PKH-2 hi /SSC hi fraction increased in a dose-dependent manner and correlated well with SA--Gal positive cell numbers after irradiation in ARO and TPC-1 cell lines (Fig. 3D). Fig.3 Continued To further confirm that SLP cells are terminally arrested, we plated ARO cells irradiated with 10Gy for colony formation assay. On day 7 we observed that along with large number of single enlarged flattened cells with increased granularity, some cells formed viable colonies. Most of SA--Gal- positive cells (Fig. 4A, B) did not incorporate BrdU, as well as percent of SA--Gal positive cells was significantly higher in the BrdU- negative population. Fig. 4 Most of SA--Gal-positive ARO cells do not synthesize DNA de novo Analysis of relationship between the number of SA--Gal positive cells after IR exposure and thyroid cancer cell line radiosensitivity We analyzed correlation between radio-sensitivity and SLP cell numbers. Radiosensitivity of thyroid cancer cell lines varies significantly (Fig.5A) even between cell lines derived from the same histological type of primary tumors. A plot of MID (represents the area under the cell survival curve) against the SA--Gal index (slope of SA--Gal frequency dose-response regression line) revealed a trend (Fig. 5B) in which more radioresistant cell lines strongly tended to show lower specific SLP yields (r=-0.93 and P=0.068). Fig. 5 Relationship between the number of SA- -Gal positive cells after IR exposure and thyroid cancer cell line radiosensitivity Conclusions: 1.IR induces SLP in normal and malignant thyroid cells. 2.Induction of TGA associated with SLP contributes to the elimination Significant proportion of SA- -Gal positive cells undergo mitotic catastrophe We observed that 689.4% of SA--Gal positive ARO cells contained multiple nuclei and 216.1% of SA-- Gal positive ARO cells were mononuclear (p<0.05). In TPC-1 cell line SA--Gal positive cells were distributed nearly equally (Fig. 2A and B) between multinuclear and mononuclear fractions five days after IR exposure (6011.2% vs. 4210.5%, respectively, p>0.05). Fig. 2 Significant proportion of SA--Gal positive cells undergo mitotic catastrophe Induction of SA- -Gal activity relationship to restricted proliferative potential of irradiated thyroid cancer cell lines To study whether IR-induced SLP cells have reduced proliferative capacity, we stained pre-irradiated viable cells with lipophilic fluorescent dye PKH-2, allowing one to discriminate populations with high and low proliferative potential. Cell growth curves and FACS analysis showed that irradiated ARO cells after a 48h delay resumed growing, although with an altered kinetics compared to untreated cells, and about 10-15% of cells failed to divide at least once after IR exposure (Fig.3A, B). Fig.3 Induction of SA--Gal activity and restricted proliferative potential of irradiated thyroid cancer cell lines Irradiation of TPC-1 cells inhibited proliferation and induced decrease in cell numbers 72h after 10Gy, which then remained nearly unchanged at 96h and 120h points of observation (Fig.3A). FACS analysis demonstrated that more then 99% of total population of TPC-1 cells virtually did not divide after IR 0 1 2 3 4 5 1 10 100 C ontrol 10G y x10 000 cells/w ell D ays Ionizing Radiation Induces Senescence-Like Phenotype Associated with Terminal Growth Arrest in Human Thyroid Cells Alexei Podtcheko 1 , Hiroyuki Namba 1 , Vladimir Saenko 2 , Akira Ohtsuru 1,3 , Dmitriy Starenki 1 , Iryna Polona 1 , Tatiana Rogounovitch 2 and Shunichi Yamashita 1 1 Department of Molecular Medicine, 2 Department of International Health and Radiation Research, Nagasaki University Graduate School of Biomedical Sciences 1-12-4, Sakamoto, Nagasaki 852-8523, Japan, 3 Takashi Nagai International Hibakusha Medical Center, Nagasaki University Hospital,Nagasaki 852-8501, Japan Introduction The term cellular senescence is used to describe a sequence of changes in cell metabolism leading to irreversible growth arrest accompanied by a distinctive set of cell phenotype traits. Process of neoplastic transformation involves a series of mutations that allow cells to bypass senescence and to acquire an unlimited proliferative potential. Some tumor cells still have an ability to enter irreversible growth arrest after being exposed to stress stimuli like DNA-damaging chemotherapeutic agents, TGF-, retinoids, HDAC inhibitors, etc. Terminally arrested cancer cell can remain metabolically active for a long period of time (more than several weeks in vitro) and show characteristic changes in morphology resembling replicative senescence in normal human cells. Senescence-like terminal growth arrest may be one of the modes of cell death following IR in a number of stromal and epithelial tumors. Radiotherapy is widely used for treatment of different forms of thyroid cancer. Until now it is not clear whether IR can induce senescent-like phenotype (SLP) and terminal growth arrest in thyroid follicular cells and tumors derived from thyroid epithelium. The aim of the present study was to evaluate whether IR induce SLP associated with terminal growth arrest in the thyroid cancer and normal cells, and if so, to evaluate whether induction of SLP could be associated with thyroid cancer cell radiosensitivity. Materials and Methods Cell lines: Human anaplastic thyroid carcinoma cell lines KTC-2 and ARO, papillary carcinoma cell lines NPA and TPC-1, and primary human thyroid epithelial culture. The induction of SLP in thyroid cells were identified by: 1) senescence associated beta- Galactosidase (SA--Gal) staining method, 2) Dual flow cytometric analysis of cell proliferation and side light scatter using vital staining with PKH-2 fluorescent dye, 3) Double labeling for 5- Bromodeoxyuridine and SA--Gal, 4) Staining for SA--Gal with consequent anti-Thyroglobulin immunohistochemistry. Results IR induces SA- -Gal activity in thyroid cell lines Cells entered the state of replicative senescence become large, flat and more granular in appearance and also positively stain for SA-- Galactosidase activity detected at pH 6.0. Similar changes could be observed in phenotypes of normal and cancer cells exposed to different DNA damaging agents, such as IR, H 2 O 2 , etc. Thyroid cell lines derived from low differentiated papillary and anaplastic thyroid carcinomas exhibited typical features of senescence-like phenotype 120h after 10Gy exposure (Fig 1A). Induction of SA--Gal activity was mostly observed in large cells with ARO KTC-2 NPA TPC-1 A Non-irradiated 10Gy/120h Primary Thyrocytes TPC-1 NPA KTC-2 ARO C 0 2 4 6 8 10 0 10 20 30 40 50 60 70 80 % -galactosidase positive cells Dose, Gy Non-irradiated 10Gy/120h ARO 10Gy S-A--Gal+ S-A--Gal - S-A--Gal+ TPC-1 10Gy A MN+ MN- 0 10 20 30 40 50 60 70 80 % o f S -A -b -G alp o sitive cells ARO TP C -1 * A 0 1 2 3 4 5 1 10 100 C on trol 10G y x10 000 cells/w ell D ays ARO, 0 Gy TPC-1, 0 Gy B 0 3 5 0 3 5 ARO, 10 Gy TPC-1, 10 Gy Events ARO/10Gy/7 days BrdU Phase- contrast Merge S-A -b-G al- S-A -b-G al+ 0 10 20 30 40 50 60 70 80 90 % o f B rd U rd -p o sitive cells B ARO/10Gy/7 days B rdU rd+ B rdU rd- 0 10 20 30 40 50 % o f S A- -G alp o sitive cells A 0 2 4 6 8 10 1 10 100 S u rvivalfractio n (% o f co n tro l) D ose,G y ARO KTC-2 NPA TPC-1 0 1 2 3 4 5 6 7 8 9 0 100 200 300 400 500 600 700 TPC NPA K TC -2 ARO M ean In activatio n D ose S -A -b eta-G alin d ex B B Fig.1 IR induces SA--Gal activity in thyroid cell lines and primary thyrocytes B 0 20 40 60 80 0 10 20 30 40 50 60 70 80 10G y 7.5G y 5G y 2.5G y 10G y 7.5G y 5G y 2.5G y % o f S -A - -G alp o sitive cells % o f S SC hi /PK H-2 hi cells TP C -1 ARO TPC-1 r=0.98, p<0.01 ARO r=0.94, p<0.01 SSC-H PKH-2 (FL-1) ARO 0 Gy 10 Gy 0 Gy 10 Gy TPC-1 D C A 0 3 5 0 3 5 PKH-2 (FL-1) days ARO TPC-1
PKH-2 hi /SSC hi fraction increased in a dose-dependent manner and correlated well with SA- -Gal positive cell numbers after irradiation in ARO and TPC-1.
Jan 12, 2016
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PKH-2hi/SSChi fraction increased in a dose-dependent manner and correlated well with SA--Gal positive cell numbers after irradiation in ARO and TPC-1 cell lines (Fig. 3D).
Fig.3 Continued
To further confirm that SLP cells are terminally arrested, we plated ARO cells irradiated with 10Gy for colony formation assay. On day 7 we observed that along with large number of single enlarged flattened cells with increased granularity, some cells formed viable colonies. Most of SA--Gal-positive cells (Fig. 4A, B) did not incorporate BrdU, as well as percent of SA--Gal positive cells was significantly higher in the BrdU-negative population.
Fig. 4 Most of SA--Gal-positive ARO cells do not synthesize DNA de novo
Analysis of relationship between the number of SA--Gal positive cells after IR exposure and thyroid cancer cell line radiosensitivity
We analyzed correlation between radio-sensitivity and SLP cell numbers. Radiosensitivity of thyroid cancer cell lines varies significantly (Fig.5A) even between cell lines derived from the same histological type of primary tumors. A plot of MID (represents the area under the cell survival curve) against the SA--Gal index (slope of SA--Gal frequency dose-response regression line) revealed a trend (Fig. 5B) in which more radioresistant cell lines strongly tended to show lower specific SLP yields (r=-0.93 and P=0.068).
Fig. 5 Relationship between the number of SA--Gal positive cells after IR exposure and thyroid cancer cell line radiosensitivity
Conclusions:
1.IR induces SLP in normal and malignant thyroid cells.
2.Induction of TGA associated with SLP contributes to the elimination of clonogenic population of thyroid cancer cells after IR and specific SLP index is associated with radiation response in vitro.
3.Further studies are warranted to evaluate exact role of radiation-induced senescence as a factor of thyroid tumor radiosensitivity and cancerogenesis in vivo.
Significant proportion of SA--Gal positive cells undergo mitotic catastrophe We observed that 689.4% of SA--Gal positive ARO cells contained multiple nuclei and 216.1% of SA--Gal positive ARO cells were mononuclear (p<0.05). In TPC-1 cell line SA--Gal positive cells were distributed nearly equally (Fig. 2A and B) between multinuclear and mononuclear fractions five days after IR exposure (6011.2% vs. 4210.5%, respectively, p>0.05).
Fig. 2 Significant proportion of SA--Gal positive cells undergo mitotic catastrophe
Induction of SA--Gal activity relationship to restricted proliferative potential of irradiated thyroid cancer cell lines
To study whether IR-induced SLP cells have reduced proliferative capacity, we stained pre-irradiated viable cells with lipophilic fluorescent dye PKH-2, allowing one to discriminate populations with high and low proliferative potential.
Cell growth curves and FACS analysis showed that irradiated ARO cells after a 48h delay resumed growing, although with an altered kinetics compared to untreated cells, and about 10-15% of cells failed to divide at least once after IR exposure (Fig.3A, B).
Fig.3 Induction of SA--Gal activity and restricted proliferative potential of irradiated thyroid cancer cell lines
Irradiation of TPC-1 cells inhibited proliferation and induced decrease in cell numbers 72h after 10Gy, which then remained nearly unchanged at 96h and 120h points of observation (Fig.3A). FACS analysis demonstrated that more then 99% of total population of TPC-1 cells virtually did not divide after IR exposure (Fig.3B).
The Growth-retarded cells with high PKH-2 fluorescence exhibit elevated 90-degree light scatter, reflecting increased cell size and granularity. Such cells also displayed reduced clonogenicity and increased SA--Gal activity. In our experiments, significant fraction of irradiated thyroid cancer cells (Fig.3C) had elevated 90-degree light scatter (SSChi) and reduced proliferative potential (PKH-2hi).
0 1 2 3 4 5
1
10
100 Control 10Gy
x10
000
cells
/wel
l
Days
Ionizing Radiation Induces Senescence-Like Phenotype Associated with Terminal Growth Arrest in Human Thyroid Cells
Alexei Podtcheko1, Hiroyuki Namba1, Vladimir Saenko2, Akira Ohtsuru1,3, Dmitriy Starenki1, Iryna Polona1, Tatiana Rogounovitch2 and Shunichi Yamashita1
1Department of Molecular Medicine, 2Department of International Health and Radiation Research, Nagasaki University Graduate School of Biomedical Sciences 1-12-4, Sakamoto, Nagasaki 852-8523, Japan, 3Takashi Nagai International Hibakusha Medical Center, Nagasaki University
Hospital,Nagasaki 852-8501, Japan
IntroductionThe term cellular senescence is used to describe
a sequence of changes in cell metabolism leading to irreversible growth arrest accompanied by a distinctive set of cell phenotype traits. Process of neoplastic transformation involves a series of mutations that allow cells to bypass senescence and to acquire an unlimited proliferative potential. Some tumor cells still have an ability to enter irreversible growth arrest after being exposed to stress stimuli like DNA-damaging chemotherapeutic agents, TGF-, retinoids, HDAC inhibitors, etc. Terminally arrested cancer cell can remain metabolically active for a long period of time (more than several weeks in vitro) and show characteristic changes in morphology resembling replicative senescence in normal human cells. Senescence-like terminal growth arrest may be one of the modes of cell death following IR in a number of stromal and epithelial tumors. Radiotherapy is widely used for treatment of different forms of thyroid cancer. Until now it is not clear whether IR can induce senescent-like phenotype (SLP) and terminal growth arrest in thyroid follicular cells and tumors derived from thyroid epithelium.
The aim of the present study was to evaluate whether IR induce SLP associated with terminal growth arrest in the thyroid cancer and normal cells, and if so, to evaluate whether induction of SLP could be associated with thyroid cancer cell radiosensitivity.
Materials and MethodsCell lines: Human anaplastic thyroid carcinoma
cell lines KTC-2 and ARO, papillary carcinoma cell lines NPA and TPC-1, and primary human thyroid epithelial culture. The induction of SLP in thyroid cells were identified by: 1) senescence associated beta-Galactosidase (SA--Gal) staining method, 2) Dual flow cytometric analysis of cell proliferation and side light scatter using vital staining with PKH-2 fluorescent dye, 3) Double labeling for 5-Bromodeoxyuridine and SA--Gal, 4) Staining for SA--Gal with consequent anti-Thyroglobulin immunohistochemistry.
ResultsIR induces SA--Gal activity in thyroid cell lines Cells entered the state of replicative senescence become large, flat and more granular in appearance and also positively stain for SA--Galactosidase activity detected at pH 6.0. Similar changes could be observed in phenotypes of normal and cancer cells exposed to different DNA damaging agents, such as IR, H2O2, etc. Thyroid cell lines derived from low differentiated papillary and anaplastic thyroid carcinomas exhibited typical features of senescence-like phenotype 120h after 10Gy exposure (Fig 1A). Induction of SA--Gal activity was mostly observed in large cells with increased granularity and flattened shape. The intensity of cellular staining for SA--Gal increased in a dose (Fig.1B) and time-dependent manner during 5 days after irradiation (data not shown). We also detected that IR induced SA--Gal activity in the primary culture of thyroid follicular cells (Fig. 1C).
ARO
KTC-2
NPA
TPC-1
ANon-irradiated 10Gy/120h
Primary Thyrocytes
TPC-1
NPA
KTC-2
ARO
C0 2 4 6 8 10
01020304050607080
%
-gal
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ARO10Gy
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BrdU Phase-contrast
Merge
S-A-b-Gal- S-A-b-Gal+0
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TPCNPA
KTC-2
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Mea
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S-A-beta-Gal index
B
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Fig.1 IR induces SA--Gal activity in thyroid cell lines and primary thyrocytes
B
0 20 40 60 80-10
01020304050607080
10Gy7.5Gy
5Gy2.5Gy
10Gy
7.5Gy
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% o
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-A-
-Gal
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% of SSChi/PKH-2hi cells
TPC-1 ARO
TPC-1r=0.98, p<0.01
AROr=0.94, p<0.01S
SC
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