Chemopreventive activity of oltipraz against induction of glandular stomach carcinogenesis in rats by N-methyl-N'-nitro-N-nitrosoguanidine [published erratum appears in Carcinogenesis 1998 May;19(5):955]

The modifying effects of oltipraz on induction of glandular stomach carcinogenesis by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were investigated in a total of 120 male 6-week-old Wistar rats, divided into six groups. Groups 1-3 (30 animals each) were given 100 p.p.m. MNNG in their drinking water for 10 weeks as an initiation treatment for gastric cancer induction and respectively fed diets supplemented with 0.04%, 0.02% and 0% oltipraz for 12 weeks, starting 1 week before and finishing 1 week after the carcinogen exposure. Groups 4-6 (10 animals each) were similarly treated without the application of MNNG. At the end of the 80th experimental week, all surviving animals were autopsied and examined histopathologically for the existence of gastric proliferative lesions. The incidence and multiplicity of adenocarcinomas were significantly (P < 0.01) lower in group 1 than in group 3. In addition, the multiplicity of atypical hyperplasias in the pyloric region was significantly (P < 0.05) decreased in group 1 as compared with the group 3 value. No gastric proliferative lesions were found in groups 4-6. In an additional short-term experiment, oltipraz significantly reduced cell proliferative activity (P < 0.01) and elevated glutathione levels (P < 0.05) in the glandular stomach mucosa of rats treated with MNNG. Thus our results clearly indicate that oltipraz can inhibit induction of proliferative glandular stomach lesions by MNNG in the rat.      

The DNA damaging drug cyproterone acetate causes gene mutations and induces glutathione-S-transferase P in the liver of female Big Blue transgenic F344 rats [published erratum appears in Carcinogenesis 1998 Apr;19(4):707]

The gestagenic and antiandrogenic drug cyproterone acetate (CPA) is mitogenic, tumorigenic and induces DNA-adducts and DNA-repair synthesis in rat liver. Thus CPA is expected to be mutagenic. However in vitro mutagenicity test systems were negative. To examine whether CPA induces mutations in rat liver, the in vivo mutation assay based on Big Blue transgenic F344 rats was employed. Single oral doses of 25, 50, 75, 100 and 200 mg CPA/kg b.w. respectively were administered to female Big Blue rats. Six weeks after treatment, liver DNA was assayed for mutations. At the highest dose, 200 mg CPA/kg b.w., the frequency of (17 +/- 4) x 10(-6) spontaneous mutations was increased to a maximum of (80 +/- 8) x 10(-6) mutations. One-hundred and 75 mg CPA/kg b.w. resulted in mutation frequencies of (35 +/- 5) and (27 +/- 5) x 10(-6), respectively. The mutation frequency at doses of 50 and 25 mg CPA/kg b.w. was similar to that of vehicle treated controls. Statistical analysis of the dose-effect relationship revealed that it was not possible to decide whether a threshold dose exists or not. DNA adducts were analyzed by the 32P-postlabelling technique. The total level of the major and the two minor adducts observed in the autoradiograms increased between doses of 25 to 75 mg CPA/kg b.w. to a maximum of approximately 12,000 +/- 3000 adducts per 10(9) nucleotides. The level did not further increase significantly with 100 and 200 mg CPA/kg b.w. After CPA treatment no preneoplastic liver foci were observed. However, single glutathione-S-transferase placental form (GST-P) positive hepatocytes were observed and the frequency was dependent on the dose. These cells are not supposed to represent initiated cells, since they occurred only transiently after 6 weeks and disappeared thereafter completely. In conclusion, our results demonstrate that CPA is mutagenic in vivo. The mutation frequency increased at high CPA doses, when the increase of the DNA adduct formation had already ceased. This suggests that the mitogenic activity of CPA is required to express the mutations.      

Induction of reactive oxygen species without 8-hydroxydeoxyguanosine formation in DNA of initiated mouse keratinocytes treated with 12-O- tetradecanoylphorbol-13-acetate [published erratum appears in Carcinogenesis 1998 Nov;19(11):2059]

Evidence for the involvement of oxidative stress in 12-O- tetradecanoylphorbol-13-acetate (TPA)-mediated tumor promotion has focused on non-initiated immune cells, tumor cell lines and non- initiated epidermis treated in vivo. This paper reports the effects of TPA on 8-hydroxydeoxyguanosine (8OHdG) formation and the generation of reactive oxygen species (ROS) in cloned initiated mouse epidermal keratinocytes in order to determine whether TPA can directly damage DNA through ROS production within the keratinocytes. Using high performance liquid chromatography with electrochemical detection (HPLC-EC), TPA did not induce 8OHdG formation in DNA of initiated keratinocytes treated under a variety of conditions. The reliability of the HPLC-EC system is demonstrated by (i) the linearity of the 8OHdG standard curve; (ii) the consistency of 8OHdG measurements in calf thymus and cellular DNA; and (iii) the dose-dependent increase in 8OHdG in DNA of initiated keratinocytes treated with UVC in the presence and absence of H2O2. Though not DNA-damaging, TPA induced a 65% increase in ROS (P < 0.05) as detected by luminol-dependent chemiluminescence. These results support a mechanism for the role of oxidative stress in tumor promotion that does not involve direct DNA damage to the keratinocyte target cell. The relationship between ROS, signal transduction and tumor promotion is discussed in light of the above results which is consistent with the role of TPA-induced ROS as second messengers in signal transduction.      

Infrequent p53 mutations in 7,12-dimethylbenz[a]anthracene–induced mammary tumors in BALB/c and p53 hemizygous mice

We conducted experiments to determine if p53 alterations, which are frequent in human breast cancers, were also common in murine mammary tumors. In 13 mammary tumors from 7,12-dimethylbenz[a]anthracene (DMBA)-treated BALB/c mice were immunohistochemically analyzed for overexpression of p53; p53 protein was not detectable. Three of the tumors were established as cell lines in vitro. p53 protein was rarely detected at passage 4 in these lines but was overexpressed by passage 8 in two of them. The p53 nucleotide sequence was shown to be wild type in one primary mammary tumor and in the two p53-overexpressing cell lines. One cell line that overexpressed p53 in vitro was implanted into BALB/c mice. The resulting tumors retained the wild-type p53 nucleotide sequence but no longer expressed detectable levels of p53 protein, suggesting that the overexpression of wild-type p53 was related to in vitro culture conditions. The effect of DMBA on mammary-tumor development was also tested in mice rendered hemizygous for p53. These mice and wild-type littermate controls had no differences in susceptibility to induction of mammary tumors by oral administration of DMBA. Furthermore, Southern blot hybridizations detected no gross alterations in the wild-type p53 allele in mammary tumors from the p53-deficient mice. Point mutation of the wild-type p53 allele was also infrequent in the DMBA-induced mammary tumors from hemizygous p53 mice; it occured in only one of seven tumors. Thus, the p53 gene is apparently not a primary target for genetic alterations in DMBA-induced mammary tumors. Next, we examined mammary tumors derived from D1 and D2 transplantable hyperplastic alveolar nodule (HAN) outgrowths, which rapidly form tumors containing Ha-ras mutations after DMBA treatment. As ras and p53 mutants can cooperate in transformation, we examined whether D1 and D2 HAN outgrowths have p53 mutations. Unlike in the DMBA-induced primary mammary tumors, nuclear p53 accumulation was observed frequently (10 of 14) in tumors that arose from D1 and D2 HAN outgrowths. Direct sequencing of the entire coding region of the p53 cDNA from six D1 and D2 tumors confirmed that the sequence was wild type. Although wild-type p53 was retained in both DMBA-induced mammary tumors and mammary tumors derived from D1 and D2 preneoplastic outgrowths, wild-type p53 overexpression was detected only in D1 and D2 tumors. Therefore, D1 and D2 tumors appear to arise by a pathway in which p53 expression is altered, whereas DMBA induction affects a different pathway that does not require such alteration. © 1994 Wiley-Liss, Inc.     

p53 mutations in benzo(a)pyrene-exposed human p53 knock-in murine fibroblasts correlate with p53 mutations in human lung tumors

Human p53 mutation spectra differ significantly from one cancer type to another. One possible reason is that carcinogenic risk factors differ, and these factors elicit distinct mutation patterns. There has been no mammalian assay, however, with which to generate mutation patterns in human p53 sequences experimentally, hampering interpretation of the human tumor spectra. We have designed a new mammalian cell assay using gene targeting technology that selects and scores human p53 gene sequence mutations in human-p53 knock-in (Hupki) murine embryonic fibroblasts (HUF) that have undergone immortalization. With the Hupki assay we examined here whether benzo(a)pyrene (BaP), a major tobacco smoke carcinogen could elicit p53 mutation patterns characterizing the human lung tumor p53 mutation spectrum. We found that, in contrast to unexposed HUFs or HUFs exposed to other carcinogenic agents, HUFs exposed to BaP acquire mutations that display major features of the human lung tumor p53 mutation spectrum: (a) predominance of G-to-T mutations, (b) unequivocal strand bias of the transversions, and (c) a mutation hotspot at codons 157 to 158. These data are consistent with the hypothesis that BaP has a direct role in causing smokers' lung tumor p53 mutations. The assay can be used to examine various hypotheses on the endogenous or exogenous factors responsible for the p53 mutations in human tumors arising in other tissues.     

Benzo[a]pyrene coated ferric oxide and aluminum oxide particles: uptake, metabolism and DNA binding in hamster pulmonary alveolar macrophages and tracheal epithelial cells in vitro [published erratum appears in Carcinogenesis 1997 May;18(5):1123]

Ferric oxide (Fe2O3) and aluminum oxide (Al2O3) particles are widely encountered in occupational settings. Benzo[a]pyrene (B[a]P), a well- characterized environmental carcinogen, is frequently adsorbed onto particles. It has been shown that B[a]P-coated Fe2O3 particles (B[a]P- Fe2O3) significantly increased lung tumors in the hamster in contrast to B[a]P-coated Al2O3 (B[a]P-Al2O3) or B[a]P alone. In order to determine the genotoxic effects of these particles on the metabolism of B[a]P, pulmonary alveolar macrophages (AM) from male Syrian golden hamsters were incubated with 5 microg (19.8 nmol) B[a]P-coated respirable size (99% < 5 microm) Fe2O3 and Al2O3 particles with loads from 0.5 to 2.0 mg. Intracellular uptake of B[a]P by AM at 24 h was higher with B[a]P-Fe2O3 than that of B[a]P alone (P < 0.05) or B[a]P- Al2O3 (P < 0.05). Total B[a]P metabolism was significantly greater in AM exposed to B[a]P-coated Fe2O3 at 1.0 and 1.5 mg than in the AM exposed to B[a]p-al2O3 (0.5, 1.0 and 1.5 mg) (P < 0.05) or B[a]P alone (P < 0.05). Similar significant differences for Fe2O3 relative to Al2O3 and B[a]P alone were also apparent for total dihydrodiols, quinones and phenolic metabolites. Co-administration of 5 microg alpha- naphthoflavone (alpha-NF, an inhibitor of cytochrome P-4501A1 and P- 4501A2) and 10(-3) M cyclohexene oxide (CO, an inhibitor of epoxide hydrolase) significantly reduced B[a]P metabolism in B[a]P-Fe2O3 (P < 0.05) and B[a]P-Al2O3 (P < 0.05) treated groups relative to B[a]P alone. AM were co-cultured with hamster tracheal epithelial cells (HTE) and treated as described above for metabolism studies to assess the DNA binding of B[a]P metabolites in the target cells, using 32P- postlabeling techniques. Two adducts were observed that had chromatographic behavior similar to 7R,8S,9S-trihydroxy-10R-(N2- deoxyguanosyl-3'-phosphate)-7,8,9,10-t etrahydrobenzo[a]pyrene [(+)- anti-BPDE-dG, adduct 1, major adduct representing 70-80% of total adducts] and 7S,8R,9R-trihydroxy-10S-(N2-deoxyguanosyl-3'-phosphate)- 7,8,9,10-t etrahydrobenzo[a]pyrene [(-)-anti-BPDE-dG, adduct 2, representing 20-30% of total adducts]. B[a]P-Fe2O3 treatment enhanced the levels of the two B[a]P-DNA adducts in the HTE compared with B[a]P- Al2O3 (P < 0.05) or B[a]P alone. The inhibitors alphaNF and CO significantly reduced total adduct levels in the HTE (P < 0.05) in the B[a]P and B[a]P-Fe2O3 treatments as well as adduct 1 and adduct 2 levels. Our data suggest that the cocarcinogenic effect of B[a]P-Fe2O3 relative to B[a]P-coated Al2O3 can be due to: (i) the enhancement of B[a]P metabolism in AM by Fe2O3 associated with the increased uptake of B[a]P; and (ii) augmentation of DNA adduct formation in epithelial cells.      

K-ras and p53 point mutations in 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced hamster lung tumors

Lung tumors were induced in Syrian golden hamsters by s.c. injectionof 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). After40 weeks lung tumor tissue was isolated. Administration of theNNK and exposure of the animals to an atmosphere of 65% oxygenresulted in a statistically significant reduction in tumor sizebut did not alter the histological tumor type or tumor incidencewhen compared with carcinogen treated animals maintained underambient air. Histologically, lung tumors had the morphologicfeatures of adenomas and adenocarcinomas with     

Analysis of K-ras p53 and c-raf-1 mutations in beryllium-induced rat lung tumors

Beryllium (Be) metal and several of its analogues have beenshown to be carcinogenic in rats. In addition, workers employedat Be processing plants have been shown to have a slight excessof lung cancer. In this study, a single inhalation exposureto Be metal produced a 64% incidence of lung tumors in the F344/Nrat. The most frequent tumor type observed was adenocarcinoma.These Be metal-induced lung carcinomas were examined for geneticalterations in the K-ras, p53, and c-raf-1 genes. DNA isolatedfrom lung neoplasms was analyzed by PCR amplification and directDNA sequence analysis, immunohistochemical analysis and Southernblot analysis. No K-ras codon 12,13 or 61 mutations were detectedin 24 lung tumors by direct sequencing. Using a more sensitiveK-ras codon 12 mutation selection assay, K-ras codon 12 GGT-GTTtransversions were detected in two of 12 adeno-carcinomas. Theseresults suggest that activation of the K-ras protooncogene isboth a rare and late event, possibly stemming from genomic instabilityduring the progression of some Be-induced rat adenocarcinomasof the lung. No mutant p53 nuclear immunoreactivity was observedin any Be-induced tumor. Because immunohistochemical analysisof the p53 protein only detects missense mutations, exons 5–8of this gene were also analyzed by direct DNA sequencing. Inorder to perform the p53 sequence analysis, it was necessaryto first characterize and sequence the/p53 intron sequencesflanking exons 5–8 and their splice sites. Details ofthis expanded intron DNA sequence information are given here.No mutations were detected within exons 5–8 of the p53gene. No rearrangement of the c-raf-1 protooncogene was detectedby Southern blot analysis. These results indicate that the mechanismsunderlying the development of Be-induced lung cancer in ratsdo not involve gene dysfunctions commonly associated with humannon-small-cell lung cancer.     

Frequent K-ras mutations and absence of p53 mutations in mucin-producing tumors of the pancreas

Mucin-producing tumors of the pancreas (MPT) are characterized by the prodcution of much mucin and a benign course after surgical treatment. We examined 16 cases of MPT and 20 cases of “common” pancreatic duct cell carcinomas (DCC) in regard to K-ras and p53 mutations. The mutations were detected by constant denaturant gel electrophoresis in combination with other techniques using PCR products amplified from the samples microdissected from the tissue sections. K-ras codon 12 mutations were identified in all MPT and in 95% of DCC. On the other hand, p53 mutations were found in four of 20 (20%) DCC, and p53 was immunocy-tochemically overexpressed in 3 of the 4 mutated cases. However, no p53 mutations and no p53 overexpression were identified in the 16 MPT. These results indicate that, although the K-ras codon 12 mutations may be almost essential for the development of both MPT and DCC, p53 mutations seemed to be involved mainly to the latters. © Wiley-Liss, Inc.     

Dietary N-acetyl-L-cysteine modulates benzo[a]pyrene-induced skin tumors in cancer-prone p53 haploinsufficient Tg.AC (v-Ha-ras) mice

Epidemiologic studies support the protective role of dietary antioxidants in preventing cancer. However, emerging evidence from clinical trials and laboratory data suggest that in some cases individual antioxidant supplements may actually exacerbate carcinogenesis. Our goal was to explore these paradoxical activities in a rodent model that possesses genotypic characteristics of human cancers. We selected the p53 haploinsufficient Tg.AC (v-Ha-ras) mouse as a model, because it contains an activated, carcinogeninducible ras oncogene and an inactivated p53 tumor suppressor gene, which are frequent genetic alterations in human cancers. These mice develop chemically induced benign and malignant skin tumors rapidly which can easily be quantified. Mice were fed basal diets with or without 3% N-acetyl-L-cysteine (NAC), a well-recognized antioxidant, prior to, during and after topical application of the carcinogen benzo[a]pyrene (64 microg/mouse) applied twice per week for 7 weeks. Tumor incidence exceeded 90% for both groups, and NAC did not reduce tumor latency. Mice fed NAC displayed a 43% reduction (P < 0.05) in tumor multiplicity and delayed the appearance of lesions (P < 0.05). Dietary NAC also significantly (P < 0.05) improved group survival by 5 weeks. Total tumor yields were reduced in both dietary groups but malignant spindle cell tumors (SCT) increased by 25% in NAC-fed mice. The v-Ha-ras oncogene and p53 protein products were clearly co-expressed in both benign and malignant lesions from both dietary groups. In summary, dietary supplementation with NAC was chemopreventive, but the marginal increase in SCT suggests a paradoxical effect.     

Breast cancer incidence and mortality in the breast cancer detection demonstration project [published errtum appears in J Natl Cancer Inst 1989 Oct 4;81(19):1513

The Breast Cancer Detection Demonstration Project (BCDDP) was a program of five annual screening examinations for breast cancer that was conducted at 29 centers in the United States. This report presents data on breast cancer incidence and mortality among the participants. A total of 283,222 women were enrolled. Our analysis is based on 55,053 white women who were 35-74 years of age at entry and who were selected for follow-up. For the first 9 years after entry, the cumulative incidence of breast cancer was estimated as 243.6 per 10,000, which is 1.34 times the expected incidence derived by use of data from the Surveillance, Epidemiology, and End Results (SEER) program. In contrast, 9-year cumulative mortality from breast cancer was only 79.6% of that expected in women with the age distribution of BCDDP participants who did not have diagnosed breast cancer at the start of observation. The ratio of observed to expected breast cancer mortality was 0.89 for women 35-49 years of age at entry, 0.76 for women 50-59 years of age at entry, and 0.74 for women 60-74 years of age at entry. Breast cancer incidence and mortality were lower for women who entered the BCDDP for routine screening than they were for women who entered for a reason such as concern about breast disease, family history of breast cancer, or a physician's recommendation. Among cases diagnosed within 5 years of entry, the 5-year case fatality attributed to breast cancer was 8.5%. Case fatality for all stages combined was greater than 50% lower for cases that were screen-detected than it was for cases that were not screen-detected. Case fatality was lower for cases diagnosed within the first 5 years of entry (which encompassed the period of screening) than it was for cases diagnosed in the sixth or seventh years.     

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.     

High frequency and heterogeneous distribution of p53 mutations in aflatoxin B1-induced mouse lung tumors.

Inactivation of the p53 tumor suppressor gene is one of the most frequent genetic alterations observed in human lung cancers. However, p53 mutations are more rarely detected in chemically induced mouse lung tumors. In this study, 62 female AC3F1 (A/J x C3H/HeJ) mice were treated with aflatoxin B1 (AFB1; 150 mg/kg i.p. divided into 24 doses over 8 weeks). At 6-14 months after dosing, mice were killed, and tumors were collected. A total of 71 AFB1-induced lung tumors were examined for overexpression of p53 protein by immunohistochemical staining. Positive nuclear p53 staining was observed in 79% of the AFB1-induced tumors, but the pattern was highly heterogeneous. In approximately 73% of the positively stained tumors, fewer than 5% of cells demonstrated positive staining; in the other 27%, between 10% and 60% of the cells stained positively, with staining localized to the periphery of the tumors in many cases. Single-strand conformational polymorphism analysis of the evolutionarily conserved regions of the p53 gene (exons 5-8) from AFB1-induced whole lung tumor DNA revealed banding patterns consistent with point mutations in 20 of 76 (26%) tumors, with 85% of the mutations in exon 7 and 15% of the mutations in exon 6. Identification of point mutations could not be confirmed by direct sequence analysis because bands representing putative mutations appeared only weakly on autoradiograms. This was presumably due to the heterogeneous nature of the DNA analyzed. Single-strand conformational polymorphism analysis of DNA from laser capture microdissected cells of paraffin-embedded AFB1-induced tumor tissue sections stained for p53 produced banding patterns consistent with point mutations in 18 of 30 (60%) DNA samples. Direct sequencing of the microdissected samples revealed mutations at numerous different codons in exons 5, 6, and 7. Of 26 mutations found in microdissected regions from adenomas and carcinomas, 9 were G:C-->A:T transitions, 11 were A:T-->G:C transitions, and 5 were transversions (2 G:C-->T:A, 2 T:A-->A:T, and 1 A:T-->C:G), whereas 1 deletion mutation was identified. The concordance between immunostaining and molecular detection of p53 alterations was 72% when laser capture microdissection was used versus 17% based on whole tumor analysis. The high mutation frequency and heterogeneous staining pattern suggest that p53 mutations occur relatively late in AFB1-induced mouse lung tumorigenesis and emphasize the value of analyzing different staining regions from paraffin-embedded mouse lung tumors.     

p53 protein accumulation and p53 gene alterations (RFLP, VNTR and p53 gene mutations) in non-invasive versus invasive human transitional bladder cancer

Eleven non-invasive and 24 invasive transitional cell bladder cancers were analysed for molecular alterations to the p53 gene and nuclear accumulation of the p53 protein. 9% (1/11) of non-invasive rumours and 21% (5/24) of invasive tumours revealed nuclear accumulation in more than 50% of the tumour cells. PCR analysis of D17S30 showed loss of heterozygosity (LOH) in invasive tumours (3/24; 12%). Two invasive tumours harboured point mutations in exon 6 and exon 7, respectively (8%). Our results indicate that p53 protein overexpression correlates with tumour progression, p53 gene mutations and LOH detected by PCR.     

Low frequency of p53 and k-ras codon 12 mutations in non-small cell lung carcinoma (NSCLC) tumors and surgical margins

AIMS AND BACKGROUND: Lung cancer is one of the most common cancers and has became a predominant cause of cancer-related death throughout the world. The k-ras codon 12 mutation, which is the most common lung cancer mutation, is found in 15 to 30% of all lung cancers. Furthermore, the p53 gene has a very important role in the biological properties of tumor cells, and it is mutated in about 50% of non-small cell lung cancers. Residual tumor cells remain in surgical margins diagnosed as tumor free by histopathological techniques, and they can play a role in forming any local recurrence. Molecular methods may be exploited that are sensitive enough to detect small numbers of tumor cells. METHODS: In the present study, we examined p53 gene mutations and k-ras codon 12 mutations from the tumor samples and surgical margins of 34 non-small-cell lung cancer patients. P53 gene mutations were analyzed by single strand conformational polymorphism analysis, heterodublex analysis and DNA sequencing. K-ras codon 12 mutations were analyzed by the mutagenic PCR-restricted fragment length polymorphism method. RESULTS: A p53 mutation was detected only in primary tumors of 3 out of 34 patients (8.82%). These mutations were clustered in exon 5. Moreover, a k-ras codon 12 mutation was detected in both the primary tumor and the surgical margin tissues of 2 out of 34 patients (5.88%). CONCLUSIONS: The detected mutation rate was low, in the range given in the literature. We think that different mechanisms related to other genes and individual genetic differences might play a role in cancer formation in our study group. We believe that molecular studies are necessary to identify biomarkers and to determine genetic alterations in histopathologically normal surgical margins.     

ras mutations in 2-amino-3-methylimidazo-[4,5-f]quinoline—induced tumors in the CDF1 mouse

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) is a very potent mutagen that is carcinogenic in rodents and nonhuman primates. IQ-induced CDF₁ mouse lung and liver tumors were examined for activated Ki-ras and Ha-ras genes, respectively. Polymerase chain reaction (PCR)—amplified target DNAs were analyzed for mutations of codons 12, 13, and 61 by single-strand conformation polymorphism (SSCP) and direct sequencing methods. All mutations were localized to codon 61 of the ras genes. Forty-nine of 54 lung tumors induced by IQ possessed activating Ki-ras mutations, as did 20 of 26 lung tumors from the vehicle-treated animals; 80% and 75% of these mutations, respectively, were A → T transversions of the second nucleotide redundant. One lung adenoma from the IQ-treated group contained a tandem duplication of the sequence corresponding to codons 50–57 of the Ki-ras gene (unpublished observations). In addition, seven of 34 IQ-induced liver tumors harbored activating Ha-ras mutations: five were C → A (G → T) transversions at the first nucleotide, and two were A → T transversions at the second nucleotide of codon 61. None of the 15 liver tumors collected from the vehicle-treated mice possessed Ha-ras mutations in codon 12, 13, or 61. These data indicate that IQ induces Ha-ras gene activation in CDF₁ mouse liver tumors. The mechanism of lung tumor induction by IQ, however, is obscured by the high frequency of Ki-ras A → T mutations observed in both the IQ-induced and spontaneous lung tumors. The different ras mutational spectra in lung and liver tumors may suggest either that two different pathways of IQ metabolism exist in these organs or that IQ contributes to CDF₁ lung tumorigenesis by a mechanism other than its direct interaction with the Ki-ras gene.     

Allelic deletions on chromosome-17 and mutations in the p53 gene in tumors metastatic to brain

Metastasis associated with tumor progression denotes more aggressive tumor behavior, more malignant histology, and worsening patient prognosis. Brain is one of the common sites to which solid tumors metastasize. Since mutations in the tumor suppressor gene p53 are associated with tumor progression to malignancy in various cancers, we examined the molecular genetic profile of the p53 gene and also analyzed allelic losses of various genes on the short arm of chromosome 17 (17p) in 10 metastatic brain tumors (4 breast, 3 renal, 1 lung, 1 esophageal, and 1 squamous cell carcinoma) and in two of the primary tumors (I breast, 1 renal) corresponding to two of the 10 metastases. Six of the 10 metastatic tumors (4/4 breast, 1/1 esophageal and 1/1 squamous cell carcinoma) contained allelic loss and/or mutations of the p53 gene. The p53 gene profile was identical in both of the primary tumors and their corresponding metastases that were examined. If borne out by a larger series of analysis on tumors, especially from breast, as well as from other organs, detection of chromosome 17/p53 alterations may be of substantial clinical significance in predicting the metastatic potential of primary tumors.     

Mutability of p53 hotspot codons to benzo(a)pyrene diol epoxide (BPDE) and the frequency of p53 mutations in nontumorous human lung

p53 mutations are common in lung cancer. In smoking-associated lung cancer,the occurrence of G:C to T:A transversions at hotspot codons, e.g., 157, 248, 249,and 273, has been linked to the presence of carcinogenic chemicalsin tobacco smoke including polycyclic aromatic hydrocarbons suchas benzo(a)pyrene (BP). In the present study, we have used a highly sensitive mutation assay to determine the p53 mutation load in nontumorous human lung and to study the mutability of p53 codons 157, 248, 249, and 250 to benzo(a)pyrene-diol-epoxide (BPDE), an active metabolite of BP in human bronchial epithelial BEAS-2B cells. We determined the p53 mutational load at codons 157, 248, 249, and 250 in nontumorous peripheral lung tissue either from lung cancer cases among smokers or noncancer controls among smokers and nonsmokers. A 5-25-fold higher frequency of GTC(val) to TTC(phe) transversions at codon 157 was found in nontumorous samples (57%) from cancer cases (n = 14) when compared with noncancer controls (n = 8; P < 0.01). Fifty percent (7/14) of the nontumorous samples from lung cancer cases showed a high frequency of codon 249 AGG(arg) to AGT(ser) mutations (P < 0.02). Four of these seven samples with AGT(ser) mutations also showed a high frequency of codon 249 AGG(arg) to ATG(met) mutations, whereas only one sample showed a codon 250 CCC to ACC transversion. Tumor tissue from these lung cancer cases (38%) contained p53 mutations but were different from the above mutations found in the nontumorous pair. Noncancer control samples from smokers or nonsmokers did not contain any detectable mutations at codons 248, 249, or 250. BEAS-2B bronchial epithelial cells exposed to doses of 0.125, 0.5, and 1.0 microM BPDE, showed G:C to T:A transversions at codon 157 at a frequency of 3.5 x 10(-7), 4.4 x 10(-7), and 8.9 x 10(-7), respectively. No mutations at codon 157 were found in the DMSO-treated controls. These doses of BPDE induced higher frequencies, ranging from 4-12-fold, of G:C to T:A transversions at codon 248, G:C to T:A transversions and G:C to A:T transitions at codon 249, and C:G to T:A transitions at codon 250 when compared with the DMSO-treated controls. These data are consistent with the hypothesis that chemical carcinogens such as BP in cigarette smoke cause G:C to T:A transversions at p53 codons 157, 248, and 249 and that nontumorous lung tissues from smokers with lung cancer carry a high p53 mutational load at these codons.     

K-ras, p53 mutations, and microsatellite instability (MSI) in gallbladder cancer

Despite the considerable progress in understanding the molecular pathology of carcinogenesis, the genetic mechanisms underlying the development and progression of gallbladder cancer (GC) are poorly understood. The survival of GC patients is generally poor. Therefore, it is very useful to define valuable prognostic factors. The most extensively studied oncogenes in gallbladder carcinogenesis are ras, commonly mutated in neoplasms of the gastrointestinal tract. K-ras oncogene is altered in a subset of gallbladder patients and mainly in those having anomalous junction of the pancreaticobiliary tract. Most of the studies of genetic abnormalities in GC have focused on p53 gene. p53 mutation/overexpression and/or LOH is present in more than 50% of gallbladder carcinomas, suggesting an important role in their pathogenesis. However, these results have not any predictive value yet. Moreover, the involvement of an alternative molecular pathway, that of microsatellite instability (MSI), is found in a limited group of GC patients. Additional research is necessary to establish its possible relation to defects of the mismatch repair (MMR) system and its proposed prognostic significance. Further elucidation of the molecular events specific to GC will help to identify novel molecular targets for the diagnosis and clinical management of the patients.