Analysis of the association between polymorphisms in MMP2, MMP3, MMP9, MMP20, TIMP1, and TIMP2 genes with white spot lesions and early childhood caries.

BACKGROUND: Matrix metalloproteinases and their inhibitors might be involved in enamel formation. AIM: This study aimed to evaluate the association between polymorphisms in MMP2, MMP3, MMP9, MMP20, TIMP1, and TIMP2 with white spot lesions (WSL) and early childhood caries (ECC). DESIGN: A cross-sectional study was performed on 786 children aged from 2 to 6 years in Brazil. After clinical evaluation, they were classified into groups with disease (the presence of WSL and/or ECC) and without disease (the absence of WSL or ECC). Genotyping of the selected polymorphisms was carried out with TaqMan real-time PCR, using genomic DNA extracted from buccal cells. Allele and genotype frequencies were compared between groups. Chi-square test, odds ratio (OR), and logistic regression were used (P ≤ 0.05). RESULTS: The dmft score was 1.3 (SD: 2.4), and 41.34% of the children have at least one caries lesion. In MMP9, the GG genotype was more frequent in the group without disease (P = 0.006). In a recessive model, WSL was associated with the marker rs1711437 in MMP20 (P = 0.019; OR = 1.20, 95% CI 1.02-1.42). The marker rs1784418 in MMP20 showed an association between the allele G distribution for the WSL group (P = 0.020; OR = 0.73, 95% CI 0.55-0.96). CONCLUSION: MMP9 and MMP20 are involved in WSL and ECC development.

Na/K-ATPase as a target for anticancer drugs: studies with perillyl alcohol.

BACKGROUND: Na/K-ATPase (NKA) is inhibited by perillyl alcohol (POH), a monoterpene used in the treatment of tumors, including brain tumors. The NKA α1 subunit is known to be superexpressed in glioblastoma cells (GBM). This isoform is embedded in caveolar structures and is probably responsible for the signaling properties of NKA during apoptosis. In this work, we showed that POH acts in signaling cascades associated with NKA that control cell proliferation and/or cellular death. METHODS: NKA activity was measured by the amount of non-radioactive Rb(+) incorporation into cultured GBM cell lines (U87 and U251) and non-tumor cells (mouse astrocytes and VERO cells). Cell viability was measured by lactate dehydrogenase levels in the supernatants of POH-treated cells. Activated c-Jun N-terminal Kinase (JNK) and p38 were assessed by western blotting. Apoptosis was detected by flow cytometry and immunocytochemistry, and the release of interleukins was measured by ELISA. RESULTS: All four cell types tested showed a similar sensitivity for POH. Perillic acid (PA), the main metabolite of POH, did not show any effect on these cells. Though the cell viability decreased in a dose-dependent manner when cells were treated with POH, the maximum cytotoxic effect of PA obtained was 30% at 4 mM. 1.5 mM POH activated p38 in U87 cells and JNK in both U87 and U251 cells as well as mouse astrocytes. Dasatinib (an inhibitor of the Src kinase family) and methyl ß-cyclodextrin (which promotes cholesterol depletion in cell membranes) reduced the POH-induced activation of JNK1/2 in U87 cells, indicating that the NKA-Src complex participates in this mechanism. Inhibition of JNK1/2 by the JNK inhibitor V reduced the apoptosis of GBM cells that resulted from POH administration, indicating the involvement of JNK1/2 in programmed cell death. 1.5 mM POH increased the production of interleukin IL-8 in the U251 cell supernatant, which may indicate a possible strategy by which cells avoid the cytotoxic effects of POH. CONCLUSIONS: A signaling mechanism mediated by NKA may have an important role in the anti-tumor action of POH in GBM cells.

Hydroxymethylbilane synthase gene mutations and polymorphisms in Brazilian families with acute intermittent porphyria.

Acute intermittent porphyria (AIP), an autosomal dominant disorder, is caused by a deficiency of hydroxymethylbilane synthase (HMBS). In the present study, we sought to establish a correlation between HMBS activity with the presence of mutations and polymorphisms. Enzyme activity was measured in red blood cells of four Brazilian unrelated AIP families (n = 124) and in blood donors (n = 80). The HMBS mutations in AIP family members were studied by PCR-SSCP followed by direct sequencing. Six intragenic SNPs (1345 G>A, 1500 T>C, 2377 C>A, 2478 A>G, 3581 A>G, and 7064 C>A) were determined by PCR-RFLP. Abnormal SSCP patterns in exons 7, 9, 12, and 15 were observed. DNA sequencing analysis revealed one nonsense mutation, R149X, two missense mutations, G111R and L338P, and one deletion, CT 730-731. All mutation carriers had lower enzyme activity. All polymorphisms, except 2377 C>A and 7064 C>A, showed no significant differences compared with previous reports. Mutation screening allowed the detection of the missense mutation, L338P, and the 730_731delCT deletion, two as yet unreported mutations in Brazilian AIP patients. Our findings also showed a high frequency of 2478 A>G and 3581 A>G polymorphism combinations suggesting that these polymorphisms contributed to enzymatic activity reduction in our study population.

The significance of p53 immunoexpression with different clones (DO-7 and PAb-240) in oral squamous cell carcinoma.

BACKGROUND/AIM: The TP53 gene is a tumor suppressor gene. Its product is a nuclear protein that regulates cell cycle arrest, apoptosis and DNA repair. Anti-p53 clones DO-7 and PAb-240 recognize the amino acid sequences 21-25 and 213-217, respectively. This study aimed to evaluate the expression of these clones and their relationship with clinicopathological features and survival analysis in oral squamous cell carcinomas (OSCC). METHODS: Information on 53 primary OSCC was collected at the Brazilian National Cancer Institute. An immunohistochemical method was applied to evaluate p53 expression (DO-7 and PAb-240). Their expression was analyzed quantitatively and correlated with clinicopathological features. Kaplan-Meier survival curves and log rank test were used. RESULTS: Immunopositivity for DO-7 was present in 64% of the cases, while 58% were positive for PAb-240. There was no correlation between immunoexpression of both antibodies and clinicopathological features or survival analysis. DO-7 expression was significantly higher (p = 0.001) than that of PAb-240. CONCLUSIONS: There were quantitative differences between the expression of the antibodies studied, which may reflect a different specificity of each one. To confirm immunohistochemical results and estimate the true prognostic role of TP53 in OSCC, it is important to perform mutation analysis.

Analysis of EGF+61A>G polymorphism and EGF serum levels in Brazilian glioma patients treated with perillyl alcohol-based therapy.

PURPOSE: Malignant gliomas are associated with alteration in EGF/EGFR signaling. Functional EGF+61A>G polymorphism is implicated with risk, recurrence, and progression of glioma. This study aimed to establish a putative association of EGF+61A>G with risk of glioma development, production of angiogenic growth factor EGF, and the response to perillyl alcohol administered by intranasal route. METHODS: The study included 83 patients with recurrent glioma enrolled in Phase I/II trial for intranasal perillyl alcohol therapy and subjects without cancer (n = 196) as control group. DNA was extracted from blood samples, EGF genotype performed with PCR-RFLP assay, and EGF circulating levels by enzyme immunoassay. Adequate statistical tests were performed to verify associations between polymorphism and glioma risk, and genotype correlation with EGF circulating levels. The log-rank test was also used to evaluate differences on patient survival. RESULTS: Patients with primary glioblastoma had high frequency of AA genotype (p = 0.037) and A allele (p = 0.037). Increased EGF circulating levels were observed in glioma patients with AA (p = 0.042), AG (p = 0.006), and AA + AG (p = 0.008) genotypes compared with GG. Patients with GG genotype showed increased but not significant (p > 0.05) survival rate, and EGF levels lower than 250 pg/mL was consistently (p = 0.0374) associated with increased survival. CONCLUSION: Presence of EGF+61A>G polymorphism in Brazilian subjects was associated with glioma risk and increased circulating EGF levels. Better response to perillyl alcohol-based therapy was observed in a group of adult Brazilian subjects with lower EGF levels.

CYP19 (TTTA)n polymorphism and breast cancer risk in Brazilian women.

A prolonged or increased exposure to endogenous estrogens associated with genetic factors are considered to be the main risk factors for breast cancer. The CYP19 gene encodes the enzyme aromatase, which catalyzes the conversion of androgens into estrogens. CYP19 alleles containing different numbers of tetranucleotide TTTA repeats in intron 4 have been associated with increased breast cancer risk. In this study we investigated, for the first time, the frequency of CYP19 (TTTA)n alleles in a South American population (n = 475) and analyzed the risk for developing breast cancer in a case-control study comprising 135 cases and 270 age-matched controls. It is shown that Brazilians possess not only the alleles identified in all the other populations studied so far (alleles containing from 7 to 13 TTTA repeats), but also the (TTTA)6 allele, that had never been described before. The (TTTA)10 allele was three times more frequent in cases when compared to controls and presented a significant positive association (p = 0.048) with breast cancer development in Brazilians.