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Mol. Cells, Vol. 1, pp. 491-4% Screening of a Specific Point Mutation in Tumor Suppressor p53 Gene of Korean Hepatocellular Carcinoma Tissues Bok-Soo Lee, Chang-Duk Jun, Young-Whan Chun, Sang-Gi Paik l , Baik-Whan Cho2, Chan ChoP, Kwon-Mook Chae4 and Hun-Taeg Chung* Department of Microbiology and Immunology, 3Department of Pathology and 4Department of Surgery, School of Medicine, Wonkwang University, Iri 570-749, Korea; IDepartment of Biology, Chungnam National University, Tafjon 305-764, Korea; 2Department of Surgery, School of Medicine, Chonbuk National University, Chunju 560- 756, Korea (Received on November 30, 1991) The point mutation at a specific site (the third base of codon 249 of exon 7) in the p53 gene was not found in the 8 hepatocellular carcinoma samples from Korean patients. This result is quite different from the report on Chinese and South African patients that showed the point mutations at the same site with the frequency of 50% in hepatocellular carcinoma sample. Even though this particular point mutation was not found in Korean samples, there might be mutations at other sites of p53 gene, because expression of mutated p53 gene was detected in the nuelei of hepatocellular carcinoma samples by using monoelonal antibodies which are specific for muta nt-type p53 proteins. Also the change of DNA content was found in the hepatocellular carcinoma samples. Originally p53 had been considered to be an oncogene, but several researchers have indicated that the wild type gene product actually functions as a tumour suppressor gene (Finlay et al., 1989; Sturzbecher et al., 1988; Wolf and Rotter, 1985). In human tumours, the short ann of chromosome 17 is often deleted or the remaining (not-deleted) p53 alleles were found to contain mutations and several reports suggest that p53 mutations play a role in the development of many common human malignancies (Baker et aI., 1989; Eliyahu et aI., 1989). Especially, hepatocellular carcinoma (HCC) is a prevalent cancer in Africa and Eastern Asia, abnormalities in the structure and expression of the p53 are frequent in HCC cell lines and allelic loses from chromosome 17p have beeen found in HCCs from China and Japan (Bressac et al., 1991). Mutations occur at specific sites (at the third base of codon 249), and this specificity could reflect exposure to a liver-specific carcinogen, being aflatoxin B1. The human p53 gene of 11 exons is 20 kilobases (kb) long, and the first and second exons are separated by an intron of 10 kb (Peter and Lionel, 1986). Baker et ai., (1990) reported that chromosome loss occurs near the mutation sites of suppressor gene in many cases. Loss of a series of chromosomes appears to occur early in the development of many cancers, with other gene changes usually coming in later (Baker et al., 1990; Marx, 1991). Mutations in the p53 gene are known to be the most common genetic alterations in diverse human cancers. Recently, two groups of researchers reported that primary hepatocel* To whom correspondence should be addressed lular carcinomas (HCC) from endemic hepatitis areas and abundant aflatoxin B had showed high frequency of point mutation at codon 249 of exon 7 in the p53 gene (Bressac et al., 1991; Hsu et aI., 1991). Korea is known as an endemic area of hepatitis and many Korean foods are also known to contain much aflatoxin. In this study, the authors tried to see wheter or not there is a specific point mutation at codon 249 of exon 7 in the p53 gene. Small fragment (110 base pairs) of the gene which contains codon 249 of exon 7 of p53 gene from the DNA of normal HCC tissue was amplified by using polymerase chain reaction. The amplified fragments from the wild type p53 gene could be digested by the restriction enzyme HaeIII. The authors screened the point mutation at the third base of codon 249 of exon 7 in the p53 gene by the electrophoresis of digested DNA segments of amplified DNA fragments from HCC tissue DNA. In addition, the nuclear phosphoprotein p53 was originally discovered in extracts of transformed cells, reacting with antiserum from animals with tumours induced by simian virus 40 (SV40). Because large T antigen is needed to maintain the transformed phenotype, it was suggested that this interaction is important for transformation (Werness et al., 1990). Wild-type p53 seems to negatively regulate cell growth and division, but overproduction or mutated p53 protein inhibits wild-type p53, either by complexing with normal p53 The abbreviations used are: HCC, hepatocellular carcinoma, PBS, phosphate-buffered saline solution; PCR, polymerase chain reaction. © 1991 The Korean Society of Molecular Biology 492 Mutations of p53 Gene in Hepatoma protein or by competitive inhibition (Reich and Levine, 1984). These recent studies suggest that mutated p53 not only relieves the tumor suppressor effect of wild-type p53 but also exerts a direct oncogenic effect (Marshall, 1991). In tumour cells, there was an increase in GI phase and then a steady elevated or slightly increasing level throughout Sand G2M phases. Also, aneuploid populations showed a uniform high p53 content (Morke and Didrik, 1991). Although it is .not a specific property of malignant cells, a high level is strongly suggestive of malignancy, and combined with DNA measurement could increase the possibility of discriminating between normal and malignant cells in tumor tissue. The authors also measured the DNA content and p53 gene product by using flow cytometer after staining the nuclei from HCC tissue by propidium iodide and fluorescein isothiocyanate-labeled monoclonal antibodies which is specific for p53 gene product. Materials and Methods DNA Preparation Paraffin-embedded tissue HCC samples were obtained from Chonbuk University. The samples were extracted with histoclear or xylene to remove paraffin and were rehydrated in a sequence of decreasing ethanol concentrations. The tissue was submerged in distilled water overnight. After discarding distilled water, 3 ml of 0.5% pepsin solution was added and incubated for I h at 37 °C, minced to tiny pieces with surgical blades. The resulting solution was then filtered through a 40 IJIIl nylon mesh, centrifuged, and washed in phosphate-buffered saline solution (PBS). The samples were dried under vacuum and added with digestion buffer (50 mM Tris-HC1, pH 8.5, 1 mM EDTA, plus 0.5% Tween 20) containing 200 fll/ ml proteinase K The samples were incubated for 3 h at 55 °C and then at 95 °c for 8 min to inactivate the protease. Any residual tissue pellet was removed by centrifuging f~r about 30 sec and collected supernatant was used for PCR. Polymerase chain reaction Amplification took place in 25-fll reaction mixtures containing I fll (100 ng) of prepared supernatant in 50 mM Tris-HCl (PH 8.3), 3 mM MgCh, 20 mM KCl, 250 f.1g borine serum albumin, each primer (P3 and R3) at 0.25 M, each dNTP at 200 /lM, and 2 units of Taq polymerase (KIST). The oligonucleotide primers were purchased from KIST. PCR reactions were carried out for 31 cycles which consisted of 94 °C (1 sec), 55 °C (1 sec), and 72 °c (5 sec). After the last cycle, the sample was left at 72 °c for an _additional 10 sec to ensure that the PCR products are fully double stranded. Analysis of mutation site Amplified PCR products were extracted with phenol, chloroform and precipitated with ethanol. After Mol. Cells the sample was dried under vacuum and resuspended in distilled water, it was digested with HaeIII for 1 h at 37 °C. The digested products were loaded onto 2% Seakem agarose (FMC) gel contating 5 f.1g/ml ethidium bromide in 1X TBE buffer. q>X174/RF HaeIII fragments (NEB) were used as size markers. Analysis of DNA ploidity Thin sections (5 f.1m) were deparaffinized in histoclear and rehydrated through a sequence of ethanol changes. The deparaffinized tissues were washed with distilled water twice. After discarding distilled water, 3 ml of 0.5% pepsin solution was added and incubated for 1 h at 37 "c, minced to tiny pieces with surgical blades. The resulting solution was then filtered through a 4O-1JIIl nylon mesh, centrifuged, and washed in PBS. After washing in PBS, 100 fll RNase I (I mg/ml, Sigma) was added and the nuclei were resuspended in staining solution (propidium iodide (PI) 25 f.1g/ml in PBS, 1 X 106 cells). The samples were analysed on an FACstar (Beckton Dickinson). The laser excitation wavelength was 488 nm at 200 mW. Green (FITC) and red (PI) fluorescence were separated by a 560 nm dichroic mirror. In addition, the green and red photomultiplier tubes (PMT) were guarded by a 530 nm bandpass and a 630 nm longpass filter, respectively. Spectral overlap between green and red signals was compensated by using the subtraction unit of the flow cytometer. At least I X 105 cells were counted for each sample. Analysis of p53 oncoprotein produced Prior to immunostaining, nuclear suspensions were prepared from paraffin-embedded tissues. The primary monoclonal antibodies (diluted to a final concentration of 2.5 f.1g antibody/20 fll) PAbl801 I, II, and III were added separately in the cell suspension (approximately 5 X 105 nuclei) in PBS with 0.5% BSA. The suspension was incubated for 30 min in a refrigerator. Following incubation, the cells were washed once in PBS with 0.1% Triton X-lOO, resuspended in 50 fll FITC-conjugated goat anti-human IgG, dilute 1:20, incubated for 30 min at room temperature. After incubation, they were again washed with PBS/Triton and resuspended in PBS. The samples were analysed on flow cytometer by the same method as the one in analysis of DNA ploidity. Results DNA content analysis of HCC samples Aneuploidy is almost always found in many human cancers, the most common cancer-related genetic change known as point mutations, allelic loss, rearrangements, deletion, and insertions. These aberrations, together with alterations of oncogenes and other tumour suppressor genes, make up the mutational network leading to malignancy. Figure I shows representative DNA histograms demonstrating a typical normal DNA diploidy (panel A), a tumour with a DNA Vol. 1 (1991) Bok-Soo Lee et af. I 500 I I A I l i 0::: ~ 0 ,·1.--:-• •""'7. ...,. t.::.J t.;:.J ~ ~ () h II I I I I I ,,I I I, 11I I " 1.. 1 \.I '. /I '~~"4!'."""'''~'' "I''.........._ ... ~" g 'I . I I I I "1"111 , j • I I I I 1 C I 1'\ I I , ' I I. , i f l {i. r ,, .1fl.. ( \ 1'1 iI Ii \. ...'........... __~ .., • , I ""'".,,\.~.J./t-<I" , , 't'4111" I I I I I I I I I I ,) ,~ .II " ...I " ! II I I I I'TT' II "r\\,..·· ·Jr.·.\¥,··,.\··,.·..V).;..__....j I I I I II I I II I I ' 200 100 I ,I I I 0 II rl 1'1 II f\ J I. II 'I ,, ',I ", 'I~ r I II It ,I I I I , ! ,"i r I ,IJI ~~ I , I ' i\ , / ~d\.'.,,,~, I I I 0 1.. 1 II \l\ r I I I I 1 I I I~ '~\\, .11 I J I I I i, /I ,I I, Il ~ "1 0 8 I I I II II I I, I I I I II II I I I 1 0 , I ,! I I~ I " ...I ~ CO ~ II q I, I~ .J, I 493 .. ,.¥,.'"'''' . •.,..,\~("'f .. ·'I-'l'~'. ,.f,.,...., . ' ., I I I I I I I I I 10 0 i I . 'j"'IV-j'"" ' t.. '·'1-' 2 00 I I I I FLUORESCENCE INTENSITY Figure 1. DNA content of nonnal and cancer cells. Nuclei were prepared from paraffin-embedded tumor tissues and stained with propidiun iodide. DNA content of each cell was measured by flow cytometer at the wavelength of 488 nm. A, diploid from nonnal tissues; B, C, and D, aneuploid from cancer tissues. aneuploidy containing hypoploidy (panel B), a abnormal DNA aneuploidy containing hyperploidy (panel C), and a tumour with DNA aneuploidy containing tetraploidy (panel D). About 60% of total HCC patients showed abnormal pattern in DNA analysis. In the case of panel C, the number of S-phase cells increased. Abscissa represents fluorescence intensity (DNA content) and ordinate shows the number of cells. The results of this analysis represent that changes of DNA content have influenced on malignancy of HCC samples. Analysis of mutant p53 protein in HCC tissues Some of the tumours, there was an increase in p53 in G 1 phase and then a steady elevated or slightly increasing level throughout Sand G 2M phases. Also, aneuploidy populations showed a high mutant p53 protein content in Figure 2. Panel A shows negative control, and panel B shows nonspecific binding to nuclear protein when only secondary antibody was used. Panel C shows positive expression of specific mutated p53 nuclear phosphoprotein. The monoclonal antibody PAb 1801 used in this study reacts only with human p53, recognizes a more stable epitope on the p53 protein and might also detect product of mutated p53 gene. The normal p53 protein is assumed to play an important role in the regulation of cell cycle, and the protein itself is strictly regulated with a very short half-life. It is also known that quiescent and dividing lymphocytes synthesize different forms of p53. The change between these two forms occors at the GO-G 1 transition. Possi~ily, this reflects a structural or posttranslational change in the protein. In many transformed cell lines and other cancers, the steady state levels of p53 are very high, and the tumorigenic efficiency seems to be related to the extent of p53 overexpression. In the present study, almost all HCC samples were p53 positive, and thus these results correlate with other findings of p53 being associated to high-grade malignancy. Screening of point mutation on specific site in p53 gene The p53 gene has a constant source of fascination 494 Mutations of p53 Gene in Hepatoma 500 kj Mol. Cells A I" o 11" III I I I I 100 200 I -------, CII I I I I I I I I I I I I I I I I 200 FLUORESCENCE INTENSITY Figure 2. Typical pattern of nuclear protein P53 expression in Korean HCCs. The nuclei prepared from paraffin-embedded tissue were stained with monoclonal antibody PAb 1801 , which recognizes human p53 mutant. This figure shows that tumor cells produce the p53 mutant. A, negative control; B, only secondary antibody used; C, PAb-II + secondary antibody used. since its discovery nearly a decade ago. Most tumours with allelic deletions contain p53 point mutations resulting in amino-acid substitutions. Such p53 gene mutations are clustered in four hot-spots which exactly coincide with the four most highly conserved regions of the gene, reflecting its functional importance and selection in the p53 protein in the development of many common human malignancies. These domains and exons 5-8 are frequently the sites of mutation. · Recently, in an area of high incidence of hepatitis B virus, and aflatoxin B which is both a mutagen (inducing G to T substitution) and a liver-specific carcinogen, the high mutation frequency (50%) was observed at a specific site in exon 7 in hepatocellular carcinoma patients. This indicates that this position is a hot spot associated with HCC development in this geographic region. For this reason, p53 gene was amplified in the exon 7 region including codon 249 known as specific mutation site, using polymerase chain reaction. Amplified products were of 110 base pairs and digested with restnctlOn enzyme HaeIII which specifically recognizes the wild type codon 249 sequence. Because the mutated codon 249 is not recognized by this enzyme, two different types of this region could be detected. Figure 3 shows results from Korean HCC samples. All amplified products had 11O-bp fragments and were digested with HaeIII yielding 75-bp and 35-bp fragments. Lanes A and E represent size markers pBR322 /BstNI and X174RF/HaeIII. Lane B shows results from normal tissues. Lanes C, D and E show results from HCC tissues. Of these lanes, the left line is the amplified product and the right one is the digested products. These results indicate that HCC patients in Korea have no specific point mutations on codon 249 of exon 7 in p53 gene. Relation to p53 mutations and DNA content in Korean hepatocellular carcinomas Table 1 shows the summarized data of mutations Vol. 1 (1991) Bok-Soo Lee et al. A B c Discussion D E bp 1,857 1,060 383 121 Figure 3. Restriction enzyme analysis of nonnal tissue, hepatocellular carcinoma tissue and tumor cell line DNAs demonstrates that codon 249 mutations did not occur. DNAs were amplified by PCR using synthetic oligonucleotide primers, which can specifically recognize DNA segment of exon 7 of p53 gene. The amplified products of 110 bp were digested with HaeIII and then separated on agarose gels. All HaeIIIdigested tumor and non-tumor DNAs yield 75- and 35-bp fragments. A, pBR322/BstNl size markers; B, nonnal tissue; C, HCC tissue; D, HepG2; E, <l>X174RF/HaeIII size markers. Table 1. p53 mutations and DNA contents in Korean hepatocellular carcinomas Patient Age Sex p53 mutation DNA ploidy I 2 3 4 63 49 35 53 WT" WT WT WT S SI M M M M M M M M Aneuploid Aneuploid Aneuploid Aneuploid Aneuploid Aneuploid Aneuploid Aneuploid Aneuploid 48 6 7 54 8 58 Cell line (HepG2) WT WT WT WT 495 aWT indicates the wild-type. on specific site in p53 gene and DNA content of Korean hepatocellular carcinomas. All samples have wild type at the third base of codon 247 on exon 7 in p53 gene, and the nuclei represent the chromosomal abnormality and the expression of mutant p53 protein. Although dietary exposure to hepatitis B virus and aflatoxin B is an epidemiologically defined risk factor for HCC in Southern Africa and China, this hot spot mutation was not found among HCC patients in Korea. The current understanding of p53 in cancer is that overproduction of a mutated p53 protein inhibits wildtype p53, either by cOIl1plexing with normal p53 protein or by competitive inhibition, but its precise function is unknown (Banks et aI., 1986; Eliyahu et al., 1985). Mutant forms of the murine p53 clones can immortalize primary rat embryo fibroblast cells and cooperate with an activated ras oncogene to transform cells. They have transforming activity and are transdominant over wild-type p53. The trans-dominant phenotype of the mutated p53 protein may be explained by its ability to oligomerize with wild-type p53, drawing it into this complex and effectively inactivating it (Halevy et ai., 1990; Herskowitz, 1987). The SV 40 large T antigen and adenovirus ElB 55-kDa proteins form complexes with p53 protein resulting in increased half-life, presumably also inactivating its normal function \" as a negative regulator of cellular growth. This change in conformation is associated with enhanced protein stability (Levine et aI., 1991; Gannon et al., 1990). There is evidence for both dominant loss-of-function mutations in transformed cells and gain-of-function mutations in tumourigenesis assays. The two are not mutually exclusive. Overexpression of the wild-type protein with mutant p53 protein or other oncogene products suppresses transformation, cell growth, and the tumourigenic potential of the cells. Thus, the ratio of mutant to wild-type p53 in a cell could be critical in regulating cell division. p53 would negatively regulate entry into S phase. The p53 protein also associates with the viral DNA replication complexes in cells infected with herpesvirus. Perhaps p53 acts in the late G 1 phase of the cell cycle to promote or prevent the assenmbly of a DNA replication-initiation complex (Diller et aI., 1990; Raycroft et al., 1990). Alternatively, p53 could act as a transactivator of gene transcription, either promoting or repressing messenger RNA synthesis. Some mutations that activate transformation affect gene transcription. Transformation-activating mutations alter the DNA-binding abilities of p53. It now needs to be determined whether the p53 DNA-binding region recognizes a specific DNA sequence. Thus p53 could have a role in regulating gene transcription, perhaps of a set of genes that effect the passage from late G 1 to S phase of the cycle (Finlay et aI., 1989; Marshall, 1991). Sequencing of the p53 gene in mammals, amphibians, birds, and fish has revealed five highly consezved dimains, four of which fall within exons 5 through 8. Domains 3, 4, and 5 are included in the two binding regions for SV40 large T antigen (Werness et al., 1990). The numerous amino acids that presumably alter the biological function of the p53 protein when substituted are dispersed among four conserved domains as well as among intervening sequences, suggesting that no single domain is responsible for maintaining p53 tumour suppressor function. Integration of hepatitis B 496 Mutations of p53 Gene in Hepatoma virus DNA is a frequent event associated with HCC. And the mutagen aflatoxin B1, which is the main aflatoxin species found in foods in Africa and China, binds preferentially to G residues in G+C-rich regions and induces G to T substitutions almost exclusively (Bressac et ai., 1991 ; Hsu et ai., 1991). Thus, aflatoxin B1 is a potent hepatocarcinogen in different species and dietary exposure to it is an epidemiologically defined risk factor for HCC, so it is possible that p53 mutations caused by aflatoxins or other environmental carcinogens might contribute to the high incidence of HCC in these areas. p53 gene mutations in HCCs were G to T transversions in seven of the 16 tumours in individuals from the region of Qidong, China, all at the third base pair position of codon 249. The mutation was not present in non-malignant cells of these individuals. Four out of 10 HCCs in individuals from a different population at high risk of liver cancer (Southern Africa) also contained G to T transversions, three of which were at codon 249. Other than the clusters of transitions at rare CpG dinucleotide sites, p53 mutations in most human cancers are dispersed over the midregion of the coding sequence (Finlay et al., 1989; Nigro et al., 1989; Hollstein et al., 1991). They found that 12 of 26 HCCs examined from two high-risk groups contained a mutation at the same codon, and other mutations as well in some cases. And so far, several oncogenes and tumour suppressor genes have been identified in a mutant form in human cancers (Nigro et ai., 1989; Hunter, 1991). The number of such genes will continue to grow, and it is also possible that genes will be identified to alter cell growth when perturbed by completely different mechanisms. Determining how these combinations disturb cellular growth control should enable us to understand the vast diversity of cancers. In this study, a point mutation at the specific site (the third base of codon 249 of exon 7) was not found in the p53 gene. These results are quite different from those which showed point mutations at the specific site at the frequency of 50 percents from Chinese and South African patients, but coincide with the results in 22 HCCs from Japanese patients. Even though the point mutation was not found, there might be mutations at other sites of p53 gene because expression of mutated p53 protein was detected by using monoclonal antibodies which are specific for mutant-type p53 protein and because DNA content was changed in the hepatocellular carcinoma samples. So, other mutations will be searched by sequencing of various amplified PCR products. References Baker, S. 1., Fearon, E. R , Nigro, 1. M., Hamilton, Mol. Cells S. R , Preisinger, A c., Jessup, 1. M., van Tuinen, P., 'Ledbetter, D. H., Barker, D. F., Nakamura, Y., White, R , and Vogelstein, 8. (1989) Science 244, 217221 Baker, S. 1., Markowitz, S., Fearon, E., Wilson, J. K v., and Vogelstein 8. (1990) Science 249, 912-925 Banks, L., Matlashewski, G., and Crawford, L. (1986) Eur. J Biochem . 159, 529-534 Bressac, B., Kew, M., Wands, J., and Ozurk, M. (1991) Nature 350, 429-431 Diller, L., Kassel, 1., Nelson, C. E., Gryka, M. 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(1991) Cell 64, 313-326 Marx, 1. (1991) Science 251, 1317 Morkve, 0., and Didrik, O. L. (1991) Cytometry 12, 438-444 Nigro, 1. M., Baker, S. 1., Preisinger, A c., Jessup, 1. M., Hosttetter, R , Cleary, K , Bigner, S. H., Davidson, N., Baylin, S., Devilee, P., Glover, T , Collins, F. S., Weston, A , Modali, R , Harris, C. c., and Vogel stein, 8. (1989) Nature 34, 705-708 Raycroft, L., Wu, H., and Lozano, G. (1990) Science 249, 1049-1051 Reich, N. c., and Levine, A 1. (1984) Nature 308, 199201 Sturzbecher, H. W., Addison, c., and Jenkins, 1. R (1988) Moi. Cell. Bioi. 8, 3740-3747 Werness, 8. A , Levine, A J., and Howley, P. M. (1990) Science 248, 76-79 Wolf, D., and Rotter, V. (1985) Proc. Nat!. Acad. Sci. U S. A. 82, 790-794