Sisko Anttila

Diagnosis of polymorphisms in carcinogen-activating and inactivating enzymes and cancer susceptibility--a review.

Up to 90% of all cancers are possibly caused by environmental factors, such as tobacco smoke, diet and occupational exposures. The majority of chemical carcinogens require metabolic activation before they interact with cellular macromolecules and can cause cancer initiation. The xenobiotic-metabolising machinery contains two main types of enzymes: the phase-I cytochromes P-450 (CYP) mediating oxidative metabolism, and phase-II conjugating enzymes. Several phase-I and phase-II genes have recently been cloned and identified in humans. Many of them show polymorphism and have been suggested to contribute to individual cancer susceptibility as genetic modifiers of cancer risk. Altered phenotypes and genotypes in the CYP subfamilies CYP1A1, CYP2D6 and CYP2E1 have been associated with tobacco smoke-induced lung cancer and other cancers. Defective glutathione S-transferase (GST) and N-acetyltransferase (NAT) enzymes have been associated with an increased risk of developing lung and bladder cancer. There are also several studies in each category in which no associations have been found. The risk of developing lung cancer is dramatically (up to 40-fold) elevated in subpopulations having simultaneously high-risk genotypes in CYP1A1 and GSTM1. There are several difficulties in this area of research. First, many of the observed restriction-fragment length polymorphisms (RFLPs) are due to mutations in introns or other silent areas of DNA, raising the possibility that any associations found between RFLPs and cancer occur only by chance. Second, biologically plausible mechanisms linking genotypes and cancer are lacking in most of the observed cases.(ABSTRACT TRUNCATED AT 250 WORDS)

Combined effect of polymorphic GST genes on individual susceptibility to lung cancer

Glutathione S‐transferases (GSTs) are known to take part in detoxification of many potentially carcinogenic compounds. Therefore, polymorphisms of the GST genes have been considered as potentially important modifiers of individual risk of environmentally induced cancers. The association between lack of glutathione S‐transferase M1 gene (GSTM1 null genotype) and susceptibility to smoking‐related lung cancer has been actively studied, with contradictory results. In contrast, little is known about the more recently found polymorphisms in GSTM3, GSTP1 and GSTT1 genes with respect to individual responses to environmental exposures. In this study, we determined the genotype distribution of all these genes, and their combinations, among 208 Finnish lung cancer patients and 294 population controls. None of the genotypes studied had a statistically significant effect on lung cancer risk, when studied separately. However, a significant association was observed for concurrent lack of the GSTM1 and GSTT1 genes and susceptibility to squamous cell carcinoma. For that cell type, the risk was more than 2‐fold when compared with that of individuals having other genotype combinations (OR = 2.3; 95% CI = 1.0–5.3; p = 0.05). Moreover, the risk was mostly attributable to patients with smoking history of 40 pack‐years or less (OR = 2.9; 95% CI = 1.1–7.7; p = 0.03). In contrast, this genotype combination did not affect the risk for other histological types of lung cancer, and the other genotype combinations had no effects on individual susceptibility to this malignancy. The overall role of GST polymorphisms in modifying the lung cancer risk may therefore be more limited than has been so far anticipated. Int. J. Cancer<0R> 77:516–521, 1998.

Identification of differentially expressed genes in pulmonary adenocarcinoma by using cDNA array

No clear patterns in molecular changes underlying the malignant processes in lung cancer of different histological types have been found so far. To identify critical genes in lung cancer progression we compared the expression profile of cancer related genes in 14 pulmonary adenocarcinoma patients with normal lung tissue by using the cDNA array technique. Principal component analyses (PCA) and permutation test were used to detect the differentially expressed genes. The expression profiles of 10 genes were confirmed by semi-quantitative real-time RT–PCR. In tumour samples, as compared to normal lung tissue, the up-regulated genes included such known tumour markers as CCNB1, PLK, tenascin, KRT8, KRT19 and TOP2A. The down-regulated genes included caveolin 1 and 2, and TIMP3. We also describe, for the first time, down-regulation of the interesting SOCS2 and 3, DOC2 and gravin. We show that silencing of SOCS2 is not caused by methylation of exon 1 of the gene. In conclusion, by using the cDNA array technique we were able to reveal marked differences in the gene expression level between normal lung and tumour tissue and find possible new tumour markers for pulmonary adenocarcinoma.

Expression of CYP2A genes in human liver and extrahepatic tissues.

Members of the human cytochrome P450 2A (CYP2A) subfamily are known to metabolize several promutagens, procarcinogens, and pharmaceuticals. In this study, the expression of the three genes found in the human CYP2A gene cluster was investigated in the liver and several extrahepatic tissues by gene-specific reverse transcriptase-polymerase chain reaction (RT-PCR). All three transcripts (CYP2A6, CYP2A7, and CYP2A13) were found to be present in liver. Quantitative RT-PCR analysis showed that CYP2A6 and CYP2A7 mRNAs were present at roughly equal levels in the liver, while CYP2A13 was expressed at very low levels. Two putative splicing variants of CYP2A7 were found in the liver. Nasal mucosa contained a low level of CYP2A6 and a relatively high level of CYP2A13 transcripts. Kidney, duodenum, lung, alveolar macrophages, peripheral lymphocytes, placenta, and uterine endometrium were negative for all transcripts. This survey gives a comprehensive picture of the expression pattern of CYP2A genes in liver and extrahepatic tissues and constitutes a basis for a search for functional CYP2A forms and their roles in chemical toxicity in liver and nasal mucosa.

Increased expression of high mobility group A proteins in lung cancer.

High mobility group A (HMGA) proteins play an important role in the regulation of transcription, differentiation, and neoplastic transformation. In this work, the expression of HMGA 1 and 2 in 152 lung carcinomas of mainly non‐small‐cell histological type has been studied by immunohistochemistry in order to evaluate their feasibility as lung cancer markers. In 17 lung cancer cases, the related bronchial epithelial changes were also studied for HMGA1 and 2 expression. RNA expression of HMGA1a and b isoforms and of HMGA2 was determined by real‐time semi‐quantitative RT‐PCR in 23 lung carcinomas. High expression of HMGA1 and HMGA2 at both mRNA and protein levels was detected in lung carcinomas, compared with normal lung tissue. Nuclear immunostaining for HMGA1 and 2 proteins also occurred in hyperplastic, metaplastic, and dysplastic bronchial epithelium. Increased nuclear expression of HMGA1 and 2 correlated with poor survival (for adenocarcinomas, HMGA1, p = 0.006; HMGA2, p = 0.05). While the expression of HMGA2 was significantly associated with cell proliferation (p = 0.008), HMGA1 expression did not show any association with proliferation or apoptotic index. Sequencing of HMGA2 transcripts from tumours with very high expression showed a normal full‐length transcript. As HMGA proteins were expressed in about 90% of lung carcinomas and their expression was inversely associated with survival, they may provide useful markers for lung cancer diagnosis and prognosis. Copyright

DNA gains in 3q occur frequently in squamous cell carcinoma of the lung, but not in adenocarcinoma

We performed a comparative genomic hybridization study on 25 samples of adenocarcinoma and 19 samples of squamous cell carcinoma of the lung to detect recurrent changes in the genetic material. DNA copy number changes were found in 16 squamous cell carcinoma samples and 17 adenocarcinoma samples. The most common changes were gains of DNA sequences in 3q (43%), 1q (34%), 8q (32%), 5p, (30%), 7p (25%), and 12p (25%). Of the squamous cell carcinoma samples with DNA copy number changes, 94% (15/16) had a gain in 3q (minimal common region of overlap q24‐qter), whereas only 24% (4/17) of the adenocarcinoma samples with DNA copy number changes showed a gain in 3q (q22‐qter) (P< 0.001). Six high‐level amplifications in 3q (q26.2‐q26.3) were detected in the squamous cell carcinoma samples but none were observed in the adenocarcinoma samples. Our results suggest that amplification of genes in 3q may be important in the tumorigenesis of squamous cell carcinoma but not necessarily of adenocarcinoma. Genes Chromosomes Cancer 22:79–82, 1998.

Differentially expressed genes in nonsmall cell lung cancer: expression profiling of cancer-related genes in squamous cell lung cancer

The expression patterns of cancer-related genes in 13 cases of squamous cell lung cancer (SCC) were characterized and compared with those in normal lung tissue and 13 adenocarcinomas (AC), the other major type of nonsmall cell lung cancer (NSCLC). cDNA array was used to screen the gene expression levels and the array results were verified using a real-time reverse-transcriptase-polymerase chain reaction (RT-PCR). Thirty-nine percent of the 25 most upregulated and the 25 most downregulated genes were common to SCC and AC. Of these genes, DSP, HMGA1 (alias HMGIY), TIMP1, MIF, CCNB1, TN, MMP11, and MMP12 were upregulated and COPEB (alias CPBP), TYROBP, BENE, BMPR2, SOCS3, TIMP3, CAV1, and CAV2 were downregulated. The expression levels of several genes from distinct protein families (cytokeratins and hemidesmosomal proteins) were markedly increased in SCC compared with AC and normal lung. In addition, several genes, overexpressed in SCC, such as HMGA1, CDK4, IGFBP3, MMP9, MMP11, MMP12, and MMP14, fell into distinct chromosomal loci, which we have detected as gained regions on the basis of comparative genomic hybridization data. Our study revealed new candidate genes involved in NSCLC.

Expression of CYP1A1, CYP1B1 and CYP3A, and polycyclic aromatic hydrocarbon‐DNA adduct formation in bronchoalveolar macrophages of smokers and non‐smokers

Variability in the expression of enzymes metabolizing carcinogens derived from cigarette smoke may contribute to individual susceptibility to pulmonary carcinogenesis. This study was designed to determine the effects of smoking and 3 major cytochrome P450 (CYP) enzymes, i.e., CYP1A1, CYP1B1 and CYP3A, which metabolize polycyclic aromatic hydrocarbons (PAH) on PAH‐DNA adduct formation in the bronchoalveolar macrophages (BAM) of 31 smokers and 16 non‐smokers. CYP protein levels were determined by immunoblotting and PAH‐DNA adduct levels by the nuclease P1 enhanced 32P‐postlabeling method. The expression of specific CYP forms was confirmed by reverse transcriptase‐polymerase chain reaction (RT‐PCR) from 10 additional samples. CYP3A protein, CYP3A5 by RT‐PCR, was detected in the majority of samples from smokers and non‐smokers. The levels of CYP3A appeared to be lower in active smokers than in ex‐smokers (p = 0.10) or never smokers (p = 0.02). CYP1A1 was not detectable by either immunoblotting or RT‐PCR. The expression of CYP1B1 was low or undetectable in most samples. The PAH‐DNA adduct levels were higher (mean 1.57/108 nucleotides) in samples from smokers compared with non‐smokers (mean 0.42/108 nucleotides, p < 0.001) and the number of adducts correlated with the number of cigarettes smoked daily (regression analysis, p < 0.001). Higher levels of adducts were detected in samples from smokers with a high level of CYP3A compared with those with a low level (regression analysis, p = 0.002). As CYP3A5 is abundant in both lung epithelial cells and BAM, its association with adduct formation suggests that this CYP form may be important in the activation of cigarette smoke procarcinogens. Int. J. Cancer 86:610–616, 2000.

Detection of mRNA encoding xenobiotic‐metabolizing cytochrome P450s in human bronchoalveolar macrophages and peripheral blood lymphocytes

Human pulmonary tissues are known to contain enzymes mediating procarcinogen activation. Peripheral blood lymphocytes and bronchoalveolar macrophages (BAMs) have been used as surrogates for the lung in studies involving cytochrome P450 (CYP) parameters, including CYP1A1 inducibility in relation to susceptibility to lung cancer. In this study, a comprehensive view of the expression patterns of xenobiotic‐metabolizing CYP forms in human BAMs and peripheral blood lymphocytes was obtained by using gene‐specific reverse transcriptase–polymerase chain reaction analysis. These patterns were compared with that in the whole lung. MRNAs of CYP2B6/7, CYP2C, CYP2E1, CYP2F1, CYP3A5, and CYP4B1 were detected in all seven BAM samples studied; however, only the mRNA of CYP2E1 was found consistently in all eight lymphocyte samples. The amounts of amplification products of CYP2B6/7, CYP2C, CYP3A5, and CYP4B1 were low and inconsistent, indicating low levels of expression in lymphocytes. Consistent with previous knowledge, mRNAs of CYP1A1, CYP2B6/7, CYP2E1, CYP2F1, CYP3A5, and CYP4B1 were detected in whole‐lung tissue. These results give an overall picture of the expression of CYP genes in the xenobiotic‐metabolizing families CYP1, CYP2, and CYP3 in BAMs, peripheral blood lymphocytes, and whole‐lung tissue and will aid in directing future studies on the respective protein products. The differences in the CYP gene expression patterns between lung and lymphocytes cast additional doubt on the use of lymphocytes as a surrogate for the lung. Mol. Carcinog. 20:224–230, 1997.

Expression of xenobiotic-metabolizing CYPs in human pulmonary tissue.

The pattern of expression of individual cytochrome P450 (CYP) forms participating in the metabolism of xenobiotics is being increasingly well characterised in the human pulmonary tissue. Recent studies using methods having increased sensitivity and specificity, such as the reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, have revealed constitutive and inducible expression of several CYP forms in different cell types of the human lung. These studies have revealed the presence of mRNA of several procarcinogen-activating CYP forms in whole lung tissue and alveolar macrophages, including CYP1A1, CYP2B6/7, CYP2E1, and CYP3A5. The results of several studies on CYP2D6 expression have yielded contradictory results. Immunohistochemical analysis shows that CYP3A5 protein is present in all lung samples studied, and is localized in the ciliated and mucous cells of the bronchial wall, bronchial glands, bronchiolar ciliated and terminal cuboidal epithelium, type I and type II alveolar epithelium, vascular and capillary endothelium, and alveolar macrophages. Also CYP3A4 protein is found in some cell types in a minority (about 20%) of lung samples. Primary cultures of freshly isolated broncho-alveolar macrophages as well as a continuously growing bronchial carcinoma cell line (A-549) are being used for CYP induction studies in our laboratory. The results indicate that CYP1 family members are inducible in these cells by polycyclic aromatic hydrocarbon (PAH) inducers, and that CYP3A5, but not CYP3A4, is present constitutively. The results of these studies indicate that several different xenobiotic-metabolizing CYPs are present in the human lung and lung-derived cell lines, possibly contributing to in situ activation of pulmonary procarcinogens. Interindividual differences in the expression of these CYPs may contribute to the risk of developing lung cancer and possibly other pulmonary diseases initiated by agents that require metabolic activation.