apoptosis as a determinant of tumor sensitivity to ... · treatment (8, 9), and a correlation...

Vol. 3, 955-961, June 1997 Clinical Cancer Research 955

Apoptosis as a Determinant of Tumor Sensitivity to Topotecan in

Human Ovarian Tumors: Preclinical in Vitro/in Vivo Studies1

Claudia Caserini, Graziella Pratesi,2

Monica Tortoreto, Barbara Bedogn#{233},

Nives Carenini, Rosanna Supino, Paola Perego,

Sabina C. Righetti, and Franco Zunino

Division of Experimental Oncology B. Istituto Nazionale Tumori. Via

Venezian I. 20133 Milan, Italy

ABSTRACT

Preclinical and clinical studies have documented the

pharmacological interest in camptothecin derivatives in thetreatment of resistant tumors. In particular, topotecan, awater-soluble derivative, exhibited promising activity in

pretreated ovarian carcinoma. The present study investi-gated the pattern of tumor response in two human ovariancarcinoma xenografts and in their cisplatin-resistant sub-

lines characterized by different mechanisms of drug resist-ance. In IGROV-1I.Ptl cells, cisplatin resistance has beenascribed to a reduced susceptibility to apoptosis as a conse-

quence of p53 mutation and inactivation of its function. In

the A2780 cisplatin-resistant subline, which retained the

wild-type p53 gene status, the development of resistance hasbeen possibly related to increased cell ability to repair drug-induced DNA damage. The in vivo results of the present

study showed that topotecan could overcome the resistance

in A2780/CP but not in IGROV-lfPtl tumor xenografts. The

pattern of tumor response following in vivo topotecan treat-ment correlated with drug ability to induce apoptosis butnot with its in vitro antiproliferative activity. The antitumorefficacy of topotecan in the four tumors reflected a different

cell response to drug-induced DNA damage, as suggested bydifferent perturbations of cell cycle progression. Indeed,only in the subline refractory to topotecan in vivo, IGROV-l/Ptl, did we observe a persistent arrest of the cells in the

S-phase, resulting in a cytostatic and not a cytotoxic effect,

since a low level of apoptosis was induced by the drug. In

conclusion, the current results support that determination of

drug-induced apoptosis is a useful predictor of tumor re-sponse to topotecan in ovarian carcinomas and suggest that

Received 8/1 3/96: revised 2/20/97: accepted 3/5/97.

The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to

indicate this fact.

I This work was partially supported by the Consiglio Nazionale delle

Ricerche (Finalized Project Applicazione Cliniche della Ricerca Onco-

logica), Associazione Italiana per Ia Ricenca sul Cancro. and Ministero

della Sanit#{225}.

2 To whom requests for reprints should be addressed. Phone: 39-2-2390626; Fax: 39-2-2390764.

p53 gene status may be a critical determinant of cell re-

sponse to topoisomerase inhibitors.

INTRODUCTION

Camptothecins are cytotoxic agents known to be DNA topo

I� inhibitors ( 1 ). The antitumor activity found in preciinicai

models (2, 3) was confirmed in clinical studies (4). Topotecan

(lO-hydroxy-9-dimethylaminomethyl-(S)-camptothecin) is a

water-soluble camptothecin analogue currently under clinical

investigation. It has been recently approved for ovarian carci-

noma treatment based on the results of a Phase III study in

cisplatin-pretreated patients (5). A lack of cross-resistance be-

tween topotecan and cisplatin in ovarian tumors had already

been documented in Phase II clinical studies (6). We previously

showed that topotecan is extremely active against i.p. growing

human ovarian tumor xenografts, including cisplatin-resistant

tumors (7).

The cellular basis of sensitivity to topo I inhibitors remains

unclear. A better understanding of the critical events involved in

drug-induced cytotoxicity may have clinical implications for

optimization of therapy with topo I inhibitors and rational de-

velopment of effective drug combinations. Although multiple

factors may contribute to the development of drug resistance of

ovarian carcinoma cells, resistance to apoptosis induction has

been proposed as a critical mechanism of drug resistance. In-

deed, apoptosis is a major mode of cell death in response to drug

treatment (8, 9), and a correlation between apoptotic death and

chemosensitivity has been documented in preclinical studies

with ovarian carcinomas ( I 0). In this regard, the status of p53,

a gene involved in cell cycle control and regulation of apoptosis

(1 1, 12), has been implicated as a critical determinant of the

response of ovarian cancer to cisplatin (13, 14).

The aim of the present investigation was to evaluate the

cytotoxic and antitumor activity of topotecan against four hu-

man ovarian tumor cell lines and their corresponding tumor

xenografts that exhibit differential response levels and different

mechanisms of resistance to cisplatin. In IGROV-l/Ptl, the

development of drug resistance has been ascribed to p.53 muta-

tion, resulting in a reduced cell ability to activate the apoptotic

response (13). In contrast, A2780/CP retained a wild-type p53

gene status. The cellular basis of reduced sensitivity of the latter

line to cisplatin is likely related to increased cell ability to repair

drug-induced DNA damage. since high levels of topo I expres-

sion were found (7).

Our results in cell cultures showed a marginal cross-resis-

tance to topotecan in the A2780 cisplatin-resistant cell line and

not in the IGROV-l/Ptl cells. However, the pattern of in vitro

3 The abbreviations used are: topo I. topoisomerase I: TW, tumorweight: TWI. tumor weight inhibition; LCK. log111 cell kill: GSH,glutathione: GST, glutathione S-transferase.

Research. on June 9, 2020. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

http://clincancerres.aacrjournals.org/

EXONS 5 6 7 8 9

Fig. I Single-strand conformation polymorphism analysis ofp53 gene

status in A2780 (Lane 2) and A2780/CP (Lane 3) cells. The comparison

to SiHa cells (Lane 1), displaying all wild-type exons, reveals no

mobility shift for all of the exons tested.

956 Topotecan and Apoptosis Induction

cell response did not reflect the in s’is’o tumor response. since

only the A2780 tumor xenograft selected for resistance to cis-

platin exhibited lack of cross-resistance to topotecan. In con-

trast, in the IGROV-i/Ptl tumor, complete cross-resistance to

topotecan was observed in t’is’o. These cells, carrying a mutant

p53 gene, were not susceptible to topotecan-induced apoptosis,

suggesting a major role for the p53-dependent pathway of

apoptosis in topotecan efficacy in ovarian tumors.

MATERIALS AND METHODS

Drugs. Topotecan (Smith-Kline Beecham Pharmaceuti-

cal, Reigate, Surrey, United Kingdom) was dissolved in water,

and cisplatin (Bristol-Meyers Squibb. Wallingford. CT) was

dissolved in saline immediately before use.

Human Tumor Cell Lines and Cytotoxicity Studies.The IGROV-l and the A2780 cell lines originated from ovarian

carcinomas oftwo untreated patients (15, 16). The resistant cell

lines, IGROV-l/Ptl and A2780/CP, were selected in our labo-

ratory in the presence of increasing cisplatin concentrations

(13). All of the cell lines were cultured in RPMI 1640 (Bio-

Whittaker, Verviers, Belgium) containing 10% FCS (Life Tech-

nologies, Inc., Gaithersburg, MD). The resistant sublines exhib-

ited karyotypic features similar to those of the parental cell lines,

but a somewhat reduced proliferation rate (doubling times 28 ±

2 versus 33 ± 1 h in the IGROV-i cell systems and 20 ± I

versus 22 ± I in the A2780 cell systems). This change was also

reflected in in s’it’o growth (see below).

For cytotoxicity studies, cells (5 X l04/ml) were seeded in

6-well plates (Costar Corporation, Cambridge, MA) and after

24 h were treated for 1 h with the drugs. Cell growth was

evaluated at 72 h after treatment by cell counting (Coulter

Electronics Ltd., Luton, United Kingdom). The results are cx-

pressed as IC50 values (drug concentration inhibiting 50% of

cell growth) and are the means of at least three independent

experiments.

In Vivo Studies. Six- to 10-week-old female athymic

Swiss nude mice were used in the study. Animals, obtained from

Charles River Italia (Caico, Italy), were maintained in laminar

air-flow rooms. Sterilized cages, bedding. and acidified water

were used for mice care. The air was kept at a temperature of

24-26#{176}C with 50% humidity. The experiments were approved

by the Institutional Committee for Animal Experimentation.

Because the IGROV-I/Pt-l tumor did not grow in the ascitic

form (7), s.c. growing tumor models were used to allow a direct

comparison of response to in vivo treatment among the four

tumor systems. Previous studies indicated a parallelism of tumor

responsiveness between s.c. and i.p. growing ovarian carcinoma

models (7).

All tumor lines originated from s.c. injection of5-I0 X l0�’

cells/flank of mice. For line maintenance and experimental

purposes. tumor fragments were grafted s.c. into both flanks of

athymic mice by a 1 3-gauge trocar. Growth of s.c. tumors was

followed by biweekly caliper measurement of tumor length and

width. TW was calculated in milligrams using the formula:

TW = width2 X length/2 according to Geran et al. ( I 7). For

chemotherapy studies, each experimental group consisted of at

least eight assessable tumors. Drugs were delivered iv. in a

volume of 10 ml/kg of body weight. Two different treatment

123 123 123 123 123

�-=� �- � =-��

schedules were used, i.e., every 7 days for 3 times (q7dX3)

or every 4 days for 3 times (q4dX3). which in our experience

represent the optimal schedules for cisplatin and topotecan,

respectively. Treatments started with just measurable tumors

(50-70 mg). The effects of drug treatments were expressed at

a specified day (see Table 1 ) as percentage of TWI in treated

versus control mice, calculated as: 100 - mean TW in

treated/mean TW in control. Moreover, the LCK produced by

the treatment was calculated from the days the tumors took to

reach a mean specified weight (see Table 1) in treated and

control mice, according to the formula: days in treated -

days in control/3.32 X tumor doubling time (calculated by

linear regression analysis of control tumor growth).

No control mouse died because of tumor burden in the

experimental frame. Death of treated mice was ascribed to lethal

toxicity and was recorded throughout all of the experimental

frame.

Statistical comparison between topotecan-treated versus

cisplatin-treated mice was evaluated by Student’s t test (two

tailed).

Biochemical Studies. The GSH level and GST activity

were assessed by processing fragments from the four tumor

lines as described previously (18). The GSH level was analyzed

according to Tietze ( 1 9), and results were expressed as nmol/mg

protein. GST activity was assayed according to Habig et a!. (20)

and was expressed as nmol/minlmg protein.

Analysis ofp53 Gene. Single-strand conformation poly-

morphism analysis was performed as described previously (21),

for exons 5-9, because they are localized in the region of the

p53 gene most often mutated. PCR-amplified exons were also

subjected to direct DNA sequencing with an Amplicycle Se-

quencing kit (Perkin-Elmer Corp., Branchburg, NJ). Each se-

quencing reaction was performed at least twice, analyzing sep-

arate amplifications. The results for IGROV-l and IGROV-l/

Ptl cells have been already reported (13). As shown in Fig. 1, no

mobility shift, for all of the exons tested, was observed for

A2780 and A2780/CP cells in comparison to the human cervical

carcinoma cell line (SiHa) carrying a wild-type p.53 gene (22).

To confirm the wild-type status of exons 5-9. all of them were

subjected to direct DNA seqencing. No mutations were re-

vealed.

Cellular Studies. Apoptosis was assessed by fluores-

cence microscopy as already described (13). Briefly. ethanol-

fixed cells were stained with propidium iodide solution (50



Clinical Cancer Research 957

Table I Efficacy of systemic topotecan on s.c. growing human ovarian tumor xenografts

Tumor line Treatment” Dose % TWI Toxic/

(Dr’ = days ± SD) schedule (1st day) Drug (mg/kg) (day) total LCK’ P’

A2780 q7d X 3 (3) Topotecan 7.5 79 (24) 0/4 1.6 <0.001

( I .9 ± 0.5) Topotecan 10 89 (24) 0/5 2. 1 <0.05

Cisplatin 6 99(24) 0/5 3.8

A2780/CP q7d X 3 (3) Topotecan 7.5 85 (24) 0/5 1.5 <0.05(3.1 ± 0.9) Topotecan 10 74 (24) 1/5 1.3 <0.05

Cisplatin 6 61 (24) 1/5 0.8IGROV-l q4d X 3 (6) Topotecan 7.5 55 (21) 0/5 1 n.s.

(3.5 ± 0.9) Topotecan 10 75 (21) 0/5 1.55 n.s.

Cisplatin S 66(21) 0/5 1.3

IGROV-l/Ptl q4d X 3 ( 15) Topotecan 10 39 (34) 1/4 0.4 n.s.

(5.1 ± 1.5) Cisplatin 5 23(34) 1/4 0.2

“ Doubling time of control tumor, calculated by linear regression analysis of the growth curve.,, Treatments started when mean TW was about 50-70 mg.‘. LCK produced by the drug and calculated to 1 g for IGROV-l lines and to 2 g for A2780 lines.(, Versus cisplatin-treated mice in the same experiment, by Student’s t test.

Table 2 Sensitivity of ovarian tumors to cisplatin and topotecan: comparison of in vitro and in viva results

Optimal TWI” (%) induced by IC501’ (�sg/ml)

Tumor model Cisplatin Topotecan Cisplatin Topotecan

A2780 99 89 1.4 0.10 ± 0.01

A2780/CP 61 85 26.0 0.50 ± 0.15

IGROV-l 66 75 4.3 0.27 ± 0.16IGROV-l/Ptl 23 39 82.0 0.25 ± 0.21

“ Data from Table 1.‘, Calculated by cell counting. The IC30 values are from a representative experiment ftr cisplatin and are the means ± SD of three independent

experiments for topotecan.

Table 3 Biochemical and molecular characterization of human ovarian tumor lines

topo I GSH content” GST activity” p53

Tumor line expression” (nmol/mg protein) (nmol/min/mg protein) status’

A2780 0.083 7.2 ± 3.3 188 ± 71 Wild-type

A2780/CP 0.150 38 ± 0.02 120 Wild-type

IGROV-l 0.027 3.9 ± 2 295 ± 59 Wild-type

IGROV-lfPtl 0.033 17.9 ± 0.8 278 Mutant

‘S By Northern blot analysis. Values were normalized to g-actin (from Ref. 7).I’ From tumor fragments (see Ref. 18).‘ By DNA single-strand conformation polymorphism analysis (from Ref. 13 and unpublished results).

gig/mI propidium iodide and 66 units/ml RNase in PBS) and

stored in the dark for 30 mm. At least 100 cells in two different

smears were examined. The percentage of apoptotic cells was

referred to the cell number of the whole population (floating +

adherent cells).

Cell cycle distribution was investigated by flow cytometric

analysis as already described (13). Briefly, at different times

after treatment, cells were detached, washed with PBS, fixed in

70% ice-cold ethanol, and stored at -20#{176}C. Cells were then

rehydrated in PBS and stained with propidium iodide solution

for 30 mm. Fluorescence intensity was determined by a FAC-

Scan flow cytometer equipped with an argon laser (Becton

Dickinson, Mountain View, CA).

RESULTS

The effects of topotecan and cisplatin on the tumor growth

of two human ovarian carcinoma xenografts, IGROV- 1 and

A2780, and their cisplatin-resistant variants are reported in

Table 1 . Cisplatin was more toxic when given according to an

“every 4 days” compared to an “every 7 days” schedule (5- and

6-mg/kg/injections were the respective maximal tolerated dos-

es). Conversely, no differences in the maximal tolerated doses

were observed for topotecan according to the two treatment

schedules investigated. Cisplatin, given according to a weekly

schedule, was very effective against the A2780 tumor, almost

completely inhibiting tumor growth (99% TWI), and much less

effective against the A2780/CP variant (6 1 % TWI and LCK

< 1). The two doses (including the maximum tolerated dose) of

topotecan investigated in the study were active on both tumor

xenografts, causing comparable LCK and tumor growth inhibi-

tion. Against the parent IGROV-l tumor, which was moderately

responsive to cisplatin (66% TWI and LCK of I .3), topotecan

was somewhat more effective than cisplatin (75% TWI and

LCK of 1 .6), although the difference was not significant. In



100’

80’

60�

40’

20�

24h

A2780 A2780/DDP

IGROV-1/Ptl �iI �-�-

IGROV-1

IIGROV-1

48h

C/)

U)01-0�00�

LL�0-JLU>Ui-I

A2780

IGROV-llPtl

A2780/DDP

IGROV-1

I80

72h

60

40

20

A2780

IGROV-1/Ptl

A2780/DDP

0

z0C)

0 0Int2

0

ccz0C)

0

52

0ccI-z0C)

B BC) 0

0

cc

z00

B B0 0


Fig. 2 Time course of apoptosis after 1 -h expo-

sure to topotecan in IGROV-l, IGROV-l/Ptl,A2780. and A2780/CP cell lines. Treated (IC50

and IC5�) and untreated (control) cells were fixed

in ice-cold ethanol at different times after drug

treatment (24. 48. and 72 h). After rehydration,

cells were stained with propidium iodide and

then analyzed with a fluorescence microscope. Atleast two smears were examined for the nuclear

morphology changes. The results presented are

the averages of the percentage of apoptotic cells

in the whole population from two independent

experiments. Bars. SE.

contrast, both drugs were inactive against the resistant IGROV-

lfPtl tumor, as documented by marginal effects on tumor

growth and LCK. Therefore, from these in viva results, topote-

can showed cross-resistance to cisplatin in the IGROV- 1/Pt 1 but

not in the A2780/CP resistant tumor line.

These findings did not reflect the pattern of antiprolifera-

tive activity of the two drugs in these cell lines in vitro, where

the cytotoxic effect of topotecan was similar in IGROV-l and

IGROV-l/Ptl cells (Table 2). Conversely, in the A2780/CP cell

system an appreciable resistance to topotecan was observed

(resistance index, 5).

Different mechanisms of resistance are likely to be in-

voived in the cisplatin-resistant phenotype of these cells. Our

results showed that the cellular response to topotecan was not



IGROV-i A2780

IGROV-1 /Ptl

0 0

A2780/Cp

72

72


Fig. 3 Flow cytometnic profiles of cell cycleperturbation after 1-h exposure to topotecan(IC8�) in IGROV-l, IGROV-lIPtl, A2780, andA2780/CP cell lines. Cells were fixed in ice-coldethanol at different times after drug treatment (0,

24, 48, and 72 h). After rehydration and pro-

pidium iodide staining, cells were analyzed with

the flow cytometer FACScan.

related to p53 status. In addition, the pattern of cell response to

the drug was not explained by topo I expression, nor by GSH

content or GST activity, since no correlation was found between

topotecan IC50 and these parameters (Table 3).

Since p53 status may influence the apoptotic response (12),

we investigated the induction of apoptosis after topotecan treat-

ment in the four cell lines (Fig. 2). The level of basal apoptosis

in control cells never exceeded 10% in all of the cell lines. At

24, 48, and 72 h after treatment, equitoxic concentrations of

topotecan (IC50 and IC80) induced a similar increase in apopto-

sis in the A2780 and A2780/CP cells. In contrast, topotecan was

a potent inducer of apoptosis only in IGROV-l but not in

IGROV-1/Ptl cells (85% versus 20% cells in apoptosis at the

respective IC80). Twenty-four and 48 h after topotecan treat-

ment, lower levels of apoptosis induction were observed in all of

the cell lines compared to the levels observed after 72 h.

Apoptosis induction was dose and time dependent in the four

ovarian carcinoma cell lines.

In an attempt to find a cellular basis for differential induc-

tion of apoptosis in the examined cell systems, perturbations of

cell cycle progression were analyzed. The effects of equitoxic

concentrations (IC80) of topotecan on cell cycle distribution are

presented in Fig. 3. The flow cytometric profiles in IGROV-1

and IGROV-lfPtl cells were quite different at all of the exam-

med time points (from 24 to 72 h after treatment): in the parental

cells, a minimal modification of the distribution along the cell

cycle was observed, with a reduction of 30-40% of cells in G�

and an increase of cells in the S-phase and G2. Conversely, in

the resistant cells, G, disappeared almost completely with an

increase in the S-phase and, to a lesser extent, in G,. A differ-

ential cell cycle perturbation after topotecan treatment (IC8�)

was observed also in the two A2780 cell systems. No modifi-

cations were observed in the A2780 cell cycle, whereas in

A2780/CP a decrease in G, and an increase in G, were observed

up to 48 h. At 72 h, cell cycle distribution was similar to that of

untreated cells.

DISCUSSION

Preclinical and clinical evidence supports the pharmaco-

logical interest of topo I inhibitors in the treatment of cisplatin-

resistant tumors. A relevant mechanism of cisplatin resistance

has been ascribed to increased DNA repair. Indirect evidence

suggests a contribution of topo I in the repair mechanism of

DNA-damaging agents, including cisplatin and ionizing radia-

tions (23, 24). Indeed, overexpression of the DNA topo 1 gene

has been reported in cispiatin-resistant cells (7, 25). In addition,

potentiation of cisplatin cytotoxicity by 9-aminocamptothecin

was found to be associated with persistence of specific cisplatin-

induced DNA lesions (26). Comparing topotecan antitumor

activity in cisplatin-sensitive and -resistant human ovarian tu-

mor xenografts, the present study showed lack of cross-resis-

tance in one of the two cisplatin-resistant tumors, A2780/CP.

High responsiveness of this tumor to topotecan treatment, in-

cluding cured mice, has already been reported in a study dealing

with local-regional treatment of the i.p. growing xenograft (7).




The lower antitumor efficacy achieved by iv. topotecan in the

present study (no cured mice were observed) indicated a more

favorable pharmacological profile for local-regional versus sys-

temic treatment with the drug. Such an observation may be

explained by a short half-life of the lactone form of the mole-

cule, which is known to be the active form (27). A clinical trial

of i.p. topotecan in ovarian carcinoma patients is suggested by

these results.

In an attempt to establish a relationship between the effi-

cacy of topo I inhibitors in cisplatin-resistant tumors and cellular

and molecular bases, we have chosen two tumor cell systems

characterized by different mechanisms of drug resistance. In

both systems, resistance to cisplatin was associated with resist-

ance to apoptosis induction. In IGROV-lfPtl cells, a markedly

reduced ability to activate apoptosis (evident at equitoxic drug

levels) has been related to inactivation of p53 function following

gene mutation in both alleles (13). In contrast, a reduction of

apoptotic response in A2780/CP cells following exposure to

cisplatin apparently did not reflect an inactivation of p53 func-

tion due to mutation of the hot spot region. In these cell lines

growing in vitro, a complete lack of cross-resistance has been

documented only in IGROV-lfPtl cells using an antiprolifera-

tive test to evaluate the pattern of cell response after a short-term

exposure to topotecan ( 1 h). However, the pattern of tumor

response after in viva treatment of the same tumor lines xc-

nografted into nude mice did not reflect the relative sensitivity

observed in vitro. Indeed, IGROV-1/Ptl tumor carrying mutant

p53 was refractory in viva to topotecan, whereas A2780/CP

tumor exhibited responsiveness comparable to that of the sen-

sitive tumor. Thus, in these models, the relative therapeutic

efficacy of topotecan reflects the differential drug ability to

activate apoptosis between resistant cells and parental cells (Fig.

2) rather than drug antiproliferative activity. The loss of wild-

type p53 function has been correlated with acquired resistance

to cisplatin (13). Based on the present findings, the presence of

the wild-type p53 gene seems to be required also for topotecan-

induced apoptosis and tumor response.

A different cell cycle control following drug exposure in

the four cell lines is possibly responsible for the lack of a

correlation between the antiproliferative activity and antitumor

efficacy of topotecan. Indeed, only minimal and transient per-

turbations of the cell cycle were observed in the treated cells

from responsive tumors, whereas a persistent accumulation in

the S-phase was detected in the resistant IGROV-1/Ptl cells up

to 72 h. A markedly different perturbation of the cell cycle

following exposure to cisplatin in resistant IGROV-1 cells (with

loss of G delay) has been ascribed to the lack of WAF-1

expression (13). Therefore, in this cell system the ability to

regulate cell cycle progression could result in cytostasis, possi-

bly providing the cell with time required for DNA repair,

because a very low level of apoptosis was induced by the drug

over 72 h. This observation may have relevant implications,

since apoptosis induction by topo I inhibitors could be a more

useful predictor of tumor responsiveness than a simple drug-

induced reduction of proliferative activity. The role of cell cycle

control and apoptosis activation as determinants of sensitivity to

topo I inhibitors has been reported for other tumor cells (28).

The current study showed that topotecan may be effective

in the treatment of cisplatin-resistant tumors. However, a lack of

cross-resistance between cisplatin and topotecan could not be

regarded as a general phenomenon, since p53 status may influ-

ence the relative sensitivity to both agents, as observed in the

IGROV-1-resistant tumor, suggesting that p.53 status may be a

critical determinant of the sensitivity of ovarian carcinoma to

DNA-damaging agents. In addition, this is the first study sup-

porting that level of apoptosis induction may be a useful pre-

dictor of tumor responsiveness in vivo. Thus, these results may

have implications for the clinical use of topo I inhibitors and for

rational development of combinations including these agents.

REFERENCES

1. Liu, L. F. DNA topoisomerase poisons as antitumor drugs. Annu.

Rev. Biochem., 58: 351-375, 1989.

2. Giovanella, B. C., Stehlin, J. S., Wall, M. E., Wani, M. C., Nicholas,

A. W., Liu, L. F., Silber, R., and Potmesil, M. DNA topoisomerase

I-targeted chemotherapy of human colon cancer in xenografts. Science

(Washington DC), 246: 1046-1048, 1989.

3. Giovanella, B. C.. Hinz, H. R., Koziciski, A. J., Stehlin, J. S., Silber,

R., and Potmesil, M. Complete growth inhibition of human cancerxenografts in nude mice by treatment with 20-(S)-camptothecin. Cancer

Res., Si: 3052-3055, 1991.

4. Slichenmyer, W. J., Rowinsky, E. K., Donehower, R. C., and

Kaufmann, S. H. The current status of camptothecin analogues as

antitumor agents. J. Nati. Cancer Inst., 85: 271-291, 1993.

5. Carmichael, J., Gordon, A., Malfetano, J., Gore, M., Spaczynski, M.,

Davidson, N., Savage, J., Clarke-Pearson, D., Hudson, I., Broom, C.,

and Ten Bokkel Huinink, W. Topotecan, a new active drug, vs paclitaxel

in advanced epithelial ovarian carcinoma: international topotecan study

group trial. Proc. Am. Soc. Clin. Oncol., 15: 283, 1996.

6. Kudelka, A. P., Tresukosol, D., Edwards, C. L., Freedman, R. S.,

Levenback, C., Chantarawiroj, P., Gonzalez de Leon, C., Kim, E. E.,

Madden, T., Wallin, B., Hord, M., Verschraegen. C.. Raber, M., andKavanagh, J. J. Phase II study of intravenous topotecan as a 5-dayinfusion for refractory epithelial ovarian carcinoma. J. Clin. Oncol., 14:

1552-1557. 1996.

7. Pratesi, G., Tortoreto, M., Cmii C., Giardini R., and Zunino, F.

Successful local regional therapy with topotecan of intraperitoneally

growing human ovarian carcinoma xenografts. Br. J. Cancer, 71: 525-

528, 1995.

8. Hickman, J. A. Apoptosis induced by anticancer drugs. Cancer

Metastasis Rev., 11: 121-139, 1992.

9. Kerr, J. F. R., Winterford, C. M., and Harmon, B. V. Apoptosis. Itssignificance in cancer and cancer therapy. Cancer (Phila.), 73: 2013-

2026, 1994.

10. Sheng Wu, G., and El-Deiry, W. S. Apoptotic death of tumor cells

correlates with chemosensitivity, independent of p53 or bcl-2. Clin.

Cancer Res., 2: 623-633, 1996.

11. Nelson, W. G., and Kastan, M. B. DNA strand-breaks: the DNA

template alterations that trigger p53-dependent DNA damage response

pathways. Mol. Cell. Biol., 14: 1815-1823, 1994.

12. Oren, M. Relationship of p53 to the control of apoptotic cell death.

Cancer Biol., 5: 221-227, 1994.

13. Perego, P., Giarola. M., Righetti, S. C., Supino, R., Caserini, C..

Delia, D., Pierotti, M. A., Miyashita, T., Reed, J. C., and Zunino, F.

Association between cisplatin resistance and mutation of p53 gene and

reduced bax expression in ovarian carcinoma cell systems. Cancer Res.,

56: 556-562, 1996.

14. Righetti, S. C., Della Torre, G., Pilotti, S., M#{233}nard,S., Ottone, F.,

Colnaghi, M. I., Pierotti, M. A., Lavarmno, C., Cornarotti, M., Oriana, S.,

Bohm, S., Bresciani, G. L., Spatti. G. B., and Zunino, F. A comparativestudy of p53 gene mutations, protein accumulation, and response to

cisplatin-based chemotherapy in advanced ovarian carcinoma. Cancer

Res., 56: 689-693, 1996.

15. B#{233}nard,J., Da Silva, J., Dc Blois, M. C., Boyer, P., Duvillard, P.,

Chiric, E., and Riou, G. Characterization of a human ovarian adenocar-




cinoma line, IGROV- 1 , in tissue culture and in nude mice. Cancer Res.,

45: 4970-4979, 1985.

16. Eva, A., Robbins, K. C., Andersen, P. R., Srinivasan, A., Tronick,S. R., Premkumar Reddy, E., Ellmore, N. W., Galen, A. T.,Lautenberger, J. A., Papas, T. S., Westin, E. H., Wong-Staal, F., Gallo,R. C., and Aaronson, S. A. Cellular genes analogues to retroviral onc

genes are transcribed in human tumour cells. Nature (Lond.), 295:

116-119, 1982.

17. Geran, R. I., Greenberg, N. H., Macdonald, M. M., Schumacher,

A. M., and Abbott B. J. Protocols for screening chemical agents andnatural products against animal tumors and other biological systems.

Cancer Chemother. Rep., 3: 1-88, 1972.

18. Pratesi, G., Dal Bo, L., Paolicchi, A., Tonarelli, P., Tongiani, R.,

and Zunino, F. The role of the glutathione-dependent system in tumor

sensitivity to cisplatin: a study of human tumor xenografts. Ann. Oncol.,

6: 283-289, 1995.

19. Tietze, F. Enzymatic method for quantitative determination of na-nogram amounts of total and oxidized glutathione. Anal. Biochem., 27:

502-522, 1969.

20. Habig, W. H., Pabst, M. J., and Jacoby, W. B. Glutathione-S-

transferase: the first enzymatic step in mercapturic acid formation.

J. Biol. Chem., 249: 7130-7139, 1974.

21. Donghi, R., Longoni, A., Pilotti, S., Michieli, P., Della Porta, G.,

and Pierotti, M. A. Gene p53 mutations are restricted to poorly differ-entiated and undifferentiated carcinomas of the thyroid gland. J. Clin.Invest., 91: 1753-1760, 1993.

22. Scheffner, M., Munger, K., Byrne, J. C., and Howley. P. M. The

state ofp53 and retinoblastoma genes in human cervical carcinoma celllines. Proc. Natl. Acad. Sd. USA, 88: 5523-5527, 1991.

23. Mattern, M. R., Hofmann, G. A., McCabe, F. L., and Johnson, R. K.

Synergistic cell killing by ionizing radiation and topoisomerase I inhib-

itor topotecan (SK&F 104864). Cancer Res., 51: 5813-5816, 1991.

24. Marchesini, R., Colombo, A., Caserini, C., Perego, P.. Supino, R.,

Capranico, G., Tronconi, M., and Zunino, F. Interaction of ionizing

radiation with topotecan in two human tumor cell lines. Int. J. Cancer,

66: 342-346, 1996.

25. Kotoh, S., Naito, S., Yokomizo, A., Kumazawa, J., Asakuno, K.,

Kohno, K., and Kuwano, M. Increased expression of DNA topoisomer-ase I gene and collateral sensitivity to camptothecin in human cisplatin-

resistant bladder cancer cells. Cancer Res., 54: 3248-3252, 1994.

26. Goldwasser, F., Valenti, M., Torres, R., Kohn, K. W., and Pommier,

Y. Potentiation of cisplatin cytotoxicity by 9-aminocamptothecin. Clin.

Cancer Res., 2: 687-693, 1996.

27. Underberg, W. J. M., Goossen, R. M. J., Smith, B. R., and Beijnen,

J. H. Equilibrium kinetics of the new experimental antitumour com-

pound SK&F 104864-A in aqueous solution. J. Pharm. Biomed. Anal.,8: 681-683, 1990.

28. Dubrez, L., Goidwasser, F., Genne, P., Pommier, Y., and Solary, E.

The role of cell cycle regulation and apoptosis triggering in determiningthe sensitivity of leukemic cells to topoisomerase I and II inhibitors.Leukemia (Baltimore), 9: 1013-1024, 1995.



1997;3:955-961. Clin Cancer Res C Caserini, G Pratesi, M Tortoreto, et al. human ovarian tumors: preclinical in vitro/in vivo studies.Apoptosis as a determinant of tumor sensitivity to topotecan in

Updated version

http://clincancerres.aacrjournals.org/content/3/6/955

Access the most recent version of this article at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://clincancerres.aacrjournals.org/content/3/6/955To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]



apoptosis as a determinant of tumor sensitivity to ... · treatment (8, 9), and a correlation...

Documents