Senescence evasion by MCF-7 human breast tumor-initiating cells - PubMed (original) (raw)
Senescence evasion by MCF-7 human breast tumor-initiating cells
Feridoun Karimi-Busheri et al. Breast Cancer Res. 2010.
Abstract
Introduction: 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.
Figures
Figure 1
Radiation response of MCF-7 monolayer and mammospheres cells. (a) Clonogenic survival assay - MCF-7 monolayer and mammospheres were trypsinized, irradiated as single-cell suspensions with increasing doses of 60Co γ-radiation, and then plated. After18 days of incubation, the colonies (consisting of more than 30 cells) were fixed, stained with crystal violet and counted. (b) Cell proliferation assay - cells were plated in a 96-well plate, irradiated and incubated for five days. Cells were exposed to 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS, inner salt) for four hours and absorbance was measured at 490 nm. Absorbance was normalized to unirradiated controls. Error bars represent the mean ± standard error of the mean from three independent experiments.
Figure 2
Relative content of reactive oxygen species in unirradiated and irradiated MCF-7 monolayer and mammosphere cells. Cells were incubated with aminophenyl fluorescein, exposed to 0, 1, 4 or 10 Gy 60Co γ-radiation, and immediately afterwards the relative fluorescence of each sample was measured by excitation at 485 nm and emission at 515 nm. Error bars represent the mean ± standard error of the mean from three independent experiments.
Figure 3
Analysis of single strand break repair in MCF-7 monolayer and mammosphere cell populations. (a) Cells were exposed to 4 Gy 60Co γ-radiation and the relative degree of single-strand breakage (SSB) was determined by alkaline single-cell gel electrophoresis (comet assay) immediately after exposure and at the times indicated after exposure. (b) The 'comets' (n of about 100) were categorized according to the NIH LISTSERV (Comet Assay Interest Group web site) in which type 1 comets display the least DNA damage and type 5 the most. The error bars represent the mean ± standard error of the mean in both panels. The comets of the unirradiated cells are labeled Cont. (c) Expression of proteins involved in SSB repair in response to ionizing radiation. Lysates were prepared from unirradiated cells and from cells harvested one hour after exposure to 1 or 10-Gy 60Co γ-radiation and analyzed by immunoblotting with antibodies against several SSB repair proteins. α-Actin served as a loading control.
Figure 4
Radiation induced H2AX phosphorylation in MCF-7 monolayer and mammosphere cells. (a) Unirradiated cells or cells exposed to 1 or 10 Gy γ-radiation and then incubated for one hour at 37°C, or (b) cells exposed to 1 Gy and incubated at 37°C for different times, were fixed, permeabilized and immunostained with an antibody to γH2AX and counter-stained with 4',6-diamidino-2-phenylindole (DAPI). H2AX phosphorylation was clearly visible as characteristic fluorescent foci in the irradiated monolayer cells, but no foci were detectable in the irradiated mammosphere cells.
Figure 5
Analysis of double strand break repair in MCF-7 monolayer and mammosphere populations. (a) Cells were exposed to 4 Gy 60Co γ-radiation and the relative degree of double-strand breakage (DSB) was determined by the comet assay under neutral conditions immediately after exposure and at the times indicated after exposure. (b) The 'comets' (n of about 100) were categorized according to the NIH LISTSERV (Comet Assay Interest Group web site) in which type 1 comets display the least DNA damage and type 5 the most. The error bars represent the mean ± standard error of the mean in both panels. The comets of the unirradiated cells are labeled Cont. (c) Expression of proteins involved in the NHEJ pathway of DSB repair in response to increasing doses of ionizing radiation. Lysates were prepared from unirradiated cells and from cells harvested one hour after exposure to 1 or 10-Gy 60Co γ-radiation and analyzed by immunoblotting with antibodies against several DSB repair proteins. Phospho-ATM and phospho-DNA-PKcs refer to phosphorylation of these proteins at Ser1981 and Ser2056, respectively. Actin served as a loading control.
Figure 6
Telomerase activity in MCF-7 monolayer and mammosphere cells. (a) Telemorase activity was determined by Telomeric Repeat Amplification Protocol (TRAP) analysis. Cell extracts were prepared from unirradiated and irradiated MCF-7 monolayer and mammospheres cells. The assay involves the addition, mediated by telomerase in the cell extract, of a number of telomeric repeats onto the 3' end of an oligonucleotide substrate, which is then subjected to amplification by PCR. When run on a 10 to 12% native polyacrylamide gel, the telomerase-extended primer appears as a ladder of 6 bp increments. The lowest band on the gel shows the 36 bp internal PCR control. (The absence of this band in the lane showing results with 10-Gy irradiated mammospheres is due to excessively high telomerase activity because amplification of the TRAP products and the internal control is semi-competitive.) The positive (+ve) control lane shows the results using a telomerase positive cell extract provided by the manufacturer of the assay kit, while a cell extract prepared from human fibroblasts (CRL 2322) served as a negative control. (b) Quantification of telomerase activity. The mean values and standard deviations were calculated from three individual determinations at each dose. The difference in activity between the monolayer and mammosphere populations at each dose were statistically significant (P < 0.05, Student's t-test).
Figure 7
Senescence-related factors in MCF-7 monolayer and mammosphere populations. (a) Expression of key proteins involved in the cellular senescence pathway in response to increasing doses of ionizing radiation. Lysates were prepared from unirradiated cells and from cells harvested one hour after exposure to 1 or 10-Gy 60Co γ-radiation and analyzed by immunoblotting with antibodies against p21 and pRb. ppRb indicates phosphorylated pRb. Actin served as a loading control. (b) Senescence was measured in unirradiated (control) cells and irradiated (4 Gy, 3 days post-irradiation) MCF-7 monolayer and mammosphere cells by staining for β-galactosidase activity. Cells (n of more than 100) were counted independently by two individuals. The error bars represent the mean ± standard error of the mean. (c) Expression of ING1 protein. Lysates were prepared as described for Figure 6a. Western blots were performed with a mixture of four monoclonal antibodies against a domain common to ING1a, ING1b and ING1c. Based on the apparent molecular weight of the observed band (about 35 kDa), the protein was identified as ING1b.
Figure 8
Expression of DNA damage/cell cycle response proteins in unirradiated and irradiated MCF-7 cells. (a) Lysates were prepared from unirradiated monolayer and mammosphere cells and from cells harvested one hour after exposure to 1 or 10-Gy 60Co γ-radiation and analyzed by immunoblotting. Phospho-Chk1 and phospho-Chk2 indicate phosphorylation of Chk1 and Chk2 at Ser345 and Thr68, respectively. Actin served as a loading control. (b) Phosphorylation of p53 at various amino acids. Phosphorylation at serines 6, 9, 37, 46, and 392 was negative (data not shown).
Comment in
- Tumor senescence and radioresistant tumor-initiating cells (TICs): let sleeping dogs lie!
Zafarana G, Bristow RG. Zafarana G, et al. Breast Cancer Res. 2010;12(4):111. doi: 10.1186/bcr2597. Epub 2010 Jul 5. Breast Cancer Res. 2010. PMID: 20619004 Free PMC article.
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