Negative regulation of mitotic promoting factor by the checkpoint kinase chk1 in simian virus 40 lytic infection.
Lytic infection of African green monkey kidney (CV-1) cells by simian virus 40 (SV40) is characterized by stimulation of DNA synthesis leading to bypass of mitosis and replication of cellular and viral DNA beyond a 4C DNA content. To define mechanisms underlying the absence of mitosis, the expression levels of upstream regulatory molecules of mitosis-promoting factor (MPF) were compared in parallel synchronized cultures of SV40-infected and uninfected CV-1 cells. The DNA replication/damage checkpoint kinase Chk1 was phosphorylated in both uninfected and SV40-infected cultures arrested at G(1)/S by mimosine, consistent with checkpoint activation. Following release of uninfected cultures from G(1)/S, Chk1 phosphorylation was lost even though Chk1 protein levels were retained. In contrast, G(1)/S-released SV40-infected cultures exhibited dephosphorylation of Chk1 in S phase, followed by an increase in Chk1 phosphorylation coinciding with entry of infected cells into >G(2). Inhibitors of Chk1, UCN-01 and caffeine, induced mitosis and abnormal nuclear condensation and increased the protein kinase activity of MPF in SV40-infected CV-1 cells. These results demonstrate that SV40 lytic infection triggers components of a DNA damage checkpoint pathway. In addition, chemical inhibition of Chk1 activity suggests that Chk1 contributes to the absence of mitosis during SV40 lytic infection.
Okubo
Eiji
E
Center for Immunology and Microbial Disease, Albany Medical College, New York 12208, USA.
Lehman
John M
JM
Friedrich
Thomas D
TD
D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002460:Cell Line; D000071877:Checkpoint Kinase 1; D002522:Chlorocebus aethiops; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D008938:Mitosis / Q000187:drug effects; D015739:Nocodazole / Q000494:pharmacology; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000502:physiology*; D013539:Simian virus 40 / Q000502:physiology*; D019311:Staurosporine / Q000031:analogs & derivatives; D014764:Viral Proteins / Q000502:physiology*
D000470:Alkaloids; D004791:Enzyme Inhibitors; D047428:Protein Kinase Inhibitors; D014764:Viral Proteins; D002110:Caffeine; C054852:7-hydroxystaurosporine; D011494:Protein Kinases; D000071877:Checkpoint Kinase 1; D019311:Staurosporine; D015739:Nocodazole