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Experimental Therapeutics |
Departments of Medicine [C. Y., M. R., J. A., G. K., S. G.], Microbiology [G. K., D. C., S. G.], Human Genetics [M. S.], Pharmacology [S. G.], and Radiation Oncology [P. D.], Virginia Commonwealth University, Medical College of Virginia, Richmond, Virginia 23298, and Department of Medicine, Boston University School of Medicine, Boston, Massachusetts [L. V.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B, STAT5, MEK/MAPK, Bcl-xL, and Akt, among others (6, 7, 8, 9, 10)
. Recently, the treatment of CML has been revolutionized by the introduction of STI571 (Gleevec, imatinib mesylate), a p.o. active tyrosine kinase inhibitor that inhibits Bcr/Abl, c-Kit, platelet-derived growth factor, and other kinases (11) . STI571 interferes with the growth of Bcr/Abl+ leukemia cells and induces apoptosis in Bcr/Abl+ leukemia cells in vitro (11 , 12) . Significantly, oral administration of STI571 to CML patients results in clinical responses in >90% patients (13) . However, the emergence of STI571 resistance in CML patients initially responsive to this agent (14) and the observation that patients in accelerated phase CML or blast crisis are less likely to respond to imatinib mesylate (15) have prompted the search for additional approaches to the treatment of this disease. Mechanisms of resistance to STI571 include diminished drug uptake, Bcr/Abl amplification, and mutations in the Bcr/Abl kinase domain, among others (16, 17, 18) . One possible approach to this problem involves the combination of STI571 with other agents that exhibit antileukemic activity. In this regard, increased activity against Bcr/Abl+ leukemic cells has been described when STI571 was combined with conventional cytotoxic drugs (19, 20, 21) , arsenic trioxide (22) , geldanamycin (23) , or tumor necrosis factor apoptosis-inducing ligand (24) . Most recently, our group has described synergistic interactions between STI571 and pharmacological MEK1/2 inhibitors (e.g., PD184351 and U0126) or the CDKI flavopiridol in Bcr/Abl+ cells, including those resistant to STI571 due to increased Bcr/Abl protein expression (25 , 26) .
HDIs, including trichostatin A, short chain fatty acids such as sodium and phenylbutyrate, SAHA, depsipeptide, MS-275, and aphicidin, among others, represent a novel class of agents that act by promoting histone acetylation, resulting in relaxation of the chromatin structure (27) . Chromatin relaxation and uncoiling permit the expression of diverse genes, including those involved in the differentiation process [e.g., p21CIP1 (28) ]. In fact, HDIs (e.g., SAHA and SB) have been shown to induce maturation in various human leukemia cell lines (29 , 30) . Under some circumstances, HDIs induce apoptosis rather than maturation in human leukemia cells (29 , 31 , 32) , although the factors that determine which response predominates remain obscure. HDIs also induce maturation in certain Bcr/Abl+ leukemia cells (e.g., K562), a phenomenon associated with diminished activation of the MAPK pathway (33) . In view of evidence that STI571 may modify the differentiation response of Bcr/Abl+ cells (34) , the possibility arose that combining STI571 with HDIs might lead to enhanced antileukemic activity. To address this issue, we have examined interactions between STI571 and clinically relevant HDIs, i.e., SB and SAHA. Here we report that coadministration of HDIs with STI571 in several CML cell lines (e.g., K562 and LAMA 84) results in disruption of multiple signaling pathways, induction of mitochondrial injury, and a dramatic potentiation of apoptosis. Moreover, this drug combination potently induces cell death in STI571-resistant Bcr/Abl+ cells displaying increased Bcr/Abl expression. Together, these findings suggest that the strategy of combining STI571 with clinically relevant HDIs warrants consideration in CML and related hematological malignancies.
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MATERIALS AND METHODS |
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4 x 105 cells/ml). Multidrug-resistant K562R cells were derived from the parental line by subculturing in progressively higher concentrations of doxorubicin as described previously (35) . They were cultured in the absence of doxorubicin before all of the experimental procedures. In addition, STI571-resistant LAMA 84 cells, designated LAMA 84-R-STI, were generated by subculturing LAMA 84 cells in progressively higher concentrations of STI571. These cells are maintained under selection pressure in medium containing 0.5 µM STI571. IC50 STI571 values for LAMA 84S and LAMA 84-R-STI are 0.3 versus 1.8 µM, respectively. For studies involving the LAMA 84R line, cells were washed free of drug and resuspended in drug-free medium 48 h before experimentation.
Reagents.
STI571 was kindly provided by Dr. Elizabeth Buchdunger (Novartis Pharmaceuticals, Basel, Switzerland) and prepared as a 10 mM stock solution in sterile DMSO (Sigma, St. Louis, MO). SB and SAHA were obtained from Calbiochem (San Diego, CA); BOC-fmk and IETD-fmk were purchased from Enzyme Products, Ltd. (Livermore, CA) and formulated in sterile DMSO before use.
Experimental Format.
Logarithmically growing cells were placed in sterile plastic T-flasks (Corning, Corning, NY), to which were added the designated drugs, and the flasks were replaced in the incubator for intervals. At the end of the incubation period, cells were transferred to sterile centrifuge tubes, pelleted by centrifugation at 400 x g for 10 min at room temperature, and prepared for analysis as described below.
Assessment of Apoptosis.
After drug exposures, cytocentrifuge preparations were stained with Wright-Giemsa and viewed by light microscopy to evaluate the extent of apoptosis (i.e., cell shrinkage, nuclear condensation, formation of apoptotic bodies, and so forth) as described previously (25)
. For these studies, the percentage of apoptotic cells was determined by evaluating 
500 cells/condition in triplicate. To confirm the results of morphological analysis, annexin V/PI staining was used. Annexin V/PI (BD PharMingen, San Diego, CA) analysis of cell death was carried out as per the manufacturer’s instructions. Analysis was carried out using a Becton Dickinson FACScan cytofluorometer (Mansfield, MA). To further confirm the morphology results, TUNEL staining was used. For TUNEL staining, cytocentrifuge preparations were obtained and fixed with 4% formaldehyde. The slides were treated with acetic acid:ethanol (1:2), stained with terminal deoxynucleotidyl transferase reaction mixture containing 1x terminal deoxynucleotidyl transferase reaction buffer (0.25 units/µl terminal deoxynucleotidyl transferase, 2.5 mM CoCl2, and 2 pmol of fluorescein-12-dUTP; Boehringer Mannheim, Indianapolis, IN), and visualized using fluorescence microscopy.
Determination of MMP (
m).
MMP was monitored using DiOC6 (36)
. For each condition, 4 x 105 cells were incubated for 15 min at 37°C in 1 ml of 40 nM DiOC6 (Calbiochem) and subsequently analyzed using a Becton Dickinson FACScan cytofluorometer with excitation and emission settings of 488 and 525 nm, respectively. Control experiments documenting the loss of m were performed by exposing cells to 5 µM carbamoyl cyanide m-chlorophenylhydrazone (Sigma; 15 min, 37°C), an uncoupling agent that abolishes the MMP.
Preparation of S-100 Fractions and Assessment of Cytochrome c Release.
U937 cells were harvested after drug treatment as described previously (37)
by centrifugation at 600 x g for 10 min at 4°C and washed in PBS. Cells (4 x 106) were lysed by incubation for 3 min in 100 µl of lysis buffer containing 75 mM NaCl, 8 mM Na2HPO4, 1 mM NaH2PO4, 1 mM EDTA, and 350 µg/ml digitonin. The lysates were centrifuged at 12,000 x g for 5 min, and the supernatant was collected and added to an equal volume of 2x Laemmli buffer. The protein samples were quantified and separated by 15% SDS-PAGE.
Immunoblot Analysis.
Immunoblotting was performed as described previously (25)
. In brief, after drug treatment, cells were pelleted by centrifugation, lysed immediately in Laemmli buffer [1x Laemmli buffer = 30 mM Tris base (pH 6.8), 2% SDS, 2.88 mM ß-mercaptoethanol, and 10% glycerol], and sonicated briefly. Homogenates were quantified using Coomassie Blue protein assay reagent (Pierce, Rockford, IL). Equal amounts of protein (20 µg) were boiled for 10 min, separated by SDS-PAGE (5% stacker and 10% resolving), and electroblotted to nitrocellulose membrane. After blocking in TBS-T (0.05%) and 5% milk for 1 h at 22°C, the blots were incubated in fresh blocking solution with an appropriate dilution of primary antibody for 4 h at 22°C. The sources of antibodies were as follows: Bcl-xL (rabbit polyclonal antibody), Santa Cruz Biotechnology; XIAP (rabbit polyclonal antibody), R&D Systems (Minneapolis, MN); Mcl-1 (mouse monoclonal antibody), PharMingen (San Diego, CA); cyclin D1 (mouse monoclonal antibody) and p21 (mouse monoclonal antibody), PharMingen; ERK1/2 (rabbit polyclonal antibody), Cell Signaling Technology (Beverly, MA); phospho-ERK1/2 (Thr202/Tyr204; rabbit polyclonal antibody), Cell Signaling Technology; JNK (rabbit polyclonal antibody), Santa Cruz Biotechnology; phospho-JNK (mouse monoclonal antibody), Santa Cruz Biotechnology; phospho-p38 MAPK (rabbit polyclonal antibody), Cell Signaling Technology; phospho-p70S6K(421/424) (rabbit polyclonal antibody), Cell Signaling Technology; phospho-STAT5, Cell Signaling Technology; pRB (mouse monoclonal antibody), PharMingen; underphosphorylated pRB (mouse monoclonal antibody), PharMingen; caspase 3 (mouse monoclonal antibody), Transduction Laboratories (Lexington, KY); PARP (C-2–10; mouse monoclonal antibody), BioMol Research Laboratories (Plymouth, MA); cytochrome c (mouse monoclonal antibody), Santa Cruz Biotechnology; AIF (mouse monoclonal antibody), Santa Cruz Biotechnology; Smac (rabbit polyclonal antibody), Upstate Biotechnology (Lake Placid, NY); caspase 8 (rabbit polyclonal antibody), PharMingen; and 
-tubulin, Calbiochem. Blots were washed three times (15 min each) in TBS-T and then incubated with a 1:2000 dilution of horseradish peroxidase-conjugated secondary antibody (Bio-Rad Laboratories, Hercules, CA) for 1 h at 22°C. Blots were again washed three times (15 min each) in TBS-T and then developed by enhanced chemiluminescence (Pierce).
Clonogenic Survival.
Effects of drug treatment on the clonogenic survival of leukemia cells were determined using a previously described clonogenic assay (36)
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Transient Transfections.
Plasmids encoding enhanced GFP under the transcriptional control of the human cytomegalovirus immediate-early promoter (pEGFP-C2) and hemagglutinin-tagged activated MEK1 (S218D/S222D in pUSEEamp) were obtained from Clontech Laboratories (Palo Alto, CA) and Upstate Biotechnology, respectively. A 1285-bp fragment containing the MEK1 cDNA was obtained by ApaI and partial EcoRI digestion and inserted in-frame into the COOH-terminal multiple cloning site of pEGFP-C2. The entire MEK1 cDNA in the fusion construct was sequenced, and the reading frame was confirmed. Log-phase K562 cells were transfected in electoporation hypo-osmolar buffer (Eppendorf) using a BTX electromanipulator 600. Twenty µg of DNA and 2.0 x 107 cells were used for each condition. After 12 h of incubation, 20–30% of the cells displayed green fluorescence. Cells transfected so as to express GFP or GFP fusion proteins were filtered through a nylon mesh to remove cell aggregates, and the GFP+ population was sorted by using a MoFLO (DakoCytomation, Fort Collins, CO) cytometer. Cells were also gated using forward and side scatter for viable cells and pulse width to remove doublets. Final purity was 95% or greater with respect to GFP expression. Viability of this population was also consistently >95%. The cells were then exposed to drugs as indicated and examined for morphological evidence of apoptosis as described above.
Statistical Analysis.
The significance of differences between experimental conditions was determined using the two-tailed Student t test. Analysis of synergism and antagonism was performed using median dose effect analysis (38)
in conjunction with a commercially available software program (Calcysyn; Biosoft, Ferguson, MO), as we have described previously (25)
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RESULTS |
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. Exposure of cells for 24 h to STI571 concentrations as high as 300 nM negligibly induced apoptosis, whereas 2.0 µM SAHA administered alone was also minimally toxic (Fig. 1A)
. However, when cells were exposed to SAHA in combination with 100 nM STI571, a clear increase in apoptosis was observed (i.e., 
20%), and for STI571 concentrations 250 nM, the large majority of cells (i.e., 75%) were apoptotic. Similarly, when cells were exposed for 24 h to 250 nM STI571 in combination with increasing concentrations of SAHA, a sharp increase in apoptosis was noted at 1.0 µM SAHA, and at SAHA concentrations 1.5 µM, the majority of cells were apoptotic (Fig. 1B)
. Median dose effect analysis of apoptosis induction over a range of STI571 and SAHA concentrations yielded CI values < 1.0, corresponding to a synergistic interaction (Fig. 1C)
. Parallel results were obtained when DNA strand breaks were assayed by TUNEL analysis (data not shown).
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. Whereas each of these agents administered individually over 48 h minimally induced apoptosis, combined treatment resulted in an increase in apoptosis that was first observed at 12 h and that reached near-maximal levels by 24 h (Fig. 2A)
. After 48 h of combined treatment, >90% of cells were apoptotic. Similar results were observed when loss of MMP (m) was monitored, although STI571 by itself was somewhat more toxic in this regard after 48 h of exposure (Fig. 2B)
. Together, these findings indicate that combined treatment with STI571 and the HDI SAHA results in early induction of mitochondrial injury and apoptosis in Bcr/Abl+ K562 cells.
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. Whereas a 24-h exposure to 250 nM STI571 or 2.0 µM SAHA alone substantially reduced clonogenicity (i.e., to 25% of control values), combined treatment resulted in a >2-log reduction in colony formation.
The effects of combined exposure of K562 cells to SAHA and STI571 for 24 h were then examined in relation to mitochondrial injury, caspase activation, and expression of apoptosis-regulatory proteins (Fig. 3)
. Whereas the individual effects of STI571 (250 nM) or SAHA (2.0 µM) were minimal, exposure of cells to a combination of these agents resulted in a striking increase in release of cytochrome c, AIF, and Smac/DIABLO into the cytosolic, S-100 cell fraction. These events were accompanied by a marked increase in caspase-9 cleavage and degradation of caspase-3, caspase-8, and PARP. Interestingly, whereas individual treatment had little effect, combined exposure to STI571 and SAHA resulted in a marked decline in levels of the Bcr/Abl protein. Thus, treatment of Bcr/Abl+ cells with a subtoxic concentration of STI571 in conjunction with the HDI SAHA resulted in a marked increase in release of proapoptotic mitochondrial proteins, activation of the caspase cascade, and reduced expression of Bcr/Abl.
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. Interestingly, exposure to SAHA alone (16 h) resulted in a clear reduction in expression of Raf, whereas STI571 had little effect (Fig. 4A)
. Coadministration of STI571 and SAHA resulted in a further diminution in Raf expression. Roughly parallel changes in levels of phospho-MEK1/2 and phospho-ERK1/2 were observed. Combined treatment with STI571 and SAHA also resulted in a marked decrease in phosphorylation of p70S6K on ERK-associated sites (421/424; Ref. 39
), as well as markedly diminished expression of phospho-STAT5, a target of Bcr/Abl (9)
. No changes in the expression of total Akt were noted, although a modest decline in phosphorylated (activated) Akt was observed in cells exposed to both SAHA and STI571. In addition, whereas STI571 and SAHA individually failed to modify expression of phospho-JNK, combined treatment resulted in a very dramatic increase in JNK activation. A slight increase in phosphorylation of p38 MAPK was observed in SAHA-treated cells, but this did not change with addition of STI571.
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. However, STI571 treatment alone induced a small decrease in expression of the antiapoptotic protein Mcl-1, as we have reported previously (25)
, whereas addition of SAHA, which by itself exerted minimal effects, resulted in a further diminution in Mcl-1 expression.
Interactions between SAHA and STI571 were then examined in relation to expression of several cell cycle-regulatory proteins (Fig. 4C)
. Treatment with SAHA resulted in a robust induction of p21CIP1, similar to effects noted in Bcr/Abl- leukemia cells (30)
. Unexpectedly, coexposure to STI-571 substantially diminished induction of p21CIP1 by SAHA. Despite this action, combined exposure of cells to SAHA and STI571 resulted in a modest increase in expression of underphosphorylated pRb accompanied by cleavage of both total and underphosphorylated protein. Lastly, K562 cells treated with both STI571 and SAHA displayed a clear reduction in levels of cyclin D1, a phenomenon previously linked to induction of apoptosis (40)
. Collectively, these findings indicate that coexposure of K562 cells to STI571 and SAHA results in perturbations in the expression of multiple signaling-, cell cycle-, and apoptosis-regulatory proteins, including down-regulation of Raf; diminished activation of MEK1/2, ERK1/2, and p70S6K; a striking activation of JNK; reduced expression of Bcr/Abl, Mcl-1, p21CIP1, and cyclin D1; and dephosphorylation/cleavage of pRb.
To assess the role of caspases in these events, K562 cells were treated for 20 h with STI571 + SAHA in the presence or absence of the pan-caspase inhibitor BOC-fmk or the caspase-8 inhibitor IETD-fmk (Fig. 5A)
. BOC-fmk markedly inhibited apoptosis, whereas IETD-fmk was minimally effective, suggesting a relatively minor role for the extrinsic pathway in STI571/SAHA-mediated lethality in these cells. However, whereas BOC-fmk was ineffective in blocking cytochrome c release into the cytosol in cells exposed to STI571 + SAHA, it largely blocked Smac/DIABLO release (Fig. 5B)
, indicating that the latter represents a secondary, caspase-dependent event. As anticipated, BOC-fmk attenuated cleavage of procaspase-3 and both total and underphosphorylated pRb (Fig. 5C)
. It also partially reversed the down-regulation of Bcr/Abl expression in STI571/SAHA-treated cells, suggesting a caspase-dependent component of this phenomenon. In contrast, BOC-fmk had little effect on down-regulation of Raf, Mcl-1, p21CIP1, or cyclin D1 by STI571/SAHA, indicating that these events are largely independent of caspase activation. In separate studies, coadministration of BOC-fmk had no effect on the actions of SAHA administered alone (data not shown).
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. Exposure of LAMA 84 cells to 1.0 µM SAHA or 200 nM STI571 alone for 24 h exerted minimal effects on cell death. However, when the agents were combined, the large majority of cells (i.e., 70%) became apoptotic, as reflected by annexin positivity. In contrast, no evidence of synergism was observed when SAHA was combined with several Bcr/Abl- leukemic cell lines, including U937, HL60, NB4, and Jurkat (Table 1)
. These findings indicate that synergistic interactions between SAHA and STI571 are restricted to human leukemic cells expressing the Bcr/Abl protein.
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Attempts were then made to establish whether such interactions could be extended to include HDIs other than SAHA. To this end, K562 and LAMA 84 cells were exposed to the indicated concentrations of STI571 in the presence or absence of SB (1 or 2 mM), after which the extent of apoptosis was assessed (Table 2A)
. As shown, coadministration of STI571 with SB resulted in a marked increase in apoptosis in both cell lines. Analogous to results obtained with SAHA, enhanced lethality was associated with increased release of cytochrome c into the cytosol; activation of procaspases-3 and -9; down-regulation of Raf, p21CIP1, Mcl-1, and cyclin D1; and a marked activation of JNK (data not shown).
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. It can be seen that coadministration of STI571 (1.0 or 1.25 µM) for 48 h, which resulted in only modest lethality in either cell line, with a minimally toxic concentration of SAHA (i.e., 1.0 or 2.0 µM) induced cell death in the majority (e.g., 65%) of K562R and LAMA 84-R-STI571 cells. Exposure of STI571-sensitive K562 and LAMA 84 cells to these STI571 concentrations for 48 h induced cell death in virtually 100% of cells (data not shown). Essentially identical results were obtained when STI571-resistant cells were exposed to STI571 in combination with SB (data not shown). Such results indicate that coadministration of STI571 with HDIs effectively increases cell death in STI571-resistant Bcr/Abl+ cells, at least in those displaying increased expression of the Bcr/Abl protein.
Finally, to assess the functional contribution of dysregulation of the Raf/MEK/MAPK axis to synergistic interactions between STI571 and HDIs in Bcr/Abl+ cells, K562 cells were transiently transfected with a vector expressing either GFP alone or a constitutively active MEK1/2/GFP fusion protein (Fig. 7)
. Purified cell populations (e.g., >95%) expressing GFP and exhibiting 96% viability were isolated using a Cytomation MoFLO cell sorter and exposed for 24 h to 250 nM STI571 ± 2.0 µM SAHA. At the end of this period, the extent of apoptosis was monitored as described above. Cells transfected with the constitutively active MEK1/2 were modestly but significantly more resistant to STI571-mediated lethality than controls transfected with GFP alone (P < 0.05), consistent with earlier reports demonstrating potentiation of STI571-induced apoptosis by pharmacological MEK1/2 inhibitors (25)
. Moreover, transient transfection of cells with mutant MEK1/2 very significantly, albeit partially, protected cells from the lethality of the SAHA/STI571 regimen (P < 0.01). These findings suggest that dysregulation of the Raf/MEK/MAPK cascade in K562 cells exposed to STI571 in conjunction with HDIs plays a significant functional role in the enhanced lethality of this drug combination, although it is likely that other factors contribute to this phenomenon.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several lines of evidence support the notion that interruption of the Raf/MEK/MAPK cascade in Bcr/Abl+ cells by HDIs contributes to the marked induction of apoptosis by the STI571/HDI regimen. For example, involvement of Raf-1 in Bcr/Abl survival signaling has been reported (46) . In addition, perturbations in MEK/MAPK have been noted in HDI-treated Bcr/Abl+ cells, although results have varied. In this context, Rivero and Adunyah (47) reported early activation of ERK in K562 cells exposed to SB, whereas Witt et al. (33) described a correlation between butyrate-induced differentiation in K562 cells and inhibition of ERK. Such disparate results could potentially reflect a biphasic temporal pattern of ERK activation/down-regulation after HDI exposure. In this regard, STI571 has been shown to oppose ERK activation in Bcr/Abl+ cells, at least at early intervals (8) . In accord with these findings, we previously observed (25) early inhibition of ERK activation in STI571-treated K562 cells, although this was followed by a late rebound to basal or elevated levels of activity. Significantly, in that study, inhibition of MEK/ERK activation in STI571-treated K562 cells (i.e., by pharmacological MEK1/2 inhibitors) resulted in a very marked degree of mitochondrial damage and apoptosis (25) . Taken together with the present finding that constitutive activation of MEK/ERK protected cells from HDI/STI571 lethality, these observations are compatible with the concept that (a) down-regulation of the Raf/MEK/ERK axis, in association with other STI571-mediated perturbations, promotes HDI-induced lethality; or (b) disruption of the Raf/MEK/ERK cytoprotective pathway lowers the threshold for STI571-mediated cell death. In support of the former possibility, we have recently observed that pharmacological MEK1/2/ERK blockade (e.g., by agents such as U0126) results in a marked potentiation of apoptosis in K562 cells exposed to HDIs.4 However, the finding that protection of HDI/STI571-treated cells by constitutive activation of MEK/ERK was incomplete indicates that other factors are involved in the lethality of this regimen.
Combined treatment with STI571 and HDIs also resulted in a striking increase in activation of the stress-related kinase JNK. Although exceptions exist, activation of stress-related kinases such as JNK and p38 MAPK generally favors cell death, whereas activation of MEK/MAPK exerts cytoprotective effects. In fact, the ratio of the net outputs of the JNK and MAPK cascades has been shown to play a key role in survival/cell death decisions (48) . Moreover, JNK activation has been implicated in cytochrome c release associated with apoptosis (49) . It is therefore tempting to speculate that the dramatic shift from MEK/MAPK to JNK signaling in Bcr/Abl+ cells exposed to the combination of STI571 and HDIs contributed to the marked potentiation of apoptosis.
In addition to disruption of the MEK1/2/ERK pathway, the possibility that dysregulation of the CDKI p21CIP1 may also play a role in synergistic interactions between STI571 and HDIs in Bcr/Abl+ cells appears plausible. For example, interference with p21CIP1 induction (e.g., in cells expressing an antisense construct; Ref. 50
) or in cells exposed to the CDKI flavopiridol (51
, 52) has been shown to promote leukemic cell apoptosis after treatment with several differentiation-inducing agents, including phorbol 12-myristate 13-acetate, bryostatin 1, and, most recently, HDIs including SB, SAHA, and ABHA (53, 54, 55)
. This phenomenon may be related to the ability of p21CIP1 to bind to and inhibit caspase-3 (56)
. The observations that acetylation of histones by HDIs specifically activates the p21CIP1 promoter (57)
and that p21CIP1 is regularly induced by HDIs in association with maturation (30)
suggest that this CDKI plays a critical role in opposing HDI-mediated cell death. The mechanism by which STI571 blocks p21CIP1 induction in HDI-treated cells is not clear but could stem from disruption of the Raf/MEK/ERK axis, which is known to operate upstream of p21CIP1 (58)
. The finding that the STI571/HDI regimen induced pRb dephosphorylation was unanticipated, given the observed down-regulation of p21CIP1, but it could reflect reduced expression of cyclin D1 and diminished cyclin-dependent kinase 4/6 activity. A hypothetical model summarizing these and other interactions between HDIs and STI571 is shown in Fig. 8
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FOOTNOTES |
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1 Supported by NIH Grants CA 63753, CA 93738, and CA 83705 and Grant 6045-03 from the Leukemia and Lymphoma Society of America. 
2 To whom requests for reprints should be addressed, at Division of Hematology/Oncology, Virginia Commonwealth University/Medical College of Virginia, MCV Station Box 230, Richmond, VA 23298. Phone: (804) 828-5211; Fax: (804) 828-8079; E-mail: stgrant{at}hsc.vcu.edu
3 The abbreviations used are: CML, chronic myelogenous leukemia; CI, combination index; HDI, histone deacetylase inhibitor; SAHA, suberoylanilide hydroxamic acid; SB, sodium butyrate; MEK, MAPK kinase; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; STAT, signal transducers and activators of transcription; pRb, retinoblastoma protein; JNK, c-Jun NH2-terminal kinase; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; MMP, mitochondrial membrane potential; GFP, green fluorescence protein; PARP, poly(ADP-ribose) polymerase; CDKI, cyclin-dependent kinase inhibitor; PI, propidium iodide; BOC-fmk, BOC-Asp(Ome)-FMK; TBS-T, Tris buffered saline-Tween-20; IETD-fmk, Z-ILe-Glu(Ome)-Thr-Asp(Ome)-FMK; ABHA, azelaic bishydroxamic acid.
4 C. Yu and S. Grant, unpublished data.
Received 9/23/02. Accepted 3/ 4/03.
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 Y. Dai, S. Chen, C. A. Venditti, X.-Y. Pei, T. K. Nguyen, P. Dent, and S. Grant Vorinostat synergistically potentiates MK-0457 lethality in chronic myelogenous leukemia cells sensitive and resistant to imatinib mesylate Blood, August 1, 2008; 112(3): 793 - 804. [Abstract] [Full Text] [PDF]
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G. Garcia-Manero, H. Yang, C. Bueso-Ramos, A. Ferrajoli, J. Cortes, W. G. Wierda, S. Faderl, C. Koller, G. Morris, G. Rosner, et al. Phase 1 study of the histone deacetylase inhibitor vorinostat (suberoylanilide hydroxamic acid [SAHA]) in patients with advanced leukemias and myelodysplastic syndromes Blood, February 1, 2008; 111(3): 1060 - 1066. [Abstract] [Full Text] [PDF]
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 J. V. Melo and C. Chuah Novel Agents in CML Therapy: Tyrosine Kinase Inhibitors and Beyond Hematology, January 1, 2008; 2008(1): 427 - 435. [Abstract] [Full Text] [PDF]
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S. M. Lee, J. H. Bae, M. J. Kim, H. S. Lee, M. K. Lee, B. S. Chung, D. W. Kim, C. D. Kang, and S. H. Kim Bcr-Abl-Independent Imatinib-Resistant K562 Cells Show Aberrant Protein Acetylation and Increased Sensitivity to Histone Deacetylase Inhibitors J. Pharmacol. Exp. Ther., September 1, 2007; 322(3): 1084 - 1092. [Abstract] [Full Text] [PDF]
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G. Dasmahapatra, N. Yerram, Y. Dai, P. Dent, and S. Grant Synergistic Interactions between Vorinostat and Sorafenib in Chronic Myelogenous Leukemia Cells Involve Mcl-1 and p21CIP1 Down-Regulation Clin. Cancer Res., July 15, 2007; 13(14): 4280 - 4290. [Abstract] [Full Text] [PDF]
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T. K. Nguyen, M. Rahmani, H. Harada, P. Dent, and S. Grant MEK1/2 inhibitors sensitize Bcr/Abl+ human leukemia cells to the dual Abl/Src inhibitor BMS-354/825 Blood, May 1, 2007; 109(9): 4006 - 4015. [Abstract] [Full Text] [PDF]
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C. Yu, B. B. Friday, J.-P. Lai, A. McCollum, P. Atadja, L. R. Roberts, and A. A. Adjei Abrogation of MAPK and Akt Signaling by AEE788 Synergistically Potentiates Histone Deacetylase Inhibitor-Induced Apoptosis through Reactive Oxygen Species Generation Clin. Cancer Res., February 15, 2007; 13(4): 1140 - 1148. [Abstract] [Full Text] [PDF]
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 A. Quintas-Cardama and J. E. Cortes Chronic Myeloid Leukemia: Diagnosis and Treatment Mayo Clin. Proc., July 1, 2006; 81(7): 973 - 988. [Abstract] [Full Text] [PDF]
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 Y. Takada, A. Gillenwater, H. Ichikawa, and B. B. Aggarwal Suberoylanilide Hydroxamic Acid Potentiates Apoptosis, Inhibits Invasion, and Abolishes Osteoclastogenesis by Suppressing Nuclear Factor-{kappa}B Activation J. Biol. Chem., March 3, 2006; 281(9): 5612 - 5622. [Abstract] [Full Text] [PDF]
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N. Gao, M. Rahmani, X. Shi, P. Dent, and S. Grant Synergistic antileukemic interactions between 2-medroxyestradiol (2-ME) and histone deacetylase inhibitors involve Akt down-regulation and oxidative stress Blood, January 1, 2006; 107(1): 241 - 249. [Abstract] [Full Text] [PDF]
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 E. J. Chung, S. Lee, E. A. Sausville, Q. Ryan, J. E. Karp, I. Gojo, W. G. Telford, M.-J. Lee, H. S. Kong, and J. B. Trepel Histone Deacetylase Inhibitor Pharmacodynamic Analysis by Multiparameter Flow Cytometry Ann. Clin. Lab. Sci., October 1, 2005; 35(4): 397 - 406. [Abstract] [Full Text] [PDF]
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 M. R. Acharya, A. Sparreboom, J. Venitz, and W. D. Figg Rational Development of Histone Deacetylase Inhibitors as Anticancer Agents: A Review Mol. Pharmacol., October 1, 2005; 68(4): 917 - 932. [Abstract] [Full Text] [PDF]
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 J. Cortes and H. Kantarjian New Targeted Approaches in Chronic Myeloid Leukemia J. Clin. Oncol., September 10, 2005; 23(26): 6316 - 6324. [Abstract] [Full Text] [PDF]
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P.-H. Tseng, H.-P. Lin, J. Zhu, K.-F. Chen, E. M. Hade, D. C. Young, J. C. Byrd, M. Grever, K. Johnson, B. J. Druker, et al. Synergistic interactions between imatinib mesylate and the novel phosphoinositide-dependent kinase-1 inhibitor OSU-03012 in overcoming imatinib mesylate resistance Blood, May 15, 2005; 105(10): 4021 - 4027. [Abstract] [Full Text] [PDF]
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K. J. Aichberger, M. Mayerhofer, M.-T. Krauth, H. Skvara, S. Florian, K. Sonneck, C. Akgul, S. Derdak, W. F. Pickl, V. Wacheck, et al. Identification of mcl-1 as a BCR/ABL-dependent target in chronic myeloid leukemia (CML): evidence for cooperative antileukemic effects of imatinib and mcl-1 antisense oligonucleotides Blood, April 15, 2005; 105(8): 3303 - 3311. [Abstract] [Full Text] [PDF]
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M. Rahmani, E. Reese, Y. Dai, C. Bauer, L. B. Kramer, M. Huang, R. Jove, P. Dent, and S. Grant Cotreatment with Suberanoylanilide Hydroxamic Acid and 17-Allylamino 17-demethoxygeldanamycin Synergistically Induces Apoptosis in Bcr-Abl+ Cells Sensitive and Resistant to STI571 (Imatinib Mesylate) in Association with Down-Regulation of Bcr-Abl, Abrogation of Signal Transducer and Activator of Transcription 5 Activity, and Bax Conformational Change Mol. Pharmacol., April 1, 2005; 67(4): 1166 - 1176. [Abstract] [Full Text] [PDF]
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A. Moldenhauer, R. C. Frank, J. Pinilla-Ibarz, G. Holland, P. Boccuni, D. A. Scheinberg, A. Salama, K. Seeger, M. A. S. Moore, and S. D. Nimer Histone deacetylase inhibition improves dendritic cell differentiation of leukemic blasts with AML1-containing fusion proteins J. Leukoc. Biol., September 1, 2004; 76(3): 623 - 633. [Abstract] [Full Text] [PDF]
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Y. Dai, M. Rahmani, X.-Y. Pei, P. Dent, and S. Grant Bortezomib and flavopiridol interact synergistically to induce apoptosis in chronic myeloid leukemia cells resistant to imatinib mesylate through both Bcr/Abl-dependent and -independent mechanisms Blood, July 15, 2004; 104(2): 509 - 518. [Abstract] [Full Text] [PDF]
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S. C. Maggio, R. R. Rosato, L. B. Kramer, Y. Dai, M. Rahmani, D. S. Paik, A. C. Czarnik, S. G. Payne, S. Spiegel, and S. Grant The Histone Deacetylase Inhibitor MS-275 Interacts Synergistically with Fludarabine to Induce Apoptosis in Human Leukemia Cells Cancer Res., April 1, 2004; 64(7): 2590 - 2600. [Abstract] [Full Text] [PDF]
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C. Yu, M. Rahmani, D. Conrad, M. Subler, P. Dent, and S. Grant The proteasome inhibitor bortezomib interacts synergistically with histone deacetylase inhibitors to induce apoptosis in Bcr/Abl+ cells sensitive and resistant to STI571 Blood, November 15, 2003; 102(10): 3765 - 3774. [Abstract] [Full Text] [PDF]
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