Senescence evasion by MCF-7 human breast tumor-initiating cells.
BACKGROUND
A subpopulation of cancer cells, tumor-initiating cells, is believed to be the driving force behind tumorigenesis and resistance to radiation and chemotherapy. The persistence of tumor-initiating cells may depend on altered regulation of DNA damage and checkpoint proteins, as well as a reduced propensity to undergo apoptosis or senescence.


METHODS
To test this hypothesis, we isolated CD24-/low/CD44+ tumor-initiating cells (as mammospheres) from MCF-7 breast cancer cells grown in adherent monolayer culture, and carried out a comprehensive comparison of cell death and DNA damage response pathways prior to and after exposure to ionizing radiation in mammospheres and monolayer MCF-7 cells. Single and double-strand break repair was measured by single-cell gel electrophoresis. The latter was also examined by phosphorylation of histone H2AX and formation of 53BP1 and Rad51 foci. Apoptosis was quantified by flow-cytometric analysis of annexin V-binding and senescence was analyzed on the basis of cellular beta-galactosidase activity. We employed the telomeric repeat amplification protocol to quantify telomerase activity. Expression of key DNA repair and cell cycle regulatory proteins was detected and quantified by western blot analysis.


RESULTS
Our data demonstrate that in comparison to the bulk population of MCF-7 cells (predominantly CD24+/CD44+), the MCF-7 mammosphere cells benefit from a multifaceted approach to cellular protection relative to that seen in monolayer cells, including a reduced level of reactive oxygen species, a more active DNA single-strand break repair (SSBR) pathway, possibly due to a higher level of expression of the key SSBR protein, human AP endonuclease 1 (Ape1), and a significantly reduced propensity to undergo senescence as a result of increased telomerase activity and a low level of p21 protein expression. No significant difference was seen in the rates of double-strand break repair (DSBR) between the two cell types, but DSBR in mammospheres appears to by-pass the need for H2AX phosphorylation.


CONCLUSIONS
Enhanced survival of MCF-7 tumor-initiating cells in response to ionizing radiation is primarily dependent on an inherent down-regulation of the senescence pathway. Since MCF-7 cells are representative of cancer cells that do not readily undergo apoptosis, consideration of senescence pathways may play a role in targeting stem cells from such tumors.
Department of Oncology, University of Alberta and Department of Experimental Oncology, Cross Cancer Institute, 11560 University Avenue, Edmonton, Alberta, Canada. fkarimi@novarx.com
Feridoun
F
Karimi-Busheri
Aghdass
A
Rasouli-Nia
John R
JR
Mackey
Michael
M
Weinfeld
D017209:Apoptosis; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000276:immunology / Q000378:metabolism / Q000473:pathology*; D051927:CD24 Antigen / Q000378:metabolism; D002453:Cell Cycle; D049109:Cell Proliferation; D016922:Cellular Senescence; D004249:DNA Damage; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000235:genetics / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D006657:Histones / Q000378:metabolism; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism; D047468:Immunoprecipitation; D000076023:Inhibitor of Growth Protein 1; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D014411:Neoplastic Stem Cells / Q000276:immunology / Q000378:metabolism / Q000473:pathology*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018874:Spheroids, Cellular; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism; D000071857:Tumor Suppressor p53-Binding Protein 1
D051927:CD24 Antigen; C468783:H2AX protein, human; D006657:Histones; D018960:Hyaluronan Receptors; C501503:ING1 protein, human; D000076023:Inhibitor of Growth Protein 1; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; C498301:TP53BP1 protein, human; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D000071857:Tumor Suppressor p53-Binding Protein 1; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase