Molecular mechanisms underlying interferon-alpha-induced G0/G1 arrest: CKI-mediated regulation of G1 Cdk-complexes and activation of pocket proteins.
(17/1897)
One prominent effect of IFNs is their cell growth-inhibitory activity. The mechanism behind this inhibition of proliferation is still not fully understood. In this study, the effect of IFN-alpha treatment on cell cycle progression has been analysed in three lymphoid cell lines, Daudi, U-266 and H9. Examination of the growth-arrested cell populations shows that Daudi cells accumulate in a G0-like state, whereas U-266 cells arrest later in G1. H9 cells are completely resistant to IFN-alpha's cell growth-inhibitory effects. The G0/G1-phase arrest is preceded by a rapid induction of the cyclin-dependent kinase inhibitors (CKIs), p21 and p15. In parallel, the activities of the G1 Cdks are significantly reduced. In addition to p21/p15 induction, IFN-alpha regulates the expression of another CKI, p27, presumably by a post-transcriptional mechanism. In the G1 Cdk-complexes, there is first an increased binding of p21 and p15 to their respective kinases. At longer exposure times, when Cdk-bound p15 and p21 decline, p27 starts to accumulate. Furthermore, we found that IFN-alpha not only suppresses the phosphorylation of pRb, but also alters the phosphorylation and expression of the other pocket proteins p130 and p107. These data suggest that induction of p21/p15 is involved in the primary IFN-alpha response inhibiting G1 Cdk activity, whereas increased p27 expression is part of a second set of events which keep these Cdks in their inactive form. Moreover, elevated levels of p27 correlated with a dissociation of cyclin E/Cdk2-p130 or p107 complexes to yield cyclin E/Cdk2-p27 complexes. In resistant H9 cells, which possess a homozygous deletion of the p15/p16 genes and lack p21 protein expression, IFN-alpha causes no detectable changes in p27 expression and, furthermore, no effects are observed on either pocket proteins in this cell line. Taken together, these data suggest that the early decline in G1 Cdk activity, subsequent changes in phosphorylation of pocket proteins, and G1/G0 arrest following IFN-alpha treatment, is not primarily due to loss of the G1 kinase components, but result from the inhibitory action of CKIs on these complexes. (+info)
Role of caspases and possible involvement of retinoblastoma protein during TGFbeta-mediated apoptosis of human B lymphocytes.
(18/1897)
In this study, we investigated the involvement of caspases in TGFbeta-induced apoptosis in human B cells. Our results show that TGFbeta-mediated nuclear fragmentation, observed in the Epstein-Barr virus-negative Burkitt's Lymphoma cell line BL41, was abolished in the presence of the tripeptide caspase inhibitor zVAD-fmk or the specific caspase-3 inhibitor DEVD-fmk. Other apoptotic manifestations such as cell shrinkage, surface phosphatidylserine expression and chromatin condensation were strongly inhibited by zVAD-fmk but only partially by DEVD-fmk. This suggests that other caspases in addition to caspase-3 control these apoptotis-associated features. Specific activation of caspase-3 during TGFbeta-induced apoptosis was demonstrated by the DEVD-fmk-sensitive expression of the active p17 subunit of caspase-3 and by in vivo cleavage of PARP. In addition, TGFbeta treatment of BL41 promoted the expression of both dephosphorylated and truncated forms of the retinoblastoma protein. Inhibition of caspase-3 activity abolished both nuclear fragmentation and expression of the truncated retinoblastoma protein, without modifying the G1 cell cycle arrest induced by TGFbeta. Our data thus demonstrate that TGFbeta-induced apoptosis of lymphoma B lymphocytes is dependent on caspase activation and involves caspase-dependent cleavage of the retinoblastoma protein. (+info)
Phenylbutyrate induces cell differentiation and modulates Epstein-Barr virus gene expression in Burkitt's lymphoma cells.
(19/1897)
Although Burkitt's lymphoma (BL) is a readily treated malignancy, recurrences, as well as disease arising in immunosuppressed patients, are notoriously resistant to conventional therapeutic approaches. The EBV is associated with a significant proportion of these lymphomas that evade immune surveillance through decreased expression of both viral and cellular antigens. Increasing the immunogenicity of BL cells may, therefore, represent a potentially beneficial therapeutic maneuver. Using in vitro models of EBV-transformed lymphoblastoid as well as BL cell lines, we demonstrate increased expression of genes coding for HLA class I and EBV latent proteins by the differentiation inducer phenylbutyrate (PB). The aromatic fatty acid also caused cytostasis associated with sustained declines in c-myc expression, a direct antitumor effect that was independent of the EBV status. We conclude, therefore, that differentiation therapy of BL with PB may lead to growth arrest with increased tumor immunogenicity in vivo. The findings may have clinical relevance because the in vitro activity has been observed with PB concentrations that are well tolerated and nonimmunosuppressive in humans, a desirable feature for the different patient populations afflicted with this disease. (+info)
Oxidative stress inhibits apoptosis in human lymphoma cells.
(20/1897)
Apoptosis and necrosis are two forms of cell death that are induced under different conditions and that differ in morphological and biochemical features. In this report, we show that, in the presence of oxidative stress, human B lymphoma cells are unable to undergo apoptosis and die instead by a form of necrosis. This was established using the chemotherapy drug VP-16 or the calcium ionophore A23187 to induce apoptosis in Burkitt's lymphoma cell lines and by measuring classical markers of apoptotic death, including cell morphology, annexin V binding, DNA ladder formation, and caspase activation. In the presence of relatively low levels of H2O2 (75-100 microM), VP-16 and A23187 were unable to induce apoptosis in these cells. Instead, the cells underwent non-apoptotic cell death with mild cytoplasmic swelling and nuclear shrinkage, similar to the death observed when they were treated with H2O2 alone. We found that H2O2 inhibits apoptosis by depleting the cells of ATP. The effects of H2O2 can be overcome by inhibitors of poly(ADP)-ribosylation, which also preserve cellular ATP levels, and can be mimicked by agents such as oligomycin, which inhibit ATP synthesis. The results show that oxidants can manipulate cell death pathways, diverting the cell away from apoptosis. The potential physiological ramifications of this finding will be discussed. (+info)
High frequency of adhesion defects in B-lineage acute lymphoblastic leukemia.
(21/1897)
Aberrant proliferation, differentiation, and/or migration of progenitors observed in various hematological malignancies may be caused by defects in expression and/or function of integrins. In this study, we have developed a new fluorescent beads adhesion assay that facilitates flow cytometric investigation of lymphocyte function-associated antigen 1 (LFA-1)- and very late activation antigen-4 (VLA-4)-mediated functional adhesion in B-lineage acute lymphoblastic leukemia (ALL) of both the CD10(-) and CD10(+) (leukemic) cell population within one blood or bone marrow sample. Surprisingly, of the 20 B-lineage ALL patients investigated, 17 contained a leukemic cell population with LFA-1- and/or VLA-4-mediated adhesion defects. Five patients contained CD10(+) cells that did not exhibit any LFA-1-mediated adhesion due to the lack of LFA-1 surface expression. The CD10(+) cells from 10 ALL patients expressed LFA-1 that could not be activated by the phorbol ester phorbol 12-myristate 13-acetate (PMA), whereas the CD10(-) cells expressed a functional LFA-1. Seven patients contained CD10(+) cells that expressed a PMA-unresponsive form of VLA-4. The PMA unresponsiveness of the integrins LFA-1 and VLA-4 expressed by the CD10(+) cells may be due to mutations in the integrins itself, in protein kinases, or in other intracellular molecules involved in integrin adhesion. These data clearly demonstrate the importance of investigating integrin function in addition to integrin surface expression. The strikingly high frequency (85%) of adhesion defects in ALL could suggest a causal relationship between integrin-mediated adhesion and B-lineage ALL. (+info)
Anomalous high p27/KIP1 expression in a subset of aggressive B-cell lymphomas is associated with cyclin D3 overexpression. p27/KIP1-cyclin D3 colocalization in tumor cells.
(22/1897)
p27 cyclin-dependent kinase inhibitor downregulation is essential for transition to the S phase of the cell cycle. Thus, proliferating cells in reactive lymphoid tissue show no detectable p27 expression. Nevertheless, anomalous high p27 expression has been shown to be present in a group of aggressive B-cell lymphomas with high proliferation index and adverse clinical outcome. This suggests that abnormally accumulated p27 protein has been rendered functionally inactive. We analyzed the causes of this anomalous presence of p27 in a group of aggressive B-cell lymphomas, including 54 cases of diffuse large B-cell lymphomas and 20 Burkitt's lymphomas. We simultaneously studied them for p27, cyclin D3, cyclin D2, cyclin D1, and cyclin E expression, because it has been stated that high levels of expression of cyclin D1 or E lead to increased p27 levels in some cell types. A statistically significant association between p27 and cyclin D3 expression was found for the group as a whole. Additionally, when dividing the cases according to the level of expression of cyclin D3 by reactive germinal centers, it was observed that cases with stronger cyclin D3 expression also show higher p27 expression. The relationship between both proteins was also shown at a subcellular level by laser confocal studies, showing that in cases with high expression of both proteins there was a marked colocalization. Additional evidence in favor of p27 sequestration by cyclin D3 was provided by coimmunoprecipitation studies in a Burkitt's cell line (Raji) showing the existence of cyclin D3/p27 complexes and the absence of CDK2/p27 complexes. These results could support the hypothesis that there are cyclin D3/p27 complexes in a subset of aggressive B-cell lymphomas in which p27 lacks the inhibitory activity found when it is bound to cyclin E/CDK2 complexes. This interaction between both proteins could lead to an abnormal nuclear accumulation, detectable by immunohistochemical techniques. (+info)
Immunophenotyping of acute lymphoblastic leukemia in pediatric patients by three-color flow cytometric analysis.
(23/1897)
Immunophenotyping of acute lymphoblastic leukemia (ALL) in children using three-color flow cytometry was carried out at Chulalongkorn Hospital, Bangkok, Thailand. Of 38 patients with acute lymphoblastic leukemia, 65.8% were identified as common ALL, 15.8% were B-ALL, and 18.4% were T-ALL. Of these 38 cases, there were 4 cases of infantile leukemia. Relapsed cases of leukemia were found most in B-ALL up to 3 out of 6 cases and to a lesser extent in T-ALL (1 of 7 cases) and c-ALL (1 of 25 cases). Our data showed the CD markers expression for common ALL (c-ALL) were CD19+/10+ (100%), CD20+ (24%), CD22+ (100%), HLA-DR+ (70.1%), and CD34+ (58.8%). CD markers expression for B-ALL were CD19+ (100%), CD20+ (33.3%), CD22+ (80%), and HLA-DR+ (80%). CD markers expression for T-ALL were CD3+ (42.9%), CD5+ (100%), CD7+ (85.7%). Myeloid aberrant expressions were found in c-ALL (25-37.5%), B-ALL (20%), and T-ALL (14.3%). The significance of the aberration is discussed. The immunophenotyping classification of ALL as c-ALL, B-ALL, and T-ALL is useful in prognosis and treatment. (+info)
Synovial Epstein-Barr virus infection increases the risk of rheumatoid arthritis in individuals with the shared HLA-DR4 epitope.
(24/1897)
OBJECTIVE: To investigate the presence of the Epstein-Barr virus (EBV) in rheumatoid arthritis (RA) synovium and its correlation with the HLA genotype in an attempt to elucidate the role of EBV in the pathogenesis of RA. METHODS: EBV DNA/RNA was investigated by polymerase chain reaction (PCR) analysis of synovial tissue from 84 patients with RA and from 81 patients with non-RA arthritis (controls) and was correlated with the patients' HLA genotype. RESULTS: EBV DNA and EBV-encoded RNA 1 transcripts were significantly more frequently present in synovial tissue from the RA patients (29 of 84) than in that from the non-RA patient controls (8 of 81). EBV DNA-positive individuals had a 5.47 times higher risk of presenting with RA than did EBV DNA-negative individuals. In HLA-DRB1*0401,0404,0405,0408-positive or shared epitope-positive patients, the risk was further increased (odds ratio for EBV and HLA-DR4 approximately 41, for EBV and the shared epitope approximately 15) compared with those who lacked both EBV DNA and RA-linked HLA genotypes. CONCLUSION: EBV seems to function as an environmental risk factor for RA, particularly in patients with the RA-linked HLA-DRB1 alleles. (+info)