Multiple polymorphisms, but no mutations, in the WAF1/CIP1 gene in human brain tumours.

The cyclin kinase inhibitor WAF1/CIP1, also termed CDKN1, mediates p53-induced cell cycle arrest in response to DNA damage. This property makes it an attractive tumour-suppressor candidate for a p53-associated tumour-suppressor gene. In order to investigate the role of WAF1/CIP1 in the pathogenesis of primary human brain tumours we performed single-stranded conformation polymorphism (SSCP) analysis and direct sequencing of exon 2 of the gene in a representative series of 158 brain tumours and corresponding blood samples. In addition, all tumours were examined for mutations in exons 5-8 of the p53 gene. Analysis of WAF1/CIP1 revealed multiple polymorphisms, the most abundant being AGC-->AGA (Ser-->Arg) at codon 31 with an allele frequency of 8.5%. Less common polymorphisms included GTG-->GGG (Val-->Gly) at codon 25, GCC-->ACC (Ala-->Thr) at codon 64, CGC-->CTC (Arg-->Leu) at codon 32, GGC-->AGC (Gly-->Ser) at codon 14 and GCG-->GTG (Ala-->Val) at codon 39 each with an allele frequency of 0.3%. These polymorphisms were all located in a conserved region of exon 2. Two of the polymorphisms were also seen in a group of 157 healthy controls indicating that WAF1/CIP1 polymorphisms do not predispose to cancer. None of the tumours included in our series showed a somatic mutation in WAF1/CIP1. All samples were also analysed for loss of heterozygosity on the short arm of chromosome 6 in the region of the WAF1/CIP1 locus. Allelic loss was observed in only one patient with a glioblastoma. Mutations in the p53 gene were found in 22 of 158 tumours. No association was found between any polymorphism of the WAF1/CIP1 gene, p53 mutations and histopathological tumour type. Our data indicate that WAF1/CIP1 mutations are probably not involved in the formation of primary human brain tumours.     

Prognostic significance of TP53 alterations in breast carcinoma.

Constant denaturant gel electrophoresis (CDGE) was used to screen 179 breast carcinomas for mutations in the conserved regions of the TP53 gene (exons 5 through 8). Mutations were found in 35 of 163 primary tumours (21%) and in 5 of 16 metastases (31%) and resided predominantly in exon 7. The majority of the mutations were G:C-->A:T transitions. Immunohistochemistry demonstrated nuclear accumulation of p53 protein in 35 of 162 primary tumours (22%) and in four of 15 metastases (27%). TP53 mutation was strongly associated with nuclear accumulation of p53 protein. In total 42 of 163 primary tumours (26%) and 5 of 16 metastases (31%) were demonstrated to contain TP53 alterations (mutation and/or nuclear protein accumulation). TP53 alteration in primary tumour was significantly associated with the following parameters: positive node status, T status > 1, negative oestrogen receptor status, negative progesterone receptor status, presence of ERBB2 gene amplification, and invasive ductal histology. Furthermore, there were statistically significant associations, independent of other prognostic factors, between TP53 alterations in primary tumour and disease-free and overall survival.     

Genetic alterations in N-bis(2-hydroxypropyl)nitrosamine-induced rat transplantable thyroid carcinoma lines: analysis of the TSH-R, G(alpha)s, ras and p53 genes

It has recently been shown that point mutations of the TSH-R or G(alpha)s genes are associated with autonomous hyperfunctioning thyroid adenomas and differentiated carcinomas. We therefore screened for mutations in the TSH-R, G(alpha)s, ras and p53 genes in nine rat transplantable thyroid carcinoma lines derived from tumors induced by DHPN as a chemical carcinogen. Mutations were identified using single- strand conformation polymorphism and DNA sequencing analysis. Point mutations in G(alpha)s codon 201 (CGC-->CAC) were detected in three lines (33%), resulting in a heterozygous alteration (Arg-->His) in the expressed G(alpha)s protein. The mean intracellular cAMP level (2.30 +/- 0.27 nmol/mg) of the three mutated cell lines was significantly increased as compared with that of the lines (1.54 +/- 0.32 nmol/mg) without the G(alpha)s mutation (P < 0.01, by paired t-test). Also, these three cell lines had an activating mutation in Ki-ras codon 12 (GGT-->GAT). One TSH-R gene mutation was found with a base substitution in codon 636 (TGC-->TGT) but no amino acid change. No p53 gene (exons 5- 8) mutations were detected in any of the cell lines analyzed. The results suggest that mutational activation of the G(alpha)s gene may play a tumorigenic role through constitutive activation of the cAMP pathway and that G-->A point mutations in the G(alpha)s and ras genes in thyroid carcinomas directly reflect interaction of the chemical carcinogen with guanine residues in DNA.      

In vitro activation of the human Harvey-ras proto-oncogene by aflatoxin B1

Activation of ras proto-oncogenes occurs frequently in vivo in chemically induced rodent tumours, including rat hepatomas induced by aflatoxin B1. This study examines the in vitro activation of a human ras gene by this mycotoxin. A plasmid containing the human Ha-ras proto- oncogene, together with a neomycin resistance gene (pECneo), was incubated in vitro with a microsomal system generating aflatoxin B1 8,9- epoxide. Subsequent transfection of the plasmid into mouse NIH 3T3 fibroblasts, followed by G418 selection and s.c. injection of surviving cells into immunodeficient mice demonstrated that the proto-oncogene had acquired transforming capacity. Although a single tumour resulted from similar treatment of incubated unconjugated plasmid, no tumours were produced by a secondary round of transfections using DNA from this tumour. Selective PCR amplification of the human Ha-ras gene in extracted tumour DNA followed by sequencing demonstrated the presence of G-->T transversions either at the first or middle base of codon 12 in tumours resulting from transfection with the aflatoxin-B1-modified pECneo plasmid, but this was not detected in the single tumour resulting from transfection with the unmodified plasmid. Thus, although a mutation in the Ha-ras gene has not been reported for human primary hepatomas occurring in aflatoxin-exposed populations, metabolically activated aflatoxin B1 is capable of mutating this proto-oncogene to its oncogenic form in vitro. No mutations were observed in codon 61. It appears that, in contrast to the frequently reported G-->T transversions in codon 249 of the p53 gene in primary hepatomas in aflatoxin-exposed humans, the failure to detect Ha-ras mutations in these tumours is not due to an inability of aflatoxin B1 to activate this proto-oncogene. The G-->T transversions observed in this study contrast with the most frequent aflatoxin B1 in vivo induced mutations, G-->A transitions in the rat Ki-ras gene. Possible mechanisms for these differences are discussed.      

Early detection of cancer-associated gene alterations in DNA isolated from rat feces during intestinal tumor-induction with 1,2-dimethylhydrazine

A highly sensitive mutation detection method was applied to reveal tarry K-ras alterations in exfoliated intestinal epithelium of Fischer-344 rats during the course of 1,2-dimethylhydrazine (DMH)-induced carcinogenesis. Ten weekly s.c. injections of DMH (50 mg/kg) in combination with consumption of a low-fiber diet resulted in 100% incidence of intestinal tumors at 20 weeks after initial DMH injection. Analysis of DNA extracted from fresh fecal samples obtained individually showed that proportion of codon 12 K-ras oncogene mutant alleles (G-->A transition at the second position of codon 12) was increased in some rats at 4 weeks and clearly in all rats at 8 weeks after initial DMH injection, i.e. much earlier than the first tumors appeared (14 weeks). A gradual increase of mutant K-ras fraction in DNA samples extracted from feces led to an extremely high level of the mutant reaching 10% of the oncogene alleles at the end of the experiment (20 weeks). K- and H-ras oncogene and p53 tumor suppressor gene mutations were analyzed in the resulting colon and duodenal tumors. 14 of 17 colon tumors had K-P as mutations (11 - G-->A transition at codon 12 second base; 3 - G-->A transition at codon 13 second base). G-->A transitions at codon 12 first base of H-ras were detected in 3 colon tumors. All 5 duodenal tumors induced in the experiment had G-->A transition at codon 12 second base of K-ras. 3 of these tumors also had H-ras mutations. No mutation was detected within exons 4-7 of p53 gene indicating that p53 alterations may not be involved in the rapid development of tumors induced by high doses of DMH. Our observations suggest that detection of K-ras mutations in stool samples are predictive of later tumor development from a very early stage.     

Hepatic levels of S-adenosylethionine and S-adenosylmethionine in rats and hamsters during subchronic feeding of DL-ethionine

The levels of S-adenosylethionine (AdoEt) and of S-adenosylmethionine(AdoMet) in the livers of weanling male rats and male and femalehamsters fed ethionine for 1–6 weeks were determined.Ethionine was fed at levels of 0,0.1, and 0.3% in the diet,and the animals were sacrificed after 0,1,3 and 6 weeks of treatment.In both species the hepatic contents of AdoEt were dependentupon the level of ethionine in the diet. For the 6-week experimentalperiod the hepatic levels of AdoEt averaged 81 µg/g liverin male hamsters fed 0.1% ethionine in the diet and 160 µg/gin those fed 0.3% ethionine; the corresponding AdoEt levelsin female hamsters were 104 and 191 µg/g liver, respectively.No marked shifts in hepatic AdoEt levels were seen in eithermale or female hamsters although a gradual rise in hepatic AdoEtfrom 145 to 233 µg/g was noted in the female hamstersreceiving 0.3% ethionine in the diet for 1–6 weeks. AdoEtlevels in the livers of rats fed 0.3% ethionine were quite variablewith values of 123, 05 and 127 µg/g liver noted at weeks1,3 and 6 respectively. In rats fed the 0.1% ethionine dietthe liver AdoEt levels dropped from 103 to 61 µg/g fromweeks 1 to 6. In animals fed the ethionine-free diet, the hepaticcontents of AdoMet were relatively constant throughout the 6-weekexperimental period, with average values of 25,17, and 29 µg/gliver respectively in the male rats, male hamsters and femalehamsters. Chronic ethionine administration always suppressedhepatic AdoMet levels. This suppression was generally greaterin animals fed the 0.1% ethionine than in those fed the 0.3%ethionine diet. Thus, the average hepatic AdoMet levels in rats,male hamsters and female hamsters receiving the 0.1% ethioninediet for 3–6 weeks were 32,18, and 45% respectively, ofthe corrdesponding AdoMet levels in control animals: however,the corresponding AdoMet levels in animals receiving the 0.3%ethionine diet were 66,42, and 62% of the respective controlvalues. Feeding 0.1% ethionine to male hamsters led to exceedinglylow levels of liver AdoMet (1.4–2.9 µg/g). No directcorrelations could be made between the effects of ethioninefeeding on the hepatic AdoEt and AdoMet levels in rats and hamstersand the previously reported differences in carcinogenicity byethionine in these species.     

A spectrum of mutations induced by crotonaldehyde in shuttle vector plasmids propagated in human cells

A spectrum of crotonaldehyde-induced mutations in the supF gene of the shuttle vector plasmid pMY189 replicated in human fibroblast cells was examined. Base sequence analysis of 104 plasmids with mutations in the supF gene revealed that the majority of the mutations were base substitutions (85%) and the rest were frameshifts (15%). A single base substitution was most frequently found (47%), while 25% had multiple base substitutions and interestingly 13% had tandem (adjacent two) base substitutions. Of the base substitution mutations, 50% were G:C-->T:A transversions and 23% were G:C-->A:T transitions. The mutations were not distributed randomly but were located at several hotspots, most of which were G:C base pairs in 5'-AAGG-3' (or 5'-CCTT-3') sequences. Production of propanodeoxyguanosine adducts may be related to such specificity in the mutation spectrum.      

Search for Ha-ras codon 61 mutations in liver tumours caused by hexachlorobenzene and Aroclor 1254 in C57BL/10ScSn mice with iron overload.

C57BL/10ScSn mice administered iron--dextran and fed the environmental pollutants hexachlorobenzene (HCB) and polychlorinated biphenyls (PCBs) develop hepatic nodules and carcinomas within 18 months. A range of lesions from the livers were analysed for the presence of mutations in the Ha-ras proto-oncogene at codon 61 using the polymerase chain reaction to amplify DNA from formalin-fixed sections, followed by oligonucleotide hybridization. Only two mutations from 23 preneoplastic and neoplastic lesions induced by HCB were detected (a focus of altered cells and a trabecular cell carcinoma). With Aroclor 1254 no mutations were detected in 28 areas at various stages of carcinogenesis analysed. Sequencing of the two mutations generated by HCB showed a C-->T transversion at the first base of codon 61 (carcinoma) and an A-->T transversion at the second base (proliferative focus). Thus, in marked contrast to some other systems of mouse liver tumour induction, hepatocarcinogenesis caused by HCB and PCBs in C57BL/10ScSn mice is an example of carcinogenesis which does not involve a high frequency of Ha-ras gene mutation at codon 61.     

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

Analyses were performed on livers and hepatocellular carcinomasfrom male Fischer 344 rats fed a choline-devoid diet, to assesswhether they carried alterations of the p53 tumor suppressorgene. The analyses consisted of immunoperoxidase staining oftissue sections with monoclonal antibodies to p53, Western blottingand cDNA sequencing. Immunostaining revealed the presence ofmutant p53 proteins in 22/27 tumors analyzed and immunoblottingin 18/20. Immunochemical evidence was obtained that occurrenceof the mutations precedes tumor development. cDNA sequencingwas performed on 11 hepatocellular carcinomas that expressedmutant p53 gene proteins. Seven were found to contain pointmutations within the 120–290 codon region of the gene,and one a microdeletion in the same region. No mutational hotspot was observed. It is concluded that mutations within thep53 gene, along with a c-myc gene amplification previously detectedin these tumors, most likely contribute to the neoplastic transformationof liver cells in this nutritional model of hepatocarcinogenesis.The results are discussed also in view of recent literatureon the presence of p53 mutations in human hepatocellular carcinomas.     

Genetic analysis of lung tumours of non-smoking subjects: p53 gene mutations are constantly associated with loss of heterozygosity at the FHIT locus.

Lung cancer is strictly associated with tobacco smoking. Tumours developed in non-smoking subjects account for less than 10% of all lung cancers and show peculiar histopathological features, being prevalently adenocarcinomas. A number of genetic data suggest that their biological behaviour may be different from that of lung tumours caused by smoking, however the number of cases investigated to date is too low to draw definitive conclusions. We have examined the status of p53 and K-ras genes and the presence of loss of heterozygosity (LOH) at the FHIT locus in a series of 35 lung adenocarcinomas that developed in subjects who had never smoked. Results were compared with those obtained in a series of 35 lung adenocarcinomas from heavy-smoking subjects. In the group of non-smoking subjects p53 mutations and LOH at the FHIT locus were present in seven (20%) cases, and the two alterations were constantly associated (P < 0.0001), whereas they were not related in the series of carcinomas caused by smoking. In tumours developed in heavy-smoking subjects, the frequency of LOH at the FHIT locus was significantly higher (P = 0.006) than in tumours from non-smoking subjects. The frequency of p53 mutations in adenocarcinomas caused by smoking was not different from that seen in non-smoking subjects. However, in the group of smoking subjects we observed mostly G:C --> T:A transversions, whereas frameshift mutations and G:C --> A:T transitions were more frequently found in tumours from non-smoking subjects. No point mutations of the K-ras gene at codon 12 were seen in subjects who had never smoked, whereas they were present (mostly G:C --> T:A transversions) in 34% of tumours caused by smoking (P = 0.002). Our data suggest that lung adenocarcinomas developed in subjects who had never smoked represent a distinct biological entity involving a co-alteration of the p53 gene and the FHIT locus in 20% of cases.     

Ethionine-induced changes in rat liver transfer RNA methylation.

We have confirmed the finding by Rajalakshmi that transfer RNA (tRNA) from livers of ethionine-treated rats can act as a substrate for homologous tRNA-methylating enzymes in vitro. This methyl-deficient tRNA from liver can be methylated in vitro by enzymes from normal or ethionine-treated rats. The in vitro inhibition of tRNA methylation that follows ethionine treatment can be at least partially relieved in vitro. The liver extracts from ethionine-treated animals contained a low-molecular-weight inhibitor of tRNA methylation. Dialysis of enzyme preparations from ethionine-treated, but not control, rats resulted in large increases in tRNA methylase activity, with either Escherichia coli or homologous tRNA's as substrate. Furthermore, the tRNA methylase activity of control rat liver enzyme extracts was greatly depressed by dialysate from liver homogenates of ethionine-treated rats. After 5 days of ethionine administration the liver tRNA methylase activities were significantly higher than those of control preparations despite the continued presence of the dialyzable inhibitor(s). The liver tRNA's from these animals were poorer methyl acceptors than those from 3-day-treated rats, although still better than tRNA's from untreated rats. These observations have been interpreted to indicate that ethionine causes the accumulation in the liver of inhibitors of tRNA methylation. Early in the course of ethionine administration, methyl-deficient tRNA can be isolated. When the period of ethionine treatment is extended, the organism attempts to maintain homeostasis by production of increased amounts of tRNA-methylating enzymes. The increased quantities of these enzymes are able to overcome, at least partially, the effects of the inhibitors and to decrease the extent to which methyl-deficient tRNA is produced.     

Enhancement of DL-ethionine-induced liver carcinogenesis in rats fed a choline-devoid diet.

The effects of a choline-devoid (CD) or a choline-supplemented (CS) diet on the induction of liver tumors in rats by DL-ethionine were investigated. Groups of male outbred Sprague-Dawley rats were fed a plain CD or a plain CS diet, or the same diets containing 0.05% DL-ethionine. Hepatocellular carcinomas developed in 50% of the rats fed the CD+ethionine diet for 14 weeks and in about 80% of the rats fed the same diet for 22-30 weeks. No hepatocellular carcinomas developed in rats fed the CS+ethionine diet, the plain CD diet, or the plain CS diet up to 30 weeks. The findings suggest that a CD diet alters the response of rat liver to DL-ethionine and leads to an early and enhanced induction of hepatocellular carcinoma.     

High frequency of p53 gene mutations in primary breast cancers in Japanese women, a low-incidence population.

The pattern of acquired mutations in the p53 tumour-suppressor gene is potentially useful for determining factors contributing to carcinogenesis in diverse populations differing in incidence and/or mortality from the disease. We previously reported differences in mutational patterns of the p53 gene in primary breast cancers from Midwest US Caucasian, African-American and Austrian women. Herein, we report 16 mutations in 27 primary breast cancers from Japanese women from Hirosaki, a population with a low incidence of breast cancer. The frequency of 59.3% of p53 mutations is the highest reported in breast cancers from a particular ethnic group thus far. A relatively high number of mutations (7/16) were heterozygous in at least some tumour cell clusters. Intergroup comparisons of the mutational pattern between this population and several other US, European and Japanese populations do not show any statistically significant differences. There were recurrent mutations at two sites, codon 273 (R --> H; three mutations), a common hotspot of mutations in breast and other cancers, and codon 183 (S --> Stop; two mutations), a very rare location for p53 mutations. These mutations were shown to be independent and presumably not in the germ line. The highest frequency of p53 mutations raises the possibility that p53 mutagenesis is a predominant factor for breast cancer development in this low-risk Japanese group, whereas in other cohorts different mechanisms are likely to account for the higher proportion of breast cancer. Further studies are needed to confirm the present observations.     

p53 gene mutation analysis in tumors of patients exposed to alpha- particles

The p53 gene was examined for point mutations in archived, alpha- radiation-associated lung and liver cancers. Lung tumors of 50 uranium miners in Germany were screened by restriction fragment length analysis for the putative hotspot mutation at codon 249 (Arg-->Met) previously detected in a significant fraction of miners from the Colorado Plateau, USA. This mutation has been proposed as a marker of radon exposure. None of the tumors we examined harbored the hotspot mutation. Five of the 50 tumors, however, did indeed harbor exon 7 mutations, as determined by subsequent mutation analysis of exon 7. These mutations were dispersed among various codons and may be attributable to heavy tobacco smoking in this cohort. In support of this interpretation, we found no mutations in exons 5-8 of the p53 gene in 13 iatrogenic liver cancers induced by injection of Thorotrast, an alpha-emitting radiocontrast agent. We propose that if the p53 tumor suppressor gene is a target for the carcinogenic action of alpha-particle radiation, loss of suppressor function may occur preferentially by mechanisms such as intrachromosomal deletions, rather than by base substitution mutations.      

p53-stabilizing Agent CP-31398 Prevents Growth and Invasion of Urothelial Cancer of the Bladder in Transgenic UPII-SV40T Mice

The high prevalence of bladder cancer and its recurrence make it an important target for chemoprevention. About half of invasive urothelial tumors have mutations in p53. We determined the chemopreventive efficacy of a p53-stabilizing agent, CP-31398, in a transgenic UPII-SV40T mouse model of bladder transitional cell carcinoma (TCC) that strongly resembles human TCC. After genotyping, six-week-old UPII-SV40T mice (n = 30/group) were fed control (AIN-76A) or experimental diets containing 150 or 300 ppm of CP-31398 for 34 weeks. Progression of bladder cancer growth was monitored by magnetic resonance imaging. At 40 weeks of age, all mice were killed; urinary bladders were collected to determine weights, tumor incidence, and histopathology. There was a significant increase in bladder weights of transgenic versus wild-type mice (male: 140.2 mg vs 27.3 mg, P < .0001; female: 34.2 mg vs 14.8 mg, P < .0001). A significant decrease in the bladder tumor weights (by 68.6–80.2%, P < .0001 in males and by 36.9–55.3%, P < .0001 in females) was observed in CP-31398-treated mice. Invasive papillary TCC incidence was 100% in transgenic mice fed control diet. Both male and female mice exposed to CP-31398 showed inhibition of invasive TCC. CP-31398 (300 ppm) completely blocked invasion in female mice. Molecular analysis of the bladder tumors showed an increase in apoptosis markers (p53, p21, Bax, and Annexin V) with a decrease in vascular endothelial growth factor in transgenic mice fed CP-31398. These results suggest that p53-modulating agents can serve as potential chemopreventive agents for bladder TCC.     

Early histological and functional alterations of ethionine liver carcinogenesis in rats fed a choline-deficient diet.

The effects of feeding a choline-deficient (CD) or a choline-supplemented diet upon the early stages of DL-ethionine carcinogenesis in rat liver were investigated. Low levels of DL-ethionine (0.05 and 0.10%) when fed with a CD diet were found to induce within 4 weeks a massive proliferation of oval cells without significant cell necrosis or presence of inflammatory cell infiltrates. The same levels of ethionine when fed with a choline-supplemented diet caused no significant histological alteration of the liver. In rats fed the CD plus ethionine diets concomitant with the proliferation of oval cells, there was a marked elevation in the content of alpha1-fetoprotein in both liver and plasma. After specific immunofluorescence staining, oval cells stained intensely for albumin and alpha1-fetoprotein. Hepatocytes stained only for albumin, and bile duct cells stained for neither albumin nor alpha1-fetoprotein. These results indicate that a diet deficient in choline markedly alters the response of rat liver to carcinogenetic doses of ethionine. Thus, ethionine hepatocarcinogenesis in rats fed a CD diet may be a useful model for the exploration of the mechanism(s) whereby a dietary factor influences hepatocarcinogenesis.     

Dietary fish oil (MaxEPA) enhances pancreatic carcinogenesis in azaserine-treated rats.

In the present study the putative chemopreventive effect of dietary fish oil (MaxEPA) on azaserine-induced pancreatic carcinogenesis in rats was investigated. Groups of rats were maintained on a semipurified low-fat (LF; 5 wt%) diet or on semipurified high-fat (HF; 25 wt%) diets containing 5 wt% linoleic acid (LA) and including 0.0, 1.2, 2.4, 4.7, 7.1 or 9.4 wt% MaxEPA. Animals fed a HF diet developed significantly higher mean numbers of atypical acinar cell nodules (AACNs), adenomas and carcinomas than animals fed a LF diet. Dietary MaxEPA caused a significant (P < 0.01) dose-related increase in mean number of AACNs (0.5 < phi < 3.0 mm). The mean number of adenomas and carcinomas remained similar among the groups. Cell proliferation was significantly lower in AACNs from animals fed HF containing 9.4% MaxEPA in comparison with HF without MaxEPA and with LF. LA levels had increased and arachidonic acid (AA) levels had decreased in blood plasma and pancreas with increasing dietary MaxEPA. Feeding MaxEPA resulted in significant decreases in 6-keto-prostaglandin (PG) F1 alpha (P < 0.05) and PGF2 alpha (P < 0.01) in non-tumorous pancreas, whereas PGE2, PGF2 alpha and thromboxane B2 (TXB2) levels were significantly (P < 0.001) higher in pancreatic tumour tissue than in non-tumorous pancreatic tissue. It is concluded that (i) dietary MaxEPA enhances dose-relatively growth of putative preneoplastic AACNs in the pancreas of azaserine-treated rats; (ii) dietary MaxEPA inhibits the conversion of LA to AA, as well as the conversion of AA to TXB2 or PGF2 alpha in non-tumorous pancreatic tissue; (iii) the high levels of PGE2, PGF2 alpha and TXB2 in pancreatic adenocarcinomas indicate a possible role for these eicosanoids in modulation of tumour growth.     

Ultraviolet-light induced p53 mutational spectrum in yeast is indistinguishable from p53 mutations in human skin cancer

Ultraviolet (UV) light has been associated with the development of human non-melanoma skin cancers (NMSC). Such cancers often exhibit mutations in the p53 tumour suppressor gene. In order to determine the UV-induced p53 mutation spectrum, a yeast expression vector that harbours a human wild-type p53 cDNA was UV-irradiated in vitro and transfected into a yeast strain that contained the ADE2 gene regulated by a p53-responsive promoter. Forty-five mutant clones contained 51 mutations. Seven mutations were tandem base pair substitutions, four of which being CC-->TT, hallmark mutations of UV mutagenesis. Eighty percent (41/51) of the mutations were single or non-tandem base pair substitutions, the majority of which (27/41) were C-->T transitions. Ninety-five percent of such mutations occurred at dipyrimidine sites. Through a rigorous statistical test, the UV-induced p53 mutation spectrum appears to differ significantly (P < 0.008) from the one induced by the antineoplastic drug chloroethyl-cyclohexyl-nitrosourea, and to be indistinguishable from the one observed in NMSC (P = 0.4). These results demonstrate that the assay allows the determination of carcinogen-specific p53 mutation fingerprints and represents a new tool for molecular epidemiology.      

K-ras mutation in colorectal cancer: relations to patient age, sex and tumour location.

DNA from 251 primary tumours obtained from 123 male and 125 female Norwegian patients with colorectal carcinoma was analysed for the presence of K-ras point mutations at codons 12 and 13. Mutations were found in 99 (39%) of the samples. The frequency of K-ras mutations was significantly related to age and sex of the patients, and to the location of the tumours (overall: P = 0.008). K-ras mutations were much less frequent in colonic tumours from male than female patients at younger ages (< 40 years, odds ratio < 0.014). The low frequency might indicate that a different, ras-independent, pathway to neoplasia is dominating in the colon of younger males. In contrast, older men had more mutations than older women (e.g. 90 years, odds ratio = 5.8). An inverse but less pronounced relationship was seen for rectal tumours. The type of mutation was found to be associated to sex of patient and location of tumour. G-->C transversions accounted for 35% of the mutations in rectal tumours from females, in contrast to only 2.5% in the rest of the material (P = 0.0005). This may indicate that there are specific carcinogens acting in this location.     

The effect of choline and methionine deficiencies on the number and volume percentage of altered hepatic foci in the presence or absence of diethylnitrosamine initiation in rat liver

The ability of methyl-deficient, amino-acid-defined diets to produce enzyme-altered foci was quantitatively determined in the livers of rats treated both with and without an initiating dose of diethylnitrosamine (DEN). Male weanling F-344 rats were fed a complete, amino-acid-defined diet for 1 week. They were then injected i.p. with a single dose of DEN (20 mg/kg body weight) and fed the complete diet for an additional week. Forty animals in each dose group were then maintained for 5-38 weeks on the complete diet (diet 1) or one of the three methyl-deficient diets customarily used in this laboratory: diet 2, devoid of methionine and choline; diet 3, devoid of methionine only; and diet 4, devoid of choline only. In diets 2 and 3, methionine was replaced by equimolar amounts of its metabolic precursor, DL-homocystine. Ten animals per group were killed 8, 12, 17, 24 and 41 weeks after DEN initiation. For 2 weeks prior to being killed, each group was maintained on the complete diet to minimize the histological abnormalities due to acute toxicity of the diets. Serial sections of the livers were obtained, stained sequentially for gamma-glutamyltranspeptidase, ATPase and glucose-6-phosphatase, and the quantitation of the focal lesions scored by these markers was carried out by quantitative stereology. The results indicated that, regardless of the enzyme marker(s) examined, there was a general correspondence between the volume and number of altered hepatic foci (AHF) formed and the previously described tumor-promoting activities of each diet. Thus, while all DEN-treated groups contained significant numbers of AHF 24 weeks after initiation, only the diet-2-fed animals displayed such foci at 8 weeks. Similarly, among the uninitiated rats, only those fed diet 2 exhibited the presence of AHF throughout the experimental period. Interestingly, the livers of uninitiated, choline-deficient rats showed a small number of AHF at 24 and 42 weeks; these foci were not observed at all in the corresponding DEN-untreated animals fed diet 3, deficient in methionine only. The results provide evidence that the carcinogenic effects of the methionine- and choline-deficient diet result more from its strongly promoting effect than from any initiating activity by the diet.