Extract
Note: Please read the complete
full text with Figures and Tables at
Introduction
Although many improvements of therapeutic techniques,
as well as new therapeutic methods, have been introduced
into cancer treatment, radiotherapy by ionizing radiation
continues to be the frontline treatment for many types of
cancers, including breast cancer[1]. Ionizing radiation is an
effective DNA-damaging agent, producing a range of lesions
in cellular DNA, including base damages, single-strand
breaks, double-strand breaks, DNA_DNA, and DNA_protein cross-links. Among them, DNA double-strand breaks
are the most important for cell death by ionizing radiation. It
is known that 2 major pathways, homologous recombination
(HR) and non-homologous end joining (NHEJ), are involved
in Double Strand Break repair[5]. The proteins that are known
to participate in such DNA repair include DNA-dependent
protein kinases (DNA-PK), Ku70 and Ku80, and X-ray-sensitive complementation group 4 (XRCC4), and DNA
ligase IV[6,7].
Tob1, a member of the Transducing Molecule of
ErbB2/B-cell Translocation G gene (Tob/BTG) family, is an
antiproliferative protein. Since the exogenous overexpression
of the Tob1 protein induces G1 arrest via the suppression of
cyclin D1, a major player in the G1 checkpoint of cell cycle
progression[8,9]. Increasing evidence suggests that Tob1
may be a new tumor suppressor gene for the following
reasons: (i) mice lacking the Tob1 gene rapidly develop
tumors in lungs, liver, and lymph
nodes[10]; (ii) the down-regulation of Tob1 expression is often observed in human
cancers[11]; and (iii) the inactivation of Tob
contributes to the progression of papillary carcinoma of the
thyroid[12], but the
increased expression of Tob1 protein suppresses tumor cell
growth and cell cycle arrest[8,9,13]. The degradation of the
Tob1 protein is through the ubiquitin_proteasome pathway
by the Skip1/Cull/F-box protein-Skp2 ubiquitin
ligase[14,15].
Enhancing the antitumor effects by combining ionizing
radiation with other agents, including gene therapy, often
allows lower doses to be used, thereby minimizing
side-effects. Tob1 is considered to play a role in mediating
radiosensitivity for the following reasons: (i) Tob1 inhibits the
expression and promoter activity of cyclin D1. Cyclin D1 is
most often overexpressed or amplified in breast cancer. Its
high expression results in a decrease in
radiosensitivity[16]; (ii) the activation
of Extracellular Signal Regulated Kinases(ERK)1 and 2 induces Tob
phosphorylation[17], while the downregulation of ERK2 is essential for the inhibition of
radiation-induced cell death[18]; and (iii) Tob1 inhibits
estrogen receptor-mediated transcriptional
activation[19]. It is known that the estrogen receptor plays a major role in breast
cancer development and mediates
radiosensitivity[20,21]. To test our hypothesis, we employed a system with the
adenovirus-mediated expression of Tob1 and found that the
enhanced expression of Tob1 by the adenovirus-mediated
expression of Tob1 increases the radiosensitivity of breast
cancer cells via increasing apoptosis and repressing DNA
repair by mediating the expression of pro-apoptotic protein
Bax and several DNA Double Strand Break repair proteins.
Materials and methods
Cell culture and irradiation Invasive
human breast cancer MDA-MB-231 cells were originally purchased from
American Type Culture Collection (Manassas, VA, USA) and
maintained in Dulbecco's modified Eagle's medium (Invitrogen,
Carlsbad, CA, USA) supplemented with 5% fetal bovine
serum, 100 unit/mL penicillin, 100 µg/mL streptomycin, and a
mixture of non-essential amino acids (Sigma_Aldrich, St
Louis, MO, USA) at 37 oC in an atmosphere
of 5% CO2. Irradiation was performed with
g-radiation (JL Shepherd Mark I Radiator, ) with a
137Cs source emitting at a fixed dose rate
of 3.5 Gy/min.
Generation of recombinant adenoviruses The
E1-deleted adenovirus-β-gal (Ad-β-gal) was obtained from
Introgen Therapeutics (Houston, TX, USA). A recombinant
adenovirus (pAd/CMV/V5-DEST; Invitrogen, USA)
containing a 1038-base pair DNA fragment encoding the complete
amino acid sequence of human Tob1 (Ad5-Tob1) between
the CMV promoter and the polyadenylation signal (TK pA)
was prepared as outlined in Figure 1. These adenoviral
vectors were propagated in 293 human embryonal kidney
cells (Invitrogen, USA) using the Stratagene MBS
mammalian transfection kit (La Jolla, CA,
USA) with a modified calcium phosphate transfection protocol. The transfected cells
were incubated at 37 oC for 7 d, then harvested and subjected
to freezing (liquid nitrogen)/thawing (in a 37
oC water bath) for 4 cycles.
The cell lysates were centrifuged at 12
000×g for 10 min at 4 oC, and the supernatant (primary virus stock)
was transferred to a fresh screw-cap mini-centrifuge tube and
stored at _80 oC. Recombinant adenoviruses were further
amplified using the same procedure; the cell lysates were
centrifuged on cesium chloride step gradients at 60
000×g for 2 h at 4 oC to separate the viruses from defective particles
and empty capsids. Recovered virus bands were dialyzed 3
times against phosphate-buffered saline (PBS). The viruses
were aliquoted in a buffer containing 10 mmol/L Tris-HCl
(pH 7.4), 10 mmol/L MgCl2, and 10%
v/v glycerol and stored at -80
oC. Under these conditions, there was no precipitation
of virus particles or loss of virus infectivity due to
inactivation or aggregation. To control the biological effect of the
virus per se, the vector, Ad5-CMV-Null, expressing no
transgene (Ad5), was constructed in a similar method, but
without the subcloned gene sequences.
For the adenovirus infection,
2.5×104 cells in each well of a sex-well dish were infected with the appropriate amount of
replication-defective adenoviruses with Ad-β-gal and
incubated with gentle shaking for 2 h at 37
oC. X-gal (1 mg/mL) was used to stain the cells for
β-gal and the cells were sustained overnight. The cells were
then fixed in 10% formalin, washed in PBS, and kept in PBS at 4
oC. Blue-stained cells were considered to be infected with
Ad-β-gal. Afterwards, fresh growth medium was added to each dish. To monitor
the Tob1 expression, the infected cells were further
incubated at 37 oC for various time-points and the Tob1 protein
was then determined by Western blot assay. To examine
radiosensitivity, the infected cells were incubated at 37
oC for 48 h before g-ray irradiation; a colony formation assay
was then performed.
Western blot assay The protein expression was detected
by Western blot assay, as previously
described[22]. The cells were harvested by trypsin and centrifugation. The cells were
washed twice with ice-cold 1×PBS and lysed with a
protein lysis buffer containing Tris-HCl [50 mmol/L
(pH 7.4)], NP-40 (1%), Na-deoxycholate (0.25%), NaCl (150 mmol/L), EDTA (1
mmol/L), phenylmethylsulphonyl fluoride (PMSF) (1
mmol/L), Na3VO4 (1 mmol/L), NaF (1 mmol/L), and an inhibitor cocktail
(Sigma_Aldrich, USA). After centrifuga-tion, the
supernatant was transferred into new tubes. The protein
concentration was determined by the Bio-Rad protein assay
(Invitro-gen, USA). 50 µg total whole cell lysates were separated on
SDS_PAGE and electroblotted onto nitrocellulose
membranes (Millipore, Billerica, MA, USA). The membranes were
then incubated in blocking solution of 5% non-fat milk in
TBS-T (20 mmol/L Tris-HCl, 150 mmol/L NaCl, and 0.1%
Tween-20), followed by overnight incubation with the
appropriate primary antibodies. After completely washing
with TBS-T and incubating with horseradish
peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology,
Santa Cruz, CA, USA) for 1 h, immunocom-plexes were
developed with an enhanced horseradish peroxidase/luminol
chemiluminescence reagent (Sigma_Aldrich, USA)
according to the manufacturer's instruction. Primary antibodies
included a mouse monoclonal anti-Tob1 antibody (4B1,
1:500 dilution) and a rabbit polyclonal anti-Ku80 antibody
(557-570, 1:500 dilution), which were purchased from
Sigma_Aldrich (USA). A mouse monoclonal anti-Ku70 antibody
(A-9, 1:1,000 dilution), a mouse monoclonal anti-DNA_PK
antibody (G-4, 1:1,000 dilution), a goat polyclonal anti-XRCC4
antibody (D-18, 1:500 dilution), an antigoat polyclonal
antibody, and a goat polyclonal anti-actin antibody (I-19,
1:2,000 dilution) were obtained from Santa Cruz
Biotechnology (USA).
Clonogenic assay Cell survival was evaluated by
clono-genic assay as detailed in a previous
study[23]. Briefly, the cells were trypsinized immediately after irradiation and
counted. Known numbers were subcultured in 100 mm
culture dishes in 2 sets of triplicates for
each dose of radiation; sufficient numbers were seeded to ensure that about 50_100
macroscopic colonies would appear in each plate of untreated
and uninfected control cells at the end of 15 d. The colonies
were then fixed, stained, and counted. Surviving fractions
were normalized by the plating efficiency of unirradiated
controls (40%_60% for MDA-MB-231). a and b (constants
determined by the linear quadratic model where
S=e_[aD+bD2] and S is the fraction of cells surviving a dose [D]) parameters,
as well as D0 (defined by slope of the terminal exponential
region of the 2-component [single-hit multitarget model]
survival curve where slope=1/D0),
Dq (quasithreshold dose), and n (extrapolation number) values were calculated for
com-parison.
DNA fragmentation gel electrophoresis Apoptosis
induction was determined by a DNA fragmentation gel
electrophoresis, as described in a previous
study[23]. The cells were collected by trypsin and centrifuged for 5 min at
13 000×g. The cells were washed with 1×PBS and
centrifuged again. The cell pellets were then lysated with 200 µL
lysis buffer [10 mmol/L Tris-HCl (pH 7.6), 100 mmol/L EDTA,
and 20 mmol/L NaCl] and centrifuged. The supernatant was
transferred into new tubes. 20 µL SDS plus 200 µL RNase A
(10 mg/mL, Sigma_Aldrich, USA) was added to each tube.
After 2 h incubation at 56 oC, 30 µL proteinase K (50 mg/mL,
Sigma_Aldrich, USA) was added to each tube. The cell
lysates were incubated at 37 oC for another 2 h. The DNA was
finally precipitated with the addition of 10
µL of 10 mol/L potassium acetate and 1 mL 100% ethanol at _80
oC for 30 min. The extracted DNA samples were centrifuged and
washed with 70% ethanol. Pure DNA were finally loaded
and run on a 1% agarose gel at 80 V in running buffer (89
mmol/L Tris-acetate, 2 mmol/L Na2EDTA, and 89 mmol/L
boric acid), stained with ethidium bromide and photographed.
The samples were run in tandem with a DNA molecular weight
ladder (Invitrogen, USA) providing molecular size markers
of 0.5 to 12 kilobase pairs. Gel photographs were evaluated
for typical ladder patterns of low molecular weight DNA
fragments in multiples of 180_200 base pairs, a hallmark of
apoptosis.
Determination of apoptosis by terminal
deoxynucleo-tidyl transferase-mediated nick end labeling assay
Terminal deoxynucleotidyl transferase-mediated nick end
labeling (TUNEL) was performed using the
APO-BRDUTM kit (Phar-Mingen, San Diego, CA, USA) to quantify the induction of
apoptosis following the manufacturer's instructions. Briefly,
the cells were fixed in 1% (w/v) paraformaldehyde in ice-cold
PBS and incubated on ice for 15 min. Then the cells were
washed twice with PBS and stored in 70%
(v/v) ethanol overnight. About
1×106 cells/treatment in duplicate, along
with positive and negative controls, were counted, pelletized,
washed twice with wash buffer, and subjected to labeling
reaction using terminal deoxynucleotidyl transferase
overnight at room temperature. At the end of
the reaction, the cells were rinsed twice before treatment with
fluorescein-labeled anti-BrdU antibody solution in the
dark for 30 min. The cells were stained with propidium iodide/RNase
solution for 30 min in the dark and analyzed by flow cytometry
(Epics XL-MCL, Beckman Coulter, Miami, FL, USA).
Host cell reactivation assay Ten thousand cells were
plated in each well of 6-well plates. The cells were infected
with Ad5-Tob1 or Ad5 at a multiplicity of infection (MOI) of
50 or 100 followed by 48 h incubation. The cells were then
infected with Ad-beta-gal
(1×103 vp/cell) that had been
irradiated with 0_4000 Gy of g-ray irradiation and
incubated for an additional 24 h. This dose of 4000 Gy was
necessitated by the small genome size of the adenoviral vector
compared with a mammalian cell. The calculations
indicated that this dose would produce about 1_2 DSBs/vector
particles. The cells were then stained with X-gal following
the procedure described above and fixed in 10% formalin.
beta-gal-positive (blue) cells were scored under high power
(×40) of a light microscope. The data are presented as the
percentage of the control.
Statistics All experiments were repeated at least 3
times. The results are expressed as the mean±SEM.
Statistical analysis was performed with 2-tailed Student's
t-test when 2 treatment regimens were compared.
Probability values of P<0.05 were considered statistically
significant.
Results
Adenovirus-mediated Tob1 gene expression increases
sensitivity of MDA-MB-231 cells to ionizing radiation
A significant infection of MDA-MB-231 cells with
adenoviral vectors was confirmed by β-gal staining (data not shown)
and the Tob1 protein expression were monitored by Western
blot assay (Figure 2A). A time-dependent increase of Tob1
protein expression was obtained following the infection of
Ad5-Tob1 at a MOI of 100 plaque-forming
units/cell, starting at 24 h and reaching the maximum at 96 h, while till higher
than uninfected control cells at 144 h. Control Ad5 infection
did not affect Tob1 expression (data not shown). Greater
than 95% transduction efficiency was detected in these cells
(data not shown).
As illustrated in Figure 2B, MDA-MB-231 showed a
typical clonogenic survival curve with a shoulder signifying
cellular repair ability. The infection of the MDA-MB-231 cells
with Ad5-Tob1 significantly increased cell susceptibility
to g-irradiation in an Ad5-Tob1 dose-dependent fashion.
For example, doses of 2, 4, and 6 Gy of g-radiation alone
killed about 28%, 56%, and 85% of cells, respectively,
while Ad5-Tob1 at the MOI of 100 enhanced cell death to
73% (P<0.01), 87% (P<0.01), and 97%
(P<0.05) at 2, 4, and 6 Gy of g-radiation, respectively. However, Ad5 (as a
negative control) did not show any significant effects on
radiosensitivity (Figure 2B). Survival curves were analyzed
using the single-hit multitarget and linear quadratic
models of cell survival. a and b (constants determined by the
linear quadratic model where
S=e_[aD+bD2] and S is the fraction of cells surviving a dose [D]) parameters as well as
D0 (defined by slope of the terminal exponential region of the
2-component [single-hit multitarget model] survival curve
where slope=1/D0), Dq (quasithreshold dose),
and n (extrapo-lation number) values are summarized in Table 1. There were
statistically significant differences in these values between
the cells infected with Ad5 and the cells infected with
AD5-Tob1 (P<0.01). Both Ad5-Tob1 and Ad5 at the MOI less than
100 did not produce any significant toxic effects in
MDA-MB-231 cells (data not shown). These results suggested
that the radiosensitizing effect of Ad5-Tob1 was mediated
by the increased expression of Tob1 and was not due to a
non-specific effect of the vector.
Ad5-Tob1 increases irradiation-induced apoptosis
In order to determine whether the increased radiosensitivity by
the adenovirus-mediated expression of Tob1 was due to
apoptosis, the TUNEL assay using the
APO-BRDUTM kit (Phoenixflow, San Diego CA, USA) was performed. The
g-ray irradiation or Ad5-Tob1 (at the MOI of 100 at 48 h
infection) alone was ineffective in inducing apoptosis in
MDA-MB-231, a cell line having a mutant p53 gene (Figure
3A). However, g-ray potently induced apoptosis in the
presence of 48 h-pre-infection of Ad5-Tob1. This degree of
sensitization appeared to be greater than additive. For
example, 6 and 8 Gy irradiation alone caused about 9% and
15% apoptosis induction, respectively, and Ad5-Tob1 alone
induced approximately 4% apoptosis. The combination,
however, induced approximately 35% and 43% of apoptotic
cells (radiation alone vs the combination of Ad5-Tob1 and
radiation, P<0.01). This synergistic effect was not observed
after the combination of Ad5 and g-ray. A significantly
increased induction of apoptosis was also seen by using the
DNA fragmentation assay in cells treated with Ad5-Tob1 (at
the MOI of 100 at 48 h pre-infection) and g-ray irradiation
(Figure 3B). These results clearly indicated that the increased
expression of Tob1 may enhance the apoptosis induction in
cells exposed to g-ray.
Ad5-Tob1 represses DNA repair Based on the large
reduction in Dq seen in the survival curves (Figure 2B), a
host cell reactivation assay was carried out to determine
whether the radiosensitizing effect of Ad5-Tob1 could be
explained as a suppression of the facility to repair DNA
damages in MDA-MB-231 cells. To do so, the cells were
accepted at 48 h-pre-infection with either Ad5-Tob1 or Ad5 at
the MOI of 100 and then infected with Ad-β-gal that had
been irradiated with g-ray irradiation at a single dose of 4000
Gy. The ability of the MDA-MB-231 cells to reactivate the
irradiated Ad-β-gal based on β-gal expression was assessed
24 h later. The results of these experiments (Figure 4),
presented as the percentage of the controls using unirradiated
Ad-β-gal, showed that MDA-MB-231 cells infected with
Ad5-Tob1 had a significantly lower ability to reactivate irradiated
Ad-β-gal compared with MDA-MB-231 cells that had either
uninfection or Ad5 infection (P<0.01). These results
indicated that an increase of Tob1 expression by the
adenovirus-mediated Tob1 gene transfer results in a reduced
capability of MDA-MB-231 to repair DNA.
Ad5-Tob1 increases Bax and decreases DNA repair
protein expression First, we determined the effect of Ad5-Tob1
on the expression of the pro-apoptotic protein Bax and the
anti-apoptotic protein Bcl-2, 2 well-known members of the
Bcl-2 gene family in apoptosis
induction[24]. As shown in Figure 5, a significant elevation of the Tob1 protein was
observed in the MDA-MB-231 cells infected with Ad5-Tob1.
Furthermore, a slight increase of Tob1 was found in response
to irradiation with g-ray, although the significance of such a
response is unclear so far. Second, exposure to g-ray caused
a dose-dependent increase of the Bax protein in the cells
infected with control Ad-5. The infection of Ad5-Tob1 alone
increased the base level of the endogenous Bax protein.
Moreover, it enhanced an increase of the g-ray-mediated Bax,
which also showed a g-ray dose-dependent promotion.
However, neither g-ray+Ad5 nor Ad5-Tob1+g-ray produced
any significant effects on the Bcl-2 protein. Third, we found
that Ad5-Tob1 not only reduced the endogenous levels of
several proteins involved in the DNA DSB repair, including
DNA-PK (Ku70 and Ku80) and XRCC4, but it also blocked
the increase of these proteins by g-ray irradiation (Figure 5).
Taken together with the in vitro DNA repair data in Figure 4,
these results indicated that the inhibition of DNA repair by
Ad5-Tob1 may be due to Ad5-Tob1 downregulation of DNA
DSB repair proteins. Further investigation is underway to
determine the vital roles of the DNA DSB repair proteins in
Tob1-mediated radiosensitivity in our laboratory.
Discussion
Tob1 is an antiproliferative protein and plays a role in
preventing cell cycle progression by downregulating the
expression and promoter activity of cyclin D1, an important
G1 checkpoint player[8,9]. Furthermore, knocking-down or
loss of the Tob1 gene in mice results in multiple tumor
development[10] and tumor
progression[11]. Based on these studies,
strategies have been developed for manipulating the Tob1
expression in the treatment of human cancer; one such
approach involves the replacement of Tob1 into tumor cells by
gene therapy[13].
The present study examined whether Tob1 might radiosensitize breast cancer cells and also the underlying
mechanism(s) by examining the repair of radiation-induced
DNA damage. Ad5-Tob1 significantly enhanced the
radiosensitivity of human breast cancer MDA-MB-231 cells in a
synergistic manner as determined based on clonogenic
survival compared with the control Ad5 vector (Figure 1).
Ad5-Tob1 also restored radiation-induced
apoptosis in MDA-MB-231 cells as shown in Figure 2. Therefore, enhanced
radiosensitivity may be due to the restoration of
radiation-induced apoptosis in some settings.
Consistent with the enhanced induction of apoptosis by
a combination of Ad5-Tob1 and g-ray, we also found that
the expression of the pro-apoptotic protein Bax, one of the
Bcl-2 family proteins, was increased in MDA-MB-231 cells
treated with Ad5-Tob1+g-ray (Figure 5). It is well known that
members of the Bcl-2 family are the most prominent
mediators of apoptosis induction by a large number of antitumor
drugs and ionizing radiation in a variety of cell types,
including cancer cells[25_28]. The Bcl-2 family consists of a growing
number of proteins, containing 4 conserved Bcl-2 homology
domains (BH, BH2, BH3, and BH4) with a transmembrane
domain and can be classified as 3
subgroups[27]: (i) the Bcl-2 subgroup, including all anti-apoptotic proteins, like Bcl-2
and Bcl-xL; (ii) the Bax subgroup, consisting of pro-apoptosis
members, like Bax and Bad; and (iii) the third subgroup,
containing BH3-only proteins, like Bid and Bim. The last
subgroup members interact with either anti-apoptotic proteins
or pro-apoptosis members and cause apoptosis promotion.
Thus, the upregulation of the pro-apoptosis protein Bax by
Ad5-Tob1 may be an important mechanism contributing to
the Adt-Tob1 promotion of radiation-mediated apoptosis,
although the anti-apoptotic protein Bcl-2 is unaltered.
Experiments to determine the possible involvement of other
Bcl-2 family members in Ad5-Tob1 radiosensitivity are
underway in our laboratory, which will provide more
information about the role of Bcl-2 family members in
Ad5-Tob1-mediated apoptosis in radiosensitivity.
DNA is a major target in damage caused by ionizing
radiation. DNA DSB are potent inducers of mutations and of
cell death by irradiation. For the survival of irradiated
mammalian cells, the repair of radiation-induced DSB is essential
and such repair apparently involves 2 mechanistically
distinct, but always overlapping, pathways: HR and NHEJ.
The repair of radiation-induced DSB can be detected by
using pulsed-field gel electrophoresis techniques, but this
approach is not sensitive enough for some types of misrepaired
lesions. Thus, we performed the assays of DNA repair by
using a host cell reactivation assay. This relatively old
approach[29] was updated to incorporate the expression of a
reporter gene as the readout;
radiation-induced DNA lesions in the reporter gene are effectively and efficiently repaired
by the host cells with complete fidelity in order for
functional gene expression to be restored. The results shown
in Figure 4 indicate that the cells infected with Ad5-Tob1
have significantly less capability to restore reporter gene
expression using an irradiated Ad-β-gal vector by more than
60% when compared with controls (uninfected and Ad5
infected). Thus, the overexpression of Tob1 in this
context sensitizes cells to ionizing radiation by suppressing their
capacity for repairing radiation-induced DNA lesions.
Consistent with these DNA repair results, we demonstrated that
the expression of certain critical proteins involved in DNA
damage repair pathways were downregulated by Ad5-Tob1
in MDA-MB-231 cells in response to ionizing radiation, as
shown in Figure 5, that is, both the endogenous base level
of DNA-PK (Ku70 and Ku80) and the XRCC4 proteins, and
the radiation-increased expressions of these proteins were
downregulated and blocked by adenovirus-mediated Tob1
gene expression. It is known that the DNA_PK, activated
by DNA ends, is a central component in the NHEJ pathway.
DNA-PK is a serine/threonine kinase that consists
of a large catalytic subunit of (DNA-dependent protein kinase
catalytic subunit, DNA-PKcs) and a DNA-targeting component Ku
(a heterodimer of Ku70 and
Ku80)[30,31]. Mammalian cells deficient in DNA-PKcs (Ku70 and Ku80) and XRCC4 exhibit
hypersensitivity to radiation, deficiencies in DNA DSB
repair and V(D)J name is too long to
writerecombination[32_34]. Thus, our present studies suggest that Tob1 may participate
in DNA DSB repair intermediates and pathways, resulting in
cellular hyposensitivity to radiation, although the interplay
of these molecular events is not clearly understood at present.
In summary, we have for first time demonstrated that the
enhanced expression of Tob1 by the
adenovirus-mediated Tob1 gene transfer augments the response of breast cancer
cells to ionizing radiation by increasing apoptosis, reducing
DNA repair ability, increasing the pro-apoptotic protein Bax,
and suppressing the expression of critical proteins in the
DNA repair pathway. A complete understanding of these
effects must await future studies. However, our present
in vitro observations underscore the need for the continued
development of strategies for sensitizing human tumor cells
to cancer radiotherapy that kill cells by using the
adenovirus-mediated expression of Tob1 in clinics.
References
1 Kufe D, Weichselbaum R. Radiation therapy: activation for gene
transcription and the development of genetic
radiotherapy-therapeutic strategies in oncology. Cancer Biol Ther 2003; 2: 326_9.
2 Robson T, Worthington J, McKeown SR, Hirst DG. Radiogenic
therapy: novel approaches for enhancing tumor radiosensitivity.
Technol Cancer Res Treat 2005; 4: 343_61.
3 Freyta SO, Kim JH, Brown SL, Barton K, Lu M, Chung M. Gene therapy
strategies to enhance the effectiveness of cancer radiotherapy.
Curr Opin Mol Ther 2004; 6: 513_24.
4 Lumniczky K, Safrany G. Cancer gene therapy: combination
with radiation therapy and the role of bystander cell killing in the
anti-tumor effect. Pathol Oncol Res 2006; 12: 118_24.
5 Jackson SP. Sensing and repairing DNA double-strand breaks.
Carcinogenesis 2002; 23: 687_96.
6 Han Z, Wang H, Hallahan DE. Radiation-guided gene therapy of
cancer. Technol Cancer Res Treat 2006; 5: 437_44.
7 Ferguson DO, Alt FW. DNA double strand break repair and
chromosomal translocation: lessons from animal models. Oncogene 2001; 20:
5572_9.
8 Bassing CH, Swat W, Alt FW. The mechanism and regulation of
chromosomal V(D)J recombination. Cell 2002; 109 (Suppl):
S45_55.
9 Matsuda S, Kawamura-Tsuzuku J, Ohsugi M, Yoshida M, Emi M,
Nakamura Y, et al. Tob, a novel protein that interacts with
p185erbB2, is associated with anti-proliferative activity.
Oncogene 1996; 12: 705_13.
10 Suzuki T, K-Tsuzuku J, Ajima R, Nakamura T, Yoshida Y,
Yamamoto T. Phosphorylation of three regulatory serines of
Tob by Erk1 and Erk2 is required for Ras-mediated cell
proliferation and transformation. Genes Dev 2002; 16: 1356_70.
11 Yoshida Y, Nakamura T, Komada M, Satoh H, Suzuki T, Tsuzuku
JK, et al. Mice lacking a transcriptional corepressor Tob are
predisposed to cancer. Genes Dev 2003; 17: 1201_6.
12 Ito Y, Suzuki T, Yoshida H, Tomoda C, Uruno T, Takamura Y,
et al. Phosphorylation and inactivation of Tob contributes to the
progression of papillary carcinoma of the thyroid. Cancer Lett
2005; 220: 237_42.
13 Yanagie H, Sumimoto H, Nonaka Y, Matsuda S, Hirose I, Hanada
S, et al. Inhibition of human pancreatic cancer growth by the
adenovirus-mediated introduction of a novel growth suppressing
gene, tob, in vitro. Adv Exp Med Biol 1998; 451: 91_6.
14 Sasajima H, Nakagawa K, Yokosawa H. Antiproliferative
proteins of the BTG/Tob family are degradated by the
ubiquitin-proteasome system. Eur J Biochem 2002; 269: 3596_604.
15 Hiramatsu Y, Kitagawa K, Suzuki T, Uchida C, Hattori T, Kikuchi
H, et al. Degradation of Tob1 mediated by SCFSkp2-dependent
ubiquitination. Cancer Res 2006; 66: 8477_83.
16 Xia F, Powell SN. The molecular basis of radiosensitivity and
chemosensitivity in the treatment of breast cancer. Semin Radiat
Oncol 2002; 12: 296_304.
17 Suzuki T, Tsuzuku JK, Ajima R, Nakamura T, Yoshida Y,
Yamamoto T. Phosphorylation of three regulatory serines of Tob by
Erk1 and Erk2 is required for Ras-mediated cell proliferation and
transformation. Genes Dev 2002; 16: 1356_70.
18 Cho HN, Lee YJ, Cho CK, Lee SJ, Lee YS. Downregulation of
ERK2 is essential for the inhibition of radiation-induced cell
death in HSP25 overexpressed L929 cells. Cell Death Differ
2002; 9: 448_56.
19 Kawate H, Wu Y, Ohnaka K, Nawata H, Takayanagi R. Tob
proteins suppress steroid hormone receptor-mediated
transcriptional activation. Mol Cell Endocrinol 2005; 230: 77_86.
20 Toillon RA, Magne N, Laios I, Lacroix M, Duvillier H, Lagneaux
L, et al. Interaction between estrogen receptor alpha, ionizing
radiation and (anti-) estrogens in breast cancer cells. Breast
Cancer Res Treat 2005; 93: 207_15.
21 Paulsen GH, Strickert T, Marthinsen AB, Lundgren S. Changes
in radiation sensitivity and steroid receptor content induced by
hormonal agents and ionizing radiation in breast cancer cells
in vitro. Acta Oncol 1996; 35: 1011_9.
22 Fan S, Wang J, Ma Y, Yuan R, Meng Q, Erdos
M, et al. Role of direct interaction in BRCA1 inhibition of estrogen receptor
activity. Oncogene 2001; 22: 77_87.
23 Fan S, Wang J, Yuan R, Rockwell S, Andres J, Zlatapolskiy
A, et al. Scatter factor protects epithelial and carcinoma cells against
apoptosis induced by DNA damaging-agents. Oncogene 1998;
17: 131_41.
24 Kim R. Recent advances in understanding the cell death
pathways activated by anticancer therapy. Cancer 2005; 103:
1551_60.
25 Walensky LD. BCL-2 in the crosshairs: tipping the balance of
life and death. Cell Death Differ 2006; 13: 1339_50.
26 Adams JM, Cory S. Life-or-death decisions by the Bcl-2 protein
family. Trends Biochem Sci 2001; 26: 61_6.
27 Thomadaki H, Scorilas A. BCL-2 family of
apoptosis-related genes: functions and clinical implications in cancer.
Crit Rev Clin Lab Sci 2006; 43: 1_67.
28 Kim R. Recent advances in understanding the cell death
pathways activated by anticancer therapy. Cancer 2005; 103:
1551_60.
29 Rainbow AJ. Repair of radiation-induced DNA breaks in human
adenovirus. Radiat Res 1974; 60: 155_64.
30 Featherstone C, Jackson SP. Ku, a DNA repair protein with
multiple cellular functions? Mutat Res 1999; 434: 3_15.
31 Tuteja R, Tuteja N. Ku autoantigen: a multifunctional
DNA-binding protein. Crit Rev Biochem Mol Biol 2000; 35: 1_33.
32 Kurimasa A, Ouyang H, Dong LJ,Wang S, Li X, Cordon-Cardo C,
et al. Catalytic subunit of DNA-dependent protein kinase:
impact on lymphocyte development and tumorigenesis. Proc Natl
Acad Sci USA 1999; 96: 1403_8.
33 Nussenzweig A, Chen C, da Costa Soares V, Sanchez M, Sokol K,
Nussenzweig MC, et al. Requirement for Ku80 in growth and
immunoglobulin V(D)J recombination. Nature (Lond) 1996; 382:
551_5.
34 Ouyang H, Nussenzweig A, Kurimasa A, Soares VC, Li X,
Cordon-Cardo C, et al. Ku70 is required for DNA repair but not for TCR
gene recombination in vivo. J Exp Med 1997; 186: 921_9.
|
