Absence of p53 mutations and various frequencies of ki-ras exon 1 mutations in rat hepatic tumors induced by different carcinogens

Mutations of p53 and Ki-ras exon 1 were investigated in rat hepatic lesions induced by four kinds of hepatocarcinogenic protocols: continuous feeding of 3′-methyl-4-dimethylaminoazobenzene (3′-Me-DAB), daily intraperitoneal injection of aflatoxin B, (AFB,), and the Soft and Farber regimen (Nature 236:701–703, 1976), in which diethylnitrosamine (DEN) or nitrosomethylurea (NMU) was used as initiating agents. DNA from microdissected tissue sections was amplified by the polymerase chain reaction (PCR) directly using primers for p53 exons 5–8 and Ki-ras exon 1 and analyzed for mutations by denaturing gradient gel electrophoresis (DGGE) or constant denaturant gel electrophoresis (CDGE). One or both of the p53 PCR primers were located within introns to prevent amplifying the p53 processed pseudogenes. In a total of 59 hepatocellular carcinomas (HCCs), no p53 aberrations were detected, indicating that p53 mutations are not very important in rat hepatic carcinogenesis. On the other hand, Ki-ras codon 12 mutations were found at low frequency in HCCs, hyperplastic foci, and cholangiof ibroses induced by 3′-Me-DAB and by AFB! but not in the lesions induced by the Solt and Farber regimen. Although Ki-ras codon 12 mutations are generally infrequent in rat hepatic tumors, their incidence thus appears to vary depending on the carcinogen used for their induction. © 1994 Wiley-Liss, Inc.     

Mutations and expression of the p53 gene in rat bladder carcinomas and cell lines

Abnormalities of the p53 gene are frequently observed in human tumors, including urinary bladder carcinoma, suggesting that p53 plays an important role in human carcinogenesis. However, its role in rat bladder carcinogenesis is unclear. We investigated p53 gene mutations and expression in rat urinary bladder carcinogenesis in vivo and in Vitro. Fifteen urothelial cell lines, including six untransformed (nontumorigenic) ones, six transformed (tumorigenic) in vitro, and three derived from tumors induced in vivo, were examined for p53 expression by immunochemical analysis and for p53 mutations; in addition, 81 rat bladders were analyzed immunohistochemically for p53 expression, and 23 rat bladder tumors were analyzed for p53 mutations. Four cell lines had mutations in the p53 gene. Two of these were missense point mutations, and the other two were splicing mutations. On the other hand, no mutations were found in the bladder tumors induced in rats. By immunoprecipitation with PAb240, which is supposed to be specific for mutant p53, we detected mutations in three of the cell lines; PAb240 did not react with wild-type p53. However, in all cell lines and in growing populations of primary cultured bladder urothelial cells, p53 expression was detected immunohistochemically or by western blotting using PAb240 or PAb 421 monoclonal antibodies. In a high percentage of transitional cell carcinomas, wild-type p53 expression was detected by immunohistochemical analysis with PAb240. These results suggest that p53 gene mutations may not occur frequently in rat bladder carcinogenesis in vivo but may occur in vitro and that p53 overexpression detected immunohistochemically is common and may be related to cell proliferation rather than to the presence of mutations in rat bladder carcinogenesis. © 1994 Wiley-Liss, Inc.     

p53 protein accumulation and gene mutations in human glioma cell lines

Mutations in, and aberrant expression of, the p53 tumor suppressor gene were assessed in 17 cell lines derived from human malignant brain tumors (glioblastoma multiforme). Ex-ons 5 through 8 were screened by single strand conformational polymorphism analysis (SSCP), followed by direct DNA sequencing. Mutations were found in 6 of 17 glioma cell lines, i.e., at a frequency similar to that found in primary malignant gliomas. Loss of the wild type allele was observed in 4 of the mutated cell lines. Two cell lines had the same mutation (CGG → TGG; Arg → Trp) in codon 248. Five of 6 mutations were transitions, 4 of which occurred at CpG dinucleotides. In one cell line a 10-bp deletion at the intron 4/exon 5 junction was found. Five of 6 glioma cell lines contained a mutation identical to that in the respective primary tumor despite prolonged in vitro culture (140-221 passages). Thus, the acquisition of p53 mutations during culture appears to be infrequent. Two cell lines derived from heterozygous tumors maintained the wild type p53 allele during long term culture. p53 protein levels were assessed by immunofluorescence cytochemistry and immunoprecipitation followed by Western blot analysis and revealed elevated levels of the p53 protein, although to a variable extent, in all cell lines with p53 mutations. A marked p53 protein accumulation was also observed in two cell lines lacking p53 mutations in exons 5 through 8, indicating that a prolonged half life of the gene product is not solely dependent on an aberrant coding sequence. The remaining cell lines had either low levels or no detectable p53 protein; one of the latter contained a gross rearrangement of the p53 gene. Our results suggest that with respect to p53 gene status, glioma cell lines usually resemble the original tumors and may, therefore, be suitable for studying the biological changes associated with p53 mutations in glial tumors.     

p53 status in spontaneous and dimethylnitrosamine—induced renal cell tumors from rats

Rats carrying the Eker tumor–susceptibility mutation (Eker rats) are predisposed to developing renal cell carcinoma. Rats heterozygous for the Eker mutation develop spontaneous multiple bilateral renal cell tumors by the age of 1 yr. In a previous study, Eker-mutation carrier and noncarrier rats were exposed to the renal carcinogen dimethylnitrosamine (DMN), and male rats carrying the Eker mutation exhibited a 70-fold increase in the induction of renal adenomas and carcinomas when compared with noncarrier rats. In this study, spontaneous and DMN-induced rat renal cell tumors (adenomas and carcinomas) were analyzed for mutations of the p53 gene by direct sequencing of cDNA polymerase chain reaction products. There were no mutations in p53 cDNA derived from renal tumors from six untreated rats. Mutations were found in one of 15 of the DMN-induced tumors: a transition at codon 140, CCT → CTT, in a renal adenoma. Additionally, seven cell lines derived from spontaneous renal cell tumors did not contain mutations in p53. The low frequency of p53 mutations (one of 21 renal cell tumors and none of seven cell lines derived from renal cell tumors) indicates that the development of both spontaneous and carcinogen-induced renal tumors involved a non–p53-dependent pathway. As p53 is infrequently mutated in human renal cell carcinomas and in rat renal mesenchymal tumors, it is likely that a tumor suppressor gene or genes other than p53 are involved in the development of renal cancer. © 1995 Wiley-Liss Inc.     

p53 gene mutational spectra in hepatocellular carcinomas induced by 2-acetylaminofluorene and N-nitroso-2-acetylaminofluorene in rats

In this study, the mutation frequencies of the p53 gene in rat hepatocellular carcinomas (HCCs) induced by N-nitroso-2-acetylaminofluorene (NO-AAF) and 2-acetylaminofluorene (AAF) were 19.23% (20 of 104) and 31.1% (33 of 106), respectively. Four noteworthy features of the mutation spectrums of the p53 gene in HCCs induced by both NO-AAF and AAF were observed: (i) There was preferential clustering of mutations at exons 5–8 in both the NO-AAF or AAF groups, (ii) Nearly all the mutations (98%) induced by NO-AAF and AAF were point mutations, (iii) A high frequency of the p53 mutations were transition mutations, and the ratios of transition to transversion in the NO-AAF and the AAF group were 13:6 and 21:12, respectively. Almost all the mutations were G→A transitions and guanosine was the major target base. (iv) The frequency and base location of p53 mutations were significantly associated with cancer cell differentiation. In poorly differentiated HCCs (58 individual tumor samples), mutations were detected in 24 of 58 samples (41.1%) and clustered mostly in exons 7 and 8 (19 of 24 samples), whereas in well-differentiated HCCs (105 individual tumor samples), the incidence of mutations was low (one of 10 in the AAF group, 17 of 95 in the NO-AAF group), and the mutations were located in exon 5 (11 of 18). The biological significance of these different mutational spectra among p53 genes deserves further investigation. © 1995 Wiley-Liss, Inc.     

Mutational analysis of three tumor suppressor genes in two models of rat hepatocarcinogenesis.

An albumin-simian virus 40 (SV40) large T-antigen (T-Ag) transgenic model and a chemically induced model of multistage hepatocarcinogenesis were created in our laboratory to study the molecular mechanisms involved in the genesis and progression of neoplasia in the rat liver. In the study presented here, these two models of rat hepatocarcinogenesis were used to perform a comparative mutational analysis of three tumor suppressor genes involved in hepatic neoplastic growth. By using polymerase chain reaction-single strand conformation polymorphism analysis and sequencing, exons 5-8 of the p53 tumor suppressor gene and a region between nt 4325 and 4479 of the rat mannose 6-phosphate/insulin-like growth factor 2 receptor (M6p/Igf2r) coding sequence were screened. The latter is homologous to the human M6P/IGF2r coding sequence which is mutated in human hepatocellular carcinoma. A complete single strand conformation polymorphism analysis of the entire coding region of the rat adenomatous polyposis coli (Apc) gene was also performed for the first time in rat tumorigenic samples. Twenty-six chemically induced rat hepatocellular carcinomas, 21 neoplasms from the livers of SV40 T-Ag animals, and five immortalized hepatic cell lines from the transgenic rats were evaluated. None of the hepatic tumors exhibited mutations in the regions analyzed. The albumin-SV40 T-Ag transgenic cell line L-60, derived from normal hepatic tissue, had two mutations in contiguous codons of exon 5 of the p53 gene: a GGT --> GTT missense transversion in codon 183 and a silent mutation in codon 184. The transversion, which may affect the DNA binding domain of the p53 protein, probably originated during cell culture and may have been positively selected because it gave a growth advantage to the mutated cells. The studied region of the M6p/Igf2r gene was not found to be mutated in these two models of rat hepatocarcinogenesis. Although M6p/Igf2r, Apc, and p53 have been shown to be mutated in a variety of human hepatic proliferative diseases, our results indicate that aberrations in these genes may not be necessary for liver carcinogenesis in the rat.     

p53 mutations in primary human lung tumors and their metastases

In a total of 26 primary human lung tumors and 60 metastases derived from them, exons 5–8 of the p53 tumor suppressor gene were analyzed by single-strand conformation polymorphism and subsequent direct DNA sequencing of amplified DNA. Mutational inactivation of the p53 gene was identified in four of five squamous cell carcinomas, three of nine adenocarcinomas, and two of nine small-cell carcinomas, the overall incidence being 35%. Point mutations occurred at a similar incidence in exons 5–8, with a preference for G←T transversions. In seven of nine cases (78%), mutations were identical in the primary tumor and all of its metastases, indicating that in lung tumors, p53 mutations usually precede metastasis and that hematogenic and lymphogenic dissemination of tumor cells to other tissues is not associated with a selection against p53 inactivation. In one case, a kidney metastasis had the same mutation as the primary squamous cell carcinoma, whereas a liver metastasis had no mutation, indicating heterogeneity of the primary lung neoplasm and selective metastasis of mutated and nonmutated tumor cells to kidney and liver, respectively. Only in one liver metastasis was a mutation identified that was neither present in the primary lung tumor nor in a kidney metastasis, suggesting that occasionally p53 mutations occur after metastatic spread. © 1994 Wiley-Liss, Inc.     

P53 mutations may be related to tumor invasiveness of human hepatocellular-carcinoma in china

A combined polymerase chain reaction (PCR) with the enzyme HaeIII restriction analysis and DNA sequencing have been employed to study the mutations at codon 249 of p53 gene in two human hepatocellular carcinoma (HCC) cell lines and 28 surgical specimens of HCC. 14 of the 28 HCC samples (50%) had p53 point mutations at codon 249. All of point mutations at codon 249 in 10 cases sequenced are AGG to AGT transversion. p53 gene mutated more frequently in invasive HCCs than that in non-invasive HCCs. This suggested that the codon 249 was a mutational hotspot of p53 gene in human HCCs in China, and p53 mutations may be related to tumor invasiveness of human HCC.     

Infrequent alterations of the p16INK4A gene in liver cancer

We examined the genomic status of the p16^INK4A (inhibitor of cyclin-dependent kinase 4 A) and cyclin-dependent kinase 4 (CDK4) genes in 62 human hepatocellular carcinomas (HCCs), 5 cholangiocellular carcinomas and 6 cell lines derived from human liver cancers. Although no samples showed the homozygous deletion of the p16^INK4A gene, we detected intragenic mutations of the p16^INK4A gene in 3 HCCs and one HCC cell line, which led to an amino-acid substitution or a frameshift. In 2 HCC samples with mis-sense mutations of the p16^INK4A gene, loss of heterozygosity on 9p22 was also detected, suggesting that the loss of function of p16 was induced during hepatocarcinogenesis. On the other hand, amplification or rearrangement of the CDK4 gene was not detected in any samples examined in this study. These results indicated that the mutations or deletions of the p16^INK4A gene are not frequent, but may play a role in a sub-set of human HCC. © 1996 Wiley-Liss, Inc.     

Murine p53 intron sequences 5-8 and their use in polymerase chain reaction/direct sequencing analysis of p53 mutations in CD-1 mouse liver and lung tumors.

Inactivating point mutations and small deletions in the p53 tumor suppressor gene have been found in human liver and lung tumor--derived cell lines and tumors. However, little evidence has been reported concerning inactivation or mutation of the p53 gene in mouse primary tumors. To examine CD-1 mouse liver and lung tumors for mutations in the p53 gene, we first sequenced p53 introns 5-8 so that polymerase chain reaction amplification and sequencing primers located within the introns could be prepared. Use of these primers prevented amplification of the mouse p53 pseudogene and allowed sequencing of exons 5-8 in their entirety as well as their intron-exon junctions. DNA isolated from CD-1 mouse tumors was amplified and directly sequenced using nested primers. Nine spontaneous hepatocellular carcinomas (HCCs) and 34 chemically induced HCCs (induced by single intraperitoneal injections of N-nitrosodiethylamine [DEN] [8 HCCs], 7,12-dimethylbenz[a]anthracene [DMBA] [8 HCCs], 4-aminoazobenzene [8 HCCs], and N-OH-2-acetylaminofluorene [10 HCCs]) were examined for mutations in exons 5-8 of the p53 gene. In addition, 12 spontaneous, 10 DMBA-induced, and 13 DEN-induced lung adenocarcinomas or adenomas were analyzed for mutations. No mutations were found in any of the tumors examined. However, a mutation was demonstrated at codon 135 in the positive-control plasmid LTRp53cG(val). The results of this study suggest that inactivation of p53 is unlikely to play a major role in murine lung or liver carcinogenesis. However, inactivation of p53 may occur at a very low frequency, or it may occur as a late event and therefore be present in only a very small number of the tumor cells, rendering it undetectable by this method. Lastly, although few p53-inactivating mutations are found outside of exons 5-8 in human tumors, it is possible that these murine tumors contained mutations outside of this region and were therefore missed by our approach.     

Frameshift mutation in codon 176 of the p53 gene in rat esophageal epithelial cells transformed by benzo[a]pyrene dihydrodiol

Mutations in the p53 tumor suppressor gene have been associated with exposure to environmental chemical carcinogens. Cultured rat esophageal epithelial cells were transformed in vitro by treatment with benzo[a]pyrene dihydrodiol (BP-DHD). A BP-DHD-transformed cell line and control cell lines were analyzed for mutations in the p53 gene and in the Ha-ras gene by single-strand conformation polymorphism analysis of polymerase chain reaction-amplified products and direct DNA sequencing. The deletion of one cytosine in codons 174–176 (TGCCCCCAC → TGCCCCAC) of the p53 gene was found only in the BP-DHD-transformed cell line. The BP-DHD-transformed cells were highly invasive and tumorigenic when transplanted into syngeneic rats, whereas control lines either were nontumorigenic or formed epithelial cysts. BP-DHD-transformed cells and control lines were negative for mutations in the Ha-ras gene. Our results suggest that the tumorigenic potential of the BP-DHD-transformed cell line is associated with a frameshift mutation in codon 176 of the p53 gene but not with mutations in the Ha-ras gene. The G/C-rich codons 174–176 in the ratp53 gene may be specific targets for BP-DHD. ©1995 Wiley-Liss, Inc.     

Screening the p53 status of human cell lines using a yeast functional assay

We have screened the p53 status of 156 human cell lines, including 142 tumor cell lines from 27 different tumor types and 14 cell lines from normal tissues by using functional analysis of separated alleles in yeast. This assay enables us to score wild-type p53 expression on the basis of the ability of expressed p53 to transactivate the reporter gene HIS3 via the p53-responsive GAL1 promoter in Saccharomyces cerevisiae. Of 142 tumor cell lines, at least 104 lines (73.2%) were found to express the mutated p53 gene: 94 lines (66.2%) were mutated in both alleles, three lines (2.1%) were heterozygous, and no p53 cDNA was amplified from seven lines (4.9%). Of the 14 cell lines originating from normal tissues, all the transformed or immortalized cell lines expressed mutant p53 only. Yeast cells expressing mutant p53 derived from 94 cell lines were analyzed for temperature-sensitive growth. p53 cDNA from eight cell lines showed p53-dependent temperature-sensitive growth, growing at 30°C but not at 37°C. Four temperature-sensitive p53 mutations were isolated: CAT→CGT at codon 214 (H214R), TAC→TGC at codon 234 (Y234C), GTG→ATG at codon 272 (V272M), and GAG→AAG (E285K). Functionally wild-type p53 was detected in 38 tumor cell lines (26.8%) and all of the diploid fibroblasts at early and late population doubling levels. These results strongly support the previous findings that p53 inactivation is one of the most frequent genetic events that occurs during carcinogenesis and immortalization. Mol. Carcinog. 19:243–253, 1997. © 1997 Wiley-Liss, Inc.     

Mutation,loss of heterozygosity,and recombination of the p53 gene in mouse forestomach tumors induced by 2-amino-3,4-dimethylimidazo[4,5-f]quinoline

2-Amino-3,4-dimethylimidazo[4,5-f]quinoline (MelQ), a food mutagen, induces forestomach tumors in CDF₁ mice. We established a polymerase chain reaction (PCR)–single-strand conformation polymorphism (SSCP) analysis system to detect mutations in the mouse p53 gene exons 2–10, which encompass all five regions conserved among species, and a system to examine loss of heterozygosity (LOH) that uses newly identified polymorphisms between BALB/c and DBA mice, the parental strains of CDF₁ mice. Four original forestomach tumors (one papilloma, two carcinomas, and one lymph-node metastasis) and four cell lines derived from four independent forestomach tumors were examined with the PCR-SSCP system and by polymorphism analysis. Of the four original tumors, the papilloma had a G→A transition at the second position of codon 171, and one carcinoma had a G→T transversion at the second position of condon 113 with loss of the wild-type allele, whereas the other two carcinomas had no detectable mutations. Of the four cell lines, two had a base substitution and LOH, and the other two had double mutations (a base substitution and a deletion). By amplification of the double mutations in a fragment, the two cell lines were shown to have four kinds of alleles, indicating induction of recombination within the p53 gene. Our results show that our PCR-SSCP analysis system is efficient for detecting p53 mutations in mouse genomic DNA and that alteration of the p53 gene plays a significant role in MelQ-induced mouse forestomach carcinogenesis. © 1995 Wiley-Liss Inc.     

p53 Abnormalities in hepatocellular carcinoma from United States patients: analysis of all 11 exons

Mutations of the tumor suppressor gene p53 have been found inhepatocellular carcinomas (HCCs) from many countries where HCCis common, but p53 mutations detected by direct sequencing inHCCs from the United States (where HCC is uncommon) have beenreported only rarely, p53 Exons 1 to 11 were analyzed by polymerasechain reaction, single-strand conformation polymorphism analysis,and direct sequencing in HCCs from 12 patients from the UnitedStates and in adjacent non-tumorous liver from 10 of them. Abnormalitiesof the p53 gene were found in five of the 12 tumors, includingmutations in exon 4 (one patient), exon 5 (one patient) andexon 8 (three patients). In all 12 tumors, a normal conformationof exon 7 was found; in five of these HCCs the absence of mutationswas confirmed by direct sequencing. Thus, the mutations of p53codon 249 that have frequently been found in HCCs from endemicareas apparently do not play a role in most HCCs in the UnitedStates. Since the mutations in codon 249 are thought to be dueto aflatoxin ingestion, the data suggest that factors otherthan aflatoxin may be responsible for the p53 abnormalitiesin the patients from the USA.     

p53 mutations as a possible predictor of response to chemotherapy in metastatic colorectal carcinomas

Although intrahepatic infusion therapy with 5-flourouracil for unresectable colorectal liver metastases may lead to improved overall survival for some patients, it is not clear why a response is not observed in others. Gene alterations in oncogenes or tumor-suppressor genes are critical events in tumor formation, and some of them could play a role in the process of drug resistance. The tumor-suppressor gene p53, which is known to trigger cell arrest or apoptosis in response to DNA damage, is found to be mutated in a wide range of human tumors. The aim of this work is to establish whether a relationship is found between p53 mutations and survival in patients undergoing adjuvant chemotheraphy for advanced Dukes' D colorectal cancers. Seventeen tumors from patients treated with 5-fluorouracil regimen via intrahepatic infusion for unresectable colorectal hepatic metastasis were considered. p53 mutations from tumor DNA were detected, after amplification by PCR of exons 5 to 8, by non-radioactive single-strand conformation polymorphism and direct DNA sequencing. Patients with mutated p53 colorectal tumors had short survival, whereas prolonged survival was associated with the presence of wild-type p53 (p = 0.019). Our data suggest that mutated p53 colorectal tumors had a weak response, or even no response, to chemotherapeutic treatment. Routine assessment of p53 status would be helpful in selecting patients with only wild-type p53 gene who have a predictably better response to chemotherapy. © 1996 Wiley-Liss, Inc.     

p21/WAF1, p53 and PCNA expression and p53 mutation status in hepatocellular carcinoma

The cyclin-dependent kinase inhibitor p21/WAF1 is regulated by p53-dependent and p53-independent pathways. In addition, p21/WAF1 binds with proliferating cell nuclear antigen (PCNA) and inhibits the action of PCNA. To investigate the possible role of p21/WAF1 in human hepatocellular carcinomas (HCCs), we examined the expression of p21/WAF1 and its relation with PCNA and p53 expression in 97 surgically resected HCCs by immunohistochemistry and with the mutation status of p53 in 26 HCCs. p53 mutation status was examined by direct DNA sequencing using 3 sets of primers covering exons 5–9. Six of the 26 tumors showed p53 point mutations and only 33% of these HCCs demonstrated p21/WAF1 expression. In contrast, 75% of HCCs without p53 mutations showed p21/WAF1 expression. Of all 97 HCCs, p21/WAF1 expression was significantly higher in the tumors than in corresponding non-tumorous liver. When the tumors were stratified into 2 groups by the median tumor p21/WAF1 score, those with higher expression were found to have a lower incidence of multiple tumor nodules (p = 0.008) and tumor microsatellite formation (p = 0.050). The tumor p21/WAF1 score was positively associated with tumor PCNA expression (p = 0.036) but not with tumor p53 expression. Thus, in HCC, expression of p21/WAF1 is in part dependent on p53 status, but a p53-independent pathway also plays a significant role in the regulation of p21/WAF1 expression. High p21/WAF1 expression is significantly associated with solitary tumor nodules and, to a lesser extent, tumor microsatellites but may not be enough to suppress tumor progression. Int. J. Cancer (Pred. Oncol.) 79:424–428, 1998. © 1998 Wiley-Liss, Inc.     

Clonality and stability of the p53 gene in human astrocytic tumor cells: Quantitative analysis of p53 gene mutations by yeast functional assay

Mutation of the p53 gene is found in about one third of astrocytic brain tumors, and expansion of tumor cell clones containing mutant p53 has been implicated in astrocytic tumor progression. However, admixture of normal cells in astrocytic tumor specimens limits the power of traditional studies of tumor cell clonality. To address this problem we have employed a yeast p53 functional assay that scores the content of mutant p53 alleles in tumors and cell lines quantitatively. We have analyzed 17 cases where matching tumor material and derived cell lines were available. The yeast assay gave >20% red (i.e., mutant p53-containing) yeast colonies in 7 out of 17 cases. One case had no mutations in the primary tumor but gave 76% red colonies in a recurrence, clearly demonstrating tumor overgrowth by a mutant clone. During early passages of cultured tumor cells, mutant p53 content increased rapidly with passage due to outgrowth of mutant clones from a heterogeneous starting population. In addition, de novo p53 mutations appeared during culture in 2 cases. This indicates that there is stronger selective pressure for mutation during the establishment of cell lines in vitro than during tumor growth in vivo. Our results demonstrate the utility of the p53 functional assay for studies of clonality and support the hypothesis of clonal progression of brain tumors in vivo. © 1996 Wiley-Liss, Inc.     

Establishment and characterization of human gastric carcinoma cell lines

We report 8 newly established gastric-carcinoma cell lines (SNU-216, 484, 520, 601, 620, 638, 668, 719) from Korean patients. Morphologic study was carried out using light and electron microscopes. CEA, αFP, and CA 19-9 and TPA in supernatant and in cell lysate were measured by radioimmunoassay. p53 and c-Ki - ras gene mutations were screened and confirmed by sequencing. The cell lines, derived from tumors with moderate differentiation, grew as a diffuse monolayer, and those from tumors with poor differentiation and minimal desmoplasia grew exclusively as non-adherent. Out of the 8 gastric-cancer cell lines, 5 had detectable levels of CEA both in supernatant and in cell lysate; there was no expression or secretion of αFP in these cells; 4 cell lines showed high levels of CA 19-9 in cell pellets. All cell lines except SNU-484 had high concentrations of TPA both in cell lysate and in supernatants. p53 mutation was found in 6 cell lines (75%): 2 (SNU-216 and SNU-668) had mutations in exon 6, and other 3 in exon 8. The c-Ki - ras mutation was found in 2 cell lines (25%), SNU-601 and SNU-668. The former showed GGT-to-GAT transition mutation at codon 12, while the latter showed CAA-to-AAA transversion mutation at codon 61. DNA profiles using restriction endonuclease Hinfl and polymorphic DNA probes ChdTC-15 and ChdTC-114 showed different unique patterns; which suggests that these cell lines are unique and not cross-contaminated. We believe that the newly characterized gastric-cancer cell lines presented in this paper will provide a useful in vitro model for studies related to human gastric cancer. Int. J. Cancer, 70:0–0, 1997. © 1997 Wiley-Liss, Inc.     

Extended lifespan and immortalization of human fibroblasts induced by x-ray irradiation

The induction of immortalization of human fibroblasts by carcinogens is a very rare process. Hybrids between immortal cells and normal fibroblasts senesce, indicating that immortal cells must lose one or more senescence genes for immortalization. To examine the possible involvement of multiple gene alterations in extended lifespan or immortalization of normal human fibroblasts, normal human fibroblasts (WHE-7 cells) and skin fibroblasts (MDAH 087 cells) derived from a Li-Fraumeni syndrome patient with a mutated p53 allele were periodically irradiated with x rays. All six unirradiated control MDAH 087 cell cultures ceased growing by 37 population doublings (PD) and senesced. In contrast, one of six MDAH 087 cultures irradiated one to three times with x rays (2 or 4 Gy at 2 Gy/min) grew continuously for over 450 PD, indicating that the cells were immortal. All 12 WHE-7 cell cultures that were irradiated under the same conditions and all 20 uniradiated control WHE-7 cultures did not become immortal. Single-stranded DNA conformation analysis and DNA sequencing revealed that no additional mutations were induced by x-ray irradiation in exons 2–10 of the p53 gene of the immortal cells (LCS-4X2 cells) and that loss of the wild-type p53 gene was necessary but not sufficient for immortalization. Karyotypic analysis and chromosome painting analysis demonstrated that a high percentage (more than 98%) of LCS-4×2 cells had lost chromosome 6. Irradiation of WHE-7 cells nine times with x rays (2 Gy at 2 Gy/min) extended the cells' lifespans but did not immortalize them. These cells (X9 cells) exhibited a nonrandom karyotypic alteration, monosomy 6, that was confirmed by loss of heterocygosity for a polymorphic dinucleotide repeat sequence on chromosome 6. DNA analysis showed that X9 cell had no mutations in exons 2–10 of the p53 gene. DNA fingerprint analysis with a multi-locus probe detected DNA rearrangements in LCS-4×2 cells and X9 cells, indicating that both cells could have mutations at a gene or genes other than the p53 gene. The results are consistent with our previous findings that cells with a mutation in one gene involved in cellular senescence (i.e., the p53 gene in Li-Fraumeni fibroblasts) are prone to immortalization. Furthermore, we conclude that immortalization of normal human fibroblasts is a multistep process involving loss or inactivation of multiple genes, such as p53 and a gene on chromosome 6. Loss of a gene on chromosome 6 without p53 alterations extends cellular lifespan without immortalizing the cells. Mol. Carcinog. 18:7–18, 1997. © 1997 Wiley-Liss, Inc.