QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Barlow, S. B. Articles by Cabral, F. Search for Related Content PubMed PubMed Citation Articles by Barlow, S. B. Articles by Cabral, F.

Paclitaxel-dependent mutants have severely reduced microtubule assembly and reduced tubulin synthesis

Steven B. Barlow^*, Manuel L. Gonzalez-Garay and Fernando Cabral

Department of Integrative Biology and Pharmacology, University of Texas Medical School, Houston, Texas 77225, USA
^* Present address: Department of Biology, San Diego State University, 5500 Campanile Drive, San Diego, CA 92182-4614, USA
Present address: Lexicon Genetics Inc., 4000 Research Forest Drive, The Woodlands, TX 77381, USA

View larger version (106K): [in a new window]   Fig. 1. Sensitivity of wild-type and mutant cell lines to paclitaxel and colcemid. Approximately 200 cells were added to replicate wells of a 24-well dish containing the indicated concentrations of paclitaxel (A) or colcemid (B) in ng/ml. The cells were allowed to grow for 7 days and then stained with methylene blue. Note that compared with wild-type (WT), mutant Tax 5-6 is resistant to paclitaxel but exhibits enhanced sensitivity to colcemid. In contrast, CV 2-8 is resistant to colcemid but more sensitive to paclitaxel. Tax 2-4 is dependent on paclitaxel for growth, while Tax 9-5 is only partially paclitaxel dependent. Strain 6H2 is a revertant of a colcemid-resistant cell line and has regained normal colcemid sensitivity.

View larger version (102K): [in a new window]   Fig. 2. Morphology of Tax 5-6 (A), Tax 9-5 (B) and Tax 18 (C) in the absence of paclitaxel. Note that Tax 5-6 cells grow normally without drug, whereas Tax 18 cells become large and multinucleated after 2 days without paclitaxel. Tax 9-5 has an intermediate phenotype with both normal (arrowhead) and abnormal (arrow) cells following 2 days of drug deprivation. Bar, 25 µm.

View larger version (62K): [in a new window]   Fig. 3. Paclitaxel sensitivity of Tax 18 and its revertant R3D. Paclitaxel concentrations are in ng/ml. Note that Tax 18 is paclitaxel dependent for growth. R3D was isolated for loss of the paclitaxel-dependent phenotype but retained significant paclitaxel resistance.

View larger version (17K): [in a new window]   Fig. 4. Effect of paclitaxel on steady state tubulin accumulation. Cells were incubated overnight (16-24 hours) in the presence of [3H]methionine and the indicated concentrations of paclitaxel to measure the amount of protein that accumulated. Following lysis in SDS, the cellular contents were mixed with a constant volume of [35S]methionine-labeled wild-type CHO extract, precipitated with acetone, and resolubilized in urea sample buffer for 2D gel analysis. Spots representing ß-tubulin and actin were excised from the gels, solubilized, and analyzed by liquid scintillation counting to determine their 3H/35S ratios. Each isotope ratio for tubulin was normalized by dividing by the isotope ratio for actin in the same sample, and the resulting values were expressed relative to untreated wild-type (WT) cells set at 100% (A) or relative to the zero concentration control for each cell line (B). The values represent the mean from 3-8 independent experiments. Representative standard deviations are shown only for WT and Tax 2-4. The inset in B shows an enlargement of the lower paclitaxel concentrations for WT, CV 2-8 and Tax 5-6.

View larger version (32K): [in a new window]   Fig. 5. Quantification of mRNA for - and ß-tubulin. Total RNA was isolated and hybridized to 32P-labeled antisense probes to -tubulin or ß-tubulin. A second antisense riboprobe to the gene encoding glyceraldehyde-phosphate dehydrogenase (GAPDH) was included in each reaction as an internal control. Following nuclease digestion, the products were separated on a polyacrylamide gel and the protected fragments were identified and quantified on a Storm phosphoimager. The results were also verified by liquid scintillation counting of bands excised from the gel. Relative amounts of tubulin message were calculated by dividing the radioactivity in the tubulin band by the radioactivity in the GAPDH band, and then expressing the results relative to WT cells set at 100%.

View larger version (28K): [in a new window]   Fig. 6. Mechanism of resistance to antimitotic drugs. Wild-type CHO cells have approximately 38% of their total tubulin in the microtubule fraction (Table 1). Alterations in tubulin that increase microtubule stability lead to increased assembly and resistance to drugs such as colcemid (Cmd) that act to destabilize microtubules. Conversely, alterations that decrease microtubule stability lead to decreased assembly and resistance to drugs such as paclitaxel (Ptx) that act to stabilize microtubules. Alterations that stabilize or destabilize microtubules too much lead to conditional lethal phenotypes such as colcemid-dependence (CmdD) or paclitaxel-dependence (PtxD), respectively. Cells with a borderline dependence phenotype (Cmd±D and Ptx±D) define the range of assembly within which microtubules function sufficiently well to allow normal cell growth. The different number of microtubules in each cell is meant to reflect qualitatively the observation that microtubule assembly increases in colcemid-resistant cells but decreases in paclitaxel-resistant cells.