Search PubMed⌕ Search

Biomedical subjects

J K Randolph

Publications and source records attributed to J K Randolph.

At least 19 recordsLinked to original sources
PubMed1998

Induction of DNA damage, inhibition of DNA synthesis, and suppression of c-myc expression by the topoisomerase I inhibitor, camptothecin, in MCF-7 human breast tumor cells.

Previous work from this laboratory has demonstrated an association between the suppression of c-myc expression and the antiproliferative activity of both topoisomerase II inhibitors and ionizing radiation in MCF-7 breast tumor cells. These findings suggested that suppression of c-myc expression could be related to the induction of DNA damage in this cell line. The present studies were designed to determine whether the inhibition of topoisomerase I (and the consequent induction of DNA strand breaks) would also result in the suppression of c-myc expression. At camptothecin concentrations of 1 microM and below, there was no detectable damage (single- or double-strand breaks) in bulk DNA or suppression of c-myc expression. At camptothecin concentrations of 5, 10, and 25 microM, where suppression of c-myc expression was observed, strand breaks in bulk DNA were also detected. These findings are consistent with the idea that suppression of c-myc expression could be a component of the DNA damage response pathway in MCF-7 breast tumor cells. In contrast to the absence of detectable damage to bulk DNA or suppression of c-myc expression at the lower concentrations of camptothecin, DNA synthesis was inhibited over the entire range of drug concentrations and demonstrated a strong correspondence with growth inhibition. These observations support the concept that growth inhibition of MCF-7 cells by camptothecin is closely related to the early suppression of DNA synthesis.

Antineoplastic Agents, Phytogenic↗
PubMed1998

Induction of DNA damage, inhibition of DNA synthesis and suppression of c-myc expression by the anthracycline analog, idarubicin (4-demethoxy-daunorubicin) in the MCF-7 breast tumor cell line.

PURPOSE: Studies were designed to elucidate the basis for the antiproliferative activity of the anthracycline antibiotic, idarubicin (4-demethoxy-daunorubicin) in MCF-7 breast tumor cells. METHODS: Growth inhibition was evaluated using the MTT tetrazolium dye assay, induction of DNA strand breaks was determined by alkaline elution, inhibition of DNA synthesis was assessed by measuring the incorporation of labelled thymidine into DNA, modulation of the expression of the c-myc oncogene was determined by Northern blotting and the induction of apoptosis was evaluated by alkaline unwinding, static field gel electrophoresis, terminal end labelling and assessment of cell morphology. RESULTS: MCF-7 cells were relatively sensitive to idarubicin, with an IC50 value for growth inhibition of approximately 0.01 microM. While DNA strand breakage was not evident below a concentration of 0.1 microM idarubicin, where growth inhibition exceeded 70%, both the inhibition of DNA synthesis and suppression of c-myc expression closely paralleled the profile of antiproliferative activity for idarubicin. Finally, while exposure to idarubicin resulted in a substantial loss of viable cells within 48-72 h, there was no morphological evidence of apoptotic body formation. The absence of apoptosis in cells exposed to idarubicin was supported by studies demonstrating the absence of DNA fragmentation using gel electrophoresis, alkaline elution and in situ DNA end-labelling assays. CONCLUSIONS: The results of these studies extend previous results from this laboratory indicating an association between suppression of c-myc expression, inhibition of DNA synthesis and growth arrest by topoisomerase II inhibitors, as well as the lack of induction of apoptotic cell death by topoisomerase II inhibitors in MCF-7 breast tumor cells.

Antibiotics, Antineoplastic↗
PubMed1997

Influence of ionizing radiation on proliferation, c-myc expression and the induction of apoptotic cell death in two breast tumour cell lines differing in p53 status.

PURPOSE: To determine the capacity of ionizing radiation to inhibit proliferation, to suppress c-myc expression and to induce apoptotic cell death in the p53 wild-type MCF-7 cell line and the p53 mutated MDA-MB231 cell line. MATERIALS AND METHODS: Growth inhibition and cell killing were determined by cell number and trypan blue exclusion. Apoptosis was assessed through cell morphology and fluorescent end-labelling. c-myc expression was monitored by Northern blotting. RESULTS: Inhibition of cell proliferation by ionizing radiation was similar in both cell lines. MDA-MB231 cells accumulated in G2 while MCF-7 cells accumulated in both the G1 and G2 phases of the cell cycle after irradiation. There was no evidence of apoptosis in either cell line. In MCF-7 cells, growth inhibition correlated closely with an early dose-dependent suppression of c-myc expression; in MDA-MB231 cells, there was no correspondence between growth inhibition and a transient, dose-independent reduction in c-myc message. CONCLUSIONS: These findings suggest that in the absence of classical apoptotic cell death, radiosensitivity is not predictably related to the p53 status of the cell. While both p53 and c-myc may be linked to the DNA damage response pathway, neither p53 nor c-myc are essential for growth arrest in response to ionizing radiation.

Apoptosis↗
PubMed1997

Ionizing radiation and teniposide increase p21(waf1/cip1) and promote Rb dephosphorylation but fail to suppress E2F activity in MCF-7 breast tumor cells.

Ionizing radiation and the topoisomerase II inhibitor, teniposide (VM-26) both increase levels of the cyclin dependent kinase inhibitor, p21(waf1/cip1) and promote dephosphorylation of the retinoblastoma tumor suppressor protein, Rb, in MCF-7 breast tumor cells, perturbations associated with suppression of the activity of the transcription factor, E2F. However, studies using an E2F binding site-luciferase reporter plasmid transfected into MCF-7 cells failed to demonstrate a reduction in E2F activity in response to VM-26 or to ionizing radiation. In contrast, E2F activity (both basal and E1A stimulated) could be suppressed by transfection with a plasmid expressing Rb, indicating that the capacity of E2F to bind to Rb and to be inactivated by Rb is functionally intact in MCF-7 cells. These findings in MCF-7 breast tumor cells suggest that E2F activity may not be directly susceptible to modulation by endogenous p21(waf1/cip1) and Rb.

Antineoplastic Agents, Phytogenic↗
PubMed1996

Growth arrest and non-apoptotic cell death associated with the suppression of c-myc expression in MCF-7 breast tumor cells following acute exposure to doxorubicin.

In the MCF-7 human breast [correction of beast] adenocarcinoma cell line, acute exposure to 1 muM doxorubicin inhibited cell proliferation by approximately 75%. Analysis of cell cycle distribution indicated that within 24 hr, the G(2)/M fraction increased more than 3-fold and the S-phase population declined by >50%. In addition to growth arrest, there was an approximately 40% reduction in the viable cell population after 72 hr. Gel electrophoretic resolution of low molecular weight DNA immediately after exposure of cells to doxorubicin failed to demonstrate "laddered" oligonucleosomal profiles associated with apoptosis. The absence of intracellular DNA fragments or release of fragmented DNA into the incubation medium was confirmed by spectrofluorophotometry over a 72 hr interval following exposure of cells to 1 muM doxorubicin. In addition, there was no evidence of the morphological features associated with apoptosis during this period. Acute exposure to 1 muM doxorubicin also produced a transient increase in c-myc message expression (within the first hour) followed by a decline to 70% of control levels within 2-4 hr. The reduction in c-myc mRNA levels was concentration dependent and corresponded closely with growth arrest (as well as with inhibition of DNA synthesis). These findings (as well as similar reports demonstrating a correspondence between reduced c-myc expression and growth inhibition by VM-26 and m-AMSA in MCF-7 cells) suggest that the down-regulation of c-myc expression may reflect perturbations in regulatory processes contributing to growth arrest in MCF-7 cells exposed to topoisomerase II inhibitors.

Breast Neoplasms↗
PubMed1995

Transcriptional down-regulation of c-myc expression in the MCF-7 breast tumor cell line by the topoisomerase II inhibitor, VM-26.

In the MCF-7 human breast tumor cell line, the topoisomerase II inhibitor, VM-26, produces a concentration dependent reduction in expression of the oncogene c-myc which parallels growth inhibition. Down-regulation of c-myc expression was examined at transcriptional and post-transcriptional levels. VM-26, at 10 microM, produced a reduction in the transcription rate of both sense and antisense strands of c-myc as determined by nuclear run-off analysis. In contrast, in the presence of the RNA synthesis inhibitor, actinomycin D, VM-26 failed to alter the half-life of the c-myc message. The capacity of VM-26 to reduce c-myc expression was not abrogated in cells pretreated with the protein synthesis inhibitor, cycloheximide (despite superinduction of c-myc expression in both control and VM-26 treated cells); this observation suggests that de novo protein synthesis may not be required to mediate the effects of VM-26 on steady state c-myc transcript levels. An extended analysis of the time course of c-myc expression demonstrated that the decline of steady state c-myc mRNA levels induced by VM-26 was biphasic, 6 h after the initial reduction in c-myc expression to approx. 30% of control levels, c-myc levels rebounded to 70% of control; after 24 h, c-myc expression declined gradually and remained at depressed levels (40% of control) at 48 and 72 h. These observations suggest that the initial transient reduction in c-myc expression associated with inhibition of transcription may represent a component of an early signalling pathway leading to growth arrest in MCF-7 breast tumor cells exposed to VM-26.

Breast Neoplasms↗
PubMed1994

Antagonism between tamoxifen and doxorubicin in the MCF-7 human breast tumor cell line.

Tamoxifen, an antiestrogen, and doxorubicin, an anthracycline antibiotic, are each utilized alone and in combination in the treatment of breast carcinoma. In view of conflicting reports relating to the interaction between these drugs, studies were undertaken to characterize the influence of tamoxifen on growth inhibition by doxorubicin in the MCF-7 breast tumor cell line in vitro. Studies combining 5 microM tamoxifen, a clinically relevant concentration, with various concentrations of doxorubicin, indicated that this drug combination produces antiproliferative effects that appear to be less than additive. Concentration-dependent growth inhibition was analyzed further using various concentrations of tamoxifen and doxorubicin by the combination index-isobologram method; this quantitative approach provided clear evidence of antagonism between these agents, a finding with potential relevance to the treatment of breast cancer.

Breast Neoplasms↗
PubMed1994

Influence of amsacrine (m-AMSA) on bulk and gene-specific DNA damage and c-myc expression in MCF-7 breast tumor cells.

In the MCF-7 human breast tumor cell line, the aminoacridine, m-AMSA, induces protein-associated DNA strand breaks consistent with inhibition of topoisomerase II. However, neither single-strand nor double-strand breaks in DNA, determined using conventional assays, show a consistent relationship with m-AMSA-induced inhibition of growth. In contrast, when DNA strand breaks are determined by alkaline unwinding under the high salt conditions of the alkaline unwinding/Southern blotting (AU/SB) assay, developed by our laboratories, damage to DNA corresponds closely with growth inhibition. The AU/SB assay, which is capable of assessing breaks within large-scale domains (upwards of 1 megabase) surrounding genes of interest, was further utilized to explore the capacity of m-AMSA to induce damage within specific genomic regions that may regulate cell growth. Regions encompassing the transcriptionally active oncogenes, c-myc and c-fos, were found to be more susceptible to m-AMSA-induced strand breaks than the region encompassing the non-transcribed alpha-satellite DNA or the genome as a whole (bulk DNA). These findings demonstrate that m-AMSA may produce more pronounced damage within specific genomic regions than in bulk DNA, m-AMSA also preferentially altered expression of the c-myc oncogene; at an m-AMSA concentration where growth was inhibited by between 70 and 80%, steady-state c-myc mRNA levels declined to approximately 10-15% of control levels within 2-3 hr; furthermore, concentration-dependent reductions in c-myc expression appeared to coincide with growth inhibition. In addition, inhibition of [3H]thymidine incorporation after 2 hr directly paralleled inhibition of growth, suggesting an early effect at the level of DNA biosynthesis, possibly related to the down-regulation of c-myc expression. It is proposed that specific lesions, e.g., in regions surrounding the c-myc gene, as well as generalized lesions in DNA may lead to growth inhibition mediated by down-regulation of the expression of select growth regulatory genes, such as c-myc.

Amsacrine↗
PubMed1994

Induction of differentiation and growth arrest associated with nascent (nonoligosomal) DNA fragmentation and reduced c-myc expression in MCF-7 human breast tumor cells after continuous exposure to a sublethal concentration of doxorubicin.

The effects on DNA integrity of continuous (72-h) exposure of human MCF-7 breast adenocarcinoma cells to 50 nM doxorubicin (a concentration which can be maintained in the plasma by continuous infusion) were characterized by bisbenzimide spectrofluorophotometry, cell flow cytometry, agarose gel electrophoresis, and neutral elution. Spectrofluorophotometry and cell flow cytometry indicated the presence of DNA fragmentation, which was maximal at 24 h. Resolution of these fragments on agarose gels failed to demonstrate "laddered" oligosomal profiles. Neutral elution analysis at 24 h indicated that doxorubicin induced fragmentation of nascent, but not mature, double-stranded DNA. Drug-treated cells exhibited endoreduplication and significant shifts in cell cycle distribution, (i.e., increased G0/G1 and G2/M fractions and a markedly reduced S-phase fraction). These alterations occurred without inhibiting the incorporation of [3H]dThd into cellular DNA; in fact, both the rate and magnitude of [3H]dThd incorporation increased progressively. Doxorubicin also produced a sustained decline in c-myc mRNA levels that paralleled both growth arrest and induction of DNA fragmentation. Ultrastructural examination revealed morphological alterations consistent with the induction of differentiation (e.g., increased lipid content and mitochondrial density, appearance of tight junctions, and secretory ducts) and further suggested the possibility of autocatalysis (e.g., lipofuschin-containing vacuoles). A gradual decline in cell number was observed, with loss of approximately 35% of the cell population after 72 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenocarcinoma↗
PubMed1994

Interference by doxorubicin with DNA unwinding in MCF-7 breast tumor cells.

The capacity of doxorubicin to inhibit topoisomerase II in the MCF-7 breast tumor cell line is supported by the induction of protein-associated single-strand breaks in DNA, as well as by interference with the decatenation activity of nuclear extracts. Doxorubicin also produces non-protein-associated DNA strand breaks (at a supraclinical concentration of 5 microM), which may indicate damage mediated via the generation of free radicals. However, no strand breaks are detected in DNA of MCF-7 cells at the IC50 for doxorubicin (approximately 0.1 microM). At doxorubicin concentrations of 0.05, 0.1, and 0.5 microM, at which growth is inhibited by approximately 15, 50, and 75%, respectively, doxorubicin interferes with radiation-induced unwinding of DNA; doxorubicin also produces a concentration-dependent inhibition of DNA synthesis that corresponds closely to growth inhibition. These studies suggest that DNA strand breaks fail to fully account for the antiproliferative activity of doxorubicin in the MCF-7 breast tumor cell line. Compromised DNA synthesis associated with interference with DNA unwinding may contribute to growth inhibition in MCF-7 cells exposed to doxorubicin.

Breast Neoplasms↗
PubMed1993

Dissociation between bulk damage to DNA and the antiproliferative activity of teniposide (VM-26) in the MCF-7 breast tumor cell line: evidence for induction of gene-specific damage and alterations in gene expression.

In the MCF-7 breast tumor cell line, induction of bulk damage to DNA (measured either as total strand breaks or as double-strand breaks) fails to correspond with the antiproliferative activity of the demethylepipodo-phyllotoxin derivative, VM-26. In contrast, VM-26 produces an early (within 2-3 h) concentration-dependent reduction in c-myc expression (and of DNA synthesis) which parallels inhibition of cell growth, suggesting the possibility of effects of VM-26 at the level of genomic regions which regulate DNA replicative function. Although VM-26 also produces a reduction in c-myc expression in K562 human leukemic cells, these alterations fail to correspond with the concentration-dependent effects on cell growth in this cell line. Utilizing the newly developed alkaline unwinding/Southern blotting assay in the MCF-7 breast tumor cell line, it was determined that VM-26 induces damage within regions surrounding the c-myc gene and the beta-globin gene which exceeds that induced in both alpha-satellite DNA and in L1 repeat sequences; damage within c-myc and beta-globin also exceeds that observed throughout the genome as a whole. These findings indicate that certain genomic regions incur preferential damage in MCF-7 cells exposed to VM-26. It appears possible that damage within such genomic regions could lead to alterations in expression of select genes associated with regulation of cellular proliferation, resulting in reduced DNA synthesis, compromised cell growth, and, ultimately, cell death.

Blotting, Southern↗
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↗