A Cdc25A antagonizing K vitamin inhibits hepatocyte DNA synthesis in vitro and in vivo.
Thioalkyl containing K vitamin analogs have been shown to be potent inhibitors of hepatoma cell growth and antagonizers of protein tyrosine phosphatase activity. We now show that they inhibit the activity of specific protein tyrosine phosphatases (PTP) in cell-free conditions in vitro, particularly the dual specificity phosphatase Cdc25A. Using primary cultures of adult rat hepatocytes that are in G0/G1 phase until stimulated into DNA synthesis by epidermal growth factor, we found that 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone or Compound 5 (Cpd 5) inhibited hepatocyte DNA synthesis and PTP activity in cell culture and in vivo after a two-thirds partial hepatectomy. We found a selective inhibition of Cdc25A activity in vitro, using both synthetic substrates and authentic cellular substrate, immunoprecipitated phospho-Cdk4. Intact Cpd 5-treated cells had decreased cellular Cdc25A activity and increased tyrosine phosphorylation of Cdk4, resulting in decreased phosphorylation of retinoblastoma (Rb). Loss of Cdk4 activity was confirmed using Cdk4 immunoprecipitates from either Cpd 5-treated or untreated cells and measuring its kinase activity using GST-Rb as target. We found a similar order of activity for inhibition of growth and Cdc25A activity using several thiol-containing analogs. Cdc25A inhibitors may thus be useful for defining biochemical pathways involving protein tyrosine phosphorylation that mediate cell growth inhibition.
Carr
Brian I
BI
Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15213, USA. carrbi@msx.upmc.edu
Wang
Ziqiu
Z
Wang
Meifang
M
Kar
Siddhartha
S
Wilcox
Craig S
CS
Rosi
Katharyn
K
Southwick
Eileen
E
Lazo
John S
JS
D000818:Animals; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases / Q000737:chemistry / Q000378:metabolism; D004261:DNA Replication / Q000502:physiology*; D022781:Hepatocytes / Q000378:metabolism*; D066298:In Vitro Techniques; D008297:Male; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins; D051381:Rats; D011916:Rats, Inbred F344; D014443:Tyrosine / Q000378:metabolism; D014812:Vitamin K / Q000037:antagonists & inhibitors*; D020687:cdc25 Phosphatases / Q000502:physiology*
D011518:Proto-Oncogene Proteins; D014812:Vitamin K; D014443:Tyrosine; C495903:Cdk4 protein, rat; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases; C517007:Cdc25a protein, rat; D020687:cdc25 Phosphatases