二羟环氧苯并芘诱导致癌相关差异表达cDNA序列的克隆

目的 克隆苯并（a）芘代谢物二羟环氧苯并芘（dihydroxyepoxy benzo pyrene,BPDE）诱导的人上皮细胞致癌相关差异表达cDNA序列。方法 以BPDE诱导恶性转化的人支气管上皮细胞株16HBE为模型,采用cDNA代表性差异分析方法,比较转化细胞及正常对照细胞间基因表达的差异,分离恶变细胞中差异表达的cDNA片段。分离的片断分别与pGEM-T载体连接并转化人JM109细菌中,对质粒DNA进行测序,以BLASTN程序进行GenBank的同源性检索。结果 克隆的13条cDNA序列中,有5条为新基因序列,已在dbest数据库中注册,其登录号分别为BG354691、BG354692、BG354693、BG354694和BG354695;8条cDNA有同源序列,分别与核糖体蛋白S23、MLN137、ACTN4、转移生长因子及G蛋白基因等有同源性。结论 克隆的13条cDNA序列可能与BPDE诱发细胞恶性转化密切相关。     

Carcinogen induced asynchronous replication of polyoma DNA is mediated by a trans-acting factor

We have demonstrated that carcinogen damage to DNA induces theproduction of cellular factors that act in trans to enhancethe asynchronous replication of polyoma viral DNA. Exposureof a polyoma virus-transformed rat cell line to benzo[a]pyrene-7,8-diol-9, 10-oxide (BPDE), the ultimate carcinogenic metaboliteof benzo[a]pyrene, led to the accumulation of heterogeneouslysized free viral DNA molecules which contain polyoma originsequences as well as cellular sequences that flank the integratedviral DNA. When the sequence gpt was linked to the polyoma earlyregion and transfected into rat cells, it underwent asynchronousreplication in response either to direct treatment of the transfectedcells with BPDE, or to fusion of untreated transfected cellswith normal cells previously exposed to BPDE. Transient arrestof the cell cycle by hydroxyurea, isoleucine deprivation ormethotrexate caused a slight enhancement of viral DNA replicationwhen compared with BPDE. Both aphidicolin, an inhibitor of DNApolymerase alpha, and 3-aminobenzamide, an inhibitor of poly[ADP]ribosyItransferase, caused marked inhibition of BPDE-induced viralDNA synthesis. The induction of a trans-acting factor in responseto damage of cellular DNA may be relevant to synergistic interactionsbetween environmental chemicals and DNA viruses in cell transformationand to the general phenomenon of gene amplification.     

Sensitivity to DNA damage induced by benzo(a)pyrene diol epoxide and risk of lung cancer: a case-control analysis

Levels of DNA adducts vary greatly in vivo, attributable to individual differences in enzymatic bioactivation of benzo(a)pyrene. We developed an assay to measure the levels of DNA adducts induced in vitro by benzo(a)pyrene diol epoxide (BPDE), a bioactivated form of benzo(a)pyrene. In this large molecular epidemiological study of lung cancer, we tested the hypothesis that the level of in vitro BPDE-induced adducts is associated with risk of lung cancer. This hospital-based case-control study included 221 newly diagnosed lung cancer cases and 229 healthy controls frequency matched on age, sex, ethnicity, and smoking status. Short-term cultured peripheral blood lymphocytes from each subject were exposed in vitro to BPDE (4 microm) for 5 h, and the 32P-postlabeling method was then used to measure BPDE-induced DNA adducts in the host cells. Overall, the patients had significantly higher levels of BPDE-DNA adducts than did the controls (mean +/- SD per 107 nucleotides, 93.2+/-89.3 for cases versus 63.7+/-61.1 for controls; P = 0.001). Univariate and multivariate logistic regression analyses were performed to calculate the crude and adjusted odds ratios and their 95% confidence intervals. When the median adduct level of controls (46/10(7) nucleotides) was used as the cutoff point, 64% of cases had higher levels (odds ratio, 2.15; 95% confidence interval, 1.39-3.33, adjusted for age, sex, ethnicity, body mass index, recent weight loss, pack-years smoked, smoking in the last 24 h, and family history of cancer). Stratified analyses showed consistently higher levels of BPDE-induced adducts in cases than in controls, regardless of subgroup of age, sex, ethnicity, body mass index, recent weight loss, pack-years smoked, smoking in the last 24 h, and family history of cancer. A significant dose-response relationship between the quartile levels of BPDE-induced DNA adducts and the risk of lung cancer was observed (trend test, P < 0.001). The significant association between the level of in vitro BPDE-induced DNA adducts and risk for lung cancer suggests that subjects very sensitive to BPDE-induced DNA damage may have a suboptimal ability to remove the BPDE-DNA adducts and so are susceptible to tobacco carcinogen exposure and, therefore, may be at increased risk of lung cancer.     

Methylated CpG dinucleotides are the preferential targets for G-to-T transversion mutations induced by benzo[a]pyrene diol epoxide in mammalian cells: similarities with the p53 mutation spectrum in smoking-associated lung cancers

A large fraction of the p53 mutations in lung cancers from smokers are G-to-T transversions, a type of mutation that is infrequent in lung cancers from nonsmokers and in most other tumors. Previous studies have indicated that there is an association between G-to-T transversion hotspots in lung cancers and sites of preferential formation of polycyclic aromatic hydrocarbon adducts along the p53 gene. p53 codons containing methylated CpG sequences are preferential targets for formation of adducts by (+/-) anti-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). To assess the role of CpG methylation in induction of mutations by BPDE, we analyzed BPDE mutagenesis in three CpG methylated target genes: a supF shuttle vector and the cII and lacI transgenes in embryonic mouse fibroblasts. After methylation of the shuttle vector at all CpG sequences, 42% of all G-to-T transversions were at CpG sites compared with 23% in unmethylated DNA. In the cII transgene, which is methylated at CpG sequences in vivo, 83 of 147 (56%) of the BPDE-induced mutations were G-to-T transversions, and 58% (48 of 83) of all G-to-T transversions occurred at methylated CpG sequences. In the lacI gene, 68% (75 of 111) of the BPDE-induced mutations were G-to-T events, and 58 of 75 (77%) of these occurred at methylated CpG sequences. The occurrence of transversion hotspots at methylated CpGs correlated with high levels of BPDE adducts formed at such sites. This situation mirrors the one in the p53 gene in lung cancers from smokers where 236 of 465 (51%) of the G-to-T transversions occurred at methylated CpG sites. These findings further strengthen a link between polycyclic aromatic hydrocarbons present in cigarette smoke and lung cancer mutations and provide evidence that mutational processes other than C-to-T transition mutations can occur selectively at methylated CpG sequences.     

胃癌及癌前病变差异表达基因的初步研究

目的:寻找胃癌、癌前病变中差异表达的基因,以确定胃癌发生前胃粘膜的分子变化方法:应用荧光mRNA差异显示技术分析胃癌(3例)、癌前病变(3例)和正常胃粘膜(3例),鉴别并分离差异表达的基因片段,进行PCR再扩增将扩增的cDNA片段克隆后测序,测序结果提交GenBank,经BLASTN软件检索以进行同源性分析.差异条带经Northern印迹验证.结果:发现2个差异表达的cDNA片段,1个在正常组织和癌前病变中高表达的cDNA片段在GenBank数据库中未找到同源序列,获得GenBank登陆号CB833297,并由UNIGENE基因库收录,为新基因片段.另1个在正常胃粘膜组织中高表达的cDNA片段,在GenBank数据库中与RP11-315A19克隆高度同源,但其功能目前尚不清楚.结论:本研究发现的2个在胃癌、癌前病变及正常胃粘膜组织中差异表达的基因,它们可能参与了胃癌的发病过程.     

In vitro BPDE-induced DNA adducts in peripheral lymphocytes as a risk factor for squamous cell carcinoma of the head and neck.

The level of DNA adducts under the same conditions of carcinogen exposure and cell proliferation reflects an integrated measure of carcinogen metabolism and DNA repair. Therefore, such DNA adduct levels have the potential to be a biomarker for susceptibility to chemical carcinogenesis. In a pilot study of 91 patients with squamous cell carcinomas of the head and neck and 115 controls who were frequency matched by age, sex, ethnicity, and smoking status, we applied a newly developed in vitro assay of benzo[a]pyrene diol epoxide (BPDE)-induced DNA adducts in short-term peripheral lymphocytes cultures. Levels of BPDE-DNA adducts were found to be significantly higher in cases than in controls (mean +/- SD, 76.8 +/- 77.4/10(7) and 47.1 +/- 48.0/10(7) nucleotides, respectively; p < 0.001). Using the median level of control values (35/10(7)) as the cut-off point, about 66% of cases were distributed above this level. Logistic regression analysis revealed that the level of BPDE-induced DNA adducts was an independent risk factor (odds ratio = 2.22; 95% confidence interval = 1.22--4.04) after adjustment for age, sex and smoking status. Further stratified analyses showed that levels of the induced adducts between cases and controls were significantly higher in both age groups, that is, younger or older than 60, as well as in both men and women. Smoking had a positive effect on the induced adducts. The highest level of induced adducts was seen in current smokers, then former smokers and non-smokers. There was a statistically significant dose--response relationship between the quartile levels of BPDE-induced DNA adducts and the risk of head and neck cancer (trend test, p = 0.003). Despite the relatively small sample size, the association of BPDE-induced DNA adducts and cancer risk suggests that this assay has the potential to complement with other biomarkers in identifying individuals at increased risk of developing tobacco-related cancers.     

GC-rich regions in genomes as targets for DNA alkylation

For many DNA-damaging agents, the extent of damage at any givenbase site is influenced by the DNA sequence surrounding thatsite. Most agents that alkylate the guanine N7 position, includingmechlorethamine (nitrogen mustard) and benzo(a)pyrene diol epoxide,alkylate oligo-guanine sequences preferentially. Since thesedata suggest that guaninecyosine(GC)-rich regions in genes couldbe preferred sites of damage by these agents, GenBank was searchedfor genes containing 30 bp sequences of > 90% GC (GC runs).While primate, rodent, other mammalian, vertebrate and animalvirus genes constituted 57% of the annotated entries, they included90% of the entries with the GC runs. In addition, the percentageof oncogenes in the group of the entries with GC runs was higherthan that in the overall database. One gene of interest containingGC runs was the human c-Ha-ras oncogene. All seven GC runs inthe c-Ha-ras gene are in the 5'flanking region, rather thanin the coding sequences. In fact, some of the GC runs are containedin Sp1-binding enhancer sequences. Gel analysis of the alkylationof cloned c-Ha-ras DNA by several carcinogenic alkylating agentsstrongly suggest that in this gene GC runs can be preferredsites of damage. These observations suggest mechanisms by whichDNA damage at sites other than oncogene coding sequences mayplay a role in carcinogenesis and/or chemotherapy.     

Nickel (II) enhances benzo[a]pyrene diol epoxide-induced mutagenesis through inhibition of nucleotide excision repair in human cells: a possible mechanism for nickel (II)-induced carcinogenesis

Nickel (II), a ubiquitous environmental and industrial contaminant, is a well-known human carcinogen, particularly in human lung cancer. Although by itself it is a weak mutagen, nickel (II) is able to significantly enhance the genotoxicity of other mutagens and carcinogens, such as polycyclic aromatic hydrocarbons (PAHs) and ultraviolet light. Certain human populations, especially cigarette smokers, are frequently exposed to both nickel (II) and PAHs. To understand the interplay of nickel (II) and PAHs in mutagenesis and human carcinogenesis, we used a shuttle vector mutagenicity assay to examine the effect of nickel (II) on (+/-) anti-7beta, 8alpha-dihydroxy-9alpha, 10alpha-epoxy-7,8,9,10-tetrahydroxybenzo[a]pyrene (BPDE)-induced mutagenesis in human cells. BPDE is an activated metabolite of benzo[a]pyrene (BP), a major carcinogen in cigarette smoke. The shuttle vector pSP189 modified with BPDE was transfected into human cells with and without nickel (II) exposure. We found that nickel (II) exposure significantly enhanced BPDE-induced mutation frequency, but did not change BPDE-induced mutational spectrum in the supF gene of pSP189 plasmids replicated in nucleotide excision repair (NER)-proficient human cells. However, the enhancing effect of nickel (II) on BPDE-induced mutation frequency was not observed in NER-deficient human XPA cells. We also found that nickel (II) exposure of human cells did not change the spontaneous mutation frequency of the supF gene in NER-proficient or NER-deficient human cells, indicating that nickel (II) did not affect the replication fidelity in human cells. Using a plasmid containing a luciferase reporter gene and a host cell reactivation assay, we have found that nickel (II) exposure greatly inhibited the repair of BPDE-DNA adducts in NER-proficient but not in NER-deficient cells. Together these results strongly suggest that nickel (II) can greatly enhance the mutagenicity and genotoxicity of PAHs by inhibiting the NER pathway in human cells, and this may constitute an important mechanism for nickel (II)-induced human carcinogenesis.     

Screening and cloning of multi-drug resistant genes in HL-60/MDR cells

ObjectiveTo screen and clone multi-drug resistance (MDR) related genes in MDR acute myeloid leukemia cells (HL-60/MDR).MethodsHL-60/MDR was established using All-Trans Retinoic Acid. With the HL-60 cells as “tester” and HL60/MDR as “driver”, the cDNA library of HL-60/MDR was established by suppression subtractive hybridization. Then 12 of the resulting subtracted cDNA clones were selected for DNA sequencing and homology analysis. The obtained expressed sequence tags (ESTs) were analyzed with the GenBank BLASTN program to identify sequence homologies to known genes.ResultsThe HL-60/MDR cells had different multi-drug resistance to six kinds of chemotherapeutic drugs. The 211 positive gene clones in differential cDNA library of HL-60/MDR cells were amplified with PCR and 46 gene clones exhibited differential expression (ratio >3). Twelve gene clones with significant differential expression (ratio >5) were screened out to homology analysis. Of these, 11 matched known genes and the rest 1 showed no significant homology to human or non-human known sequences. It was named as gene clone HA117.ConclusionsThis effort provides the partial list of genes differential expressed in HL-60/MDR cells and a novel gene HA117 was found to be related to MDR. Identification of these genes contributes to our understanding of MDR development, and potentially provides candidate target genes to overcome MDR.     

Detection of genetic alterations in esophageal squamous cell carcinomas and adjacent normal epithelia by comparative DNA fingerprinting using inter-simple sequence repeat PCR.

In this study, we screened 19 esophageal squamous cell carcinomas (ESCCs) for the detection of genetic alterations using inter-simple sequence repeat PCR, a DNA fingerprinting approach. Three simple repetitive unanchored primers representing tri- and tetranucleotide repeats [(GTG)(5), (GACA)(4), and (GATA)(4)] were used, and evidence of gains and losses of chromosomal sequences were detected in all tumors (19 of 19 cases) for at least one of the primers. In 13 of these cases, apparently normal marginal epithelia adjacent to the tumors were also collected and examined. Eight of the 13 (62%) patients showed matching somatic mutations in the marginal epithelia adjacent to the tumors. Five of these 8 (63%) marginal epithelial samples were histologically normal, two were dysplastic, and one had extremely rare tumor cells. In 3 of these 13 (23%) cases, the profile bands were also seen to quantitatively increase in intensity, progressing from normal epithelia to marginal epithelia to tumors. Ten profile bands showing gains and one profile band showing loss in tumors compared with the corresponding normal epithelia were cloned, and their origins were determined by sequencing. The DNA sequence of one of the profile bands showing gain in the tumor could be matched to an expressed sequence tag sequence that has been mapped to the 7q22 region, a genomic amplification novel to ESCC. The sequence of the other profile band showing gain in the tumor could be matched to a nonexonic sequence of chromosome 20, whereas the sequences of the remaining profile bands could not be matched with any known sequences after comparison with the genomic sequence data in the European Molecular Biology Laboratory and GenBank databases. The bona fide nature of the gains or losses of 11 profile bands in the original cases was confirmed by direct genomic PCR amplification. The frequencies of these specific gene alterations in tumors were then analyzed in a total of 60 ESCCs, which included 41 additional cases of ESCC. Significantly, 26 of 60 (43%) tumors showed the DNA amplification for the expressed sequence tag sequence of chromosome 7, whereas the frequency of other individual gene alterations ranged from 7% to 15%. It is concluded that the inter-simple sequence repeat PCR strategy is adequate for the detection of somatic mutations in tumors, most of which are quantitative alterations in anonymous genomic sequences. This approach is also suitable for detection of somatic mutations preceding the onset of morphologically detectable neoplasia in ESCC.     

BPDE-induced lymphocytic 3p21.3 aberrations may predict head and neck carcinoma risk

BACKGROUND: Tobacco exposure is an established risk factor for head and neck squamous cell carcinoma (HNSCC). Benzo[alpha]pyrene diol expoxide (BPDE), a main metabolic product of the tobacco smoke constituent benzo[alpha]pyrene, induces chromosomal aberrations at specific loci. Chromosomal aberrations in peripheral blood lymphocytes (PBLs) induced by BPDE may reflect individuals' genetic susceptibility to tobacco carcinogens. METHODS: This study was designed to detect BPDE-induced aberrations in PBLs at locus 3p21.3 in cultured lymphocytic cells. Our hypothesis is that the presence of BPDE-induced 3p21.3 aberrations is a biomarker of an individual's genetic susceptibility and that individuals with these aberrations are at an increased risk for HNSCC. PBL cultures from 52 cases and 54 controls were treated with 2 microM BPDE for 24 hours before the 3p21.3 aberrations were assessed by fluorescence in situ hybridization. One thousand lymphocyte interphases were scored for each sample. RESULTS: We found that BPDE-induced chromosome 3p21.3 aberrations occurred more frequently in cases (mean: 31.4 per 1000 cells) than in controls (mean: 22.1 per 1000 cells; P < 0.001). However, when 6q27 was selected as a control locus, no such difference was observed (P = 0.545). When the 75th percentile value of induced aberrations in the controls was used as a cutoff point to classify 3p21.3 BPDE-induced sensitivity, 30 of the 52 cases (57.69%) and only 14 of the 54 controls (25.93%) were sensitive to BPDE exposure. This approach resulted in an odds ratio of 4.8 (95% confidence interval: 1.87-12.28) for HNSCC risk associated with BPDE-induced 3p21.3 aberrations. There was also a dose-response relationship between the number of BPDE-induced aberrations at 3p21.3 and risk for HNSCC. CONCLUSIONS: The results from this study demonstrated that 3p21.3 may be a specific molecular target of tobacco carcinogens and that BPDE sensitivity at this locus may reflect an individual's genetic susceptibility to HNSCC.     

Risk assessment of renal cell carcinoma using alkaline comet assay

BACKGROUND: DNA damage induced by mutagens has been associated with an individual's susceptibility to cancer. METHODS: In the current study, which involved 193 renal cell carcinoma (RCC) patients and 193 controls, DNA damage before mutagen induction (baseline), after benzo(alpha)pyrene dio epoxide (BPDE) treatment, and after gamma-radiation induction were assayed by comet assay in peripheral blood lymphocytes. Olive tail moments were used as DNA damage parameters. The 5 variables that were analyzed for their associations with RCC risk were baseline, BPDE-induced, gamma-radiation-induced, net BPDE-induced (BPDE-induced subtract baseline), and net gamma-radiation-induced (gamma-radiation-induced subtract baseline) Olive tail moments. RESULTS: Significantly higher Olive tail moments were observed in cases compared with controls at baseline (1.95 vs 1.65; P = .008), after BPDE induction (3.10 vs 2.38; P < .001), and after gamma-radiation induction (4.25 vs 3.47; P < .001). The net BPDE-induced and gamma-radiation-induced DNA damage was also found to be significantly higher in cases compared with controls (P < .001 for both mutagens). Using the 75th percentile Olive tail moments in the controls as the cutoff point, the authors found that high levels of baseline DNA damage, BPDE-induced DNA damage, and gamma-radiation-induced DNA damage were associated with significantly increased risks of RCC, with odds ratios of 1.96 (95% confidence interval [95% CI], 1.26-3.06), 2.70 (95% CI, 1.72-4.23), and 3.13 (95% CI, 1.99-4.92), respectively. Similarly, net BPDE-induced and net gamma-radiation-induced DNA damages were also found to be significantly associated with elevated risks of RCC. CONCLUSIONS: The results of the current study suggest that both baseline and mutagen-induced DNA damages assessed by comet assay are associated with an increased risk of RCC.     

应用抑制消减杂交技术筛选人肾癌差异表达新基因

背景与目的：认识肾癌差异表达基因有助于阐明肾癌发生,发展的分子机制。但至今有关肾癌差异基因尤其肾癌特异相关基因的研究仍不令人满意,本实验应用抑制消减杂交技术筛选入肾癌组织与正常肾组织间差异表达的新基因,以期克隆出新的肾癌特异相关基因。方法：以肾癌组织mRNA为检测对象（Tester)。正常肾组织mRNA为驱赶者（Driver)。构建cDNA消减文库,随机挑取文库克隆进行酶切及测序,所得结果在GenBank中做同源性比对分析。对感兴趣的片段进行电子定位确定其在染色体的位置,用Northern blot,半定量RT-PCR方法检测新基因在肾癌组织与正常肾组织中的差异表达。结果：文库包含414个阳性克隆;随机挑取280个克隆提取质粒并酶切分析,其中265个有插入片段;将其中80个克隆进行测序,初步显示28、158、170、249号4个克隆为新基因片段,电子定位表明上述4个基因分别位于染色体21q^22,4q^15.3,9q^34,22q^11.2。已在GenBank中登录（BM181083,BI784487,BI863835,BI863386）,对其中28、170号克隆用Northern杂交,半定量RT-PCR检测,证实新基因在肾癌组织中表达较正常肾组织显著增高。结论：抑制消减杂交技术是筛选,克隆肾癌差异表达新基因的有效手段;筛选到的新基因片段为进一步克隆其全长,研究基因功能提供了实验基础。     

Sensitivity to benzo(a)pyrene diol-epoxide associated with risk of breast cancer in young women and modulation by glutathione S-transferase polymorphisms: a case-control study.

Mounting epidemiological evidence suggests that smoking may play a role in the etiology of breast cancer. Because smoking-related DNA adducts are detectable in both normal and malignant breast tissues, we hypothesized that breast cancer patients may be sensitive to tobacco-induced carcinogenesis, and this sensitivity could be modulated by variants of metabolic genes. To test this hypothesis, we evaluated benzo(a)pyrene diol-epoxide (BPDE)-induced mutagen sensitivity and polymorphisms of GSTM1 and GSTT1 in a pilot case-control study of breast cancer. Short-term cell cultures were established from blood samples of 100 female breast cancer patients and 105 healthy controls. After 5 h of in vitro exposure to 4 microM of BPDE, we harvested the lymphocytes for cytogenetic evaluation and recorded and compared the frequency of BPDE-induced chromatid breaks between cases and controls. We used a multiplex PCR-based assay to simultaneously detect polymorphisms of GSTM1 and GSTT1 from genomic DNA. We performed univariate and multivariate logistic regression analyses and calculated odds ratios (OR) and 95% confidence intervals (CIs). Cases had a significantly higher frequency of chromatid breaks than did controls (P < 0.0001). The level of chromatid breaks greater than the median value of controls was associated with a >3-fold increased risk of breast cancer [adjusted odds ratio (ORadj) = 3.11; 95% CI = 1.72-5.64]. The risk was more pronounced in those who were < 45 years (ORadj = 4.79; 95% CI = 1.87-12.3), ever-smokers (ORadj = 5.55; 95% CI = 1.85-16.6), alcohol drinkers (ORadj = 4.64; 95% CI = 1.70-12.7), and those who had the GSTT1 null variant (ORadj = 8.01; 95% CI = 1.16-55.3). These data suggest that sensitivity to BPDE-induced chromosomal aberrations may contribute to the risk of developing breast cancer, and such sensitivity may be modulated by both genetic and environmental factors. Larger studies are needed to confirm our findings.     

Chinese medicinal herbs modulate mutagenesis, DNA binding and metabolism of benzo[a]pyrene 7,8-dihydrodiol and benzo[a]pyrene 7,8-dihydrodiol-9,10-epoxide.

Oldenlandia diffusa(OD) and Scutellaria barbata (SB) have been used in traditional Chinese medicine for treating liver, lung and rectal tumors. In this study, the effects of aqueous extracts of these two herbs on benzo[a]pyrene 7,8-dihydrodiol. (BaP 7,8-DHD) and benzo[a]pyrene 7,8-dihydrodiol-9,10-epoxide (BPDE)-induced mutagenesis using Salmonella typhimurium TA100 as the bacterial tester strain and rat liver 9000 x g supernatant (S9) as the metabolic activation system were assessed. We also determined the effects of these two herbs on BaP 7,8-DHD and BPDE binding to calf thymus DNA. Organosoluble metabolites of BaP 7,8-DHD and water-soluble conjugates of BaP 7,8-DHD and BPDE were analyzed by high-performance liquid chromatography (HPLC) and alumina column liquid chromatography. Mutagenesis assays revealed that these two herbs produced a significant concentration-dependent inhibition of histidine-independent (His+) revertants induced by BaP 7,8-DHD and BPDE. OD and SB also inhibited BPDE-induced mutagenesis in a concentration-dependent manner in the absence of S9. SB had a greater inhibitory effect than OD. SB significantly inhibited BaP 7,8-DHD and BPDE binding to DNA while OD significantly enhanced DNA binding of both compounds. OD and SB inhibited the formation of organosoluble metabolites of BaP 7,8-DHD and decreased the formation of water-soluble conjugates of BaP 7,8-DHD and BPDE. However, the fraction of the total radioactivity in the water-soluble conjugates present as sulfate and glutathione was increased by OD and SB. Glucuronide fraction was decreased. The results of this study affirm our previous work suggesting that these two Chinese medicinal herbs possess antimutagenic properties and further suggest that they act as blocking agents through a scavenging mechanism.     

Attenuation of BPDE-induced p53 accumulation by TPA is associated with a decrease in stability and phosphorylation of p53 and downregulation of NFkappaB activation: role of p38 MAP kinase

DNA damage caused by benzo[a]pyrene (B[a]P) or other polynuclear hydrocarbons (PAHs) induce p53 protein as a protective measure to eliminate the possibility of mutagenic fixation of the DNA damage. 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibits p53 response induced by B[a]P and other DNA-damaging agents and may cause tumor promotion. The molecular mechanism of attenuation of B[a]P-induced p53 response by TPA is not known. We investigated the effect of TPA on p53 response in (+/-)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE)-treated mouse epidermal JB6(P(+)) Cl 41 cells. BPDE treatment induced p53 accumulation which was attenuated significantly by TPA. Cells treated with BPDE and TPA showed increased ratio of Mdm2 to p53 proteins in p53 immunoprecipitate and decreased p53 life span compared to BPDE-treated cells indicating p53 destabilization by TPA. TPA also inhibited BPDE-induced p53 phosphorylation at serine15. Activation of both ERKs and p38 MAPK by BPDE and attenuation of BPDE-induced p53 accumulation by U0126 or SB202190, specific inhibitor of MEK1/2 or p38 MAPK, indicate the role of ERKs and p38 MAPK in p53 accumulation. Interestingly, TPA potentiated BPDE-induced activation of ERKs whereas p38 MAPK activation was significantly inhibited by TPA, suggesting that inhibition of p38 MAPK is involved in p53 attenuation by TPA. Furthermore, SB202190 treatment caused decreased p53 stability and inhibition of phosphorylation of p53 at serine15 in BPDE-treated cells. We also observed that TPA or SB202190 attenuated BPDE-induced nuclear factor kappa B (NFkappaB) activation in JB6 Cl 41 cells harboring NFkappaB reporter plasmid. To our knowledge this is the first report that TPA inhibits chemical carcinogen-induced NFkappaB activation. Interference of TPA with BPDE-induced NFkappaB activation implicates abrogation of p53 function which has been discussed. Overall, our data suggest that abrogation of BPDE-induced p53 response and of NFkappaB activation by TPA is mediated by impairment of the signaling pathway involving p38 MAPK.     

人高、低转移肺大细胞癌细胞系的mRNA差异显示研究

目的 通过研究人高、低转移肺大细胞癌细胞系基冈差异表达谱，克隆与肿瘤转移相关的基因。方法 应用mRNA差异显示技术(DDRT-PCR)对高、低转移人肺大细胞癌细胞系的mRNA进行差异显示，切胶回收差异表达cDNA，克隆测序后与GenBank数据库进行Blast比对。结果 获得差异表达cDNA50条，其中已知功能基因18条，末知功能基因16条，DNA来源16条，EST4条。结论 人高、低转移肺大细胞癌细胞系的基因表达差异较大，转移的过程受基因的表达与调控的影响。应用DDRT-PCR技术有单找到调控肿瘤转移的基因，为肿瘤的诊断和治疗提供新的线索。