ER-Alpha-cDNA As Part of a Bicistronic Transcript Gives Rise to High Frequency, Long Term, Receptor Expressing Cell Clones

Within the large group of Estrogen Receptor alpha (ERα)-negative breast cancer patients, there is a subgroup carrying the phenotype ERα−, PR−, and Her2−, named accordingly “Triple-Negative” (TN). Using cell lines derived from this TN group, we wished to establish cell clones, in which ERα is ectopically expressed, forming part of a synthetic lethality screening system. Initially, we generated cell transfectants expressing a mono-cistronic ERα transcription unit, adjacent to a separate dominant selectable marker transcription unit. However, the yield of ERα expressing colonies was rather low (5–12.5%), and only about half of these displayed stable ectopic ERα expression over time. Generation and maintenance of such cell clones under minimal exposure to the ERα ligand, did not improve yield or expression stability. Indeed, other groups have also reported grave difficulties in obtaining ectopic expression of ERα in ERα-deficient breast carcinoma cells. We therefore switched to transfecting these cell lines with pERα-IRES, a plasmid vector encoding a bicistronic translation mRNA template: ERα Open Reading Frame (ORF) being upstream followed by a dominant-positive selectable marker (hygroR) ORF, directed for translation from an Internal Ribosome Entry Site (IRES). Through usage of this bicistronic vector linkage system, it was possible to generate a very high yield of ERα expressing cell clones (50–100%). The stability over time of these clones was also somewhat improved, though variations between individual cell clones were evident. Our successful experience with ERα in this system may serve as a paradigm for other genes where ectopic expression meets similar hardships

pCDNA3-ERα transfectants of MDA-MB-231: Assaying stability of ERα trans-activity via the dual luciferase assay.

<p>For each time point, measured in days from the received time of individual clones covering a 60 mm plate, cells were plated in 24-well tissue culture plates at 50–70% density, and grown in DMEM supplemented with 5% FCS. Twenty four hours later, cells were transiently co-transfected with a p2xERE-pS2-luc plasmid together with a pRNL-TK plasmid. Forty eight hours after transfection cells were lysed whereby <i>firefly</i> and <i>Renilla</i> luciferase activities were measured and normalized to the positive control, MCF-7.</p

pERα-IRES MDA-MB-231 transfectants: Dual-Luciferase reporter assay for ERα activity.

<p>Technical details as in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031977#pone-0031977-g006" target="_blank">Fig. 6</a>.</p

pCDNA3-ERα transfectants of MDA-MB-231: Responsiveness to ligand.

<p>MDA-MB-231 derived clones were seeded in 60 mm dishes and grown for 24 hrs under three conditions: DMEM supplemented with 5% FCS, phenol red-free DMEM supplemented with 5% CSS, and phenol red-free DMEM supplemented with 5% CSS and 2×10⁻⁸ M E₂. The top panel shows the 66 KDa ERα protein detected with the anti-hERα antibody. The bottom panel shows the 57 KDa α-tubulin protein within the same blot after stripping the anti-hERα antibody and re-probing with the anti-α-tubulin antibody.</p

Characterization of ectopically expressed RNAs by long range RT-PCR.

<p>MDA-MB-231 parental cell line (231-parental), its pcDNA3-ERα stable transfectant (ERα-2), and its ERα-IRES stable transfectants (ERα-IRES-5 and ERα-IRES-3) were analyzed for expression of ERα–harboring transcript (1.8 kb), Hygromycin B resistance gene-containing transcript (1.0 kb), and ERα-IRES-Hygro^R fused transcript (3.2 kb), by RT followed by long range PCR amplification. pERα-IRES DNA served as a PCR positive control for the ERα cDNA primers (1.8 kb), the Hygromycin B resistance gene ORF primers (1.0 kb), and the 5′ sense ERα primer plus 3′ antisense Hygro^R fused ORFs primers (3.5 kb). <b>A</b> First four lanes from left contain the ERα cDNA primers; lanes 5–8 the 5′ sense ERα primer together with the 3′ antisense Hygro^R gene primer. <b>B.</b> The 5′ sense ERα primer together with the 3′ antisense Hygro^R gene primer. <b>C.</b> Lanes 1 and 2 from left, the ERα primers. Lanes 3 and 4 the Hygro^R gene primers. Primer sequences are detailed in the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031977#s2" target="_blank">Methods</a>” section.</p

pERα-IRES MDA-MB-231 transfectants: ERα expression via Western immunoblot analysis.

<p><b>A.</b> MDA-MB-231 clones selected for Hygromycin B resistance were lysed and ERα expression was tested by Western immunoblot analysis. ERα positive MCF-7 cell line was used as a positive control. MDA-MB-231 parental cell-line represented the negative control. <b>B.</b> Representation of ERα steady state expression values. The values of ERα expression were normalized to tubulin expression, with MCF7 value being one unit.</p

pCDNA3-ERα transfectants of MDA-MB-231: Testing Response to ERα ligand via the Dual-Luciferase reporter assay.

<p>Clones were plated in 24-well tissue culture plates at 50–70% density under the three growth conditions mentioned in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031977#pone-0031977-g004" target="_blank">Fig. 4</a>. Twenty four hours later cells were transiently co-transfected with a p2xERE-pS2-luc plasmid together with a pRNL-TK plasmid. Forty eight hours after transfection cells were lysed whereby <i>firefly</i> and <i>Renilla</i> luciferase activities were measured and compared to the positive control, MCF-7. MDA-MB-231 parental cell-line was used as a negative control. The presented values were normalized to that of MCF-7 cells seeded in DMEM supplemented with 5% FCS.</p

Map of pIRES-ERα bicistronic plasmid.

<p>The vector contains a single mammalian transcription unit initiating from the CMV immediate early promoter and terminating with an SV40 derived polyA addition fragment.</p

pERα-IRES MDA-MB-435 (A) & GILM2 transfectants (B): ERα activity over time.

<p>Technical details as in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031977#pone-0031977-g006" target="_blank">Fig. 6</a>. ERα values were normalized to the values obtained at each time point with MCF-7 cells, taken as the 100%.</p

pERα-IRES MDA-MB-435 transfectants: Western immunoblot analysis of ERα protein.

<p><b>A.</b> MDA-MB-435 cell clones selected for Hygromycin B resistance were lysed and ERα expression was tested by Western immunoblot analysis. ERα positive MCF-7 cell line was used as a positive control. MDA-MB-435 parental cell-line represented the negative control. <b>B.</b> Representation of ERα steady state expression values. The values of ERα expression were normalized to α tubulin expression in the cells.</p