Tumor suppressive role of the α-isoform of transcriptional repressor PRDM1 in the pathogenesis of NK-cell malignancies

Abstract

NK cell lymphoma is one of the cellular malignancies that arise from lymphocytes. Due to its rarity and aggressiveness the detailed molecular pathogenesis of NK cell lymphoma remains to be discovered. There are recent studies showing that the master regulator of B-cell differentiation into plasma cells, the Positive Regulatory Domain containing 1, with ZNF domain(PRDM1) has tumor suppressive function not only in diffuse large B-cell lymphoma (DLBCL), but also in NK cell lymphoma. The PRDM1 has two isoforms, αand β, where the former one is a functional isoform and the latter is a defective isoform with shortened and disrupted positive regulatory domain formed from transcription of internal promoter.

By semi -quantitative RT-PCR, PRDM1-αexpression was found to be absent in 80% (4/5) NK cell lines while present in the normal NK cells. Loss of PRDM1 expression suggests its role as tumor suppressor. In order to study the tumor suppressive role of the αisoform of PRDM1, short-hairpin RNA (shRNA) with isoform specific sequence is used to knockdown the expression of PRDM1-αin NK cell lines. Western blot result showed about 40% decrease of PRDM1-αprotein after knockdown. Retroviral infection of the NK cell lines, NKYS and YT which have endogenous α-isoforms of expression, for the delivery of the shRNA was done and were subsequently subjected to in vitro functional analyses including MTS assay, colony formation assay, cell viability test and cell cycle analysis to determine potential effect of the loss of PRDM1-αon the NK cell lines.

The PRDM1-αprotein isoform is expected to be able to repress excessive growth of NK cell line. When this isoform is inactivated, the NK cell lines are expected to proliferate significantly than the negative control counterpart in functional analyses. However in this study only YTcell line showed significant proliferation advantage in MTS and colony formation assay after the knockdown of PRDM1-α by shRNA. Cell viability assays and cell cycle analyses failed to show significant changes in both NK cell lines and yet even showed inhibitory effect after the knockdown of the gene. Ectopic expression of PRDM1-αby retroviral infection was done in KHYG cell line to further evaluate its tumor suppressive function. Apoptotic assay on the KHYG cells with ectopic expression of PRDM1-αwas performed and percentage of cells with late apoptosis was found to be significantly higher in this cell line. This suggests that one of the mechanisms for PRDM1-αto act as tumor suppressor is via the apoptosis pathway which in turn promotes the cell death.

Future studies will be made to further investigate the effects of knockdown of PRDM-1αby designing another shRNA sequence which knockdown the expression of gene by at least 50% and to further investigate the role of PRDM1-αinthe pathogenesis NK cell lymphomaby proliferation assays, colony formation assay and cell cycle analysis.