PU.1-Induced Growth Arrest and Apoptosis in Classical Hodgkin Lymphoma Cells

Abstract Abstract 2628 PU.1 is an Ets family transcription factor, which is essential for differentiation of both myeloid and lymphoid lineage cells. We have previously shown that PU.1 is down-regulated in various myeloma cell lines and myeloma cells from a subset of myeloma patients. In such cell lines, the promoter and the upstream regulatory element (URE) located in 17 kb 5'-upstream of the PU.1 gene are highly methylated. Furthermore, conditionally expressed PU.1 induces both cell growth arrest and apoptosis in PU.1-low to -negative myeloma cell lines, U266 and KMS12PE. Therefore, we concluded that the down-regulation of PU.1 is necessary for myeloma cell growth. In another B cell malignancy, classical Hodgkin lymphoma, it has been reported that PU.1 is also down-regulated through methylation of its promoter. To evaluate whether down-regulation of PU.1 is essential for growth of classical Hodgkin lymphoma cells, we conditionally expressed PU.1 in two classical Hodgkin lymphoma cell lines, L428 and KMH2, using the tet-off system (designated as L428tetPU.1 and KMH2tetPU.1 cells, respectively). Up-regulation of PU.1 by tetracycline removal induced complete growth arrest in L428tetPU.1 and KMH2tetPU.1 cells. Annexin V staining revealed that up-regulation of PU.1 induced apoptosis in both cell lines. Furthermore, BrdU staining analysis revealed that PU.1 induced G0/G1 arrest in those cells. L428tetPU.1 and KMH2tetPU.1 cells expressing PU.1 showed morphological changes that included the enlargement cytosol and the appearance of various sizes of vacuoles. We next injected L428tetPU.1 and KMH2tetPU.1 cells to immunodeficiency mice (Rag2−/− Jak3−/− bulb/c) subcutaneously. Tumor formation was observed in all those mice with continuous administration of tetracycline (0.5 g/l) in the drinking water. After enlargement of tumor to 1–2 cm diameter, we removed tetracycline in half of the mice. Tetracyclin withdrawal resulted in tumor regression or stable disease, whereas all the mice continuously receiving tetracycline had continuous tumor growth and finally died. These data strongly suggest that PU.1 induced growth arrest and apoptosis of classical Hodgkin lymphoma cells both in vitro and vivo. We next performed DNA microarray analysis to compare gene expression levels of L428tetPU.1 cells before and after PU.1 expression to elucidate the mechanisms of growth arrest and apoptosis induced by PU.1. Among genes related to cell cycle and apoptosis, p21 (CDKN1A) was highly up-regulated in L428tetPU.1 cells after PU.1 induction, and this was also confirmed by mRNA and protein levels. Finally, to clarify the role of p21 up-regulation by PU.1, we stably introduced p21 siRNA in L428tetPU.1 cells. Such stably expressed p21 siRNA rescued L428tetPU.1 cells from growth arrest induced by PU.1, suggesting that the growth arrest in L428tetPU.1 cells by PU.1 should be at least partially dependent on p21 up-regulation. These data suggested that up-regulation of PU.1 by demethylation agents and/or HDAC inhibitors might serve as a possible treatment modality for classical Hodgkin lymphoma. Disclosures: No relevant conflicts of interest to declare.