Dlk1 Up Regulated Might Be an Important Event in Human MDS.

Abstract

The diagnosis of myelodysplastic syndrome (MDS) is made largely on the dysplastic morphology of BM cells from aspiration or biopsies. Prognosis scored by IPSS is depending on the percentage of marrow myeloblasts and the clonal cytogenetic abnormalities. To expand the understanding of genetic defects in hematopoietic cells of MDS in hope of finding novel genes correlated to pathogenesis and provide possible diagnostic marker for MDS, we have applied microarray to analyze the clinical samples from MDS patients. Total RNAs of CD34+ cells from 8 patients ( 2 RAEBt,2 RAEB,2 RA,1 RAS,1 CAA ) and one healthy people were extracted followed by a double in vitro transcription to circumvent the limited number of CD34+ cells. Following a modified Affymetrix target amplification protocol. Biotinylated cRNA was synthesized from 50 ng total RNA by double-round amplification and hybridized to an Human Genome U133 Plus 2.0 Array (Affymetrix). From the expression profile of 18404 different genes, we revealed that DNTT,MLL3,IL1R2,MAPK1,IGLL1 were down regulated while EGR-1, Rap1GAP or MAF were up regulated compared with normal controls. Most notably, Dlk1 was up regulated in MDS, while down regulated in AML and normal. By real-time RT-PCR we confirmed that in BMNCs the median levels of Dlk1 transcript in patients with RA and RAS were 2.55 (range, 0.00–23.7), RAEB and RAEBt were 8.24(range, 2.01–18.44), AML were 1.88 (range, 0.12–5.13), and other patients were 0.37(range, 0.00–1.79), respectively. The abundance of Dlk1 mRNA in MNCs from most MDS patients was markedly greater than that in the MNCs from others (P <0.05 ). Dlk1 expression in RAEB and RAEBt is markedly higher than AML (P <0.05 ) Forced expression of Dlk1 in transfected K562 cells resulted in faster growth than control cells, affected apoptosis induced by As2O3. and reduced the G2 arrested cells induced by TPA. By using the same experimental system we found that forced expression of Dlk1 can increase the mRNA levels of HES1 and p21WAF1 transcript variant 1. To elucidate the mechanisms we analyzed the levels of phosphorylated-p38 and p38 in Dlk1 transfected K562 cells treated with TPA. Dlk1 inhibited p38 phosphorylation while expression of p38 kept no change. These results support further investigation on the role of Dlk1 in abnormal hematopoiesis in MDSheterogeneous cell component. Diagnosis is currently depending on the dysplastic morphology of.