Mechanism of Sensitizaron of Chinese Hamster Ovary Cells to Melphalan by Hypoxie Treatment with Misonidazole1

Misonidazole (MISO) has previously been shown to enhance the antitumor effect of many chemotherapeutic drugs, in partic ular the bifunctional alkylating agents, in vivo without similarly increasing normal tissue toxicity. The presence of hypoxic cells in solid tumors may account for the preferential effect, since chemosensitization in vitro only occurs when cells are exposed to MISO under hypoxic conditions, i.e., conditions where reduc tion of MISO and ultimately toxicity can occur. Mechanisms for the enhancement of melphalan (L-PAM) toxicity were investi gated in vitro using Chinese hamster ovary suspension culture cells. A 2-hr hypoxic treatment with 5 mw MISO, which is not toxic itself, sensitized the cells to both a subsequent or previous 1-hr L-PAM exposure under aerated conditions. The resultant dose reduction factors at the 1% survival level were 4.0 when the MISO treatment was given immediately before L-PAM and 1.9 when MISO was given after L-PAM. Although depletion of glutathione (GSH) occurs during hypoxic pretreatments with MISO, simulating the same magnitude of GSH depletion with the GSH depleter, diethylmaleate, gave a dose reduction factor of only 1.4, and GSH depletion would not be expected to contribute to the sensitization which occurs when MISO is given after LPAM. In addition to the role of GSH depletion, the following chemosensitization mechanisms were investigated: (a) increased drug uptake and binding; and (b) increased formation of DMA interstrand cross-links which are believed to represent the lethal lesion for bifunctional alkylating agents. L-PAM binding studies indicated that the total binding of [14C]L-PAM to cellular macromolecules was increased by a factor of 1.4 in MISO-pretreated and diethylmaleate-pretreated cells having the same GSH defi ciency. Although the increase in binding was sufficient to account for the sensitization to L-PAM when GSH is depleted with diethylmaleate, the results of the binding study eliminate the possibility of increased drug uptake or binding accounting for more than a small component (1.4 versus 4.0) of the chemosen sitization effect achieved with MISO pretreatment. The technique of alkaline elution was used to study the effects of MISO pre treatment on DNA cross-linking. Kinetic studies indicated that DNA-DNA cross-links were maximal at 12 hr after L-PAM treat ment and that MISO pretreatment had no effect on the rate of removal of cross-links. The major effect of MISO was to increase the yield of cross-links at any given dose of L-PAM, resulting in a dose-response curve for cross-links with a slope 3.9 times steeper than that of the control. We conclude that enhanced efficiency of DNA cross-link formation can account, in a quanti tative manner, for the sensitization to L-PAM by prior treatment with MISO in vitro. INTRODUCTION MISO,3 a 2-nitroimidazole compound currently in clinical trials as an oxygen-mimetic radiosensitizer of hypoxic tumor cells (7, 13,49), has been shown to enhance the effectiveness of L-PAM, cyclophosphamide, and several nitrosoureas (1,3-bis(2-chloroethyl)-1 -nitrosourea and 1-(2-chloroethyl)-3-cyclohexyl-1 -nitrosourea) against murine solid tumors without similar increases in normal tissue toxicity (1, 6, 15, 21, 32, 34, 35). Although pharmacokinetic effects (39, 50) and alterations in the repair of potentially lethal damage (20, 21, 34) have been implicated in this MISO-induced chemosensitization in vivo under some con ditions, it is unlikely that such mechanisms can account for all of the data (16, 32). One possible explanation for the preferential effect of MISO in solid tumors when a therapeutic gain is noted is the presence of hypoxic cells, since it has been shown that pretreatment of cells with MISO under hypoxic conditions in vitro increases their sensitivity to several chemotherapeutic agents, while similar pretreatments under aerated conditions do not (29, 37). Also, it appears that only a transient period of hypoxia is necessary, since treatment with the second agent need not be under hypoxic conditions, and the chemosensitization effect remains for up to 6 hr after MISO is removed and oxygen is introduced (30, 36, 37, 40). Prolonged exposure of mammalian cells to nitroimidazole com pounds such as MISO under hypoxic conditions ultimately leads to cell death (13). The preferential toxicity of MISO in hypoxic mammalian cells appears to be related to the O2-sensitive reduc tion of the nitro group (41) and its associated effects in the cell, e.g., the accumulation and macromolecular binding of reduced MISO metabolites (45), GSH depletion (3, 47), alteration of glycolysis (5, 46), alteration in cell membranes (31), and a timedependent decrease in O2 utilization rates (43). It is likely that some of these events leading to the expression of MISO cytotoxicity are responsible for rendering the cell more sensitive to chemotherapeutic drugs in vitro, since chemosensitization is noted for the same treatment conditions but usually precedes the expression of overt toxicity (29, 36, 37). The lethal action of many bifunctional alkylating agents is believed to be a consequence of their ability to form DNA interstrand cross-links (8, 9, 51, 53). Although the mechanistic details and reaction rates vary for different drugs, cross-link formation appears to involve 2 steps: an initial alkylation of a nucleophilic site on one strand of DNA; and a slower second reaction with the opposite strand that can occur in the absence of free drug (12, 17). Cross-link repair also appears to involve 2 1This work was supported by Research Grant CA 15201 and Training Grant CA 09302 from the United States National Cancer Institute. 2To whom requests for reprints should be addressed. Received November 1,1982; accepted April 11,1983. 3The abbreviations used are: MISO.misonidazole; L-PAM, melphalan (L-phenylalanine mustard); GSH, reduced glutathione; DEM,, diethylmaleate; CHO, Chinese hamster ovary; TCA, trichloroacetic acid; PBS, phosphate-buffered saline (0.15 M NaCI-0.7 mm KH2PO«-4.3rnw K2HPO«); SDS, sodium dodecyl sulfate; CF, cross link factor.