Genetic polymorphisms of phase I and phase II metabolic enzymes as modulators of lung cancer susceptibility.

OBJECTIVES: Tobacco exposure remains the main etiologic factor for lung cancer (LC). Interactions between environment and individual genetic profile are particularly important for this disease. The aim of this study was to evaluate the contribution of CYP1A1*2A, CYP1A1*2C, CYP2D6*4, GSTP1, GSTM1, GSTT1 and NAT2 polymorphisms for the susceptibility to LC in a Portuguese population considering their demographic and clinical characteristics. MATERIALS AND METHODS: A total of 200 LC and 247 controls subjects from the Centre of Portugal were studied. Clinical and demographic characteristics were collected from clinical files and by individual questionnaires. Polymorphisms of CYP1A1*2A, CYP1A1*2C, CYP2D6*4, GSTP1, GSTM1, GSTT1 and NAT2 were genotyped using PCR-RFLP, PCR multiplex, ARMS and real time. RESULTS: Gender, family history of cancer, smoke cessation and alcohol consumption were independent risk factors (p < 0.05). Associations found between phases I and II genes and LC population reveal a sex dependent distribution. Logistic regression analysis demonstrates that enhanced activation by CYPs, associated by reduced or loss of function of phase II enzymes, can lead to a greater risk. GSTP1 and NAT2 polymorphisms studied have a significant contribution for the histological tumour types and the presence of metastases, at time of diagnosis, respectively, when males with smoking habits were considered. CONCLUSION: Multiple interactions between environment and individual characteristics are clearly associated to this disease. Variants of the detoxification genes may act synergistically contributing to this disease and modifying the risk posed by smoking and sex. The GSTT1*0 and GSTP1 (Ile462Val) might contribute to the malignant phenotype through different mechanisms.

The CTLA4 +49 A/G polymorphism is not associated with susceptibility to type 1 diabetes mellitus in the Portuguese population.

CTLA4 genetic polymorphisms have been associated with type 1 diabetes. We genotyped 207 patients and 249 controls for the most frequently investigated polymorphism of the CTLA4 gene (+49A/G (rs231775)). No significant differences were observed, suggesting that this polymorphism is not strongly associated with type 1 diabetes in the Portuguese population.

Combined GSTM1 and GSTT1 null genotypes are associated with a lower risk of papillary thyroid cancer.

Individual susceptibility to cancer is influenced by polymorphisms of genes encoding drug-metabolizing enzymes such as the glutathione S-transferases (GST). The null polymorphisms of the GSTM1 and GSTT1 genes have been associated to a modified risk of several cancers but studies of thyroid cancer have produced conflicting results. The aim of this study was to investigate the relationship between these polymorphisms and the risk of papillary thyroid cancer (PTC). A total of 188 patients with PTC and 247 controls were genotyped using a PCR-based assay. Odds ratios (OR) and 95% confidence intervals (CI) for each homozygous null genotype were determined. The frequency of each of the GSTM1 and GSTT1 null genotypes did not differ significantly between patients and controls (OR=0.83, 95%CI: 0.56-1.21; p=0.328; and OR=0.66, 95%CI: 0.39-1.12; p=0.123, respectively), but the frequency of individuals that had the combined GSTM1 null/GSTT1 null genotypes was significantly lower in the patient group (OR=0.50, 95%CI: 0.26-0.97; p=0.040). The GSTM1 null genotype was associated with a lower risk of advanced cancer stages (III/IV) (OR=0.50, 95%CI: 0.26-0.96; p=0.036) and the GSTT1 null genotype was associated with a lower risk of the follicular variant of PTC (OR=0.31, 95%CI: 0.10-0.97; p=0.044). These results suggest that GSTM1 and GSTT1 null genotypes are weak, yet possible, modifiers of the risk of PTC. This protective effect may be due to a role of the GSTM1 and GSTT1 encoded enzymes in the metabolic activation of putative thyroid carcinogens or in other pathways involved in thyroid carcinogenesis.

Soluble VCAM-1 and E-selectin in breast cancer: relationship with staging and with the detection of circulating cancer cells.

In breast cancer, the correct evaluation of cancer dissemination is essential to establish prognosis and treatment choices. This study analyses the relationship between circulating levels of soluble VCAM-1 and E-selectin and the presence of circulating cancer cells in breast cancer patients. Plasma levels of VCAM-1 and E-selectin were measured by enzyme-linked immunosorbent assay (ELISA). The presence of circulating cancer cells was diagnosed using a RT nested-PCR assay detecting the cancer specific transcript, epidermal growth factor receptor variant III (EGFRvIII) mRNA. Blood samples were collected from 64 patients divided in three groups: group A of 11 women selected for neoadjuvant chemotherapy; group B of 13 women with metastatic disease and group C, with 40 women having completed their treatment at least one year ago and with no evidence of relapse. The mutant transcript was detected in 45.5% of patients from group A, in 61.5% of patients from group B and in none of the group C patients. For both VCAM-1 and E-selectin, plasma levels increased with disease staging and with the presence of EGFRvIII mRNAin peripheral blood. The differences were statistically significant (p < 0.025) when group C was compared with all patients from group B, with patients from group B with EGFRvIII positive results or with all patients with EGFRvIII positive results. Increased plasma levels of VCAM-1 and E-selectin are associated with advanced stage of breast cancer and with the presence of circulating cancer cells. The combined analysis of these parameters may contribute to a more accurate evaluation of cancer dissemination.

[Molecular characterisation of a kindred with MEN2A and clinical implications]. / Caracterização molecular de uma família com nem2a e suas implicações clínicas.

INTRODUCTION: MEN2A is an autossomal dominant cancer syndrome characterised by the presence of medullary thyroid cancer, pheochromocytoma and primary hyperparathyroidism. Germline mutations of the RET protooncogene constitute the molecular defect and can be identified in affected individuals. Genetic screening of family members at risk allows early diagnosis and preventive measures before the appearance of the disease. We present a family with several members affected with MEN2A, their molecular characterisation and the clinical implications of genetic testing. POPULATION AND METHODS: We studied 18 members distributed among three generations of a family of which four members were clinically affected with MEN2A and cutaneous lichen amyloidosis. RET gene mutations were screened for in affected individuals and their offspring by PCR-RFLP techniques. RESULTS: Genetic testing revealed a point mutation at codon 634 (TGC>TGG), in the heterozygous state, in all affected individuals. The same mutation was also found in a five years old asymptomatic child which after total thyroidectomy showed to have multifocal medullary thyroid carcinoma. DISCUSSION: Genetic screening is the most suitable method for pre-symptomatic diagnosis of MEN2A allowing an efficient and early identification of individuals who will later develop the disease. These can be monitored more closely and be submitted to a prophylactic thyroidectomy before the appearance of medullary thyroid carcinoma. The ideal moment for this intervention is still under discussion although the results of this study suggest that it should be undertaken before the age of five.

Genetic polymorphism of CYP2D6, GSTM1 and NAT2 and susceptibility to haematological neoplasias.

Xenobiotic-metabolizing enzymes constitute an important line of defence against a variety of carcinogens. Many are polymorphic, constituting the basis for the wide inter-individual variation in metabolic capacity and possibly a source of variation in the susceptibility to chemical-induced carcinogenesis. The aim of this study was to determine the existence of any association between the main genetic polymorphisms of cytochrome P450 2D6 (CYP2D6), glutathione S-transferase M1 (GSTM1) and N-acetyltransferase 2 (NAT2) and an altered risk for haematological neoplasias. A total of 160 patients and 128 controls were genotyped by means of PCR-RFLP-based assays. Mutated alleles comprising CYP2D6*4, GSTM1*0, NAT2*5A, *5B, *5C, *6 and *7 were analysed along with the wild-type alleles. The results showed a higher frequency of CYP2D6 extensive metabolizers carrying two functional alleles in the leukaemia group, when compared with controls (76.6 versus 57.0%, P = 0.008). No differences were found in the case of Hodgkin and non-Hodgkin lymphomas. Analysis of the GSTM1 and NAT2 polymorphisms failed to show an association with any of the neoplasias, although a near significant increase in fast acetylators was also found in the leukaemia group (50.0 versus 35.9%, P = 0.06). The results suggest an association of extensive metabolism with an increased risk for leukaemia, possibly by an increase in the metabolic activation of chemical carcinogens or linkage to another cancer-causing gene. Opposite findings presented in other studies may reflect geographical differences in the type of environmental carcinogens to which different populations are exposed.

N-acetyltransferase genotypes in the Portuguese population.

Fast and slow acetylator phenotypes differ in their respective frequencies among different populations. A polymerase chain reaction-restriction fragment length polymorphism based genotyping assay was used to determine the frequency of the most important NAT2 polymorphisms in a group of 128 Portuguese individuals. The results showed that slow acetylators represented 64.1% of the group, and the frequencies of NAT2*4, *5A, *5B, *5C, *6 and *7 alleles were 0.211, 0.031, 0.379, 0.023, 0.328 and 0.027, respectively. These values are similar to those presented in other studies in Caucasians. The data obtained may be useful in epidemiological studies of the influence of acetylator polymorphisms on carcinogenesis or other environmental caused diseases.

Calcium stores in electropermeabilized HL-60 cells before and after differentiation.

Non-induced HL-60 cells (N-IND) and HL-60 cells induced to differentiate with 2 microM retinoic acid (IND) were electropermeabilized with electrical discharges, and the intracellular Ca2+ stores were measured in each type of cell. Both N-IND and IND cells accumulate Ca2+ in the presence of ATP after electropermeabilization. The Ca2+ is stored in at least two different compartments; accumulation in one of the compartments is inhibited by oligomycin and CCCP, and it is not releasable by Ins(1,4,5)P3. The maximal accumulation of Ca2+ by the Ins(1,4,5)P3 sensitive pool is about 0.3 nmol/10(6) cells and 0.9 nmol/10(6) cells for the N-IND and for the IND cells, respectively, and the half-maximal value occurs at a free Ca2+ concentration of 0.23 microM and 0.63 microM, respectively. The oligomycin + CCCP sensitive pool hardly accumulates any Ca2+ at this level of free Ca2+, but at higher free [Ca2+] (greater than microM) its maximal capacity is 80-100-fold higher than the Ins(1,4,5)P3-sensitive pool (about 17-18 nmol/10(6) cells). It is concluded that at physiological free Ca2+ concentrations, the non-mitochondrial Ca2+ pool is regulating the intracellular free Ca2+ in N-IND and IND HL-60 cells, and that this Ca2+ pool can be mobilized by Ins(1,4,5)P3. Furthermore, the capacity of this pool increases about 3-fold when the cells are induced to differentiate with retinoic acid.