Association of p53 codon72 Arg>Pro polymorphism with susceptibility to nasopharyngeal carcinoma: evidence from a case-control study and meta-analysis.

Tumor suppressor p53 is a critical player in the fight against cancer as it controls the cell cycle check point, apoptotic pathways and genomic stability. It is known to be the most frequently mutated gene in a wide variety of human cancers. Single-nucleotide polymorphism of p53 at codon72 leading to substitution of proline (Pro) in place of arginine (Arg) has been identified as a risk factor for development of many cancers, including nasopharyngeal carcinoma (NPC). However, the association of this polymorphism with NPC across the published literature has shown conflicting results. We aimed to conduct a case-control study for a possible relation of p53 codon72 Arg>Pro polymorphism with NPC risk in underdeveloped states of India, combine the result with previously available records from different databases and perform a meta-analysis to draw a more definitive conclusion. A total of 70 NPC patients and 70 healthy controls were enrolled from different hospitals of north-eastern India. The p53 codon72 Arg>Pro polymorphism was typed by polymerase chain reaction, which showed an association with NPC risk. In the meta-analysis consisting of 1842 cases and 2330 controls, it was found that individuals carrying the Pro allele and the ProPro genotype were at a significantly higher risk for NPC as compared with those with the Arg allele and the ArgArg genotype, respectively. Individuals with a ProPro genotype and a combined Pro genotype (ProPro+ArgPro) also showed a significantly higher risk for NPC over a wild homozygote ArgArg genotype. Additionally, the strength of each study was tested by power analysis and genotype distribution by Hardy-Weinberg equilibrium. The outcome of the study indicated that both allele frequency and genotype distribution of p53 codon72 Arg>Pro polymorphism were significantly associated with NPC risk. Stratified analyses based on ethnicity and source of samples supported the above result.

C/EBPbeta-mediated transcriptional regulation of bcl-xl gene expression in human breast epithelial cells in response to cigarette smoke condensate.

In earlier studies, we have shown that cigarette smoke condensate (CSC), a surrogate for cigarette smoke, is capable of transforming the spontaneously immortalized human breast epithelial cell line, MCF10A. These transformed cells displayed upregulation of the anti-apoptotic gene, bcl-xl. Upregulation of this gene may impede the apoptotic pathway and allow the accumulation of DNA damage that can lead to cell transformation and carcinogenesis. In the present study, we have determined the mechanism of CSC-mediated transcriptional upregulation of bcl-xl gene expression in MCF10A cells. We cloned the human bcl-xl promoter (pBcl-xLP) and identified putative transcription factor binding sites. Sequential deletion constructs that removed the putative cis-elements were constructed and transfected into MCF10A cells to determine the CSC-responsive cis-element(s) on the pBcl-xLP. Gel-shift, super-shift and chromatin immunoprecipitation analysis confirmed that CCAAT/enhancer-binding protein (C/EBPbeta) specifically bound to a C/EBP-binding site on the pBcl-xLP in vitro and in vivo. Additionally, overexpression of C/EBPbeta-LAP2 stimulated pBcl-xLP activity and Bcl-xL protein levels, which mimicked the conditions of CSC treatment. Our results indicate that C/EBPbeta regulates bcl-xl gene expression in MCF10A cells in response to CSC treatment; therefore, making it a potential target for chemotherapeutic intervention of cigarette smoke-induced breast carcinogenesis.

Cigarette smoke condensate-induced level of adenomatous polyposis coli blocks long-patch base excision repair in breast epithelial cells.

Our previous studies have shown that treatment with cigarette smoke condensate (CSC) transforms normal breast epithelial cell line, MCF-10A. In the present study, the mechanism of CSC-induced transformation of breast epithelial cells was examined. We first determined whether benzo[a]pyrene (B[a]P)- and CSC-induced levels of APC are capable of inhibiting long-patch base excision repair (LP-BER) since our earlier studies had shown that an interaction of APC with DNA polymerase beta (pol-beta) blocks strand-displacement synthesis. With the use of a novel in vivo LP-BER assay, it was demonstrated that increased and decreased APC levels in different breast cancer cell lines were associated with a decrease or increase in LP-BER activity, respectively. The effect of APC on LP-BER in malignant and pre-malignant breast epithelial cell lines was produced by either overexpression or knockdown of APC. Furthermore, it was shown that the decreased LP-BER in B[a]P- or CSC-treated pre-malignant breast epithelial cells is associated with an increased level of APC and decreased cell growth. Our results suggest that the decreased growth allows cells to repair the damaged DNA before mitosis, and failure to repair damaged DNA has the potential to transform pre-malignant breast epithelial cells.

Effect of dextrans on cryopreservation of goat cauda epididymal spermatozoa using a chemically defined medium.

Experiments were carried out to investigate the cryoprotecting potential of dextrans (ranging from 10 to 2000 kDa) using a synthetic model system developed recently in this laboratory. Goat spermatozoa from the cauda epididymidis were extracted in a chemically defined medium (modified Ringer's solution) and assayed for motility using a phase-contrast microscope. The sperm cells were subjected to a freezing protocol in a computer-controlled biofreezer (cooling at 0.25 degrees C min(-1) to 5 degrees C; 5 degrees C min(-1) to -20 degrees C; 20 degrees C min(-1) to -100 degrees C) and plunged into liquid nitrogen. The frozen cells were thawed rapidly at 37 degrees C in a thermostatic waterbath. In the absence of dextran, all the spermatozoa lost their motility. The cryoprotecting efficacy of each dextran was found to be biphasic in nature. Initially, as the concentration of dextran was increased, the recovery of sperm motility also increased and reached an optimum value; however, with further increases in dextran concentration, the recovery of motility decreased sharply. Of all the sugar polymers tested, 10 kDa dextran showed the highest cryoprotecting efficacy, whereas the 2000 kDa sugar polymer was the least active. Dextrans of 10, 40, 73, 173, 252, 500 and 2000 kDa offered maximum cryorecovery of forward motility to the extent of approximately 23%, 21%, 19%, 18%, 16%, 15% and 8%, respectively. Optimum concentrations of these dextrans for cryoprotection of sperm motility were 8.42, 2.50, 1.09, 0.37, 0.31, 0.10 and 0.04 mmol l(-1), respectively. It thus appears that each dextran has a characteristic cryoprotection profile. The data show that the cryoprotecting efficacy and optimum cryoprotecting concentrations of dextrans are inversely related to their molecular mass. Dextran also served as a significant cryoprotectant in the presence of glycerol (0.87 mol l(-1)) and dimethyl sulphoxide (0.76 mol l(-1)), which are well known cryoprotectants; the action of the combined cryoprotectants was almost additive. The presence of glycerol or glycerol plus dimethyl sulphoxide caused a significant reduction (from 8.42 to 6.27 mmol l(-1)) in the optimum concentration of dextran. In the presence of the three cryoprotectants, recovery of sperm motility was as high as 58% (forward motility) and 60% (total motility).

Effect of amino acids on goat cauda epididymal sperm cryopreservation using a chemically defined model system.

Studies were carried out to analyze the cryoprotecting efficacy of several amino acids by use of a chemically defined synthetic medium (modified Ringer's solution) and goat cauda epididymal sperm as the model system. Motile goat cauda sperm dispersed in the synthetic medium were subjected to a freezing protocol in a computer-controlled bio-freezer, cooling 0.25 degrees C x min(-1) to 5 degrees C, 5 degrees C x min(-1) to -20 degrees C, and 20 degrees C x min(-1) to -100 degrees C, prior to being plunged into liquid nitrogen. In the absence of amino acids, sperm cells completely lost their flagellar motility. Of all the amino acids tested, l-alanine showed maximal cryoprotection potential. l-Alanine at 135 mM offered optimum cryoprotection potential: recovery of sperm forward motility and total motility were 14 +/- 2% and 19 +/- 2%, respectively. l-Glutamine, l-proline, and glycine at optimum concentration (100-150 mM) cryopreserved approx. 11-17% total motility of the sperm cells, whereas amino acids such as l-arginine, l-lysine, and l-histidine offered little cryoprotection (0-5%) to the cells. Increasing the amino acid concentration beyond the optimum level sharply decreased the recovery of the sperm motility, which therefore showed a biphasic cryoprotection profile. Addition of amino acids enhanced (approx. 7-10%) the cryoprotection efficacy of the well-known cryoprotectants glycerol and a combination of glycerol and dimethyl sulfoxide. The presence of glycerol caused a marked reduction (from 100-150 mM to 20-70 mM levels) in the optimal cryoprotective concentration of the amino acids. The combined cryoprotecting action of glycerol, dimethyl sulfoxide, and amino acids provided motility recovery as high as 52%. The observation that amino acids and dimethyl sulfoxide had an additive effect in augmenting the cryoprotecting potential of glycerol suggests that the mechanism of their action is different from that of glycerol. This cocktail of cryoprotectants may be useful for cryopreservation of semen of various species.

Development of a simple sperm cryopreservation model using a chemically defined medium and goat cauda epididymal spermatozoa.

This investigation was carried out to develop a simple sperm cryopreservation model using a chemically defined synthetic medium (modified Ringer's solution) and mature goat cauda epididymal sperm as the model system. Rates of cooling, freezing, and maximum freezing temperature were manipulated with the help of a computer-controlled programmable biofreezer. Highly motile goat cauda sperm dispersed in a modified Ringer's solution was subjected to the freezing protocol: cooling 0.25 degrees C min(-1) to 5 degrees C, 5 degrees C min (-1) to -20 degrees C, 20 degrees C min(-1) to -100 degrees C, prior to plunging into liquid nitrogen. In the absence of any cryoprotective agent, all of the spermatozoa lost their motility. Addition of glycerol (0.22 to 0.87 M) caused a dose-dependent increase of sperm motility recovery. The highest recovery of forward and total motility was (32 and 35%, respectively) at 0.87 M. Further increase of the glycerol concentration caused a marked decrease in motility. Changes in the cooling rate particularly before and during freezing had a notable effect on the sperm motility recovery. There was no or low recovery (0-18%) of sperm motility when the cells were transferred directly to liquid nitrogen from the initial two cooling stages. The data demonstrate the importance of all of the cooling stages in the cryopreservation of the cells. Like glycerol, dimethyl sulfoxide (Me(2)SO) and ethylene glycol also showed a dose-dependent increase in motility recovery as well as a biphasic curve of cryoprotection. At optimal concentrations, dimethyl sulfoxide (1.00 M) and ethylene glycol (1.29 M) were effective in recovering sperm motility to the extent of 20 and 13%, respectively. Thus these reagents have markedly lower cryoprotection potential than glycerol.