Miguel Aguilar-Santelises

Bcl-2, Bax and p53 expression in B-CLL in relation to in vitro survival and clinical progression.

Our previous data have shown that isolated leukemic cells from progressive chronic lymphocytic leukemia (B‐CLL) patients respond to growth stimulation in vitro and express high levels of p53, immunoreactive with the configuration‐specific antibody PAb 240. We have now analyzed the in vitro survival of B‐CLL cells in relation to Bcl‐2, Baxα and p53 expression and compared this with the clinical progression of the disease. Leukemic cells from patients with progressive disease demonstrated higher in vitro survival, compared with non‐progressive B‐CLL and normal B cells. All cells were sensitive to treatment with a combination of glucocorticoid and cAMP. Bcl‐2 protein levels were not related to clinical progression, as measured by flow cytometry. Competitive PCR showed that Bcl‐2 mRNA was over‐expressed in most of the B‐CLL samples and that p53 mRNA expression was similar between B‐CLL groups and normal values and thus not related to clinical progression. However, since Baxα expression was lower in progressive than in non‐progressive patients, the Bcl‐2/Baxα ratio at the mRNA level was significantly higher in the progressive group. Our data suggest that the Bcl‐2/Baxα ratio is important for the regulation of B‐CLL cell survival, that p53 over‐expression in progressive B‐CLL is the result of post‐transcriptional modifications and that a directed PKA activation may potentiate the cytolytic effect of glucocorticoids in vivo.

Alterations in Bcl-2/Bax protein levels in platelets form part of an ionomycin-induced process that resembles apoptosis.

Platelets are physiologically anucleated cells, derived from megakaryocytes, that undergo vesiculation and transformation into small particles when they are stimulated in vitro by ionomycin and other agents. Electron microscopy images suggest a similarity to apoptosis in cells with nuclei, which ends with cell disintegration and formation of apoptotic bodies. By PCR, we have demonstrated mRNA expression of bcl‐2, baxα and p53 in highly purified non‐stimulated platelets. A side‐scatter shift and a decrease in the Bcl‐2/Bax protein ratio were observed by flow cytometry analysis after stimulation with ionomycin. The ionomycin‐induced modifications were inhibited by the calpain I inhibitor calpeptin and, less effectively, by VAD‐cmk, a broad‐spectrum caspase inhibitor. However, caspase 3‐like activity was very low, with only a twofold increase after ionomycin stimulation, as measured by the cleavage of the fluorogenic peptide substrate DEVD‐AMC. Our data indicate that platelets may constitute a natural model for the analysis of cytoplasmic events in apoptosis.

Organotin compounds decrease in vitro survival, proliferation and differentiation of normal human B lymphocytes

Organotin compounds (OTC) are organometallic compounds with vast industrial and agriculture applications that give rise to ubiquitous environmental contamination. OTC are immunotoxic, but most studies have been performed in rodents and almost exclusively focused on T cell immunity. Humans can be exposed to OTC by inhalation, absorption, and consumption of contaminated food and water. To analyse the effects of OTC in human immune tissue, we isolated B cells from tonsils and exposed them to five OTC at various concentrations, during in vitro culture. Non-stimulated B cells were killed by 100 nM of all tested OTC after 8 h in vitro culture, under sub-optimal conditions, except TET. OTC also decreased the proliferation of tonsillar B lymphocytes stimulated with Staphylococcus aureus Cowan 1 (SAC) and IL-2, when present at 100 nM and higher concentrations. IgM secretion was reduced in stimulated cell cultures exposed to 100 nM dibutyltin chloride (DBT). Accordingly, increased phosphatidylserine exposure demonstrated that 100 nM TPT and DBT induced B cells to die by apoptosis. These data indicate that human B cells are diminished in their capacity to survive, proliferate and differentiate in the presence of OTC in vitro.

Serum levels of helper factors (IL-1α, IL-1β and IL-6), T-cell products (sCD4 and sCD8), sIL-2R and β2-microglobulin in patients with B-CLL and benign B lymphocytosis

Abstract Chronic B-lymphocytic leukemia (B-CLL) cells may be regulated by immune functions. In an attempt to analyze such functions, helper factors (IL-1α, IL-1β and IL-6), T-cell products (sCD4 and sCD8) and sIL-2R and β 2 -microglobulin were measured in serum of patients at different stages of the disease. Patients were classified as having monoclonal lymphocytosis of undetermined significance (MLUS), stable or progressive B-CLL respectively. A significant, but modest, increase of IL-1α was found in B-CLL as well as in MLUS patients whereas IL-6 levels were increased in MLUS only. sCD8 levels were increased both in MLUS and B-CLL but augmented sCD4 concentrations were found statistically significant only in progressive B-CLL. β 2 -microglobulin and sIL-2R were related to the extent of the monoclonal B-cell fraction. The data indicate an increased T-suppressor activity in both MLUS and B-CLL patients and a selective increase of helper T-cell activity in progressive B-CLL. A possible immunoregulatory influence of helper T cells on disease progression is discussed.

BCL-2 delay apoptosis and PARP cleavage induced by NO donors in GT1-7 cells

BCL-2 is a negative regulator of cell death in several systems. Here we report that bcl-2 expression protects against apoptosis induced by nitric oxide (NO) donors in GT1-7 hypothalamic cells. BCL-2 significantly inhibited neuronal death caused by 200 mM S-nitrosocysteine (SNOC), 200 mM S-nitroso-N-acetyl-penicillamine (SNAP), or 1 mM 3-morpholinosydnonimine (SIN-1). To explore further the protective mechanism(s) elicited by bcl-2 expression, we investigated whether BCL-2 could prevent NO-induced cleavage of poly- ADP-ribose-polymerase (PARP), which is a substrate for interleukin-1b converting enzyme (ICE)-like proteases in apoptosis. Formation of 85 and 25 kDa PARP fragments elicited by NO donors was inhibited in cells over-expressing bcl-2.

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.

Inhibitors of arachidonic acid metabolism reduce DNA and nuclear fragmentation induced by TNF plus cycloheximide in U937 cells.

U937 human myeloid leukemia cells are induced to apoptosis by tumour necrosis factor (TNF) plus cycloheximide (CHX). We have analysed the effect of various inhibitors of the arachidonic acid (AA) metabolism on several features of this process. The formation of high molecular weight and oligonucleosomal DNA fragments as well as nuclear fragmentation were reduced by inhibitors of 5-lipoxygenase (BWA4C and BWB70C), 5-LO activating protein (MK-886), and cytosolic PLA2 (AACOCF3). None of these agents blocked the morphological changes detected by microscopy or flow cytometry, phosphatidylserine exposure on the cell surface or Caspase 3-like activation. AA also induced nuclear fragmentation at a concentration of 1 – 20 μM. However, the mechanisms by which these inhibitors act, remain unexplained since there was no 5-LO expression in the U937 cells and no AA release followed their stimulation with TNF plus CHX.

Higher T-cell imbalance and growth factor receptor expression in B-cell chronic lymphocytic leukemia (B-CLL) as compared to monoclonal B-cell lymphocytosis of undetermined significance (B-MLUS)

The surface marker phenotype of lymphocytes derived from 12 patients with B-CLL was compared to that of lymphocytes from 10 patients with an other monoclonal but clinical benign form of B-cell proliferative disorder termed monoclonal B-cell lymphocytosis of undetermined significance (B-MLUS). A panel of well characterized monoclonal antibodies was used for the surface marker determinations. The mean total number of B cells (CD20) was 8.5 x 10(9)/1 in B-MLUS as compared to 44 x 10(9)/1 in B-CLL (p less than 0.001). B-CLL had a greater imbalance in T-cell subpopulations than B-MLUS and healthy controls. Total numbers of CD3+, CD8+ cells as well as cells expressing the NK-related antigens (CD16, Leu-7) and IL-2 receptor (CD25) bearing lymphocytes were statistically significant higher in B-CLL than in B-MLUS. Analyses of B-cell enriched populations showed that B-CLL represented B cells of an early maturation stage, whereas B cells from B-MLUS were more mature as judged by the loss of the CD21 surface marker. A larger fraction of B cells in B-CLL compared to B-MLUS exhibited a higher activation stage as revealed by the expression of the CD21, CD25 and CD35 structures as well as the FMC7 antigen.

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.

Low IL-1 beta production in leukemic cells from progressive B cell chronic leukemia (B-CLL).

In vitro production of IL-1 beta by cells from 32 patients with benign monoclonal lymphocytosis of undetermined significance (MLUS) and B cell chronic lymphocytic leukemia (B-CLL) was investigated. Normal B lymphocytes (2 x 10(6)) secreted approximately 5 ng/ml of IL-1 beta during 24 h and approximately ten times more after stimulation with Staphylococcus aureus, strain Cowan 1 (SAC). When patients were studied, a loss of IL-1 beta production was found in leukemic cells from progressive disease. Cells from MLUS patients secreted near normal levels of IL-1 beta and responded to SAC stimulation, whereas cells from patients with progressive B-CLL produced no, or little IL-1 beta, and did not respond to SAC. Loss of IL-1 beta production in progressively growing B-CLL may be related to an increased malignant character of these cells. This is discussed in relation to the immunogenicity of the leukemic cells and their capacity to differentiate.