The effect of Helicobacter pylori infection on levels of DNA damage in gastric epithelial cells.

BACKGROUND: Helicobacter pylori infection leads to an increased risk of developing gastric cancer. The mechanism through which this occurs is not known. We aimed to determine the effect of H. pylori and gastritis on levels of DNA damage in gastric epithelial cells. METHODS: Epithelial cells were isolated from antral biopsies from 111 patients. DNA damage was determined using single cell gel electrophoresis and the proportion of cells with damage calculated before and 6 weeks after eradication of H. pylori. Cell suspensions generated by sequential digestions of the same biopsies were assayed to determine the effect of cell position within the gastric pit on DNA damage. RESULTS: DNA damage was significantly higher in normal gastric mucosa than in H. pylori gastritis [median (interquartile range) 65% (58.5-75.8), n = 18 and 21% (11.9-29.8), n = 65, respectively, p <.001]. Intermediate levels were found in reactive gastritis [55.5% (41.3-71.7), n = 13] and H. pylori negative chronic gastritis [50.5% (36.3-60.0), n = 15]. DNA damage rose 6 weeks after successful eradication of H. pylori[to 39.5% (26.3-51.0), p =.007] but was still lower than in normal mucosa. Chronic inflammation was the most important histological factor that determined DNA damage. DNA damage fell with increasing digestion times (r = -.92 and -.88 for normal mucosa and H. pylori gastritis, respectively). CONCLUSIONS: Lower levels of DNA damage in cells isolated from H. pylori infected gastric biopsies may be a reflection of increased cell turnover in H. pylori gastritis. The investigation of mature gastric epithelial cells for DNA damage is unlikely to elucidate the mechanisms underlying gastric carcinogenesis.

A potential mechanism for fumonisin B(1)-mediated hepatocarcinogenesis: cyclin D1 stabilization associated with activation of Akt and inhibition of GSK-3beta activity.

Fumonisin B(1) (FB(1)) is a worldwide corn contaminant and has been epidemiologically linked to the high incidence of human esophageal cancer in South Africa and China. FB(1) is hepatocarcinogenic in rats by an unknown mechanism. Inhibition of ceramide synthase and disruption of membrane phospholipids have been shown to be mechanisms of toxicity. Here we show overexpression of cyclin D1 protein in both preneoplastic and neoplastic liver specimens obtained from a long-term feeding study of FB(1) in rats. In rats fed FB(1) short-term, cyclin D1 protein levels in liver were increased up to five-fold in a dose-responsive manner. Northern blot analysis demonstrated no increase in mRNA levels of cyclin D1. 2D electrophoresis of cyclin D1 protein in FB(1)-treated samples showed a distinct pattern of migration (presence of less negatively charged form of the protein) that differed from controls. Recently, it has been shown that phosphorylation of cyclin D1 by glycogen synthase kinase 3beta (GSK-3beta) on a single threonine residue (Thr-286) positively regulates proteosomal degradation of cyclin D1. In FB(1)-treated samples we detected GSK-3beta phosphorylated on serine 9; activated protein kinase B (Akt) appears to be responsible for this activity-inhibiting phosphorylation. These findings suggest that overexpression of cyclin D1 results from stabilization due to a lack of phosphorylation mediated by GSK-3beta. We also observed an increase in cyclin dependent kinase 4 (Cdk4) complexes with cyclin D1 in FB(1)-treated samples; additionally, elevated Cdk4 activity was shown by increased phosphorylation of the retinoblastoma protein. In summary, the activation of Akt leads to increased survival, inhibition of GSK-3beta activity and post-translational stabilization of cyclin D1, all events responsible for disruption of the cell cycle G(1)/S restriction point in hepatocytes. This is the first report suggesting the mechanism by which FB(1) acts as a carcinogen.

In vivo DNA damage in gastric epithelial cells.

A number of risk factors have been linked epidemiologically with gastric cancer, but studies of DNA damage in gastric epithelial cells are limited. The comet assay is a simple technique for determining levels of DNA damage in individual cells. In this study, we have validated the comet assay for use in epithelial cells derived directly from human gastric biopsies, determined optimal conditions for biopsy digestion and investigated the effects of oxidative stress and digestion time on DNA damage. Biopsies taken at endoscopy were digested using combinations of pronase and collagenase, ethylenediaminetetra-acetic acid (EDTA) and vigorous shaking. The resultant cell suspension was assessed for cell concentration and epithelial cell and leukocyte content. A score for DNA damage, the comet %, was derived from the cell suspension, and the effect of various digestion conditions was studied. Cells were incubated with H(2)O(2) and DNA damage was assessed. Pronase and collagenase provided optimum digestion conditions, releasing 1. 12x10(5) cells per biopsy, predominantly epithelial. Of the 23 suspensions examined, all but three had leukocyte concentrations of less than 20%. The comet assay had high inter-observer (6.1%) and inter-assay (4.5%) reproducibility. Overnight storage of the biopsy at 4 degrees C had no significant effect on DNA migration. Comet % increased from a median of 46% in untreated cells to 88% in cells incubated for 45 min in H(2)O(2) (p=0.005). Serial 25-min digestions were performed on biopsies from 13 patients to release cells from successively deeper levels in the crypt. Levels of DNA migration were significantly lower with each digestion (r=-0.94, p<0.001), suggesting that DNA damage is lower in younger cells released from low in the gastric crypt. The comet assay is a reproducible measure of DNA damage in gastric epithelial cells. Damage accumulates in older, more superficial cells, and can be induced by oxidative stress.

Microsatellite instability is infrequent in azoxymethane-induced rat intestinal tumors: An assessment by capillary electrophoresis.

A rat model of colon cancer in which tumors are induced by azoxymethane (AOM) is frequently used to study putative environmental agents that may modify the risk of human colon cancer development. In order to evaluate the usefulness of this model for human risk assessment, a comparison of the molecular changes associated with tumorigenesis in the rat model with those in human colon cancer is desirable. Microsatellite instability (MSI), an alteration in length of short repetitive DNA sequences associated with defective DNA mismatch repair, is an important molecular characteristic of many human colon tumors. Intestinal tumors were induced in male Fischer 344 rats injected with 15 mg/kg body wt AOM in four weekly doses. Thirteen intestinal tumors were examined for MSI at 10 different microsatellite loci, using a capillary electrophoresis (CE) method for accurate assessment of DNA length. This method was shown to have a resolution of 1 bp for a 140-bp PCR product and to be capable of detecting one mutant sequence within a background of 10 wild-type sequences. The CE method also readily distinguished a known MSI-positive human tumor sample from its matching control sample. Among the 13 rat intestinal tumors examined, only one had MSI, which was present at only a single locus. We conclude that, unlike sporadic human colon tumors in which 15-30% of tumors have MSI (usually at multiple loci), MSI is very rare in AOM-induced rat intestinal tumors.

Effect of peanut oil and randomized peanut oil on cholesterol and oleic acid absorption, transport, and distribution in the lymph of the rat.

Peanut oil was shown to be atherogenic in cholesterol-fed rats, rabbits, and monkeys. However, after randomization, a process in which the fatty acids in peanut oil are randomly rearranged, its atherogenicity was significantly reduced in cholesterol-fed rabbits and monkeys. The mechanism for this effect remains unknown. This study was designed to investigate whether the absorption, transport and distribution of dietary cholesterol and oleic acid in the lymph were altered in the presence of peanut oil or randomized peanut oil. Previous investigators collected lymph through the mesenteric duct for 6 h and analyzed lymph for cholesterol. In the present study, lymph fluids were collected at timed intervals for up to 8 h and then at 24 h via the thoracic duct. Cholesterol and oleic acid (fatty acid) were estimated not only in the whole lymph but also in lymph lipoprotein fractions and in major lipid fractions. A 24-h lymph collection will enhance accuracy as short-term fluctuations in lipid absorption will not affect the results. Thoracic duct lymph collection is quantitative compared to mesenteric duct lymph collection, which provides only a fraction of the total lymph. Rats were given a lipid emulsion containing either peanut oil or randomized peanut oil. The emulsion also contained cholesterol, oleic acid, and sodium taurocholate in saline and was given through a duodenal catheter. Results show that absorption, transport, and distribution of cholesterol and oleic acid in the lymph fluids were similar in both dietary groups. These results suggest that the atherogenicity of peanut oil may be due to other events taking place subsequent to the release of cholesterol-containing chylomicrons and very low density lipoprotein by the small intestinal epithelial cells into the blood or may be due to the triglyceride structure itself.

Polymerase chain reaction-single-strand conformation polymorphism analysis for the VHL gene in chemically induced kidney tumors of rats using intron-derived primers.

von Hippel-Lindau (VHL) gene mutations occur throughout three exons including the exon-intron boundaries in human VHL disease-associated and sporadic renal cell carcinomas. To explore the possible role of the VHL gene in chemically induced rat kidney tumors originating from various cell types, more than 150 bp of Fischer 344 and Noble rat VHL intron sequences flanking the three exons was determined by dideoxy sequencing. Five primer sets were selected for polymerase chain reaction amplification of the coding regions of rat VHL exons 1-3 and the exon-intron boundaries. Tissues from 10 renal eosinophilic epithelial tumors induced by N-nitrosoethyl(2-hydroxyethyl)amine, 10 nephroblastomas induced by N-nitroso-N-ethylurea, and seven renal mesenchymal tumors induced by N-nitrosomethyl(methoxymethyl)amine were examined for VHL mutations by polymerase chain reaction-single-strand conformation polymorphism analysis. No mutation was detected in any tumor type, indicating that VHL mutations are not involved in the pathogenesis of rat kidney tumors arising from the distal region of the renal tubules, the metanephric blastema, or stromal tissues of the cortex.

Detection of mutant p53 in clam leukemia cells.

Leukemia in the soft-shell clam, Mya arenaria, is characterized by tumor cells which are detected initially in the hemolymph. This disease is much more common in clams inhabiting polluted waters, suggesting an environmental component to its pathogenesis. In this study, leukemia cells were identified using a murine monoclonal antibody, 1E10, which recognizes a leukemia-specific protein expressed by tumor cells. Mutant p53 protein was detected using a murine monoclonal antibody (PAb 240) which reacts with mutant p53. Using immunofluorescence, the reactivity of clam cells to the 1E10 antibody was evaluated along with mutant p53 protein reactivity. Reverse transcriptase-polymerase chain reactions followed by sequence analyses were utilized to examine clams with hemocytes reacting with the p53 antibody for possible p53 gene mutations. Mutant p53 protein was expressed by tumor cells from five animals with advanced disease (in which greater than 90% of cells reacted with 1E10). A C-->G transversion was detected at the end of exon 6 from two of the five animals that reacted with both the mutant p53 antibody and 1E10. This substitution changes the amino acid of this codon from proline to alanine. Overall, our results suggest that environmentally induced alterations in p53 can contribute to the pathogenesis of leukemia in soft-shell clams inhabiting polluted water and/or sediment.

Mutated and wild-type p53 expression and HPV integration in proliferative verrucous leukoplakia and oral squamous cell carcinoma.

The frequencies of overexpression and mutation in the p53 tumor suppressor gene were examined in proliferative verrucous leukoplakia and oral squamous cell carcinoma with immunohistochemistry and single-strand conformation polymorphism analysis of DNA fragments amplified by polymerase chain reaction. Ten samples each of normal oral mucosa, proliferative verrucous leukoplakia, and squamous cell carcinoma were immunostained with antibodies against p53 protein; 8 of 10 cases of proliferative verrucous leukoplakia cases and 7 of 10 cases of oral squamous cell carcinoma were positive for p53 protein. Minimal staining was observed in normal oral tissues. The quantified labeling indexes demonstrated a range that corresponded to lesion progression. Single-strand conformation polymorphism analysis revealed p53 gene mutations within exons 5 to 8 in 40% (4 of 10) of the squamous cell carcinoma samples. Two of the 4 mutated squamous cell carcinoma samples lacked p53 expression. No p53 mutations were detected in proliferative verrucous leukoplakia tissues. Human papillomavirus 16 was identified in 2 of 7 p53 positive oral squamous cell carcinoma samples. Human papillomavirus 16 and 18 were identified in two of eight p53 positive proliferative verrucous leukoplakia samples. One p53 negative squamous cell carcinoma sample was positive for human papillomavirus 16 and had a mutation in exon 6 of the p53 gene. Human papillomavirus infection along with p53 expression plays a yet to be defined role in the pathogenesis of a limited number of cases of proliferative verrucous leukoplakia and squamous cell carcinoma. p53 immunohistochemistry, p53 gene mutations, and human papillomavirus infection prevalence do not provide a means to differentiate between leukoplakia and carcinoma and do not provide a predictive test for progression of leukoplakia to carcinoma.

Lack of p53 and ras mutations in Helicobacter hepaticus-induced liver tumors in A/JCr mice.

Helicobacter hepaticus is a recently discovered bacterium that invades mouse liver causing chronic active hepatitis followed by development of preneoplastic hepatocellular foci, hepatocellular adenomas and carcinomas. This establishes a unique animal model for study of the mechanisms of cancer development due to a chronic bacterial infection. A possible mechanism of bacteria-associated tumorigenesis is mutation of oncogenes or tumor suppressor genes. Since mutations in ras oncogenes have been widely detected in a variety of chemically induced and spontaneous mouse liver tumors and specific mutations in the p53 tumor suppressor gene have been associated with human bladder cancers attributed to chronic schistosomal infection, we studied exons 1 and 2 of the N-, K- and H-ras genes and exons 5-8 of the p53 gene for the presence of point mutations in 25 liver tumors from 10 naturally infected A/JCr mice, ranging in age from 16 to 24 months. The 20 adenomas and five carcinomas varied in size from 0.1 to 2.3 cm and arose in livers characterized by a wide assortment of pathological profiles, including hepatitis, inflammation, hyperplasia, hypertrophy, leukocyte infiltration, necrosis and focal phenotypic alteration. DNA samples extracted from formalin-fixed paraffin-embedded tissues were screened by PCR/SSCP analysis and showed no mutations in the analyzed genes. Complete absence of mutations in ras genes in 25 mouse liver tumors is unusual. Other genes may be targeted or H. hepaticus infection causes liver cancer through other pathways than direct damage to DNA.

K-ras codon 12 and 61 point mutations in bromodeoxyuridine- and N-nitrosomethylurea-induced rat renal mesenchymal tumors.

The mutagenic thymidine analog bromodeoxyuridine (BrdUrd) may incorrectly incorporate opposite deoxyguanine in DNA, then pair with deoxyadenosine during subsequent replication. It appears to preferentially target the 3'-G of 5'-NGGN-3' sequences in mammalian cells in culture to induce G-->A transitions. Ras genes should therefore be vulnerable to activation by mutation at glycine codons 12 (GGT) and/or 13 (GGC) by misincorporation of BrdUrd. There is limited evidence that BrdUrd may be carcinogenic or co-carcinogenic in rats: three renal mesenchymal tumors, a tumor known to be associated with activating mutations in the c-K-ras-2 oncogene, were reported in 87 rats treated with BrdUrd alone, while N-nitrosomethylurea (NMU) alone or NMU + BrdUrd resulted in incidences of 12/52 and 26/76, respectively, against a zero incidence in untreated rats. We analyzed renal mesenchymal tumors from rats treated with BrdUrd for mutations in K-ras exons 1 and 2 and compared the prevalence and spectrum of mutations with those found in comparable tumors induced with NMU. DNAs from 22 paraffin-embedded renal mesenchymal tumors from rats treated 12-15 months earlier with BrdUrd (three specimens) or NMU (11 specimens) or both agents sequentially (eight specimens) were amplified by PCR. The base sequence of codons 12-13 and 59-63 of K-ras was determined by the dideoxynucleotide method. Sequencing results were confirmed by allele-specific oligonucleotide hybridization. Two of three tumors that appeared in rats given BrdUrd alone contained both a codon 12 GGT-->GAT transition and a codon 61 CAA-->CTA transversion. One tumor induced by NMU alone also showed a codon 12 GGT-->GAT mutation, while only wild type sequence could be demonstrated in the codon 12-13 region in the remaining ten such tumors. Three NMU-induced tumors also showed codon 61 CAA-->CTA mutations, while the remaining tumors had wild type sequence. While the GGT-->GAT transitions identified in tumors from BrdUrd-treated rats are consistent with BrdUrd mutagenesis by misincorporation, the co-occurrence of CAA-->CTA transversions, the overall low prevalence of mutations, and the lack of any difference in mutation spectrum between tumors induced by NMU and those that occurred in BrdUrd-treated rats suggests that in both groups the mutations that did occur did not result from a direct effect of either agent.

Transplacental mutagenicity of cisplatin: H-ras codon 12 and 13 mutations in skin tumors of SENCAR mice.

Cisplatin is an anticancer agent sometimes used in pregnant women. It is also a potent initiator of skin tumors in mice when administered transplacentally. For characterization of the transplacental mutagenicity of cisplatin, tumors initiated in fetal skin by cisplatin or 7,12-dimethylbenz[a]anthracene (DMBA) and promoted by postnatal 12-O-tetradecanoyl-phorbol-13-acetate (TPA) were analyzed for H-ras mutations by 'cold' single-strand conformation polymorphism analysis and direct sequencing. The expected high incidence of exon II codon 61 mutations (20/20) was found in transplacental DMBA-initiated tumors, with no exon I change. By contrast, 6/10 cisplatin tumors had seven mutations in codons 12 or 13 of exon I, all at GpG dinucleotides. Four of these were unique codon 13 GGC --> GTC changes, significantly different from the DMBA group and from historical TPA-only controls. The activation of codons 12 and 13 by cisplatin is in accord with the known in vitro preference of cisplatin for GpG sites for intrastrand cross-linking adduct formation. These results provide the first evidence that cisplatin can act transplacentally to cause specific mutations in fetal skin that are not seen in skin tumors caused by treatment of adult skin with this agent. This is evidence for unique molecular fetal carcinogenic pathways and underscores concern about human fetal risk due to maternal cisplatin treatment.

Histogenesis and the role of p53 and K-ras mutations in hepatocarcinogenesis by glyceryl trinitrate (nitroglycerin) in male F344 rats.

Glyceryl trinitrate (GTN) was previously reported to induce hepatocellular carcinoma (HCC) in rats after prolonged feeding. The present experiments were undertaken to evaluate the histogenesis and molecular biology of these tumors and the possible role of nitric oxide (NO), a GTN metabolite, in their development. Male F344 rats received a single i.g. intubation of GTN (1.2 g/kg) at 6 weeks of age and/or a diet containing 1% GTN from 8 weeks of age until necropsy, i.e. for up to 78 weeks. Some animals were subjected to 2/3 partial hepatectomy (PH) at 9 weeks of age. Five sequential sacrifices (14, 32, 52, 78 and 84 weeks of age) were performed. No liver tumors developed in control rats or in rats that received GTN only by a single i.g. intubation, even when intubation was followed by PH. Preneoplastic foci, mainly of clear cell and mixed cell type (identified as positive for glutathione S-transferase placental form) were found from 14 weeks of age in rats receiving GTN in the diet. Focal eosinophilic areas (atypical foci) composed of atypical hepatocytes that often extended into the veins were observed beginning at 52 weeks of age. Some mixed hepatocholangiocellular adenomas and carcinomas arose in eosinophilic lesions. HCCs were seen beginning at 78 weeks of age, but only in rats receiving dietary GTN. Incidence of HCC in the latter animals was 50-75%. Most HCCs were well differentiated. The carcinogenic effect of GTN given in the diet was not affected by prior intubation of a large single dose followed by PH. No p53 mutations were found in 18 tumors but K-ras point mutations, all within codon 12, were found in 8/18 tumors, mostly those with cholangiocellular elements. These were first or second position G-->T transversions or second position G-->A transitions. While these mutation types have also been commonly seen in bacteria after NO-related DNA damage, the fact that tumors arose only on prolonged feeding of this potently bioactive agent at massive doses seems consistent with a more complex mechanism involving multiple (i.e. genetic and/or epigenetic) factors in carcinogenesis by GTN.

The significance of B-cell clonality in gastric lymphoid infiltrates.

The significance of the demonstration of a clonal B-cell population in gastric lymphoid infiltrates was investigated by analysis of immunoglobulin heavy chain (IgH) gene rearrangements using sensitive polymerase chain reactions, employing fluorescently labelled primers to target the FR3 and FR1 regions. Tissue blocks were studied showing different histological features (high-grade lymphoma, low-grade lymphoma, and chronic gastritis) from 12 gastrectomies for primary gastric lymphoma, together with blocks showing chronic gastritis from 13 cases of gastric adenocarcinoma and biopsies from 33 patients with active Helicobacter-associated chronic gastritis. Clonal IgH gene rearrangements were detected in lymphoma samples from eight of the gastrectomies for lymphoma (67 per cent). In four of these eight specimens, clonal rearrangements were also detectable in the samples showing only chronic gastritis. Three of 28 (11 per cent) informative biopsies showing active Helicobacter-associated chronic gastritis had detectable clonal populations. Clonal rearrangements were also demonstrated in two of eight (25 per cent) informative blocks showing chronic gastritis from eight gastrectomies for adenocarcinoma. It is concluded that the detection of a clonal population in a suspicious lymphoid infiltrate does not confirm the diagnosis of lymphoma, nor does the absence of such a population imply benignity.

Mutation in the p53 tumor suppressor gene in rat esophageal papillomas induced by N-nitrosomethylbenzylamine.

Mutations in the p53 tumor suppressor gene have been implicated in the pathogenesis of a wide variety of human neoplasms. The location and types of p53 gene mutation can reflect exposure of humans to certain types carcinogenic agents. Much less is known about the role of p53 mutational inactivation in rodent tumors. Using both 'Hot' (radioactive) and 'Cold' (non-radioactive) single strand conformation polymorphism (SSCP) analyses, the present study of analyzed exons 5-8 and the exon-intron junction of the p53 gene from rat esophageal papillomas induces by N- nitrosomethylbenzylamine (NMBA) for mutations. Nine of 30 (30%) esophageal papillomas contained SSCP mobility shifts, principally within exons 5 and 7. These positive SSCP findings were further validated by direct DNA sequencing analysis. Eight of the nine mutations were G:C-->A:T transitions in codons 131, 149, 153, 242 (2), 243, 248, and the 5 end of intron 7. None of these G:C-->A:T mutations occurred at the CpG sites. The other mutation was a frameshift mutation in codon 176. The G:C-A:T transitions observed in this study are consistent with the documented formation of O(6)-methylguanine adducts in DNA N-nitroso compounds. These results suggest that point mutations of the p53 gene are involved in the development of approximately one-third of NMBA-induced rat esophageal papillomas. 'Hot' and 'Cold' SSCP methods were equally sensitive for the identification of mutations in the rat p53 gene.

Effect of sesame oil on serum and liver lipid profiles in the rat.

In our previous study (Satchithanandam, S., Reicks, M., Calvert, R.J., Cassidy, M.M. and Kritchevsky, D. (1993) J. Nutr. 123, 1852-1858), we found that the absorption of lymphatic cholesterol by rats fed diets containing 24% sesame oil was about 50% less than that by rats fed the control diet containing no sesame oil. The effect of sesame oil on serum cholesterol levels was not determined at that time. In the present study, three groups of male Wistar rats (75-100 g) were fed a control diet or a diet containing 12 or 24% sesame oil. To increase serum cholesterol levels, 1% cholesterol and 0.5% cholic acid were added to each diet. After rats were fed for 4 weeks, total cholesterol, LDL-cholesterol, HDL-cholesterol, and triglyceride levels were measured in the serum. Liver weight and cholesterol and triglyceride levels were determined. Liver cholesterol levels were significantly lower in rats fed the 24% sesame oil diet, and the liver lipid level was significantly higher in the 24% sesame oil-fed group, compared with levels in the group fed the control diet. Liver weights and esterified cholesterol and liver triglyceride levels were not significantly different among the groups. Levels of serum total cholesterol and LDL-cholesterol were significantly lower in rats fed the 24% sesame oil diet, compared with levels in the control group. Serum triglyceride and HDL-cholesterol levels did not differ significantly among the groups. The mechanism by which a diet containing 24% sesame oil reduces levels of serum and liver cholesterol, liver LDL cholesterol, and liver lipids is not known. However, the high degree of unsaturation (85%) of sesame oil and the presence of linoleic acid may be important factors.

Cloning and sequence of a processed p53 pseudogene from rat: a potential source of false 'mutations' in PCR fragments of tumor DNA.

We describe here the nucleotide (nt) sequence of a p53 processed pseudogene (psi-gene) from the normal F344 rat genome. Exon-derived primers were utilized to amplify and clone a 1447-bp polymerase chain reaction (PCR) product corresponding to the coding regions of exons 2-11 of the functional gene. This psi-gene is a cDNA-like sequence possessing 87% homology with the functional rat p53. We have also partially characterized two additional and distinctly different putative rat p53 psi-genes, focussing on the sequences surrounding the reported rat p53 mutational hot spots of codons 202R and 211R within exon 6/7. Each of these three psi-gene sequences contained various single- and/or double-nt substitutions, small deletions and insertions that distinguish them from p53. One substitution, 211R CGG-->CAG, found both in the cloned psi-gene and in one of the partially characterized, putative psi-genes, corresponded precisely with the sequence that has been reported as a mutation at one of the hot spots. Co-amplification of one or more of the p53 psi-genes with portions of the functional p53 is likely, if exon-based primers are utilized for PCR amplification of rat p53. Consequently, psi-gene sequences are potential sources of sequence variations that can be misidentified as somatic cell mutations by direct sequencing of inappropriately generated PCR products.

Lack of p53 point mutations in chemically induced mouse hepatoblastomas: an end-stage, highly malignant hepatocellular tumor.

Inactivation of the p53 tumor suppressor gene appears to be an important event in the progression of many types of human neoplasms; however its role in rodent experimental tumorigenesis is controversial. Previous studies have shown that a wide array of chemically induced and spontaneous mouse liver tumors lack p53 mutations within the evolutionarily conserved regions of exons 5-8. However, since p53 inactivation in human neoplasms occurs relatively late in tumor progression, it is possible that the mouse liver tumors evaluated previously were not suitably advanced to incur p53 aberrations. In the present study, we examined an end-stage, highly malignant embryonal mouse liver tumor known as the hepatoblastoma (HB) for p53 mutations utilizing the highly sensitive 'cold' single-strand conformation polymorphism (SSCP) technique. In addition, several of the HBs were examined by direct nucleotide sequencing. No aberrations of the p53 gene were detected within exons 5-8 of any of the 16 HBs examined. These results confirm that the p53 gene plays a minimal role in the development or malignant progression of hepatocellular tumors in mice.