nucleic acid molecule of claim 1.

4. Isolated nucleic acid molecules that are up-regulated in response to p53 induction in a cultured cell, comprising part or all of one or more sequences selected from the group consisting of SEQ ID NOS. 1, 2, 4, 5, 6, 7, 8 and 9.

5. An isolated nucleic acid molecule that is down-regulated in response to p53 induction in a cultured cell, which comprises part or all of SEQ ID NO: 10.

6. A method for isolating protein coding sequences corresponding to any one or more of SEQ ID NOS : 1-10, which comprises: a) assembling a hybridization reaction mixture in which is dispensed on or more of the isolated nucleic acid molecules in a single stranded form, and a test sample that comprises the corresponding protein coding sequence, in a single- stranded form, under conditions enabling hybridization of the isolated nucleic acid molecule and the protein sequence, thereby forming a double-stranded nucleic acid molecule; b) separating the double-stranded molecule comprising the isolated nucleic acid and the protein coding sequence; and c) cloning the protein coding sequence .

7. An isolated protein produced by expression of the protein coding sequences isolated by the method of claim 6.

8. Antibodies immunologically specific for part or all of the protein of claim 7.

9. A recombinant DNA molecule comprising a nucleic acid molecule selected from the group consisting of a) an isolated nucleic acid molecule having a sequence selected from the group consisting of SEQ ID NOS: 1-10; and b) or a protein coding sequence produced by the method of claim 6 ; operably linked to a vector for transforming cells .

10. Cells transformed with the recombinant DNA molecule of claim 8.

11. An oligonucleotide between about 10 and about 100 nucleotides in length, which specifically hybridizes with a pre-determined portion of one or more Of SEQ ID NOS: 1-10.