Michael S. Orr

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.

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 μM. While DNA strand breakage was not evident below a concentration of 0.1 μM 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.

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.

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.

Radiosensitization of HL-60 human leukaemia cells by bryostatin-1 in the absence of increased DNA fragmentation or apoptotic cell death

Ionizing radiation produced a dose-dependent reduction in the proliferative capacity of HL-60 human promyelocytic leukaemia cells. A small percentage of the cell population demonstrated morphological evidence of apoptosis at 24h following radiation doses of > or = 5 Gy (i.e. 8% at 5 Gy and 16% at 10 Gy respectively) and produced a laddered oligonucleosomal pattern of DNA fragments by static-field gel electrophoresis. The antiproliferative effects of 1 and 2.5 Gy ionizing radiation were significantly enhanced by preincubating cells with bryostatin-1 at a concentration (10 nM) and time frame (24h) associated with down-regulation of total cellular protein kinase C (PKC) activity. Potentiation by bryostatin-1 of the radiation effect on proliferation was not associated with a concomitant increase in internucleosomal DNA fragmentation, in the fraction of cells exhibiting apoptotic morphology, or in the extent of radiation-induced single- or double-strand breaks in bulk DNA. Staurosporine, a potent but nonspecific inhibitor of PKC, was ineffective in altering the radiosensitivity of HL-60 cells or the degree of DNA fragmentation induced by ionizing radiation. These findings indicate that bryostatin 1 increases the sensitivity of human myeloid leukaemic cells to low radiation doses without enhancing DNA fragmentation or apoptosis, and that this capacity may involve factors other than, or in addition to, down-modulation of PKC activity.

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.