Serine/threonine protein phosphatase type 1gamma1 is required for the completion of cytokinesis in human A549 lung carcinoma cells.
In lower eukaryotic organisms, the loss of serine/threonine protein phosphatase type 1 (PP1) results in growth arrest after the onset of mitosis. In humans, four highly homologous isoforms of PP1 (PP1alpha, PP1delta, PP1gamma1, and PP1gamma2) have been identified. Determining the roles of these phosphatases, however, has proven difficult due to the lack of subtype-specific inhibitors. In this study, we developed chimeric antisense 2'-O-(2-methoxy)ethylphosphothioate oligonucleotides targeting human PP1gamma1 that specifically inhibit PP1gamma1 gene expression. Two potent antisense oligonucleotides (ISIS 14435 and 14439; IC(50) approximately 50 nM) were then employed to elucidate the cellular functions of PP1gamma1 during cell cycle progression. In A549 cells, the inhibition of PP1gamma1 expression resulted in a dose-dependent inhibition of cellular proliferation, with growth arrest occurring after approximately 36-48 h, when PP1gamma1 mRNA expression was inhibited by >85%. Fluorescence-activated cell sorter analysis revealed that ISIS 14435/14439-induced growth arrest was associated with an increase in the number of cells containing 4N DNA. Immunostaining of treated cells revealed that the inhibition of PP1gamma1 expression had no apparent effect on the formation of mitotic spindles. However, decreased expression was associated with the failure of cell division in a late stage of cytokinesis and the formation of dikaryons.
Cheng
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Department of Biochemistry and Molecular Biology, University of South Alabama, Mobile, Alabama 36688, USA.
Dean
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Honkanen
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D002277:Carcinoma / Q000201:enzymology*; D002455:Cell Division / Q000187:drug effects / Q000502:physiology*; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D006801:Humans; D015151:Immunoblotting; D008175:Lung Neoplasms / Q000201:enzymology*; D008856:Microscopy, Fluorescence; D018715:Microscopy, Video; D008954:Models, Biological; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000502:physiology*; D020033:Protein Isoforms / Q000502:physiology; D013997:Time Factors; D014407:Tumor Cells, Cultured
D016376:Oligonucleotides, Antisense; D020033:Protein Isoforms; D010749:Phosphoprotein Phosphatases