Stilbene 5c, a microtubule poison with vascular disrupting properties that induces multiple modes of growth arrest and cell death.

The stilbene derivative, cis-3,4',5-trimethoxy-3'-aminostilbene (stilbene 5c), is a potentially potent antitumor agent that acts via binding to the colchicine-binding site in tubulin. The current studies were designed to investigate the effectiveness of stilbene 5c against the HCT-116 human colon cancer cell line and B16/F10 melanoma cells as well as human endothelial cell tube formation and tumor perfusion. Stilbene 5c produced a time-dependent decrease in cell viability in both cell lines and the capacity of the cells to proliferate was not restored upon removal of the drug. Treatment with stilbene 5c also promoted both senescence and autophagy in both cell lines. TUNEL and annexin 5 staining indicated that apoptosis also occurs in stilbene 5c-treated HCT-116 cells, but not in B16/F10 melanoma cells. DAPI staining revealed morphological changes in the cell nuclei (binucleated and micronucleated cells) indicative of mitotic catastrophe in HCT-116 cells but not in the B16/F10 melanoma cells. p53-null HCT-116 cells demonstrated a similar growth arrest/cell death response to stilbene as p53-wild type HCT-116 cells. Stilbene 5c also completely inhibited human endothelial cell tube formation on Matrigel, consistent with potential anti-angiogenic actions. Using a new method developed for monitoring the pharmacodynamic effects of stilbene 5c in vivo, we found that a single injection of stilbene 5c reduced tumor perfusion by 65% at 4h, returning to baseline by 24h, while subsequent daily injections of stilbene 5c produced progressively larger reductions and smaller rebounds. This work indicates that stilbene 5c could potentially be effective against melanoma and colon cancer through the promotion of multiple modes of growth arrest and cell death coupled with anti-angiogenic and antivascular actions.

p53-Dependent accelerated senescence induced by ionizing radiation in breast tumour cells.

Ionizing radiation has been reported to promote accelerated or premature senescence in both normal and tumour cell lines. The current studies were designed to characterize the accelerated senescence response to radiation in the breast tumour cell in terms of its dependence on functional p53 and its relationship to telomerase activity, telomere lengths, expression of human telomerase reverse transcriptase (hTERT, the catalytic subunit of telomerase) and human telomerase RNA (hTR, the RNA subunit of telomerase), as well as the induction of cytogenetic aberrations. Studies were performed in p53 wild-type MCF-7 cells, MCF-7/E6 cells with attenuated p53 function, MDA-MB231 cells with mutant p53 and MCF-7/hTERT cells with constitutive expression of hTERT. Telomerase activity was measured by the telomeric repeat amplification protocol (TRAP assay), telomere lengths by the terminal restriction fragment (TRF) assay, hTR and hTERT expression by reverse transcriptase-polymerase chain reaction (RT-PCR), senescence by beta-galactosidase staining, and apoptosis by TdT-mediated d-UTP-X nick-end labelling (TUNEL assay). Widespread and extensive expression of beta-galactosidase, a marker of cellular senescence, was evident in MCF-7 breast tumour cells following exposure to 10 Gy of ionizing radiation. Radiation did not suppress expression of either hTERT or hTR, alter telomerase activity or induce telomere shortening. Senescence arrest was also observed in irradiated MCF-7/hTERT cells, which have elongated telomeres due to the ectopic expression of the catalytic component of telomerase. In contrast to MCF-7 cells, irradiated MDA-MB231 breast tumour cells and MCF-7/E6 cells failed to senesce and instead demonstrated a delayed apoptotic cell death. Irradiation produced chromosome end associated abnormalities, including end-to-end fusions (an indicator of telomere dysfunction) in MCF-7 cells, MCF-7/hTERT cells, as well as in MCF-7/E6 cells. When cells were maintained in culture following irradiation, proliferative recovery was evident exclusively after senescence while the cell lines which responded to radiation by apoptosis continued to decline in cell number. Accelerated senescence in response to ionizing radiation is p53 dependent and associated with telomer dysfunction but is unrelated to changes in telomerase activity or telomere lengths, expression of hTERT and hTR. In the absence of functional p53, cells are unable to arrest for an extended period, resulting in apoptotic cell death while accelerated senescence in cells expressing p53 is succeeded by proliferative recovery.

Suppression of c-myc expression and c-Myc function in response to sustained DNA damage in MCF-7 breast tumor cells.

The topoisomerase II inhibitors teniposide (VM-26), doxorubicin, and amsacrine (m-AMSA), as well as ionizing radiation, induce a transient suppression of c-myc mRNA, which correlates with growth inhibition of MCF-7 breast tumor cells. To further assess the involvement of c-mvc in the DNA damage-induced signal transduction pathways of the breast tumor cell, we determined the influence of sustained DNA damage on c-myc expression, c-Myc protein levels and c-Myc function. Continuous exposure of MCF-7 breast tumor cells to VM-26 induced DNA strand breaks that were sustained for at least 9 hr. DNA strand breakage was accompanied by a decline in c-myc transcripts and c-Myc protein levels by >90% after VM-26 exposure for 24 hr. The activity of a transcriptional target of the c-Myc protein, ornithine decarboxylase, was reduced by approximately 75% within 9 hr of DNA damage, in parallel to the declines in c-myc mRNA and protein levels. Extended exposure to VM-26 resulted in an initial loss of approximately 35% of the cell population followed by the death of additional cells such that by 72 hr only 50% of the cells were viable. Although apoptosis was evident 72 hr after initiating drug exposure [based on cell cycle analysis, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assays, and an assessment of cell morphology], the primary phase of cell killing, which occurred during the first 24 hr was non-apoptotic. These studies indicate that non-apoptotic pathways can also mediate cell death in the breast tumor cell and support the role of c-myc expression, c-Myc protein, and c-Myc function as elements of the DNA damage response pathway in the breast tumor cell.

A non-calcemic sulfone version of the vitamin D(3) analogue seocalcitol (EB 1089): chemical synthesis, biological evaluation and potency enhancement of the anticancer drug adriamycin.

Novel side-chain diene sulfones 5, analogues of the natural hormone 1alpha,25-dihydroxyvitamin D(3) (calcitriol, 1), were designed to incorporate some of the therapeutically most favorable structural features of the Leo Pharmaceutical Company's drug candidate diene EB 1089 (seocalcitol, 4) and of the Hopkins' non-calcemic side-chain sulfone analogues 2 and 3. Synthesis of diene sulfones 5 features selective Swern oxidation of a primary silyl ether in the presence of a secondary silyl ether (9-->10) and Horner-Wadsworth-Emmons aldehyde addition by a 1-phosphonyl-3-sulfonyl stabilized carbanion regiospecifically at the 1-position to form E,E-diene sulfone 11. Sulfone diene analogue 5a with natural 1alpha,3beta-diol functionality, but not its diastereomer 5b with unnatural A-ring stereochemistry, is antiproliferative in vitro toward murine keratinocytes and malignant melanoma cells, as well as toward MCF-7 human breast cancer cells. Combining diene sulfone 5a with the currently used anticancer drug adriamycin (ADR) caused a noteworthy 3-fold enhancement of ADR antiproliferative potency in MCF-7 cells. Sulfone diene analogue 5a is weakly active transcriptionally in MCF-7 and ROS 17/2.8 cells, binds poorly but measurably to the vitamin D receptor (VDR), and desirably is non-calcemic in vivo at a daily dose (7 days) of 10 microg/kg of rat body weight.

The vitamin D3 analog, ILX-23-7553, enhances the response to adriamycin and irradiation in MCF-7 breast tumor cells.

UNLABELLED: Ionizing radiation and the anthracycline antibiotic, Adriamycin, generally fail to promote a primary apoptotic response in experimental breast tumor cell lines. Similarly, the primary response of breast tumor cells to vitamin D3 (1,25(OH)2D3) and vitamin D3 analogs such as EB 1089 is growth inhibition. Previous studies have demonstrated that pretreatment of MCF-7 breast tumor cells with vitamin D3 or EB 1089 can increase sensitivity to both Adriamycin and irradiation. PURPOSE: The capacity of the vitamin D3 analog, ILX 23-7553, to enhance the antiproliferative and cytotoxic effects of Adriamycin or irradiation and to promote apoptosis in MCF-7 breast tumor cells was assessed in the present study. RESULTS: Pretreatment of MCF-7 cells with ILX 23-7553 followed by Adriamycin or irradiation decreased viable cell numbers by 97% and 93%, respectively. Cell numbers were reduced by 56%, 74% and 75% by ILX 23-7553, Adriamycin and irradiation alone. Pretreatment with ILX 23-7553 also shifted the dose response curve for clonogenic survival, increasing sensitivity to Adriamycin 2.5-fold and sensitivity to radiation fourfold. In addition, ILX 23-7553 pretreatment conferred sensitivity to Adriamycin- or irradiation-induced DNA fragmentation and resulted in morphological changes indicative of apoptotic cell death in MCF-7 cells. Statistical analysis demonstrated that ILX 23-7553 interacts additively and not synergistically with both Adriamycin and irradiation. CONCLUSIONS: ILX 23-7553 enhances the effects of Adriamycin and irradiation in MCF-7 breast tumor cells by decreasing viable cell numbers, reducing clonogenic survival and inducing apoptotic cell death. Current studies are focused on elucidating the mechanisms underlying the induction of apoptosis as well as understanding the nature of the interactions between ILX 23-7553 and Adriamycin or irradiation.

Rb dephosphorylation and suppression of E2F activity in human breast tumor cells exposed to a pharmacological concentration of estradiol.

This report characterizes the influence of a pharmacological concentration of estradiol on growth arrest and cell death in MCF-7 breast tumor cells, with a focus on elements of the Rb-E2F cell-cycle regulatory pathway. Continuous exposure of MCF-7 breast tumor cells to 100 microM estradiol produces a marked reduction in the G1 and S phase populations and a corresponding increase in the G2/M population within 24 h; after 48 h, accumulation of cells in G1 becomes evident while after 72 h the cells appear to be equally distributed between the G1 and G2/M phases. The accumulation of cells in G1 is temporally associated with dephosphorylation of the Rb protein and suppression of E2F activity. Estradiol also produces an initial burst of cell death with loss of approximately 40% of the tumor cell population within 24 h; however, there is no tangible evidence for the occurrence of apoptosis based on terminal transferase end-labeling of DNA, DNA fragmentation analysis by alkaline unwinding, cell-cycle analysis or cell morphology. In addition to the lack of caspase-3 in MCF-7 cells, the absence of apoptosis could be related, at least in part, to the fact that estradiol promotes a rapid reduction in levels of the E2F-1 and Myc proteins. Overall, these studies are consistent with the concept that alterations in the levels and/or activity of the E2F family of proteins as well as proteins interacting with the E2F family may influence the nature of the antiproliferative and cytotoxic responses of the breast tumor cell.

Growth arrest and cell death in the breast tumor cell in response to ionizing radiation and chemotherapeutic agents which induce DNA damage.

Breast tumor cells are relatively refractory to apoptosis in response to modalities which induce DNA damage such as ionizing radiation and the topoisomerase II inhibitor, adriamycin. Various factors which may modulate the apoptotic response to DNA damage include the p53 status of the cell, levels and activity of the Bax and Bcl-2 families of proteins, activation of NF-kappa B, relative levels of insulin like growth factor and insulin-like growth factor binding proteins, activation of MAP kinases and PI3/Akt kinases, (the absence of) ceramide generation and the CD95 (APO1/Fas) signaling pathway. Prolonged growth arrest associated with replicative senescence may represent an alternative and reciprocal response to DNA-damage induced apoptosis that is p53 and/or p21waf1/cip1 dependent while delayed apoptosis may occur in p53 mutant breast tumor cells which fail to maintain the growth-arrested state. Clearly, the absence of an immediate apoptotic response to DNA damage does not eliminate other avenues leading to cell death and loss of self-renewal capacity in the breast tumor cell. Nevertheless, prolonged growth arrest (even if ultimately succeeded by apoptotic or necrotic cell death) could provide an opportunity for subpopulations of breast tumor cells to recover proliferative capacity and to develop resistance to subsequent clinical intervention.

The vitamin D3 analog EB 1089 enhances the antiproliferative and apoptotic effects of adriamycin in MCF-7 breast tumor cells.

Exposure of MCF-7 breast tumor cells to the vitamin D3 analog, EB 1089 enhances the response to adriamycin. Clonogenic survival studies indicate that EB 1089 shifts the dose-response curve for sensitivity to adriamycin by approximately six-fold in p53 wild-type MCF-7 cells; comparative studies in MCF-7 cells with a temperature-sensitive dominant negative p53 mutation show less than a two-fold shift in adriamycin sensitivity in the presence of EB 1089. The combination of EB 1089 with adriamycin also promotes apoptotic cell death in the p53 wild-type MCF-7 cells but not in the MCF-7 cells expressing mutant p53. EB 1089 treatment blocks the increase in p21waf1/cip1 levels induced by adriamycin and interferes with induction of MAP kinase activity by ionizing radiation, effects which could be related to the capacity of EB 1089 to promote secretion of insulin-like growth factor binding protein. Taken together with our previous findings that EB 1089 enhances breast tumor cell sensitivity to ionizing radiation, there studies further support the concept that vitamin D3 analogs could have utility in combination with conventional chemotherapy and/or radiotherapy in the treatment of breast cancer.

Influence of topoisomerase II inhibitors and ionizing radiation on growth arrest and cell death pathways in the breast tumor cell.

Promotion of apoptosis (which is frequently dependent on functional p53) is thought to be critical for the effectiveness of chemotherapy or radiotherapy. Studies in this as well as other laboratories have demonstrated that breast tumor cells are relatively refractory to apoptosis in response to modalities that induce DNA damage. This report describes our efforts to understand the basis for the absence of an apoptotic response to adriamycin and ionizing radiation in the breast tumor cell based on alterations in cell-cycle and apoptotic regulatory proteins. We also report on the permissive effects of Vitamin D3 and the Vitamin D3 analog EB 1089 in the promotion of apoptosis in p53-wild-type cells. Our studies suggest that regulation of apoptosis in the breast tumor cell may require modulation of signaling events other than or in addition to the p53-dependent DNA damage response.

Estradiol enhances liposome-mediated uptake, preferential nuclear accumulation and functional expression of exogenous genes in MDA-MB231 breast tumor cells.

Exposure of p53 mutated estrogen-receptor-negative MDA-MB231 human breast tumor cells to a pharmacological concentration of estradiol enhances liposome-mediated uptake and expression of SV-40 luciferase. Unexpectedly, the effect of estradiol on SV-40 expression is evident even when estradiol exposure occurs after the initial uptake phase; this suggests that estradiol may influence gene expression by mechanisms other than increasing gene uptake alone, such as altering the intracellular distribution of the gene. We determined that while uptake of SV-40 luciferase is increased only three-fold by estradiol, there is a 30-fold increase in the nuclear/cytoplasmic ratio of the gene. In order to demonstrate that the influence of estradiol on gene uptake and expression is translated into a functional response, the effects of estradiol on the function of an exogenous gene, in this case the apoptotic function of p53, were assessed in the p53 mutated MDA-MB231 breast tumor cell. While liposome-mediated delivery of CMV-p53 alone was ineffective in promoting cell death, incubation with estradiol and the liposomal p53 complex resulted in a two-fold increase in cell killing over that observed in cells transfected with the corresponding mock vector (empty vector for p53). Evidence that cell killing was occurring through apoptosis included apoptotic body formation, cell shrinkage and an increase in fluorescence after terminal transferase end-labeling. The capacity of estradiol to promote apoptosis in MDA-MB231 cells by a p53-liposome complex is likely to be related to the preferential redistribution of the gene from the cytoplasm to the nucleus which could occur during both the uptake and post-uptake phases. Consequently, although direct effects on gene expression, and the stability of message and protein cannot be ruled out, the predominant effect of estradiol in this experimental system appears to be to influence DNA translocation from the cytoplasm to the cell nucleus.

The vitamin D3 analog EB 1089 enhances the response of human breast tumor cells to radiation.

Previous studies from this laboratory as well as others have demonstrated that breast tumor cells fail to undergo primary apoptosis in response to agents which induce DNA damage such as ionizing radiation and the topoisomerase II inhibitor adriamycin. Similarly, the primary response of breast tumor cells to vitamin D(3) [1,25-(OH)(2)-D(3)] and its analogs such as EB 1089 is growth inhibition, with apoptosis occurring in only a small fraction of the cell population. The possibility that the combination of vitamin D(3) compounds with radiation might promote cell death (i.e. through a differentiation stimulus plus DNA damage) was investigated by exposing both TP53 (formerly known as p53) wild-type and TP53 mutated breast tumor cells to 1,25-(OH)(2)-D(3) or EB 1089 for 48 h prior to irradiation. This combination resulted in enhanced antiproliferative effects in the TP53 wild-type MCF-7 cells based on both a clonogenic assay and the determination of numbers of viable cells. The combination of EB 1089 with radiation increased DNA fragmentation based on both the terminal transferase end-labeling (TUNEL) and bisbenzamide spectrofluorometric assays, suggesting the promotion of apoptosis. The observed increase in DNA fragmentation was not due to an enhancement of the extent of initial DNA damage induced by radiation. These findings suggest that vitamin D compounds may be useful in combination with radiation in the treatment of breast cancer.

A critical evaluation of the mechanisms of action proposed for the antitumor effects of the anthracycline antibiotics adriamycin and daunorubicin.

The mechanisms responsible for the antiproliferative and cytotoxic effects of the anthracycline antibiotics doxorubicin (Adriamycin) and daunorubicin (daunomycin) have been the subject of considerable controversy. This commentary addresses the potential role of DNA synthesis inhibition, free radical formation and lipid peroxidation, DNA binding and alkylation, DNA cross-linking, interference with DNA strand separation and helicase activity, direct membrane effects, and the initiation of DNA damage via the inhibition of topoisomerase II in the interaction of these drugs with the tumor cell. One premise underlying this analysis is that only studies utilizing drug concentrations that reflect the plasma levels in the patient after either bolus administration or continuous infusion are considered to reflect the basis for drug action in the clinic. The role of free radicals in anthracycline cardiotoxicity is also discussed.

Sustained enhancement of liposome-mediated gene delivery and gene expression in human breast tumour cells by ionizing radiation.

PURPOSE: To investigate whether irradiation improves the delivery and expression of liposome-DNA complexes in human breast tumour cells. MATERIALS AND METHODS. MDA-MB231 and MCF-7 human breast tumour cells were transfected with a liposomal SV40-luciferase complex and irradiated immediately after, at 24h after or 24h prior to transfection and in the presence or absence of serum. The amount of luciferase plasmid in the cell was evaluated after extraction by the Hirt procedure, while luciferase expression was measured using a luminescence assay. RESULTS: Ionizing radiation enhanced the liposome-mediated delivery and expression of the SV40-luciferase transgene in MDA-MB231 breast tumour cells both in the absence and presence of serum as well as in MCF-7 breast tumour cells. Improved transgene delivery and expression was observed at a clinically relevant dose of 2 Gy, and was dose-dependent over a dose range of 2-10 Gy. The effects of irradiation on transgene expression were observed with irradiation immediately prior to exposure of the cells to the liposome-transgene complex, with irradiation up to 24 h before or up to 24 h after initiation of exposure. CONCLUSIONS: Irradiation at 24 h prior to exposure of breast tumour cells to the liposome-transgene complex appears to be the optimal approach for enhancing transgene delivery and expression. These findings suggest that ionizing radiation could promote the utility of gene therapy in the treatment of breast cancer.

Enhancement of liposomal gene delivery in human breast cancer cells by dimethyl sulfoxide.

Non-toxic concentrations ( 1%) of dimethyl sulfoxide (DMSO) enhance the liposomal delivery of DNA to both MCF-7 and MDA-MB-231 human breast tumor cells. Uptake of SV-40-luciferase was enhanced in MCF-7 cells by 14-fold while uptake of CMV-beta-galactosidase was increased 10-fold. In MDA-MB-231 cells, uptake of SV-40-luciferase was increased by approximately 70%. A mixture of ethanol and polyethylene glycol (45:55) at a concentration of 1% produced less pronounced improvements in transgene delivery to MCF-7 cells (a 70% increase in SV-40-luciferase uptake and a 4-fold increase in CMV-beta-galactosidase uptake) but no improvement in SV-40-luciferase gene delivery to MDA-MB-231 cells. These studies suggest that select pharmaceutical adjuvants which dissolve clinically useful drugs may have promise as non-toxic vehicles for improving transgene delivery. However, the relative effectiveness of these adjuvants is likely to vary depending on both the nature of the gene being delivered as well as the tumor cell which is the target for uptake of the exogenous gene.

Estradiol enhances gene delivery to human breast tumor cells.

The influence of estradiol on the delivery of plasmid DNA to estrogen receptor positive MCF-7 human breast cancer cells was studied by the use of a reporter assay and by histochemical staining. Continuous exposure to estradiol enhanced the lipofectamine-mediated delivery of both pSV40-luciferase and pCMV beta-galactosidase in a concentration-dependent manner. Estradiol increased both the amount of pCMV beta-galactosidase per cell and the total fraction of cells competent to receive the transgene. The efficiency of transgene delivery to MCF-7 cells was further improved by repeating the transfection procedure in the presence of estradiol. Although overall gene uptake was reduced in control cells when studies were performed at room temperature (as opposed to 37 degrees C), potentiation of gene uptake by estradiol was maintained. At a concentration of 100 microM, estradiol also enhanced delivery of the transgene to estrogen receptor negative MDA-MB-231 breast tumor cells, indicating that the potentiating effects of estradiol are not mediated through the estrogen receptor. These studies are the first to raise the possibility that gene delivery to breast tumor cells can be improved by estradiol in single- or repeated-treatment regimens.

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.

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.

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.

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.