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      Identification of G1-Regulated Genes in Normally Cycling Human Cells

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          Abstract

          Background

          Obtaining synchronous cell populations is essential for cell-cycle studies. Methods such as serum withdrawal or use of drugs which block cells at specific points in the cell cycle alter cellular events upon re-entry into the cell cycle. Regulatory events occurring in early G1 phase of a new cell cycle could have been overlooked.

          Methodology and Findings

          We used a robotic mitotic shake-off apparatus to select cells in late mitosis for genome-wide gene expression studies. Two separate microarray experiments were conducted, one which involved isolation of RNA hourly for several hours from synchronous cell populations, and one experiment which examined gene activity every 15 minutes from late telophase of mitosis into G1 phase. To verify synchrony of the cell populations under study, we utilized methods including BrdU uptake, FACS, and microarray analyses of histone gene activity. We also examined stress response gene activity. Our analysis enabled identification of 200 early G1-regulated genes, many of which currently have unknown functions. We also confirmed the expression of a set of genes candidates ( fos, atf3 and tceb) by qPCR to further validate the newly identified genes.

          Conclusion and Significance

          Genome-scale expression analyses of the first two hours of G1 in naturally cycling cells enabled the discovery of a unique set of G1-regulated genes, many of which currently have unknown functions, in cells progressing normally through the cell division cycle. This group of genes may contain future targets for drug development and treatment of human disease.

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          Most cited references53

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          Identification of genes periodically expressed in the human cell cycle and their expression in tumors.

          The genome-wide program of gene expression during the cell division cycle in a human cancer cell line (HeLa) was characterized using cDNA microarrays. Transcripts of >850 genes showed periodic variation during the cell cycle. Hierarchical clustering of the expression patterns revealed coexpressed groups of previously well-characterized genes involved in essential cell cycle processes such as DNA replication, chromosome segregation, and cell adhesion along with genes of uncharacterized function. Most of the genes whose expression had previously been reported to correlate with the proliferative state of tumors were found herein also to be periodically expressed during the HeLa cell cycle. However, some of the genes periodically expressed in the HeLa cell cycle do not have a consistent correlation with tumor proliferation. Cell cycle-regulated transcripts of genes involved in fundamental processes such as DNA replication and chromosome segregation seem to be more highly expressed in proliferative tumors simply because they contain more cycling cells. The data in this report provide a comprehensive catalog of cell cycle regulated genes that can serve as a starting point for functional discovery. The full dataset is available at http://genome-www.stanford.edu/Human-CellCycle/HeLa/.
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            Minireview: Cyclin D1: normal and abnormal functions.

            Cyclin D1 encodes the regulatory subunit of a holoenzyme that phosphorylates and inactivates the retinoblastoma protein and promotes progression through the G1-S phase of the cell cycle. Amplification or overexpression of cyclin D1 plays pivotal roles in the development of a subset of human cancers including parathyroid adenoma, breast cancer, colon cancer, lymphoma, melanoma, and prostate cancer. Of the three D-type cyclins, each of which binds cyclin-dependent kinase (CDK), it is cyclin D1 overexpression that is predominantly associated with human tumorigenesis and cellular metastases. In recent years accumulating evidence suggests that in addition to its original description as a CDK-dependent regulator of the cell cycle, cyclin D1 also conveys cell cycle or CDK-independent functions. Cyclin D1 associates with, and regulates activity of, transcription factors, coactivators and corepressors that govern histone acetylation and chromatin remodeling proteins. The recent findings that cyclin D1 regulates cellular metabolism, fat cell differentiation and cellular migration have refocused attention on novel functions of cyclin D1 and their possible role in tumorigenesis. In this review, both the classic and novel functions of cyclin D1 are discussed with emphasis on the CDK-independent functions of cyclin D1.
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              Mammalian cells cycle without the D-type cyclin-dependent kinases Cdk4 and Cdk6.

              Cdk4 and Cdk6 are thought to be essential for initiation of the cell cycle in response to mitogenic stimuli. Previous studies have shown that Cdk4 is dispensable for proliferation in most cell types, an observation attributed to a putative compensatory role by Cdk6. Cdk6-null mice are viable and develop normally although hematopoiesis is slightly impaired. Embryos defective for Cdk4 and Cdk6 die during the late stages of embryonic development due to severe anemia. However, these embryos display normal organogenesis and most cell types proliferate normally. In vitro, embryonic fibroblasts lacking Cdk4 and Cdk6 proliferate and become immortal upon serial passage. Moreover, quiescent Cdk4/Cdk6-null cells respond to serum stimulation and enter S phase with normal kinetics although with lower efficiency. These results indicate that D-type cyclin-dependent kinases are not essential for cell cycle entry and suggest the existence of alternative mechanisms to initiate cell proliferation upon mitogenic stimulation.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2008
                15 December 2008
                : 3
                : 12
                : e3943
                Affiliations
                [1 ]Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, Florida, United States of America
                [2 ]The Channing Laboratory, Brigham and Women's Hospital and Department of Medicine, Harvard Medical School, Boston, Massachusetts, United States of America
                [3 ]Department of Genetics, Norris Cotton Cancer Center, Dartmouth Medical School, Hanover, New Hampshire, United States of America
                [4 ]Department of Biological Science, Florida State University, Tallahassee, Florida, United States of America
                [5 ]Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire, United States of America
                [6 ]Department of Statistics, Florida State University, Tallahassee, Florida, United States of America
                Georgia Institute of Technology, United States of America
                Author notes

                Conceived and designed the experiments: MJB KEA MLW MH. Performed the experiments: MJB KEA MLW HWB MH. Analyzed the data: MJB KEA MLW HWB DLM MH. Contributed reagents/materials/analysis tools: MJB KEA AB MLW HWB DLM MH. Wrote the paper: MJB MLW MH.

                Article
                08-PONE-RA-06266R1
                10.1371/journal.pone.0003943
                2600614
                19079774
                c766907f-fab9-45ad-841f-074167a3ddaa
                Beyrouthy et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                : 7 September 2008
                : 18 November 2008
                Page count
                Pages: 10
                Categories
                Research Article
                Cell Biology/Cell Growth and Division
                Cell Biology/Gene Expression
                Computational Biology/Genomics
                Genetics and Genomics/Bioinformatics
                Molecular Biology/Bioinformatics
                Oncology/Gynecological Cancers

                Uncategorized
                Uncategorized

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