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Biochemistry and Biophysics |
B through the IKK Complex by the Topoisomerase Poisons SN38 and Doxorubicin
Institut National de la Santé et de la Recherche Médicale U526, Activation des Cellules Hématopoïétiques, Physiologie de la Survie et de la Mort Cellulaires et Infections Virales [V. Bo., V. Bu., J. F. P.] and Institut National de la Santé et de la Recherche Médicale U364, Immunologie Cellulaire et Moléculaire [A. L.], IFR 50 Génétique et Signalisation Moléculaires. Faculté de Médecine Pasteur, 06107 Nice cedex 02, France, and Centre Antoine Lacassagne, Laboratoire d’Oncopharmacologie, 06050 Nice cedex 1, France [N. M., J-L. F., G. M.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B is involved in the regulation of cell survival. NFB is activated in many malignant tumors and seems to play a role in the resistance to cytostatic treatments and escape from apoptosis. We have studied the effects on NFB activation of two topoisomerase poisons and DNA damaging agents that are used in chemotherapy: SN38 (7-ethyl-10-hydroxycamptothecin), the active metabolite of CPT11, and doxorubicin. In HeLa cells, both drugs activate NFB using a preexisting pathway that requires a functional IB-specific kinase complex, IB-specific kinase activation, IB-
phosphorylation, and degradation. Blocking NFB activation by stable expression of a mutant super-repressor IB- molecule sensitized HeLa cells to the apoptotic actions of drugs and tumor necrosis factor. RNase protection assay analysis demonstrate that NFB is involved in the regulation of a complex pattern of gene activation and repression during the cellular response of HeLa cells to topoisomerase poisons. The blockade of NF-B activation seems to shift the death/survival balance toward apoptosis. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B (5)
, which, besides well-known functions in the control of immunity and inflammation (6)
, now seems to be an important player in the regulation of cell survival through the induction of antiapoptotic genes (7)
. A constitutive NFB activity has been observed in many cancer types and was shown to contribute to cell survival and protection against apoptosis induced by DNA-damaging agents (reviewed in Ref. 8
). In addition, the blockade of NFB activation increased the sensitivity of malignant cells to the proapoptotic effect of TNF or, more dramatically, to treatments with drugs or radiation (9)
. The failure to activate NFB in response to ionizing radiation results in cell apoptosis (10)
. Therefore, NFB has been proposed as a therapeutic target to increase the efficiency of actual treatments of cancer (11)
. In most cells, Rel/NFB proteins are rendered inactive in the cytoplasm by inhibitory subunits of the IB family and activated by a large number of stimuli arising from membrane receptors (i.e., TNF, IL1; reviewed in Ref. 5
). After receptor engagement, IKKs (IKK and IKKß) that reside in a high-molecular weight, multiprotein complex, the IKK signalsome, are activated to phosphorylate IB molecules on two NH2-terminal serines (32 and 36 for IB-). This phosphorylation targets IB- for polyubiquitination and subsequent degradation in situ by the 26S subunit of the proteasome to liberate NFB. NFB subunits then can translocate to the nucleus to activate target gene expression (reviewed in Ref. 12
).
NFB is activated by UV (13)
or 
-ray irradiation (14)
or cytotoxic drugs (5)
. However, it is not clear whether these conditions use a signaling pathway(s) from the nucleus to the cytoplasm or directly generate cytoplasmic events. Topoisomerases are essential for cell survival and crucial for every aspects of DNA metabolism. They are a target of choice for several drugs used in various cancer treatments. Topoisomerases control the degree of supercoiled DNA (15)
, and whereas type I is crucial for transcription (16)
, type II is necessary for DNA replication (17)
. In both situations, the enzymes create transient single-strand breaks in DNA, and the poisons prevent the religation of the strand breaks. SN38, a powerful active metabolite of CPT-11, targets topoisomerase I activity (18)
, whereas doxorubicin and daunorubicin of the anthracycline family are topoisomerase type II poisons (17)
.
The aim of the present study was to define the mechanisms of NFB activation by the topoisomerase poisons SN38 and doxorubicin in HeLa cells. Studying the effect of topoisomerase poisons on NFB activation and dissecting this signaling pathway is a prerequisite to a better understanding of the effects of DNA damage on cellular functions and to the identification of crucial cellular steps, the targeting of which could help to increase the efficiency of cancer treatments. We show here that SN38 and doxorubicin activate NFB in a pathway that requires a functional IKK complex, IKK activation, IB- phosphorylation, and degradation. This activation of NFB protects cells from the apoptotic effects of the drugs. Moreover, we have studied the effects of SN38 and doxorubicin on the expression of several genes that are important for the control of the balance between survival and death.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B- rabbit polyclonal (C15), anti-p50 rabbit polyclonal, anti-p65 rabbit polyclonal, and anti phospho-IB antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). The IKK-expressing vectors were kindly provided by Frank Mercurio (19) . SN38 was kindly provided by Rhône Poulenc Rorer. TNF was from PeproTech (Rocky Hill, NJ). Doxorubicin was from Pharmacia & Upjohn SA (St-Quentin-en-Yvelines, France). PMA was from Sigma Chemical Co.-Aldrich (L’Isle d’Abeau, FR).
Cell Culture.
HeLa cells were maintained in DMEM containing 50 units/ml penicillin, 50 µg/ml streptomycin, 1 mM sodium pyruvate, 2 mM glutamine, and 10% FCS (Life Technologies, Inc., Cergy Pontoise, France). The 70Z/3 murine pre-B cell line and its NEMO-defective variant have been described previously (20)
and were maintained in RPMI 1640 supplemented with 10% FCS and 50 µM ß-mercaptoethanol.
Luciferase Assays.
HeLa cells were transiently transfected by the classical calcium phosphate method, as described previously (21)
with 0.5 µg of a luciferase reporter gene controlled by a minimal thymidine kinase promoter and six reiterated B sites (B6 thymidine kinase luc). Forty-eight h after transfection, cells were stimulated for 18 h as indicated.
Cells were harvested and lysed in 200 µl of lysis reporter buffer (Promega). Soluble extracts were assayed for luciferase activity on a luminometer. Luciferase activity was normalized to protein amount.
EMSA.
Total cellular extracts were prepared in Totex lysis buffer [20 mM HEPES (pH 7.9), 350 mM NaCl, 20% glycerol, 1% NP40, 1 mM MgCl2, 0.5 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 1 mM PMSF, and 10 µg/ml aprotinin ]. Supernatants from 15,000 x g, 15 min centrifugation, were collected.
For mobility shift assay, a NFB probe was used consisting of a synthetic double-stranded oligonucleotide containing the B binding site of the immunoglobulin promoter (5'-GATCCAAGGGACTTTCCATG-3'). The end-labeled probe (T4 kinase) was incubated with Totex extracts samples (10 µg) for 20 min at 37°C. Complexes were separated by electrophoresis on a 5% nondenaturing polyacrylamide gel in 0.5 x Tris-borate EDTA. Dried gels were subjected to autoradiography.
For supershift assay, antibodies against p65 or p50 or non immune serum were added 10 min before the labeled probe.
Western Blot Analysis.
After stimulation, HeLa cells were solubilized in lysis buffer [50 mM HEPES (pH7.4), 150 mM NaCl, 20 mM EDTA, 100 µM NaF, 10 mM Na3VO4, 1 mM PMSF, and 0.5% NP40] and incubated for 30 min at 4°C. Lysates were centrifuged at 10,000 x g for 10 min at 4°C, and supernatants were adjusted with concentrated SDS sample buffer. Proteins were separated by SDS/PAGE and transferred to Immobilon membranes (Millipore, Bedford, MA) in a Tris (20 mM), glycine (150 mM) and methanol (20%) buffer at 55 V for 4 h at 4°C.
IB- was detected with polyclonal antibodies (Santa Cruz Biotechnology) in saturation buffer [50 mM Tris (pH 7.5), 50 mM NaCl, 0.15% Tween 20, and 5% skim milk) and with a secondary peroxidase-conjugated antirabbit antibody. Proteins were visualized with Amersham enhanced chemiluminescence detection.
Immunoprecipitation and Kinase Assay.
Whole extracts from 1.2 x 106 HeLa cells treated or not as indicated were prepared in lysis buffer and precleared by incubation with nonimmune serum and protein A-Sepharose. Then lysates were incubated for 3 h at 4°C with a 1:100 dilution of anti-IKKß antiserum and IPs were collected by adding 15 µl of a 50% slurry protein A-Sepharose/PBS for 1 h at 4°C and centrifugation. IPs were washed twice in lysis buffer 0.5% NP40, once in STOP and once in kinase buffer [20 mM HEPES (pH 7.5), 10 mM MgCl2, 100 µM Na3VO4, 20 mM ß glycerophosphate, 2 mM DTT, and 50 mM NaCl complete without EDTA protease inhibitors cocktail]. Samples were incubated for 30 min at 30°C in kinase buffer with 2.5 µCi [-32P]ATP and 0.5 µg of recombinant IB- as substrate. Reactions were stopped by the addition of SDS sample buffer, and samples were boiled and resolved on 10% SDS-PAGE. Dried gels were autoradiographed on X-OMAT AR films (Kodak, Rochester, NY).
Determination of Subdiploid DNA Content.
HeLa cells were stimulated as indicated and harvested by centrifugation. Cell cycle analysis was performed by quantifying the DNA content using propidium iodide staining, according to Vindelov et al. (22)
.
Generation of HeLa Expressing IB- WT or IB-SA.
The cDNA for WT IB- or serines 32 and 36 to alanines IB- mutant were a kind gift of Patrick A. Baeuerle and were subcloned in pEF6/myc-His A (Invitrogen, Carlsbad, CA). HeLa cells were transfected with 10 µg of plasmid using the Tfx 20 reagent (Promega, Madison, WI). Forty-eight h after transfection, cells were split into fresh medium containing 10 µg/ml of blasticidin until blasticidin-resistant colonies emerged. The expression of transfected IB- was monitored by Western blot using anti-myc antibodies, and the inhibitory effect of the mutated protein on NFB activation was checked by EMSA.
Measurement of Caspase Activity.
Cells were stimulated as indicated and then incubated for 30 min at 4°C in lysis buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 20 mM EDTA, 0.2% Triton X-100, 1 mM PMSF, and 10 µg/ml aprotinin). Lysates were cleared at 10,000 x g for 15 min at 4°C. One hundred µg of protein were incubated in a 96-well plate with 0.2 mM Ac-DEVD-pNA for various times at 37°C. The caspase activity is measured at 410 nm in the presence or absence of 1 µM of N-acetyl-Asp-Glu-val-Asp-CHO. The specific caspase activity is expressed in Ac-pNA cleavage or released absorbance.
RPA.
After stimulation, cells were harvested, and total RNA was isolated using the TriPure reagent (Boehringer, Mannheim, Germany). Multiprobe template sets (hAPO-1c, hAPO2c, and hAPO5) were from PharMingen (San Diego, CA). The 32P-labeled riboprobes were hybridized for 18 h with 5 µg of RNA. The hybridized RNA was digested with RNase, and remaining RNase-protected probes were purified, resolved on denaturing polyacrylamide gels, imaged on a STORM 840 (Molecular Dynamics, Amersham Pharmacia Biotech, Freiburg, Germany), and quantified using the ImageQuant software (Molecular Dynamics). The expression of each mRNA species was calculated after equalization according to the expression level of the two housekeeping genes L32 and GAPDH.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B by SN38 and Doxorubicin.B activation, were analyzed on HeLa cells. After different treatments with the drugs, HeLa cells were harvested, and the binding of NFB to DNA was visualized by EMSA. No activation of NFB could be detected in unstimulated cells (Fig. 1, A, B, and D
, Lanes 1, and C, Lane 2). SN38 treatment for 2 h induced a dose-dependent activation of NFB (Fig. 1A
, Lanes 2–6), with a maximum at 10 µM (Lane 5). The activation was slightly lower than that observed after TNF stimulation (Lane 7). At 10 µM, SN38 induced a rapid activation of NFB, which was detectable after 1 h (Fig. 1B
, Lane 3) and lasted until 3 h (Lane 5). The upper complex corresponding to NFB binding was competed by an excess of B-unlabeled probe, but not by an unrelated double-strand oligonucleotide (data not shown).
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B (Fig. 1, C and D)
, with a maximal effect at 5 µM (Fig. 1C
, Lane 5) after 2 h (Fig. 1D
, Lane 4).
The ability of SN38 and doxorubicin to induce NFB-dependent gene activation was then examined in luciferase assays. HeLa cells were transfected with a reporter construct in which the luciferase gene is placed under a 6-fold repetition of a B binding site and then treated for 18 h with different concentrations of SN38 or doxorubicin. Luciferase accumulation was measured as indicated in "Materials and Methods." As shown in Fig. 1E
, SN38 and doxorubicin induced B-transcription of the luciferase gene in a dose-dependent manner. Taken together, the results showed that SN38 and doxorubicin are powerful activators of NFB in HeLa cells.
Effect of CHX on NFB Activation.
NFB activation by SN38 and doxorubicin could be either a direct mechanism or mediated through the secondary production of NFB activators such as TNF- and IL1ß. To distinguish between these possibilities, protein neosynthesis was blocked by pretreatment of HeLa cells with CHX before drug stimulation. As shown in Fig. 1E
, CHX had no effect on NFB activation induced by two doses (5 and 10 µM) of SN38 (compare Lanes 3 and 5 with Lanes 2 and 4) nor by two doses (1 and 5 µM) of doxorubicin (compare Lanes 7 and 9 with Lanes 6 and 8). Thus, activation of NFB by topoisomerase inhibitors does not require synthesis of a protein intermediate.
Serines 32 and 36 on IB- Are Required for SN38 and Doxorubicin-induced NFB Activation.
Engagement of the classical NFB activation pathway requires phosphorylation of IB- on serines 32 and 36 with subsequent degradation to allow the release of active NFB.
Luciferase assays were performed in the presence of plasmids coding for WT IB- or a serines 32–36 to alanines-mutated, dominant-negative version of IB-. As shown in Fig. 2A
, expression of WT IB- only slightly decreased SN38- or TNF-induced activation, but had no effect on doxorubicin-induced activation. By contrast, the double serine mutant of IB- totally inhibited TNF, SN38, or doxorubicin-induced NFB activation.
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B-.B- after drug treatment. HeLa cells were treated or not with CHX for 1 h before stimulation by topoisomerase poisons. The cellular levels of IB- were visualized by Western blotting. SN38 induced a rapid decrease in IB- levels after 1 h that lasted for 3 h (Fig. 2B
, top panel, Lanes 2–4). The decrease was amplified by CHX pretreatment which prevented IB- resynthesis (top panel, Lanes 6–8). The same results were observed with doxorubicin, except that degradation of IB- was slower and delayed compared with SN38. Degradation was visible only after 2 h (bottom panel, Lane 3). CHX pretreatment strongly amplified the effect of doxorubicin (bottom panel, Lanes 6–8).
Involvement of IKKs in Drug-induced NFB Activation.
Phosphorylation and degradation of IB- is regulated by two specific kinases, IKK and IKKß (12)
. We investigated whether these kinases could be involved in NFB activation induced by SN38 and doxorubicin. HeLa cells were left untreated or stimulated with the DNA-damaging drugs for 15 min. The IKK complex was then precipitated from cell lysates with specific IKKß antibodies, and IPs were subjected to in vitro kinase assay in the presence of recombinant IB- (0.5 µg). The results displayed on Fig. 2C
show that SN38 (Lane 4) and, to a lesser extent, doxorubicin (Lane 6) induced phosphorylation of IKKß (top panel) as well as of IB- (bottom panel). No kinase activity could be detected in unstimulated cells (Lane 2) nor in precipitates with nonimmune serum (Lanes 1, 3, and 5). The stimulatory conditions used (SN38 and doxorubicin) did not affect the amount of cellular IKK, IKKß, and NEMO proteins (three bottom panels).
Phosphorylation of IB- was then studied using a phospho-specific IB- antibody. Cells were pretreated or not with the proteasome inhibitor ALLN before stimulation with SN38 doxorubicin or TNF for the indicated times. ALLN was used to stabilize the phosphoform of IB-. In the presence of ALLN, SN38 and doxorubicin as well as TNF-induced phosphorylation of IB- that was detectable after 30 min and remained stable over 2 h (Fig. 2D)
. The levels of total IB- decreased upon stimulation and were slightly stabilized by ALLN pretreatment.
Activation of NFB by Drugs Requires a Functional IKK Complex.
The IKK are part of a high-molecular weight protein complex whose integrity and functionality is guarantied by a scaffold component NEMO/IKK. In a gene reporter experiment, cDNAs for dominant negative forms of IKKs mutated within the ATP acceptor site (IKKKM and IKKßKM) were transfected in HeLa cells, and NFB activation was measured in a luciferase assay. Expression of IKKKM or IKKßKM totally inhibited NFB activation induced by SN38, doxorubicin or, as control, TNF (Fig. 3A)
. These experiments highlight the importance of the IKK proteins in the effect of topoisomerase poisons.
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B activation (20)
. This is shown on Fig. 3B
. Stimulation by PMA (Lanes 2 and 10) leads to NFB activation in 70Z/3 cells but not in the 1.3E2 variant (Lanes 6 and 15). Similarly, SN38 and doxorubicin induced a dose-dependent NFB activation in the parental (respectively, Lanes 3, 4, and 11–13) but not in the NEMO-deficient variant (Lanes 7, 8, and 16–18).
Altogether, these results demonstrate that the DNA-damaging drugs SN38 and doxorubicin require an intact and functional IKK complex for activation of NFB.
Regulation of Drug-induced Apoptosis by NFB.
In some but not all tumor cell lines, activation of NFB prevents apoptosis. We have stably transfected HeLa cells with WT or dominant-negative forms (IB-SA, mutated on the two regulatory NH2-terminal serines 32 and 36) of IB-. The stable expression of IB-SA but not of WT IB- completely blocked NFB activation by various stimuli (TNF, IL1, PMA, SN38, and doxorubicin) as observed in EMSA experiments (data not shown). We then studied the sensibility of stable clones to drug-induced apoptosis. Cell death was visualized by quantification of either caspase 3 activity and of the number of cells with a DNA content <2 N (sub-G1 population). As shown on Fig. 4A
, stimulation of WT IB--expressing cells with SN38, doxorubicin, and TNF for 8 h led to increases in caspase 3 activity (respectively 1.4-, 2.5-, and 1.8-fold) that were potentiated by expression of the dominant inhibitor IB-SA (2.2-, 3-, and 2.9-fold). The blockade of NFB activation also potentiated the number of apoptotic cells visualized as sub-G1 cells after stimulation by SN38 and TNF (Fig. 4B)
. The apoptotic effect of doxorubicin could not be evaluated by this method, likely because of an interference of the drug with propidium iodide staining.
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B by drugs or TNF decreased the efficiency of these compounds to induce cell death.
Expression of Life and Death-regulatory Genes by DNA-damaging Drugs.
The main effect of these drugs is to kill cancerous cells by apoptosis. We thus analyzed by RPA their effects on the expression of several genes that are involved in the control of cell death or survival. To evaluate the contribution of NFB to the actions of the drugs, we used the stable transfectants of HeLa that express either WT or a dominant-negative form of IB- (IB-SA). After stimulation of HeLa cells, RNA was prepared and hybridized to specific probes. The relative expression of the mRNA species, expressed in arbitrary units, was calculated after quantification of each band and normalization according to the expression levels of the housekeeping genes L32 and GAPDH. SN38 and doxorubicin induced a rapid decrease (1.3–2-fold) in the expression of the antiapoptotic genes coding for Bcl-w, Bcl-xl, Mcl-1 (Fig. 5)
. Used as a control, TNF increased the levels of these mRNA species (1.1–1.8-fold). When activation of NFB was blocked by expression of the IB- repressor, TNF could no longer stimulate Bcl-w, Bcl-xl, and Mcl-1 gene expression. Moreover, in these conditions, SN38 rather had a up-regulatory effect (1.2-fold) on Bcl-w and Bcl-xl expression. TNF had no significant effect on the expression of the genes for the proapoptotic mitochondrial proteins Bad, Bax, and Bak. On the contrary, SN38 and doxorubicin slightly decreased their expression levels (1.3–1.4-fold). In the absence of NFB activation, these mRNA species were either up-regulated (1.1-fold) by SN38 or still decreased (1.3–1.5-fold) by doxorubicin. The drugs were found to stimulate the expression of the genes for the inhibitors of apoptosis c-IAP-1, c-IAP-2, XIAP, and TRPM-2, although with a lower efficiency compared with TNF (average, 1.1–1.4-fold versus 1.1–1.5-fold). When NFB was blocked, the drugs had a rather inhibitory effect (1.4–2.2-fold). In particular, the high-level expression of TRPM2 completely collapsed with a basal level decreased by 2.8-fold compared with WT IB--expressing cells, and no stimulatory effects were observed.
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B-SA super repressor-expressing cells both drugs slightly stimulated expression of caspase 8 (1.1-fold). SN38 and doxorubicin stimulated transcription of the caspase 9 gene (3-fold) in an NFB-dependent manner. Although TNF had no effect on caspase 8 expression and other caspases tested (caspases 1–7 and 10, not shown), its small activatory effect on caspase 9 expression (1.2-fold) was potentiated in the absence of NFB activation (2.8-fold). Student’s t test analysis shows that the differences are statistically significant, with Ps between (*, P < 0.2; ** P < 0.1; and ***, P < 0.05).
On the whole, the results show that NFB is involved in a complex pattern of gene activation and repression during the cellular response of HeLa cells to topoisomerase poisons. The blockade of NFB activation seems to shift the death/survival balance toward apoptosis.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B activation after production of DNA damage by the topoisomerase poisons SN38 and doxorubicin. Both compounds induced a rapid and dose-dependent activation of NFB. This is the consequence of stimulation of the IKK kinases that phosphorylate and inactivate the inhibitory subunit IB-. Drug-induced NFB activation measured by binding of the factor to DNA or activation of transcription could be blocked by the dominant-negative form of the IKK or by a dominant-negative mutant of IB-. Moreover, disruption of the integrity of the IKK complex by the absence of the NEMO component prevents activation of NFB by the drugs. These results demonstrate that topoisomerase-targeting agents activate NFB through mobilization and stimulation of the classical IKK complex.
The primary site of action of DNA-damaging drugs is in the nucleus. It has been shown by other investigations that camptothecin requires binding to topoisomerase I (23)
whereas mitoxantrone, an anthracenedione, must bind topoisomerase II (24)
to activate NFB. We have excluded an indirect action of SN38 and doxorubicin through the synthesis of an autocrine NFB activator, because their effects could not be blocked by a protein synthesis inhibitor. Therefore, the two compounds likely mobilize a preexisting signaling pathway that starts from the nucleus at the level of DNA strand breaks to end up in the cytosol at the IKK complex. Several proteins have been proposed to map in the NFB-signaling pathway mobilized by DNA damage. One interesting candidate is ATM, a member of the high molecular weight PI3-kinase-like family, which is mutated in ataxia-telangiectasia patients (25)
, characterized by immunodeficiency and extreme radiosensitivity (26)
. NFB activation by ionizing radiation is defective in ataxia-telangiectasia cell lines (27
, 28)
or in tissues or from ATM null (-/-) mice (28)
. Activation of NFB subsequent to DNA damage was also shown to depend on ATM expression (29)
.
DNA-PK has also been implicated in the activation of NFB after DNA damage, however the results seem more conflicting. On one side, no NFB activation could be detected after irradiation in DNA-PK-negative cell lines derived from a human glioma biopsy (30)
. On the contrary, recently it has been reported clearly that separate invalidation of the three subunit of DNA-PK, the catalytic subunit PKCs and the DNA binding subunits Ku 70 and Ku 80 did not prevent NFB activation by irradiation (28)
. It remains possible that the type (single- or double-strand breaks) or by the extent of the lesions to the DNA, together with the production or not of reactive oxygen intermediates, could have different requirements for ATM versus DNA-PK.
NFB is now recognized as an important player in the regulation of cell survival. Inactivation of the p65 NFB (relA) gene results in embryonic death after massive liver apoptosis (31)
. Many malignant tumors display constitutive NFB activation that allows malignant cells to escape growth regulation by apoptosis (32)
. This constitutive NFB activation is caused by genetic alterations affecting nfkb/ikb genes or to unrestrained IKK stimulation (reviewed in (8)
. The survival power of NFB could be demonstrated in various cell lines by the expression of a super-repressor mutant form of the inhibitory subunit IB-, which become more susceptible to apoptosis induced by TNF, cytotoxic drugs, or -rays (9
, 31
, 33)
. Using HeLa cells stably transfected with a dominant negative mutant of IB-, we could confirm that activation of NFB by SN38 and doxorubicin lowered the efficiency of the drugs to induce apoptosis. This phenomenon has also been described in vivo, inasmuch as the expression of the IB- repressor in a mouse model of tumor formation with injected human fibrosarcoma cells decreased the chemoresistance to CPT and SN38 of the resulting tumors (11)
. In some models however, NFB does not regulate or even promotes apoptosis (34, 35, 36)
. It would be most important to elucidate which cellular event regulates the cellular response to NFB modulation.
NFB controls a wide set of genes, among which several are interesting candidates for regulators of apoptosis (5)
. Differential screening approaches on various cellular models have allowed the identification of several antiapoptotic genes (37, 38, 39, 40)
: TRAF1 and TRAF2 adapters likely act by inducing a constitutive signaling to NFB to strengthen transcription of the antiapoptotic gene (38)
; c-IAP-1 and c-IAP-2 bind and block caspase activation (41)
; Bcl-x and Bfl1 of the Bcl-2 family control mitochondrial homeostasis and prevent release of cytochrome c and subsequent activation of caspase 9 (42)
.
HeLa cells expressing the IB-SA repressor were an exquisite model to study the implication of NFB on gene transcription during drug-induced DNA damage. An RPA analysis of several genes involved in cell death/survival was performed in the present study and revealed the existence of a complex network of gene activation and repression. Interestingly, SN38 and doxorubicin seemed to exert both pro- and antiapoptotic actions. Proapoptotic effects result, at least in part, from the down-regulation of molecules Bcl-w, Bclx-l, Mcl-1 and the up-regulation of caspase 9 expression. On the other hand, antiapoptotic actions were mediated via down-regulation of Bad, Bax, Bak, and stimulation of c-IAP-1, c-IAP-2, XIAP, and TRPM2 expression. The blocking of NFB activation leads to an inversion of the effects of the drugs on gene expression that could account for the increased cell death observed in these conditions. Notably, SN38 and doxorubicin failed to up-regulate c-IAP-1, c-IAP-2, XIAP, and TRPM2 gene expression and stimulated caspase 8 as well as Bad, Bax, and Bak mRNA species. This assay only addresses the expression of mRNA and therefore must be completed by a quantitative study of the relevant proteins to strengthen these observations. Deciphering the signaling pathways as well as identification of the target genes involved in the antiapoptotic actions of NFB can be an interesting approach for the improvement of anticancer treatments through new drugs design.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by institutional grants from Institut National de la Santé et de la Recherche Médicale, Grant 9332 from the Association pour la Recherche contre le Cancer, La Ligue Nationale contre le Cancer, La Ligue contre le Cancer comité des Bouches du Rhône, and La Fondation de France comité Leucémies. 
2 To whom requests for reprints should be addressed, at INSERM U526, Faculté de Médecine, Pasteur, 06107 Nice cedex 02, France. Phone: 33-493-377-676; Fax: 33-493-817-852; E-mail: peyron{at}unice.fr
3 The abbreviations used are: SN38, 7-ethyl-10-hydroxycamptothecin; NFB, nuclear factor B; TNF, tumor necrosis factor; IL, interleukin; IKK, IB-specific kinase; PMA, phorbol 12-myristate 13-acetate; NEMO, NFB-essential modulator; CPT11, 7-ethyl-10-[4-(1-piperidino)-1piperidino]carbonyloxycamptothean; IP, immunoprecipitate; PMSF, phenylmethylsulfonyl fluoride; WT, wild type; RPA, RNase protection assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EMSA, electrophoretic mobility shift assays; CHX, cycloheximide; Ac-DEVD-pNA, acetyl-Asp-Glu-Val-Asp-p-nitroanilide; ALLN, N-acetyl-Leu-Leu-NorLencinal.
Received 4/10/01. Accepted 8/31/01.
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REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
B transcription factors. Oncogene, 18: 6853-6866, 1999.[Medline]
B, and Rel proteins. evolutionarily conserved mediators of immune responses. Annu. Rev. Immunol., 16: 225-260, 1998.[Medline]
B to the rescue. Trends Genet., 15: 229-235, 1999.[Medline]
and cancer therapy-induced apoptosis: potentiation by inhibition of NF-B. Science, 274: 784-787, 1996.B results in apoptosis induced by radiation. Radiat. Res., 149: 596-601, 1998.[Medline]
B. Nat. Med., 5: 412-417, 1999.[Medline]
B activity. Annu. Rev. Immunol., 18: 621-663, 2000.[Medline]
B. J. Clin. Investig., 88: 691-695, 1991.
B Kinases essential for NF-B activation. Science (Wash. DC), : 286-290, 1997.
B kinase complex essential for NF-B activation. Cell, 93: 1231-1240, 1998.[Medline]
B activation by camptothecin. A linkage between nuclear DNA damage and cytoplasmic signaling events. J. Biol. Chem., 275: 9501-9509, 2000.B activation in HL60 cells. J. Biol. Chem., 275: 25231-25238, 2000.B. Oncogene, 17: 1821-1826, 1998.[Medline]
B following DNA damage. Oncogene, 18: 2261-2271, 1999.[Medline]
B following DNA damage. Biochem. Biophys. Res. Commun., 247: 79-83, 1998.[Medline]
B. Nature, 376: 167-170, 1995.[Medline]
B transcription factors. Oncogene, 18: 6910-6924, 1999.[Medline]
-induced apoptosis by NF-B. Science (Wash. DC), 274: 787-789, 1996.B, cancer, and apoptosis. Biochem. Pharmacol., 60: 1085-1089, 2000.[Medline]
B in p53-mediated programmed cell death. Nature, 404: 892-897, 2000.[Medline]
B and 26S proteasome in apoptotic cell death induced by topoisomerase I and II poisons in human non-small cell lung carcinoma. J. Biol. Chem., 276: 8029-8036, 2001.B control. Proc. Natl. Acad. Sci. USA, 94: 10057-10062, 1997.B antiapoptosis induction of TRAF1 and TRAF2 ans c-IAP1 and c-IAP2 to suppress caspase-8 activation. Science (Wash. DC), 281: 1680-1683, 1998.-induced apoptosis. Genes Dev., 13: 382-387, 1999.This article has been cited by other articles:
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  S. Fan, Q. Meng, J. J. Laterra, and E. M. Rosen Role of Src Signal Transduction Pathways in Scatter Factor-mediated Cellular Protection J. Biol. Chem., March 20, 2009; 284(12): 7561 - 7577. [Abstract] [Full Text] [PDF]
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 B. B. Aggarwal, R.V. Vijayalekshmi, and B. Sung Targeting Inflammatory Pathways for Prevention and Therapy of Cancer: Short-Term Friend, Long-Term Foe Clin. Cancer Res., January 15, 2009; 15(2): 425 - 430. [Abstract] [Full Text] [PDF]
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 L. Shkreta, U. Froehlich, E. R. Paquet, J. Toutant, S. A. Elela, and B. Chabot Anticancer drugs affect the alternative splicing of Bcl-x and other human apoptotic genes Mol. Cancer Ther., June 1, 2008; 7(6): 1398 - 1409. [Abstract] [Full Text] [PDF]
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 J. G. Izzo, U. Malhotra, T.-T. Wu, R. Luthra, A. M. Correa, S. G. Swisher, W. Hofstetter, K.S. C. Chao, M.-C. Hung, and J. A. Ajani Clinical Biology of Esophageal Adenocarcinoma after Surgery Is Influenced by Nuclear Factor-{kappa}B Expression Cancer Epidemiol. Biomarkers Prev., June 1, 2007; 16(6): 1200 - 1205. [Abstract] [Full Text] [PDF]
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J. G. Izzo, A. M. Correa, T.-T. Wu, U. Malhotra, C. K.S. Chao, R. Luthra, J. Ensor, A. Dekovich, Z. Liao, W. N. Hittelman, et al. Pretherapy nuclear factor-{kappa}B status, chemoradiation resistance, and metastatic progression in esophageal carcinoma. Mol. Cancer Ther., November 1, 2006; 5(11): 2844 - 2850. [Abstract] [Full Text] [PDF]
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X. Dolcet, D. Llobet, M. Encinas, J. Pallares, A. Cabero, J. A. Schoenenberger, J. X. Comella, and X. Matias-Guiu Proteasome Inhibitors Induce Death but Activate NF-{kappa}B on Endometrial Carcinoma Cell Lines and Primary Culture Explants J. Biol. Chem., August 4, 2006; 281(31): 22118 - 22130. [Abstract] [Full Text] [PDF]
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 C Montagut, I Tusquets, B Ferrer, J M Corominas, B Bellosillo, C Campas, M Suarez, X Fabregat, E Campo, P Gascon, et al. Activation of nuclear factor-{kappa} B is linked to resistance to neoadjuvant chemotherapy in breast cancer patients. Endocr. Relat. Cancer, June 1, 2006; 13(2): 607 - 616. [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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 Y. Takada, H. Ichikawa, V. Badmaev, and B. B. Aggarwal Acetyl-11-Keto-beta-Boswellic Acid Potentiates Apoptosis, Inhibits Invasion, and Abolishes Osteoclastogenesis by Suppressing NF-{kappa}B and NF-{kappa}B-Regulated Gene Expression. J. Immunol., March 1, 2006; 176(5): 3127 - 3140. [Abstract] [Full Text] [PDF]
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 J. G. Izzo, U. Malhotra, T.-T. Wu, J. Ensor, R. Luthra, J. H. Lee, S. G. Swisher, Z. Liao, K.S. C. Chao, W. N. Hittelman, et al. Association of Activated Transcription Factor Nuclear Factor {kappa}B With Chemoradiation Resistance and Poor Outcome in Esophageal Carcinoma J. Clin. Oncol., February 10, 2006; 24(5): 748 - 754. [Abstract] [Full Text] [PDF]
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 W. C. Ho, K. M. Dickson, and P. A. Barker Nuclear Factor-{kappa}B Induced by Doxorubicin Is Deficient in Phosphorylation and Acetylation and Represses Nuclear Factor-{kappa}B-Dependent Transcription in Cancer Cells Cancer Res., May 15, 2005; 65(10): 4273 - 4281. [Abstract] [Full Text] [PDF]
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 C. Frelin, V. Imbert, E. Griessinger, A.-C. Peyron, N. Rochet, P. Philip, C. Dageville, A. Sirvent, M. Hummelsberger, E. Berard, et al. Targeting NF-{kappa}B activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells Blood, January 15, 2005; 105(2): 804 - 811. [Abstract] [Full Text] [PDF]
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C. G. Broustas, P. C. Gokhale, A. Rahman, A. Dritschilo, I. Ahmad, and U. Kasid BRCC2, a Novel BH3-like Domain-containing Protein, Induces Apoptosis in a Caspase-dependent Manner J. Biol. Chem., June 18, 2004; 279(25): 26780 - 26788. [Abstract] [Full Text] [PDF]
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Y. Li, S. Gazdoiu, Z.-Q. Pan, and S. Y. Fuchs Stability of Homologue of Slimb F-box Protein Is Regulated by Availability of Its Substrate J. Biol. Chem., March 19, 2004; 279(12): 11074 - 11080. [Abstract] [Full Text] [PDF]
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 G. R. Panta, S. Kaur, L. G. Cavin, M. L. Cortes, F. Mercurio, L. Lothstein, T. W. Sweatman, M. Israel, and M. Arsura ATM and the Catalytic Subunit of DNA-Dependent Protein Kinase Activate NF-{kappa}B through a Common MEK/Extracellular Signal-Regulated Kinase/p90rsk Signaling Pathway in Response to Distinct Forms of DNA Damage Mol. Cell. Biol., March 1, 2004; 24(5): 1823 - 1835. [Abstract] [Full Text] [PDF]
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 L. Boyer, S. Travaglione, L. Falzano, N. C. Gauthier, M. R. Popoff, E. Lemichez, C. Fiorentini, and A. Fabbri Rac GTPase Instructs Nuclear Factor-{kappa}B Activation by Conveying the SCF Complex and IkB{alpha} to the Ruffling Membranes Mol. Biol. Cell, March 1, 2004; 15(3): 1124 - 1133. [Abstract] [Full Text] [PDF]
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 J van der Heijden, M C de Jong, B A C Dijkmans, W F Lems, R Oerlemans, I Kathmann, C G Schalkwijk, G L Scheffer, R J Scheper, and G Jansen Development of sulfasalazine resistance in human T cells induces expression of the multidrug resistance transporter ABCG2 (BCRP) and augmented production of TNF{alpha} Ann Rheum Dis, February 1, 2004; 63(2): 138 - 143. [Abstract] [Full Text] [PDF]
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 S. Solier, A. Lansiaux, E. Logette, J. Wu, J. Soret, J. Tazi, C. Bailly, L. Desoche, E. Solary, and L. Corcos Topoisomerase I and II Inhibitors Control Caspase-2 Pre-Messenger RNA Splicing in Human Cells Mol. Cancer Res., January 1, 2004; 2(1): 53 - 61. [Abstract] [Full Text] [PDF]
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 G. M. Hur, J. Lewis, Q. Yang, Y. Lin, H. Nakano, S. Nedospasov, and Z.-g. Liu The death domain kinase RIP has an essential role in DNA damage-induced NF-kappa B activation Genes & Dev., April 1, 2003; 17(7): 873 - 882. [Abstract] [Full Text] [PDF]
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J. S. Russell and P. J. Tofilon Radiation-induced Activation of Nuclear Factor-kappa B Involves Selective Degradation of Plasma Membrane-associated Ikappa Balpha Mol. Biol. Cell, October 1, 2002; 13(10): 3431 - 3440. [Abstract] [Full Text] [PDF]
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