Biomedical subjects

Gerd P Pfeifer

Publications and source records attributed to Gerd P Pfeifer.

At least 55 records · Page 3Linked to original sources

PubMed2004

G-to-T transversions and small tandem base deletions are the hallmark of mutations induced by ultraviolet a radiation in mammalian cells.

Ultraviolet A (UVA) radiation received from the sun and from the widespread use of tanning beds by populations residing in areas of northern latitude represents a potential risk factor for human health. The genotoxic and cancer-causing effects of UVA have remained controversial. A mutagenic role for UVA based on DNA damage formation by reactive oxygen species as well as by generation of photoproducts such as cyclobutane pyrimidine dimers (CPDs) has been suggested. Here, we investigated the mutagenicity of UVA in relation to its DNA damaging effects in transgenic Big Blue mouse embryonic fibroblasts. We determined the formation of a typical oxidative DNA lesion, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG), and of CPDs, as well as quantified the induction of mutations in the cII transgene in cells irradiated with a 2000 W UVA lamp. UVA irradiation at a dose of 18 J/cm(2) produced significant levels of 8-oxo-dG in DNA (P < 0.03) but did not yield detectable CPDs. UVA irradiation also increased the cII mutant frequency almost 5-fold over background (P < 0.01) while showing moderate cytotoxicity (70% cell viability). UVA-induced mutations were characterized by statistically significant increases in G-to-T transversions and small tandem base deletions (P = 0.0075, P = 0.008, respectively) relative to spontaneously derived mutations. This mutational spectrum differs from those previously reported for UVA in other test systems; however, it corresponds well with the known spectrum of mutations established for oxidative base lesions such as 8-oxo-dG. We conclude that UVA has the potential to trigger carcinogenesis owing to its mutagenic effects mediated through oxidative DNA damage.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed2004

The scaffold protein CNK1 interacts with the tumor suppressor RASSF1A and augments RASSF1A-induced cell death.

The connector enhancer of KSR (CNK) is a multidomain scaffold protein discovered in Drosophila, where it is necessary for Ras activation of the Raf kinase. Recent studies have shown that CNK1 also interacts with RalA and Rho and participates in some aspects of signaling by these GTPases. Herein we demonstrate a novel aspect of CNK1 function, i.e. reexpression of CNK1 suppresses tumor cell growth and promotes apoptosis. As shown previously for apoptosis induced by Ki-Ras(G12V), CNK1-induced apoptosis is suppressed by a dominant inhibitor of the mammalian sterile 20 kinases 1 and (MST1/MST2). Immunoprecipitates of MST1 endogenous to LoVo colon cancer cells contain endogenous CNK1; however, no association of these two polypeptides can be detected in a yeast two-hybrid assay. CNK1 does, however, bind directly to the RASSF1A and RASSF1C polypeptides, constitutive binding partners of the MST1/2 kinases. Deletion of the MST1 carboxyl-terminal segment that mediates its binding to RASSF1A/C eliminates the association of MST1 with CNK1. Coexpression of CNK1 with the tumor suppressive isoform, RASSF1A, greatly augments CNK1-induced apoptosis, whereas the nonsuppressive RASSF1C isoform is without effect on CNK1-induced apoptosis. Overexpression of CNK1-(1-282), a fragment that binds RASSF1A but is not proapoptotic, blocks the apoptosis induced by CNK1 and by Ki-Ras(G12V). Thus, in addition to its positive role in the proliferative outputs of active Ras, the CNK1 scaffold protein, through its binding of a RASSF1A.MST complex, also participates in the proapoptotic signaling initiated by active Ras.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Methylation of the retinoid response gene TIG1 in prostate cancer correlates with methylation of the retinoic acid receptor beta gene.

Methylation of CpG islands and associated gene silencing may lead to malignant progression, but the mechanisms of CpG island methylation in cancer are unknown. The tazarotene-induced gene 1 (TIG1), also known as retinoid acid (RA) receptor-responsive 1 gene was first identified as an RA-responsive gene and was shown to be downregulated in prostate cancer. Here, we show that this downregulation is caused by the methylation of the promoter and CpG island of TIG1. TIG1 was methylated in 26 of 50 (52%) primary prostate cancers, but was not methylated in normal tissues or benign hyperplasias. Three of four tumors that metastasized, five of six that were poorly differentiated and all that were assigned a Gleason score higher than 8 (7/7) were methylated in the promoter of TIG1. The samples with peripheral invasion were more frequently methylated (21/32, 66%) than tissues without peripheral invasion (5/18, 28%). In addition, Gleason 7-10 cancers (21/30, 70%) were significantly more frequently methylated compared with Gleason 4-6 cancers (4/18, 22%) (P<0.01). The retinoic acid receptor beta (RAR-beta) gene was frequently methylated as well (42/50, 84%). When TIG1 showed methylation, RAR-beta was also methylated (25/26 samples). In almost all samples where RAR-beta was not methylated, TIG1 was also in an unmethylated state (14/15 samples). The methylation of TIG1 and RAR-beta was positively correlated (r=0.35; P=0.017). It is possible that the methylation of the retinoid response gene TIG1 occurred in response to the methylation and inactivation of RAR-beta. These observations may contribute to our understanding of mechanistic events leading to CpG island methylation in cancer.

Carcinoma↗

PubMed2004

Biological consequences of 8-methoxypsoralen-photoinduced lesions: sequence-specificity of mutations and preponderance of T to C and T to a mutations.

Psoriatic patients undergoing psoralen plus ultraviolet radiation (PUVA) therapy are susceptible for squamous cell carcinoma and melanoma of the skin. To investigate the etiological relevance of PUVA for these diseases, we performed mutation spectrometry on the cII transgene in mouse embryonic fibroblasts treated with a single or split PUVA dose (PUVA-I or PUVA-II, respectively). Both treatments were significantly mutagenic as they increased the cII mutant frequency up to 3.7-fold over background, and produced different mutational spectra from that derived spontaneously (p<0.01), but not from one another. The signature of induced mutations, i.e., T to C transitions and T to A transversions with significant site-specificities, i.e., adjacent to T bases at the 3'-neighboring side and to pyrimidines at the 5'-neighboring side, was more pronounced after PUVA-II treatment. Also, the overall mutations occurring at T bases with the same site-specificities were more prevalent after PUVA-II treatment. The characteristic PUVA-induced mutations predominate in the p53 mutational spectrum in controlled in vivo test systems or in high-dose PUVA-treated patients, and also are easily recognizable in the overall PUVA-treated patients. We conclude that PUVA-induced mutagenesis is initiated by PUVA-I treatment and subsequently, augmented by PUVA-II treatment, leaving a unique mutational signature on the cII transgene. The signature mutations of PUVA are discernible in the p53 mutational spectrum in PUVA-treated patients but complex exposure to other therapeutic/environmental carcinogens also leads to the frequent occurrence of other types of mutations in this population.

Animals↗

PubMed2004

Parent-of-origin-specific binding of nuclear hormone receptor complexes in the H19-Igf2 imprinting control region.

Parent-of-origin-specific expression of the mouse insulin-like growth factor 2 gene (Igf2) and the closely linked H19 gene located on distal chromosome 7 is regulated by a 2.4-kb imprinting control region (ICR) located upstream of the H19 gene. In somatic cells, the maternally and paternally derived ICRs are hypo- and hypermethylated, respectively, with the former binding the insulator protein CCCTC-binding factor (CTCF) and acting to block access of enhancers to the Igf2 promoter. Here we report on a detailed in vivo footprinting analysis-using ligation-mediated PCR combined with in vivo dimethyl sulfate, DNase I, or UV treatment-of ICR sequences located outside of the CTCF binding domains. In mouse primary embryo fibroblasts carrying only maternal or paternal copies of distal chromosome 7, we have identified five prominent footprints specific to the maternal ICR. Each of the five footprinted areas contains at least two nuclear hormone receptor hexad binding sites arranged with irregular spacing. When combined with fibroblast nuclear extracts, these sequences interact with complexes containing retinoic X receptor alpha and estrogen receptor beta. More significantly, the footprint sequences bind nuclear hormone receptor complexes in male, but not female, germ cell extracts purified from fetuses at a developmental stage corresponding to the time of establishment of differential ICR methylation. These data are consistent with the possibility that nuclear hormone receptor complexes participate in the establishment of differential ICR methylation imprinting in the germ line.

Animals↗

PubMed2003

Enhancement of the mutagenicity of benzo(a)pyrene diol epoxide by a nonmutagenic dose of ultraviolet A radiation.

We investigated the effects of single and combined exposures to two ubiquitous environmental carcinogens, polycyclic aromatic hydrocarbons and UVA radiation, in Big Blue mouse embryonic fibroblasts. We quantified the cytotoxicity, DNA adduct formation, and induction of mutations in the cII transgene in cells treated with a single agent or combinations of agents in both direct and reverse order. Mapping of DNA adducts by terminal transferase-dependent PCR showed the preferential formation of bulky adducts at identical nucleotide positions along the cII gene after treatment with the prototype polycyclic aromatic hydrocarbon, benzo(a)pyrene diol epoxide [B(a)PDE], or B(a)PDE plus UVA radiation treatments but not after UVA irradiation alone. The cII mutant frequency determined by a lambda phage-based mutation detection system was not increased significantly by UVA irradiation (1.7-fold over background; P < 0.3); however, B(a)PDE alone or in combinations with UVA radiation significantly increased the cII mutant frequency (P < 0.001). The highest cII mutant frequency was induced by the treatment with B(a)PDE followed by UVA irradiation, which was more than the added mutant frequencies of the two agents individually (>12.2-fold versus <7.6-fold over background; P < 0.01). In support of these findings, DNA sequencing analyses showed that the mutational spectra induced by B(a)PDE alone or combined with UVA radiation were significantly different from those derived spontaneously (P < 0.0001) or by UVA irradiation (P < 0.0005). The signature of mutations produced by B(a)PDE i.e., "G-->T + C-->A" transversions, was significantly enhanced when the B(a)PDE treatment was followed by UVA irradiation (47% versus 65%; P < 0.01). Also, the methylated CpG dinucleotide-targeted overall mutations specifically induced by B(a)PDE were increased after the subsequent UVA irradiation (43% versus 51%, respectively). Such enhancements in the mutational signature of B(a)PDE were most pronounced within the preferential DNA adduction sites along the cII gene after the treatment with B(a)PDE plus UVA radiation, which suggests that the primary B(a)PDE adducts are converted to more mutagenic species on UVA irradiation. We conclude that UVA radiation at a nonmutagenic dose has an enhancing effect on the mechanism by which B(a)PDE induces mutations.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed2003

Control of microtubule stability by the RASSF1A tumor suppressor.

The RAS association domain family 1A (RASSF1A) gene is silenced by DNA methylation in over 50% of all solid tumors of different histological types. However, the biochemical function of the RASSF1A protein is unknown. We show that RASSF1A colocalizes with microtubules in interphase and decorates spindles and centrosomes during mitosis. RASSF1A has a strong cytoprotective activity against the microtubule-destabilizing drug nocodazole, and against cold-treatment in vivo. Conversely, loss of RASSF1 in RASSF1-/- mouse embryonic fibroblasts renders the cells more sensitive to nocodazole-induced depolymerization of microtubules. The domain required for both microtubule association and stabilization was mapped to a 169 amino-acid fragment that contains the RAS association domain. Overexpression of RASSF1A induces mitotic arrest at metaphase with aberrant mitotic cells reminiscent of such produced by the microtubule-stabilizing drug paclitaxel (taxol), including monopolar spindles, or complete lack of a mitotic spindle. Altered microtubule stability in cells lacking RASSF1A is likely to affect spindle assembly and chromosome attachment, processes that need to be carefully controlled to protect cells from genomic instability and transformation. In addition, knowledge of the microtubule-targeting function of RASSF1 may aid in the development of new anticancer drugs.

Animals↗

PubMed2003

Human thymine DNA glycosylase (TDG) and methyl-CpG-binding protein 4 (MBD4) excise thymine glycol (Tg) from a Tg:G mispair.

The repair enzymes thymine DNA glycosylase (TDG) and methyl-CpG-binding protein 4 (MBD4) remove thymines from T:G mismatches resulting from deamination of 5-methylcytosine. Thymine glycol, a common DNA lesion produced by oxidative stress, can arise from oxidation of thymine or from oxidative deamination of 5-methylcytosine, and is then present opposite adenine or opposite guanine, respectively. Here we have used oligonucleotides with thymine glycol incorporated into different sequence contexts and paired with adenine or guanine. We show that TDG and MBD4 can remove thymine glycol when present opposite guanine but not when paired with adenine. The efficiency of these enzymes for removal of thymine glycol is about half of that for removal of thymine in the same sequence context. The two proteins may have evolved to act specifically on DNA mismatches produced by deamination and by oxidation-coupled deamination of 5-methylcytosine. This repair pathway contributes to mutation avoidance at methylated CpG dinucleotides.

5-Methylcytosine↗

PubMed2003

Weak yet distinct mutagenicity of acrylamide in mammalian cells.

BACKGROUND: Despite concern raised with the announcement that common heating processes such as frying introduce acrylamide, a known rodent carcinogen, into food, the mutagenicity of acrylamide in mammalian DNA is controversial. METHODS: Big Blue mouse embryonic fibroblasts, which carry a lambda phage cII transgene, were treated with acrylamide. Formation of DNA adducts was determined by terminal transferase-dependent polymerase chain reaction. Mutational events were detected with a lambda phage-based mutagenesis assay and expressed as the frequency of the number of mutant cII plaques per total number of plaques screened. Mutations were confirmed by DNA sequence analysis. All statistical tests were two-sided. RESULTS: In vitro treatment of the cells with acrylamide at millimolar concentrations induced DNA adducts along the cII gene. Treatment with acrylamide at micromolar concentrations increased the frequency of mutations in the cII gene up to twofold relative to control treatment (13.8 x 10(-5), 95% confidence interval [CI] = 12.3 to 15.3 x 10(-5) versus 6.9 x 10(-5), 95% CI = 6.5 to 7.3 x 10(-5), df = 2, 21; P<.001; ANOVA). The specificity of acrylamide in inducing cII gene mutations was shown by a statistically significantly different mutational spectrum from that in control-treated cells, with an excess of G --> C transversions and A --> G transitions (P =.024; Adams and Skopek test). Although some of the frequently mutated sites in the cII gene co-localized with sites of preferential DNA adduct formation, there was no direct relationship. CONCLUSION: Acrylamide had distinct mutagenicity in transgenic mouse embryonic fibroblast cells, which might potentially be ascribed to its DNA adduct-inducing property. Whether acrylamide has the same effects on human cells is yet to be determined.

Acrylamide↗

PubMed2003

Frequent RASSF1A promoter hypermethylation and K-ras mutations in pancreatic carcinoma.

Recently, we have characterized the Ras association domain family 1A gene (RASSF1A) at the segment 3p21.3, which is frequently lost in variety of human cancers and epigenetically inactivated in many types of primary tumors, such as lung, breast, kidney, prostate and thyroid carcinomas. Here, we investigated the methylation status of the RASSF1A CpG island promoter in the pathogenesis of pancreatic cancer. RASSF1A hypermethylation was detected in 29 out of 45 (64%) primary adenocarcinomas, in 10 out of 12 (83%) endocrine tumors and in eight out of 18 (44%) pancreatitis samples. In seven out of eight pancreas cancer cell lines, RASSF1A was silenced and was retranscribed after treatment with 5-aza-2'-deoxycytidine. Additionally, we analysed the aberrant methylation frequency of cell cycle inhibitor p16(INK4a) and K-ras gene mutations in the pancreatic samples. p16 inactivation was detected in 43% of adenocarcinomas, in 17% of neuroendocrine tumors, in 18% of pancreatitis and in 63% of pancreas cancer cell lines. K-ras mutations were detected in 16 out of 45 (36%) primary adenocarcinomas. Pancreatic adenocarcinomas with K-ras mutation have significantly less RASSF1A methylation and vice versa (P=0.001, chi(2) test). In conclusion, our data indicate that inactivation of the RASSF1A gene is a frequent event in pancreatic cancer and suggest an inverse correlation between RASSF1A silencing and K-ras activation.

Codon↗

PubMed2003

On the origin of G --> T transversions in lung cancer.

G-->T transversions in the TP53 gene are more common in lung cancers from smokers than in any other cancer except for hepatocellular carcinomas linked to aflatoxin. The high frequency of G-->T transversions in lung cancer has been attributed to the mutagenic action of cigarette smoke components, in particular polycyclic aromatic hydrocarbons (PAH). In a recent review [Mutat. Res. 508 (2002) 1-19], Rodin and Rodin have questioned the direct mutagenic action of PAH-like compounds and have suggested that other factors, such as selection of pre-existing endogenous mutations by smoke-induced stress, can better explain the excess of G-->T transversions in lung tumors. Their two main arguments against an involvement of PAH are that smoking may inhibit the repair of G-->T primary lesions on the non-transcribed strand and that lung cancer cell lines show a higher frequency of G-->T transversions than primary lung tumors suggesting that these mutations are not related to smoking. We illustrate here that both of these suggestions are incompatible with available evidence and that the abundance and sequence specificity of G-->T transversions in lung tumors is best explained by a direct mutagenic action of PAH compounds present in cigarette smoke.

DNA Damage↗

PubMed2003

Inactivation of RAS association domain family 1A gene in cervical carcinomas and the role of human papillomavirus infection.

Recently, we have identified a new putative tumor suppressor gene, RASSF1A (Ras association domain family 1A gene), located at human chromosome 3p21.3, the segment that is often lost in many types of human cancers. The RASSF1A promoter was shown to be frequently hypermethylated in various epithelial tumors, including small cell lung, breast, bladder, prostate, gastric, and renal cell carcinomas. In this study, we have analyzed the methylation status of the RASSF1A gene in primary human cervical cancers and in eight cervical cancer cell lines. The RASSF1A promoter is hypermethylated in 4 of 42 (= 10%) of squamous cell carcinomas, in 4 of 19 (= 21%) of adenosquamous carcinomas, and in 8 of 34 (= 24%) of cervical adenocarcinomas. Although in adenocarcinomas, methylation of RASSF1A and presence of human papillomavirus (HPV) type 16 or 18 sometimes coexisted, not a single case of HPV-16/18-positive squamous cell carcinomas had RASSF1A methylation. Similarly, in all eight analyzed cervical cell lines, RASSF1A inactivation and HPV infection were mutually exclusive (Fisher's exact test; P = 0.0357): two HPV-negative cervical cancer cell lines had a methylated and silenced RASSF1A promoter (C-33A and HT-3), whereas the other six HPV-positive lines expressed RASSF1A mRNA (ME 180, MS751, SiHa, C-4I, HeLa, and CaSki). For cervical tumors and cell lines combined, the Pearson's chi(2) test (chi(2) = 3.99; P <or= 0.05) indicates a borderline-significant reverse correlation between inactivation of RASSF1A and the presence of high-risk HPVs. Our data imply that the presence of HPVs in cervical carcinomas alleviates the requirement for RASSF1A inactivation and suggests that these two events may engage the same tumorigenic pathway.

DNA Methylation↗

PubMed2003

Epigenetic inactivation of RAS association domain family protein 1 (RASSF1A) in malignant cutaneous melanoma.

Recent findings have shown the inactivation of a Ras effector homologue gene referred to as the Ras association domain family 1 (RASSF1) gene, which is a potential human tumor suppressor gene located on chromosome 3p21.3. Hypermethylation of the CpG island promoter region of a major alternative transcript of this gene, RASSF1A, has been suggested to play a key role in pathogenesis of various carcinomas. There is limited analysis of inactivation of RASSF1A in tumors other than carcinomas. Hypermethylation of two regions of the RASSF1A CpG island was investigated in metastatic cutaneous melanomas using methylation-specific PCR; region 1 is located upstream, and region 2 is located within the first exon (1alpha) of the open reading frame of the RASSF1A transcript. Eleven melanoma cell lines and 44 melanoma tumors were examined. Methylation of RASSF1A CpG island promoter region 1 was detected in 7 (64%) cell lines and 18 (41%) tumors, and methylation of region 2 was detected in 9 (82%) cell lines and 22 (50%) tumors. Overall, RASSF1A gene hypermethylation was detected in 55% of the melanoma tumors. No methylation was detected in normal skin tissues or healthy donor lymphocytes. All cell lines that showed methylation at promoter region 1 were also methylated at promoter region 2. Hypermethylation of both CpG island regions correlated with no expression of the RASSF1A gene. RASSF1A transcripts could be reexpressed in cell lines after treatment with 5'-aza-2'-deoxycytidine. Our findings indicate that the RASSF1A gene is turned off in a significant number of melanomas and that CpG promoter region hypermethylation may play a role in the transcriptional inactivation of the RASSF1A gene in malignant melanoma.

Base Sequence↗

PubMed2003

Frequent epigenetic inactivation of the RASSF1A gene in hepatocellular carcinoma.

Aberrant promoter methylation is a fundamental mechanism of inactivation of tumor suppressor genes in cancer. The Ras association domain family 1A gene (RASSF1A) is frequently epigenetically silenced in several types of human solid tumors. In this study, we have investigated the expression and methylation status of the RASSF1A gene in hepatocellular carcinoma (HCC). In two HCC cell lines (HepG2 and Hep3B) RASSF1A was inactivated and treatment of these cell lines with a DNA methylation inhibitor reactivated the transcription of RASSF1A. The methylation status of the RASSF1A promoter region was analysed in 26 primary liver tissues including HCC, hepatocellular adenoma (HCA), liver fibrosis, hepatocirrhosis. Out of 15, 14 (93%) HCC were methylated at the RASSF1A CpG island and hypermethylation was independent of hepatitis virus infection. RASSF1A was also methylated in two out of two fibrosis and in three (75%) out of four cirrhosis; the latter carries an increased risk of developing HCC. Additionally, we analysed the methylation status of p16(INK4a) and other cancer-related genes in the same liver tumors. Aberrant methylation in the HCC samples was detected in 71% of samples for p16, 25% for TIMP3, 17% for PTEN, 13% for CDH1, and 7% for RARbeta2. In conclusion, our results demonstrate that RASSF1A and p16(INK4a) inactivation by methylation are frequent events in hepatocellular carcinoma, but not in HCA, which is in contrast to HCC without cirrhosis, viral hepatitis, storage diseases, or genetic background. Therefore, this study gives additional evidence against a progression of adenoma to carcinoma in the liver. Thus, RASSF1A hypermethylation could be useful as a marker of malignancy and to distinguish between the distinct forms of highly differentiated liver neoplasm.

Base Sequence↗

PubMed2003

Deamination of 5-methylcytosines within cyclobutane pyrimidine dimers is an important component of UVB mutagenesis.

UVB mutagenesis is characterized by an abundance of C --> T and 5-methylcytosine --> T transitions at dipyrimidine sequences. It is not known how these mutations might arise. One hypothesis is that UV-induced mutations occur only after deamination of the cytosine or 5-methylcytosine within the pyrimidine dimer. It is not clear how methylation of cytosines at the 5-position influences deamination and how this affects mutagenesis. We have now conducted experiments with a CpG-methylated supF shuttle vector that was irradiated with UVB and then incubated at 37 degrees C to allow time for deamination before passage through a human cell line to establish mutations. This led to a significantly increased frequency of CC --> TT mutations and of transition mutations at 5'-PymCG-3' sequences. A spectrum of deaminated cis-syn cyclobutane pyrimidine dimers in the supF gene was determined using the mismatch glycosylase activities of MBD4 protein in combination with ligation-mediated PCR. Methylation at the C-5 position promoted the deamination of cytosines within cis-syn cyclobutane pyrimidine dimers, and these two events combined led to a significantly increased frequency of UVB-induced transition mutations at 5'-PymCG-3' sequences. Under these conditions, the majority of all supF mutations were transition mutations at 5'-PymCG-3', and they clustered at several mutational hot spots. Exactly these types of mutations are frequently observed in the p53 gene of nonmelanoma skin tumors. This particular mutagenic pathway may become prevalent under conditions of inefficient DNA repair and slow proliferation of cells in the human epidermis.

5-Methylcytosine↗

PubMed2003

Simulated sunlight and benzo[a]pyrene diol epoxide induced mutagenesis in the human p53 gene evaluated by the yeast functional assay: lack of correspondence to tumor mutation spectra.

Many mutations in the p53 gene destroy the transcriptional transactivation function of the p53 protein. This function of p53 can be determined in a yeast assay using a p53 responsive reporter gene. The yeast assay could hold promise for the identification of mutagens implicated in human cancer if the p53 mutational spectra obtained with this assay would match human tumor mutation data. Ultraviolet (UV) light from the sun and polycyclic aromatic hydrocarbons, such as benzo[a]pyrene, are strongly implicated in the spectrum of p53 mutations found in human non-melanoma skin cancers and smoking-associated lung cancers, respectively. We have used these two model mutagens to assess the feasibility of using the p53 yeast assay in cancer epidemiology. After treatment of CpG methylated p53 DNA with a solar UV simulator or with benzo[a]pyrene diol epoxide (BPDE), the modified p53 sequences were assayed in yeast for mutational outcome. As expected, BPDE produced predominantly G to T transversions and simulated sunlight produced mostly C to T transitions at dipyrimidine sites in the p53 coding sequence. However, the preferentially mutated p53 sequences (hotspots) in the yeast assay were completely different from those in the mutational spectra found in human lung and skin cancers. The data indicate that this assay is not a reliable measurement of p53 mutagenesis in human tissues and that, perhaps, transcriptional activation is not the primary function of p53 in tumor suppression.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed2002

Mutations induced by 1,3-butadiene metabolites, butadiene diolepoxide, and 1,2,3,4-diepoxybutane at the Hprt locus in CHO-K1 cells.

Butadiene (BD) is an important industrial chemical that is classified as a probable human carcinogen. Butadiene diolepoxide (BDE) and 1,2,3,4-diepoxybutane (DEB) are metabolites of carcinogenic BD and contain the DNA-reactive one and two epoxides, respectively. In this study, the mutation frequencies and mutation spectra that are induced by BDE and DEB have been investigated at the hprt locus in CHO-K1 cells. The BDE- and DEB-treated CHO-K1 cells were allowed to grow for several days, then seeded in a medium that contained 6-thioguanine in order to select the hprt mutants. BDE exhibited the mutagenic activity at concentrations that were approximately 100-times higher than DEB. The mutation spectra for BDE and DEB were determined by a reverse transcription-polymerase chain reaction of hprt mRNA, which was followed by automatic DNA sequencing of the PCR products. The mutational spectrum for BDE was exon deletions (16/41), G x C --> A x T transitions (11/41), and A x T --> G x C transitions (5/41). The mutational spectrum for DEB was exon deletions (15/39), G x C --> A x T transitions (11/39), and A x T --> T x A transversions (5/39). The most common base substitution that was induced by both BDE and DEB was G x C --> A x T transitions. The sites of the single base substitutions that were induced by BDE and DEB were guanine and adenine, which was consistent with the DNA adduct profiles. The high frequencies of the exon deletions by each metabolite occurred in the regions of exons 2, 3, or 4. These data indicate that BDE and DEB are mutagenic carcinogens by forming DNA adducts at the site of adenine and guanine, and inducing large exon deletions and single base substitutions.

Animals↗

PubMed2002

Tobacco smoke carcinogens, DNA damage and p53 mutations in smoking-associated cancers.

It is estimated that cigarette smoking kills over 1 000 000 people each year by causing lung cancer as well as many other neoplasmas. p53 mutations are frequent in tobacco-related cancers and the mutation load is often higher in cancers from smokers than from nonsmokers. In lung cancers, the p53 mutational patterns are different between smokers and nonsmokers with an excess of G to T transversions in smoking-associated cancers. The prevalence of G to T transversions is 30% in smokers' lung cancer but only 12% in lung cancers of nonsmokers. A similar trend exists, albeit less marked, in laryngeal cancers and in head and neck cancers. This type of mutation is infrequent in most other tumors aside from hepatocellular carcinoma. At several p53 mutational hotspots common to all cancers, such as codons 248 and 273, a large fraction of the mutations are G to T events in lung cancers but are almost exclusively G to A transitions in non-tobacco-related cancers. Two important classes of tobacco smoke carcinogens are the polycyclic aromatic hydrocarbons (PAH) and the nicotine-derived nitrosamines. Recent studies have indicated that there is a strong coincidence of G to T transversion hotspots in lung cancers and sites of preferential formation of PAH adducts along the p53 gene. Endogenously methylated CpG dinucleotides are the preferred sites for G to T transversions, accounting for more than 50% of such mutations in lung tumors. The same dinucleotide, when present within CpG-methylated mutational reporter genes, is the target of G to T transversion hotspots in cells exposed to the model PAH compound benzo[a]pyrene-7,8-diol-9,10-epoxide. As summarized here, a number of other tobacco smoke carcinogens also can cause G to T transversion mutations. The available data suggest that p53 mutations in lung cancers can be attributed to direct DNA damage from cigarette smoke carcinogens rather than to selection of pre-existing endogenous mutations.

Carcinogens↗