Format

Send to

Choose Destination
J Cell Physiol. 1988 Aug;136(2):341-7.

Altered molecular properties of tubulin in a multidrug-resistant variant of Chinese hamster cells selected for resistance to vinca alkaloids.

Author information

1
Memorial Sloan-Kettering Cancer Center, New York, New York 10021.

Abstract

The basis for the markedly altered intracellular binding of [3H]vincristine in a multidrug-resistant variant (DC-3F/VCRd-5L) of Chinese hamster lung cells (DC-3F) was investigated. Binding of [3H]vincristine by protein in cytosol derived from each cell type exhibited a differing requirement for GTP in MgCl2 containing buffer of low-ionic strength. Binding of [3H]vincristine occurred to cytosolic protein derived from both variant and parental DC-3F cells, but after removal of GTP, binding only occurred to cytosolic protein from parental cells regardless of the presence of added GTP. Although binding by cytosolic protein from parental DC-3F cells did not require GTP, the addition of 0.1 mM GTP increased by two-fold the rate and extent of binding. When cytosol from variant and parental DC-3F cells was incubated with low concentrations of [3H]vincristine in high-ionic strength buffer and analyzed by molecular-sieve HPLC, most of the protein binding [3H]vincristine in parentally derived cytosol eluted as Mr 110,000-115,000 daltons, corresponding to that for dimeric tubulin. The same binding species was not detected in cytosol derived from variant cells. However, these same fractions derived with both parental and variant cytosols contained tubulin as shown by SDS-PAGE and immunoblotting. A smaller peak of [3H]vincristine binding and an amount of tubulin equal to that found in later fractions were found in the void volume during the same HPLC elution runs with cytosol from both variant and parental DC-3F cells. Evidence was also obtained for differences between parental and variant DC-3F cells in beta-tubulin isoforms following isoelectric focusing and immunoblotting. Parental-cell cytosol contains a single isoform of beta-tubulin. However, in variant cell cytosol the same isoform and, in addition, three more basic isoforms were found. These alterations in [3H]vincristine binding and in isoform compositions of beta-tubulin in variant versus parental DC-3F cells may have importance in regard to vincristine resistance in DC-3F cells.

PMID:
3045133
DOI:
10.1002/jcp.1041360218
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Wiley
Loading ...
Support Center