Publications (14)71.19 Total impact
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Article: MEK1/2 inhibitors promote Ara-C-induced apoptosis but not loss of Deltapsi(m) in HL-60 cells.
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ABSTRACT: The effects of pharmacologic MEK1/2 inhibitors on ara-C-mediated mitochondrial injury, caspase activation, and apoptosis have been examined in HL-60 leukemic cells. Coadministration of subtoxic concentrations of the MEK1/2 inhibitors U0126 (20 microM), PD98059 (40 microM), or PD184352 (10 microM) with 10-100 microM ara-C (6 h) potentiated apoptosis (i.e., by approx twofold), and pro-caspase 3, pro-caspase 8, Bid, and PARP cleavage. Unexpectedly, MEK1/2 inhibitors failed to enhance ara-C-mediated loss of mitochondrial membrane potential (DeltaPsi(m)), but instead induced substantial increases in cytosolic release of cytochrome c and Smac/DIABLO. U0126/ara-C-mediated apoptosis and pro-caspase 3 activation, but not cytochrome c or Smac/DIABLO release, were blocked by the pan-caspase inhibitor ZVAD-fmk. Together, these findings indicate that potentiation of ara-C-mediated lethality in HL-60 cells by MEK1/2 inhibitors involves enhanced cytosolic release of cytochrome c and Smac/DIABLO but not discharge of DeltaPsi(m), implicating activation of an apoptotic pathway that differs, at least with respect to the nature of the accompanying mitochondrial injury, from that triggered by ara-C alone.Biochemical and Biophysical Research Communications 10/2001; 286(5):1011-8. · 2.48 Impact Factor -
Article: Evidence of a functional role for the cyclin-dependent kinase-inhibitor p21WAF1/CIP1/MDA6 in promoting differentiation and preventing mitochondrial dysfunction and apoptosis induced by sodium butyrate in human myelomonocytic leukemia cells (U937).
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ABSTRACT: The impact of dysregulation of the cyclin-dependent kinase inhibitor p21WAF1/CIP1/MDA6 has been examined in U937 human monocytic leukemia cells in relation to cell cycle arrest and differentiation following treatment with the histone deacetylase inhibitor sodium butyrate (SB). Cells stably transfected with a p21WAF1/CIP1/MDA6 antisense construct, in marked contrast to their wild-type counterparts, failed to up-regulate p21WAF1/CIP1/MDA6, undergo G1 arrest, or express the maturation marker CD11b when exposed to 1 or 3 mM SB. However, antisense-expressing cells were significantly more susceptible to SB-mediated mitochondrial injury and apoptosis, manifested by increased cytosolic translocation of cytochrome c, activation of pro-caspase 3, and degradation of PARP. Dysregulation of p21WAF1/CIP1/MDA6 did not modify the extent of SB-induced histone acetylation, but did result in cleavage of p27KIP1, Bcl-2 and pRb, as well as diminished levels of full-length underphosphorylated pRb. Finally, dysregulation of p21WAF1/CIP1/MDA6 did not modify SB-mediated down-regulation of E2F-1 or c-Myc, but was associated with enhanced down-regulation of cyclins D1 and E. Together, these findings indicate that in U937 leukemia cells, p21WAF1/CIP1/MDA6 plays a critical functional role in SB-mediated G1 arrest and maturation, and suggest that cells displaying dysregulation of this CDKI respond to SB by engaging a default apoptotic program.International Journal of Oncology 08/2001; 19(1):181-91. · 2.40 Impact Factor -
Article: Pharmacological inhibitors of the mitogen-activated protein kinase (MAPK) kinase/MAPK cascade interact synergistically with UCN-01 to induce mitochondrial dysfunction and apoptosis in human leukemia cells.
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ABSTRACT: Interactions between the checkpoint abrogator UCN-01 and several pharmacological inhibitors of the mitogen-activated protein kinase (MAPK) kinase (MEK)/MAPK pathway have been examined in a variety of human leukemia cell lines. Exposure of U937 monocytic leukemia cells to a marginally toxic concentration of UCN-01 (e.g., 150 nM) for 18 h resulted in phosphorylation/activation of p42/44 MAPK. Coadministration of the MEK inhibitor PD184352 (10 microM) blocked UCN-01-induced MAPK activation and was accompanied by marked mitochondrial damage (e.g., cytochrome c release and loss of DeltaPsi(m)), caspase activation, DNA fragmentation, and apoptosis. Similar interactions were noted in the case of other MEK inhibitors (e.g., PD98059; U0126) as well as in multiple other leukemia cell types (e.g., HL-60, Jurkat, CCRF-CEM, and Raji). Coadministration of PD184352 and UCN-01 resulted in reduced binding of the cdc25C phosphatase to 14-3-3 proteins, enhanced dephosphorylation/activation of p34(cdc2), and diminished phosphorylation of cyclic AMP-responsive element binding protein. The ability of UCN-01, when combined with PD184352, to antagonize cdc25C/14-3-3 protein binding, promote dephosphorylation of p34(cdc2), and potentiate apoptosis was mimicked by the ataxia telangectasia mutation inhibitor caffeine. In contrast, cotreatment of cells with UCN-01 and PD184352 did not substantially increase c-Jun-NH(2)-terminal kinase activation nor did it alter expression of Bcl-2, Bcl-x(L), Bax, or X-inhibitor of apoptosis. However, coexposure of U937 cells to UCN-01 and PD184352 induced a marked increase in p38 MAPK activation. Moreover, SB203580, which inhibits multiple kinases including p38 MAPK, partially antagonized cell death. Lastly, although UCN-01 +/- PD184352 did not induce p21(CIP1), stable expression of a p21(CIP1) antisense construct significantly increased susceptibility to this drug combination. Together, these findings indicate that exposure of leukemic cells to UCN-01 leads to activation of the MAPK cascade and that interruption of this process by MEK inhibition triggers perturbations in several signaling and cell cycle regulatory pathways that culminate in mitochondrial injury, caspase activation, and apoptosis. They also raise the possibility that disrupting multiple signaling pathways, e.g., by combining UCN-01 with MEK inhibitors, may represent a novel antileukemic strategy.Cancer Research 08/2001; 61(13):5106-15. · 7.86 Impact Factor -
Article: The cyclin-dependent kinase inhibitor (CDKI) flavopiridol disrupts phorbol 12-myristate 13-acetate-induced differentiation and CDKI expression while enhancing apoptosis in human myeloid leukemia cells.
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ABSTRACT: Interactions between the cyclin-dependent kinase inhibitor (CDKI) flavopiridol (FP) and phorbol 12-myristate 13-acetate (PMA) were examined in U937 human leukemia cells in relation to differentiation and apoptosis. Simultaneous, but not sequential, exposure of U937 cells to 100 nM FP and 10 nM PMA significantly increased apoptosis manifested by characteristic morphological features, mitochondrial dysfunction, caspase activation, and poly(ADP-ribose) polymerase cleavage while markedly inhibiting cellular differentiation, as reflected by diminished plastic adherence and CD11b expression. Enhanced apoptosis in U937 cells was associated with an early caspase-independent increase in cytochrome c release and accompanied by a substantial decline in leukemic cell clonogenicity. Moreover, PMA/FP cotreatment significantly increased apoptosis in HL-60 promyelocytic leukemia cells and in U937 cells ectopically expressing the Bcl-2 protein. In U937 cells, coadministration of FP blocked PMA-induced expression and reporter activity of the CDKI p21WAF/CIP1 and triggered caspase-mediated cleavage of the CDKI p27KIP1. Coexposure to FP also resulted in a more pronounced and sustained activation of the mitogen-activated protein kinase kinase/extracellular signal-regulated protein kinase cascade after PMA treatment, although disruption of this pathway by the mitogen-activated protein kinase kinase 1 inhibitor U0126 did not prevent potentiation of apoptosis. FP accelerated PMA-mediated dephosphorylation of the retinoblastoma protein (pRb), an event followed by pRb cleavage culminating in the complete loss of underphosphorylated pRb (approximately Mr 110,000) by 24 h. Finally, gel shift analysis revealed that coadministration of FP with PMA for 8 h led to diminished E2F/pRb binding compared to the effects of PMA alone. Collectively, these findings indicate that FP modulates the expression/activity of multiple signaling and cell cycle regulatory proteins in PMA-treated leukemia cells and that such alterations are associated with mitochondrial damage and apoptosis rather than maturation. These observations also raise the possibility that combining CDKIs and differentiation-inducing agents may represent a novel antileukemic strategy.Cancer Research 04/2001; 61(6):2583-91. · 7.86 Impact Factor -
Article: Evidence of a functional role for the cyclin-dependent kinase inhibitor p21CIP1 in leukemic cell (U937) differentiation induced by low concentrations of 1-beta-D-arabinofuranosylcytosine.
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ABSTRACT: The functional role of the cyclin-dependent kinase inhibitor (CDKI) p21CIP1 in differentiation of human myelomonocytic leukemia cells (U937) exposed to low concentrations of the antimetabolite 1-beta-D-arabino-furanosylcytosine (ara-C) was examined utilizing a cell line stably expressing a p21CIP1 antisense construct. Continuous exposure to 50 nM ara-C led to marked induction of p21CIP1 at 48-72 h in empty-vector control cells but not in their antisense-expressing counterparts (p21AS/F4 and B8). Such treatment induced expression of the myelomonocytic differentiation marker CD11b in approximately 35% of control cells, but no evidence of maturation was noted in antisense-expressing lines. However, antisense-expressing cells exposed to low concentrations of ara-C exhibited a reciprocal increase in apoptosis, manifested by the appearance of cells with classic morphologic features and hypodiploid quantities of DNA, reduced mitochondrial membrane potential (deltapsim), an increase in cytochrome c release into the cytosol, cleavage/activation of procaspases-9 and -3, and degradation of PARP and p27Kip1. Whereas empty-vector control cells exposed to 50 nM ara-C exhibited a decline in Bcl-2 expression, dephosphorylation of pRb, and an initial accumulation in S-phase, antisense-expressing cells did not. However, c-Myc down-regulation induced by low concentrations of ara-C was, if anything, more complete in antisense-expressing cells. Exposure of control but not antisense-expressing cells to ara-C led to phosphorylation/activation of MAP kinase at 24 h; moreover, the specific MEK/MAP kinase inhibitor PD98059 enhanced low-dose ara-C-mediated apoptosis only in wild-type cells. Lastly, exposure to 50 nM ara-C for 72 h resulted in detectable levels of cytoplasmic p21CIP1, a phenomenon associated with resistance to apoptosis, only in empty vector controls. Collectively, these findings demonstrate a functional role for p21CIP1 in leukemic cell maturation induced by low concentrations of ara-C. They also indicate that, as in the case of more conventional differentiation-inducers such as phorbol esters, disruption of the p21CIP1 response after exposure to low concentrations of the cytotoxic drug ara-C prevents leukemic cells from engaging a maturation program, but instead directs them along an apoptotic pathway.Differentiation 09/2000; 66(1):1-13. · 2.81 Impact Factor -
Article: Inhibition of the mitogen activated protein kinase pathway potentiates radiation-induced cell killing via cell cycle arrest at the G2/M transition and independently of increased signaling by the JNK/c-Jun pathway.
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ABSTRACT: The ability of low dose ionizing radiation (2 Gy) to modulate the activities of the mitogen activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK1) cascades in human monocytic leukemia (U937/pREP4) cells and in cells over-expressing dominant negative c-Jun (TAM67) (U937/TAM67) was investigated. Radiation exposure caused prolonged ( approximately 1 h) MAPK activations in U937 cells. In contrast, low dose irradiation weakly modulated JNK1 activity in these cells. Inhibition of the MAPK pathway by use of the specific MEK1/2 inhibitor (10 microM PD98059) in both U937/pREP4 and U937/TAM67 cells prior to radiation exposure permitted strong prolonged radiation-induced activations of JNK1. Expression of TAM67 decreased the ability of radiation to cause apoptosis compared to control transfected cells. However, combined MEK1/2 inhibition and radiation exposure in both cell types caused a large decrease in suspension culture growth and a large increase in apoptosis, when compared to either treatment alone. Reduced proliferation after combined irradiation and PD98059 treatment in both cell types correlated with prolonged cell cycle arrest in G2/M phase. Prolonged growth arrest was abolished when MEK1/2 inhibitor was removed 6 h following irradiation, which was associated with a reduction in apoptosis. The ability of MEK1/2 inhibition to cause prolonged G2/M growth arrest was reduced in U937 cells stably transfected with a p21Cip-1/WAF1 antisense construct (U937/p21AS). This data correlated with an enhancement of radiation-induced apoptosis and a reduced ability of MEK1/2 inhibition to potentiate apoptosis. Collectively our data demonstrate that inhibition of MEK1/2 function increases the radiation sensitivity of U937 cells, independently of c-Jun function, and decreases the ability of these cells to recover from the radiation-induced G2/M cell cycle checkpoint arrest. In addition, our data also demonstrate that the ability of MEK1/2 inhibition to potentiate radiation-induced cell death in U937 cells in part requires an ability of cells to express low levels of p21Cip-1/WAF1.International Journal of Oncology 03/2000; 16(2):413-22. · 2.40 Impact Factor -
Article: Induction of apoptosis in U937 human leukemia cells by suberoylanilide hydroxamic acid (SAHA) proceeds through pathways that are regulated by Bcl-2/Bcl-XL, c-Jun, and p21CIP1, but independent of p53.
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ABSTRACT: Determinants of differentiation and apoptosis in myelomonocytic leukemia cells (U937) exposed to the novel hybrid polar compound SAHA (suberoylanilide hydroxamic acid) have been examined. In contrast to hexamethylenbisacetamide (HMBA), SAHA-related maturation was limited and accompanied by marked cytoxicity. SAHA-mediated apoptosis occurred within the G0G1 and S phase populations, and was associated with decreased mitochondrial membrane potential, caspase-3 activation, PARP degradation, hypophosphorylation/cleavage of pRB, and down-regulation of c-Myc, c-Myb, and B-Myb. Enforced expression of Bcl-2 or Bcl-XL inhibited SAHA-induced apoptosis, but only modestly potentiated differentiation. While SAHA induced the cyclin-dependent kinase inhibitor p21CIP1, antisense ablation of this CDKI increased, rather than decreased, SAHA-related lethality. In contrast, conditional expression of wild-type p53 failed to modify SAHA actions, but markedly potentiated HMBA-induced apoptosis. Finally, SAHA modestly increased expression/activation of the stress-activated protein kinase (SAPK/JNK); moreover, SAHA-related lethality was partially attenuated by a dominant-negative c-Jun mutant protein (TAM67). SAHA did not stimulate mitogen-activated protein kinase (MAPK), nor was lethality diminished by the specific MEK/MAPK inhibitor PD98059. These findings indicate that SAHA potently induces apoptosis in human leukemia cells via a pathway that is p53-independent but at least partially regulated by Bcl-2/Bcl-XL, p21CIP1, and the c-Jun/AP-1 signaling cascade.Oncogene 12/1999; 18(50):7016-25. · 6.37 Impact Factor -
Article: Bryostatin 1 enhances paclitaxel-induced mitochondrial dysfunction and apoptosis in human leukemia cells (U937) ectopically expressing Bcl-xL.
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ABSTRACT: The effects of the protein kinase C (PKC) activator and down-regulator bryostatin 1 were examined with respect to paclitaxel-induced apoptosis and antiproliferative activity in human myeloid leukemia cells (U937) displaying enforced expression of the anti-apoptotic protein Bcl-xL. Overexpression of Bcl-xL blocked various aspects of paclitaxel-mediated apoptosis, including caspase-3 activation, degradation of poly(ADP-ribose) polymerase (PARP), loss of mitochondrial membrane potential (Delta Psim), and release of cytochrome c. However, subsequent (but not prior) exposure of paclitaxel-treated U937/Bcl-xL cells (500 nM; 6 h) to bryostatin 1 (10 nM; 15 h) restored the extent of apoptosis, caspase activation, and mitochondrial damage to levels approximating those in paclitaxel-treated empty-vector control cells (U937/Neo). Potentiation of paclitaxel-induced apoptosis by bryostatin 1 in U937/Bcl-xL cells occurred primarily in the G2M cell population, and was associated with alterations in Bcl-xL gel mobility and a reduction in paclitaxel-mediated stimulation of CDK1 activity. Enhancement of paclitaxel-induced apoptosis by bryostatin 1 in Bcl-xL overexpressors was accompanied by a corresponding reduction in clonogenic potential. In contrast to its effects on apoptosis, bryostatin 1 failed to restore paclitaxel-mediated increases in free Bax levels in U937/Bcl-xL cells. Lastly, the actions of bryostatin 1 were mimicked by a pharmacologic inhibitor of the MEK1/MAP kinase pathway (PD98059), but not by SB203580, an inhibitor of p 38 MAP kinase. Moreover, sequential exposure of both U937/Neo or/Bcl-xL cells to paclitaxel followed by bryostatin 1 or PD98059 was associated with a net reduction in MAP kinase activity. Collectively, these findings indicate that protection against paclitaxel-mediated mitochondrial dysfunction and apoptosis in human U937 leukemia cells conferred by Bcl-xL overexpression can be substantially overcome by bryostatin 1 and possibly other agents that interrupt the MAP kinase signal transduction pathway.Leukemia 11/1999; 13(10):1564-73. · 9.56 Impact Factor -
Article: Induction of apoptosis and differentiation by fludarabine in human leukemia cells (U937): interactions with the macrocyclic lactone bryostatin 1.
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ABSTRACT: We have examined interactions between the purine nucleoside analog fludarabine (9-beta-arabinofuranosyl-2-fluoroadenine) and the macrocyclic lactone bryostatin 1 in the human monocytic leukemic cell line U937. Fludarabine exerted dose-dependent effects on U937 cell viability and growth which were associated with both induction of apoptosis, as well as cellular maturation. Incubation of cells with bryostatin 1 (10 nM; 24 h) after, but not before a 6-h exposure to 10 microM fludarabine resulted in a modest but significant increase in apoptosis, and was associated with greater than a 1 log reduction in clonogenicity. Subsequent exposure to bryostatin 1 also increased the percentage of fludarabine-treated cells displaying differentiation-related features (eg plastic adherence, CD11b positivity) compared to cells exposed to fludarabine alone. Bryostatin 1 did not increase the retention of the active fludarabine metabolite, F-ara-ATP, nor did it increase 3H-F-ara-A incorporation into DNA. Despite its capacity to trigger cellular maturation, fludarabine exposure (either with or without bryostatin 1) failed to induce the cyclin-dependent kinase inhibitors (CDKls) p21WAF1/CIP1 and p27KIP1. Nevertheless, dysregulation of p21 (resulting from stable transfection of cells with a p2lWAF1/CIP1 antisense construct) reduced fludarabine-mediated differentiation, while inducing a corresponding increase in apoptosis. Enforced expression of Bcl-2 partially protected cells from fludarabine-related apoptosis, an effect that was overcome, in part, by subsequent exposure of cells to bryostatin 1. Interestingly, Bcl-2-overexpressing cells were as or in some cases, more susceptible to differentiation induction by fludarabine (+/- bryostatin 1) than their empty vector-containing counterparts. Collectively, these results indicate that the antiproliferative effects of fludarabine toward U937 leukemic cells involve both induction of apoptosis and cellular maturation, and that each of these processes may be enhanced by bryostatin 1.Leukemia 08/1999; 13(7):1046-55. · 9.56 Impact Factor -
Article: Loss of the bcl-2 phosphorylation loop domain increases resistance of human leukemia cells (U937) to paclitaxel-mediated mitochondrial dysfunction and apoptosis.
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ABSTRACT: The impact of ectopic expression of an N-terminal phosphorylation loop deletant Bcl-2 protein (Bcl-2Delta32-80) on the response of U937 monoblastic leukemia cells to paclitaxel was examined. In contrast to recent findings in HL-60 cells (Fang et al., Cancer Res. 58, 3202, 1998), U937 cells overexpressing Bcl-2Delta32-80 were significantly more resistant than those overexpressing full-length protein to caspase-3 and -9 activation, PARP degradation, and apoptosis induced by paclitaxel (500 nM; 18 h). Bcl-2Delta32-80 was also more effective than its full-length counterpart in opposing paclitaxel-mediated mitochondrial dysfunction, e.g., loss of mitochondrial membrane potential (Deltapsim) and cytochrome c release into the cytoplasm. Enhanced resistance of U937/Bcl-2Delta32-80 cells to paclitaxel was observed primarily in the G2M population. Together, these findings demonstrate that deletion of the Bcl-2 phosphorylation loop domain increases resistance of U937 leukemia cells to paclitaxel-mediated mitochondrial damage and apoptosis and suggest that factors other than, or in addition to, phosphorylation contribute to Bcl-2-related cytoprotectivity against paclitaxel in this model system.Biochemical and Biophysical Research Communications 06/1999; 259(1):67-72. · 2.48 Impact Factor -
Article: Dysregulation of the cyclin-dependent kinase inhibitor p21WAF1/CIP1/MDA6 increases the susceptibility of human leukemia cells (U937) to 1-beta-D-arabinofuranosylcytosine-mediated mitochondrial dysfunction and apoptosis.
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ABSTRACT: The effects of dysregulation of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 on the apoptotic response of U937 monocytic leukemia cells to 1-beta-D-arabinofuranosylcytosine (ara-C) were examined. After a 6-h exposure to 1 microM ara-C, cells stably transfected with a p21WAF1/CIP1 antisense construct were significantly more sensitive to the induction of classic apoptotic morphology, DNA fragmentation, caspase-3 activation, poly(ADP-ribose) polymerase degradation, and underphosphorylation of the retinoblastoma protein (pRb) than their empty-vector counterparts. Enhanced susceptibility of antisense-expressing cells to ara-C was accompanied by a corresponding reduction in clonogenic and suspension culture growth. The increased sensitivity of these cells to ara-C-mediated lethality could not be attributed to cytokinetic perturbations, nor did ara-CTP formation or (ara-C)DNA incorporation differ significantly between the cell lines. Moreover, synchronization of p21 antisense-expressing cells in S-phase by aphidicolin block resulted in a further increase in ara-C-mediated apoptosis, suggesting enhanced drug sensitivity of the S-phase cell fraction. After exposure to ara-C, p21 antisense-expressing cells displayed a greater decline in mitochondrial membrane potential (deltapsi(m)) and generation of reactive oxygen species than their empty-vector counterparts, as well as early potentiation (e.g., within 2-4 h) of cytochrome c release into the cytosolic S-100 fraction. Lastly, ara-C-mediated increases in mitogen-activated protein kinase activity over basal levels were attenuated in p21 antisense-expressing cells. Collectively, these findings indicate that dysregulation of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 increases the susceptibility of U937 human leukemia cells to ara-C-related lethality, and this phenomenon occurs as a relatively early event that is independent of cell cycle or pharmacodynamic factors and is associated with mitochondrial perturbations implicated in activation of the apoptotic protease cascade.Cancer Research 04/1999; 59(6):1259-67. · 7.86 Impact Factor -
Article: Evidence of a functional role for the cyclin-dependent kinase inhibitor p21(WAF1/CIP1/MDA6) in the reciprocal regulation of PKC activator-induced apoptosis and differentation in human myelomonocytic leukemia cells.
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ABSTRACT: The functional role of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) in leukemic cell G1 arrest, differentiation, and apoptosis induced by two PKC activators (PMA and bryostatin 1) was examined using antisense-expressing lines [U937/p21AS(F4) and U937/p21AS(B8)]. Following incubation with 10 nM PMA (24 h), antisense-expressing cells displayed induction of p27(KIP1) but not of p21, whereas empty vector-containing cells (U937/pREP4) exhibited induction of both p21 and p27. Antisense-expressing cells were impaired in G1 arrest, dephosphorylation of the retinoblastoma protein, dephosphorylation and reduction in activity of cyclin-dependent kinase 2, and acquisition of differentiated features (e.g., plastic adherence). Bryostatin 1 induced p27 but not p21 in control cells and was less effective than PMA in initiating G1 arrest and related events. Nevertheless, disruption of p21 expression abrogated the effects of bryostatin 1 on cell cycle arrest and cellular maturation. Dysregulation of p21 did not, however, modify PMA- or bryostatin 1-mediated down-regulation of c-Myc protein. Unexpectedly, disruption of p21 failed to attenuate the net reduction in viable cell number following PMA or bryostatin 1 treatment inasmuch as impaired differentiation was accompanied by a lowered threshold for PMA- and bryostatin 1-induced apoptosis. Inhibition of p21 expression also promoted PMA- and bryostatin 1-mediated loss of mitochondrial transmembrane potential (DeltaPsim ) and release of cytochrome c into the cytosol. Together, these findings demonstrate a critical functional role for p21 in regulating myelomonocytic leukemic cell G1 arrest and differentiation following exposure to two PKC activators exhibiting disparate patterns of activity. They also suggest that following treatment with these agents, dysregulation of p21 prevents leukemic cells from engaging a normal differentiation program through a c-Myc-independent mechanism, and instead directs cells along an apoptotic pathway.Experimental Cell Research 11/1998; 244(1):105-16. · 3.58 Impact Factor -
Article: Effects of bryostatin 1 and calcium ionophore (A23187) on apoptosis and differentiation in human myeloid leukemia cells (HL-60) following 1-beta-D-arabinofuranosylcytosine exposure.
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ABSTRACT: The goal of the present study was to determine whether partial restoration of the differentiation-inducing capacity of the PKC activator bryostatin 1 by the calcium ionophore A23187 is accompanied by enhancement of apoptosis in ara-C-pretreated human leukemia cells. When HL-60 cells were exposed to ara-C (10 or 100 microM;6 h) followed by bryostatin 1 alone (10 nM; 24 h), no increase in apoptosis was noted. In contrast, subsequent exposure of ara-C-pretreated cells to A23187 (250 nM; 24 h) increased apoptosis by approximately 100%. When ara-C-pretreated cells were incubated with A23187 and bryostatin 1, no further potentiation of cell death (compared to cells exposed to A23187 alone) was observed. Nevertheless, the combination of bryostatin 1 and A23187 substantially increased inhibition of clonogenicity in cells preincubated with ara-C (e.g., by > or = 2 logs). This effect was associated with morphological and functional evidence (i.e., plastic adherence) of enhanced leukemic cell maturation. The differentiating capacity of the combination of bryostatin 1 and A23187 was significantly weaker than that of the phorbol diester, PMA (10 nM), and unaccompanied (at 24 h) by induction of the cyclin-dependent kinase inhibitors (CDKIs) p21WAF1/CIP1 and p27KIP1. However, the extent of apoptosis was comparable in cells exposed to ara-C followed by PMA or bryostatin 1 + A23187, suggesting that differentiation per se is not solely responsible for enhancement of cell death in ara-C-pretreated cells. Coadministration of bryostatin 1 and the organotellurium compound AS101, which mimics the actions of A23187 in some systems, after ara-C also led to enhanced antiproliferative effects which were unaccompanied by an increase in apoptosis. Finally, exposure of cells to ara-C followed by other differentiation-inducing agents, including dimethylsulfoxide and sodium butyrate also resulted in increases in cell death in this cell line. These findings indicate that the inability of bryostatin 1 to potentiate apoptosis in ara-C-pretreated HL-60 cells may involve factors other than an inadequate differentiation stimulus. They also suggest that loss of leukemic self-renewal capacity following exposure to cytotoxic and differentiation-inducing agents may involve mechanisms other than, or in addition to, potentiation of apoptosis, particularly cellular maturation.International Journal of Oncology 04/1998; 12(4):927-34. · 2.40 Impact Factor -
Article: Modulation of protein kinase C activity and calcium-sensitive isoform expression in human myeloid leukemia cells by bryostatin 1: relationship to differentiation and ara-C-induced apoptosis.
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ABSTRACT: Previous studies have shown that pretreatment of human myeloid leukemia cells (HL-60) with the protein kinase C (PKC) activator bryostatin 1 potentiates ara-C-induced apoptosis. To test the hypothesis that this capacity stems from down-regulation of PKC activity and/or Ca2+-dependent (group-I; cPKC) isoform expression, comparisons were made between the effects of this agent and the stage-2 tumor promoter mezerein under conditions favoring either cellular differentiation or drug-induced apoptosis. Twenty-four-hour pretreatment of HL-60 cells with 10 nM bryostatin 1, which does not induce differentiation in this cell line, led to a profound reduction in membrane and cytosolic PKC activity, decreased expression of cPKC isoforms (alpha, betaI, betaII, gamma), and a marked increase in ara-C induced apoptosis. In contrast, 10 nM mezerein, which induces HL-60 cell differentiation, was less effective in down-regulating membrane and cytosolic PKC activity as well as alpha, betaI, and gamma cPKC isoform expression, and failed to potentiate ara-C-related apoptosis. The effects of bryostatin 1 were dominant to those of mezerein, in that the combination resulted in down-regulation of PKC activity and expression and potentiation of ara-C-induced apoptosis, but not cellular maturation. However, coadministration of the Ca2+ ionophore A23187 (250 nM) restored bryostatin 1's differentiating ability while antagonizing its capacity to augment apoptosis, despite failing to reverse bryostatin 1-induced down-regulation of PKC activity and cPKC isoform expression. Furthermore, pretreatment of differentiation-responsive monocytic leukemia cells (U937) with bryostatin 1 substantially reduced PKC activity and cPKC isoform expression, but exerted minimal effects on ara-C-related apoptosis. In contrast, exposure of U937 cells to bryostatin 1 after ara-C dramatically increased apoptosis, a phenomenon that did not occur in differentiation-unresponsive HL-60 cells. Collectively, these observations indicate that down-regulation of total assayable PKC activity and cPKC expression by bryostatin 1 are insufficient, by themselves, to account for potentiation of leukemic cell apoptosis, at least under conditions in which differentiation occurs. They also provide further evidence that a reciprocal and highly schedule-dependent relationship exists between leukemic cell differentiation and drug-induced apoptosis.Experimental Cell Research 11/1996; 228(1):65-75. · 3.58 Impact Factor
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1998–2001
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Virginia Commonwealth University
- School of Medicine
Richmond, VA, USA -
Richmond VA Medical Center
Richmond, VA, USA
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