5 or 15 5 gestation days Importantly, the age of the embryos see

5 or 15.5 gestation days. Importantly, the age of the embryos seems to have an effect on the properties of the cells [30]. In the present work we investigated the effect of the cellular microenvironment in young vs old RECs in response to combined treatment with FPTase inhibitors and CDK inhibitors. Material VX-680 supplier and Methods Plasmids pLTRp53cGval135, comprising a chimera of mouse p53 cDNA and genomic DNA (generous gift of Dr. M. Oren), has been previously referred to as pLTRp53cG [6]. It encodes a mutant protein harboring a substitution from alanine to valine

at the amino acid in position 135. The plasmids pVV2, bearing the neomycin resistance sequence, and pVEJB coding for a mutated human c-Ha-Ras gene cloned into pVVJ were used. Cell Clones PD0332991 clinical trial The transformed rat cell clones were established as previously described in detail [40] using primary Fisher rat embryo cells (RECs). RECs were obtained from embryos isolated at 13.5 (y) and 15.5 (o) gestation days. Cells were grown at basal temperature (37°C) in DMEM supplemented with 10% FCS in an atmosphere of 7.5% CO2. For experiments dealing with a change of the conformational state of p53 protein, cells grown at basal temperature,

were shifted to 32°C for indicated periods of time. Drugs Olomoucine (OLO) and roscovitine (ROSC) were prepared as 50 mM stock solution in DMSO according to the published procedure [14]. Aliquots of the stock solution were stored until use at -20°C. Furthermore, L-744,832 [(2 S)-2-[[(2 S)-2-[(2 S,3 S)-2-[(2R)-2-amino-3-mercaptopropyl]amino]-3-methylpentyl]oxy]-1-oxo-3-phenylpropyl]amino]-4-(methylsulfonyl)-butanoic acid 1-methylethyl ester ] an inhibitor of protein farnesyltransferase (FTI) from Alexis Biochemicals (Lausen, Switzerland) was used. The stock solution of L-774,832

was prepared in DMSO. Aliquots of stock solutions were protected from light and stored until use at -20°C. Cell Treatment After plating, cells were cultivated at a basal temperature of 37°C for 24 h. Then drugs were added to a final concentration as indicated. Quisqualic acid Cells were incubated continuously for 24 h or 48 h, or alternatively, after 24 h treatment medium was changed and cells were post-incubated (p. i.) in a drug-free medium for a further 24 h or 48 h. In some experiments cells were shifted to 32°C and kept there for at least 24 h prior to the onset of treatment to allow p53 to adopt wt conformation. Determination of Population Doubling Time To determine the kinetics of the proliferation of distinct cell clones, cells were plated into PD of 6 cm diameter. For each time point two PDs were used. Immortalized cells were plated at a medium density (2 × 105) and transformed cells at a lower density (0.5 × 105/PD). Cells were cultivated at a basal temperature for 5 days. PDs were collected in 12 h intervals, suspended in a defined volume of medium and were counted in a cell counter (CASY). Cell number was CBL0137 in vitro determined in at least two distinct aliquots of cell suspension collected from each PD.

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