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Biomedical subjects

R Clarke

Publications and source records attributed to R Clarke.

At least 235 records · Page 13Linked to original sources
PubMed1986

The effects of Mg2+ ions or EDTA on nuclear integrity and apparent subcellular distribution of unoccupied oestrogen receptors in breast cancer cells.

Homogenisation and fractionation of cells in the presence of Mg2+ or EDTA resulted in unoccupied oestrogen receptor being recovered in the particulate fraction. Nuclei were partially purified by pelleting at 100,000 g through 41% and 44% (w/w) sucrose (in buffer containing Mg2+ or EDTA), plasma membranes being collected from the top of the 41% barrier. In Mg2+-prepared fractions, both 5'-nucleotidase and unoccupied receptor were distributed between plasma membrane, partially-pure nuclei and mitochondrial/microsomal pellets. Lactate dehydrogenase was not a significant contaminant of particulate fractions. In EDTA fractions, the majority of binding activity was in the partially-pure nuclei (which were extensively disrupted) and mitochondrial/microsomal pellets. Little or no binding was found in the EDTA-prepared plasma membranes which were amorphous in appearance. Mg2+-prepared nuclei, freed of membranous contamination by pelleting through 1.8 M sucrose, were intact by electron microscopy but had no 5'-nucleotidase or unoccupied receptor. These data suggest that recovery of receptor in partially-pure nuclei during fractionation is not caused by trapping of cytosolic protein but rather by redistributed nuclear receptor having become bound to adhering plasma membrane fragments during homogenisation. Implications for the study of cell-free systems are discussed.

Breast Neoplasms↗
PubMed1986

Effect of cytotoxic drugs on estrogen receptor expression and response to tamoxifen in MCF-7 cells.

We have examined the effects of a 24-h exposure to clinically achievable concentrations of adriamycin, melphalan, 5-fluorouracil, and vincristine on the estrogen binding capacity of MCF-7 human breast cancer cells using a whole cell binding assay. Adriamycin (0.018 to 1.8 microM), melphalan (0.1 to 5 microM), 5-fluorouracil (0.077 to 15.4 microM), and vincristine (0.01 to 1 nM) reduce the estrogen binding capacity in a dose dependent manner. The rate of protein synthesis is reduced following exposure to 5-fluorouracil but not following exposure to adriamycin, melphalan, or vincristine. The rate of cell proliferation, influx of the ligand, and the Kd of remaining estrogen receptor are unaltered following drug exposure. These drugs may, therefore, be inducing a nonspecific reduction in the rate of receptor recycling and/or synthesis. Vincristine (1 nM) abolished estrogen receptor expression but following removal of the drug receptor levels did not reach that expressed in untreated cells for at least 48 h. Prior exposure to vincristine (1 nM) reduced the antiproliferative effects of tamoxifen (2 microM) toward MCF-7 cells.

Antineoplastic Agents↗
PubMed1985

The effect of the rate of cell proliferation on the synthesis of methotrexate poly-gamma-glutamates in two human breast cancer cell lines.

The synthesis of methotrexate poly-gamma-glutamates by the MDA-MB-436 and MCF-7 human breast cancer cell lines is highly dependent on the rate of cell growth. Slowly proliferating cells accumulate methotrexate to the same extent as rapidly proliferating cells but convert a lower percentage of the drug to polyglutamate forms. The longest polyglutamate-derivatives of methotrexate are generally only synthesized when the cells are doubling rapidly. The MDA-MB-436 cells exhibit a biphasic response of doubling time and polyglutamation to increasing initial cell number. Extremes of cell density are associated with long doubling times and reduced polyglutamate synthesis. MCF-7 cells show increasing doubling time and decreasing polyglutamate synthesis in response to increasing initial cell number.

Breast Neoplasms↗
PubMed1985

The effect of leucovorin on the synthesis of methotrexate poly-gamma-glutamates in the MCF-7 human breast cancer cell line.

The modulating effects of leucovorin on the synthesis of methotrexate (MTX) polyglutamates in the MCF-7 human breast cancer cell line have been investigated using a paired-ion high performance liquid chromatography (HPLC) system. Leucovorin decreased the intracellular level of MTX and profoundly affected polyglutamate synthesis irrespective of whether it was administered with or after MTX. Inhibition of MTX polyglutamate synthesis was also observed when concentrations of leucovorin too low to affect intracellular levels of MTX were employed. Leucovorin did not promote efflux of MTX from the MCF-7 cells and did not affect the distribution of the retained drug amongst the various polyglutamate forms.

Breast Neoplasms↗
PubMed1985

Oestrogen receptor status and the response of human breast cancer cell lines to a combination of methotrexate and 17-beta oestradiol.

We have investigated the modifying influence of 17-beta oestradiol (E2), on the cytotoxicity of methotrexate (MTX) towards two cell lines derived from human breast carcinoma. E2 (10(-7) M-10(-6) M) significantly reduced the antimetabolic effects of the drug towards an E2 non-responsive cell line, MDA-MB-436, whilst potentiating the action of MTX in an E2 responsive line, MCF-7. Similarly, E2 (10(-6) M) partially reversed the anti-proliferative effects of MTX in the MDA-MB-436 line and potentiated growth inhibition in the E2 responsive cells. This potentiation was not observed if E2 was replaced by the less biologically active alpha-isomer. In both cell lines pharmacological concentrations of the E2 reduced intracellular levels of MTX achieved during a 48 h treatment period. The latter finding is consistent with the ability of E2 to protect MDA-MB-436 cells from the action of MTX. Potentiation of the effects of MTX towards MCF-7 cells occurs despite reduced intra-cellular drug levels.

Breast Neoplasms↗
PubMed1985

Growth hormone-releasing factor analogue (hGRF1-29NH2): immunoreactive-GRF plasma levels after intravenous and subcutaneous administration.

A homologous radioimmunoassay (RIA) system for human growth hormone-releasing factor 1-29NH2 (hGRF 1-29-NH2) was developed and applied to the measurement of immunoreactive (IR) concentrations of the peptide in anaesthetized rats to determine some of its pharmaco-kinetics after i.v. and s.c. administration. Analysis of the biphasic disappearance curve of IR-hGRF-1-29NH2 from plasma after i.v. injection (10 micrograms) gave values for the half-lives of the initial distribution phase (alpha) and for the elimination phase (beta) of 1.9 +/- 0.2 min and 10.4 +/- 0.2 min respectively. There was rapid uptake of the peptide from the site of s.c. injection but comparison of areas under the plasma IR-hGRF1-29NH2/time curves showed that the estimated total amount in the circulation after s.c. injection was only 4% of that obtained after i.v. administration. A large degree of degradation of the peptide is indicated either at the site of injection or during transfer to plasma; this susceptibility to rapid breakdown is reflected in the short half-life of the peptide in the circulation. Therefore the measurement of the above parameters is a prerequisite when assessing potency of GRF analogues in-vivo and when implementing suitable dosage regimens for clinical purposes.

Animals↗
PubMed1983

Metallothionein and the development of the mottled disorder in the mouse.

Copper accumulates in kidney tissue of mottled (Mo) mice largely in association with a low MW cytosol protein, and the reduced copper levels in neonatal mutant liver are largely the result of a reduction in the amount of copper associated with this same protein. On the basis of ion-exchange chromatographic profile, heat stability, absence of a 280nm absorption peak, and the binding of Cd109 and Zn65 the protein mutants in the kidney is identified as metallothionein (MT). Amino acid analysis, however, failed to confirm this, and it is suggested that the high copper content of the mutant protein results in its oxidative degradation during purification, even when normal anaerobic precautions are taken. Estimates of thionein protein content of tissues from mutant and normal mice demonstrated that the levels are significantly elevated in both young and adult mutant kidney and depressed in young mutant liver, in parallel therefore with the changes in tissue copper levels. In adult mutant liver tissue, however, thionein levels are significantly raised, even though tissue copper content is normal. The synthesis and degradation of MT was examined in some detail. Incorporation of S35-cysteine in kidney MT was significantly raised in both young and adult mutant mice, while in adult tissue the rate of degradation of MT was significantly depressed. The elevated kidney MT levels arise therefore in young mutant mice from an increased rate of synthesis and in adult mice from the combined effects of increased synthesis and reduced degradation.

Animals↗