Int. J. Cancer: 66,342-346 (1996) Publication of the International Union Against Cancer Publication de I’Union Internationale Contre 18 Cancer I

0 1996 Wiley-Liss, Inc.

INTERACTION OF IONIZING RADIATION WITH TOPOTECAN IN TWO HUMAN TUMOR CELL LINES Renato MARCHESINI’, Ambrogio COLOMBO~, Claudia CASERINI~, Paola PER EGO^, Rosanna SUPINO~, Giovanni CAPRANICO~, Marco TRONCONI~ and Franco ZUNIN02.3

Diviswns of ‘Health Physics and 2Experimental Oncology B, Istituto Nazionale Tumori, Milan, Italy.

The effect of topotecan, a topoisomerase I inhibitor, on ionizing radiation-induced qtotoxici was studied in 2 human tumor cell lines characterized by a Xfferent expression of the target enzyme. The cytotoxicity of topotecan alone or in combination with radiation was assessed in exponentially grow- ing non-small-cell lung cancer (H460) and glioblastoma (GBM) cells using the colony-forming assay. An isobologram method was used to evaluate the treatment interaction. An apparent supra-additive effect in cell killing following drug-radiation- combined treatment was observed only in CBM cells exposed to topotecan for 24 hr. In the case of H460 cells, interaction varied from a strong infra-additive effect at low radiation doses to a slight supra-additive effect when cells were exposed to radia- tion doses greater than 3 Gy. Northern blot analysis indicated that topoisomerase I expression in H460 cells was 8-fold higher than that of GBM cells. Although the H460 cell line exhibited an increased sensitivity to topotecan, only in the GBM cell line (which expressed a lower level of topoisomerase I) did the drug potentiate the radiation cytotoxicity. The observation that the radiosensitization by topotecan was related to topoisomerase I level is consistent with a putative role of the enzyme in processes involved in the repair of radiation damage. It is conceivable that the modulation of enzyme function results in an effective reduction of cellular capability for repair of radia- tion damage only if the enzyme is not over-expressed. Although a precise role of topoisomerase I in the cellular response to ionizing radiations (in particular, in DNA repair) remains to be documented, such results suggest the potential interest of topoisomerase I inhibitorr in combination with radiation thempy for tumors expressing low topoisomerase I levels. o 1996 Wley-Liss, Inc.

Radiation therapy is an effective trcatmcnt modality for local control of solid tumors. The approach plays an important palliative role in inoperable tumors, including lung cancer and CNS tumors. Radioresistance represents a major limitation to therapeutic efficacy. A variety of agents have been evaluated as modulators to overcome the radioresistance of tumor cells. The combination of radiation therapy with cytotoxic agents is a promising approach to enhance radiosensitivity and may have clinical relevance (Devine et al., 1991). The mechanism of sensitization by cytotoxic drugs has been related to inhibition of repair of potentially lethal damage and/or to accumulation of cells in a radiosensitive phase of the ccll cycle. A number of anti-tumor agents have been examined for interaction with radiation, including alkylating agents, cisplatin, 5-fluorouracil and taxol. Camptothecin analogs have been shown to enhance the lethal effects of ionizing radiation (Boothman et al., 1989, 1992; Mattern et al., 1991; Hennequin et al., 1994; Roffler et al., 1994). Such agents are topoisomerase I inhibitors and exert their cytotoxic effects by stabilizing a transient intermediate complex between enzyme and DNA (cleavable complex), which is formed during the enzyme reaction (Potmesil, 1994). Indirect evidence supports a role for topoisomerase I in DNA repair (Boothman et al., 1989). Cells selected for resistance to camptothecin and defective in topoisomerase 1 are hypersensi- tive to ionizing radiation (Mattern ef al., 1991). Increased expression of topoisomerase I has been found in cisplatin- resistant cell lines (Kotoh et al., 1994; Pratesi et al., 1995).

In the present study, cytotoxic interactions between topote- can, a camptothecin analog, and ionizing radiation were

studied in 2 human tumor cell lines, including a non-small-cell lung cancer (non-SCLC) (H460) and a glioblastoma (GBM line). These cell systems were selected because they are characterized by markedly different expressions of the target enzyme. Our results indicate a synergistic interaction only in the cell line expressing a low level of topoisomerase I, thus supporting a rolc of this enzyme in repair of radiation damage.

MATERIAL AND METHODS Cell lines and drugs

H460 (human lung carcinoma cell line) and GBM (human multiform glioblastoma cell line) (Perego et al., 1994) cells were cultured in monolayer in RPMI 1640 plus 10% heat- inactivated FCS. Topotecan (10-hydroxy-9-dimethylamino- methyl-(S)-camptothecin) is a water-soluble analog of campto- thecin. It was supplied by SmithKline Beecham (King of Prussia, PA) as powder and dissolved in distilled water to obtain a 1 mM stock solution. The solution was stored at 4°C and diluted in saline at the required concentrations before experiments. Treatments

To assess the toxicity of topotecan on H460 and GBM cell lines, 500 cells/dish were seeded in 6-cm-diameter Petri dishes. Twenty-four hours later, cells were exposed to different concentrations of topotecan for different times. At the end of treatments, cells were washed twice with cold Hanks’ balanced salt solution, then complete medium was added and cells were incubated at 37°C in a 5% C 0 2 atmosphere to allow colony formation. After about 10 days, colonies were fixed with methanol, stained with methylene blue and microscopically counted. Control values for untreated cells were about 150 and 100 colonies for H460 and GBM cell lines, respectively.

For combined treatments, topotecan was always given imme- diately before irradiation and maintained in culture medium for different times (30 min, 1 or 24 hr). This schedule was employed since, for optimal radiosensitization by topotecan, the drug is required to be in contact with the cells immediately after irradiation (Mattern et al., 1991). To avoid variability related to time-lapse of drug addition following exposure to radiation, the drug was added immediately before irradiation. Previous experiments indicated that pre-treatment with topo- tecan did not affect radiation cytotoxicity. Topotecan concen- tration ranged from 4 to 500 nM for the H460 cell line and from 16 nM to 10 p,M for the GBM cell line. Radiation exposure was performed with an IBL 437 C (13’Cs) irradiator (ORIS, Gif-sur-Yvette, France) at a dose rate of 9 Gy/min. In each experiment, the surviving fraction was evaluated in comparison to the control cells. All experiments were done in triplicate. At least 2 independent experiments were performed

3T0 whom correspondence and reprint requests should be sent, at Division of Experimental Oncology B, lstituto Nazionale Tumori, Via Venezian 1,20133 Milan, Italy. Fax: (39)2-239-0764.

Received: August 18,1995 and in revised form January 12,1996.

INTERACTION BETWEEN ?-RAYS AND TOPOTECAN

for each point, and data points are mean values obtained by averaging results from each experiment.

Northern blotting analysis Total RNA was purified from exponentially growing cells by

cell lysis with guanidine isothiocyanate and centrifugation in a cesium chloride gradient (Davis et al., 1988). An aliquot of 20 pg total RNA was electrophoresed in a formaldehyde- containing 1% agarose gel and transferred onto a nylon membrane. A 0.7-kb human topoisomerase I cDNA fragment was purified from the plasmid pGEM-CDI, kindly provided by Dr. L. Liu (Piscataway, NJ). A fragment (about 10 kb) of human y-actin cDNA was derived from plasmid pSP64.

DNA probes were 32P-labeled with a random primer kit (Amersham, Aylesbury, UK). Pre-hybridization was carried out for at least 4 hr at 42°C in 50% formamid, 5 x SCC, 5 x Denhardt's solution, 0.2% SDS and 50 pg denaturated salmon sperm. Hybridization was performed for 16-18 hr at 42°C in the same buffer containing 10% dextran sulfate and 32P- labeled DNA probes. Final washes of filters were performed at 50°C for 20 min in 0.5 x SSC and 0.1% SDS. The expression level was evaluated by measuring radioactivity with a phosphor- imager (model 425; Molecular Dynamics, Sunnyvale, CA).

SDS-PAGE and Western blotting Trypsinized cells were washed twice in cold PBS and

pelleted. Cells were lysed on ice in 40 pL/106 cells of sample buffer (0.125 M Tris-HCL [pH 6.81, 5% SDS, 1 mM PMSF, 12.5 pg/ml leupeptin, 10 yg/ml aprotinin and 10 pg/ml pepstatin) and sonicated. Protein concentration was measured with the BCA Protein Assay Reagent (Pierce, Rockford, IL). SDS-PAGE was performed according to the procedure of Laemmli (1970). Different aliquots of cell extracts (12,40 and 120 pg) were run on a 7.5% SDS-polyacrylamide gel and then transferred onto a 0.45-pm nitrocellulose membrane (Bio- Rad, Richmond, CA). Topoisomerase expression was detected by a serum derived from a patient affected by Xeroderma pigmentosum, which is known to contain a specific anti- topoisomerase I antibody. As an internal standard, 30 pl of solution of topoisomerase I purified from P388 cells was used. Reactive bands were revealed by ['251] protein A (Amersham). Normalization was carried out with respect to the expression of actin, which was revealed on the same blot by a rabbit anti-actin antibody. Image quantitation was performed with the phosphor-imager (Molecular Dynamics).

Data processing Results of combined treatments were analyzed according to

the method described by Steel and Peckham (1979). The method allows evaluation of an additivity envelope against which the combined treatment data points are compared. Briefly, points to the left of the envelope imply positive interaction between agents (enhancement effect), points to the right imply negative interaction (inhibition or antagonism) and points within the envelope imply that the agents are acting by independent mechanisms. According to the method suggested by Hennequin et al. (1994), a fractional supra-additivity (or infra-additivity) was then calculated for each value of the cell surviving fraction after combined treatment. The calculated fraction represents, for a selected cumulative radiation dose, the percentage by which the radiation dose should be de- creased (or increased) to obtain the same cell survival follow- ing radiation treatment alone.

343

RESULTS

Cell sensitivity to topotecan was determined by colony- forming assay following different exposure times. IC5,, values were deduced from linear regression analysis of dose-response

FIGURE 1 -Northern blot analysis of topoisomerase I gene expression in H460 and GBM cell lines. Total RNA (20 Fg) was electrophoresed in formaldehyde-containing 1 %-agarose el, trans- ferred to a nylon membrane and h bridized with 32!-labeled human topoisomerase I cDNA probe (pgem-4-DI). The filter was then stripped and rehybridized with a human y-actin probe derived from the plasmid pSPM.

curves. After 24-hr exposure to topotecan, IGo values were 0.02 and 0.038 pM for H460 and GBM cell lines, respectively. A major difference between cell lines was observed in the range of concentrations causing a cell kill greater than 80% (data not shown). In the case of GBM cells, a plateau was found at topotecan concentrations above 0.02 pM, correspond- ing to a residual survival of about 10%. The differential cytotoxic effect of topotecan was more pronounced following a short exposure to the drug. ICso values were 0.4 pM for the H460 cell line (30-min exposure) and 2.4 pM for the GBM cell line (1-hr exposure). Topotecan did not influence colony dimensions in either cell line.

Northern blotting analysis revealed that the levels of topoi- somerase I expression were markedly higher in H460 than in GBM cells (Fig. 1). In 2 independent experiments, the ratio of topoisomerase expression (normalized to y-actin expression) between the 2 cell lines was 7.95 2 2.05. This marked difference was confirmed by Western blot analysis (Fig. 2), indicating a substantially higher (at least 4-fold) level of topoisomerase I protein in H460 than in GBM cells.

In spite of the quite different expressions of topoisomerase I, GBM and H460 cell lines exhibited a comparable response to ionizing radiation. An inverse relationship between radiosen- sitivity and expression of topoisomerase I has been described in cell systems with acquired resistance to camptothecin (Mattern et al., 1991). Such a relation could not be generalized and presumably could not be applied to cells of different tumor origins.

Survival curves of H460 cells following exposure to radiation with or without concomitant exposure to topotecan in different experimental conditions are shown in Figure 3. Exposure to radiation alone at a dose of 4 Gy reduced cell growth to 95%. y-ray-treated cells grew more slowly; thus, colonies were smaller than control colonies. When irradiation was per- formed in the presence of topotecan, the effect of combined treatment was dependent on drug concentration and exposure time. A short exposure time to 50 nM topotecan produced an apparently antagonistic effect (Fig. 3a). In contrast, lower drug concentrations (16 and 25 nM) at a prolonged exposure (24 hr) caused an enhanced killing effect (Fig. 3b). Isobologram analysis of cell survival in combined treatments is shown in Figure 4. Degrees of interaction were similar in the different experimental conditions, varying from infra-additivity at low

344 MARCHESINI ETAL.

40

- Supra addltlvlty (96)

0 7

RCURE 2 - Western blot analysis of topoisomerase I protein in H460 cells (lanes 1-3: 12,40 and 120 kg of rotein, res ectively) and in GBM cells (lanes 4-6: 12, 40 an8120 kg o! protein, respectively). Lane 7: Purified topoisomerase I from P388 cells. An anti-actine antibody was used for normalization and image quanti- tation.

1

:E rn O ' l

' I 0.01 I I

0 1 a 3 4

Radiation dose (Gy)

0.01 I I

0 1 a 3 4

7"j Supra additivity (96) T 1

c

0 1 2 3 4

Radiation dose (Gy)

FIGURE 4 - Isobologram analysis of H460 cell survival in com- bined treatment. (a) Thirty-minute exposure to 100 nM topotecan; (b) 24-hr exposure to 16 nM topotecan; (c) 24-hr exposure to 25 nM topotecan. The solid line was calculated from survival curve parameters. Points, experimental data. Bars: SD.

I

0 1 2 3 4

Radiation dose (Gy)

FIGURE 3 - Radiation survival curves of the H460 cell line with or without (W) combined treatment with topotecan. (a) Thir minute exposure to topotecan at 50 nM (A) and 100 nM (0); zj 24-hr exposure to topotecan at 16 nM (A) and 25 nM (0). Bars: SD.

radiation doses to additivity and then to a slight supra-additive effect at doses greater than 3 Gy.

Figure 5 shows survival curves for the GBM cell line. Following a short (1 hr) exposure (Fig. 5a), a merely additive effect of topotecan and radiation was evidcnt at a concentra-

tion of 2 FM, whereas no interaction was observed at 0.8 kM. Combined treatment with a long exposure to topotecan (24 hr) at subtoxic concentrations (Fig. 56) increased the slope of the survival curve with respect to radiation treatment alone, which suggests, according to the linear-quadratic model, that single lethal events were increased.

Isobologram analysis of GBM cell survival in combined treatments is shown in Figurc 6. With a short exposure (1 hr), the interaction was mainly additive at 2 p M topotecan, whereas at a longer exposure time (24 hr), a fractional supra-additivity of about 20% was apparent, which became more evident when the concentration was increased from 4 to 20 nM.

345 INTERACTION BETWEEN 7- RAYS AND TOPOTECAN

401 Supra additivity (96) I 0

40

Infra additivity (%) 1 0 1 2 3 4 6 0 1 a 3 4 5

T T Supra additivity (%) 1

0.011 ' I . 8 ' I 1 I , I 0 1 a 3 4 5

Radiation dose ( G y )

FIGURE 5 - Radiation survival curves of the GBM cell line with or without (m) combined treatment with topotecan. (a) One-hour exposure to topotecan 0.8 p M (A) and 2 pM (+); (6) 24-hr exposure to topotecan 4 nM (A) and 20 nM (+). Bars: SD.

DISCUSSION

The H460 cell line was more sensitive to topotecan than GBM cells, following a short and a long exposure. ICso values for the GBM cell line were 2 -C 6-fold higher than for the H460 cell line at long and short exposures, respectively. It is noteworthy that the choice of different exposure times (30 min and 1 hr for H460 and GBM, respectively) was based merely on a compromise to treat the 2 cell lines at topotecan concentra- tions resulting in a relatively similar surviving fraction. In these cell systems, the pattern of cell response to topotecan was consistent with the level of expression of the target enzyme. However, differential sensitivity is dependent on time of exposure to the drug and, following a 24-hr exposure, the difference was less marked than expected on the basis of a markedly different expression (8-fold higher in H460 cells) of topoisomerase I and of proliferation rate (doubling time, approximately 22-25 hr and 70 hr for H460 and GBM cells, respectively). Thus, the results confirm previous observations indicating a lack of precise correlations among topoisomerase I gene expression, proliferation rate and cell sensitivity to camptothecin in a variety of human cell lines of different tumor types (Perego et al., 1994b). In addition, our present results indicated that, in spite of the different expressions of topoisom- erase I, GBM and H460 cell lines exhibited a comparable radiosensitivity. Nevertheless, the shoulder on the survival curve, which raises when the repair system is operative, was slightly more marked in the case of H460 cells, suggesting that accumulation of sublethal lesions could occur in H460 cells at a lower dose than in GBM cells. Moreover, since GBM cells had a doubling time longer than H460 cells, it is reasonable to

20 41 Additivity

Infm additivity (%)

0 1 2 3 4 6

I F 4o

Supra additivity (%) T

"1 Additivity I i , , , , , , . , , , , , , , , , , , , , , , ,T"

~~

Infra additivity (%)

0 1 2 3 4 9

Radiation dose (Gy)

FIGURE 6 - Isobologram analysis of GBM cell survival in com- bined treatment. (a) One-hour exposure to 2 pM topotecan; (b) 24-hr exposure to 4 nM topotecan; (c) 24-hr exposure to 20 nM topotecan. The solid line was calculated from survival curve parameters. Points, experimental data. Bars: SD.

expect a different distribution of cell populations in the radiosensitive phases. In contrast to observations with the cell lines used in this study, camptothecin-resistant cell lines characterized by a reduced topoisomerase I content have been reported to be hypersensitive to ionizing radiation (Mattern et al., 1991). The biochemical and molecular basis of differential radiosensitivities among various human cancer cell lines re- mains unclear. Our results could be rationalized in terms of the involvement of multiple factors as determinants of radiosen- sitivity. Cellular ability to repair DNA damage produced by ionizing radiation may play a primaly role in relative radiosen- sitivity.

In spite of the comparable radiosensitivity of GBM and H460 cell lines, a supra-additive interaction between topote-

346 MARCHESIN1 ETAL.

can and radiation could be found in GBM cells, which expressed a low level of topoisomerase I expression and exhibited a reduced sensitivity to topotecan. Supra-additive effects could be detected following a prolonged exposure to the topoisomerase I inhibitor. Short-term exposure to the drug was ineffective in potentiating the cytotoxicity of radiation, at least at radiation doses useful in clinical practice. A plausible explanation for a supra-additive interaction in GBM cells under these conditions may be that only a persistent inhibition of topoisomerase I function results in an effective impairment of cellular capability for repair of potentially lethal damage. In these conditions, a potentiation of radiation-induced cytotoxic- ity could be achieved as a consequence of a low level of topoisomerase I expression. This interpretation is also consis- tent with findings indicating that camptothecin (Beidler and Cheng, 1995) and ionizing radiation (Boothman et al., 1994) induce a post-transcriptional down-regulation of topoisomer- ase I in drug-treated cells. Lack of a synergistic interaction in H460 cells between radiation and drug supports a different cellular capability for repair of potentially lethal damage. It is conceivable that an effective and persistent inhibition of

enzyme activity was not achieved in H460 cells as a conse- quence of the high level of topoisomerase I expression. Additional experiments on enzyme activity and enzyme-DNA complex formation following drug or combined treatment are needed to support this interpretation. In conclusion, if an inverse correlation between topoisomer-

ase I expression and the ability of topotecan to potentiate the lethal effects of radiation is confirmed in other cell systems, topoisomerase I inhibitors could be of potential clinical inter- est as radiosensitizers in the treatment of tumors expressing low levels of the target enzyme.

ACKNOWLEDGEMENTS

This work was partially supported by the Associazione Italiana per la Ricerca sul Cancro, by the Minister0 della Sanita and by the Consiglio Nazionale delle Ricerche (Final- ized Project “Applicazioni Cliniche della Ricerca Onco- logica”). The authors thank Ms. S. Tinelli for purification of murine topoisomerase I and Ms. L. Zanesi, Mr. M. Azzini and Ms. B. Johnston for editorial assistance.

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