Pim kinases are essential for survival of Myc-induced lymphoma cells in vitro


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Linus Plym Forshell (1), Tacha Zi Plym Forshell (1), Jonas Nilsson (1)
Department of Molecular Biology, Umeå, Sweden (1), Department of Molecular Biology, Umeå, Sweden (2), Department of Molecular Biology, Umeå, Sweden (3)

Background

The Pim kinase family consists of three serine/threonine kinases, Pim1-3. Elevated levels of Pim kinases are implicated in human tumorigenesis of liver, pancreas, colon, kidney, prostate and the hematopoietic system. Studies in transgenic mice have shown that Pim1 or Pim2 overexpression, in collaboration with the oncogene Myc, accelerates tumor progression and that deletion of the kinases slows down lymphomagenesis. Surprisingly, however, little work has addressed the importance of Pim kinases, and especially Pim3, in maintenance of lymphomas.

Method

We have analyzed expression of Pim kinases in spontaneously developed Myc-induced lymphomas by qRT-PCR. Since we found that all Pim kinases are more or less expressed we have treated Myc overexpressing cell lines originating from the same mouse model with an inhibitor targeting all three Pim kinases. Inhibitor-treated cells were subjected to western blot to monitor levels of phosphorylation of the surrogate marker Bad, a pro-apoptotic protein that is inhibited by Pim kinases. Flow cytometry was used to monitor cell cycle changes and apoptosis. Cell viability was determined by trypan blue staining and cell proliferation was determined by a 3H-Thymidine incorporation assay.

Results

In the cell lines tested, cell proliferation is strongly reduced by the Pim kinase inhibitor. Western Blot shows a rapid reduction in the amount of phosphorylated Bad. Flow cytometry show an increase in cell death as evident by cells with a sub-G1 DNA content and caspase-3 positive cells. This is further supported by a trypan blue viability assay.

Conclusion

We show that inhibition of the Pim kinase family reduces cell proliferation and initiates cell death in vitro. Ongoing experiments aim to clarify the mechanism behind this cell death and to investigate the efficacy of this inhibitor in mouse models.

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