There are several recent reports that certain mutations in critical transcription factors could be associated with leukemogenesis; however, it remains unclear whether such mutations could be linked to the pathogenesis of multiple myeloma (MM). In the present study, we attempted to determine whether mutations in transcription factors could be found in the patients with MM, utilizing MM cell lines and freshly isolated MM cells, purified with CD138 antibody and magnetic beads. When we looked at the expression of Blimp-1, XBP-1, NF-kB (p65 and p50), and PU.1 using semi-quantitative RT-PCR, all except PU.1 were found expressed in all MM cell lines and freshly isolated MM cells examined. All the PCR products were sequenced, but no significant mutations were identified. As examined with RT-PCR, PU.1 was found completely downregulated in 6 of 8 MM cell lines, while only faint to no expression of PU.1 was seen in 12 of 41 freshly isolated MM cells. As PU.1 message was sequenced, no mutations were found in 29 PU.1-positive MM cells or 2 MM cell lines that had relatively high PU.1 expression. Considering that PU.1 is essential for myeloid and lymphoid differentiation and that mice with null mutation in the PU.1 gene have no granulocytes, monocytes/macrophages or B lymphocytes, together with a recent report by others that AML developed in mice with PU.1 downregulation by 20%, we asked whether PU.1 downregulation might be associated with the oncogenesis or maintenance of MM. To examine the effects of PU.1 expression on MM cells, we generated tet-off conditional PU.1 expression model of PU.1-negative U266 MM cell line. Surprisingly the induction of PU.1 expression resulted in cell cycle arrest and apoptosis of those cells. Cell cycle analysis using flow cytometry revealed that PU.1-expressing U266 cells were in the G1 arrest. May-Giemsa staining revealed that such cells were swollen, enlarged, and vacuolous. These changes were completely reverted when the expression of PU.1 was silenced by tetracycline addition. There was no change in expression levels of cell cycle-related genes (p27, cyclin D1, cyclin E) and apoptosis-related genes (Bcl-2, Bcl-XL, and A1), while caspase3 cleavage was observed by 72 hr following PU.1 induction. We are currently examining the mechanism(s) of PU.1 downregulation in MM cells and a downstream cascade of PU.1 expression during cell death.