Markedly decreased binding of vincristine to tubulin in vinca alkaloid-resistant Chinese hamster cells is associated with selective overexpression of alpha and beta tubulin isoforms.
Vinca alkaloids are among a number of cytotoxic agents which target tumor cell microtubules. Studies described herein document the basis for one form of acquired resistance to these plant alkaloids involving an alteration of tubulin in a variant (DC-3F/VCRd-5L) of DC-3F Chinese hamster cells. Our results revealed a markedly decreased binding of [(3)H]vincristine (VCR) to tubulin extracted from this variant compared to tubulin extracted from wild-type DC-3F cells. This was quantitated as a 10- to 15-fold decrease in on-rate in the presence of GTP for the [(3)H]VCR associating with tubulin in cell-free cytosol and a 10-fold increase in off-rate for GTP-dependent dissociation of the [(3)H]VCR-tubulin complex. Quantitative RT-PCR and nucleotide sequencing of poly(A)(+) RNA also carried out with variant and wild-type DC-3F cells documented a different pattern of relative expression, but no base pair differences in the open reading frame of the three alpha and beta tubulin isoforms detected in each cell type. This was accounted for by selective overexpression of one alpha tubulin (alphaII) and two beta tubulin (betaI and betaIV) isoforms in the variant cells. These results would appear to provide an underlying basis for the large decrease in [(3)H]VCR binding by tubulin in these variant Chinese hamster cells and a major component of their acquired resistance to this vinca alkaloid.
Sirotnak
F M
FM
Program of Molecular Pharmacology and Experimental Therapeutics, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA. sirotnaf@mskcc.org
Danenberg
K D
KD
Chen
J
J
Fritz
F
F
Danenberg
P V
PV
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D006224:Cricetinae; D017931:DNA Primers / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics; D015870:Gene Expression; D007700:Kinetics; D011485:Protein Binding; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014404:Tubulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014748:Vinca Alkaloids / Q000494:pharmacology*; D014750:Vincristine / Q000378:metabolism*
D017931:DNA Primers; D020033:Protein Isoforms; D014404:Tubulin; D014748:Vinca Alkaloids; D014750:Vincristine