Xenografts of human rhabdomyosarcoma (RMS) have been derived that differ in their degree of sensitivity to Vinca alkaloids. Lines Rh12 and Rh18 demonstrated, respectively, high and moderate sensitivity to vincristine (VCR), but showed little responsiveness to vinblastine (VLB) in vivo. Rh18/ VCR-3, a subline of Rh18 selected for resistance to VCR under in situ conditions, was insensitive to further challenge with VCR. Resistance was associated with elimination of the agent in a biphasic manner, whereas sensitivity to VCR corresponded to very prolonged drug retention in sensitive neoplastic tissues. The initial half-times for drug retention in tumors in vivo (t 1 2α) correlated with the degree of sensitivity of tumors to Vinca alkaloids, decreasing t 1 2α being associated with decreased sensitivity. A single binding species was observed when membrane-free supernatant fractions were incubated at 37° for 15 min with 10.4 nM [3H]VCR and analyzed by gel filtration HPLC. The protein eluted with a retention time of 57 min and corresponded to a molecular weight (Mr) of approximately 113,000 daltons, agreeing very closely with the Mr of dimeric tubulin (≈110,000 daltons). Two fractions were collected and eluted on a one-dimensional denaturing gel. Proteins were transferred subsequently to nitrocellulose and probed with an 125I-labeled monoclonal antibody specific for β-tubulins. Only the fraction containing bound [3H]VCR contained tubulin. Estimates for the dissociation constants (Kd) for the binding affinity of VCR and VLB in crude, membrane-free supernatant fractions from RMS xenografts were obtained by computer curve fitting using a mathematical binding model. Data fitted a two-site binding model, with Kd values for the high-affinity site ranging from 61 to 160 nM, and for the low-affinity site, from 42 to 94 μM. At physiologically achievable drug concentrations, the relationship between binding affinity, drug retention and tumor sensitivity was examined further. A close relationship was apparent between the Kd values for VCR in Rh12, Rh18 and Rh18/VCR-3 tumor supernatant fractions and VLB in Rh12 preparations, and 1 2α values for drug retention. Prolonged drug retention correlated with a low binding constant. As 1 2α decreased, binding affinity also decreased, as demonstrated by an increase in the Kd value. Consequently, the tightness of drug binding in tumors also correlated with the degree of sensitivity of the xenografts to Vinca alkaloids. Data strongly support the contention that intrinsic resistance to VLB and acquired resistance to VCR are related to the affinity of drug binding in RMS under the in situ conditions of tumor growth. Small differences in drug concentration and Kd values (<4-fold) have accounted for the loss of drug sensitivity in vivo in these human tumor lines and may relate to the level of drug resistance that is observed clinically.
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