The mutational signature of alpha-hydroxytamoxifen at Hprt locus in Chinese hamster cells

The anti-oestrogen tamoxifen is very effective in the treatment and prevention of breast cancer. Tamoxifen is not a pure antagonist, but possesses weak oestrogenic activity that may contribute to a slightly increased risk of endometrial cancer. Whilst this can be incorporated into risk-benefit analysis for the use of the drug, residual concerns exist over the exact mechanism of formation of these tumours. Tamoxifen is a potent hepatocarcinogen in the rat, probably via a genotoxic mechanism. Whilst tamoxifen does not appear to cause liver tumours in humans, DNA adducts have been found in endometrial tissue of women receiving the drug. Hence, there is still a need to establish the mechanism of formation of these tumours. We have therefore determined the molecular nature of mutations induced in vitro by alpha-hydroxytamoxifen, the putative proximate genotoxic metabolite, in a mammalian cell line (V79-rHSTa) with stable expression of rat hydroxysteroid sulfotransferase a, which catalyses the further metabolism of alpha-hydroxytamoxifen to its ultimate genotoxic product. DNA sequence alterations were examined at the Hprt gene in 50 mutant clones. Simple base substitutions, mainly GC-->TA transversions, predominated. However, single G:C base pair deletions and partial/complete exon skippings were also observed. All but one of the mutations involved guanine bases on the non-transcribed strand, probably indicating preferential repair of alpha-hydroxytamoxifen-induced guanine adducts from the transcribed strand. Nearest neighbour analysis of the mutations (on the non-transcribed strand) indicated that thymines (20/40) followed by guanines (13/40) were the most frequent 5' neighbours, with adenines or guanines the most frequent 3' neighbours. Many of the mutations occurred at TTGA/G sequences. Three mutational hot spots accounted for 11 GC-->TA transversions and another site for two single G:C base pair deletions. A search for these characteristic mutations in tumour-related genes of treated rats and humans should help in understanding the mechanism(s) of tamoxifen-induced carcinogenicity.     

Loss of heterozygosity and point mutation at Aprt locus in T cells and fibroblasts of Pms2-/- mice

Mice null for the Pms2 mismatch repair (MMR) gene exhibit a predisposition to lymphoma, microsatellite repeat instability, and failure of spermatogenesis. To study the role of Pms2 in the maintenance of in vivo genomic integrity in somatic cells, we characterized Aprt mutations in T cells and fibroblasts of 129 x C3H Pms2-/-Aprt+/- mice. The spontaneous frequency of DAP-resistant T lymphocytes, as a consequence of APRT-deficiency, was increased threefold. Point mutation, which accounted for less than 20% of the DAP(r) mutant clones in Pms2+/+ mice, was predominant in the mutant T cell clones from Pms2-/- mice. These point mutations were predominantly TA to CG transitions. Fibroblasts of Pms2-/- mice exhibited only a modest increase in the frequency of clones with point mutations, such that mitotic recombination was still the primary cause of APRT deficiency. Thus, the mutator phenotype as a consequence of PMS2 deficiency is tissue-dependent, which may be related to the tissue-specific tumor proneness of Pms2-/- mice.     

The tumour promoter TPA does not affect mutation to ouabain resistance in L5178Y mouse lymphoma cells

Following treatment with ultraviolet light or ethyl methane-sulphonate mouse lymphoma L5178Y cells were cultured for different periods in the presence of non-toxic levels of the tumour promoter 12-O-tetra-decanoylphorbol-13-acetate (TPA). There were no significant changes in either the spontaneous or induced frequencies of ouabain-resistant variants recovered after TPA treatment. These results confirm that any effect of TPA on mutagenesis may be by modifying metabolic cooperation and the recovery of resistant cells.     

Cytotoxic and mutagenic response of mismatch repair-defective human cancer cells exposed to a food-associated heterocyclic amine

The cytotoxic and mutagenic effects of 2-amino-1-methyl-6-phenylimidazo-[4,5-b]-pyridine (PhIP), a food-associated heterocyclic amine, were measured in three human cancer cell lines possessing different mismatch repair (MMR) defects and in matched cell lines corrected for the MMR deficiencies by specific chromosome transfer. Cells deficient in MMR were more resistant to PhIP-induced cytotoxicity and displayed approximately 3-fold more induced mutations at the hypoxanthine-guanine phosphoribosyl transferase locus. These results suggest that defects in MMR carried by patients with hereditary nonpolyposis colorectal cancer syndrome may result in enhanced sensitivity to certain dietary and environmental carcinogens such as PhIP.     

Reduced apoptosis and increased deletion mutations at Aprt locus in vivo in mice exposed to repeated ionizing radiation

Exposure to ionizing radiation (IR) is a risk factor for carcinogenesis because it is a mutagen. However, a single 4-Gy whole body X-ray exposure only induced a modest increase of mutations at the Aprt reporter gene locus in mouse T cells. Intriguingly, when the same dose of IR was given in a fractionated protocol (1 Gy x 4 at weekly intervals), there was a strong induction of Aprt mutations in T cells. Many of these were mutations that arose via interstitial deletions inclusive of Aprt or by intragenic deletions. We hypothesized that the weekly fractionated X-ray exposures select for somatic cells with reduced p53 expression and/or reduced apoptosis, which, in turn, may have facilitated the accumulation of interstitial deletions, as in p53-deficient mice. We indeed found that splenocytes of mice with three previous exposures (1 Gy x 4 in total) were more resistant to X-ray-induced apoptosis than those of mice exposed to X-rays for the first time (1 Gy total). Thus, repeated X-ray radiation selects for reduced apoptosis in vivo. However, this reduced apoptosis is p53-independent, because p53 induction and the up-regulation of genes downstream of p53, such as Bax and p21, were similar between the 1-Gy and 1 Gy x 4 groups. Reduced apoptosis probably allows the generation of more mutations, particularly deletion mutations. Because both reduced apoptosis and increased somatic mutation are risk factors for carcinogenesis, they may contribute to the paradigm in which different radiation exposure schemes are varied in their efficiency in inducing lymphomagenesis.     

Induction of mutation to ouabain resistance and of malignant transformation by chemicals in cloned mouse M2 fibroblasts

Malignant transformation and mutation to ouabain-resistanceis induced in cloned M2 mouse fibroblasts in vitro by treatmentwith N-methyl-N'-nitro-N-nitrosoguanidine and two derivativesof polycyclic aromatic hydrocarbons, the 3, 4-diol derived from7-methylbenz[a]anthracene and the 7, 8-diol-9, 10-oxide derivedfrom benzo[a]pyrene. Transformation is an event more frequentthan mutation, the ratio of transformation to mutation frequenciesvaried between 24 and 1118. Another hydrocarbon derivative,the 9, 10-diol-7, 8-oxide derived from ben-zo[a]pyrene, didinduce ouabain-resistance without causing transformation. Inducedouabain-resistant clones did not behave like transformants,and transformants did not exhibit ouabain resistance. The significanceof these findings is discussed.     

In vitro transformation of fetal brain cells from CDF rats exposed in utero to N-ethyl-N-nitrosourea: morphologic and immunologic studies

Inbred CDF rats received 250 mg N-ethyl-N-nitrosourea (ENU)/kg body weight in monosodium phosphate buffer (pH 5.5) during late pregnancy (18-21 days). Control rats received only buffer. One hour after ENU or buffer administration, the fetuses were delivered and fetal brain cells (FBC) were put in long-term tissue culture. Cell growth consisted of monolayers of glial, fibroblastic, and epithelial-like cells. FBC from buffer-treated rats could not be subcultured for more than 13-15 passages; only one culture was maintained up to the 26th passage. FBC from ENU-treated rats showed in vitro malignant transformation beginning at the 8th passage (100 days). Injection of these cells sc into irradiated or neonatally thymectomized syngeneic rats induced small palpable tumors that regressed completely by 15-28 days. Small nonprogressively growing subcutaneous tumors in normal rats, however, were obtained only after the 19th in vitro passage (200 days). These transformed FBC appeared highly antigenic. Tumor-associated antibody was demonmmune adherence assays. Rats immunized with irradiated malignant FBC showed rising tumor antibody titers. The antibody was absorbed by ENU-exposed FBC but not by normal fetal or adult syngeneic brain cells. We concluded that ENU-exposed cells become malignant when grown in vitro for at least 8 passages (100 days) but grow in vivo in X-irradiated or neonatally thymectomized rats only. These antigenic and tumorigenic clones may be selected in vitro, where they grow readily in the absence of immune resistance.     

Colostrinin decreases spontaneous and induced mutation frequencies at the hprt locus in Chinese hamster V79 cells

Colostrinin (CLN), a uniform mixture of low-molecular weight, proline-rich polypeptides, induces neurite outgrowth of pheochromocytoma cells, extends the lifespan of diploid fibroblast cells, inhibits beta amyloid-induced apoptosis and resulted in improved cognitive function when administered to Alzheimer's patients. Here we investigated CLN's antimutagenic activity in cells stressed oxidatively or exposed to chemical or physical agents. Our data show that CLN did not alter cell cycle kinetics and cloning efficiency, while it inhibited the development of spontaneous mutations at the coding region of the hypoxanthine phosphoribosyl-transferase (hprt) gene in Chinese hamster V79 cells. In a dose-dependent manner, CLN lowered reactive oxygen species (ROS)-induced frequency of cells resistant to 6-thioguanine (6-TG) to nearly background level. Likewise, CLN decreased the frequency of methyl methanesulfonate- or mitomycin C-induced mutations in V79 cells. Notably, CLN (at 100, 250, and 500 ng per ml concentrations) decreased UVA-induced mutation frequency, while only the highest dose of CLN also decreased significantly the number of UVB-induced 6-TG-resistant mutant cells. Similar results were obtained using cell cultures of human origin. Overall, our data show that CLN significantly lowers the mutation frequency that develops spontaneously or is induced by ROS, chemical and physical agents. CLN itself has no mutagenic activity. Therefore, CLN may be used in human therapies systemically and/or locally for the prevention of diseases associated with sequence alterations in genomic and mitochondrial DNA.     

Mutagenicity of butadiene and its epoxide metabolites: II. Mutational spectra of butadiene, 1,2-epoxybutene and diepoxybutane at the hprt locus in splenic T cells from exposed B6C3F1 mice

The mutagenic potential and mutational spectra of butadiene(BD), 1,2-epoxybutene (EB), and diepoxybutane (DEB) were determinedin splenic T cells from exposed B6C3F1 mice. Mice exposed byinhalation to 625 p.p.m. BD for 2 weeks displayed an averagehprt^– mutation frequency of 6.2 x10^–6 compared to1.2x10^–6 in controls. Mice were also given three dailyi.p. doses of 60, 80 and 100 mg EB/kg or 7, 14 and 21 mg DEB/kg.Average hprt^– frequencies of 5.4x10^–6, 4.lx10^–6and 8.6x10^–6 were seen in the EB groups, respectively,while average frequencies of 4.6x10^–6, 9.4x10^–6and 13x10^–6 were seen in the DEB groups. DNA sequencingrevealed that approximately half of the mutations induced invivo by BD, EB and DEB were frameshift mutations. A +1 frameshift‘hotspot’ in six consecutive guanine bases in exon3 was observed with all three compounds. The remaining mutationsproduced by BD, EB and DEB were transition and transversionmutations at both AT and GC base pairs. Base pair substitutionsinduced by BD were biased in favor of mutation at AT base pairs.The mutational spectra produced by BD, EB and DEB were verysimilar to that observed previously with ethylene oxide, suggestingthat these epoxide agents may be working through a similar mutagenicmechanism.     

The correlation between DNA adducts and chromosomal aberrations in the target organ of benzidine exposed, partially-henatectomized mice

An experimental system was developed to test the associationbetween benzidine—DNA adduct levels and chromosome aberrationsin the target organ, the liver, of mice. A 2/3 partial hepatectomywas performed (0 h), then the animals were treated with benzidine(0, 7.8, 19.5, 38.2 or 97.8 mg/kg, i.p.) and an agar-coated50 mg 5-bromodeoxyuridine tablet was implanted subcutaneously(58 h). Colcemid was given at 4 mg/kg i.p. (70 h), and the animalswere sacrificed 2 h later. The liver from each animal was divided,with portions allocated for cytogenetics and DNA adduct analysis.DNA adducts were analyzed with the ³²P-postlabeling technique.DNA adduct and chromosomal aberration data were available ona total of 43 animals. Benzidine was shown to be a potent clastogenin liver, the target organ, as opposed to its reported weakactivity in the bone marrow. A linear dose response was demonstratedfor benzidine—DNA adducts found in the liver. The correlationbetween adduct levels and aberrations in individual animalswas 0.43 (P < 0.05). However, most of the residual variancewas due to four outlying cases. When these cases were removedfrom the data set and the analysis repeated, the linear correlationcoefficient increased to 0.74. When the data were analyzed bydose groups, the correlation was 0.91. These data support thehypothesis that carcinogen—DNA adducts are responsiblefor the induction of chromosomal aberrations, and perhaps othergenotoxic events, induding neoplasia.     

Loss of heterozygosity at the RB locus is frequent and correlates with muscle invasion in bladder carcinoma.

Studies of second, non-ocular tumours in surviving retinoblastoma patients and their families have reported a higher than expected incidence and lower age at diagnosis of bladder tumours. This suggests that RB mutations may predispose to bladder cancer. To determine whether this gene is involved in the development of sporadic bladder tumours we have examined 162 bladder tumours for evidence of structural alterations to the RB gene. Ninety-four patients were informative with one or more intragenic RB probes, and 28 of these (29%) showed loss of heterozygosity (LOH). Of these, two tumours showed homozygous deletions with the 5' intragenic probe p123M1.8. The probe p68RS2.0, which recognizes a variable number of tandem repeats site in intron 17 of the RB gene, detected new alleles in 5 of 162 tumours, one of which also showed LOH at another polymorphic site within the gene. The 28 tumours with RB LOH were screened with the RB cDNA probes pR3.8 and pR0.9, which revealed two homozygous deletions and one rearrangement. The tumours with RB LOH were also screened for loss of three markers which flank RB, D13S1 which maps proximal and D13S2 and D13S3 which map distal to RB on chromosome 13q. Two tumours showed retention of heterozygosity for flanking markers on one side of RB and another for markers on both sides. These results suggest that RB is the target gene on 13q in these bladder tumours. When RB loss was compared with tumour grade and stage, an association between high tumour grade and RB loss (0.005 greater than P greater than 0.001) and between muscle invasion and RB loss (P greater than 0.001) was found. Twenty-six of the 28 tumours with LOH were muscle-invasive. This represents 56% of invasive tumours. Only 2/48 (4%) superficial tumours showed RB allele loss, and one of these has progressed rapidly to invasive disease. These results show that LOH at the RB locus is a frequent genetic event in bladder tumours and may identify a subset of more aggressive tumours.     

Correlation between O6-methylguanine-DNA-methyltransferase activity and resistance of human cells to the cytotoxic and mutagenic effect of N-methyl-N''-nitro-N-nitrosoguanidine

Cells from Gardner's syndrome (GS) and familial polyposis coli(FP) patients, persons with a hereditary predisposition to coloncancer, were compared to those of normal persons for sensitivityto the cytotoxic and mutagenic action of N-methyl-N'-nitro-N-nitrosoguanidine(MNNG), a model compound chosen because methylating agents havebeen implicated in colon carcinogenesis. FP cell line GM2355and GS cell lines 2938 and GM3948 exhibited normal sensitivityto the cytotoxic and mutagenic effects of MNNG. In contrast,GS cell line GM3314 and cells from an apparently normal fetusGM0011 showed extreme sensitivity to the killing and mutageniceffect of this alkylating agent. To determine if the resistanceof the various cell lines to MNNG correlated with their abilityto remove methyl groups from the O⁶-position of guanine, wemeasured their O⁶-methylguanine-DNA methyltransferase (MT) activity.The resistant cell lines exhibited normal levels of MT; thesensitive strains showed virtually nondetectable levels of thisactivity. We also compared fibroblasts from a xeroderma pigmentosum(XP) patient (XP12BE, complementation group A), an SV40 virus-transformedXP cell line (XPIZROSV) and a normal cell line transformed bythis virus (GM637) for their response to the cytotoxic and mutageniceffect of MNNG and for MT activity. XP12BE cells showed normalsensitivity and a normal level of MT; GM637 cells showed anintermediate level of sensitivity and a reduced level of MTactivity; XP12ROSV cells were extremely sensitive to the cytotoxicand mutagenic effect of MNNG and showed virtually nondetectablelevels of MT activity. The MT did not remove methyl groups fromO⁴-methylthymine. These results suggest that O⁶-methylguanineand/or any other adduct repaired by the methyltransferase, isa potentially cytotoxic and mutagenic lesion. They also indicatethat the predisposition to colon cancer of FP and GS patientsis not necessarily correlated with an increased sensitivityof their fibroblasts to mutations induced by methylating carcinogens.     

The mutational specificity of simulated sunlight at the aprt locus in rodent cells

To elucidate the nature of sunlight mutagenesis in mammaliancells, the mutational specificity of simulated solar light (SSL)has been established at the Chinese hamster ovary adenine phosphoribosyltransferase(aprt) locus. Among a collection of 36 independent SSL-inducedmutations, the majority were single or tandem double C     

Clinical implications of T790M mutation in patients with acquired resistance to EGFR tyrosine kinase inhibitors

Objectives

Although T790M mutation is considered to be the major mechanism of acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in patients with non-small cell lung cancer (NSCLC), its clinical implication remains undetermined.

Methods

Post-progression tumor specimens were prospectively collected for T790M mutation analysis in NSCLC patients with acquired resistance to initial EGFR TKIs. Clinical features were compared between patients with and without T790M.

Results

Out of 70 cases, 36 (51%) were identified to have T790M mutation in the rebiopsy specimen. There was no difference in the pattern of disease progression, progression-free survival for initial TKIs (12.8 and 11.3 months), post-progression survival (14.7 and 14.1 months), or overall survival (43.5 and 36.8 months) in patients with and without T790M. In total, 34 patients received afatinib after post-progression biopsy as a subsequent treatment, and the response rate was 18%. The median progression-free survival for afatinib was 3.7 months for the entire group, and 3.2 and 4.6 months for the groups with and without T790M, respectively (P = 0.33).

Conclusions

The identification of T790M as acquired resistance mechanism was clinically feasible. Although T790M had no prognostic or predictive role in the present study, further research is necessary to identify patients with T790M-mutant tumors who might benefit from newly developed T790M-specific TKIs.     

Resistance to 6-thioguanine in mismatch repair-deficient human cancer cell lines correlates with an increase in induced mutations at the HPRT locus

Although the resistance to the cytotoxic response of certain DNA damaging agents has been well characterized in cells deficient in mismatch repair, little is known about how such resistance affects mutagenesis. Using human cancer cell lines defective in mismatch repair (MMR) and complementary cell lines in which the MMR defects were corrected by chromosome transfer, we present the cytotoxic effect and the mutagenic response at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus following exposure to the chemotherapeutic agent, 6-thioguanine (6-TG). Upon exposure to 6-TG, there was a differential cytotoxic response. The MMR-deficient cells were resistant to 6-TG exposure up to 5 microM, whereas the MMR-proficient cell lines were significantly more sensitive at the same levels of exposure. Furthermore, the mutagenic response at HPRT induced by 6-TG was substantially increased in the MMR-deficient lines relative to the MMR- proficient cell lines. These findings support the notion that cytotoxicity to 6-TG is mediated through functional MMR and that resistance to the cytotoxic effects of 6-TG is directly associated with an increase in induced mutations in MMR-defective cells. These data suggest that the use of 6-TG as a chemotherapeutic agent may result in the selection of MMR-defective cells, thereby predisposing the patient to an increased risk for developing secondary tumors.      

Mislocalization of death receptors correlates with cellular resistance to their cognate ligands in human breast cancer cells

Multiple clinical trials are ongoing to evaluate the potential antitumor activity of human TNF variants, Fas ligand (FasL), TNF-related apoptosis inducing ligand (TRAIL) and its agonistic antibodies. These drug products act through the death receptors (DRs) TNF receptor 1 (TNFR1), Fas/CD95, DR4 (TRAIL-R1) and/or DR5 (TRAIL-R2), respectively. Therefore, characterization of the level and localization of DR expression in cancer cells is important for DR-targeted therapy. In this study, we examined the subcellular distribution of the four DRs in a panel of 10 human breast cancer cell lines by western blots and flow cytometry and 50 human breast tumors by immunohistochemistry. Despite their total protein expressions, the DRs were found to be absent on the surface of some cell lines. Consistent with this result, all four DRs were found to be mostly expressed in the cytoplasm and/or the nucleus of primary breast tumors (n=50). We further determined the growth inhibition activity (GI50) of the death ligands, recombinant human TNFα, FasL and TRAIL, and found a correlation with the subcellular localization of the corresponding DRs. These results demonstrate an aberrant expression of the death receptors in breast cancer cells, and suggest that the lack of surface DRs appears to be predictive of tumor resistance to DR-targeted therapies.     

Map kinase activation correlates with K-ras mutation and loss of heterozygosity on chromosome 6 in alveolar bronchiolar carcinomas from B6C3F1 mice exposed to vanadium pentoxide for 2 years

Previous work showed a correlation between K-ras mutation and loss of heterozygosity (LOH) on chromosome 6 in the region of K-ras in lung carcinomas from B6C3F1 mice. We hypothesized that mitogen-activated protein kinase (MAPK) would be activated only in those lung neoplasms with both K-ras mutation and LOH. As MAPK activity can be correlated directly with signal detection using antibodies to phosphorylated MAPK, we were able to analyze lung carcinomas from B6C3F1 mice for the presence or absence of MAPK activity by western analysis. Vanadium pentoxide-induced mouse lung carcinomas, which had been shown to have a high frequency of K-ras mutations and LOH on chromosome 6 and for which frozen tumor tissue was available, were used for this study. Total MAPK expression levels were similar between normal lung and lung carcinomas. Phospho-MAPK was elevated in five of six lung carcinoma samples examined in which K-ras mutations and chromosome 6 LOH were identified and in four of five carcinomas with K-ras mutations that lacked LOH. Phospho-MAPK was undetectable or weakly expressed in seven carcinomas examined without K-ras mutations and in normal lung. By immunohistochemistry three K-ras positive/LOH negative samples exhibited multifocal areas of nuclear and cytoplasmic staining for phospho-MAPK. Large amounts of non-staining fibroblasts, lymphocytes and macrophages were also observed in these tumors. Two of these lung carcinomas were microdissected and chromosome 6 LOH was detected in regions of phospho-MAPK positive cells. These results suggest that MAPK is activated during vanadium pentoxide-induced B6C3F1 mouse lung tumorigenesis following K-ras mutation and loss of the wild-type K-ras allele.     

Loss of heterozygosity at the RB locus correlates with loss of RB protein in primary malignant neuro-endocrine lung carcinomas

RB protein expression and loss of heterozygosity (LOH) in the RB gene were studied in 77 primary lung carcinomas of all histological types. RB protein expression was studied by immunohistochemistry with 3 anti-RB antibodies, and was found altered in 23/29 (79%) neuro-endocrine (NE) carcinomas and in 18/48 (37%) non-NE carcinomas. RB gene allele status was studied with 3 probes detecting RFLP in RB locus. Fifty-five patients were informative, and loss of heterozygosity was detected in 29 (52%) of the corresponding tumors with 1 of the 3 probes used; 89% of the informative NE carcinomas, excluding careinoids, and only 13% of the non-NE carcinomas exhibited LOH and loss of RB-protein expression. LOH at the RB locus was strongly correlated with the absence of RB protein in malignant NE carcinomas, and this association was strongly correlated with the neuro-endocrine phenotype. Inactivation of the RB protein in primary NE carcinomas, excluding carcinoids, therefore seems to imply in the majority of cases the mutation of one allele and loss of the remaining allele of the Rfl gene, leading to loss of RB-protein expression. In contrast, RB-protein expression was independent of allele status in non-NE carcinomas and carcinoids.