Potentiation of radiation sensitivity in breast tumor cells by the vitamin D3 analogue, EB 1089, through promotion of autophagy and interference with proliferative recovery.

1,25-Dihydroxyvitamin D(3) and vitamin D(3) analogues, such as EB 1089, potentiate the response to ionizing radiation in breast tumor cells. The current studies address the basis for this interaction by evaluating DNA damage and repair, the effect of interference with reactive oxygen generation, the involvement of p53 and caspase-3, signaling through c-myc, as well as the induction of senescence and multiple modes of cell death. EB 1089 failed to increase the extent of radiation-induced DNA damage or to attenuate the rate of DNA repair. The reactive oxygen scavengers N-acetyl-l-cysteine and reduced glutathione failed to protect the cells from the promotion of cell death by EB 1089 and radiation. Whereas MCF-7 cells expressing caspase-3 showed significant apoptosis with radiation alone as well as with EB 1089 followed by radiation, EB 1089 maintained its ability to confer susceptibility to radiation-induced cell killing, in large part by interference with proliferative recovery. In contrast, in breast tumor cells lacking p53, where radiation promoted extensive apoptosis and the cells failed to recover after radiation treatment, EB 1089 failed to influence the effect of radiation. EB 1089 treatment interfered with radiation-induced suppression of c-myc; however, induction of c-myc did not prevent senescence by radiation alone or radiation-induced cell death promoted by EB 1089. EB 1089 did not increase the extent of micronucleation, indicative of mitotic catastrophe, induced by radiation alone. However, EB 1089 did promote extensive autophagic cell death in the irradiated cells. Taken together, these studies suggest that the effect of EB 1089 treatment on the radiation response is related in part to enhanced promotion of autophagic cell death and in part to interference with the proliferative recovery that occurs with radiation alone in p53 wild-type breast tumor cells.

Influence of p53 and caspase 3 activity on cell death and senescence in response to methotrexate in the breast tumor cell.

The influence of p53 function and caspase 3 activity on the capacity of the antifolate, methotrexate, to promote senescence arrest and apoptotic cell death was investigated in breast tumor cells. In p53 wild-type, but caspase 3 deficient MCF-7 breast tumor cells, death of approximately 40% of the cell population was observed immediately after acute exposure to 10 microM methotrexate (the IC80 value for a 2 h drug exposure). There was no evidence of either DNA fragmentation, a sub G0 population or morphological alterations indicative of apoptosis; however, PARP cleavage was detected. Cell death was succeeded by growth arrest for at least 72 h--where arrest was characterized by expression of the senescence marker, beta-galactosidase. The response to methotrexate in MCF-7/E6 cells with attenuated p53 function was also primarily growth arrest--but lacking characteristics of senescence. In contrast, MCF-7 cells which expressed caspase 3 demonstrated a gradual and continuous loss of cell viability and unequivocal morphological evidence of apoptosis. DNA fragmentation indicative of apoptosis was also detected after exposure to methotrexate in p53 mutant MDA-MB231 breast tumor cells which also express caspase 3. Methotrexate-induced both p53 and p21waf1/cip1 in MCF-7 cells within 6 h; however, no significant DNA strand breakage was evident before 18 h, suggesting that the induction of p53 reflects a response to cellular stress other than DNA damage, such as nucleotide depletion. Overall, these studies suggest that the nature of the cellular response to methotrexate depends, in large part, on p53 and caspase function. p53 appears to be required for methotrexate-induced senescence, but not apoptosis, caspase 3 is required for DNA fragmentation and the morphological changes associated with apoptosis, while neither p53 nor caspase 3 are required for methotrexate-induced growth arrest. Furthermore, the senescence phenotype may occur in the absence of direct DNA damage.

Cytotoxicity, DNA strand breakage and DNA-protein crosslinking by a novel transplatinum compound in human A2780 ovarian and MCF-7 breast carcinoma cells.

Cisplatin, cis-[PtCl2(NH3)2], is commonly utilized in various combination chemotherapy protocols for the treatment of both ovarian and breast cancer while the corresponding trans isomer is therapeutically inactive. This work describes efforts to elucidate the cellular mechanism of action of a novel trans-platinum compound, trans-(dichloroamminethiazole)platinum(II) (ATZ), which demonstrates antiproliferative and cytotoxic effects against both MCF-7 human breast and A2780 human ovarian carcinoma cells in culture. A2780 cells were approximately twofold more sensitive to ATZ than MCF-7 cells in both cell growth and clonogenic survival assays. Dye exclusion studies revealed a 50-70% loss in cell viability within the first 12 h of drug treatment in both cell lines. This initial wave of cell death was succeeded by a prolonged interval of growth arrest during which a small fraction of apoptotic cells was detected. Binding of ATZ to DNA, as estimated by atomic absorption spectroscopy, was similar for the two cell lines and was almost completely reversed 24 h after drug removal. ATZ also induced DNA strand breakage as well as DNA-protein crosslinking during the initial 12 h period when the bulk of cell death was evident. However, neither the extent of DNA strand breakage nor that of DNA protein crosslinking was sufficient to explain the different drug sensitivity in the two cell lines. At 24 and 48 h after exposure of MCF-7 cells to high concentrations of ATZ, the formation of DNA-topoisomerase I complexes is detected, coincident with a high degree of apoptosis. These studies suggest that ATZ has the capacity to interfere with topoisomerase I in the tumor cell, a function not evident in cis-platinum-based drugs.

Potentiation of cell killing by fractionated radiation and suppression of proliferative recovery in MCF-7 breast tumor cells by the Vitamin D3 analog EB 1089.

A senescence-like growth arrest succeeded by recovery of proliferative capacity was observed in MCF-7 breast tumor cells exposed to fractionated radiation, 5 x 2 Gy. Exposure to EB 1089, an analog of the steroid hormone 1alpha, 25 dihydroxycholecalciferol (1alpha, 25 dihydroxy Vitamin D(3); calcitriol), prior to irradiation promoted cell death and delayed both the development of a senescent phenotype and the recovery of proliferative capacity. EB 1089 also reduced clonogenic survival over and above that produced by fractionated radiation alone and further conferred susceptibility to apoptosis in MCF-7 cells exposed to radiation. In contrast, EB 1089 failed to enhance the response to radiation (or to promote apoptosis) in normal breast epithelial cells or BJ fibroblast cells. EB 1089 treatment and fractionated radiation additively promoted ceramide generation and suppressed expression of polo-like kinase 1. Taken together, these data indicate that EB 1089 (and 1alpha, 25 dihydroxycholecalciferol or its analogs) could selectively enhance breast tumor cell sensitivity to radiation through the promotion of cell death, in part through the generation of ceramide and the suppression of polo-like kinase.