Showing papers in "Carcinogenesis in 2001"

XRCC1. XRCC3. XPD gene polymorphisms, smoking and (32)P-DNA adducts in a sample of healthy subjects

Abstract: DNA repair genes have an important role in protecting individuals from cancer-causing agents. Polymorphisms in several DNA repair genes have been identified and individuals with non-dramatic reductions in the capacity to repair DNA damage are observed in the population, but the impact of specific genetic variants on repair phenotype and cancer risk has not yet been clarified. In 308 healthy Italian individuals belonging to the prospective European project EPIC, we have investigated the relationship between DNA damage, as measured by (32)P-DNA adduct levels, and three genetic polymorphisms in different repair genes: XRCC1-Arg399Gln (exon 10), XRCC3-Thr241Met (exon 7) and XPD-Lys751Gln (exon 23). DNA adduct levels were measured as relative adduct level (RAL) per 10(9) normal nucleotides by DNA (32)P-post-labelling assay in white blood cells from peripheral blood. Genotyping was performed by PCR-RFLP analysis. The XRCC3-241Met variant was significantly associated with higher DNA adduct levels, whereas XRCC1-399Gln and XPD-751Gln were associated with higher DNA adduct levels only in never-smokers. XRCC3-241Met homozygotes had an average DNA adduct level of 11.44 +/- 1.48 (+/-SE) compared with 7.69 +/- 0.88 in Thr/Met heterozygotes and 6.94 +/- 1.11 in Thr/Thr homozygotes (F = 3.206, P = 0.042). Never-smoking XRCC1-399Gln homozygotes had an average DNA adduct level of 15.60 +/- 5.42 compared with 6.16 +/- 0.97 in Gln/Arg heterozygotes and 6.78 +/- 1.10 in Arg/Arg homozygotes (F = 5.237, P = 0.007). A significant odds ratio (3.81, 95% CI 1.02-14.16) to have DNA adduct levels above median value was observed for XPD-751Gln versus XPD-751Lys never-smoking homozygotes after adjustment for several confounders. These data show that all the analysed polymorphisms could result in deficient DNA repair and suggest a need for further investigation into the possible interactions between these polymorphisms, smoking and other risk factors.

A reappraisal of the potential chemopreventive and chemotherapeutic properties of resveratrol.

Abstract: Resveratrol, a phytoalexin found in grapes and wines, has been reported to exhibit a wide range of pharmacological properties and is believed to play a role in the prevention of human cardiovascular disease (the so-called 'French paradox'). This molecule may also play a major role in both cancer prevention and therapy. In this review article we summarize the recent advances that have provided new insights into the molecular mechanisms underlying the promising properties of resveratrol. These include cyclooxygenase, nitric oxide synthase and cytochrome P450 inhibition, as well as cell cycle effects, apoptosis modulation and hormonal activity.

Green tea polyphenol (-)-epigallocatechin-3-gallate treatment of human skin inhibits ultraviolet radiation-induced oxidative stress

Abstract: The use of naturally occurring botanicals with substantial antioxidant activity to afford protection to human skin against UV damage is receiving increasing attention. The green tea constituent (-)-epigallocatechin-3-gallate (EGCG) is a potent antioxidant and has shown remarkable preventive effects against photocarcinogenesis and phototoxicity in mouse models. In this study we have investigated the effects of topical application of EGCG, the major polyphenol present in green tea, to human skin before UV irradiation on UV-induced markers of oxidative stress and antioxidant enzymes. Using immunohistochemistry and analytical enzyme assays, we found that application of EGCG (mg/cm(2) skin) before a single UV exposure of 4x minimal erythema dose (MED) markedly decreases UV-induced production of hydrogen peroxide (68-90%, P < 0.025-0.005) and nitric oxide (30-100%, P < 0.025-0.005) in both epidermis and dermis in a time-dependent manner. EGCG pretreatment also inhibits UV-induced infiltration of inflammatory leukocytes, particularly CD11b(+) cells (a surface marker of monocytes/macrophages and neutrophils), into the skin, which are considered to be the major producers of reactive oxygen species. EGCG treatment was also found to inhibit UV-induced epidermal lipid peroxidation at each time point studied (41-84%, P < 0.05). A single UV exposure of 4x MED to human skin was found to increase catalase activity (109-145%) and decrease glutathione peroxidase (GPx) activity (36-54%) and total glutathione (GSH) level (13-36%) at different time points studied. Pretreatment with EGCG was found to restore the UV-induced decrease in GSH level and afforded protection to the antioxidant enzyme GPx. Further studies are warranted to study the preventive effects of EGCG against multiple exposures to UV light of human skin.

Patterns of p53 G-->T transversions in lung cancers reflect the primary mutagenic signature of DNA-damage by tobacco smoke.

Abstract: It is unquestionable that the major cause of lung cancer is cigarette smoking p53 mutations are common in lung cancers from smokers but less common in non-smokers A large fraction of the p53 mutations in lung cancers are G-->T transversions, a type of mutation that is infrequent in other tumors aside from hepatocellular carcinoma Previous studies have indicated that there is a good correlation between G-->T transversion hotspots in lung cancers and sites of preferential formation of polycyclic aromatic hydrocarbon (PAH) adducts along the p53 gene The origin of p53 mutations in lung cancer has been questioned by recent reports suggesting that there are no significant differences in p53 mutation spectra between smokers and non-smokers and between lung cancers and non-lung cancers [SN Rodin and AS Rodin (2000) Human lung cancer and p53: The interplay between mutagenesis and selection P:roc Natl Acad Sci USA, 97, 12244-12249] We have re-assessed these issues by using the latest update of the p53 mutation database of the International Agency for Research on Cancer (14 051 entries) as well as recent data from the primary literature on non-smokers We come to the conclusion that the p53 mutation spectra are different between smokers and non-smokers and that this difference is highly statistically significant (G-->T transversions are 30 versus 10%; P T transversions in lung cancers but are almost exclusively G-->A transitions in non-lung cancers Our data reinforce the notion 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

Mechanisms of N-acetylcysteine in the prevention of DNA damage and cancer, with special reference to smoking-related end-points

Abstract: Although smoking cessation is the primary goal for the control of cancer and other smoking-related diseases, chemoprevention provides a complementary approach applicable to high risk individuals such as current smokers and ex-smokers. The thiol N-acetylcysteine (NAC) works per se in the extracellular environment, and is a precursor of intracellular cysteine and glutathione (GSH). Almost 40 years of experience in the prophylaxis and therapy of a variety of clinical conditions, mostly involving GSH depletion and alterations of the redox status, have established the safety of this drug, even at very high doses and for long-term treatments. A number of studies performed since 1984 have indicated that NAC has the potential to prevent cancer and other mutation-related diseases. N-Acetylcysteine has an impressive array of mechanisms and protective effects towards DNA damage and carcinogenesis, which are related to its nucleophilicity, antioxidant activity, modulation of metabolism, effects in mitochondria, decrease of the biologically effective dose of carcinogens, modulation of DNA repair, inhibition of genotoxicity and cell transformation, modulation of gene expression and signal transduction pathways, regulation of cell survival and apoptosis, anti-inflammatory activity, anti-angiogenetic activity, immunological effects, inhibition of progression to malignancy, influence on cell cycle progression, inhibition of pre-neoplastic and neoplastic lesions, inhibition of invasion and metastasis, and protection towards adverse effects of other chemopreventive agents or chemotherapeutical agents. These mechanisms are herein reviewed and commented on with special reference to smoking-related end-points, as evaluated in in vitro test systems, experimental animals and clinical trials. It is important that all protective effects of NAC were observed under a range of conditions produced by a variety of treatments or imbalances of homeostasis. However, our recent data show that, at least in mouse lung, under physiological conditions NAC does not alter per se the expression of multiple genes detected by cDNA array technology. On the whole, there is overwhelming evidence that NAC has the ability to modulate a variety of DNA damage- and cancer-related end-points.

Amino acid substitution variants of APE1 and XRCC1 genes associated with ionizing radiation sensitivity

Abstract: DNA repair gene family;Exposure to ionizing radiation (IR) has been linked to cancers it functions in the HRR pathway for repairing double-strandof the thyroid, breast and lung as well as leukemia (1). Ionizing breaks, which plays important roles in maintaining genomeradiation exposure damages cellular DNA in many ways, stability (30).

The chemistry and biology of aflatoxin B1: from mutational spectrometry to carcinogenesis

Abstract: Dietary exposure to aflatoxin B(1) (AFB(1)) is associated with an increased incidence of hepatocellular carcinoma (HCC), especially in populations in which exposure to hepatitis B virus (HBV) is a common occurrence. Most HCC samples from people living where HBV is prevalent have one striking mutational hotspot: a GC-->TA transversion at the third position of codon 249 of the p53 gene. In this review, the chemical reaction of an electrophilic derivative of aflatoxin with specific DNA sequences is examined, along with the types of mutations caused by AFB(1) and the sequence context dependence of those mutations. An attempt is made to assign the source of these mutations to specific chemical forms of AFB(1)-DNA damage. In addition, epidemiological and experimental data are examined regarding the synergistic effects of AFB(1) and HBV on HCC formation and the predominance of one hotspot GC-->TA transversion in the p53 gene of affected individuals.

Genetic polymorphisms in DNA repair genes and risk of lung cancer

Abstract: Polymorphisms in DNA repair genes may be associated with differences in the repair efficiency of DNA damage and may influence an individual's risk of lung cancer. The frequencies of several amino acid substitutions in XRCC1 (Arg194Trp, Arg280His and Arg399Gln), XRCC3 (Thr241Met), XPD (Ile199Met, His201Tyr, Asp312Asn and Lys751Gln) and XPF (Pro379Ser) genes were studied in 96 non-small-cell lung cancer (NSCLC) cases and in 96 healthy controls matched for age, gender and cigarette smoking. The XPD codon 312 Asp/Asp genotype was found to have almost twice the risk of lung cancer when the Asp/Asn + Asn/Asn combined genotype served as reference [odds ratio (OR) 1.86, 95% confidence interval (CI), 1.02-3.40]. In light cigarette smokers (less than the median of 34.5 pack-years), the XPD codon 312 Asp/Asp genotype was more frequent among cases than in controls and was associated with an increased risk of NSCLC. Compared with the Asn/Asn carriers, the OR in light smokers with the Asp/Asn genotype was 1.70 (CI0.35 0.43-6.74) and the OR in those with the Asp/Asp genotype was 5.32 (CI0.35-21.02) (P trend = 0.01). The 312 Asp/Asp genotype was not associated with lung cancer risk in never-smokers or heavy smokers (>34.5 pack-years). The XPD-312Asp and -751Lys polymorphisms were in linkage disequilibrium in the group studied; this finding was further supported by pedigree analysis of four families from Utah. The XPD 312Asp amino acid is evolutionarily conserved and is located in the seven-motif helicase domain of the RecQ family of DNA helicases. Our results indicate that these polymorphisms in the XPD gene should be investigated further for the possible attenuation of DNA repair and apoptotic functions and that additional molecular epidemiological studies are warranted to extend these findings.

Cerivastatin, an inhibitor of HMG-CoA reductase, inhibits the signaling pathways involved in the invasiveness and metastatic properties of highly invasive breast cancer cell lines: an in vitro study.

Abstract: Cerivastatin is used in the treatment of hypercholesterolemia to inhibit 3-hydroxy 3-methylglutaryl coenzyme A reductase and thus prevent the synthesis of cholesterol precursors, such as farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), responsible, respectively, for translocation of Ras and Rho to the cell membrane, a step required for their cell signaling, leading to cell proliferation and migration. Recently, it has been suggested that non lipid-related effects of statins could play a beneficial role in cancer therapy. In this study, we have investigated the mechanisms by which statins inhibit cancer and the types of cancers which could benefit from this therapy. In MDA-MB-231 cells, an aggressive breast cancer cell line with spontaneous activation of Ras and NFkappaB and overexpression of RhoA, cerivastatin induced inhibition of both cell proliferation and invasion through Matrigel. This anti-proliferative effect was related to G(1)/S arrest due to an increase in p21(Waf1/Cip1). The anti-invasive effect was observed from 18 h and could be explained by RhoA delocalization from the cell membrane, resulting in disorganization of the actin fibers and disappearance of focal adhesion sites. The importance of RhoA inactivation in both these inhibitory effects was proved by their reversion by GGPP but not by FPP. Moreover, cerivastatin was also shown to induce inactivation of NFkappaB, in a RhoA inhibition-dependent manner, resulting in a decrease in urokinase and metalloproteinase-9 expression, two proteases involved in cell migration. The participation of Ras inactivation is considered a subsidiary mechanism for the effects of cerivastatin, as they were not rescued by FPP. Prolonged treatment of MDA-MB-231 cells with high doses of cerivastatin induced a loss of cell attachment. Interestingly, the effect of cerivastatin was considerably lower on poorly invasive MCF-7 cells. In conclusion, our results suggest that cerivastatin inhibits cell signaling pathways involved in the invasiveness and metastatic properties of highly invasive cancers.

Re: Henderson,B.E. and Feigelson,H.S. (2000) hormonal carcinogenesis. Carcinogenesis, 21, 427-433.

Abstract: and postmenopausal hyperandrogenia and the risk of breast I partly agree with the authors because some recent data cancer. These data also suggest that association of androgen suggest that, apart from the mechanism mentioned, the with breast cancer is independent of estrogen, but the impact association of androgen with breast cancer is independent of of androgen on breast cancer, of course, warrants further estrogen. Xie et al. (2,3) have shown that testosterone affects investigation (particularly in young patients because of the the stroma of the gland, probably through a paracrine action poor prognosis compared with older patients) just to define of epithelial cells, and hence, the stroma may promote carcino- the strategy of chemoprevention and treatment of this cohort genesis in a reciprocal fashion, shortening the latency time of of patients. carcinogenesis in mammary gland. In vitro studies (4) on some human breast tumor cell lines also support the role of androgens References in breast carcinogenesis via stimulation of cell proliferation as 1. Henderson,B.E. and Feigelson,H.S. (2000) Hormonal carcinogenesis. direct activation of estrogen receptor alpha. Analysis of DNA Carcinogenesis, 21, 427–433. 2. Xie,B., Tsao,S.W. and Wong,Y.C. (1999) Sex hormone-induced mammary sequence from exon 1 of the androgen receptor gene suggests carcinogenesis in female Noble rats: the role of androgens. Carcinogenesis,

Etiology and chemoprevention of esophageal squamous cell carcinoma

Abstract: Squamous cell carcinoma (SCC) of the human esophagus has a multifactorial etiology involving several environmental and/or genetic factors. Current modalities of therapy for this disease offer poor survival and cure rates. Although a number of approaches could be undertaken to reduce the occurrence of esophageal SCC, including changes in lifestyle and improved nutrition, such approaches are not easily implemented. Chemoprevention offers a viable alternative that is likely to be effective against this disease. Clinical investigations in areas of high incidence of esophageal SCC have shown that primary chemoprevention of this disease is feasible, if potent inhibitors are identified. Studies in the Fischer 344 rat model of nitrosamine-induced tumorigenesis have proven valuable in understanding the biology of esophageal SCCs and help identify surrogate end-point biomarkers and putative agents that can be useful in human chemoprevention studies. Several compounds that inhibit tumor initiation by suspected human esophageal carcinogens have been identified using this model. These include diallyl sulfide, isothiocyanates and several polyphenolic compounds. Novel biomarkers, including nuclear/nucleolar morphometry using computer-assisted image analysis of preneoplastic lesions, have been developed to measure efficacy of chemopreventive agents against esophageal SCC. The identification of single agents that inhibit the progression of dysplastic lesions, however, has proven difficult. Results from a food-based approach suggest that the use of freeze-dried berry preparations can affect both initiation and promotion/progression of esophageal SCC in an animal model. These observations provide valuable information for future studies on chemoprevention of cancers of the esophagus in a clinical setting. Given the complex etiology of esophageal SCC, it is felt that the most effective chemoprevention strategies would include agents that reduce mutational events associated with carcinogen exposure in combination with agents that inhibit the progression of intraepithelial dysplasia to invasive cancer.

Synergistic mechanisms in carcinogenesis by polycyclic aromatic hydrocarbons and by tobacco smoke: a bio-historical perspective with updates

Abstract: B[a]P (benzo[a]pyrene) has been used as a prototype carcinogenic PAH since its isolation from coal tar in the 1930's. One of its diol epoxides, BPDE-2, is considered its ultimate carcinogen on the basis of its binding to DNA, mutagenicity and extreme pulmonary carcinogenicity in newborn mice. However, BPDE-1 has a similar binding to DNA and mutagenicity but it is not carcinogenic. In addition, BPDE-2 is a weak carcinogen relative to B[a]P when repeatedly applied to mouse skin, the conventional assay site. Its carcinogenicity is increased when applied once as an initiator followed repeatedly by a promoter. This indicates a major role for promotion in carcinogenesis by PAHs. Promotion itself is a 2-stage process, the second of which is selective propagation of the initiated cells. Persistent hyperplasia underlies selection by promoters. The non-carcinogenicity of BPDE-1 has yet to be resolved. PAHs have long been considered the main carcinogens of cigarette smoke but their concentration in the condensate is far too low to account by themselves for the production of skin tumors. The phenolic fraction does however have strong promotional activity when repeatedly applied to initiated mouse skin. Several constituents of cigarette smoke are co-carcinogenic when applied simultaneously with repeated applications of PAHs. Catechol is co-carcinogenic at concentrations found in the condensate. Since cigarette smoking involves protracted exposure to all the smoke constituents, co-carcinogenesis simulates its effects. Both procedures, however, indicate a major role for selection in carcinogenesis by cigarette smoke. That selection may operate on endogenous mutations as well as those induced by PAHs. There are indications that the nicotine-derived NNK which is a specific pulmonary carcinogen in animals contributes to smoking-induced lung cancer in man. Lung adenoma development by inhalation has been induced in mice by the gas phase of cigarette smoke. The role of selection has not been evaluated in either of these cases.

Alcohol and aldehyde dehydrogenase gene polymorphisms and oropharyngolaryngeal, esophageal and stomach cancers in Japanese alcoholics

Abstract: Alcohol dehydrogenase-2 (ADH2) and aldehyde dehydrogenase-2 (ALDH2) gene polymorphisms play roles in ethanol metabolism, drinking behavior and esophageal carcinogenesis in Japanese; however, the combined influence of ADH2 and ALDH2 genotypes on other aerodigestive tract cancers have not been investigated. ADH2/ALDH2 genotyping was performed on lymphocyte DNA samples from Japanese alcoholic men (526 cancer-free; 159 with solitary or multiple aerodigestive tract cancers, including 33 oropharyngolaryngeal, 112 esophageal, 38 stomach and 22 multiple primary cancers in two or three organs). After adjustment for age, drinking and smoking habits, and ADH2/ALDH2 genotypes, the presence of either ADH2*1/2*1 or ALDH2*1/2*2 significantly increased the risk for oropharyngolaryngeal cancer [odds ratios (ORs), 6.68 with ADH2*1/2*1 and 18.52 with ALDH2*1/2*2] and esophageal cancer (ORs, 2.64 and 13.50, respectively). For patients with both ADH2*1/2*1 and ALDH2*1/2*2, the risks for oropharyngolaryngeal and esophageal cancers were enhanced in a multiplicative fashion (OR = 121.77 and 40.40, respectively). A positive association with ALDH2*1/2*2 alone was observed for stomach cancer patients who also had oropharyngolaryngeal and/or esophageal cancer (OR = 110.58), but it was not observed for those with stomach cancer alone. Furthermore, in the presence of ALDH2*1/2*2, the risks for multiple intra-esophageal cancers (OR = 3.43) and for esophageal cancer with oropharyngolaryngeal and/or stomach cancer (OR = 3.95) were higher than the risks for solitary intra-esophageal cancer and for esophageal cancer alone, but these tendencies were not observed for ADH2*1/2*1 genotype. Alcoholics' population attributable risks due to ADH2/ALDH2 polymorphisms were estimated to be 82.0% for oropharyngolaryngeal cancer and 63.9% for esophageal cancer.

Association between manganese superoxide dismutase (MnSOD) gene polymorphism and breast cancer risk

Abstract: Superoxide dismutases play a key role in the detoxification of superoxide radicals and thus protect cells from damage induced by free radicals. Within mitochondria manganese superoxide dismutase (MnSOD) provides a major defence against oxidative damage by reactive oxygen species. Polymorphism in the mitochondrial targeting sequence of MnSOD has recently been associated with risk of breast cancer. We examined this in a study population consisting of 483 breast cancer cases and 482 controls, all of Finnish Caucasian origin. Odds ratios (OR) and 95% confidence intervals (95% CIs) were estimated by unconditional logistic regression. MnSOD genotypes containing the variant A allele were found to be associated with a 1.5-fold (95% CI 1.1-2.0) increased risk of breast cancer compared with those with the homozygous wild-type genotype (MnSOD VV). This finding supports the proposal that MnSOD genotypes may modify individual breast cancer risk.

Growth inhibitory effects of diallyl disulfide on human breast cancer cell lines.

Abstract: Diallyl disulfide (DADS) is an oil-soluble organosulfur compound found in garlic. The effect of synthetic DADS on the growth of estrogen receptor (ER)-positive (KPL-1 and MCF-7) and -negative (MDA-MB-231 and MKL-F) human breast cancer cell lines was examined. In an in vitro MTT assay, regardless of ER status, DADS at an IC(50) of 1.8-18.1 microM after 72 h incubation caused inhibition of growth in all four cell lines examined. Growth inhibition was due to apoptosis as seen by the appearance of a sub G1 fraction. In MDA-MB-231 cells, the apoptosis cascade comprised up-regulation of Bax protein (142%), down-regulation of Bcl-X(L) protein (38%) and activation of caspase-3 (438%) compared with controls. In an in vivo assay by orthotopic (right thoracic mammary fat pad) transplantation of KPL-1 cells in female nude mice, intraperitoneal injection of 1 or 2 mg DADS three times a week from the day of tumor cell inoculation until the end of the experiment (after 35 days) caused growth retardation and 43% reductions in primary tumor weight, respectively, compared with DADS-untreated mice without apparent side effects. Cell proliferation as evaluated by proliferating cell nuclear antigen (PCNA)-labeling in transplanted tumor of DADS-untreated mice was 59.6%, and 1 and 2 mg DADS-treated mice was 44.6 and 44.5%, respectively. In MDA-MB-231 cells, DADS antagonized the effect of linoleic acid (LA), a potent breast cancer cell stimulator (at DADS = 1.8 microM and LA > or = 6.5x10(2) microM concentration), and synergized the effect of eicosapentaenoic acid (EPA), a potent breast cancer cell suppressor (at DADS >3 x 10(-3) microM and EPA > 6.3 x 10(-1) microM concentration). Thus, DADS could be a promising anticancer agent for both hormone-dependent and -independent breast cancers, and may harmonize with polyunsaturated fatty acids known as modulators of breast cancer cell growth.

Promoter methylation of the DNA repair gene MGMT in astrocytomas is frequently associated with G : C-->A : T mutations of the TP53 tumor suppressor gene

Abstract: O(6)-Methylguanine-DNA methyltransferase (MGMT) is a repair protein that specifically removes promutagenic alkyl groups from the O(6) position of guanine in DNA. Repair of O(6)-alkylguanine adducts by tumour cells has been implicated in drug resistance since it reduces the cytotoxicity of alkylating chemotherapeutic agents. We assessed promoter methylation of the MGMT gene in astrocytic brain tumours by methylation-specific PCR. MGMT promoter methylation was detected in 26 of 54 (48%) low-grade diffuse astrocytomas (WHO grade II) and in 12 of 16 (75%) of secondary glioblastomas (WHO grade IV) that had progressed from low-grade astrocytomas. The frequency of MGMT methylation was significantly lower in primary (de novo) glioblastomas (13 of 36, 36%, P = 0.0155). The majority of low-grade astrocytomas with MGMT methylation (24/26, 92%) contained a TP53 mutation, whereas only 11 out of 28 (39%) cases without MGMT methylation carried a TP53 mutation (P A:T transition mutations at CpG sites were significantly more frequent in low-grade astrocytomas with MGMT methylation (15/26, 58%) than in those without (3/28, 11%, P = 0.0004). These results suggest that loss of MGMT expression as a result of promoter methylation, which frequently occurs at an early stage in the pathway leading to secondary glioblastomas, appears to be associated with increased frequency of TP53 mutations, in particular G:C --> A:T transitions.

Mechanisms of tolerance to DNA damaging therapeutic drugs.

Abstract: The cytotoxic effect of many anticancer drugs relies on their ability to damage DNA. Drug resistance can be associated with the ability to remove potentially lethal DNA lesions. DNA damage tolerance offers an alternative route to resistance. In a drug-tolerant cell, persistent DNA damage has become uncoupled from cell death. Tolerance to some DNA damaging drugs is accompanied by inactivation of the cell's DNA mismatch repair pathway. This is widely acknowledged as the mechanism underlying resistance to methylating agents and to 6-thioguanine which produce structurally similar types of DNA damage. Defects in mismatch repair are also associated with resistance to numerous drugs that produce a wide variety of structurally diverse DNA lesions. Here I consider possible mechanisms by which mismatch repair might influence drug resistance and the extent to which loss of mismatch repair might be considered to confer a multidrug resistance phenotype.

Dose-dependent effect of dietary meat on endogenous colonic N-nitrosation

Abstract: Human male volunteers were studied in a metabolic facility whilst they were fed randomized controlled diets. In eight volunteers there was a significant increase in faecal apparent total N:-nitroso compounds (ATNC) and nitrite excretion (P A transitions in codons 12 and 13 of K-ras. Endogenous NOC formation, combined with prolonged transit times in the gut, may explain the epidemiological associations between high meat/low fibre diets and colorectal cancer risk.

DNA hypermethylation is a mechanism for loss of expression of the HLA class I genes in human esophageal squamous cell carcinomas

Abstract: The three human leukocyte antigen (HLA) class I antigens, HLA-A, HLA-B and HLA-C, play important roles in the elimination of transformed cells by cytotoxic T cells. Frequent loss of expression of these antigens at the cell surface has been observed in many human cancers. Various mechanisms for post-transcriptional regulation have been proposed and tested but the molecular mechanisms for transcriptional regulation are not clear. We show by immunohistochemistry that the HLA class I antigens are absent in 26 of 29 (89%) samples of human esophageal squamous cell carcinomas (ESCC). Eleven of the 26 ESCC samples lost mRNA expression for at least one of the HLA genes, as shown by RT-PCR. DNA from the 29 pairs of ESCC and neighboring normal epithelium were examined for CpG island hypermethylation, homozygous deletion, microsatellite instability (MSI) and loss of heterozygosity (LOH). DNA from normal epithelial tissues had no detectable methylation of the CpG islands of any of these gene loci. Thirteen of 29 ESCC samples (45%) exhibited methylation of one or more of the three HLA loci and six samples (21%) exhibited methylation of all three loci. The HLA-B gene locus was most frequently methylated (38%). HLA-B mRNA expression in an ESCC cell line, where HLA-B was hypermethylated and did not express mRNA, was activated after treatment with 5-aza-2'-deoxycytidine. Homozygous deletion of these three gene loci was not observed. Relatively low rates of LOH and MSI were observed for the microsatellite markers D6S306, D6S258, D6S273 and D6S1666, close to the HLA-A, -B and -C loci, although a high ratio of LOH was observed at a nearby locus (represented by the markers D6S1051 and D6S1560), where the tumor suppressor gene p21(Waf1) resides. A strong correlation between genetic alterations and mRNA inactivation was observed in the ESCC samples. Our results indicate that HLA class I gene expression was frequently down-regulated in ESCC at both the protein and mRNA levels and that hypermethylation of the promoter regions of the HLA-A, -B and -C genes is a major mechanism of transcriptional inactivation.

Activation of the PPAR pathway induces apoptosis and COX-2 inhibition in HT-29 human colon cancer cells

Abstract: The gamma isoform of the peroxisome proliferator-activated receptor (PPARgamma) is a nuclear receptor that regulates adipocyte differentiation. Recently it has been shown to be expressed in human colonic mucosa and cancer, but its role in colon carcinogenesis and progression is still unclear. We demonstrate that activation of PPARgamma by ciglitazone (cig), a selective PPARgamma ligand, induces HT-29 human colon cancer cells to undergo apoptosis. Treatment with cig also down-regulates expression of cyclooxygenase-2 (COX-2) protein. Simultaneous exposure of cells to cig and 9-cis-retinoic acid (9-cis-RA), a ligand for retinoid X receptor, results in an increased apoptotic effect and increased inhibition of COX-2 expression, compared with cells treated with either cig or 9-cis-RA alone. As COX-2 is overexpressed in human colon cancer and has been implicated in augmenting invasiveness and tumorigenecity, the ability of PPARgamma activation to decrease COX-2 expression and induce apoptosis suggests that the PPARgamma pathway may be considered as a therapeutic target for colon cancer.

A Western-style diet induces benign and malignant neoplasms in the colon of normal C57Bl/6 mice

Abstract: Decreased dietary intakes of calcium, vitamin D and folic acid have been suggested as risk factors for human colon cancer. We previously fed a Western-style diet (WD) containing reduced calcium, vitamin D and increased fat content to normal C57/Bl6 mice: hyperproliferation, hyperplasia and whole crypt dysplasias developed in the colon following WD administration. Utilizing the same diet, we now also decreased the levels of several nutrients that are required for biochemical reactions involving methyl group inadequacy, i.e. folic acid, methionine, choline and vitamin B(12). Dietary levels of these nutrients were reduced to nutrient-density levels approximating those consumed by large segments of human Western populations. This further modification of the WD resulted in adenoma and carcinoma development in normal mouse colon (P < 0.04 compared with AIN-76A diet). The results indicate, for the first time, that a semi-purified rodent diet designed to mimic the human Western diet can induce colonic tumors in normal mice without carcinogen exposure.

Base excision repair in a network of defence and tolerance.

Abstract: Survival of a species depends on balanced generation of genetic variation but at the same time on the protection of the genome from changes that cause disease and fitness reduction. DNA repair pathways limit mutations but do not totally eliminate them. In fact, some DNA repair pathways are error prone. DNA repair thus has a central function not only in protecting the genome, but also in the generation of genetic diversity. Expression of DNA repair proteins is subject to a delicate balance where both too few and too many of a type may result in increased cytotoxicity and/or mutation (1–4). DNA repair is integrated with transcription, replication, cell cycle control and apoptosis in complex networks (5), a full discussion of which is beyond the scope of this article, and our abilities. While DNA repair is an ancient and conserved defence mechanism, various pathways, and additions to basic pathways, have evolved during different time periods (6). Furthermore, the pathways have overlapping specificities and function as back-up systems for each other. Thus, cytotoxic and mutagenic abasic sites (AP sites) may be dealt with by the relatively accurate mechanisms nucleotide excision repair (NER) (7,8), base excision repair (BER) and recombination repair, as well as by highly error-prone translesion DNA synthesis (TLS) (Figure 1; 9). In addition, different enzymes may substitute for each other in a specific pathway, or variants of a pathway. Conditions that govern the selection of a pathway in each case are not well understood. Furthermore, components of DNA repair systems, such as error-prone DNA polymerases, may also cause untargeted mutations at sites where no apparent damage is present. In this commentary we will address functional aspects of BER and different complementary functions in the light of recent discoveries with regard to mutations, cancer, evolution and ageing. In summary, recent results from different DNA glycosylasedeficient mice have failed to demonstrate strongly increased mutation rates, increased cancer frequencies or other severely altered phenotypes. This may be due to overlap in functions between DNA glycosylases, as well as repair by alternative

Gene–environment interaction between an aldehyde dehydrogenase-2 (ALDH2) polymorphism and alcohol consumption for the risk of esophageal cancer

Abstract: Aldehyde dehydrogenase-2 (ALDH2) degrades acetaldehyde metabolized from ethanol. Its encoding gene ALDH2 has a functional polymorphism: ALDH2 Glu487LYS: An association between this polymorphism and esophageal cancer among alcoholics has been reported. To further evaluate the gene-environment interaction, a hospital-based case-control study was conducted. Cases were 102 patients with histologically confirmed esophageal cancer and controls were 241 non-cancer outpatients of Aichi Cancer Center. ALDH2 genotypes were examined by a PCR-CTPP method developed in our laboratory, which does not require a digestion stage. Logistic regression analysis was employed for estimation of relative risk and gene-environment interaction. The allele frequency for ALDH2 Lys487 was 0.28, consistent with previous reports. The age, sex, smoking and drinking status adjusted odds ratio for the ALDH2 Glu/Lys and Lys/Lys genotypes as compared with the Glu/Glu genotype was 3.43 (95% CI 1.74-6.75). The odds ratio for heavy drinking was 49.6 (14.5-169.4) among Lys487 carriers and 7.84 (2.77-22.2) for the Glu/Glu genotype. The gene-environment interaction between alcohol drinking and the ALDH2 Lys487 allele was 6.84 (2.39-19.6), whereas no significant interaction was obtained with smoking status. Although limited because of its prevalent case-control design, our study revealed a strong gene-environment interaction between ALDH2 polymorphism and heavy alcohol consumption. Taking the observed high risk of esophageal cancer in association with the ALDH2 Lys487 allele into consideration, reducing alcohol intake may be most protective among Lys487 allele carriers of this polymorphism.

Hepatocyte growth factor signalling stimulates hypoxia inducible factor-1 (hif-1) activity in hepg2 hepatoma cells

Abstract: Hepatocyte growth factor (HGF), a multifunctional cytokine of mesenchymal origin, activates the DNA binding of hypoxia inducible factor-1 (HIF-1) in the HepG2 cell line: the activated complex contained the inducible alpha subunit. An increased expression of HIF-1alpha (mRNA and nuclear protein levels) was observed. To investigate the molecular basis of the HIF-1 response under this non-hypoxic condition, we evaluated first the expression of putative target genes. We found a time-dependent increase in steady-state mRNA levels of heme oxygenase and urokinase plasminogen activator at 4 h, followed by that of urokinase receptor at 10 h. The enhanced expression of these genes might confer the invasive phenotype, since HGF is a proliferative and scatter factor. Second, we examined some aspects of HIF-1 activity regulation in HGF-treated cells with the following findings: (i) the activation of HIF-1 DNA binding was prevented by proteasome blockade, probably because stabilization of the cytosolic alpha-subunit protein level is not sufficient to generate a functional form: also under these conditions nuclear protein level of HIF-1alpha did not increase; (ii) N-acetylcysteine, a free radical scavenger, strongly decreased HIF-1 activation suggesting a role of reactive oxygen species in this process; (iii) the thiol reducing agent dithiothreitol was ineffective. Third, consistent with these data, N-acetylcysteine reduced the stimulatory effect of HGF on stress kinase activities, while p42/44 mitogen activated kinase (MAPK) was unmodified, suggesting an involvement of c-Jun-N-terminal kinase (JNK) and p38 MAPK in HIF-1 activation. Finally, LY 294002 induced the blockade of phosphatidylinositol 3-kinase (PI3K), one of the principal transducers of HGF/Met receptor signalling, prevented the enhancement of HIF-1 DNA binding and JNK activity, but the inhibition of p42/44 MAPK phosphorylation with PD 98059 was ineffective. In conclusion, we suggest that HGF triggers a signal transduction cascade involving PI3K and ultimately activates HIF-1.

Dietary genistin stimulates growth of estrogen-dependent breast cancer tumors similar to that observed with genistein

Abstract: The estrogenic soy isoflavone, genistein, stimulates growth of estrogen-dependent human breast cancer (MCF-7) cells in vivo. Genistin is the glycoside form of genistein and the predominant form found in plants. It is generally believed that genistin is metabolized to the aglycone genistein in the lower gut. However, it is unclear if the rate of metabolism of genistin to genistein is sufficient to produce a level of genistein capable of stimulating estrogen-dependent breast cancer cell growth. Our hypothesis was that dietary genistin would stimulate tumor growth similar to that observed with genistein in athymic mice. To test this hypothesis, genistin or genistein was fed to athymic mice containing xenografted estrogen-dependent breast tumors (MCF-7). Mice were fed either genistein at 750 p.p.m. (parts per milllion) or genistin at 1200 p.p.m., which provides equal molar concentrations of aglycone equivalents in both diets. Tumor size was measured weekly for 11 weeks. At completion of the study, half of the animals per treatment group were killed and tumors collected for evaluation of cellular proliferation and estrogen-responsive pS2 gene expression. Incorporation of bromo-deoxyuridine into cellular DNA was utilized as an indicator of cellular proliferation. Dietary genistin resulted in increased tumor growth, pS2 expression and cellular proliferation similar to that observed with genistein. The remaining mice were switched to diets free of genistin and genistein. When mice were placed on isoflavone free diets, tumors regressed over a span of 9 weeks. Next, we examined how effectively and where metabolism of genistin to genistein occurred in the digestive tract. We present evidence that demonstrates conversion of genistin to its aglycone form genistein begins in the mouth and then continues in the small intestine. Both human saliva and the intestinal cell-free extract from mice converted genistin to genistein. In summary, the glycoside genistin, like the aglycone genistein, can stimulate estrogen-dependent breast cancer cell growth in vivo. Removal of genistin or genistein from the diet caused tumors to regress.

Age-related and tissue-specific accumulation of oxidative DNA base damage in 7,8-dihydro-8-oxoguanine-DNA glycosylase (Ogg1) deficient mice.

Abstract: Mutations that influence the repair of oxidative DNA modifications are expected to increase the steady-state (background) levels of these modifications and thus create a mutator phenotype that predisposes to malignant transformation. We have analysed the steady-state levels and repair kinetics of oxidative DNA modifications in cells of homozygous ogg1(-/-) null mice, which are deficient in Ogg1 protein, a DNA repair glycosylase that removes the miscoding base 8-hydroxyguanine (8-oxoG) from the genome. Oxidative purine modifications including 8-oxoG were quantified by means of an alkaline elution assay in combination with Fpg protein, the bacterial functional analogue of Ogg1 protein. In primary hepatocytes of adult ogg1(-/-) mice aged 9-12 months, the steady-state level of the lesions was 2.8-fold higher than in wild-type control mice. In contrast, no difference between ogg1(-/-) and wild-type mice was observed in splenocytes, spermatocytes and kidney cells. In hepatocytes of ogg1(-/-) mice, but not in wild-type controls, the steady-state levels increased continuously over the whole lifespan. No significant accumulation of the oxidative base modifications was observed in ogg1(-/-) fibroblasts in culture when they were kept confluent for 8 days. Both in confluent and proliferating ogg1(-/-) fibroblasts, the global repair of additional oxidative base modifications induced by photosensitization was 4-fold slower than in wild-type cells. The results suggest that the consequences of an Ogg1 defect are restricted to slowly proliferating tissues with high oxygen metabolism such as liver, because of a back-up mechanism for the repair of 8-oxoG residues that is independent of transcription and replication.

p53 protein at the hub of cellular DNA damage response pathways through sequence-specific and non-sequence-specific DNA binding.

Abstract: Our environment contains physical, chemical and pathological agents that challenge the integrity of our DNA. In addition to DNA repair, higher multicellular organisms have evolved multiple pathways of response to damage including programmed cell death-apoptosis. The p53 protein appears to sense multiple types of DNA damage and coordinate with multiple options for cellular response. The p53 protein activities depend upon its DNA binding. Specific p53 protein post-translational modifications are required for efficient sequence-specific binding and transcriptional activities. Non-sequence-specific DNA binding may involve a wide spectrum of p53 proteins and predominate as DNA damage is more severe or p53 protein is more highly induced. p53 protein is not strictly required for DNA damage sensing and repair. Rather, p53 protein may govern an apoptosis checkpoint through competition with DNA repair proteins for non-sequence-specific binding to exposed single-stranded regions in the DNA duplex. This model provides a framework for testing mechanisms of p53-mediated apoptosis dependent upon the p53 protein modification state, the level of p53 protein accumulation, the level of DNA damage and the capacity of the damaged cell to repair.

Characterization of a vitamin D receptor knockout mouse as a model of colorectal hyperproliferation and DNA damage

Abstract: The vitamin D receptor knockout (VDR-KO) mouse presents with a skeletal phenotype typical for complete lack of genomic 1,25-dihydroxycholecalciferol effects. Our previous data from human colorectal tissue suggest that the steroid hormone and its receptor may have protective function against tumour progression. In order to investigate the relevance of the vitamin D system for pre-malignant site-directed changes in the colon, we characterized the amount and site-specific distribution of the VDR along the large intestine in wild-type (WT), heterozygote (HT) and KO mice. We also evaluated expression of proliferating cell nuclear antigen (PCNA), of cyclin D1 and the levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a marker of oxidative stress. In colon ascendens, proliferative cells were dispersed all along the crypt and expression levels of all three markers were high in WT mice. A decrease of VDR expression did not affect expression significantly. In colon descendens, however, fewer proliferative cells were solely located in the lower third of the crypt, and an inverse relationship between VDR reduction, PCNA positivity and cyclin D1 expression was found in HT and KO mice. In parallel to enhanced proliferation a highly significant increase of 8-OHdG positivity occurred. Therefore, the sigmoid colon of VDR-KO mice, fed on an appropriate lactose/calcium-enriched diet to alleviate impaired calcium homeostasis-related phenotypic changes, is an excellent model for investigating induction and prevention of pre-malignant changes in one of the hotspots for human colorectal cancer incidence.

Green tea catechins partially protect DNA from (.)OH radical-induced strand breaks and base damage through fast chemical repair of DNA radicals.

Abstract: The catechins, (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG) and (-)-epigallocate-chin gallate (EGCG) are believed to be active constituents of green tea accounting for the reported chemoprevention of certain cancers. The molecular mechanisms by which the measured low concentrations (ca. micromolar) of catechins in humans can reduce the incidence of carcinogenesis is not clear. Using an in vitro plasmid DNA system and radiolytically generating reactive oxygen species (ROS) under constant scavenging conditions, we have shown that all four catechins, when present at low concentrations, ameliorate free radical damage sustained by DNA. A reduction in both prompt DNA single-strand breaks and residual damage to the DNA bases, detected by subsequent incubation with the DNA glycosylases formamidopyrimidine (FPG), endonuclease III (EndoIII) and 5' AP endonuclease exonuclease III (ExoIII), was observed. EGCG was found to be the most active of the catechins, with effects seen at micromolar concentrations. Combined fast-reaction chemistry studies support a mechanism of electron transfer (or H-atom transfer) from catechins to ROS-induced radical sites on the DNA. These results support an antioxidant role for catechins in their direct interaction with DNA radicals.

Esophageal adenocarcinoma: a review and perspectives on the mechanism of carcinogenesis and chemoprevention.

Abstract: The incidence rate of esophageal adenocarcinoma (EAC) has increased sharply in the past 30 years. Many risk factors have been identified and gastroesophageal reflux disease (GERD) is the most important one. Columnar-lined esophagus, resulting from GERD, is recognized as a key precursor lesion of EAC. In this article, we review the studies on EAC in humans and animal models. We propose that the pathogenesis of EAC is mainly driven by inflammation and oxidative stress, which are augmented by iron overload. The overproduction of prostaglandin E2 and leukotriene B4 and overexpression of their receptors are believed to be major factors in exacerbating inflammation and oxidative stress. Based on this mechanistic understanding, antioxidants, inhibitors of arachidonic acid metabolism enzymes and receptor antagonists of certain eicosanoids are proposed as potential chemopreventive agents for EAC in future studies.