Pit-1 overexpression in the human breast adenocarcinoma MCF-7 cell line
ABSTRACT: The aim of the study is to evaluate Pit-1-induced genes in the MCF-7 cell line The Pit-1 transcription factor (also known as POU1F1) plays a critical role in cell differentiation during organogenesis of the anterior pituitary in mammals and is a transcriptional activator for pituitary gene transcription. Increased expression of Pit-1 has been reported in human tumorigenic breast cells. Here, we found that Pit-1 overexpression or knockdown in human breast cancer cell lines induced profound phenotypic changes in the expression of proteins involved in cell proliferation, apoptosis, and invasion. In immunodeficient mice, Pit-1 overexpression induced tumoral growth and promoted metastasis in lung. In patients with invasive ductal carcinoma of the breast and node-positive tumors elevated expression of Pit-1 was significantly and independently associated with the occurrence of distant metastasis. These findings suggest that Pit-1 could help to make a more accurate prognosis in patients with node positive breast cancer and may represent a new therapeutic target (Journal of Clinical Investigation 2010, 120:4289-4302) MCF-7 cells were transfected with the pcDNA3 (control, two samples as condition, named C1 and C2) or the pcDNA3-Pit-1 overexpression vector (two samples as condition, named 1+ and 2+) for 48 hours.
Project description:EpCAM is frequently overexpressed in human invasive breast cancer. We reported EpCAM overexpression to be an independent prognostic marker for poor overall survival in node-positive breast cancer. We used microarrays in order to investigate changes of the transcriptome on EpCAM gene overexpression in human breast cancer cells Hs578T cells were found to express only very little EpCAM mRNA and protein in comparison to established and well characterized breast cancer cell lines such as MCF-7 or SK-BR-3. Hs578T cells were stably transfected with the EpCAM cDNA containing construct pIRESpuro3_EpCAM and the respective empty vector control.
Project description:TGFB2-AS1 is a long non-coding RNA which is induced by ΤGFβ signaling. In order to assess the importance of TGFB2-AS1 on the regulation of gene expression, we performed an AmpliSeq transcriptomic array in human keratinocytes (HaCaT), which stably over-express TGFB2-AS1 or control pcDNA3 empty vector. In addition, cells were stimulated with TGFβ1 for 24 hours, in order to observe the effects of TGFB2-AS1 on gene expression, downstream of TGFβ signaling. RNA from the following four conditions was used in this experiment: 1) pcDNA3, 2) pcDNA3+TGFβ1, 3) pcDNA3-TGFB2-AS1, 4) pcDNA3-TGFB2-AS1+TGFβ1. Biological triplicates were used per condition.
Project description:EpCAM is frequently overexpressed in human invasive breast cancer. We reported EpCAM overexpression to be an independent prognostic marker for poor overall survival in node-positive breast cancer. We used microarrays in order to investigate changes of the transcriptome on EpCAM gene overexpression in human breast cancer cells Overall design: Hs578T cells were found to express only very little EpCAM mRNA and protein in comparison to established and well characterized breast cancer cell lines such as MCF-7 or SK-BR-3. Hs578T cells were stably transfected with the EpCAM cDNA containing construct pIRESpuro3_EpCAM and the respective empty vector control.
Project description:Gadd45a is a stress-induced protein that causes skeletal muscle atrophy. The goal of these studies was to determine the effects of Gadd45a overexpression on mRNA levels in mouse skeletal muscle. For additional details see Ebert et al, Stress-Induced Skeletal Muscle Gadd45a Expression Reprograms Myonuclei and Causes Muscle Atrophy. JBC epub. June 12, 2012. Tibialis anterior (TA) muscles from muscle-specfic ATF4 knockout mice (ATF4 mKO) were transfected with either 20 mg empty plasmid (pcDNA3) (left TA) or 20 mg pCMV-FLAG-Gadd45a (right TA) and harvested 7 days later. mRNA levels in Gadd45a-transfected muscles were normalized to levels in control transfected muscles.
Project description:Prop1 controls the expression of genes besides Pit1 that are important for cell mgration, survival and differentiation in the mouse and human pituitary gland. Microarray analysis was used to compare pituitary RNA from newborn Prop1 and Pit1 mutants and their wild type littermates. Total RNA was collected and pooled from Prop1df/df, Porp1+/+, Pit1dw/dw, and Pit+/+ at the age of P1. 5 pools per genotype were hybridized to the Affymetrix Mouse Genome 430 2.0 array.
Project description:Human breast cancer cell line MCF-7 is usually sensitive to chemotherapy drug BMS-554417, an insulin receptor (IR) and insulin-like growth factor receptor (IGFR) inhibitor. However, through step-wise increase in BMS-554417 doses in culture media, we were able able to screen and select a single MCF-7 clone that is BMS-554417 resistant. It is cross resistant to BMS-536924. This new line of MCF-7 cells was named as MCF-7R4. The transcriptome profiling of both MCF-7 and MCF-7R4 was performed using Affymetrix HG-U133 plus2.0 GeneChip arrays. Five replicates of MCF-7 and five replicates of MCF-7R4 were profiled.
Project description:N-α-acetyltransferase 10 protein (Naa10p, also called ARD1), the catalytic subunit of N-acetyltransferase A, is a critical regulator of cell death and proliferation. Naa10p is also shown to regulate cancer metastasis by inhibiting cell motility, however its role in cancer metastasis is not fully understood. In this study, we found that high expression of Naa10p is positively correlated with the survival of patients with breast cancer, while negatively correlated with lymph node metastasisr. Naa10p inhibits breast cancer cell migration and invasion in vitro and decreases the xenograft growth and metastasis in nude mice. Microarray screening revealed that Naa10p down-regulates expression of several pro-invasive genes, which was validated by qRT-PCR analysis. To gain an insight into Naa10p’s inhibitory effect on cancer metastasis, we performed microarray analysis with RNA samples extracted from Naa10p-silenced MCF-7 (MCF-7SA) and control cells (MCF-7NC).