calpain and Leukemia--Lymphocytic--Chronic--B-Cell

Topics: Calcium; Calpain; Cortactin; Focal Adhesion Protein-Tyrosine Kinases; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Phosphorylation; Receptors, Antigen, B-Cell

B-cell Chronic Lymphocytic Leukemia (B-CLL) is the most common hematological disorder among middle-aged/elderly people in the Western countries. We have shown earlier that B-CLL cells exhibit elevated total amount and available activity of µ-calpain, belonging to a family of ubiquitous, strongly Ca-dependent proteases, involved in the control of proliferation and apoptosis. In this study we attempted to estimate a potential clinical value of μ-calpain in relation to B-CLL clinical staging in patients with extremely high lymphocytosis and studied the molecular mechanisms associating calpain activity with clinical progress of the disease. We observed significant correlations between the amounts of intracellular μ-calpain and clinical staging of the disease, with RAI stage 1 corresponding to the highest calpain amounts in the leukemic cells. There was also a positive, statistically significant correlation between the amount of μ-calpain and phosphorylated (p)ZAP-70 in B-CLL lymphocytes. Calpain activity in the B-CLL cells is associated with decreased activities of pro-apoptotic caspases -3 and -9, and reciprocally with an increased amount of anti-apoptotic Bcl-2. Together, all of these findings make calpain activity in B-CLL cells a promising target modifying the properties of these cells and facilitating therapy. Finally, the proportion of CD19+ B cells with elevated μ-calpain and pZap-70 was markedly reduced in patients after successful therapy.

Topics: Aged; Aged, 80 and over; Apoptosis; B-Lymphocytes; Calpain; Case-Control Studies; Caspase 3; Caspase 9; Cells, Cultured; Disease Progression; Female; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Male; Middle Aged; Neoplasm Staging; Oligopeptides; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; ZAP-70 Protein-Tyrosine Kinase

Evading apoptosis is a hallmark of B-cell chronic lymphocytic leukemia (CLL) cells and an obstacle to current chemotherapeutic approaches. Inhibiting histone deacetylase (HDAC) has emerged as a promising strategy to induce cell death in malignant cells. We have previously reported that the HDAC inhibitor MGCD0103 induces CLL cell death by activating the intrinsic pathway of apoptosis. Here, we show that MGCD0103 decreases the autophagic flux in primary CLL cells. Activation of the PI3K/AKT/mTOR pathway, together with the activation of caspases, and to a minor extent CAPN1, resulting in cleavage of autophagy components, were involved in MGCD0103-mediated inhibition of autophagy. In addition, MGCD0103 directly modulated the expression of critical autophagy genes at the transcriptional level that may contribute to autophagy impairment. Besides, we demonstrate that autophagy is a pro-survival mechanism in CLL whose disruption potentiates cell death induced by anticancer molecules including HDAC and cyclin-dependent kinase inhibitors. In particular, our data highlight the therapeutic potential of MGCD0103 as not only an inducer of apoptosis but also an autophagy suppressor in both combination regimens with molecules like flavopiridol, known to induce protective autophagy in CLL cells, or as an alternative to circumvent undesired immunomodulatory effects seen in the clinic with conventional autophagy inhibitors.

Topics: Aged; Aged, 80 and over; Autophagy; Benzamides; Calpain; Cell Survival; Female; Flavonoids; Histone Deacetylase Inhibitors; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Male; Middle Aged; Phosphatidylinositol 3-Kinases; Piperidines; Proto-Oncogene Proteins c-akt; Pyrimidines; TOR Serine-Threonine Kinases; Transcription, Genetic

Gossypol, a cottonseed extract derivative, acts as a BH3-mimetic, binding to the BH3 pocket of antiapoptotic proteins and displacing pro-death partners to induce apoptosis. However, knowledge on the molecular underpinnings of its downstream effects is limited. Since chronic lymphocytic leukemia (CLL) cells express high levels of antiapoptotic proteins that act as a survival mechanism for these replicationally quiescent lymphocytes, we investigated whether gossypol induces apoptosis in these cells and what mechanism underlies gossypol-mediated cytotoxicity. Gossypol induced cell death in a concentration- and time-dependent manner; 24-hour incubation with 30 microM gossypol resulted in 50% cell death (median; range, 10%-80%; n = 47) that was not abrogated by pan-specific caspase inhibitor. Starting at 4 hours, the mitochondrial outer membrane was significantly permeabilized (median, 77%; range, 54%-93%; n = 15). Mitochondrial outer membrane permeabiliztaion (MOMP) was concurrent with increased production of reactive oxygen species (ROS); however, antioxidants did not abrogate gossypol-induced cell death. Mitochondrial membrane permeabilization was also associated with loss of intracellular adenosine triphosphate (ATP), activation of BAX, and release of cytochrome c and apoptosis-inducing factor (AIF), which was translocated to the nucleus. Blocking AIF translocation resulted in a decreased apoptosis, suggesting that AIF contributes to gossypol-mediated cytotoxicity in CLL lymphocytes.

Topics: Acetylcysteine; Adenosine Triphosphate; Amino Acid Chloromethyl Ketones; Antioxidants; Apoptosis; Apoptosis Inducing Factor; bcl-2-Associated X Protein; BH3 Interacting Domain Death Agonist Protein; Biological Transport, Active; Calpain; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; Dipeptides; Gossypol; Humans; In Vitro Techniques; Leukemia, Lymphocytic, Chronic, B-Cell; Mitochondrial Membranes; Molecular Mimicry; Reactive Oxygen Species; Superoxides

Studies to investigate signal transduction pathways that support viability and prevent apoptosis of chronic lymphocytic leukemia cells (CLL) were initiated as a result of microarray cDNA analyses which revealed expression of genes whose products regulate cell cycle progression. Immunoblots revealed translation of several genes including caspases, cyclin D1, and the PI3-kinase dependent, survival kinase, Akt. Akt was found to be activated. Inhibition of PI3-kinase with specific inhibitor, LY294002, led to the induction of apoptosis that was caspase 8 dependent, but independent of Akt as LY294002 did not depress a high basal level of Akt activity found in CLL cells. Phosphorylation of Akt was maintained, enzymatic activity undiminished, and phosphorylation of substrates sustained. Caspases, however were activated, PARP cleaved and DNA fragmented. Caspase inhibitors revealed that initiator caspase 8 was required for classic apoptosis when PI3-kinase was inhibited, and specific activity assays demonstrated its early activation. GSK-3beta a kinase regulated via PI3-kinase dependent, down-stream kinases, was responsible for regulating cyclin D1 levels in CLL cells, but neither GSK-3beta nor calpain was responsible for induction of apoptosis, or activation of executioner caspase 3, following LY294002 treatment. PI3-kinase mediated protection against caspase activation in CLL B-cells therefore is not mediated through classic Akt survival pathways. The data further support the hypothesis that signal transducing, membrane associated receptors triggered by extrinsic factors, maintain CLL leukemic B-cell survival in vivo by preventing caspase activation.

Topics: Apoptosis; Calpain; Caspase 8; Caspase Inhibitors; Caspases; Cell Cycle; Cell Survival; Chromones; Cyclin D1; Enzyme Activation; Enzyme Inhibitors; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Morpholines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Signal Transduction; Tumor Cells, Cultured

Decreased susceptibility to apoptosis and impaired proliferative control are thought to be responsible for prolonged life span and accumulation of chronic lymphocytic leukemia (B-CLL) cells. The activity of calpains (calcium-dependent, neutral proteases, active in the cells responding to signals inducing a rise of cytoplasmic Ca(++)) is involved in the regulation of apoptosis of some cell types by interaction with caspase-3. This work verifies the hypothesis of the abnormal activity of calpains and its role in reduced apoptosis of the B-CLL cells. Casein zymography, reverse transcriptase-polymerase chain reaction, and Western blotting were used for identification and quantification of the activity and expression of calpains in B-CLL cells and purified normal B lymphocytes. The activity and expression of mu-calpain (requiring micromolar Ca(++) for activation) are significantly higher in the leukemic than in nonmalignant cells. Contrarily, the activity and expression of m-calpain (requiring millimolar Ca(++)) as well as the expression of calpastatin (an endogenous inhibitor of calpains) are unchanged or reduced in the B-CLL lymphocytes. Correspondingly, the activity of caspase-3 is many times lower in the B-CLL cells than in normal B lymphocytes. Inhibition of overexpressed mu-calpain in living B-CLL cells in vitro results in doubling of the proportion of the cells undergoing spontaneous apoptosis. This observation suggests a possible role for calpains in longer survival of the B-CLL cells and may open new therapeutic possibilities.

Topics: Adult; Aged; Aged, 80 and over; Apoptosis; Calcium; Calpain; CD3 Complex; Enzyme Activation; Female; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Male; Middle Aged; Signal Transduction