Tamoxifen functions as a molecular agonist inducing cell cycle-associated genes in breast cancer cells. (49/67632)

Tamoxifen is a widely used breast cancer therapeutic and preventative agent. Although functioning as an estrogen antagonist at the cellular level, transcriptional profiling revealed that at the molecular level, tamoxifen functions largely as an agonist, virtually recapitulating the gene expression profile induced in breast cancer cells by estrogen. Remarkably, tamoxifen induces transcription factors and genes involved in promoting cell cycle progression including fos, myc, myb, cdc25a, cyclins E and A2, and stk15 with kinetics that paralleled that of cells cycling in response to estrogen, even though tamoxifen-treated cells are not transiting through the cell cycle. Induction of cell cycle-associated genes was specific for tamoxifen, and did not occur with raloxifene. However, cyclin D1 was a key estrogen-induced gene not expressed in response to tamoxifen or raloxifene but constitutively expressed in tamoxifen-resistant cells.  (+info)

Efficient gene transfer to hepatoblastoma cells through asialoglycoprotein receptor and expression under the control of the cyclin A promoter. (50/67632)

Specific gene delivery into hepatoma cells by liposomes and specific gene expression under the control of the cyclin A promoter were examined in HepG2 cells, a hepatoblastoma cell line that overexpresses cyclin A. A plasmid carrying the luciferase gene under the cyclin A promoter sequence was condensed with poly-L-lysine and encapsulated into anionic asialofetuin-labeled liposomes (AF-liposomes), which were preferentially taken up by hepatocytes through the action of the asialoglycoprotein receptor (AgpR). AF-liposomes delivered plasmids to the hepatoma cells by receptor-mediated endocytosis through the AgpR, and transgene expression could be achieved under the control of the cyclin A promoter. Furthermore, a fusogenic lipid, DOPE, as a liposomal component was required for the enhancement of transfection efficiency of AF-liposomes.  (+info)

Adherens junctions and tight junctions are regulated via different pathways by progastrin in epithelial cells. (51/67632)

Adhesion between neighbouring epithelial cells is a crucial and tightly controlled process. In the gastrointestinal tract, the integrity of cell-cell contacts is essential for the regulation of electrolyte absorption and for the prevention of tumour metastasis. We recently showed that migration of the gastric epithelial cell line IMGE-5 is stimulated by the nonamidated form of the hormone gastrin(17). Here, we examine the effect on cell-cell adhesion of the prohormone progastrin, the concentration of which is increased in the plasma of patients with colorectal carcinoma. Progastrin induced the dissociation of both tight junction (TJ) and adherens junction (AJ) complexes in IMGE-5 cells. In progastrin-secreting DLD-1 human colorectal carcinoma cells, expression of an antisense gastrin construct restored membrane localisation of zonula occludens-1 (ZO-1), occludin, beta-catenin and E-cadherin. This restoration was reversed by treatment with exogenous progastrin. Endogenous or exogenous progastrin also increased the paracellular flux of mannitol, and induced cell migration of several gastrointestinal cell lines. In addition, progastrin enhanced Src tyrosine kinase activity and induced a spatial delocalisation of protein kinase C alpha. Using dominant-negative mutants and pharmacological inhibitors, we showed that the stimulation of Src kinase activity was essential for the regulation of TJs. By contrast, the dissociation of AJs involved phosphatidylinositol 3-kinase, partly through the formation of a complex with protein kinase C alpha. We conclude that separate pathways mediate the disruption of AJs and TJs by progastrin. Either pathway may contribute to the co-carcinogenic role of this prohormone in colorectal carcinoma.  (+info)

Antisense candidates against protein kinase C-alpha designed based on phylogenesis and simulant structure of mRNA. (52/67632)

AIM: To optimize the antisense drug design by the combined method of phylogenetic analysis and secondary structure prediction and to get ideal candidates. METHODS: The phylogenetic analysis and the secondary structure simulation were performed by computer. Oligodeoxynucleotides (ODN) were designed against the full-conserved blocks with low local reaction free energy of protein kinase C (PKC)-alpha mRNA. The in vitro effects of ODN were evaluated by human A549 lung carcinoma cells and mouse B16-BL6 melanoma cells, the expression of target mRNA was detected by in situ hybridization and RT-PCR. The in vivo effects of ODN were also evaluated by models of A549 xenografts in nude mice and B16 melanoma in mice. RESULTS: Three ODN had significantly lower IC50 values than that of ISIS3521, the positive control, on A549 cells in vitro. Five ODN inhibited the growth of B16-BL6 cells with IC50 <100 nmol/L, while IC50 of ISIS3521 was >200 nmol/L. In situ hybridization and RT-PCR showed that the best candidate AP1261 inhibited the expression of PKC-alpha at mRNA level in a dose-dependent manner. AP1261 inhibited the growth of A549 and B16 tumors in vivo at 0.005-0.5 mg.kg(-1).d(-1). The inhibitory rate of AP1261 on A549 tumors was greater than that of ISIS3521 at the same dose. ISIS3521 did not affect the growth of B16 tumors. CONCLUSION: AP1261 may be of value as an antitumor agent or adjuvant and the combined method of phylogenetic analysis and secondary structure prediction is a potential helpful tool for antisense drug design.  (+info)

Environmental factors affecting transcription of the human L1 retrotransposon. II. Stressors. (53/67632)

Retrotransposons have clearly molded the structure of the human genome. The reverse transcriptase coded for by long interspersed nuclear elements (LINEs) accounts for 35% of the human genome, with 8-9 x 10(5) copies of the most common human LINE element, L1Hs. Retrotransposons cycle through an RNA intermediate with transcription as the rate limiting step. Because various retrotransposons have been demonstrated to be induced by environmental stimuli, we investigated the response of the L1Hs promoter to various agents. L1Hs promoter activity was analyzed by transfecting an L1Hs-expressing cell line with plasmids containing one of two L1Hs promoters fused to the LacZ reporter gene. L1Hs promoter activity was then monitored with a beta-galactosidase assay. Treatment with UV light and heat shock resulted in a small increase in beta-galactosidase activity from one promoter, while treatment with tetradecanoylphorbol 13-acetate resulted in small increases in beta-galactosidase activity from both promoters. No increase in beta-galactosidase activity was observed after exposure to X-rays or hydrogen peroxide.  (+info)

Statistics of the Comet assay: a key to discriminate between genotoxic effects. (54/67632)

The alkaline Comet assay is a widely used single cell gel electrophoresis technique for the quantification of DNA strand breaks, crosslinks and alkali-labile sites induced by a series of physical and chemical agents. DNA migration in an electric field, supposed proportional to strand breakage, is a proposed estimation of genotoxicity. Breaks are quantified from geometric and fluorescence measurements by image analysis of comet-shaped DNA, often reported parameters being tail DNA and tail moment. Although a variety of statistical approaches have been used in the literature, most of these do not take into account the distribution patterns of comet data. In order to investigate a methodology for statistically demonstrating a comet effect, two different experiments, a reproducibility study and a trend analysis, were undertaken on a murine lymphoma cell line (P388D1) photodynamically stressed after induction of porphyrins with delta-aminolaevulinic acid. This treatment results in significant heterogeneity of DNA damage, producing values ranging from 0 to 100% tail DNA in the same sample. The comparison of distribution curves for stressed and non-stressed samples shows that none of the application conditions are verified, either for parametric tests (which require normal distributions), or non-parametric tests (which assume essentially similar distributions). Meaningful statistics (median and 75th percentile) were consequently extracted from repeated experiments and found suitable for comparing stress conditions in an ANOVA and in a trend analysis; the 75th percentile is theoretically more sensitive but tends to more rapidly saturate at extensive stress levels. We conclude that a trend analysis of median comet metrics from repeated experiments at different stress levels is certainly an efficient way to statistically demonstrate a genotoxic effect. Whether the considered comet parameter is tail DNA or tail moment had no influence on the conclusions of our experiments, which were carried up to stress levels leading to a median 70% tail DNA.  (+info)

Altered glycosylation pattern allows the distinction between prostate-specific antigen (PSA) from normal and tumor origins. (55/67632)

Prostate-specific antigen (PSA) is a glycoprotein secreted by prostate epithelial cells. PSA is currently used as a marker of prostate carcinoma because high levels of PSA are indicative of a tumor situation. However, PSA tests still suffer from a lack of specificity to distinguish between benign prostate hyperplasia and prostate cancer. To determine whether PSA glycosylation could provide a means of differentiating between PSA from normal and tumor origins, N-glycan characterization of PSA from seminal fluid and prostate cancer cells (LNCaP cell line) by sequencing analysis and mass spectrometry was carried out. Glycans from normal PSA (that correspond to low and high pI PSA fractions) were sialylated biantennary complex structures, half of them being disialylated in the low pI PSA fraction and mostly monosialylated in the high pI PSA. PSA from LNCaP cells was purified to homogeneity, and its glycan analysis showed a significantly different pattern, especially in the outer ends of the biantennary complex structures. In contrast to normal PSA glycans, which were sialylated, LNCaP PSA oligosaccharides were all neutral and contained a higher fucose content. In 10-15% of the structures fucose was linked alpha1-2 to galactose, forming the H2 epitope absent in normal PSA. GalNAc was increased in LNCaP glycans to 65%, whereas in normal PSA it was only present in 25% of the structures. These carbohydrate differences allow a distinction to be made between PSA from normal and tumor origins and suggest a valuable biochemical tool for diagnosis and follow-up purposes.  (+info)

Glycosylation of human pancreatic ribonuclease: differences between normal and tumor states. (56/67632)

Characterization of the N-glycans from human pancreatic ribonuclease (RNase 1) isolated from healthy pancreas and from pancreatic adenocarcinoma tumor cells (Capan-1 and MDAPanc-3) revealed completely different glycosylation patterns. RNase 1 from healthy cells contained neutral complex biantennary structures, with smaller amounts of tri- and tetraantennary compounds, and glycans with poly-N-acetyllactosamine extensions, all extensively fucosylated. In contrast, RNase 1 glycans from tumor cells (Capan-1) were fucosylated hybrid and complex biantennary glycans with GalNAc-GlcNAc antennae. RNase 1 glycans from Capan-1 and MDAPanc-3 cells also contained sialylated structures completely absent in the healthy pancreas. Some of these features provide distinct epitopes that were clearly detected using monoclonal antibodies against carbohydrate antigens. Thus monoclonal antibodies to Lewis(y) reacted only with normal pancreatic RNase 1, whereas, in contrast, monoclonal antibodies to sialyl-Lewis(x) and sialyl-Lewis(a) reacted only with RNase 1 secreted from the tumor cells. These glycosylation changes in a tumor-secreted protein, which reflect fundamental changes in the enzymes involved in the glycosylation pathway, open up the possibility of using serum RNase 1 as a tumor marker of pancreatic adenocarcinoma.  (+info)