Characterization of benzo[a]pyrene-initiated mouse skin papillomas for Ha-ras mutations and protein kinase C levels

The frequency and spectrum of Ha-ras mutations in benzo[a]pyrene (B[a]P)-initiated/12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted CD-1 mouse skin papillomas were characterized by amplifying high molecular weight papilloma DNA using the polymerase chain reaction (PCR) followed by direct DNA sequencing. Analysis of 10 individual B[a]P-initiated early emergence papillomas indicated that 90% contained a Ha-ras mutation. Twenty percent of these papillomas contained a GGA-->GTA transversion in the 12th codon, 50% contained a GGC-->GTC transversion in the 13th codon and 20% contained a CAA-->CTA transversion in the 61st codon. A characteristic of 7,12-dimethylbenz[a]anthracene (DMBA)-initiated papillomas, which contain an A-->T mutation in the 61st codon of Ha-ras, is that they exhibit a constitutive decrease in both protein kinase C (PKC) activity and PKC alpha and beta 2 isozyme levels when compared to epidermis. In the present study we found that total PKC activity, as well as PKC alpha and beta 2 isoforms, were markedly decreased in B[a]P-initiated early emergence papillomas and that this decrease was also accompanied by an altered subcellular distribution of PKC activity. The particulate/cytosolic (P/C) ratio of PKC activity in the epidermis was 0.39, whereas the P/C ratio in the papillomas was 0.77. These results demonstrate that B[a]P-initiated/TPA-promoted papillomas exhibit a high incidence of specific ras mutations and that PKC levels are constitutively decreased in these papillomas, indicating that an activated ras gene is associated with and may contribute to the observed decrease in PKC levels.     

XPA-deficiency in hairless mice causes a shift in skin tumor types and mutational target genes after exposure to low doses of U.V.B

Xeroderma pigmentosum (XP) patients with a defect in the nucleotide excision repair gene XPA, develop tumors with a high frequency on sun-exposed areas of the skin. Here we describe that hairless XPA-deficient mice also develop skin tumors with a short latency time and a 100% prevalence after daily exposure to low doses of U.V.B. Surprisingly and in contrast to U.V.B.-exposed repair proficient hairless mice who mainly develop squamous cell carcinomas, the XPA-deficient mice developed papillomas with a high frequency (31%) at a U.V. dose of 32 J/m2 daily. At the highest daily dose of 80 J/m2 mainly squamous cell carcinomas (56%) and only 10% of papillomas were found in XPA-deficient hairless mice. p53 gene mutations were examined in exons 5, 7 and 8 and were detected in only 3 out of 37 of these skin tumors, whereas in tumors of control U.V.B.-irradiated wild type littermates this frequency was higher (45%) and more in line with our previous data. Strikingly, a high incidence of activating ras gene mutations were observed in U.V.B.-induced papillomas (in 11 out of 14 tumors analysed). In only two out of 14 squamous cell carcinomas we found similar ras gene mutations. The observed shift from squamous cell carcinomas in wild type hairless mice to papillomas in XPA-deficient hairless mice, and a corresponding shift in mutated cancer genes in these tumors, provide new clues on the pathogenesis of chemically- versus U.V.B.-induced skin carcinogenesis.     

p53 mutations in hepatocellular carcinomas induced by a choline-devoid diet in male Fischer 344 rats

Analyses were performed on livers and hepatocellular carcinomas from male Fischer 344 rats fed a choline-devoid diet, to assess whether they carried alterations of the p53 tumor suppressor gene. The analyses consisted of immunoperoxidase staining of tissue sections with monoclonal antibodies to p53, Western blotting and cDNA sequencing. Immunostaining revealed the presence of mutant p53 proteins in 22/27 tumors analyzed and immunoblotting in 18/20. Immunochemical evidence was obtained that occurrence of the mutations precedes tumor development. cDNA sequencing was performed on 11 hepatocellular carcinomas that expressed mutant p53 gene proteins. Seven were found to contain point mutations within the 120-290 codon region of the gene, and one a microdeletion in the same region. No mutational hot spot was observed. It is concluded that mutations within the p53 gene, along with a c-myc gene amplification previously detected in these tumors, most likely contribute to the neoplastic transformation of liver cells in this nutritional model of hepatocarcinogenesis. The results are discussed also in view of recent literature on the presence of p53 mutations in human hepatocellular carcinomas.     

K-ras and p53 mutations are an independent unfavourable prognostic indicator in patients with non-small-cell lung cancer

We examined 159 consecutive cases of non-small-cell lung cancer (NSCLC) for a mutation at codon 12 of the K-ras gene and for a mutation of the p53 gene occurring in exons 5-8. Eleven (6.9%) had mutations of the K-ras (ras+) and 57 (35.8%) had mutations of the p53 (p53+). There were 95 cases (59.7%) with ras- p53-, seven cases (4.4%) with ras+/p53-, 53 cases (33.3%) with ras-/p53+ and four cases (2.5%) with ras+/p53+. The ras+ group had a worse prognosis than the ras group in all cases and in 107 early-stage cases (stage I-II, P<0.05). The p53+ group had a worse prognosis in 107 early-stage cases (P<0.01), but there was no statistically significant difference when 52 advanced-stage cases (stage III-IV) or all patients were considered. Both ras and p53 mutations were unfavourable prognostic factors in 94 cases with adenocarcinoma, but there was no statistical significance in 57 cases with squamous cell carcinoma. According to Cox's model, the pathological stage, ras mutation and p53 mutation were found to be independent prognostic factors. Our results suggest that ras and p53 mutations were independent unfavourable prognostic markers especially in the early stage of NSCLC or in adenocarcinoma.     

High mutation frequency in ras genes of skin tumors isolated from DNA repair deficient xeroderma pigmentosum patients

Xeroderma pigmentosum (XP) patients are clinically characterized by a very high incidence of skin cancers on exposed skin, at an early age. XP cells in vitro are strongly deficient in excision-repair and highly mutagenized by UV light. We were, therefore, interested in measuring mutation frequency and in determining mutation spectra in patients' tumors exposed to UV lesions. We chose to look at oncogene activation in skin tumors with the idea that more mutations, particularly of the ras gene family, would be found in XP tumors where lesions remain unrepaired compared to normal individuals. Our results clearly show that more than a 2-fold significantly higher mutation frequency (50%) of the ras genes was found in XP in contrast to control tumors (22%). The majority of the mutations were found at codon 12 of all three ras genes with a preponderance for N-ras in XP samples. The mutation spectra indicate that all mutations found were located opposite pyrimidine-pyrimidine sequences which represent a hot spot for UV-induced DNA lesions. Most of the mutations were of the type expected from studies performed in vitro with model systems. This high mutation frequency in XP was accompanied by a very high level of Ha-ras and c-myc gene amplification and rearrangement. All these data are consistent with a fundamental role of unrepaired UV-induced DNA lesions as an initiating event in human skin tumors on exposed parts of the body.     

Telomerase is not an epidermal stem cell marker and is downregulated by calcium

An understanding of the basic mechanisms responsible for the pathogenesis of liver neoplasms is needed in order to develop better therapeutic strategies. The present study utilized a pharmacogenetic mouse model to assess the role of cytochrome P4501A1 (Cyp1a1) in modulating genetic damage to oncogenic and tumor suppressor loci following in utero exposure to the polycyclic aromatic hydrocarbon, 3-methylcholanthrene (MC). Analysis of the Ha-ras, Ki-ras, INK4a and p53 genes was carried out with lysates from paraffin-embedded liver tissue from transplacentally-treated mice. The lysates were subjected to DNA amplification by the PCR technique followed by allele-specific oligonucleotide hybridization screening and SSCP analysis. All of the 26 neoplasms screened (23 hepatocellular carcinomas, two hepatocellular adenomas and one sarcoma) exhibited a GGC-->CGC (GLY13-->ARG13) transversion at the Ki-ras gene locus. None of the tumors had Ki-ras mutations at codon 12 of exon 1. Approximately 12% (3/26) of the liver tumors exhibited point mutations in exon 1 of the INK4a gene, with each of the three tumors exhibiting two point mutations. Analysis of exon 2 of the INK4a gene showed the presence of a CCG-->CTG (PRO73-->LEU73) transition in two of the 26 neoplasms. No mutations were found in exons 1 or 2 of the Ha-ras gene, or in exons 5-8 of the p53 gene. Analysis of tumor RNAs showed overexpression of Ha-ras, cip1 and c-jun in approximately 38% of the liver tumor samples. The results of this study suggest that mutagenic damage to oncogenes and tumor suppressor genes may be critical factors in mediating transplacentally-induced liver tumorigenesis. The fact that Ki-ras mutations were found in all of the tumors suggests that mutation at this gene locus may be an early event in liver tumor pathogenesis, while mutation in tumor suppressor genes may occur later during tumor progression. These combined results are consistent with the pathogenesis of cancer in humans.     

Ha-ras mutations in N-nitrosomorpholine-induced lesions and inhibition of hepatocarcinogenesis by antisense sequences in rat liver

To evaluate the application of Ha-ras mRNA antisense oligonucleotide therapy for liver tumors, we examined the frequency and types of mutation in codon 61 of the Ha-ras oncogene in preneoplastic lesions and hepatocellular carcinomas induced by N-nitrosomorpholine (NNM) in rats. Thirty-seven percent of preneoplastic lesions and 50% of hepatocellular carcinomas contained mutations, mostly CAA-CTA and CAA-AAA transversions. We also investigated the effects on NNM-induced lesions of an antisense oligonucleotide directed against a point mutation (CAA-CTA) in codon 61 of Ha-ras mRNA. In this experiment, Sprague-Dawley rats were given free access to water containing NNM for 8 weeks and received twice-weekly i.p. injections of a mutated Ha-ras antisense oligonucleotide with a 5' phosphorothioate linkage or a sense oligonucleotide in oligonucleotide-liposome complexes. At week 16, rats that had received the mutated Ha-ras antisense oligonucleotides had significantly fewer and smaller preneoplastic lesions positive for glutathione-S-transferase, placental type, and had smaller hepatocellular carcinomas than rats that had received the sense oligonucleotide. Mean cellular fluorescence in the liver was found to increase with higher doses of mutated, fluorescein-isothiocyanate-labeled antisense or sense oligonucleotides. Moreover, mutated Ha-ras antisense oligonucleotide decreased the expression of mutated Ha-ras mRNA (CAA-CTA). Our findings indicate that mutated Ha-ras antisense oligonucleotide significantly inhibits hepatocarcinogenesis in rats and could be an effective therapy against liver tumors.     

Mutations of K-ras oncogene and absence of H-ras mutations in squamous cell carcinomas of the lung

Mutations of the K-ras gene have been implicated in the pathogenesis of human lung adenocarcinomas. In most studies published so far, squamous cell lung cancers harbored ras mutations only exceptionally or no mutations were detected at all. We have examined 141 lung tumor DNA samples for mutations in codons 12, 13, and 61 of K-ras and H-ras oncogenes. A large panel of 118 squamous cell carcinomas was included in the study. For K-ras codon 12, we used a sensitive two-step PCR-restriction fragment length polymorphism method which detects <1% of mutated DNA in the sample. K-ras mutations were found in 17 tumors (12%; 14 in codon 12 and 3 in codon 13). Among 19 adenocarcinomas, mutation was revealed in 7 samples (37%). Of these, one sample harbored two point mutations in codon 12. Nine mutational events were found in squamous cell carcinomas (8%, one adenosquamous carcinoma included, all in codon 12). Of four large cell carcinomas, one contained a mutation. Mutant-enriched PCR products harboring mutations were directly sequenced. Fifteen mutational events were G-->T transversions or G-->A transitions, one was a G-->C transition, and one sample revealed a frameshift deletion of one G from codon 12. Similar mutational spectrum was found in both squamous cell carcinomas and adenocarcinomas, suggesting similar carcinogenic pathways in both histological types of the tumor. The presence of mutations did not correlate with the stage of the disease. Moreover, we analyzed all samples for mutations in codons 12, 13, and 61 of the H-ras gene. We found only one mutation in codon 12. Thus, H-ras mutations apparently play an inferior role in lung carcinogenesis. We conclude that mutations of the K-ras oncogene can play a role in the development of not only lung adenocarcinomas but also of a subset (about 8%) of squamous cell carcinomas.     

Gastric adenocarcinoma producing neuron-specific enolase

Analysis of H-, K- and N-ras genes for point mutations by PCR-SSCP and direct sequencing of 46 oral SCCs that were previously analyzed for p53 mutations revealed that 9 (20%) had point mutations in either the H-ras or the N-ras. A novel mutation at codon 59 (GCC-ACC) of H-ras thus far reported only in v-H-ras of Harvey murine sarcoma virus was observed in a tumor of the cheek. Majority (8/9) of these mutations were observed in H-ras, one in N-ras and none in K-ras. This study indicated that the ras gene mutation was relatively high in oral cancers associated with tobacco chewing and the ras and p53 mutational events seem to be independent and mutually exclusive.     

Missed opportunities for prevention: smoking cessation counseling and the competing demands of practice

Activating mutations in and expression of the Ha-ras gene were examined in benign and malignant female Sprague-Dawley rat mammary gland tumors induced by the heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and promoted by a diet high in polyunsaturated fat. Ha-ras mutations were detected in codons 12 and 13 by selective polymerase chain reaction amplification of mutated sequences and nucleotide sequencing. The percentage of Ha-ras mutations in carcinomas from PhIP-treated rats was significantly higher in rats on a low-fat diet than in rats on a high-fat diet (82% (nine of 11) vs 26% (seven of 27), respectively, P < 0.01). In addition, whereas 56% of the carcinomas with Ha-ras mutations from rats on a low-fat diet carried double Ha-ras mutations, none of the carcinomas from rats on a high-fat diet had double mutations. Ha-ras mutations were also detected in benign tumors (largely adenomas) induced by PhIP in rats on different diets; two of eight and three of four benign tumors examined from rats on low-fat and high-fat diets, respectively, had Ha-ras mutations, suggesting that activating Ha-ras mutations alone are not sufficient for PhIP-induced tumors to become malignant. No differences were observed in the level of Ha-ras mRNA expression in the different groups. In our animal model, a high-fat diet increased the incidence and percentage of malignant PhIP-induced mammary gland tumors yet decreased the percentage of carcinomas showing Ha-ras mutations. Thus, the complement of genetic alterations associated with PhIP-induced mammary gland carcinogenesis is probably altered by the level of dietary fat.     

Detection and clinical correlations of ras gene mutations in human ovarian tumors

In epithelial ovarian neoplasms K-ras codon 12 gene mutations show a wide variation fluctuating between 4-39% in invasive carcinomas and 20-48% in borderline malignant tumors. In this study, we showed the pattern of point mutations in codon 12 of the K-ras, H-ras and N-ras genes, using polymerase chain reaction restriction fragment length polymorphism analysis in 74 tissue specimens of Greek patients with epithelial ovarian tumors. K-ras and H-ras gene mutations were detected in 11/48 (23%) and 3/48 (6%) cases with primary invasive ovarian carcinomas, respectively, while N-ras gene mutations were not found. No mutation of K-, H- and N-ras genes was detected in 23 ovarian cystadenomas. In 1 out of 3 borderline ovarian tumors (33%) we found an H-ras gene mutation. The prevalence of mutations in K-ras gene was 1/8 (13%) in mucinous, 7/29 (24%) in serous, 1/3 (33%) in endometrioid and 2/8 (25%) in clear-cell adenocarcinomas and in H-ras gene 1/8 (13%) in mucinous and 2/29 (7%) in serous adenocarcinomas. Analysis of the results revealed no significant correlation between ras gene mutations and clinicopathological parameters or clinical outcome of this primary invasive ovarian carcinoma population. Our present data suggest that ras gene mutations in invasive ovarian carcinomas occur in 29% of Greek patients and are not associated with the differentiation of the epithelial cells or the response of patients to adjuvant platinum-based chemotherapy.     

Altered WAF1 genes do not play a role in abnormal cell cycle regulation in breast cancers lacking p53 mutations

Seventy-five to 80% of breast cancers are negative for p53 gene mutations. We have investigated the possibility that altered WAF1 genes provide an alternative mode of cell cycle disruption in these tumors. DNA from a total of 85 primary breast tumors and cell lines from both the United States and Australia were examined for WAF1 and p53 mutations. With the exception of one primary tumor containing the polymorphic codon 31 (AGC-->AGA), no missense mutations in the WAF1 gene were found in 33 primary tumors or in the 19 cell lines from the United States. By contrast, 2 of 33 tumors from Australia contained tumor-specific missense mutations in the WAF1 gene, while an additional six cases contained the AGC-->AGA polymorphic 31st codon in the WAF1 gene. The p53 mutation frequency in the Australian cohort (18%) was found to be similar to that reported by us (Glebov et al., Cancer Res., 54: 3703-3709, 1994; Runnebaum et al., Proc. Natl. Acad. Sci. USA, 88: 10657-10661, 1991) in the tumors of United States patients (13%) with sporadic breast cancer. Thus, mutations in the WAF1 gene are rare in tumors with or without p53 mutations, suggesting that except in a minor population of breast cancer patients of Caucasian origin, cell cycle dysregulation by mutated p53 or WAF1 genes may not contribute to breast tumor initiation or progression.     

Alterations of p53 gene and Ha-ras gene are independent events in solar keratosis and squamous cell carcinoma

In order to clarity the multiple-step progression from solar keratosis to squamous cell carcinoma, aberrations of the p53 gene (exons 2-11) and ras genes (exons 1 and 2) in solar keratosis and squamous cell carcinoma were investigated by polymerase chain reaction and single-strand conformation polymorphism analysis. In a series of Japanese patients, eight of 27 (30%) samples of solar keratosis and three of six (50%) samples of squamous cell carcinoma showed structural abnormalities in the p53 gene. Only one solar keratosis (4%) showed a point mutation in the Ha-ras gene but not in the p53 gene. Among these cases, no mutation of ras genes could be detected in squamous cell carcinoma. Simultaneous mutation of ras genes and the p53 gene was not detected in any cases of either solar keratosis or squamous cell carcinoma. It is concluded that aberrations of the p53 gene and ras genes are induced through independent processes of ultraviolet irradiation in the course of carcinogenic change from solar keratosis to squamous cell carcinoma.     

Combined effects of insulin treatment and adipose tissue-specific agouti expression on the development of obesity

BACKGROUND & AIMS: Infantile and childhood liver tumors have been found in 0.42% of individuals with a germline mutation in the adenomatous polyposis coli (APC) gene. This study analyzed a hepatocellular adenoma of a 2-year-old child at risk for familial adenomatous polyposis to identify genetic alterations in hepatic tumors initiated by APC germline mutations. METHODS: Mutation screening was performed for the APC gene (protein truncation test and DNA sequence analysis), p53 gene (complementary DNA cloning and sequencing), and members of the Ras gene family (complementary DNA sequence analysis). RESULTS: Both the mother and child had a germinal CGA-->TGA transition at codon 1451 leading to an Arg1451Ter stop mutation in the APC gene. Loss of the wild-type APC allele as a second hit revealed hemizygosity of the inherited mutation in the tumor. Furthermore, a CGC-->CAC transition in the p53 gene of the adenoma resulted in an Arg-->His missense mutation in codon 175. No loss of heterozygosity was detected at the p53 locus. Ras gene mutations were not found. CONCLUSIONS: Biallelic inactivation of APC gene and p53 mutation are early events in hepatocellular tumorigenesis. Additional reports will confirm whether inherited APC gene mutations between codon 1444 and 1578 increase the risk for hepatic tumors.     

Chronic ultraviolet exposure-induced p53 gene alterations in Sencar mouse skin carcinogenesis model

Alterations of the tumor suppresser gene p53 have been found in ultraviolet radiation (UVR) related human skin cancers and in UVR-induced murine skin tumors. However, links between p53 gene alterations and the stages of carcinogenesis induced by UVR have not been clearly defined. We established a chronic UVR exposure-induced Sencar mouse skin carcinogenesis model to determine the frequency of p53 gene alterations in different stages of carcinogenesis, including UV-exposed skin, papillomas, squamous-cell carcinomas (SCCs), and malignant spindle-cell tumors (SCTs). A high incidence of SCCs and SCTs were found in this model. Positive p53 nuclear staining was found in 10/37 (27%) of SCCs and 12/24 (50%) of SCTs, but was not detected in normal skin or papillomas. DNA was isolated from 40 paraffin-embedded normal skin, UV-exposed skin, and tumor sections. The p53 gene (exons 5 and 6) was amplified from the sections by using nested polymerase chain reaction (PCR). Subsequent single-strand conformation polymorphism (SSCP) assay and sequencing analysis revealed one point mutation in exon 6 (coden 193, C-->A transition) from a UV-exposed skin sample, and seven point mutations in exon 5 (codens 146, 158, 150, 165, and 161, three C-->T, two C-->A, one C-->G, and one A-->T transition, respectively) from four SCTs, two SCCs and one UV-exposed skin sample. These experimental results demonstrate that alterations in the p53 gene are frequent events in chronic UV exposure-induced SCCs and later stage SCTs in Sencar mouse skin.     

Clonal analysis of bilateral breast cancer

Forty-nine pairs of bilateral breast tumors (41 synchronous and 8 asynchronous cases) were examined for X-chromosome inactivation status and p53 mutations to address the issue of their clonality. Among 12 cases that were informative for the trinucleotide repeat polymorphism in exon 1 of the androgen receptor gene on the X chromosome, 3 cases were found to have different alleles of the locus inactivated in the right and left breast tumors, indicating that the two tumors arose from distinct transformed cells. Thirteen tumors (13%) from 11 women (22%) contained somatic mutations in exons 5-8 of the p53 gene. In two cases, both breast tumors harbored p53 mutations, but the specific mutations were not identical. Seven synchronous and two asynchronous cases had p53 mutations in one tumor only. A germ line p53 mutation at codon 248, one of the most common p53 mutations in Li-Fraumeni syndrome, was observed in one case. Immunohistochemical analysis of p53 protein with a monoclonal antihuman p53 antibody showed concordant positivity between the right and left tumors in three bilateral breast cancer cases. Our results suggest that at least some bilateral breast tumors originate from distinct cells, but that some bilateral breast tumors may be related through a common p53 abnormality.     

Detection of K-ras mutations in lung carcinomas: relationship to prognosis

The K-ras mutation is one of the most common genetic alterations found in human lung cancer. To evaluate the prognostic value of ras gene alterations in lung cancer in a U.S. population, we have screened 173 human lung tumors, which included 127 adenocarcinomas, 37 squamous carcinomas, and 9 adenosquamous carcinomas, for mutations in the K-ras gene using the combination of the PCR and denaturing gradient gel electrophoresis. Forty-three tumors contained K-ras mutations. Of these, 41 were identified among the adenocarcinomas (32%), 1 among the squamous carcinomas (2.7%), and 1 among the adenosquamous carcinomas (11%). Forty of these mutations were found in codon 12 and consisted of 24 G to T transversions, 12 G to A transitions, 2 G to C transversions, and 1 double GG to TT mutation. Two other G to T transversions were found in codon 13, and 1 A to C transversion was found in codon 61. The data showed that gender did not seem to affect the incidence and the types of the K-ras mutations or amino acid changes. Examination of the mutations in adenocarcinomas in relation to overall survival showed no difference in adenocarcinomas with K-ras mutations compared with K-ras-negative adenocarcinomas. However, the substitution of the wild-type GGT (glycine) at codon 12 with a GTT (valine) or a CGT (arginine) showed a strong trend (P = 0.07) toward a poorer prognosis compared with wild-type or other amino acid substitutions. Substitution of the wild-type glycine for aspartate (GAT) showed a strong trend (P = 0.06) for a better outcome than the valine or arginine substitution. Although these trends will require larger patient populations for verification, these data suggest that the prognostic significance of K-ras mutations may depend on the amino acid substitution in the p21(ras) protein.     

Molecular epidemiology of breast cancers in northern and southern Japan: the frequency, clustering, and patterns of p53 gene mutations differ among these two low-risk populations

Comparison of acquired mutations in the p53 tumor suppressor gene can illuminate factors contributing to carcinogenesis among cancer cohorts. Japan has an ethnically homogeneous population with a low incidence of breast cancer. Previously we reported an unusual frequency, allelic status, and clustering of mutations in breast cancers from the northern part of the main Japanese island. To extend these findings, exons 2-11 and adjacent intronic sequences were analysed in tumors of women from northern (Hokkaido) and southern (Tokushima) Japan. The frequency of breast cancers with p53 gene mutations in the Hokkaido group is the highest reported (81%) while that in Tokushima (28%) is similar to most other populations. Thirteen of the 19 mutations (68.4%) in the Hokkaido cohort were heterozygous, an unusually high frequency for p53 mutations in any tumor type. There were three missense mutations at codon 175, a known hotspot for alterations in the p53 gene, and three missense mutations at codon 179, a rare site for p53 changes. In addition, the patterns of p53 gene mutation differed between the two Japanese cohorts (P=0.04). The multiple differences in acquired p53 mutations suggest unsuspected biological differences among breast cancers in northern and southern Japan. In addition, the high frequency of p53 mutations in breast cancers from Hokkaido predicted a poorer prognosis for this population which was confirmed on examination of mortality data.     

Transforming growth factor-beta receptors on human endometrial cells: identification of the type I, II, and III receptors and glycosyl-phosphatidylinositol anchored TGF-beta binding proteins

BACKGROUND: Mutation of the p53 tumor suppressor gene is the most commonly found genetic alteration in human cancer. The E6 gene product of human papillomavirus (HPV) 16 and 18 can inactivate the p53 protein by promoting its degradation. Because most HPV-positive cervical carcinoma cell lines contain wild-type p53 whereas HPV-negative cell lines have point mutations in the p53 gene, a major role in the development of HPV-negative cervical cancer has been attributed to p53. Recent studies, however, have observed no consistent presence of p53 mutation in HPV-negative primary cervical carcinomas. The MDM2 oncogene, which forms an autoregulatory loop with the wild-type p53 protein, has been found amplified in a high percentage of human sarcomas, thus abolishing the antiproliferative function of p53. METHODS: Forty-three primary cervical carcinomas and 10 autopsy-derived distant metastases from one patient were examined for p53 mutation and MDM2 amplification. These tumors had been selected from 238 cervical cancers that had been HPV-typed by Southern blot hybridization and polymerase chain reaction as a representative sample for their HPV status and their clinicopathologic characteristics. Seventeen of the cases had a remarkably good or poor clinical outcome. Human papillomavirus DNA sequences had been detected in 30 of these 43 primary tumors and 13 were negative for HPV by both methods. p53 mutation in the highly conserved exons 5-8 was studied by single-strand conformation polymorphism analysis and direct sequencing. MDM2 amplification was analyzed by Southern blot hybridization. RESULTS: Only two missense point mutations and one nucleotide sequence polymorphism were detected: a TAC-->TGC transition in codon 234 in exon 7, resulting in a Tyr-->Lys substitution, a CGT-->TGT transition in codon 273 in exon 8, resulting in an Arg-->Cys substitution and a polymorphism (CGA-->CGG) in codon 213 in exon 6. Both tumors revealing the point mutations were HPV-negative carcinomas. Amplification of the MDM2 gene was observed in 1 of the 53 specimens tested. CONCLUSIONS: In contrast to data derived from cultured cervical carcinoma cell lines and primary sarcomas, these results indicate that p53 mutation and amplification of the MDM2 oncogene are rare even in HPV-negative primary cervical carcinomas. However, to the authors; knowledge, this is the first observation of MDM2 amplification in humans outside sarcomas and neuroepithelial tumors.     

Analysis of genetic disorders of cancer of the rectum: differences in relation to cancer of the colon

AIMS AND METHODS: We studied the mechanisms of colon and rectal carcinogenesis by analysing in a series of 83 rectal tumors the prevalence of the two tumor types characteristic of colon cancer, i.e., the LOH+ type, defined by p53 and APC mutations (studied by DGGE and protein truncation assay), and the RER+ type, which is characterized by the instability of some mononucleotide repeat microsatellites (Bat 25 and Bat 26). Additionally, we analyzed the occurrence of Ki-Ras mutations (direct sequencing). RESULTS: Only one tumor turned out to be RER+. Moreover, in 59% of the tumor cases mutations were found in p53, essentially affecting codon 175. The APC and Ki-Ras genes were found to be mutated in 40 and 26% of the rectal tumors, respectively. In 18 tumors (21%) none of the genes studied were mutated. CONCLUSIONS: The RER+ phenotype is rare among rectal tumors, which are essentially LOH+. In these LOH+ tumors the p53 gene is more frequently mutated than in colorectal tumors with the same phenotype. Mutations in the APC and Ki-Ras genes, on the other hand, are less frequent in rectal tumors. Tumors with the RER- and LOH- phenotype may develop as a result of a third carcinogenesis model which must be defined.