Point Mutation, Allelic Loss and Increased Methylation of c-Ha-Ras Gene in Human Hepatocellular Carcinoma

NORIO OGATA, TOMOTERU KAMIMUFtA AND HITOSHI ASAKURA Third Department of Internal Medicine, Niigata University School of Medicine, 1 Asahi-machi, Niigata, 951, Japan

Somatic alterations of the c-Ha-rus gene were ex- amined in 21 Japanese patients with hepatocellular carcinoma. Restriction endonuclease analysis by double digestion with Map1 and HpuII revealed that DNAs from two of 21 hepatocellular carcinoma tissues were affected by nucleotide substitution at the twelfth amino acid codingsequence of the c-Ha-rus gene. DNAs from cirrhotic noncancerous liver tissue, but not leukocytes, of one of these patients possessed the mutation, whereas DNAs from noncirrhotic liver tissue and leukocytes of the other patient did not. In one of the nine patients harboring heterozygosity for c-Ha- rus-related BumHI-fragments, the loss of one allele was demonstrated as a somatic change not only in DNA from the tumor tissue but also in DNA from the cirrhotic nontumorous tissue. In two of the 19 patients comparatively examined for digestion patterns of c-Ha- rus locus with H’II and MspI, extensive methylation was observed as a somatic modification in both DNAs from the tumor and the cirrhotic nontumorous tissues. These results thus indicate that the genetic lesions affecting the c-Ha-rua gene do occur in human hepa- tocellular carcinoma and probably serve as one of the multiple steps in the process of hepatic carcinogenesis. (HEPATOLOGY 1991; 13:31-37.)

Hepatocellular carcinoma (HCC) is a common neo- plasm in Japan and in other Asian and African countries. Epidemiological surveys have shown that persistent infection with HBV is closely linked to the occurrence of HCC (11, and molecular analyses have demonstrated that the HBV genome is integrated into chromosomes of HCC (2,3). However, since the integration of HBV DNA alone does not lead to the development of HCC (4,5) and not all HCCs are related to HBV infection, it has been hypothesized that functional changes of cellular genes also may have some significance for the progression of HCC (6).

Among several types of gene alterations that have been considered to play important roles in malignant

Received January 30, 1990; accepted July 26, 1990. This work was partly supported by a Grant-in-Aid for Scientific Research

from the Ministry of Education, Science and Culture of Japan. Address reprint requests to: Norio Ogata, M.D., National Institute of Allera,

and Infectious Diseases, Building F, Room 201, National Institutes of Health, Bethesda, MD 20892.

31/1/26313

processes, the activation of cellular oncogenes has been analyzed most extensively (7-9). The rus oncogene family, c-Ha-ras, c-Ki-rus and N-rus, acquires the capacity to transform cells either by single point muta- tions, resulting in amino acid substitutions at positions 12, 13,59 and 61 in its coding Mr. 21,000 proteins (p21) (10-16) or by overexpression of the normal rus p21 (17, 18). Many types of human malignancies have been revealed to harbor such alterations (19-21). As for human HCC, the incidences of activation for the N-rus gene and the c-Ki-rus gene have been reported (22,23); however, positive results on that for the c-Ha-rus gene are lacking.

One of the other gene alterations that has been considered to be involved in neoplastic events is the inactivation of tumor suppressor genes (7, 24, 25). The short arm of human chromosome 11 (l lp) , where the c-Ha-rus gene resides, harbors this kind of gene(s) of which inactivation induces tumorigenic expression (26, 27). In certain types of human malignancies, this locus has been documented to be affected by alterations that could inactivate gene functions, such as structural loss (28-31) or increased methylation (32). A recent report has documented that specific deletions in chromosomes l l p and 13q occur in the tumor tissues in human HCC (33).

In the present study, we investigated Japanese pa- tients with HCC for the occurrence of point mutations at the twelfth codon, the existence of allelic loss and the state of hypermethylation of the c-Ha-rus gene using restriction endonuclease analyses. The results showed that these three alterations did occur somatically not only in HCC cells but also in matched hepatocytes in cirrhotic nontumorous parts of liver tissue.

MATERIALS AND METHODS Tissue Samples. HCC tissues from 21 Japanese patients

were obtained through surgical resection. Noncancerous liver tissues were also obtained during surgery with the HCC tissues from 19 of the 21 patients.

Immediately after the liver resection, the tumor and the nontumorous portions were dissected carefully and cut into small pieces. A part of each tissue specimen was examined histologically to confirm the diagnoses of cancerous and noncancerous tissues and to inspect the degree of contami- nation of stromal and blood cells. The remainder of the tissue

31

32 OGA'I'A, KAMIMURA AND ASAKURA I IEPATOLOGY

TABLE 1. Summary of HCC patients positive for somatic alterations of c-Ha-ras gene

Integration Point Allelic Increased Histology of of HBV DNA mutation loss methylat ion

nontumorous Case Sex Age tissue T N T N T N T N

--

- - - - - 1 F 27 CPH + + t 7 F 43 LC f + + Homo - -

t - ~ + + - - 11 M 60 LC + 14 M 56 LC 16 M 62 LC - Homo 4 +

- i + - -

- -

T ~ tumor tissue; N = nontumorous tissue; CPII = chronic persistent hepatitis; LC = liver cirrhosis; Homo = homozygote of c-Ha-ras locus.

specimen was stored at -80°C until DNA extraction. HCC tissues with few stromal and blood cells and noncancerous liver tissues without discernible metastasis were used for DNA analyses. Blood samples were obtained from eight of the 21 tumor-bearing patients after surgery, and peripheral leuko- cytes were purified by a dextran sedimentation method. Normal liver tissues were obtained at autopsy from five patients who did not have liver diseases.

DNA Purification. Histologically proven cancerous and noncancerous tissues and peripheral leukocytes were digested with lysis buffer containing 0.01 mol/L Tris hydrochloride, 200 kg/ml proteinase K and 1% SDS. High molecular weight DNAs were purified by means of repeated extraction with phenol and chloroform followed by precipitation in 70% ethanol.

Blot Hybridization. Fifteen micrograms of DNA was com- pletely digested with restriction endonucleases (Takara, Kyoto, Japan). Digestion with BumHI was performed as recommended by the supplier. Digestion with MspI and/or HpuII was carried out by adding each enzyme in the amount of 10 units/yg DNA. The digested DNA samples were size- fractionated in agarose gels of appropriate concentrations (see "Results"), denatured, neutralized and transferred to nylon membranes (Dupont Gene Screen Plus, Boston, MA) (34). The filters were prehybridized in 5 x SSC (1 x SSC is 0.15 mol/L NaCl and 0.015 mol/L sodium citrate), 5 x Denhardt's so- lution (50 x Denhardt's solution is l% Ficoll, l% BSA and 1% polyvinylpyrolidone), 1% SDS, 50% formamide and 100 g / m l sonicated salmon sperm DNA for 16 hr at 42" C. Hybridization was performed in the same solution with a radiolabeled probe and 10% dextran sulfate added for 24 h r at 42" C (35). The unreacted probe was removed from the filter by washing three times for 10 min at room temperature with 2 x SSC and 1% SDS and five times for 30 min at 65" C with 0.1 x SSC and 1% SDS. The membranes were then exposed to x-ray film at -70" C for autoradiography. The intensities of the bands resulting from the autoradiograms were quantified by densi- tometric scan (JOYCE-LOEBL Chromoscan 200, Gateshead, UK).

Probe Preparation. The human c-Ha-rus 8.3 kilobase (kb) pair recombinant clone pP1 (12) was donated by the Japan Cancer Research Resource Bank. For the detection of point mutations at codon 12 of the c-Ha-rus gene, 602 base pair (bp) SmuI fragment spanning from the first exon to the second exon was electroeluted and used as a probe. For the analyses of the allelic loss and the methylation state of c-Ha-rus locus, full length 8.3 kb BarnHI fragment was used as a probe. The radiolabeling reaction was performed with [a - 32P] - dCTP to a specific activity of 5 x 10' to 1 x lo9 cpm/p,g of DNA using a multiprime labeling system (Amersham, Little Chal- font, UK).

RESULTS Detailed information of HCC patients positive for

c-Ha-ras alterations is summarized in Table 1. Restriction Endonuelease Analysis of c-Ha-ras Gene

at Codon 12. Since an alteration at codon 12 of the human c-Ha-ras gene leads to the loss of a restriction site for HpaIIIMspI digestion (361, DNA samples har- boring lesions at this position should generate an abnormal 411 bp band by HpaIIIMspI digestion when using the 602 bp SmaI fragment as a probe, whereas DNA specimens without alterations at this position should demonstrate normal 355 bp and 56 bp bands (13). In this study, DNAs double digested with HpuII and MspI were electrophoresed in a 2% agarose gel to separate a 41 1 bp band and a 355 bp band; a 56 bp band was missed under these experimental conditions (Fig. 1).

Of the 21 patients, DNAs from HCC tissues of Patient 1 (Fig. 1, case 1, lane T) and Patient 7 (Fig. 1, case 7, lane T) demonstrated a 411 bp band together with a normal 355 bp band, indicating that the c-Ha-rus gene of the HCC tissues from these two patients underwent alter- ations affecting the twelfth amino acid of its p21 coding sequence (Table 1). The other 19 patients failed to yield abnormal fragments as shown representatively in Figure 1 (cases 3, 12, and 19). Interestingly, in Patient 7, DNA from noncancerous liver tissue, for which the histological diagnosis was cirrhosis, also demonstrated a 411 bp band (Fig. 1, case 7, lane N), indicating the occurrence of a nucleotide alteration similar to that from the cancerous tissue, whereas Patient 1, in whom nontumorous liver tissue was histologically diagnosed as chronic persistent hepatitis, showed only a 355 bp band (Fig. 1, case 1, lane N). DNAs from peripheral leukocytes of these two patients gave only a 355 bp band (Fig. 1, case 1 and case 7, lane W). Thus the alterations at the twelfth codon of c-Ha-rus gene observed in the two HCCs and one nontumorous liver tissue were shown to be the consequence of somatic changes.

Densitometric scan of the bands in Patient 1 demon- strated greater density for the mutated fragment than for the normal one in the tumor tissue (Fig. 2, panel A) and showed no intensity signal for the mutated fragment in the nontumorous tissue (Fig. 2, panel B). In Patient 7, almost equal density was observed between the normal and the mutated bands from the tumor tissue (Fig. 2, panel C), whereas the mutated fragment

Vol. 13, No. 1, 1991 ALTERATIONS OF c-Ha-Ras GENE IN HCC 33

FIG. 1. Restriction endonuclease analysis of c-Ha-ras gene at codon 12 in patients with HCC. DNA samples were double digested with MspI and HpaII and hybridized to a 602 bp SmaI fragment covering portions of the first and second exons of c-Ha-ras gene. T, N and W indicate DNA from HCC tissue, nontumorous tissue and peripheral leukocytes, respectively. Molecular weight markers shown on the left are HpaII fragments of pUC 19 plasmid.

from the nontumorous tissue was obviously less intense than the normal one (Fig. 2, panel D).

Loss of Heterozygosity a t c-Ha-ras Locus. The c-Ha-ras gene shows polymorphism by BamHI digestion caused by the variable cycle of the tandemly reiterated 28 bp sequence located at the 3' end (36). With BarnHI digestion, the DNA from individuals who are homo- zygous for this locus shows a single c-Ha-rus-related fragment, whereas DNA from heterozygous individuals yields two such fragments (28). Electrophoresis on a 0.7% agarose gel revealed that nine patients carried heterozygosity at this locus (Fig. 3).

Among these nine patients possessing two BamHI fragments, DNA from HCC tissue of Patient 14 (Fig. 3, case 14, lane T) repeatedly demonstrated a difference in the intensity between two allelic restriction fragments of 8.3 kb and 6.6 kb. Also, DNA from noncancerous cirrhotic liver tissue from the same patient (Fig. 3, case 14, lane N) exhibited different intensities between the two bands, although the degree was lower than that from HCC tissue; whereas in the DNA from peripheral leukocytes of the same patient, the two allelic fragments showed nearly equal intensities (Fig. 3, case 14, lane W). The results of band intensity were confirmed by densi- tometric scan (Fig. 4). These findings indicate that the loss of one c-Ha-rus allele took place somatically in HCC and cirrhotic nontumorous liver tissues of Patient 14. DNAs from the other eight heterozygous patients did not show significant differences in the intensities of the two alleles in tumorous or nontumorous liver tissues as representatively shown in Figure 3, cases 13 and 5.

State of Methylation at c-Ha-ras Locus. The c-Ha-ras gene contains many CCGG sites (36) of which methy-

M A

C

B N

I t

D

N

FIG. 2. Densitometric scan resulting from Southern blot analysis demonstrated in Figure 1, of the twelfth codon c-Ha-ras fragments of cases 1 and 7: mutated (M) and normal (N). (A) DNA from HCC tissue of case 1. (B) DNA from noncancerous, noncirrhotic liver tissue of case 1. (C) DNA from HCC tissue of case 7. (D) DNA from noncancerous cirrhotic liver tissue of case 7.

lation can be assessed by digestion with MspI and HpaII because both MspI and HpaII recognize the CCGG sequence, but HpaII will not cut the sequence when the internal cytosine is methylated (37). A 1.6% agarose gel allowed us to compare signals from HpaII- digested DNA with those from MspI-digested DNA (Fig. 5).

Of the 19 patients harboring various patterns of signals with HpaII digestion, DNAs from both HCC and cirrhotic nontumorous liver tissues of Patient 11 (Fig. 5, case 11, lanes T and N) and Patient 16 (Fig. 5, case 16, lanes T and N) characteristically failed to demonstrate HpaII bands, ranging from 6.0 to 1.0 kb, which could be observed in DNAs from the cancerous and noncancerous liver tissues of the other 17 patients as representatively shown in Figure 5 (cases 8, 4 and 15). DNAs from matched peripheral leukocytes of the former two pa- tients yielded bands ranging from 6.0 to 1.0 kb by HpaII digestion (Fig. 5, cases 11 and 16, lane W). When normal liver DNAs from five individuals were examined for the methylation patterns, all DNAs showed, similar to DNAs from the latter 17 HCC patients, bands spanning from 6.0 to 1.0 kb after HpaII digestion (Fig. 6). These results strongly suggest that in Patient 11 and Patient 16 the c-Ha-rus locus of both HCC and cirrhotic non- tumorous liver tissues was affected by extensive meth- ylation associated with the development of HCC as a somatic modification.

DISCUSSION In this report, histological studies of HCC and

nontumorous liver tissues allowed us to examine DNA signals that reflect the genetic features of parenchymal cells, although the results obtained from nontumorous tissues might have been slightly influenced by a very small population of contaminated stromal or blood cells.

34 HEPATOLOGY OGATA, KAMIMURA AND ASAKURA

A 6 C

FIG. 3. Southern blot analysis for the loss of c-Ha-rus allele in patients with HCC. Paired DNA samples from HCC tissue fT), nontumorous liver tissue (N) and peripheral leukocytes W) of the same patient were digested with BumHI and hybridized to a 8.3 kb BumHI fragment of c-Ha-ras gene. Molecular weight markers indicated are Hind111 fragments of hc1857 DNA.

Activation of the ras gene family is considered to work in a dominant manner in neoplastic processes of many types of human malignancies (7, 19-21). However, in human HCC, a limited number of studies has been made on the incidence of rus-activation without positive results for c-Ha-ras activation (22, 23).

Using restriction endonuclease analysis, we showed that the twelfth codon of the c-Ha-ras gene was affected by nucleotide alteration in two of 21 HCC tissues (9.5%). The absence of such a change in matched peripheral leukocytes clearly indicates that this genetic alteration was not transmitted in the germ line. Thus, it is strongly suggested, although not confirmed by sequencing of the locus, that the activation of the c-Ha-rus gene might occur in these HCC cells by point mutation at the twelfth amino acid coding sequence. Our results, together with the findings of Tsuda et al. (231, who reported a very low incidence of point mutations at the twelfth codon of c-Ki-ras gene (3.3%) and negative results for N-rus gene, indicate that rus activation by point mutation a t the twelfth codon may be a rare event in the progression of HCC. However, Gu et al. (22) have demonstrated a relatively high incidence (36.4%) of detecting N-ras gene in NIH 3T3 transformants in which DNA from HCC tissues or HCC cell lines was introduced. Therefore other mechanisms of rus activation, such as point mutations at different positions or overexpression, still remain to be clarified (14-18). It is noteworthy that one of the mutation-bearing patients, Patient 7, also carried the mutated allele in cirrhotic nontumorous liver tissue,

FIG. 4. Densitometric scan of a high molecular weight (H) and a low molecular weight fL) c-Ha-rus alleles, resulting from Southern blot analysis of case 14 shown in Figure 4. (A) DNA from HCC tissue. (B) DNA from noncancerous cirrhotic liver tissue. (C) DNA from pe- ripheral leukocytes.

indicating that the mutational activation of the c-Ha-ras gene might occur in the nonmalignant hepatocytes. In this patient, both the cancerous and the noncancerous tissues possessed two restriction fragments, mutated and normal. These two fragments gave almost equal intensities in the tumor tissue, whereas the mutated fragment in the nontumorous tissue possessed a weaker signal than the normal one. These findings raise at least two possibilities concerning the allelic zygosity for mutation and cellular clonality. First, the mutation- positive cells were of clonal origin harboring heterozy- gosity for the lesion, one mutated and the other normal; hepatocytes being affected by the alteration on one allele might be exclusively selected and eventually undergo malignant growth, producing HCC tissue with the monoclonal cells. Second, the mutation-positive cells were heterogeneous for the lesion, carrying the mu- tation homozygosity and heterozygosity; during the progression of the malignancy, a shift toward homozy- gosity of the mutated allele might occur, resulting in a predominant population of homozygous cells in HCC tissue. The other mutation-harboring patient, Patient 1, did not yield the mutated fragment in noncirrhotic liver tissue, suggesting that the mutational activation might be specific for HCC cells in this case. In the tumor tissue of this patient, the signal for the mutated allele was more intense than that for the normal one, supporting the second possibility mentioned above that HCC cells might be heterogeneous for mutational zygosity.

Inactivation of normal sequences has also been pos- tulated to be involved in neoplastic events, possibly by allowing the tumorigenic expression of recessively working genes (7). Actually, in certain types of human malignancies, the loss of the c-Ha-ras gene on chro- mosome l l p has been documented (25, 28-31) and recently several genes on chromosome 1 l p including c-Ha-ras have been reported to be concomitantly af- fected by hypermethylation (32). Wang and Rogler (33) have reported a high percentage of detecting simple deletions in chromosome l l p and 13q in human HCC tissues and proposed that recessive genes may work also in the progression of HCC.

Vol. 13, No. 1, 1991 ALTERATIONS OF c-Ha-Rus GENE IN HCC 35

Case 1 1 16 8 4 1 5 -- --- T N W T N W T N T N T N ---- - - CL_-. -_L_

6 . 6 - 4 . 4 - 2.3 - 2 . 0 - 1 . 1 -

0 . 6 -

kb

FIG. 5. Southern blot analysis for the methylation of c-Ha-ras locus in patients with HCC. Paired DNA samples from HCC tissue (TI, nontumorous liver tissue (N) and peripheral leukocytes (W) of the same patients were digested with MspI (left lane of each sample) and with HpaII (right lane of each sample), respectively, and hybridized to a 8.3 kb BamHI fragment of c-Ha-ras gene. Molecular weight markers indicated are Hind111 or Sac1 fragments of hc1857 DNA.

In the present study, we demonstrated that one of nine HCC patients (11.1%) harbored the deletion of one BurnHI-related c-Ha-ras allele as a somatic change. Interestingly, again, the cirrhotic nontumorous liver tissue of this patient also showed the allelic loss. In each tissue, the allelic loss was not complete, and the degree of the loss was greater in the tumor tissue than in the nontumorous tissue. These results suggest a selection process in cells exhibiting the alteration during the development of HCC, resulting in the great majority of HCC cells having the lesion. We, herein, also showed that the c-Ha-rus locus in two of 19 HCC patients (10.5%) was extensively methylated in both tumor and cirrhotic nontumorous liver tissues. The emergence of small HpaII fragments of hundreds of base pairs in these tissues suggests that the whole extent of the c-Ha-rus gene might not be hypermethylated or that the degree of methylation might vary among cells. Taken together, it has thus been implicated that the structural or func- tional inactivation of the c-Ha-ras gene on chromosome l l p might be involved in the development of some HCCs probably from the early stages of its progression. Although the precise role of this gene inactivation is not yet clear, one possibility may be, as has been hypothe- sized in several kinds of malignancies (7,28-33), that the inactivation of tumor suppressor gene(s) on chro- mosome l l p (27) leads to the tumorigenic expression of the altered allele of recessive gene(s). Further studies on the other genes on chromosome l l p together with expression analyses would afford more information about whether the suppressed loci are the same or not between the allelic loss-harbored tissues and the in- creased methylation-carrying tissues.

One of the most intriguing findings in this report seems to be the existence of each somatic alteration of

A

- B C

,- -

6 . 6 - 2.3 - 2 . 0 - a

V ' 1 . 1 -

0 . 6 -

kb

D E

iF

FIG. 6 . Southern blot analysis for the methylation pattern of c-Ha-ras locus in normal liver tissues. DNA specimens from five individuals (A, B, C, D and E) were digested with MspI (lefl lane of each sample) and HpaII (right lane of each sample), respectively, and hybridized to a 8.3 kb BumHI fragment of c-Ha-ras gene. Molecular weight markers are the same as in Figure 5.

the c-Ha-ras gene in cirrhotic liver tissues. This suggests that the ras alterations may not work at the final step but serve as one of multiple steps during the progression of HCC. Similar findings of rus point mutations in nonmalignant cells have been reported in patients with other neoplasms (38-401, and increased methylation of genes on chromosome l l p has been documented in human T cell lymphotropic virus-infected cells early

36 OGATA, KAMIMURA AND ASAKURA HEPATOLOGY

during the transformation process (32). Based on the mode of integration of HBV DNA into chromosomes, it has been postulated that hepatocytes and HCC cells carrying the integrated viral DNA undergo clonal growth (41). We also have indicated polyclonal growth of hepatocytes for liver tissue reconstruction, which be- comes prominent in cirrhotic liver tissues (42). The two patients harboring the point mutations of c-Ha-rus gene (Patients 1 and 7) were positive for integrated HBV DNA, and their modes indicated clonal growth of hepatocytes containing the integrants in the cirrhotic tissues from Patient 7, whereas little, if any, clonal growth of hepatocytes was seen in the noncirrhotic tissue from Patient 1 (data not shown) (42). Thus it is tempting to suggest a parallelism between the c-Ha-rus- mutated cells and HBV-integrated cells in their profiles of clonal growth. Although our patient possessing allelic loss of the c-Ha-ras gene (Patient 14) did not carry the integrated HBV DNA, a correlation of chromosome 1 l p deletions with HBV DNA integration has been strongly proposed (33), and one of our two patients affected by increased methylation of the c-Ha-rus gene (Patient 11) was positive for HBV DNA integration. Taken together, it seems interesting to investigate the causal rela- tionship between the effects of “extrinsic mutagens” such as HBV DNA integration and the alterations of c-Ha-rus gene for understanding the multistep mo- lecular mechanism of HCC development.

In conclusion, the presented data, although at a low incidence, demonstrate the possible significance of so- matic alterations of c-Ha-ras gene for the progression of HCC as in other human malignancies. It is well known that many human HCCs arise from chronic liver diseases, especially cirrhosis. Therefore investigations of other unresolved genetic alterations in the progressive sequence from chronic liver diseases to HCC are req- uisite. Fu et al. (43) have found that the strong expression of insulin-like growth factor I1 on chro- mosome l l p is associated with the progression of HCC from its early stages in woodchuck models.

Acknowledgments: We wish to thank Dr. Keisuke Yoshida at the First Department of Surgery, Dr. Ryo Kominami at the First Department of Biochemistry, Niigata University School of Medicine; and Dr. Koichi Shibasaki at the Department of Internal Medicine, Nippon Dental University, for their generous support of this work.

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