Effects of thrombopoietin, interleukin-3 and the kinase inhibitor K-252a on growth and polyploidization of the megakaryocytic cell line M-07e.
Thrombopoietin (TPO) is a recently cloned growth and differentiation factor implicated in megakaryocytopoiesis. Here, we show that TPO, interleukin-3 (IL-3) and, at least in short-term assays, also interferon gamma (IFN gamma) induced proliferation in acute myeloid leukemia (AML-M7)-derived M-07e cells. The Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway was activated after stimulation with any of the three cytokines. Thus, the TPO-receptor (TPO-R) MPL was tyrosine phosphorylated after a short-term stimulation with TPO, followed by tyrosine phosphorylation of STAT 3 and STAT 5, but not of STAT 1. IL-3 and IFN gamma induced phosphorylation of STAT 5 or STAT 1, respectively, without affecting the other STATs. As STATs are thought to regulate proliferation by modulating expression of inhibitors of cyclin-dependent kinases (Cdk), we analyzed p21 and p27 expression after stimulation with TPO or IL-3. In contrast to the constitutively low p21 expression, p27 mRNA levels were high in synchronized, cytokine-deprived cells in G0/1 phase. Stimulation with TPO or IL-3 induced a rapid decrease of p27 mRNA. The phosphorylation cycle of the retinoblastoma protein (Rb) was inversely correlated with the level of p27 mRNA. Hyperphosphorylation of Rb was detectable 9 h after onset of stimulation, concomitantly with the decrease of p27 mRNA and shortly before transition of the cells into S phase. As phosphorylation of Rb is a key event for transition of cells into S phase, our observations support the notion of p27 being an important regulator during cytokine-induced proliferation. Whether the JAK/STAT pathway is directly involved in p27 expression or not, remains to be elucidated. The JAK inhibitor AG-490 blocked cytokine-induced STAT 5 phosphorylation and proliferation of M-07e cells in a dose-dependent manner. Although these data indicate a role for the JAK/STAT pathway in cytokine-induced proliferation, a direct influence on the p27 mRNA downregulation has to be confirmed. The second major effect of TPO, polypoidization, could not be observed in M-07e cells. Even long-term culture with TPO did not induce endomitosis in these cells. However, polyploidization could be brought about by the kinase inhibitor K-252a. After 3 days of exposure to this reagent, 17% of the originally mononucleated cells contained two to five nuclei. K-252a-induced polykaryon formation was not preceded by STAT 5 phosphorylation. Thus, K-252a did not mimic TPO stimulation at the early steps of the signaling chain. Taken together, our experiments confirm a role for the JAK/STAT pathway in cytokine-induced proliferation; TPO and IL-3 induce downregulation of the Cdk inhibitor p27, hyperphosphorylation of Rb and subsequently transition of the cells into S phase; the kinase inhibitor K-252a induces polyploidization of M-07e cells, but this effect is independent of STAT 5 phosphorylation.
Quentmeier
H
H
DSMZ-German Collection of Microorganisms and Cell Cultures, Department of Human and Animal Cell Cultures, Braunschweig.
Zaborski
M
M
Drexler
H G
HG
D002227:Carbazoles / Q000494:pharmacology*; D018797:Cell Cycle Proteins; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors; D016213:Cyclins / Q000235:genetics; D004247:DNA / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D026121:Indole Alkaloids; D007377:Interleukin-3 / Q000494:pharmacology*; D008533:Megakaryocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000235:genetics; D008894:Milk Proteins; D009363:Neoplasm Proteins; D010766:Phosphorylation; D011123:Polyploidy; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D018121:Receptors, Cytokine; D053628:Receptors, Thrombopoietin; D011994:Recombinant Proteins / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050794:STAT1 Transcription Factor; D050796:STAT3 Transcription Factor; D050799:STAT5 Transcription Factor; D013926:Thrombopoietin / Q000494:pharmacology*; D015534:Trans-Activators / Q000378:metabolism; D025521:Tumor Suppressor Proteins
C493956:CDKN1A protein, human; D002227:Carbazoles; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D026121:Indole Alkaloids; D007377:Interleukin-3; D008869:Microtubule-Associated Proteins; D008894:Milk Proteins; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D018121:Receptors, Cytokine; D053628:Receptors, Thrombopoietin; D011994:Recombinant Proteins; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D050799:STAT5 Transcription Factor; D015534:Trans-Activators; D025521:Tumor Suppressor Proteins; C082218:MPL protein, human; D050760:Cyclin-Dependent Kinase Inhibitor p27; D004247:DNA; D013926:Thrombopoietin; C049985:staurosporine aglycone; D011493:Protein Kinase C; D018844:Cyclin-Dependent Kinases