Inorganic arsenic is a major environmental contaminant associated with an increased risk of human skin cancer. Arsenic modulates cellular signaling pathways that affect diverse processes such as cell proliferation, differentiation, and apoptosis, including genotoxic damage. The p53 protein plays a central role in mediating stress and DNA damage responses, leading to either growth arrest or apoptosis. Several signal transduction pathways activated under a plethora of stressing conditions increase p53 protein levels. To further understand the molecular mechanisms involved in the arsenic mode of action, we explored the effects of this metalloid on the activation of the phosphatidyl inositol 3-kinase (PI3K)/Ca2+/diacylglicerol dependent protein kinase/protein kinase B (PKB) signaling cascade and its repercussion in p53 activation in two epithelial cell types: primary normal human keratinocytes cultures (NHK) and the carcinoma-derived C33-A cell line. Although in both cell systems arsenic leads to an increase in p53 and its binding to DNA, the final outcome is different. In NHK, arsenic triggers a sustained activation of the PI3K/PKB/glycogen synthase kinase-3 beta pathway, driving the cell into a cell-differentiated stage in which the proliferation signals are turned down. In sharp contrast, in C33-A cells, arsenic leads to a transient increase in p53 followed by a drastic reduction in its nuclear levels and an increase in cell proliferation. These findings favor the notion that p53-stage and transcriptional abilities are important to understand modifications in the proliferation-differentiation balance, an equilibrium that is severely impaired by arsenic.
Rhesus monkey kidney MA104 cells are a polarized epithelium with some unusual characteristics, including a resistance to ouabain, although their Na+-K+-ATPase has normal affinity with this drug. This work suggests that MA104 cells have high expression of functionally P-glycoprotein in their membranes. This was established using four complementary methods to investigate the expression and function of P-glycoprotein in these cells. MA104 cells were strongly resistant to vincristine, which could be reversed by three know n P-glycoprotein modulators; verapamil, cyclosporin A and trifluoperazine. In addition, MA104 cells accumulate little rhodamine 123, and the incubation with verapamil increased this accumulation. The mdr1-mRNA was detected by reverse transcription-polymerase chain reaction and a subcloned 283-bp product was identified. Its nucleotide sequence was compared with the related region of hum an mdr1, showing a high identity (96% ) between the two sequences. The expression of P-glycoprotein in the cell membrane was observed by Western blot and immunofluorescence. The results taken together suggest that MA104 cells intrinsically have a high expression of functionally P-glycoprotein in their membranes
Glutamate is involved in gene expression regulation in neurons and glial cells through the activation of a diverse array of signaling cascades. In Bergmann glia, Ca2+ -permeable alpha-hydroxy-5-methyl-4-isoazole-propionic acid (AMPA) receptors become tyrosine phosphorylated after ligand binding and by these means form multiprotein signaling complexes. Of the various proteins that associate to these receptors, the phosphatidylinositol 3-kinase (PI-3K) deserves special attention since D3-phosphorylated phosphoinositides are docking molecules for signaling proteins with a pleckstrin homology domain. In order to characterize the role of PI-3K in AMPA receptors signaling, in the present report we analyze the involvement of the serine/threonine protein kinase B in this process. Our results demonstrate an augmentation in protein kinase B phosphorylation and activity after glutamate exposure. Interestingly, the effect is independent of Ca2+ influx, but sensitive to Src blockers. Our present findings broaden our current knowledge of glial glutamate receptors signaling and their involvement glutamatergic neurotransmission.
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