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 Qiagen’s 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.
Project description: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. Keywords: Cancer associated risk factor 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 Qiagen’s 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.
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 µg 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 63°C. 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 ≤ 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 ≤ 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.
Project description:Genomic aberrations of Cyclin D1 (CCND1) and CDK4 in neuroblastoma indicate that dysregulation of the G1 entry checkpoint is an important cell cycle aberration in this pediatric tumor. Here we report that analysis of Affymetrix expression data of primary neuroblastic tumors shows an extensive over-expression of Cyclin D1 and CDK4 which correlates with histological subgroups and prognosis respectively. Immunohistochemical analysis demonstrated an over-expression of Cyclin D1 in neuroblasts and a low Cyclin D1 expression in all cell types in ganglioneuroma. This suggests an involvement of G1 regulating genes in neuronal differentiation processes which we further evaluated using RNA interference against Cyclin D1 and its kinase partner CDK4 in several neuroblastoma cell lines. This resulted in pRb pathway inhibition as shown by an almost complete disappearance of CDK4 specific pRb phosphorylation; reduction of E2F transcriptional activity and a decrease of Cyclin A protein levels. The Cyclin D1 and CDK4 knock-down resulted in a significant reduction in cell proliferation, a G1 specific cell cycle arrest and moreover an extensive neuronal differentiation. Affymetrix microarray profiling of siRNA treated cells revealed a shift in expression profile towards a neuronal phenotype. Several new potential downstream players are identified. We conclude that neuroblastoma functionally depend on over-expression of G1 regulating genes to maintain their undifferentiated phenotype. Experiment Overall Design: The Cell line IMR-32 at time point 0 and transiently transfected with siRNA against GFP, Cyclin D1 and CDK4 at time point 48 hours. All experiments are biological triplicates.
Project description:We analyzed gene expression profiles of myeloma cells belonging to the group of bas prognosis RPMI 8226 and LP1 expressing either the GFP protein or a cyclin D1-GFP fusion protein Two cell lines RPMI 8226 and LP1. Two clones from each cell line GFP and cyclin D1-GFP. Four biological replicates independently grown and harvested. One replicate per array.
Project description:The CCND1 gene, which is frequently overexpressed in cancers, encodes the regulatory subunit of a holoenzyme that phosphorylates the retinoblastoma protein (pRb). It is known that cyclin D1 regulates ERα transactivation using heterologous reporter systems, the significance of this observation to E2 dependent gene activation is unknow. E2 stimulated MCF7 cells treated with cyclin D1 siRNA in order to analyze the genes regulated by estradiol in a cyclin D1 dependent manner. Hormone deprived MCF7 cells were treated with cyclin D1 siRNA or control siRNA and stimulated with E2 or vehicle Four separate 10cm plates of MCF7 cells treated with control siRNA were compared to four 10cm plates of MCF7 cells treated with cyclin D1 siRNA. 2 plates in each group treated with vehicle and two plates treated with E2.
Project description:Analysis of mammary glands from tet-inducible(rtTA) transgenic mice expressing cyclin D1 using Affymetrix Mouse Gene 1.0 ST GeneChip arrays. MMTV-rtTA transgenic mice (MMTV-Mouse Mammary Tumor Virus promoter) were cross-mated to cyclin D1 transgenic mice under control of tet operon. 8-week-old tetracycline-inducible cyclin D1/rtTA bi-transgenic pregnant female mice (12 days postcoitus) were treated with doxycycline through drinking water supplementation at a final concentration of 2 mg/ml. Control mice were rtTA transgenics alone and treated in the same manner. After 7 days of doxycycline treatment, the mice were sacrificed and mammary glands taken for RNA isolation. Results provide insight into the in vivo gene expression pattern regulated by cyclin D1 through acute induction. Analysis of mammary glands from MMTV-cyclin D1/WT and MMTV-cyclin D1/KE using Affymetrix Mouse 430A v2.0 GeneChip arrays. Cyclin D1 point mutant, cyclin D1/KE K112E (K112E) contains a lysine to glutamine substitution at amino acid position 112. cyclin D1. The cyclin D1/KE mutant fails to induce cyclin D1-dependent kinase activity. Female MFD1, MFD1-KE, and WT mice were monitored twice weekly, up to 760 days, for the development of palpable tumors. Those developing palpable tumors were sacrificed within a week of tumor detection. Tumors were dissected and portions snap frozen for RNA isolation. Results provide insight into the in vivo gene expression pattern regulated by cyclin D1 that is kinase independent. Two separate control mice were positive for MMTV-rtTA transgene compared to 3 separate cyclin D1/rtTA bitransgenic female mice and 3 separate cyclin D1 KE mutant/rtTA bitransgenic female mice (Mouse Gene 1.0 ST arrays). Three separate control WT FvBmice were compared to three MMTV-cyclin D1/WT and 3 MMTV-cyclin D1/KE mice (Mouse 430A v2.0 arrays).
Project description:H9 human pluripotent stem cells were grown in chemically defined media with Activin A and FGF2. Four 10cm plates with 70% confluency were used per sample. Cells were cross-linked by adding a final concentration of 1% PFA into culture media and incubated for 10 min at RT for Cyclin D1 ChIP. ChIP-seq experiment was performed in duplicate.
Project description:We have characterized gene expression changes in HeLa cells following long term depletion of Cyclin T2 or Cyclin T1 using shRNA HeLa cells were transduced with VSV-G pseudotyped lentiviral vectors expressing shRNA against either Cyclin T2 or Cyclin T1. As a control, cells were transduced with shRNA vector with four nucleotides mismatch in the Cyclin T1 mRNA that has been previously shown to have minimal effects on mRNA expression levels. The vectors have a GFP reporter that can be used to estimate transduction efficiency. Five days post-transduction, cells were harvested and total RNA extracted. Transcriptional profiling was carried out on these RNA samples. Two independent biological replicate experiments were carried out in this analysis and the xpression values normalized by GC-RMA and averaged. The following comparisons were made: MM to Cyclin T2 and MM to Cyclin T1.
Project description:To study the oncogenic potential of cyclin D1b in the context of mature B cells we generated several cell clones derived from LP-1 MM cell line expressing either cyclin D1b, Myc or cyclin K oncogenes. Transcriptomic analysis allowed us to describe several mechanisms of cyclin D1b- and K-mediated oncogenesis. Three-condition experiment: LP-1cl1, LP-1K and LP-1D1b. Multiple myeloma LP-1 cell lines. Biological replicates: 4 control (LP-1cl1), 4 transfected with cyclin K (Lp-1K) and 4 transfected with cyclin D1b (LP-1D1b) independently grown and harvested. One replicate per array.
Project description:It is well known that deregulation of chromatin modifiers, such as histone acetylases and methylases, causes malignancies. However, the possible role of histone phosphorylation in carcinogenesis has not yet been elucidated. Here, we found that histone phosphorylation by itself can be the causal event in carcinogenesis. First, we found that histone H2A T120 is phosphorylated in human cancer cell lines and proved that this phosphorylation is catalyzed by hVRK1. By knocking down VRK1, cyclin D1 was found to be downregulated by loss of H2A T120 phosphorylation and increased H2A K119 ubiquitylation of its promoter region, resulting in impaired cell growth. In human cancer tissues, we found that histone H2A is hyperphosphorylated, with upregulated cyclin D1. Mechanistically, histone H2A T120 phosphorylation and histone H2A K119 ubiquitylation, which repress transcription, are mutually inhibitory, suggesting that histone phosphorylation indirectly activates chromatin. Furthermore, mutated H2A T120D, which mimics phosphorylation, causes elevated H3K4 methylation in the same nucleosome. Subsequently, H3K4R, which functionally mimics H3K4 methylation, increases H3 S10 phosphorylation in the same nucleosome. Finally, both VRK1 and the H2A T120D mutant histone transformed NIH/3T3 cells. This suggests that histone H2A T120 phosphorylation by hVRK1 causes inappropriate gene and protein expression, including upregulated cyclin D1, resulting in carcinogenesis. Overall design: mRNA profiles of the five NIH/3T3 cell lines expressing wild-type as well as mutant H2A T120D were generated by deep sequencing using illumina MiSeq.