Cancer Poster Session



Materials & Methods


Discussion & Conclusion



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Accumulation of genetic alterations and their significance in each primary human cancer and cell line.

Contact Person: Yoshinori Murakami (ymurakam@ncc.go.jp)


Most human tumors develop and progress toward malignancy through accumulation of multiple genetic alterations including activation of oncogenes and inactivation of tumor suppressor genes [1-4]. The proteins which are encoded by these genes interact with one another and construct elaborated networks involved in cell growth regulation such as signal transduction, cell cycle control and programmed cell death [1,4]. Over- or under expression and abnormal structures of these proteins are involved in oncogenesis. Patterns of genetic alterations are different not only among types of tumors, but also among tumors of the same organ from different patients. Thus, understanding of the genetic alterations accumulated in tumors in each patient is essential to determine the molecular mechanisms underlying tumorigenesis. To detect genetic changes including small alterations such as point mutations, we developed a method of single-strand conformation polymorphism (SSCP) analysis [5]. This method was applied to analyses of DNAs from the same set of surgical specimens of human tumors including non-small cell lung cancers, pancreatic cancers, hepatocellular carcinomas and gliomas. Studies of the DNA from each type of human cancer cell lines could also provide useful information about the multiple genetic alterations accumulation of which resulted in tumorigenesis. This article provides an overview of the mutated genes accumulated in tumors from a variety of patients and also in a number of human cancer cell lines detected both by ourselves and by other groups using a variety of DNA analysis methods including SSCP.

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Materials and Methods

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].

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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].

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Discussion and Conclusion

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].

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Murakami, Y.; Sekiya, T.; (1998). Accumulation of genetic alterations and their significance in each primary human cancer and cell line.. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Available at URL http://www.mcmaster.ca/inabis98/cancer/murakami0563/index.html
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