Expression and function of prostate-apoptosis-response-gene-4 in lymphatic cells.

Inhibition of apoptosis contributes to the pathogenesis of lymphatic malignancies. In particular, the elevated expression of Bcl-2 is considered to be a marker of poor prognosis, since increased levels of Bcl-2 confer longevity as well as chemoresistance. After demonstrating an inverse expressional pattern of Bcl-2 and prostate-apoptosis-response-4 (Par-4) in ex vivo cells of patients suffering from acute lymphatic leukemia (ALL) as well as a deregulated expression of Par-4 in acute and chronic lymphatic neoplasias, the molecular mechanisms underlying these results were investigated. Thus, it was demonstrated that in neoplastic lymphatic cells Par-4 exerts a proapoptotic role augmenting chemosensitivity by down-regulating Bcl-2, promoting disruption of mitochondrial membrane potential and enforcing caspase-activation. Moreover, Par-4 enables cells to circumvent inhibition of the central executioner caspase-3 by alternative activation of caspases following a decrease in expression levels of inhibitors of apoptosis proteins (IAP)

Synergistic effects of chemotherapeutic drugs in lymphoma cells are associated with down-regulation of inhibitor of apoptosis proteins (IAPs), prostate-apoptosis-response-gene 4 (Par-4), death-associated protein (Daxx) and with enforced caspase activation.

Cytotoxic drugs mediate apoptotic tumor cell death by influencing key regulator proteins of programmed cell death. In clinical practice cytotoxic drug combinations are desired to potentiate tumor cell kill and to minimize side effects. Nevertheless, the molecular mechanisms underlying synergistic and antagonistic effects on tumor cells are still poorly understood. In order to elucidate these molecular mechanisms we established models of synergistic and antagonistic drug combinations within the same lymphoma cell lines. By combination index method we demonstrated that bendamustine in combination with either doxorubicin or mitoxantrone caused antagonistic effects on disruption of mitochondrial membrane potential as well as on the rate of apoptosis. In contrast the combination of bendamustine with cladribine acted synergistically on these parameters. By using the IC(50) (dosages causing 50% rate of apoptosis) the synergistic effect of the combination of bendamustine and cladribine was associated with an enhanced mitochondrial release of cytochrome c and Smac/DIABLO, by down-regulation of x-linked inhibitor of apoptosis (XIAP), cIAP1, Par-4 and Daxx as well as by a significantly increased activation of caspases-3, -6, -7, -8 and -9. At the same rate of apoptosis (IC(50)), the antagonistic combinations did not increase the release of cytochrome c or Smac/DIABLO, nor down-regulate the expression of XIAP, cIAP1, Par-4 and Daxx, nor increase the activation of caspases. The role of down-regulation of IAPs and of enforced caspase activation for synergism in this model was supported by the observation, that broad spectrum inhibition of caspases re-established expression of XIAP. Our study is the first to outline the molecular alterations caused by synergistic and antagonistic drug combinations within the same lymphoma cell model. The above described mechanisms were already assessable at a point where the effects of synergistic or antagonistic combinations could not yet be discriminated quantitatively by the level of apoptosis rate of the lymphoma cells

Prostate apoptosis response gene-4 (par-4) abrogates the survival function of p185(BCR-ABL) in hematopoietic cells

Objective Prostate apoptosis response gene-4 (par-4) is deregulated in acute and chronic lymphatic leukemia. Given its pro-apoptotic role in neoplastic lymphocytes and evidence that par-4 antagonizes oncogenic Ras in solid tumors, we hypothesized that par-4 may act as a tumor suppressor impairing transformation induced by p185BCR-ABL. Materials and Methods The capacity of par-4 to interfere with factor independence induced by p185BCR-ABL and V12ras was evaluated by analysis of factor-independent growth of p185BCR-ABL/ par-4 and V12ras/par-4 tranduced cells. The expression of par-4 and p185BCR-ABL by the respective constructs was controlled by Western blot analysis. Activated Ras was detected by pull-down assay in the cell clones expressing p185BCR-ABL in the absence and presence of par-4. Results Expression of p185BCR-ABL causes factor independence, signifying a conversion toward a transformed phenotype in hematopoietic precursors. We demonstrate that par-4 completely abolishes factor independence induced by p185BCR-ABL and partially abrogates factor independence caused by activated V12ras. Evaluating the underlying molecular mechanisms, we show that par-4 hinders activation of oncogenic Ras and causes concomitant disruptions of p185BCR-ABL-mediated signaling. Conclusion We provide the first evidence that par-4 exhibits an antitransforming capacity by antagonizing p185BCR-ABL-induced factor-independent proliferation in hematopoietic cells

Prostate-apoptosis-response-gene-4 increases sensitivity to TRAIL-induced apoptosis.

The capacity of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) to preferentially induce apoptosis in malignant cells while sparing normal tissues renders it an attractive therapeutic agent. Nevertheless, the molecular determinants governing sensitivity towards TRAIL remain to be defined. Acknowledging the previously demonstrated deregulation of prostate-apoptosis-response-gene-4 (par-4) in ex vivo cells of patients suffering from acute and chronic lymphatic leukemia, we here tested the hypothesis that expression of par-4 influences sensitivity to TRAIL. Evaluating this hypothesis we show, that par-4-transfected T-lymphoblastic Jurkat cells exhibit a considerably increased rate of apoptosis upon incubation with an agonistic TRAIL-antibody as compared to their mock-transfected counterparts. Defining the underlying molecular mechanisms we provide evidence, that par-4 enhances sensitivity towards TRAIL by employing crucial members of the extrinsic pathway. Thus, par-4-overexpressing Jurkat clones show an enforced cleavage of c-Flip(L) together with an increased activation of the initiator caspases-8 and -10. In addition, expression of par-4 enables cells to down-regulate the inhibitor-of-apoptosis proteins cIAP-1, cIAP-2, XIAP and survivin with a concomitantly enhanced activation of the executioner caspases-6 and -7. Supporting the crucial role of caspase-8 in par-4-promoted apoptosis we demonstrate that inhibition of caspase-8 considerably reduces TRAIL-induced apoptosis in par-4 and mock-transfected Jurkat clones and reverses the described molecular changes. In conclusion, we here provide first evidence that expression of par-4 in neoplastic lymphocytes augments sensitivity to TRAIL-induced cell death and outline the responsible molecular mechanisms, in particular the crucial role of caspase-8 activation

In the erythroleukemic cell line HEL Prostate-apoptosis-response-gene-4 (par-4) fails to down-regulate Bcl-2 and to promote apoptosis.

In a variety of malignant cells Prostate-apoptosis-response-gene-4 (Par-4) exhibits a pro-apoptotic influence sensitizing these cells to apoptosis-inducing agents by downregulating expression of Bcl-2. Considering the crucial role of Bcl-2 in the development of chemoresistance of acute myeloid leukemia (AML) cells, we here assessed the potential of Par-4 to down-regulate Bcl-2 and to induce apoptosis in the erythroleukemic cell line HEL. Testing a potential pro-apoptotic role of Par-4 upon incubation with various conventional chemotherapeutic drugs, novel agents such as the signal transduction inhibitor STI 571 and the histone deacetylase (HDAC)- inhibitor trichostatin A (TSA), as well as with the experimental substances Fas and TRAIL, we provide evidence that in the erythroleukemic cell line HEL expression of Par-4 is not sufficient to sensitize to any of these pro-apoptotic stimuli. We further demonstrate that--in contrast to previous reports in non-AML cells--Par-4 expression in HEL cells leads to an upregulation of Bcl-2. Moreover, Par-4-positive HEL cells exhibit a decreased level of the proapoptotic protein Bax as compared to Par-4- negative cells. In addition, Par-4 increases the expression of Daxx--whose downregulation is associated with augmented chemosensitivity--as well as expression of the procaspases-8, -9 and -10, whereas the levels of the procaspases-3 and -7 remain unaltered. In conclusion we here demonstrate that in the erythroleukemic cell line HEL--in contrast to other cell types Par-4 fails to promote apoptosis and outline the underlying molecular mechanisms

In bcr-abl-positive myeloid cells resistant to conventional chemotherapeutic agents, expression of Par-4 increases sensitivity to imatinib (STI571) and histone deacetylase-inhibitors.

In a variety of malignant cells the prostate-apoptosis-response-gene-4 (Par-4) induces increased sensitivity towards chemotherapeutic agents by down-regulating anti-apoptotic B-cell lymphoma-gene 2 (Bcl-2). Hypothesizing that Par-4 also influences apoptosis in myeloid cell lines, we tested this hypothesis by stably transfecting bcr-abl transformed-K562 cells with a Par-4-expressing vector. Here we demonstrate that over-expression of Par-4 in K562 cells up-regulates expression levels of Bcl-2 and death-associated protein (Daxx). Upon treatment with different chemotherapeutic agents, Fas- or TRAIL agonistic antibodies, Par-4-positive cells did not exhibit an increased rate of apoptosis as compared to Par-4-negative control cells. However, incubation with histone deacetylase (HDAC)-inhibitors Trichostatin A (TSA) and LAQ824 or the tyrosinkinase inhibitor Imatinib (STI571) increased the rate of apoptosis in Par-4-positive K562 cells. Assessing the underlying molecular mechanisms for the Par-4-induced response to HDAC-inhibitors and STI571 we provide evidence, that these effects are associated with a down-regulation of Daxx, enforced activation of caspases and enhanced cleavage of cellular inhibitor of apoptosis (cIAP)-1 and -2

In lymphatic cells par-4 sensitizes to apoptosis by down-regulating bcl-2 and promoting disruption of mitochondrial membrane potential and caspase activation.

Inhibition of apoptosis is a hallmark of malignancies of the hematopoetic system. Previous studies in nonhematopoetic cells demonstrated that the prostate-apoptosis-response-gene-4 (Par-4) is up-regulated in cells undergoing programmed cell death and that Par-4 exerts its proapoptotic effect by down-regulating Bcl-2. After showing the aberrant expressional pattern of Par-4 in neoplastic lymphocytes as well as demonstrating inverse expressional patterns of Par-4 and Bcl-2 in malignant cells of patients suffering from acute lymphocytic leukemia, we assessed the functional consequences of Par-4 overexpression during apoptosis in Jurkat T lymphocytes. We show that in lymphatic cells Par-4 overexpression decreases the level of Bcl-2, whereas Bax, the proapoptotic counterpart of Bcl-2, retains unaltered levels. Moreover, Par-4 overexpression is accompanied by cleavage of poly(ADP-ribose) polymerase (PARP). Despite these effects, overexpression of Par-4 alone is not sufficient to induce apoptosis but markedly increases the rate of apoptosis on treatment with different chemotherapeutic agents. On chemotherapeutic treatment Par-4 overexpression enhances disruption of mitochondrial membrane potential, PARP-cleaving activity, as well as activation of caspase-3. The hypothesis of caspase-dependency of Par-4-promoted apoptosis is additionally supported by demonstrating complete abrogation of programmed cell death after pretreatment with a broad spectrum caspase-inhibitor. On inhibition of caspase-3 overexpression of Par-4 enables lymphatic cells to alternatively activate caspases-9, -6, and -7 by diminishing the influence of the inhibitors of apoptosis proteins (IAPs) cIAP1 and XIAP. Our study is the first to identify Par-4 as a proapoptotic protein in lymphatic cells, outlining a model of action evaluating the role of Bcl-2/Bax, as well as demonstrating the impact of Par-4 expression on PARP cleavage, disruption of mitochondrial membrane potential, caspase activation, and interactions with inhibitors of apoptosis proteins