Characterization of PIM kinases and cancer stem cell features in trophoblast cells

Mary, Stella GND

The proviral insertion in murine (PIM) lymphoma proteins are a serine /threonine kinase family composed of three isoforms: PIM1, PIM2 and PIM3 which play a critical role in the control of the cell survival, proliferation, homing, migration, apoptosis inhibition, micro-environmental signaling and drug resistance. In the human immortalized first trimester extravillous trophoblast cell line HTR8/SVneo and in the choriocarcinoma cell line JEG-3, we have analyzed the mRNA expression of PIM1, PIM2 and PIM3 by quantitative real time-PCR, and their protein expression by Western blotting and immunocytochemistry. We have further investigated the expression kinetics after stimulation with LIF. We have inhibited PIM kinases by using SGI-1776 at different doses, subsequently, we have studied the kinetics of cell function by cell viability, proliferation and apoptosis (BAD, BCL-XL, (cleaved) PARP and CASP3) and also the expression and potential PIM target c-MYC. Additionally, apoptosis and necrosis was tested by flow cytometry by annexin V and propidium iodide binding. The major results are that all analyzed PIM kinases are expressed in both cell lines and further increased upon stimulation with LIF. Inhibition of PIM kinases significantly reduces viability and proliferation and induces apoptosis as assessed by increased cleaved PARP and annexin V/propidium iodide binding. Simultaneously, phosphorylation of c-MYC was reduced. We have observed that the HTR8/SVneo cells forming spheroids expressed the stem cell surface marker CD44+ve high/CD34+ve low /CD24+ve low. Upon LIF stimulation these markers showed a decreased expression. The stemness-related transcription factors NANOG and CDX2 were constitutively expressed. Upon OSM treatment the expression level of NANOG is increased and that of CDX2 decreased. Hence, we conclude that HT8/SVneo cells contain a fraction or side-population that expresses stemness related factors.

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Mary, Stella: Characterization of PIM kinases and cancer stem cell features in trophoblast cells. 2016.

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