Lyda M. Osorio

Primary HIV-1 infection sets the stage for important B lymphocyte dysfunctions

Objectives:To investigate the effects of primary HIV-1 infection (PHI) and of two antiretroviral therapies [highly active antiretroviral therapy (HAART) or reverse transcriptase inhibitors (RTI)] on activation, differentiation and survival of B cells. Methods:Naive and memory B cells from three groups [PHI (31), chronic infection (26) and healthy donors (12)] were studied for surface expression of Fas, LAIR-1, CD70, intracellular expression of Bcl-2 and spontaneous apoptosis. Fluorescence activated cell sorting (IgD+IgM+CD19+CD27+) and short-term cell culture to analyse induction of CD25 on B cells were performed in five patients with PHI. Patients with PHI were sampled at baseline, and after 1 and 6 months of therapy. Results were analysed by parametric and non-parametric tests and by mathematical modelling. Results:In PHI, B cells were significantly decreased; naive and memory B lymphocytes showed a high degree of activation, manifested by hypergammaglobulinaemia, altered expression of Fas and LAIR-1, and high rate of spontaneous apoptosis. Antiretroviral treatment improved the activation/differentiation status of B cells, reduced apoptosis to levels comparable to those in healthy individuals and restored the ability of B cells to respond to T cell-dependent activation. B cells showed slightly better recovery in patients taking HAART than in those taking RTI. Decreased IgM-positive memory B cells and lower induction of CD25 expression on B cells upon T cell activation at diagnosis of PHI was shown in five patients tested. These parameters normalized after 6 months of therapy. Conclusion:B cell dysfunctions found in chronic HIV-1 infection appear during PHI and initiation of antiretroviral therapy early during infection may help to preserve the B cell compartment.

Sensitization to TRAIL-induced apoptosis and modulation of FLICE-inhibitory protein in B chronic lymphocytic leukemia by actinomycin D

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent activator of the cell death pathway and exerts tumoricidal activity in vivo with minimal toxicity. In order to investigate the therapeutic potential of TRAIL in B chronic lymphocytic leukemia (B-CLL) we have analyzed the expression of TRAIL receptors (TRAIL-Rs) in leukemic cells from B-CLL patients and their in vitro sensitivity to apoptosis induced by recombinant human TRAIL. We have found TRAIL-R1 and -R2 death receptor, and TRAIL-R3 and -R4 decoy receptor mRNA expression in most of the 57 B-CLL patients studied (R1 82%, R2 100%, R3 96% and R4 82%). TRAIL-R1 and R2 proteins were expressed on the surface and within the cells, whereas R3 and R4 decoy receptors were almost exclusively expressed in the cytoplasm. Despite TRAIL death receptor expression, B-CLL cells were relatively resistant to induction of apoptosis by recombinant human TRAIL (300 ng/ml). However, the susceptibility to TRAIL-induced apoptosis was increased by treatment of B-CLL cells with actinomycin D (Act D). Western blot analysis showed higher constitutive expression of the long form of FLICE-inhibitory protein (FLIPL) in B-CLL as compared to normal tonsillar B cells. Act D treatment down-regulated both long and short FLIP expression, which was correlated with the increase in B-CLL sensitivity to TRAIL. Although the surface TRAIL death receptor expression was up-regulated both by cell culture and by Act D treatment, the changes were not correlated with a gain in susceptibility to TRAIL. In addition, neither decoy receptors nor Bcl-2 expression were affected by Act D. Our findings suggest the possible involvement of FLIP in regulating TRAIL-mediated apoptosis in B-CLL.

High expression of Bfl‐1 contributes to the apoptosis resistant phenotype in B‐cell chronic lymphocytic leukemia

In order to identify regulatory genes involved in the development of an apoptosis‐resistant phenotype in patients with chemotherapy refractory B‐cell chronic lymphocytic leukemia (B‐CLL) expression of apoptosis‐regulating genes in B‐CLL cells was quantified using cDNA arrays and RT‐PCR. Data were obtained from and compared between 2 groups of B‐CLL patients with either nonprogressive, indolent, previously untreated disease and with leukemic cells sensitive to in vitro fludarabine‐induced apoptosis, referred to as sensitive B‐CLL (sB‐CLL) or with progressive, chemotherapy refractory disease and with leukemic cells resistant to in vitro fludarabine‐induced apoptosis, referred to as resistant B‐CLL (rB‐CLL). By performing a supervised clustering of genes that most strongly discriminated between rB‐CLL vs. sB‐CLL a small group of genes was identified, where bfl‐1 was the strongest discriminating gene (p < 0.05), with higher expression in rB‐CLL. A group of apoptosis‐regulating genes were modulated during induction of apoptosis by serum deprivation in vitro in a similar manner in all cases studied. However, bfl‐1 was preferentially downregulated in sB‐CLL as compared to rB‐CLL (p < 0.05). We conclude that bfl‐1 may be an important regulator of B‐CLL apoptosis, which could contribute to disease progression and resistance to chemotherapy, and as such represent a future potential therapeutic target.

Upregulation of bfl-1 is a potential mechanism of chemoresistance in B-cell chronic lymphocytic leukaemia

B-cell chronic lymphocytic leukaemia (B-CLL) is characterised by the progressive accumulation of monoclonal CD5+ B cells. In a previous study, we have analysed the expression profile of apoptosis-regulating genes using a cDNA-based microarray and found overexpression of the antiapoptotic bcl-2 family member, bfl-1, in B-CLL cells with an apoptosis-resistant phenotype. In this study, bfl-1 mRNA levels have been determined by competitive PCR in an extended population of B-CLL patients to characterise its role in disease progression and development of chemoresistance. bfl-1 levels were significantly higher in patients with no response (NR) to last chemotherapy than in patients responding (partial response (PR)) to last chemotherapy (P<0.05) and in patients who had not required treatment (P<0.05). We found no correlation between bfl-1 mRNA levels and disease progression, IGHV mutational status or other clinical parameters. In addition, bfl-1 mRNA levels were inversely correlated with apoptotic response to in vitro fludarabine treatment of B-CLL cells. Specific downregulation of bfl-1 using siRNA induced apoptosis in resistant cells. Our data suggest that bfl-1 contributes to chemoresistance and might be a therapeutic target in B-CLL.

Expression and transcriptional regulation of functionally distinct Bmf isoforms in B-chronic lymphocytic leukemia cells

Bmf is a BH3-only Bcl-2 family member that is normally sequestered to myosin V motors by binding to the dynein light chain 2 (DLC2). Certain damage signals release Bmf, which then binds prosurvival Bcl-2 proteins and triggers apoptosis. Here, two novel isoforms of human Bmf, Bmf-II and Bmf-III, were identified and cloned from cDNA derived from B-chronic lymphocytic leukemia (B-CLL) cells. Bmf-II and Bmf-III were characterized as two splice variants, lacking the BH3 domain but retaining the DLC2 binding domain. Bmf (here called Bmf-I) expression in HeLa cells induced apoptosis and reduced colony formation in contrast to Bmf-II and Bmf-III, which had no effect on apoptosis and instead increased colony formation. While bmf-I mRNA was expressed in many cell types, expression was higher in B lymphoid cells and bmf-II and bmf-III were mainly detected in B-CLL and normal B cells. bmf-I mRNA was upregulated in normal and leukemic B cells, while bmf-III mRNA was downregulated only in B-CLL cells by serum deprivation. We show that Bmf is regulated by transcriptional activation and alternative splicing and conclude that the relative levels of Bmf isoforms may have a role in regulating growth and survival in B cells and leukemic B-CLL cells.

c‐Myc‐dependent etoposide‐induced apoptosis involves activation of Bax and caspases, and PKCdelta signaling

The c‐Myc transcription factor is a key regulator of cell proliferation, differentiation, and apoptosis. While deregulation of myc induces programmed cell death, defects in the apoptotic program facilitate Myc‐driven tumor development. We have treated c‐Myc inducible mouse cells and rat fibroblasts with different c‐myc status with cytotoxic drugs to explore the effect of c‐Myc on drug‐induced apoptosis. We found that c‐Myc overexpression potentiated etoposide‐, doxorubicin‐, and cisplatin‐induced cell death in mouse fibroblasts. In addition, these drugs provoked a strong apoptotic response in c‐Myc‐expressing cells, but a weak apoptosis in c‐myc null Rat1 cells. In contrast, staurosporine‐induced apoptosis was c‐Myc‐independent, confirming a functional apoptotic pathway in c‐myc null cells. Apoptosis was paralleled by c‐Myc‐dependent Bax‐activation after etoposide and doxorubicin treatment, but not after cisplatin administration. All three drugs induced higher caspase activation in c‐Myc expressing cells than in c‐myc null cells. Furthermore, etoposide treatment of c‐Myc expressing cells resulted in PKCδ cleavage, while inhibition of PKCδ reduced etoposide‐induced apoptosis and prevented Bax activation. Taken together, these findings suggest that Bax and caspase activation, together with PKCδ signaling are involved in c‐Myc‐dependent etoposide‐induced apoptosis.

Lipoprotein lipase is differentially expressed in prognostic subsets of chronic lymphocytic leukemia but displays invariably low catalytical activity

Lipoprotein lipase (LPL) expression has been shown to correlate with IGHV mutational status and to predict outcome in chronic lymphocytic leukemia (CLL). We here investigated the prognostic impact of LPL expression in relation to other prognostic markers including IGHV3-21 usage in 140 CLL patients. Additionally, we studied the catalytic activity of LPL in CLL cells. A significant difference in LPL mRNA expression was detected in IGHV unmutated compared to mutated CLL patients (p<0.001). However, the poor-prognostic mutated/stereotyped IGHV3-21 patients did not differ from other mutated CLL cases. Clinical outcome was significantly different in CLL cases with high versus low LPL expression (p<0.001), and LPL expression exceeded mutation status/IGHV3-21 usage as an independent prognostic marker. Finally, LPL protein expression correlated significantly with mRNA expression and was higher in IGHV unmutated versus mutated CLL (p=0.018), although the majority of synthesized protein was catalytically inactive indicating a non-catalytical function in CLL.

Increased serum levels of soluble Fas in progressive B-CLL.

Abstract: Clinical progression of B‐cell chronic lymphocytic leukemia (B‐CLL) depends on survival and accumulation of leukemic cells, regulated in part by physical cell contact and soluble molecules. Here we have studied the Fas/FasL system in relation to clinical progression in B‐CLL. Serum levels of soluble Fas (sFas) and FasL (sFasL) were determined by ELISA in 43 progressive and 40 non‐progressive B‐CLL patients and in 21 control individuals. Correlation between sFas serum levels and clinical progression, stage and survival were statistically analyzed. We found high levels of sFas in B‐CLL sera correlated with disease progression (p<0.01). In addition, higher sFas levels were found in patients in stages II, III and IV in comparison to patients in stage 0 (p<0.05, p<0.01, p<0.03, respectively). Survival was significantly shorter for patients with 6 ng/ml sFas serum levels, although a multivariate analysis did not show sFas to be a significant independent prognostic factor. Fresh B‐CLL cells showed only low levels of membrane expression, which were not correlated to sFas levels in serum. In vitro activation of B‐CLL cells increased Fas expression, as reported earlier, and induced cells to release sFas into the supernatant. In conclusion, our results indicate that sFas in serum may be a useful parameter for the prediction of clinical progression in B‐CLL.

Cytokine expression in B-CLL in relation to disease progression andin vitro activation

Earlier, we reported an association between lowin vitro andin vivo IL-1 and IL-6 production, decreased IL-1β and IL-10 mRNA expression and B cell chronic lymphocytic leukemia (B-CLL) disease progression. We have now further investigated cytokine mRNA transcription in B-CLL cells and cytokine serum levels in B-CLL patients. Reverse transcriptase polymerase chain reaction (RT-PCR) amplification of tumor necrosis factor (TNFα), IFNγ, IL-6 and BCGF was equally often seen in non-progressive and progressive patients. However, 4 out of 23 non-progressive cases expressed mRNA for IL-12 while no IL-12 expression was seen in 15 progressive patients. No IL-12 was found in sera or supernatants fromin vitro stimulated B-CLL cells, whereas TNFα and IL-10 were detected in sera from 51 and 31 of 65 B-CLL patients, respectively. TNFα values were significantly high in sera from patients in stages III and IV with disease progression. TNFα and IL-10 were also detected in culture supernatants fromin vitro stimulated B-CLL cells, whereas IFNγ was undetectable in these cultures and rarely positive in serum. Although further investigations are required, our data and that from previous reports indicate that B-CLL-derived cytokines are involved in B-CLL disease progression.

The involvement of epithelial Fas in a human model of graft versus host disease.

Background. Graft-versus-host disease (GVHD) is an important complication occurring after hematopoietic stem-cell transplantation (HSCT). Animal model studies have shown the involvement of the Fas (APO-1/CD95)/Fas-Ligand pathway in GVHD pathogenesis, but its association with cutaneous GVHD in human remains to be established. Methods. In the present study, Fas involvement in skin damage was assessed using a human skin explant model of GVHD. Fas and FasL expression were measured by immunohistochemistry and blockade of Fas pathway was investigated using an antagonistic anti-human Fas monoclonal antibody. In addition, levels of soluble Fas (sFas) were determined in the serum of patients receiving allogeneic HSCT with and without GVHD. Results. The results showed that Fas up-regulation in the epithelium of human skin explants correlated with graft-versus-host reaction (GVHR) in the skin explant model (P<0.001). Decreased GVHR grades were observed by using a Fas blocking monoclonal antibody. Levels of sFas were increased post-HSCT (P<0.001) but rather than being associated with the severity of GVHD, sFas levels differed with the conditioning treatments the patients received before the HSCT. Conclusions. Higher GVHR grades were associated with increased Fas expression in the epithelium of the skin explants. In addition, by blocking Fas-mediated apoptosis, the GVHR grades were decreased. Our study thus shows the involvement of Fas in cutaneous GVHD damage, and supports the potential use of Fas as a therapeutic target.