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ABSTRACT: Antiproliferative effects of proteasome inhibitors are suggested to be primarily due to effects on nuclear factor-kappaB (NF-kappaB)-dependent pathways and the induction of apoptosis. The objective of this study was to elucidate the mechanistic basis for the antiproliferative effects of the proteasome inhibitor, bortezomib, in human clear cell renal cell cancer cells (CCRCC).
von Hippel Lindau (VHL) mutation/methylation status and cytotoxic response to bortezomib was determined in a panel of CCRCC cell lines. Effects on target protein/gene expression and the role of p53 in bortezomib-mediated cytotoxicity, inhibition of proteasome activity, survivin transcript and protein expression as well as induction of p21 expression was determined in CCRCC that differed in their intrinsic sensitivity to bortezomib.
VHL status was not associated with cytotoxic response to bortezomib treatment. Cytotoxicity in cell lines that differed in intrinsic sensitivity to bortezomib correlated with sustained inhibition of proteasome activity, survivin expression and induction of p21 expression. Stable down-regulation of p53 expression by siRNA led to attenuation of bortezomib effects, survivin down-regulation and p21 induction, suggesting that cellular effects are p53-dependent.
These results demonstrate that the antiproliferative effects of bortezomib in CCRCC cells are VHL independent and dependent on pathways regulated by p53.
Anticancer research 09/2009; 29(8):2961-9. · 1.73 Impact Factor
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ABSTRACT: We previously reported that phosphorylation of topoisomerase (topo) IIalpha at serine-1106 (Ser-1106) regulates enzyme activity and sensitivity to topo II-targeted drugs. In this study we demonstrate that phosphorylation of Ser-1106, which is flanked by acidic amino acids, is regulated in vivo by casein kinase (CK) Idelta and/or CKIepsilon, but not by CKII. The CKI inhibitors, CKI-7 and IC261, reduced Ser-1106 phosphorylation and decreased formation of etoposide-stabilized topo II-DNA cleavable complex. In contrast, the CKII inhibitor, 5,6-dichlorobenzimidazole riboside, did not affect etoposide-stabilized topo II-DNA cleavable complex formation. Since, IC261 specifically targets the Ca(2+)-regulated isozymes, CKIdelta and CKIepsilon, we examined the effect of down-regulating these enzymes on Ser-1106 phosphorylation. Down-regulation of these isozymes with targeted si-RNAs led to hypophosphorylation of the Ser-1106 containing peptide. However, si-RNA-mediated down-regulation of CKIIalpha and alpha' did not alter Ser-1106 phosphorylation. Furthermore, reduced phosphorylation of Ser-1106, observed in HRR25 (CKIdelta/epsilon homologous gene)-deleted Saccharomyces cerevisiae cells transformed with human topo IIalpha, was enhanced following expression of human CKIepsilon. Down-regulation of CKIdelta and CKIepsilon also led to reduced formation of etoposide stabilized topo II-DNA cleavable complex. These results provide strong support for an essential role of CKIdelta/epsilon in phosphorylating Ser-1106 in human topo IIalpha and in regulating enzyme function.
Nucleic Acids Research 12/2008; 37(2):382-92. · 8.03 Impact Factor
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Susan A J Vaziri,
Jason Hill,
Kenichi Chikamori, Dale R Grabowski,
Nagio Takigawa,
Mamta Chawla-Sarkar,
Lisa R Rybicki,
Andrei V Gudkov,
Tarek Mekhail,
Ronald M Bukowski,
Mahrukh K Ganapathi,
Ram Ganapathi
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ABSTRACT: Proteasome inhibition following DNA damage results in the synergistic induction of apoptosis via a nuclear factor-kappaB-independent mechanism. In this study, we identify the role of p53 in mediating apoptosis by the sequence-specific treatment involving the DNA-damaging, topoisomerase I-targeting drug SN-38 followed by the proteasome inhibitor PS-341 (SN-38-->PS-341). The p53-dependent sensitization of DNA damage-induced apoptosis by PS-341 is accompanied by persistent inhibition of proteasome activity and increased cytosolic accumulation of p53, including higher molecular weight forms likely representing ubiquitinated species. In contrast, pretreatment with PS-341 followed by treatment with SN-38 (PS-341-->SN-38), which leads to an antagonistic interaction, results in transient inhibition of proteasome activity and accumulation of significantly lower levels of p53 localized primarily to the nucleus. Whereas cells treated with PS-341-->SN-38 undergo G2 + M cell cycle arrest, cells treated with SN-38-->PS-341 exhibit a decreased G2 + M block with a concomitant increase in the sub-G1 population. Decreased accumulation of cells in the G2 + M phase of the cell cycle in SN-38-->PS-341-treated cells compared with PS-341-->SN-38-treated cells correlates with enhanced apoptosis and reduced expression of two p53-modulated proteins, 14-3-3sigma and survivin, both of which play critical roles in regulating G2 + M progression and apoptosis. The functional role of 14-3-3sigma or survivin in regulating the divergent function of p53 in response to SN-38-->PS-341 and PS-341-->SN-38 treatment in inducing apoptosis versus G2 + M arrest/DNA repair, respectively, was confirmed by targeted down-regulation of these proteins. These results provide insights into the mechanisms by which inhibition of proteasome activity modulates DNA damage-induced apoptosis via a p53-dependent pathway.
Molecular Cancer Therapeutics 01/2006; 4(12):1880-90. · 5.23 Impact Factor
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Kenichi Chikamori, Dale R Grabowski,
Michael Kinter,
Belinda B Willard,
Satya Yadav,
Ruedi H Aebersold,
Ronald M Bukowski,
Ian D Hickson,
Anni H Andersen,
Ram Ganapathi,
Mahrukh K Ganapathi
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ABSTRACT: Topoisomerases alter DNA topology and are vital for the maintenance of genomic integrity. Topoisomerases I and II are also targets for widely used antitumor agents. We demonstrated previously that in the human leukemia cell line, HL-60, resistance to topoisomerase (topo) II-targeting drugs such as etoposide is associated with site-specific hypophosphorylation of topo II alpha. This effect can be mimicked in sensitive cells treated with the intracellular Ca(2+) chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM). Here we identify Ser-1106 as a major phosphorylation site in the catalytic domain of topo II alpha. This site lies within the consensus sequence for the acidotrophic kinases, casein kinase I and casein kinase II. Mutation of serine 1106 to alanine (S1106A) abrogates phosphorylation of phosphopeptides that were found to be hypophosphorylated in resistant HL-60 cells or sensitive cells treated with BAPTA-AM. Purified topo II alpha containing a S1106A substitution is 4-fold less active than wild type topo II alpha in decatenating kinetoplast DNA and also exhibits a 2-4-fold decrease in the level of etoposide-stabilized DNA cleavable complex formation. Saccharomyces cerevisiae (JN394t2-4) cells expressing S1106A mutant topo II alpha protein are more resistant to the cytotoxic effects of etoposide or amsacrine. These results demonstrate that Ca(2+)-regulated phosphorylation of Ser-1106 in the catalytic domain of topo II alpha modulates the enzymatic activity of this protein and sensitivity to topo II-targeting drugs.
Journal of Biological Chemistry 05/2003; 278(15):12696-702. · 4.77 Impact Factor
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Kenichi Chikamori, Dale R. Grabowski,
Michael Kinter,
Belinda B. Willard,
Satya Yadav,
Ruedi H. Aebersold,
Ronald M. Bukowski,
Ian D. Hickson,
Anni H. Andersen,
Ram Ganapathi,
Mahrukh K. Ganapathi
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ABSTRACT: Topoisomerases alter DNA topology and are vital for the maintenance of genomic integrity. Topoisomerases I and II are also
targets for widely used antitumor agents. We demonstrated previously that in the human leukemia cell line, HL-60, resistance
to topoisomerase (topo) II-targeting drugs such as etoposide is associated with site-specific hypophosphorylation of topo
IIα. This effect can be mimicked in sensitive cells treated with the intracellular Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA-AM). Here we identify Ser-1106 as a major phosphorylation site in the catalytic domain of topo IIα.
This site lies within the consensus sequence for the acidotrophic kinases, casein kinase I and casein kinase II. Mutation
of serine 1106 to alanine (S1106A) abrogates phosphorylation of phosphopeptides that were found to be hypophosphorylated in
resistant HL-60 cells or sensitive cells treated with BAPTA-AM. Purified topo IIα containing a S1106A substitution is 4-fold
less active than wild type topo IIα in decatenating kinetoplast DNA and also exhibits a 2–4-fold decrease in the level of
etoposide-stabilized DNA cleavable complex formation.Saccharomyces cerevisiae (JN394t2-4) cells expressing S1106A mutant topo IIα protein are more resistant to the cytotoxic effects of etoposide or amsacrine.
These results demonstrate that Ca2+-regulated phosphorylation of Ser-1106 in the catalytic domain of topo IIα modulates the enzymatic activity of this protein
and sensitivity to topo II-targeting drugs.
Journal of Biological Chemistry 04/2003; 278(15):12696-12702. · 4.77 Impact Factor
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ABSTRACT: Activation of signaling pathways following DNA damage induced by topoisomerase (topo) poisons can lead to cell death by apoptosis. NF-kappaB, a major regulator of the stress response and a negative regulator of apoptosis is often activated following treatment with topoisomerase poisons. Since activation of NF-kappaB is generally considered to relay an anti-apoptotic signal, inactivation of this signaling molecule is considered to represent an important strategy to improve therapeutic efficacy. Although this strategy seems to be effective in some model systems, our results in human non-small cell lung cancers differed. In this review we will discuss the role of NF-kappaB in mediating topoisomerase poison-induced DNA damage and apoptosis and the consequence of inhibiting its activity. Newer insights about the importance of proteasome inhibitors and anti-apoptotic genes in topoisomerase poison-induced signaling mechanisms leading to apoptosis will also be reviewed. The knowledge obtained from these studies may be useful for translation to a clinical setting for development of more effective therapeutic strategies.
Current Pharmaceutical Design 02/2002; 8(22):1945-58. · 3.87 Impact Factor
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ABSTRACT: The cell cycle phase-dependent induction of DNA damage and apoptosis by etoposide (VP-16) and its modulation by 1-[N,O-bis(1,5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl]-4-piperazine (KN-62), an inhibitor of calcium-calmodulin-dependent enzymes, were examined in sensitive (HL-60/S) and VP-16-resistant (HL-60/DOX0.05) HL-60 cells. Cells from exponential-phase cultures were enriched by centrifugal elutriation into G1, S, and G2+M fractions. Modulation of VP-16-induced apoptosis by KN-62 in HL-60/S cells was apparent only in the S phase at the ic50 concentration. However, in the HL-60/DOX0.05 cells, significant (P < 0.001) potentiation of VP-16-induced apoptosis by a non-cytotoxic concentration of 2 μM KN-62 was apparent in cells in the G1, S, and G2+M phases, as well as over the entire concentration range tested. VP-16-induced apoptosis and its potentiation by a non-cytotoxic concentration of 2 μM KN-62 were correlative with drug-stabilized DNA cleavable complex formation based on a band depletion assay. In agreement with the results on apoptosis in the resistant HL-60/DOX0.05 cells, the enhanced depletion of the α and β isoforms of topoisomerase II by VP-16 + KN-62 was observed in G1, S, and G2+M cells. Results suggest that the effects of KN-62 in reversing resistance are based on its role as a potent sensitizer of VP-16-induced DNA damage and apoptosis in a cell cycle phase-independent manner.
Biochemical Pharmacology 02/2001; · 4.70 Impact Factor
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ABSTRACT: The potentiation of topoisomerase (topo)-I-induced apoptosis by proteasome inhibitors is dependent on the treatment sequence, but not on NF-kappaB. In this study, alternate mechanisms modulating apoptosis induced with the topo I-targeting drug, SN-38, when followed by the proteasome inhibitor bortezomib (PS-341) were investigated.
Human non-small cell lung carcinoma (NSCLC-3) cells transfected with a control vector (NSCLC-3/neo) or a vector containing dominant negative IkappaBalpha (NSCLC-3/mIkappaBalpha) were treated with SN-38 for 1 h followed by PS-341 for 4 h (SN-38 --> PS-341), or with either drug alone. The functional role of the anti-apoptotic protein survivin was tested using NSCLC-3 transfected with myc-tagged wild-type (NSCLC-3/myc-survivin), or dominant negative mutant T34A survivin (NSCLC-3/myc-T34A).
In NSCLC-3/neo or NSCLC-3/mIkappaBalpha cells, treatment with SN-38 --> PS-341 led to down-regulation of the survivin transcript and protein, enhanced apoptosis and reduced (> 3-fold) survival compared to SN-38 or PS-341 alone. In contrast to the cells transfected with wild-type survivin, or the control NSCLC-3/neo, those cells transfected with mutant survivin and treated with SN-38 --> PS-341 exhibited enhanced caspase 9 activity (> 2-fold), caspase 3 (> 2- to 3-fold) activity and cytotoxicity compared to the NSCLC-3/neo cells.
In contrast to inhibition of NF-kappaB activity, down-regulation of the anti-apoptotic survivin was correlated with modulation of the sequence-dependent synergistic effects of PS-341 in SN-38-induced apoptosis.
Anticancer research 26(3A):1869-76. · 1.73 Impact Factor
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ABSTRACT: We previously demonstrated that KN-62, an inhibitor of calcium calmodulin-dependent enzymes, sensitizes human leukemia HL-60 cells resistant to topoisomerase II-targeting drugs. The objective of this study was to determine pathways of apoptosis downstream of DNA damage induced by KN-62 co-treatment with VP-16.
HL-60/Y/DOX0.05 cells were treated with VP-16, KN-62, or VP-16 + KN-62. Following treatment, cells were assayed for c-IAP1, c-IAP2 and XIAP protein expression, as well as caspase activation, cytochrome c release and PARP cleavage.
Baseline c-IAP1 protein levels were 2-fold higher in HL60 cells selected for resistance to doxorubicin compared to the parent sensitive line. VP-16 and KN-62 co-treatment was associated with caspase activation via the mitochondrial pathway and significant reductions (p = 0.002) in c-IAP1 protein expression but not with c-IAP2 or XIAP.
These data suggest that KN-62 co-treatment sensitizes doxorubicin-resistant cells to VP-16-induced apoptosis by enhancing caspase activity and reducing c-IAP1 expression.
Anticancer research 23(5A):3657-61. · 1.73 Impact Factor
