Human malignant breast carcinomas show abundant Fra-1 and c-Fos expression co-localizing with the ER marker calnexin.

<p>Expression of Fra-1 (<b>A</b>), c-Fos (<b>B</b>), the ER marker calnexin, and the nuclear marker of proliferating cells PCNA was examined by triple labeling in malignant human breast tumor specimens (n = 210) and non-pathological samples (n = 37). Six representative samples of invasive ductal carcinomas from a tissue array immunostained for Fra-1 (A, green) or c-Fos (B, green), calnexin (red) and PCNA (blue) and three non-pathological samples (bottom row) are shown for each onco-protein examined. Note that all actively proliferating cells (PCNA positive cells) show abundant Fra-1 and c-Fos expression which in all cases is observed co-localizing with the ER marker as evidenced by the yellow color of the merged, triple-labeled images.</p

MDA-MB231 and MCF7 cells show abundant Fra-1 and c-Fos expression, which co-localizes with the ER marker calnexin.

<p>Expression of Fra-1 (upper left columns), c-Fos (lower left columns) and the ER marker calnexin (middle columns) are shown in MDA-MB231 cells (<b>A</b>), and MCF7 cells (<b>B</b>). Cells were immunostained for Fra-1 (top panels, green) or c-Fos (lower panels, green) and for calnexin (red). The top rows of each panel show quiescent cells (–FBS) while those in the second row correspond to growing cells (+FBS). The last column of each panel is the merge image of the previous two columns. Yellow color evidences Fra-1/ER (top panels) or c-Fos/ER (lower panels) co-localization sites. Clear co-localization is evidenced for both Fra-1 and c-Fos with the ER marker calnexin as determined by confocal immunofluorescence analysis. (<b>C</b>). Fra-1 (upper panel) and c-Fos (lower panel) content was determined by WB in total homogenate (TH) and in the microsomal (MF) and supernatant (SF) fractions obtained after centrifuging MDA-MB231 (left panel) or MCF7 (right panel) cells at 100,000×g for 1 h. In addition, Fra-1 and c-Fos content was examined in MF and SF obtained after stripping MF with 1M KCl prior to centrifugation. Results shown are from one representative experiment out of three performed with essentially the same results.</p

Schematic representations of c-Fos and Fra-1 and comparison of their Basic Domains (BD).

<p>c-Fos is a 380 amino acid (aa) protein whereas Fra-1 contains 271 aa. The BD of c-Fos spans from aa 139 to 159 whereas that of Fra-1 spans from aa 107 to aa 127. Comparison of both BDś shows that of the 12 basic aa that each contain (schematized in bold letters), these are highly homologous showing only two conservative substitutions of the basic aa that are underlined in the scheme. c-Fos contains a C-terminal trans-activating domain (TAD) that is not present in Fra-1.</p

Phospholipid synthesis is activated in Fra-1 or c-Fos-containing human breast tumor biopsies as compared to non-pathological specimens.

<p>(<b>A</b>) Phospholipid labeling capacity was determined in TH prepared from the 5 non-pathological (white columns) and 8 tumor specimens (black columns) shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053211#pone-0053211-g007" target="_blank">Figure 7</a>. Results are the mean cpm incorporated into phospholipids/mg protein ± SD of 2 experiments performed in triplicate with each sample. (<b>B</b> and <b>C</b>) Black columns show the phospholipid labeling capacity (mean cpm incorporated into phospholipids/mg TH protein ± SD of 2 experiments performed in triplicate) of tumor samples #5 (<b>B</b>) and #6 (<b>C</b>) in TH (TTH), MF (TMF), 1M KCl-stripped MF (TMF+1M KCl) and stripped MF plus 1.5 ng of recombinant Fra-1 or 1 ng of c-Fos/µg of TH. White columns (NTH) show the phospholipid labeling capacity of pooled TH from the 5 non-pathological samples shown in (A). *: p<0.01.</p

Fra-1 and c-Fos expression in malignant breast tumor biopsies and non-pathological samples.

<p>(<b>A</b>). Fra-1 (first row), c-Fos (second row) and PCNA content was determined by WB in total homogenate prepared from 5 normal (N1 to N5) and 8 tumor samples (T1 to T8) randomly selected from the collection of breast tissue samples. Note the abundant Fra-1, c-Fos and PCNA expression in all tumor samples contrasting with the very low or undetectable expression levels in the non-pathological ones. Tubulin was used as a loading control (lower panel). (<b>B</b>) TH, MF and SF obtained as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053211#pone-0053211-g002" target="_blank">Figure 2</a> from tumors #5 (left panel) and #6 (right panel) were examined for Fra-1 and c-Fos content. In addition, Fra-1 and c-Fos content was examined in MF and SF obtained after stripping MF with 1M KCl prior to centrifugation. Results shown are from one representative experiment out of three performed with essentially the same results.</p

Nuclear AP-1-Fra-1/c-Fos and cytoplasmic Fra-1/c-Fos are required at early and late stages of cell proliferation, respectively.

<p>(<b>A</b>) MDA-MB231 cells were cultured+or – NLSP that blocks AP-1 nuclear import, in the presence or the absence of anti-Fra-1 or anti-c-Fos antibodies added at the indicated times (0 h or 16 h) after FBS addition and examined for proliferation. Controls were performed that received FITC-IgG antibody (last column). Proliferation was determined as indicated under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053211#s2" target="_blank">Materials and Methods</a> and expressed as arbitrary fluorescent units of DNA. Results are the mean ± SD of 3 experiments performed in quintuplicate; 10,000 cells were plated for each proliferation assay. Note that nuclear import of Fra-1/c-Fos-AP-1 is required only during the first 16 h after FBS addition whereas cytoplasmic Fra-1/c-Fos are required at all time points (compare column 5 with columns 6,7 or 8). (<b>B</b>) TH form MDA-MB231 cells cultured as in (A) were assayed for phospholipid synthesis capacity. Results are the mean cpm incorporated into phospholipids/mg protein ± SD of 3 experiments performed in triplicate. *: p<0.001.</p

Both Fra-1 and c-Fos are capable of sustaining cell proliferation.

<p>(<b>A</b>). MDA-MB231 cells non-transfected (first column), control-transfected (second column) or transfected to block the expression of c-Fos (third column), or of Fra-1 (fourth column), or of both proteins (last column) were examined for cell proliferation. Proliferation was determined as indicated under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053211#s2" target="_blank">Materials and Methods</a> and expressed as arbitrary fluorescent units of DNA. Results are the mean ± SD of 3 experiments performed in sextuplicate; 10,000 cells were plated for each proliferation assay. *: p<0.001. (<b>B</b>) WB determination of the expression of Fra-1 (top row), c-Fos (second row), PCNA (third row) and Tubulin used as a loading control (bottom row) of the samples used in (A) to determine cell proliferation.</p