Taxol, Radiation, and Oncogenic Transformation1

The novel antineoplastic drug taxol has been shown to be active clini cally against several types of human tumors. With improvement in treat ment strategy and the number of long-term survivors increasing, a ques tion that needs to be addressed is the potential carcinogenic effect of the treatment in the induction of second malignancies. We show here that when tested using an in vitro assay for oncogenic transforming potential, taxol is ineffective in focus induction at doses significantly higher than those used in the clinic. However, taxol enhances the oncogenic potential of -y-rays in a synergistic fashion. The fact that taxol blocks cells at the <.j/M phases of the cell cycle may account for this interaction since (¡,/M are relatively radiosensitive phases of the cell cycle. INTRODUCTION Taxol, a novel antineoplastic drug extracted from the bark of Taxus breviolia, has been shown to be active clinically against various hu man tumors, including advanced ovarian cancer, breast cancer, and malignant melanoma (1-3). The discovery of the possible antitumor effect of taxol can be dated back to the late 1960s, when a crude extract of bark from the western yew tree was shown to be cytotoxic against a range of murine tumors. It had been known since the Middle Ages that the leaves and bark of the yew were toxic to domestic animals. Later studies identified taxol as the active component of the bark extract (4). Schiff et al. (5, 6) first demonstrated that taxol functioned through its ability to stabilize microtubules and promoted microtubule assembly. As a result, the drug can selectively block cells in the G2 and M phases of the cell cycle. Recent studies by Tishler et al. (l, 8) showed that taxol enhanced the radiosensitivity of a grade 3 astrocytoma cell line in a dose-dependent manner. Thereby, this drug shows promise as an adjunct to radiotherapy as well as showing activity alone as a chemotherapy agent. As anticancer therapy improves and the number of long-term sur vivors increases, a question that always arises for a new treatment agent is its potential oncogenicity. To some extent, the development of a second malignancy is the price of success, since a patient must be a long-term survivor, with the original tumor controlled before a second malignancy can become evident. The incidence of second malignan cies induced by radiotherapy and chemotherapy is well documented in patient studies (9, 10). In addition, a variety of in vitro assays of oncogenic transformation have been used to compare the oncogenic potential of the many classes of anticancer agents available. Most known chemotherapy agents can induce malignant transformation in vitro (11, 12). Due to the long latency for cancer induction in humans by either radiation or chemical agents and the large population base required to compare the carcingoenic potential of these compounds, model systems that give quick and meaningful answers are clearly needed. In the present study, the effects of taxol on cell growth kinetics and oncogenic transforming potency, either alone or in com bination with -y-irradiation, were assessed using the mouse C3H 10T1/2 cell system. Received 10/9/92; accepted 2/2/93. The costs of publication of this article were defrayed in pan by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported by National Cancer Institute Grant CAI2536. 2 To whom requests for reprints should be addressed, at Center for Radiological Research, College of Physicians & Surgeons, Columbia University, VC-11-218, 630 West 168th Street, New York, NY 10032. MATERIALS AND METHODS The C3H 10T1/2 mouse embryo fibroblast cell line was used for these studies (13). Cells were maintained in Eagle's basal medium supplemented with 10% heat-inactivated fetal bovine serum (Biofluid, Rockville, MD) and 25 ug/ml gentamycin. Cells were routinely cultured in 75-cm2 flasks and incubated in 5% CO2-air at 37°C.Only cells from passages 8 through 12 were used in these studies. The spontaneous transforming frequency for this batch of cells, expressed as the fraction of dishes with foci, ranged from (0.01 to 0.03. Taxol (NSC 125973) was obtained from the National Cancer Institute drug program by Dr. P. Schiff of the Department of Radiation Oncology at Columbia University, and the drug was made available for the present study through a continuing collaboration. The drug was dissolved in dimethyl sulfoxide at a stock concentration of 1 mm. Working concentrations were prepared fresh from the frozen stock just before use. To determine cytotoxicity, asynchronous C3H 10T1/2 cells growing in 25 cm2 flasks were treated with graded doses of taxol at 37°Cfor 24 h under aerated conditions. A 24-h treatment protocol was chosen for the present studies to correlate with the population doubling time of 10T1/2 cells of —¿ 18-20 h. In experiments where radiation was involved, the drug-treated cultures were irradiated with y-rays from a '"Cs irradiator at an absorbed dose rate of 118 cGy/min. After treatment, the cultures were washed twice with buffered salt solution, trypsinized off the dishes, counted with a Coulter elec tronic counter, and replated in 100-mm-diameter dishes for colony formation. Cultures were incubated for 10-12 days; after that time they were fixed with formaldehyde and stained with Giemsa, and the number of colonies was counted. To determine the effects of taxol on the growth kinetics of C3H 10T1/2 cells, exponentially growing cells were plated at 5 x IO4cells/60-mm-diameter Petri dish in 4 ml of growth medium. After overnight incubation, the cultures were treated with graded doses of taxol for a 24-h period. At the end of the treatment, all cultures were washed twice with buffered salt solution and replenished with fresh medium. For each data point, 3 replicate dishes of cells from each treatment group were trypinized, and the total number of cells per dish was determined. For the transformation assay, taxoland/or radiation-treated cultures were trypsinized and replated in 100-mm-diameter dishes at a density such that approximately 200-400 viable cells would survive the treatments (14, 15). All treated and control cultures were maintained for a total of 6 weeks, and the medium was changed every 10 days. The cultures were then fixed, stained, and scored for type II and III transformed foci as described previously (16, 17). All dishes were coded and scored independently twice. Transformation frequencies were expressed as fractions of dishes with foci. Overall, 6 separate experiments involving a total of 2800 dishes were included in these studies.