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

Establishment of a Chemical Synthetic Lethality Screen in Cultured Human Cells

The synthetic lethality screen is a powerful genetic method for unraveling functional interactions between proteins in yeast. Here we demonstrate the feasibility of a chemical synthetic lethality screen in cultured human cells, based in part on the concept of the yeast method. The technology employs both an immortalized human cell line, deficient in a gene of interest, which is complemented by an episomal survival plasmid expressing the gene of interest, and the use of a novel double-label fluorescence system. Selective pressure imposed by any one of several synthetic lethal metabolic inhibitors prevented the spontaneous loss of the episomal survival plasmid. Retention or loss over time of this plasmid could be sensitively detected in a blind test, while cells were grown in microtiter plates. Application of this method should thus permit high throughput screening of drugs, which are synthetically lethal with any mutant human gene of interest, whose normal counterpart can be expressed. This usage is particularly attractive for the search of drugs, which kill malignant cells in a gene-specific manner, based on their predetermined cellular genotype. Moreover, by replacing the chemicals used in this example with a library of either DNA oligonucleotides or expressible dominant negative genetic elements, one should be able to identify synthetic lethal human genes

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

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

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-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