|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Blood, Vol. 95 No. 7 (April 1), 2000:
pp. 2378-2385
NEOPLASIA
From the Austin Research Institute, Austin Hospital,
Heidelberg, Victoria, Australia.
Multidrug resistance (MDR) is often characterized by the expression
of P-glycoprotein (P-gp), a 170-kd ATP-dependent drug efflux protein.
As well as effluxing xenotoxins, functional P-gp can confer resistance
to caspase-dependent apoptosis induced by a range of different stimuli,
including Fas ligand, tumor necrosis factor, UV irradiation, and serum
starvation. However, P-gp-positive cells remain sensitive to
caspase-independent death induced by cytotoxic T-cell granule proteins,
perforin, and granzyme B. It is, therefore, possible that agents that
induce cell death in a caspase-independent manner might circumvent
P-gp-mediated MDR. We demonstrated here that hexamethylene bisacetamide
(HMBA) induced equivalent caspase-independent cell death in both
P-gp-positive and -negative cell lines at concentrations of 10 mmol/L
and above. The HMBA-induced death pathway was marked by release of
cytochrome c from the mitochondria and reduction of Bcl-2 protein
levels. In addition, we show that functional P-gp specifically inhibits the activation of particular caspases, such as caspases-8 and -3, whereas others, such as caspase-9, remain unaffected. These studies
greatly enhance our understanding of the molecular cell death events
that can be regulated by functional P-gp and highlight the potential
clinical use of drugs that function via a caspase-independent pathway
for the treatment of MDR tumors.
(Blood. 2000;95:2378-2385)
Multidrug resistance (MDR) is a major clinical problem
in treating human cancer and is often characterized by the expression of the MDR1 gene product, P-glycoprotein (P-gp).1 A
member of the adenosine triphosphate-binding cassette transporter
family, P-gp has traditionally been thought to confer resistance to
chemotoxins by actively effluxing them from the cell.2,3
However, recent reports by our group and others4-6 have
indicated that, in addition to its role as an efflux pump, P-gp
regulates programmed cell death mediated by antineoplastic agents,
serum starvation, UV irradiation, and ligation of the cell surface
death receptors Fas and tumor necrosis factor (TNF) receptor. Common to
these diverse apoptotic stimuli is their dependence on the activation of caspases to initiate cell death. We, therefore, hypothesized that
the successful treatment of P-gp+ MDR tumors might be
enhanced using chemotherapeutic agents that can function in absence of
caspase activation.
There are now at least two molecular pathways leading to
caspase-dependent apoptosis (Figure 1). The
best-defined pathway involves ligation of death receptors, typically
members of the TNF superfamily such as Fas and TNF receptor, at the
cell surface resulting in sequential activation of proximal caspases
such as caspase-8 and downstream effector caspases such as
caspase-3.7-9
The second, less well-defined caspase-dependent pathway involves the
disruption of the mitochondrial transmembrane potential ( In addition to caspase-dependent apoptosis, there is now sufficient
evidence to suggest that apoptosis can occur in the absence of active
caspases. Caspase-independent cell death has been shown to be induced
by perforin and granzyme B,19 anti-CD2,20 and the protein kinase C inhibitor staurosporine.20,21 In the
absence of caspase activation, cytoplasmic events related to apoptosis, such as cell shrinkage, cytoplasmic condensation, and loss of mitochondrial membrane integrity and exposure of phosphatidylserine, are still induced by these stimuli.19-21
Caspase-independent cell death has been shown to be inhibited by
Bcl-2,22,23 suggesting that a loss of mitochondrial
function is central to this death process.
Work by our group and others4-6 has demonstrated that
functional P-gp can confer resistance to a wide range of
caspase-dependent apoptotic stimuli, such as ligation of cell-surface
death receptors, serum starvation, and UV irradiation. We demonstrated
that functional P-gp-inhibited activation of caspase-3 following Fas
ligation and that this inhibitory effect could be reversed using P-gp
antagonists, such as specific anti-P-gp monoclonal antibodies (mAbs) or
the pharmacological inhibitor verapamil.4 Many
chemotherapeutic drugs, such as doxorubicin and vincristine, function
in a caspase-dependent manner;4,5 therefore, P-gp may play
a dual role in regulating cell death induced by these stimuli (i) by
removing the toxins from the cell and (ii) by
inhibiting the activation of caspases. Importantly, P-gp does not offer
cells protection from death induced by lytic concentrations of the
pore-forming protein perforin5 or by a combination of
granzyme B and sublytic concentrations of perforin,4,5
which together can function in a caspase-independent manner.4,5,19 We, therefore, reasoned that drugs that were not P-gp substrates and could mediate caspase-independent cell death
might be clinically useful for the treatment of MDR. Given that
caspase-independent cell death is comparatively poorly understood, and
few cell-death stimuli have been shown to function in a
caspase-independent manner, we undertook a search for a
chemotherapeutic agent that could kill P-gp-expressing cells by this mechanism.
Hexamethylene bisacetamide (HMBA) is a hybrid polar compound
that can induce terminal differentiation of transformed
cells24,25 and has been used clinically to treat patients
with acute myeloid leukemia and myelodysplastic syndrome.26
Terminal differentiation induced by HMBA correlates with a dramatic
decrease in cdk4-dependent kinase activity and hypophosphorylation of
the retinoblastoma tumor suppressor protein.27
Interestingly, HMBA also induced activation of the JAK/STAT signal
transduction pathway via JAK2 and STAT5 but suppressed activation via
SHC and GRB2.28 Although hypophosphorylation of
retinoblastoma tumor suppressor protein and the related family member
p107 is clearly involved in causing cells to arrest in the G1 phase of
the cell cycle, it is unclear what role HMBA-mediated modulation of
signaling pathways triggered by stimulation of cytokine and/or growth
factor receptors plays in inducing cellular differentiation. A previous
report29 indicated that HMBA could induce cell death in
P-gp-expressing cells; however, no data were shown and the mechanism of
cell death in P-gp-expressing cells was not explored. We report here
that HMBA can induce cell death in P-gp-expressing human tumor cells,
and, in agreement with our previous reports, HMBA induced activation of
caspase-3 in P-gp- but not P-gp+ cells.
However, in the absence of caspase activation, HMBA could still induce
cell death that was marked by the caspase-independent release of
cytochrome c from the mitochondria to the cytosol and a reduction in
Bcl-2 levels. Importantly, overexpression of Bcl-2 inhibited
HMBA-mediated cell death. Furthermore, we illustrate that
P-gp-expressing cells have a reduced capacity to activate caspases
involved in death receptor-mediated apoptosis, such as caspases-8
and-3, but not caspase-9, which is activated following release of
cytochrome c from the mitochondria.
Cell culture
Viability assays
Clonogenic assays Colony assays were performed on cells treated for 24-72 hours with various apoptotic stimuli as previously described.5DNA fragmentation Cells (1 × 106) were washed with phosphate-buffered saline (PBS) and pelleted by centrifugation. Genomic DNA was prepared as previously described.32 DNA (10-15 µg) was run on a 2% agarose gel for 2 hours. The gel was stained in ethidium bromide (1 µg/mL) for 15 minutes and washed for 1 hour in water.TUNEL assay Cells (2 × 105) treated with various apoptotic stimuli were assayed by TUNEL (Boehringer Mannheim) as previously described.33Propidium iodide (PI) staining Cells (2 × 105) treated with various apoptotic stimuli were washed with PBS and fixed in 50% ice-cold EtOH/PBS for 20 minutes on ice. Cells were washed with PBS and incubated in PI solution (69 mmol/L PI/388 mmol/L Na Citrate/100 µg/mL Rnase A) for 15 minutes at 37°C. Cells were immediately analyzed by FACscan flow cytometer (Becton Dickinson).Western blot analysis Cells (2 × 105) were lysed in 50 mL of ice-cold Nonidet P-40 lysis buffer as previously described.4 Protein determinations were performed using the Bradford reaction.34 Proteins (10-20 µg) were separated on SDS-12% or SDS-15% polyacrylamide gels and electroblotted onto nylon membranes. Blots were probed with anti-human caspase-3 mAb (Transduction Laboratories, Lexington, KY), anti-human FLICE mAb (gift from Marcus Peter, University of Heidelberg, Germany), anti-human caspase-9 (gift from Xiaodong Wang, Howard Hughes Medical Institute and Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX; and Douglas Green, La Jolla Institute for Allergy and Immunology, San Diego, CA), anti-human Bcl-2 mAb (David Huang, WEHI, Melbourne Australia), anti-human PARP (Boehringer Mannheim), or anti-human a-tubulin mAb (Sigma, St. Louis, MO) and visualized by enhanced chemiluminescence (Amersham, UK).Cytochrome c release Cells (2 × 107) treated for 3 days with 10 or 15 mmol/L HMBA were washed twice in ice-cold PBS. Cytosolic lysates were prepared as previously described.21 Cytosolic proteins were separated on a SDS-15% polyacrylamide gel and transferred onto nylon membranes. Blots were probed with anti-human cytochrome c mAb (PharMingen, San Diego, CA; clone 7H8.2C12) and visualized as above. The same blots were reprobed with anti- -tubulin to determine that
equal amounts of protein were present in each well. Cytospin slides of
CEM cells treated for 24-72 hours with 10 and 15 mmol/L HMBA in the
presence or absence of 40 µmol/L ZVAD-fmk or ZFA-fmk were stained for
cytochrome c as previously described35 and visualized by
confocal microscopy.
Immunofluorescence and analysis of apoptotic nuclei Cytospin slides of 1 × 105 cells treated for varying times with 0-20 mmol/L HMBA were fixed in EtOH/acetic acid (6/1) (vol/vol) for 5 minutes. Slides were stained with 10 ng/mL Hoechst 33 258 for 5 minutes and washed twice in citrate buffer pH 5.5. Cells were visualized by confocal and fluorescence microscopy, and 450-500 cells per sample were counted and apoptotic nuclei scored.
P-gp does not protect tumor cells from HMBA-mediated death P-gp+ and P-gp- CEM, LoVo, and K562 cell lines were used to examine the effects of HMBA on cell viability. CEM cell lines were cultured in the presence of 0-20 mmol/L HMBA for 24-72 hours, and cell death was initially determined by trypan blue exclusion assays. HMBA-induced equivalent cell death in a dose- and time-dependent manner in both P-gp+ and P-gp- CEM (Figure 2A) and LoVo (Figure 2B) cell lines. However, the effects of HMBA on cell viability were cell specific. P-gp+ and P-gp- K562 cells treated with HMBA did not die (Figure 2C), but rather underwent G1 cell cycle arrest as measured by PI staining for DNA content (data not shown), consistent with previous reports24 that HMBA induces growth arrest in this erythroid cell line. As controls for drug sensitivity, P-gp- CEM, LoVo, and K562 cells were effectively killed by the chemotherapeutic agent, doxorubicin (100-200 ng/mL), whereas P-gp+ cells were resistant to doxorubicin-induced death (Figure 2A-C). To evaluate viability by a more sensitive method, and to correlate trypan blue staining with cell death, clonogenic assays were employed. HMBA treatment of P-gp+ or P-gp- CEM cells resulted in a dose-dependent decrease in colony formation (Figure 2D). P-gp+ CEM cells survived doxorubicin-mediated apoptosis, whereas P-gp- CEM cells were sensitive to doxorubicin-induced cell death.
HMBA induces nuclear morphological changes consistent with apoptosis To characterize the biochemical and morphological changes in cells undergoing HMBA-induced cell death, CEM cells were cultured in the presence or absence of 10 mmol/L HMBA for 24-48 hours and subsequently assayed for classical hallmarks of apoptosis, such as chromatin condensation, DNA laddering, and cleavage of the DNA repair enzyme PARP.36-38 Treatment of P-gp+ and P-gp- CEM cells with HMBA resulted in DNA fragmentation as evidenced by the formation of DNA ladders (Figure 3A) and by TUNEL staining (Figure 3B). In addition, CEM cells treated with HMBA and subsequently stained with Hoechst 33 258 displayed chromatin condensation indicative of apoptotic nuclei (Figure 3C). Apoptotic nuclei appeared within 6 hours after treatment with 10 mmol/L HMBA, and the number of apoptotic cells increased with time (>15% at 24 hours, >25% at 48 hours). In addition, PARP was cleaved in a dose-dependent manner in both P-gp+ and P-gp- CEM cells (Figure 3D).
HMBA can induce caspase-independent cell death As HMBA induced many of the cell death events indicative of caspase-dependent apoptosis, we sought to determine whether this chemotherapeutic agent could still kill CEM cells in the absence of caspase activation. To examine if HMBA-induced death was caspase-independent, CEM cells were pretreated with the universal caspase inhibitor ZVAD-fmk or the control inhibitor ZFA-fmk and then cultured for 48 hours with 10 mmol/L HMBA. P-gp+ and P-gp- CEM cells treated with HMBA in the presence of ZVAD-fmk displayed equivalent cell death when compared with controls treated with HMBA alone, indicating that HMBA-induced cell death was caspase-independent (Figure 4A). By comparison, P-gp- CEM cells treated for 48 hours with 5 ng/mL vincristine in the presence of ZVAD-fmk were protected from cell death, indicating that, as previously demonstrated,5 vincristine-induced cell death was dependent on active caspases (Figure 4A). As expected, P-gp+ CEM cells were resistant to caspase-dependent cell death induced by vincristine (Figure 4A). These control experiments with vincristine as the apoptotic mediator also serve to confirm the effectiveness of the caspase inhibitory ZVAD-fmk peptide over the 48-hour time course of the experiment. These data were confirmed by clonogenic assays with equivalent HMBA-induced cell death in the presence of ZVAD-fmk and ZFA-fmk (data not shown).
HMBA induces activation of caspase-9 As we had previously determined that caspase-3 activation was inhibited in P-gp+ cells, we were surprised to find that the caspase substrate PARP was equivalently cleaved in both P-gp+ and P-gp- cells. Therefore, we determined which caspases were activated following HMBA treatment and potentially responsible for the observed PARP cleavage and apoptotic nuclear events. Caspase-3 was activated by HMBA in P-gp- cells (Figure 5A, top left panel) but not in P-gp+ cells (Figure 5A, top right panel). Similarly, and consistent with our earlier report,4 caspase-3 was activated following Fas ligation only in P-gp- but not P-gp+ CEM cells (Figure 5B, top panel). Inhibition of caspase-3 activation in P-gp-expressing CEM cells can be reversed by the P-gp inhibitor verapamil and mAbs to P-gp4, indicating that these cells do not express a mutated form of caspase-3 but rather that expression of functional P-gp inhibits caspase-3 activation. We then investigated whether key "activator" caspases upstream of caspase-3, such as caspase-8, typically involved in receptor-mediated cell death,7-9 or caspase-9, central to the mitochondrial death pathway,14,15 were activated during HMBA-induced death. Western analyses for caspases-8 and -9 were performed on lysates from cells treated with HMBA (Figure 5A). Even after 72 hours, no evidence of caspase-8 activation was observed in either P-gp+ or P-gp- cells treated with as much as 15 mmol/L HMBA (Figure 5A, middle panel). However, treatment with anti-Fas mAb resulted in a reduction in pro-caspase-8 and the appearance of active caspase-8 in P-gp- but not P-gp+ CEM cells (Figure 5B, middle panel), demonstrating for the first time that P-gp can inhibit caspase-8 activation. As with caspase-3, caspase-8 could be activated in Fas-ligated P-gp+ CEM cells following treatment with either specific antagonistic anti-P-gp mAbs or with verapamil (data not shown). In contrast, HMBA induced the typical cleavage and auto-activation39 of caspase-9 to p37, p15, and p13 subunits in a dose-dependent manner in both P-gp+ and P-gp- CEM cells (Figure 5A, bottom panel), indicating that the cell death pathway that involves disruption of the mitochondrial membrane might be functioning in HMBA-mediated cell death and that the activation of this caspase is not affected by P-gp expression.
HMBA stimulates the loss of Bcl-2 and release of mitochondrial cytochrome c Activation of caspase-9 can be mediated via the interaction of mitochondrial cytochrome c with Apaf 1 in the presence of dATP/ATP in the cytosol.14,15 We, therefore, examined whether cytochrome c was released from the mitochondria during HMBA-mediated death. Cytochrome c was present in all cytosolic extracts of P-gp+ and P-gp- CEM cells treated with HMBA (Figure 6A) but was not detected in untreated control cells. In addition, HMBA-induced cytochrome c release was caspase-independent as it was not inhibited by addition of ZVAD-fmk (Figure 6A). These results were confirmed by confocal microscopy on cells treated as above and stained by immunofluorescence for mitochondrial and cytosolic cytochrome c (data not shown).
Overexpression of Bcl-2 inhibits HMBA-induced release of cytochrome c and apoptosis To further investigate the role of Bcl-2 in HMBA-induced cell death, we examined the effects of overexpression of Bcl-2 in CEM cells treated with HMBA. CEM cells overexpressing human Bcl-2 and parental CEM cells were treated with 10 mmol/L HMBA for 24-72 hours, and cell death was determined by trypan blue exclusion (Figure 7A). After just 24 hours of treatment with 10 mmol/L HMBA, parental CEM cells underwent apoptosis (Figure 7A); however, Bcl-2 overexpressing cells were protected against HMBA-induced apoptosis at concentrations from 10 mmol/L (Figure 7A) to 30 mmol/L for as long as 72 hours (data not shown). Clonogenic assays were performed on cells treated for 72 hours with 0-15 mmol/L HMBA. As illustrated in Figure 7B, CEM-Bcl-2 cells formed significant numbers of colonies at HMBA concentrations as high as 15 mmol/L. These findings are consistent with an earlier report that overexpression of Bcl-2 inhibits HMBA-mediated cell death.29 In addition, cytochrome c was detected in cytosolic lysates from CEM cells treated for 72 hours with 10 mmol/L HMBA but was not present in lysates from CEM-Bcl-2 cells treated in the same manner (Figure 7C), illustrating that the overexpression of Bcl-2 can completely block HMBA-mediated cytochrome c release.
P-gp-expressing, MDR T-cell leukemia, and colon carcinoma cell lines were shown to undergo cell death induced by HMBA, a potent differentiation agent of some hemopoietic cell lineages. P-gp+ and P-gp- cells treated with HMBA displayed classical morphological changes consistent with apoptosis, including chromatin condensation, DNA fragmentation, and membrane damage. Nuclear events, such as DNA fragmentation and chromatin condensation induced by HMBA treatment, were dependent on activation of caspases. However, in the absence of caspase activation, HMBA still mediated a decrease in Bcl-2 protein levels, cytochrome c release, cell membrane damage, and subsequent cell death. Although HMBA could activate caspases such as caspase-9 in both P-gp+ and P-gp- cells, consistent with our previous findings, activation of caspase-3 was inhibited in P-gp+ cells. In addition, we have now established that P-gp can affect activation of caspase-8 following ligation of cell surface Fas. We have previously shown that addition of P-gp-inhibitory antibodies MRK-16 or UIC2, or pharmacological inhibitor verapamil, can allow the Fas-mediated activation of caspase-3 in the same P-gp-positive cells used in this study,4,5 demonstrating that functional P-gp is affecting caspase-3 activation and there are not endogenous inhibitors of caspases in P-gp-expressing cells. We have demonstrated that HMBA activates two death-inducing pathways (Figure 1): (i) a caspase-dependent pathway that mediates nuclear apoptotic events and (ii) a caspase-independent cell death pathway involving a decrease in Bcl-2 protein levels, mitochondrial damage, and cytochrome c release that could be inhibited by the overexpression of Bcl-2. To date, few examples of caspase-independent death stimuli have been described, and this mechanism of cell death remains poorly understood. Those stimuli which have been described to induce caspase-independent cell death include CTL granule proteins,19 viral proteins,40 anti-CD2,20 gangliosides,41 staurosporine,20,21 and the Bcl-2 family member Bax.42 HMBA appears to be the first chemotherapeutic drug shown to induce this novel form of cell death.
We thank Joseph Trapani, Vivien Sutton, and Sarah Russell for their critical review of this manuscript, and David Huang, Jerry Adams, Beni Wolf, Douglas Green, Xiaodong Wang, Carsten Scaffidi, Peter Krammer, and Marcus Peter for reagents.
Submitted September 13, 1999; accepted December 2, 1999.
Supported by project grants from the NH&MRC and the Anti-Cancer Council, Victoria.
R.W.J. is an R.D. Wright Fellow of the National Health and Medical Research Council of Australia (NH&MRC); M.J.S. is a Principal Research Fellow, NH&MRC; and A.A.R. is currently supported by the Dr Laurence LeWinn Post-graduate Research Scholarship.
Reprints: Ricky W. Johnstone, The Austin Research Institute, Austin Hospital, Studley Rd, Heidelberg 3084, Victoria, Australia; e-mail: r.johnstone{at}ari.unimelb.edu.au.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
1. Gottesman M, Pastan I. Biochemistry of multidrug resistance mediated by the multidrug transporter. Annu Rev Biochem. 1993;62:385[Medline] [Order article via Infotrieve]. 2. Higgins CF. ABC transporters: from microorganisms to man. Annu Rev Cell Biol. 1992;8:67. 3. Doige CA, Ames FG. ATP-dependent transport systems in bacteria and humans: relevance to cystic fibrosis and multidrug resistance. Annu Rev Microbiol. 1993;47:291[Medline] [Order article via Infotrieve].
4.
Smyth MJ, Krasovskis E, Sutton VR, Johnstone RW.
The drug efflux protein, P-glycoprotein, additionally protects drug-resistant cells from multiple forms of caspase-dependent apoptosis.
Proc Natl Acad Sci U S A.
1998;95:7024
5.
Johnstone RW, Cretney E, Smyth MJ.
P-glycoprotein protects leukemia cells against caspase-dependent, but not caspase-independent, cell death.
Blood.
1999;93:1075 6. Robinson LJ, Roberts WK, Ling TT, Lamming D, Sternberg SS, Roepe PD. Human MDR1 protein overexpression delays the apoptotic cascade in Chinese hamster ovary fibroblasts. Biochemistry. 1997;36:11,169[Medline] [Order article via Infotrieve]. 7. Boldin MP, Goncharov TM, Goltsev YV, Wallach D. Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1- and TNF receptor-induced cell death. Cell. 1996;85:803[Medline] [Order article via Infotrieve]. 8. Muzio M, Chinnaiyan AM, Kischkel FC, et al. FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex. Cell. 1996;85:872.
9.
Srinivasula SM, Ahmad M, Fernades-Alnemri T, Litwack G, Alnemri ES.
Molecular ordering of the Fas-apoptotic pathway: the Fas/APO-1 protease Mch5 is a CrmA-inhibitable protease that activates multiple Ced-3/ICE-like cysteine proteases.
Proc Natl Acad Sci U S A.
1996;93:14,486 10. Liu X, Kim CN, Yang J, Jemmerson R, Wang X. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell. 1996;86:147[Medline] [Order article via Infotrieve].
11.
Kluck RM, Bossy-Wetzel E, Green DR, Newmeyer DD.
The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis.
Science.
1997;275:1132
12.
Reed JC.
Cytochrome c: can't live with it 13. Susin SA, Lorenzo HK, Zamzami N, et al. Molecular characterization of mitochondrial apoptosis-inducing factor. Nature. 1999;397:441[Medline] [Order article via Infotrieve]. 14. Li P, Nijhawan D, Budihardjo I, et al. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell. 1997;91:479[Medline] [Order article via Infotrieve]. 15. Zou H, Henzel WJ, Liu X, Lutschg A, Wang X. Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3. Cell. 1997;90:405[Medline] [Order article via Infotrieve]. 16. Li H, Zhu H, Xu C, Yuan J. Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell. 1997;94:491. 17. Luo X, Budihardjo I, Zou H, Slaughter C, Wang X. Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell. 1997;94:481.
18.
Yang J, Liu X, Bhalla K, et al.
Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked.
Science.
1997;275:1129
19.
Trapani JA, Jans DA, Jans PJ, Smyth MJ, Browne KA, Sutton VR.
Efficient nuclear targeting of granzyme B and the nuclear consequences of apoptosis induced by granzyme B and perforin are caspase-dependent, but cell death is caspase-independent.
J Biol Chem.
1997;273:27934
20.
Deas O, Dumont C, MacFarlane M, et al.
Caspase-independent cell death induced by anti-CD2 or staurosporine in activated human peripheral T lymphocytes.
J Immunol.
1997;161:3375 21. Bossy-Wetzel E, Newmeyer DD, Green DR. Mitochondrial cytochrome c release in apoptosis occurs upstream of DEVD-specific caspase activation and independently of mitochondrial transmembrane depolarization. EMBO J. 1998;17:37[Medline] [Order article via Infotrieve].
22.
Okuno S, Shimizu S, Ito T, et al.
Bcl-2 prevents caspase-independent cell death.
J Biol Chem.
1998;273:34,272 23. Sutton VR, Vaux DL, Trapani JA. Bcl-2 prevents apoptosis induced by perforin and granzyme B, but not that mediated by whole cytotoxic lymphocytes. J Immunol. 1997;158:783[Abstract].
24.
Richon VM, Russo P, Venta-Perez G, Cordon-Cardo C, Rifkind RA, Marks PA.
Two cytodifferentiation agent-induced pathways, differentiation and apoptosis, are distinguished by the expression of human papillomavirus 16 E7 in human bladder carcinoma cells.
Cancer Res.
1997;57:2789
25.
Marks PA, Richon VM, Kiyokawa H, Rifkind RA.
Inducing differentiation of transformed cells with hybrid polar compounds: a cell cycle-dependent process.
Proc Natl Acad Sci U S A.
1994;91:10,251
26.
Andreef M, Stone R, Michaeli J, et al.
Hexamethylene bisacetamide in myelodysplastic syndrome and acute myelogenous leukemia: a phase II clinical trial with a differentiation-inducing agent.
Blood.
1992;80:2604 27. Kiyokawa H, Richon VM, Rifkind RA, Marks PA. Suppression of cyclin-dependent kinase 4 during induced differentiation of erythroleukemia cells. Mol Cell Biol. 1994;11:7195.
28.
Yamashita T, Wakao H, Miyajima A, Asano S.
Differentiation inducers modulate cytokine signaling pathways in a murine erythroleukemia cell line.
Cancer Res.
1998;58:556
29.
Siegel DS, Zhang X, Feinman R, et al.
Hexamethylene bisacetamide induces programmed cell death (apoptosis) and down-regulates BCL-2 expression in human myeloma cells.
Proc Natl Acad Sci U S A.
1998;95:162 30. Zalcberg JR, Hu XF, Wall DM, et al. Cellular and karyotypic characterization of two doxorubicin resistant cell lines isolated from the same parental human leukemia cell line. Int J Cancer. 1994;57:522[Medline] [Order article via Infotrieve]. 31. Broggini M, Grandi M, Ubezio P, Geroni C, Giuliani FC, D'Incalci M. Intracellular doxorubicin concentrations and drug-induced DNA damage in a human colon adenocarcinoma cell line and in a drug-resistant subline. Biochem Pharmacol. 1988;37:4423[Medline] [Order article via Infotrieve]. 32. Herr I, Wilhelm D, Bohler T, Angel P, Debatin K-M. Activation of CD95 (APO-1/Fas) signaling by ceramide mediates cancer therapy-induced apoptosis. EMBO J. 1997;16:6200[Medline] [Order article via Infotrieve]. 33. Trapani JA, Jans P, Smyth MJ, et al. Perforin-dependent nuclear entry of granzyme B precedes apoptosis, and is not a consequence of nuclear membrane dysfunction. Cell Death Diff. 1998;5:488[Medline] [Order article via Infotrieve]. 34. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248[Medline] [Order article via Infotrieve].
35.
MacDonald G, Shi L, Vande Velde C, Lieberman J, Greenberg AH.
Mitochondria-dependent and -independent regulation of Granzyme B-induced apoptosis.
J Exp Med.
1999;189:131 36. Martin SJ, Green DR. Protease activation during apoptosis: death by a thousand cuts? Cell. 1995;82:349[Medline] [Order article via Infotrieve]. 37. Skalka M, Matyasova J, Cejkova M. The sensitivity of chromatin from thymuses and spleens of irradiated mice to alkaline solutions. FEBS Lett. 1976;72:271. 38. Wyllie AH. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature. 1980;284:555[Medline] [Order article via Infotrieve]. 39. Srinivasula SM, Ahmad M, Fernandes-Alnemri T, Alnemri ES. Autoactivation of procaspase-9 by Apaf-1-mediated oligomerization. Mol Cell. 1998;7:949.
40.
Lavoie JN, Nguyen M, Marcellus RC, Branton PE, Shore GC.
E4orf4, a novel adenovirus death factor that induces p53-independent apoptosis by a pathway that is not inhibited by zVAD-fmk.
J Cell Biol.
1998;140:637
41.
De Maria R, Lenti L, Malisan F, et al.
Requirement for the GD3 ganglioside in CD95- and ceramide-induced apoptosis.
Science.
1997;277:1652
42.
Miller TM, Moulder KL, Knudson CM, et al.
Bax deletion further orders the cell death pathway in cerebellar granule cells and suggests a caspase-independent pathway to cell death.
J Cell Biol.
1997;139:205
43.
Janicke RU, Ng P, Sprengart ML, Porter AG.
Caspase-3 is required for
44.
Eischen CM, Kottke TJ, Martins LM, et al.
Comparison of apoptosis in wild-type and Fas-resistant cells: chemotherapy-induced apoptosis is not dependent on Fas/Fas ligand interactions.
Blood.
1997;90:935
45.
Byrd JC, Shinn C, Waselenko JK, et al.
Flavopiridol induces apoptosis in chronic lymphocytic leukemia cells via activation of caspase-3 without evidence of bcl-2 modulation or dependence on functional p53.
Blood.
1998;92:3804 46. Hafid-Medheb K, Poindessous-Jazat V, Augery-Bourget Y, Hanania N, Robert-Lezenes J. Bcl-XL induction during terminal differentiation of friend erythroleukemia cells correlates with delay in apoptosis and loss of proliferative capacity but not haemoglobinization. Cell Death Differ. 1999;6:166[Medline] [Order article via Infotrieve].
47.
Richon VM, Webb Y, Merger R, et al.
Second generation hybrid polar compounds are potent inducers of transformed cell differentiation.
Proc Natl Acad Sci U S A.
1996;93:5705
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  M. M. Shareef, B. Brown, S. Shajahan, S. Sathishkumar, S. M. Arnold, M. Mohiuddin, M. M. Ahmed, and P. M. Spring Lack of P-Glycoprotein Expression by Low-Dose Fractionated Radiation Results from Loss of Nuclear Factor-{kappa}B and NF-Y Activation in Oral Carcinoma Cells Mol. Cancer Res., January 1, 2008; 6(1): 89 - 98. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Han, L. Li, S. Qiu, Q. Lu, Q. Pan, Y. Gu, J. Luo, and X. Hu Shikonin circumvents cancer drug resistance by induction of a necroptotic death Mol. Cancer Ther., May 1, 2007; 6(5): 1641 - 1649. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Turella, G. Filomeni, M. L. Dupuis, M. R. Ciriolo, A. Molinari, F. De Maria, M. Tombesi, M. Cianfriglia, G. Federici, G. Ricci, et al. A Strong Glutathione S-Transferase Inhibitor Overcomes the P-glycoprotein-mediated Resistance in Tumor Cells: 6-(7-NITRO-2,1,3-BENZOXADIAZOL-4-YLTHIO)HEXANOL (NBDHEX) TRIGGERS A CASPASE-DEPENDENT APOPTOSIS IN MDR1-EXPRESSING LEUKEMIA CELLS J. Biol. Chem., August 18, 2006; 281(33): 23725 - 23732. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Kelly, N. J. Waterhouse, E. Cretney, K. A. Browne, S. Ellis, J. A. Trapani, and M. J. Smyth Granzyme M Mediates a Novel Form of Perforin-dependent Cell Death J. Biol. Chem., May 21, 2004; 279(21): 22236 - 22242. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J. Peart, K. M. Tainton, A. A. Ruefli, A. E. Dear, K. A. Sedelies, L. A. O'Reilly, N. J. Waterhouse, J. A. Trapani, and R. W. Johnstone Novel Mechanisms of Apoptosis Induced by Histone Deacetylase Inhibitors Cancer Res., August 1, 2003; 63(15): 4460 - 4471. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z.-H. Miao and J. Ding Transcription Factor c-Jun Activation Represses mdr-1 Gene Expression Cancer Res., August 1, 2003; 63(15): 4527 - 4532. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Poulaki, C. S. Mitsiades, A. M. Joussen, A. Lappas, B. Kirchhof, and N. Mitsiades Constitutive Nuclear Factor-{kappa}B Activity Is Crucial for Human Retinoblastoma Cell Viability Am. J. Pathol., December 1, 2002; 161(6): 2229 - 2240. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. W. Johnstone, K. M. Tainton, A. A. Ruefli, C. J. Froelich, L. Cerruti, S. M. Jane, and M. J. Smyth P-glycoprotein Does Not Protect Cells against Cytolysis Induced by Pore-forming Proteins J. Biol. Chem., May 11, 2001; 276(20): 16667 - 16673. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. A. Ruefli, M. J. Ausserlechner, D. Bernhard, V. R. Sutton, K. M. Tainton, R. Kofler, M. J. Smyth, and R. W. Johnstone The histone deacetylase inhibitor and chemotherapeutic agent suberoylanilide hydroxamic acid (SAHA) induces a cell-death pathway characterized by cleavage of Bid and production of reactive oxygen species PNAS, September 11, 2001; 98(19): 10833 - 10838. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2000 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
m), release of cytochrome c, and
subsequent activation of caspase-9. In addition, disruption of
can't live without it.
Cell.
1997;91:559