Grade-dependent effects on cell cycle progression and apoptosis in response to doxorubicin in human bladder cancer cell lines.

Doxorubicin is an important component of combination therapy for muscle-invasive urinary bladder cancer. Treatment with this topoisomerase II poison is able to interfere with cell cycle progression and lead to cancer cell death. Using FACS analysis, Western immunoblotting and semi-quantitative RT-PCR, we studied the effects of doxorubicin on cell cycle progression and apoptosis, and also explored the possibility of using groups of genes as biomarkers of prognosis and/or response to doxorubicin treatment in human urinary bladder cancer cells. Doxorubicin induced dose-dependent G2/M and/or G1/S cell cycle arrest, followed by grade- and dose-dependent reduction in the amount of the cytosolic trimeric form of FasL, activation of Caspase-8, Caspase-9, Caspase-3, cleavage of PARP, Lamin A/C, Bcl-XL/S and interestingly Hsp90, and finally cell death. Data presented here also suggest the use of the expression patterns of Cyclin-E2, Cyclin-F, p63, p73, FasL, TRAIL, Tweak, Tweak-R, XAF-1, OPG and Bok genes for identification of the differentiation grade, and Cyclin-B2, GADD45A, p73, FasL, Bik, Bim, TRAIL, Fas, Tweak-R, XAF-1, Bcl-2, Survivin, OPG, DcR2 and Bcl-XL genes for the detection of response to doxorubicin in human bladder cancer cells.

Cloning and functional characterization of the 5' regulatory region of ovine Hormone Sensitive Lipase (HSL) gene.

Hormone Sensitive Lipase (HSL) catalyzes the rate-limiting step in the mobilization of fatty acids from adipose tissue, thus determining the supply of energy substrates in the body. HSL enzymatic activity is increased by adrenergic agonists, such as catecholamines and glucagons, which induce cyclic AMP (cAMP) intracellular production, subsequently followed by the activation of Protein Kinase A (PKA) and its downstream signaling cascade reactions. HSL constitutes the critical enzyme in the modulation of lipid stores and the only component being subjected to hormonal control in terms of the recently identified Adipose Triglyceride Lipase (ATGL). In order to acquire detailed knowledge with regard to the mechanisms regulating ovine HSL (ovHSL) gene transcription activity, we initially isolated and cloned the 5' proximal and distal promoter regions through a genome walking approach, with the utilization of the already characterized ovHSL cDNAs. As evinced by BLAST analysis and a multiple alignment procedure, the isolated genomic fragment of 2.744 kb appeared to contain the already specified 5'-untranslated region (5'-UTR), which was interrupted by a relatively large intron of 1.448 kb. Regarding the upstream remaining part of 1.224 kb, it was demonstrated to represent a TATA-less promoter area, harboring several cis-regulatory elements that could be putatively recognized by relatively more general transcription factors, mainly including Stimulating protein 1 (Sp1), CCAAT-box Binding Factors (CBFs), Activator Protein 2 (AP2) and Glucocorticoid Receptor (GR), as well as other cis-acting regions denominated as Insulin Response Element (IRE), Glucose Response Element (GRE), Fat Specific Element (FSE) and cAMP Response Element (CRE), which could likely function in a nourishment (i.e. glucose)-/hormone-dependent fashion. When different genomic fragments were directionally (5' to 3') cloned into a suitable reporter vector upstream of a promoter-less luciferase gene and transiently transfected into 3T3-L1 (mouse fibroblasts) as well as T24 (human bladder cancer) cell lines, strong promoter activities were unambiguously detected, with the -140/+18 nucleotide sequence bearing the highest transcriptional response, thus indicating that the 1.224 kb 5' flanking region, isolated by genome walking, veritably contains the ovHSL gene promoter. Of particular significance are the observations that the functional promoter fragments could trigger the transcriptional activity of luciferase gene only under high concentration of glucose conditions in both cell lines.

DNA damage and repair capacity in lymphocytes from obstructive sleep apnea patients.

Obstructive sleep apnea (OSA) syndrome is a respiratory disease that is linked to heart attacks and high blood pressure. In the present study, we used the Comet assay to compare basal DNA damage and DNA damage induction by hydrogen peroxide, ethanol, and gamma-irradiation in lymphocytes from 35 OSA patients and 35 controls. We also measured the apoptosis and necrosis produced by these agents and the ability of the lymphocytes to repair the induced DNA damage. It was found that lymphocytes isolated from OSA patients had higher basal levels of DNA damage and were more sensitive to the effects of the DNA-damaging agents than lymphocytes from controls. OSA patients also had a reduced capacity to repair the DNA damage induced by the three agents, but apoptosis and necrosis were similar in OSA patients and the controls.

DNA damage and repair efficiency in lymphocytes from schizophrenic patients.

In the present study we examined schizophrenic patients' lymphocytes sensitivity to the effects of external factors, such as hydrogen peroxide and gamma-irradiation and also their repair efficiency with the comet assay. Our results did no show any difference in basal levels of DNA damage between schizophrenic and normal populations. The slightly increased sensitivity of the schizophrenic population to the externally induced DNA damage compared to controls was not statistically significant. Also the small reduction in the DNA repair efficiency in schizophrenics in comparison to normal population was found to be not statistically significant. Finally, patients with heritable predisposition to schizophrenia did not show any difference in their response from the other schizophrenics.

DNA damage in a human population affected by chronic psychogenic stress.

The effects of chronic psychogenic stress on the expression of DNA damage and cellular response to the damage were investigated. Using the comet assay, basal DNA damage was found to be similar in lymphocytes of both affected and non-affected populations (n = 30 in both groups). The induction of DNA damage in lymphocytes by external factors (H2O2 and gamma-irradiation), was also investigated. In these studies, cells were treated with 50, 100 and 150 microM H2O2 for 5 minutes or with 0.8, 2.5 and 4.2 Gy gamma-rays. A significant difference was found between the chronically stressed and the control populations, indicating the enhanced sensitivity of the former population. Cells were also held for 2 hours after the treatment, allowing time for the cells to deal with the induced DNA damage. Based on the level of residual DNA strand breaks, cells from the stressed population had more breaks than the controls. Gender does not alter these findings. In conclusion, our data indicate that cells from the stressed population were more sensitive to the induction of DNA damage and had higher level of residual damage. Therefore, stress conditions may cause the affected individuals to be susceptible to environmental mutagenic agents.

A study on the effects of seasonal solar radiation on exposed populations.

In the present study the effects of seasonal solar radiation (summer and winter) on exposed populations of two different age groups (20-25 and 40-55 years old) were investigated. In addition, the effects of external factors, such as hydrogen peroxide (H(2)O(2)) and gamma-irradiation, as well as the repair efficiency of human lymphocytes from these populations, was also evaluated. Our results show that the amount of DNA damage appears to be influenced by the exposure to solar radiation, with the summer exposure being the most damaging. Age was also found to be a significant factor, with the older population being more susceptible to solar radiation than the younger one. Season does not appear to affect the sensitivity to external DNA-damaging agents, while age does. Age was also found to have an effect on the DNA repair capacity of the examined populations.