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.

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 p185(BCR-ABL). MATERIALS AND METHODS: The capacity of par-4 to interfere with factor independence induced by p185(BCR-ABL) and V12ras was evaluated by analysis of factor-independent growth of p185(BCR-ABL)/ par-4 and V12ras/par-4 transduced cells. The expression of par-4 and p185(BCR-ABL) by the respective constructs was controlled by Western blot analysis. Activated Ras was detected by pull-down assay in the cell clones expressing p185(BCR-ABL) in the absence and presence of par-4. RESULTS: Expression of p185(BCR-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 p185(BCR-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 p185(BCR-ABL)-mediated signaling. CONCLUSION: We provide the first evidence that par-4 exhibits an antitransforming capacity by antagonizing p185(BCR-ABL)-induced factor-independent proliferation in hematopoietic cells.

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.

Expression of ZAP-70 protein correlates with disease stage in chronic lymphocytic leukemia and is associated with, but not generally restricted to, non-mutated Ig VH status.

The mutational status of immunoglobulin variable region genes (Ig VH) is a well established prognostic parameter in chronic lymphocytic leukemia (CLL). Recently, a subset of genes with a characteristic expression profile correlating with the mutational status of B-CLLs has been identified. One of the overexpressed genes in the prognostically unfavorable group of CLL patients with unmutated Ig VH genes encodes for the protein tyrosine kinase ZAP-70, which is physiologically involved in T-cell signaling. Since ZAP-70 has been described to be prognostically relevant in CLL, we analyzed the possible relationship of its expression to the mutational status of Ig VH genes as well as to other prognostic factors in CLL and indolent lymphomas. The mutational status of Ig VH genes was analyzed by seminested PCR, direct sequencing and comparison with the sequences of the EMBL databases in 60 samples of patients with B-CLL and 18 samples of patients with indolent B-cell malignancies. ZAP-70 protein expression was assessed in all samples by immunoblotting and for semiquantitative analysis the ratio of ZAP-70 to tubulin expression was calculated. ZAP-70 protein was found to be expressed in all investigated B-cell malignancies. Expression levels varied within a wide range in each entity. The highest mean level of ZAP-70 expression was observed in unmutated B-CLLs, however, with broad expression variability. High levels of ZAP-70 expression correlated with higher stage Binet B or C and with unmutated Ig VH genes. Overall survival rates estimated by Kaplan-Meier curves did not differ among patients with high or low ZAP-70 expression. We conclude that ZAP-70 is associated with the mutational status of Ig VH genes, but this expression pattern is not present in all individual cases. Furthermore, high levels of ZAP-70 correlated with Binet stages B or C indicating an involvement of ZAP-70 in mechanisms promoting growth of B-CLL cells.

Expression of Daxx sensitizes Jurkat T-cells to the apoptosis-inducing effect of chemotherapeutic agents.

BACKGROUND: The role of Daxx, in particular its ability to promote or hinder apoptosis, still remains controversial. In order to elucidate the functional relevance of Daxx in the extrinsic signaling of malignant lymphocytes Jurkat T-cells were stably transfected with a Daxx-expressing vector or with the respective Daxx-negative control vector. RESULTS: Assessing first the impact of Daxx expression on the rate of proliferation we demonstrate that overexpression of Daxx alone is not sufficient to alter proliferation in neoplastic lymphocytes. Nevertheless, expression of Daxx down-regulates anti-apoptotic Bcl-2 and up-regulates pro-apoptotic BID. In addition, Daxx-overexpressing Jurkat cells exhibit a decreased expression of the pro-caspase-8, -10, -9 and -3 and a concomitant increase of the inhibitors of apoptosis proteins survivin, XIAP, cIAP-1 and -2. We further demonstrate, that upon incubation with various chemotherapeutic agents these Daxx-induced molecular alterations sensitize Jurkat T-cells to the apoptosis-inducing effects of specific chemotherapeutic agents. CONCLUSIONS: We here outline the molecular changes elicited by Daxx on major components of the apoptotic cascade of malignant lymphocytes and demonstrate the capacity of Daxx to sensitize these cells to the apoptosis-inducing effect of various chemotherapeutic agents.

Folinic acid antagonizes methotrexate-induced differentiation of monocyte progenitors.

OBJECTIVE: The anti-inflammatory action of low-dose methoxetrate (MTX) in the treatment of rheumatoid arthritis (RA) appears to be partially impaired by folate supplementation. Here we investigated whether a folate excess impairs monocyte differentiation, a putative anti-inflammatory action of low-dose MTX. METHODS: Monocyte differentiation of U937 promonocytic cells was assessed by CD11b and CD14 immunostaining and fluorescent absorbent cell sorting (FACS) analysis. Cell proliferation and viability were determined by cell counts and trypan-blue staining, respectively. Nuclear apoptosis was assessed by 7-actinomycin staining. Cells were treated with 10(-10)-10(-6) M MTX in the presence or absence of folinic acid. Exposure to 1,25-OH-vitamine D(3) and TGF-beta served as a positive control of monocyte differentiation in U937 cells. RESULTS: Low-dose MTX-induced monocyte differentiation was marginal when compared with 1,25-OH-D(3) + TGF-beta treatment. Low-dose MTX inhibited cell proliferation, induced apoptosis, and reduced cell viability. All the antiproliferative, cytotoxic, and monocyte differentiating effects of MTX were completely reversed by folinic acid. CONCLUSIONS: Monocyte differentiation is part of the folate-dependent MTX actions.

Expression of interleukin-18 receptor in fibroblast-like synoviocytes.

An excess of the proinflammatory substance IL-18 is present in joints of patients with rheumatoid arthritis (RA), and expression of IL-18 receptor (IL-18R) regulates IL-18 bioactivity in various cell types. We examined the expression of IL-18R alpha-chain and beta-chain and the biologic effects of IL-18 in fibroblast-like synoviocytes (FLS) after long-term culture. The presence of both IL-18R chains was a prerequisite for IL-18 signal transduction in FLS. However, all FLS cultures studied were either resistant or barely responsive to IL-18 stimulation as regards cell proliferation, expression of adhesion molecules ICAM-1 and vascular cell adhesion molecule (VCAM)-1, and the release of interstitial collagenase and stromelysin, IL-6 and IL-8, prostaglandin E2, or nitric oxide. We conclude that the presence of macrophages or IL-18R+ T cells that can respond directly to IL-18 is essential for the proinflammatory effects of IL-18 in synovitis in RA.

Prostate apoptosis response gene-4 sensitizes neoplastic lymphocytes to CD95-induced apoptosis.

Evaluating the functional consequences of prostate apoptosis response gene-4 (par-4) expression in CD95-induced apoptosis of neoplastic lymphocytes, we demonstrate that par-4 increases apoptosis by upregulating the CD95 receptor on the cell surface and--with a concomitant decrease of the FLICE-like inhibitory protein (FLIP)--by promoting cleavage of the initiator caspases-8 and -10. This results in an enforced activation of the executioner caspases-6, -7, and -3 as well as in an activation of the mitochondrial pathway. Upon inhibition of caspase-8, overexpression of par-4 enables Jurkat cells to maintain a higher sensitivity to CD95-induced apoptosis by downregulating cIAP-2 and XIAP and by enforcing activation of the initiator caspase-10 as well as of the executioner caspases-6, -7, and -3.

Upon drug-induced apoptosis expression of prostate-apoptosis-response-gene-4 promotes cleavage of caspase-8, bid and mitochondrial release of cytochrome c.

Par-4 functions as a tumor suppressor antagonizing the transforming capacity and the resistance of malignant cells towards apoptotic stimuli. After demonstrating that par-4 promotes apoptosis by activating signaling of the intrinsic pathway of apoptosis, we hypothesized that par-4 also impacts on key molecules of the extrinsic pathway without the requirement of a receptor/ligand interaction. Here, we provide first evidence, that expression of par-4 increases cleavage of caspase-8, truncation of Bid and its translocation to the mitochondria, resulting in an augmentation of cytochrome c and AIF efflux into the cytosol, effects par-4-positive cells are able to retain to a higher extent than par-4-negative cells upon inhibition of caspase-3 activation.

Gamma-catenin contributes to leukemogenesis induced by AML-associated translocation products by increasing the self-renewal of very primitive progenitor cells.

Acute myeloid leukemia (AML) is characterized by the block of differentiation, deregulated apoptosis, and an increased self-renewal of hematopoietic precursors. It is unclear whether the self-renewal of leukemic blasts results from the cumulative effects of blocked differentiation and impaired apoptosis or whether there are mechanisms directly increasing self-renewal. The AML-associated translocation products (AATPs) promyelocytic leukemia/retinoic acid receptor alpha (PML/RAR alpha), promyelocytic leukemia zinc finger (PLZF)/RAR alpha (X-RAR alpha), and AML-1/ETO block hematopoietic differentiation. The AATPs activate the Wnt signaling by up-regulating gamma-catenin. Activation of the Wnt signaling augments self-renewal of hematopoietic stem cells (HSCs). Therefore, we investigated how AATPs influence self-renewal of HSCs and evaluated the role of gamma-catenin in the determination of the phenotype of HSCs expressing AATPs. Here we show that the AATPs directly activate the gamma-catenin promoter. The crucial role of gamma-catenin in increasing the self-renewal of HSCs upon expression of AATPs is demonstrated by (i) the abrogation of replating efficiency upon hindrance of gamma-catenin expression through RNA interference, and (ii) the augmentation of replating efficiency of HSCs upon overexpression of gamma-catenin itself. In addition, the inoculation of gamma-catenin-transduced HSCs into irradiated recipient mice establishes the clinical picture of AML. These data provide the first evidence that the aberrant activation of Wnt signaling by the AATP decisively contributes to the pathogenesis of AML.