Genetic changes observed in cancer cells include amplification, rearrangement, loss of genes, single-base substitution and deletion or insertion of nucleotides. Conventional Southern blotting can detect gene amplification, rearrangement or deletion. However, it is not effective for the detection of small DNA changes such as single-base substitutions and deletion or insertion of nucleotides. Various methods, especially polymerase chain reaction (PCR) - based methods, have been developed and used to detect these small DNA changes: i.e. direct sequencing of the genomic DNA [17], digestion of DNA fragments with restriction endonucleases, allele-specific oligonucleotide (ASO) hybridization [18], allele specific amplification [19,20] RNAse A mismatch cleavage [21,22] and denaturing gradient gel electrophoresis (DGGE) [23,24]. Each of these methods, however, has several disadvantages in terms of the detection of mutations either in efficiency or in convenience. Combined use of PCR and SSCP analysis could overcome these disadvantages to some extent [5,25].

I. Altered regulation of cell growth by genetic changes
Numerous pathways for the control of cell growth have been demonstrated in normal cells, where the products of oncogenes and tumor suppressor genes are involved in several independent cascades or within the same cascade [1-4].

p16 interacts with CDK4 and inhibits its activity, while CDK4 associates with cyclin D1 to inactivate pRB resulting in cell cycle progression. Thus p16, CDK4/cyclin D1 and pRB share, at least in part, the same cascade (the RB cascade). In fact, inactivation of the tumor suppressor p16 or RB genes, or amplification of the proto-oncogenes CDK4 or cyclin D1 were demonstrated in various types of human cancer [4,6].

p53, p21 and MDM2 protein are also known to form a network, where p53 can induce p21 production for the induction of cell cycle arrest, while MDM2 can bind and inactivate p53. Inactivation of the tumor suppressor gene p53 as well as amplification of the mdm2 gene have been reported in various human cancers [7-9]. The gene encoding p21 is also a possible tumor suppressor gene [10].

RAF protein has been identified as the downstream effector of the signal transduction pathways for RAS protein the genes encoding which, i.e. the K-, H- or N-ras genes, are often activated by point mitations in human cancers [11]. Thus, the raf gene is a possible proto-oncogene because activating rearrangement or mutation could enhance the RAS cascade [12,13]. AKT2 was shown to be activated by the signal from the platelet-derived growth factor receptor (PDGFR)[14], while oncogenic activation occurs by amplification of the gene in a subset of tumors [15,16].

Thus, aberrations of genes and their accumulation in each type of cancer cell should be examined from the view-point of the altered regulation pathways of cell growth.

II. Multiple genetic alterations in human tumors.
We have been analyzing alterations in various genes in the same set of surgical tumor specimens and in human cancer cell lines from several organs, including the lung, pancreas, liver and bain. The results of analyses of surgical specimens as well as those from studies on cell lines by both ourselves and other groups are summarized in Tables 1 to 4.

(1) Non-small cell lung cancers
We analyzed mutation of the ras, p53, RB, p21, raf and c-erbB2 genes in human non-small cell lung cancers (NSCLCs) by PCR-SSCP. As summarized in Table 1A, mutations in the ras and p53 genes were observed in 22 (19%) and 60 (52%) of 115 NSCLC tumors, respectively, while only 9 tumors carried both aberrations [26,27]. These results suggested that mutations in the p53 and ras genes are frequent but independent events in NSCLC tumors. Moreover, alterations in these two genes were not restricted to any particular clinical stage or to histological differentiation [26-29]. On the other hand, DNA analyses demonstrated that most cell lines established from NSCLCs carried mutations in both K-ras and p53 genes, suggesting that cells with both genetic alterations could be selected preferentially to grow in vivo (Table 1B).

In connection with the RAS cascade, aberration of the raf gene was analyzed in the same set of 115 NSCLC tumors. However, no mutation was observed in the raf gene, suggesting that the oncogenic activation of RAS protein might not be replaced by RAF activation in NSCLC tumors [30].

With regard to the p53 cascade in NSCLCs, absence of mutations in the p21 gene in 67 tumors suggested that dysfunction of p21 was not equivalent to that of p53. Multiple disorders caused by p53 inactivation including disruption of apoptotic pathways could be important in the development of this type of tumor [31].

Inactivation of the RB gene was detected in only 2 of 43 NSCLC tumors [32,33]. A subset of NSCLC cell lines also carried a mutated RB gene [34]. Inactivation of the p16 gene was frequently observed in NSCLC cell lines indicating that disruption of the p16 - cyclin D1/CDK4 - pRB cascade is involved in the majority of these tumors [32-35]. It should be noted that alterations of the p16 and RB genes are mutually exclusive 8 (Table 1B). Six cell lines carrying mutated p16 genes expressed normal RB proteins. On the other hand, in the cell line NCI-H596 with the wild-type p16 gene, homozygous deletion of the RB gene was observed. In contrast, no amplification of the cyclin D1 or CDK4 genes was detected [36].

No possible activating mutations in the transmembrane domain of c-erbB-2 protein were detected in NSCLCs tumors [37]. While most of the NSCLC cells showed multiple genetic alterations, a large cell lung cancer cell line, Lu99, did not carry mutations in any of the genes analyzed [34,38].

(2) Pancreatic cancers
Mutation of the K-ras gene, inactivation of the p53, p16, DPC4, RB, BRCA2 and p15 genes and amplification of the AKT2 and c-myc genes are known to be involved in human pancreatic cancers [39]. Rozenblum et al. reported comprehensive analyses of first-passage xenografts from 42 primary pancreatic cancers and demonstrated the accumulation of multiple genetic alterations in these samples [40]. We also detected frequent mutations at codon 12 of the K-ras and p53 genes, relatively rare inactivation of the APC gene and no mutation of the p21 gene by SSCP analysis (Table 2A) [31,41,42,43]. The incidence of K-ras mutation (14/27, 52%) in our SSCP analysis of primary pancreatic cancers was lower than those in other reports [44,45]. We confirmed the lack of mutations by sequence analysis of all the 12 tumors which showed no mobility shift. Contamination by normal cells in tumor specimens did not seem to be a severe problem because some of the tumors without K-ras mutation showed the mobility shift on the SSCP analysis of the p53 gene [Table 2A, K. Yashima, unpublished results]. These findings in primary pancreatic cancers as well as the presence of pancreatic cancer cell lines without K-ras mutation suggested that a significant proportion of pancreatic cancers do not require the activated K-ras gene.

We also demonstrated amplification of the AKT2 gene in a subset of pancreatic tumors and cell lines [16]. Since the AKT2 gene encodes a cytoplasmic serine-threonine kinase which is activated by PDGF and overexpression of PDGF has been reported in pancreatic cancers, aberrations in the cascade in which AKT2 is involved may play an important role in pancreatic carcinogenesis [14].

The combined results of studies on genetic alterations in pancreatic cancer cell lines indicated that multiple genetic alterations were accumulated in the majority of the cells (Table 2B). As observed in NSCLC cell lines, inactivation of the p16 gene is frequently observed in pancreatic cancer cells and could be complementary to the aberration of the RB gene, as 5 cell lines carrying the mutated p16 gene did not show any abnormalities in the RB gene (Table 2B). The only exception was a Capan-2 cell line carrying aberrations in both the p16 and RB genes. In this cell line, inactivation of p16 and pRB may act synergistically in malignant cell growth.

DPC4 and p15 proteins are involved in a growth inhibitory cascade from the signals of transforming growth factor beta. The genes for these proteins were also inactivated in a subset of pancreatic cancer cell lines [46]. However, homozygous deletion of the p15 gene was always accompanied by that of the adjacent p16 gene and no mutation specific to the p15 gene was detected. These observations indicate that inactivation of the p15 gene might be an accessory event in pancreato-carcinogenesis.

It is noteworthy that no alterations were observed in the genes analyzed in a small subset of pancreatic cancers, even in advanced stages including two cell lines, SW979 and SW850, or the xenograft QGP-1.

(3) Hepatocellular carcinomas
Chronic hepatitis and liver cirrhosis resulting from chronic infection by hepatitis C virus (HCV) and hepatitis B virus (HBV) are the major etiological factors of hepatocellular carcinoma (HCC) in Japan [47]. We have demonstrated that p53 and RB gene inactivation are frequent events in HCCs [28,48,49]. As shown in Table 3A, RB mutation was observed in well-differentiated adenocarcinomas, while that of the p53 gene was not, suggesting involvement of the RB mutation in an earlier stage than that of the p53 mutation in multi-stage carcinogenesis of HCCs. Functional inactivation of p16 protein and amplification of the cyclin D1 gene reported by other groups indicated that disruption of the p16 - cyclin D1/CDK4 - pRB cascade was also important in HCCs [50-52]. However, neither amplification of the mdm2 gene nor mutation of the p21 gene was detected in the same set of HCCs, suggesting that disorder of the pathways specifically induced by p53 such as those of apoptosis might play a pivotal role in HCC formation [Y. Ohyama, Y. Murakami, unpublished results].

Overview of the results of genetic analyses in HCC cell lines confirmed the finding that inactivation of p53, RB and p16 genes were the major genetic alterations. Several reports suggested that HBX protein could associate with and inactivate p53 at least at a very early stage of HCC formation [53]. Observations in two cell lines, HuH1 and HuH2, supported this suggestion (Table 3B). However, some HCC cell lines show both inactivation of the p53 gene and integration of the HBX gene [54]. Furthermore, in some cell lines, neither of these aberrations were observed. Mutation of the ras gene was quite rare in HCCs, while it was observed in the hepatoblastoma cell line HepG2 [55].

(4) Gliomas
We have demonstrated p53 mutation, homozygous deletion of p16, CDK4 amplification as well as LOH at polymorphic loci on chromosomes 11q and 10p in advanced type human gliomas [56-60]. In glioma cell lines, inactivation of the p16 gene is observed frequently, while aberrations of other genes involved in the RB cascade such as mutation of the RB gene or amplification of the cyclin D1 or CDK4 gene were not detected in cells with p16 alterations [61]. Mutations of the PTEN and p53 genes were also observed at high frequency in glioma cells, while mutation of the ras gene has not been reported in gliomas [61-63].

The results of extensive studies on aberrations of oncogenes and tumor suppressor genes in the same set of human tumors and cancer cell lines were summarized [101]. In primary tumors, accumulation of multiple genetic alterations was observed characteristically in advanced cases. In addition to small DNA changes, loss or gain of chromosomal fragments was often detected in advanced cancers. The frequency of mutations was higher in cancer cell lines than that in primary tumors, probably because most of the cancer cell lines were derived from advanced tumors. Alternatively, cells carrying genetic alterations such as the p53 gene may be selected to grow continuously in culture or some additional changes might occur during growth in vivo. The genetic changes observed in each type of cancer cell line reflected those in primary tumors quite well.

These multiple genetic changes can be classified into a number of groups based on the function of their products in the signal transduction cascades of cell growth. These include the p16 - cyclin D1/CDK4 - pRB, MDM2 - p53 - p21 or RAS - RAF cascades [1,4]. Comprehensive survey of genetic changes in human tumors and cell lines demonstrated that alterations in a different cascade could be independent, while those within the same cascade were often complementary and mutually exclusive as observed in esophageal cancers [6]. These observations suggest that aberration of only one of the members in a cascade could cause its disfunction resulting in tumorigenesis. However, in some cancer cells multiple disorders even within the same cascade were observed, suggesting synergistic effects on malignant progression [4].

It is noteworthy that a subset of primary tumors or cell lines did not show any alterations in the genes analyzed even in the advanced stages. This observation suggested the involvement of completely different pathways in oncogenesis. Disruption of different sets of genes such as those of TGF beta receptor II or BAX protein carrying special DNA sequences targeted for mutation by defective DNA repair enzymes might be involved in such tumors [40,64,65].

1. R.A. Weinberg, How cancer arises, Scientific American, 275 (1996) 62-70.
2. Nishimura, S., Sekiya, T, Human cancer and cellular oncogenes. Biochem. J. 243 (1987) 313-327. [3] W.K. Cavenee, R.L. White, The genetic basis of cancer. Scientific American, 272 (1995) 72-79.
3. L. Zhu, G.H. Enders, C.L. Wu, M.A. Starz, K.H. Moberg, J.A. Lees, N. Dyson, E. Harlow, Growth suppression by members of the retinoblastoma protein family, Cold Spring Harbor Symposia on Quantitative Biology. 59 (1994) 75-84.
4. M. Orita, H. Iwahana, H. Kanazawa, K. Hayashi, T. Sekiya, Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc. Natl. Acad. Sci. USA, 86 (1989) 2766-8409.
5. W. Jiang, Y.J. Zhang, S.M. Kahn, M.C. Hollstein, R.M. Santella, S.H. Lu, C.C. Harris, R. Montesano R., I.B. Weinstein, Altered expression of the cyclin D1 and retinoblastoma genes in human esophageal cancer, Proc. Natil. Acad. Sci. USA 90 (1993) 9026-9030.
6. M. Hollstein, D. Sidransky, B. Vogelstein, C.C. Harris, p53 mutations in human cancers, Science 253 (1991) 49-53.
7. J.D. Oliner, K.W. Kinzler, P.S. Meltzer, D.L. George, B. Vogelstein, Amplification of a gene encoding a p53-associated protein in human sarcomas, Nature 358 (1992) 80-83.
8. F.S. Leach, T. Tokino, P. Meltzer, M. Burrell, M. J.D. Oliner, S. Smith, D.E. Hill, D. Sidransky, K.W. Kinzler, B. Vogelstein, p53 Mutation and MDM2 amplification in human soft tissue sarcomas,. Cancer Res. 53 (1993) 2231-2234.
9. W.S. el-Deiry, T. Tokino T., V.E. Velculescu, D.B. Levy, R. Parsons, J.M. Trent, D. Lin, W.E. Mercer, K.W. Kinzler, B. Vogelstein, B., WAF1, a potential mediator of p53 tumor suppression, Cell, 75 (1993) 817-825.
10. M.R. Smith, S.J. DeGudicibus, D.W. Stacey, DW. Requirement for c-ras proteins during viral oncogene transformation, Nature 320 (1986) 540-543.
11. M. Fukui, T. Yamamoto, S. Kawai, K. Maruo, K. Toyoshima, Detection of a raf-related and two other transforming DNA sequences in human tumors maintained in nude mice, Proc. Natl. Acad. Sci.USA 82 (1985) 5954-5958.
12. F. Ishikawa, F. Takaku, K. Hayashi, M. Nagao, T. Sugimura, Activation of rat c-raf during transfection of hepatocellular carcinoma DNA, Proc. Natl. Acad. Sci. USA 83 (1986) 3209-3212.
13. T.F. Franke, S.I. Yang, T.O. Chan, K. Datta, A. Kazlauskas, D.K. Morrison, D.R. Kaplan, P.N. Tsichlis, The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase, Cell 81 (1995) 727-736.
14. J.Q. Cheng, B. Ruggeri, W.M. Klein, G. Sonoda, D.A. Altomare, D.K. Watson, J.R. Testa, Amplification of AKT2 in human pancreatic cells and inhibition of AKT2 expression and tumorigenicity by antisense RNA, Proc. Natl. Acad. Sci. USA, 93 (1996) 3636-3641.
15. W. Miwa, J. Yasuda, Y. Murakami, K. Yashima, K. Sugano, T. Sekine, A. Kono, S. Egawa, K. Yamaguchi, Y. Hayashizaki, T. Sekiya, Isolation of DNA sequences amplified at chromosome 19q13.1-q13.2 including the AKT2 locus in human pancreatic cancer, Biochem. Biophys. Res. Commun. 225 (1996) 968-974.
16. V. Murray, Improved double-stranded DNA sequencing using the linear polymerase chain reaction, Nucleic Acids Res. 17 (1989) 8889.
17. B.J. Conner, A.A. Reyes, C. Morin, K. Itakura, R.L. Teplitz, R.B. Wallace, Detection of sickel cell bS-globin allele by hybridization with synthetic oligonucleotides, Proc. Natl. Acad. Sci. USA, 80 (1983) 278-282.
18. S.S. Sommer, A.R. Groszbach, C.D. Bottema, PCR amplification of specific alleles (PASA) is a general method for rapidly detecting known single-base changes, Biotechniques 12 (1992) 82-87.
19. S. Ye, S. Humphries, F. Green, Allele specific amplification by tetra-primer PCR, Nucl. Acids Res. 20 (1992) 1152.
20. R.M. Myers, Z. Larin, T. Maniatis, Detection of single base substitutions by ribonuclease cleavage at mismatches in RNA:DNA dupexes, Science, 230 (1985) 1242-1246.
21. E. Winter, F. Yamamoto, C. Almoguera, M.A. Perucho, Methosd to detect and characterize point mutations in transcribed genes: Amplification and overexpression of the mutant c-Ki-ras allele in human tumor cells, Proc. Natl. Acad. Sci. USA, 82 (1985) 7575-7579.
22. V.C. Sheffield, D.R. Cox, D.R. Lerman, R.M. Myers, Attachment of a 40-base-pair G+C-rich sequence (GC-clamp) to genomic DNA fragments by the polymerase chain reaction results in improved detection of single-base changes. Proc. Natl. Acad. Sci. USA, 86 (1989) 232-236.
23. E.S. Abrams, S.E. Murdaugh, L.S. Lerman, Comprehensive detection of single base changes in hman genomic DNA using denaturing gradient gel electrophoresis and a GC clamp, Genomics, 7 (1990) 463-475.
24. M. Orita, Y. Suzuki, T. Sekiya, K. Hayashi, Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction, Genomics, 5 (1989) 874-879.
25. Y. Suzuki, M. Orita, M. Shiraishi, K. Hayashi, T. Sekiya, Detection of ras gene mutations in human lung cancers by single-strand conformation polymorphism analysis of polymerase chain reaction products, Oncogene 5 (1990) 1037-1043.
26. Y. Kishimoto, Y. Murakami, M. Shiraishi, K. Hayashi, T. Sekiya. Aberrations of the p53 tumor suppressor gene in human non-small cell carcinomas of the lung, Cancer Res., 52 (1992) 4799-4804.
27. Y. Murakami, Y. Suzuki, Y. Kishimoto, S. Hirohashi, K. Hayashi, T. Sekiya, T, Detection of DNA aberrations in human cancers by single-strand conformation polymorphism analysis of polymerase chain reaction products, Tohoku J. Exp. Medicine, 168 (1992) 247-255.
28. T. Sekiya, Detection of mutant sequences by single-strand conformation polymorphism analysis, Mutation Research, 288 (1993) 79-83. [30] W. Miwa, J. Yasuda, K. Yashima, R. Makino, T. Sekiya, Absence of activating mutations of the RAF1 protooncogene in human lung cancer, Biol. Chem. Hoppe-Seyler, 375 (1994) 705-709.
29. T. Shimizu, W. Miwa, S. Nakamori, O. Ishikawa, Y. Konishi, T. Sekiya, Absence of a mutation of the p21/WAF1 gene in human lung and pancreatic cancers, Jpn. J Cancer Res. 87 (1996) 275-278.
30. R. Sachse, Y. Murakami, M. Shiraishi, K. Hayashi, T. Sekiya, DNA aberrations at the retinoblastoma gene locus in human squamous cell carcinomas of the lung, Oncogene, 9 (1994) 39-47.
31. K. Tamura, X. Zhang, Y. Murakami, S. Hirohashi, H.J. Xu, S.X. Hu SX, W.F. Benedict, T. Sekiya. Deletion of three distinct regions on chromosome 13q in human non-small-cell lung cancer, Int. J Cancer, 74 (1997) 45-49.
32. Y. Murakami, M. Katahira, R. Makino, K. Hayashi, S. Hirohashi, T. Sekiya, Inactivation of the retinoblastoma gene in a human lung carcinoma cell line detected by single-strand conformation polymorphism analysis of the polymerase chain reaction product of cDNA, Oncogene 6 (1991) 37-42.
33. T. Shimizu, T. Sekiya, Loss of heterozygosity at 9p21 loci and mutations of the MTS1 and MTS2 genes in human lung cancers, Int. J Cancer, 63 (1995) 616-620.
34. A. Okamoto, D.J. Demetrick, E.A. Spillare, K. Hagiwara, S.P. Hussain, W.P. Bennett, K. Forrester, B. Gerwin, M. Serrano, D.H. Beach, C.C. Harris, Mutations and altered expression of p16INK4 in human cancer, Proc. Natl. Acad. Sci. USA 91 (1994) 11045-11049.
35. R. Sachse, Y. Murakami, M. Shiraishi, K. Hayashi, T. Sekiya, Absence of activating mutations in the transmembrane domain of the c-erbB-2 protooncogene in human lung cancer, Jpn. J Cancer Res. 83 (1992) 1299-1303.
36. Y. Murakami, K. Hayashi, T. Sekiya, Detection of aberrations of the p53 alleles and the gene transcript in human tumor cell lines by single-strand conformation polymorphism analysis, Cancer Res., 51 (1991) 3356-3361.
37. Y. Murakami, Genetic alteraions in human pancreatic cancer, J Hep Bil Pancr Surg. 4 (1997) 283-290.
38. E. Rozenblum, M. Schutte, M. Goggins, S.A. Hahn, S. Panzer, M. Zahurak, S.N. Goodman, A. Taylor, R.H. Hruban, C.J. Yeo, S.E. Kern, Tumor-suppressive pathways in pancreatic carcinoma, Cancer Res. 57 (1997) 1731-1734.
39. S. Nakamori, K. Yashima, Y. Murakami, O. Ishikawa, H. Ohigashi, S. Imaoka, S. Yaegashi, Y. Konishi, T. Sekiya. Association of p53 gene mutations with short survival in pancreatic adenocarcinoma. Jpn. J Cancer Res. 86 (1995) 174-181.
40. T. Sekiya, Y. Murakami, K. Hayashi, Detection of DNA aberrations in human cancers by single-strand conformation polymorphism analysis of the polymerase chain reaction. Int. J. Pancreatol. 13 (1993) 69-78.
41. K. Yashima, S. Nakamori, Y. Murakami, A. Yamaguchi, K. Hayashi, O. Ishikawa, Y. Konishi, T. Sekiya. Mutations of the adenomatous polyposis coli gene in the mutation cluster region: comparison of human pancreatic and colorectal cancers, Int. J Cancer 59 (1994) 43-47.
42. C. Almoguera, D. Shibata, K. Forrester, J. Martin, N. Arnheim, M. Perucho M, Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes, Cell 53 (1988) 549-554.
43. V.T. Smit, A.J. Boot, A.M. Smits, G.J. Fleuren, C.J. Cornelisse, J.L. Bos, KRAS codon 12 mutations occur very frequently in pancreatic adenocarcinomas, Nucl. Acids Res. 16 (1988) 7773-7782.
44. S.A. Hahn, M. Schutte, A.T. Hoque, C.A. Moskaluk, L.T. da Costa, E. Rozenblum, C.L. Weinstein, A. Fischer, C.J. Yeo, R.H. Hruban, S.E. Kern, a candidate tumor suppressor gene at human chromosome 18q21.1, Science 271 (1996) 350-353.
45. N. Okada, G. Ohshio, K. Yamaki, T. Imamura, M. Imamura, Elevated serum c-erbB-2 protein levels in patients with pancreatic cancer: correlation to metastasis and shorter survival, Oncology 52 (1995) 392-396.
46. Y. Murakami, K. Hayashi, S. Hirohashi, T. Sekiya. Aberrations of the tumor suppressor p53 and retinoblastoma genes in human hepatocellular carcinomas, Cancer Res., 51 (1991) 5520-5525.
47. X. Zhang, H.J. Xu, Y. Murakami, R. Sachse, K. Yashima, S. Hirohashi, S.X. Hu, W.F. Benedict, T. Sekiya, Deletions of chromosome 13q, mutations in Retinoblastoma 1, and retinoblastoma protein state in human hepatocellular carcinoma. Cancer Res. 54 (1994) 4177-4182.
48. Y-.J., Zhang, W. Jiang, C.J. Chen, C.J. Amplification and overexpression of cyclin D1 in human hepatocellular carcinoma. Biochem. Biophys. Res. Commun. 196 (1993) 1010-1016.
49. N. Nishida, Y. Fukuda, T. Komeda, Amplification and overexpression of the cyclin D1 gene in aggresive human hepatocellular carcinoma. Cancer Res., 54 (1994) 3107-3110.
50. A.M. Hui, M. Sakamoto, Y. Kanai, Y. Ino, M. Gotoh, J. Yokota, S. Hirohashi S, Inactivation of p16INK4 in hepatocellular carcinoma, Hepatology, 24 (1996) 575-579.
51. H. Ueda, S.J. Ullrich, J.D. Gangemi, Functional inactivation but not structural mutation of p53 causes liver cancer. Nature Genet. 9 (1995) 41-47.
52. C.A. Richards, S.A. Short, S.S. Thorgeirsson, B.E. Huber BE, Characterization of a transforming N-ras gene in the human hepatoma cell line Hep G2: additional evidence for the importance of c-myc and ras cooperation in hepatocarcinogenesis, Cancer Res. 50 (1990) 1521-1527.
53. I.C. Hsu, T. Tokiwa, W. Bennett, R.A. Metcalf, J.A. Welsh, T. Sun, C.C. Harris, p53 gene mutation and integrated hepatitis B viral DNA sequences in human liver cancer cell lines, Carcinogenesis. 14 (1993) 987-992.
54. S. Mashiyama, Y. Murakami, T. Yoshimoto, T. Sekiya, K. Hayashi, Detection of p53 gene mutations in human brain tumors by single-strand conformation polymorphism analysis of polymerase chain reaction products, Oncogene 6 (1991) 1313-1318.
55. Y. Sonoda, T. Yoshimoto, T. Sekiya, Homozygous deletion of the MTS1/p16 and MTS2/p15 genes and amplification of the CDK4 gene in glioma, Oncogene. 11 (1995) 2145-2149.
56. Y. Sonoda, M. Iizuka, J. Yasuda, R. Makino, T. Ono, T. Kayama, T. Yoshimoto, T. Sekiya T, Loss of heterozygosity at 11p15 in malignant glioma, Cancer Res. 55 (1995) 2166-2168.
57. Y. Sonoda, Y. Murakami, T. Tominaga, T. Kayama, T. Yoshimoto, T. Sekiya, Deletion mapping of chromosome 10 in human glioma, Jpn. J Cancer Res. 87 (1996) 363-367.
58. H. Kon, Y. Sonoda, T. Kumabe, T. Yoshimoto, T. Sekiya, Y. Murakami. Structural and functional evidence for the presence of tumor suppressor genes on the short arm of chromosome 10 in human gliomas. Oncogene 16 (1998) 257-263.
59. M. Katahira, Y. Murakami, K. Hayashi, T. Sekiya, T. Detection of aberrations of the p53 gene but not the RB gene in human brain tumors by single-strand conformation polymorphism analysis of polymerase chain reaction products. J. Tokyo Women Medical Collage 62 (1992) 206-213.
60. J. Li, C. Yen, D. Liaw, K. Podsypanina, S. Bose, S.I. Wang, J. Puc, C. Miliaresis, L. Rodgers, R. McCombie, S.H. Bigner, B.C. Giovanella, M. Ittmann, B. Tycko, H. Hibshoosh, M.H. Wigler, R. Parsons, PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer, Science 275 (1997) 1943-1947.
61. D. Liaw, D.J. Marsh, J. Li, P.L. Dahia, S.I. Wang, Z. Zheng, S. Bose, K.M. Call, H.C. Tsou, M. Peacocke, C. Eng, R. Parsons, Germline mutations of the PTEN gene in Cowden disease, an inherited breast and thyroid cancer syndrome, Nature Genet. 16 (1997) 64-67.
62. S. Markowitz, J. Wang, L. Myeroff, R. Parsons, L. Sun. J. Lutterbaugh, R.S. Fan, E. Zborowska, K.W. Kinzler, B. Vogelstein, Inactivation of the type II TGF-beta receptor in colon cancer cells with microsatellite instability, Science 268 (1995) 1336-1338.
63. N. Rampino, H. Yamamoto, Y. Ionov, Y. Li, H. Sawai, J.C. Reed, M. Perucho M, Somatic frameshift mutations in the BAX gene in colon cancers of the microsatellite mutator phenotype, Science 275 (1997) 967-969.
64. J.Y. Chen, W.D. Funk, W.E. Wright, J.W. Shay, J.D. Minna, Heterogeneity of transcriptional activity of mutant p53 proteins and p53 DNA target sequences, Oncogene 8 (1993) 2159-2166.
65. M. Ramet, K. Castren, K. Jarvinen, K. Pekkala, T. Turpeenniemi-Hujanen, p53 protein expression is correlated with benzo[a]pyrene-DNA adducts in carcinoma cell lines. Carcinogenesis. 16(9):2117-2124, 1995 Sep.
66. R. Alemany, S. Ruan, M. Kataoka, P.E. Koch, T. Mukhopadhyay, R.J. Cristiano, Growth inhibitory effect of anti-K-ras adenovirus on lung cancer cells, Cancer Gene Therapy. 3 (1996) 296-301.
67. R. Alemany, S. Ruan, M. Kataoka, P.E. Koch, T. Mukhopadhyay, R.J. Cristiano, J.A. Roth, W.W. Zhang, Growth inhibitory effect of anti-K-ras adenovirus on lung cancer cells, Cancer Gene Therapy 3 (1996) 296-301.
68. D.M. Valenzuela, J. Groffen, Four human carcinoma cell lines with novel mutations in position 12 of c-K-ras oncogene, Nucleic Acids Res. 14 (1986) 843-852.
69. T. Mitsudomi, J. Viallet, J.L. Mulshine, R.I. Linnoila, J.D. Minna, A.F. Gazdar, Mutations of ras genes distinguish a subset of non-small-cell lung cancer cell lines from small-cell lung cancer cell lines, Oncogene 6 (1991) 1353-1362.
70. K. Shimizu, D. Birnbaum, M.A. Ruley, O. Fasano, Y. Suard, L. Edlund, E. Taparowsky, M. Goldfarb, M. Wigler, Structure of the Ki-ras gene of the human lung carcinoma cell line Calu-1, Nature 304 (1983) 497-500.
71. Y. Taya, K. Hosogai, S. Hirohashi, Y. Shimosato, R. Tsuchiya, N. Tsuchida, M. Fushimi, T. Sekiya, S. Nishimura, EMBO Journal 3 (1984) 2943-2946.
72. M.A. Hawk, K.T. Cesen, J.C. Siglin, G.D. Stoner, R.J. Ruch, Cancer Lett. 109 (1996) 217-222.
73. M. Reiss, D.E. Brash, T. Munoz-Antonia, J.A. Simon, A. Ziegler, V.F. Vellucci, Z.L. Zhou, Status of the p53 tumor suppressor gene in human squamous carcinoma cell lines, Oncol. Res. 4 (1992) 349-357.
74. G.I. Shapiro, J.E. Park, C.D. Edwards, L. Mao, A. Merlo, D. Sidransky, M.E. Ewen, B.J. Rollins, Multiple mechanisms of p16INK4A inactivation in non-small cell lung cancer cell lines, Cancer Res. 55 (1995) 6200-6209.
75. M. Fukumoto, D.H. Shevrin, I.B. Roninson, Analysis of gene amplification in human tumor cell lines, Proc. Natl. Acad. Sci. USA, 85 (1988) 6846-6850.
76. K. Yoshimoto, M. Shiraishi, S. Hirohashi, S. Morinaga, Y. Shimosato, T. Sugimura, T. Sekiya, Rearrangement of the c-myc gene in two giant cell carcinomas of the lung, Jpn. J. Cancer Res. 77 (1986) 731-735.
77. D.J. Capon, E.Y. Chen, A.D. Levinson, P.H. Seeburg, D.V. Goeddel, Complete nucleotide sequences of the T24 human bladder carcinoma oncogene and its normal homologue, Nature 302 (1983) 33-37.
78. M.S. Greenblatt, A.P. Grollman, C.C. Harri, Deletions and insertions in the p53 tumor suppressor gene in human cancers: confirmation of the DNA polymerase slippage/misalignment model, Cancer Res. 56 (1996) 2130-2136.
79. A.J. Levine, J. Momand, C.A. Finlay, The p53 tumour suppressor gene, Nature 351 (1991) 453-456.
80. T.A. Lehman, W.P. Bennett, R.A. Metcalf, J.A. Welsh, J. Ecker, R.V. Modali, S. Ullrich, J.W. Romano, E. Appella, J.R. Testa et al. p53 mutations, ras mutations, and p53-heat shock 70 protein complexes in human lung carcinoma cell lines, Cancer Res. 51 (1991) 4090-4096.
81. S.M. Bodner, J.D. Minna, S.M. Jensen, D. D'Amico, D. Carbone, T. Mitsudomi, J. Fedorko, D.L. Buchhagen, M.M. Nau, A.F. Gazdar et al., Expression of mutant p53 proteins in lung cancer correlates with the class of p53 gene mutation, Oncogene, 7 (1992) 743-749.
82. T. Nobori, K. Miura, D.J. Wu, A. Lois, K. Takabayashi, D.A. Carson, Deletions of the cyclin-dependent kinase-4 inhibitor gene in multiple human cancers, Nature 368 (1994) 753-756.
83. G. Berrozpe, J. Schaeffer, M.A. Peinado, F.X. Real, M. Perucho, Comparative analysis of mutations in the p53 and K-ras genes in pancreatic cancer, Int. J Cancer, 58 (1994) 185-191.
84. M. Naumann, N. Savitskaia, C. Eilert, A. Schramm, H. Kalthoff, W. Schmiegel, Frequent codeletion of p16/MTS1 and p15/MTS2 and genetic alterations in p16/MTS1 in pancreatic tumors, Gastroenterology 110 (1996) 1215-1224.
85. H. Yamada, T. Yoshida, H. Sakamoto, M. Terada, T. Sugimura, Establishment of a human pancreatic adenocarcinoma cell line (PSN-1) with amplifications of both c-myc and activated c-Ki-ras by a point mutation, Biochem. Biophys. Res. Commun. 140 (1986) 167-173.
86. B. Ruggeri, S.Y. Zhang, J. Caamano, M. DiRado, S.D. Flynn, A.J. Klein-Szanto, Human pancreatic carcinomas and cell lines reveal frequent and multiple alterations in the p53 and Rb-1 tumor-suppressor genes, Oncogene. 7 (1992) 1503-1511.
87. C. Caldas, S.A. Hahn, L.T. da Costa, M.S. Redston, M. Schutte, A.B. Seymour, C.L. Weinstein, R.H. Hruban, C.J. Yeo, S.E. Kern, Frequent somatic mutations and homozygous deletions of the p16 (MTS1) gene in pancreatic adenocarcinoma, Nature Genet. 8 (1994) 27-32.
88. L. Huang, T.L. Goodrow, S.Y. Zhang, A.J. Klein-Szanto, H. Chang, B.A. Ruggeri, Deletion and mutation analyses of the P16/MTS-1 tumor suppressor gene in human ductal pancreatic cancer reveals a higher frequency of abnormalities in tumor-derived cell lines than in primary ductal adenocarcinomas, Cancer Res. 56 (1996) 1137-1141.
89. Q. Liu, Y.X. Yan, M. McClure, H. Nakagawa, F. Fujimura, A.K. Rustgi, MTS-1 (CDKN2) tumor suppressor gene deletions are a frequent event in esophagus squamous cancer and pancreatic adenocarcinoma cell lines, Oncogene 10 (1995) 619-622.
90. M. Schutte, R.H. Hruban, L. Hedrick, K.R. Cho, G.M. Nadasdy, C.L. Weinstein, G.S. Bova, W.B. Isaacs, P. Cairns, H. Nawroz, D. Sidransky, R.A. Casero, P.S. Meltzer, S.A. Hahn, S.E. Kern, DPC4 gene in various tumor types, Cancer Res. 56 (1996) 2527-2530.
91. M. Goggins, M. Schutte, J. Lu, C.A. Moskaluk, C.L. Weinstein, G.M. Petersen, C.J. Yeo, C.E. Jackson, H.T. Lynch, R.H. Hruban, S.E. Kern, Germline BRCA2 gene mutations in patients with apparently sporadic pancreatic carcinomas, Cancer Res. 56 (1996) 5360-5364.
92. S.A. Hahn, M. Schutte, A.T. Hoque, C.A. Moskaluk, L.T. da Costa, E. Rozenblum, C.L. Weinstein, A. Fischer, C.J. Yeo, R.H. Hruban, S.E. Kern, DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1, Science, 271 (1996) 350-353.
93. B. Bressac, K.M. Galvin, T.J. Liang, K.J. Isselbacher, J.R. Wands, M. Ozturk, Abnormal structure and expression of p53 gene in human hepatocellular carcinoma, Proc. Natl. Acad. Sci. USA, 87 (1990) 1973-1977.
94. Y.J. Zhang, W. Jiang, C.J. Chen, C.S. Lee, S.M. Kahn, R.M. Santella, I.B. Weinstein, Amplification and overexpression of cyclin D1 in human hepatocellular carcinoma, Biochem. Biophys. Res. Commun. 196 (1993) 1010-1016.
95. C. Gomez-Manzano, J. Fueyo, A.P. Kyritsis, P.A. Steck, J.A. Roth, T.J. McDonnell, K.D. Steck, V.A. Levin, W.K. Yung, Adenovirus-mediated transfer of the p53 gene produces rapid and generalized death of human glioma cells via apoptosis, Cancer Res. 56 (1996) 694-699.
96. T. Ohtsubo, X. Wang, A. Takahashi, K. Ohnishi, H. Saito, C.W. Song, T. Ohnishi, p53-dependent induction of WAF1 by a low-pH culture condition in human glioblastoma cells, Cancer Res. 57 (1997) 3910-3913.
97. J. He, J.R. Allen, V.P. Collins, M.J. Allalunis-Turner, R. Godbout, R.S. Day 3rd., C.D. James, CDK4 amplification is an alternative mechanism to p16 gene homozygous deletion in glioma cell lines, Cancer Res. 54 (1994) 5804-5807.
98. J. Fueyo, C. Gomez-Manzano, W.K. Yung, G.L. Clayman, T.J. Liu, J. Bruner, V.A. Levin, A.P. Kyritsis, Adenovirus-mediated p16/CDKN2 gene transfer induces growth arrest and modifies the transformed phenotype of glioma cells, Oncogene 12 (1996) 103-110.
99. Murakami, Y. & Sekiya, T. Accumulation of genetic alterations and their significance in each primary human cancer and cell line. Mutation Resarch, 400, 421-437, 1998.