Articles in PresS. Am J Physiol Cell Physiol (December 17, 2014). doi:10.1152/ajpcell.00321.2014
1
The topoisomerase II catalytic inhibitor ICRF-193 preferentially targets
2
telomeres that are capped by TRF2
3
Lianxiang Chen1#, Xiaowei Zhu1#, Yaru Zou1, Jun Xing1, Eric Gilson2, Yiming Lu1*
4
and Jing Ye1*
5
1
6
Medicine; Pôle Sino-Français de Recherches en Sciences du Vivant et Génomique,
7
Shanghai Ruijin Hospital, Shanghai 200025, China. 2.Institut for Research on Cancer
8
and Aging, Nice (IRCAN), Nice University, CNRS UMR7284/INSERM U1081,
9
Faculty of Medicine, Nice, France; department of Medical Genetics, Archet 2
10
Hospital, CHU of Nice, France; Pôle Sino-Français de Recherches en Sciences du
11
Vivant et Génomique, Shanghai Ruijin Hospital, Shanghai 200025, China
Shanghai Ruijin Hospital affiliated to Shanghai Jiaotong University, School of
12 13
# These authors contributed equally to this work
14 15
Running Title: TOP II inhibitor regulates telomere function
16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
*Address correspondence to: Dr. Jing Ye Shanghai Ruijin Hospital Shanghai Jiaotong University School of Medicine 197 Ruijin Second Road (Rm. 1110, Building 11) Luwan District, Shanghai, China Phone: 86-13585625087; Fax: 86-21 64671676; Email: [email protected] or Dr. Yiming Lu Shanghai Ruijin Hospital Shanghai Jiaotong University School of Medicine 197 Ruijin Second Road (Rm. 1110, Building 11) Luwan District, Shanghai, China Phone: 86 21 64335180; Fax: 86 21 64335180; Email: [email protected]
Copyright © 2014 by the American Physiological Society.
34
ABSTRACT
35
The increased level of chromosome instability in cancer cells is not only a driving
36
force for oncogenesis but also can be the Achille’s heel of the disease since many
37
chemotherapies kill cells by inducing a non tolerable rate of DNA damage. A wealth
38
of published evidence showed that telomere stability can be more affected than the
39
bulk of the genome by several conventional anti-neoplasic drugs. In the present study,
40
HT1080 cell lines compromised for either TRF2 or POT1 were treated with ICRF-193
41
(3uM, 24H) or Bleomycin (1uM, 24H). DNA damage was assayed by combining
42
telomeric DNA staining of a (CCCTAA) n PNA probe with immuno-fluorescence of
43
53BP1 in order to score the rate of telomeres colocalizing with 53BP1 foci. We found
44
that ICRF-193, but not bleomycin, leads to DNA damage preferentially at telomeres,
45
which can be rescued by TRF2 inhibition. POT1 inhibition exacerbates telomere
46
dysfunction induced by ICRF-193. Thus, ICRF-193 induces damage at telomere
47
properly capped by TRF2 but not by POT1. These findings are expected to broaden
48
our view on the mechanism by which conventional therapeutic molecules act to
49
eliminate cancer cells and how to use TRF2 and POT1 level as surrogate markers for
50
anti-topoisomerase II sensitivity.
51 52 53
Keywords: Topoisomerase II, ICRF-193, telomere, TRF2, POT1
54
INTRODUCTION
55
Telomeres are key features of chromosome termini that preserve genome integrity(5) .
56
In many organisms, their protective functions depend upon at least two pathways. The
57
first relies on telomerase, which can compensate for replicative erosion(8); the second
58
relies on the binding to telomeric DNA of the shelterin protein complex composed of
59
six polypeptides (TRF1, TRF2, RAP1, Tin2, TPP1 and Pot1)(11). Three of the
60
shelterin components recognize directly telomeric DNA; TRF1 and TRF2 bind
61
telomeric DNA duplexes, while Pot1 binds single-stranded 3’ overhangs. Among the
62
shelterin subunits, TRF2(3) is at the heart of the molecular events that maintain
63
telomere integrity in mammals by preventing at telomeres different events normally
64
occurring during DNA damage response : activation of ATM pathway(15),
65
non-homologous
66
Moreover, TRF2 helps folding the telomeric DNA into t-loop, which might contribute
67
to telomere protection by masking the 3’ overhang (1, 12).
end-joining
(NHEJ)(2)
and
homologous
recombination(9).
68
A correct function of telomere seemingly displays antagonistic functions as far as
69
tumorigenesis is concerned(7). On one hand, reactivation of telomerase in cancer cells
70
is crucial for tumour progression. On the other hand, telomere shortening cooperates
71
with p53 deficiency to favour carcinogenesis in aged mice(16). TRF2 appears also to
72
play an important role in oncogenesis as it is up-regulated in several human tumors
73
(4,14) and has been found to exert oncogenic properties when overexpressed in mouse
74
keratinocytes(6). Recently, we showed that the high level of TRF2 found in cancer
75
cells activates the expression of positively regulates the transcription of HS3ST4, a
76
gene encoding an heparan sulfate (glucosamine) 3-O-sulphotransferase that is
77
involved in the recruitment of NK-cells, revealing that TRF2 upregulation can have
78
oncogenic properties through cell non-autnomous mechanisms (4).
79
Inhibition of telomerase provides an interesting “universal” strategy for cancer
80
therapy: immunological and pharmacological inhibition of telomerase is currently
81
used in a series of clinical protocols. However, with many but not all telomerase
82
therapeutic approaches, growth arrest has been observed only when telomeres reach a
83
critically short length. An alternative approach, although still poorly developed, is to
84
target key components of telomere structure such as the use of G4 ligand(17).
85
Interestingly, although DNA damages induced by drugs used in conventional
86
chemotherapy are usually believed to occur stochastically in the genome, a wealth of
87
evidence indicate that telomere stability is more affected than the bulk of the genome
88
by several genotoxic molecules (e.g. cisplatin, spindle poisons as well as
89
anti-topoisomerase I and II and replication inhibitors) (13). For instance, hydroxyurea
90
(HU) is a chemotherapeutic agent commonly used for various malignancies and
91
hematological disorders, including chronic myelogenous leukemia and sickle cell
92
anemia. Chronic, low-level treatment with HU preferentially decreased the rate of
93
telomere DNA synthesis and dissociated TRF2 from telomere DNA. All these indicate
94
that a broad spectrum of genotoxic drugs can have specific DNA damage at telomeres
95
either by binding directly to the DNA structure, or by occupancy the interactive
96
domain of telomeres to specific telomeric nucleoproteins.
97
Collectively, these findings linking conventional chemotherapies to telomeres
98
open new avenues for innovative combinations of chemotherapy drugs, for the use of
99
telomere parameters as surrogate markers for therapy response and toxicity as well as
100
for the introduction of future drugs against telomerase and telomere structural
101
components. Therefore, there is a need for a precise understanding of the impact of
102
genotoxic drugs on telomere functions. In this context, this work was designed to
103
investigate the effects of the topoisomerase II catalytic inhibitor ICRF-193 (meso-2,
104
3-bis (2, 6-dioxopiperazin-4-yl) butane) on telomere functions and conversely to
105
study the impact of specific telomere dysfunctions on ICRF-193 effects. We found
106
that dysfunctional telomeres as a consequence of TRF2 inhibition, but not of POT1
107
inhibition, are less sensitive to ICRF-193 than control ones. This might be due to the
108
fact that TRF2 has seemingly opposed roles regarding telomere topology: it can create
109
topological barriers, rendering telomeres more sensitive to topoisomerase depletion,
110
and it can cooperate with topoisomerase II to prevent replicative damage at
111
telomeres(19). These findings are expected to broaden our view on the mechanism by
112
which conventional therapeutic molecules act to eliminate cancer cells and how to use
113
TRF2 and POT1 level as surrogate markers for anti-topoisomerase II sensitivity.
114
115
MATERIALS AND METHODS
116
Cell Culture, Transfections and Lentiviral or retrovirus production and
117
infections
118
Human Embryonic Kidney 293 phoenix cells and HT1080 fibrosacoma cells were
119
grown in DMEM with 10% fetal calf serum and penicillin-streptomycin at 37ºC.
120
Transfections of plasmid DNA were performed with lipofectamine 2000 reagent
121
(Invitrogen) as described by the protocol. Empty vector or expressing TRF2∆B∆M or
122
shPOT1 vector, were produced by transient transfection of Human Embryonic Kidney
123
293 Phenix cell lines with two other packaging plasmids, p8.91 and pVSVg. 48 h and
124
72 h post transfection, the infectious supernatant was collected and applied to the
125
target cells. The efficiency of infection was determined by flow cytometry analysis of
126
GFP expression. The stable HT1080 cell line with POT1 knockdown induced by
127
shPOT1 was selected by100nM puromycin for 3 three weeks. HT1080 cells treated
128
with ICRF-193 (3uM, 24H), or Bleomycin (1uM, 24H) before performing further
129
experiments.
130 131
Plasmid constructs and siRNAs
132
TRF2∆B∆M was subcloned in pWPIR-GFP lentiviral vector upstream of the IRES in
133
order to allow individual translation of the bicistronic mRNA containing both
134
myc-TRF2∆B∆M and GFP. pLKO.1-puro lentivirus vectors expressing shPOT1 or
135
empty vector as a control were purchased from Sigma.
136 137
Immuno- fluorescence detection
138
Slides were either fixed by MeOH at -20°C, or by 4% formaldehyde at room
139
temperature10 to 15 min, then incubated 1h with blocking buffer (0,8x PBS, 50mM
140
NaCl, 0,5% Triton X100, 3% milk),
followed by 1h of incubation at 37°C with lst
141
antibody Rabbit polyclonal to 53BP1 (NOVUS BIOLOGICALS – NB 100-305) in
142
blocking buffer. Cells were then washed with 0.8xPBS, 50 mM NaCl, 0.1% Trition
143
X-100, followed by Donkey polyclonal anti mouse ALEXA488 (A21202;
144
MOLECULAR PROBES) and Donkey polyclonal anti rabbit ALEXA555 (A-31572;
145
MOLECULAR PROBES) . After washing with 0.8xPBS, 50 mM NaCl, 0.1% Trition
146
X-100, the nucleus was labelled with DAPI and/or TOTO3.
147 148
Combined immuno-fluorescence detection and PNA or DNA fluorescence in situ
149
hybridisation
150
Cells were permeabilised in Triton X-100 buffer (20 mM Tris-HCl pH 8.0, 50 mM
151
NaCl, 3 mM MgCl2, 0.5% Triton X-100 and 300 mM sucrose) at room temperature
152
(RT) for 5 min, followed by 15 min fixation in 3% (para) formaldehyde and 2%
153
sucrose, then again permeabilized in Triton X-100 buffer at RT for 10 min. Cells were
154
blocked in 2% BSA in PBS overnight at 4°C. Then followed IF steps shown above.
155
After final washes, slides were fixed for 2 min. at RT in 4% (para) formaldehyde,
156
followed by dehydration through an ethanol series (50%, 75% and 100%, 5 min. each).
157
Fluorochrome-coupled PNA probe (AATCCC)
158
PNA probe/µl, dissolved in 70% formamide, 10 mM Tris pH 7.2, 1% Blocking
159
Reagent (ROCHE BIOCHEMICALS) first denaturing at 80°C for 3 min, then
160
hybridisation for 2h at RT. Alternatively, slides were hybridised either with the
161
digoxigenin-labeled ITS probe (8ng/µl in 50% formamide / 2xSSC / 10% dextrane
162
sulphate) or with a probe labeling the subtelomeric region of the long arm of
163
chromosome 4 (TelVysion DNA Probes, VYSIS). Washes were the following: 2x15
164
min. at RT in 70% formamide, 10 mM Tris pH 7.2, 3x5 min. in 50 mM Tris pH 7.5,
was then applied to the slides (0.3 ng
165
150 mM NaCl，0.05% Tween-20. Finally, slides were counterstained with DAPI,
166
mounted in VECTASHIELD (VECTOR LABORATORIES).
167 168
Microscopy.
169
Images of metaphase preparations and Cytokinesis-Block micronucleus cytome assay
170
were recorded on an AxioPlan microscope from ZEISS, equipped with a
171
Plan-Apochromat 63x, NA 1.4, oil immersion lens and a cooled CCD camera
172
(CoolSNAP HQ, PHOTOMETRICS). Image acquisition, processing and analysis
173
software were from MetaMorph (MOLECULAR DEVICES).
174
Confocal images were made with the ZEISS LSM-510 confocal scanning laser
175
microscope. Optical sections were recorded at an interval of less than 200nm, with a
176
Plan-Apochromat 63x, NA 1.4, oil immersion lens. Excitation wavelengths were
177
488nm, 543nm and 633nm for the FITC-labeled PNA and DNA probes, for the
178
Alexa555-labeled secondary antibodies and for the TOTO3-stained DNA respectively.
179
Co-localisations were scored in each optical section by scrolling through the z-stack.
180
The appearance of the images was improved by whole-image operations with
181
Photoshop (ADOBE).
182 183
Statistical analysis
184
According to the results of univeriate test, continuous normal variables were
185
expressed as mean value±SD. Categorical variables were expressed as percentage;
186
comparison between these groups were analyzed by Chi-Square test. Parametric
187
variables of normal distribution were analyzed either by 2-tail T-test, or by F test of
188
ANVOA followed by Duncan test for each two group comparison. Results were
189
considered significant at p
1
The topoisomerase II catalytic inhibitor ICRF-193 preferentially targets
2
telomeres that are capped by TRF2
3
Lianxiang Chen1#, Xiaowei Zhu1#, Yaru Zou1, Jun Xing1, Eric Gilson2, Yiming Lu1*
4
and Jing Ye1*
5
1
6
Medicine; Pôle Sino-Français de Recherches en Sciences du Vivant et Génomique,
7
Shanghai Ruijin Hospital, Shanghai 200025, China. 2.Institut for Research on Cancer
8
and Aging, Nice (IRCAN), Nice University, CNRS UMR7284/INSERM U1081,
9
Faculty of Medicine, Nice, France; department of Medical Genetics, Archet 2
10
Hospital, CHU of Nice, France; Pôle Sino-Français de Recherches en Sciences du
11
Vivant et Génomique, Shanghai Ruijin Hospital, Shanghai 200025, China
Shanghai Ruijin Hospital affiliated to Shanghai Jiaotong University, School of
12 13
# These authors contributed equally to this work
14 15
Running Title: TOP II inhibitor regulates telomere function
16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
*Address correspondence to: Dr. Jing Ye Shanghai Ruijin Hospital Shanghai Jiaotong University School of Medicine 197 Ruijin Second Road (Rm. 1110, Building 11) Luwan District, Shanghai, China Phone: 86-13585625087; Fax: 86-21 64671676; Email: [email protected] or Dr. Yiming Lu Shanghai Ruijin Hospital Shanghai Jiaotong University School of Medicine 197 Ruijin Second Road (Rm. 1110, Building 11) Luwan District, Shanghai, China Phone: 86 21 64335180; Fax: 86 21 64335180; Email: [email protected]
Copyright © 2014 by the American Physiological Society.
34
ABSTRACT
35
The increased level of chromosome instability in cancer cells is not only a driving
36
force for oncogenesis but also can be the Achille’s heel of the disease since many
37
chemotherapies kill cells by inducing a non tolerable rate of DNA damage. A wealth
38
of published evidence showed that telomere stability can be more affected than the
39
bulk of the genome by several conventional anti-neoplasic drugs. In the present study,
40
HT1080 cell lines compromised for either TRF2 or POT1 were treated with ICRF-193
41
(3uM, 24H) or Bleomycin (1uM, 24H). DNA damage was assayed by combining
42
telomeric DNA staining of a (CCCTAA) n PNA probe with immuno-fluorescence of
43
53BP1 in order to score the rate of telomeres colocalizing with 53BP1 foci. We found
44
that ICRF-193, but not bleomycin, leads to DNA damage preferentially at telomeres,
45
which can be rescued by TRF2 inhibition. POT1 inhibition exacerbates telomere
46
dysfunction induced by ICRF-193. Thus, ICRF-193 induces damage at telomere
47
properly capped by TRF2 but not by POT1. These findings are expected to broaden
48
our view on the mechanism by which conventional therapeutic molecules act to
49
eliminate cancer cells and how to use TRF2 and POT1 level as surrogate markers for
50
anti-topoisomerase II sensitivity.
51 52 53
Keywords: Topoisomerase II, ICRF-193, telomere, TRF2, POT1
54
INTRODUCTION
55
Telomeres are key features of chromosome termini that preserve genome integrity(5) .
56
In many organisms, their protective functions depend upon at least two pathways. The
57
first relies on telomerase, which can compensate for replicative erosion(8); the second
58
relies on the binding to telomeric DNA of the shelterin protein complex composed of
59
six polypeptides (TRF1, TRF2, RAP1, Tin2, TPP1 and Pot1)(11). Three of the
60
shelterin components recognize directly telomeric DNA; TRF1 and TRF2 bind
61
telomeric DNA duplexes, while Pot1 binds single-stranded 3’ overhangs. Among the
62
shelterin subunits, TRF2(3) is at the heart of the molecular events that maintain
63
telomere integrity in mammals by preventing at telomeres different events normally
64
occurring during DNA damage response : activation of ATM pathway(15),
65
non-homologous
66
Moreover, TRF2 helps folding the telomeric DNA into t-loop, which might contribute
67
to telomere protection by masking the 3’ overhang (1, 12).
end-joining
(NHEJ)(2)
and
homologous
recombination(9).
68
A correct function of telomere seemingly displays antagonistic functions as far as
69
tumorigenesis is concerned(7). On one hand, reactivation of telomerase in cancer cells
70
is crucial for tumour progression. On the other hand, telomere shortening cooperates
71
with p53 deficiency to favour carcinogenesis in aged mice(16). TRF2 appears also to
72
play an important role in oncogenesis as it is up-regulated in several human tumors
73
(4,14) and has been found to exert oncogenic properties when overexpressed in mouse
74
keratinocytes(6). Recently, we showed that the high level of TRF2 found in cancer
75
cells activates the expression of positively regulates the transcription of HS3ST4, a
76
gene encoding an heparan sulfate (glucosamine) 3-O-sulphotransferase that is
77
involved in the recruitment of NK-cells, revealing that TRF2 upregulation can have
78
oncogenic properties through cell non-autnomous mechanisms (4).
79
Inhibition of telomerase provides an interesting “universal” strategy for cancer
80
therapy: immunological and pharmacological inhibition of telomerase is currently
81
used in a series of clinical protocols. However, with many but not all telomerase
82
therapeutic approaches, growth arrest has been observed only when telomeres reach a
83
critically short length. An alternative approach, although still poorly developed, is to
84
target key components of telomere structure such as the use of G4 ligand(17).
85
Interestingly, although DNA damages induced by drugs used in conventional
86
chemotherapy are usually believed to occur stochastically in the genome, a wealth of
87
evidence indicate that telomere stability is more affected than the bulk of the genome
88
by several genotoxic molecules (e.g. cisplatin, spindle poisons as well as
89
anti-topoisomerase I and II and replication inhibitors) (13). For instance, hydroxyurea
90
(HU) is a chemotherapeutic agent commonly used for various malignancies and
91
hematological disorders, including chronic myelogenous leukemia and sickle cell
92
anemia. Chronic, low-level treatment with HU preferentially decreased the rate of
93
telomere DNA synthesis and dissociated TRF2 from telomere DNA. All these indicate
94
that a broad spectrum of genotoxic drugs can have specific DNA damage at telomeres
95
either by binding directly to the DNA structure, or by occupancy the interactive
96
domain of telomeres to specific telomeric nucleoproteins.
97
Collectively, these findings linking conventional chemotherapies to telomeres
98
open new avenues for innovative combinations of chemotherapy drugs, for the use of
99
telomere parameters as surrogate markers for therapy response and toxicity as well as
100
for the introduction of future drugs against telomerase and telomere structural
101
components. Therefore, there is a need for a precise understanding of the impact of
102
genotoxic drugs on telomere functions. In this context, this work was designed to
103
investigate the effects of the topoisomerase II catalytic inhibitor ICRF-193 (meso-2,
104
3-bis (2, 6-dioxopiperazin-4-yl) butane) on telomere functions and conversely to
105
study the impact of specific telomere dysfunctions on ICRF-193 effects. We found
106
that dysfunctional telomeres as a consequence of TRF2 inhibition, but not of POT1
107
inhibition, are less sensitive to ICRF-193 than control ones. This might be due to the
108
fact that TRF2 has seemingly opposed roles regarding telomere topology: it can create
109
topological barriers, rendering telomeres more sensitive to topoisomerase depletion,
110
and it can cooperate with topoisomerase II to prevent replicative damage at
111
telomeres(19). These findings are expected to broaden our view on the mechanism by
112
which conventional therapeutic molecules act to eliminate cancer cells and how to use
113
TRF2 and POT1 level as surrogate markers for anti-topoisomerase II sensitivity.
114
115
MATERIALS AND METHODS
116
Cell Culture, Transfections and Lentiviral or retrovirus production and
117
infections
118
Human Embryonic Kidney 293 phoenix cells and HT1080 fibrosacoma cells were
119
grown in DMEM with 10% fetal calf serum and penicillin-streptomycin at 37ºC.
120
Transfections of plasmid DNA were performed with lipofectamine 2000 reagent
121
(Invitrogen) as described by the protocol. Empty vector or expressing TRF2∆B∆M or
122
shPOT1 vector, were produced by transient transfection of Human Embryonic Kidney
123
293 Phenix cell lines with two other packaging plasmids, p8.91 and pVSVg. 48 h and
124
72 h post transfection, the infectious supernatant was collected and applied to the
125
target cells. The efficiency of infection was determined by flow cytometry analysis of
126
GFP expression. The stable HT1080 cell line with POT1 knockdown induced by
127
shPOT1 was selected by100nM puromycin for 3 three weeks. HT1080 cells treated
128
with ICRF-193 (3uM, 24H), or Bleomycin (1uM, 24H) before performing further
129
experiments.
130 131
Plasmid constructs and siRNAs
132
TRF2∆B∆M was subcloned in pWPIR-GFP lentiviral vector upstream of the IRES in
133
order to allow individual translation of the bicistronic mRNA containing both
134
myc-TRF2∆B∆M and GFP. pLKO.1-puro lentivirus vectors expressing shPOT1 or
135
empty vector as a control were purchased from Sigma.
136 137
Immuno- fluorescence detection
138
Slides were either fixed by MeOH at -20°C, or by 4% formaldehyde at room
139
temperature10 to 15 min, then incubated 1h with blocking buffer (0,8x PBS, 50mM
140
NaCl, 0,5% Triton X100, 3% milk),
followed by 1h of incubation at 37°C with lst
141
antibody Rabbit polyclonal to 53BP1 (NOVUS BIOLOGICALS – NB 100-305) in
142
blocking buffer. Cells were then washed with 0.8xPBS, 50 mM NaCl, 0.1% Trition
143
X-100, followed by Donkey polyclonal anti mouse ALEXA488 (A21202;
144
MOLECULAR PROBES) and Donkey polyclonal anti rabbit ALEXA555 (A-31572;
145
MOLECULAR PROBES) . After washing with 0.8xPBS, 50 mM NaCl, 0.1% Trition
146
X-100, the nucleus was labelled with DAPI and/or TOTO3.
147 148
Combined immuno-fluorescence detection and PNA or DNA fluorescence in situ
149
hybridisation
150
Cells were permeabilised in Triton X-100 buffer (20 mM Tris-HCl pH 8.0, 50 mM
151
NaCl, 3 mM MgCl2, 0.5% Triton X-100 and 300 mM sucrose) at room temperature
152
(RT) for 5 min, followed by 15 min fixation in 3% (para) formaldehyde and 2%
153
sucrose, then again permeabilized in Triton X-100 buffer at RT for 10 min. Cells were
154
blocked in 2% BSA in PBS overnight at 4°C. Then followed IF steps shown above.
155
After final washes, slides were fixed for 2 min. at RT in 4% (para) formaldehyde,
156
followed by dehydration through an ethanol series (50%, 75% and 100%, 5 min. each).
157
Fluorochrome-coupled PNA probe (AATCCC)
158
PNA probe/µl, dissolved in 70% formamide, 10 mM Tris pH 7.2, 1% Blocking
159
Reagent (ROCHE BIOCHEMICALS) first denaturing at 80°C for 3 min, then
160
hybridisation for 2h at RT. Alternatively, slides were hybridised either with the
161
digoxigenin-labeled ITS probe (8ng/µl in 50% formamide / 2xSSC / 10% dextrane
162
sulphate) or with a probe labeling the subtelomeric region of the long arm of
163
chromosome 4 (TelVysion DNA Probes, VYSIS). Washes were the following: 2x15
164
min. at RT in 70% formamide, 10 mM Tris pH 7.2, 3x5 min. in 50 mM Tris pH 7.5,
was then applied to the slides (0.3 ng
165
150 mM NaCl，0.05% Tween-20. Finally, slides were counterstained with DAPI,
166
mounted in VECTASHIELD (VECTOR LABORATORIES).
167 168
Microscopy.
169
Images of metaphase preparations and Cytokinesis-Block micronucleus cytome assay
170
were recorded on an AxioPlan microscope from ZEISS, equipped with a
171
Plan-Apochromat 63x, NA 1.4, oil immersion lens and a cooled CCD camera
172
(CoolSNAP HQ, PHOTOMETRICS). Image acquisition, processing and analysis
173
software were from MetaMorph (MOLECULAR DEVICES).
174
Confocal images were made with the ZEISS LSM-510 confocal scanning laser
175
microscope. Optical sections were recorded at an interval of less than 200nm, with a
176
Plan-Apochromat 63x, NA 1.4, oil immersion lens. Excitation wavelengths were
177
488nm, 543nm and 633nm for the FITC-labeled PNA and DNA probes, for the
178
Alexa555-labeled secondary antibodies and for the TOTO3-stained DNA respectively.
179
Co-localisations were scored in each optical section by scrolling through the z-stack.
180
The appearance of the images was improved by whole-image operations with
181
Photoshop (ADOBE).
182 183
Statistical analysis
184
According to the results of univeriate test, continuous normal variables were
185
expressed as mean value±SD. Categorical variables were expressed as percentage;
186
comparison between these groups were analyzed by Chi-Square test. Parametric
187
variables of normal distribution were analyzed either by 2-tail T-test, or by F test of
188
ANVOA followed by Duncan test for each two group comparison. Results were
189
considered significant at p
