Stability of drug-induced tubulin rings by fluorescence correlation spectroscopy.
Fluorescence correlation spectroscopy (FCS) was applied to investigate the stability of tubulin rings that result from the interaction of alpha beta-tubulin dimers with three vinca domain-binding peptides--cryptophycin 1, hemiasterlin, and dolastatin 10. These peptides inhibit tubulin polymerization into microtubules and, instead, induce the formation of single-walled tubulin rings of 23.8 nm mean diameter for cryptophycin and 44.6 nm mean diameter for hemiasterlin and dolastatin, as revealed by electron microscopy on micromolar drug-tubulin samples. However, the hydrodynamic diameter and the apparent number of fluorescent particles, determined from analysis of FCS measurements obtained from nanomolar drug-tubulin samples, indicate variation in the stability of the rings depending on the drug and the tubulin concentration. Cryptophycin-tubulin rings appear to be the most stable even with tubulin concentration as low as 1 nM, whereas hemiasterlin-tubulin rings are the least, depolymerizing even at relatively high concentrations (100 nM). In contrast, the dolastatin-tubulin rings demonstrate an intermediate level of stability, depolymerizing significantly only at tubulin concentrations below 10 nM. We also compare the stability results with those of cytotoxicity measurements taken on several cell lines and note a rough correlation between the cytotoxicity of the drugs in cell cultures and the stability of the corresponding drug-induced rings.
Boukari
Hacène
H
Laboratory of Integrative and Medical Biophysics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA. boukarih@mail.nih.gov
Nossal
Ralph
R
Sackett
Dan L
DL
D000818:Animals; D000970:Antineoplastic Agents / Q000633:toxicity*; D002417:Cattle; D047630:Depsipeptides; D019281:Dimerization; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D020128:Inhibitory Concentration 50; D036103:Nanotechnology / Q000379:methods; D009842:Oligopeptides / Q000633:toxicity; D017239:Paclitaxel / Q000633:toxicity; D010456:Peptides, Cyclic / Q000633:toxicity; D011108:Polymers / Q000737:chemistry; D012235:Rhodamines / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014404:Tubulin / Q000737:chemistry* / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014747:Vinblastine / Q000633:toxicity
D000970:Antineoplastic Agents; D047630:Depsipeptides; D005456:Fluorescent Dyes; D009842:Oligopeptides; D010456:Peptides, Cyclic; D011108:Polymers; D012235:Rhodamines; D014404:Tubulin; C088419:cryptophycin; D014747:Vinblastine; C467070:hemiasterlin; C064570:dolastatin 10; D017239:Paclitaxel