Human CDC25C ELISA Matched Antibody Pair

Venenum bufonis has been used as an antitumor drug in China for many years. Bufotalin, as an active component of Venenum bufonis, has been proven to exhibit antitumor effects in cancer types. In the present study, the effect of bufotalin on the human melanoma skin cancer cell line A375 was analyzed using MTT and colony formation assays. Bufotalin significantly inhibited the proliferation and colony formation of A375 cells. Further studies demonstrated that bufotalin significantly upregulated the protein levels of ATM serine/threonine kinase and Chk2, downregulated CDC25C protein expression, and subsequently inhibited CDK1 expression, leading to cell cycle arrest at the G2/M phase of the A375 cells. Furthermore, bufotalin significantly increased BAX expression levels, decreased BCL-2 expression, and then upregulated apoptosis-related proteins, caspase-3/-9, followed by A375 cell apoptosis. Taken together, these results show that bufotalin induces cell cycle arrest at the G2/M phase and cell apoptosis, resulting in the inhibition of A375 cell proliferation, thereby suggesting that bufotalin may be utilized in melanoma treatment.

Chk1 phosphorylation by the PI3-like kinases ATR and ATM is critical for its activation and its role in prevention of premature mitotic entry in response to DNA damage or stalled replication. The breast and ovarian tumor suppressor, BRCA1, is among several checkpoint mediators that are required for Chk1 activation by ATM and AIR. Previously we showed that BRCA1 is necessary for Chk1 phosphorylation and activation following ionizing radiation. BRCA1 has been implicated in S-phase checkpoint control yet its mechanism of action is not well characterized. Here we report that BRCA1 is critical for Chk1 phosphorylation in response to inhibition of replication by either cisplatin or hydroxyurea. While Chk1 phosphorylation of S317 is fully dependent on BRCA1, additional proteins may mediate S345 phosphorylation at later time points. In addition, we show that a subset of phosphorylated Chk1 is released from the chromatin in a BRCA1-dependent manner which may lead to the phosphorylation of Chk1 substrate, Cdc25C, on S216 and to S-phase checkpoint activation. Inhibition of Chk1 kinase by UCN-01 or expression of Chk1 phosphorylation mutants in which the serine residues were substituted with alanine residues abrogates BRCA1-dependent cell cycle arrest in response replication inhibition. These data reveal that BRCA1 facilitates Chk1 phosphorylation and its partial chromatin dissociation following replication inhibition that is likely to be required for S-phase checkpoint signaling. (C) 2012 Elsevier Ltd. All rights reserved.