Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of mouse 3T3 cells transduced with cyclin D1 splice variants cyclin D1a and D1b

ABSTRACT: Cyclin D1 is an important cell cycle regulator but in cancer its overexpression also increases cellular migration mediated by p27KIP1 stabilization and RhoA inhibition. Recently, a common polymorphism at the exon 4-intron 4 boundary of the human cyclin D1 gene within a splice donor region was associated with an altered risk of developing cancer. Altered RNA splicing caused by this polymorphism gives rise to a variant cyclin D1 isoform termed cyclin D1b, which has the same N-terminus as the canonical cyclin D1a isoform but a distinct C-terminus. Analysis was performed of mouse cyclin D1 knockout 3T3 cells infected with splice variants of cyclin D1. 3T3 cells transduced with retroviral vectors expressing each cyclin D1 isoform were processed for expression analysis. Experiment Overall Design: Three Mouse Embryonic Fibroblasts cell lines obtained from littermate cyclin D1 knockout E14 embyos were serial passaged to obtain cyclin D1 knockout (D1KO) 3T3 cells. Each of the D1KO 3T3 cell lines were infected using the Murine Stem Cell Virus (MSCV) expressing splice variants of cyclin D1; cyclin D1a/ cyclin D1b or GFP control to give triplicate sample sets. 7 days post infection total RNA from each sample was extracted using Trizol and further purified using Qiagenâs RNeasy Kit. Preparation of biotinylated cRNA and hybridization to oligonucleotide arrays (Affymetrix mouse genome genechip 430 2.0) were performed in conjuction with Pestell lab and the Nucleic Acid Core Facility at Thomas Jefferson University. Mouse 430 2.0 genechip contains 39,000 transcripts. Gene chips were scanned and analyzed using Robust Multi-array Average (RMA) algorithm.

ORGANISM(S): Mus musculus

SUBMITTER: Mathew Casimiro

PROVIDER: E-GEOD-9161 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The cyclin D1 oncogene encodes the regulatory subunit of a holoenzyme that phosphorylates and inactivates the Rb protein and promotes progression through G1 to S phase of the cell cycle. Several prostate cancer cell lines and a subset of primary prostate cancer samples have increased cyclin D1 protein expression. However, the relationship between cyclin D1 expression and prostate tumor progression has yet to be clearly characterized. This study examined the effects of manipulating cyclin D1 expression in either human prostatic epithelial or stromal cells using a tissue recombination model. The data showed that overexpression of cyclin D1 in the initiated BPH-1 cell line increased cell proliferation rate, but did not elicit tumorigenicity in vivo. However, overexpression of cyclin D1 in Normal Prostate Fibroblasts (NPF) that were subsequently recombined with BPH-1 did induce malignant transformation of the epithelial cells. The present study also showed that recombination of BPH-1 + cyclin D1 overexpressing fibroblasts (NPF cyclin D1) resulted in permanent malignant transformation of epithelial cells (BPH-1 NPF-cyclin D1 cells) similar to that seen with Carcinoma Associated Fibroblasts (CAFs). Microarray analysis showed that the expression profiles between CAFs and NPF cyclin D1 cells were highly concordant including cyclin D1 upregulation. These data indicated that the tumor-promoting activity of cyclin D1 may be tissue-specific. Keywords: cyclin D1; stromal-epithelial interactions; prostate cancer; cDNA microarray NPF cyclin D1 cells were generated from NPFs, which were isolated from two different patient samples; CAFs were isolated from two different patient samples as well. RNA was isolated from NPFs, CAFs, and NPF cyclin D1 cells using total RNA isolation kit (Qiagen). Custom spotted cDNA microarrays were constructed as previously described (True, L., Coleman, I., Hawley, S., Huang, C. Y., Gifford, D., Coleman, R., Beer, T. M., Gelmann, E., Datta, M., Mostaghel, E., Knudsen, B., Lange, P., Vessella, R., Lin, D., Hood, L., and Nelson, P. S. A molecular correlate to the Gleason grading system for prostate adenocarcinoma. Proc Natl Acad Sci U S A, 103: 10991-10996, 2006) using a non-redundant set of 6,700 prostate-derived cDNA clones identified from the Prostate Expression Data Base (PEDB), a public sequence repository of expressed sequence tag data derived from human prostate cDNA libraries. Total RNA was amplified through one round of linear amplification using the MessageAmp aRNA kit (Ambion, Austin, TX). Sample quality and quantification was assessed by agarose gel electrophoresis and absorbance at A260. Cy3 and Cy5 labeled cDNA probes were made from 4 M-BM-5g of amplified RNA. Two NPF cyclin D1 and two CAF samples (labeled with Cy3) were hybridized head-to-head with an NPF control sample labeled with Cy5. Probes were hybridized competitively to microarrays under a coverslip for 16 h at 63M-BM-0C. Fluorescent array images were collected for both Cy3 and Cy5 by using a GenePix 4000B fluorescent scanner, and image intensity data were gridded and extracted using GenePix Pro 4.1 software. Differences in gene expression between NPF cyclin D1/NPF and CAF/NPF groups were determined using a two-sample t-test with Significance Analysis of Microarrays (SAM) software (http://www-stat.stanford.edu/_tibs/SAM/) with a False Discovery Rate (FDR) of M-bM-^IM-$ 10% considered significant (Tusher, PNAS, 2001). Similarities in gene expression between NPF cyclin D1/NPF and CAF/NPF groups were determined using a one-sample t-test in SAM with an FDR of M-bM-^IM-$ 0.1% considered significant. These results were reduced to unique genes by eliminating all but the highest scoring clones for each gene. A Pearson correlation coefficient was calculated in Excel to assess the strength of the linear relationship between NPF cyclin D1/NPF and CAF/NPF average log2 ratios.

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Transcription profiling of mouse 3T3 cells transduced with cyclin D1 splice variants cyclin D1a and D1b

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