Adriamycin-induced Senescence in Breast Tumor Cells Involves Functional p53 and Telomere Dysfunction

Lynne W Elmore,Catherine W Rehder,Xu Di,Patricia A Mcchesney,Colleen K Jackson-Cook,David A Gewirtz,Shawn E Holt

doi:10.1074/jbc.m205477200

Abstract

Direct experimental evidence implicates telomere erosion as a primary cause of cellular senescence. Using a well characterized model system for breast cancer, we define here the molecular and cellular consequences of adriamycin treatment in breast tumor cells. Cells acutely exposed to adriamycin exhibited an increase in p53 activity, a decline in telomerase activity, and a dramatic increase in beta-galactosidase, a marker of senescence. Inactivation of wild-type p53 resulted in a transition of the cellular response to adriamycin treatment from replicative senescence to delayed apoptosis, demonstrating that p53 plays an integral role in the fate of breast tumor cells treated with DNA-damaging agents. Stable introduction of hTERT, the catalytic protein component of telomerase, into MCF-7 cells caused an increase in telomerase activity and telomere length. Treatment of MCF-7-hTERT cells with adriamycin produced an identical senescence response as controls without signs of telomere shortening, indicating that the senescence after treatment is telomere length-independent. However, we found that exposure to adriamycin resulted in an overrepresentation of cytogenetic changes involving telomeres, showing an altered telomere state induced by adriamycin is probably a causal factor leading to the senescence phenotype. To our knowledge, these data are the first to demonstrate that the mechanism of adriamycin-induced senescence is dependent on both functional p53 and telomere dysfunction rather than overall shortening.

Highlights

Most normal somatic cells continually shorten their telomeres after each cell division because of incomplete replication at the end of linear chromosomes [1, 2]
Inactivation of wild-type p53 resulted in a transition of the cellular response to adriamycin treatment from replicative senescence to delayed apoptosis, demonstrating that p53 plays an integral role in the fate of breast tumor cells treated with DNA-damaging agents
Replicative senescence in normal human cells involves the action of the tumor suppressors p53 and pRB, presumably in response to the DNA damage signal elicited by shortened telomeres and/or telomere dysfunction

Summary

EXPERIMENTAL PROCEDURES

Materials—RPMI 1640 medium and trypsin-EDTA (0.5% trypsin, 5.3 mM EDTA) were obtained from Invitrogen. Following overnight incubation at 37 °C, the cells were washed twice with PBS, and the percentage of positively stained cells was determined after counting three random fields of 100 cells each. As a positive control for ␤-galactosidase expression, MCF-7 cells were exposed to 200 nM retinoic acid for 4 days and stained. Western Analysis for p53 and p21waf-1 Proteins—MCF-7 cells (parental, HPV-16 E6, and pLXSN) were treated with 1 ␮M adriamycin for 2 h, washed with PBS, and cultured for an additional 2 h prior to lysing in a standard radioimmune precipitation assay buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 100 mM dithiothreitol, and protease inhibitors). Metaphase spreads were scored for chromosomal findings from both the MCF-7-hTERT cell cultures before and after adriamycin treatment. The frequency of the types of chromosomal abnormalities seen in the cells with and without adriamycin treatment were compared using a contingency chi-square test with an ␣ Ͻ 0.05 significance level

RESULTS

Chromatid Chromosome

DISCUSSION