TGFβ regulates the expression and activities of G2 checkpoint kinases in human myeloid leukemia cells

Transforming Growth Factor-beta (TGFO) is known to be a negative regulator of G1 cyclin/cdk activity. It is not clear whether TGF beta has any effect on G2 checkpoint kinases. We have found that TGF beta downregulated the expression of several G2 checkpoint kinases including cdc2, cyclin B1, and cdc25c without causing cell accumulation in G2/M phases in two human leukemia cell lines. The inhibition was time-dependent with a maximal inhibition being observed by 24 h for cyclin B1 and cdc2 and by 48 h for cdc25c. The inhibition was not a result of G1 arrest but a direct effect of TGF beta which downregulates their expression at mRNA level. In proliferating cells, there was a significant formation of cdc2-pRb complexes, which was decreased to 30% of control levels by 48 h after initiating TGF beta treatment. Cdc2 showed a marked kinase activity on GST-Rb protein in proliferating cells detected by in vitro kinase assay, which was downregulated in response to TGF beta. In addition, TGF beta caused a rapid and transient dephosphorylation of cdc2 (Tyr15) and cdc25c (Ser216) for about 2-3 h before a dramatic decrease of both molecules by 48 h. Taken together, our data suggest that TGF beta has a direct inhibitory effect on G2 checkpoint kinases, which is regulated at mRNA level. The transient activation of cdc2 and cdc25c and subsequent inhibition of cdc2, cyclin 131, and cdc25c could amplify TGF-induced GI arrest and growth inhibition. (c) 2007 Elsevier Ltd. All rights reserved.