Expression of oncomodulin does not lead to the transformation or immortalization of mammalian cells in vitro

1989 ◽  
Vol 94 (3) ◽  
pp. 517-525
Author(s):  
A.M. Mes-Masson ◽  
S. Masson ◽  
D. Banville ◽  
L. Chalifour
Keyword(s):  
Mammalian Cells ◽  
Mouse Kidney ◽  
Kidney Cells ◽  
T Antigens ◽  
Primary Mouse ◽  
Specific Antisera ◽  
Growth Properties ◽  
Metallothionein Promoter ◽  
Specific Mrna

A recombinant plasmid (pMTONCO) containing the coding sequences for rat oncomodulin under the direction of the metallothionein promoter was constructed. pMTONCO was co-transfected with the pSV2-NEO plasmid into primary mouse kidney cells or Rat-1 cells using the calcium phosphate technique and stable transformants were isolated after selection with G418. Transcription from the metallothionein promoter was inducible with heavy metals and produced an oncomodulin-specific mRNA. The presence of oncomodulin protein in stable cell lines was verified by immunoprecipitation with specific antisera. While a plasmid encoding the polyomavirus T-antigens was able to prolong the life-span of primary mouse kidney cells in culture, no equivalent activity was noted when the pMTONCO plasmid was used to transfect primary cells. When expressed in Rat-1 cells, oncomodulin did not affect the growth properties of these cells, nor did it predispose cells to higher frequencies of oncogenic transformation to a viral oncogene. We conclude that oncomodulin is neither an immortalizing nor transforming agent in vitro.

scholarly journals Replication Past the -Radiation-Induced Guanine-Thymine Cross-Link G[8,5-Me]T by Human and Yeast DNA Polymerase

2010 ◽  
Vol 2010 ◽  
pp. 1-10 ◽  
Author(s):  
Paromita Raychaudhury ◽  
Ashis K. Basu
Keyword(s):  
Dna Polymerase ◽  
Mammalian Cells ◽  
Translesion Synthesis ◽  
Kidney Cells ◽  
Equal Frequency ◽  
Cross Link ◽  
Dominant Mutation ◽  
Human Embryonic Kidney Cells ◽  
Radiation Induced

-Radiation-induced intrastrand guanine-thymine cross-link, G[8,5-Me]T, hinders replicationin vitroand is mutagenic in mammalian cells. Herein we reportin vitrotranslesion synthesis of G[8,5-Me]T by human and yeast DNA polymerase (hPol and yPol ). dAMP misincorporation opposite the cross-linked G by yPol was preferred over correct incorporation of dCMP, but further extension was 100-fold less efficient for :A compared to :C. For hPol , both incorporation and extension were more efficient with the correct nucleotides. To evaluate translesion synthesis in the presence of all four dNTPs, we have developed a plasmid-based DNA sequencing assay, which showed that yPol was more error-prone. Mutational frequencies of yPol and hPol were 36% and 14%, respectively. Targeted was the dominant mutation by both DNA polymerases. But yPol induced targeted in 23% frequency relative to 4% by hPol . For yPol , targeted and constituted 83% of the mutations. By contrast, with hPol , semi-targeted mutations (7.2%), that is, mutations at bases near the lesion, occurred at equal frequency as the targeted mutations (6.9%). The kind of mutations detected with hPol showed significant similarities with the mutational spectrum of G[8,5-Me]T in human embryonic kidney cells.

A Scanning Electron-Microscope Study of the Surface Features of Mammalian Cells In Vitro

1972 ◽  
Vol 11 (1) ◽  
pp. 233-247
Author(s):  
GISELE M. HODGES ◽  
MARJORIE D. MUIR
Keyword(s):  
Cell Surface ◽  
Mammalian Cells ◽  
Physiological State ◽  
Culture Conditions ◽  
Kidney Cells ◽  
Scanning Electron Microscope Study ◽  
Cell Strains ◽  
Scanning Electron ◽  
Morphology Of Surface

The cell surface aspects and the density, distribution and morphology of surface cytoplasmic projections (microprocesses) of HeLa, BHK and baby mouse kidney cells, maintained under different culture conditions, have been compared using scanning electron microscopy. No significant differences were noted in the surface aspects of these cell strains. Comparison of the cell strains, under identical culture conditions, showed variations from one strain to another in the density and morphology of the cell surface microprocesses which may reflect basic physiological differences between the strains. For a given cell strain, variations were observed in response to changes in culture conditions indicative that the presence of surface microprocesses is related to the physiological state of the cell.

The Localization of Trypsin in Cultured Mammalian Cells

1973 ◽  
Vol 12 (3) ◽  
pp. 887-902
Author(s):  
GISELE M. HODGES ◽  
D. C. LIVINGSTON ◽  
L. M. FRANKS
Keyword(s):  
Mammalian Cells ◽  
Cultured Cells ◽  
Intracellular Localization ◽  
Mouse Kidney ◽  
Kidney Cells ◽  
Intracellular Uptake ◽  
Primary Event ◽  
Temperature Dependent ◽  
Surface Localization ◽  
Cell Surface Localization

The localization of trypsin in HeLa and CBM17 baby mouse kidney cells was studied using fluorescent and electron-microscope autoradiographical techniques. Intracellular uptake of trypsin, as well as cell surface localization, was demonstrated by the use of direct FITC- and tritium acetylated-labelled trypsin and immunoreactive procedures. Intracellular penetration of the enzyme was temperature dependent and while evident at 37 and 25°C was negligible at 4°C. Higher proteolytic activity could be demonstrated in the supernatants from disrupted trypsin-treated cells than in supernatants from disrupted PBS-treated cultures. Treatment of trypsin with serum, whilst depressing enzyme protease activity, did not modify intracellular uptake of the enzyme and intracellular localization of trypsin persisted in cultured cells for up to about 48 h. The results, while not accounting for the primary event in cell alteration following exposure to trypsin, clearly suggest that consideration must be given to the fact that intracellular penetration of the enzyme may affect certain intrinsic processes of the cell.

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