J. Microbiol. Biotechnol. (2012), 22(3), 431–435http://dx.doi.org/10.4014/jmb.1110.10061First published online December 17, 2011pISSN 1017-7825 eISSN 1738-8872

Cancer-Specific Induction of Adenoviral E1A Expression by Group IIntron-Based Trans-Splicing Ribozyme

Won, You-Sub and Seong-Wook Lee*

Department of Molecular Biology, Institute of Nanosensor and Biotechnology, Dankook University, Yongin 448-701, Korea

Received: October 18, 2011 / Accepted: November 16, 2011

In this study, we describe a novel approach to achieve

replicative selectivity of conditionally replicative adenovirus

that is based upon trans-splicing ribozyme-mediated

replacement of cancer-specific RNAs. We developed a

specific ribozyme that can reprogram human telomerase

reverse transcriptase (hTERT) RNA to induce adenoviral

E1A gene expression selectively in cancer cells that

express the RNA. Western blot analysis showed that the

ribozyme highly selectively triggered E1A expression in

hTERT-expressing cancer cells. RT-PCR and sequencing

analysis indicated that the ribozyme-mediated E1A

induction was caused via a high fidelity trans-splicing

reaction with the targeted residue in the hTERT-

expressing cells. Moreover, reporter activity under the

control of an E1A-dependent E3 promoter was highly

transactivated in hTERT-expressing cancer cells. Therefore,

adenovirus containing the hTERT RNA-targeting trans-

splicing ribozyme would be a promising anticancer agent

through selective replication in cancer cells and thus

specific destruction of the infected cells.

Keywords: Adenovirus, E1A, oncolytic virus, hTERT, RNA

replacement, trans-splicing ribozyme

Cancer remains one of the top causes of death in humans.

A number of strategies including surgery, radiation, and

chemotherapeutic approaches have been used for cancer

treatment; however, most of them are accompanied by

significant side effects and the lack of controlled trials [6].

Use of replicative viral reagents represents a new approach

to the neoplastic disease. Targeted tumor cell killing by the

viral agent is achieved by direct consequence of the viral

replication. Several conditionally oncolytic viruses that

selectively infect or replicate in cancer cells, but leave

normal cells, have been identified. Some are naturally

attenuated viral strains that more effectively infect or

replicate in cancer cells. Others are genetically modified to

mediate cytotoxicity from the oncolytic effects [1, 4, 10].

However, this antitumor agent is largely limited to specific

gene-deficient tumors and frequently harbors low in vivo

efficacy. Additionally, tissue-specific promoters used in

these viral vectors caused limitations in their antitumor

spectra to specific cancer types [11].

Tetrahymena group I intron could target and cleave a

substrate RNA and trans-splice an exon attached at its 3'

end onto the cleaved target RNA in mammalian cells as

well as in bacteria. The trans-splicing ribozyme has been

developed to revise mutant transcripts associated with

several human genetic and malignant diseases [13, 14, 16].

Moreover, we demonstrated that the ribozyme could

selectively induce antiviral gene activities in HCV RNA-

containing cells through the specific RNA replacement of

the HCV RNA [15]. Furthermore, we have recently shown

that the ribozymes could replace cancer-specific transcripts

such as human telomerase reverse transcriptase (hTERT)

RNA or cytoskeleton-associated protein 2 transcript with

RNA exerting cytotoxic activity, and thus the ribozymes

could specifically regress cancer cells expressing the target

RNA [2, 7, 9, 12, 17]. This RNA replacement approach

would be a more attractive approach for cancer therapy

because it should inhibit or reduce the expression of the

target RNA and simultaneously produce therapeutic gene

activity selectively in the target RNA-associated cancer

cells.

In this study, we propose a new conditionally oncolytic

adenovirus whose selective replication in cancers can

be regulated at the post-transcriptional level. To this end,

we constructed a hTERT-targeting trans-splicing ribozyme

harboring the adenoviral E1A gene that stimulates S-phase

entry and transactivates both viral and cellular genes that

are critical for a productive viral infection [3]. We investigated

whether the specific ribozyme can induce expression of the

E1A gene, effectively and selectively, in hTERT-expressing

cancer cells through the targeted RNA replacement.

*Corresponding authorPhone: +82-31-8005-3195; Fax: +82-31-8005-4058;E-mail: SWL0208@dankook.ac.kr

432 Won and Lee

Moreover, we verified the selective trans-activity of E1A

protein by the ribozyme in the target RNA-expressing

cells.

MATERIAL AND METHODS

Construction of Trans-Splicing Ribozyme

Expression vectors encoding for hTERT-specific trans-splicing

ribozymes under the control of the cytomegalovirus (CMV) promoter

were constructed as previously described [12]. Briefly, the Rib21AS

ribozyme targeting U21 on the hTERT RNA was generated to

harbor the extended internal guide sequence, which includes as an

extension of the P1 helix, an additional 6-nt-long P10 helix, and a

325-nt-long antisense sequence complementary to the downstream

region of the targeted hTERT RNA uridine. The cDNA as a 3' exon

encoding for the adenoviral E1A open reading frame gene (orf) or

adenoviral 5'UTR plus orf gene was inserted into the NruI/BamHI

site downstream of the modified group I intron expression construct,

and designated pCMV-Rib21AS-E1A and pCMV-Rib21AS-UTR-

E1A, respectively. The pCMV-E1A and pCMV-UTR-E1A vectors

encoding the E1A orf and 5'UTR plus E1A orf controlled by the

CMV promoter, respectively, were generated as controls.

Cell Culture

SK-OV3 (human ovarian cancer cell line), MCF7 (human breast

cancer cell line), HeLa (human cervical cancer cell line), HCT116

(human colorectal cancer cell line), Hep3B (human hepatocarcinoma

cell line), AGS (human gastric cancer cell line), HT-29 (human

colorectal cancer cell line), IMR90 (telomerase-negative normal

human lung fibroblast), SK-Lu1 (telomerase-negative human lung

adenocarcinoma), and THLE3 (SV40 large T antigen immortalized

but nontumorigenic normal liver cells) were purchased from ATCC

(Manassas, VA, USA). The SK-OV3, HeLa, HCT116, AGS, and

HT-29 cells were maintained in DMEM supplemented with 10%

heat-inactivated FBS (Jeil Biotech Services Inc., Daegu, Korea). The

Hep3B, SK-Lu1, and IMR90 cells were cultured in EMEM with

10% FBS. MCF7 cells were grown in RPMI 1640 with 10% FBS.

The THLE3 cells were cultured in bronchial/tracheal epithelial cell

growth medium (Cambrex, East Rutherford, NJ, USA) with 10%

FBS, 6.5 ng triiodothyromine/ml, 50 µg gentamycin/ml, and 50 ng

amphotericin-B/ml. The transfection reagent for each cell was as

follows: SK-OV3, MCF7, Hep3B, AGS, HT-29, IMR90, and THLE3

cells, ExGen 500 (Fermentas, Hanover, MD, USA); HeLa, HCT116,

Lipofectamine (Invitrogen, Carlsbad, CA, USA); SK-Lu1, DMRIE-

C (Invitrogen).

Western Blot Analysis

Cells were washed in PBS, scraped in 100 µl of lysis buffer (HEPES

50 mM, NaCl 150 mM, PMSF 0.1 mM, NaVanadate 1.0 mM, Triton

X-100 1.0%, glycerol 10.0%, pH 7.4) and solubilized for 60 min at

4oC and centrifuged for fractionation. Total protein was quantitated

using a Bio-Rad DC assay kit (Bio-Rad, Hercules, CA, USA), resolved

by 10% SDS-PAGE, and then transferred to a polyvinylidene

difluoride membrane (Millipore, Bedford, MA, USA). The membrane

was blocked in 5% non-fat milk in PBS containing 0.1% Tween-20

for 1 h at room temperature and probed with antibodies specific to

E1A (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or actin

(Santa Cruz Biotechnology). Subsequently, the membrane was

incubated with HRP-conjugated secondary antibody for 1 h at room

temperature. Finally, the membrane was developed by the enhanced

chemiluminescence detection method (Amersham Phamacia, Cardiff,

UK).

RT-PCR Analysis

For the analysis of trans-splicing products in cells, total RNA was

isolated from ribozyme vector-transfected cells 24 h after transfection

with guanidine isothiocyanate supplemented with 20 mM EDTA.

RNA (5 µg) was reverse transcribed with an oligo(dT) primer in the

presence of 10 mM L-argininamide, and the resulting cDNA was

amplified with a 5' primer specific for the hTERT RNA (5'-GGG

GAATTCAGCGCTGCGTCCTGCT-3') and with a 3' primer specific

for the 3' exon E1A sequence (5'-CCCAAGCTTTCGTCACTGG

GTGGAAAGCC-3'). The amplified cDNA was then reamplified

with a 5' primer specific for the hTERT RNA and with a nested 3'

primer specific for the E1A sequence (5'-CCCAAGCTTCCGGTAC

AAGGTTTGGCAT-3'). The amplified cDNA was then cloned and

sequenced. For the internal control, cDNAs were amplified with

GAPDH primers (5'-TGACATCAAGAAGGTGGTGA-3' and 5'-

TCCACCACCCTGTTGCTGTA-3').

Luciferase Assay

The reporter gene construct encoding firefly luciferase under the

E1A-dependent E3 promoter was transiently co-transfected with

pCMV-Rib21AS-E1A or pCMV-E1A along with phRL-TK (Promega,

Madison, WI, USA), which encodes Renilla luciferase under the TK

promoter and is used for normalization of transfection efficiency.

Twenty-four hours after transfection, cells were lysed and luciferase

activity was determined by chemiluminescence in a FB12 Luminometer

(Berthold Detection System, Oak Ridge, TN, USA) using the dual-

luciferase reporter assay system (Promega).

RESULTS

Design of Adenoviral E1A Gene Expressing Trans-

Splicing Ribozyme

We modified a ribozyme (designated Rib21AS), which

was directed at U21 on the hTERT RNA, to contain an

extension of the P1 helix, a 6-nt-long addition of the P10

helix, and a 325-nt-long antisense sequence that was

complementary to the downstream region of the targeted

uridine of the hTERT RNA. This modification enhanced

the specificity and efficacy of group I-based trans-splicing

ribozyme activity in cells [12]. A set of ribozymes were

constructed to harbor adenoviral E1A orf with or without

5'UTR as 3' exon (Fig. 1).

Selective Induction of E1A Gene Expression in hTERT-

Positive Cell Lines by the Specific Ribozyme

To observe whether the modified ribozymes could react

with and trans-splice the endogenous hTERT RNA and

thus induce the expression of E1A proteins selectively in

hTERT(+) cells, we transfected expression vectors encoding

the specific ribozymes into variant hTERT-positive or

-negative cells and assessed and compared the quantity of

CANCER-SPECIFIC E1A BY TRANS-SPLICING RIBOZYME 433

E1A production in the cells (Fig. 2). E1A proteins were

efficiently produced through the transfection of ribozyme

expression vector pCMV-Rib21AS-E1A or pCMV-Rib21AS-

UTR-E1A (Fig. 2A, lanes 4 or 5, respectively, and Fig. 2C)

in hTERT(+) cells. Inclusion of 5'UTR in the 3' exon of the

ribozyme improved the E1A production. Neither transfection

of the control vector nor pCMV-Rib21AS-Fluc, however,

produced any E1A proteins in the cells (Fig. 2A, lanes 2

and 3). Noticeably, little E1A proteins could be produced

with the transfection of the ribozyme expression vectors in

hTERT(-) cells (Fig. 2B, lanes 4 and 5, and Fig. 2C).

Trans-Splicing Reaction in hTERT-Positive Cancer

Cells by the Specific Ribozyme

We determined whether the ribozyme-mediated selective

E1A expression in hTERT(+) cancer cells is due to a

specific and high-fidelity trans-splicing reaction with

hTERT RNA in those cells. Ribozyme transfection generated

Fig. 1. Schematic diagram of the modified E1A-expressing trans-splicing ribozyme, Rib21AS-E1A. Potential base parings between the ribozyme and the hTERT RNA are depicted with vertical lines. Arrows represent the 5' and 3' splicing sites.

Fig. 2. The selective induction of adenoviral E1A gene expression by the specific ribozyme in the hTERT-expressing cells. Western blot analysis of E1A expression in the hTERT-positive (A) or -negative cells (B), which were transiently transfected with vector control (lane 2),

pCMV-Rib21AS-Fluc (lane 3), pCMV-Rib21AS-E1A (lane 4), pCMV-Rib21AS-UTR-E1A (lane 5), pCMV-E1A (lane 6), or pCMV-UTR-E1A (lane 7).

Whole cell lysates from 293 cells were loaded as control for endogenous E1A expression (lane 1). The actin level was assessed for internal control. (C) The

amount of E1A protein in each vector-transfected cell was normalized to the amount of actin protein. E1A expression in hTERT(+) or (-) cells transfected

with each vector was then quantitated as a percentage of endogenous E1A level in 293 cells. The expressed values represent the means with standard

deviation of five independent determinants.

434 Won and Lee

trans-spliced products in hTERT(+) AGS cells (Fig. 3A,

lanes 3 and 4) but not in an RNA sample isolated from

mock-transfected AGS cells mixed with ribozyme-transfected

SK-Lu 1 cells which are hTERT(-) (Fig. 3A, lanes 5 and

6). This result indicates that the amplified product

observed in the ribozyme-transfected cells was generated

through the trans-splicing reaction specifically inside the

hTERT(+) cells, but neither during RNA manipulation nor

via nonspecific reaction. Sequence analyses of the trans-

spliced products verified that the ribozyme accurately

targeted U21 of the hTERT RNA and spliced its 3' exon

into the target RNA in the cells (Fig. 3B and 3C).

Selective Induction of E3 Promoter-Controlled Reporter

Expression in hTERT-Positive Cell Lines by the

Specific Ribozyme

The E1A gene inserted into the 3' exon of the ribozyme

contains the full E1A gene sequence. Therefore, the trans-

spliced E1A protein provided by the ribozyme should be

functional selectively in hTERT(+) cells. In order to verify

the function of produced E1A in cells, we monitored the

level of firefly luciferase expression under the control of

the adenoviral E3 promoter whose activity is induced by

the adenoviral E1A protein (Fig. 4A) [8]. Transfection of

the ribozyme expression vector efficiently induced firefly

luciferase activity in all of tested hTERT(+) cancer cells,

the level of which is comparable to the level in cells

transfected with control pCMV-E1A vector (Fig. 4B). In

sharp contrast, the specific ribozyme negligibly induced

reporter gene activity in hTERT(-) cells when compared

with vector control. These results suggest that the specific

trans-splicing ribozyme selectively produced functional

E1A protein in hTERT(+) cancer cells.

DISCUSSION

In this study, we developed a hTERT-targeting trans-

splicing ribozyme, which can selectively induce adenoviral

E1A gene expression in hTERT(+) cancer cells. The E1A

expression occurred mainly through RNA replacement via

highly specific trans-splicing reaction with the targeted

hTERT RNA in the cells. Importantly, induction of the

adenoviral E1A gene by the ribozyme activated E1A-

dependent E3 promoter activity selectively in hTERT(+)

cancer cells with efficiency comparable to that associated

with the strong CMV promoter-derived E1A gene expression.

Cancer-specific expression of adenoviral E1A with

tumor- or tissue-specific promoters has been proposed as a

scheme to engineer conditionally oncolytic adenovirus

[11]. However, the above approach could be limited owing

to leaky specificity of the specific promoters when inserted

into the viral genome [18]. Recently, posttranscriptional

control of the adenoviral E1A expression by using normal

tissue-specific microRNA was proposed as an alternate

specific and safer virotherapy strategy against cancer [5].

Fig. 3. Trans-splicing reaction with hTERT RNA by the specific ribozyme in cells. (A) RT-PCR amplification of trans-spliced RNA products generated in cells. AGS cells were mock transfected or transfected with control vector orribozyme-expression vector (pCMV-Rib21AS-E1A or pCMV-Rib21AS-UTR-E1A). Mock-transfected AGS cells were mixed with SK-Lu 1 cellstransfected with pCMV-Rib21AS-E1A (Mix, lane 5) or pCMV-Rib21AS-UTR-E1A (Mix, lane 6). The amplified trans-spliced (T/S) RNA productand GAPDH RNA in each transfectant cell are presented. (B, C) A representative sequence of the trans-spliced transcripts resulting fromtransfection with pCMV-Rib21AS-E1A (B) or pCMV-Rib21AS-UTR-E1A (C) into AGS cells. The expected sequence around the splicingjunction is represented with an arrow, with the ribozyme recognition sequence in hTERT RNA boxed and the uridine at position 21 circled.

CANCER-SPECIFIC E1A BY TRANS-SPLICING RIBOZYME 435

However, we could not completely exclude the possibility

of cytotoxicity in normal cells due to sequestering of the

normal microRNA pathway. Moreover, systems using

specific promoter or microRNA control would be useful

only in specific tumor types. In contrast, regulation of E1A

expression via the trans-splicing ribozyme system will be

safer, because it occurs through exogenously introduced

catalytic activity of the ribozyme, but not inherent cellular

machinery. Additionally, the ribozyme could be a more

potent anticancer tool because of its ability of specific

induction of E1A activity combined with the capability of

target inhibition. Noticeably, telomerase activity is highly

elevated in most cancers. Therefore, the adenovirus equipped

with the hTERT-specific ribozyme developed here will be

useful as a conditionally replicative oncolytic virus with

high efficacy and safety for diverse cancer types.

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Fig. 4. Functional analysis of trans-spliced E1A protein by theribozyme in cells. (A) Schematic illustration of E1A-expressing ribozyme constructs and

E1A-inducible E3 promoter reporter system. (B) Transactivity of the E1A

protein in hTERT(+) and (-) cell lines. Cells were cotransfected with the

reporter construct along with control or E1A-expressing ribozyme

expression vector. The firefly luciferase activities were quantified and then

expressed relative to those by CMV promoter-driven E1A. Averages of

three independent determinants are shown with bars with standard deviations.