Post-transcriptional regulation of Rad51c by miR-222 contributes cellular transformation.

DNA repair inhibition has been described as an essential event leading to the initiation of carcinogenesis. In a previous study, we observed that the exposure to metal mixture induces changes in the miR-nome of the cells that was correlated with the sub-expression of mRNA involved in processes and diseases associated with metal exposure. From this analysis, one of the miRNAs that shows changes in its expression is miR-222, which is overexpressed in various cancers associated with exposure to metals. In silico studies showed that a possible target for the microRNA-222 could be Rad 51c, a gene involved in the double-stranded DNA repair. We could appreciate that up-regulation of miR-222 reduces the expression both gene and as a protein expression of Rad51c by RT-PCR and immunoblot, respectively. A luciferase assay was performed to validate Rad51c as miR-222 target. Neutral comet assay was performed in order to evaluate DNA double-strand breaks under experimental conditions. Here, we demonstrate that miR-222 up-regulation, directly regulates Rad51c expression negatively, and impairs homologous recombination of double-strand break DNA repair during the initiation stage of cell transformation. This inhibition triggers morphological transformation in a two-stage Balb/c 3T3 cell assay, suggesting that this small RNA acts as an initiator of the carcinogenesis process.

As-Cd-Pb Mixture Induces Cellular Transformation via Post-Transcriptional Regulation of Rad51c by miR-222.

BACKGROUND/AIMS: Exposure to heavy metals is today a threat to society. The understanding of the molecular processes related to diseases related to exposure to metals mixture involve changes in the expression of microRNAs. Changes on microRNAs expression may alter several cellular processes, among them, DNA repair inhibition has been described as an essential event leading to the initiation of metal-induced carcinogenesis. METHODS: We evaluate the miR-222 expression in the two-stage transformation Balb/c 3T3 cell assay treated with As-Cd-Pb mixture. RESULTS: We could appreciate that up-regulation of miR-222 reduces the expression both gene and as a protein expression of Rad51c by RT-PCR and immunoblot, respectively. CONCLUSION: Here, we demonstrate that the mixture of As-Cd-Pb at epidemiologically relevant concentrations induces miR-222 up-regulation, which directly negatively regulates Rad51c expression and impairs homologous recombination of DNA during the initiation stage of cell transformation. This inhibition triggers morphological transformation in a murine two-stage Balb/c 3T3 cell assay, suggesting that this small RNA acts as an initiator of the carcinogenesis process.

Hydrogen Peroxide-Induced DNA Damage and Repair through the Differentiation of Human Adipose-Derived Mesenchymal Stem Cells.

Human adipose-derived mesenchymal stem cells (hADMSCs) are recognized as a potential tool in cell tissue therapy because of their capacity to proliferate and differentiate in vitro. Several studies have addressed their use in regenerative medicine; however, little is known regarding their response to DNA damage and in particular to the reactive oxygen species (ROS) that are present in the microenvironment of implantation. In this study, we used the ROS-inducing agent hydrogen peroxide to explore the responses of (1) hADMSCs and (2) derived terminally differentiated adipocytes to oxidatively generated DNA damage. Using single cell gel electrophoresis, a dose-related increase was found for both DNA breaks and oxidative lesions (formamidopyrimidine DNA glycosylase-sensitive sites) upon exposure of hADMSCs to hydrogen peroxide. DNA repair capacity of hADMSCs was affected in cells exposed to 150 and 200 µM of hydrogen peroxide. An increase in the basal levels of DNA breaks and oxidative DNA lesions was observed through adipocyte differentiation. In addition, hydrogen peroxide-induced DNA damage increased through adipocyte differentiation; DNA repair capacity also decreased. This study is the first follow-up report on DNA repair capacity during adipogenic differentiation. Remarkably, in terminally differentiated adipocytes, DNA breakage repair is abolished while the repair of DNA oxidative lesions remains efficient.

Human Papillomavirus Types 16 and 18 Early-expressed Proteins Differentially Modulate the Cellular Redox State and DNA Damage.

Oxidative stress has been proposed as a risk factor for cervical cancer development. However, few studies have evaluated the redox state associated with human papillomavirus (HPV) infection. The aim of this work was to determine the role of the early expressed viral proteins E1, E2, E6 and E7 from HPV types 16 and 18 in the modulation of the redox state in an integral form. Therefore, generation of reactive oxygen species (ROS), concentration of reduced glutathione (GSH), levels and activity of the antioxidant enzymes catalase and superoxide dismutase (SOD) and deoxyribonucleic acid (DNA) damage, were analysed in epithelial cells ectopically expressing the viral proteins. Our research shows that E6 oncoproteins decreased GSH and catalase protein levels, as well as its enzymatic activity, which was associated with an increase in ROS production and DNA damage. In contrast, E7 oncoproteins increased GSH, as well as catalase protein levels and its activity, which correlated with a decrease in ROS without affecting DNA integrity. The co-expression of both E6 and E7 oncoproteins neutralized the effects that were independently observed for each of the viral proteins. Additionally, the combined expression of E1 and E2 proteins increased ROS levels with the subsequent increase in the marker for DNA damage phospho-histone 2AX (γH2AX). A decrease in GSH, as well as SOD2 levels and activity were also detected in the presence of E1 and E2, even though catalase activity increased. This study demonstrates that HPV early expressed proteins differentially modulate cellular redox state and DNA damage.

HPV+ cervical carcinomas and cell lines display altered expression of caspases.

OBJECTIVE: Loss of expression of apoptotic regulatory proteins in many neoplasias might result in defective or delayed apoptosis, thus facilitating tumor growth or survival. We analyzed here, the basal expression of precursors of apoptotic Caspases in normal cervical epithelium, HPV+ cervical tumor samples and HPV+ tumor-derived cell lines. METHODS: Expression of initiator and effector Caspases was analyzed by immunochemistry in normal cervical epithelium and three types of cervical tumors (squamous cell carcinoma, adenocarcinoma and adenosquamous cell carcinoma) whereas expression of Caspases in HeLa, SiHa and CaSki cells was by immunofluorescence, Western blot and RT-PCR. Besides, the effect of the HPV-16 E6/E7 oncogenes on Caspases expression in cervical cells was evaluated by transfecting C33-A (HPV-) cells. RESULTS: Expression of Caspases 3 and 9 was undetectable in adenocarcinoma and adenosquamous cell carcinoma, respectively. Whereas in squamous cell carcinoma, the expression of Caspases was similar those observed in normal samples. Expression of Caspases 3 and 6 was low in HeLa and CaSki cells, while Caspase 8 was low in SiHa and it was not detected in C33-A cells. All Caspases were detected in the cytoplasm and nucleus of the cells. We did not observe an effect of the E6/E7 oncogenes on the expression of Caspases in C33-A cell. CONCLUSION: Our results showed a differential expression of several Caspases in carcinoma samples and cell lines, suggesting multiple alterations of the Caspase pathways in cervical cancer.

Abnormal distribution of E-cadherin and beta-catenin in different histologic types of cancer of the uterine cervix.

OBJECTIVE: The goal of this study was to analyze the cellular distribution and possible alterations of beta-catenin and E-cadherin proteins in different histologic types of uterine cervical cancer and precursor lesions, compared to normal controls. METHODS: We performed an immunochemical staining analysis of the cellular distribution of beta-catenin and E-cadherin proteins in biopsy samples from 20 normal exocervical squamous epithelium, 43 premalignant lesions, and a large series of 126 invasive tumors of different histologic types that included 68 squamous carcinomas, 31 adenosquamous carcinomas, and 27 adenocarcinomas. Statistical significance was evaluated by the chi-square or Fisher's Exact test. RESULTS: We observed beta-catenin abnormally distributed in the cytoplasm of 62% of premalignant lesions and more than 70% of invasive cancers, statistically significant when compared with normal tissue (P < 0.05). Similarly, we found that E-cadherin exhibit a significant abnormal distribution in the cytoplasm of 58% of premalignant lesions (P < 0.05) and in more than 71% of squamous carcinoma and adenosquamous carcinoma when compared with normal tissue (P < 0.05). We found no differences in the distribution of E-cadherin between adenocarcinomas compared with control samples. Interestingly, we found that both, beta-catenin and E-cadherin, were absent in the membrane of nearly 40% premalignant lesions. Nuclear staining of beta-catenin was rarely seen in any cases, contrary to what has been reported for this and other neoplasias. CONCLUSION: Our findings indicate that cellular alterations of both beta-catenin and E-cadherin are frequent in tumors of the uterine cervix of different histologic types, and support a role for these proteins in cervical cancer development.