scholarly journals Characterization of a mutant rat kangaroo cell line with alterations in the cell cycle and DNA repair

2000 ◽  
Vol 23 (3) ◽  
pp. 689-694
Author(s):  
E.N. Miyaji ◽  
R.T. Johnson ◽  
C.S. Downes ◽  
E. Eveno ◽  
M. Mezzina ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Excision Repair ◽  
Uv Light ◽  
Cell Metabolism ◽  
Cell Cycle Control ◽  
Pyrimidine Dimers ◽  
Dna Replication And Repair ◽  
Increased Sensitivity

Using a positive selection system for isolating DNA replication and repair related mutants, we isolated a clone from a rat kangaroo cell line (PtK2) that has increased sensitivity to UV light. Characterization of this clone indicated normal post-replication repair after UV irradiation, and normal removal rates of cyclobutane pyrimidine dimers and pyrimidine(6-4)pyrimidone photoproducts by excision repair. However, this cell line has decreased ability to make early incisions on damaged DNA, possibly indicating a defect in preferential repair of actively transcribed genes, and a slower cell proliferation rate, including a longer S-phase. This phenotype reinforces the present notion that control of key mechanisms in cell metabolism, such as cell cycle control, repair, transcription and cell death, can be linked.

scholarly journals Characterization of a novel HHV-8-positive cell line reveals implications for the pathogenesis and cell cycle control of primary effusion lymphoma

Leukemia ◽  
2000 ◽  
Vol 14 (7) ◽  
pp. 1301-1309 ◽  
Author(s):  
A Carbone ◽  
AM Cilia ◽  
A Gloghini ◽  
D Capello ◽  
L Fassone ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Primary Effusion Lymphoma ◽  
Cell Cycle Control

A novel role for DNA photolyase: binding to DNA damaged by drugs is associated with enhanced cytotoxicity in Saccharomyces cerevisiae

1994 ◽  
Vol 14 (12) ◽  
pp. 8071-8077
Author(s):  
M E Fox ◽  
B J Feldman ◽  
G Chu
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Uv Radiation ◽  
Excision Repair ◽  
Yeast Cells ◽  
Repair System ◽  
Dna Photolyase ◽  
Yeast Saccharomyces Cerevisiae ◽  
Pyrimidine Dimers ◽  
Increased Sensitivity ◽  
Nitroquinoline Oxide

DNA photolyase binds to and repairs cyclobutane pyrimidine dimers induced by UV radiation. Here we demonstrate that in the yeast Saccharomyces cerevisiae, photolyase also binds to DNA damaged by the anticancer drugs cis-diamminedichloroplatinum (cis-DDP) and nitrogen mustard (HN2) and by the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Surprisingly, mutations in photolyase were associated with resistance of yeast cells to cis-DDP, MNNG, 4-nitroquinoline oxide (4NQO), and HN2. Transformation of yeast photolyase mutants with the photolyase gene increased sensitivity to these agents. Thus, while the binding of photolyase to DNA damaged by UV radiation aids survival of the cell, binding to DNA damaged by other agents may interfere with cell survival, perhaps by making the lesions inaccessible to the nucleotide excision repair system.

scholarly journals Quantitative Characterization of a Mitotic Cyclin Threshold Regulating Exit from Mitosis

2005 ◽  
Vol 16 (5) ◽  
pp. 2129-2138 ◽  
Author(s):  
Frederick R. Cross ◽  
Lea Schroeder ◽  
Martin Kruse ◽  
Katherine C. Chen
Keyword(s):  
Cell Cycle ◽  
Budding Yeast ◽  
Cell Cycle Control ◽  
Predictive Ability ◽  
Eukaryotic Cell ◽  
Model Predictions ◽  
Mitotic Cyclin ◽  
Constitutive Overexpression

Regulation of cyclin abundance is central to eukaryotic cell cycle control. Strong overexpression of mitotic cyclins is known to lock the system in mitosis, but the quantitative behavior of the control system as this threshold is approached has only been characterized in the in vitro Xenopus extract system. Here, we quantitate the threshold for mitotic block in budding yeast caused by constitutive overexpression of the mitotic cyclin Clb2. Near this threshold, the system displays marked loss of robustness, in that loss or even heterozygosity for some regulators becomes deleterious or lethal, even though complete loss of these regulators is tolerated at normal cyclin expression levels. Recently, we presented a quantitative kinetic model of the budding yeast cell cycle. Here, we use this model to generate biochemical predictions for Clb2 levels, asynchronous as well as through the cell cycle, as the Clb2 overexpression threshold is approached. The model predictions compare well with biochemical data, even though no data of this type were available during model generation. The loss of robustness of the Clb2 overexpressing system is also predicted by the model. These results provide strong confirmation of the model's predictive ability.

scholarly journals Cell cycle analysis of the activity, subcellular localization, and subunit composition of human CAK (CDK-activating kinase).

1994 ◽  
Vol 127 (2) ◽  
pp. 467-478 ◽  
Author(s):  
J P Tassan ◽  
S J Schultz ◽  
J Bartek ◽  
E A Nigg
Keyword(s):  
Cell Cycle ◽  
Somatic Cell ◽  
Cell Cycle Control ◽  
Gel Filtration ◽  
Multiprotein Complex ◽  
Cyclin Dependent Kinases ◽  
Critical Residue ◽  
Cdk Activating Kinase

The activity of cyclin-dependent kinases (cdks) depends on the phosphorylation of a residue corresponding to threonine 161 in human p34cdc2. One enzyme responsible for phosphorylating this critical residue has recently been purified from Xenopus and starfish. It was termed CAK (for cdk-activating kinase), and it was shown to contain p40MO15 as its catalytic subunit. In view of the cardinal role of cdks in cell cycle control, it is important to learn if and how CAK activity is regulated during the somatic cell cycle. Here, we report a molecular characterization of a human p40MO15 homologue and its associated CAK activity. We have cloned and sequenced a cDNA coding for human p40MO15, and raised specific polyclonal and monoclonal antibodies against the corresponding protein expressed in Escherichia coli. These tools were then used to demonstrate that p40MO15 protein expression and CAK activity are constant throughout the somatic cell cycle. Gel filtration suggests that active CAK is a multiprotein complex, and immunoprecipitation experiments identify two polypeptides of 34 and 32 kD as likely complex partners of p40MO15. The association of the three proteins is near stoichiometric and invariant throughout the cell cycle. Immunocytochemistry and biochemical enucleation experiments both demonstrate that p40MO15 is nuclear at all stages of the cell cycle (except for mitosis, when the protein redistributes throughout the cell), although the p34cdc2/cyclin B complex, one of the major purported substrates of CAK, occurs in the cytoplasm until shortly before mitosis. The absence of obvious changes in CAK activity in exponentially growing cells constitutes a surprise. It suggests that the phosphorylation state of threonine 161 in p34cdc2 (and the corresponding residue in other cdks) may be regulated primarily by the availability of the cdk/cyclin substrates, and by phosphatase(s).

scholarly journals Molecular and Cellular Characterization of Two Patient-Derived Ductal Carcinoma in Situ (DCIS) Cell Lines, ETCC-006 and ETCC-010

2020 ◽  
Author(s):  
Julia Samson ◽  
Kellie Dean
Keyword(s):  
Breast Cancer ◽  
Cell Cycle ◽  
Cell Line ◽  
Cell Lines ◽  
Ductal Carcinoma ◽  
Breast Cancer Cell Lines ◽  
Breast Epithelial Cell ◽  
Anchorage Independent Growth

Abstract Background: Currently it is unclear how in situ breast cancer progresses to invasive disease; therefore, a better understanding of the events that occur during the transition to invasive carcinoma is warranted. Here we have conducted a detailed molecular and cellular characterization of two, patient-derived, ductal carcinoma in situ (DCIS) cell lines, ETCC-006 and ETCC-010. Methods: Human DCIS cell lines, ETCC-006 and ETCC-010, were compared against a panel of cell lines including the immortalized, breast epithelial cell line, MCF10A, breast cancer cell lines, MCF7 and MDA-MB-231, and another DCIS line, MCF10DCIS.com. Cell morphology, hormone and HER2/ERBB2 receptor status, cell proliferation, survival, migration, anchorage-independent growth, indicators of EMT, cell signalling pathways and cell cycle proteins were examined using immunostaining, immunoblots, and quantitative, reverse transcriptase PCR (qRT-PCR), along with clonogenic, wound-closure and soft agar assays. RNA sequencing (RNAseq) was used to provide a transcriptomic profile. Results: ETCC-006 and ETCC-010 cells displayed notable differences to another DCIS cell line, MCF10DCIS.com, in terms of morphology, steroid-receptor/HER status and markers of EMT. The ETCC cell lines lack ER/PR and HER, form colonies in clonogenic assays, have migratory capacity and are capable of anchorage-independent growth. Despite being isogenic, less than 30% of differentially expressed transcripts overlapped between the two lines, with enrichment in receptor tyrosine kinase pathways and DNA replication/cell cycle programs. Conclusions: For the first time, we provide a molecular and cellular characterization of two, patient-derived DCIS cell lines, ETCC-006 and ETCC-010, facilitating future investigations into the molecular basis of DCIS to invasive ductal carcinoma transition.

Redox factor-1 contributes to the regulation of progression from G0/G1 to S by PDGF in vascular smooth muscle cells

2003 ◽  
Vol 285 (2) ◽  
pp. H804-H812 ◽  
Author(s):  
Tongrong He ◽  
Neal L. Weintraub ◽  
Prabhat C. Goswami ◽  
Papri Chatterjee ◽  
Dawn M. Flaherty ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Smooth Muscle ◽  
Dna Binding ◽  
Cell Cycle Progression ◽  
Redox Regulation ◽  
Excision Repair ◽  
Chemical Reduction ◽  
Cell Cycle Control ◽  
S Phase ◽  
Nuclear Extracts

Redox factor-1 (Ref-1/APE), a multifunctional DNA base excision repair and redox regulation enzyme, plays an important role in oxidative signaling, transcription factor regulation, and cell cycle control. We hypothesized that Ref-1 plays a regulatory role in smooth muscle cell (SMC) proliferation induced by PDGF. Ref-1 antisense oligodeoxynucleotides (AODN), which diminished the level of Ref-1 protein in SMCs by ∼50%, inhibited PDGF-BB (composed of the homodimer of B-polypeptide chain)-induced [3H]thymidine incorporation compared with control oligodeoxynucleotides. Ref-1 AODN inhibited PDGF-BB-induced S phase entry by ∼63%, which was overcome by overexpression of Ref-1 by adenoviral-mediated gene transfer. Overexpression of Ref-1 alone without PDGF enhanced SMC entry into the S phase. Furthermore, decreasing Ref-1 protein by treatment of SMCs with Ref-1 AODN, or by immunodepletion of Ref-1 from nuclear extracts, inhibited PDGF-BB-induced activator protein-1 (AP-1) DNA binding activity. Chemical reduction restored the AP-1 DNA binding in Ref-1-depleted nuclear extracts. These results suggest that Ref-1 contributes to the regulation of PDGF-BB-stimulated cell cycle progression from G0/G1 to S in SMCs, with one of the possible steps being redox-regulation of AP-1 by Ref-1 protein.

Molecular Mechanisms of Cell Cycle Control in the Mouse Y1 Adrenal Cell Line

2004 ◽  
Vol 30 (4) ◽  
pp. 503-509 ◽  
Author(s):  
Érico T. Costa ◽  
Fábio L. Forti ◽  
Kátia M. Rocha ◽  
Miriam S. Moraes ◽  
Hugo A. Armelin
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Molecular Mechanisms ◽  
Cell Cycle Control ◽  
Adrenal Cell

scholarly journals Myb-Related Fission Yeast cdc5p Is a Component of a 40S snRNP-Containing Complex and Is Essential for Pre-mRNA Splicing

1999 ◽  
Vol 19 (8) ◽  
pp. 5352-5362 ◽  
Author(s):  
W. Hayes McDonald ◽  
Ryoma Ohi ◽  
Natalia Smelkova ◽  
David Frendewey ◽  
Kathleen L. Gould
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Fission Yeast ◽  
Genetic Interaction ◽  
Cell Cycle Control ◽  
Mrna Splicing ◽  
Splicing Factor ◽  
Immunoaffinity Purification ◽  
Snrnp Protein

ABSTRACT Myb-related cdc5p is required for G2/M progression in the yeast Schizosaccharomyces pombe. We report here that all detectable cdc5p is stably associated with a multiprotein 40S complex. Immunoaffinity purification has allowed the identification of 10 cwf (complexed with cdc5p) proteins. Two (cwf6p and cwf10p) are members of the U5 snRNP; one (cwf9p) is a core snRNP protein. cwf8p is the apparent ortholog of the Saccharomyces cerevisiaesplicing factor Prp19p. cwf1 + is allelic to theprp5 + gene defined by the S. pombesplicing mutant, prp5-1, and there is a strong negative genetic interaction between cdc5-120 andprp5-1. Five cwfs have not been recognized previously as important for either pre-mRNA splicing or cell cycle control. Further characterization of cwf1p, cwf2p, cwf3p, and cwf4p demonstrates that they are encoded by essential genes, cosediment with cdc5p at 40S, and coimmunoprecipitate with cdc5p. We further show that cdc5p associates with the U2, U5, and U6 snRNAs and that cells lackingcdc5 + function are defective in pre-mRNA splicing. These data raise the possibility that the cdc5p complex is an intermediate in the assembly or disassembly of an active S. pombe spliceosome.

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