GTT Transversion Research Articles

Dietary phytate lowers K-ras mutational frequency, decreases DNA-adduct and hydroxyl radical formation in azoxymethane-induced colon cancer.

Objective(s):Dietary phytate is known to protect against azoxymethane (AOM)-induced preneoplastic lesions. The present study was designed to determine whether dietary phytate affects mutation frequency in colon epithelial cells challenged with azoxymethane in vivo, through lowering the formation of O6-methyl guanosine (O6-MeG) and 8-hydroxy deoxyguanosine (8-OHdG) adducts.Materials and Methods:We used Fisher F344 rats induced with AOM for 20 weeks and undertook 1% or 2% phytate supplementation for subsequent 16 weeks to monitor the mutation frequencies of one of the candidate genes, K-ras, along with DNA adduct load.Results:Dietary phytate significantly suppressed aberrant crypt foci formation and effectively inhibited colon tumor formation in a dose-dependent manner. DNA sequencing results demonstrated that 60% of the colon tumors from AOM-treated and control diet fed animals showed GGT to GAT transition and 40% of the tumors showed GGT to GTT transversion at codon 12, along with 18% of the tumors showing GGC to CGC transversion at codon 13. Phytate supplementation at 1 and 2% lowered the frequency of GGT > GAT to 30 and 10%, respectively. Phytate supplementation also nullified the codon 13 mutations. No mutations were observed at codon 61 in any of the experimental groups.Conclusion:The lowered frequency of K-ras mutations correlated with decreased formation of hydroxyl radicals, O5-meG and 8-OH-dG levels in phytate-supplemented animals with lowered tumor burden.

Unilateral follicular variant of papillary thyroid carcinoma with unique KRAS mutation in struma ovarii in bilateral ovarian teratoma: a rare case report

BackgroundStruma ovarii (SO) is a rare form of ovarian mature teratoma in which thyroid tissue is the predominant element. Because of its rarity, the differential diagnosis between benign and malignant SO has not been clearly defined. It is believed that malignant transformation of SO has similar molecular features with and its prognosis corresponds to that of malignant tumors originating in the thyroid.Case presentationWe report 35-year-old woman with bilateral ovarian cysts incidentally detected by ultrasound during the first trimester of pregnancy. Four months after delivery of a healthy child without complication she was admitted to the hospital for acute abdominal pain. Laparoscopic left adnexectomy was performed initially in a regional hospital; right cystectomy was done later in a specialized clinic. Intraoperative frozen section and a final pathology revealed that the cyst from the left ovary was composed of mature teratomatous elements, normal thyroid tissue (>50%) and a non-encapsulated focus of follicular variant of papillary thyroid carcinoma (PTC).Normal and cancerous thyroid tissues were tested for BRAF and RAS mutations by direct sequencing, and for RET/PTC rearrangements by RT-PCR/Southern blotting. A KRAS codon 12 mutation, the GGT → GTT transversion, corresponding to the Gly → Val amino acid change was identified in the absence of other genetic alterations commonly found in PTC.ConclusionTo the best of our knowledge, this is the first time this mutation is described in a papillary thyroid carcinoma arising in struma in the ovarii. This finding provides further evidence that even rare mutations specific for PTC may occur in such tumors. Molecular testing may be a useful adjunct to common differential diagnostic methods of thyroid malignancy in SO.

KRAS Mutation Analysis Helps to Differentiate Between Pulmonary Metastasis from Colon Adenocarcinoma In Situ and Primary Lung Adenocarcinoma

A 69-year-old woman never-smoker was noted to have a solitary 4-cm lesion in the left upper lobe on routine preoperative chest x-ray before elective hip replacement. Chest computed tomography redemonstrated the spiculated 4 3.3-cm lesion. Positron emission tomography-computed tomography confirmed that the lesion was fluorodeoxyglucose-avid with no other sites of fluorodeoxyglucose avidity (Figure 1). The patient underwent a core needle biopsy of the lung lesion showing a cytokeratin (CK) 20 and caudal type homeobox transcription factor 2–positive adenocarcinoma, which was negative for CK7 and thyroid transcription factor-1. The expected immunohistochemical markers for a primary lung adenocarcinoma are CK7 and thyroid transcription factor-1, whereas the usual markers for a primary colorectal adenocarcinoma are CK20 and caudal type homeobox transcription factor 2. The patient underwent a video-assisted thoracoscopic lobectomy and mediastinal lymphadenectomy revealing a 3.8-cm adenocarcinoma with the same histologic (Figure 2A) and immunophenotype (Figure 3) as the previous biopsy. One inferior mediastinal lymph node (level 9L) was also involved with tumor, and levels 5, 10L, 11L, and 12L nodes were negative for disease. A colonoscopy, upper endoscopy, and capsule video enteroscopy did not show any evidence of a primary tumor. Seven years before the discovery of the lung mass, she had a 2-cm sigmoid adenoma with areas of high-grade dysplasia (Figure 2B) and carcinoma in situ resected endoscopically in a piecemeal fashion. Her resected lung tumor was sent for genetic testing and found to harbor a Kirsten ras sarcoma viral oncogene homolog (KRAS) gene codon 12 GGT to GTT (glycine to valine) missense mutation (KRAS G12V). The tumor was wild-type for the epidermal growth factor receptor gene. Given there was no evidence of primary colorectal tumor on endoscopy, the archived, paraffin-embedded sigmoid adenoma was also genotyped and found to carry the same KRAS codon 12 GTT transversion. This suggested that the lung tumor was a late recurrence from the original KRAS mutant sigmoid carcinoma in situ. She was subsequently found to have multiple new pulmonary lesions.

Targeted deletion of the genes encoding NTH1 and NEIL1 DNA N-glycosylases reveals the existence of novel carcinogenic oxidative damage to DNA

We have generated a strain of mice lacking two DNA N-glycosylases of base excision repair (BER), NTH1 and NEIL1, homologs of bacterial Nth (endonuclease three) and Nei (endonuclease eight). Although these enzymes remove several oxidized bases from DNA, they do not remove the well-known carcinogenic oxidation product of guanine: 7,8-dihydro-8-oxoguanine (8-OH-Gua), which is removed by another DNA N-glycosylase, OGG1. The Nth1 −/− Neil1 −/− mice developed pulmonary and hepatocellular tumors in much higher incidence than either of the single knockouts, Nth1 −/− and Neil1 −/− . The pulmonary tumors contained, exclusively, activating GGT → GAT transitions in codon 12 of K-ras of their DNA. Such transitions contrast sharply with the activating GGT → GTT transversions in codon 12 of K-ras of the pathologically similar pulmonary tumors, which arose in mice lacking OGG1 and a second DNA N-glycosylase, MUTY. To characterize the biochemical phenotype of the knockout mice, the content of oxidative DNA base damage was analyzed from three tissues isolated from control, single and double knockout mice. The content of 8-OH-Gua was indistinguishable among all genotypes. In contrast, the content of 4,6-diamino-5-formamidopyrimidine (FapyAde) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) derived from adenine and guanine, respectively, were increased in some but not all tissues of Neil1 −/− and Neil1 −/− Nth1 −/− mice. The high incidence of tumors in our Nth1 −/− Neil1 −/− mice together with the nature of the activating mutation in the K-ras gene of their pulmonary tumors, reveal for the first time, the existence of mutagenic and carcinogenic oxidative damage to DNA which is not 8-OH-Gua.

Expression and alteration of ras and p53 proteins in patients with lung carcinoma accompanied by idiopathic pulmonary fibrosis.

The ras oncogene and the p53 tumor suppressor gene play important roles in the carcinogenic process of lung carcinoma. The authors evaluated whether alterations of the ras and p53 proteins may contribute to the development of lung carcinoma in patients with idiopathic pulmonary fibrosis (IPF) and whether such alterations may explain the high incidence of lung carcinoma among patients with IPF. Lung tissues were obtained from 35 patients who had IPF without complications of lung carcinoma and from 36 patients who had IPF with complications of lung carcinoma. Altered expression of ras and p53 proteins was evaluated by immunohistochemistry, and mutations of both genes were evaluated by polymerase chain reaction-single strand conformation polymorphism and sequencing analyses. The frequency of expression of ras protein in type II alveolar pneumocytes was significantly greater in lung tissues from patients with IPF who had lung carcinoma compared with lung tissues from patients with IPF who did not have lung carcinoma (75% vs. 40%, respectively; P < 0.01). K-ras point mutation in codon 12 (GGT to GTT transversion) was detected in lung tissue with interstitial pneumonia, in which ras protein was overexpressed in type II alveolar pneumocytes obtained from 2 of 41 patients with IPF complicated by lung carcinoma, causing amino acid substitution (Gly to Val) in both patients. A p53 mutation was detected in three of six lung tissue samples from patients who had IPF lung with positive p53 immunoreactivity, and multiple mutations were detected in two samples. Expression of ras protein in type II alveolar pneumocytes and mutation in the codon 12 of K-ras gene in lung tissue may contribute to the induction of lung carcinoma in patients with IPF. Furthermore, the presence of multiple mutations in the p53 gene may explain the high incidence lung carcinoma in patients with IPF.

Heterogeneity of mutant versus wild-type Ki-ras in primary and metastatic colorectal carcinomas, and association of codon-12 valine with early mortality.

The point mutations occurring in codons 12 and 13 of Ki-ras in 78 patients with colorectal carcinoma (31 Dukes' A and B, 21 Dukes' C, and 26 Dukes' D) have been determined by allele-specific oligonucleotide hybridization and sequencing. Duplicate samples of invasive primary carcinoma, adjacent normal tissue, and available lymph node and liver metastases from the same patients were microdissected from paraffin sections. There were no differences in the mutation rate between primary carcinomas and secondary deposits: 26 of 78 (33 per cent) primary carcinomas, 10 of 32 (31 per cent) lymph node metastases, and 10 of 26 (38 per cent) liver metastases. Multiple sampling revealed frequent heterogeneity within carcinomas: 9 of 26 primaries with Ki-ras mutations also contained areas of carcinoma with only the wild-type gene, implying that Ki-ras mutation, even when present in a colonic carcinoma, may not have been necessary for establishing the malignant phenotype. Also, 2 of 26 (8 per cent) Dukes' D patients had a mutation in their primary carcinoma but none in liver metastases and 6 of 47 (13 per cent) Dukes' C and D patients had mutations in liver or lymph node metastases but none in the primary carcinoma. Such heterogeneity may modify the effectiveness of novel therapies targeting mutant Ki-ras function, such as farnesyltransferase inhibition. Mutation of codon 12 from GGT (glycine) to GTT (valine) was more prevalent in primary and metastatic deposits of Dukes' C/D carcinomas (P = 0.01) than in primary carcinomas from Dukes' A/B patients. Mutations of codon 12 to GAT, AGT, GCT and codon 13 GGC to GAC were also found, but no correlation with carcinoma aggressiveness was apparent. Follow-up of 71/78 patients (up to 12 years) revealed decreased overall survival (P = 0.001) in patients with the GGT to GTT transversion in codon 12, even when the analysis was restricted to Dukes' D cases, supporting the suggestion that this mutation may confer a more aggressive phenotype in colorectal carcinoma.

Comparison of ras activation in prostate carcinoma in Japanese and American men

BACKGROUND Comparative studies of point mutations in K-, N-, and H-ras oncogenes were performed on prostate carcinoma from Japanese and American patients to clarify the racial difference. METHODS We probed for mutations in 70 Japanese and 31 American specimens using polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis and immunohistochemistry for ras p21. RESULTS Within the 70 Japanese specimens, eight mutations in codon 12 of K-ras (five GGT→GTT transversions and three GGT→GAT transitions) and one mutation in codon 12 of the N-ras gene (a GGT→GTT transversion) were confirmed, whereas the American samples yielded only one definable mutation, a GGT→GAT transition, in codon 12 of K-ras. CONCLUSIONS The frequency of ras gene mutations in clinical carcinoma in Japanese men was higher than that in American men. It is suggested that there may be fundamental differences in the etiology of prostate carcinoma in Japan and the United States, perhaps based on genetics and/or environmental factors. Prostate 30:53–57, 1997 © 1997 Wiley-Liss, Inc.

K-ras activation andras p21 expression in latent prostatic carcinoma in Japanese men

Twenty-three clinically silent prostatic carcinomas discovered in Japanese men at autopsy were surveyed for ras proto-oncogene mutations by mutation-specific oligonucleotide probe hybridization after polymerase chain reaction (PCR) amplification from a section of formalin-fixed, paraffin-embedded tissue. Six of the 22 that were satisfactory amplified contained activating point mutations in codon 12 of K-ras, a significantly higher frequency than has been reported in patients with clinically advanced disease in the United States. Of the six cases with activating point mutations in codon 12 of K-ras, one had a GGT----GAT transition, four had GGT----GTT transversions, and one had both GGT----GAT and GGT----GTT mutations. Sections from the same tissues were immunohistochemically stained with an anti-ras p21 antibody. Carcinoma cells stained for ras p21 to some degree in 13 cases. Immunohistochemically detectable expression of p21 was always focal and was not necessarily associated with K-ras mutation. K-ras oncogene activation in prostatic carcinoma appears to merit additional study as a significant event in the pathogenesis of this neoplasm.

Analysis of ras gene mutations in rainbow trout liver tumors initiated by aflatoxin B1.

The suspect human hepatocarcinogen aflatoxin B1 (AFB1) is a well-known potent initiator of hepatic tumors in rainbow trout (Oncorhynchus mykiss). Both hepatocellular carcinomas and mixed hepatocellular/cholangiocellular carcinomas are induced by AFB1 in trout, with the mixed form predominating. We previously isolated two c-ras genes from trout liver cDNA, and in the present study we analyzed DNA from 14 AFB1-induced trout liver tumors for point mutations in exon 1 of both genes. Using the polymerase chain reaction (PCR) and oligonucleotide hybridization methods, a high proportion (10/14) of the AFB1-initiated tumor DNAs showed evidence of activating point mutations in the trout c-Ki-ras gene. Of the 10 mutant ras genotypes, seven were codon 12 GGA----GTA transversions, two were codon 13 GGT----GTT transversions, and one was codon 12 GGA----AGA transition. Nucleotide sequence analysis of cloned PCR products from four of these tumor DNAs provided definitive evidence for two codon 12 GGA----GTA mutations, one codon 12 GGA----AGA mutation, and one codon 13 GGT----GTT mutation, in complete agreement with the oligonucleotide hybridization results. No mutations were detected in exon 1 of a second trout ras gene also expressed in liver, nor in DNA from control livers. This is the first report of experimentally induced ras gene point mutations in a lower vertebrate fish model. The results indicate that the hepatocarcinogen AFB1 induces c-Ki-ras gene mutations in trout similar to those in rat liver tumors.