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(1/29112) Expression of Bcl-2 protein is decreased in colorectal adenocarcinomas with microsatellite instability.

Bcl-2 is known to inhibit apoptosis and is thought to play a role in colorectal tumour development. Studies of the promoter region of bcl-2 have indicated the presence of a p53 responsive element which downregulates bcl-2 expression. Since p53 is commonly mutated in colorectal cancers, but rarely in those tumours showing microsatellite instability (MSI), the aim of this study was to examine the relationship of bcl-2 protein expression to MSI, as well as to other clinicopathological and molecular variables, in colorectal adenocarcinomas. Expression of bcl-2 was analysed by immunohistochemistry in 71 colorectal cancers which had been previously assigned to three classes depending upon their levels of MSI. MSI-high tumours demonstrated instability in three or more of six microsatellite markers tested, MSI-low tumours in one or two of six, and MSI-null in none of six. Bcl-2 expression in tumours was quantified independently by two pathologists and assigned to one of five categories, with respect to the number of cells which showed positive staining: 0, up to 5%; 1, 6-25%; 2, 26-50%; 3, 51-75%; and 4, > or =76%. Bcl-2 negative tumours were defined as those with a score of 0. Bcl-2 protein expression was tested for association with clinicopathological stage, differentiation level, tumour site, age, sex, survival, evidence of p53 inactivation and MSI level. A significant association was found between bcl-2 expression and patient survival (P = 0.012, Gehan Wilcoxon test). Further, a significant reciprocal relationship was found between bcl-2 expression and the presence of MSI (P = 0.012, Wilcoxon rank sum test). We conclude that bcl-2 expressing colorectal cancers are more likely to be MSI-null, and to be associated with improved patient survival.  (+info)

(2/29112) Growth inhibition of breast cancer cells by Grb2 downregulation is correlated with inactivation of mitogen-activated protein kinase in EGFR, but not in ErbB2, cells.

Increased breast cancer growth has been associated with increased expression of epidermal growth factor receptor (EGFR) and ErbB2 receptor tyrosine kinases (RTKs). Upon activation, RTKs may transmit their oncogenic signals by binding to the growth factor receptor bound protein-2 (Grb2), which in turn binds to SOS and activates the Ras/Raf/MEK/mitogen-activated protein (MAP) kinase pathway. Grb2 is important for the transformation of fibroblasts by EGFR and ErbB2; however, whether Grb2 is also important for the proliferation of breast cancer cells expressing these RTKs is unclear. We have used liposomes to deliver nuclease-resistant antisense oligodeoxynucleotides (oligos) specific for the GRB2 mRNA to breast cancer cells. Grb2 protein downregulation could inhibit breast cancer cell growth; the degree of growth inhibition was dependent upon the activation and/or endogenous levels of the RTKs. Grb2 inhibition led to MAP kinase inactivation in EGFR, but not in ErbB2, breast cancer cells, suggesting that different pathways might be used by EGFR and ErbB2 to regulate breast cancer growth.  (+info)

(3/29112) The cytoskeletal network controls c-Jun expression and glucocorticoid receptor transcriptional activity in an antagonistic and cell-type-specific manner.

The physical and functional link between adhesion molecules and the cytoskeletal network suggests that the cytoskeleton might mediate the transduction of cell-to-cell contact signals, which often regulate growth and differentiation in an antagonistic manner. Depolymerization of the cytoskeleton in confluent cell cultures is reportedly sufficient to initiate DNA synthesis. Here we show that depolymerization of the cytoskeleton is also sufficient to repress differentiation-specific gene expression. Glutamine synthetase is a glia-specific differentiation marker gene whose expression in the retinal tissue is regulated by glucocorticoids and is ultimately dependent on glia-neuron cell contacts. Depolymerization of the actin or microtubule network in cells of the intact retina mimics the effects of cell separation, repressing glutamine synthetase induction by a mechanism that involves induction of c-Jun and inhibition of glucocorticoid receptor transcriptional activity. Depolymerization of the cytoskeleton activates JNK and p38 mitogen-activated protein kinase and induces c-Jun expression by a signaling pathway that depends on tyrosine kinase activity. Induction of c-Jun expression is restricted to Muller glial cells, the only cells in the tissue that express glutamine synthetase and maintain the ability to proliferate upon cell separation. Our results suggest that the cytoskeletal network might play a part in the transduction of cell contact signals to the nucleus.  (+info)

(4/29112) Activation of c-Jun N-terminal kinase 1 by UV irradiation is inhibited by wortmannin without affecting c-iun expression.

Activation of c-Jun N-terminal kinases (JNKs)/stress-activated protein kinases is an early response of cells upon exposure to DNA-damaging agents. JNK-mediated phosphorylation of c-Jun is currently understood to stimulate the transactivating potency of AP-1 (e.g., c-Jun/c-Fos; c-Jun/ATF-2), thereby increasing the expression of AP-1 target genes. Here we show that stimulation of JNK1 activity is not a general early response of cells exposed to genotoxic agents. Treatment of NIH 3T3 cells with UV light (UV-C) as well as with methyl methanesulfonate (MMS) caused activation of JNK1 and an increase in c-Jun protein and AP-1 binding activity, whereas antineoplastic drugs such as mafosfamide, mitomycin C, N-hydroxyethyl-N-chloroethylnitrosourea, and treosulfan did not elicit this response. The phosphatidylinositol 3-kinase inhibitor wortmannin specifically blocked the UV-stimulated activation of JNK1 but did not affect UV-driven activation of extracellular regulated kinase 2 (ERK2). To investigate the significance of JNK1 for transactivation of c-jun, we analyzed the effect of UV irradiation on c-jun expression under conditions of wortmannin-mediated inhibition of UV-induced stimulation of JNK1. Neither the UV-induced increase in c-jun mRNA, c-Jun protein, and AP-1 binding nor the activation of the collagenase and c-jun promoters was affected by wortmannin. In contrast, the mitogen-activated protein kinase/ERK kinase inhibitor PD98056, which blocked ERK2 but not JNK1 activation by UV irradiation, impaired UV-driven c-Jun protein induction and AP-1 binding. Based on the data, we suggest that JNK1 stimulation is not essential for transactivation of c-jun after UV exposure, whereas activation of ERK2 is required for UV-induced signaling leading to elevated c-jun expression.  (+info)

(5/29112) Insertion of excised IgH switch sequences causes overexpression of cyclin D1 in a myeloma tumor cell.

Oncogenes are often dysregulated in B cell tumors as a result of a reciprocal translocation involving an immunoglobulin locus. The translocations are caused by errors in two developmentally regulated DNA recombination processes: V(D)J and IgH switch recombination. Both processes share the property of joining discontinuous sequences from one chromosome and releasing intervening sequences as circles that are lost from progeny cells. Here we show that these intervening sequences may instead insert in the genome and that during productive IgH mu-epsilon switch recombination in U266 myeloma tumor cells, a portion of the excised IgH switch intervening sequences containing the 3' alpha-1 enhancer has inserted on chromosome 11q13, resulting in overexpression of the adjacent cyclin D1 oncogene.  (+info)

(6/29112) Molecular mechanisms of thyroid hormone-stimulated steroidogenesis in mouse leydig tumor cells. Involvement of the steroidogenic acute regulatory (StAR) protein.

Using a mouse Leydig tumor cell line, we explored the mechanisms involved in thyroid hormone-induced steroidogenic acute regulatory (StAR) protein gene expression, and steroidogenesis. Triiodothyronine (T3) induced a approximately 3.6-fold increase in the steady-state level of StAR mRNA which paralleled with those of the acute steroid response ( approximately 4.0-fold), as monitored by quantitative reverse transcriptase-polymerase chain reaction assay and progesterone production, respectively. The T3-stimulated progesterone production was effectively inhibited by actinomycin-D or cycloheximide, indicating the requirement of on-going mRNA and protein synthesis. T3 displayed the highest affinity of [125I]iodo-T3 binding and was most potent in stimulating StAR mRNA expression. In accordance, T3 significantly increased testosterone production in primary cultures of adult mouse Leydig cells. The T3 and human chorionic gonadotropin (hCG) effects on StAR expression were similar in magnitude and additive. Cells expressing steroidogenic factor 1 (SF-1) showed marginal elevation of StAR expression, but coordinately increased T3-induced StAR mRNA expression and progesterone levels. In contrast, overexpression of DAX-1 markedly diminished the SF-1 mRNA expression, and concomitantly abolished T3-mediated responses. Noteworthy, T3 augmented the SF-1 mRNA expression while inhibition of the latter by DAX-1 strongly impaired T3 action. Northern hybridization analysis revealed four StAR transcripts which increased 3-6-fold following T3 stimulation. These observations clearly identified a regulatory cascade of thyroid hormone-stimulated StAR expression and steroidogenesis that provides novel insight into the importance of a thyroid-gonadal connection in the hormonal control of Leydig cell steroidogenesis.  (+info)

(7/29112) Non-steroidal anti-inflammatory drug-induced apoptosis in gastric cancer cells is blocked by protein kinase C activation through inhibition of c-myc.

Apoptosis plays a major role in gastrointestinal epithelial cell turnover, ulcerogenesis and tumorigenesis. We have examined apoptosis induction by non-steroidal anti-inflammatory drugs (NSAIDs) in human gastric (AGS) cancer cells and the role of protein kinase C (PKC) and apoptosis-related oncogenes. After treatment with aspirin or indomethacin, cell growth was quantified by MTT assay, and apoptosis was determined by acridine orange staining, DNA fragmentation and flow cytometry. The mRNA and protein of p53, p21waf1/cip1 and c-myc was detected by Northern and Western blotting respectively. The influence of PKC on indomethacin-induced apoptosis was determined by co-incubation of 12-O-tetradecanoylphorbol 13-acetate (TPA). The role of c-myc was determined using its antisense oligonucleotides. The results showed that both aspirin and indomethacin inhibited cell growth and induced apoptosis of AGS cells in a dose- and time-dependent manner, without altering the cell cycle. Indomethacin increased c-myc mRNA and protein, whereas p53 and p21wafl/cip1 were unchanged. Down-regulation of c-myc by its antisense oligonucleotides reduced apoptosis induction by indomethacin. TPA could inhibit indomethacin-induced apoptosis and accumulate cells in G2/M. Overexpression of c-myc was inhibited by TPA and p21waf1/cip1 mRNA increased. In conclusion, NSAIDs induce apoptosis in gastric cancer cells which may be mediated by up-regulation of c-myc proto-oncogene. PKC activation can abrogate the effects of NSAIDs by decreasing c-myc expression.  (+info)

(8/29112) p53 status of newly established acute myeloid leukaemia cell lines.

We analysed the status of the p53 gene and protein in eight newly established acute myeloid leukaemia (AML) cell lines representing blast cells of either de novo leukaemia patients in first remission or patients with relapsed and chemotherapy-resistant disease causing their death. There were no mutations in the p53 gene in any of the cell lines as analysed by single-strand conformation polymorphism of amplified exons 5-8. However, the p53 protein was clearly and consistently expressed in all of these cell lines, as shown by immunohistochemistry, Western blotting and flow cytometry. The consistently expressed p53 protein was located in both the nucleus and the cytoplasm of all the cell lines and, as shown by flow cytometry, it was mostly in a conformation typical of the mutated protein. These AML cell lines offer a tool for studying the production and function of the p53 protein and its possible role in cell cycle regulation and chemoresistance as well as in the regulation of apoptosis in AML.  (+info)