Reporter assays with the rat caIII gene promoter show repressed activity, demonstrating that Evi1 either directly or indi-rectly modulates transcription of this gene in Rat1 cells.. Toge
Trang 1apoptosis in Evi1 transformed Rat1 fibroblasts due to
repression of carbonic anhydrase III
P Roy1, E Reavey1, M Rayne1, S Roy1, M Abed El Baky1, Y Ishii2and C Bartholomew1
1 Department of Biological & Biomedical Sciences, Glasgow Caledonian University, City Campus, Glasgow, UK
2 Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan
Introduction
Multiple mechanisms have been proposed for Evi1’s
contribution to cancer progression, including enhanced
cell proliferation, impaired differentiation and evasion
of apoptosis [1] Evasion of apoptosis has been
observed in both haemopoietic and epithelial cells with
a variety of agents and suggests that Evi1 is a survival
factor For example, either deregulated or enforced
expression of Evi1 has been shown to protect HEK293, HEC-1B and Jurkat cells from UV-induced apoptosis [2], U937 cells from tumour necrosis factor-a-induced apoptosis [2], SiHa cells from interferon-a-induced apoptosis [3] and both rat intestinal epithe-lial cells and HT-29 cells from transforming growth factor-b (TGFb)- and paclitaxel-induced apoptosis [4]
Keywords
apoptosis; carbonic anhydrase III; Evi1;
H2O2
Correspondence
C Bartholomew, Department of Biological &
Biomedical Sciences, Glasgow Caledonian
University, City Campus, Cowcaddens
Road, Glasgow G4 OBA, UK
Fax: +44 141 331 3208
Tel: +44 141 331 3213
E-mail: c.bartholomew@gcal.ac.uk
(Received 18 August 2009, revised
9 November 2009, accepted 16 November
2009)
doi:10.1111/j.1742-4658.2009.07496.x
EVI1 is a nuclear zinc finger protein essential to normal development, which participates in acute myeloid leukaemia progression and transforms Rat1 fibroblasts In this study we show that enforced expression of Evi1 in Rat1 fibroblasts protects from paclitaxel-induced apoptosis, consistent with previously published studies Surprisingly, however, these cells show increased sensitivity to hydrogen peroxide (H2O2)-induced apoptosis, dem-onstrated by elevated caspase 3 catalytic activity This effect is caused by a reduction in carbonic anhydrase III (caIII) production caIII transcripts are repressed by 92–97% by Evi1 expression, accompanied by a similar reduc-tion in caIII protein Reporter assays with the rat caIII gene promoter show repressed activity, demonstrating that Evi1 either directly or indi-rectly modulates transcription of this gene in Rat1 cells Targeted knock-down of caIII alone, with Dicer-substrate short inhibitory RNAs, also increases the sensitivity of Rat1 fibroblasts to H2O2, which occurs in the absence of any other changes mediated by Evi1 expression Enforced expression of caIII in Evi1-expressing Rat1 cells reverts the phenotype, restoring H2O2 resistance Together these data show that Evi1 represses transcription of caIII gene expression, leading to increased sensitivity to
H2O2-induced apoptosis in Rat1 cells and might suggest the basis for the development of a novel therapeutic strategy for the treatment of leukae-mias and solid tumours where EVI1 is overexpressed
Abbreviations
AKT, protein kinase B; caIII, carbonic anhydrase III; DCF-DA, 2¢-7¢-dichloroflourescene diacetate; DMEM, Dulbecco’s modified Eagle’s medium; DsiRNA, Dicer-substrate short inhibitory RNA; FACS, fluorescent activated cell sorter; H2O2, hydrogen peroxide;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; PI3K, phosphoinositide-3-kinase; ROS, reactive oxygen species;
RTQ, real-time quantitative; siRNA, short inhibitory RNA; TGFb, transforming growth factor-b.
Trang 2Variant forms of Evi1 are also antiapoptotic,
protect-ing murine acute myeloid leukaemia cells that have
been treated with arsenic trioxide [5]: in this case the
agent actually targets degradation of AML1⁄ EVI1 in
order to induce programmed cell death Recent
evi-dence also supports a survival role in nonpathological
conditions, as mice require Evi1 to maintain adequate
numbers of haemopoietic stem cells [6] and this is also
consistent with a more general requirement for Evi1
for cell survival during murine development [7]
Evi1 is a 145 kDa nuclear protein member of the
cys2his2 zinc finger family [8] It possesses multiple
domains that have been identified by both sequence
homology and functional activity, including: two
dis-tinct zinc finger motifs of seven and three repeating
units at the N-terminus and towards the C-terminus,
respectively [8]; a central repressor domain [9] and a
C-terminal acidic domain [8] These domains have been
shown to interact with other molecules, including DNA
[10,11] and proteins [1] and are responsible for
mediat-ing Evi1 inhibition of apoptosis Interactions of various
molecules with these motifs enable Evi1 to impair or
activate particular signalling pathways, including TGFb
[12,13], c-Jun N-terminal kinase (JNK) [2] and
phos-phoinositide-3-kinase⁄ protein kinase B (PI3K ⁄ AKT)
[4] Intervention of critical signalling molecules are the
basis for Evi1-mediated enhanced cell survival
A number of agents have been used to study the
impact of Evi1 on apoptosis in cells, including UV
light, tumour necrosis factor-a, TGFb, interferon-a,
arsenic trioxide and taxol (paclitaxel) Hydrogen
per-oxide (H2O2) also induces apoptosis, but the impact of
Evi1 expression on its apoptotic-inducing capability
has not been investigated previously Either
exoge-nously supplied or endogenous H2O2 generate reactive
oxygen species (ROS), which if unchecked cause
oxida-tive stress, resulting in damaged cellular DNA, lipids
and proteins that interfere with cell function To
com-bat oxidative stress, complex antioxidant defence
mechanisms have evolved to protect cells from
oxidative injury Established antioxidants include the
enzymatic systems catalase, superoxide dismutase,
glu-tathione peroxidases and peroxiredoxin III and
nonen-zymatic systems including vitamins C, E and B2,
coenzyme Q10, glutathione and carotene [14] If the
amount of ROS exceeds the capacity of the
antioxi-dant machinery, then oxidative stress occurs [15]
The enzyme carbonic anhydrase III (caIII) (EC
4.2.1.1) is also thought to protect cellular proteins
from oxidation [16] Carbonic anhydrases are a family
of 15 distinct isozymes that catalyse the reversible
con-version of H2O + CO2 and H++ HCO3) [17] caIII
is unique, very abundant in liver, skeletal muscle and
adipocytes and unlike other members of this family has low hydratase catalytic activity [18] The function
of caIII is unknown, but it is suggested that it has an antioxidant function and it has been shown to protect cells from H2O2-induced apoptosis [19,20]
In this study, we investigated H2O2-induced apoptosis in Rat1 cells expressing an Evi1 transgene Surprisingly, we found that Evi1 expression increases sensitivity to H2O2-mediated apoptosis in complete contrast to the protective effect of other apoptosis-inducing agents Increased sensitivity is primarily due
to the transcriptional downregulation of caIII gene expression mediated by Evi1
Results
Evi1-expressing Rat1 fibroblasts are resistant to taxol-induced apoptosis
Independent, stable populations of Rat1 cells express-ing murine Evi1 were generated by infection with the p50M5.6neo retrovirus (Fig 1A), produced by tran-sient transfection of EcoPak2 cells, and designated 5.61 and 5.62 Empty vector Neo1 and Neo2 cells were similarly created with the p50MX-neo retrovirus Evi1 expression was confirmed by western blot analysis with a-Evi1 (1806), detecting a 145 kDa protein in 5.61 and
o n A
R T L
p50M5.6neo
35 kDa
α-gapdh
A
B
Fig 1 Schematic representation of the murine Evi1-expressing recombinant retroviral vector p50M5.6neo and production of Evi1 in virus-infected Rat1 fibroblasts (A) Viral long terminal repeats (LTR), the murine Evi1 gene, including the N-terminal and C-terminal zinc finger domains (black boxes), repressor domain (grey box) and acidic domain (striped box), the Neo gene (neo) and splice donor (SD) and splice acceptor (SA) sites for the production of
subgenom-ic transcripts for the expression of neo (B) Western blot analysis
of whole cell protein extracts derived from the indicated cell lines and populations using a-Evi1 (1806) and a-gapdh (6C5) antibodies The positions of 145 and 35 kDa Evi1 and gapdh proteins are shown.
Trang 35.62 cells that is absent from Neo1, Neo2 and parental
Rat1 cells (Fig 1B)
Previous studies have shown that Evi1 is a survival
factor, protecting cells from apoptosis induced by a
variety of agents To determine if Evi1 also protects
Rat1 cells from apoptosis, we treated our panel of cell
populations with paclitaxel (taxol) Apoptosis was
monitored by measuring caspase 3 catalytic activity
The results showed that taxol (1 lm, 16 h) induced
sig-nificantly higher caspase 3 catalytic activity in Rat1,
Neo1 and Neo2 cells than in Evi1-expressing 5.61 and
5.62 cells (Fig 2) Taxol induced caspase 3 activity in
all cell populations, but to a much lesser extent in 5.61
and 5.62 cells These data show that Evi1 protects
Rat1 cells from taxol-induced apoptosis, consistent
with previous studies in other cell types
Rat1 fibroblasts expressing Evi1 have increased
sensitivity to H2O2-induced apoptosis
Although previous studies have shown that Evi1
pro-tects cells from a variety of inducers of apoptosis, the
effects of H2O2 have not yet been examined Rat1 cells
and 5.61 cells were exposed to various
concen-trations of H2O2and cell viability monitored by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide
(MTT) assay The results showed that cell viability
was reduced in a H2O2dose-dependent manner in both
Rat1 and 5.61 cells (Fig 3A) Surprisingly, the
viabil-ity of 5.61 cells was significantly less than Rat1 cells at
each concentration of H2O2(Fig 3A) The viability of
the entire panel of cell populations was examined by
MTT assay following H2O2 (750 lm) treatment and this confirmed that ectopic Evi1 expression decreased survival in Rat1 cells (Fig 3B) This was supported by the dramatic morphological change observed in H2O2 -treated 5.61 cells compared with parental Rat1 or empty vector control Neo cells treated with the same concentration of reagent (Fig 3C)
The morphological changes observed in 5.61 cells treated with H2O2 resembled apoptosis Therefore, we examined caspase 3 activation in cultures of the cell populations The results showed that H2O2 induced caspase 3 catalytic activity in all cell populations exam-ined, but the level of activation was significantly greater in 5.6 cell populations compared with parental Rat1 and vector control cells (Fig 3D)
Evi1 represses expression of the potential antioxidant caIII in Rat1 cells
Previous studies have shown that arsenic trioxide induces apoptosis in leukaemia cells by degrading the Evi1 fusion protein, AML1⁄ EVI1 [5] H2O2-induced Evi1 degradation could reduce cell survival in the pres-ence of this agent Therefore, the stability of Evi1 transgene expression in 5.61 cells was examined by western blot analysis (a-Evi1, 1806) following H2O2 treatment for 4, 10 and 16 h However, the abundance
of Evi1 protein remained unchanged during this time period, confirming that H2O2had no effect on protein stability (Fig 4), eliminating this mechanism
Recently, we used microarray technology to identify Evi1-mediated induction and repression of gene tran-scripts in Rat1 cells (E R Reavey & C Bartholomew, unpublished results) Inspection of these data revealed transcriptional repression of caIII, which encodes a protein that has previously been shown to protect cells from H2O2-induced apoptosis [19,20]
The microarray data were confirmed by real-time quantitative RT-PCR (RTQ-RT-PCR) using total cel-lular RNA from Rat1 and derivative Neo and 5.6 cells These data showed that caIII mRNA transcripts were repressed by 92–97% in 5.61 and 5.62 cells rela-tive to Rat1 and empty vector control cells Neo1 and Neo 2 (Fig 5A) Western blot analysis with a-caIII (E-19) confirmed that caIII protein levels were also dramatically reduced in 5.61 and 5.62 cells (Fig 5B), consistent with the RTQ-RT-PCR data
caIII gene promoter activity is repressed by Evi1
in Rat1 cells
To determine if Evi1-mediated caIII repression occurs
at the level of gene transcription, reporter assays were
160 000
180 000
200 000
***
80 000
100 000
120 000
140 000
0
20 000
40 000
60 000
Fig 2 Histogram showing relative caspase 3 catalytic activity of
the indicated cell lines and populations in the absence (grey
col-umns) or presence of 1 l M paclitaxel for 16 h (black columns) The
columns represent the mean of an experiment performed in
qua-druplicate and error bars the standard deviation ***P £ 0.0002.
Trang 4performed A pGL3-basic vector containing )1485 to
+55 of rat caIII gene promoter sequence [21],
desig-nated p-1485⁄ +55 caIII luc, was created (Y Ishii,
unpublished data) and transfected into Rat1, Neo and
5.6 cells, together with the control vector pRLCMV
The activity of p-1485⁄ +55 caIII luc in the various
cell types, normalized for pRLCMV activity, is shown
in Fig 6A The results showed that the caIII gene
promoter had at least 10-fold greater transcriptional
activity in Rat1, Neo1 and Neo2 cells compared with
the Evi1-expressing 5.61 and 5.62 cells In contrast, the
activity of a minimal thymidine kinase gene promoter
construct (pGL2tkluc), normalized for pRLCMV, was
similar in all cell types examined (Fig 6B) These
results show that Evi1 specifically repressed the
transcriptional activity of the caIII gene promoter in
Rat1 cells
caIII knockdown alone enhances H2O2-induced apoptosis in Rat1 cells and enhanced caIII expression is protective
Short inhibitory RNAs (siRNA) were used to deter-mine if repression of caIII gene expression alone
0 20 40 60 80 100 120
Neo 1 Neo 2 5.61 cells 5.62 cells
Neo 1 Neo 2 5.61 cells 5.62 cells
0
20
40
60
80
100
120
UT 100 150 200 250 500 750
H2O2 (µM)
Rat1 Neo2 5.61 cells
750 M
H2O2
Untreated
0
20 000
40 000
60 000
80 000
100 000
120 000
140 000
Rat1 Neo1 Neo2
5.61 cells 5.62 cells
Rat1 Neo1 Neo2
5.61 cells 5.62 cells Relative caspase 3 activity (luminescence, RLU)
**
***
C
D
Fig 3 Cell viability and caspase 3 catalytic activity of cell lines and populations following 16 h treatment with H2O2 (A) Histogram of the percentage viability (MTT assay) of untreated cells (UT) and cells treated with the indicated concentration of H2O2for 16 h Grey columns are Rat1 cells and black columns are 5.61 cells The columns represent the mean of an experiment performed in quadruplicate and error bars the standard deviation (B) Histogram of the percentage viability (MTT assay) of the indicated untreated (black columns) and 16 h of
750 l M H2O2treated (grey columns) cell lines and populations The columns represent the mean of an experiment performed in quadrupli-cate and error bars the standard deviation **P £ 0.002 (C) Photographs showing the morphology of either untreated or treated (16 h
750 l M H 2 O 2 ) indicated cell lines and populations (D) Histogram showing relative caspase 3 catalytic activity of the indicated cell lines and populations in the absence (grey columns) or presence of 750 l M H2O2for 16 h (black columns) The columns represent the mean of an experiment performed in quadruplicate and error bars the standard deviation ***P < 0.0001.
UT 4 h 10 h 16 h UT 4 h 10 h 16 h
5.61 cells Neo1
α-Evi1 α-gapdh
145 kDa
35 kDa
Fig 4 Western blot analysis of whole cell protein extracts derived from Neo1 and 5.61 cell populations following treatment for 0 (UT),
4, 10 and 16 h with 750 l M H2O2using a-Evi1 (1806) and a-gapdh (6C5) antibodies The positions of 145 kDa Evi1 and 35 kDa gapdh proteins are shown.
Trang 5tizes Rat1 cells to H2O2 Both RTQ-RT-PCR and
wes-tern blot analysis (a-caIII) were used to identify an
effective Dicer-substrate siRNA (DsiRNA) that
inhib-ited both caIII mRNA gene transcripts (98%
reduc-tion, Fig 7A, 10 nm siRNA1) and caIII protein
(Fig 7B, 10 nm siRNA1) when transfected into Rat1
cells A control, nonspecific DsiRNA had no effect on
either caIII mRNA or caIII protein when transfected
into Rat1 cells at the same concentration (Fig 7A,B,
Non sp siRNA)
The effect of caIII siRNA1 on H2O2 sensitivity in
transfected Rat1 cells was then investigated by
moni-toring caspase 3 catalytic activity siRNA1 transfected
Rat1 cells treated with 750 lm H2O2 for 16 h had at
least double the caspase 3 activity observed with
750 lm H2O2-treated untransfected and nonspecific
DsiRNA transfected Rat1 control cells (Fig 7C) caIII
knockdown with a second distinct siRNA (siRNA3)
produced the same phenotype (Fig S1) The results
show that H2O2induced caspase 3 catalytic activity in
all cells, but the level of activation was significantly
greater in Rat1 cells transfected with a caIII-specific
siRNA (Fig 7C) Furthermore, caIII knockdown only
sensitized Rat1 cells to H2O2 treatment and had no effect upon apoptosis induced by treatment with taxol (Fig S2)
caIII expression was restored in 5.61 cells to deter-mine if the sensitivity to H2O2 treatment could be reverted Rat1 cells were transiently transfected with a caIII expression vector (pRC-sport 6caIII), which sig-nificantly increased cellular levels of the caIII protein (Fig 8A) The increased levels of caIII protein in 5.61 cells protected them from H2O2 treatment, compared with untreated or empty vector control transfected cells, as determined by measuring caspase 3 catalytic activity (Fig 8B)
Finally, we measured intracellular levels of ROS to determine if the basal oxidized state varied between Rat1 and 5.61 cells Cells labelled with 2¢-7¢-dichloro-flourescene diacetate (DCF-DA) were examined by fluorescent automatic cell sorter (FACS) The results
2.00
***
1.20
1.60
0.40
0.80
0.00
Neo1 Neo2 5.61 cells 5.62 cells
Rat1 Neo1 Neo2 5.61 cells
α-caIII α-gapdh
35 kDa
27 kDa
A
B
Fig 5 caIII gene expression in Rat1 cells and derivative cell
popu-lations (A) Histogram of caIII mRNA levels normalized for gapdh
mRNA relative to normalized caIII mRNA in Rat1 cells, determined
by RTQ-RT-PCR The columns are the mean of an experiment
per-formed in quadruplicate and the error bars the standard deviation.
(B) Western blot analysis of whole cell protein extracts using a-caIII
(E-19) and a-gapdh (6C5) antibodies The positions of 27 kDa caIII
and 35 kDa gapdh proteins are shown ***P < 0.0001.
B A
Fig 6 Reporter assays showing the activity of the caIII and mini-mal herpes simplex virus thymidine kinase (HSV tk) gene promot-ers in Rat1 and derivative cells (A) Histogram of caIII gene promoter firefly luciferase reporter activity (p-1485 ⁄ +55 caIII luc) normalized for cytomegalovirus (CMV) immediate early enhancer ⁄ promoter renilla luciferase reporter activity (pRLCMV) The columns are the mean of an experiment performed in quadruplicate and error bars the standard deviation ***P < 0.0001 (B) Histogram of HSV tk gene promoter firefly luciferase reporter activity (pGL2tkluc) normalized for pRLCMV The columns are the mean of an experi-ment performed in quadruplicate and error bars the standard deviation.
Trang 6showed that the mean fluorescence of 5.61 cells was
significantly higher than that observed in Rat1 and
Neo1 cells (Fig 9) This shows that ROS were elevated
in 5.61 cells, consistent with the observed reduction in caIII expression
Discussion
We show here for the first time that ectopic expression
of Evi1 sensitizes Rat1 cells to H2O2-induced apopto-sis This represents the first description that Evi1 can actually stimulate cell death Previous studies with a variety of agents have shown that Evi1 protects cells from apoptosis and functions as a survival factor, pro-viding one of multiple suggested roles that contribute
to the development and progression of leukaemia Consistent with this view, we also show that Evi1 pro-tects Rat1 cells from apoptosis induced by at least one
of these agents (taxol) and therefore probably also acts
as a survival factor in these cells Evi1-mediated pro-tection from taxol-induced apoptosis in rat intestinal epithelial cells and colon cancer cells (HT-29) is due to
UT Non sp siRNA siRNA
α-caIII α-gapdh
35 kDa
27 kDa
0
0.2
0.4
0.6
0.8
1
1.2
1.4
**
Non sp siRNA 1
siRNA
0
100 000
200 000
300 000
400 000
500 000
600 000
700 000
800 000
900 000
UT Non sp
siRNA
siRNA 1
UT
750 µ M
***
A
B
C
Fig 7 DsiRNA-mediated knockdown of caIII mRNA and protein
and enhanced caspase 3 catalytic activity in Rat1 cells (A)
Histo-gram of caIII mRNA levels normalized for gapdh mRNA relative to
normalized caIII mRNA in untreated Rat1 cells, determined by
QRT-PCR in Rat1 cells transfected for 48 h with 10 n M of either a
non-specific siRNA (Non sp siRNA) or a caIII-non-specific siRNA (siRNA
10 n M ) The columns are the mean of an experiment performed in
quadruplicate and the error bars the standard deviation.
**P £ 0.0041 (B) Western blot analysis of whole cell extracts
derived from Rat1 cells, transfected as described in (A), with a-caIII
(E-19) and a-gapdh (6C5) antibodies The positions of 27 kDa caIII
and 35 kDa gapdh proteins are shown (C) Relative caspase 3
cata-lytic activity in Rat1 cells transfected as described in (A), either
treated (black columns) or untreated (grey columns) with 750 l M
H2O2for 16 h The columns are the mean of an experiment
per-formed in quadruplicate and error bars the standard deviation.
***P < 0.0001.
α-caIII α-gapdh
35 kDa
27 kDa
1400 000
1600 000
1800 000
800 000
1000 000
1200 000
***
200 000
400 000
600 000
***
0
B A
Fig 8 5.61 cell protection from H2O2-induced caspase 3 catalytic activity by ectopic expression of caIII (A) Western blot analysis of whole cell extracts derived from untransfected 5.61 cells (UT), empty vector transfected 5.61 cells (pRC CMV) and caIII expres-sion vector transfected cells (pRC-sport6caIII) with a-caIII (E-19) and a-gapdh (6C5) antibodies The positions of 27 kDa caIII and
35 kDa gapdh proteins are shown (B) Relative caspase 3 catalytic activity in untransfected 5.61 cells (grey columns) and 5.61 cells transfected with pRC CMV (white columns) or pRC-sport6caIII (black columns) with (H 2 O 2 ) or without (UT) 750 l M H 2 O 2 treat-ment ***P £ 0.0007.
Trang 7stimulation of PI3K and its downstream effector AKT
[4] The same mechanism probably also operates in
Rat1 cells, but was not examined in this study
Sensitization to H2O2-induced apoptosis, determined
by caspase 3 catalytic activity, was seen in both
Evi1-expressing Rat1 cells (5.6 cells) and in caIII knockdown
Rat1 cells Furthermore, Evi1-expressing Rat1 cells had
a 90% reduction in caIII gene transcripts and protein
Together, these results confirm that Evi1-mediated
stim-ulation of H2O2-induced cell death is due to the reduc-tion in cellular levels of the caIII protein
The results presented here suggest that caIII protects Rat1 cells from H2O2-derived ROS and therefore acts
as an antioxidant However, the biological activity of caIII is an enigma Unlike other products of this gene family, caIII has very low catalytic activity and so it is unlikely that it functions in hydrating carbon dioxide [18] Furthermore, knockout mice, deficient in caIII, have normal growth, development and lifespan under laboratory conditions, suggesting that the protein is not essential [22] However, several recent observations suggest that caIII is an important antioxidant, consis-tent with the observations here caIII is highly abun-dant in skeletal muscle, a tissue of high oxygen consumption and antioxidant activity Microarray analysis of skeletal muscle of wild-type and caIII-defi-cient knockout mice revealed that caIII has a possible role in the glutathione-mediated antioxidative system [23] This is supported by biochemical evidence show-ing that caIII undergoes rapid reversible S-glutathiola-tion or irreversible oxidation in mildly and exhaustively stressed muscle, respectively [23]: in the presence of glutathione, glutathione peroxidases restore reversibly S-glutathiolated caIII This mechanism would explain both the protective effect of ectopic caIII expression observed in NIH3T3 cells [20] and the increased sensitivity of caIII knockdown Rat1 or Evi1-expressing Rat1 cells exposed to H2O2
Several possibilities exist to explain the caIII repres-sion effect observed in the present study Abundant caIII gene transcripts and protein occur in Rat1 cells, which, like all fibroblasts examined (C Bartholomew, unpublished results), normally express low levels of endogenous evi1 Therefore, caIII gene expression and protein production normally occur efficiently in the presence of evi1 in Rat1 cells The simplest explanation
is that merely elevated cellular levels of Evi1 are suffi-cient to repress caIII transcription Consistent with this, previous studies have shown that the abundance
of Evi1 is crucial to 32Dcl3 granulocyte differentiation [24], suggesting that differential changes in gene expression must occur that are dependent on the quan-tity of cellular levels of the Evi1 protein However, other possibilities exist Multiple naturally occurring evi1 isoforms occur and it might be that it is the relative increase in the abundance of the Evi1 full-length form [25] in Rat1 cells (5.6 cells), observed here, that signifi-cantly represses caIII gene expression It is possible that only some isoforms of Evi1 repress caIII expression, whereas perhaps other forms either have no effect or the opposite effect to the full-length form Some studies have shown that the MDS1⁄ EVI1 isoform has
Rat1 MFI –250
ROS levels
Neo1 MFI –144
ROS levels
5.61 cells MFI –905
ROS levels
10 0 10 1 10 2 10 3 10 4
10 0 10 1 10 2
FL1-H
FL1-H
10 3 10 4
100
100
101
10 1
102
102
FL1-H
103
10 3
104
104
10 5
10 0
10 1
102
10 3
104
105
10 0
101
102
10 3
10 4
105
M1
M1
M1
Fig 9 Histogram of fluorescence intensity (x-axis) versus cell
num-ber (y-axis) of indicated DCF-DA-labelled cells analysed by FACS.
The mean fluorescence intensity (MFI) for the region designated
M1 is shown for each cell type.
Trang 8the opposite effect of the full-length form For example,
it is reported that the MDS1⁄ EVI1 isoform enhances
the growth inhibitory effects of TGFb [26], whereas the
full-length form blocks this response [12]
caIII is a very abundant protein, particularly in liver,
muscle and adipocytes However, very little is known
about its transcriptional regulation Transcription of the
rat caIII gene is inhibited by the aryl hydrocarbon
recep-tor ligand 3-methylchlanthrene in hepatocytes and in
the livers of rats fed an ethanol-supplemented diet
[21,27] Human CAIII mRNA is induced in muscle of
athletes training under hypoxic conditions [28] One
study has been conducted with the caIII gene promoter,
with a preliminary analysis of an active 2.8 kb human
CAIII gene promoter in myogenic cells and a significant
loss of activity upon deletion to)715 bp [29]
Reporter assays with the caIII gene promoter
()1485 ⁄ +55) showed that this region contains strong
promoter activity in Rat1 cells, consistent with a
previ-ous analysis of the human promoter [29] This region
also has the cis-regulatory elements necessary for
Evi1-mediated transcriptional repression
Evi1-mediated repression could be caused by binding
directly to promoter sequences Previous studies with
artificial promoter reporter constructs have shown that
Evi1 can function as a DNA-binding transcriptional
repressor protein [9] Evi1 binds several corepressor
molecules, CtBP, the histone methyltransferase
SUV39H1 and the histone deacetylase HDAC1
[30–32], each of which mediates transcriptional
repres-sion However, to date no genes that are direct targets
for transcriptional repression have been identified
Furthermore, very few genes have been identified that
are directly regulated and induced by Evi1; GATA2
being the best characterized [6,33] Inspection of the
)1485 ⁄ +55 rat nucleotide sequence for Evi1
protein-binding sites with matinspector software revealed
mul-tiple potential sites This suggests that caIII may be a
direct target for Evi1-mediated transcriptional
repres-sion, although binding and biological activity of any of
these motifs require experimental investigation
Alternatively, repression of caIII gene expression
could be indirect Evi1 has been shown to interact with a
number of transcription factors, including PU.1,
RUNX1, GATA1, E2F1 and SMAD3 [12,34–37] and
signalling molecules such as JNK and PI3K⁄ AKT [2,4]
to inhibit their biological activities Therefore, it remains
possible that Evi1 might repress caIII gene expression
by interacting with a transcription factor or by
inhibit-ing a signallinhibit-ing pathway that is normally required for
the high level of expression observed in Rat1 cells
Inspection of the caIII )1485 ⁄ +55 promoter region
with matinspector software showed several potential
binding sites for E2F family proteins, but not for any of the other transcription factors Evi1 has been shown to interact with The precise mechanism by which Evi1 represses caIII gene expression awaits a more detailed analysis of the)1485 ⁄ +55 gene promoter region These data show that Evi1 represses transcription of caIII in Rat1 cells (5.6 cells) and as a consequence these cells are vulnerable to oxidative stress This raises the possibility that Evi1 regulates caIII in other cell types and if the caIII protein is an important antioxidant, then they too would be vulnerable to oxidative stress
EVI1 is overexpressed in some human neoplasias, including acute myeloid leukaemias [38] and hepatocel-lular carcinoma [4] CAIII is also very abundant in nor-mal liver and presumably is an important antioxidant in this tissue Interestingly, CAIII expression is reduced in human hepatocellular carcinoma [39], although it is not known which tumours overexpress EVI1 EVI1 might
be responsible for CAIII repression in some cases and perhaps different mechanisms operate in others CAIII expression in haemopoietic cells and leukaemia cells has not been described There is some evidence that CAIII might operate as an antioxidant in erythrocytes in cer-tain anaemias [40], suggesting that it might be important
in protecting haemopoietic cells from oxidative stress It would be interesting to assess the expression level of CAIII in normal haemopoetic cells and leukaemia cells
to determine if it is reduced in these neoplasias and if levels are inversely proportional to EVI1 expression If this is the case, then tumours overexpressing EVI1 might
be vulnerable to therapeutic agents that induce oxidative stress
Materials and methods
Preparation of plasmid DNA Plasmids p50MX-neo, p50M5.6neo, pGL2tkluc, pBluescript KSII, pCMVcar3 (I.M.A.G.E Id 4195712) and pRLCMV have all been described previously [9] and were obtained from Promega UK (Southampton, UK), Stratagene (La Jolla, CA, USA) and Source Bioscience, geneservice (Cam-bridge, UK) The construction of pGL3-caIII ()1485 ⁄ +55) has not been published (Y Ishii, unpublished data) Plas-mid DNAs were prepared by affinity chromatography using NucleobondPC500EF gravity flow columns according to the manufacturer’s instructions (Macherey-Nagal, Du¨ren, Germany)
Cell culture Rat1 and EcoPak2 cells were cultured in complete medium comprising Dulbecco’s modified Eagle’s medium (DMEM,
Trang 9Lonza Group, Basel, Switzerland, BE12-604F)
supple-mented with 5% newborn calf serum (Sigma-Aldrich,
Poole, UK, N4637) or 10% fetal calf serum (Lonza
Group, DE14-801F), respectively, and 2.5 mm glutamine,
50 lgÆmL)1 penicillin, 50 unitsÆmL)1 streptomycin (Lonza
Group, BE17-605E and BE17-603E), 37C, 5% CO2 For
retrovirus production, EcoPak2 cells (Clontech-Takara Bio
Europe, Saint-Germain-en-Laye, France) were plated on
collagen (Sigma-Aldrich, C38671) coated dishes and
tran-siently transfected with either p50M5.6-neo or p50MX-neo
using the calcium phosphate coprecipitate method described
previously [41] Virus was harvested and used to infect
Rat1 fibroblasts, as described previously [9]; infected cells
were selected in complete medium supplemented with
50 lgÆmL)1 G418 (Invitrogen, Paisley, UK) For paclitaxel
and H2O2treatment, cells were incubated in complete
med-ium supplemented with either 1 lm paclitaxel
(Sigma-Aldrich, T7191) or 100–750 lm H2O2 (Sigma-Aldrich,
21676) for 16 h
Western blotting
Protein extracts, SDS⁄ PAGE and western blotting were
performed as described previously [9] with either a-caIII
(Santa Cruz Biotechnology, Santa Cruz, CA, USA, E-19),
a-Evi1 (1806) or a-gapdh (Fitzgerald Industries, North
Acton, MA, USA, 6C5) and diluted 1⁄ 200 or 1 ⁄ 5000 (1806
and 6C5) Appropriate horseradish peroxidase-conjugated
anti-goat (Sigma-Aldrich, A5420), anti-rabbit
(Sigma-Aldrich, A9169) or anti-mouse (Sigma-(Sigma-Aldrich, A9044) IgG
secondary antibodies were used at 1⁄ 5000 dilutions and
detection was performed by enhanced chemiluminescence
(Pierce, Rockford, IL, USA, 32209)
DNA-mediated transfection and reporter assays
Rat1 cells and derivatives were transfected using Fugene6
(Roche Diagnostics, Mannheim, Germany, 11815091001)
For reporter assays, cells were transfected with recombinant
pGL3-caIII ()1485 ⁄ +55) and pRLCMV plasmid DNAs
Constant DNA concentrations were maintained with
pBlue-script KSII Cells (5· 103
) were incubated with a 1 : 6 ratio DNA : FuGENE6, prepared as described by the
manufac-turer, in 96-well plates (Costar, New York, NY, USA,
3917) for 48 h Cells were lysed, and luciferase activity
determined using the dual-luciferase reporter assay system
(Promega, TM046) in a Fluostar OPTIMA luminometer
(BMG LABTECH, Offenburg, Germany)
Oligonucleotides
Gene-specific oligonucleotides were designed using primer
expresssoftware version 3.0 (Applied Biosystems),
synthe-sized and supplied by Eurogentec (Seraing, Belgium):
5¢ rat caIII: ccgggactattggacctacca 3¢ rat caIII: cagtagcagccacacaatgca 5¢ HEX, 3¢ TAMRA rat caIII probe: cttcaccacgccaccctgc gag
5¢ rat gapdh: gggcagcccagaacatca 3¢ rat gapdh: ccgttcagctctgggatgac 5¢ 6-FAM, 3¢ TAMRA rat gapdh probe: ccctgcatccactgg tgctgcc
Preparation of total cellular RNA, cDNA synthesis and RTQ-RT-PCR
RNA was prepared from semiconfluent cultures of cells using the Trizol method (Invitrogen, 1559-026) Total cellular RNA (1 lg) was used to synthesize cDNA with Supermix III first-strand strand synthesis for QPCR according to the man-ufacturer’s instructions (Invitrogen, 11752) Five per cent of the cDNA reaction was used for RTQ-PCR using the ABso-lute QPCR mix (ABgene, Epsom, UK, AB-4136), gene-spe-cific oligonucleotide primers and dual-labelled probes, 95C,
15 min followed by 40 cycles 95C, 30 s, 60 C, 30 s in an OPTICON 2 DNA engine (MJ Research, Watertown, MA, USA)
The efficiency of the RTQ-PCR reactions was calculated using the formula efficiency =)1 + 10()1 ⁄ slope) against the standard curve of each assay over a gradient of template concentration with each gene The efficiencies for caIII and gapdhprimers⁄ probes were 90 and 101%, respectively Rela-tive expression levels between caIII and gapdh were deter-mined using the arithmetic comparative 2)DDCt method [42] and were determined relative to caIII in Rat1 cells (calibrator)
Caspase 3 assay Cells were incubated in 96-well dishes (Costar 3917), trea-ted with various agents and apoptosis determined using the Caspase 3⁄ 7-Glo assay according to the manufacturer’s instructions (Promega, G8090), measuring luminescence with a Fluostar OPTIMA luminometer (BMG LABTECH)
MTT assays MTT assays were performed on cells grown in 96-well tissue culture plates following treatment with H2O2 Cells were treated with 500 lgÆmL)1MTT (Sigma-Aldrich, M5655) in complete medium, 37C, 5% CO2, 1 h The medium was removed and replaced with 100 lL dimethylsulfoxide (Sigma-Aldrich, 472301) and absorbance measured at
570 nm using an MRX plate reader (Dynatech Laboratories, Guernsey Channel Island, UK) The final absorbance was determined by subtracting the absorbance of treated wells lacking cells The formazan concentration was determined using the formula: c (formazan concentration; lm) = A (absorbance)⁄ e (extinction coefficient) 1 (path length)
Trang 10Knockdown of rat caIII
Rat caIII knockdown was achieved in Rat1 cells using
Tri-FECTa DsiRNA Kit RNC.RNAI.NO19292.9 (Integrated
DNA Technologies, Leuven, Belgium) SiRNA1 (5¢-CCA
UUGAACUGCAUACUAAAGACAT-3¢, 5¢-AUGUCUU
UAGUAUGCAGUUCAAUGGGU-3¢) was found to be
the most effective and used for these studies The control
DsiRNA sequence used was (5¢-CUUCCUCUCUUUCUC
UCCCUUGUGA-3¢, 5¢ UCACAAGGGAGAGAAAGA
GAGGAAGGA-3¢) In total, 1 · 105 Rat1 cells per well
were seeded in a 12-well tissue culture plate in compete
medium and incubated, 37C, 5% CO2 Twenty-four hours
later the medium was removed and replaced with 600 lL
compete medium After 1 h, 1.5 lL Silentfect (BioRad,
Hercules, CA, USA, 170-3360) in 50 lL DMEM was mixed
with 50 lL DMEM containing DsiRNA and added to the
cells, giving a final DsiRNA concentration of 10 nm The
cells were incubated, 37C, 5% CO2, for 48 h prior to
isolation of whole cell protein extracts or treatment with
H2O2or 24 h for isolation of total cellular RNA
ROS assay
The ROS assay was performed by labelling cells with
DCF-DA [43] Cells grown in complete medium were
labelled for 30 min with 20 lm DCF-DA (Sigma-Aldrich,
35845), 37C, 5% CO2 The cells were trypsinized, pelleted
and washed three times with ice-cold phosphate-buffered
saline (Lonza Group, BE17-516F), then analysed for
fluorescence by FACS (FACSCaliber, Becton Dickinson,
Oxford, UK)
Acknowledgements
This work was funded by a Glasgow Caledonian
University PhD studentship and Overseas Research
Student Award Scheme (PR) and in part by the
Leukaemia Research Fund (CB, 08018)
References
1 Weiser R (2007) The oncogene and developmental
regu-lator EVI1: expression, biochemical properties, and
bio-logical functions Gene 396, 346–357
2 Kurokawa M, Mitani K, Yamagata T, Takahashi T,
Izutsu K, Ogawa S, Moriguchi T, Nishida E, Yazaki Y
& Hirai H (2000) The evi-1 oncoprotein inhibits c-Jun
N-terminal kinase and prevents stress-induced cell
death EMBO J 19, 2958–2968
3 Buonamici S, Li D, Mikhail FM, Sassano A, Platanias
LC, Colamonici O, Anastasi J & Nucifora G (2005)
EVI1 abrogates interferon-alpha response by
selectively blocking PML induction J Biol Chem 280,
428–436
4 Liu Y, Chen L, Ko TC, Fields AP & Thompson EA (2006) Evi1 is a survival factor which conveys resistance
to both TGFbeta- and taxol-mediated cell death via PI3K⁄ AKT Oncogene 25, 3565–3575
5 Shackelford D, Kenific C, Blusztajn A, Waxman S & Ren R (2006) Targeted degradation of the AML1⁄ MD-S1⁄ EVI1 oncoprotein by arsenic trioxide Cancer Res
66, 11360–11369
6 Yuasa H, Oike Y, Iwama A, Nishikata I, Sugiyama D, Perkins A, Mucenski ML, Suda T & Morishita K (2005) Oncogenic transcription factor Evi1 regulates hematopoietic stem cell proliferation through GATA-2 expression EMBO J 24, 1976–1987
7 Hoyt PR, Bartholomew C, Davis AJ, Yutzey K, Gamer
LW, Potter SS, Ihle JN & Mucenski ML (1997) The Evi1 proto-oncogene is required at midgestation for neural, heart, and paraxial mesenchyme development Mech Dev 65, 55–70
8 Morishita K, Parker DS, Mucenski ML, Jenkins NA, Copeland NG & Ihle JN (1988) Retroviral activation
of a novel gene encoding a zinc finger protein in IL-3-dependent myeloid leukemia cell lines Cell 54, 831–840
9 Bartholomew C, Kilbey A, Clark AM & Walker M (1997) The Evi-1 proto-oncogene encodes a transcrip-tional repressor activity associated with transformation Oncogene 14, 569–577
10 Delwel R, Funabiki T, Kreider BL, Morishita K & Ihle
JN (1993) Four of the seven zinc fingers of the Evi-1 myeloid-transforming gene are required for sequence-specific binding to GA(C⁄ T)AAGA(T ⁄ C)AAGATAA Mol Cell Biol 13, 4291–4300
11 Funabiki T, Kreider BL & Ihle JN (1994) The carboxyl domain of zinc fingers of the Evi-1 myeloid transform-ing gene binds a consensus sequence of GAAGAT-GAG Oncogene 9, 1575–1581
12 Kurokawa M, Mitani K, Irie K, Matsuyama T, Takahashi T, Chiba S, Yazaki Y, Matsumoto K & Hirai H (1998) The oncoprotein Evi-1 represses TGF-beta signalling by inhibiting Smad3 Nature 394, 92–96
13 Izutsu K, Kurokawa M, Imai Y, Maki K, Mitani K & Hirai H (2001) The corepressor CtBP interacts with Evi-1 to repress transforming growth factor beta signal-ing Blood 97, 2815–2822
14 Ames BN, Shigenaga MK & Hagen TM (1993) Oxidants, antioxidants, and the degenerative diseases of aging Proc Natl Acad Sci USA 90, 7915–7922
15 Wu WS (2006) The signalling of ROS in tumor progres-sion Cancer Metastasis Rev 25, 695–705
16 Wistrand PJ (2002) Carbonic anhydrase III in liver and muscle of male rats Purification and properties Ups J Med Sci 107, 77–88
17 Lehtonen J, Shen B, Vihinen M, Casini A, Scozzafava
A, Supuran CT, Parkkila AK, Saarnio J, Kivela¨ AJ,