DETECTION OF HUMAN P58 PROTEIN KINASE AND ITS DNA FOR DIAGNOSIS OF CERTAIN CANCERS

Abstract: A method of diagnosing cancers associated with the absence of the gene encoding human p58 protein, such as neuroblastomas, ductal carcinomas of the breast, malignant melanoma, Merkel cell carcinoma or endocrine neoplasia is set forth. In one embodiment, cells, especially in metaphase, are prepared from a tissue to be examined. There is then performed an in situ hybridization with a labelled probe including a DNA sequence which is either complementary to the p58 protein kinase gene or to a portion of the p58 kinase gene which is long enough to identify such gene, if present. Determination of the presence of the absence of the gene on the number 1 chromosome indicates that a cell is cancerous or, at the very least, potentially cancerous. In a second embodiment, antibodies specific to p58 protein kinase are employed for its detection. Kits including a probe or antibody as discussed above are also set forth.

Description: DETECTION OF HUMAN P58 PROTEIN KINASE AND ITS DNA FOR DIAGNOSIS OF CERTAIN CANCERS BACKGROUND OF THE INVENTION 1. Field of the Invention This invention relates to a method and to a kit for detecting a genetic mutation associated with a cancer and, more particularly, to a method and a kit using a labelled probe to detect a gene whose absence is associated with certain types of cancers. The invention further relates to a method and to a kit for detecting a genetic mutation associated with a cancer using a labelled antibody to detect a protein whose absence is associated with certain types of cancers. 2. Background of the Invention The ability of a cell to reproduce by cell division is one of the most fundamental processes of all living matter. Abnormalities of these processes are often involved in the growth of transformed cells. Therefore, a thorough understanding of the events involved in normal cell division is crucial to understanding transformed cell growth. To this end, several groups have identified a cell division control protein kinase gene superfamily and, in particular, the human cell division control (CDC)-related gene family which contains at least four members, including the mitotic protein kinase p34 cdc2 (Lee and Nurse, 1987; Hanks, 1987; Hanks et al, 1988; Bunnell et al., 1990a; Johnson and Smith, 1991). The protein kinase p34 cdc2 has been shown to be a crucial component of a normal mitotic promoting factor complex, which includes other protein components such as the cyclin regulatory subunits. In particular, the p34 edc2 protein kinase has been shown to be required for both the G 2 - to S-phase transition in the cell cycle. A number of studies have demonstrated that the p34 cdc2 protein kinase is the catalytic subunit of starfish, clam, and Xenopus oocyte maturation-promoting factor (MPF) (Arion et al., 1988; Draetta et al., 1988; Dunphy et al., 1988; Gutier et al., 1988) and its somatic cell mitotic counterpart (Draetta and Beach, 1988; Langan et al., 1989; Brizuela et al., 1989). Interaction of the p34 c<ic2 kinase catalytic subunit with a cyclin regulatory subunit results in an active protein kinase complex that phosphorylates a number of cellular substrates that play important roles in regulating the physical processes of mitosis (Moreno et al, 1989; Norbury and Nurse, 1989). The exact function(s) of the other members of the p34 cdc2 -related protein kinase gene family is unknown, but it is clear that this family of genes is much larger than first anticipated. We previously identified a CDC-related protein kinase, p58, that is structurally and functionally related to p34 cdc2 (Bunnel et al., Elevated Expression of a p58 protein kinase leads to change in the CHO cell cycle. Proc. Natl. Acad. Sci. USA 87: 7467-1990). Abnormal expression of the p58 protein kinase in eukaryotic cells leads to alterations in the ability of these cells to traverse the cell cycle, as well as increased mitotic abnormalities, suggesting that p58 is a negative regulator of normal cell cycle progression (Bunnell et al., 1990a,b). We have also found that the p58 gene is well conserved evolutionarily, that its expression is regulated during murine embryogenesis, and that its activity is coordinately regulated with p34 cdc2 during the cell cycle (Kidd et al, 1991; Xiang et al., in preparation for publication). The p34 cdc2 protein kinase has been localized to human chromosome 10 (Spurr et al., 1988). However, the locations of the other members of the human CDC- related protein kinase gene family have to date remained unknown. It was postulated that knowledge of the locations of these cell cycle genes on human chromosomes may provide important information regarding the possible mechanisms that underlie cellular transformation. In this regard, it has recently been demonstrated that a human cyclin- related gene (potentially a cell cycle regulatory subunit) corresponds to the translocation breakpoint for the bell-linked candidate oncogene or human chromosome 11 (Motokura et al, 1991). Previously, a human cyclin A gene, which is a known cell cycle regulatory subunit, was identified as a locus interrupted by hepatitis B virus integration associated with hepatocellular carcinoma (Wang et al, 1990). These results suggest that some members of the cell division control gene family (regulatory and/or catalytic subunits) may be associated with the transformed phenotype. Thus, the cDNA clone of p - δS^ 2 has been both isolated and characterized. The protein pSδ"* 2 exhibits a 68% homology to the human p34 cdc2 protein. The p58 protein is 436 amino acids in size, is apparently regulated by phosphorylation and Ca 2+ calmodulin, and is localized to the cytoplasm and nucleus of both rapidly dividing and differentiated cells. Expression of the corresponding mRNA is regulated during the cell cycle, peaking during S-phase. Elevated expression of the CDC-related kinase in eukaryotic cells leads to an apparent block of the cell cycle at late telophase and abnormal cytokinesis. Additionally, the expression of the p58 gene has been found to be regulated via sequences within its 3' untranslated region and protein results in apparent changes in DNA replication. It was also shown that a severalfold increase in the expression of the p58 kinase alters normal progression of cells through the cell cycle and dramatically increases the frequency of mitotic abnormalities. It was suggested that alterations in the level of endogenous p58 can affect the cell cycle, with too little p58 allowing cells to proceed through the cell cycle more rapidly, whereas too much p58 inhibits cell cycle progression. The results were viewed as being consistent with the possibility that p58 kinase acts as a negative regulator of some component of the cell cycle pathway. It was concluded that the altered cell cycle phenotype and mitotic abnormalities associated with these overexpressors suggest that elevated expression of the p58 kinase interferes with the late events associated with normal mitosis. While the above information concerning the p58 kinase protein has been of academic interest in view of its apparent role in the regulation of cell cycles, much has remained unknown including its precise location in the genome, its relationship to other protein kinases, and the effects that expression or lack of expression of such protein would have in vivo. Thus, although potential applications for a cell cycle regulator such as p58 have been postulated by workers in the art, the art has heretofore lacked the understanding of the protein, the gene encoding the protein, and the role of each in vivo. SUMMARY AND OBJECTS OF THE INVENTION In view of the potential applications of a cell cycle regulator, as discussed above, it should be apparent that there exists a need in the art for further characterization of human p58 protein kinase leading ultimately to use of such kinase in applications where regulation of a cell cycle is relevant, such as human cancer. It is, therefore, a primary objective of the present invention to fulfill that need by localizing the expressed p58 gene so as to obtain a further understanding of the potential role of these genes in normal, as well as transformed, cell growth. More particularly, it is object of the present invention to localize the human p58 protein to a particular chromosome and to use such knowledge to diagnose certain types of cancers which have been found to be associated with the absence of the gene encoding human p58 protein from the genome. It is another object of the present invention to provide a method for diagnosing cancers associated with the absence of the gene encoding human p58 protein which uses labelled probes or antibodies in a test which is reliable. Yet another object of the present invention is to provide a method for diagnosing cancers associated with the absence of the gene encoding human p58 protein which can be carried out easily by a technician using, for example, a pre-prepared kit. Using a combination of somatic cell hybrid DNA panels, fluorescent in situ hybridization (FISH) with the corresponding chromosomal gene, and chromosome microdissection followed by nested polymerase chain reaction (PCR) amplification, we have localized the gene encoding human p58 protein to the telomeric region of the short arm of chromosome 1 (1 p36). A sequence-related gene of unknown nature was also found to map to human chromosome 15. We have further found that formation of certain tumors is associated with the loss of this region of chromosome 1 and whose phenotype is associated with potential tumor suppressor genes. The localization and characterization of the human p58 protein kinase gene in the genome, combined with the observation that the absence of such gene is indicative of certain types of cancers, has allowed us to develop of method of diagnosing such cancers using either labelled oligonucleotide probes complementary to at least a portion of the gene or labelled antibodies which recognize the expressed protein. Therefore, in a first aspect, the present invention relates to a method of diagnosing a cancer associated with the absence of the gene encoding human p58 protein comprising the steps of; (a) preparing cells, such as metaphase cells from a tissue to be examined; (b) performing in situ hybridization with a labelled probe including a DNA sequence which is either complementary to the p58 protein kinase gene or to a portion of the p58 protein kinase gene which is long enough to identify such gene, if present; and (c) determining the presence or absence the of gene on chromosome 1. Included among the cancers associated with the absence of the gene encoding human p58 protein are neuroblastoma, ductal carcinoma of the breast, malignant melanoma, Merkel cell carcinoma or endocrine neoplasia. In a second aspect, the present invention relates to a method for diagnosing cancer comprising reacting cells with an antibody directed to the p58 protein kinase and detecting the presence or absence of specific binding of the antibody to the cells. In a third aspect, the present invention relates to kits for diagnosing a cancer associated with the absence of the gene encoding human p58 protein comprising a labelled probe including a DNA sequence complementary to the human p58 protein kinase gene or to a portion of the p58 protein kinase gene which is long enough to identify such gene, and a solution suitable for obtaining hybridization of the probe to a number 1 human chromosome. In a fourth aspect, the present invention relates to kits for diagnosing a cancer associated with the absence of the gene encoding human p58 protein kinase gene comprising an antibody directed to human p58 protein kinase and a means of detecting the presence or absence of specific binding of the antibody to the p58 protein kinase. With the foregoing as well as other objects, advantages and features of the invention that will become hereinafter apparent, the nature of the invention may be more clearly understood by reference to the following detailed description of the invention, the appended claims and the drawings. BRIEF DESCRIPTION OF THE DRAWING Figure 1 depicts restriction endonuclease digestions of human p58 chromosomal gene cosmid 2A. One microgram of cosmid DNA was digested with the indicated enzyme, electrophoresed on a 1 % agarose-TAE gel, and Southern transferred. The blot was then hybridized with a 1.5-kb EcoRI fragment from the cDNA containing the entire p58 protein kinase open reading frame (ORF). The arrows on the left indicate BamHI fragments of interest 6.5 and 0.8 kb in size. The sizes of lambda HindHT molecular weight markers are shown on the right; Figure 2 depicts a human-mouse somatic cell hybrid analyses. Total genomic DNA (12 l) was digested with either BamHI (A) or EcoRI (B), electrophoresed on 0.8% agarose-TAE gels, and Southern transferred. The blots were then hybridized with the same p58 cDNA probe as used in Fig. 1. The lanes correspond to the following DNAs: lane 1, GM/NA09925; lane 2, GM/NA09926; lane 3, GM/NA09927; lane 4, GM/NA09928; lane 5, GM/NA09929; lane 6, GM/NA09930A; lane 7, GM/NA09931; lane 8, GM/NA09932; lane 9, GM/NA09933; lane 10, GM/NA09934; lane 11, GM/NA09935A; lane 12, GM/NA09936; lane 13, GM/NA09937; lane 14, GM/NA09938; lane 15, GM/NA09940; lane 16, GM/NA10324; lane 17, GM NA10567; lane 18, GM/NAO10611: H. human: C. Chinese hamster: M, mouse. The arrows on the right of the panels indicate fragments of interest. The lambda Hindm molecular weight size markers are indicated on the left; Figure 3 depicts the hybridization of the human p58 protein kinase gene to human metaphase chromosomes, [A] A human metaphase spread hybridized to the p58 protein kinase gene (cosmid 2A) suppressed with human DNA. A human chromosome 1- specifϊc centromere probe was also included in the reaction. In situ hybridization and detection were performed as described by Lichter and colleagues. The arrows indicate the p58 hybridizing regions detected by biotin-labelled p58 cosmid 2A. fB] The same human metaphase chromosomes spread shown in A after Giemsa handing. The arrows indicate the position of the biotin-labelled p58 cosmid 2A sequences shown in A on the same Giemsa-handed chromosomes; Figure 4 depicts human chromosome 1 idiogram showing localization of the p58 protein kinase gene. The arrow on the left shows the location of all signals in all in situ metaphase spreads using the p58 cosmid 2A as a probe. The arrows on the right demark the location of the microdissection points for the nested PCR amplification experiment; Figure 5 depicts localization of the p58 protein kinase gene on human chromosome 1. Nested PCR amplification was performed using primers specific for the p58 protein kinase cosmid 2A gene. Templates for the reactions were as follows: lane 1, microdissected chromosome 1 fragment corresponding to 1 pter; lane 2, microdissected chromosome 1 fragment corresponding to 1 qter: lane 3, negative control containing no DNA template: lane 4, total human genomic DNA. MW indicates the 123-bp molecular weight standard ladder. Sizes of the MW standards are given on the left, while the size of the expected p58 gene product (342bp) is denoted on the right. DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS As is set forth in greater detail below, the present inventors have now isolated and localized the chromosomal gene encoding p58 protein to the human gene map. The gene contains 13 exons and 12 introns, spans approximately 12 kb of DNA, and is localized to the telomeric end of human chromosome 1, lp36. This region of chromosome 1 is commonly deleted in numerous tumors that are neural crest in origen, including malignant melanoma and neuroblastoma. The phenotype of these tumors suggests that the gene deleted in these tumors normally suppresses tumor formation, much like the retinoblastoma gene product and the p53 oncoprotein. The isolation of this CDC- related protein kinase gene allowed us to test the hypothesis that this gene is involved in neuroblastoma and malignant melanoma. The p58 cosmid clone, containing the corresponding gene from human chromosome lp36, is used in fluorescent in situ hybridization (FISH) studies with a control cosmid for a single copy sequence on the long arm, lq, to determine the frequency of p58 gene deletion in these tumors. Concomitantly, DNA from the same chromosome preparations can be examined for mutations in the p58 coding region by polymerase chain reaction (PCR) amplification of the DNA and subsequent DNA sequence analysis of the various exonic regions. MATERIALS AND METHODS DNA Probes and Somatic Cell Samples The human chromosomal gene corresponding to the previously reported 3.5-kb p58 mRNA was isolated from a human chromosomal DNA cosmid library. The corresponding human p58 cDNA has been reported and its sequence is known (Bunnell et al, 1990a). The murine-human somatic cell panel number 1 and murine, human, and Chinese hamster control DNAs were obtained from the NIGMS Human Genetic Mutant Cell Repository. Restriction endonuclease digestions were performed according to manufacturer's specifications and Southern blot analysis was performed as previously described (Bunnell et al., 1990a,b). DNA probes were labeled by random oligonucleotide primer incorporation of [α- 32 P]dCTP (Amersham, > 3000 Ci/mmol) as described previously (Kidd et al., 1991). In Situ Hybridization In situ hybridization of the human p58 cosmid 2 A probe to human metaphase chromosomes was performed using a modification of the technique of Lichter and colleagues (Lichter et al., 1988, 1990). Nick-translation was performed using the entire human p58 cosmid and bio-16-UPT (Bethesda Research Labs). The labeled probe was separated from the reaction by centrifugation through a Sephadex G-50 quick spin column (Boehringer Mannheim). Slides were first pretreated with RNase A, dehydrated, and then denatured at 70°C in 2X SSC and 70% formamide. Slides were hybridized overnight at 37°C in Oncor Hybrisol VI in the presence of the labeled probe. Excess repetitive sequence human DNA (Blockit, Oncor) was included in the hybridization mixture as well as human chromosome 1-specific α satellite DNA probe (Oncor). Probe detection was accomplished using fluorescein-labeled avidin at 37°C for 20 min. Amplification and chromosome staining were performed as specified by Oncor. Photographs were taken using a Leitz fluorescent microscope and Ektachrome 800/1600 film. Slides were then destained and Giemsa banded according to Cannizzaro and Emanuel (1984). Chromosome Microdissection and PCR The nested PCR primers were generated based on the position of intron/exon junctions surrounding nucleotides 527 to 804 of the human p58 cDNA (Bunnell et al., 1990a; Eipers, Lahti and Kidd, in preparation). The sequence of the primers are as follows: Primer 1 5' TGAGTGCCCTGGGCTTGG 3' Primer 2 5' TTCCTTGGCACCAAGCAG 3' Primer 3 5' GGGTCCACATGGGAGCCT 3' Primer 4 5' CTCTGGGGCGCGGTACCA 3' Primers 3 and 4 are nested inside the region amplified by primers 1 and 2. A portion of the sequences of primers 1 and 3 is derived from the adjacent intronic sequence of the p58 chromosomal gene. Inclusion of intronic DNA sequences in the PCR probe design will also help to ensure that this p58 locus is, indeed, the gene being analyzed and limits the possibility of interference from the related p58 sequence on human chromosome 15. Chromosome microdissection was performed on a Giemsa-banded human metaphase chromosome spread as described previously with some modifications (Han et al, 1991). Freshly prepared metaphase chromosome spreads were baked at 80 °C for 30 min and G- banded using standard cytogenetic technique. Chromosome microdissection was performed on an Olympus microscope under x750 magnification with a long working distance objective (x50). The microscope has a rotable stage so that the best cutting angle can easily be obtained. The dissected chromosome fragments were delivered into 50 μl of PCR reaction mixture [67 mM Tris-HCl, pH 8.8, 6.7 m MgCl 2 , 170 μl/ml BSA, 16.6 mM (NH 4 ) 2 SO 4 , 10% DMSO, 0.02 mM dNTP, and 100 ng each of the primers]. Primers 1 and 2 were used for the first round PCR of 20 cycles (94°C 1 min, 50°C 2 min. and 72° C 3 min); 5 μl of the PCR product was then added as template for the second round PCR reaction using nested primers 3 and 4. Fifteen microliters of the final product were separated by 1.5% TBE agarose gel electrophoresis and visualized by ethidium bromide staining. RESULTS Human-Mouse Somatic Cell Hybrid Analysis To demonstrate that a subset of the bands normally seen on a human genomic Southern blot using the entire coding sequence of the human p58 cDNA as a probe corresponded to a single locus, one of the isolated human cosmid clones (p58 cos 2A) was digested with several restriction endonucleases and analyzed (Fig. 1). Four distinct BamHI fragments (6.5, 4.1, 0.8, and 0.192 kb) and two distinct EcόSl fragments (9.7 and 2.4 kb) were observed (Fig. 1). The 0.192 kb BamHI fragment corresponds to a 0.192 bp BamHI fragment found in the coding region of the reported human cDNA (Bunnel et al., 1990a), plus an additional 75-bp intron found in this region, and indicates that this portion of the chromosomal gene is interrupted by a small intron. The observed cosmid restriction fragments of 6.5 and 0.8 kb are highlighted since they clearly segregate with a single chromosome locus on the somatic cell panel. This human cosmid p58 genomic locus has been sequenced and found to contain exons that are absolutely identical to the reported human p58 cDNA (Eipers, Lahti, and Kidd, in preparation; Bunnell et al., 1990a). When total human genomic DNA digested with BamHI is probed with the same p58 cDNA sequence, restriction fragments of 21, 6.5, 4.1, and 0.8 kb were observed (Fig. 2A). Only the 21-kb BamHI fragment is not found in the .BαmHI digest of the p58 cosmid isolate shown in Fig. 1. The 6.5-, 4.1-, and 0,8-kb BamHI fragments are all found in human-mouse somatic cell hybrids containing human chromosome 1 (Fig. 2A, lanes 1, 2, 3 and 9; Table 1), while the 21-kb BamHI fragment segregates with a separate locus corresponding to human chromosome 15 (Fig. 2A, lanes 1, 2, 3, 4, 9, 10, 13, and 18; Table 1), The 0.192-kb BamHI fragment is too small to be observed and resolve reliably by Southern blot analysis of total genomic DNA. However, its presence was verified by other techniques (see below). The apparent 6.5-kb BamHI band in lane 6 of Fig. 2A is an anomaly, most likely due to incomplete digestion of DNA. The 4.1- and 0.8-kb BamHI fragments are not concomitantly observed in this lane. To further demonstrate that the cosmid containing the p58 chromosomal gene shown in Fig. 1 segregates with human chromosome 1, the same human-mouse somatic cell DNAs were digested with Ec Bl and analyzed with the coding region of the p58 cDNA (Fig. 2B). Once again, the characteristic 9.7-kb EcoRI fragment found in the p58 cosmid is found only in lanes of the gel corresponding to hybrids containing human chromosome 1 (fig. 2B, lanes 1, 2, 3 and 9; Table 1). An 8.0-kb human EcoRI fragment corresponding to the presumed p58-related locus on chromosome 15 cannot be analyzed in this blot due to the presence of a related 8.0-kb EcoRI p58 murine genomic restriction fragment (Fig. 2B). The 2.4-kb EcoRI fragment corresponding to the remainder of the p58 cosmid shown in Fig. 1 also cannot be analyzed in this blot, once again due to the presence of a related 2.4-kb EcoRI p58 murine genomic fragment (Fig. 2B). Thus, the human-mouse somatic cell DNA analyses strongly suggest that the p58 chromosomal gene corresponding to the reported human p58 cDNA sequence is localized to human chromosome 1 (0% discordance), while a highly related p58 sequence appears to be localized to human chromosome 15 (0% discordance). In Situ Analysis To further substantiate the localization of the expressed p58 locus to human chromosome 1, in situ localization was performed using the entire p58 cosmid clone shown in Fig. 1. This type of analysis also helped us to regionally localize the expressed p58 gene on chromosome 1. Hybridization was specific to both homologs of chromosome 1 near a telomere with no significant background in 20 metaphase spreads analyzed by FISH (Fig. 3 and 4). In fact, 78 of 80 sister chromatids showed hybridization at a chromosomal telomere with the biotinylated p58 cosmid probe. The use of a contiguous 35- to 40-kb fragment of human DNA allows the unambiguous TABLE 1 Syniciiy or human p58 in 18 humnn-rodent somatic cell hybrids Human chromosome p58 Rearrang Hybrid p58 related 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X " Y chromoso GM/NΛ09925 + + + + - + + + + + - - - + - + I - + + + + - + - G /NA09926 + I 4- 4- 4- 4- - 4- 4- 4- - 4- - - 4- 4- 4- - 4- 4- 4- 4- - - - G /NΛ09927 4- 4- + 4- 4- 4- - 4- 4- 4- - 4- - - 4- 4- 4- - 4 4 1- 4 - - - G /NΛ09928 - I - 4- 4- - 4- 4 - 4- «- * - -» - - I I GM/NΛ09929 - - . . + + . + . + . - + + - + - - + - - ) - - - - G /NΛ09930Λ - - . + + . . - + - - - - + + + - - f + - + i i GM/NΛ0993I - - . . . - + - + - - + - + - -, - - + - - -1 I - - - GM/NΛ099J2 - - . . . + + + . + . - + + - - - - -» - - - ι - - - GM/NΛ09933 + 4 4- - + - + + + + - - - + + + + - 4 ■ ♦ -I I I GM/NΛ09934 - 4 - 4- - - + + - + - - + + - - + - + f- - 4- f- - - - GM/NΛ09935Λ - - . . . + . + + + + . . + -) . - + » . . GM/NΛ0 < >936 • - -* - - - -*- - - → - * - ' » - - GM/NΛ09937 - 4 + + - + + + - - - + - 4 I - + ♦ G /NΛ09938 - - - - - + + + + . . . + + . + . - + . - ι • » i G /NΛ09940 - »• . - + . - - + .« ι- - 4- G /NΛI0324 - - 4 - R:2l' . G /NΛ10587 - - GM/NΛI06II - - 4 d(l3) Λ % Discordant, 58 0 22 39 28 44 39 33 33 28 27 50 56 17 44 22 28 61 22 II 33 56 33 28 32 % Discordant, p58 related 22 22 28 39 44 28 33 II SO 39 61 56 39 44 0 50 39 22 22 44 56 44 50 30 Note. Chromosomes were scored positive if they were present in 2096 cells. " NΛ10611 is I human/hamster hybrid; nil others are hu an /mouse hybrids. _ 46, XX, -0, 4 der(fl)l(6:2I). " _ " 46,XY.I(J:16)((|13:2;(|I3). 46.XX.drl(!1). determination of its location and is highly specific for the DNA sequence corresponding to this large contiguous DNA fragment (Lichter et al, 1988; 1990). Potential repetitive DNA sequences contained in the cosmid clone are effectively competed from hybridizing to similar repetitive DNA sequences spread throughout the human genome by inclusion of excess repetitive sequence human DNA (Blockit, Oncor). A human chromosome 1- specific α satellite DNA probe was included in the hybridization mixture to confirm the identify of chromosome 1 on the metaphase spread. Hybridization with the human chromosome 1-specific α satellite DNA alone did not produce a signal at a telomere (data not shown). Conversely, hybridization with the p58 cosmid alone repeatedly produced the same signal only at a chromosome 1 telomere (data not shown). The same metaphase spreads examined for hybridization of the p58 clone were destained and Giemsa banded. In every spread examined, it was shown that the p58 clone hybridized to the chromosome 1 short (p) arm near the telomere at band p36. These results corroborate the somatic cell hybrid localization of the p58 gene to chromosome 1 and further refine the localization to near the telomere at lp36. Chromosome Microdissection and Nested PCR Amplication As a third, and final, method of verifying the chromosomal localization of the expressed p58 kinase gene we employed direct amplification of the target sequence within a single microdissected Giemsa-banded chromosome 1 arm using PCR (Han et al., 1991). One advantage of this method is that it allows one to definitively map a specific gene, for which DNA sequence is known, to a defined chromosome fragment obtained from a metaphase spread. Sufficient knowledge of the p58 chromosomal gene structure allowed us to use this methodology by designing a set of four nested PCR oligomer primers (see Materials and Methods) that would produce a 273-bp product if the gene were present (Eipers and Kidd, unpublished). This 275-bp fragment contains p58 coding sequences as well as intronic sequences that are unique to this p58 chromosomal gene locus. A Giemsa-banded metaphase spread of human chromosomes was used for microdissection of chromosome 1 as described previously (Han et al, 1991). The telomeric ends of both the long (lq) and short (lp) arms of chromosome 1, as well as total human genomic DNA and a negative control, were used as substrates for the nested PCR analyses (see Fig. 3). Only DNA from the telomeric end of the short arm of chromosome 1 (Fig. 5, lane 1), and from total human genomic DNA (Fig. 5, lane 4) produced the expected 273-bp PCR product. DNA isolated from the telomeric region of the long arm of chromosome 1 (Fig. 5, lane 2) or no DNA template (Fig. 5, lane 3) failed to produce a PCR product. Thus, the expressed p58 chromosomal gene was once again assigned to the distal short art of chromosome 1 on the basis of this set of experiments. Diagnosis of Cancers: After preparing the metaphase cells and screening them with a labelled probe, such as in the preferred embodiment described above, it is determined whether a hybridization occurs. If such hybridization does not occur, then it will be known that the cell is potentially cancerous. More specifically, the present inventors have now found that there are certain tumors associated with the absence of the human p58 protein kinase gene from the number 1 chromosome and that these tumors may be detected using this correlation and appropriately labeled oligonucleotide probes complementary to the gene (or a sufficiently long segment of the gene) or labelled antibodies generated from the protein kinase or, at the very least, the antigenic determinative portion thereof. In stating that a sufficiently long segment of the gene may be employed is meant that depending on the particular source being screened and the conditions employed a person skilled in the art will select polynucleotide probes of varying lengths to insure against false positives. The selection of an polynucleotide of suitable length is within the skill of a person skilled in the art. Included among the cancers which may be detected in accordance with the present invention are neuroblastomas, ductal carcinomas of the breast, malignant melanomas, Merkel cell carcinomas or endocrine neoplasias. Labelled antibodies are prepared in accordance with techniques known in the art. Such antibodies may be prepared from protein purified to homogeneity from a natural source or, alternatively, may be obtained recombinantly. Generation of the antibodies from the naturally occurring protein is described in Bunnell et al PNAS 87, 1990. With respect to recombinantly produced protein, it will be appreciated that for the purposes of generating antibodies, it is only necessary that the transformed host, such as E. Coli express the antigenic determinative portion of the protein. Indeed, the present inventors have found that incorporation of the entirety of the p58 kinase gene into an E. Coli host results in the expression of a product which is difficult to recover. Accordingly, the present invention further encompasses a transformed host wherein only the antigenic determinative portion of p58 kinase is inserted into an appropriate host. The antigenic determinative portion of the protein has been found to correspond to a region of the p58 protein from amino acid 1 to 74. The actual preparation of vectors to include the antigenic determinative portion of the protein as well as the transformation of a suitable host were carried out in accordance with techniques well known in the art. Finally, the present invention contemplates kits including either probes or antibodies as well as other reagents required for diagnosis of a patient. Such kits, including pre-prepared probe or antibody in combination with other reagents would facilitate application of the method such that a technician with minimal training would be able to cany out the diagnosis. A kit is contemplated including a probe as described above, optionally a label such as biotin for detection with fluorescently labelled avidin, and the reagents necessary to prepare a metaphase cells for screening. Although only preferred embodiments of the invention are specifically illustrated and described above, it will be appreciated that many modifications and variations of the present invention are possible in light of the above teachings and within the purview of the appended claims without departing from the spirit and intended scope of the invention.

Claims: WHAT IS CLAIMED IS 1. A method of diagnosing a cancer associated with the absence of the gene encoding human p58 protein comprising the steps of; (a) preparing cells from a tissue to be examined; (b) performing in situ hybridization with a labelled probe including a DNA sequence which is either complementary to the p58 protein kinase gene or to a portion of the p58 protein kinase gene which is long enough to identify such gene, if present; and (c) determining the presence or absence the of gene on chromosome 1. 2. The method according to Claim 1 wherein said cells from a tissue are metaphase cells. 3. The method according to Claim 1, wherein the cancer is neuroblastoma, ductal carcinoma of the breast, malignant melanoma, Merkel cell carcinoma or endocrine neoplasia. 4. The method according to Claim 1, wherein the probe is fluorescently labelled. 5. A method for diagnosing cancer comprising reacting cells with an antibody directed to p58 protein kinase and detecting the presence or absence of specific binding of the antibody to the cells. 6. The method according to Claim 5 wherein said cancer is neuroblastoma, ductal carcinoma of the breast, malignant melanoma, Merkel cell carcinoma or endocrine neoplasia. 7. A kit for diagnosing a cancer associated with the absence of the gene encoding human p58 protein comprising a labelled probe including a DNA sequence complementary to the human p58 protein kinase gene or to a portion of the p58 protein kinase gene which is long enough to identify such gene, and a solution suitable for obtaining hybridization of the probe to a number 1 human chromosome. 8. A kit for diagnosing a cancer associated with the absence of the gene encoding human p58 protein kinase gene comprising an antibody directed to human p58 protein kinase and a means of detecting the presence or absence of specific binding of the antibody to the p58 protein kinase. 9. The kit according to Claim 5, wherein the probe is labelled with biotin and is detected with flureoscently labelled avidin. [received by the International Bureau on 02 Feburary 1993 (02.02.93); original claims 1 and 9 amended; other claims unchanged (2 pages)] WHAT IS CT TMF.D TS 1. A method of diagnosing a cancer associated with the absence of the gene encoding human p58 protein kinase comprising the steps of; (a) preparing cells from a tissue to be examined; (b) perfoπning in situ hybridization with a labelled probe including a DNA sequence which is either complementary to the p58 protein kinase gene or to a portion of the p58 protein kinase gene which is long enough to identify such gene, if present; and (c) determining the presence or absence of the gene on chromosome 1. 2. The method according to Claim 1 wherein said cells from a tissue are metaphase cells. 3. The method according to Claim 1, wherein the cancer is neuroblastoma, ductal carcinoma of the breast, malignant melanoma, Merkel cell carcinoma or endocrine neoplasia. 4. The method according to Claim 1 , wherein the probe is fluorescendy labelled. 5. A method for diagnosing cancer comprising reacting cells with an antibody directed to p58 protein kinase and detecting the presence or absence of specific binding of the antibody to the cells. 6. The method according to Claim 5 wherein said cancer is neuroblastoma, ductal carcinoma of the breast, malignant melanoma, Merkel cell carcinoma or endocrine neoplasia. 7. A kit for diagnosing a cancer associated with the absence of the gene encoding human p58 protein comprising a labelled probe including a DNA sequence complementary to the human p58 protein kinase gene or to a portion of the p58 protein kinase gene which is long enough to identify such gene, and a solution suitable for obtaining hybridization of the probe to a number 1 human chromosome. 8. A kit for diagnosing a cancer associated with the absence of the gene encoding human p58 protein kinase gene comprising an antibody directed to human p58 protein kinase and a means of detecting the presence or absence of specific binding of the antibody to the p58 protein kinase. 9. The kit according to Claim 5, wherein the antibody is labelled with biotin and is detected with fluorescently labelled avidin.

Inventors: KIDD VINCENT J, KIDD, VINCENT, J.
Applicants: UAB RESEARCH FOUNDATION, THE UAB RESEARCH FOUNDATION
Application year: 1992
Main classification: C12Q1
IPC codes: A61K39, C12Q1, G01N33