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

• Published in: Biochemical pharmacology Vol. 68; no. 9; pp. 1699 - 1708
• Main Authors: Hattangadi, Deepali K., DeMasters, Gerald A., Walker, Teneille D., Jones, Kara R., Di, Xu, Newsham, Irene F., Gewirtz, David A.
• Format: Journal Article
• Language: English
• Published: New York, NY Elsevier Inc 01-11-2004; Elsevier Science
• Subjects: Antineoplastic agents; Apoptosis; Apoptosis - drug effects; Apoptosis - physiology; Biological and medical sciences; Breast Neoplasms - pathology; Breast tumor; Caspase 3; Caspases - physiology; Cellular Senescence - drug effects; Cellular Senescence - physiology; Cyclin-Dependent Kinase Inhibitor p21; Cyclins - biosynthesis; DNA Damage - drug effects; Drug Screening Assays, Antitumor; General aspects; Humans; Medical sciences; Methotrexate; Methotrexate - pharmacology; p53; Pharmacology. Drug treatments; Senescence; Tumor Cells, Cultured; Tumor Suppressor Protein p53 - physiology; Caspase 3; Senescence; Methotrexate; Breast tumor; Apoptosis; p53; Antineoplastic agent; Enzyme; Cysteine endopeptidases; Caspase; In vitro; Antifolate; Mammary gland diseases; Peptidases; TP53 Gene; Antimetabolic; Cell death; Hydrolases; Tumor cell; Tumor suppressor gene
• ISSN: 0006-2952; 1873-2968
• DOI: 10.1016/j.bcp.2004.06.033

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
μM methotrexate (the IC
80 value for a 2
h drug exposure). There was no evidence of either DNA fragmentation, a sub G
0 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 p21
waf1/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.