Role of CTLA4 in the Proliferation and Survival of Chronic Lymphocytic Leukemia

<div><p>Earlier, we reported that CTLA4 expression is inversely correlated with CD38 expression in chronic lymphocytic leukemia (CLL) cells. However, the specific role of CTLA4 in CLL pathogenesis remains unknown. Therefore, to elucidate the possible role of CTLA4 in CLL pathogenesis, CTLA4 was down-regulated in primary CLL cells. We then evaluated proliferation/survival in these cells using MTT, ³H-thymidine uptake and Annexin-V apoptosis assays. We also measured expression levels of downstream molecules involved in B-cell proliferation/survival signaling including STAT1, NFATC2, c-Fos, c-Myc, and Bcl-2 using microarray, PCR, western blotting analyses, and a stromal cell culture system. CLL cells with CTLA4 down-regulation demonstrated a significant increase in proliferation and survival along with an increased expression of STAT1, STAT1 phosphorylation, NFATC2, c-Fos phosphorylation, c-Myc, Ki-67 and Bcl-2 molecules. In addition, compared to controls, the CTLA4-downregulated CLL cells showed a decreased frequency of apoptosis, which also correlated with increased expression of Bcl-2. Interestingly, CLL cells from lymph node and CLL cells co-cultured on stroma expressed lower levels of CTLA4 and higher levels of c-Fos, c-Myc, and Bcl-2 compared to CLL control cells. These results indicate that microenvironment-controlled-CTLA4 expression mediates proliferation/survival of CLL cells by regulating the expression/activation of STAT1, NFATC2, c-Fos, c-Myc, and/or Bcl-2.</p></div

Microenvironmental influence on the expression of CTLA4 and associated molecules.

<p><b>Panel A:</b> Supervised cluster analyses of expression of CTLA4 and associated molecules among CLL cells from three different <i>in vivo</i> microenvironments such as peripheral blood (PB-CLL, n = 20), bone marrow (BM-CLL, n = 18) and lymph nodes (LN-CLL, n = 15) using microarray analyses. <b>Panel B:</b> Validation of the differentially expressed genes among PB-CLL (n = 12), BM-CLL (n = 12), and LN-CLL (n = 12) cells using real-time PCR analyses. <b>Panel C:</b> Upregulation of CTLA4, c-Myc, and Bcl-2 in CLL cells (n = 6) co-cultured <i>in vitro</i> with stromal cells including OMA-AD and HMEC cells.</p

Upregulation of STAT1/phospho-STAT1, NFATC2, c-Fos/phospho-c-Fos, and c-Myc in CTLA4-downregulated CLL (low CD38/high CTLA4) cells as determined by RT-PCR, real-time PCR, and/or Western blotting.

<p><b>Panels A:</b> Semi-quantitative RT-PCR showing that downregulation of CTLA4 by AS in CLL cells leads to up-regulation of STAT1, NFATC2, c-Fos, and c-Myc in CLL. <b>Panel B:</b> Real-time PCR results from four patient samples showing a significantly higher expression of c-Myc in CTLA4-downregulated CLL cells compared to control CLL cells or CLL cells treated with irrelevant AS. <b>Panel C–D:</b> Western blot results showing up-regulation and quantification of the expression of STAT1 and its phosphorylation, c-Fos and its phosphorylation, NFATC2, and c-Myc at the protein level in CTLA4 down-regulated CLL patient samples (n = 3). *p indicates the statistical difference between control CLL cells and CTLA4-downregulated CLL cells. β-Actin was used as a control.</p