Inhibition of epidermal growth factor receptor expression by RNA interference in A549 cells

AIM: To investigate the biological features of A549 cells in which epidermal growth factor (EGF) receptors expression were suppressed by RNA interference (RNAi). METHODS: A549 cells were transfected using short small interfering RNAs (siRNAs) formulated with Lipofectamine 2000. The EGF receptor numbers were determined by Western blotting and flowcytometry. The antiproliferative effects of sequence specific double stranded RNA (dsRNA) were assessed using cell count, colony assay and scratch assay. The chemosensitivity of transfected cells to cisplatin was measured by MTT. RESULTS: Sequence specific dsRNA-EGFR down-regulated EGF receptor expression dramatically. Compared with the control group, dsRNA-EGFR reduced the cell number by 85.0 %, decreased the colonies by 63.3 %, inhibited the migration by 87.2 %, and increased the sensitivity of A549 to cisplatin by four-fold. CONCLUSION: Sequence specific dsRNA-EGFR were capable of suppressing EGF receptor expression, hence significantly inhibiting cellular proliferation and motility, and enhancing chemosensitivity of A549 cells to cisplatin. The successful application of dsRNA-EGFR for inhibition of proliferation in EGF receptor overexpressing cells can help extend the list of available therapeutic modalities in the treatment of non-small-cell lung carcinoma (NSCLC).

Role of smoking in global and regional cancer epidemiology: current patterns and data needs

Although smoking is widely recognized as a major cause of cancer, there is little information on how it contributes to the global and regional burden of cancers in combination with other risk factors that affect background cancer mortality patterns. We used data from the American Cancer Society's Cancer Prevention Study II (CPS-II) and the WHO and IARC cancer mortality databases to estimate deaths from 8 clusters of site-specific cancers caused by smoking, for 14 epidemiologic subregions of the world, by age and sex. We used lung cancer mortality as an indirect marker for accumulated smoking hazard. CPS-II hazards were adjusted for important covariates. In the year 2000, an estimated 1.42 (95% CI 1.27-1.57) million cancer deaths in the world, 21% of total global cancer deaths, were caused by smoking. Of these, 1.18 million deaths were among men and 0.24 million among women; 625,000 (95% CI 485,000-749,000) smoking-caused cancer deaths occurred in the developing world and 794,000 (95% CI 749,000-840,000) in industrialized regions. Lung cancer accounted for 60% of smoking-attributable cancer mortality, followed by cancers of the upper aerodigestive tract (20%). Based on available data, more than one in every 5 cancer deaths in the world in the year 2000 were caused by smoking, making it possibly the single largest preventable cause of cancer mortality. There was significant variability across regions in the role of smoking as a cause of the different site-specific cancers. This variability illustrates the importance of coupling research and surveillance of smoking with that for other risk factors for more effective cancer prevention. (C) 2005 Wiley-Liss, Inc.

The Australian mortality decline: cause-specific mortality 1907-1990

This review describes the changes in composition of mortality by major attributed cause during the Australian mortality decline this century. The principal categories employed were: infectious diseases, nonrheumatic cardiovascular disease, external causes, cancer,'other' causes and ill-defined conditions. The data were age-adjusted. Besides registration problems (which also affect all-cause mortality) artefacts due to changes in diagnostic designation and coding-are evident. The most obvious trends over the period are the decline in infectious disease mortality (half the decline 1907-1990 occurs before 1949), and the epidemic of circulatory disease mortality which appears to commence around 1930, peaks during the 1950s and 1960s, and declines from 1970 to 1990 (to a rate half that at the peak). Mortality for cancer remains static for females after 1907, but increases steadily for males, reaching a plateau in the mid-1980s (owing to trends in lung cancer); trends in cancers of individual sites are diverse. External cause mortality declines after 1970. The decline in total mortality to 1930 is associated with decline in infection and 'other' causes, Stagnation of mortality decline in 1930-1940 and 1946-1970 for males is a consequence of contemporaneous movements in opposite directions of infection mortality (decrease) and circulatory disease and cancer mortality (increase). In females, declines in infections and 'other' causes of death exceed the increase in circulatory disease mortality until 1960, then stability in all major causes of death to 1970. The overall mortality decline since 1970 is a consequence of a reduction in circulatory disease,'other' cause, external cause and infection mortality, despite the increase in cancer mortality (for males).

Allele and genotype frequencies of polymorphic cytochromes P4502D6, 2C19 and 2E1 in Aborigines from Western Australia

The polymorphisms of the important xenobiotic metabolizing enzymes CYP2D6, CYP2C19 and CYP2E1 have been studied extensively in a large number of populations and show significant heterogeneity in the frequency of different alleles/genotypes and in the prevalence of the extensive and poor metabolizer phenotypes, Understanding of inter-ethnic differences in genotypes is important in prediction of either beneficial or adverse effects from therapeutic agents and other xenobiotics. Since no data were available for Australian Aborigines, we investigated the frequencies of alleles and genotypes for CYP2D6, CYP2C19 and CYP2E1 in a population living in the far north of Western Australia. Because of its geographical isolation, this population can serve as a model to study the impact of evolutionary forces on the distribution of different alleles for xenobiotic metabolizing enzymes. Twelve CYP2D6 alleles were analysed, The wild-type allele *1 was the most frequent (85.8%) and the non-functional alleles (*4, *5, *16) had an overall frequency of less than 10%. Only one subject (0.4%) was a poor metabolizer for CYP2D6 because of the genotype *5/*5, For CYP2C19, the frequencies of the *1 (wild-type) and the non-functional (*2 and *3) alleles were 50.2%, 35.5% and 14.3%, respectively. The combined CYP2C19 genotypes (*2/*2, *2/*3 or *3/*3) correspond to a predicted frequency of 25.6% for the CYP2C19 poor metabolizer phenotype, For CYP2E1, only one subject had the rare c2 allele giving an overall allele frequency of 0.2%. For CYP2D6 and CYP2C19, allele frequencies and predicted phenotypes differed significantly from those for Caucasians but were similar to those for Orientals indicating a close relationship to East Asian populations. Differences between Aborigines and Orientals in allele frequencies for CYP2D6*10 and CYP2E1 c2 may have arisen through natural selection, or genetic drift, respectively, Pharmacogenetics 11:69-76 (C) 2001 Lippincott Williams & Wilkins.

Tolerance or immunity to a tumor antigen expressed in somatic cells can be determined by systemic proinflammatory signals at the time of first antigen exposure

Mice transgenic for the E7 tumor Ag of human papillomavirus type 16, driven from a keratin 14 promoter, express E7 in keratinocytes but not dendritic cells. Grafted E7-transgenic skin is not rejected by E7-immunized mice that reject E7-transduced transplantable tumors. Rejection of recently transplanted E7-transgenic skin grafts, but not of control nontransgenic grafts or of established E7-transgenic grafts, is induced by systemic administration of live or killed Listeria monocytogenes or of endotoxin. Graft recipients that reject an E7 graft reject a subsequent E7 graft more rapidly and without further L. monocytogenes exposure, whereas recipients of an E7 graft given without L. monocytogenes do not reject a second graft, even if given with L. monocytogenes. Thus, cross-presentation of E7 from keratinocytes to the adaptive immune system occurs with or without a proinflammatory stimulus, but proinflammatory stimuli at the time of first cross-presentation of Ag can determine the nature of the immune response to the Ag. Furthermore, immune effector mechanisms responsible for rejection of epithelium expressing a tumor Ag in keratinocytes are different from those that reject an E7-expressing transplantable tumor. These observations have implications for immunotherapy for epithelial cancers.

Relationships between non-occupational cadmium exposure and expression of nine cytochrome P450 forms in human liver and kidney cortex samples

This study was undertaken to assess associations between age, gender, cigarette smoke and non-workplace cadmium exposure, and liver pathology and inter-individual variation in cytochrome P450 (CYP) expression in human tissues. Autopsy specimens of twenty-eight Queensland residents whose ages ranged from 3 to 89 years were analyzed for the presence of nine CYP protein isoforms by immunoblotting. All subjects were Caucasians and their liver cadmium contents ranged from 0.11 to 3.95 kg/g wet weight, while their kidney cadmium contents were in the range of 2 to 63 mug/g wet weight. CYP1A2, CYP2A6, CYP2D6, CYP3A4, and CYP3A5 were detected in liver but not in kidney, and CYP1A1 and CYP1B1 were not found in liver or kidney. Lowered liver CYP2C8/19 protein contents were found to be associated with liver pathology. Importantly, we show elevated levels of CYP2C9 protein to be associated with cadmium accumulation in liver. No mechanism that explains this association is apparent, but there are two possibilities that require further study. One is that variation in CYP2C9 protein levels may be, in part, attributed to an individual's non-workplace exposure to cadmium, or an individual's CYP2C9 genotype may be a risk factor for cadmium accumulation. A positive correlation was found between liver CYP3A4 protein and subject age. Levels of liver CYPIA2 protein, but not other CYP forms, were increased in people more exposed to cigarette smoke, but there was no association between CYPIA2 protein and cadmium. CYP2A6 protein was found in all liver samples and CYP2A6 gene typing indicated the absence of CYP2A6 null allele (CYP2A6(D)) in this sample group, confirming very low prevalence of homozygous CYP2A6(D) in Caucasians. CYP2A6 gene types W/W, WIC, and CIC were not associated with variations in liver microsomal CYP2A6 protein. CYP2D6 protein was absent in all twenty-five kidney samples tested but was detectable in liver samples of all but two subjects, indicating the prevalence of the CYP2D6 null allele (CYP2D6(D)) in this sample group to be about 7%, typical of Caucasian populations. (C) 2001 Elsevier Science Inc. All rights reserved.

Metabolic activation of polycyclic aromatic hydrocarbons and other procarcinogens by cytochromes P450 1A1 and P4501B1 allelic variants and other human cytochromes P450 in Salmonella typhimurium NM2009

A variety of polycyclic aromatic hydrocarbons and their dihydrodiol derivatives, arylamines, heterocyclic amines, and nitroarenes, were incubated with cDNA-based recombinant (Escherichia coli or Trichoplusia ni) systems expressing different forms of human cytochrome P450 (P450 or CYP) and NADPH-P450 reductase using Salmonella typhimurium, tester strain NM2009, and the resultant DNA damage caused by the reactive metabolites was detected by measuring expression of umu gene in the cells. Recombinant (bacterial) CYP1A1 was slightly more active than any of four CYP1B1 allelic variants, CYP1B1*1, CYP1B1*2, CYP1B1*3, and CYP1B1*6, in catalyzing activation of chrysene-1,2-diol, benz[a]anthracene-trans-1,2-, 3,4-, 5,6-, and 8,9-diol, fluoranthene-2,3-diol, dibenzo[a]pyrene, benzo[c]phenanthrene, and dibenz[a,h]anthracene and several arylamines and heterocyclic amines, whereas CYP1A1 and CYP1B1 enzymes had essentially similar catalytic specificities toward other procarcinogens, such as (+)-, (-)-, and (+/-)-benzo[a]pyrene-7,8-diol, 5-methylchrysene-1,2-diol, 7,12-dimethylbenz[a]anthracene-3,4-diol, dibenzo[a,l]pyrene-11,12-diol, benzo[b]fluoranthene-9,10-diol, benzo[c]chrysene, 5,6-dimethylchrysene-1,2-diol, benzo[c]phenanthrene-3,4-diol, 7,12-dimethylbenz[a]anthracene, benzo[a]pyrene, 5-methylchrysene, and benz[a]anthracene. We also determined activation of these procarcinogens by recombinant (T. ni) human P450 enzymes in S. typhimurium NM2009. There were good correlations between activities of procarcinogen activation by CYP1A1 preparations expressed in E. coli and T. ni cells, although basal activities with three lots of CYP1B1 in T. ni cells were very high without substrates and NADPH in our assay system. Using 14 forms of human P450S (but not CYP1B1) (in T. ni cells), we found that CY1P1A2, 2C9, 3A4, and 2C19 catalyzed activation of several of polycyclic aromatic hydrocarbons at much slower rates than those catalyzed by CYP1A1 and that other enzymes, including CYP2A6, 2B6, 2C8, 2C18, 2D6, 2E1, 3A5, 3A7, and 4A11, were almost inactive in the activation of polycyclic aromatic hydrocarbons examined here.

Primary Bartholin gland carcinoma: a report of seven cases

This study reviews our experience with 7 patients with primary Bartholin gland cancer (BGC) treated at the Queensland Gynaecological Cancer Centre (QCGC) and compares this with previously published data. A retrospective clinicopathologic review of all patients with primary BGC treated at QCGC from 1988 to 2000 was performed. Of the 7 patients treated, all underwent primary surgery and 5 of the 7 patients received radiotherapy postoperatively. All patients presented with a local swelling or a lump. Two had associated discharge and 2 had associated pain. Of the 7 patients, 2, 3 and 2 respectively were classified as having Stage IB, II or III disease. Five of the 7 patients had squamous cell carcinoma (SCC), one had adenoid-cystic carcinoma and 1 had a small-cell neuroendocrine cancer of the Bartholin gland. None of the patients with SCC developed recurrent disease. The patient with adenoid-cystic carcinoma experienced local recurrences at 4 years and again at 5 years and 3 months. Nine years after primary treatment she was diagnosed with pulmonary metastases. The patient with small-cell neuroendocrine cancer of the Bartholin gland was considered tumour-free after operation. Thorough imaging, including a CT scan of her chest, abdomen and pelvis showed no evidence of disease. She died 1 year and three months after diagnosis from disseminated pulmonary disease. We present the first report, of small cell neuroendocrine cancer of the Bartholin gland. Therapeutic principles in the management of vulval cancer at other sites appear to be appropriate for management of BGC.

The design of hazard risk assessment matrices for ranking occupational health risks and their application in mining and minerals processing

Two hazard risk assessment matrices for the ranking of occupational health risks are described. The qualitative matrix uses qualitative measures of probability and consequence to determine risk assessment codes for hazard-disease combinations. A walk-through survey of an underground metalliferous mine and concentrator is used to demonstrate how the qualitative matrix can be applied to determine priorities for the control of occupational health hazards. The semi-quantitative matrix uses attributable risk as a quantitative measure of probability and uses qualitative measures of consequence. A practical application of this matrix is the determination of occupational health priorities using existing epidemiological studies. Calculated attributable risks from epidemiological studies of hazard-disease combinations in mining and minerals processing are used as examples. These historic response data do not reflect the risks associated with current exposures. A method using current exposure data, known exposure-response relationships and the semi-quantitative matrix is proposed for more accurate and current risk rankings.

Immunohistochemistry versus microsatellite instability testing in phenotyping colorectal tumors

Purpose: To compare microsatellite instability (MSI) testing with immunohistochemical (IHC) detection of hMLH1 and hMSH2 in colorectal cancer. Patients and Methods: Colorectal cancers from 1, 144 patients were assessed for DNA mismatch repair deficiency by two methods: MSI testing and IHC detection of hMLH1 and hMSH2 gene products. High-frequency MSI (MSI-H) was defined as more than 30% instability of at least five markers; low-level MSI (MSI-L) was defined as 1% to 29% of loci unstable. Results: Of 1, 144 tumors tested, 818 showed intact expression of hMLH1 and hMSH2. Of these, 680 were microsatellite stable (MSS), 27 were MSI-H, and 111 were MSI-L. In all, 228 tumors showed absence of hMLH1 expression and 98 showed absence of hMSH2 expression: all were MSI-H. Conclusion: IHC in colorectal tumors for protein products hMLH1 and hMSH2 provides a rapid, cost-effective, sensitive (92.3%), and extremely specific (100%) method for screening for DNA mismatch repair defects. The predictive value of normal IHC for an MSS/MSI-L phenotype was 96.7%, and the predictive value of abnormal IHC was 100% for an MSI-H phenotype. Testing strategies must take into account acceptability of missing some cases of MSI-H tumors if only IHC is performed. (C) 2002 by American Society of Clinical Oncology.

Variation in coumarin 7-hydroxylase activity associated with genetic polymorphism of cytochrome P450 2A6 and the body status of iron stores in adult Thai males and females

The relationships between catalytic activity of cytochrome P450 2A6 (CYP2A6), polymorphism of CYP2A6 gene, gender and levels of body iron stores were analysed in a sample group of 202 apparently healthy Thais, aged 1947 years. Eleven individuals were found to have high activity of CYP2A6, judged by the relatively large amounts (11.2-14.6 mg) of 7-hydroyxcoumarin (7-OHC) excreted 3 h following administration of 15 mg of coumarin. Ten individuals, however, did not excrete any 7-OHC. Of these 10, four were found to have no CYP2A6 gene (whole gene deletion; CYP2A6*4 allele). The frequency of the CYP2A6 alleles; *1A, *1B and *4 in the whole sample group was 52, 40 and 8% while the frequency of the CYP2A6 gene types; *1A/* 1A, *1A/* 1B, *1B/* 1B, *1A/* 4, *1BI* 4, *4/* 4 was 29, 41, 16, 7, 5 and 2%. Subjects having CYP2A6* 1A/* 1B gene-type group were found to have higher rates of coumarin 7-hydroxylation compared with those of the CYP2A6* 1B/* 1B and CYP2A6* 1A/* 4 gene types. The inter-individual variability in CYP2A6 catalytic activity was therefore attributed in part to the CYP2A6 genetic polymorphism. Variation in CYP2A6 activity in this sample group was not associated with gender but, interestingly, it did show an inverse association with plasma ferritin; an indicator of body iron stores. Higher rates of coumarin 7-hydroxylation were found in individuals with low body iron stores (plasma ferritin < 20 μg/l) compared with subjects having normal body iron store status. Subjects (n = 16) with iron overload (plasma ferritin > 300 mug/l) also tended to have elevated rates of coumarin 7-hydroxylation. These results suggest an increased CYP2A6 expression in subjects who have excessive body iron stores. Further investigations into the underlying factors that may lead to increased expression of CYP2A6 in association with abnormal body iron stores are currently in progress in our laboratory. Pharmacogenetics 12:241-249 (C) 2002 Lippincott Williams Wilkins.