FASEB J 06

7/29/2019 FASEB J 06

1/13

The FASEB Journal FJ Express Full-Length Article

Inhibition of poly(ADP-ribose) polymerase preventsirinotecan-induced intestinal damage and enhancesirinotecan/temozolomide efficacy against colon

carcinomaLucio Tentori,* Carlo Leonetti, Marco Scarsella, Alessia Muzi,* Emanuela Mazzon,

Matteo Vergati,* Olindo Forini,* Rena Lapidus, Weizheng Xu,

Annalisa Susanna Dorio,* Jie Zhang, Salvatore Cuzzocrea, and Grazia Graziani*,1

*Department of Neuroscience, University of Rome Tor Vergata, Rome, Italy; ExperimentalClinical Laboratory, Institute for Cancer Research Regina Elena, Rome, Italy; Department ofClinical and Experimental Medicine and Pharmacology, University of Messina and Centro NeurolesiBonino-Pulejo (IRCCS), Messina, Italy; and MGI Pharma, Baltimore, Maryland, USA

ABSTRACT Poly(ADP-ribose) polymerase (PARP) in-

hibitors enhance the antitumor activity of the topoisom-erase I inhibitor irinotecan (CPT-11), which is used totreat advanced colorectal carcinoma. Since PARP inhibi-tors sensitize tumor cells also to the methylating agenttemozolomide (TMZ) and clinical trials are evaluatingCPT-11 in combination with TMZ, we tested whether thePARP inhibitor GPI 15427 (10-(4-methyl-piperazin-1-ylm-ethyl)-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one) in-creases the efficacy of CPT-11 TMZ against coloncancer. Moreover, due to the ability of PARP inhibitors toavoid cell death consequent to PARP-1 overactivation, weevaluated whether oral administration of GPI 15427 pro-vides protection from the dose-limiting intestinal toxicity

of CPT-11. The results of colony formation assay indi-cated that GPI 15427 increased the antiproliferative ef-fects (combination index

7/29/2019 FASEB J 06

2/13

7/29/2019 FASEB J 06

3/13

bated at 4C for 30 min (22). Various amounts of cell extractswere incubated with 10 g of calf thymus DNA previouslylabeled with N-[3H]-methyl-N-nitrosourea (GE Healthcare,Milan, Italy; 18 Ci/mmol). O6-alkylguanine DNA alkyltrans-ferase activity was determined by measuring the transfer of[3H]-methyl groups from methylated DNA to O6-alkylgua-nine DNA alkyltransferase and expressed as fmol of methylgroups per mg of proteins in cell extract.

N-methylpurine DNA glycosylase activity assay

Cells (1107) were sonicated at 4C in 0.1 ml buffer I (50 mMTris-HCl, 3 mM dithiothreitol, and 2 mM EDTA, pH 8.3),

with freshly added 1 mM 4-(2-aminoethyl)-benzene-sulfonylfluoride hydrochloride. Various amounts of cell extracts wereincubated with 15 g of freshly dissolved calf thymus DNAmethylated by N-[3H]methyl-N-nitrosourea in a total volumeof 100 l of buffer II (20 mM Tris-HCl, 1 mM dithiothreitol,60 mM NaCl, and 1 mM EDTA, pH 8). N-methylpurine DNAglycosylase activity was expressed as fmol of methylpurinesreleased per mg of proteins.

PARP activity assay

Cells (5106) were lysed in 0.5 ml of a buffer containing 0.1%Triton X, 50 mM Tris-HCl pH 8, 0.6 mM EDTA, 14 mM-mercaptoethanol, 10 mM MgCl2, and protease inhibitors.Proteins (25 g) were incubated with 2 Ci 32P-NAD (GEHealthcare), 10 M NAD, 50 mM Tris-HCl, 10 mM MgCl2,14 mM -mercaptoethanol, 10 g nuclease-treated salmontestes DNA. PARP activity was expressed as fmol of 32P-NAD/g of protein (13).

For analysis of GPI 15427 on cellular PARP, intact cells(5105 tumor cells) were treated with the inhibitor andpermeabilized with digitonin (0.1 mg/ml) in the presence of0.25 Ci 3H-NAD (PerkinElmer, Milan, Italy) (23).

For in vivoPARP inhibition, peripheral blood lymphocytesPBL, from untreated or GPI 15427 (40 mg/kg per os)-treatedmice (8/group), were separated from whole blood by Ficoll-

Hypaque density gradient, permeabilized and incubated with3H-NAD in the presence of 5 g palindromic deoxyoligo-nucleotide (CGGAATTCCG).

Northern and Western blot analysis

Northern blot analysis was performed using total RNA (15g) and cDNA probes corresponding to the DNA bindingdomain of PARP-1 (kindly provided by Prof. AlexanderBurkle, University of Konstanz, Germany) or to glyceralde-hyde-3-phosphate dehydrogenase (GAPDH). Western blotanalysis was performed with monoclonal antibodies directedagainst PARP-1 (BD Biosciences, Milan, Italy), breast cancerresistance protein (Chemicon, Temecula, CA, USA), andactin (Sigma-Aldrich). Signals were quantified using a Kodakdensitometer (Rochester, NY, USA).

Colony formation assay

Cells were seeded in triplicate into 6-well plates (2102/well)and, after overnight incubation, treated with GPI 15427 (2M), TMZ (Schering-Plough, Kenilworth, NJ, USA; 311000M) or SN-38 (Alexis, Florence, Italy; 0.15 nM). When usedin combination with TMZ, SN-38 was added to cultures 1 hafter TMZ exposure. For experiments to assess the ability ofGPI 15427 to enhance the antiproliferative effects of TMZ orSN-38, the PARP inhibitor was added to cell cultures 15 minbefore the anticancer drugs. Cells were cultured to allow

colony formation, and after 1014 days colonies were fixedand stained with rhodamine B basic violet 10 (Sigma-Al-drich). Only colonies comprising 50 cells were scored assurvival colonies. Chemosensitivity was evaluated in terms ofIC50, i.e., the concentration of the drug capable of inhibitingcolony-forming ability by 50%.

To evaluate whether the combination TMZSN-38 orTMZSN-38GPI 15427 was synergic, tumor cells were ex-posed to TMZ or SN-38 alone or in combination at fixedequipotent ratios (corresponding to 1, 0.5, 0.2, 0.1 the IC50for each drug) in the absence or presence of a nontoxic dose

of GPI 15427 (2 M). The dose-effect curves were analyzed bythe median-effect method of Chou and Talalay using theCalcuSyn Software (Biosoft, Cambridge, UK). The combina-tion index (CI) indicates a quantitative measure of the degreeof drug interaction in terms of synergistic (CI1), additive(CI1), or antagonistic effect (CI1). Graphs were gener-ated plotting CI as a function of the fraction of cells affected(Fa) by the dose of the anticancer drugs.

In vivo studies of antitumor activity

HT-29 or LoVo cells (1106) were inoculated intramuscle(i.m.) in male athymic cluster of differentiation CD-1 mice(nu/nu genotype, 8/group). Tumors were measured with

caliper and treatment started when nodules reached 200400mm3. Xenograft growth was monitored by measuring tumornodules every 24 days for 3 wk.

TMZ was dissolved in dimethyl-sulfoxide, diluted in saline(0.5 mg/ml) and administered intraperitoneally (i.p.) at 10mg/kg/day 5 days and CPT-11 (Campto, Aventis, Milan,Italy) i.p at 4 mg/kg/day 5 days, doses corresponding to1/15 of the maximum tolerated dose of TMZ GPI 15427 perosand 1/10 of the maximum tolerated dose of CPT-11 GPI15427 per os. GPI 15427 was dissolved in 70 mM PBS withoutpotassium and administered by oral gavage once a day (40mg/kg) for 5 days, 1 h before TMZ or CPT-11. Since thesynergistic effect deriving from TMZ and CPT-11 combina-tion is schedule dependent (4), CPT-11 was administered 1 hafter TMZ. Treatment was repeated for two cycles. Control

mice were always treated with vehicles.Toxicity was assessed on the basis of apparent drug-relateddeaths (within 7 days after the last treatment) and net body wtloss [i.e., (initial wt-lowest wt)/initial wt100%].

All procedures involving animal care were performed incompliance with national and international guidelines (Eu-ropean Economy Community Council Directive 86/109,OLJ318, Dec. 1, 1987).

Myelosuppression assay

Athymic nu/nu mice (6/group) received vehicle, TMZ (100mg/kg/day5 days), GPI 15427 (40 mg/kg/day5 days) orGPI 15427 TMZ. Three mice per group were sacrificed onday 8 or 15 and whole blood was analyzed for complete bloodcount.

Intestinal toxicity assay

Wistar rats (10/group) were treated i.p. with CPT-11 (30mg/kg/day3 days) (24) and received vehicle or two oraldoses of GPI 15427 (40 mg/kg) 30 min before and 1 h aftereach CPT-11 treatment. The day after the last treatment, rats

were anesthetized with urethane (1.4 g/kg/i.p.) and jejunumwas collected. A 0.5 cm segment of intestine was fixed in 10%buffered formalin and embedded into paraffin wax. Serial 7m sections were mounted on silane-coated glass slides.Sections were dewaxed in xylene, rehydrated in graded

E3PARP INHIBITOR FOR CHEMOSENSITIZATION/PROTECTION

7/29/2019 FASEB J 06

4/13

ethanol, stained with hematoxylin/eosin, and analyzed usinglight microscopy (Zeiss, Milan Italy).

For analysis of (ADP-ribose) polymers by immunohisto-chemistry, sections were dewaxed, permeabilized with 0.1%(w/v) Triton X-100 in PBS for 20 min, and incubatedovernight with an anti-(ADP-ribose) polymers Ab (Alexis, 1:50in PBS, v/v). Specific labeling was detected with a biotin-conjugated goat anti-rabbit IgG and avidin-biotin peroxidasecomplex (DBA, Milan, Italy). Densitometry of (ADP-ribose)polymers staining was performed as described previouslyusing an Imaging Densitometer (AxioVision, Zeiss, Milan,

Italy) and a computer program (25). In particular, thedensitometry analysis of immunocytochemistry photographs(n5) was carried out in section in which the jejunum wasoriented in order to observe all the mucosa villi. In this typeof section it is possible to evaluate the presence/absence ofthe positive staining.

Terminal deoxynucleotidyltransferase (TdT)-mediated uri-dine triphosphate (UTP) end labeling (TUNEL) assay wasperformed using a commercially available kit (Apotag, HRPkit, DBA).

For diarrhea assessment all animals were checked threetimes daily and diarrhea was recorded. The severity of diar-rhea was scored as follows: 0, no diarrhea; 1, mild diarrhea(staining of anus); 2, moderate diarrhea (staining over top ofthe legs and lower abdomen); 3, severe diarrhea (staining

over legs and higher abdomen, often associated with contin-ual oozing).

Statistical analysis

For tumor xenografts, the results were analyzed by 1-wayANOVA for multiple comparisons. Residuals were examinedfor normality and homogeneity of variance. Because the P

value for the ANOVA Fstatistic was 0.001, we calculated the95% confidence intervals for the differences between thegroups using the post-test Bonferroni multiple comparisonsmethod (Primer of Biostatistics Statistical Software Program,McGraw-Hill Medical). A P value of 0.05 was considered

significant.Students t tests was used for the other statistical analyses.All statistical analyses were two-sided and results were consid-ered to be statistically significant at P 0.05.

RESULTS

GPI 15427 inhibits the activity of colon cancers withdifferent levels of PARP-1

Cell lines were analyzed for PARP-1 activity (which

accounts for 90% of the cellular poly(ADP-ribosyl)a-tion activity) and for the expression of the correspond-ing protein or transcript. The results indicate thatactivity correlated with expression of PARP-1 proteinand that LoVo and HCT-116 showed the highest levelof PARP-1, whereas HT-29 and HCT-116 Chr3 thelowest (Fig. 1). HT-29 and HCT-116 Chr3 linesshowed also the lowest levels of PARP-1 transcript(Fig. 2). Cells were then exposed to graded concen-trations of the PARP inhibitor GPI 15427 and assayedfor PARP-1 activity 1 h later. The results show thatGPI 15427 inhibited PARP-1 activity in all cell lines,

with comparable IC50s (range of mean values: 7487nM, Table 2), indicating high and uniform penetra-tion of the drug.

Figure 1. PARP-1 activity (A) and expression (B) in humancolon cancer cell lines. A) Total cellular PARP-1 activity wasmeasured in cell extracts obtained from colon cancer lines inthe presence of nuclease-treated salmon testes DNA and32P-NAD. PARP activity was expressed as fmol 32P-NAD/gof protein and the results of five independent experiments

were presented as Box and Whisker plots. The results ofstatistical analysis by Students t test are as follows: HT-29 vs.Lovo, P 0.0001; HT-29 vs. HCT-15, P 0.0003; HT-29 vs.HCT-116 Chr3, P 0.19. Confidence intervals (95%) were as

follows: LoVo, 508579; HT-29, 112168; HCT-15, 321423;HCT-116, 523647; HCT-116 Chr3, 148218. B) Cell lysates(50 g) from the indicated colon cancer cell lines wereelectrophoresed and analyzed for the expression of PARP-1or actin. The signals were quantified by densitometric scan-ning of the autoradiograms and normalized in relation toactin. Histograms represent the mean values of the ratiosbetween optical densities (O.D.) of PARP-1 and those of actinobtained from 3 different experiments. Bars: sd values.

E4 Vol. 20 August 2006 TENTORI ET AL.The FASEB Journal

7/29/2019 FASEB J 06

5/13

Recovery of PARP-1 activity on GPI 15427 treatmentwas assessed exposing LoVo cells, which express highlevels of PARP-1, to 2 M GPI 15427 (a concentrationequivalent to the peak plasma concentration reachedin animals treated per os with 40 mg/kg) (16). Theresults of three independent experiments indicate that

a 15 min treatment with GPI 15427 induced 90 2%inhibition of PARP-1 activity, which persisted un-changed 24 h later when GPI 15427 was not removedfrom culture medium (905% inhibition). On theother hand, activity largely recovered within 24 h whenthe inhibitor was removed from culture (234% inhi-bition). Comparable results were obtained in HT-29cells, characterized by low levels of PARP-1 (data notshown).

GPI 15427 increases the antiproliferative activity ofCPT-11 and TMZ combination in colon cancer cellswith different chemosensitivity profiles

It has been demonstrated that MR deficiency is associ-ated with resistance to TMZ and increased sensitivity toCPT-11 (911). All the cell lines used for this study,with the exception of HT-29 and HCT-116 Chr3, arecharacterized by MR functional defects (Table 1).Analysis of the expression of breast cancer resistanceprotein, an ATP binding cassette transporter, which is

regarded as an important determinant of resistance tocamptothecins (26), reveals that HT-29 expressedhigher levels of this protein with respect to the othercell lines. Analysis of O6-alkylguanine DNA alkyltrans-ferase, as an indicator of cell ability to repair O6-methylguanine, and of N-methylpurine DNA glycosy-lase, which repairs N-methylpurines, indicates thatHT-29 and LoVo cells showed the highest levels ofO6-alkylguanine DNA alkyltransferase and N-methylpu-rine DNA glycosylase activity (Fig. 3).

Evaluation of sensitivity to SN-38 reveals that HT-29was the most resistant, in accordance with breast cancerresistance protein expression and MR proficiency. The

MR-proficient HCT-116 Chr3 was more resistant thanits MR-deficient counterpart, consistent with a protec-tive role of MR against camptothecin cytotoxicity. Mostcell lines were resistant to TMZ either due to MRdeficiency (HCT-15, HCT-116, LoVo) or to elevatedO6-alkylguanine DNA alkyltransferase activity (HT-29).The MR-proficient HCT-116 Chr3 line was 3-fold moresensitive than its MR-deficient counterpart. In all celllines, 2 M GPI 15427 significantly enhanced theantiproliferative effects of SN-38 (25-fold) or TMZ (5-to 16-fold) (Fig. 4).

Since TMZ and CPT-11 combination is currentlyevaluated in clinical trials, we then investigated the

influence on tumor cell growth of the combinationTMZ SN-38 in the presence or not of the PARPinhibitor GPI 15427. Cells were treated with TMZ andSN-38 in combination at fixed equipotent ratios se-lected on the basis of the IC50 values of each cell line,as described in Materials and Methods. Figure 5 showsthe results of the median effect analysis using CalcusynSoftware. The CI values indicate that the combinationTMZ SN-38 was highly synergistic in LoVo cells andantagonistic in HCT-116 Chr3 cells; the addition of GPI15427 always potentiated the antiproliferative effect ofthe drug combination.

Figure 2. PARP-1 transcript in human colon cancer cell lines.Expression of PARP-1 was evaluated by Northern blot analysis.The hybridization signals were quantified by densitometricscanning of the autoradiograms and normalized in relation toGAPDH, actin or ethidium bromide staining of rRNA. Histo-grams represent the mean value of the ratios between theoptical densities (O.D.) of PARP-1 and those of GAPDH, actinor ethidium bromide staining for each cell line. Bars: sd

values. The results are representative of one of three differentexperiments.

TABLE 2. Inhibitory effects of GPI 15427 on PARP-1 activity

Cell lines IC50 (nM)a 95% Confidenceinterval

HT-29 87 8291LoVo 74 6084HCT-15 86 7696HCT-116 74 6585HCT-116 Chr3 79 7088

a The ability of GPI 15427 to inhibit PARP activity of coloncancer lines was assessed in cells permeabilized with digitonin and inthe presence of3H-NAD. Values represent the means of GPI 15427IC50s calculated from 3 independent experiments. GPI 15427 IC50 onpurified PARP-1 (100 ng, Alexis) was 36 nM (95% confidenceinterval: 2844) in accordance with previous studies (13).


7/29/2019 FASEB J 06

6/13

Oral administration of GPI 15427 enhances theantitumor efficacy of TMZCPT-11 in HT-29 andLoVo xenografts

To examine whether GPI 15427 was capable ofinhibiting PARP in vivo, mice were treated with 40mg/kg GPI 15427 per os, a dose capable of increasingthe antitumor efficacy of TMZ (16), and 1 h or 24 hlater PBL were analyzed for PARP activity. The resultsindicate that GPI 15427 inhibited PARP activity ofPBL by60% [untreated control: fmol 23,3 (95%confidence intervals: 1728), GPI 15427: fmol 9,7

Figure 3. Analysis of breast cancer resistance protein expres-sion and O6-alkylguanine DNA alkyltransferase or N-meth-

ylpurine DNA glycosylase activity in human colon cancer celllines. Cell lysates (50 g) from the indicated colon cancer celllines were electrophoresed and analyzed for the expression ofbreast cancer resistance protein (BCRP) or actin (upperpanel). O6-alkylguanine DNA alkyltransferase (AGT) activityis expressed as fmol of methyl groups per mg of total proteinand the results of five independent experiments were pre-sented as Box and Whisker plots (middle panel). The results

of statistical analysis by Students ttest are as follows: HT-29 vs.Lovo, P 0.36; LoVo or HT-29 vs. HCT-15, HCT-116, orHCT-116 Chr3, P 0.0001. Confidence intervals (95%) wereas follows: LoVo, 675755; HT-29, 707775; HCT-15, 491563;HCT-116, 196214; HCT-116 Chr3, 140177. N-methylpurineDNA glycosylase (MPG) activity is expressed as fmol ofmethylpurines released per mg of protein and the results of 5independent experiments were presented as Box and Whis-ker plots (lower panel). The results of statistical analysis byStudents ttest are as follows: HT-29 vs. LoVo, P 0.08; LoVoor HT-29 vs. HCT-15 or HCT-116 or HCT-116 Chr3, P 0.0001. Confidence intervals (95%) were as follows: LoVo,454546; HT-29, 531639; HCT-15, 274356; HCT-116, 260 322; HCT-116 Chr3, 167225.

Figure 4. Chemosensitivity of colon cancer cells to TMZ orSN-38 as single agents or in combination with the PARPinhibitor GPI 15427. Chemosensitivity of colon cancer cells toSN-38 or TMZ as single agents or in combination with 2 MGPI 15427 was assessed by colony formation assay and theresults were expressed as IC50. Only colonies comprising 50cells were scored as survival colonies. Data are means fromthree independent experiments; bars: sd. The results ofstatistical analysis by Students t test of the differences insensitivity to SN-38 are as follows: HT-29 vs. LoVo, P 0.04; vs.HCT-15, P 0.03; vs. HCT-116 P 0.0001; vs. HCT-116 Chr3,P 0.04. GPI 15427 significantly enhanced the antiprolifera-tive effects of SN-38 or TMZ in all cell lines (P0.0001).


7/29/2019 FASEB J 06

7/13

(95% confidence intervals: 510), P0.0007], sug-gesting that the compound was bioavailable andpharmacologically active. At 24 h activity completely

recovered.Toxicity studies determined the five daily administra-tions of 40 mg/kg/day GPI 15427 per os10 mg/kg/day/i.p. TMZ 4 mg/kg/day/i.p. CPT-11 as themaximum tolerated dose of the 3 drug combination.Among the colon cancer cell lines analyzed in vitro,HT-29 and LoVo were selected for the in vivo studies.HT-29 is a highly aggressive tumor with a doubling timeof 9 days and is the most resistant to SN-38 in vitro.From colon cell lines with higher PARP activities, LoVowas chosen because of its high resistance to SN-38.Moreover, both lines are resistant to TMZ due to highO6-alkylguanine DNA alkyltransferase levels (HT-29) or

to MR deficiency (LoVo). In HT-29 xenograft model,TMZ CPT-11 did not inhibit tumor growth. It isnoteworthy that GPI 15427 significantly enhanced theantitumor effect of TMZ CPT-11 combination (Fig. 6and Table 3). On the other hand, treatment with GPI15427 did not significantly affect tumor growth inhibi-tion in the groups treated with TMZ or CPT-11. InLoVo xenografts the combination of TMZ CPT-11was more effective than in HT-29 xenografts, resultingin a statistically significant delay in tumor growth. Alsoin this model, GPI 15427 significantly enhanced growthinhibition induced by CPT-11 TMZ, increasing the

growth delay by 12 days with respect to treatment withCPT-11 TMZ and causing tumor regression in allanimals (Table 3).

Figure 5. Synergistic effect of GPI 15427 on TMZand SN-38 combination in colon cancer cell lines.Cells were treated with TMZ SN-38 (diamondsymbols) at fixed molar ratios (1, 0.5, 0.2, 0.1times the IC50 concentration for each drug) or

with TMZ SN-38 and 2 M GPI 15427 (squaresymbols). Colony-forming ability was evaluatedafter 1014 days. Combination index (CI)-frac-tion affected (Fa) plots of interactions betweenTMZ and SN-38 or TMZ, SN-38 and GPI 15427

were generated by computer analysis using Cal-cuSyn software Version 2.0. The combinationindex (CI) indicates a synergistic (CI1), addi-tive (CI1) or antagonistic effect (CI1).

Figure 6. GPI 15427 per os increases the antitumor activity ofCPT-11 and TMZ combination in resistant and highly prolif-erating HT-29 colon cancer cells. Symbols represent themeans of tumor nodule volumes determined in 8 animals foreach group every 3 days. Bars: sd The tumor growth curvecorresponding to the group treated with GPI 15427 as a singleagent was omitted since it overlaps with that of the controlgroup. Differences between the three drug combination andall the other groups were statistically significant starting fromday 10 onward (P0.05, using the post-test Bonferroni mul-tiple comparisons method). Treatment produced animalbody wt loss 10% of original wt. All mice recovered theinitial body wt 2 wk after treatment.


7/29/2019 FASEB J 06

8/13

Protective effect of PARP inhibitor on CPT-11-induced jejunum damage and delayed diarrhea

We then investigated whether oral administration ofthe PARP inhibitor GPI 15427 might attenuate CPT-11

toxicity, using a well-established rat model for evaluat-ing CPT-11-induced intestinal mucosa damage (24).Rats were treated with GPI 15427 (40 mg/kg/q23days) together with 30 mg/kg/q 3 days CPT-11, adose previously used to assess the efficacy of othercompounds in preventing CPT-11 intestinal damage(24). Histological analysis of jejunum sections from ratstreated with CPT-11 showed severe intestinal injury(Fig. 7A). Necro-inflammatory debris in small foci wasdetected within the epithelial cell layer together withfocal loss of cells. Numerous neutrophils were observedamong epithelial cells and edematous lamina propria.Additionally, some areas with an increased inflamma-

tory reaction characterized by lymphatic infiltrationwere detected (Fig. 7B). The severity of mucosa damagewas also demonstrated by the presence of apoptoticcells (Fig. 7C) and mitotic figures (Fig. 7D). Conversely,the sections of jejunum from rats treated with GPI15427 CPT-11 or with vehicle showed no significanthistological alterations (Fig. 7E, F).

To assess whether intestinal damage was associatedwith PARP-1 activation, jejunum sections were analyzedfor the presence of poly(ADP-ribosyl)ated proteins byimmunohistochemical staining with an anti-(ADP-ri-bose) polymers Ab. Jejunum sections from CPT-11-

treated rats showed positive staining for (ADP-ribose)polymers in the damaged areas (Fig. 8A) mainly local-ized in epithelial cells (Fig. 8B), in inflammatory cells(Fig. 8C), and in the surrounding area (Fig. 8D).Densitometry analysis indicated that 6.1 0.21% of

total jejunum tissue area positively stained with anti-(ADP-ribose) polymers Ab (Fig. 8D), whereas only0.5 0.09% of total jejunum tissue area was found tobe positive in the jejunum of rats treated with GPI15427 CPT-11 (Fig. 8E). No positive (ADP-ribose)polymers staining was found in the jejunum of ratstreated with vehicle (Fig. 8F).

To test whether the tissue damage was associated withapoptosis, we measured TUNEL-like staining in thejejunum tissue. Almost no apoptotic cells were detect-able in the jejunum tissue of vehicle-treated rats (Fig.9A), while sections from CPT-11-treated rats showed amarked appearance of dark brown apoptotic cells (Fig.

9B, B1), characterized by chromatin compaction intouniformly dense masses in perinuclear membrane,formation of apoptotic bodies and membrane bleb-bing (see particles B1). No apoptotic cells or fragmentswere found in the jejunum of rats treated with GPI15427 CPT-11 (Fig. 9C).

The ability of GPI 15427 to protect animals fromdelayed diarrhea was also investigated. In animals thatreceived CPT-11 mild to moderate diarrhea developedafter 24 h. Oral administration of GPI 15427 reducedseverity of delayed diarrhea (Fig. 10). No diarrhea wasrecorded in control animals.

TABLE 3. GPI 15427 per os increases the antitumor activity of CPT-11 and TMZ combination in drug-resistant LoVo and HT-29colon cancer cellsa

LoVob (treatment) T-C (days)c Nadir weight loss (%)d Regressionse Nadir inhibition (%)

GPI 15427 0 0 0/6 0TMZ (10 mg/kg) 3.2 0 0/6 14(day 6)CPT-11 (4 mg/kg) 2.7 0 0/6 18(day19)GPI 15427 TMZ 7 8 1/6 39(day13)GPI 15427 CPT-11 6.7 0 0/6 34(day29)TMZ CPT-11 14.9 2 1/6 43(day43)

GPI 15427 TMZ CPT-11 26 8 6/6 56(day43)

HT-29b (treatment) T-C (days)c Nadir weight loss (%)d Regressionse Nadir inhibition (%)

GPI 15427 0 0 0/6 0TMZ (10 mg/kg) 3.4 3 0/6 14(day 6)CPT-11 (4 mg/kg) 5 3 0/6 16(day10)GPI 15427 TMZ 3.6 5 0/6 21(day 6)GPI 15427 CPT-11 5 5 0/6 17(day10)TMZ CPT-11 5.6 6 0/6 20(day14)GPI 15427 TMZ CPT-11 10.4 7 2/6 57(day24)

aValues represent the maximum percentage of tumor growth inhibition in the different treatment groups calculated with respect tovehicle-treated controls. The day after tumor challenge in which the nadir of tumor growth inhibition was observed is shown in parentheses.

b In vivo doubling time of LoVo and HT-29 cells was 18 and 9 days, respectively.c Xenograft response was assessed by growth delay, calculated as TC, which represents the difference in days between the median time for

tumors of treated (T) and control (C) animals to reach a volume twice that of the volume at the time of initial treatment. In both HT-29 andLoVo xenograft models, differences between the 3 drug combination and all the other groups were statistically significant (P0.05, using thepost-test Bonferroni multiple comparisons method). In LoVo xenograft, the difference between TMZ CPT-11 and TMZ or CPT-11 wasstatistically significant (P0.05, using the post-test Bonferroni).

d Body weight loss was calculated as follows: initial weight-lowest weight/initial weight 100%.e Tumor regression was defined as a reduction in the tumor volume for at least 2 consecutive measurements.


7/29/2019 FASEB J 06

9/13

GPI 15427 did not exacerbate myelotoxicity inducedby full dose of TMZ

We evaluated whether PARP inhibition exacerbates

myelosuppression induced by TMZ, the dose-limitingside effect of this drug in patients. Mice were treatedwith GPI 15427 per os40 mg/kg/day 5 days 100mg/kg TMZ and complete blood count analyzed onday 8 and 15 post-initiation of treatment. TMZ treat-ment caused a 78% and 60% decline of WBC on days8 and 15, respectively (Table 4), whereas GPI 15427had no effect on WBC. Coadministration of TMZ andGPI 15427 did not enhance the myelosuppressiveeffects of TMZ. No changes in red blood cells,hemoglobin (Hb), or platelets were observed (datanot shown).

DISCUSSION

In the present study we demonstrate for the first timethat oral administration of GPI 15427, a recently devel-

Figure 8. Protective effects of GPI 15427 on CPT-11-induced(ADP-ribose) polymers formation. Analysis of jejunum sec-tions obtained from CPT-11-treated rats showed positivestaining for (ADP-ribose) polymers, a product of PARP-1activation, in the damaged areas (A) mainly localized inepithelial cell (B) as well as in inflammatory cells (C). Inaddition, some positive staining for (ADP-ribose) polymers

was found in the surrounding area of the jejunum sectionsobtained from CPT-11-treated rats (D). In contrast, signifi-cantly no positive (ADP-ribose) polymer staining was found inthe jejunum of rats treated with GPI 15427 CPT-11 (E). Nostaining for (ADP-ribose) polymers in jejunum tissues was ob-tained in vehicle-treated rats (F). Figure is representative of atleast 3 experiments performed on different experimental days.

Figure 7. Protective effects of GPI 15427 on CPT-11-induced jejunum damage. The histological analysis of

jejunum sections stained with hematoxylin/eosin from ratstreated with CPT-11 showed severe intestinal injury (A). Inparticular, there was necro-inflammatory debris in smallfoci within the epithelial cell layer with focal loss of cells;numerous neutrophils were observed among epithelialcells and edematous lamina propria as well as lymphaticinfiltration was detected (B). The severity of mucosa damage

was also demonstrated by the presence of apoptotic cells (C)and mitotic figures (D). Conversely, the sections of jejunumfrom rats treated with GPI 15427 CPT-11 (E) demonstratedno histological alterations or presence of apoptotic cellsor/and mitotic figures. No histological alterations were found inthe tissue from vehicle-treated rats (F). Figure is representativeof at least 3 experiments performed on different experimentaldays.


7/29/2019 FASEB J 06

10/13

oped PARP inhibitor, enhances the antitumor activityof CPT-11 and TMZ used in combination against coloncancer. It is noteworthy that GPI 15427 provides pro-tection from intestinal damage induced by CPT-11 anddid not exacerbate myelotoxicity of TMZ.

The cell lines tested showed different pattern of

susceptibility to CPT-11 and TMZ, mainly due to breastcancer resistance protein expression, O6-alkylguanineDNA alkyltransferase levels, or MR functional status.MR plays an important role in the maintenance ofgenomic stability due to its involvement in the postrep-licative repair. Germline mutations of MR genes causesusceptibility to hereditary nonpolyposis colorectal can-cer, which accounts for 5% of colorectal cancers, andMR defects or microsatellite instability occur in 15%of sporadic colorectal tumors (27,28). Besides beinginvolved in genomic surveillance, MR strongly influ-ences tumor susceptibility or resistance to a number of

anticancer drugs (29). All MR-deficient colon cancerlines were resistant to TMZ, with IC50s 7- to 15-foldhigher than the plasma peak concentration reached inpatients (60 M). Resistance to TMZ of the MR-proficient HT-29 line is instead due to high O6-alkyl-guanine DNA alkyltransferase activity. In regard toN-methylpurine DNA glycosylase levels and sensitivityto methylating agents, conflicting results have beenreported. In fact, embryonic stem cells derived fromN-methylpurine DNA glycosylase knockout mice weremore sensitive than wild-type (WT) cells, whereas N-methylpurine DNA glycosylase-deficient bone marrowcells were more resistant (30,31). Moreover, overex-pression of N-methylpurine DNA glycosylase was asso-ciated with increased sensitivity to TMZ, suggesting thatimbalanced BER process might generate toxic interme-diates (32). Our results indicate that the ability torepair N-methylpurines does not seem to substantiallyaffect susceptibility to TMZ. In fact, HCT-15 and HCT-116, which are both MR deficient and tolerant toO6-methylguanine, possess different sensitivity to TMZeven though they have similar N-methylpurine DNA

glycosylase activity.MR-deficient lines were more sensitive to SN-38 thanthe MR-proficient HT-29 line and HCT-116 was moresensitive than the MR-proficient counterpart HCT-116Chr3. Hypersensitivity of MR-deficient tumors to topo-isomerase I inhibitor has been attributed to reduceddouble-strand break repair by nonhomologous end-joining (10, 11). Moreover, the endogenous expressionof breast cancer resistance protein, a membrane trans-porter known to confer resistance to camptothecins,

Figure 10. Protective effects of GPI 15427 on irinotecan-induced delayed diarrhea. The severity of diarrhea was eval-uated by observers blinded to the treatment groups andscored as follows: 0, no diarrhea; 1, mild diarrhea (staining ofanus); 2, moderate diarrhea (staining over top of the legs andlower abdomen); 3, severe diarrhea (staining over legs andhigher abdomen, often associated with continual oozing). Nodiarrhea was recorded in control animals. On the contrary, inanimals that received CPT-11, no diarrhea was observed inthe first 24 h; however, mild to moderate diarrhea developedafter this time point. Oral administration of GPI 15427reduced severity of delayed diarrhea. Data are means se. of10 rats for each group. *P 0.01 vs. CPT-11.

Figure 9. Protective effects of GPI 15427 on CPT-11-inducedapoptosis analyzed by TUNEL assay. Almost no apoptotic cells

were detectable in the jejunum tissue of vehicle-treated rats(A). On the contrary, sections from CPT-11-treated ratstissues demonstrated a marked appearance of dark brownapoptotic cells and intercellular apoptotic fragments (B, B1)associated with a specific apoptotic morphology, character-ized by the compaction of chromatin into uniformly densemasses in perinuclear membrane, the formation of apoptoticbodies as well as membrane blebbing (see insert, B1). On thecontrary, no apoptotic cells or fragments were found in the

jejunum of rats treated with GPI 15427 CPT-11 (C). D)Thepositive staining in the Kit positive control tissue (normalrodent mammary gland). Figure is representative of at least 3experiments performed on different experimental days.


7/29/2019 FASEB J 06

11/13

contributes to the resistant phenotype of HT-29 (26,33).It has been reported that PARP-1 might participate in

colorectal carcinogenesis, since its expression wasfound to be increased in carcinomas with respect to thecorresponding normal intestinal epithelium. Moreover,PARP-1 expression was highest in undifferentiated nor-mal cells of intestinal crypts (34). The cell lines testedin the present study differed in PARP-1 levels, with thelowest level being expressed by the moderately differ-entiated HT-29 (35). In all cell lines the results ofcolony formation assay indicated that inhibition ofPARP by GPI 15427 significantly increased sensitivity to

both SN-38 and TMZ. The entity of the chemosensitiz-ing effect was not substantially influenced by endoge-nous PARP-1 levels. In fact, GPI 15427 induced a similarsensitization to TMZ or SN-38 in LoVo and HT-29 cells,which possess different PARP-1 levels. In regard toTMZ, the most pronounced effect was observed inHCT-15 cells, which showed the highest level of resis-tance to the drug. In HT-29 cells, GPI 15427 enhancedTMZ antiproliferative activity by 5-fold, whereas O6-benzylguanine, a selective inhibitor of O6-alkylguanineDNA alkyltransferase currently under investigation as achemosensitizer in combination with TMZ, increasedthe cytostatic effect of TMZ barely by 1.2-fold (36). In

addition, O6-alkylguanine DNA alkyltransferase inhibi-tors cannot be utilized for chemosensitization to TMZof MR-deficient tumors.

Treatment with TMZ, followed by SN-38, inducedsynergistic antiproliferative effects mainly in LoVocells; this effect was confirmed in vivo, since the anti-cancer drug combination significantly delayed cellgrowth with respect to treatment with the single agents.It has been reported that O6-methylguanine induced byN-methyl-N-nitro-N-nitrosoguanidine increases topo-isomerase I cleavage complexes (37). Our data indicatethat the synergistic antiproliferative effect exerted byTMZ CPT-11 seems to be independent of O6-meth-

ylguanine, since all cell lines express moderate/highlevels of O6-alkylguanine DNA alkyltransferase and areconsistent with those previously reported in rhabdo-myosarcoma and neuroblastoma models (3). In HCT-116 Chr3, the TMZ and SN-38 combination resulted inantagonist effect likely due to tumor cell sensitivity toTMZ. It can be hypothesized that TMZ-induced growtharrest would prevent cells from entering S phase, thuscounteracting the toxicity of topoisomerase I inhibi-tors, which requires DNA duplication and occurs whenreplication fork encounters a cleavable complex.

Inhibition of PARP markedly increased the antipro-

liferative effects of the combination TMZ SN-38 in allcell lines. Moreover, the in vivoresults indicate that thispotentiation was particularly evident with the highlyaggressive HT-29 line, which possesses a doubling time2-fold shorter than LoVo and was less responsive toTMZ CPT-11. The results obtained in vivo alsoindicate that oral administration of GPI 15427 signifi-cantly inhibited PARP activity of PBL, suggesting thatthe compound is well absorbed and pharmacologicallyactive.

In the experimental in vivo model used in thepresent study, TMZ or CPT-11 did not inhibit tumorgrowth nor was their activity significantly potentiated

when used in combination with the PARP inhibitor.The apparent discrepancy with the in vitro resultsshowing the ability of GPI 15427 to enhance theantiproliferative activity of TMZ or CPT-11 combinedwith the PARP inhibitor is likely due to the low doses ofchemotherapeutic agents used in vivo.

A major concern with the use of biomodulators ofresistance is the increase of toxicity of chemotherapytoward normal tissues. In regard to CPT-11, its dose-limiting toxicity is diarrhea, which is of two types: acuteand delayed-onset. Acute diarrhea occurs within 24 h, isthe result of the cholinergic activity of CPT-11, and isprevented or rapidly suppressed by atropine. Delayed-

onset diarrhea occurs 24 h after drug administrationand can be grade 3 (severe) or grade 4 (life-threaten-ing) in up to 40% of the patients (38). The currentstrategy to treat delayed diarrhea consists in a high doseof the mu-opiate receptor agonist loperamide or soma-tostatin analog octreotide, but the success of theseapproaches is often limited (39). Delayed diarrhea hasbeen attributed to SN-38, generated from CPT-11 byintestinal carboxylesterases or from the SN-38 glucuro-nide present in bile by mucosal and bacterial -gluco-ronidase. However, the mechanism by which this mol-ecule provokes mucosa injury in the small intestine isstill uncertain (24). In the present study we demon-

strate that CPT-11 doses that generate high SN-38concentrations induce PARP-1 overactivation with ADP-ribose polymer formation, which contributes to intesti-nal damage. Oral administration of GPI 15427 preventsADP-ribose polymer accumulation and protects epithe-lial cells from cell death. In fact, an extensive synthesisof (ADP-ribose) polymers is known to deplete thecellular pools of NAD and ATP leading to cell death.PARP inhibition would spare cells from energy loss,preventing metabolic failure and providing cytoprotec-tion (40). Indeed, GPI 15427 reduced inflammation ofjejunum and severity of delayed diarrhea in animals

TABLE 4. Effect of GPI 15427 and TMZ administration on white blood cells (WBC) in nude mice

WBC (1103/l)

Vehicle TMZ alone GPI 15427 alone Combination

Day 8 5.85 1.125 1.22 0.39l 6.55 0.85 2.29 1.17Day 15 8.62 3.17 3.44 0.49 7.37 1.98 2.99 1.84


7/29/2019 FASEB J 06

12/13

receiving CPT-11 at doses higher than those used incombination with TMZ. The involvement of PARP-1 ininflammatory diseases, including acute and chronicinflammation of the gut, and the protective effect ofPARP inhibitors have been demonstrated in variousexperimental models (41). Besides the involvement inPARP-mediated cellular suicidal pathway, PARP-1 en-hances the activities of NF-B and activating proteinAP-1, key transcription factors regulating the expres-sion of inflammatory mediators and adhesion mole-cules (41). In the present model, the PARP inhibitoralso prevents inflammation as indicated by the preser-vation of the intestinal architecture, with the absence ofedema and lymphatic infiltration.

Reduction of CPT-11 toxicity by PARP inhibitorappears in some ways in contrast to the enhancement ofantitumor efficacy exerted by GPI 15427 in combina-tion with TMZ CPT-11. This dual role of PARPinhibitor is due to the fact that PARP-1 may act as asurvival factor, favoring DNA repair when the damage ismoderate, or as a cell death mediator in the presence ofextensive DNA damage that triggers PARP-1 overactiva-

tion. In this case, oral doses of GPI 15427 allowimmediate achievement of sufficient PARP inhibitionin the gut wall just where it is required to prevent mucosaldamage. On the other hand, chemosensitization inducedat the tumor level by GPI 15427 in combination withlow doses of CPT-11 TMZ relies on inhibition of thePARP activity that is needed for DNA repair.

GPI 15427 did not exacerbate myelotoxicity inducedby full doses of TMZ. This might be due to thecomplete recovery of PARP function when GPI 15427 isno longer available. On the contrary, when TMZ iscombined in vivowith O6-benzylguanine, an increase ofmyelotoxicity was reported, which required a marked

reduction of TMZ dosing (42). Indeed, when HT-29 orLoVo cells were treated with O6-benzylguanine O6-alkylguanine DNA alkyltransferase activity did not re-cover after 24 h (data not shown). Upon interactionwith O6-benzylguanine, O6-alkylguanine DNA alkyl-transferase is rapidly degraded by the proteasome, andthis contributes to induce a long-lasting depletion ofO6-alkylguanine DNA alkyltransferase since the proteinhas to be resynthesized (43). The ability of GPI 15427to prevent CPT-11 intestinal toxicity but not TMZ-induced myelotoxicity might be attributed to thehigher concentrations of the PARP inhibitor reached inthe gut by means of oral administration with respect to

those reached in bone marrow following absorptionand distribution of the compound.

In conclusion, these data indicate that GPI 15427sensitizes tumors to methylating agents and topoisom-erase I poison combination, representing a novel strat-egy to enhance the efficacy and reduce toxicity ofchemotherapy in colon cancer. Since PARP inhibitorshave recently entered phase I-II clinical trials in com-bination with TMZ, our findings on the combination ofPARP inhibitor with TMZ CPT-11 will provide therational basis for the development of new clinicalprotocols.

This work was supported by grants from the Italian Ministryof Education and Research Fondo per gli Investimenti dellaRicerca di Base (FIRB) to G.G. and Programmi di Ricercascientifica di rilevante Interesse Nazionale (PRIN) projectsto G.G. and L.T. J.Z., W.X., and R.L. work for MGI PHARMA,

which is developing PARP inhibitors for cancer treatment.

REFERENCES

1. Douillard, J. Y., Cunningham, D., Roth, A. D., Navarro, M.,James, R. D., Karasek, P., Jandik, P., Iveson, T., Carmichael, J.,Alakl, M., et al. (2000) Irinotecan combined with fluorouracilcompared with fluorouracil alone as first-line treatment formetastatic colorectal cancer: a multicentre randomised trial.Lancet 355, 10411047

2. Rasheed, Z. A., and Rubin, E. H. (2003) Mechanisms of resis-tance to topoisomerase I-targeting drugs. Oncogene 22, 72967304

3. Houghton, P. J., Stewart, C. F., Cheshire, P. J., Richmond, L. B.,Kirstein, M. N., Poquette, C. A., Tan, M., Friedman, H. S., andBrent, T. P. (2000) Antitumor activity of temozolomide com-bined with irinotecan is partly independent of O6-methylgua-nine-DNA methyltransferase and mismatch repair phenotypesin xenograft models. Clin. Cancer Res. 6, 41104118

4. Patel, V. J., Elion, G. B., Houghton, P. J., Keir, S., Pegg, A. E.,Johnson, S. P., Dolan, M. E., Bigner, D. D., and Friedman, H. S.(2000) Schedule-dependent activity of temozolomide plusCPT-11 against a human central nervous system tumor-derivedxenograft. Clin. Cancer Res. 6, 41544157

5. Jones, S. F., Gian, V. G., Greco, F. A., Miranda, F. T., Shipley,D. L., Thompson, D. S., Hainsworth, J. D., Toomey, M. A.,Willcutt, N. T., and Burris, H. A., 3rd. (2003) Trial of irinotecanand temozolomide in patients with solid tumors. Oncology 17,4145

6. Wagner, L. M., Crews, K. R., Iacono, L. C., Houghton, P. J.,Fuller, C. E., McCarville, M. B., Goldsby, R. E., Albritton, K.,Stewart, C. F., and Santana, V. M. (2004) Phase I trial oftemozolomide and protracted irinotecan in pediatric patientswith refractory solid tumors. Clin. Cancer Res. 10, 840848

7. Reardon, D. A., Quinn, J. A., Rich, N. J., Desjardins, A.,Vredenburgh, J., Gururangan, S., Sathornsumetee, S., Badrud-doja, M., McLendon, R., Provenzale, J., et al. (2005) Phase I trial

of irinotecan plus temozolomide in adults with recurrent malig-nant glioma. Cancer 104, 14781486

8. Tentori, L., and Graziani, G. (2004) Temozolomide: an updateon pharmacological strategies to increase its antitumor activity.Med. Chem. Rev.-Online. 1, 144150

9. DAtri, S., Tentori, L., Lacal, P. M., Graziani, G., Pagani, E.,Benincasa, E., Zambruno, G., Bonmassar, E., and Jiricny, J.(1998) Involvement of the mismatch repair system in temozo-lomide-induced apoptosis. Mol. Pharmacol. 54, 334341

10. Jacob, S., Aguado, M., Fallik, D., and Praz, F. (2001) The role ofthe DNA mismatch repair system in the cytotoxicity of thetopoisomerase inhibitors camptothecin and etoposide to hu-man colorectal cancer cells. Cancer Res. 61, 65556562

11. Magrini, R., Bhonde, M. R., Hanski, M. L., Notter, M., Scherubl,H., Boland, C. R., Zeitz, M., and Hanski, C. (2002) Cellulareffects of CPT-11 on colon carcinoma cells: dependence on p53and hMLH1 status. Int. J. Cancer 101, 2331

12. Tentori, L., Leonetti, C., Scarsella, M., dAmati, G., Portarena,I., Zupi, G., Bonmassar, E., and Graziani, G. (2002) Combinedtreatment with temozolomide and poly(ADP-ribose) polymer-ase inhibitor enhances survival of mice bearing hematologicmalignancy at the central nervous system site. Blood 99, 22412244

13. Tentori, L., Leonetti, C., Scarsella, M., DAmati, G., Vergati, M.,Portarena, I., Xu, W., Kalish, V., Zupi, G., Zhang, J., andGraziani, G. (2003) Systemic administration of GPI 15427, anovel poly(ADP-ribose) polymerase-1 inhibitor, increases theantitumor activity of temozolomide against intracranial mela-noma, glioma, lymphoma. Clin. Cancer Res. 9, 53705379

14. Miknyoczki, S. J., Jones-Bolin, S., Pritchard, S., Hunter, K., Zhao,H., Wan, W., Ator, M., Bihovsky, R., Hudkins, R., Chatterjee, S.,et al. (2003) Chemopotentiation of temozolomide, irinotecan,


7/29/2019 FASEB J 06

13/13

and cisplatin activity by CEP-6800, a poly(ADP-ribose) polymer-ase inhibitor. Mol. Cancer Ther. 2, 371382

15. Calabrese, C. R., Almassy, R., Barton, S., Batey, M. A., Calvert,A. H., Canan-Koch, S., Durkacz, B. W., Hostomsky, Z., Kumpf,R. A., Kyle, S., et al. (2004) Anticancer chemosensitization andradiosensitization by the novel poly(ADP-ribose) polymerase-1inhibitor AG 14361. J. Natl. Cancer Inst. 96, 5667

16. Tentori, L., Leonetti, C., Scarsella, M., Vergati, M., Xu, W.,Calvin, D., Morgan, L., Tang, Z., Woznizk, K., Alemu, C., et al.(2005) Brain distribution and efficacy as chemosensitizer of anoral formulation of PARP-1 inhibitor GPI 15427 in experimentalmodels of CNS tumors. Int. J. Oncol. 26, 415422

17. Schreiber, V., Ame, J. C., Dolle, P., Schultz, I., Rinaldi, B.,Fraulob, V., Menissier-de Murcia, J., and de Murcia, G. (2002)Poly(ADP-ribose) polymerase-2 (PARP-2) is required for effi-cient base excision DNA repair in association with PARP-1 andXRCC1. J. Biol. Chem. 277, 2302823036

18. Yung, T. M., Sato, S., and Satoh, M. S. (2004) Poly(ADP-ribosyl)ation as a DNA damage-induced post-translational mod-ification regulating poly(ADP-ribose) polymerase-1 topoisomer-ase I interaction. J. Biol. Chem. 279, 3968639696

19. Malanga, M., and Althaus, F. R. (2004) Poly(ADP-ribose) reac-tivates stalled DNA topoisomerase I and induces DNA strandbreak resealing. J. Biol. Chem. 279, 52445248

20. Tentori, L., and Graziani, G. (2005) Chemopotentiation byPARP inhibitors in cancer therapy. Pharmacol. Res. 52, 2533

21. Ha, H. C., and Snyder, S. H. (1999) Poly(ADP-ribose) polymer-ase is a mediator of necrotic cell death by ATP depletion. Proc.Natl. Acad. Sci. U. S. A. 96, 1397813982

22. Tentori, L., Vergati, M., Muzi, A., Levati, L., Ruffini, F., Forini,O., Vernole, P., Lacal, P. M., and Graziani, G. (2005) Genera-tion of an immortalized human endothelial cell line as a modelof neovascular proliferating endothelial cells to assess chemo-sensitivity to anticancer drugs. Int. J. Oncol. 27, 525535

23. Bakondi, E., Bai, P., Szabo, E. E., Hunyadi, J., Gergely, P., Szabo,C., and Virag, L. (2002) Detection of poly(ADP-ribose) polymer-ase activation in oxidatively stressed cells and tissues usingbiotinylated NAD substrate. J. Histochem. Cytochem. 50, 9198

24. Fittkau, M., Voigt, W., Holzhausen., H. J., and Schmoll, H. J.(2004) Saccharic acid 1.4-lactone protects against CPT-11-in-duced mucosa damage in rats. J. Cancer Res. Clin. Oncol. 130,388394

25. Cuzzocrea, S., Misko, T. P., Costantino, G., Mazzon, E., Micali,A., Caputi A. P., Macarthur, H., and Salvemini, D. (2000)Beneficial effects of peroxynitrite decomposition catalyst in a rat

model of splanchnic artery occlusion and reperfusion. FASEB J.14, 1061107226. Rajendra, R., Gounder, M. K., Saleem, A., Schellens, J. H., Ross,

D. D., Bates, S. E., Sinko, P., and Rubin, E. H. (2003) Differen-tial effects of the breast cancer resistance protein on the cellularaccumulation and cytotoxicity of 9-aminocamptothecin and9-nitrocamptothecin. Cancer Res. 63, 32283233

27. Rowley, P. T. Inherited susceptibility to colorectal cancer.(2005) Annu. Rev. Med. 56, 539554

28. Herman, J. G., Umar, A., Polyak, K., Graff, J. R., Ahuja, N., Issa,J. P., Markowitz, S., Willson, J. K., Hamilton, S. R., Kinzler, K. W.,et al. (1998) Incidence and functional consequences of hMLH1promoter hypermethylation in colorectal carcinoma. Proc. Natl.Acad. Sci. U. S. A. 95, 68706875

29. Fedier, A., and Fink, D. (2004) Mutations in DNA mismatchrepair genes: implications for DNA damage signaling and drugsensitivity. Int. J. Oncol. 24, 10391047

30. Engelward, B. P., Weeda, G., Wyatt, M. D., Broekhof, J. L., deWit, J., Donker, I., Allan, J. M., Gold, B., Hoeijmakers, J. H., andSamson, L. D. (1997) Base excision repair deficient micelacking the Aag alkyladenine DNA glycosylase. Proc. Natl. Acad.Sci. U. S. A. 94, 1308713092

31. Roth, R. B., and Samson, L. D. (2002) 3-Methyladenine DNAglycosylase-deficient Aag null mice display unexpected bonemarrow alkylation resistance. Cancer Res. 62, 656660

32. Trivedi, R. N., Almeida, K. H., Fornsaglio, J. L., Schamus, S., andSobol, R. W. (2005) The role of base excision repair in the

sensitivity and resistance to temozolomide-mediated cell death.Cancer Res. 65, 63946400

33. Doyle, L. A., and Ross, D. D. (2003) Multidrug resistancemediated by the breast cancer resistance protein breast cancerresistance protein (ABCG2). Oncogene 22, 73407358

34. Idogawa, M., Yamada, T., Honda, K., Sato, S., Imai, K., andHirohashi, S. (2005) Poly(ADP-ribose) polymerase-1 is a com-ponent of the oncogenic T-cell factor-4/beta-catenin complex.Gastroenterology 128, 19191936

35. Fogh, J., and Trempe, G. (1975) New human tumour cell lines.In Human tumour cells in vitro(Fogh, J., ed) pp. 115141, PlenumPress, New York, U. S. A.

36. Wedge, S. R., Porteus, J. K., May, B. L., and Newlands, E. S.(1996) Potentiation of temozolomide and BCNU cytotoxicity byO(6)-benzylguanine: a comparative study in vitro. Br. J. Cancer73, 482490

37. Pourquier, P., Waltman, J. L., Urasaki, Y., Loktionova, N. A.,Pegg, A. E., Nitiss, J. L., and Pommier, Y. (2001) TopoisomeraseI-mediated cytotoxicity of N-methyl-N-nitro-N-nitrosoguani-dine: trapping of topoisomerase I by the O6-methylguanine.Cancer Res. 61, 5358

38. Saliba, F., Hagipantelli, R., Misset, J. L., Bastian, G., Vassal, G.,Bonnay, M., Herait, P., Cote, C., Mahjoubi, M., Mignard, D., andCvitkovic, E. (1998) Pathophysiology and therapy of irinotecan-induced delayed-onset diarrhea in patients with advanced colo-rectal cancer: a prospective assessment. J. Clin. Oncol. 16,27452751

39. Benson, A. B., 3rd, Ajani, J. A., Catalano, R. B., Engelking, C.,Kornblau, S. M., Martenson, J. A., Jr., McCallum, R., Mitchell,

E. P., ODorisio, T. M., Vokes, E. E., and Wadler, S. (2004)Recommended guidelines for the treatment of cancer treat-ment-induced diarrhea. J. Clin. Oncol. 22, 29182926

40. Mazzon, E., Dugo, L., Li, J. H., Di Paola, R., Genovese, T.,Caputi, A. P., Zhang, J., and Cuzzocrea, S. (2002) GPI 6150, aPARP inhibitor, reduces the colon injury caused by dinitroben-zene sulfonic acid in the rat. Biochem. Pharmacol. 64, 327337

41. Cuzzocrea, S. (2005) Shock, inflammation and PARP. Pharma-col. Res. 52, 7282

42. Quinn, J. A., Desjardins, A., Weingart, J., Brem, H., Dolan, M. E.,Delaney, S. M., Vredenburgh, J., Rich, J., Friedman, A. H.,Reardon, D. A., et al. (2005) Phase I trial of temozolomide plusO6-benzylguanine for patients with recurrent or progressivemalignant glioma. J. Clin. Oncol. 23, 71787187

43. Xu-Welliver, M., and Pegg, A. E. (2002) Degradation of thealkylated form of the DNA repair protein, O(6)-alkylguanine-

DNA alkyltransferase. Carcinogenesis 23, 82383044. Umar, A., Boyer, J. C., Thomas, D. C., Nguyen, D. C., Risinger,J. I., Boyd, J., Ionov, Y., Perucho, M., and Kunkel, T. A. (1994)Defective mismatch repair in extracts of colorectal and endo-metrial cancer cell lines exhibiting microsatellite instability.

J. Biol. Chem. 269, 143671437045. Gibson, N. W., Hartley, J. A., Barnes, D., and Erickson, L. C.

(1986) Combined effects of streptozotocin and mitozolomideagainst four human cell lines of the Mer phenotype. CancerRes. 46, 49954998

46. Papadopoulos, N., Nicolaides, N. C., Liu, B., Parsons, R., Len-gauer, C., Palombo, F., DArrigo, A., Markowitz, S., Willson,J. K., Kinzler, K. W., et al. (1995) Mutations of GTBP ingenetically unstable cells. Science 268, 19151917

47. Papadopoulos, N., Nicoladesi, N. C., Wei, Y. F., Ruben, S. M.,Carter, K. C., Rosen, C. A., Haseltine, W. A., Fleischmann, R. D.,Fraser, C. M., Adams, M. D., et al. (1994) Mutation of a MutL

homolog in hereditary colon cancer. Science 263, 1625162948. Koi, M., Umar, A., Chaulan, D. P., Cherian, S. P., Carethers,

J. M., Kunkel, T. A., and Boland, C. R. (1994) Human chromo-some 3 corrects mismatch deficiency and microsatellite instabil-ity and reduces N-methyl-N-nitrosoguanidine tolerance in co-lon tumor cells with homozygous hMLH1 mutation. Cancer Res.54, 43084312

Received for publication February 16, 2006.Accepted for publication March 31, 2006.


FASEB J 06

Documents

Transcript of FASEB J 06