Identification and characterization of WSG, a fusion gene associated with the proliferation of the WI-38 VA13 cells

It is well known that human fibroblasts can be immortalized using simian virus 40 (SV40) T antigen. However, the mechanisms of the SV40-immortalization processes remain unclear. In the present study, the authors identified and characterized a fusion gene, WSG (WI-38 VA13 Specific Gene), which has an integrated sequence of SV40 and chromosome 16p13. WSG is only detectable in WI-38 VA13 cells and not in other human cell lines or tissues. Transient transfection of the constructed pEGFP-WSG certified the WSG localization at the nuclear of HeLa cells. The growth assays and the knockdown experiment indicate that WSG is involved in the WI-38 VA13 cell proliferation. These results support potential capacities of WSG to be a candidate gene involved in proliferation of the WI-38 VA13 cells.

Keywords: fusion gene; proliferation; WI-38 VA13; WSG

Normal human diploid fibroblasts divide only a limited number of times before they enter cellular senescence, an irreversible growth arrest state. Senescence is defined operationally as the cessation of cellular proliferation. Repeated passaging of normal human somatic cells in culture, exposure to oxidative conditions, or activation of oncogenes can cause cells to gradually reduce their proliferation rate and enter replicative senescence accompanied by continued metabolic activity [[1]]. Several morphological and physiological characteristics of the senescent phenotype include a flattened morphology, enlarged cell size, diminished DNA replication, and positive β-galactosidase staining at neutral pH (SA-β-GAL) [[2]].

Simian virus 40 (SV40) has been widely used as a model system for mammalian cell replication and gene expression. Expression of SV40 T antigen leads to change the cell morphology, growth, and life span. SV40-induced immortalization of human cells proceeds via overcoming 2 stages. The translation product of SV40 T antigen is known to function as a viral oncoprotein binding to p53 (TP53) and retinoblastoma (RB)-1 proteins, binding to the respective domains, therefore inhibiting their tumor-suppressing function [[3]]. Senescence occurs at mortality stage 1 (M1), also termed the Hayflick limit. M1 appears to require functional p53 and retinoblastoma protein (pRB); inactivation of these cell cycle regulators, for example, by viral oncogenes such as SV40 T antigen allows cells to bypass the M1 checkpoint and acquire an extended life span of approximately 20 additional postdivision days (PDs) [[4]]. This extended life span is limited and ends at mortality stage 2 (M2). By overcoming both the mortality stages (M1 and M2), cells acquire immortality [[5]].

It is now generally regarded that the integration of SV40 into the human genome is a random event [[6]]. Many of the randomly integrated SV40 genes are frequently lost from the human genome during the immortalization processes of the cells [[7]]. Several reports [[8]] showed that SV40 stably integrates into different chromosomal regions in different SV40-immortalized cell lines, but into only one chromosomal region per cell lines. These suggestions indicate that although integration of SV40 into several chromosomal sites is possible, the integration into a specific chromosome region may be necessary for the immortalization of the cells [[12]]. However, it is regarded that not all the genomic aberrations are related to the oncogenic transformation [[13]].

The WI-38 VA13 (subclone 2RA) cell line is the SV40-mediated transformant of WI-38, which is a line of normal human lung fibroblasts [[14]]. We previously constructed the cDNA library of the up-regulated clones in WI-38 VA13 [[15]]. From this screening, we discovered a cDNA fragment that had a single match with chromosome 16 genome sequence. In the present study, we identified the full sequence and characterize a novel gene with a unique transcript made by SV40 large T antigen and chromosome 16p13 sequence; this gene is specifically detectable in WI-38 VA13 cells. We name this gene WSG (WI-38 VA13 Specific Gene).

The cell lines WI-38, RWPE-1, CCD-18Co, Hs 677.St, AGS, K-562 and SVG p12 were purchased from the American Type Culture Collection (Manassas, VA, USA). WI-38 VA13, DU-145, KM1214, CCD-986sk, WM-266–4, NCI-H596, HeLa, SK-N-SH, A172, and WI-26 VA4 cells were obtained from the Korean Cell Line Bank (Seoul, South Korea). Senescent WI-38 cells were obtained by repeated passaging in culture until it reached senescence. WI-38, CCD-18Co, WM-266–4, and SVG p12 cells were propagated in minimal essential medium containing Earle's salts, 2 mM l-glutamine, and 0.1 mM nonessential amino acids (EMEM), supplemented with 1 mM sodium pyruvate. WI-38 VA13, AGS, DU-145, NCI-H596, HeLa, SK-N-SH, and A172 cells were cultured in RPMI-1640; Hs 677.St, K-562, KM1214, CCD-986sk, and WI-26 VA4 cells were maintained in Dulbecco's modified Eagle's Medium (DMEM); RWPE-1 cells were cultured in keratinocyte serum-free medium; all media were supplemented with 10% fetal bovine serum (FBS), streptomycin (100 μg/mL), and penicillin (100 U/mL). All cell lines were cultured at 37°C in a humidified 5.0% CO2 incubator. Tissue samples were obtained from the Korea Lung Tissue Bank assigned and supported by the Korea Science and Engineering Foundation in the Ministry of Science and Technology. Tissue samples were homogenized in TRI REAGENT (Molecular Research Center, OH, USA) and stored at −80°C.

A 477-bp RsaI-RsaI fragment was obtained by suppression subtractive hybridization. To isolate the full coding region of WSG, 5′-RACE was performed using the BD SMART RACE cDNA Amplification Kit (Clontech) according to the manufacturer's instructions. Gene-specific primers were 5′-ATCCA-CCAACCAGCCTCTCCCTATCTTC-3′ for the first polymerase chain reaction (PCR) and 5′-CTAC-ATGTGTGAGCACAAATATAATAACTCCC-3′ for the nested PCR. The 3′-RACE product did not exceed the size of the original cDNA fragment. The final PCR product was cloned into pGEM-T Easy Vector System I (Promega Corp.;, WI, USA) and bidirectionally sequenced.

The sequence of WSG was analyzed using T7 promoter and an ABI PRISM 3730XL Analyzer (Macrogen, Seoul, South Korea). The nucleic acid sequence was searched against the NCBI Blast database and the longest putative ORF (open reading frame) in WSG was predicted by DNASTAR (Seoul National University, South Korea). The WoLF PSORT II (http://wolfpsort.org) server was used to predict the subcellular localization of the WSG protein.

Total RNA was extracted from the cell lines and tissue samples using TRI REAGENT (Molecular Research Center). The ratio of A260 and A260/A280 of samples were determined by using ND-1000 Spectrophotometer (NanoDrop Technologies). RNA (10 μg per lane) was separated by electrophoresis in a 1.0% agarose gel containing 2.2 M formaldehyde and then transferred to BrightStar-Plus, a positively charged nylon membrane (Ambion, TX, USA). The membrane was hybridized with a [α-32P]dCTP cDNA probe of WSG using the random priming method. The blot was hybridized at 68°C for 2 hours in ExpressHyb Hybridization Solution (Clontech Laboratories, Mountain View, CA, USA) and washed with 2× SSC/0.05% sodium dodecyl sulfate (SDS) twice at room temperature for 15 minutes and 0.1× SSC/0.1% SDS twice at 68°C for 15 minutes. Subsequently, the membrane was exposed to radiographic film at −80°C for 2 days, followed by autoradiography.

Full-length cDNA was synthesized using the SuperScript First-Strand Synthesis System (Invitrogen, CA, USA). All cDNAs were normalized with 18S rRNA sense (5′-TACCTACCTGGTTGATCCTG-3′) and antisense (5′-GGGTTGGTTTTGATC-TGATA-3′) primers. The following primers were used for the amplification of WSG by PCR: 5′-CTCTGAGCTATTCCAGAAG-3′ (sense primer) and 5′-GCTGTGAACATTCAAGATCC-3′ (antisense pri- mer). PCR was carried out in a total volume of 25 μL, using TaKaRa Ex Taq (TAKARA BIO; Shiga, Japan), under the following conditions: 300 seconds at 94°C for initial denaturation, followed by 35 cycles of 94°C for 40 seconds (denaturation), 55°C for 30 seconds (annealing), and 72°C for 50 seconds (extension), with a final extension step at 72°C for 300 seconds. Aliquots (7 μL) of the PCR products were analyzed on 1% agarose gels. RT-PCR was performed at least 3 separate times.

Plasmids (C-terminal pEGFP-WSG) were constructed by using the following primers: 5′- CC-CTCGAGGGATGCCATCTAGTGA-3′ (the underline indicates the Xho1 restriction site) and 5′- CGGAATTCCCCCTCACACATGTAGAG-3′ (the underline indicates the EcoR1 restriction site). Constructs were transiently transfected into HeLa cells using WelFect-EX Plus (WelGENE, Daegu, South Korea) and visualized with a fluorescent microscope (Nikon Eclipse TE300) at 24 hours after transfection. The nuclear dye Bisbenzimide H 33258 (Hoechst 33258) (Sigma-Aldrich, MO, USA) was used to stain the DNA and visualize the nucleus.

To suppress the endogenous expression of WSG, we synthesized siRNA duplex corresponding to the target sequence described in Figure 1. The WSG-siRNA was generated by annealing the following sequences: sense, 5′-UGAGUACAGUGUGCACAUGTTdT-dT-3′; antisense, 5′-CAUGUGCACACUGUACU-CAdTdT-3′. WSG-siRNA duplex was synthesized by Bioneer Corporation (Daejeon, South Korea). A siRNA duplex targeting enhanced green florescent protein (EGFP) (sense, 5′-GUUCAGCGTGTCC-GGCGAGdTdT-3′; antisense, 5′-CUCGCCGG-CCACGCTGAACdTdT-3′) was used as a negative control. WI-38 VA13 cells (1 × 105 cells) were cultured in plates to give 60% to 80% confluence at the time of transfection. Transfection of the siRNA duplex (final concentration, 150 nM) was performed using WelFect-EX Plus (WelGENE) according to the manufacturer's instruction. Harvested cells were counted daily for 3 days using a hemocytometer after trypan blue staining exclusion.

Graph: FIGURE 1 Nucleotide and putative amino acid sequences of the WSG. The stop codon (TAG) is shown as an asterisk (*), the integration site on WSG is marked with ▾, and the target region for the WSG-siRNA is boxed. The underlined nucleotides near the 3′ end correspond to a polyadenylation signal (AATAAA), and the arrows are the primer sequences used to confirm the mRNA expression of WSG. The putative nuclear localization signals (NLS) are shaded. The accession number for this sequence is FJ208848.

WI-38 VA13 cells (1 × 105 cells) were plated on 6-well plates. Untreated cells were harvested daily for 5 days without treatment of siRNA. To prepare the WSG re-introduction experiment, the expression of WSG was repressed by WSG-siRNA. After knockdown of WSG from the WI-38 VA13 cells, WSG was re-introduced in the cells at day 3 using WelFect-EX Plus (WelGENE). We used pEGFP-WSG for transient transfection. Harvested cells were counted daily for 5 days using a hemocytometer after trypan blue staining exclusion.

In previous study [[15]], we detected a partial sequence using suppression subtractive hybridization that showed sequence similarity only with chromosome 16 in the BLAST program. Using 5′- and 3′-RACE technique, we determined the full sequence of this fragment. The nucleic acid sequence and deduced amino acid sequence of the full-length cDNA are show in Figure 1. The full length is 2492 bp in length with an ORF of 1488 nucleotides (nt) coding for 495 amino acids.

The 5′-RACE analysis showed that this gene is made by the integration of a partial sequence of the SV40 T antigen (2026-bp) and chromosome 16p13 (466-bp), and results in a generation of a putative amino acid sequence. The sequence of the ORF includes the connected point of SV40 T antigen and chromosome 16p13 (Figure 2).

Graph: FIGURE 2 A schematic representation of the WSG. Integration of partial SV40 large T antigen (from bp 1 to 2062) into chromosome 16p13 (from bp 2063 to 2492) generates a full-length WSG of 2492 bp with a 1488-bp length ORF.

Because this fusion gene was isolated from WI-38 VA13 cells, we designated it as WSG (WI-38 VA13 Specific Gene; refer to GenBank accession no. FJ208848).

We analyzed the WSG mRNA expression level in several normal, cancerous, and SV40-immortalized cell lines. As shown in Figure 3A, the results of the Northern blot demonstrate that WSG is specifically detectable in WI-38 VA13 cells, whereas the expression of WSG was undetectable in senescent cells or other SV40-immortalized cells. Similar to the result of Figure 3A, the RT-PCR result shows that WSG is expressed only in the WI-38 VA13 cell line (Figure 3B). Furthermore, WSG was neither expressed in the normal lung tissues nor the corresponding lung cancer tissues (adenocarcinoma and squamous cell carcinoma) (Figure 3C). Therefore, we provisionally concluded that WSG is expressed only in the WI-38 VA13 cells, which means it is a WI-38 VA13 cell–specific gene.

Graph: FIGURE 3 Expression analysis of the WSG mRNA. (A) Northern blot analysis was performed on senescent lung (WI-38), SV40-immortalized lung (WI-38 VA13), SV40-immortalized lung (WI-26 VA4), and SV40-immortalized brain (SVG p12) cells. (B) RT-PCR analysis was performed on various immortalized cells, normal cells, and cancerous cells. Cell lines on the left panel are senescent lung (WI-38), SV40-immortalized lung (WI-38 VA13), SV40-immortalized lung (WI-26 VA4), SV40-immortalized brain (SVG p12), cervix adenocarcinoma (HeLa), chronic myelogenous leukemia (K562), neuroblastoma (SK-N-SH), and glioblastoma (A172). Cell lines on the right panel are pairs of normal and cancer cell lines. Lung N (WI-38), C (adenosquamous, NCI-H596); prostate N (RWPE-1), C (adenocarcinoma, DU-145); colon N (CCD-18Co), C (carcinoma, KM1214); stomach N (Hs 677.St), C (adenocarcinoma, AGS); skin N (CCD-986sk), C (melanoma, WM-266–4). N and C indicate normal and cancerous cells, respectively. (C) RT-PCR analysis displayed null expression of WSG in normal lung and non-small-cell lung cancer (NSCLCs) tissues from cancer patients. We examined four pairs of each: normal and adenocarcinoma tissues (left panel) and normal and squamous cell carcinoma (right panel). N and T indicate normal and tumor tissues, respectively.

The amino acid sequence contains 2 putative nuclear localization sequences (NLSs) at amino acid positions 18 to 24 (PKKKRKV) and 309 to 315 (PKKRYWL) (Figure 1). When we transiently transfected the HeLa cells with the pEGFP-WSG, the signal corresponded with that of the nuclear staining dye (Hoechst 33258) (Figure 4). Consistent with the WoLF PSORT II prediction [[16]], we concluded that WSG localize to the nuclear of cells.

Graph: FIGURE 4 Subcellular localization of WSG in HeLa cells via fluorescence microscopy. HeLa cells were transiently transfected with (A) pEGFP as a control, and (B) pEGFP-WSG. Compared to the ubiquitous expression of EGFP within the cells (A), EGFP-WSG was specifically localized to the nuclear of cells. Arrows indicate the nuclear localization of EGFP-WSG. Fluorescent microscope was used to visualize the cells. Hoechst 33258 was used for nuclear staining (C: pEGFP-transfected cells; D: pEGFP-WSG–transfected cells).

To study the function of WSG on the cell proliferation, we overexpressed WSG in WI-38 VA13 cells using pEGFP-WSG. We used G418 (Geneticin) to select the cells expressing EGFP-WSG. We could not detect a significant growth acceleration of WI-38 VA13 cells for 6 days (data not shown).

We also analyzed the WSG knockdown effect on WI-38 VA13 cells using samm interfering RNA (siRNA) technique. The WSG-siRNA was designed to include both SV40 T antigen and chromosome 16p13 to specifically target the integrated linkage of WSG (Figure 1). We found out that the mRNA expression was significantly decreased after the transfection of WSG-siRNA (Figure 5A). Corresponding to this result, fewer cells were observed in WSG-siRNA–treated cells at day 3, as shown in Figure 5B. To quantify the effect of WSG-knockdown on cell growth, we treated WI-38 VA13 cells with WSG-siRNA or EGFP-siRNA, and compared the proliferation rates for 3 days. The number of GFP-siRNA–treated cells increased comparably to the untreated WI-38 VA13 cells, whereas the growth of WSG-siRNA–treated cells was relatively repressed (Figure 5C). At day 3, the number of WSG-siRNA–treated cells had increased to 19.48 (± 0.55) × 104, which was only 45.3% of that of control siRNA-treated cells (42.98 (± 1.38) × 104). Significant growth retardation was shown in the WSG-siRNA–transfected cells.

Graph: FIGURE 5 WSG promotes the proliferation of WI-38 VA13 cells. (A) WI-38 VA13 cells were treated with siRNAs and RT-PCR was performed to analyze the suppression of WSG. G and S indicate GFP-siRNA–treated cells and WSG-siRNA–treated cells, respectively. (B) The phase-contrast photographs were taken on day 3. (C) Effect of WSG-siRNA on proliferation of WI-38 VA13 cells. Cell numbers were counted daily for 3 days. The results are expressed as the mean ± SD of 6 independent cultures. (D) After suppressing the WSG expression, WSG was re-introduced in WI-38 VA13 cells at day 3 (marked with asterisk). The proliferation rate of the WSG re-introduced cells was compared with the native cells.

In order to confirm the effect of WSG to the proliferation of WI-38 VA13 cells, we re-introduced WSG into the WSG-siRNA–treated cells at day 3. The cells with WSG re-introduction had their proliferation capability (see the growth rate) restored similar to that of the native cells (Figure 5D).

Most immortal cells maintain their telomere length during an infinite number of cell divisions, commonly through activation of telomerase, a ribonucleoprotein enzyme that catalyzes the addition of telomeric DNA repeats to the 3′ end of chromosomal DNA, and subsequently prevents the loss of telomeric sequences upon cell divisions [[17]]. However, recent studies using telomerase-negative immortal cells [[18]] and human keratinocyte or mammary epithelial cells [[20]] indicate that telomerase activation alone is not sufficient for immortalization. Silencing or functional interruption of certain critical genes associated with replicative senescence, such as p16INK4a and pRB, are thought to accompany immortality [[21]]. In fact, alterations that inhibit tumor suppressor genes in the p53 and pRB pathways are common in cancer [[22]]. Numerous studies in cell culture systems have demonstrated that the simian virus 40 (SV40) T antigen [[23]] and human papillomavirus type 16 (HPV-16) E6 or E7 [[25]] genes can immortalize somatic cells. Therefore, maintenance of telomeres and telomerase activity, a balance between oncogenes and tumor suppressor genes, and infection of viral oncogenes are all involved in the immortalization processes [[27]].

Genetic changes resulting in the proliferation of cells include the activation of proto-oncogenes as well as the inactivation of tumor suppressor genes [[28]]. However, the mechanism of how WSG is involved in the proliferation of WI-38 VA13 cells is unknown. Genetic analysis of infinite division in human cells suggests that immortal cells can be classified into at least 4 complementation groups, and SV40-immortalized cells are categorized into a single distinct group [[29]]. Although many genes involved in the proliferation or immortalization processes were identified, the entire mechanism remains mysterious.

In this study, we identified and characterized a fusion gene from WI-38 VA13 cells, which are SV40-immortalized lung fibroblasts. The generation of WSG seems to be an uncommon case among the SV40-immortalized cells. Unlike the strong expression of WSG in WI-38 VA13 cells, no signal was detected in WI-26 VA4 (SV40-immortalized lung fibroblasts) or SVG p12 (SV40-immortalized brain fibroblasts). Sequence analysis shows that WSG is a fusion transcript generated by the integration of SV40 T antigen into chromosome 16p13. WoLF PSORT II, a useful tool for nuclear localization sequence (NLS) prediction, yielded 2 putative monopartite NLSs, which may explain why this protein is localized to the nuclear. The growth rate of the WSG-overexpressed WI-38 VA13 cells did not increase significantly, whereas its knockdown reduced the proliferation of WI-38 VA13 cells. After the re-introduction of WSG, the proliferation rate of the WSG-siRNA–treated WI-38 VA13 cells was restored. We applied WSG to the WI-38 fibroblasts or other normal type cells expecting to observe cell growth. However, WSG showed no influence to other normal cells. These facts provide evidence that although WSG is not the major factor for the cell growth, it may play role as a contributor in the proliferation of WI-38 VA13 cells.

Yano et al. characterized a DNA sequence at the SV40 integration site in a human fibroblast cell line VA13 immortalized by SV40 [[30]]. Suggestion was given that the viral genome integrated at a single site of the chromosome. The 367 nucleotides on the 3′ end of WSG have 98% similarity with this DNA sequence which both locates on 16p13. This abnormal chromosome site (16p13) was also studied in a SV40-transformed human bronchial epithelial cell line, BEAS-2B [[31]]. Taken together, it seems that a specific chromosomal region (16p13) is vulnerable to the integration of SV40, and WSG is generated through this integration.

In conclusion, we identified and characterized a fusion gene, WSG (WI-38 VA13 Specific Gene), generated by the integration of the SV40 T antigen. We found out that WSG plays role in the proliferation of WI-38 VA13 cells. We are now investigating the signal pathway about how WSG affects the proliferation of the WI-38 VA13 cells. Although complex regulation system controls cellular proliferation, diverse functions of WSG in the proliferation process can be imagined, and these are currently under investigation.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.