(A) The DUP5-1 minigene shown here contains three exons and two introns derived from human β-globin gene and is essentially identical to previously described version 46 except that vector backbone is pcDNA 3

1/Myc-His(-) C. The middle exon is a chimera of β-globin exon 1 and 2. The name of each minigene and exon and intron sizes are indicated. Minigene βg-ΔiE7-βg is a derivative of DUP5-1 in which the middle exon 2 is replaced by exon 7 of CEACAM1. Minigene βg-E7-βg was created by digesting pDUP5-1 with ApaI and BglII and inserting PCR amplified CEACAM1 fragment that contains exon 7 and the flanking introns 6 and 7 (thick line) except first 150 bp in intron 6 and last 94 bp in intron 7. (B and C) RT-PCR analysis of MDA-MB468 and ZR75 cells transfected with the indicated minigenes or empty vector. -RT represents omission of reverse transcriptase during cDNA synthesis. The data shown is representative of three independent experiments. Bar diagrams represent the mean ± SD of at least three independent experiments. *< 0.001 versus DUP5-1.<p><b>Copyright information:</b></p><p>Taken from "Altered splicing of CEACAM1 in breast cancer: Identification of regulatory sequences that control splicing of CEACAM1 into long or short cytoplasmic domain isoforms"</p><p>http://www.molecular-cancer.com/content/7/1/46</p><p>Molecular Cancer 2008;7():46-46.</p><p>Published online 28 May 2008</p><p>PMCID:PMC2490704.</p><p></p

(A) Total RNAs from the indicated cell lines were isolated and subjected to RT-PCR as described in Figure 2

The position of CEACAM1 long and short cytoplasmic domain splice variants is indicated on right. (B) Histogram depicting CEACAM1-S/CEACAM1-L (S:L) ratio in a logarithmic bar graph quantified with NIH ImageJ program. Data represent the mean ± SD of at least three independent experiments.<p><b>Copyright information:</b></p><p>Taken from "Altered splicing of CEACAM1 in breast cancer: Identification of regulatory sequences that control splicing of CEACAM1 into long or short cytoplasmic domain isoforms"</p><p>http://www.molecular-cancer.com/content/7/1/46</p><p>Molecular Cancer 2008;7():46-46.</p><p>Published online 28 May 2008</p><p>PMCID:PMC2490704.</p><p></p

(A) CEACAM1 splicing pattern in ten breast cancer specimens (1–10) and two normal-appearing (N1 and N2) breast tissues is shown

See Figure 2 and experimental section for details. (B) Histogram representing the quantification of CEACAM1S/L ratio as determined in Figure 2. *< 0.007 versus normal.<p><b>Copyright information:</b></p><p>Taken from "Altered splicing of CEACAM1 in breast cancer: Identification of regulatory sequences that control splicing of CEACAM1 into long or short cytoplasmic domain isoforms"</p><p>http://www.molecular-cancer.com/content/7/1/46</p><p>Molecular Cancer 2008;7():46-46.</p><p>Published online 28 May 2008</p><p>PMCID:PMC2490704.</p><p></p

(A) Schematic diagram of RT-PCR strategy for the identification of CEACAM1 long and short cytoplasmic domain splice variants

Binding sites for the forward and reverse primers and the expected size of amplified fragments are shown. The PCR amplified products were separated by electrophoresis on a 2.5% agarose gel. M, refers to 100 bp DNA ladder (NEB). (B) Histogram shows CEACAM1-S/CEACAM1-L (S:L) ratio quantified with NIH ImageJ program.<p><b>Copyright information:</b></p><p>Taken from "Altered splicing of CEACAM1 in breast cancer: Identification of regulatory sequences that control splicing of CEACAM1 into long or short cytoplasmic domain isoforms"</p><p>http://www.molecular-cancer.com/content/7/1/46</p><p>Molecular Cancer 2008;7():46-46.</p><p>Published online 28 May 2008</p><p>PMCID:PMC2490704.</p><p></p

(A) Schematic representation of CEACAM1 minigene or chimeras cloned into pcDNA 3

1/Myc-His(-) C. Open or shaded boxes are exons and lines represent introns. The name of each minigene is indicated on right. The size of middle exon in all minigenes is identical to exon 7 of CEACAM1. In CAM 6-βg-8 the entire middle exon is replaced by 53 bp of •-globing exon 2. In minigenes CAM 6-E7.1•g-8 and CAM 6-E7.2•g-8, the first and the last 20 bp (E7.1 and E7.2 regions) of CEACAM1 exon 7 are replaced by •-globin exon 2 sequences. The forward (T7) and reverse priming sites (BGH) are indicated. (B and C) RT-PCR analysis of RNA derived from MDA-MB468 and ZR75 cells transiently transfected with indicated minigenes or empty vector. -RT represents omission of reverse transcriptase during cDNA synthesis. The data shown is representative of three independent experiments. Bar diagrams represent the mean ± SD of at least three independent experiments. *< 0.001 and **< 0.01 versus CAM 6-7-8.<p><b>Copyright information:</b></p><p>Taken from "Altered splicing of CEACAM1 in breast cancer: Identification of regulatory sequences that control splicing of CEACAM1 into long or short cytoplasmic domain isoforms"</p><p>http://www.molecular-cancer.com/content/7/1/46</p><p>Molecular Cancer 2008;7():46-46.</p><p>Published online 28 May 2008</p><p>PMCID:PMC2490704.</p><p></p

HP1β Is a Biomarker for Breast Cancer Prognosis and PARP Inhibitor Therapy

<div><p>Members of the heterochromatin protein 1 family (HP1α, β and γ) are mostly associated with heterochromatin and play important roles in gene regulation and DNA damage response. Altered expression of individual HP1 subtype has profound impacts on cell proliferation and tumorigenesis. We analyzed the expression profile of HP1 family by data mining using a published microarray data set coupled with retrospective immunohistochemistry analyses of archived breast cancer biospecimens. We found that the patient group overexpressing <i>HP1β</i> mRNA is associated with poorly differentiated breast tumors and with a significantly lower survival rate. Immunohistochemical staining against HP1α, HP1β and HP1γ shows that respective HP1 expression level is frequently altered in breast cancers. 57.4 - 60.1% of samples examined showed high HP1β expression and 39.9 - 42.6 % of examined tumors showed no or low expression of each HP1 subtype. Interestingly, comparative analysis on HP1 expression profile and breast cancer markers revealed a positive correlation between the respective expression level of all three HP1 subtypes and Ki-67, a cell proliferation and well-known breast cancer marker. To explore the effect of individual HP1 on PARP inhibitor therapy for breast cancer, MCF7 breast cancer cells and individually HP1-depleted MCF7 cells were treated with PARP inhibitor ABT-888 with or without carboplatin. Notably, HP1β-knockdown cells are hypersensitive to the PARP inhibitor ABT-888 alone and its combination with carboplatin. In summary, while increased HP1β expression is associated with the poor prognosis in breast cancer, compromised HP1β abundance may serve as a useful predictive marker for chemotherapy, including PARP inhibitors against breast cancer.</p></div

<i>HP1β</i> message abundance is associated with survival of breast cancer patients.

<p>A microarray database of 295 breast cancer patients (NKI-295 dataset) was analyzed and <i>HP1β</i> message signals were investigated. <i>A</i>. Kaplan-Meier analyses indicated that <i>HP1β</i> mRNA abundance is inversely correlated with both disease-free survival (DFS) and overall survival (OS) for breast cancer patients. <i>B</i>. Group of high <i>HP1β</i> expressors is associated with aggressive and poorly differentiated breast cancers. Low or high <i>HP1β</i> message abundance are denoted from microarray database from the public domain [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121207#pone.0121207.ref023" target="_blank">23</a>].</p

Distinct SB cell markers.

<p><b>A) Flow cytometry of the SB mixture.</b> Beads were purchased from Spherotech, Inc. and examined by flow cytometry to determine the size range standards (a). Using these ranges as a reference, the SB cells were estimated to be 2–6 µm in diameter. Flow cytometry for the hPB and hBM samples before separation (b) and the layer containing only the SB mixture after separation (d). The addition of red blood cell (RBC) lysis buffer revealed that G6 contained RBCs (c). G1 gating represents the background of the staining buffer (e). G2 gating contained particles that were <1 µm in size; most of these were microparticles and microvesicles. G3 contained three major populations (>1 µm): SB cells (G4), RBCs (G6), and white blood cells (WBCs) (G5). The WBCs (G5) were larger than 6–7 µm and have nuclei. <b>B) Flow cytometry of the SB cells.</b> The G4 region was further divided into P1 and P2. (a) P1 gating represents the platelet population. Nearly all of the cells in this gate were CD9+. (b) P2 gating represents the SB cells. We found that 68.3% of these cells were SYTO+, indicating chromosome structure. (c) Lgr5, a stem cell marker, was expressed by 32% of the P2 population. Black: staining with Lgr5; red: staining with the isotype control. <b>C) Flow cytometry of RBCs in the SB cell mixture.</b> G6 represents the RBC location. The RBCs were CD235a+. <b>D) Flow Cytometry of VSELs and BLSCs in the SB cell mixture.</b> Few cells expressed CD66e, a marker of BLSCs (left) and CD133, a marker of VSELs (right), in the SB mixture.</p

Levels of HP1 protein in breast cancer tissues are heterogeneous.

<p><i>A</i>. Normal skin and breast tissues were stained with an anti-HP1β antibody (<i>upper panel</i>). Representative examples of breast cancer sample staining by an anti-HP1β antibody (<i>lower panel</i>). Depending on expression level and subcellular localization of HP1β, cancer samples were classified to three groups. <i>B</i>. Comparison of expression HP1 subtypes in breast cancer samples. Three sets of 190 breast cancer tumors were stained with individual subtypes anti-HP1 antibodies, anti-HP1α, HP1β or HP1γ (Abcam antibodies: ab77256, ab10478, ab10480). IHC staining patterns were compared and IHC staining scores were determined as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121207#pone.0121207.s001" target="_blank">S1 Fig</a>. Scale bars: 100 μm. HP1-High indicates the group of tumors with abundant expression of all three HP1 subtypes. HP1-Low is the group with no or low expression of all three HP1 subtypes. HP1-Mixed group of cancer samples are high level expression with one or two HP1 subtypes. Representative images are shown.</p

Positive correlation of HP1 and Ki-67 levels in breast cancer.

<p>Levels of HP1α, HP1β and HP1γ IHC signal were positively correlated with Ki-67 levels in breast cancer patients. 0 indicates no or low expression and 1 denotes high expression of respective HP1 subtype, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121207#pone.0121207.g002" target="_blank">Fig. 2</a>, and Ki-67 level, respectively.</p