Synergistic Activity of Deguelin and Fludarabine in Cells from Chronic Lymphocytic Leukemia Patients and in the New Zealand Black Murine Model.

B-cell chronic lymphocytic leukemia (CLL) remains an incurable disease, and despite the improvement achieved by therapeutic regimes developed over the last years still a subset of patients face a rather poor prognosis and will eventually relapse and become refractory to therapy. The natural rotenoid deguelin has been shown to induce apoptosis in several cancer cells and cell lines, including primary human CLL cells, and to act as a chemopreventive agent in animal models of induced carcinogenesis. In this work, we show that deguelin induces apoptosis in vitro in primary human CLL cells and in CLL-like cells from the New Zealand Black (NZB) mouse strain. In both of them, deguelin dowregulates AKT, NFκB and several downstream antiapoptotic proteins (XIAP, cIAP, BCL2, BCL-XL and survivin), activating the mitochondrial pathway of apoptosis. Moreover, deguelin inhibits stromal cell-mediated c-Myc upregulation and resistance to fludarabine, increasing fludarabine induced DNA damage. We further show that deguelin has activity in vivo against NZB CLL-like cells in an experimental model of CLL in young NZB mice transplanted with spleen cells from aged NZB mice with lymphoproliferation. Moreover, the combination of deguelin and fludarabine in this model prolonged the survival of transplanted mice at doses of both compounds that were ineffective when administered individually. These results suggest deguelin could have potential for the treatment of human CLL.

Expression of human leukocyte antigen-G in systemic lupus erythematosus.

The purpose of this study was to examine the expression of human leukocyte antigen-G (HLA-G) in patients with systemic lupus erythematosus (SLE) and its relation with interleukin-10 (IL-10) production. The study included 50 female SLE patients and 59 healthy female donors. HLA-G expression in peripheral blood and cutaneous biopsies was determined by flow cytometry and immunohistochemistry, respectively. Soluble HLA-G (sHLA-G) and IL-10 were quantified in serum samples by enzyme-linked immunosorbent assay. SLE patients presented with serum sHLA-G and IL-10 levels significantly higher than that observed in controls (median [interquartile range (IQR)] = 43.6 U/ml [23.2-150.2] vs 26.84 U/ml [6.0-45.2], p = 0.004; and 1.4 pg/ml [0-2.3] vs 0 pg/ml [0-1.5], p = 0.01, respectively). But no correlation was observed between sHLA-G and both IL-10 levels and the disease activity index for SLE patients. The expression of membrane HLA-G in peripheral lymphocytes from SLE patients was low, but higher than in controls (median [IQR] = 1.5% [0.6-1.8] and 0.3% [0.2-0.8], respectively; p = 0.02). Finally, these findings were in accordance with the weak expression of HLA-G in skin biopsies. Despite the fact that patients present higher levels of HLA-G than healthy controls, which suggests a possible relevance of this molecule in SLE, it seems not to be related to IL-10 production or disease activity.

Association of the microsatellite in the 3' untranslated region of the CD154 gene with rheumatoid arthritis in females from a Spanish cohort: a case-control study.

CD40-CD154 interaction is an important mediator of inflammation and has been implicated in T helper type 1-mediated autoimmune diseases including rheumatoid arthritis (RA). Linkage studies have shown association of markers in the proximity of the CD154 gene. In the present work we investigated whether specific allele variants of the microsatellite in the 3' UTR of the CD154 gene might modulate the risk of RA. The study, in a case-control setting, included 189 patients and 150 healthy controls from the Canary Islands, Spain. The 24CAs allele was less represented in female patients than in controls (0.444 in controls versus 0.307 in patients, P = 0.006, odds ratio (OR) 0.556, 95% confidence interval (CI) 0.372 to 0.831) but not in males (0.414 versus 0.408), and only when homozygous (P = 0.012; OR 0.35, 95% CI 0.16 to 0.77). We also verified that CD154 association with RA was independent of human leukocyte antigen (HLA) phenotype. A further functional study showed that after stimulation anti-CD3, CD154 mRNA was more stable in CD4+ T lymphocytes from patients with RA bearing the 24CAs allele (mRNA half-life 208 minutes) than in patients without the 24CAs allele (109 minutes, P = 0.009). However, a lower percentage of CD154+CD4+ T lymphocytes was seen in freshly isolated peripheral blood mononuclear cells from patients carrying 24CAs alleles (mean 4.28 versus 8.12; P = 0.033), and also in CD4+ T lymphocytes stimulated with anti-CD3 (median 29.40 versus 47.60; P = 0.025). These results were concordant with the smaller amounts of CD154 mRNA isolated from stimulated T lymphocytes with 24CAs alleles. The CD154 microsatellite therefore seems to affect the expression of the gene in a complex manner that implies not only mRNA stability. These data suggest that the CD154 microsatellite contributes to the regulation of mRNA and protein expression, although further studies will be necessary to elucidate its role in disease predisposition.

CD5 does not regulate the signaling triggered through BCR in B cells from a subset of B-CLL patients.

CD5 is a transmembrane protein expressed on all T lineage cells and a subset of B cells. It is known that CD5 is physically associated with the T-cell receptor and B-cell receptor (BCR), inhibiting the signaling triggered by both of them. CD5 is also characteristic of B-chronic lymphocytic leukemia (B-CLL) B cells, although its implication in the development of this lymphoproliferative disorder has not been studied. In the present study, we examined the effect of CD5 in apoptosis, cell viability and global protein tyrosine phosphorylation mediated by BCR in B cells from B-CLL patients. As opposed to tonsil B cells, we did not observe an increase in the apoptotic or viability signals induced by anti-immunoglobulin M or SAC/interleukin-2 when CD5 was dissociated from BCR in leukemic cells of the majority of patients. We also observed that CD5 did not regulate the BCR-induced phosphotyrosine pattern in B-CLL B cells. These findings suggest that CD5 does not inhibit properly the BCR-mediated signaling in leukemic cells. This defect in inhibiting the BCR might contribute to the enhanced survival of B-CLL B cells.

CD5 provides viability signals to B cells from a subset of B-CLL patients by a mechanism that involves PKC.

B-chronic lymphocytic leukaemia (B-CLL) is a heterogeneous disease characterized by an accumulation of B lymphocytes expressing CD5. To date, the biological significance of this molecule in B-CLL B cells remains to be elucidated. In this study, we have analysed the functional consequences of the binding of an anti-CD5 antibody on B-CLL B cells. To this purpose, we have measured the percentage of viability of B-CLL B cells in the presence or in the absence of anti-CD5 antibodies and also examined some of the biochemical events downstream the CD5-signalling. We demonstrate that anti-CD5 induces phosphorylation of protein tyrosine kinases and protein kinase C (PKC), while no activation of Akt/PKB and MAPKs is detected. This signalling cascade results in viability in a group of patients in which we observe an increase of Mcl-1 levels, whereas the levels of bcl-2, bcl-x(L) and XIAP do not change. We also report that this pathway leads to IL-10 production, an immunoregulatory cytokine that might act as an autocrine growth factor for leukaemic B cells. Inhibition of PKC prevents the induction of Mcl-1 and IL-10, suggesting that the activation of PKC plays an important role in the CD5-mediated survival signals in B cells from a subset of B-CLL patients.

Polymorphism in the CD5 gene promoter in B-cell chronic lymphocytic leukemia and mantle cell lymphoma.

Despite the low incidence of microsatellite instability (MSI) in lymphoid malignant neoplasms, it has been reported that the CD5 promoter MSI was relatively frequent among B-cell chronic lymphoproliferative disorders. We studied the presence of MSI in the CD5 promoter in 134 cases of B-cell chronic lymphocytic leukemia (B-CLL) and 47 of mantle cell lymphoma (MCL) by comparing the pattern of microsatellite repeats on autologous germline and tumor DNA samples. Microsatellite alterations were not observed in any case. However, the allele distribution of this polymorphism showed a higher frequency of the 18 CA allele (0.585) in MCL cases (P = .026; odds ratio [OR], 1.75; 95% confidence interval [CI], 1.07-2.87) and of the 19 CA allele (0.179) in B-CLL cases (P = .005; OR, 2.26; 95% CI, 1.27-4.01) compared with control cases (0.442 and 0.087, respectively). This suggests that although MSI seems not to be involved in the pathogenesis of these 2 lymphoid malignant neoplasms, the polymorphic CD5 promoter is associated with increased susceptibility to these disorders.