title: Silencing CDK4 radiosensitizes breast cancer cells by promoting apoptosis.
abstract: BACKGROUND
  The discovery of molecular markers associated with various breast cancer subtypes has greatly improved the treatment and outcome of breast cancer patients. Unfortunately, breast cancer cells acquire resistance to various therapies. Mounting evidence suggests that resistance is rooted in the deregulation of the G1 phase regulatory machinery.
  
  
  METHODS
  To address whether deregulation of the G1 phase regulatory machinery contributes to radiotherapy resistance, the MCF10A immortalized human mammary epithelial cell line, ER-PR-Her2+ and ER-PR-Her2- breast cancer cell lines were irradiated. Colony formation assays measured radioresistance, while immunocytochemistry, Western blots, and flow cytometry measured the cell cycle, DNA replication, mitosis, apoptosis, and DNA breaks.
  
  
  RESULTS
  Molecular markers common to all cell lines were overexpressed, including cyclin A1 and cyclin D1, which impinge on CDK2 and CDK4 activities, respectively. We addressed their potential role in radioresistance by generating cell lines stably expressing small hairpin RNAs (shRNA) against CDK2 and CDK4. None of the cell lines knocked down for CDK2 displayed radiosensitization. In contrast, all cell lines knocked down for CDK4 were significantly radiosensitized, and a CDK4/CDK6 inhibitor sensitized MDA-MB-468 to radiation induced apoptosis. Our data showed that silencing CDK4 significantly increases radiation induced cell apoptosis in cell lines without significantly altering cell cycle progression, or DNA repair after irradiation. Our results indicate lower levels of phospho-Bad at ser136 upon CDK4 silencing and ionizing radiation, which has been shown to signal apoptosis.
  
  
  CONCLUSIONS
  Based on our data we conclude that knockdown of CDK4 activity sensitizes breast cancer cells to radiation by activating apoptosis pathways.
authors.affiliation: Department of Radiation Oncology, Emory University School of Medicine, Atlanta, USA.
authors.forename: Katie R
authors.initials: KR
authors.lastname: Hagen
authors.affiliation: Department of Radiation Oncology, Emory University School of Medicine, Atlanta, USA.
authors.forename: Xiangbin
authors.initials: X
authors.lastname: Zeng
authors.affiliation: Department of Radiation Oncology, Emory University School of Medicine, Atlanta, USA.
authors.forename: Mi-Young
authors.initials: MY
authors.lastname: Lee
authors.affiliation: Department of Radiation Oncology, Emory University School of Medicine, Atlanta, USA.
authors.forename: Shannon
authors.initials: S
authors.lastname: Tucker Kahn
authors.affiliation: Department of Radiation Oncology, Emory University School of Medicine, Atlanta, USA.
authors.forename: Mary Kathryn
authors.initials: MK
authors.lastname: Harrison Pitner
authors.affiliation: Department of Radiation Oncology, Emory University School of Medicine, Atlanta, USA.
authors.forename: Sandra S
authors.initials: SS
authors.lastname: Zaky
authors.affiliation: Department of Biostatistics and Bioinformatics, Emory University School of Public Health, Atlanta, USA.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Department of Medical Oncology, Emory University School of Medicine, Atlanta, USA.
authors.forename: Ruth M
authors.initials: RM
authors.lastname: O'Regan
authors.affiliation: Department of Radiation Oncology, Emory University School of Medicine, Atlanta, USA.
authors.forename: Xingming
authors.initials: X
authors.lastname: Deng
authors.affiliation: Department of Radiation Oncology, Emory University School of Medicine, Atlanta, USA.
authors.forename: Harold I
authors.initials: HI
authors.lastname: Saavedra