scholarly journals Chfr acts with the p38 stress kinases to block entry to mitosis in mammalian cells

2004 ◽  
Vol 166 (4) ◽  
pp. 507-516 ◽  
Author(s):  
Takahiro Matsusaka ◽  
Jonathon Pines
Keyword(s):  
Cell Lines ◽  
Mammalian Cells ◽  
Proteasome Activity ◽  
Chromosomal Damage ◽  
Stress Kinases ◽  
Curr Biol ◽  
G2 Phase ◽  
Transformed Cell Lines ◽  
Recent Demonstration ◽  
Microtubule Poisons

Entry into mitosis in vertebrate cells is guarded by a checkpoint that can be activated by a variety of insults, including chromosomal damage and disrupting microtubules (Rieder, C.L., and R.W. Cole. 1998. J. Cell Biol. 142:1013–1022; Rieder, C.L., and R.W. Cole. 2000. Curr. Biol. 10:1067–1070). This checkpoint acts at the end of interphase to delay cells from entering mitosis, causing cells in prophase to decondense their chromosomes and return to G2 phase. Here, we show that in response to microtubule poisons this “antephase” checkpoint is primarily mediated by the p38 stress kinases and requires the Chfr protein that is absent or inactive in several transformed cell lines (Scolnick, D.M., and T.D. Halazonetis. 2000. Nature. 406:430–435) and lung tumors (Mizuno, K., H. Osada, H. Konishi, Y. Tatematsu, Y. Yatabe, T. Mitsudomi, Y. Fujii, and T. Takahashi. 2002. Oncogene. 21:2328–2333). Furthermore, in contrast to previous reports, we find that the checkpoint requires ubiquitylation but not proteasome activity, which is in agreement with the recent demonstration that Chfr conjugates ubiquitin through lysine 63 and not lysine 48 (Bothos, J., M.K. Summers, M. Venere, D.M. Scolnick, and T.D. Halazonetis. 2003. Oncogene. 22:7101–7107).

scholarly journals Introduction and recovery of a selectable bacterial gene from the genome of mammalian cells

1982 ◽  
Vol 2 (8) ◽  
pp. 966-976
Author(s):  
M L Breitman ◽  
L C Tsui ◽  
M Buchwald ◽  
L Siminovitch
Keyword(s):  
Molecular Weight ◽  
Cell Line ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Dna Molecules ◽  
High Molecular Weight Dna ◽  
Bacterial Gene ◽  
E Coli ◽  
Transformed Cell ◽  
Transformed Cell Lines

The simian virus 40 (SV40)-pBR322 recombinant, pSV2, carrying the origin of SV40 replication and the gpt gene of Escherichia coli, has been stably introduced into Chinese hamster ovary hprt- cells. All gpt-transformed cell lines were found to contain one or more insertions of pSV2 sequences exclusively associated with high-molecular-weight DNA. Additional analyses showed that at least one integrated copy in each cell line retained an intact gpt gene and flanking SV40 sequences required for expression of xanthine-guanine phosphoribosyltransferase. Most cell lines contained pSV2 sequences which had integrated with partial sequence duplication. Upon fusion with COS-1 cells, a simian cell line permissive for autonomous pSV2 replication, most gpt-transformed cell lines produced low-molecular-weight DNA molecules related to pSV2. The majority of these replicating DNAs were indistinguishable from the original transfecting plasmid in both size and restriction enzyme cleavage pattern. In addition, the recovered DNA molecules were able to confer ampicillin resistance to E. coli and to transform mouse L cells and Gpt- E. coli to a Gpt+ phenotype. These studies indicate that all of the genetic information carried by this SV40-plasmid recombinant can be introduced into and retrieved from the genome of mammalian cells.

scholarly journals Regulation of the CDK-related protein kinase PCTAIRE-1 and its possible role in neurite outgrowth in Neuro-2A cells

2002 ◽  
Vol 115 (17) ◽  
pp. 3479-3490 ◽  
Author(s):  
Ralph Graeser ◽  
Julian Gannon ◽  
Randy Y. C. Poon ◽  
Thierry Dubois ◽  
Alastair Aitken ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Neurite Outgrowth ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Neuroblastoma Cell ◽  
Brain Extract ◽  
Related Protein ◽  
Transformed Cell Lines

PCTAIRE-1 is a CDK-related protein kinase found in terminally differentiated cells in brain and testis, and in many immortalised and transformed cell lines. Bacterially expressed PCTAIRE is completely inactive as a protein kinase, but is a very good substrate for protein kinase A (PKA),which phosphorylates a total of four sites in the N-terminus of PCTAIRE-1. Phosphorylation of one of these sites, Ser119, generates a 14-3-3 binding site, which is functional in vitro as well as in vivo. Mutation of another PKA site, Ser153, to an alanine residue generated an activated kinase in transfected mammalian cells. This activity was comparable to that of CDK5 activated by a bacterially expressed, truncated version of p35nck,p21. Gel filtration analysis of a brain extract suggested that monomeric PCTAIRE-1 was the active species, implying that PCTAIRE-1 may not be a true CDK, in that it does not require a partner (cyclin-like) subunit for kinase activity. Finally, we found that various forms of PCTAIRE-1 transfected into neuroblastoma cell lines could either promote or inhibit neurite outgrowth,suggesting a potential role for the PCTAIRE-1 gene product in the control of neurite outgrowth.

scholarly journals Introduction and recovery of a selectable bacterial gene from the genome of mammalian cells.

1982 ◽  
Vol 2 (8) ◽  
pp. 966-976 ◽  
Author(s):  
M L Breitman ◽  
L C Tsui ◽  
M Buchwald ◽  
L Siminovitch
Keyword(s):  
Molecular Weight ◽  
Cell Line ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Dna Molecules ◽  
High Molecular Weight Dna ◽  
Bacterial Gene ◽  
E Coli ◽  
Transformed Cell ◽  
Transformed Cell Lines

The simian virus 40 (SV40)-pBR322 recombinant, pSV2, carrying the origin of SV40 replication and the gpt gene of Escherichia coli, has been stably introduced into Chinese hamster ovary hprt- cells. All gpt-transformed cell lines were found to contain one or more insertions of pSV2 sequences exclusively associated with high-molecular-weight DNA. Additional analyses showed that at least one integrated copy in each cell line retained an intact gpt gene and flanking SV40 sequences required for expression of xanthine-guanine phosphoribosyltransferase. Most cell lines contained pSV2 sequences which had integrated with partial sequence duplication. Upon fusion with COS-1 cells, a simian cell line permissive for autonomous pSV2 replication, most gpt-transformed cell lines produced low-molecular-weight DNA molecules related to pSV2. The majority of these replicating DNAs were indistinguishable from the original transfecting plasmid in both size and restriction enzyme cleavage pattern. In addition, the recovered DNA molecules were able to confer ampicillin resistance to E. coli and to transform mouse L cells and Gpt- E. coli to a Gpt+ phenotype. These studies indicate that all of the genetic information carried by this SV40-plasmid recombinant can be introduced into and retrieved from the genome of mammalian cells.

scholarly journals Differences in Extracellular Vesicle Protein Cargo Are Dependent on Head and Neck Squamous Cell Carcinoma Cell of Origin and Human Papillomavirus Status

Cancers ◽  
2021 ◽  
Vol 13 (15) ◽  
pp. 3714
Author(s):  
Christine Goudsmit ◽  
Felipe da Veiga Leprevost ◽  
Venkatesha Basrur ◽  
Lila Peters ◽  
Alexey Nesvizhskii ◽  
...  
Keyword(s):  
Squamous Cell Carcinoma ◽  
Cell Carcinoma ◽  
Head And Neck ◽  
Cell Lines ◽  
Squamous Cell ◽  
Extracellular Vesicle ◽  
Neck Squamous Cell Carcinoma ◽  
Cytokeratin 19 ◽  
Oral Keratinocytes ◽  
Transformed Cell Lines

To identify potential extracellular vesicle (EV) biomarkers in head and neck squamous cell carcinoma (HNSCC), we evaluated EV protein cargo and whole cell lysates (WCL) from HPV-positive and -negative HNSCC cell lines, as well as normal oral keratinocytes and HPV16-transformed cells. EVs were isolated from serum-depleted, conditioned cell culture media by polyethylene glycol (PEG) precipitation/ultracentrifugation. EV and WCL preparations were analyzed by LC-MS/MS. Candidate proteins detected at significantly higher levels in EV compared with WCL, or compared with EV from normal oral keratinocytes, were identified and confirmed by Wes Simple Western protein analysis. Our findings suggest that these proteins may be potential HNSCC EV markers as proteins that may be (1) selectively included in EV cargo for export from the cell as a strategy for metastasis, tumor cell survival, or modification of tumor microenvironment, or (2) representative of originating cell composition, which may be developed for diagnostic or prognostic use in clinical liquid biopsy applications. This work demonstrates that our method can be used to reliably detect EV proteins from HNSCC, normal keratinocyte, and transformed cell lines. Furthermore, this work has identified HNSCC EV protein candidates for continued evaluation, specifically tenascin-C, HLA-A, E-cadherin, EGFR, EPHA2, and cytokeratin 19.

scholarly journals Plasma Treatment of Fish Cells: The Importance of Defining Cell Culture Conditions in Comparative Studies

2021 ◽  
Vol 11 (6) ◽  
pp. 2534
Author(s):  
Henrike Rebl ◽  
Claudia Bergemann ◽  
Sebastian Rakers ◽  
Barbara Nebe ◽  
Alexander Rebl
Keyword(s):  
Plasma Treatment ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Atmospheric Pressure Plasma ◽  
Fish Farming ◽  
Skin Lesions ◽  
Atmospheric Plasma ◽  
Fish Cell ◽  
Farmed Fish ◽  
Fish Cells

The present study provides the fundamental results for the treatment of marine organisms with cold atmospheric pressure plasma. In farmed fish, skin lesions may occur as a result of intensive fish farming. Cold atmospheric plasma offers promising medical potential in wound healing processes. Since the underlying plasma-mediated mechanisms at the physical and cellular level are yet to be fully understood, we investigated the sensitivity of three fish cell lines to plasma treatment in comparison with mammalian cells. We varied (I) cell density, (II) culture medium, and (III) pyruvate concentration in the medium as experimental parameters. Depending on the experimental setup, the plasma treatment affected the viability of the different cell lines to varying degrees. We conclude that it is mandatory to use similar cell densities and an identical medium, or at least a medium with identical antioxidant capacity, when studying plasma effects on different cell lines. Altogether, fish cells showed a higher sensitivity towards plasma treatment than mammalian cells in most of our setups. These results should increase the understanding of the future treatment of fish.

scholarly journals Poly-L-ornithine-mediated transformation of mammalian cells.

1987 ◽  
Vol 7 (6) ◽  
pp. 2286-2293 ◽  
Author(s):  
V C Bond ◽  
B Wold
Keyword(s):  
Cell Lines ◽  
Mammalian Cells ◽  
High Efficiency ◽  
Protein Product ◽  
Marker Genes ◽  
Recipient Mouse ◽  
L Cells ◽  
Dna And Rna ◽  
Selectable Marker Genes

Poly-L-ornithine has been used to introduce DNA and RNA into mammalian cells in culture. Ornithine-mediated DNA transfer has several interesting and potentially useful properties. The procedure is technically straightforward and is easily applied to either small or large numbers of recipient cells. The efficiency of transformation is high. Under optimal conditions, 1 to 2% of recipient mouse L cells take up and continue to express selectable marker genes. DNA content of transformants can be varied reproducibly, yielding cells with just one or two copies of the new gene under one set of conditions, while under a different set of conditions 25 to 50 copies are acquired. Cotransformation and expression of physically unlinked genes occur at high efficiency under conditions favoring multiple-copy transfer. Polyornithine promotes gene transfer into cell lines other than L cells. These include Friend erythroleukemia cells and NIH 3T3 cells. Both are transformed about 1 order of magnitude more efficiently by this procedure than by standard calcium phosphate products. However, the method does not abolish the large transformation efficiency differences between these cell lines that have been observed previously by other techniques. (vi) mRNA synthesized in vitro was also introduced into cells by this method. The RNA was translated resulting in a transient accumulation of the protein product.

scholarly journals Molecular analysis of ethyl methanesulfonate-induced reversion of a chromosomally integrated mutant shuttle vector gene in mammalian cells.

1988 ◽  
Vol 8 (10) ◽  
pp. 4185-4189 ◽  
Author(s):  
J A Greenspan ◽  
F M Xu ◽  
R L Davidson
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Dna Sequences ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Shuttle Vector ◽  
Ethyl Methanesulfonate ◽  
Wild Type ◽  
Single Base ◽  
Type Sequence

The molecular mechanisms of ethyl methanesulfonate-induced reversion in mammalian cells were studied by using as a target a gpt gene that was integrated chromosomally as part of a shuttle vector. Murine cells containing mutant gpt genes with single base changes were mutagenized with ethyl methanesulfonate, and revertant colonies were isolated. Ethyl methanesulfonate failed to increase the frequency of revertants for cell lines with mutant gpt genes carrying GC----AT transitions or AT----TA transversions, whereas it increased the frequency 50-fold to greater than 800-fold for cell lines with mutant gpt genes carrying AT----GC transitions and for one cell line with a GC----CG transversion. The gpt genes of 15 independent revertants derived from the ethyl methanesulfonate-revertible cell lines were recovered and sequenced. All revertants derived from cell lines with AT----GC transitions had mutated back to the wild-type gpt sequence via GC----AT transitions at their original sites of mutation. Five of six revertants derived from the cell line carrying a gpt gene with a GC----CG transversion had mutated via GC----AT transition at the site of the original mutation or at the adjacent base in the same triplet; these changes generated non-wild-type DNA sequences that code for non-wild-type amino acids that are apparently compatible with xanthine-guanine phosphoribosyltransferase activity. The sixth revertant had mutated via CG----GC transversion back to the wild-type sequence. The results of this study define certain amino acid substitutions in the xanthine-guanine phosphoribosyltransferase polypeptide that are compatible with enzyme activity. These results also establish mutagen-induced reversion analysis as a sensitive and specific assay for mutagenesis in mammalian cells.

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