Conditionally Expressed PU.1 Transactivates TRAIL Gene and Induces Apoptosis in Myeloma Cell Lines.

PU.1 is an Ets family transcription factor, which is important for differentiation of both myeloid and lymphoid lineages. In the Friend leukemia model, the failure of PU.1 down-regulation in erythroblasts reportedly results in differentiation arrest, leading to erythroleukemia. In mice conditionally knocked-out of the 3.5 kb length of enhancer region located in14 kb 5′ upstream of the PU.1 gene, PU.1 is down-regulated in myeloid cells and B cells to 20% of that of wild type, and such mice develop acute myeloid leukemia and CLL-like diseases. Since the 3.5 kb enhancer region contains a suppressor region for PU.1 expression in T cells, such mice ectopically express PU.1 in T cells and develop T cell lymphoma. Thus, the failure of proper expression of PU.1 in certain differentiation stages for certain cell lineages appears to result in hematological malignancies.

We recently reported that human 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′ upstream of the PU.1 gene which is homologous to that in14 kb 5′ upstream of murine PU.1 gene. Conditionally expressed PU.1 induced cell growth arrest and apoptosis of two PU.1 low-negative myeloma cell lines, U266 and KMS12PE, suggesting that the down-regulation of PU.1 is essential for myeloma cell growth. We have also reported that PU.1 is expressed in normal plasma cells and PU.1 is down-regulated in myeloma cells of certain myeloma patients, who appear to have poor prognosis. In the present study, to elucidate the mechanisms of the cell growth arrest and apoptosis in PU.1-conditionally expressing myeloma cells, we performed DNA microarray analysis to compare gene expression levels before and after PU.1 expression, utilizing Illumina Sentrix^® Human-6 Expression BeadChip. Of 47,296 genes, 479 genes were up-regulated (>2fold) and 1,697 genes down-regulated (<0.5 fold), either 1 or 3 days after PU.1 expression in U266 cells. Among cell-cycle related genes, p21^WAF1/CIP1 was found up-regulated in U266 cells, which was confirmed at protein levels. Among apoptosis related genes, TRAIL was highly up-regulated in both U266 and KMS12PE cell lines. Stably expressed siRNA for TRAIL inhibited apoptosis of PU.1-expressing U266 cells, suggesting that TRAIL may have a crucial role in the PU.1- induced apoptosis. We subsequently examined how TRAIL was up-regulated in such PU.1-expressing myeloma cells. We performed chromatin immunoprecipitation assay and found that PU.1 directly binds to the promoter region of the TRAIL gene in U266 cells. We also determined the PU.1 binding site using electrophoretic mobility shift assays. An introduction of mutations in the PU.1 binding site abolished the binding. We also subcloned the TRAIL promoter and exon1 5′ non-coding sequence into pGL4.26 vector and performed reporter assays. Induction of PU.1 by removal of tetracycline in U266 cells induced 4-fold up-regulation of luciferase activity compared to that without PU.1 expression (28.7-fold compared to that with pGL4.26 vector alone) and mutations in the PU.1 binding site completely abolished the up-regulation. Taken together, we hereby conclude that PU.1 can directly transactivate the TRAIL gene in myeloma cells, leading to apoptosis.