Radiation-inducible promoter mediated PUMA Gene in the the treatment of Tca8113

doi:10.4236/ojst.2011.14030

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Open Journal of Stomatology, 2011, 1, 195-201

doi:10.4236/ojst.2011.14030 Published Online December 2011 (http://www.SciRP.org/journal/ojst/

OJST

Published Online December 2011 in SciRes. http://www.scirp.org/journal/OJST

Radiation-inducible promoter mediated PUMA gene in the the

treatment of Tca8113

Dong-Sheng Yu1, We i Zhao1, Jia-Xian Luo1, Bing Liu1, Xiao-Lin Wu2

1Institute of Stomatological Research, Sun Yat-sen University, Guangzhou, China;

2Department of Stomatology, The Conde S. Januário Hospital Centre, Macao, China.

Email: [email protected]

Received 12 August 2011; revised 20 September 2011; accepted 3 October 2011.

ABSTRACT

Objective: The aim of this study is to explore the

therapeutic effect of the PUMA gene mediated by ra-

diation-inducible promoters in the treatment of ton-

gue squamous cell carcinoma. Methods: Recombi-

nant pcDNA3.1 (+)/E-PUMA was constructed, in

which the PUMA gene was mediated by a synthetic

radiation inducible promoter. The recombined plas-

mids were transfected into the Tca8113 cell and xeno-

grafts of human tongue squamous carcinoma in na-

ked mice respectively. After 24 h, the tumors were tr-

eated with 3 Gy of irradiation to upregulate the PU-

MA gene expression. PUMA mRNA was detected by

RT-PCR. Proliferating cell nuclear antigen (PCNA)

and apoptosis were detected by immunohistochemi-

cal method and in situ end-labeling (ISEL) respecti-

vely. The data were analyzed using the SPSS11.0 soft-

ware package for chi-square test. Results: Compared

with the control group, the comparative survival rate

of Tca8113 cells in the PUMA/IR group was markedly

decreased and the xenografts were signifycantly sup-

pressed. Up-regulation of PUMA gene expression was

observed in the Tca8113 cells and in the xenografts

after irradiation. The apoptosis indices of the Tca-

8113 cells and xenograft with irradiation were mar-

kedly higher than those without irradiation. At the

same time, the proliferation indices of the Tca8113

cells and xenografts with irradiation were markedly

lower than those without irradiation. Conclusions:

radiation-inducible promoters can serve as molecular

switches to improve the expression of PUMA gene in

tongue squamous cell carcinoma both in vivo and in

vitro. Low-dose induction radiation can significantly

improve therapeutic efficiency.

Keywords: Blast; Graph Theory; Redundant Sequences;

CD-HIT

1. INTRODUCTION

The p53 up-regulated modulator of apoptosis (PUMA)

was newly discovered in 2001, which possessed power-

ful pro-apoptotic effect as a member of the Bcl-2 family

and the key protein during the apoptosis caused by ra-

diation [1-3]. Although its specific mechanism for in-

ducing apoptosis needs further explanation, the pro-

apoptotic function has achieved rather good results, be-

coming a promising new target in gene therapy [4-6].

Radiation-inducible promoters can regulate and control

the downstream therapeutic gene expression, a new stra-

tegy for radiation-gene therapy founded in recent years

[7,8]. It combines traditional radiation treatment with

gene therapy, which may reduce radiation dosage and in-

creases efficacy of gene therapy [9,10]. Human tongue

squamous cell carcinoma is the most common type of

neoplasm in the oral cavity [11,12]. Little is known

about the role of PUMA in the targeted treatment of hu-

man tongue squamous cell carcinoma. This research adopt-

ed radiation-inducible promoters to mediate PUMA gene

for treating human tongue squamous cell carcinoma in

vivo or in vitro, to observe the synergistic effect of radia-

tion and to explore a new policy for the targeted therapy

of tongue squamous cell carcinoma.

2. MATERIALS AND METHODS

2.1. Cells and Cell Culture

Tongue squamous carcinoma cells (China Center for Ty-

pe Culture Collection Wuhan University Hubei China)

were cultured in RPMI 1640 (GIBCO, Carlsbad, CA,

USA) culture medium supplemented with 10% fetal calf

serum (GIBCO), 50 U/ml penicillin G, and 50 µg/ml

streptomycin. Subculture occurred weekly (seeded at 5 ×

105 cells/ml), with feeding with complete medium twi-

ce a week. All cells were cultured in a humidified 5%

CO2 environment at 37˚C.

2.2. Animals

BALB/c naked mice were provided by the Experimen-

D.-S. Yu et al. / Open Journal of Stomatology 1 (2011) 195-201

196

tal Animal Center of Sun Yat-sen University. Animal ex-

periments were performed with the permission from the

Animal Ethical Commission of Sun Yat-sen University,

China.

2.3. pcDNA (+)–3.1/E-PUMA Construction

All restriction and modifying enzymes were supplied by

Promega or Roche and were used according to the ma-

nufacturer’s instructions. DNA isolation and purification

were carried out using the appropriate kit from Qiagen

(Crawley, UK). Nhe I and Hind III were used for conduct-

ing double digestion of pMD18-T-E, producing DNA frag-

ments containing radiation inducible promoter sequences.

The six-CArG elements enhancer (E6) was cloned using

the complementary single-strand-edoligodeoxyribo-nu-

cleotides pair: 5’-AGATCTGC TAGC(CCATATAA-GG)6-

TCGCGAA-3’ and 5’-T CGCGA(CCTTATATGG)6GC-

TAGCAGATCTA-3’, which were synthesized by the Son-

gon Company, Shanghai, China. Linkers were produced

by mixing 0.05 nmol of each oligonucleotide in 5 µl vo-

lumes, heating them at 55˚C for 5 min and then cooling

them to room temperature. They were inserted into PU-

MA upstream of the plasmid pcDNA (+)3.1-PUMA, ob-

taining the eukaryotic expression plasmid pcDNA (+)3.1-

E-PUMA,which carries the radiation inducible promo-

ter mediated PUMA gene.

2.4. The Killing Effect of PUMA Gene Mediated

by Radiation-Inducible Promoter in Vitro

Grouping. Tca8113 cells were divided into four groups

as follows:

Control group: no intervention; PUMA group: PUMA

gene transfection only; IR group: irradiation only; PU-

MA/IR group: PUMA gene transfection and irradiation.

Transfection of DNA. Tca8113 cells were transfected

with Lipofectamine (GIBCO) by modifying the manu-

facturer’s procedure based on our previous study [7,8].

Tca8113 cells (2 × 105) were seeded into a six-well plate

at 2 × 105 cells per well, and conventionally maintained

for 24 h. A total of 24 wells of Tca8113 cells were pre-

pared to be transfected. Two micrograms of plasmid

DNA (10 µL) was mixed with 90 µL of serum-free me-

dium. This mixture was then incubated with a mixture of

15 µL lipofectamine (2 mg/mL) and 85 µL serum-free

medium for 45 min at room temperature. Subsequently,

the DNA-Lipofectamine complex (200 µL) was added to

each well.

Radiation procedure. After transfection for 24 h, the

transfected cells were irradiated by linear accelerator

(Primvs, Siemens, Germany) at a dose of 3 Gy. We cho-

se the radiation dose of 3 Gy based on our previous re-

search [8]. The monolayers of the transfected cells in the

control group were also mock-irradiated and subse qu-

ently treated identically as with the irradiated monolay-

ers of transfected cells.

Microculture tetrazolium assay. A micro-culture te-

trazolium (MTT) was used to assess cell viability. All ex-

periments were performed in triplicate. The results were

expressed as percentages of the control, which was con-

sidered 100%.

RNA preparation and RT-PCR. After IR for 48 h,

total mRNA from transfected Tca8113 cells was ex-

tracted using an RNeasy Mini Kit (Qiagen, USA) ac-

cording to the manufacturer’s in structions. RT-PCR was

carried out using a reverse transcription kit (Promega,

USA) and human β-actin gene was used as the internal

control. The PUMA gene primers were as follows: 5’-T-

GTCGAATAAACGCTTTACAAAC-3’ (forward) and 5’-

AACGTTTGTAATG ATGG CTTCTG-3’ (reverse). The

β-actin primers were as follows: 5’-GGTCGGAGTCA-

ACGGATTTGGTCG-3’ (forward) and 5’-CCTCCGAC-

GCCTGCTTCAC CAC-3’ (reverse). The transcript lev-

els were normalized according to transcription of the β-

actin internal control, and the products were resolved by

agarose gel electrophoresis. The intensity was quantified

by image-analysis software (NIH Image; Apple, Cuper-

tino, CA, USA).

Detection of apoptosis by flow cytometry. After trans-

fection for 48 h, both adherent and floating cells were

harvested and centrifuged. The cell pellets were washed

with phosphate-buffered saline (PBS) and then fixed

with overnight with 70% ethanol. After incubation in

phosphate-citric acid, the cells were resuspended in PBS

containing propidium iodide (10 µg/mL) and RNase (10

µg/mL) .The cells were quantified with a FACScan flow

cytometer (Becton Dickinson). Salmon sperm DNA (ss-

DNA) was used as the DNA toxicity control.

2.5. Tumor Inhibition Effect in Vivo

Establishment of transplantable tumor model. The

freeze-stored Tca8113 cells were revived in an incubator

with 5% CO2 at 37˚C, and subcultured in RPMI-1640

containing 100 mL/L fetal bovine serum. The trypsin

method was utilized to collect the cancer cells. Naked

mice 6 to 8 weeks old were given subcutaneous inject-

tions at 6 × 106 inoculated tumor cells for each mouse.

When the diameters of transplanted tumor reached 8 mm,

the treatments were begun.

Plasmid transfection and radiation therapy. The

mice with transplanted tumors were randomly divided

into four groups with 10 mice for each group: (A) con-

trol group, (B) transfection group, (C) radiation group,

and (D) transfection and radiation group. Then, 2 μg of

the plasmid pcDNA3.1 (+)/E-PUMA was dissolved into

100 μL of serum-free RPMI-1640 culture medium. At

the same time, 80 μL serumfree RPMI-1640 culture me-

D.-S. Yu et al. / Open Journal of Stomatology 1 (2011) 195-201 197

dium was used to dilute 20 μL Lipofectamine. Both of

them were mixed and shaken well, and another 800 μL

of serum-free RPMI-1640 culture medium was added

into the mixture. This prepared miscible liquid was used

for intratumoral injection (100 μL/mouse). After 24 h

transfection, groups C and D were exposed to 3 Gy of

radiation via a linear accelerator.

Description of tumor growth curve. Vernier caliper was

used to measure maximum of the tumors and maximum

transverse diameter of tumors (b) every four days, and

the maximum tumor size of multiple tumors were re-

corded. Tumor size was calculated using the formula V =

1/2(a × b2). On the 20th day of transfection, the animals

were euthanized and tumor growth curves were con-

structed.

RNA preparation and RT-PCR. After transfection

for 96 h, the naked mice were sacrificed and the tumor

tissues were subsequently collected. RT-PCR was em-

ployed as in the in vitro experiment described above.

Detect PCNA expression. The proliferating cell nu-

clear antigen (PCNA) was detected with immunohisto-

chemical kit (Boster, Wuhan, China) according to the

manufacturer’s protocol: Briefly, the tumor specimens

were conventionally fixed, embedded, sectioned, depar-

affinized, incubated for 15 min at RT in 3% H2O2 diluted

in methanol; add diluted primary antibody and incubate

overnight at 4˚C; The sections were then washed three

times with buffer solution, secondary antibodies (rabbit

anti-rat monoclonal antibodies 1/75 dilution) were added,

and the specimens were incubated for 30 min at room

temperature. ABC reagent was added and the sections

were incubated for 30 min and immersed in hematoxylin

for 90 s for counterstaining. Primary antibodies were

replaced with PBS for the blank control whereas oral

mucosal cells were used for the positive control.

Proliferation indices (PIs) were calculated by observ-

ing the proportion of positively staining cells. Staining

was considered positive when the cell nuclei had been

stained brown. The number of positive cells and total

observed cells was counted under light microscopy

(×400 magnification) with a 10 × 10 grid. The observers

randomly selected distinct fields during three separate

evaluations in each slide. Ten sections were used to

evaluate every group. PIs were calculated using the fol-

lowing formula: PI = (positive ly staining cells/total ob-

served cells) × 100%.

In situ end-labeling (ISEL) detects apoptosis. Apop-

tosis was detected by in situ end-labeling (ISEL) with an

apoptosis labeling kit (Boster, Wuhan, China) according

to the manufacturer’s protocol. Tumor specimens were

conventionally fixed, embedded, sectioned, and deparaf-

finized. Endogenous peroxidases were then blocked by

immersing the slides in 3% H2O2 for 30 min at room

temperature. The slides were incubated in ISEL solution

in an 18˚C ice bath for 2 h. ABC solution was then ap-

plied and the slides were incubated at room temperature

for 30 min. DAB was subsequently applied for 5 min at

room temperature. Counterstaining was done by immer-

sion in hematoxylin for 60 s. Finally, the slides were

rehydrated and topped with coverslips. For the blank

control, the DIG-dUTP was replaced with TBS solution,

whereas oral mucosal cells were used in the positive

control.The calculation of AI is the same as that of PI.

2.6. Statistical Analysis

All experiments, as well as the measurements on each

experiment, were run in triplicate. The experimental data

are presented as means ±s.d. Statistical analyses were

performed by a one-way ANOVA and chi-square test us-

ing SPSS software 11.0 (SPSS Inc., Chicago, IL, USA).

A P value of 0.05 was considered statistically signify-

cant.

3. RESULTS

3.1. Inhibition of Tca8113 Growth Cells in Vitro

The variability of Tca8113 cells was detected by MTT

assay. Figure 1 shows that Tca8113 cells grew rapidly in

the blank control group. The Tca8113 cells in the PUMA

group and the IR group were significantly inhibited com-

pared with the control group. Furthermore, the growth of

Tca8113 cells in the PUMA/IR group was significantly

more inhibited than in any other group.

3.2. Expression of PUMA mRNA in Transfected

Tca8113 Cells

After IR for 48 h, the PUMA expression was detected by

RT-PCR. Figure 2 shows that the mRNA levels in the

PUMA/IR group (0.782 ± 0.035) were significantly

Figure 1. Tca8113 cell growth in vitro. The

growth of Tca8113 cells in the PUMA/IR

group was significantly inhibited compared

with any other group. There was significant

difference between the PUMA group and

the PUMA/IR group.

D.-S. Yu et al. / Open Journal of Stomatology 1 (2011) 195-201

198

1900 bp

750 bp

Figure 2. PUMA transcription after trans-

fection into Tca8113 cells. Lines M, DNA

maker; Line 1, product of β-actin (750

bp); Line 2, product of PUMA gene in

the PUMA/IR group; Line 3, product of

PUMA gene in the PUMA group.

higher than those in the PUMA group (0.527 ± 0.013, P

< 0.01). This result suggests that the synthetic radiation

inducible promoters combined with IR up-regulated

PUMA gene expression in the transfected Tca8113 cells.

3.3. FACScan Analysis of Cells Apoptosis

After IR for 48 h, the percentage of cell apoptosis in the

PUMA/IR group was about (26.32 ± 2.32)%, which is

significantly higher than the (16.65 ± 1.44)% in the

PUMA group (P < 0.05) and the (9.76 ± 0.85)% in the IR

group (P < 0.01) (Figure 3). These results indicate that

IR marked enhanced the anticancer ability of PUMA ge-

ne therapy in vitro.

3.4. Tumor Gr owth Curve

The transplanted tumor growth curves of the naked mice

from different groups are shown in Figure 4. PUMA

gene transfection and 3Gy irradiation exposure have cer-

tain inhibitory effects on tumor growth, but are obvi-

ously lower than that of the radiation gene therapy.

ContrPUMA IRPUMA/IR

Apoptosis rate (%

)

Figure 3. Cells apoptosis in different group were analyzed by

flow cytometry 48 h after irradiation. The apoptosis index was

highest (26.32 ± 2.32)% in the PUMA⁄IR group, which was

significantly higher than (16.65 ± 1.44) in the PUMA group (P

< 0.05) and (9.76 ± 0.85)% in the IR group (P < 0.01).

Figure 4. The growth curves of tumors in naked

mice. Tumor growth was fast in the blank con-

trol group, but significantly depressed in the IR,

PUMA and PUMA⁄IR groups. Furthermore,

there was a significant difference between the

PUMA group and PUMA/IR group.

3.5. PUMA mRNA Expression in the Xenografts

PUMA gene mRNA expression is merely detected in the

control group and the IR group. The PUMA gene frag-

ment was detected in both the PUMA group and the

PUMA/IR group (Figure 5), but the band intensity of

the former is significantly higher than that of the latter.

Computer image analyzer scans of the electrophoretic

band grey values were compared with the β-actin grey

value and their ratios are shown in Ta b l e 1 . The differ-

ences between the two groups are significant (P < 0.05).

Radiation treatment increases PUMA expression level.

3.6. PCNA Staining and ISEL Labeling Results

The PCNA staining and ISEL labeling results are shown

in Figures 6(a)-(d) and Figure 6(A)-(D), whereas the PI

and AI results are in Table 2.

4. DISCUSSION

Apoptosis is a gene-determined active cell death. It plays

a key role in the normal development of multi-cellular

organisms. The occurrence of tumor is closely linked with

2000 bp

750 bp

1000 bp

500 bp

250 bp

M 1 2 3 4 5

Figure 5. Agarose gel electrophoresis of RT-

PCR products M: DNA marker; 1: control group;

2: radiation group; 3: tranfection and radiation

group; 4: tranfection group.

D.-S. Yu et al. / Open Journal of Stomatology 1 (2011) 195-201

199

Table 1. Comparison of grey level of agarose gel electrophore-

sis.

Group Mean (χ ± s)

PUMA group 0.826 ± 0.081

PUMA/IR group 1.031 ± 0.096

Table 2. The AIs and PIs in the different groups (χ ± s%).

Group PI (%) AI (%)

control 43.54 ± 6.42 8.76 ± 1.51

IR 35.26 ± 5.31a 12.33 ± 3.11a

PUMA 33.44 ± 4.32a 4.76 ± 4.12a

PUMA/IR 25.68 ± 5.43ab 28.98 ± 5.22ab

aCompared with control, P < 0.05; bCompared with PUMA, P < 0.05.

abnormal apoptosis [13-15]. Although many p53-regu-

lated genes participate in apoptosis, PUMA is currently

considered one of the key genes in the process [1,2,16].

PUMA belongs to the BH-3 subfamily of the bcl-2 pro-

tein family. The BH-3 structural domain and its position

in the mitochondria are indispensable in the induction of

apoptosis and inhibition of cell growth [1,17]. PUMA in-

duces apoptosis through p53-dependent and non-p53 de-

pendent pathways. Differing from other target spots, PU-

MA not only regulates expression through common p53

responsive elements located in its promoter, but also in-

duces expression by non-p53 dependent mechanisms,

such as glucocorticoids, removal of growth factors, ki-

nase inhibitors and phorbol esters [18,19]. While study-

ing the mechanism of myoblast apoptosis during the dif-

ferentiation and development process of the skeletal mu-

scle, Shaltouki et al. [20] found that PUMA can directly

mediate the release of cytochrome C through a non-p53-

dependent pathway, thereby activating Caspase-9 and

causing myoblast apoptosis. The function of PUMA is

unaffected by p53 expression. In experiments by Wang

[21], there was no correlation between p53 expression

and the inhibition of proliferation and the induction of

apoptosis in esophageal cancer cell lines, including KY-

SE-150, KYSE-410, KY SE-510, and YES-2. Exoge-

nous transfection of PUMA into human melanoma cell

systems causes apoptosis regardless of p53 expression.

Hideaki reported that extensive apoptosis occurred in the

expression mutation and wild characteristic of four PU-

MA-transfected cell lines [22]. Wang also found that the

inhibition of proliferation and induction of apoptosis by

Ad-PUMA in esophageal cancer cell lines were better

than that of Ad-p53 [21]. Apoptosis induction by PUMA

is independent of p53 expression, and its effect is better

than that of p53, which makes it a potential target for

gene therapy.

PUMA is a key protein in apoptosis, and radiation

damage can result in PUMA activation, leading to apop-

tosis through a series of complex mechanisms [23] Al-

though its specific induction mechanism requires further

explanation, the application of the pro-apoptotic function

of p53 in anti-tumor experiments has achieved fairly

Figure 6. Expression of PCNA and apoptosis cells in different group tumors. Expression of

PCNA (SABC, ×400) in the: (a) control group; (b) IR group; (c) PUMA group; (d)

PUMA/IR group. Apoptotic cells (ISEL, ×400) in the: (A) control group; (B) IR radiation

group; (C) PUMA group; (D) PUMA/IR group.

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D.-S. Yu et al. / Open Journal of Stomatology 1 (2011) 195-201

200

good results. Yu et al. found that PUMA gene transfec-

tion could enhance the sensitivity of lung cancer cells to

radiotherapy [24].

There may be serious side effects in patients receiving

radiotherapy. Decreasing the occurrence of these side ef-

fects will be beneficial for cancer patients. PUMA gene

transfection can promote the apoptosis of tumor cells

and enhance its sensitivity to radiotherapy [25,26]. Cur-

rently, methods to increase PUMA-targeted expression

in tumors are the largest obstacle in the application of

PUMA in clinical practice. A new radiation-gene strat-

egy that uses radiation inducible promoters that stimu-

late downstream gene expression to construct control

and regulation systems for therapeutic gene expression

has been adopted in the current research [7,27,28]. This

system is a typical exogenous regulation mechanism that

can selectively kill tumor cells while protecting normal

structures from damage [7,29]. With the development of

radiotherapy techniques such as intensity modulation

radiated therapy and three-dimensional conformal radio

therapy, the radiation range can be adjusted to the target

areas by regulating the output dosage of various areas in

the radiation field [30,31].

To educe synergistic effects by integrating various

therapeutic strategies together is a trend in current gene

therapy [31,32]. In the current study, we demonstrated

that PUMA gene expression in tongue squamous cell

carcinoma was significantly upregulated at the transcri-

ptional level by treatment with radiation-inducible pro-

moters following 3 Gy of IR. We also proved that the

combination of PUMA gene therapy system with 3 Gy

of IR could significantly inhibit the growth of tumor

cells by promoting apoptosis and reducing proliferation

in vivo and in vitro. Although these results are not yet

applicable for the treatment of humans, they indicate that

radiation inducible promoters can serve as molecular

switches for regulating the expression of the PUMA ge-

ne and that low-dose radiation can significantly improve

therapeutic targeting efficiency in treatment of tongue

squamous cell carcinoma.

5. ACKNOWLEDGEMENTS

This study was supported by the National Natural Sciences Foundation

of China (Grant No. 3097 3340), the Guangdong Sciences and Tech-

nology Project (Grant No. 2008B030301113 and 2011B050400030),

and the Guangdong Natural Sciences Foundation (Grant No. 915100-

890100 0187 and S2011020003247).

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