JVI Accepts, published online ahead of print on 13 August 2014 J. Virol. doi:10.1128/JVI.02107-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
1
Inhibition of breast cancer cell proliferation through disturbance of the
2
Calcineurin/NFAT pathway by Human Herpesvirus-6B U54 tegument
3
protein
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Mathieu Iampietroa, Annie Gravela, Louis Flamanda,b #
a
Division of infectious and immune diseases, CHU de Quebec Research Center, Quebec
city, Canada; b Department of Microbiology, Infectious Diseases and Immunology, Faculty of Medicine, Laval University, Quebec city, Canada.
Running Title: HHV-6B U54 protein inhibits breast cancer progression # Address correspondence to:
Louis Flamand PhD MBA Room T1-49 Division of infectious and immune diseases CHU de Quebec Research Center, Quebec city, Canada G1V 4G2 Tel: 418-525-4444 (x46164) Fax: 418-654-2765 Email:[email protected]
Abstract: 69 words Text: 1029 words
38
1
39
ABSTRACT
40
Nuclear factor of activated T cell (NFAT) proteins are key regulators involved in multiple
41
physiological mechanisms such as immune response or cell growth. Selective calcineurin /NFAT
42
inhibitors already demonstrated their capacity to decrease NFAT-dependent cancer cell
43
progression, particularly in breast cancer. In this study, we report a role for the human
44
herpesvirus 6B (HHV-6B) U54 tegument protein in inhibiting MCF-7 breast cancer cell line
45
proliferation by inhibiting NFAT activation.
46
RESULTS
47
The NFAT transcription factors include five elements (NFAT1-NFAT5) (4, 12) in which
48
four of them are regulated by Ca2+ (NFAT1-NFAT4) (6, 7). In resting cells, NFAT proteins are
49
hyperphosphorylated and remain cytoplasmic. Upon cell stimulation and rises in intracellular
50
Ca2+ concentrations, the Ca2+/calmodulin-dependent serine phosphatase calcineurin (CaN) (10)
51
becomes active, recruits and dephosphorylates NFAT exposing a nuclear localization signal
52
allowing NFAT to migrate to the nucleus and enhance the expression of several genes (5, 13, 27,
53
29).
54
Disorders of CaN/NFAT pathway cause disturbances in adaptative immune responses,
55
cell differentiation or cell proliferation (15). Selective inhibitors known to prevent CaN/NFAT
56
interaction (30) and subsequent NFAT activation include drugs such as cyclosporine A (CsA)
57
(21) and FK506 (Tacrolimus) (9), inhibitory peptides (1, 2) or proteins expressed by pathogens
58
such as A238L protein of African swine fever virus (24, 25). Recently, Iampietro et al identified
59
the HHV-6B U54 tegument protein as being capable of inhibiting NFAT activation and
60
subsequent IL-2 gene expression, pointing out for a role of the U54 protein in immune evasion
2
61
(17). Considering that the functions of NFAT extend beyond the development of the adaptive
62
immune response, we evaluated the effects of U54 expression of in the proliferation of cells
63
whose growth is NFAT-dependent.
64
Breast cancer is the leading cause of cancer death in women worldwide (16), and is
65
caused by disturbance of NFAT activity (18, 32) by promoting cell transformation, proliferation,
66
invasion and tumor angiogenesis (20, 23). Relative expression of NFAT members in MCF-7 cells
67
is presented under table 1. We used the MCF-7 breast cancer cell line to test the possible
68
inhibitory effects of HHV-6B U54 protein on cell proliferation. To achieve this goal, MCF-7
69
cells (2x105) were transfected with expression vectors 4TO, 4TO-U54 (encoding WT U54), 4TO-
70
U54mut (IT296-297AA mutant with reduced NFAT inhibitory potential), 4TO-U11 (encoding
71
WT U11) and NFAT-Luc reporter plasmids, as described in Iampietro et al (17). After 48h, cells
72
were stimulated or not with TPA 25 ng/ml and ionomycin 0.5μM (TPA/ionomycin) to induce
73
CaN/NFAT pathway for an additional 24h. Luciferase activity was determined and normalized
74
with protein content (n=4) as described in Iampietro et al (17). Treatment with TPA/ionomycin
75
activated endogenous NFAT resulting in a 5x fold increase (P
76
while in cells expressing U54 showed a 70% reduction in luciferase activity (P
77
treated with CsA 5μg/ml were used as positive control. Expression of U54mut or U11, a second
78
HHV-6 tegument protein, had marginal effects on reporter activity. Protein expression was
79
monitored by western blot analysis with beta-actin as loading controls. Next, we wanted to
80
determine whether the U54 inhibitory activity would translate in a physiological effect such as
81
reduced cell growth. We transfected 293T and MCF-7 cells (1x105) with the same plasmids as
82
above and cultured the cells for 96h. CsA or FK506 5 μg/ml were used as positive inhibitory
83
controls. Transfection efficiencies were determined for several wells (n=6) using a GFP reporter
0.0001) in luciferase activity 0.0001). Cells
3
84
vector and found to be equivalent (not shown). Cells were counted every 24h for four days using
85
an automatic cellometer auto T4 cell counter (Nexcelcom, Lawrence, MA). After 72h and 96h,
86
4TO-transfected MCF-7 cells grew approximately 4.5x and 7.5x, respectively (P
87
(figure 2A). Cells transfected with 4TO-U54mut or 4TO-U11 showed proliferation equivalent as
88
4TO control cells. In contrast, at 72 and 96h post-transfection, MCF-7 proliferation was
89
significantly inhibited by U54 (figure 2A). Similar results were obtained with FK-506 (figure
90
2B). 293T cells, which do not rely on NFAT for proliferation (used as controls), were not
91
affected by CsA or U54 expression (figure 2C). Protein expression was monitored by western
92
blot analysis. We next determined how the U54 protein would cause NFAT inactivation, leading
93
to cell growth inhibition. To highlight this mechanism, we analyzed the phosphorylation status of
94
ectopically expressed NFAT1 detected with an antibody detecting the hyperphosphorylated forms
95
(140 kDa) of NFAT1. MCF-7 cells (1,5x105) were transfected with 4TO, 4TO-U54, 4TO-U11,
96
4TO-U54mut and REP-NFAT1 plasmids. After 48h, cells were stimulated or not with
97
TPA/ionomycin for 10 min. Cells pretreated with CsA 10 μg/ml were used as a positive control
98
for inhibition of NFAT dephosphorylation. As shown in figure 3, under resting condition,
99
NFAT1
was
hyperphosphorylated,
as
expected.
In
control-transfected
0.001) (n=4)
cells
(4TO),
100
TPA/ionomycin induced the dephosphorylation of NFAT1, as U11 and U54mut expressing cells.
101
In contrast, in the presence of U54 or CsA, NFAT1 remained hyperphosphorylated (figure 3A).
102
Densitometric analyses were used to quantify the relative levels of NFAT1 phosphorylation
103
(figure 3B).
104
Lastly, we studied whether U54 would impact the expression of a gene, such as COX-2,
105
whose expression partly depends on NFAT and whose role in cancer progression is well
106
documented (8, 28, 31). Under the same conditions tested in figure 3, we evaluated COX-2
4
107
mRNA levels following a 24h stimulation with TPA/ionomycin. A 12-14x fold increase in COX-
108
2 mRNA was recorded in 4TO, U11 and U54mut transfected cells (P
109
expressing cells or cells treated with CsA showed significantly reduced COX-2 mRNA. (P
110
0.0001) (n=4) (figure 4A). Protein expression was monitored by western blot analysis. These
111
results confirm the capacity of HHV-6B U54 protein to abrogate NFAT activation and
112
subsequent MCF-7 breast cancer cell line proliferation.
0.001) (n=4) while U54
113
In a previous work, we identified HHV-6B U54 tegument protein as a new viral protein
114
inhibiting the CaN/NFAT pathway (17), likely favoring immune escape and enhancing viral
115
infection. In this study, we have extended these observations by highlighting the capacity of U54
116
to inhibit breast cancer cell growth. Cancer immunotherapy protocols have demonstrated some
117
effectiveness against virally-induced disorders (14) such as in EBV post transplant
118
lymphoproliferative disorders (3, 22) but remain a big challenge against solid tumors such as
119
breast cancer. Immunodominant epitopes derived from the HHV-6B U54 protein have been
120
described recently (11, 26). Coupled to our observation that U54 can inhibit cancer cell growth,
121
expression of U54 in breast cancer cells could give a double advantage: first by liming the cancer
122
cell proliferations and second, by “marking” them for anti-U54 CD8 T cell recognition and
123
destruction. However, before such therapy can be envisionned, several more studies are needed
124
to identify a reliable gene therapy approach allowing the specific expression of U54 in breast
125
cancer cells.
126
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Miskin, J. E., C. C. Abrams, L. C. Goatley, and L. K. Dixon. 1998. A viral mechanism for inhibition of the cellular phosphatase calcineurin. Science 281:562-5.
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Rao, A., C. Luo, and P. G. Hogan. 1997. Transcription factors of the NFAT family: regulation and function. Annu Rev Immunol 15:707-47.
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Serfling, E., F. Berberich-Siebelt, S. Chuvpilo, E. Jankevics, S. Klein-Hessling, T.
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Twardzik, and A. Avots. 2000. The role of NF-AT transcription factors in T cell
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Sieber, M., and R. Baumgrass. 2009. Novel inhibitors of the calcineurin/NFATc hub -
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2005. Akt blocks breast cancer cell motility and invasion through the transcription factor
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NFAT. Mol Cell 20:539-50.
211 212 213 214 215 216 217 218 219 220 221 9
222 223 224
LEGENDS
225
Table 1: Relative expression of NFAT transcripts in MCF7 and Jurkat cell lines
226
Expression of NFAT transcripts in MCF7 and Jurkat cell lines was evaluated by RT-PCR assay.
227
MCF7 and Jurkat cells were lysed using Qiazol lysis reagent. RNA was extracted and RT was
228
performed to obtain cDNAs that were tested with specific primers to evaluate expression of
229
mRNAa. Primers used are NFAT1 forward 5’- CGA AGA AGA GCC GAA TGC AC -3’ and
230
NFAT1 reverse 5’- AGA AAC TTC TGC GGC CCT AC -3’, NFAT2 forward 5’- CAC TCC
231
TGC TGC CTT ACA CA -3’ and NFAT2 reverse 5’- AAG ATG CGA GCA TGC GAC TA -3’,
232
NFAT3 forward 5’- CGG CCT CTA AGA GAG GTT GA -3’ and NFAT3 reverse 5’- CCT CCT
233
TTT CCT CCC CGA AC -3’ while primers used for GAPDH are described in Jaworska et al(19).
234
Figure 1: U54 inhibits endogenous NFAT transcriptional activity in MCF-7 cells
235
Transcriptional activity of endogenous-expressed NFAT factors was evaluated in MCF-7 cells by
236
luciferase assay. A) MCF-7 cells were transfected with 4TO, 4TO-U54, 4TO-U11, 4TO-U54mut
237
and NFAT-Luc reporter plasmids. As positive control, we pretreated 4TO condition with CsA.
238
Luciferase activity was determined and normalized to protein content (n=4) Western blot analysis
239
confirmed the expression of proteins of interest for each condition tested. Beta-actin was included
240
as loading control.
241
Figure 2: U54 specifically reduces MCF-7 breast cancer cells growth
242
A-B) MCF-7 cells and –C) 293T cells were transfected with 4TO, 4TO-U54, 4TO-U11 and 4TO-
243
U54mut plasmids. As control, we pretreated 4TO condition with CsA (A-C) or FK506 (B). Cells 10
244
were harvested and evaluated at 24h, 48h, 72h and 96h following transfection by cell counting
245
assay (n=4). Western blot analysis confirmed the expression of proteins of interest for each
246
condition tested. Beta-actin was included as loading control.
247
Figure 3: U54 inhibits dephosphorylation of NFAT1 protein.
248
NFAT1 dephosphorylation was evaluated in MCF-7 cells by Western blot assay. A) MCF-7 cells
249
were transfected with 4TO, 4TO-U54, 4TO-U11, 4TO-U54mut and REP-NFAT1 plasmids. As
250
positive control, 4TO transfected cells we pretreated with CsA. Western blot analyses were
251
performed by blotting each condition with a phospho specific anti-NFAT1 antibody (140 kDa)
252
(upper panel). Western blot analysis confirmed expression of U54, U54mut or U11 proteins
253
(middle panels). Beta-actin was included as loading control. B) Densitometric analysis of P-
254
NFAT1 was performed following western blot analysis. The P-NFAT1 level in resting cells
255
(4TO) was set at 100%. Following TPA/ionomycin stimulation, P-NFAT1 levels were compared
256
to their respective resting control (one representative experiment of three independent
257
experiments).
258
Figure 4: U54 reduces COX-2 gene transcription in MCF-7 cell line
259
A) MCF-7 cells were transfected with 4TO, 4TO-U54, 4TO-U11 and 4TO-U54mut plasmids for
260
48h before being stimulated or not with TPA/ionomycin for an additional 24h. RNA was
261
extracted and COX-2 mRNA levels determined by real-time RT-QPCR. The primers used for
262
COX-2 detection were COX-2 forward 5’- TGC ATT CTT TGC CCA GCA CT -3’ and COX-2
263
reverse 5’- AAA GGC GCA GTT TAC GCT GT -3’. The primers for GAPDH, used for
264
normalization were previously described (19). B) Western blot analysis confirmed the expression
265
of U54 and U54mut proteins. Beta-actin was included as loading control.
11
266 267 268 269 270 271 272 273 274
Table 1: Relative expression of NFAT1, 2, 3 in MCF7 and Jurkat T cells.
275 276
- denotes absence of expression; + denotes basal expression level; ++ denotes moderate
277
expression level; +++ denotes high expression level.
278
12
Figure 2 A
B
!
!
!
!
C
!
!
1
Inhibition of breast cancer cell proliferation through disturbance of the
2
Calcineurin/NFAT pathway by Human Herpesvirus-6B U54 tegument
3
protein
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Mathieu Iampietroa, Annie Gravela, Louis Flamanda,b #
a
Division of infectious and immune diseases, CHU de Quebec Research Center, Quebec
city, Canada; b Department of Microbiology, Infectious Diseases and Immunology, Faculty of Medicine, Laval University, Quebec city, Canada.
Running Title: HHV-6B U54 protein inhibits breast cancer progression # Address correspondence to:
Louis Flamand PhD MBA Room T1-49 Division of infectious and immune diseases CHU de Quebec Research Center, Quebec city, Canada G1V 4G2 Tel: 418-525-4444 (x46164) Fax: 418-654-2765 Email:[email protected]
Abstract: 69 words Text: 1029 words
38
1
39
ABSTRACT
40
Nuclear factor of activated T cell (NFAT) proteins are key regulators involved in multiple
41
physiological mechanisms such as immune response or cell growth. Selective calcineurin /NFAT
42
inhibitors already demonstrated their capacity to decrease NFAT-dependent cancer cell
43
progression, particularly in breast cancer. In this study, we report a role for the human
44
herpesvirus 6B (HHV-6B) U54 tegument protein in inhibiting MCF-7 breast cancer cell line
45
proliferation by inhibiting NFAT activation.
46
RESULTS
47
The NFAT transcription factors include five elements (NFAT1-NFAT5) (4, 12) in which
48
four of them are regulated by Ca2+ (NFAT1-NFAT4) (6, 7). In resting cells, NFAT proteins are
49
hyperphosphorylated and remain cytoplasmic. Upon cell stimulation and rises in intracellular
50
Ca2+ concentrations, the Ca2+/calmodulin-dependent serine phosphatase calcineurin (CaN) (10)
51
becomes active, recruits and dephosphorylates NFAT exposing a nuclear localization signal
52
allowing NFAT to migrate to the nucleus and enhance the expression of several genes (5, 13, 27,
53
29).
54
Disorders of CaN/NFAT pathway cause disturbances in adaptative immune responses,
55
cell differentiation or cell proliferation (15). Selective inhibitors known to prevent CaN/NFAT
56
interaction (30) and subsequent NFAT activation include drugs such as cyclosporine A (CsA)
57
(21) and FK506 (Tacrolimus) (9), inhibitory peptides (1, 2) or proteins expressed by pathogens
58
such as A238L protein of African swine fever virus (24, 25). Recently, Iampietro et al identified
59
the HHV-6B U54 tegument protein as being capable of inhibiting NFAT activation and
60
subsequent IL-2 gene expression, pointing out for a role of the U54 protein in immune evasion
2
61
(17). Considering that the functions of NFAT extend beyond the development of the adaptive
62
immune response, we evaluated the effects of U54 expression of in the proliferation of cells
63
whose growth is NFAT-dependent.
64
Breast cancer is the leading cause of cancer death in women worldwide (16), and is
65
caused by disturbance of NFAT activity (18, 32) by promoting cell transformation, proliferation,
66
invasion and tumor angiogenesis (20, 23). Relative expression of NFAT members in MCF-7 cells
67
is presented under table 1. We used the MCF-7 breast cancer cell line to test the possible
68
inhibitory effects of HHV-6B U54 protein on cell proliferation. To achieve this goal, MCF-7
69
cells (2x105) were transfected with expression vectors 4TO, 4TO-U54 (encoding WT U54), 4TO-
70
U54mut (IT296-297AA mutant with reduced NFAT inhibitory potential), 4TO-U11 (encoding
71
WT U11) and NFAT-Luc reporter plasmids, as described in Iampietro et al (17). After 48h, cells
72
were stimulated or not with TPA 25 ng/ml and ionomycin 0.5μM (TPA/ionomycin) to induce
73
CaN/NFAT pathway for an additional 24h. Luciferase activity was determined and normalized
74
with protein content (n=4) as described in Iampietro et al (17). Treatment with TPA/ionomycin
75
activated endogenous NFAT resulting in a 5x fold increase (P
76
while in cells expressing U54 showed a 70% reduction in luciferase activity (P
77
treated with CsA 5μg/ml were used as positive control. Expression of U54mut or U11, a second
78
HHV-6 tegument protein, had marginal effects on reporter activity. Protein expression was
79
monitored by western blot analysis with beta-actin as loading controls. Next, we wanted to
80
determine whether the U54 inhibitory activity would translate in a physiological effect such as
81
reduced cell growth. We transfected 293T and MCF-7 cells (1x105) with the same plasmids as
82
above and cultured the cells for 96h. CsA or FK506 5 μg/ml were used as positive inhibitory
83
controls. Transfection efficiencies were determined for several wells (n=6) using a GFP reporter
0.0001) in luciferase activity 0.0001). Cells
3
84
vector and found to be equivalent (not shown). Cells were counted every 24h for four days using
85
an automatic cellometer auto T4 cell counter (Nexcelcom, Lawrence, MA). After 72h and 96h,
86
4TO-transfected MCF-7 cells grew approximately 4.5x and 7.5x, respectively (P
87
(figure 2A). Cells transfected with 4TO-U54mut or 4TO-U11 showed proliferation equivalent as
88
4TO control cells. In contrast, at 72 and 96h post-transfection, MCF-7 proliferation was
89
significantly inhibited by U54 (figure 2A). Similar results were obtained with FK-506 (figure
90
2B). 293T cells, which do not rely on NFAT for proliferation (used as controls), were not
91
affected by CsA or U54 expression (figure 2C). Protein expression was monitored by western
92
blot analysis. We next determined how the U54 protein would cause NFAT inactivation, leading
93
to cell growth inhibition. To highlight this mechanism, we analyzed the phosphorylation status of
94
ectopically expressed NFAT1 detected with an antibody detecting the hyperphosphorylated forms
95
(140 kDa) of NFAT1. MCF-7 cells (1,5x105) were transfected with 4TO, 4TO-U54, 4TO-U11,
96
4TO-U54mut and REP-NFAT1 plasmids. After 48h, cells were stimulated or not with
97
TPA/ionomycin for 10 min. Cells pretreated with CsA 10 μg/ml were used as a positive control
98
for inhibition of NFAT dephosphorylation. As shown in figure 3, under resting condition,
99
NFAT1
was
hyperphosphorylated,
as
expected.
In
control-transfected
0.001) (n=4)
cells
(4TO),
100
TPA/ionomycin induced the dephosphorylation of NFAT1, as U11 and U54mut expressing cells.
101
In contrast, in the presence of U54 or CsA, NFAT1 remained hyperphosphorylated (figure 3A).
102
Densitometric analyses were used to quantify the relative levels of NFAT1 phosphorylation
103
(figure 3B).
104
Lastly, we studied whether U54 would impact the expression of a gene, such as COX-2,
105
whose expression partly depends on NFAT and whose role in cancer progression is well
106
documented (8, 28, 31). Under the same conditions tested in figure 3, we evaluated COX-2
4
107
mRNA levels following a 24h stimulation with TPA/ionomycin. A 12-14x fold increase in COX-
108
2 mRNA was recorded in 4TO, U11 and U54mut transfected cells (P
109
expressing cells or cells treated with CsA showed significantly reduced COX-2 mRNA. (P
110
0.0001) (n=4) (figure 4A). Protein expression was monitored by western blot analysis. These
111
results confirm the capacity of HHV-6B U54 protein to abrogate NFAT activation and
112
subsequent MCF-7 breast cancer cell line proliferation.
0.001) (n=4) while U54
113
In a previous work, we identified HHV-6B U54 tegument protein as a new viral protein
114
inhibiting the CaN/NFAT pathway (17), likely favoring immune escape and enhancing viral
115
infection. In this study, we have extended these observations by highlighting the capacity of U54
116
to inhibit breast cancer cell growth. Cancer immunotherapy protocols have demonstrated some
117
effectiveness against virally-induced disorders (14) such as in EBV post transplant
118
lymphoproliferative disorders (3, 22) but remain a big challenge against solid tumors such as
119
breast cancer. Immunodominant epitopes derived from the HHV-6B U54 protein have been
120
described recently (11, 26). Coupled to our observation that U54 can inhibit cancer cell growth,
121
expression of U54 in breast cancer cells could give a double advantage: first by liming the cancer
122
cell proliferations and second, by “marking” them for anti-U54 CD8 T cell recognition and
123
destruction. However, before such therapy can be envisionned, several more studies are needed
124
to identify a reliable gene therapy approach allowing the specific expression of U54 in breast
125
cancer cells.
126
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NFAT. Mol Cell 20:539-50.
211 212 213 214 215 216 217 218 219 220 221 9
222 223 224
LEGENDS
225
Table 1: Relative expression of NFAT transcripts in MCF7 and Jurkat cell lines
226
Expression of NFAT transcripts in MCF7 and Jurkat cell lines was evaluated by RT-PCR assay.
227
MCF7 and Jurkat cells were lysed using Qiazol lysis reagent. RNA was extracted and RT was
228
performed to obtain cDNAs that were tested with specific primers to evaluate expression of
229
mRNAa. Primers used are NFAT1 forward 5’- CGA AGA AGA GCC GAA TGC AC -3’ and
230
NFAT1 reverse 5’- AGA AAC TTC TGC GGC CCT AC -3’, NFAT2 forward 5’- CAC TCC
231
TGC TGC CTT ACA CA -3’ and NFAT2 reverse 5’- AAG ATG CGA GCA TGC GAC TA -3’,
232
NFAT3 forward 5’- CGG CCT CTA AGA GAG GTT GA -3’ and NFAT3 reverse 5’- CCT CCT
233
TTT CCT CCC CGA AC -3’ while primers used for GAPDH are described in Jaworska et al(19).
234
Figure 1: U54 inhibits endogenous NFAT transcriptional activity in MCF-7 cells
235
Transcriptional activity of endogenous-expressed NFAT factors was evaluated in MCF-7 cells by
236
luciferase assay. A) MCF-7 cells were transfected with 4TO, 4TO-U54, 4TO-U11, 4TO-U54mut
237
and NFAT-Luc reporter plasmids. As positive control, we pretreated 4TO condition with CsA.
238
Luciferase activity was determined and normalized to protein content (n=4) Western blot analysis
239
confirmed the expression of proteins of interest for each condition tested. Beta-actin was included
240
as loading control.
241
Figure 2: U54 specifically reduces MCF-7 breast cancer cells growth
242
A-B) MCF-7 cells and –C) 293T cells were transfected with 4TO, 4TO-U54, 4TO-U11 and 4TO-
243
U54mut plasmids. As control, we pretreated 4TO condition with CsA (A-C) or FK506 (B). Cells 10
244
were harvested and evaluated at 24h, 48h, 72h and 96h following transfection by cell counting
245
assay (n=4). Western blot analysis confirmed the expression of proteins of interest for each
246
condition tested. Beta-actin was included as loading control.
247
Figure 3: U54 inhibits dephosphorylation of NFAT1 protein.
248
NFAT1 dephosphorylation was evaluated in MCF-7 cells by Western blot assay. A) MCF-7 cells
249
were transfected with 4TO, 4TO-U54, 4TO-U11, 4TO-U54mut and REP-NFAT1 plasmids. As
250
positive control, 4TO transfected cells we pretreated with CsA. Western blot analyses were
251
performed by blotting each condition with a phospho specific anti-NFAT1 antibody (140 kDa)
252
(upper panel). Western blot analysis confirmed expression of U54, U54mut or U11 proteins
253
(middle panels). Beta-actin was included as loading control. B) Densitometric analysis of P-
254
NFAT1 was performed following western blot analysis. The P-NFAT1 level in resting cells
255
(4TO) was set at 100%. Following TPA/ionomycin stimulation, P-NFAT1 levels were compared
256
to their respective resting control (one representative experiment of three independent
257
experiments).
258
Figure 4: U54 reduces COX-2 gene transcription in MCF-7 cell line
259
A) MCF-7 cells were transfected with 4TO, 4TO-U54, 4TO-U11 and 4TO-U54mut plasmids for
260
48h before being stimulated or not with TPA/ionomycin for an additional 24h. RNA was
261
extracted and COX-2 mRNA levels determined by real-time RT-QPCR. The primers used for
262
COX-2 detection were COX-2 forward 5’- TGC ATT CTT TGC CCA GCA CT -3’ and COX-2
263
reverse 5’- AAA GGC GCA GTT TAC GCT GT -3’. The primers for GAPDH, used for
264
normalization were previously described (19). B) Western blot analysis confirmed the expression
265
of U54 and U54mut proteins. Beta-actin was included as loading control.
11
266 267 268 269 270 271 272 273 274
Table 1: Relative expression of NFAT1, 2, 3 in MCF7 and Jurkat T cells.
275 276
- denotes absence of expression; + denotes basal expression level; ++ denotes moderate
277
expression level; +++ denotes high expression level.
278
12
Figure 2 A
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