Genomic changes elicited in gemcitabine-treated renal cancer cells associated with increased sensitivity to cytolysis by IL-2 stimulated human peripheral blood lymphocytes

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Previously, we reported that in vitro pretreatment of the human renal cell cancer (RCC) cell line, 769P, with sublethal doses of gemcitabine (GZ) under clinically achievable conditions, significantly increased tumor sensitivity to lysis by IL2-stimulated human peripheral blood lymphocytes (PBL). IL2 was chosen for this investigation as the currently most active agent for the treatment of human RCC. Its’ clinical activity is thought to result from induction and upregulation of cytolytic activity with broad tumor specificity in lymphocytes from both NK and T cell populations. The present study utilized genomic and phenotypic analysis to investigate changes induced by GZ in RCC cells in an attempt to understand the mechanism(s) responsible for improved tumor sensitivity to lysis. Expression of selected genes known to modulate tumor recognition, adhesion, and lytic sensitivity was assessed in media and GZ-treated 769P cells using the Agilent genomic expression microarray platform. The cells employed for these analyses were found to exhibit cytolytic sensitivity to IL2 activated PBL of 1032 and 1710 lytic units for media-treated and GZ-treated cells respectively. With respect to tumor-associated recognition molecules, GZ treatment (20 uM for 24 hours) increased mRNA expression for the Major Histocompatibility Class I-related molecule, MR-1 by 7 fold and expression of HLA-A, B, E and F by 1.5-3 fold compared to cells cultured under equivalent conditions in media alone. Similarly, expression of mRNA for the adhesion molecules, ICAM-1, Necl-2 and MCAM was upregulated in GZ-treated cells by 6.5, 2 and 2 fold respectively. GZ also doubled the expression of mRNA for the death receptors, FAS and TRAIL-receptor 2. Increased expression of the death receptor protein FAS by > 2 fold on GZ-treated RCC cells at 24 hours was confirmed by flow cytometry. However, flow cytometry failed to show quantitative changes in expression of HLA-A, B, and C molecules on GZ-treated cells. Results of this study indicate that treatment of RCC cells with sublethal concentrations of GZ capable of significantly increasing tumor lytic sensitivity to IL2-activated PBL, is associated with changes in the expression of mRNA for molecules that mediate tumor recognition, adhesion, and apoptotic signaling. It appears that the fidelity of mRNA translation for the death receptor, FAS, is maintained in GZ-treated cells whereas this is not the case with mRNA for MHC Class I molecules, known to provide inhibitory signals to NK cells. Whether this reflects post-transcriptional or post-translational events is currently under investigation. Nevertheless, the failure to increase expression of Class I molecules on GZ-treated cells which express increased amounts of FAS may be a key mechanism whereby GZ pretreatment improves the sensitivity of RCC cells to IL2-activated human PBL.