Submitted April 21, 2008
Accepted October 7, 2008
Stroma-dependent apoptosis in clonal hematopoietic precursors correlates with expression of PYCARD
Andrew J Mhyre, A. Mario Marcondes, Emily Y Spaulding, and H Joachim Deeg*
Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, United States
Department of Pathology, University of Washington, Seattle, WA, United States
Department of Medicine, University of Washington, Seattle, WA, United States
* Corresponding author; email: jdeeg{at}fhcrc.org.
The role of the marrow microenvironment in the pathophysiology of Myelodysplastic Syndromes (MDS) remains controversial. Using stroma/hematopoietic cell co-cultures, we investigated the effects of stroma-derived signals on apoptosis sensitivity in hematopoietic precursors. The leukemia-derived cell line, KG1a, is resistant to pro-apoptotic ligands. However, when co-cultured with the human stroma cell line, HS5 (derived from normal marrow), and exposed to TNF
, KG1a cells showed Caspase-3 activation and induction of apoptosis. Apoptosis was contact dependent. Identical results were obtained in co-culture with primary stroma. Gene expression profiling of KG1a cells identified co-culture-induced upregulation of various genes involved in apoptosis, including PYCARD. Suppression of PYCARD expression in KG1a by miRNA interfered with apoptosis. Knockdown of TNF receptor 1 (R1) or R2 in HS5 cells had no effect. However, knockdown of R1 in KG1a cells prevented TNF-induced apoptosis, while apoptosis was still induced by TRAIL. Primary CD34+ cells from MDS marrow, when co-cultured with HS5 and TNF, also underwent apoptosis. In contrast, no apoptosis was observed in CD34+ cells from the marrow of healthy donors. These data indicate that stroma may convey not only protective effects on hematopoietic cells, but, dependent upon the milieu, may also facilitate apoptosis.
