Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Overexpression of SERTAD3, a putative oncogene located within the 19q13 amplicon, induces E2F activity and promotes tumor growth


The amplified region of chromosome 19q13.1–13.2 has been associated with several cancers. The well-characterized oncogene AKT2 is located in this amplicon. Two members of the same gene family (SERTAD1 and SERTAD3) are also located within this region. We report herein the genomic structure and potential functions of SERTAD3. SERTAD3 has two transcript variants with short mRNA half-lives, and one of the variants is tightly regulated throughout G1 and S phases of the cell cycle. Overexpression of SERTAD3 induces cell transformation in vitro and tumor formation in mice, whereas inhibition of SERTAD3 by small interfering RNA (siRNA) results in a reduction in cell growth rate. Furthermore, luciferase assays based on E2F-1 binding indicate that SERTAD3 increases the activity of E2F, which is reduced by inhibition of SERTAD3 by siRNA. Together, our data support that SERTAD3 contributes to oncogenesis, at least in part, via an E2F-dependent mechanism.


Genetic and chromosomal abnormalities are common features associated with cancer development and progression (Lengauer et al., 1998; Sieber et al., 2003). Amplified chromosomal regions, known as amplicons, are revealed as either homogenously staining regions or as double minutes (Barker, 1982; Alitalo et al., 1983; Lengauer et al., 1998).

Specific gene amplifications have been implicated in a wide variety of cancers. Examples of known amplicons in cancer include chromosome 17q12 containing ErbB-2 (Ross and Fletcher, 1999; Luoh, 2002), chromosome 11q13 containing CCND1 and EMS1 (Ormandy et al., 2003; Zaharieva et al., 2003), chromosome 12.13–14 containing the p53 suppressor MDM2 (Oliner et al., 1992; Reifenberger et al., 1993) and N-Myc amplification on chromosome 2p24 in neuroblastomas (Shiloh et al., 1986; Schneider et al., 1992; Hiemstra et al., 1994).

One region of recurrent gene amplification is located on chromosome 19q. In particular, amplification of chromosome 19q13 has been linked to different types of cancers including pancreatic carcinoma (Miwa et al., 1996; Curtis et al., 1998; Hoglund et al., 1998), ovarian carcinoma (Thompson et al., 1996; Bicher et al., 1997; Wang et al., 1999), breast cancer (Muleris et al., 1995) and other cancers (Marchio et al., 1997; Petersen et al., 1997; Beghini et al., 2003), supporting that genes included in this amplicon can participate in tumor formation.

SERTAD1, a putative oncogene, is found close to AKT2 within the 19q13.1–13.2 amplicon (Tang et al., 2002). SERTAD1 is a transcription factor known to inhibit p16INK4a activity, interact with E6 of HPV 16 and interact with proline hydroxylase domain (PHD)-containing proteins such as TIF1β (Sugimoto et al., 1999; Hsu et al., 2001; Gupta et al., 2003). In addition to SERTAD1, we have identified a novel gene that is located on chromosome 19q13.1 in tandem with SERTAD1 (Figure 1a). This gene, which we named SERTAD3 (RBT1 or Replication protein A Binding Transactivator), is a member of the SERTAD family of transcription factors (Cho et al., 2000). There is significant homology between all of the family members, especially between SERTAD3 and SERTAD1 (31% identity and 46% similarity; Figure 1b). The location of SERTAD3, along with its overexpression in several cancers and the high similarity to SERTAD1, suggests an oncogenic role for this protein.

Figure 1

Genomic organization of SERTAD genes. (a) The SERTAD3 and SERTAD1 genes are located in tandem on chromosome 19 within the 19q13.1–13.2 amplicon in close proximity to AKT2. All genes containing a SERTA domain have an intron in their 5′ UTR close to the translational start site (ATG). Splicing donor and acceptor sites are indicated. (b) Members of the SERTAD family have a high degree of sequence homology. SERTAD3 and SERTAD1 share the highest homology with 31% identity and 46% similarity. (c) All members of the SERTAD family contain the same protein- and DNA-binding motifs including the cyclin A-binding motif, SERTA domain, PHD-bromo interacting domain and C-terminal transcriptional activation domain.

Herein, we demonstrate that members of this family share binding domains and have similar genomic structures. SERTAD3 has two transcript variants with distinct regulation patterns. SERTAD3 overexpression in non-transformed cells led to oncogenic transformation, both in vitro and in vivo. Conversely, SERTAD3 inhibition led to a marked decrease in cell growth. Furthermore, SERTAD3 is found to stimulate E2F-1 activity. These results support that SERTAD3 promotes oncogenesis at least in part via induction of E2F-1 transcriptional activity.


Homology of SERTA domain-containing proteins

The SERTA domain, an 47-residue motif named for the first proteins that were discovered with this motif (SEI-1, RBT1 and TARA) (Calgaro et al., 2002), is thought to be important for protein–protein interactions. The SERTAD3 gene is localized at chromosome 19q13.1–13.2 in tandem with SERTAD1, approximately 15 kb apart and in proximity to the AKT2 oncogene. SERTAD3 and SERTAD1 are members of a larger family that includes SERTAD2/TRIP-Br2 and CDCA4/HEPP (Abdullah et al., 2001; Calgaro et al., 2002).

The SERTAD family shares a similar, unusual genomic structure. All members have intronless coding regions yet contain one intron in their 5′ untranslated regions (UTRs) (Figure 1a). Furthermore, the splice acceptor site is always found within 7 bp of the ATG start codon. TARA, the Drosophila homologue of mammalian SERTAD genes, also contains one intron in its 5′ UTR (Calgaro et al., 2002). Members of the SERTAD family show significant homology (Figure 1b), especially within protein domains such as the SERTA domain. It has been shown that most of the SERTA domain in SERTAD1 is included in its cyclin-dependent kinase 4 (CDK4)-binding site and heptad repeat region (Sugimoto et al., 1999). Other common protein motifs can be identified in SERTAD homologues including the cyclin A-binding site, PHD-bromo interacting domain and C-terminal activation domain (Figure 1c).

Characterization of SERTAD3

To determine whether genes homologous to SERTAD3 existed in other species, a similarity search was carried out using the SERTAD3 amino-acid sequence. The full-length protein (196 aa) was BLASTed against other species. The sequences of several mammalian SERTAD3 proteins were aligned using ClustalW (Figure 2a), revealing a high degree of conservation ranging from 83% identity (rat) to 98% identity (rhesus monkey).

Figure 2

Genomic structure of SERTAD3. (a) Alignment of mammalian SERTAD3 protein. The amino-acid sequence is highly conserved (83–98%), especially within the protein- and DNA-binding domains such as the SERTA domain (residues 26–73 in human SERTAD3). (b) The coding region (591 bp) is shown in black whereas UTRs are shown in gray. Exon 3 is comprised of the terminal 7 bp of the 5′ UTR, the coding region and the 3′ UTR. TV1 (NM_013368) contains exons 2+3, whereas TV2 (NM_203344) contains exons 1+3.

Unlike other genes in the SERTAD family, SERTAD3 has two distinct transcript variants differing only in their 5′ UTR. Both variants contain identical coding regions and 3′ UTRs that are found in a single exon (exon 3). Transcript variant 1 (TV1, NM_013368) includes a 276 bp 5′ UTR from exon 2. There is a 240 bp intron separating exons 2 and 3 that is excised during the transcription of TV1. Transcript variant 2 (TV2, NM_203344) consists of 162 bp 5′ UTR from exon 1 spliced with exon 3. The genomic organization of SERTAD3 is illustrated in Figure 2b. Nucleotide sequences from exons 1 and 2 (TV2 and TV1 5′ UTRs, respectively) were then BLASTed and the resulting matches aligned using ClustalW. There was a significant similarity between the 5′ UTR of TV2 and the SERTAD3 5′ UTR of other mammalian species, especially the region directly upstream of the splice site (76–93% identity, data not shown). No significant similarity to other species was found with the TV1 5′ UTR.

Cell cycle analysis of SERTAD3 transcript variants

Most genes that play an important role in cell survival and proliferation are tightly regulated (Alarcon-Vargas and Ronai, 2004). SERTAD1 is differentially expressed throughout the G1 and S phases (Sugimoto et al., 1999); so we investigated the regulation of SERTAD3 gene expression throughout the cell cycle.

Primary human fibroblasts (CRL2097) were synchronized through serum starvation. Total RNA was collected at various time points and transcriptional levels were tested using reverse transcriptase–polymerase chain reaction (RT–PCR). Synchronization was confirmed by cell cycle analysis (Figure 3a) and by examining the levels of cyclin A and CDK4 using RT–PCR (Figure 3b). As expected, cyclin A expression increases dramatically as cells enter S phase, whereas CDK4 expression remains constant throughout the cell cycle.

Figure 3

Expression of SERTAD3 throughout G1/S phases. Primary human fibroblasts (CRL2097) were synchronized by serum starvation, released with 10% FBS and collected from 0 to 32 h. Exponentially growing fibroblasts were also harvested (Log). (a) Synchronization was validated by cell cycle analysis. The number of cells within each phase is plotted as a percentage of the total number. (b) The expression of cyclin A and CDK4 was examined using semi-quantitative RT–PCR to validate synchronization. GAPDH was used as an internal control. A negative RT–PCR control (–) is included. (c) SERTAD3 TV1 expression is regulated throughout G1 and S phases, peaking at 2 h after release from serum starvation. (d) Stability of SERTAD3 mRNA. MCF-7 cells were treated with either 10 μg/ml actinomycin-D (i) to inhibit mRNA production or ethanol control (ii). RNA was harvested and the abundance of SERTAD3 transcripts was measured using RT–PCR. GAPDH was used as an internal control. Levels of both transcripts decrease significantly after 2 h of actinomycin-D treatment, whereas GAPDH levels are not significantly reduced after 24 h.

We then examined the RNA levels of SERTAD3. A significant difference was found when looking at the levels of SERTAD3 transcript variants. TV1 expression is almost undetectable when the fibroblasts are released but then is quickly induced, peaking 2 h post-release (Figure 3c). There is another substantial increase in TV1 expression as cells enter S phase that was seen in independent studies. However, TV2 expression remains unchanged throughout G1 and S phases (data not shown).

The expression pattern of SERTAD3 TV1 throughout the cell cycle is highly similar to its family member SERTAD1 (Sugimoto et al., 1999), exhibiting a peak in both RNA and protein levels 2 h after release from G0/G1. As these two genes are located in tandem on chromosome 19q13.1–q13.2, there is reason to believe that SERTAD3 TV1 and SERTAD1 share common regulatory mechanisms. SERTAD3 TV2 is constitutively expressed throughout the cell cycle and thus appears to be regulated differently.

Based on the regulation of SERTAD3 throughout the cell cycle, we examined the stability of SERTAD3 transcript variants in CRL2097 cells as well as MCF-7 cells because these breast cancer cells express a high level of SERTAD3. Cells were treated with actinomycin-D and the RNA was harvested after 2, 4, 8 and 24 h post-treatment. Semiquantitative RT–PCR was used to compare mRNA levels of TV1, TV2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We find that both transcript variants of SERTAD3 have similar short mRNA half-lives in MCF-7 cells (Figure 3d); there was a 50% decrease of initial transcripts after 2 h and a decrease of over 90% after 8 h. Similar results were obtained when using CRL2097 cells (data not shown), confirming that the rapid turnover of SERTAD3 is not cell type-specific. The tight regulation of SERTAD3 at the levels of both transcription and mRNA stability suggests that this protein is important within the cell and may have important functions in survival or tumorigenesis.

Overexpression of SERTAD3 in NIH3T3 cells causes cellular transformation

Normal (non-transformed) cells express low amounts of SERTAD3 compared to cancer cells (Cho et al., 2000). To address the impact of SERTAD3 on oncogenic cell transformation, we overexpressed SERTAD3 in a polyclonal population of non-transformed NIH3T3 cells (NIH3T3SERTAD3) (Figure 4a). Cells were grown to confluence and serum starved. Upon serum stimulation, the NIH3T3vector cells remain in G0/G1, likely as a result of contact inhibition whereas NIH3T3SERTAD3 resume cell cycle progression (Figure 4b) showing that NIH3T3SERTAD3 cells are not contact inhibited. Furthermore, we performed a soft agar colony formation assay and found that NIH3T3SERTAD3 induced colony formation in agar compared to non-transformed control cells (Figure 4c and inset).

Figure 4

Cellular Transformation in NIH3T3. (a) Western blot of protein extracts from cells stably transfected with empty pBabe vector (NIH3T3vector) or pBabe-SERTAD3 (NIH3T3SERTAD3). Protein extracts (100 μg) were loaded in each lane and probed with rabbit anti-SERTAD3 antibody. (b) Contact inhibition of confluent NIH3T3 cells. Parental cells and cells overexpressing SERTAD3 were serum starved at 100% confluence for 48 h before release. The cell cycle distribution was determined by flow cytometry. NIH3T3SERTAD3 cells were able to continue through the cell cycle. (c) Growth in soft agar 9 days subsequent to plating. Insert represents the average colonies formed by NIH3T3vector and NIH3T3SERTAD3 cells. (d) Tumor growth in Balb/c Nu/Nu mice. Eight mice were injected with either NIH3T3vector or NIH3T3SERTAD3 exponentially growing fibroblasts of which four representative mice are shown. All mice (4/4) injected with NIH3T3SERTAD3 cells formed tumor nodules whereas no tumors were formed with NIH3T3vector cells (0/4). (e) Graphical representation of tumor growth over time.

To assess the potential of NIH3T3SERTAD3 to form tumors, control and SERTAD3-overexpressing NIH3T3 were injected subcutaneously into the flanks of nude mice. Interestingly, NIH3T3SERTAD3 cells formed large tumors whereas control mice injected with parental NIH3T3 did not (Figure 4d and e).

SERTAD3 siRNA inhibits cell proliferation

To further establish the connection between SERTAD3 expression and cell proliferation, endogenous SERTAD3 was knocked down using SERTAD3 siRNA. RT–PCR and Western blot analysis confirmed that SERTAD3 transcript variants were efficiently inhibited in MCF-7 cells (Figure 5b and insert). However, the expression of other SERTAD family members remained unchanged (Figure 5b).

Figure 5

SERTAD3 siRNA inhibits MCF-7 growth. (a) SERTAD3 siRNA knocks down SERTAD3 transcripts. MCF-7 cells were treated with either non-targeting siRNA (control) or SERTAD3 siRNA in 100 and 150 nM concentrations. SERTAD3 mRNA was harvested after 48 h and the abundance of SERTAD3 transcripts was measured using RT–PCR. GAPDH was used as an internal control. Both transcripts are knocked down significantly when using the two siRNA concentrations. (b) Expression of the SERTAD family was determined in SERTAD3 siRNA- and control siRNA-treated cells. No knockdown was observed in other SERTAD members. Insert represents Western blot analysis of SERTAD3 in control and siRNA MCF7 cells using a SERTAD3 polyclonal antibody. GAPDH was used as an internal control. (c) Effect of SERTAD3 siRNA on MCF-7 cell growth. MCF-7 cells were treated with either non-targeting siRNA (control) or SERTAD3 siRNA in 100 and 150 nM concentrations. The number of cells was counted every 24 h. Results are shown as the mean±s.e.m.

Following siRNA transfection, the effect of SERTAD3 knockdown on cell growth was studied in MCF-7 cells by counting cells every 24 h. The number of cells in both treatments was compared to both non-transfected cells and those transfected with non-targeting siRNA (control). Efficient SERTAD3 knockdown was confirmed by RT–PCR and Western blot analysis (Figure 5b and insert). The growth of MCF-7 cells is directly proportional to the level of SERTAD3 (Figure 5c). After 3 days in culture, there is a twofold difference in growth between control-treated cells and those transfected with the lower SERTAD3 siRNA concentration, and a fourfold difference when comparing control-treated cells with those transfected with the highest SERTAD3 siRNA concentration. Inhibition of cell proliferation was also seen with a pancreatic cell line (PANC-1) with amplification of 19q13 (Curtis et al., 1998) after expression of SERTAD3 siRNA (not shown).

SERTAD3 expression is correlated with E2F-1 transcriptional activity

Members of the SERTAD family share significant homology in their respective cyclin A-binding, heptad repeat, PHD-bromo interaction and C-terminal activation domains (Figure 1c). The cyclin A-binding and hydrophobic heptad repeat domains (zipper) of SERTAD proteins are similar to those found in members of the E2F family of transcriptional activators. We reasoned that SERTAD3 regulates the cell cycle, at least in part, via modulation of E2F activity.

To explore this hypothesis, the impact of SERTAD3 on E2F-regulated genes was examined using a luciferase reporter plasmid containing four consecutive E2F-1-binding sites with dyad symmetry. This vector (pLuc-(E2F)4) was coexpressed in NIH3T3 cells with pCMV-E2F1 and either pCMV-SERTAD3 or pCMV-SERTAD1. As shown in Figure 6a, expression of exogenous E2F-1 in NIH3T3 increases reporter gene activity. Consistent with a role for SERTAD3 and its homologue SERTAD1 to increase E2F-1 activity, E2F reporter activity is higher in cells overexpressing SERTAD3 or SERTAD1 compared to control cells.

Figure 6

Transcriptional assays. (a) Stimulation of E2F transcriptional activity in NIH3T3 cells. E2F activity was assayed using a luciferase reporter plasmid containing four consensus E2F-1-binding sites (pLuc-(E2F)4). Transfection of plasmid quantities is shown in nanograms. Note that exogenous addition of SERTAD3 or SERTAD1 increases the activity of E2F. (b) Effect of SERTAD3 siRNA on E2F activity in MCF-7 cells. Transcriptional activity of E2F is decreased fourfold in cells co-transfected with 150 nM SERTAD3 siRNA. (c) Synchronized MCF-7 cells were transfected with either SERTAD3 siRNA or control siRNA, and the protein levels of E2F-1 throughout G1 phase were measured using Western blot. SERTAD3 knockdown decreased E2F-1 accumulation in MCF-7 cells compared to control. β-Actin was used as a loading control.

The connection between E2F-1 activity and SERTAD3 expression was further tested using MCF-7 cells and SERTAD3 siRNA. As expected, cells containing the pLuc-(E2F)4 reporter vector show a high level of luciferase activity when expressing exogenous E2F-1. However, when these cells are co-transfected with SERTAD3 siRNA (S2), the luciferase activity decreases fourfold (Figure 6b). In addition, Western blot analysis of synchronized MCF-7 cells treated with either SERTAD3 siRNA or control siRNA demonstrates that SERTAD3 knockdown decreases E2F-1 accumulation in G1 phase (Figure 6c). These experiments support that SERTAD3 potentiates E2F-1 transcriptional activity, and this may contribute to cell transformation.


We previously reported that SERTAD3 (RBT1), a new member of the SERTAD family of transcription factors, has a higher expression in cancer cells (Cho et al., 2000). Here, we describe the localization of SERTAD3 within the 19q13.1–13.2 amplicon located next to its family member SERTAD1. One of the two SERTAD3 transcript variants (TV1) is tightly regulated throughout G1 and S phases of the cell cycle. The pattern of expression is identical to that of SERTAD1 (Sugimoto et al., 1999), and the fact that these two genes are located in tandem suggests common mechanisms of gene regulation.

All members of the SERTAD family contain several motifs believed to play a role in protein–protein interactions and DNA binding; the SERTA domain is the most highly conserved region among SERTAD members. Of significance to this study, the cyclin A-binding domain found in these proteins is similar to the one found in E2F proteins (Cho et al., 2000). The PHD-interacting domain, also found in AKT2 and other regulatory proteins (Bienz, 2006), could also be an important protein–protein interacting motif.

The oncogenic nature of SERTAD3 was demonstrated in mouse fibroblasts that stably overexpress SERTAD3 and show transformed behavior in vitro and in nude mice where cells with SERTAD3 overexpression formed large tumors. Furthermore, the growth rate of MCF-7, a breast cancer cell line, is diminished when knocking down the levels of SERTAD3, lending further evidence that this protein confers a growth advantage to cells.

Overexpression of E2F-1 has been shown to cause cellular transformation in rodent cells (Singh et al., 1994; Yang and Sladek, 1995). SERTAD3 overexpression may cause transformation in NIH3T3 fibroblasts in part through modulation of the E2F-1 pathway. Although the normal physiological functions of SERTAD3 have not been fully defined, we speculate that one of its roles involves the modulation of the E2F-1 transcription factor. In support of this hypothesis, the results of luciferase assays have clearly shown that NIH3T3 cells transfected with SERTAD3 have significantly greater E2F-1 activity in comparison to parental cells. Furthermore, knocking down SERTAD3 levels in MCF-7 cells with siRNA causes a reduction in both E2F transcriptional activity and accumulation. Ongoing studies will further establish the connections between SERTAD3 and E2F-regulated genes.

In summary, we have shown that the amplified region of chromosome 19q13.1–13.2 contains SERTAD3 and SERTAD1, two highly conserved homologues. Furthermore, both its function and regulation indicate an important role for this protein in cellular growth and proliferation. Taken together, this information suggests that overexpression of SERTAD3 due to the amplification of chromosome 19q13.1–13.2 can promote oncogenesis, likely through E2F activation.

Materials and methods

Cell lines

NIH3T3 mouse fibroblasts, primary human fibroblasts (CRL2097), MCF-7 breast carcinoma cells and HEK293GP cells were obtained from the American Type Culture Collection (Manassas, VA, USA). NIH3T3, CRL2097 and MCF-7 cells were cultured in Dulbecco's modified Eagle's medium, minimum essential medium and Roswell's Park Memorial Institute 1640 medium, respectively, supplemented with 10% fetal bovine serum (FBS). All cell lines were maintained in culture at 37°C in an atmosphere of 5% CO2.

Plasmids and generation of cells stably expressing SERTAD3

pBabe-puro (Morgenstern and Land, 1990), a murine leukemia virus-based retroviral vector, was used to transduce the SERTAD3 gene. The cDNA for wild-type SERTAD3 (NM_013368) was derived by PCR using Pfu turbo (Stratagene, La Jolla, CA, USA), cloned into pBabe via BamHI and EcoRI restriction sites, and confirmed by DNA sequence analysis. The insert was also excised from SERTAD3-pBabe and cloned into pcDNA3.1zeo (Invitrogen, Carlsbad, CA, USA) and pFLAG-CMV2 (Kodak, New Haven, CT, USA). pBabe or SERTAD3-pBabe vector was transfected into the packaging cell line 293GP using Lipofectamine (Gibco BRL, Gaithersburg, MD, USA). Viral supernatants (500 ml each of SERTAD3-pBabe and pBabe) were collected at 24 and 48 h after transfection. Viral titer was determined using PA18G-BHK-21 cells and puromycin selection. The concentrated viral stocks had titers of approximately 109 infectious units per ml. NIH3T3 cells were infected with SERTAD3-pBabe or pBabe and then subjected to selection in the same medium containing puromycin (2 μg/ml). Cells were selected in puromycin for 1 week with one change of medium in between. Cells were subsequently pooled, maintained under selection and characterized.

Synchronization of cells in G0/G1

Synchronization of CRL2097, NIH3T3 and MCF-7 cells was achieved through serum starvation (0.02% FBS) for 60 h. The cells were released with 10% FBS and collected after 0–32 h. Cells in an exponential (log) phase of growth were also collected for comparison.

Flow cytometry

Synchronization of CRL2097 and NIH3T3 fibroblasts in G0/G1 was achieved as mentioned above and analysed by flow cytometry as previously described (Loignon and Drobetsky, 2002) using a FACScan flow cytometer equipped with CellFit software (Beckton Dickinson, San Jose, CA, USA).

RNA Isolation

TRIzol was used to extract total RNA from cells according to the instructions of the manufacturer. RNA pellets were washed twice with 70% ethanol, resuspended in 40 μl RNase-free H2O and stored at −80°C. RNA quantity and quality were assessed on an Eppendorf BioPhotometer.


Semiquantitative RT–PCR was performed using the QIAGEN One-Step RT–PCR kit and 0.1 μg. RNA in a PTC-100 thermal cycler (MJ Research, Waltham, MA, USA) under the following conditions: 50°C for 30 min, 94°C for 15 min, followed by cycling 94°C for 45 s, 59–61°C for 45 s and 72°C for 45 min, and a final step of 10 min at 72°C. Primers used in this study are listed in Table 1.

Table 1 Primers for RT–PCR

Stability of SERTAD3 mRNA

MCF-7 and CRL2097 cells were plated in 60 mm dishes at a confluency of 25% (1.5 × 106 cells) and cultured for 24 h. Cells were treated with either 10 μg/ml actinomycin-D dissolved in 100% ethanol or ethanol alone as a negative control. RNA was harvested at 0, 2, 4, 8 and 24 h post-treatment. mRNA stability was measured using semiquantitative RT–PCR.

siRNA transfections

SERTAD3 siRNA was designed by Dharmacon (Lafayette, CO, USA) using their SMARTselection design algorithm (siRNA sequence is GGC AUG UCC UCA UCC AUA A). Transfections were carried out according to the manufacturer's instructions with final siRNA concentrations of 100–150 nM. The cells were harvested 24–48 h post-transfection. Non-targeting siRNA no. 1 was used as a negative control.

Western blotting

Protein analysis by Western blot was performed as described previously (Loignon and Drobetsky, 2002) using rabbit anti-SERTAD3 antibody (Cho et al., 2000) and mouse anti-E2F1 SC-250X (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at dilutions of 1:200, and mouse anti-β-actin (Sigma, St Louis, MO, USA) at a dilution of 1:4000.

Tumor induction in nude mice

Exponentially growing cells were suspended in phosphate-buffered saline (106 cells per 0.1 ml) and injected subcutaneously into the flanks of Balb/c Nu/Nu mice. Tumor volumes were measured every second or third day by external measurement. Tumor volumes were estimated using the equation: volume=π/6(length × width2).

Transfections and luciferase assay

Mammalian cell transient transfections were performed using either Lipofectamine or Lipofectamine 2000 reagent according to the manufacturer's recommendations and based on a protocol we described earlier (Yen et al., 2002). Relative luciferase activity was calculated and reported as a ratio between firefly luciferase and Renilla luciferase activity. Measurements were repeated three times and are shown as the mean±s.e.m.


  1. Abdullah JM, Jing X, Spassov DS, Nachtman RG, Jurecic R . (2001). Cloning and characterization of Hepp, a novel gene expressed preferentially in hematopoietic progenitors and mature blood cells. Blood Cells Mol Dis 27: 667–676.

    CAS  Article  Google Scholar 

  2. Alarcon-Vargas D, Ronai Z . (2004). c-Jun-NH2 kinase (JNK) contributes to the regulation of c-Myc protein stability. J Biol Chem 279: 5008–5016.

    CAS  Article  Google Scholar 

  3. Alitalo K, Schwab M, Lin CC, Varmus HE, Bishop JM . (1983). Homogeneously staining chromosomal regions contain amplified copies of an abundantly expressed cellular oncogene (c-myc) in malignant neuroendocrine cells from a human colon carcinoma. Proc Natl Acad Sci USA 80: 1707–1711.

    CAS  Article  Google Scholar 

  4. Barker PE . (1982). Double minutes in human tumor cells. Cytogenetics 5: 81–94.

    CAS  Article  Google Scholar 

  5. Beghini A, Magnani I, Roversi G, Piepoli T, Di Terlizzi S, Moroni RF et al. (2003). The neural progenitor-restricted isoform of the MARK4 gene in 19q13.2 is upregulated in human gliomas and overexpressed in a subset of glioblastoma cell lines. Oncogene 22: 2581–2591.

    CAS  Article  Google Scholar 

  6. Bicher A, Ault K, Kimmelman A, Gershenson D, Reed E, Liang B . (1997). Loss of heterozygosity in human ovarian cancer on chromosome 19q. Gynecol Oncol 66: 36–40.

    CAS  Article  Google Scholar 

  7. Bienz M . (2006). The PHD finger, a nuclear protein-interaction domain. Trends Biochem Sci 31: 35–40.

    CAS  Article  Google Scholar 

  8. Calgaro S, Boube M, Cribbs DL, Bourbon H-M . (2002). The Drosophila gene taranis encodes a novel trithorax group member potentially linked to the cell cycle regulatory apparatus. Genetics 160: 547–560.

    CAS  PubMed Central  Google Scholar 

  9. Cho JM, Song DJ, Bergeron J, Benlimame N, Wold MS, Alaoui-Jamali MA . (2000). SERTAD3, a novel transcriptional co-activator, binds the second subunit of replication protein A. Nucleic Acids Res 28: 3478–3485.

    CAS  Article  Google Scholar 

  10. Curtis LJ, Li Y, Gerbault-Seureau M, Kuick R, Dutrillaux AM, Goubin G . (1998). Amplification of DNA sequences from chromosome 19q13.1 in human pancreatic cell lines. Genomics 53: 42–55.

    CAS  Article  Google Scholar 

  11. Gupta S, Takhar PP, Degenkolbe R, Koh CH, Zimmermann H, Yang CM . (2003). The human papillomavirus type 11 and 16 E6 proteins modulate the cell-cycle regulator and transcription cofactor TRIP-Br1. Virology 317: 155–164.

    CAS  Article  Google Scholar 

  12. Hiemstra JL, Schneider SS, Brodeur GM . (1994). High-resolution mapping of the N-myc amplicon core domain in neuroblastomas. Prog Clin Biol Res 385: 51–57.

    CAS  Google Scholar 

  13. Hoglund M, Gorunova L, Andren-Sandberg A, Dawiskiba S, Mitelman F, Johansson B . (1998). Cytogenetic and fluorescence in situ hybridization analyses of chromosome 19 aberrations in pancreatic carcinomas: frequent loss of 19p13.3 and gain of 19q131–13.2. Genes Chromosomes Cancer 21: 8–16.

    CAS  Article  Google Scholar 

  14. Hsu SI, Yang CM, Sim KG, Hentschel DM, O'Leary E, Bonventre JV . (2001). TRIP-Br: a novel family of PHD zinc finger- and bromodomain-interacting proteins that regulate the transcriptional activity of E2F-1/DP-1. EMBO J 20: 2273–2285.

    CAS  Article  Google Scholar 

  15. Lengauer C, Kinzler KW, Vogelstein B . (1998). Genetic instabilities in human cancers. Nature 396: 643–649.

    CAS  Article  Google Scholar 

  16. Loignon M, Drobetsky EA . (2002). The initiation of UV-induced G(1) arrest in human cells is independent of the p53/p21/pRb pathway but can be attenuated through expression of the HPV E7 oncoprotein. Carcinogenesis 23: 35–45.

    CAS  Article  Google Scholar 

  17. Luoh SW . (2002). Amplification and expression of genes from the 17q11 approximately q12 amplicon in breast cancer cells. Cancer Genet Cytogenet 136: 43–47.

    CAS  Article  Google Scholar 

  18. Marchio A, Meddeb M, Pineau P, Danglot G, Tiollais P, Bernheim A et al. (1997). Recurrent chromosomal abnormalities in hepatocellular carcinoma detected by comparative genomic hybridization. Genes Chromosomes Cancer 18: 59–65.

    CAS  Article  Google Scholar 

  19. Miwa W, Yasuda J, Murakami Y, Yashima K, Sugano K, Sekine T et al. (1996). Isolation of DNA sequences amplified at chromosome 19q13.1–q13.2 including the AKT2 locus in human pancreatic cancer. Biochem Biophys Res Commun 225: 968–974.

    CAS  Article  Google Scholar 

  20. Morgenstern JP, Land H . (1990). Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res 18: 3587–3596.

    CAS  Article  Google Scholar 

  21. Muleris M, Almeida A, Gerbault-Seureau M, Malfoy B, Dutrillaux B . (1995). Identification of amplified DNA sequences in breast cancer and their organization within homogeneously staining regions. Genes Chromosomes Cancer 14: 155–163.

    CAS  Article  Google Scholar 

  22. Oliner JD, Kinzler KW, Meltzer PS, George DL, Vogelstein B . (1992). Amplification of a gene encoding a p53-associated protein in human sarcomas. Nature 358: 80–83.

    CAS  Article  Google Scholar 

  23. Ormandy CJ, Musgrove EA, Hui R, Daly RJ, Sutherland RL . (2003). Cyclin D1, EMS1 and 11q13 amplification in breast cancer. Breast Cancer Res Treat 78: 323–335.

    CAS  Article  Google Scholar 

  24. Petersen I, Langreck H, Wolf G, Schwendel A, Psille R, Vogt P et al. (1997). Small-cell lung cancer is characterized by a high incidence of deletions on chromosomes 3p, 4q, 5q, 10q, 13q and 17p. Br J Cancer 75: 79–86.

    CAS  Article  Google Scholar 

  25. Reifenberger G, Liu L, Ichimura K, Schmidt EE, Collins VP . (1993). Amplification and overexpression of the MDM2 gene in a subset of human malignant gliomas without p53 mutations. Cancer Res 53: 2736–2739.

    CAS  Google Scholar 

  26. Ross JS, Fletcher JA . (1999). The HER-2/neu oncogene: prognostic factor, predictive factor, and target for therapy. Semin Cancer Biol 9: 125–138.

    CAS  Article  Google Scholar 

  27. Schneider SS, Hiemstra JL, Zehnbauer BA, Taillon-Miller P, Le Paslier DL, Vogelstein B et al. (1992). Isolation and structural analysis of a 12-megabase N-myc amplicon from a human neuroblastoma. Mol Cell Biol 12: 5563–5570.

    CAS  Article  Google Scholar 

  28. Shiloh Y, Korf B, Kohl NE, Sakai K, Brodeur GM, Harris P et al. (1986). Amplification and rearrangement of DNA sequences from the chromosomal region 2p24 in human neuroblastomas. Cancer Res 46: 5297–5301.

    CAS  Google Scholar 

  29. Sieber OM, Heinimann K, Tomlinson IP . (2003). Genomic instability – the engine of tumorigenesis? Nat Rev Cancer 3: 701–708.

    CAS  Article  Google Scholar 

  30. Singh P, Wong SH, Hong W . (1994). Overexpression of E2F-1 in rat embryo fibroblasts leads to neoplastic transformation. EMBO J 13: 3329–3338.

    CAS  Article  Google Scholar 

  31. Sugimoto M, Nakamura T, Ohtani N, Hampson L, Hampson IN, Shimamoto A et al. (1999). Regulation of CDK4 activity by a novel CDK4-binding protein, p34(SEI-1). Genes Dev 13: 3027–3033.

    CAS  Article  Google Scholar 

  32. Tang TC, Sham JS, Xie D, Fang Y, Huo KK, Wu QL et al. (2002). Identification of a candidate oncogene SEI-1 within a minimally amplified region at 19q13.1 in ovarian cancer cell lines. Cancer Res 62: 7157–7161.

    CAS  Google Scholar 

  33. Thompson FH, Nelson MA, Trent JM, Guan XY, Liu Y, Yang JM et al. (1996). Amplification of 19q13.1–q13.2 sequences in ovarian cancer. G-band, FISH, and molecular studies. Cancer Genet Cytogenet 87: 55–62.

    CAS  Article  Google Scholar 

  34. Wang ZJ, Churchman M, Campbell IG, Xu WH, Yan ZY, McCluggage WG et al. (1999). Allele loss and mutation screen at the Peutz–Jeghers (LKB1) locus (19p13.3) in sporadic ovarian tumors. Br J Cancer 80: 70–72.

    CAS  Article  Google Scholar 

  35. Yang XH, Sladek TL . (1995). Overexpression of the E2F-1 transcription factor gene mediates cell transformation. Gene Exp 4: 195–204.

    CAS  Google Scholar 

  36. Yen L, Benlimame N, Nie Z, Xiao D, Wang T, Al Moustafa A et al. (2002). Differential regulation of tumor angiogenesis by distinct ErbB homo- and heterodimers. Mol Biol Cell 13: 4029–4044.

    CAS  Article  Google Scholar 

  37. Zaharieva BM, Simon R, Diener PA, Ackermann D, Maurer R, Alund G et al. (2003). High-throughput tissue microarray analysis of 11q13 gene amplification (CCND1, FGF3, FGF4, EMS1) in urinary bladder cancer. J Pathol 201: 603–608.

    CAS  Article  Google Scholar 

Download references


We thank Dr Masataka Sugimoto (Paterson Institute for Cancer Research) for donating a FLAG-tagged SERTAD1 expression vector and Dr Marie Classon (Massachusetts General Hospital Cancer Center, USA) for donating the E2F luciferase reporter plasmid (pLuc-(E2F)4) and the E2F-1 expression vector. This work was supported by the Canadian Breast Cancer Research Alliance and in part by the Canadian Institutes for Health Research. M Alaoui-Jamali is an FRSQ Scholar and Dundi and Lyon Sachs Distinguished Scientist. H Darwish was supported by NSERC.

Author information



Corresponding author

Correspondence to M A Alaoui-Jamali.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Darwish, H., Cho, J., Loignon, M. et al. Overexpression of SERTAD3, a putative oncogene located within the 19q13 amplicon, induces E2F activity and promotes tumor growth. Oncogene 26, 4319–4328 (2007).

Download citation


  • 19q13
  • cell cycle
  • oncogenesis
  • E2F

Further reading


Quick links