In Situ Selection of a Human Rhabdomyosarcoma Resistant to Vincristine with Altered β-Tubulins¹

In order to simulate more closely conditions in which resistance to vincristine (VCR) is selected in human solid tumors, a human rhabdomyosarcoma grown as a xenograft in immune-deprived mice has been selected for resistance in situ. Karyotype analysis showed the resistant line, HxRh18/VCR-3, to have a diploid modal number, with no apparent translocations, whereas the predominant population in the parental, sensitive HxRh18 xenograft demonstrated a modal number near-tetraploid with many marker chromosomes. From the rapid rate at which resistance was selected and from karyotypic evidence, data strongly suggest that HxRh18/VCR-3 was a subpopulation within the parent tumor. When grown in the same host, HxRh18/VCR-3 tumors accumulated less drug, and the rate of [³H]VCR loss was 5-fold greater than in HxRh18 tumors. Thus, accumulation and retention of [³H]VCR in HxRh18/VCR-3 resistant tumors was identical to that of [³H]vinblastine (VLB) in HxRh18 xenografts. HxRh18 xenografts are intrinsically resistant to VLB. Analysis by high-performance liquid chromatography of [³H]VCR:protein complexes in HxRh18 cytosols indicated one binding species (M_r 95,000 to 116,000), probably the tubulin heterodimer. Of interest was the observation that β-tubulin species, identified on Western blots by monoclonal antibody, differed in these tumors. In HxRh18/VCR-3, less acidic β-tubulins of HxRh18 were decreased or absent, with three additional more acidic isoforms present in the resistant line. As vincristine may bind to the β-subunit of tubulin, this may have importance to vincristine resistance in vivo.