Genome-wide analysis of impact of culture conditions on gene expression of primary leukemic cells from chronic lymphocytic leukemia (CLL)

Proliferation and survival of CLL cells is highly dependent on the interaction of the tumor cells with different populations of accessory cells, incl. mesenchymal bone marrow stromal cells. In this project interactions between CLL cells and transformed bone-marrow derived fibroblast cell lines were studied. Gene expression was analyzed in CLL cells that were cocultured for different time periods with human (HS-5) or murine (M2-10B4) transformed fibroblasts, as compared to CLL cells from the same patient sample that were cultured in DMEM medium or conditioned medium in which HS-5 cells had been growing. Total RNA obtained from primary CLL cells, right after CD19 sorting (control) or after being cultured for 30 min, 1 h, 2, h, 3 h, 4 h, 6 h, 8h, 24 h or 48 h in DMEM, in conditioned medium from HS-5 cells, or co-cultured with HS-5 or M2-10B4 cells. In addition, total RNA was extracted from CLL cells that were cultured in DMEM (control) or cocultured with HS-5 or M2-10B4 cells for 24 h, then washed and cultured in DMEM for further 24 h. Total RNA was also also extracted for reference from M2-10B4 cells, as well as from HS-5 cells that were cultured alone, or after they had been cocultured with patient CLL cells for 6 or 24 h.