adenosine-5--o-(3-thiotriphosphate) and Carcinoma

Primary cell cultures of human colorectal carcinomas were established and characterized immunocytochemically. In the isolated cancer cells intracellular Ca2+ concentrations ([Ca2+]i) were measured by the fura-2 method. Stimulation with either extracellular ATP or UTP caused a biphasic rise of [Ca2+]i in a dose-dependent manner and cross-desensitization between both nucleotides was observed. The rank order of potency was ATP >== UTP > ATP-gamma-S > ADP > adenosine which is characteristic for a P2U-receptor subtype. Selective agonists of P1-, or P2X- purinoceptors had no effect on [Ca2+]i. The initial rise in [Ca2+]i was independent of extracellular calcium [Ca2+]e, whereas the second phase was not observed under [Ca2+]e-free conditions suggesting a capacitative Ca2+-entry-mechanism. Intracellular Ca2+ mobilization was proven by use of the Ca2+-ATPase inhibitor thapsigargin. P2U-specific mRNA could be detected by RT-PCR in both colorectal tumor tissues and in the human colorectal cancer cell line HT 29. In HT 29 cells, the hydrolysis-resistant ATP analog ATP-gamma-S inhibited cell proliferation and, also, induced apoptosis in a dose-dependent manner. Thus, human colorectal cancer cells express functional P2U-receptors which may play a role in the regulation of cell proliferation and apoptosis.

Topics: Adenosine Triphosphate; Aged; Apoptosis; Calcium; Carcinoma; Cell Division; Colorectal Neoplasms; Dose-Response Relationship, Drug; Female; HT29 Cells; Humans; Male; Polymerase Chain Reaction; Purinergic P1 Receptor Agonists; Purinergic P2 Receptor Agonists; Receptors, Purinergic P2; Receptors, Purinergic P2Y2; RNA, Messenger; Tumor Cells, Cultured; Uridine Triphosphate

The p53 tumor suppressor protein is activated in cells in response to DNA damage and prevents the replication of cells sustaining genetic damage by inducing a cell cycle arrest or apoptosis. Activation of p53 is accompanied by stabilization of the protein, resulting in accumulation to high levels within the cell. p53 is normally degraded through the proteasome following ubiquitination, although the mechanisms which regulate this proteolysis in normal cells and how the p53 protein becomes stabilized following DNA damage are not well understood. We show here that p53 can also be a substrate for cleavage by the calcium-activated neutral protease, calpain, and that a preferential site for calpain cleavage exists within the N terminus of the p53 protein. Treatment of cells expressing wild-type p53 with an inhibitor of calpain resulted in the stabilization of the p53 protein. By contrast, in vitro or in vivo degradation mediated by human papillomavirus E6 protein was unaffected by the calpain inhibitor, indicating that the stabilization did not result from inhibition of the proteasome. These results suggest that calpain cleavage plays a role in regulating p53 stability.

Topics: Adenosine Triphosphate; Adenylyl Imidodiphosphate; Amino Acid Sequence; Breast Neoplasms; Calpain; Carcinoma; Chelating Agents; Cysteine Proteinase Inhibitors; Edetic Acid; Humans; Molecular Sequence Data; Mutation; Oncogene Proteins, Viral; Papillomaviridae; Repressor Proteins; Tumor Cells, Cultured; Tumor Suppressor Protein p53