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J K Randolph

Publications and source records attributed to J K Randolph.

8 recordsLinked to original sources
PubMed1992

Components of intrinsic drug resistance in the rat hepatoma.

A carcinogen-transformed rat hepatoma cell line (Reuber H-35) was utilized as a model system for investigation of the biochemical factors which may limit the effectiveness of chemotherapy in intrinsically resistant tumors such as hepatocellular carcinoma. Northern blotting demonstrated expression of mRNA coding for the P-170 membrane-glycoprotein associated with the multi-drug resistance phenotype, while Western blotting identified the P-170 glycoprotein in the hepatoma cell membrane. Consistent with these observations, tumor cell sensitivity to the vinca alkaloids, vincristine and vinblastine, to the anthracycline antibiotics, Adriamycin and daunorubicin, and to the demethylepipodophyllotoxin derivative, VM-26, was enhanced by continuous incubation in the presence of the calcium channel antagonist, verapamil. Verapamil produced a minimal change in cell sensitivity to the demethylepipodophyllotoxin derivative, VP-16, and to the aminoacridine, m-AMSA. Relatively high detoxification potential via the glutathione metabolic pathway was also observed in the hepatoma cell. The capacity of topoisomerase II in nuclear extracts from the hepatoma cell to mediate cleavable complex formation stimulated by VM-26, VP-16 and m-AMSA appeared to be at least comparable to, if not greater than that from drug-sensitive HL-60 cells, suggesting that drug resistance may not occur at the level of this enzyme. Consistent with findings in a number of tumor cell lines resistant to antineoplastic drugs, the antiproliferative activity of the topoisomerase II inhibitors VM-26, VP-16 and m-AMSA appeared to be dissociable from the induction of DNA strand breaks, suggesting that such lesions in DNA may fail to fully account for the antiproliferative activity of these agents in the hepatoma cell.

Animals↗
PubMed1990

Biochemical lesions in DNA associated with the antiproliferative effects of mitoxantrone in the hepatoma cell.

The H-35 rat hepatoma cell was markedly more sensitive to the anthracenedione mitoxantrone (IC50, 0.05 microM) than to the anthracycline antibiotics daunorubicin (IC50, 0.5 microM) and Adriamycin (IC50, 2.5 microM). In the rat hepatoma cell, mitoxantrone inhibited DNA and protein syntheses, with minimal effects on RNA synthesis. In contrast to daunorubicin, mitoxantrone induced both DNA strand breaks and DNA-protein cross links. The capacity of mitoxantrone to induce more extensive DNA cleavage than anthracycline antibiotics such as daunorubicin may be related to the sustained cellular retention of mitoxantrone (62% of accumulated drug) as compared to that for daunorubicin (32% of accumulated drug). Protein-associated DNA cleavage is likely to be one of the primary lesions contributing to the antiproliferative activity of mitoxantrone in the hepatoma cell, although marked growth inhibition was observed without corresponding alterations in DNA integrity.

Animals↗
PubMed1990

Precise, easy measurement of glass pipet tips for microinjection or electrophysiology.

We describe a simple procedure to measure precisely the tip diameter of glass micropipets. This procedure can improve the precision of microinjection and some electrophysiological measurements due to their extreme sensitivity to the pipet's exact geometric dimensions. The technique is nondestructive and readily carried out as a brief, extra step in these applications. Digital measurements of the threshold pressures for gas bubbling from (gas filled) pipets submerged in methanol were used to calculate the corresponding inner tip diameters using the LaPlace equation. Direct measurements of the inner tip diameters (from 0.2-5.0 mu) using scanning electron microscopy confirmed the validity of this indirect, simple procedure.

Electrophysiology↗
PubMed1989

Enhanced sensitivity of the rat hepatoma cell to the daunorubicin analogue 4-demethoxydaunorubicin associated with induction of DNA damage.

The H-35 rat hepatoma, a cell line which is relatively resistant to the classical anthracycline antibiotics such as Adriamycin [the concentration of drug which inhibits cell proliferation by 5090 (IC50) = 2.5 microM] and daunorubicin (IC50 of 0.5 microM), is markedly more sensitive to the 4-demethoxydaunorubicin derivative, idarubicin (IC50 of 0.025 microM). In contrast to daunorubicin, which has previously been shown to inhibit hepatoma cell proliferation in the absence of perceptible DNA cleavage, idarubicin induces concentration-dependent DNA damage which may account for its enhanced capacity to inhibit proliferation of the rat hepatoma. Free radical scavengers fail to interfere with inhibition of cell proliferation induced by idarubicin. Damage to the cell membrane or alterations in mitochondrial integrity do not appear to represent components of idarubicin toxicity in this tumor cell line. Inhibition of DNA synthesis by idarubicin parallels inhibition of cell growth; however, sensitivity of DNA synthesis to idarubicin is significantly less than that for cell proliferation (IC50 values of 0.5 microM and 0.025 microM, respectively). It is postulated that the antiproliferative effects of idarubicin in the H-35 rat hepatoma model may be a consequence of alterations in DNA integrity which ultimately result in the inhibition of cellular biosynthetic processes.

Animals↗
PubMed1989

Expression of protein-associated DNA damage in the alkaline elution assay in the absence of enzymatic deproteinization.

In the alkaline elution assay, expression of protein-associated DNA damage induced by topoisomerase II antagonists is facilitated by proteinase K digestion of the drug-stabilized topoisomerase-II-DNA complex. In the absence of this enzymatic deproteinization step, drug-induced DNA strand breaks are masked by the binding of the topoisomerase-II-DNA complex to the synthetic filter from which DNA is eluted subsequent to alkaline denaturation. In this manuscript, we report that as the number of cells lysed on the filter is increased, binding of the topoisomerase-II-DNA complex to the filter is compromised, permitting expression of DNA damage in the absence of enzymatic deproteinization.

Animals↗
PubMed1985

Induction of methotrexate release from rat hepatocytes in suspension by alpha-adrenergic agents: involvement of calcium and metabolic energy.

Hepatocytes in suspension which have accumulated [3H]methotrexate release the antifolate compound into the medium upon exposure to alpha-adrenergic agents. In the presence of metabolic poisons, such as sodium azide, dinitrophenol, or dicumarol, the release of methotrexate is attenuated, indicating that integrity of the cellular metabolic apparatus is required for response to the hormonal stimulus. In the presence of millimolar concentrations of the organic acid, probenecid, release of cellular methotrexate may be reduced (1 mM probenecid) or eliminated (2 mM probenecid), suggesting the involvement of a "membrane carrier." Microtubule poisons such as vincristine, vinblastine, and griseofulvin do not modify epinephrine + isobutyl methyl xanthine (IBMX)-induced release of methotrexate. The involvement of calcium in release of methotrexate from the hepatocyte is substantiated by a dose-dependent response to the calcium ionophore, A23187, in the presence of calcium, with a lack of response in the absence of calcium. These effects of A23187 are not related to inhibition of methotrexate influx. Other putative "calcium antagonists" such as tetracaine, neomycin sulfate, and 3,4,5-trimethoxybenzoic acid [8-(diethylamino)octyl ester], do not interfere with epinephrine + IBMX-induced release of [3H]methotrexate, suggesting that these agents may not be effective probes of calcium flux in the liver cell.

1-Methyl-3-isobutylxanthine↗
PubMed1979

Formation of methotrexate polyglutamates in rat hepatocytes.

Polyglutamate derivatives of [3H]methotrexate (MTX) were detected in freshly isolated rat hepatocytes in suspension within 15 min after exposure to the folate analog. The rate of polyglutamate synthesis remained constant for at least one hr, and the polyglutamate derivatives accounted for an increasing proportion of the intracellular radiolabel with time. After initial exposure to 1 micron [3H]MTX, polyglutamate derivatives of Mtx continued to be synthesized even after the extracellular [3H]-MTX concentration had been reduced 20-fold. Prolonged exposure of hepatocytes in primary culture to 1 micron [3H]MTX resulted in the formation of longer-chain polyglutamate derivatives of MTX. The present studies demonstrate another important biosynthetic capacity of the freshly isolated hepatocyte and suggest the usefulness of this system for studying the mechanism of, and controlling factors in, the synthesis of polyglutamate derivatives of MTX. The ramifications of the formation of MTX polyglutamates on drug cytotoxicity in general and hepatotoxicity in particular are considered.

Animals↗