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[Cancer Research 60, 4238-4244, August 1, 2000]
© 2000 American Association for Cancer Research

Tumor Biology

Increased Resistance to Anticancer Therapy of Mouse Cells Lacking the Poly(ADP-ribose) Polymerase Attributable to Up-Regulation of the Multidrug Resistance Gene Product P-Glycoprotein1

Gabriele Wurzer, Zdenko Herceg and Józefa Wsierska-Gadek2

Institute of Cancer Research, University of Vienna, A-1090 Vienna, Austria [G. W., J. W-G.], and International Agency for Research on Cancer, 69372 Lyon, France [Z. H.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mouse embryo fibroblasts lacking poly(ADP-ribose) polymerase (PARP)-1 express a barely detectable level of wild-type (wt) p53 protein. Doxorubicin at concentrations activating wt p53 in normal mouse embryo fibroblasts failed to induce it in mutant cells. wt p53 was only activated in response to a 10-fold higher doxorubicin dose. Treatment with higher doxorubicin concentrations was cytotoxic for normal but not for PARP-1 -/- cells. The latter was also resistant to other anticancer agents. The increased resistance of mutant cells to drugs resembled a unique phenomenon known as multidrug resistance (MDR). Interestingly, the MDR gene product P-glycoprotein was clearly up-regulated in PARP-1-deficient cells as compared with normal counterparts. Pretreatment with verapamil reversed the MDR phenotype.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PARP3 -1 represents the major cellular poly(ADP-ribosyl)ating activity. It was originally described in 1966 (1) as the first member of the multienzyme family. Recently, other PARP enzymes have been identified (2 , 3) . PARP-1, a nuclear zinc-finger enzyme, is a highly sensitive detector of DNA damage appearing after {gamma}-irradiation or treatment with cytotoxic or genotoxic agents (for review, see Refs. 4, 5, 6 ). DNA breakage stimulates the enzyme activity up to 200-fold, resulting in transient formation of poly(ADP-ribose) chains covalently coupled to acceptor proteins (4 , 5) . Remarkably, a few proteins directly involved in DNA metabolism and regulation of chromatin structure, such as Topos, DNA ligases, or DNA polymerases, have been described as targets for poly(ADP-ribosyl)ation (5) . PARP-1 possesses at least three distinct structural domains that are closely related to their functions (6) . The biological role of PARP-1 in DNA repair and DNA recombination was postulated several years ago. However, its exact function in these processes was not fully understood. Recently, PARP-1 was identified as an integral component of a multienzyme complex acting in base excision repair (7) . It has been shown that PARP-1 activity is necessary for the in vivo formation of a functional complex with XRCC1 protein and is additionally essential for recognition of DNA lesions. PARP-1 has also been shown to be involved in apoptosis (8 , 9) . Several lines of evidence indicate that PARP-1 cleavage, which prevents depletion of cellular NAD+ and ATP pools, may play a key role in determining whether cells undergo apoptosis or necrosis (10) . To understand the physiological function of PARP better, new approaches have been developed, such as antisense expression and trans-dominant negative regulation of PARP-1. Recently, mice lacking PARP-1 have been generated by gene disruption and homologous recombination (11 , 12) . They show normal fetal and neonatal development but exhibit inherent genomic instability and are extremely sensitive to {gamma}-rays (11 , 13) . Recently, a role of PARP-1 in controlling telomere length and chromosomal stability has been demonstrated (14) . Surprisingly, PARP-1 gene disruption rendered mice resistant to experimentally induced diabetes (15 , 16) and cerebral ischemia (17) . Moreover, the inactivation of the PARP-1 gene affected the constitutive expression of wt p53 (18 , 19) . Interestingly, only the RS form of wt p53 protein was reduced to a barely detectable level in consequence of an ~8-fold shortening of a half-life (19 , 20) , whereas alternatively spliced p53 remained unchanged. Treatment with doxorubicin at concentrations activating RS p53 in normal MEFs failed to induce wt p53 protein in PARP knock-out cells. We therefore decided to explore this surprising observation and tested increasing doxorubicin concentrations for their capacity to induce p53 response. We simultaneously monitored the cytotoxic action. As expected, doxorubicin at higher concentrations was strongly cytotoxic for normal MEFs and only negligibly elevated the p53 level. Surprisingly, the viability of PARP-1 -/- cells remained unaffected, even after treatment with higher doxorubicin concentrations. Remarkably, fibroblasts lacking PARP-1 also exhibited reduced sensitivity in response to other anticancer drugs. The increased resistance of PARP-1-deficient cells to anticancer therapy was attributable to up-regulation of the MDR gene product P-gp. Pretreatment with a specific MDR modulator reversed the MDR phenotype. Interestingly, reconstitution of cells lacking the PARP-1 gene with the human counterpart partially restored cell susceptibility to anticancer drugs. Our results identify the mechanism of the increased resistance of PARP-1-deficient cells to chemotherapy and could offer an explanation for the lack of the development of streptozocin-induced diabetes in PARP-1-deficient mice (15 , 16) .


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells.
Mice lacking PARP-1 were generated by homologous recombination (12 , 13) . Immortalized MEFs were obtained from PARP-1 +/+ (A-19) and from PARP-1 -/- (A-11 and A-12) mice. Cells were grown in DMEM supplemented with 10% FCS in an atmosphere of 7.5% CO2. PARP-1 -/- cells (clone A-11) were reconstituted with human PARP-1. An eukaryotic expression construct containing full-length human cDNA under the control of SV-40 promoter was used to generate stable cell lines expressing exogenous PARP-1. Cells were positively selected with hygromycin. Resistant clones (A-11/wt2 and A-11/wt3) were isolated and characterized by Southern blot analysis.

Antibodies.
Anti-p53 antibodies PAb421 and PAb240 were obtained from Oncogene Research Products (Cambridge, MA.). Anti-mouse double minute 2 (mdm-2) antibodies (SGM-14) were from DAKO A/S (Glostrup, Denmark). Monoclonal anti-PARP-1-antibodies (C2–10) were from Dr. G. Poirier. Anti-NF-{kappa}B antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Topo II{alpha} antibodies were from NeoMarkers (Union City, CA), and anti-Topo IIß antibodies were from Biotrends (Cologne, Germany). For immunodetection of P-gp protein, three different anti-P-gp antibodies were used: C-19 from Santa Cruz, clone 265/F4 from Neomarkers, and clone JSB-1 from Monosan (Uden, the Netherlands). Antiactin antibodies (clone C4) were from ICN (Aurora, Ohio). Appropriate secondary antibodies linked to horseradish peroxidase, Cy-2, or Cy-3 were from Amersham International (Little Chalfont, Buckinghamshire, England).

Drugs.
Doxorubicin hydrochloride was from Farmitalia Carlo Era AG (Zug, Switzerland). 5-FU, cytarabine, verapamil hydrochloride, probenecid, and VP-16 were from Sigma; LMB was from Novartis. TNF-{alpha} and actinomycin D were from Boehringer (Mannheim, Germany).

Cell Treatment.
Cells were treated for the indicated periods of time with various anticancer drugs [doxorubicin (0.1–10 µg/ml), with VP-16, 5-FU, cisplatin, and cytarabine], with 100 nM LMB (21) , an inhibitor of protein export, with modulators of MDR [verapamil (10 µM) and probenecid (1 mg/ml)], or with TNF-{alpha} (5 ng/ml) alone or in combination with actinomycin D (1 µg/ml) and lactacystin (20 µM).

Indirect IF Microscopy.
Cells grown on coverslips were rinsed with PBS, fixed in ice-cold methanol-acetone (3:2) for 20 min, and washed with PBS. The cells were incubated with anti-p53 antibodies at appropriate concentrations, and the immune complexes were detected by incubation with secondary antibodies covalently coupled to Cy-2 (Amersham International). To visualize nuclei, preparations were additionally stained with the fluorescent dye 4',6-diamidino-2-phenylindole.

Drug Cytotoxicity in Vivo.
Sensitivity of cells to drugs was assessed using a microtiter plate colorimetric assay, which measures the ability of viable cells to cleave the tetrazolium salt (WST-1) to a water-soluble purple formazan. Cells were plated at the appropriate density (10,000 cells/well) in a 96-well microtiter plate. Twenty-four h after plating, cells were exposed to various concentrations of distinct drugs for 4 h. After treatment, the medium was removed and replaced with 200 µl of drug-free medium, and plates were incubated for a further 70 h. At this time, 20 µl of WST-1 were added to each well. After a 4-h incubation at 37°C, plates were shaken for 5 min. Absorbance was measured at 450 nm and 690 nm on a Labsystem Multiskan microtiter plate reader. Each column represents the mean ± SD (bars) of three separate experiments, each performed in triplicate.

[3 H]Daunomycin Uptake.
Cells were incubated with 0.1 µM [3 H]daunomycin (NEN Life Science Products, Boston, MA; with or without 10 µM verapamil hydrochloride or 1 mM probenecid) up to 6 h at 37°C. The incubation was terminated by six rapid washing steps with cold PBS with or without verapamil or probenecid. Then the cells were lysed in buffer containing 1% SDS, and accumulated radioactivity was determined by liquid scintillation counting. Data (mean cpm ± SD) of [3 H]daunomycin uptake were calculated from three separate experiments.

Determination of Topo II Unknotting Activity.
Topo II activity was determined using the decatenation assay of kinetoplast kDNA. The reaction mixture contained 50 mM Tris/HCl (pH 7.4), 120 mM KCl, 10 mM MgCl2, 0.5 mM DTT, 0.5 mM EDTA, 1 mM ATP, and 200 ng of kDNA (TopogGEN Inc., Columbus, Ohio). The reaction started by the addition of a high salt nuclear extract was performed at 30°C and was terminated after 20 min by the addition of 2 µl of solution containing 10% SDS, 5% bromphenol blue, and 30% glycerol. The samples were separated on 1% agarose gels at 6 V/cm for 3 h in Tris-borate EDTA buffer. DNA was stained with ethidium bromide. As the control, decatenated DNA marker (TopogGEN Inc.) representing either circular monomer or covalently closed circular product was used.

Immunoblotting.
Proteins dissolved in reduced SDS-sample buffer were separated on SDS-polyacrylamide gels and transferred electrophoretically onto PVDF (Amersham International). Equal loading of proteins was confirmed by Ponceau S staining. Blots were incubated with specific primary antibodies, and the immune complexes were detected autoradiographically using appropriate peroxidase-conjugated secondary antibodies and enhanced chemiluminescent detection reagent ECL+ (Amersham International). In some cases, blots were stripped and used for several sequential incubations.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Lack of p53 Induction in PARP-1-deficient Cells in Response to Doxorubicin.
Treatment of normal mouse fibroblasts or the human breast carcinoma cell line MCF-7 with 0.2 µg/ml (0.35 µM) doxorubicin resulted in an induction of wt p53 protein in a time-dependent manner (Fig. 1Citation ). A marked elevation of the basal level of p53 protein already occurred in normal MEFs after 1 h. Moreover, during the following few hours, the enhancement of p53 level was accompanied by the up-regulation of its responsive genes, such as mdm-2 or GADD-45 (19) . In contrast, PARP-1-deficient cells failed to activate p53 protein upon doxorubicin treatment at this dose. To establish whether the absence of p53 signals was attributable to the detection limit of the immunoblotting or lack of PAb421 immunoreactivity, we have examined both possibilities. The monitoring of p53 expression at a single cell level by IF microscopy (Fig. 2Citation ) revealed strong staining of nuclei of control- and doxorubicin-treated wt MEFs by PAb421. In contrast, PARP-1 -/- cells did not give any characteristic nuclear signals even after doxorubicin treatment. However, inhibition of nuclear export by LMB (21) resulted in a considerable accumulation of PAb421 immunoreactive wt p53 in the nuclei of PARP-1-deficient cells, thereby showing that the absence of p53 signals even after doxorubicin application was not attributable to lack of PAb421 reactivity, which is characteristic for the latent p53 form, but was a consequence of impaired p53 inducibility in PARP-1 knock-out cells. Testing of primary embryo fibroblasts and established immortalized cell lines showed the same results.



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  		Fig. 1. Lack of p53 induction in cells lacking PARP-1 in response to doxorubicin treatment. Normal and PARP-1 -/- MEFs, the human breast carcinoma MCF-7, and human osteosarcoma Saos-2 cells were treated with 0.2 µg/ml (0.35 µM) doxorubicin for indicated times. B, PARP KO cells were also treated with increasing doxorubicin concentrations for 24 h. Total cell lysates (20 µg protein/lane) were separated on 10% SDS gels and transferred onto the PVDF membrane. Immunoblotting was performed with anti-p53 PAb421 antibody and subsequently with anti-PARP-1 C-2–10 and antiactin antibody. Saos-2 cells were used as the p53-negative control.

 


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  		Fig. 2. Different p53 staining patterns in normal and PARP-deficient MEFs by indirect IF. Cells growing on slides were treated with 0.2 µg/ml doxorubicin for 6 h or incubated with 100 nM LMB for 20 h. Then the cells were fixed and immunostained with anti-p53 PAb421 antibody. Nuclei were visualized with 4',6-diamidino-2-phenylindole.

 
Generation of Mouse Cell Clones Expressing Human PARP-1 Gene.
To ensure that the observed biological differences between normal and PARP-1-deficient cell lines are a consequence of the inactivation of PARP-1 gene and not attributable to some unknown effects, we decided to reconstitute the PARP-1 null phenotype cells (A-11) with the human counterpart. After selection, some clones that expressed human PARP-1 were established. This was confirmed by Southern and Western blotting and additionally by determination of the enzymatic activity.

High Doxorubicin Concentration Is Necessary to Activate p53 in PARP-1 Null Phenotype Cells.
Testing of higher doxorubicin doses revealed that the 10-fold doxorubicin concentration (2 µg/ml) was necessary to induce RS wt p53 in PARP-1-deficient cells (Fig. 1BCitation ). Even at this doxorubicin concentration, the kinetics of p53 activation were clearly delayed as compared with normal MEFs. At first, a very weak PAb421 immunoreactive signal appeared 3 h after treatment and reached the highest intensity after a further 3 h. Once accumulated, p53 protein declined after longer doxorubicin treatment. Interestingly, in reconstituted mouse cells, wt p53 protein already accumulated after 3 h even in response to lower doxorubicin dose.

Increased Resistance of PARP-1-deficient MEFs to Doxorubicin.
It became apparent that normal and PARP-1-deficient mouse fibroblasts show differential susceptibility to doxorubicin. We therefore tested the cytotoxic effect of this anticancer drug. Cell survival was determined with WST-1 colorimetric assay. As expected, doxorubicin strongly affected the viability of normal MEFs. The concentration of doxorubicin that reduced the cell survival to 50% of untreated controls (IC50) was 1 µg/ml and 5 µg/ml for PARP-1 +/+ and PARP-1 KO cells, respectively (Fig. 3Citation ). Remarkably, human PARP-1 reconstitution rendered mutant cells doxorubicin-sensitive (IC50, 3 µg/ml).



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  		Fig. 3. Cytotoxic action of doxorubicin on wt MEFs. Twenty-four h after plating, cells were treated for 4 h with increasing doxorubicin concentrations. After the medium change, cells were maintained at 37°C for 70 h. Then 10 µl of WST-1 were added. After 4-h incubation, absorbance at 450 and 690 nm was measured. The advantage of this assay is that the application of water soluble dye allows the direct measurement of absorbance in a microtiter plate and eliminates additional steps that are necessary in other assays and can lead to undesired loss of sample. •, {blacksquare}, and {blacktriangledown}, mean ± SD (bars) of the three separate experiments, each performed in triplicate.

 
Expression and Activity of Topo II Is Not Altered in PARP-1 KO Cells.
Low susceptibility of cells to chemotherapeutic agents is often multifactorial and may be connected with altered activity of Topos. Therefore, we tested the level of both Topo II enzymes in total cell lysates. No difference in the levels of Topo II{alpha} and IIß was observed between normal and PARP-1-deficient MEFs (Fig. 4 and CCitation ). Equal protein loading was confirmed by sequential incubation of the blot with antiactin antibodies. Moreover, mutant and normal cells exhibited comparable activity of Topo II as measured by decatenation of kinetoplast DNA (Fig. 4BCitation ).



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  		Fig. 4. Normal level of Topos II{alpha} and IIß in PARP-1 -/- cells. Total cell lysates (25 µg of protein/lane) were separated on 8% SDS gels and transferred onto the PVDF membrane. Immunoblotting was performed with anti-Topo IIß (A) or with anti-Topo II{alpha} (C) antibody and subsequently with anti-PARP-1 C-2–10 and antiactin antibody. B, decatenating Topo II activity in high salt nuclear extracts (10 µg of protein) was determined using kDNA (TopoGEN Inc.) as a substrate. The samples were separated on 1% agarose gel. DNA was stained with ethidium bromide. As a positive control, Topo II pretreated kDNA was loaded.

 
MDR of PARP-1-deficient MEFs.
The increased resistance of PARP-1 null phenotype cells to doxorubicin and its at least partial regression after reconstitution with the PARP-1 gene was intriguing. We therefore decided to examine the effect of other anticancer agents differing in their mode of action. As shown in Table 1Citation , normal mouse fibroblasts were susceptible to the treatment with drugs, such as cytarabine and cisplatin. The corresponding IC50 values were 3.7 µg/ml and 30 µM, respectively. On the other hand, these drugs were only negligibly cytotoxic for PARP-1-deficient cells. The opposite effect was observed on in vivo treatment with VP-16. Normal mouse cells showed low sensitivity to this drug, whereas cells lacking PARP-1 were very sensitive and died after VP-16 treatment at a high concentration (IC50, 13 µg/ml). The diminished cytotoxicity of several anticancer drugs on PARP-1 -/- cells resembled the biological phenomenon known as MDR. The MDR is associated with decreased intracellular drug accumulation and correlates most frequently with the increased expression of MDR genes. The drug resistance can be reversed by some specific MDR modulators.


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  		Table 1 Effect of different anticancer drugs on viability of wt and PARP-1 KO fibroblasts

Twenty-four h after plating, cells were treated for 4 h with drugs at concentrations ranging from 0.1 µg to 100 µg/ml. After medium change, cells were maintained at 37°C for 70 h. Then 10 µl of WST-1 were added, and absorbance at 450 and 690 nm was measured. IC50 was determined from three separate experiments; each drug concentration was tested in triplicate.

 
[3 H]Daunomycin Uptake.
To explore the mechanism of the MDR of PARP-1-deficient cells, we have examined the uptake of [3 H]daunomycin, a drug closely related to doxorubicin. We have determined an early and late uptake of radioactively labeled drug. The measurement of the early uptake after brief exposure to a drug reflects the capacity of naive cells to transport the drug. However, an estimation of the late uptake is indicative as to whether the drug is retained in the cell or whether the product of the MDR gene that is thought to act as an ATP-dependent efflux pump was activated to keep the intracellular drug concentration below cytotoxic levels. As shown in Fig. 5Citation , intracellular [3 H]daunomycin concentration was only slightly reduced in PARP-1 -/- cells and amounted to 80% of that found in the normal counterpart after 1-h incubation. However, after 6-h incubation or preincubation with doxorubicin, the intracellular level of labeled daunomycin in PARP -/- cells decreased markedly and reached only 25% of that determined in the normal MEFs. In contrast to PARP-1-deficient cells, normal MEFs and PARP-1-reconstituted cells accumulated more radioactively labeled drug after prolonged incubation. These results indicate that the drug treatment led to enhanced activation of the efflux pump in mutant but not in wt or reconstituted MEFs.



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  		Fig. 5. Early and late [3H]daunomycin uptake in normal and mutant MEFs. Twenty-four h after plating, 2 µM [3H]daunomycin was added to a final concentration of 0.1 µM. The incubation with [3H]daunomycin alone or in combination with 10 µM verapamil hydrochloride or with 1 mM probenecid was performed for 1 h and for 6 h at 37°C. The incubation was terminated by six rapid washing steps with cold PBS with or without verapamil or probenecid. Then the cells were lysed in buffer containing 1% SDS, and accumulated radioactivity was determined by liquid scintillation counting. Data (mean cpm ± SD) of [3H]daunomycin uptake were calculated from three separate experiments.

 
Effect of MDR Modulators on Doxorubicin Retention in PARP-1 -/- Cells.
To further characterize the mechanism of the MDR of PARP-1-deficient cells, we tested the effect of two specific modulators: verapamil, a calcium antagonist, and probenecid, an inhibitor of organic anion transporter. Interestingly, pretreatment of PARP-1-deficient cells with the MDR modulator verapamil enhanced the drug retention (Fig. 5Citation ), whereas probenecid (known as an MRP reversor) did not elevate doxorubicin accumulation. This substantiates the assumption that up-regulated P-gp activity was responsible for the impaired drug accumulation in PARP-1 -/- cells.

Expression of P-gp Protein in Mouse Cells.
The basal level of P-gp protein was determined in untreated mouse cell lines by immunoblotting. As shown in Fig. 6ACitation , normal MEFs expressed only negligible amounts of P-gp proteins, whereas the P-gp level was significantly elevated in PARP-1 -/- cells. The enhanced expression of P-gp protein in PARP-1 KO cells was partially abolished after reconstitution of the cells with human PARP-1. Equal protein loading was confirmed by sequential incubation of the blot with antiactin antibody. The specificity of the immunodetection by monoclonal anti-P-gp antibody was proved using a plasma membrane fraction isolated from human chemoresistant HL-60 cells expressing up-regulated P-gp protein. Fractionation of untreated mutant cells revealed that P-gp protein was randomly distributed between the cytosol, nucleus, and plasma membrane (Fig. 6BCitation ). However, doxorubicin treatment resulted in a rapid P-gp recruitment in the plasma membrane. A subsequent immunoblotting with anti-NF-{kappa}B antibody revealed that NF-{kappa}B failed to translocate from the cytosol into the nucleus of PARP-1 -/- cells under the treatment, indicating a lack of NF-{kappa}B activation in response to brief doxorubicin treatment.



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  		Fig. 6. P-gp protein expression in mouse fibroblasts. Proteins (20 of µg protein/lane) were separated electrophoretically on 8% SDS gel and transferred onto the membrane. Immunoblotting was performed with monoclonal anti-P-gp antibody JSB-1 from Monosan and subsequently with antiactin or with anti-NF-{kappa}B antibodies. A, total cell lysates from untreated cells. B, fractionation of control and doxorubicin-treated PARP -/- cells.

 
Inhibition of P-gp Activity Renders Mutant Cells Susceptible to Anticancer Drugs.
To ensure that enhanced P-gp activity in PARP-1 -/- cells conferred the MDR, we tested the effect of combined treatment on the cell survival. As expected, verapamil combined with doxorubicin abolished the resistance of cells lacking PARP to anticancer drugs, and cells became drug-sensitive (Fig. 7Citation A). Administration of probenecid did not affect the susceptibility of mutant cells to doxorubicin. Interestingly, verapamil-mediated inhibition of P-gp resulted in a rapid activation of p53 protein in response to a low dose of doxorubicin (Fig. 7BCitation ).



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  		Fig. 7. Increased doxorubicin cytotoxicity after pretreatment of PARP-1-deficient cells with MDR modulator verapamil. Twenty-four h after plating, cells were treated with doxorubicin alone or in combination with verapamil or probenecid for 4 h. Then medium was changed, and cells were maintained at 37°C for 70 h to perform WST-1 assay or were lysed in radioimmunoprecipitation assay buffer. A, increased doxorubicin cytotoxicity. B, strong induction of wt p53. Total cell lysates (20 µg/lane) were separated on 10% SDS gel. Equal protein loading was confirmed by incubation with antiactin antibodies.

 
Rapid p53 Activation in PARP-1 KO Cells by Stress Stimuli Not Involving MDR.
In vivo treatment with TNF-{alpha} resulted in a rapid activation of wt p53 in PARP-1-deficient cells. p53 protein was already detected in total cell lysate after 4-h incubation. The kinetics and extent of p53 induction in cells possessing and lacking PARP-1 were very similar. It was accompanied by increased expression (Fig. 8Citation ) and translocation of NF-{kappa}B into the nucleus, reflecting the activation of its transcriptional activity (not shown). The inhibition of proteasome activity by lactacystin partially prevented the induction of NF-{kappa}B in PARP -/- cells (A-12) (22) . This was accompanied by diminished p53 expression, indicating that NF-{kappa}B-mediated activation of p53 is exerted not only by its stabilization. These data show that impaired inducibility of wt p53 is not a general feature of PARP-1-deficient cells but strongly depends on the type of stress stimuli and the pathway of their transduction.



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  		Fig. 8. Rapid wt p53 induction in PARP -/- cells after TNF-{alpha} treatment. Cells were treated for 4 h with TNF-{alpha} + actinomycin D. In some assays, lactacystin (CE = 20 µM) was included. Total cell lysates (20 µg/lane) were separated on 10% SDS gel. p53 protein was visualized by incubation with anti-p53 antibody PAb421.

 
Loss of p53-mediated Negative Regulation of P-gp in PARP-1 -/- Cells.
What might be the reason for the enhanced expression of P-gp protein in PARP-1 -/- cells? P-gp is known to be negatively regulated by wt p53 protein. In view of our previous observation that wt p53 protein is reduced to a barely detectable level in cells lacking functional PARP-1, the loss of p53-mediated negative regulation of P-gp would offer an attractive and logical explanation of our results. We therefore investigated whether ectopically expressed wt p53 or mutant p53 would affect cell sensitivity to doxorubicin. Indeed, transfection of wt p53 additionally sensitized to doxorubicin normal (not shown) but not PARP-1 -/- cells. The attendant analysis of p53 expression by immunoblotting revealed an enhancement of PAb421-reactive p53 band in normal fibroblasts, whereas no PAb421 p53 signal could be detected in PARP-1 -/- cells. On the other hand, after transfection of cells with transforming p53 mutants (132Phe or 135Val), p53 signals reactive with PAb421 also appeared in cells lacking PARP-1. This indicates that not only endogenous but also ectopically expressed RS wt p53 is extremely unstable in the absence of functionally active PARP-1.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
One of the major problems in the therapy of many human malignancies is the development of resistance to chemotherapeutic agents (23) . Analysis of the phenotype of drug-resistant cancer cells has allowed their classification into two groups: cells exhibiting single compound resistance or resistance to a single class of drugs possessing the same mechanism of action, and cells broadly resistant to many different agents. The latter phenomenon is known as MDR (23, 24, 25) . Present knowledge of the means by which cells become multidrug-resistant indicates several different mechanisms, including interference with programmed cell death, increased DNA repair capacity, alterations in Topo II activity, and the elevated expression of energy-dependent pump systems that extrude anticancer drugs from treated cells. Two such pump systems have been detected: MDR encoding the multidrug transporter, and MRP encoding MDR-associated protein, the glutathione conjugate transporter. Both are energy-dependent transporters that belong to a multigene family of ATP-binding cassette proteins. The mammalian MDR genes encode membrane-linked glycoproteins (P-gps) of ~Mr 170,000, which recognize and transport many different substrates, such as doxorubicin, daunomycin, vinblastine, vincristine, and actinomycin D (26) .

The anthracycline antibiotic doxorubicin is one of the most useful antineoplastic agents, displaying a broad range of clinical activity against solid tumors as well as against hematopoietic malignancies. However, doxorubicin is generally ineffective in the treatment of cancer of the colon and rectum. The inactivity is generally attributed to intrinsic resistance to the cytotoxic effects of doxorubicin.

In the present study, we observed the increased resistance of cells lacking PARP-1 to doxorubicin treatment. In these cells, a 10-fold doxorubicin concentration was required for induction of p53 response, and IC50 for doxorubicin was about 5-fold higher than that in normal counterparts. PARP-1-deficient cells also exhibited a decreased sensitivity to other anticancer agents, such as cytarabine and cisplatin, but not to 5-FU or VP-16. The latter agent was even more cytotoxic for cells lacking PARP-1 than for their wt counterparts.

To elucidate the cause of the MDR exhibited by PARP-1-deficient cells, we addressed the question as to which of the known mechanisms may primarily contribute to the development of the drug resistance. Because PARP-1 -/- cells have been previously shown to possess diminished DNA repair capacity and normal apoptotic response to cytotoxic agents, both pathways seemed to be ruled out. We have therefore focused our attention on mechanisms involving alteration of Topo II and transmembrane drug transporters. No differences in the enzymatic activity and in the basal level of Topos II{alpha} and IIß could be detected between normal, mutant, and reconstituted MEFs. These results seem to contradict the previous observation of Canitrot et al. (27) who reported the decreased expression and activity of Topo IIß in PARP-1 -/- cells. However, careful examination of experimental protocols shows that the data of both studies cannot be directly compared. Canitrot et al. (27) determined the level and activity of Topo IIß in low salt nuclear extracts. We performed the corresponding analysis using total nuclear proteins for immunoblotting and high salt nuclear extracts for measurement of enzymatic activity. The use of high salt concentrations during the extraction procedure was necessary to solubilize the Topo IIß. As a component of the nuclear scaffold, this is poorly soluble.

We present several lines of evidence that the MDR appearing in cells lacking PARP-1 is primarily based on the up-regulation of P-gp protein. Comparison of the early and late uptake of [3 H]daunomycin shows a markedly reduced accumulation of the drug in the mutant cells, implicating activation of a mechanism based on an efflux pump. Remarkably, pretreatment of cells lacking PARP-1 with the MDR modulator verapamil but not probenecid resulted in an increase of [3 H]daunomycin accumulation, with concomitant doxorubicin susceptibility of the cells. The selective activity of verapamil strongly supported the assumption that induced P-gp protein conferred chemoresistance on PARP-1-deficient cells. Indeed, mutant cells express a higher level of P-gp than their parental counterparts. Fractionation of control and doxorubicin-treated PARP-1 -/- cells revealed that P-gp randomly distributed in untreated controls was rapidly recruited in the plasma membrane under brief doxorubicin treatment. Moreover, cells lacking PARP-1 conferred resistance to some other drugs, which are known substrates of P-gp, such as daunomycin, cytarabine, and cisplatin, whereas no resistance could be observed to other anticancer agents, such as VP-16 or 5-FU, which are poor substrates for P-gp. Relatively low chemoresistance to cisplatin is not surprising because this drug enters the cell by two routes (25) . It is a substrate for the transporter system and is also able to enter cells by passive diffusion.

The impaired inducibility of wt p53 observed after doxorubicin is not a general feature of PARP-1-deficient cells, but it greatly depends on the type of stress stimuli. In response to other agents that are transduced by pathways not involving the drug transporter system, such as TNF-{alpha}, the wt p53 response was induced with the same kinetics and to a similar extent as the normal counterparts.

The expression of P-gp protein is known to be negatively regulated by wt p53 protein (28 , 29) . The dramatic reduction of wt p53 protein in the absence of functional PARP seems to be responsible for the loss of negative regulation of P-gp and contributes to the development of MDR. Our data could offer an explanation for the lack of development of experimentally generated diabetes in PARP-1-deficient mice. One cannot exclude that the intracellular level of streptozocin in PARP-1 -/- mice was reduced as compared to normal counterparts.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. B. Wolff-Winiski (Novartis Forschungsinstitut GmbH, Vienna, Austria) for providing us with LMB, Dr. M. Oren for murine p53 plasmids encoding different p53 mutants, and to Drs. E. Elbling and W. Berger for a kindly gift of a P-gp positive control. We thank Maria Eisenbauer for the cultivation of cells and Paul Breit for preparation of photomicrographs. We also thank Christian Balcarek and Silvia Magyar for excellent technical assistance.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by a grant from the Herzfelder’sche Familienstiftung. Back

2 To whom requests for reprints should be addressed, at Institute of Cancer Research, Borschkegasse 8a, A-1090 Vienna, Austria. Phone: 43-1-4277-65247; Fax: 43-1-4277-9651 or 65194. E-mail: Jozefa.Antonia.Gadek-Wesierski{at}univie.ac.at Back

3 The abbreviations used are: PARP, poly(ADP-ribose) polymerase; IF, immunofluorescence; LMB, leptomycin B; MEF, mouse embryo fibroblast; RS, regularly spliced; wt, wild-type; MDR, multidrug resistance; P-gp, P-glycoprotein; NF-{kappa}B, nuclear factor {kappa}B; Topo, topoisomerase; 5-FU, 5-fluorouracil; VP-16, etoposide; PVDF, polyvinylidene difluoride membrane; TNF-{alpha}, tumor necrosis factor {alpha}; WST-1. Back

Received 3/13/00. Accepted 5/31/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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