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Steven Grant

Publications and source records attributed to Steven Grant.

At least 91 records · Page 5Linked to original sources
PubMed2003

Enforced expression of the tumor suppressor p53 renders human leukemia cells (U937) more sensitive to 1-[beta-D-arabinofuranosyl]cytosine (ara-C)-induced apoptosis.

The effects of enforced expression of p53 on the sensitivity of p53(-/-) human monocytic leukemia cells (U937) to apoptosis following exposure to the S-phase-specific antimetabolite 1-[beta-D-arabinofuranosyl]cytosine (ara-C) were examined. Cells were stably transfected with a plasmid containing a chimeric DNA construct encoding a temperature-sensitive p53 variant (135(ala-->val)), which transactivates at 32 degrees but is non-functional at 37 degrees. A significant reduction in the S-phase population was observed in ptsp53 mutants incubated at 32 degrees. Nevertheless, while vector controls did not exhibit differential sensitivity to ara-C at 32 degrees versus 37 degrees, temperature-sensitive p53 mutants displayed a significant increase in apoptosis at the permissive temperature. This was not accompanied by increased ara-CTP formation, DNA incorporation of [3H]ara-C, or altered expression of Bcl-2 or Bax. Enhanced sensitivity was associated with increased mitochondrial injury (e.g. cytochrome c release), caspase activation, and loss of clonogenic survival. Significantly, ptsp53 cells synchronized in S phase were markedly more sensitive to ara-C-mediated mitochondrial injury and apoptosis at 32 degrees, indicating that wild-type p53 specifically enhances the susceptibility of this subpopulation to ara-C lethality. Consistent with these results, transient transfection of human wild-type p53 cDNA rendered parental U937 cells more sensitive to ara-C-mediated cell death. Collectively, these findings indicate that p53 expression renders S-phase U937 cells more susceptible to ara-C-mediated mitochondrial dysfunction, cytochrome c release, apoptosis, and loss of clonogenic survival without enhancing ara-C metabolism. Such findings raise the possibility that loss of functional p53 activity allows leukemia cells to circumvent ara-C lethality.

Antimetabolites, Antineoplastic↗
PubMed2003

Synergistic antileukemic interactions between 17-AAG and UCN-01 involve interruption of RAF/MEK- and AKT-related pathways.

Interactions between the protein kinase C (PKC) and Chk1 inhibitor UCN-01 and the heat shock protein 90 (Hsp90) antagonist 17-AAG have been examined in human leukemia cells in relation to effects on signal transduction pathways and apoptosis. Simultaneous exposure (30 hours) of U937 monocytic leukemia cells to minimally toxic concentrations of 17-AAG (eg, 400 nM) and UCN-01 (eg, 75 nM) triggered a pronounced increase in mitochondrial injury (ie, loss of mitochondrial membrane potential [Deltapsim]; cytosolic release of cytochrome c), caspase activation, and apoptosis. Synergistic induction of apoptosis was also observed in other human leukemia cell types (eg, Jurkat, NB4). Coexposure of human leukemia cells to 17-AAG and the PKC inhibitor bisindolylmaleimide (GFX) did not result in enhanced lethality, arguing against the possibility that the PKC inhibitory actions of UCN-01 are responsible for synergistic interactions. The enhanced cytotoxicity of this combination was associated with diminished Akt activation and marked down-regulation of Raf-1, MEK1/2, and mitogen-activated protein kinase (MAPK). Coadministration of 17-AAG and UCN-01 did not modify expression of Hsp90, Hsp27, phospho-JNK, or phospho-p38 MAPK, but was associated with further p34cdc2 dephosphorylation and diminished expression of Bcl-2, Mcl-1, and XIAP. In addition, inducible expression of both a constitutively active MEK1/2 or myristolated Akt construct, which overcame inhibition of ERK and Akt activation, respectively, significantly attenuated 17-AAG/UCN-01-mediated lethality. Together, these findings indicate that the Hsp90 antagonist 17-AAG potentiates UCN-01 cytotoxicity in a variety of human leukemia cell types and suggest that interference with both the Akt and Raf-1/MEK/MAP kinase cytoprotective signaling pathways contribute to this phenomenon.

Alkaloids↗
PubMed2003

Histone deacetylase inhibitors promote STI571-mediated apoptosis in STI571-sensitive and -resistant Bcr/Abl+ human myeloid leukemia cells.

Interactions between the Bcr/Abl kinase inhibitor STI571 (Gleevec, imatinib mesylate) and histone deacetylase inhibitors (HDIs) have been examined in STI571-sensitive and -resistant Bcr/Abl(+) human leukemia cells (K562 and LAMA 84). Cotreatment of K562 cells with 250 nM imatinib mesylate and 2.0 micro M suberoylanilide hydroxamic acid (SAHA) for 24 h, exposures that were minimally toxic alone, resulted in a marked increase in mitochondrial damage (e.g., cytochrome c, Smac/DIABLO, and apoptosis-inducing factor release), caspase activation, and apoptosis. Similar events were observed in other Bcr/Abl(+) cells (i.e., LAMA 84), and in cells exposed to STI571 in combination with the HDI sodium butyrate. Coexposure of cells to HDIs in conjunction with STI571 resulted in multiple perturbations in signaling and cell cycle-regulatory proteins, including down-regulation of Raf, phospho-mitogen-activated protein kinase kinase (MEK), phospho-extracellular signal-regulated kinase (ERK), phospho-Akt, phospho-signal transducers and activators of transcription 5, cyclin D1, and Mcl-1, accompanied by dephosphorylation and cleavage of retinoblastoma protein and a striking increase in phosphorylation of c-Jun NH(2)-terminal kinase. Coexposure of Bcr/Abl(+) cells to STI571 also blocked SAHA-mediated induction of p21(CIP1) and resulted in down-regulation of Bcr/Abl protein expression. STI571 and SAHA also interacted synergistically to induce apoptosis in STI571-resistant K562 and LAMA 84 cells that display increased Bcr/Abl protein expression. Lastly, inducible expression of a constitutively active MEK1/2 construct significantly attenuated SAHA/STI571-mediated apoptosis in K562 cells, implicating disruption of the Raf/MEK/ERK axis in synergistic antileukemic effects of this drug combination. Together, these findings indicate that combined exposure of Bcr/Abl(+) cells to the kinase inhibitor STI571 and HDIs leads to diverse perturbations in signaling and cell cycle-regulatory proteins, associated with a marked increase in mitochondrial damage and cell death. They also raise the possibility that this strategy may be effective in some Bcr/Abl(+) cells that are resistant to STI571 through increased Bcr/Abl expression.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2003

The lethal effects of pharmacological cyclin-dependent kinase inhibitors in human leukemia cells proceed through a phosphatidylinositol 3-kinase/Akt-dependent process.

The impact of disruption of the PI3K (phosphatidylinositol 3-kinase) pathway on the response of human leukemia cells to pharmacological cyclin-dependent kinase (CDK) inhibitors has been examined. Exposure of U937 monocytic leukemia cells to minimally toxic concentrations of flavopiridol (FP), roscovitine, or CGP74514A for 3 h in conjunction with the PI3K inhibitor LY294002 (abbreviated LY in the article) resulted in a marked decrease in Akt phosphorylation. Coexposure of cells to LY and CDK inhibitors also resulted in an early (i.e., within 3 h) and striking increase in mitochondrial damage [e.g., cytochrome c, second mitochondria-derived activator of caspases/direct inhibitor of apoptosis (IAP)-binding protein with low isoelectric point (Smac/DIABLO), and apoptosis-initiating factor (AIF) release], caspase activation, and apoptosis. Similar interactions were observed in a variety of other leukemia cell types (e.g., HL-60, Jurkat, Raji, and NB4). Apoptosis, induced by FP/LY, was substantially blocked by ectopic expression of Bcl-2, but to a considerably lesser extent by dominant-negative caspase-8. FP-induced apoptosis was not enhanced by agents that inhibited protein kinase (PK) A (H89), PKC (GFX), mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK1/2; U0126), p38 MAP kinase (MAPK; SB202190), m-target of rapamycin (TOR; rapamycin), or ataxia-telangiectasia mutation (ATM; caffeine), whereas the PI3K inhibitor wortmannin exerted effects similar to those of LY. The dramatic potentiation of CDK inhibitor-induced apoptosis by LY was accompanied by diminished Bad phosphorylation, induction of Bcl-2 cleavage, and down-regulation of X-linked IAP (XIAP) and Mcl-1. Cells exposed to CDK inhibitors + LY also exhibited reduced phosphorylation of glycogen synthase kinase (GSK)-3, forkhead transcription factor (FKHR), p70(S6K), and ERK, but increased activation of p34(cdc2) and p38 MAPK. LY/CDK inhibitor-treated cells also displayed diminished pRb dephosphorylation on CDK2- and CDK4-specific sites, retinoblastoma protein cleavage, and down-regulation of cyclin D(1). Inducible expression of constitutively active (myristolated) Akt significantly, albeit partially, attenuated apoptosis in Jurkat leukemia cells treated with either FP alone or the combination of FP and LY. Finally, cotreatment with LY and FP resulted in a dramatic increase in apoptosis in primary leukemic blasts obtained from a patient with acute myeloblastic leukemia. Together, these findings suggest that the PI3K/Akt pathway plays a major role in regulating the apoptotic response of human leukemia cells to pharmacological CDK inhibitors and raise the possibility that combined interruption of CDK- and PI3K-related pathways may represent a novel therapeutic strategy in hematological malignancies.

2-Aminopurine↗
PubMed2003

Induction of tumor necrosis factor by bryostatin 1 is involved in synergistic interactions with paclitaxel in human myeloid leukemia cells.

Interactions between the protein kinase C (PKC) activator/down-regulator bryostatin 1 and paclitaxel have been examined in human myeloid leukemia cells (U937) and in highly paclitaxel-resistant cells ectopically expressing a Bcl-2 phosphorylation loop-deleted protein (Delta Bcl-2). Treatment (24 hours) of wild-type cells with paclitaxel (eg, 5 to 20 nM) in combination with 10 nM bryostatin 1 induced a marked increase in mitochondrial damage (eg, cytochrome c and Smac/DIABLO [second mitochondria-derived activator of caspases/direct IAP binding protein with low pI] release), caspase activation, Bid cleavage, and apoptosis; moreover, bryostatin 1 circumvented the block to paclitaxel-induced mitochondrial injury and apoptosis conferred by ectopic expression of the loop-deleted protein. Coadministration of tumor necrosis factor (TNF) soluble receptors, or ectopic expression of CrmA or dominant-negative caspase-8, abrogated potentiation of paclitaxel-induced mitochondrial injury and apoptosis by bryostatin 1, implicating the extrinsic apoptotic pathway in this process. Similar events occurred in HL-60 leukemia cells. Potentiation of paclitaxel-induced apoptosis in wild-type and mutant cells by bryostatin 1 was associated with increases in TNF-alpha mRNA and protein and was mimicked by exogenous TNF-alpha. Coadministration of the selective PKC inhibitor GFX (1 microM) blocked the increase in TNF-alpha mRNA levels and apoptosis in bryostatin 1/paclitaxel-treated cells. Lastly, synchronization of cells in G(2)M increased their sensitivity to TNF-alpha-associated lethality. Collectively, these findings indicate that in U937 cells, bryostatin 1 promotes paclitaxel-mediated mitochondrial injury and apoptosis, and circumvents resistance to cell death conferred by loss of the Bcl-2 phosphorylation domain, through the PKC-dependent induction of TNF-alpha. They further suggest that this process is amplified by paclitaxel-mediated arrest of cells in G(2)M, where they are more susceptible to TNF-alpha-induced lethality.

Antineoplastic Agents↗
PubMed2003

The use of cyclin-dependent kinase inhibitors alone or in combination with established cytotoxic drugs in cancer chemotherapy.

Cyclin-dependent kinase (CDK) inhibitors are small molecule inhibitors of the kinases required for the orderly progression of cells, both normal and neoplastic, through the cell cycle. Because cell cycle dysregulation is such a common occurrence in neoplasia, the search for agents that might block cell cycle traverse has been the focus of intense interest. These efforts have led to the identification of a broad array of compounds that interfere directly with the function of CDKs. Two of these agents (flavopiridol and UCN-01) have now entered the clinical arena, and others are scheduled to do so in the near future. In preclinical studies, CDK inhibitors have shown the ability not only to block neoplastic cell proliferation, but also to induce, through a variety of mechanisms, programmed cell death. The latter capacity may stem from the diverse effects that CDK inhibitors exert on multiple kinases and apoptotic regulatory molecules. In addition, there is abundant preclinical evidence that CDK inhibitors can potentiate, generally in a dose- and sequence-dependent manner, the anti-tumor effects of many established cytotoxic agents. In clinical studies in humans, flavopiridol and UCN-01 have been shown to be tolerable, although clear evidence of single agent activity or enhancement of the efficacy of established agents has not yet emerged. This may reflect a failure to optimize drug schedules/pharmacokinetics, or to identify the critical molecular targets of these agents. Finally, in recent years, a rationale has emerged for combining CDK inhibitors with other molecularly targeted agents (i.e. differentiation-inducers and signal transduction modulators). Current research has basically two goals: (a). to identify CDK inhibitor concentrations and schedules that inhibit the growth of and induce apoptosis in specific tumor cell types; and (b). to establish a rational basis for combining CDK inhibitors with more conventional cytotoxic agents to enhance antitumor efficacy. This review gives a brief summary of such efforts, with an emphasis on agents and combinations that are in or near clinical development.

Animals↗
PubMed2003

Cyclin-dependent kinase inhibitors.

Cell-cycle dysregulation is one of the cardinal characteristics of neoplastic cells. For this reason, small molecule inhibitors targeting cyclin-dependent kinases (CDKs), of which flavopiridol is a prototype, have been the focus of extensive interest in cancer therapy. In addition to inhibiting cell-cycle progression, these agents exhibit a variety of other activities, including the induction of cell death. Recently, several novel mechanisms of action have been ascribed to the CDK inhibitor flavopiridol, including interference with transcription, most likely through disruption of P-TEFb (i.e. the CDK9/cyclin T complex), and induction of apoptosis, possibly a consequence of downregulation of various anti-apoptotic proteins. It has also been observed that combining CDK inhibitors with either conventional cytotoxic drugs or novel signal transduction modulators dramatically promotes neoplastic cell death in a variety of preclinical models. Efforts are underway to uncover inhibitors that selectively target specific CDKs and to develop these as a new generation of antitumour drugs. For all of these reasons, it is likely that interest in CDK inhibitors as antineoplastic agents will continue for the foreseeable future.

Animals↗
PubMed2003

Regulation of p21 and p27 expression by the hepatitis B virus X protein and the alternate initiation site X proteins, AUG2 and AUG3.

BACKGROUND: The hepatitis B virus X gene has three in-frame start codons encoding the pX, AUG2 and AUG3 proteins. The AUG2 and AUG3 genes are 5'-truncated in respect to the full-length pX gene; however, all three genes terminate at the same stop codon. The activity of pX as an oncogene is well characterized; however, less is known about the AUG2 and AUG3 proteins. METHODS: The effects of pX, AUG2 and AUG3 on p21Cip,1/WAF,1/MDA6 and p27Kip-1 cyclin kinase inhibitor (CKI) protein expression, and the impact they have on proliferation, were investigated in CHO K-1 cells. CHO K-1 cells were chosen because they can be transfected at 100% efficiency. RESULTS: p21- and p27-luciferase reporter expression is modulated by increasing doses of the hepatitis B X proteins. At low concentrations of pX or AUG2, p21- and p27-luciferase activity was increased, and at high concentrations, p21- and p27-luciferase activity was decreased. Expression of the AUG3 gene showed a different profile: it was increasingly stimulatory with dose for both promoters. Western blot analyses demonstrated that p21 and p27 protein levels were modulated as predicted based on data generated in the promoter-luciferase experiments. Tritiated thymidine labeling of DNA showed biphasic kinetics of incorporation in the presence of varying pX and AUG2 concentrations, whereas labeling decreased with AUG3 concentration. The growth inhibitory effect of pX expression was reduced by antisense ablation of either p21 or p27. CONCLUSIONS: The relative expression level of pX, AUG2, and AUG3 impacts on CKI expression and cell proliferation. Our findings may explain why divergent effects of pX expression on growth have been observed by different groups, which may be related to relative pX expression levels.

Animals↗
PubMed2003

Bryostatin 1 and UCN-01 potentiate 1-beta-D-arabinofuranosylcytosine-induced apoptosis in human myeloid leukemia cells through disparate mechanisms.

The effects of the PKC activator and down-regulator bryostatin 1 and the PKC and Chk1 inhibitor 7-hydroxystaurosporine (UCN-01) were compared with respect to potentiation of 1-beta-D-arabinofuranosylcytosine (ara-C)-induced apoptosis in human myelomonocytic leukemia cells (U937). Whereas bryostatin 1 and UCN-01 both markedly enhanced ara-C-induced mitochondrial injury (e.g., cytochrome c and Smac/DIABLO release, loss of mitochondrial membrane potential), caspase activation, and apoptosis, ectopic expression of an N-terminal loop-deleted Bcl-2 mutant protein protected cells from ara-C/UCN-01- but not ara-C/bryostatin 1-mediated lethality. Conversely, ectopic expression of CrmA or dominant-negative caspase-8 abrogated potentiation of ara-C-mediated apoptosis by bryostatin 1 but not by UCN-01. Exposure of cells to ara-C and bryostatin 1 (but not UCN-01) resulted in sustained release of tumor necrosis factor (TNF) alpha; moreover, potentiation of ara-C lethality by bryostatin 1 (but not by UCN-01) was reversed by coadministration of TNF soluble receptors or the selective PKC inhibitor bisindolylmaleimide (1 microM). Finally, similar events were observed in the human promyelocytic leukemia cell line HL-60. Together, these findings suggest that potentiation of ara-C lethality in human myeloid leukemia cells by bryostatin 1 but not UCN-01 involves activation of the extrinsic, receptor-mediated apoptotic pathway, and represents a consequence of bryostatin 1-mediated release of TNF-alpha. They also argue that the mechanism by which bryostatin 1 promotes ara-C-induced mitochondrial injury, caspase activation, and apoptosis involves factors other than or in addition to PKC down-regulation or modulation of Bcl-2 phosphorylation status.

Alkaloids↗
PubMed2003

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) promotes mitochondrial dysfunction and apoptosis induced by 7-hydroxystaurosporine and mitogen-activated protein kinase kinase inhibitors in human leukemia cells that ectopically express Bcl-2 and Bcl-xL.

Previous studies have demonstrated that cotreatment with mitogen activated-protein kinase kinase (MEK) 1/2 inhibitors (e.g., PD184352) and the checkpoint abrogator 7-hydroxystaurosporine (UCN-01) dramatically induces apoptosis in a variety of human leukemia and multiple myeloma cell types. The purpose of this study was to evaluate the roles of Bcl-2 family members and the relative contribution of the intrinsic mitochondrial versus the extrinsic receptor-related apoptotic pathways to MEK inhibitors/UCN-01-induced leukemic cell death. Cotreatment of U937 cells with PD184352 and UCN-01 resulted in the activation of procaspase-3, -9, and -8 as well as Bid cleavage. PD184352/UCN-01-induced mitochondrial dysfunction and apoptosis were both substantially attenuated in cells ectopically expressing Bcl-2, an N-terminal phosphorylation loop-deleted mutant Bcl-2, or Bcl-xL, but not in cells expressing dominant-negative (DN) caspase-8, cytokine response modifier A (cowpox virus-encoded antiapoptotic protein), or DN Fas-associated death domain. Coadministration of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) or TNF-alpha substantially increased MEK inhibitors (e.g., PD184352 or U0126)/UCN-01-induced mitochondrial dysfunction, activation of procaspase-8 and Bid, and apoptosis in Bcl-2- and Bcl-xL-overexpressing cells but not in those in which the extrinsic pathway was interrupted. Together, these findings suggest that the MEK inhibitors/UCN-01 regimen primarily induces leukemic cell apoptosis by engaging the intrinsic, mitochondrial apoptotic pathway and that resistance to these events conferred by increased expression of certain antiapoptotic Bcl-2 family members can be overcome, at least in part, by coadministration of TRAIL and other agents that activate the extrinsic apoptotic cascade.

Apoptosis↗
PubMed2003

Bile acid regulation of C/EBPbeta, CREB, and c-Jun function, via the extracellular signal-regulated kinase and c-Jun NH2-terminal kinase pathways, modulates the apoptotic response of hepatocytes.

Previously, we have demonstrated that deoxycholic acid (DCA)-induced signaling of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in primary hepatocytes is a protective response. In the present study, we examined the roles of the ERK and c-Jun NH(2)-terminal kinase (JNK) pathways, and downstream transcription factors, in the survival response of hepatocytes. DCA caused activation of the ERK1/2 and JNK1/2 pathways. Inhibition of either DCA-induced ERK1/2 or DCA-induced JNK1/2 signaling enhanced the apoptotic response of hepatocytes. Further analyses demonstrated that DCA-induced JNK2 signaling was cytoprotective whereas DCA-induced JNK1 signaling was cytotoxic. DCA-induced ERK1/2 activation was responsible for increased DNA binding of C/EBPbeta, CREB, and c-Jun/AP-1. Inhibition of C/EBPbeta, CREB, and c-Jun function promoted apoptosis following DCA treatment, and the level of apoptosis was further increased in the case of CREB and c-Jun, but not C/EBPbeta, by inhibition of MEK1/2. The combined loss of CREB and c-Jun function or of C/EBPbeta and c-Jun function enhanced DCA-induced apoptosis above the levels resulting from the loss of either factor individually; however, these effects were less than additive. Loss of c-Jun or CREB function correlated with increased expression of FAS death receptor and PUMA and decreased expression of c-FLIP-(L) and c-FLIP-(S), proteins previously implicated in the modulation of the cellular apoptotic response. Collectively, these data demonstrate that multiple DCA-induced signaling pathways and transcription factors control hepatocyte survival.

Amino Acid Sequence↗
PubMed2003

Stress and radiation-induced activation of multiple intracellular signaling pathways.

Exposure of cells to a variety of stresses induces compensatory activations of multiple intracellular signaling pathways. These activations can play critical roles in controlling cell survival and repopulation effects in a stress-specific and cell type-dependent manner. Some stress-induced signaling pathways are those normally activated by mitogens such as the EGFR/RAS/PI3K-MAPK pathway. Other pathways activated by stresses such as ionizing radiation include those downstream of death receptors, including pro-caspases and the transcription factor NFKB. This review will attempt to describe some of the complex network of signals induced by ionizing radiation and other cellular stresses in animal cells, with particular attention to signaling by growth factor and death receptors. This includes radiation-induced signaling via the EGFR and IGFI-R to the PI3K, MAPK, JNK, and p38 pathways as well as FAS-R and TNF-R signaling to pro-caspases and NFKB. The roles of autocrine ligands in the responses of cells and bystander cells to radiation and cellular stresses will also be discussed. Based on the data currently available, it appears that radiation can simultaneously activate multiple signaling pathways in cells. Reactive oxygen and nitrogen species may play an important role in this process by inhibiting protein tyrosine phosphatase activity. The ability of radiation to activate signaling pathways may depend on the expression of growth factor receptors, autocrine factors, RAS mutation, and PTEN expression. In other words, just because pathway X is activated by radiation in one cell type does not mean that pathway X will be activated in a different cell type. Radiation-induced signaling through growth factor receptors such as the EGFR may provide radioprotective signals through multiple downstream pathways. In some cell types, enhanced basal signaling by proto-oncogenes such as RAS may provide a radioprotective signal. In many cell types, this may be through PI3K, in others potentially by NFKB or MAPK. Receptor signaling is often dependent on autocrine factors, and synthesis of autocrine factors will have an impact on the amount of radiation-induced pathway activity. For example, cells expressing TGFalpha and HB-EGF will generate protection primarily through EGFR. Heregulin and neuregulins will generate protective signals through ERBB4/ERBB3. The impact on radiation-induced signaling of other autocrine and paracrine ligands such as TGFbeta and interleukin 6 is likely to be as complicated as described above for the ERBB receptors.

Animals↗
PubMed2003

Protein kinase C-dependent activation of the tumor necrosis factor receptor-mediated extrinsic cell death pathway underlies enhanced apoptosis in human myeloid leukemia cells exposed to bryostatin 1 and flavopiridol.

Interactions between the protein kinase C activator bryostatin 1 and the cyclin-dependent kinase (CDK) inhibitor flavopiridol (FP) have been examined in human myeloid leukemia cells (U937 and HL-60). Previous studies have demonstrated synergistic induction of apoptosis in leukemic cells exposed to the potent differentiation-inducer phorbol 12-myristate 13-acetate (PMA) in conjunction with FP [L. Cartee et al., Cancer Res., 61: 2583-2591, 2001]. Although bryostatin 1 (10 nM) is a very weak inducer of differentiation compared with PMA in these cells, coadministration of a minimally toxic concentration of FP (100 nM) did not promote bryostatin 1-related maturation but instead caused a marked increase in mitochondrial damage (e.g., cytochrome c release; loss of Deltapsi(m)), caspase activation, poly(ADP-ribose) polymerase cleavage, and apoptosis. Bryostatin 1/FP-induced apoptosis was significantly diminished in cells ectopically expressing dominant-negative Fas-associated death domain or by coadministration of tumor necrosis factor (TNF)-alpha soluble receptors, implicating the extrinsic pathway in bryostatin 1/FP actions. Enhanced apoptosis in bryostatin 1/FP-treated cells was accompanied by down-regulation of Mcl-1 and a sustained increase in TNF-alpha release. The selective protein kinase C inhibitor GFX blocked TNF-alpha and cytochrome c release in bryostatin 1/FP-treated cells and attenuated apoptosis. Finally, coadministration of bryostatin 1 (or PMA) with FP induced a marked increase in apoptosis in U937 cells ectopically expressing an NH(2)-terminal phosphorylation loop-deleted Bcl-2 protein, which are otherwise highly resistant to FP-mediated lethality. Taken together, these findings suggest that synergistic induction of apoptosis by bryostatin 1 and FP does not stem from disruption of the leukemic cell maturation process but instead results from enhanced release of TNF-alpha and activation of the extrinsic apoptotic cascade, culminating in cell death.

Antineoplastic Agents↗
PubMed2003

MDA-7 (interleukin-24) inhibits the proliferation of renal carcinoma cells and interacts with free radicals to promote cell death and loss of reproductive capacity.

The median survival of metastatic renal cell carcinoma (RCC) is 12 months, and the majority of treatment options are palliative. MDA-7 (interleukin-24), when expressed via a recombinant replication defective adenovirus, Ad.mda-7, has profound antiproliferative and cytotoxic effects in a wide variety of tumor cells but not in nontransformed cells. The studies in this study examined the impact of MDA-7 on RCC proliferation and survival. RCC lines (A498 and UOK121N), but not primary renal epithelial cells, were resistant to adenoviral infection that correlated with a lack of coxsackievirus and adenovirus receptor expression. Additional studies were performed using purified preparations of bacterially synthesized glutathione S-transferase (GST)-MDA-7 protein. GST-MDA-7, but not GST, caused a dose-dependent inhibition of RCC proliferation but not of primary renal epithelial cells. Clinically achievable concentrations of the novel therapeutic agent arsenic trioxide (0.5-1 micro M) were found to have little effect on RCC growth. However, the combination of GST-MDA-7 and arsenic trioxide resulted in a greater than additive reduction in cell growth that correlated with a large increase in tumor cell death. The free radical scavenger N-acetyl cysteine abolished the potentiating effect of arsenic trioxide. Although pro-caspase 3, poly(ADP-ribose) polymerase, and Bcl-(XL) levels, as well as nucleosomal DNA integrity, were reduced by combined treatment, cell killing was predominantly nonapoptotic. Combined treatment of RCC lines with GST-MDA-7 and arsenic trioxide also resulted in a substantial reduction in clonogenic survival compared with either treatment individually. Collectively, these findings demonstrate that MDA-7 protein, in combination with agents that generate free radicals, may have potential in the treatment of RCC.

Acetylcysteine↗
PubMed2003

Simultaneous activation of the intrinsic and extrinsic pathways by histone deacetylase (HDAC) inhibitors and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) synergistically induces mitochondrial damage and apoptosis in human leukemia cells.

Interactions between histone deacetylase (HDAC) inhibitors and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), also known as Apo2 ligand, were examined in human leukemia cells (e.g., U937, Jurkat, and HL-60). Simultaneous exposure of cells to 100-ng/ml TRAIL with either 1-mM sodium butyrate or 2- micro M suberoylanilide hydroxamic acid resulted in a striking increase in leukemic cell mitochondrial damage, caspase activation, and apoptosis. Lethal effects were significantly diminished in U937 cells ectopically expressing dominant-negative caspase-8, dominant-negative Fas-associated death domain, CrmA (receptor pathway), or Bcl-2 or Bcl-X(L) (mitochondrial pathway). Analysis of mitochondrial events in U937 cells exposed to TRAIL/HDAC inhibitors revealed enhanced Bid activation and Bax translocation, loss of mitochondrial membrane potential, and cytoplasmic release of cytochrome c, Smac/DIABLO, and apoptosis-inducing factor. No changes were observed in expression of FLICE-like inhibitory protein, TRAIL receptors, or reactive oxygen species generation. TRAIL/HDAC inhibitor-induced apoptosis triggered caspase-dependent cleavage of p21(WAF1/CIP1); moreover, enforced expression of a nuclear localization signal deletant form of p21(WAF1/CIP1) significantly diminished lethality. Lastly, p27(KIP1), pRb, X-linked inhibitor of apoptosis, and Bcl-2 displayed extensive proteolysis. These findings indicate that coadministration of TRAIL with HDAC inhibitors synergistically induces apoptosis in human myeloid leukemia cells and provide further evidence that simultaneous activation of the extrinsic and intrinsic pathways in such cells leads to a dramatic increase in mitochondrial injury and activation of the caspase cascade.

Apoptosis↗
PubMed2003

Apoptosis regulators.

Over the last decade, a great deal of attention has been directed at elucidating the role of apoptosis regulators in governing survival decisions in neoplastic cells, particularly those of hematopoietic origin. A major focus of this work has involved investigation of the function of pro- and anti-apoptotic members of the BCL-2 family, and the relationship between these proteins and mitochondrial integrity. Currently, these proteins can be classified into two broad categories: those that modulate mitochondrial function and those that regulate the activation of caspases responsible for activation and execution of the apoptotic cascade. Within the first category, certain proteins (e.g., BCL-2, BCL-xL) act to preserve mitochondrial integrity by preventing loss of mitochondrial membrane potential and/or release of pro-apoptotic proteins such as cytochrome C into the cytosol. Other proapoptotic proteins (e.g., BAX, BAK, BIM) promote release of cytochrome C. These proteins are therefore primarily involved in regulation of the intrinsic, mitochondrial apoptotic pathway. Within the second category, proteins such as the inhibitors of apoptosis proteins (e.g., XIAP) or FLIP block the activation of caspases, particularly those involved in engagement of the receptor-related, extrinsic apoptotic pathway. Cross-talk between the intrinsic and extrinsic pathways exists. For example, the BH3-domain only protein BID is cleaved by the activation of pro-caspase-8 through the extrinsic pathway, and translocates to the mitochondrion to promote cytochrome C release. Apoptosis is also regulated by various signal transduction pathways, possibly through post-translational modifications in BCL-2 family proteins. For example, phosphorylation of BCL-2 through a JNK-dependent mechanism has been postulated to contribute to apoptosis induced by the taxane class of cytotoxic agents. Finally, attempts to modulate apoptotic pathways with small molecules have recently received much attention. For example, small molecule inhibitors of BCL-2 or mimetics of SMAC/DIABLO, which opposes the actions of XIAP, have recently been shown to promote the antineoplastic activity of conventional cytotoxic agents. It is likely that an improved understanding of apoptosis regulation will lead to new insights into neoplastic transformation, and may also provide important leads for the development of novel antileukemic strategies.

Animals↗
PubMed2002

Combined treatment with the checkpoint abrogator UCN-01 and MEK1/2 inhibitors potently induces apoptosis in drug-sensitive and -resistant myeloma cells through an IL-6-independent mechanism.

The effects of combined exposure to the checkpoint abrogator UCN-01 and pharmacologic MEK1/2 inhibitors were examined in human multiple myeloma (MM) cell lines. Treatment of RPMI8226, NCI-H929, and U266 MM cells with a minimally toxic concentration of UCN-01 (150 nM) for 24 hours resulted in mitogen-activated protein (MAP) kinase activation, an effect that was blocked by coadministration of the MEK1/2 inhibitor PD184352. These events were accompanied by enhanced activation of p34(cdc2) and a marked increase in mitochondrial damage (loss of DeltaPsim; cytochrome c and Smac/DIABLO (direct IAP binding protein with low pI) release), poly(ADP-ribose) polymerase (PARP) cleavage, and apoptosis. PD184352/UCN-01 also dramatically reduced clonogenic survival in each of the MM cell lines. In contrast to As(2)0(3), apoptosis induced by PD184352/UCN-01 was not blocked by the free-radical scavenger N-acetyl-L-cysteine. Whereas exogenous interleukin 6 substantially prevented dexamethasone-induced lethality in MM cells, it was unable to protect them from PD184352/UCN-01-induced apoptosis despite enhancing Akt activation. Insulinlike growth factor 1 (IGF-1) also failed to diminish apoptosis induced by this drug regimen. MM cell lines selected for a high degree of resistance to doxorubicin, melphalan, or dexamethasone, or displaying resistance secondary to fibronectin-mediated adherence, remained fully sensitive to PD184352/UCN-01-induced cell death. Finally, primary CD138(+) MM cells were also susceptible to UCN-01/MEK inhibitor-mediated apoptosis. Together, these findings suggest that simultaneous disruption of cell cycle and MEK/MAP kinase signaling pathways provides a potent stimulus for mitochondrial damage and apoptosis in MM cells, and also indicate that this strategy bypasses the block to cell death conferred by several other well-described resistance mechanisms.

Alkaloids↗