Microarray and pathway analysis reveals decreased CDC25A and increased CDC42 associated with slow growth of BCL2 overexpressing immortalized breast cell line.

Bcl-2 is an anti-apoptotic protein that is frequently overexpressed in cancer cells but its role in carcinogenesis is not clear. We are interested in how Bcl-2 expression affects non-cancerous breast cells and its role in the cell cycle. We prepared an MCF10A breast epithelial cell line that stably overexpressed Bcl-2. We analyzed the cells by flow cytometry after synchronization, and used cDNA microarrays with quantitative reverse-transcription PCR (qRT-PCR) to determine differences in gene expression. The microarray data was subjected to two pathway analysis tools, parametric analysis of gene set enrichment (PAGE) and ingenuity pathway analysis (IPA), and western analysis was carried out to determine the correlation between mRNA and protein levels. The MCF10A/Bcl-2 cells exhibited a slow-growth phenotype compared to control MCF10A/Neo cells that we attributed to a slowing of the G(1)-S cell cycle transition. A total of 363 genes were differentially expressed by at least two-fold, 307 upregulated and 56 downregulated. PAGE identified 22 significantly changed gene sets. The highest ranked network of genes identified by IPA contained 24 genes. Genes that were chosen for further analysis were confirmed by qRT-PCR, however, the western analysis did not always confirm differential expression of the proteins. Downregulation of the phosphatase CDC25A could solely be responsible for the slow growth phenotype in MCF10A/Bcl-2 cells. Increased levels of GTPase Cdc42 could be adding to this effect. PAGE and IPA are valuable tools for microarray analysis, but protein expression results do not always follow mRNA expression results.

The relationship between apoptosis and high-mobility group protein 1 release from murine macrophages stimulated with lipopolysaccharide or polyinosinic-polycytidylic acid.

High-mobility group protein 1 (HMGB1) is a nonhistone nuclear protein whose function depends on cellular location. Inside the cell, HMGB1 modulates a variety of important cellular processes, including transcription, whereas outside the cell, HMGB1 acts as a cytokine that can promote inflammation and mediate sepsis and arthritis in animal models. In in vitro studies, proinflammatory molecules such as LPS, lipoteichoic acid, polyinosinic-polycytidylic acid (poly(I:C)), TNF-alpha, and type I and II IFNs can induce HMGB1 release from macrophages. Although these agents can activate cells, they can also induce apoptosis under certain circumstances. Therefore, because of evidence that apoptotic as well as necrotic cells can contribute to HMGB1-mediated events in sepsis, we have investigated the relationship between apoptosis and HMGB1 release in macrophages and other cells. In these experiments, using RAW 264.7 cells as a model, LPS and poly(I:C) caused HMGB1 release into the medium whereas CpG ODN failed to induce this response. With both LPS and poly(I:C), the extent of HMGB1 release correlated with the occurrence of apoptosis as measured by caspase 3 activation, lactate dehydrogenase release, and TUNEL staining. Similar results were obtained with primary murine macrophages as well as human Jurkat T cells. For Jurkat cells, poly(I:C) and NO donors induced apoptosis as well as HMGB1 release. Together, these results indicate that HMGB1 release from macrophages is correlated with the occurrence of apoptosis and suggest that these processes reflect common mechanisms and can occur concomitantly.

Pro-thrombotic and pro-oxidant effects of diet-induced hyperhomocysteinemia.

Elevated plasma homocysteine levels are associated with the risk of atherosclerosis and arterial and venous thrombosis. We have previously demonstrated that rabbits rendered hyperhomocysteinemic by parenteral administration of homocysteine develop a dysfibrinogenemia that is associated with the formation of fibrin clots that are abnormally resistant to fibrinolysis. We suggested that this acquired dysfibrinogenemia contributes to the thrombotic tendency in hyperhomocysteinemia. However, it was possible that the homocysteine-associated dysfibrinogenemia was an artifact of the parenteral administration model. Therefore, the goals of the current study were to develop a diet-induced model of homocysteinemia in rabbits and determine whether a dysfibrinogenemia and evidence of oxidative stress develop in this model as they do when homocysteine is injected. We found that rabbits fed a diet severely deficient in folate and mildly deficient in choline develop mild hyperhomocysteinemia: 14.8+/-4.0 microM in deficient rabbits compared to 9.0+/-1.7 microM in controls. The deficient rabbits also develop evidence of oxidant stress: increased lipid peroxidation in liver, impaired mitochondrial enzyme activities in liver and elevated caspase-3 levels in plasma. Most importantly, the deficient rabbits also develop a dysfibrinogenemia characterized by increased resistance to fibrinolysis. We believe that this dietary model of homocysteinemia is clinically relevant and reproduces many features associated with hyperhomocysteinemia in previous work using in vitro and in vivo models. Our findings suggest that an acquired dysfibrinogenemia could play a role in the increased risk of atherothrombotic disease in mildly hyperhomocysteinemic human subjects.

The extracellular release of HMGB1 during apoptotic cell death.

High mobility group box 1 protein (HMGB1) is a non-histone nuclear protein with dual function. Inside the cell, HMGB1 binds DNA and regulates transcription, whereas outside the cell, it serves as a cytokine and mediates the late effects of LPS. The movement of HMGB1 into the extracellular space has been demonstrated for macrophages stimulated with LPS as well as cells undergoing necrosis but not apoptosis. The differential release of HMGB1 during death processes could reflect the structure of chromatin in these settings as well as the mechanisms for HMGB1 translocation. Since apoptotic cells can release some nuclear molecules such as DNA to which HMGB1 can bind, we therefore investigated whether HMGB1 release can occur during apoptosis as well as necrosis. For this purpose, Jurkat cells were treated with chemical inducers of apoptosis (staurosporine, etoposide, or camptothecin), and HMGB1 release into the medium was assessed by Western blotting. Results of these experiments indicate that HMGB1 appears in the media of apoptotic Jurkat cells in a time-dependent manner and that this release can be reduced by Z-VAD-fmk. Panc-1 and U937 cells treated with these agents showed similar release. In addition, HeLa cells induced to undergo apoptosis showed HMGB1 release. Furthermore, we showed using confocal microscopy that HMGB1 and DNA change their nuclear location in Jurkat cells undergoing apoptosis. Together, these studies indicate that HMGB1 release can occur during the course of apoptosis as well as necrosis and suggest that the release process may vary with cell type.