The bicistronic vector pRF was constructed by inserting the firefly luciferase (FL or FLUC) gene from the plasmid pGL-6 (Beyotime Institute of Biotechnology, Haimen, China) into the linker region of the plasmid pRL-SV4 (Promega Corporation, Madison, WI, USA) downstream of the Renilla luciferase (RL or RLUC) gene. To insert the FLUC fragment, a polymerase chain reaction (PCR) was performed on the pGL-6 plasmid with primers FLUCF 5′-GCTCTAGACTCGAGCATATGGAATTCGGCGCGCCCATGGAAGATGCCAAAAACATTAAGAAGGGCCC-3′ (forward) and FLUCR 5′-AAATATGCGGCCGCTTACACGGCGATCTTGCCGCCCTT-3′ (reverse). The PCR was conducted in a 50-µl reaction mixture containing 2X PrimeSTAR Max Premix (Takara Biotechnology Co., Ltd., Dalian, China), 0.2 µM forward and reverse primers, and 200 ng genomic DNA, which was purchased from the Beyotime Institute of Biotechnology. The PCR was conducted at 98°C for 3 min, followed by 32 cycles of 98°C for 1 min, 55°C for 10 sec and 72°C for 1 min, and 72°C for 5 min. The amplified product was digested with Bsp119 and XbaI, and subsequently cloned into pRL-SV40, thus generating pRF. In this vector, the two cistrons were separated by ~20 base pairs (bp) of the intercistronic linker region containing several unique endonuclease restriction sites. The expression of bicistronic mRNA is driven by a simian virus (SV) 40 promoter. The full length 5′-UTR complementary DNA (cDNA) of ATF2, nuclear respiratory factor (NRF) and XIAP were obtained by gene synthesis (Sangon Biotech Co., Ltd., Shanghai, China), while serial truncations of ATF2 were produced by PCR amplification with forward and reverse primers (Table I) containing EcoRI and NdeI endonuclease restriction sites, respectively. The PCR was conducted in a 50-µl reaction mixture containing 2X PrimeSTAR Max Premix, 0.2 µM forward and reverse primers, and 200 ng genomic DNA. The PCR was conducted at 98°C for 3 min, followed by 32 cycles of 98°C for 1 min, varying annealing temperatures (as indicated in Table I) and 72°C for 10 sec, and 72°C for 5 min. All these 5′-UTR cDNAs were each inserted immediately upstream of the translation start codon of the FLUC gene in the dual luciferase vector pRF at the EcoRI and NdeI sites.

The HEK293 cell line (Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China) was cultured in Dulbecco's modified Eagle medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), while the Bel7402, HCT-8 and NIH-3T3 cell lines (Type Culture Collection of the Chinese Academy of Sciences) were cultured in RPMI 1640 medium (Gibco; Thermo Fisher Scientific, Inc.) with 10% (vol/vol) fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), penicillin (100 U/ml) and streptomycin (100 µg/ml). All cells were kept at 37°C in a humidified atmosphere with 5% CO₂. Transient transfection was performed using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.)according to the manufacturer's protocol. The cell media were replaced with complete culture media at 6 h post-transfection, and 24–48 h later, the cells were rinsed twice with phosphate-buffered saline, and cell extracts were then prepared using 5X Passive Lysis Buffer (Promega Corporation).

FLUC and RLUC activities were measured using the Dual-Luciferase Reporter Assay System (Promega Corporation) according to the manufacturer's protocol, with the exception that only 100 µl of each reagent was used. The signals were measured with a luminometer.

Total RNA was isolated from the cells using TRIzol reagent (Beyotime Institute of Biotechnology), and was reverse transcribed into cDNA using oligo(dT)18 primer (Takara Biotechnology Co., Ltd.) and PrimeScript Reverse Transcriptase (Takara Biotechnology Co., Ltd.). PCR was next performed using specific primers, which were designed according to the corresponding sequences of the ATF2 5′-UTR mRNA: ATF2F 5′-GCACGTAATGACAGTGTCATTGT-3′ (forward) and ATF2R 5′-ATGTGGGCTGTGCAGTTTGTG-3′ (reverse). The PCR was conducted in a 50-µl reaction mixture containing 2X PrimeSTAR Max Premix, 0.2 µM forward and reverse primers, and 250 ng total RNA. The PCR was conducted at 98°C for 3 min, followed by 32 cycles of 98°C for 1 min, 51.6°C for 15 sec and 72°C for 18 sec, and 72°C for 5 min. Upon electrophoresis on a 1.5% agarose gel, the PCR products were visualized with ethidium bromide staining under UV light. DNA bands were analyzed using ImageJ software (NIH, Bethesda, MD, USA).

Whole-cell proteins were obtained using radioimmunoprecipitation assay buffer containing 1 mM phenylmethane sulfonyl fluoride. The same quantity of total protein was loaded onto each lane of the gel, and the proteins were electrophoresed on 8% polyacrylamide gels containing 0.1% sodium dodecyl sulfate. The resolved proteins were semi-dry transferred to a polyvinylidene fluoride membrane. The membrane was blocked with 0.5% bovine serum albumin (Beyotime Institute of Biotechnology) at room temperature for ~3 h, prior to be incubated with the corresponding primary antibodies [anti-ATF2 (1:5,000; ab32061; Abcam, Cambridge, MA, USA) or anti-β-actin (1:1,000; AA128; Beyotime Institute of Biotechnology), which was used as an internal reference] at room temperature for 2 h. Next, the membrane was incubated with the secondary antibody Goat Anti-Rabbit HRP (IgG H&L) (1:3,000; ab6721; Abcam) at room temperature for 1.5–2.0 h. Finally, the antigen-antibody complexes were visualized by enhanced chemiluminescence (Thermo Fisher Scientific, Inc.).

Each experiment was performed ≥3 times. All experimental data were analyzed using GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA, USA) and presented as mean values ± standard error of the mean. Statistical analysis was performed using the Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

ATF2 exhibited very low expression in Bel7402 cells (Fig. 1A). Notably, when Bel7402 cells were treated with increasing concentrations (0.01, 0.2 and 0.4 µg/ml) of two chemotherapeutic drugs (ADM and PTX), ATF2 protein expression was induced following treatment with ADM and PTX (Fig. 1A) in a dose-dependent manner. To demonstrate whether the ATF2 induction changed with the exposure time to the drugs, ATF2 protein expression was detected at various times following 0.4-µg/ml ADM and PTX treatment for 2, 4, 8, 12 and 24 h. Fig. 1B reveals that ATF2 protein expression is increased in a time-dependent manner following treatment with ADM and PTX. Next, the mechanism by which these drugs increased the levels of ATF2 in Bel7402 cells was examined. Treatment of Bel7402 cells with either ADM or PTX did not result in any significant change (P=0.552) in ATF2 mRNA levels (Fig. 1C), indicating that ADM/PTX-induced ATF2 expression occurs at the translational level.

Since by searching the ATF2 gene in the human genome database (http://www.ncbi.nlm.nih.gov/nuccore/NM_001880.3) it was observed that the ATF2 5′-UTR is generally long (ATF2, 299 nt) and rich in GC, the possibility that the ATF2 5′-UTR contains an IRES that could regulate ATF2 protein synthesis following treatment with chemotherapeutic drugs was examined. To evaluate whether ATF2 translation was initiated from an IRE, the ATF2 5′-UTR was inserted between the two cistrons of a bicistronic vector, in which the first cistron encodes the RLUC gene and the second cistron encodes the FLUC gene (Fig. 2A). The first cistron is proximal to the mRNA's cap structure, and therefore, is expected to be translated by the conventional cap-dependent translation mode. The second cistron is distal from the cap site; therefore, it is expected to undergo translation only upon insertion of an IRES element between the two cistrons (27). The bicistronic vectors pR-ATF2-F were transfected into HEK293 Bel7402 and HCT-8 human cells, and into murine NIH-3T3 cells. The pR-XIAP-F and pR-NRF-F vectors, which contain the well-characterized cellular XIAP and NRF IRES, were transfected as positive controls, while pRF was used as a negative control. The RL and FL activities were determined as aforementioned described. The IRES activity was calculated as the ratio of FLUC to RLUC normalized to that of pRF-transfected cells. As indicated in Fig. 2B, the ATF2 IRES was observed to be active in the HEK293, Bel7402 and HCT-8 cell lines, but not in the NIH-3T3 cell line. The ATF2 IRES activity was different in each cell line, being high in Bel7402 and HEK293 cells (~5-fold higher than the XIAP IRES activity). These results suggest that a putative IRES element was present in the 5′-UTR of the ATF2 mRNA. The difference in the levels of IRES activity observed in these cell lines is probably due to intrinsic differences between these cell types.

To exclude the possibility that the expression of the second cistron in these two bicistronic mRNAs could be derived from monocistronic RNAs due to the presence of a cryptic promoter in the 5′-UTR, pR-ATF2-F-basic plasmids were generated, in which the ATF2 5′-UTR was inserted in the promoterless pRF-basic vector upstream of the RLUC coding sequence. The HEK293 and Bel7402 cell lines were each transfected with the pRF plasmid (a vector containing the SV40 early promoter and serving as a positive control), the promoterless pRF-basic plasmid and the pR-ATF2-F-basic plasmid. Luciferase activities were measured as aforementioned described. Fig. 2C indicates that the FLUC activity measured in pR-ATF2-F-basic-transfected cells is comparable to that measured in cells transfected with the pRF-basic plasmid, and is ~350-fold lower than the activity produced by pR-ATF2-F. This result clearly illustrated that the ATF2 5′-UTR was devoid of promoter activity.

To further identify the core regions that promote the internal initiation of translation, serial truncations of the ATF2 5′ leader sequence were created (Fig. 3A). Subsequently, the activities of the inserted sequences were assayed as aforementioned described. The ability of these truncated sequences to promote the translation of the mRNA was also compared with that of pRF. Deletions of 30 nt of the 5′ end of ATF2 resulted in a marked decrease in IRES activity, and further deletion of 52, 75 and 142 nt of the 5′ end of ATF2 led to a similar reduction. Notably, at the 3′ end of ATF2, deletion of 70, 140 and 210 nt did not produce a significant decrease (P=0.148), since the IRES activity remained almost the same as that of the full-length ATF2 5′-UTR (Fig. 3B). These data indicated that the 5′ end of ATF2 is important for positioning the IRES in context with the translational start site, and the core IRES region of ATF2 contains 30 bases and resided between nt −299 and −269. The results were the same when different cells were transfected. These results demonstrated that the full length of the ATF2 5′-UTR contributes to its maximal IRES activity, and the 5′ 30 nt of the ATF2 5′ leader region are essential for its IRES activity.

It has been reported that, for certain mRNAs, IRES-mediated initiation of protein translation is activated during conditions such as cellular stress and apoptosis (28). In the present study, ATF2 protein expression in Bel7402 cells was induced under treatment with ADM and PTX (Fig. 1). To investigate whether the ATF2 IRES activity is also affected by cellular stress, the ATF2 activity was examined in Bel7402 cells treated with two chemotherapeutic drugs (ADM and PTX), which induce a stress condition in cells. Gene transfections and reporter assays were performed to determine if ADM and PTX induced ATF2 IRES activity. Bel7402 cells were transfected with the pRF plasmid, containing the full-length fragment of the ATF2 5′-UTR sequence, and then treated with ADM and PTX. It was observed that the FLUC activity in transfected cells treated with ADM and PTX was higher than that of untreated cells (Fig. 4). Furthermore, RT-PCR analyses of mRNA expression of FLUC were performed in cells transfected with pRL-ATF2-FL following treatment with ADM and PTX. It was observed that FLUC mRNA expression was not altered by drugs-induced cellular stress (Fig. 5), suggesting that ADM and PTX induced ATF2 IRES activity.