Fernando Cabral - Academia.edu (original) (raw)

Papers by Fernando Cabral

Research paper thumbnail of A Mechanism of Cellular Resistance to Drugs that Interfere with Microtubule Assembly

Annals of The New York Academy of Sciences, 1986

Research paper thumbnail of Purification and characterization of a transformation-dependent protein secreted by cultured murine fibroblasts

Biochemistry, 1981

The major excreted protein (MEP) of transformed mouse fibroblasts has been purified, and monospec... more The major excreted protein (MEP) of transformed mouse fibroblasts has been purified, and monospecific antisera against it have been prepared. Synthesis and secretion of this protein have previously been shown to be stimulated by transformation or treatment with tumor-promoting phorbol esters, but its function is still not known [Gottesman, M. M. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 2767-2771; Gottesman, M. M., & Sobel, M. E. (1980) Cell (Cambridge, Mass.) 19, 449-4551. The purified protein shows charge heterogeneity by twedimensional gel electrophoresis; the major intracellular and extracellular species have a molecular weight of 35 000 and a pZ of 6.8-7.3. The purified secreted protein Support by the Monsanto Corporation during the early phases of this research is gratefully acknowledged. 'Present address: Division of Endocrinology, University of Texas Medical School, Houston, TX 77025.

Research paper thumbnail of Isolation of Chinese Hamster Ovary Cell Mutants Requiring the Continuous Presence of Taxol for Cell Divison

Chinese hamster ovary (CHO) cell mutants resistant to the cytotoxic effects of taxol and requirin... more Chinese hamster ovary (CHO) cell mutants resistant to the cytotoxic effects of taxol and requiring the drug for normal growth were isolated in a single step. One of these mutant cell lines, Tax-18, fails to divide in the absence of taxol; instead, the cells become larger, rounder, flatter, and multinucleated. Analysis by flow cytometry indicates that during taxol deprivation there is an accumulation of cells in G2 + M phase but that the cells are able to leak through the block in the absence of cell division and further increase their DNA content beyond the tetraploid amount. This interpretation is confirmed by karyotype analysis and by time-lapse studies that show cells rounded for mitosis two to five times longer than in wild-type cultures or in Tax-18 cultures grown in taxol. The cells finally attempt to undergo cytokinesis, fail, and spread out again, but as larger cells than before. Tax-18 has a normal growth rate and morphology when grown in taxol even at concentrations three to five times below the selecting concentration of the drug. The cells, however, have increased sensitivity to microtubule-disrupting drugs such as colcemid, griseofulvin, and D20. The mutation for taxol auxotrophy behaves recessively in somatic cell hybridization experiments, and the phenotypic reversion rate is ~10 -5 in a nonmutagenized population. Both ~-and ~-tubulin are present in apparently normal amounts and with normal electrophoretic mobilities on twodimensional gels. The results suggest that Tax-18 lacks a factor necessary for mitosis and that taxol may be able to substitute for this factor.

Research paper thumbnail of Isolation of a Taxol-Resistant Chinese Hamster Ovary Cell Mutant That has an Alteration in alpha -tubulin

Proceedings of The National Academy of Sciences, 1981

Taxol is a plant alkaloid that has antimitotic activity and appears to stabilize microtubules Nat... more Taxol is a plant alkaloid that has antimitotic activity and appears to stabilize microtubules Nature (London) 277, 665-667]. Taxolresistant cells were selected from a population of UV-mutagentreated Chinese hamster ovary cells by a single-step procedure. These mutants have normal morphologies and growth rates but are 2-to 3-fold more resistant to the toxic effects of the drug than the wild-type parent. One out of 20 mutants screened by two-dimensional electrophoresis for chemical alterations in tubulin had an "extra" spot with a more acidic isoelectric point than a-tubulin. This extra spot was shown to be an electrophoretic variant a-tubulin by its copurification with tubulin in crude microtubule-containing preparations and by one-dimensional peptide mapping. The a-tubulin mutant was found to be temperature sensitive for growth, and this property was used as the basis for the selection of revertants. Seventeen temperature-resistant revertants of the a-tubulin mutant were selected for their ability to grow at 400C and three of these revertants were found to have simultaneously lost their taxol resistance and the electrophoretic variant ei-tubulin. These results provide evidence that an alteration in a-tubulin can confer taxol resistance on a mammalian cell line and suggest that a-tubulin is essential for cell viability.

Research paper thumbnail of A Mechanism of Cellular Resistance to Drugs that Interfere with Microtubule Assembly

Annals of The New York Academy of Sciences, 1986

Research paper thumbnail of Purification and characterization of a transformation-dependent protein secreted by cultured murine fibroblasts

Biochemistry, 1981

The major excreted protein (MEP) of transformed mouse fibroblasts has been purified, and monospec... more The major excreted protein (MEP) of transformed mouse fibroblasts has been purified, and monospecific antisera against it have been prepared. Synthesis and secretion of this protein have previously been shown to be stimulated by transformation or treatment with tumor-promoting phorbol esters, but its function is still not known [Gottesman, M. M. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 2767-2771; Gottesman, M. M., & Sobel, M. E. (1980) Cell (Cambridge, Mass.) 19, 449-4551. The purified protein shows charge heterogeneity by twedimensional gel electrophoresis; the major intracellular and extracellular species have a molecular weight of 35 000 and a pZ of 6.8-7.3. The purified secreted protein Support by the Monsanto Corporation during the early phases of this research is gratefully acknowledged. 'Present address: Division of Endocrinology, University of Texas Medical School, Houston, TX 77025.

Research paper thumbnail of Isolation of Chinese Hamster Ovary Cell Mutants Requiring the Continuous Presence of Taxol for Cell Divison

Chinese hamster ovary (CHO) cell mutants resistant to the cytotoxic effects of taxol and requirin... more Chinese hamster ovary (CHO) cell mutants resistant to the cytotoxic effects of taxol and requiring the drug for normal growth were isolated in a single step. One of these mutant cell lines, Tax-18, fails to divide in the absence of taxol; instead, the cells become larger, rounder, flatter, and multinucleated. Analysis by flow cytometry indicates that during taxol deprivation there is an accumulation of cells in G2 + M phase but that the cells are able to leak through the block in the absence of cell division and further increase their DNA content beyond the tetraploid amount. This interpretation is confirmed by karyotype analysis and by time-lapse studies that show cells rounded for mitosis two to five times longer than in wild-type cultures or in Tax-18 cultures grown in taxol. The cells finally attempt to undergo cytokinesis, fail, and spread out again, but as larger cells than before. Tax-18 has a normal growth rate and morphology when grown in taxol even at concentrations three to five times below the selecting concentration of the drug. The cells, however, have increased sensitivity to microtubule-disrupting drugs such as colcemid, griseofulvin, and D20. The mutation for taxol auxotrophy behaves recessively in somatic cell hybridization experiments, and the phenotypic reversion rate is ~10 -5 in a nonmutagenized population. Both ~-and ~-tubulin are present in apparently normal amounts and with normal electrophoretic mobilities on twodimensional gels. The results suggest that Tax-18 lacks a factor necessary for mitosis and that taxol may be able to substitute for this factor.

Research paper thumbnail of Isolation of a Taxol-Resistant Chinese Hamster Ovary Cell Mutant That has an Alteration in alpha -tubulin

Proceedings of The National Academy of Sciences, 1981

Taxol is a plant alkaloid that has antimitotic activity and appears to stabilize microtubules Nat... more Taxol is a plant alkaloid that has antimitotic activity and appears to stabilize microtubules Nature (London) 277, 665-667]. Taxolresistant cells were selected from a population of UV-mutagentreated Chinese hamster ovary cells by a single-step procedure. These mutants have normal morphologies and growth rates but are 2-to 3-fold more resistant to the toxic effects of the drug than the wild-type parent. One out of 20 mutants screened by two-dimensional electrophoresis for chemical alterations in tubulin had an "extra" spot with a more acidic isoelectric point than a-tubulin. This extra spot was shown to be an electrophoretic variant a-tubulin by its copurification with tubulin in crude microtubule-containing preparations and by one-dimensional peptide mapping. The a-tubulin mutant was found to be temperature sensitive for growth, and this property was used as the basis for the selection of revertants. Seventeen temperature-resistant revertants of the a-tubulin mutant were selected for their ability to grow at 400C and three of these revertants were found to have simultaneously lost their taxol resistance and the electrophoretic variant ei-tubulin. These results provide evidence that an alteration in a-tubulin can confer taxol resistance on a mammalian cell line and suggest that a-tubulin is essential for cell viability.