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C M Weghorst

Publications and source records attributed to C M Weghorst.

At least 37 records · Page 2Linked to original sources
PubMed1997

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.

Adenoma↗
PubMed1997

Mutation and downregulation of the transforming growth factor beta type II receptor gene in primary squamous cell carcinomas of the head and neck.

In the present study, we analyzed 28 squamous cell carcinomas of the head and neck (SCCHN) for mutations in the coding region of TbetaR-II using 'Cold' SSCP and automatic DNA sequencing analyses. Twenty-one percent (6/28) of the SCCHN examined contained TbetaR-II mutations compared with patient-matched normal tissues. These alterations included five missense mutations (A:T-->G:C transitions in codons 250, 401, 448 and 488, and a G:C-->T:A transversion in codon 373), and a 38-bp deletion between nucleotides 1825 to 1862. In addition to these code-altering mutations, one case exhibited a silent mutation (A:T-->G:C transition in codon 451) and three cases contained one of two potential population polymorphisms (codons 354 and 389). In contrast to colon and gastric cancers exhibiting microsatellite instability (MI) or replication errors (RER+), no 'indirect' frameshift mutations were identified within a 10-bp polyadenine repeat present in the TbetaR-II coding sequence. All of the mutations in the present study occurred within the highly conserved serine/threonine kinase domain and represent the first report of such 'direct' TbetaR-II mutations in primary human tumors. In addition, we analyzed a subset of SCCHN and corresponding normal samples for TbetaR-II mRNA expression using semi-quantitative multiplex RT-PCR. Expression of TbetaR-II was decreased by 24% to 74% in 20 of 23 SCCHN (87%) compared with patient-matched normal tissues. Taken together, the results from this study suggest that alterations in the nucleic acid sequence and mRNA expression of TbetaR-II are prevalent events in the development of SCCHN, which may deregulate cell cycle control.

Carcinoma, Squamous Cell↗
PubMed1997

Molecular characterization of mutations in the hprt gene of normal human skin keratinocytes treated with N-ethyl-N-nitrosourea: influence of O6-alkylguanine alkyltransferase.

O6-Alkylguanine-DNA alkyltransferase (AGT) is responsible for repairing the O6-alkylguanine lesion in DNA. There is wide variation in the levels of AGT between organ and cell types, which appears to correlate with cell and tissue type sensitivity to the mutagenic and carcinogenic effects of alkylating agents. In order to investigate the role of AGT in modulating the frequency and types of mutations induced in one type of normal human parenchymal cells, we examined the types and frequency of mutations in the hypoxanthine (guanine) phosphoribosyltransferase (hprt gene in 116 mutants derived from two N-ethyl-N-nitrosourea (ENU)-treated normal human skin keratinocyte cell lines. O6-Benzylguanine (O6-BZ; 5 microM x 2 hours) was used to specifically inhibit AGT activity before ENU treatment (0 to 5 mM x 1 hour). O6-BZ increased both the cytotoxic and mutagenic effects of ENU by 1.8- and 3- to 5-fold, respectively. In both treatment groups, most of the mutations were base substitutions (72%). The proportion of GC to AT transitions in the O6-BZ group (14/31) was twice that in the group treated with ENU alone, consistent with the loss of AGT activity in these cells. There was no strand specificity of GC to AT and AT to GC transitions in both groups. Base transversions accounted for 28% of total base substitutions. A lower than expected proportion of AT to TA transversions were observed in both cell lines, which decreased in the O6-BZ pretreated group. A strand bias was observed for GC to TA and AT to TA transversions. Most of the G to A and G to T base substitutions had one or more purines flanking 3' to the mutated deoxyguanosines. There were more deletion mutants with the deletion of exon 1, 4, 6, and 8 in the BZ group than in the control group. These data, characterizing the mutational spectra of ENU in normal human keratinocytes treated in vitro, indicate that GC to AT and AT to GC transition mutations predominate in these cells depleted or not depleted of AGT.

Cell Line↗
PubMed1996

Overexpression of cyclin D1 enhances gene amplification.

Defects in cell cycle control and increased genomic instability, including gene amplification, often occur during cancer development. Cyclin D1 plays a pivotal role in G1, and this gene is frequently amplified and overexpressed in several types of human cancer. This study demonstrates that ectopic overexpression of cyclin D1 in a rat liver epithelial cell line markedly increased the yield of cells containing amplified copies of the CAD gene. This effect was associated with a loss of G1-S checkpoint control, although the cyclin D1-overexpressing cells had a normal p53 gene. The capacity of cyclin D1 to enhance gene amplification may contribute to the process of genomic instability during tumor development.

Animals↗
PubMed1996

RET mutation screening in MEN2 patients and discovery of a novel mutation in a sporadic medullary thyroid carcinoma.

RET germline mutations were found to predispose to the development of three variants of multiple endocrine neoplasia type 2, MEN2A, MEN2B, and familial medullary thyroid carcinoma (FMTC). We have screened for RET mutations at exons 10, 11, 13, and 16 in leukocyte DNA extracted from 37 individuals, and have identified RET germline mutations in 12 affected individuals from 9 unrelated families. No RET germline mutation was found in 19 individuals with apparent sporadic diseases. We have also screened for RET mutations at exons 10, 11, and 16 in tumor DNA extracted from 13 freshly frozen medullary thyroid carcinomas (MTC). RET mutation was detected in every tumor, either inherited or sporadic, indicating that RET plays an important role in the development of both inherited and sporadic MTC. We initially screened for RET mutations by direct DNA sequencing of the genomic PCR products amplified from patients' leukocyte or tumor DNA. Recently, we utilized the "Cold SSCP" method, nonradioactive single-stranded conformation polymorphism analysis, to screen for RET mutations and have identified a novel mutation, a 6-bp deletion preceding the cysteine-634, in a sporadic MTC.

Adolescent↗
PubMed1996

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.

Animals↗
PubMed1996

Absence of p16/MTS1 gene mutations in human prostate cancer.

The tumor suppressor gene p16/MTS1, located on chromosome 9p21, is a cell cycle regulatory gene which is frequently altered in human cancers. The role of this gene in prostate cancer is unknown. To determine the frequency of deletions and point mutations of p16/MTS1 in human prostate cancer, we examined 18 cancer and matched benign and hyperplastic tissue specimens. Deletions of p16/MTS1 were detected by semi-quantitative multiplex polymerase chain reaction in which a portion of exon 2 of the p16/MTS1 gene and a control marker, the glyceraldehyde 3-phosphate dehydrogenase gene, were amplified simultaneously. 'Cold' single-stranded conformational polymorphism (SSCP) analysis was performed to examine exons 1 and 2 of the p16/MTS1 gene for point mutations. Our data indicate no evidence for intragenic homozygous deletion in the prostate tumors. One prostate tumor and matched benign tissue showed mobility shifts. Direct DNA sequencing of the SSCP positive samples showed a G --> A transition in codon 140 which would result in an amino acid change from alanine to threonine. Our results indicate that deletions and point mutations in the p16/MTS1 gene are rare and do not play a major role in human prostate carcinogenesis.

Aged↗
PubMed1996

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.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1996

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.

Animals↗
PubMed1995

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.

Animals↗
PubMed1995

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.

Animals↗
PubMed1995

Mutations of the K-ras and p53 genes in gastric adenocarcinomas from a high-incidence region around Florence, Italy.

K-ras and p53 gene mutations in intestinal-type gastric carcinomas from a high-incidence area around Florence, Italy, were studied by single strand conformation polymorphism and DNA sequencing analysis. Single-strand conformation polymorphism analysis of K-ras indicated aberrant bands in 13 of 34 cases. Sequencing revealed point mutations in 7 (including two at a previously unreported site in codon 11), a significantly higher frequency than reported in countries other than Japan. No K-ras mutations were identified in stage III tumors. Single-strand conformation polymorphisms in p53 exons 5-8 occurred in 30 of 34 cases, with mutations identifiable by direct sequencing in 65% of the cases. Of these, 91% were base substitutions, a value similar to that usually reported, but the percentage of G:C to A:T transitions (90% in this study, 89% in all published European cases combined) differed significantly from that in Oriental cases (48%). The percentage of A:T to G:C transitions was greater in Oriental (22%) than European cases (2%), as was also true for transversions (30% in Oriental tumors, 9% in European tumors). The frequency of mutations at CpG sites (14%) varied significantly from the 67% in cases from a neighboring region in Italy. Helicobacter pylori infection was established in 19 cases and was somewhat more common in cases lacking a p53 mutation.

Adenocarcinoma↗
PubMed1995

Gender-dependent differences in hepatic tumor promotion in diethylnitrosamine initiated infant B6C3F1 mice by alpha-hexachlorocyclohexane.

Chronic exposure of B6C3F1 mice to phenobarbital (PB), subsequent to a single initiating dose of diethylnitrosamine (DENA) at 15 d of age, has been previously shown to inhibit hepatic tumorigenesis in male mice, while promoting hepatic tumor formation in female mice (Weghorst & Klaunig, 1989). In the present study, the effects of another hepatic tumor promoter, alpha-hexachlorocyclohexane (alpha-HCH), in similarly initiated B6C3F1 mice was investigated. Male and female mice received a single intraperitoneal (ip) injection of either DENA or saline at 15 d of age. Beginning at 28 d of age, the mice received either alpha-HCH in the diet (250 ppm) or untreated basal diet. Like PB, alpha-HCH inhibited hepatic tumorigenesis in male mice, while promoting hepatic tumor formation in female mice following chronic exposure. In an additional experiment, already formed preneoplastic hepatic foci in male and female B6C3F1 mice were examined for their responsiveness to the induction of DNA synthesis by alpha-HCH treatment. The mice received a single ip injection of DENA at 15 d of age to induce hepatocellular foci. Beginning at 24 wk of age, mice received either basal diet or diet containing 250 ppm alpha-HCH for 7 consecutive d. DNA synthesis was assessed by continuous [3H]thymidine infusion via subcutaneously implanted osmotic minipumps. In female mice treated with alpha-HCH, DNA synthesis in hepatocellular foci was increased substantially compared to untreated females. In contrast, male mice receiving alpha-HCH showed no increase in DNA synthesis in hepatocellular foci from that seen in non-alpha-HCH-treated males. Based on these results, we postulate that the gender-dependent differences in hepatic tumorigenesis observed in B6C3F1 mice initiated during infancy may be related to chemical tumor promoter modulation of the normal hormonal environment, or to differences in the ability of hepatocellular foci to respond to the induction of DNA synthesis by the tumor promoter.

Animals↗
PubMed1995

Alteration of the tumor suppressor gene p53 in a high fraction of hormone refractory prostate cancer.

PURPOSE: We studied the role of p53 tumor suppressor gene alteration in prostate cancer progression by demonstrating a difference in abnormal p53 findings between early and hormone refractory disease. MATERIALS AND METHODS: The study included p53 immunohistochemistry of 26 archival transurethral resection specimens from patients with radiation recurrent and hormone refractory disease, 27 untreated primary tumors and 8 untreated metastatic lesions. p53 mutation analysis of tumor deoxyribonucleic acid (DNA) from microdissected specimens was done by cold single strand conformational polymorphism and DNA sequencing. RESULTS: Elevated p53 protein was present in 16 of 17 hormone refractory specimens (94%), 4 of 8 untreated metastatic tumors (50%) and 6 of 27 primary untreated tumors (22%). DNA analysis of representative specimens with elevated p53 confirmed p53 gene alterations in 9 of 11 cases (82%). CONCLUSIONS: Our study revealed a clear progression of increased p53 alteration from untreated primary to hormone refractory disease (p < 0.00005).

Base Sequence↗
PubMed1995

Increased p53 protein does not correlate to p53 gene mutations in microdissected human testicular germ cell tumors.

PURPOSE: To determine if primary testicular germ cell tumors that overexpress p53 tumor suppressor gene protein have p53 gene mutations. MATERIALS AND METHODS: We examined 30 primary testicular tissues from 26 patients representing two groups. Group one consisted of eleven cases (6 nonseminomatous germ cell tumors and 5 seminomas) in which tissue samples for DNA analysis were microdissected from paraffin block regions with elevated immunohistochemical staining for p53 protein. Group two consisted of 19 testis tumor tissues which had been fresh frozen and were chosen to correspond to archival tissue specimens exhibiting elevated levels of p53 protein. The DNA was extracted from these tissues and subjected to exon specific amplification by polymerase chain reaction (PCR) and cold single-strand conformation polymorphism (Cold SSCP) analysis. RESULTS: In these cases with elevated p53 protein, no p53 gene exon 5-8 mutations were detected except 1 seminoma with a codon 140 silent mutation (no protein alteration). CONCLUSIONS: Testicular tumors appear to exhibit elevated levels of wild-type p53 protein, the significance of which is yet to be elucidated.

Base Sequence↗
PubMed1994

Low incidence of point mutations detected in the p53 tumor suppressor gene from chemically induced rat renal mesenchymal tumors.

Previous studies have shown renal mesenchymal tumors (RMTs) induced in rats by a single intrarenal injection of nickel subsulfide and iron are more pleomorphic and metastatically aggressive than RMTs induced by a single ip injection of methyl(methoxymethyl)nitrosamine (DMN-OMe). While both RMT types contain high levels of K-ras activation, the specific mutational spectra within codon 12 of K-ras are quite different. Nickel subsulfide and iron-induced tumors exhibited codon 12 GGT-->GTT transversions exclusively, while DMN-OMe RMTs showed a wide array of codon 12 mutations, as well as mutations within codons 61 and 63 [K. G. Higinbotham, J. M. Rice, B. A. Diwan, K. S. Kasprzak, C. D. Reed, and A. O. Perantoni, Cancer Res., 52: 4747-4751, 1992; K. G. Higinbotham, J. M. Rice, and A. O. Perantoni, Mol. Carcinog., 5: 136-139, 1992]. In an effort to further correlate carcinogen-specific molecular events in renal tumors, we investigated the p53 tumor suppressor gene in RMTs induced by these two carcinogens for the presence of point mutations. The evolutionarily conserved portion of the coding region of the gene, including part of exon 4 through exon 10, was surveyed for point mutations utilizing single-strand conformation polymorphism and chemical cleavage of mismatches analyses. None (0 of 10) of the nickel subsulfide and iron-induced RMTs and only 1 of 10 DMN-OMe-induced tumors that were evaluated contained point mutations within this portion of the p53 gene. Direct sequencing of the one single-strand conformation polymorphism and chemical cleavage of mismatches-"positive" DMN-OMe-induced RMT revealed a GCC-->GTC (Ala-->Val) transition in codon 345 within exon 10. These results suggest that the different tumorigenic phenotypes exhibited by these two RMTs are not the result of specific mutations or patterns of mutations within the portion of the p53 gene examined and that the mutated p53 tumorigenic pathway, whereby p53 plays a major role in many human neoplasms, does not function in RMTs induced by either agent.

Animals↗