Primary and Compensatory Roles for RB Family Members at Cell Cycle Gene Promoters That Are Deacetylated and Downregulated in Doxorubicin-Induced Senescence of Breast Cancer Cells

Abstract

When treated with DNA-damaging chemotherapy agents, many cancer cells, in vivo and in vitro, undergo a terminal growth arrest and acquire a senescence-like phenotype. We investigated the molecular basis for this in breast cancer cells following a 2-hour treatment with 1 μM doxorubicin. Treated cells arrested in G₁ and G₂ phases of the cell cycle, with concomitant reductions in S-phase and G₂-M regulatory genes. p53 and p21 protein levels increased within hours after treatment and were maintained for 5 to 6 days but were reduced 8 days posttreatment, though the cells remained growth arrested. Levels of p130 rose after drug treatment, and it was the primary RB family member recruited to the S-phase promoters cyclin A and PCNA and G₂-M promoters cyclin B and cdc2, remaining present for the entire 8-day time period. In contrast, p107 protein and promoter occupancy levels declined sharply after drug treatment. RB was recruited to only the PCNA promoter. In MCF-7 cells with p130 knockdown, p107 compensated for p130 loss at all cell cycle gene promoters examined, allowing cells to retain the growth arrest phenotype. Cells with p130 and p107 knockdown similarly arrested, while cells with knockdown of all three family members failed to downregulate cyclin A and cyclin B. These results demonstrate a mechanistic role for p130 and compensatory roles for p107 and RB in the long-term senescence-like growth arrest response of breast cancer cells to DNA damage.

We acknowledge Johanna Echigo for assistance in screening p130 knockdown clones, Stefan Taubert and the Bruno Amati lab for technical advice and correspondence on the chromatin immunoprecipitations, Charles Thomas of the UTHSCSA/SACI FACS core facility for help with flow cytometry studies, James R. Smith for helpful scientific discussions, and Sandra N. Garcia for critical reading of the manuscript.

This work was supported by a grant from the Department of Defense Breast Cancer Research Program (DAMD17-03-1-0324) (J.G.J.) and NIA PO1AG2752 and the Ellison Medical Foundation (O.M.P.-S.).