Expression of Genes of Potential Importance in the Response to Chemotherapy and DNA Repair in Patients with Ovarian Cancer

GYNECOLOGIC ONCOLOGY ARTICLE NO.

65, 130–137 (1997)

GO964609

Expression of Genes of Potential Importance in the Response to Chemotherapy and DNA Repair in Patients with Ovarian Cancer1 A. M. CODEGONI,* M. BROGGINI,* M. R. PITELLI,† M. PANTAROTTO,* V. TORRI,* C. MANGIONI,†

AND

M. D’INCALCI*,2

*Department of Oncology, Istituto di Ricerche Farmacologiche ‘‘Mario Negri,’’ via Eritrea 62, 20157 Milan, Italy; and †Ospedale San Gerardo, Universita` di Milano, Monza, Milan, Italy Received August 5, 1996

The expression of different genes potentially involved in DNA repair and in cell responses to chemotherapy was evaluated in 33 previously untreated ovarian cancer patients. In biopsies of the same patients the expression of repair genes O6-methylguanine DNA methyltransferase (MGMT), 3-methyladenine DNA glycosylase (MAG), ERCC1, MDR-1, DNA topoisomerase I, DNA topoisomerase IIa, and glutathione S-transferase-p (GST-p) was assessed by Northern blot analysis. No direct statistical correlation was found between the expression of these genes and the response to chemotherapy (mainly platinum-based with or without doxorubicin and cyclophosphamide). Univariate analysis showed a weak negative correlation (P Å 0.037) between the expression of ERCC1 and mortality, whereas no statistically significant correlation was found for other parameters. The MDR-1 gene encoding for the Pglycoprotein P-170 was mostly undetectable in these patients (as assessed by Northern blotting), whereas relatively high levels of MAG and MGMT were found in the majority of patients. A statistically significant correlation was found between the expression of DNA topoisomerase I and the expression of either ERCC1 (P Å 0.0026) or GST-p (P Å 0.0279). q 1997 Academic Press

INTRODUCTION

The prognosis of patients with ovarian cancer is still poor. Since the introduction of platinum-based chemotherapy, which increased the response rate, overall survival has not improved significantly, with only 20% of patients surviving at 5 years [1, 2]. The success of treatment is, among other factors, limited by drug resistance that the tumors can develop during treatment or that is intrinsic to the malignancies. Studies using cultured tumor cell models or in vivo animal systems suggest that a variety of mechanisms can contribute to drug resistance. Decreased drug accumulation, due to overexpression of MDR-1 coding for P-glycoprotein (P1 This work was performed in memory of Nerina and Mario Mattioli and was partially supported by the Italian Association for Cancer Research. 2 To whom correspondence should be sent. Fax: *39-2-3546277.

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0090-8258/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.

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170), and increased detoxification through glutathione and glutathione S-transferase (GST) are mechanisms which prevent drugs from damaging DNA [3, 4]. MDR-1 gene expression in normal and tumor ovarian tissues is reportedly low, although ovarian cancers are often resistant to doxorubicin, a drug whose activity is sensitive to the presence of P-170 [5]. Bourhis et al. observed increased transcripts in 3 of 10 ovarian cancer patients treated with doxorubicin [6] and Arao et al., who analyzed MDR-1 gene expression by RT-PCR in 52 cases of ovarian cancer, confirmed the low expression of MDR-1 and the lack of association with clinical outcome [7]. The GST enzymes are a family of proteins implicated in the resistance to a number of drugs including cis-diamminedichloro platinum (DDP) and its analogues [8]. The p subtype (GST-p) is predominant in ovarian tumors and its levels were increased in tumor cell lines resistant in vitro to DDP and alkylating agents [8, 9]. Some questions persist regarding GST-p expression and response to chemotherapy in patients with ovarian cancer. A relationship has been reported between high levels of GST-p, worse prognosis, and resistance to chemotherapy [10]. Other studies, however, did not support these findings and could not find any real relationship between GST-p expression and survival or response to chemotherapy [9, 11, 12]. Topoisomerases are a class of enzymes that participate in breakage and rejoining of DNA strands and have been identified as the intracellular target of several anticancer drugs [13, 14]. Cell lines that express high levels of topoisomerases are more sensitive to the cytotoxic effects of topoisomerase poisons [15, 16]. No differences in the levels of topoisomerase I (TOPO I) and topoisomerase IIa (TOPO IIa) and their activity have been found in untreated ovarian cancer patients or patients treated with DDP and/or cyclophosphamide [17]. Very little information is available on other enzymes involved in DNA repair after treatment with DNA-damaging agents such as O6-methylguanine DNA methyltransferase

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(MGMT) and 3-methyladenine DNA glycosylase (MAG) or proteins involved in nucleotide excision repair (NER). Lee et al. [18] suggested that in ovarian tumors the resistance to alkylating agents may be related to the high cellular expression of MGMT protein, found in tumor biopsies. In another study in 20 ovarian cancer patients [19] a positive correlation was observed between high tumor MGMT activity and poor initial response to DDP-based therapy. The expression of MAG is related to resistance to alkylating agents [20, 21] but no information is available on the expression of this glycosylase in human ovarian cancer. An inverse correlation between the response to DDP-based chemotherapy and expression of ERCC1, an enzyme involved in the mechanism of NER [22], was reported in 28 ovarian cancer patients [23, 24]. Resistance to chemotherapy is probably related to a large number of factors which are involved in the uptake and detoxification of drugs as well as in the repair of druginduced lesions. This study was designed to investigate, in ovarian cancers in the same tumor biopsy, the levels of expression of several genes involved in the mechanism of drug sensitivity and resistance, such as MDR-1, MGMT, MAG, GST-p, ERCC1, TOPO I, and TOPO IIa. MATERIALS AND METHODS

Patients We analyzed 33 ovarian cancer patients. Fresh tumor tissues were obtained at first laparatomy before any other treatment, from April 1992 to June 1993. After surgery patients received platinum-based chemotherapy either alone or in standard combinations. The treatment groups included DDP, carboplatin (CBDCA), and cyclophosphamide–doxorubicin–DDP. The stage and the histological grading of the primary tumors were defined according to the FIGO criteria. Patients were classified after initial surgery as having unresectable residual disease õ2, 2–5, or ú5 cm. The minimal followup was 6 months. Table 1 summarizes the patients’ characteristics. A clinically complete remission was defined as complete disappearance of all clinically detectable disease for at least 1 month. A partial response was defined as a 50% or greater reduction of the largest lesion without evidence of any new lesion. Processing of Tumor Samples The tissues were freed of necrotic, hemorrhagic, and connective tissue; minced; and stored at 0807C in cryotubes (Nunc) until processed. Total RNA was extracted by the guanidium isothiocyanate method [25]. All extraction procedures were done at 47C.

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RNA Analysis Total cellular RNA (10 mg/lane) was size-fractionated by electrophoresis through 1% agarose–formaldehyde gel, then transferred to a nylon filter (Gene Screen Plus Dupont) by capillary blotting. Filters were baked for 2 hr at 807C and prehybridized for at least 2 hr with 50% formamide, 10% dextran sulfate, and 1% sodium dodecyl sulfate (SDS) in 1 M NaCl at 427C. The probes used to detect mRNA were plasmid pBShTOP2 (containing the entire cDNA) for TOPO IIa), the 1.3-kb EcoRI/SalI fragment of pcDR1.3 plasmid containing the human MDR-1 cDNA, the 0.85-kb BamHI–SalI fragment from YEpGAL1-hTOP1 plasmid for TOPO I, the 725-bp EcoRI fragment of clone 11 for GST-p, the 1.2-kb BamHI fragment isolated from PUC9-MPG for MAG, the 0.8-kb EcoRI fragment from pKT100 for MGMT, the 1.1kb EcoRI/HindIII fragment from pE12-12 for ERCC1, and the 1.3-kb PstI insert of a-actin. The probes were labeled with [32P]dCTP using a RediPrime kit (Amersham). Hybridization was done at 427C in the same solution used for prehybridization with 0.5–1 1 106 cpm/ml labeled probe; blots were washed twice with 21 SSC at room temperature and once with 21 SSC–1% SDS for 30 min at 657C. Membranes were exposed to X-ray film at 0807C. The mRNAs were quantitated by scanning the autoradiographs with a densitometer and comparing the intensity of the different mRNAs with a-actin mRNA which was used to normalize the amount of RNA loaded. For comparison, total RNA extracted from SKOV-3 cell line was loaded on each gel. For each gene the values were transformed to scores according to the expression in relation to actin, and the values are reported in Table 2. Ratios between 0 and 0.01 for all the genes evaluated were assigned the sign 0; / was assigned to values between 0.1 and 1 for MAG and MGMT, 0.1–0.5 for TOPO IIa, 0.1–1.5 for TOPO I, 0.1– 2.5 for GST-p, and 0.1–2 for ERCC1. The sign // was assigned to values greater than 1 for MAG and MGMT, 1.5 for TOPO I, 0.5–1.5 for TOPO IIa, 3–6 for ERCC1, and 2.5–10 for GST-p, and /// was assigned to values greater than 6 for ERCC1, 10 for GST-p, and 1.5 for TOPO IIa. For MDR-1 the patients were divided into two groups: 0, not expressing and /, low expressing. DNA Index Analysis DNA index was evaluated using flow cytometry. Frozen primary tumors were thawed directly at 377C. Automatic disaggregation was accomplished by placing the tissue fragments into a disposable mechanical disaggregation cup (Medicans) in 2 ml PBS plus 10% fetal calf serum. Rotation speed was around 80 rpm for 15 sec after which the cell suspension was filtered through a 50-mesh nylon filter. The

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TABLE 1 Patients’ Clinical and Pathological Characteristics Patient

Age

Stage

Grade

Histologic type

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

58 50 65 31 50 31 54 57 50 37 42 68 60 52 75 68 54 62 31 61 54 51 60 58 47 54 50 54 77 41 40 53 46

III IIIc IIIc IIIc III III IIIc IIIc IV IIIa IIIc IIIc IIIc IIIc IIIc IV IIIc IIIc IIIc IIIc IIIc IV IIIc IIIc IIIc III IIc IIIc IIIc IIIc IIIc IIIc IIIc

3 3 3 2 3 2 3 3 3 3 3 3 3 3 3 3 2 2 2 3 3 3 3 2 2 3 2 3 3 3 3 2 3

Und Ser Ser End Ser Muc Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Muc Ser Ser Ser Ser Ser Und End Ser Und Ser Ser Ser Ser

Residual disease õ2 õ2 õ2 õ2 2–5 õ2 ú5 ú5 2–5 2–5 2–5 ú5 õ2 ú5 ú5 2–5 õ2 ú5 ú5 2–5 ú5 ú5 ú5 ú5 2–5 2–5 õ2 2–5 2–5 õ2 ú5 õ2 õ2

cm cm cm cm cm cm cm cm cm cm cm cm cm cm cm cm cm cm cm cm cm cm cm cm cm cm cm cm cm cm cm cm cm

Therapy DDP–CBDCA DDP–CAP DDP–CAP DDP–CAP CAP CAP CAP DDP–CAP DDP–CAP CBDCA CAP DDP–CAP CBDCA CAP CAP CAP CBDCA CBDCA DDP CBDCA CBDCA DDP DDP CBDCA DDP DDP DDP DDP DDP DDP–CAP CAP CAP CBDCA

Note. Abbreviations used: Und, undifferentiated; Ser, serous; Muc, mucinous; End, endometrioid; DDP, cisplatin; CAP, cyclophosphamide, doxorubicin, cisplatin; CBDCA, carboplatin.

cell suspension was stained with propidium iodide as described [26]. Ploidy was expressed as DNA index, representing the ratio between the G1 peak of cancer cells and the G0/G1 peak of normal leukocytes used as standard. Statistical Analysis Correlations among gene expression, DNA ploidy, histological type, residual tumor, and response to chemotherapy were estimated using the Kendall t correlation coefficient. Logistic regression analysis [27] was used to evaluate the extent to which the probability of a complete or partial response depended on variables under investigation. Time to death was calculated from the day of first surgery to the date the patient was last seen for follow-up or to the date of death. Kaplan and Meier’s method [28] was used to estimate survival curves. The Cox model [29] was used for assessing the relationship between gene expression and survival.

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RESULTS

Table 1 summarizes the patients’ clinical and pathological characteristics. Of the 33 patients from whom tumor specimens were considered in our analysis, the majority (84.8%) had stage III ovarian carcinoma, with only 3 stage IV and 1 stage II. The median age at surgery was 54 years (range 31–77); 78.8% had a serous histological type tumor. The 33 tumor specimens obtained at surgery were analyzed for their DNA content. Table 2 reports the DNA index: of 33 cases analyzed 13 were diploid and the others were aneuploid (all hyperdiploid). Table 2 also reports the patients’ clinical responses to chemotherapy, evaluated by standard criteria, the survival, and the relative expression of the mRNA for the genes evaluated in this study. The mRNA values are reported as the ratio between the expression of the mRNA examined and the expression of the

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TABLE 2 mRNA Expression, DNA Index, and Clinical Response in 33 Ovarian Cancer Patients Patient

Clinical response

Survival time

DNA index

MAG

MGMT

ERCC1

GST-p

TOPO I

TOPO IIa

MDR-1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

CR PR PR CR PR NR PR CR CR PR CR NR NR NR CR CR CR NR NR NR PR NR PR CR CR CR CR PR CR PR PR PR NR

3.2 3.3 3.1 3.2 3.5 0.4 3.2 3.6 3.9 3.7 3.6 0.4 3.5 1.1 0.5 2.6 3.1 1.1 3.2 0.2 1.0 0.6 3.0 1.0 2.8 0.7 2.8 1.4 2.8 2.6 2.0 1.0 1.4

1 1 1.36 1 1.5 1.2 1 1.43 1.87 1 1.6 1 1.85 1.63 1.64 1.2 1.81 1.37 1 1 1.62 1 1.4 1.28 1 1.8 1.3 1 1.5 1 1.3 1.2 1

0 // 0 / // 0 / / / / / / / / / / / / / ND / / / / // / / 0 / / / ND /

// /// // / // / / / / / / / / // // / // / 0 // // / / / 0 0 // // // // // ND /

ND ND /// // ND / // /// // // // /// // ND /// /// // / / / ND ND / / 0 / ND / / / 0 ND //

/ /// // // // / / / / / / / / / / / / / 0 / / / / / / / / / / / / / /

ND ND // // ND // / // // // // // / ND // // // / / / ND ND // / // / ND / / / / ND /

0 // // 0 0 // 0 0 0 0 // // // // /// // // // 0 // 0 // // / / / 0 0 / 0 0 / /

0 0 0 0 0 0 0 0 0 / 0 0 0 0 0 0 0 0 / 0 0 0 0 0 0 0 0 0 / 0 0 ND 0

Note. Abbreviations used: CR, complete response; PR, partial response; NR, no response; ND, not determined; 0, no expression; /, expressed; //, moderate expression; ///, very high expression. For relative levels see Materials and Methods.

mRNA for a-actin gene used to normalize loading. The relative abundances of the different mRNAs were widely distributed among the patients. All the genes examined were measurable in terms of mRNA by the Northern blot technique. We examined the level of expression of one gene and another. A statistically significant positive correlation was found between the expression of TOPO I and either ERCC1 (P Å 0.0026) or GST-p (P Å 0.0279). Univariate analysis showed no significant correlations between the level of expression of any gene investigated and response to chemotherapy (Table 3). No significant correlation was found either between FIGO grade or aneuploidia and the level of expression of the examined genes (data not shown). Table 4 shows that a statistically significant negative correlation exists between the expression of ERCC1 and mortality.

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Multivariate analysis confirmed the negative correlation between ERCC1 expression and mortality. Figure 1 shows the survival of patients with different levels of ERCC1. In order to represent two groups evenly distributed, the median ERCC1 expression value was used as cutoff. DISCUSSION

The mechanism of action of anticancer drugs is only partially elucidated. It is still difficult to explain why a certain drug is specifically active against one human tumor but not against others. Most of the information on the molecular pharmacology of anticancer drugs has been obtained in cancer cell lines and there appear to be correlations between some of the biological or biochemical characteristics of tumor cells and their sensitivity to certain drugs. The drugs that are most effective in ovarian cancer are Pt(II) coordination

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TABLE 3 Correlation between the Level of Expression of the Examined Genes and Probability of Response to Chemotherapy (Logistic Model) Gene

N

CR / PR

NR

MAG MGMT ERCC1 GST-p TOPO I TOPO IIa

31 32 25 33 25 33

23 23 18 24 18 24

8 9 7 9 7 9

Odds ratio (95% CI) 1.05 1.72 1.24 1.78 1.72 0.34

(0.22–5.07) (0.78–3.78) (0.83–1.87) (0.63–5.03) (0.62–4.73) (0.08–1.52)

P value 0.480 0.180 0.293 0.276 0.295 0.158

Note. The expressions of the various genes were considered continuous values in the model. The value of odds ratio can be interpreted as the ratio of the odds of not achieving a response of an increase of one unit in the corresponding level of expression of each gene. Assigned reference level is 1. The higher the odds ratio the greater the probability of not achieving a response.

complexes, i.e., DDP or CBDCA, alkylating agents such as cyclophosphamide, taxanes, and anthracyclines [2, 30–33]. Therefore it is important to characterize the biochemical and biological features of human ovarian cancer that may be related to the sensitivity or resistance to these drugs. A first consideration is that MDR-1 is not frequently expressed in ovarian cancer, so resistance to doxorubicin or to taxanes could be due to other mechanisms. Since P-glycoprotein, encoded by the MDR-1 gene, is involved in the transport of many natural products which are under early clinical investigation, the absence of expression of MDR1 suggests these novel compounds are worth investigating against ovarian cancer. On the other hand other transport proteins such as MRP or LRP, possibly involved in multidrug resistance and not related to the expression of P-glycoprotein, have been recently discovered [34, 35]. Therefore further studies are needed to ascertain whether these proTABLE 4 Correlation between the Level of Expression of the Examined Genes and Mortality Gene

N

Events

MAG MGMT ERCC1 GST-p TOPO I TOPO IIa

31 32 25 33 25 33

15 16 12 17 12 17

Hazard ratio (95% CI) 0.50 0.88 0.64 0.52 0.53 1.08

(0.13–2.00) (0.64–1.19) (0.42–0.97) (0.24–1.10) (0.26–1.09) (0.40–2.91)

P value 0.329 0.392 0.037 0.085 0.085 0.885

Note. The expressions of the various genes were considered continuous values in the model. The value of hazard ratio can be interpreted as the ratio of the hazards of an increase of one unit in the corresponding level of expression of each gene. Assigned reference level is 1. The higher the hazard ratio the greater the probability of dying.

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FIG. 1. Survival curves of patients with different expression of ERRC1 mRNA. Patients were divided into two groups according to the expression of ERCC1 (lower or higher than 2.75).

teins, possibly involved in non-P-glycoprotein-associated multidrug resistance, play a role in the mechanism of resistance of ovarian cancer to anticancer drugs. As regards the expression of genes encoding proteins involved in the DNA-repair mechanisms, all those examined were expressed at widely differing levels in different ovarian carcinomas. The majority of tumors expressed MGMT. To our knowledge no previous studies on large series of ovarian cancer have been published on the levels of this DNA-repair protein. There were indications that MGMT was highly expressed in human ovarian cancer. Tagliabue et al. reported that in two ovarian cancer xenografts the levels of MGMT were much higher than in some other tumors [36], and some reports in human cancer included some ovarian cancers with relatively high levels of this enzyme [18, 19, 37, 38]. The finding that human ovarian cancers express high levels of MGMT may explain why chloroethylating and methylating agents are inactive, since the efficient repair of O6-alkylguanine is one mechanism of resistance to these drugs [39, 40]. To our knowledge the expression of MAG in human ovarian cancer biopsies has never been reported before. The available information is limited even for cancer cell lines [21]. The lack of data means we cannot compare the levels of expression of MAG in ovarian and other human tumors. However, in several cases the levels of expression are lower than those previously found in cancer cell lines. The importance of the expression of MAG in the sensitivity to alkylating agents is still debatable [20, 21, 41–43]. It seems that only when there are very low levels of endogenous MAG, transfection of cells with the bacterial Tag gene encoding for MAG or with the mammalian gene under the control of an inducible promoter can confer resistance to alkylating agents [20, 21, 41]. The two cases in which we found no expression of MAG seemed particularly interesting as to our knowledge there

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are no reported cases of mammalian cells which do not express MAG. Therefore we reexamined MAG gene mRNA by PCR: tumors which appeared negative for MAG by Northern blot analysis showed a minimal expression with the more sensitive PCR method. However, the presence of MAG mRNA might have been due to slight contamination by normal cells in the tumor biopsy specimen, which is practically inevitable. Further studies using in situ hybridization techniques are required on this point to assess the expression of MAG in the neoplastic cells as well as in the normal cells present in the biopsy specimen. If there are tumor cells that do not express MAG, drugs might be designed to methylate N-3-adenine selectively, e.g., a DNA minor groove binder with a methylating moiety, which would be selectively toxic for the cancer cells. ERCC1 has been reported to play a major role in the response to DDP or nitrogen mustards [44]. Mutants which do not express the functional ERCC1 protein are several times more susceptible to these drugs than parental cell lines with normal ERCC1 [45, 46]. Therefore one could expect that the higher the levels of expression of ERCC 1, the less susceptible were the tumors to DDP- or CBDCA-based therapies. Instead the opposite was found. Patients who showed higher tumor expression of ERCC1 had a higher probability of response and of longer survival. A tentative explanation is the following: the enzymes which take part in nucleotide excision repair work in concert, therefore if the increase of one of these enzymes is not associated with the increase of all the others, the efficacy of the process does not increase. If, for example, there is an increase in ERCC1 and 04, which cut DNA 5* of the DNA lesion, without an increase in all the other enzymes which are involved in the replacement of the damaged DNA, the uncoordinated reactions may even worsen the efficiency of the repair. This hypothesis is supported by the observation that cells which overexpress ERCC1 following transfection with the ERCC1 gene become more susceptible to the cytotoxicity of DNA-damaging agents than control cells which express lower levels of ERCC1 [46]. The finding that higher expression of ERCC1 is related to a higher probability of response is in contrast with the data reported by Dabholkar [23, 47]. The different results may be due to the fact that the levels of ERCC1 mRNA evaluated in our study could not adequately reflect the levels of the functional ERCC1 protein. Dabholkar et al. [23] have in fact recently reported that in ovarian cancer a large proportion of ERCC1 mRNA may exist as alternatively spliced species without exon 8, which is essential for the functional ability of ERCC1 protein to make the 5*-DNA incision. For TOPO I and TOPO IIa gene expression, too, there was broad variability. No correlation was observed between the tumor levels of TOPO IIa and response to chemotherapy.

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However, only 50% of patients received doxorubicin, a drug acting as topoisomerase II inhibitor and for which, in cancer cell lines in vitro, correlations have been reported between its levels and drug sensitivity [14, 15]. Some variability was also found in the levels of TOPO I in different cases of ovarian carcinoma. Knowledge of the levels of topoisomerase I is of potential interest in view of the recent report of activity of the topoisomerase I inhibitor topotecan in ovarian cancer [48]. Further studies are needed to clarify whether the tumor expression of TOPO I and possibly also the level of enzymatic activity are correlated to the response to TOPO I inhibitors. If a correlation is found it will then be possible to rationally select patients potentially sensitive to TOPO I inhibitors. As regards GST-p and response to therapy, there was a trend which was actually opposite to what was expected. In fact nonresponders appeared to have low tumor levels of GST-p. The importance of GST-p in the resistance to DNAinteracting drugs is controversial, and some studies have reported a lack of correlation between the level or activity of this enzyme and sensitivity to DDP [9, 12]. An interesting observation is that a positive correlation exists between the expression of TOPO I and either ERRC1 or GST-p expression. This finding deserves further investigation in larger series of ovarian cancer as well as in other tumors as it might suggest that there is a common mechanism of regulation of the expression of these proteins. ACKNOWLEDGMENTS We thank Dr. Ivana Scovassi for the generous gift of anti-TOPO II a antibodies and we thank the following for the gifts of cDNA used as probes for mRNA analysis: Dr. J. Wang for TOPO IIa, Dr. P. Benedetti for TOPO I, Dr. Kaina for MGMT and MAG, Dr. J. Moscow for GSTp, and Dr. J. Hoeijmakers for ERCC1. The authors also thank Angelo Tinazzi for his assistance in statistical analysis and Maura Montani for preparing the manuscript.

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