Microarray Analysis of PU.1-Induced Growth Arrest and Apoptosis of Myeloma Cell Lines.

PU.1 is an Ets family transcription factor, which is important for differentiation of granulocytes, monocytes/macrophages, and B cells. In the Friend leukemia model, it is reported that the failure of PU.1 down-regulation in erythroblasts reportedly results in differentiation arrest that leads to erythroleukemia. In conditional knockout mice of the 3.5 kb length of enhancer region located in14 kb 5′ of the PU.1 gene, PU.1 is down-regulated in myeloid cells and B cells down to 20% of that of wild type, and such mice develop acute myeloid leukemia and CLL-like disease. In addition, a deletion of the 3.5 kb enhancer region, which also contains the suppressor region for PU.1 in T cells, results in ectopic expression of PU.1 in T cells, which leads to T cell lymphoma in those mice. Taken together, the failure of up-regulation or down-regulation of PU.1 in certain differentiation stages for each lineage appears to cause differentiation arrest and hematological malignancies. We recently reported that PU.1 is down-regulated in a majority of myeloma cell lines through the methylation of the promoter and enhancer region located in17 kb 5′ of human PU.1 gene which is homologous to that in14 kb 5′ of murine PU.1 gene. Conditionally expressed PU.1 induced cell growth arrest and apoptosis of those PU.1 low-negative myeloma cell lines, U266 and KMS12PE, suggesting that down-regulation of PU.1 is necessary for myeloma cell growth. In addition, we reported that PU.1 is expressed in normal plasma cells and PU.1 is down-regulated in myeloma cells of some myeloma patients. Myeloma patients with low-to-negative PU.1 expression (lower 25th percentile of PU.1 expression level distribution among 30 patients we examined) may have poor prognosis compared to those with high PU.1 expression, although more patient samples have to be examined to define the significance of the relationship of PU.1 expression levels and prognosis. To elucidate the mechanisms of PU.1 induced cell growth arrest and apoptosis of myeloma cells, we next performed DNA microarray analysis to compare gene expression levels before and after PU.1 induction. We utilized Illumina Sentrix® Human-6 Expression BeadChip. Of 47296 genes, 479 genes were up-regulated (>2fold) and 1697 genes down-regulated (<0.5 fold) either day 1 or 3 after PU.1 induction in U266 cells. Among apoptosis related genes, TRAIL was highly up-regulated in both U266 and KMS12PE cell lines. Stably expressed siRNA for TRAIL partially inhibited apoptosis of U266 cells expressing PU.1, suggesting that TRAIL is related to PU.1 induced cell death of U266 cells. Among cell-cycle related genes, p21^WAF1/CIP1 was found up-regulated in U266 cells, which was confirmed with protein levels. We are now examining the roles of the observed up-regulated genes in both U266 and KMS12PE myeloma cell lines.