| It has been showed that bone marrow microenvironment, known as the hematopoietic microenvironment, plays critical role in the maintenance and differentiation of normal hematopoietic progenitors. Growth and differentiation of most types of hematopoietic cells in vivo require direct contact with bone stromal cells (BMSCs). However, the molecular mechanisms of the interaction between stromal cells and hematopoietic cells are not well defined. Nevertheless, it has been shown that stromal cells produce a variety of growth factors, and in some cellular systems, it has been observed that direct cell-cell contact is needed for cell growth and differentiation. Since leukemic cells originate from their normal counterparts and also reside within the bone marrow microenvironment, it is likely that stromal cells influence the proliferation and apoptosis of leukemic cells. Previous studies reported that direct interaction with stromal cells and cytokines increases the survival of leukemic cell lines during chemotherapy drugs exposure, emphasizing the role of stromal cells in the maintenance of leukemic cells. This interaction may play a role in the resistance of residual, post-chemotherapy leukemia cells to chemotherapeutic agents, a problem that remains a major hurdle in the treatment of leukemia. These cells that give rise to such leukemia cell regrowth must therefore originate from a compartment of cells that is relatively protected from cytotoxic agents. This has been suggested in previous studies in which stromal cells were shown to prevent spontaneous or induced apoptosis in AML, ALL, and CLL cells. But the molecular mechanisms are not well defined.In present study, the role of normal and leukemic bone marrow stromal cells on the survival of acute lymphoblastic leukemia (ALL) cell line Jurkat cells exposed to chemotherapeutic agent DNR was investigated. Normal and leukemic BMSCs were isolated with Percoll. And BMSCs were cocultured with Jurkat cells in vitro. Apoptosis of Jurkatcells stained with Annexin V/PI were detected by flow cytometry. Cell cycle distribution of Jurkat cells was analyzed by flow cytometry after cell stained with PI. Suppression subtractive hybridization (SSH) method was employed to establish subtracted cDNA library of differentially expressed genes in Jurkat cells coculturing with leukemic BMSCs during DNR exposure in vitro. The cDNA fragments were sequenced and analyzed too. The mRNA expression levels of CR6 interacting factor 1 (CRIF1) gene and Gadd45 were analyzed with RT-PCR.We found that the increase of apoptotic Jurkat cells percentage was in direct proportion to DNR doses within 0.1-2.0umol/L as well as exposure period. The percentage of apoptotic Jurkat cells coculturing with normal BMSCs induced by 0.5 |imol/L DNR is lower than those of Jurkat cells maintained in suspension (P<0.05). The percentage of apoptotic Jurkat cells coculturing with leukemic BMSCs is lower than those of Jurkat cells coculturing with normal BMSCs induced by 0.5 umol/L DNR (.PO.05). Jurkat cells coculturing with BMSCs were demonstrated increased in the Go/Gi phase. The percentage of Go/Gi phase is similar in Jurkat cells coculturing with normal and leukemic BMSCs (,P>0.05). Based on the investigation of the protection of leukemic BMSCs on Jurkat cell line, the differentially expressed gene cDNA library of Jurkat cells coculturing with leukemic BMSCs in vitro was developed using SSH. Primary screening was done by reverse northern hybridization. 30 up-regulated and 22 down-regulated cDNA fragments were isolated and sequenced. Analysis and comparison were performed in GenBank using BLAST. Among these clones, most of them showed high homology (more than 86%) with known genes, and up-regulated clones showed obviously similar to histone 1 H2a, COX5B (cytochrome c oxidase subunit Vb), CRIF1 (CR6 interacting factor 1), Galectin-1), NADH dehydrogenase 1, Ribosomal protein LI8a, a down-regulated gene is Ribosomal protein Sll. These known genes are related to cell cycle regulation, cellular apoptosis and energy metabolism. The information of differentially expressed genes in Jurkat cells implied that its secretion function, cell cycle and apoptosis were affected by leukmic BMSCs.Concerning the importance of CRIF1 and Gadd45 on the survival of Jurkat cells coculturing with leukemic BMSCs during chemotherapeutic agent DNR exposure, we examined the expression of CRIF1 and Gadd45 in mRNA level. Post cocultured withleukemic BMSCs, the expression of CRIFl and Gadd45 in Jurkat cells in mRNA level is significantly higher than that of the control. This indicates that high mRNA levels of CRIFl and Gadd45 might be significantly associated with the Go/Gi arrest of Jurkat cells during DNR exposure. Our datas suggest that Go/Gi arrest, which is partially induced by CRIFl and Gadd45, plays a key role in the survival of stromal-supported Jurkat cells during DNR exposure in vitro.
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