Evaluation of endogenous cyclin A1 and VEGF expression in breast cancer cell lines.

<p>(A) Cyclin A1 mRNA levels are assessed in T47D, BT549, MCF-7, MDA-MB-468, MDA-MB231 and Cama-1 cell lines by semiquantitative RT-PCR and a representative picture is shown (upper panel). Quantification of cyclin A1 mRNA level is shown and mean ± SD represents three independent experiments (lower panel). (B) Western blot analysis of levels of cyclin A1 protein in the cell lines as indicated. A representative picture is shown (upper panel). Quantification of cyclin A1 protein level is shown and mean ± SD represents three independent experiments (lower panel). (C) VEGF mRNA levels are assessed in the indicated cell lines by semiquantitative RT-PCR and a representative picture is shown (upper panel). Quantification of cyclin A1 protein level is shown and mean ± SD represents three independent experiments (lower panel). (D) ELISA assay of VEGF secretion in breast cancer cell lines as indicated. Mean ± SD represents three independent experiments.</p

Long-term effect of elevated level of cyclin A1 on growth and angiogenesis phenotype of xenograft tumors <i>in mice.</i>

<p>MCF-7 cells stable expressing pcDNA–cyclin A1 or pcDNA vectors were subcutaneous implanted into female nude mice with E2 supplementation. (A, B) Representative microphotographs of xenograft tumor sections stained with Haematoxylin and Eosin are shown. (C, D) Representative pictures show the xenograft tumors stained with antibody against human CD31, the CD31 positive vessels are indicated. The control tumor “control-pcDNA” and cyclin A1 expressing tumors “cyclin A1-pcDNA” are indicated. (E) Growth curves of the two groups of xenograft tumors are indicated. The time is indicated in x-axis and tumor volume in mm³ is indicated in y-axis. (F) Quantification of the tumor vascularizations in cyclin A1 expressing xenograft tumors “cyclin A1-pcDNA” and in control xenograft tumors “control-pcDNA”. The numbers of CD31-positive blood vessels in the central vs. edge regions of the tumor areas are shown. P values are indicated. Mean ± SD represents three independent experiments. (G–N) Xenograft tumors from “cyclin A1-pcDNA” and “control-pcDNA” groups were immunostained with antibodies against VEGF, VEGFR1, ER-α and Ki67. The representative microphotographs are shown.</p

Effect of cyclin A1 expression on tumor invasion is associated with its effect on VEGF expression in MCF-7 cells.

<p>(A) Evaluation of cyclin A1 and VEGF expression in metastatic lesions from lymph nodes from patients with breast cancer metastasis using immunohistochemical analysis. Representative pictures show the cancer cells are strongly positive to cyclin A1 and VEGF expression. Upper panels represent cores at 20x magnificantion and lower panels represent the higher magnification (40x) of the selected areas. (B) MCF-7 cells that were transfected with cyclin A1pCMS-EGFP or pCMS-EGFP vectors were applied on the Matrigel-coated invasion chamber and were assessed after 48 or 72 hours. Data in graphs are the mean ± SD represents two independent experiments, each performed in duplicates. P value is indicated. (C) MDA-MB-231 cells transfected with cyclin A1pCMS-EGFP or pCMS-EGFP were applied on the Matrigel-coated invasion chamber and were analyzed after 48 or 72 hours. Data in graphs are the mean of two independent experiments, each performed in duplicate, p=0.002 for 48 h and p=0.02 for 72 h. (D) Cell cycle distribution of the cells that were transfected with cyclin A1pCMS-EGFP or pCMS-EGFP. Data in graphs are the mean ± SD represents three independent experiments from flow cytometry analysis. The percentage of cells at onset of each cell cycle phase is indicated. (E) Western blot analysis shows the levels of cyclin D1 and CDK1 protein expression in the cells that were transfected with cyclin A1pCMS-EGFP or pCMS-EGFP. (F) Representative picture shows the VEGF mRNA expression in the cells transfected with cyclin A1pCMS-EGFP or pCMS-EGFP (upper panel). Quantification of VEGF mRNA level in the samples is indicated. P value is shown (lower panel). (G) ELISA assay of VEGF secretion in the cells transfected with cyclin A1pCMS-EGFP or pCMS-EGFP. Mean ± SD represents three independent experiments (lower panel). Breast cancer cell lines used for these studies are T47D, MCF-7 and MDA-MB231 as indicated.</p

The effect of cyclin A1 on growth and vascularization of tumor xenografts <i>in mice.</i>

<p>MCF-7 cells transfected with cyclin A1pCMS–EGFP or pCMS-EGFP vectors were subcutaneous implanted into female nude mice with E2 supplementation. (A, B) Representative microphotographs of xenograft tumor sections stained with Haematoxylin and Eosin are shown. The control tumor “pCMS-control” and cyclin A1 expressing tumor “pCMS-cyclin A1” are indicated. (C, D) Representative pictures show the xenograft tumors stained with antibody against human CD31, the CD31 positive vessels are indicated. (E) Growth curves of the two groups of xenograft tumors. The control tumor “pCMS-control” and cyclin A1 expressing tumor “pCMS-cyclin A1” are indicated. The time is indicated in x-axis and tumor volume in mm³ is indicated in y-axis. (F) Quantification of the tumor vascularizations in cyclin A1 expressing xenograft tumors “pCMS-cyclin A1” and in control xenograft tumors “pCMS-control”. The numbers of CD31-positive blood vessels in the central vs. edge regions of the tumor areas are shown. P values are indicated. Mean ± SD represents three independent experiments.</p

Evaluation of the association between cyclin A1 and ER-α estrogen signaling and the regulation of VEGF expression.

<p>(A) -Evaluation of the effect of estrogen on mRNA expression of cyclin A1, VEGF and ER-α, in T47D, MCF-7 and MDA-MB231 cells using semi-quantitative RT-PCR analysis. The three cell lines mentioned above were cultured in charcoal stripped medium (CSS) or in CSS medium containing E2 at 5 µM for additional 48 hours as indicated. (B) The effect of E2 treatment or cyclin A1 overexpression alone or in combination on VEGF mRNA level was determined in MCF-7 cells. Cells that were transfected with cyclin A1pCMS-EGFP or pCMS-EGFP vectors in the absence or presence of E2 are indicated. (C) Evaluation of the effects of Tamoxifen (Tam), E2, and E2 in combination with Tamoxifen (E2+Tam) on VEGF protein expression in MCF-7 cells. MCF-7 cells were transfected with cyclin A1 vector or control vector as indicated. Data in graphs below are the mean ± SD represents two independent experiments. (D) Immunoblot analysis data obtained in MDA-MB-231 which were treated using the same conditions as mentioned in (C). (E) Immunoprecipitation analysis (IP) shows physical interaction between cyclin A1 and ER-αin MCF-7 cells that were transfected with cyclin A1pCMS-EGFP or pCMS-EGFP vectors. ER-α antibody was used in IP to pull down the immunocomplexes and subsequent Westernblot was performed using cyclin A1 or ER-α antibodies to detect the immunocomplexes as indicated. The input was used as controls as indicated. (F) Evaluation of ER-α mRNA expression in the cells that were transfected with A1pCMS-EGFP or pCMS-EGFP vectors. The representative picture is shown in the upper panel. Quantification of ER-α mRNA level is shown in the lower panel and mean ± SD represents three independent experiments. (G) Western blot analysis shows the expression level of ER-α protein in the cells that were transfected with A1pCMS-EGFP or pCMS-EGFP vectors. Breast cancer cell lines used for these studies are T47D, MCF-7 and MDA-MB231 as indicated.</p

Epigenetic silencing of miR-26A1 in chronic lymphocytic leukemia and mantle cell lymphoma: Impact on EZH2 expression

<p>Downregulation of miR26A1 has been reported in various B-cell malignancies; however, the mechanism behind its deregulation remains largely unknown. We investigated miR26A1 methylation and expression levels in a well-characterized series of chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). From 450K methylation arrays, we first observed miR26A1 (cg26054057) as uniformly hypermethylated in MCL (n = 24) (all >75%), while CLL (n = 18) showed differential methylation between prognostic subgroups. Extended analysis using pyrosequencing confirmed our findings and real-time quantitative PCR verified low miR26A1 expression in both CLL (n = 70) and MCL (n = 38) compared to normal B-cells. Notably, the level of miR26A1 methylation predicted outcome in CLL, with higher levels seen in poor-prognostic, IGHV-unmutated CLL. Since EZH2 was recently reported as a target for miR26A1, we analyzed the expression levels of both miR26A1 and <i>EZH2</i> in primary CLL samples and observed an inverse correlation. By overexpression of miR26A1 in CLL and MCL cell lines, reduced EZH2 protein levels were observed using both Western blot and flow cytometry. In contrast, methyl-inhibitor treatment led to upregulated miR26A1 expression with a parallel decrease of EZH2 expression. Finally, increased levels of apoptosis were observed in miR26A1-overexpressing cell lines, further underscoring the functional relevance of miR26A1. In summary, we propose that epigenetic silencing of miR26A1 is required for the maintenance of increased levels of EZH2, which in turn translate into a worse outcome, as shown in CLL, highlighting miR26A1 as a tumor suppressor miRNA.</p