Biomedical subjects

J C Reed

Publications and source records attributed to J C Reed.

At least 199 records · Page 11Linked to original sources

PubMed1998

Bcl-2 family proteins and mitochondria.

The Bcl-2 family of proteins plays a pivotal role in regulating cell life and death. Many of these proteins reside in the outer mitochondrial membrane, oriented towards the cytosol. Cytoprotective Bcl-2 family proteins such as Bcl-2 and Bcl-XL prevent mitochondrial permeability transition pore opening and release of apoptogenic proteins from mitochondria under many circumstances that would otherwise result in either apoptosis or necrosis. In contrast, some pro-apoptotic members of this family such as Bax can induce these destructive changes in mitochondria in both mammalian cells and when expressed exogenously in yeast. The mechanisms by which Bcl-2 family proteins control cell life and death remain elusive, but may include both the ability to form ion channels or pores in membranes and physical interactions with a variety of proteins implicated in apoptosis regulation.

Animals↗

PubMed1998

Expression and location of Hsp70/Hsc-binding anti-apoptotic protein BAG-1 and its variants in normal tissues and tumor cell lines.

BAG-1 is a multifunctional protein that blocks apoptosis and interacts with several types of proteins, including Bcl-2 family proteins, the kinase Raf-1, certain tyrosine kinase growth factor receptors, and steroid hormone receptors, possibly by virtue of its ability to regulate the Hsp70/Hsc70 family of molecular chaperones. Two major forms of the human and mouse BAG-1 proteins were detected by immunoblotting. The longer human and mouse BAG-1 proteins (BAG-1L) appear to arise through translation initiation at noncanonical CTG codons located upstream of and in-frame with the usual ATG codon used for production of the originally described BAG-1 protein. Immunoblotting experiments using normal tissues revealed that BAG-1L is far more restricted in its expression and is present at lower levels than the more prevalent BAG-1 protein. Human but not mouse tissues also produce small amounts of an additional isoform of BAG-1 of intermediate size (BAG-1M) that probably arises through translation initiation at yet another site involving an ATG codon. All three isoforms of human BAG-1 (BAG-1, BAG-1M, and BAG-1L) retained the ability to bind Hsc70. Subcellular fractionation and immunofluorescence confocal microscopy studies indicated that BAG-1L often resides in the nucleus, consistent with the presence of a nuclear localization sequence in the NH2-terminal unique domain of this protein. In immunohistochemical assays, BAG-1 immunoreactivity was detected in a wide variety of types of cells in normal adult tissues and was localized to either cytosol, nucleus, or both, depending on the particular type of cell. In some cases, cytosolic BAG-1 immunostaining was clearly associated with organelles resembling mitochondria, consistent with the reported interaction of BAG-1 with Bcl-2 and related proteins. Furthermore, experiments using a green fluorescence protein (GFP)-BAG-1 fusion protein demonstrated that overexpression of Bcl-2 in cultured cells can cause intracellular redistribution of GFP-BAG-1, producing a membranous pattern typical of Bcl-2 family proteins. The BAG-1 protein was found at high levels in several types of human tumor cell lines among the 67 tested, particularly leukemias, breast, prostate, and colon cancers. In contrast to normal tissues, which only rarely expressed BAG-1L, tumor cell lines commonly contained BAG-1L protein, including most prostate, breast, and leukemia cell lines, suggesting that a change in BAG-1 mRNA translation frequently accompanies malignant transformation.

Amino Acid Sequence↗

PubMed1998

Interaction of BAG-1 with retinoic acid receptor and its inhibition of retinoic acid-induced apoptosis in cancer cells.

BAG-1 (also known as RAP46) is an anti-apoptotic protein, which has been shown previously to interact with a number of nuclear hormone receptors, including receptors for glucocorticoid, estrogen, and thyroid hormone. We show here that BAG-1 also interacts with retinoic acid receptor (RAR). Gel retardation assays demonstrated that in vitro translated BAG-1 protein could effectively inhibit the binding of RAR but not retinoid X receptor (RXR) to a number of retinoic acid (RA) response elements (RAREs). A glutathione S-transferase-BAG-1 fusion protein also specifically bound RAR but not RXR. Interaction of BAG-1 and RAR could also be demonstrated by yeast two-hybrid assays. In transient transfection assays, co-transfection of BAG-1 expression plasmid inhibited the transactivation activity of RAR/RXR heterodimers but not RXR/RXR homodimers. When stably expressed in breast cancer cell lines, BAG-1 inhibited binding of RAR/RXR heterodimer to a number of RAREs and suppressed RA-induced growth inhibition and apoptosis. In addition, RA-induced suppression of Bcl-2 expression was abrogated by overexpression of BAG-1. These results demonstrate that BAG-1 can regulate retinoid activities through its interaction with RAR and suggest that elevated levels of BAG-1 protein could potentially contribute to retinoid resistance in cancer cells.

Animals↗

PubMed1998

Changes in c-Jun but not Bcl-2 family proteins in p53-dependent apoptosis of mouse cerebellar granule neurons induced by DNA damaging agent bleomycin.

Tumor suppressor gene p53 is a critical regulator of the cellular response to DNA damage. To examine the function of p53 in postmitotic CNS neurons, we cultured cerebellar granule cells from 15-day-old wild type and p53-deficient mice, and analyzed changes of protein expression in apoptosis elicited by DNA damage. When cerebellar granule cells from wild type mice were treated with bleomycin, a DNA strand-break inducing agent, neuronal death occurred. In contrast, cells from p53-deficient mice were resistant to bleomycin-induced neuronal death. Furthermore, cells from p53 heterozygous mice showed an intermediate resistance between wild type and p53-deficient mice. These results show that p53 is required for the bleomycin-induced cerebellar granule cell death. To examine which proteins are involved in this apoptosis, we examined changes in protein levels of the Bcl-2 family, including Bcl-2, Bcl-X and Bax. The relative amounts of these proteins did not change after bleomycin treatment, suggesting that the changes in the levels of these Bcl-2 family proteins are not necessary for apoptosis in this system. In contrast, the levels of c-Jun protein significantly increased 6 h after treatment with bleomycin in wild type but not in p53-deficient cerebellar granule cells. These results raise the possibility that c-Jun is required for p53-dependent neuronal apoptosis induced by bleomycin.

Animals↗

PubMed1998

p53-inducible human homologue of Drosophila seven in absentia (Siah) inhibits cell growth: suppression by BAG-1.

The Drosophila seven in absentia (sina) gene is required for R7 photoreceptor cell formation during Drosophila eye development, where it functions within the Ras/Raf pathway and targets other proteins for degradation via associations with a ubiquitin-conjugating enzyme. Recently, a mammalian sina homologue was reported to be a p53-inducible gene in a myeloid leukemia cell line. To explore the function of human SINA-homologous (Siah) proteins, expression plasmids encoding Siah-1A were transiently transfected into 293 epithelial cells and GM701 fibroblast cells, resulting in growth arrest without induction of apoptosis. We discovered that BAG-1, a ubiquitin-like Hsp70/Hsc70-regulating protein, is a negative regulator of Siah-1A. Siah-1A was identified as a BAG-1-binding protein via yeast two-hybrid methods. Specific interaction of BAG-1 with Siah-1A was also demonstrated by in vitro binding experiments using glutathione S-transferase fusion proteins and co-immunoprecipitation studies. Siah-1A-induced growth arrest in 293 and GM701 cells was abolished by co-transfection of wild-type BAG-1 with Siah-1A but not by a C-terminal deletion mutant of BAG-1 that fails to bind Siah-1A. Over-expression of BAG-1 significantly inhibited p53-induced growth arrest in 293 cells without preventing p53 transactivation of reporter gene plasmids. BAG-1 also prevented growth arrest following UV-irradiation-induced genotoxic injury without interfering with accumulation of p53 protein or p21(waf-1) expression. BAG-1 functions downstream of p53-induced gene expression to inhibit p53-mediated suppression of cell growth, presumably by suppressing the actions of Siah-1A. We suggest that Siah-1A may be an important mediator of p53-dependent cell-cycle arrest and demonstrate that Siah-1A is directly inhibited by BAG-1.

Amino Acid Sequence↗

PubMed1998

Modulation of Bcl-2 protein levels by an intracellular anti-Bcl-2 single-chain antibody increases drug-induced cytotoxicity in the breast cancer cell line MCF-7.

Extensive experimental evidence suggests that Bcl-2 promotes cell survival by preventing the onset of apoptosis induced by a variety of stimuli. In addition, Bcl-2 expression has been correlated with resistance and poor response to chemotherapy in a number of cell types. Therefore, this protein represents a logical target for gene therapy strategies designed to achieve selective gene product ablation. In this study, we have developed an approach based upon intracellular expression of single-chain antibodies (sFvs) to achieve modulation of Bcl-2 protein levels in target cells. Using a transient expression system, we show that this intracellular anti-Bcl-2 sFv mediates specific reduction of Bcl-2 levels. This effect significantly enhances drug-mediated cytotoxicity in Bcl-2-overexpressing tumor cells, whereas transfection of the anti-Bcl-2 sFv did not affect the growth rate of the tumor cell lines. This method thus represents a novel and efficient way to selectively abrogate the activity of Bcl-2.

Antibodies, Monoclonal↗

PubMed1998

BAG-1L protein enhances androgen receptor function.

BAG-1 is a regulator of heat shock protein (Hsp) 70/Hsc70 family proteins that interacts with steroid hormone receptors. The recently identified BAG-1 long (BAG-1L) protein, an isoform of BAG-1 that arises from translation initiation at a noncanonical CUG codon, was co-immunoprecipitated with androgen receptors (AR) from LNCaP prostate cancer cells and other cell lysates, whereas the shorter originally identified BAG-1 and BAG-1M (RAP 46) proteins were not. BAG-1L, but not BAG-1 or BAG-1M (RAP46), also markedly enhanced the ability of AR to transactivate reporter gene plasmids containing an androgen response element (ARE) in PC3 prostate cancer and other cell lines. A C-terminal region deletion mutant of BAG-1L failed to co-immunoprecipitate with AR and functioned as a trans-dominant inhibitor of BAG-1L, impairing AR-induced transactivation of ARE-containing reporter plasmids. In addition, BAG-1L significantly reduced the concentrations of 5alpha-dihydrotestosterone (DHT) required for AR activity but did not induce ligand-independent transactivation. BAG-1L also markedly improved the ability of AR to transactivate reporter genes when cells were cultured with DHT in combination with the anti-androgen cyproterone acetate. The effects of BAG-1L on AR could not be explained by detectable alterations in the DHT-induced translocation of AR from cytosol to nucleus, nor by BAG-1L-induced increases in the amounts of AR protein. These findings implicate BAG-1L in the regulation of AR function and may have relevance to mechanisms of prostate cancer resistance to hormone-ablative and anti-androgen therapy.

Alternative Splicing↗

PubMed1998

Acidic pH promotes dimerization of Bcl-2 family proteins.

Several members of the apoptosis-regulating Bcl-2 family of proteins can homo- or heterodimerize with each other at neutral pH and can also form ion channels in synthetic membranes at low pH. The effects of low pH on dimerization among these proteins, however, have not heretofore been examined. Surface plasmon resonance was used to examine the kinetics of dimerization as a function of pH between the anti-apoptotic protein Bcl-XL (applied in the mobile phase) and three other members of the Bcl-2 family: Bcl-2, Bax, and Bid (immobilized on biosensor chips). In all cases, the relative affinity of dimerization was substantially increased at pH 4.0 compared to pH 7.0-7.4, ranging from a approximately 10-fold enhancement for Bcl-XL/Bcl-XL homodimers to >60-fold for Bcl-XL/Bid heterodimers. Comparison of the apparent association (ka) and dissociation (kd) rates at neutral and acidic pH revealed that the major contributor to increased affinity at low pH was a decreased rate of dimer dissociation. Thus, low pH stabilizes homo- and heterodimeric complexes comprised of Bcl-XL and these other Bcl-2 family proteins. At pH 4.0, the circular dichroism spectra of Bcl-XL and Bax were essentially unchanged relative to pH 7.0-7.4, indicating a complete retention of alpha-helical secondary structure at low pH and excluding gross denaturation of the proteins. Size-exclusion chromatography and bisANS (4,4'-dianilino-1, 1'-binaphthyl-5,5'-disulfonic acid) labeling studies provided indirect evidence that Bcl-XL may undergo conformational changes at low pH. The findings are discussed with respect to the mechanisms of ion-channel formation by Bcl-2 family proteins and the putative molten globule state that has been proposed for these and structurally similar proteins.

Amino Acid Sequence↗

PubMed1998

Expression of apoptosis-regulating proteins in chronic lymphocytic leukemia: correlations with In vitro and In vivo chemoresponses.

B-cell chronic lymphocytic leukemia (B-CLL) represents a neoplastic disorder caused primarily by defective programmed cell death (PCD), as opposed to increased cell proliferation. Defects in the PCD pathway also contribute to chemoresistance. The expression of several apoptosis-regulating proteins, including the Bcl-2 family proteins Bcl-2, Bcl-XL, Mcl-1, Bax, Bak, and BAD; the Bcl-2-binding protein BAG-1; and the cell death protease Caspase-3 (CPP32), was evaluated by immunoblotting using 58 peripheral blood B-CLL specimens from previously untreated patients. Expression of Bcl-2, Mcl-1, BAG-1, Bax, Bak, and Caspase-3 was commonly found in circulating B-CLL cells, whereas the Bcl-XL and BAD proteins were not present. Higher levels of the anti-apoptotic protein Mcl-1 were strongly correlated with failure to achieve complete remission (CR) after single-agent therapy (fludarabine or chlorambucil) (P = .001), but the presence of only seven CRs among the 42 patients for whom follow-up data were available necessitates cautious interpretation of these observations. Higher levels of the anti-apoptotic protein BAG-1 were also marginally associated with failure to achieve CR (P = .04). Apoptosis-regulating proteins were not associated with patient age, sex, Rai stage, platelet count, hemoglobin (Hb) concentration, or lymph node involvement, although higher levels of Bcl-2 and a high Bcl-2:Bax ratio were correlated with high numbers (>10(5)/microL) of white blood cells (WBC) (P = .01; .007) and higher levels of Bak were weakly associated with loss of allelic heterozygosity at 13q14 (P = .04). On the basis of measurements of apoptosis induction by fludarabine using cultured B-CLL specimens, in vitro chemosensitivity data failed to correlate with in vivo clinical response rates (n = 42) and expression of the various apoptosis-regulating proteins. Although larger prospective studies are required before firm conclusions can be reached, these studies show the expression in B-CLLs of multiple apoptosis-regulating proteins and suggest that the relative levels of some of these, such as Mcl-1, may provide information about in vivo responses to chemotherapy. In vitro chemosensitivity data, however, do not appear to be particularly useful in predicting responses in B-CLL.

Adult↗

PubMed1998

Bax directly induces release of cytochrome c from isolated mitochondria.

Bax is a pro-apoptotic member of the Bcl-2 protein family that resides in the outer mitochondrial membrane. It is controversial whether Bax promotes cell death directly through its putative function as a channel protein versus indirectly by inhibiting cellular regulators of the cell death proteases (caspases). We show here that addition of submicromolar amounts of recombinant Bax protein to isolated mitochondria can induce cytochrome c (Cyt c) release, whereas a peptide representing the Bax BH3 domain was inactive. When placed into purified cytosol, neither mitochondria nor Bax individually induced proteolytic processing and activation of caspases. In contrast, the combination of Bax and mitochondria triggered release of Cyt c from mitochondria and induced caspase activation in cytosols. Supernatants from Bax-treated mitochondria also induced caspase processing and activation. Recombinant Bcl-XL protein abrogated Bax-induced release of Cyt c from isolated mitochondria and prevented caspase activation. In contrast, the broad-specificity caspase inhibitor benzyloxycarbonyl-valinyl-alaninyl-aspartyl-(0-methyl)- fluoromethylketone (zVAD-fmk) and the caspase-inhibiting protein X-IAP had no effect on Bax-induced release of Cyt c from mitochondria in vitro but prevented the subsequent activation of caspases in cytosolic extracts. Unlike Ca2+, a classical inducer of mitochondrial permeability transition, Bax did not induce swelling of mitochondria in vitro. Because the organellar swelling caused by permeability transition causes outer membrane rupture, the findings, therefore, dissociate these two events, implying that Bax uses an alternative mechanism for triggering release of Cyt c from mitochondria.

Animals↗

PubMed1998

The permeability transition pore complex: a target for apoptosis regulation by caspases and bcl-2-related proteins.

Early in programmed cell death (apoptosis), mitochondrial membrane permeability increases. This is at least in part due to opening of the permeability transition (PT) pore, a multiprotein complex built up at the contact site between the inner and the outer mitochondrial membranes. The PT pore has been previously implicated in clinically relevant massive cell death induced by toxins, anoxia, reactive oxygen species, and calcium overload. Here we show that PT pore complexes reconstituted in liposomes exhibit a functional behavior comparable with that of the natural PT pore present in intact mitochondria. The PT pore complex is regulated by thiol-reactive agents, calcium, cyclophilin D ligands (cyclosporin A and a nonimmunosuppressive cyclosporin A derivative), ligands of the adenine nucleotide translocator, apoptosis-related endoproteases (caspases), and Bcl-2-like proteins. Although calcium, prooxidants, and several recombinant caspases (caspases 1, 2, 3, 4, and 6) enhance the permeability of PT pore-containing liposomes, recombinant Bcl-2 or Bcl-XL augment the resistance of the reconstituted PT pore complex to pore opening. Mutated Bcl-2 proteins that have lost their cytoprotective potential also lose their PT modulatory capacity. In conclusion, the PT pore complex may constitute a crossroad of apoptosis regulation by caspases and members of the Bcl-2 family.

Animals↗

PubMed1998

IAPs block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition of distinct caspases.

Inhibitor of apoptosis (IAP) gene products play an evolutionarily conserved role in regulating programmed cell death in diverse species ranging from insects to humans. Human XIAP, cIAP1 and cIAP2 are direct inhibitors of at least two members of the caspase family of cell death proteases: caspase-3 and caspase-7. Here we compared the mechanism by which IAPs interfere with activation of caspase-3 and other effector caspases in cytosolic extracts where caspase activation was initiated by caspase-8, a proximal protease activated by ligation of TNF-family receptors, or by cytochrome c, which is released from mitochondria into the cytosol during apoptosis. These studies demonstrate that XIAP, cIAP1 and cIAP2 can prevent the proteolytic processing of pro-caspases -3, -6 and -7 by blocking the cytochrome c-induced activation of pro-caspase-9. In contrast, these IAP family proteins did not prevent caspase-8-induced proteolytic activation of pro-caspase-3; however, they subsequently inhibited active caspase-3 directly, thus blocking downstream apoptotic events such as further activation of caspases. These findings demonstrate that IAPs can suppress different apoptotic pathways by inhibiting distinct caspases and identify pro-caspase-9 as a new target for IAP-mediated inhibition of apoptosis.

Apoptosis↗

PubMed1998

A single BIR domain of XIAP sufficient for inhibiting caspases.

The inhibitor of apoptosis proteins (IAPs) constitute an evolutionarily conserved family of homologous proteins that suppress apoptosis induced by multiple stimuli. Some IAP family proteins, including XIAP, cIAP-1, and cIAP-2, can bind and directly inhibit selected caspases, a group of intracellular cell death proteases. These caspase-inhibiting IAP family proteins all contain three tandem BIR domains followed by a RING zinc finger domain. To determine the structural basis for caspase inhibition by XIAP, we analyzed the effects of various fragments of this IAP family protein on caspase activity in vitro and on apoptosis suppression in intact cells. The RING domain of XIAP failed to inhibit the activity of recombinant caspases-3 or -7, whereas a fragment of XIAP encompassing the three tandem BIR domains potently inhibited these caspases in vitro and blocked Fas (CD95)-induced apoptosis when expressed in cells. Further dissection of the XIAP protein demonstrated that only the second of the three BIR domains (BIR2) was capable of binding and inhibiting these caspases. The apparent inhibition constants (Ki) for BIR2-mediated inhibition of caspases-3 and -7 were 2-5 nM, indicating that this single BIR domain possesses potent anti-caspase activity. Expression of the BIR2 domain in cells also partially suppressed Fas-induced apoptosis and blocked cytochrome c-induced processing of caspase-9 in cytosolic extracts, whereas BIR1 and BIR3 did not. These findings identify BIR2 as the minimal caspase-inhibitory domain of XIAP and indicate that a single BIR domain can be sufficient for binding and inhibiting caspases.

Animals↗

PubMed1998

Granzyme release and caspase activation in activated human T-lymphocytes.

Recently it has been reported that caspase-3 activation occurs in stimulated T-lymphocytes without associated apoptosis (Miossec, C., Dutilleul, V., Fassy, F., and Diu-Hercend, A. (1997) J. Biol. Chem. 272, 13459-13462). To explore this phenomenon, human peripheral blood lymphocytes (PBLs) were stimulated with mitogenic lectins or anti-CD3 antibody, and the proteolytic processing of different caspases and caspase substrates was analyzed by immunoblotting. Proteolytic processing of caspases-3 and -7 and the caspase substrates poly(ADP-ribose) polymerase, GDP dissociation inhibitor, and PKCdelta was observed when PBLs were activated in vitro, and lysates were prepared using RIPA buffer which contains 1% Nonidet P-40, 0.5% deoxycholate, and 0.1% SDS. In contrast, when a lysis buffer containing 2% SDS was used, the caspases remained in their zymogen pro-forms, and no proteolytic processing of caspase substrates was detected. Moreover, in experiments using intact cells and a cell-permeable fluorigenic caspase substrate, no caspase activity was observed in activated T-cells, whereas it was clearly detected when PBLs were treated with the apoptosis-inducing anticancer drug etoposide. Since the granzyme B is a direct activator of caspase-3 and its expression is induced following T-cell activation, we tested the effects of anti-GraB, an engineered serpin that specifically inhibits GraB. When the activated T-lymphocytes were lysed in RIPA buffer containing anti-GraB, no proteolytic processing or activation of caspase-3 was observed, strongly suggesting that release of GraB or similar proteases from their storage sites in cytotoxic granules during the lysis procedure is responsible for caspase activation. These findings demonstrate that T-cells do not process caspases upon activation and caution about the method of cell lysis used when studying granzyme-expressing cells.

Caspase 3↗

PubMed1998

BCL-2 expression correlates with lower proliferative activity in the intermediate- and high-grade non-Hodgkin's lymphomas: an Eastern Cooperative Oncology Group and Southwest Oncology Group cooperative laboratory study.

An inverse relationship between BCL-2 expression and cell cycle transition has been suggested by recent studies in murine models. To investigate the clinical relevance of these laboratory studies, a group of 116 paraffin-embedded non-Hodgkin's lymphoma (NHL) biopsy specimens (Working Formulation Groups D-H, and J) from a cooperative group study of cellular DNA content were analyzed for the 14;18 translocation using polymerase chain reaction (PCR)-based methods and, if sufficient tissue remained, for BCL-2 and BAX expression by immunohistochemistry. The results of these studies were then compared with the results of the previously performed flow cytometric analysis of ploidy and proliferative activity (S-phase-fraction). BCL-2 expression was inversely associated with proliferative activity (P = .001; n = 41), but there was no association between staining for Bax and %S-phase. Ploidy was not associated with either BCL-2 or BAX expression. The t(14;18) was detected in 21 of the 54 cases in which PCR-amplifiable DNA was recovered; 20 of these occurred at the major breakpoint region and 1 at the minor breakpoint region. High levels of BCL-2 or BAX expression occurred independently of t(14;18). There was no association between t(14;18) and either ploidy or proliferative activity. The inverse relationship between BCL-2 expression and proliferative activity in the intermediate- and high-grade NHLs is consistent with recent studies suggesting that Bcl-2 both retards entry into the cell cycle and inhibits apoptosis.

Cell Division↗

PubMed1998

Elevated expression of the apoptotic regulator Mcl-1 at the time of leukemic relapse.

Bcl-2, Bcl-xL, and Mcl-1 are three related intracellular polypeptides that have been implicated as negative regulators of apoptosis. In contrast, the partner protein Bax acts as a positive regulator of apoptosis. Based on the observation that all four of these polypeptides are expressed in a variety of acute myelogenous leukemia (AML) and acute lymphocytic leukemia (ALL) cell lines, cellular levels of these polypeptides were examined by immunoblotting in bone marrow samples harvested from 123 adult AML patients and 36 adult ALL patients before initial antileukemic therapy. Levels of Bcl-2, Mcl-1, Bcl-xL, and Bax each varied over a more than 10-fold range in different pretreatment leukemia specimens. When the 54 AML and 23 ALL samples that contained greater than 80% malignant cells were examined in greater detail, it was observed that pretreatment levels of Bcl-2 and Mcl-1 correlated with each other (R = .44, P < .001 for AML and R = .79, P < .0001 for ALL). In addition, a weak negative correlation between Bax expression and age was observed in AML samples (R = -0.35, P < .02) but not ALL samples. There was no relationship between pretreatment levels of these polypeptides and response to initial therapy. However, examination of 19 paired samples (the first harvested before chemotherapy and the second harvested 23 to 290 days later at the time of leukemic recurrence) revealed a greater than or equal to twofold increase in Mcl-1 levels in 10 of 19 pairs (7 of 15 AML and 3 of 4 ALL) at recurrence. In contrast, 2 of 19 pairs contained twofold less Mcl-1 at the time of recurrence. Approximately equal numbers of samples showed twofold increases and decreases in Bcl-2 (5 increases, 3 decreases) and Bcl-xL (1 increase, 4 decreases) at recurrence. Bax levels did not show a twofold decrease in any patient. these results, coupled with recent observations that cells overexpressing Mcl-1 are resistant to a variety of chemotherapeutic agents, raise the possibility that some chemotherapeutic regimens might select for leukemia cells with elevated levels of this particular apoptosis inhibitor.

Adult↗

PubMed1998

BCL-X expression in multiple myeloma: possible indicator of chemoresistance.

Because murine myeloma plasma cells and normal human lymph node plasma cells express BCL-X, we evaluated BCL-X expression in malignant human plasma cells. BCL-X expression was detected in several human myeloma cell lines, as well as in CD38-sorted bone marrow cells obtained from some patients. Only the antiapoptotic long form of BCL-X (BCL-X-L), was detected. Because BCL-X-L expression can protect tumor cells from apoptotic death induced by chemotherapeutic agents, we tested the clinical relevance of expression in 55 archival bone marrow biopsies. The biopsies were stained by immunohistochemistry, and BCL-X expression was correlated with the subsequent response to treatment. BCL-X expression in malignant plasma cells strongly correlated with decreased response rates in patient groups treated with either melphalan and prednisone or vincristine, Adriamycin, and dexamethasone. Response rates were 83-87% in non-BCL-X-expressing cases and 20-31% in BCL-X-expressing cases. In addition, BCL-X expression was more frequent in specimens taken from patients at relapse (77%), when compared to those at initial diagnosis (29%). Further support for the association of drug resistance with BCL-X-L expression came from studies of the 8226 dox-40 cell line. This line, which expresses p-glycoprotein and serves as a model of multidrug resistance in multiple myeloma cells, demonstrated an up-regulated expression of BCL-X-L, which was relatively specific, in that BCL-2 or BAX expression was not altered. In addition, dox-40 cells demonstrated a generalized resistance to apoptosis that was induced by several different agents. These results indicate that malignant plasma cells can express BCL-X-L and that such expression may be a marker of chemoresistant disease.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed1998

Bc1-2, Raf-1 and mitochondrial regulation of apoptosis.

Raf-1 kinase was shown to bind via its catalytic domain (Cat) to Bcl-2 in a BH4 domain-dependent manner. Using a green fluorescent protein (GFP)-Raf-1 (Cat) fusion protein, Bcl-2 but not Bcl-2(delta BH4) was found to target Raf-1 to mitochondria in cells. Targeting Raf-1 (Cat) to mitochondrial membranes by fusing with the transmembrane domain of an outer mitochondrial membrane protein protected cells from apoptosis and resulted in phosphorylation of BAD protein, whereas plasma-membrane targeted Raf-1 failed to phosphorylate BAD and did not protect against cell death. Moreover, a Bcl-2 binding protein, BAG-1, was shown to not only bind Raf-1, but also increase the activity of this kinase through a protein-protein interaction. The findings suggest that Bcl-2 targets Raf-1 to mitochondria, allowing this kinase to contribute to cellular survival by phosphorylating BAD or possibly other protein substrates in the vicinity of Bcl-2.

Animals↗