Fibroblast-serum data

Circadian oscillation profiles of clock genes are induced in several mammalian peripheral culture cells by serum shock (Balsalobre et al 1998). To elucidate whether serum induces the circadian expression of human clock genes in normal human diploid fibroblasts, we applied RT-PCR ELISA methods to detect RNA levels of clock genes in WI-38 cells after serum stimulation (Fig. 3). Since WI-38 cells in culture invariably undergo senescence after a finite number of doublings, we selected young WI-38 cells. The RT-PCR-ELISA data are expressed as amounts (Moles) of corresponding cDNA plasmids in... 

Levels of HA that cells deposit must respond to various physiological states including growth phase,239 confluence, inversely related to cell density in both fibroblasts,240 and keratinocytes,241 mitosis and cell detachment from the substratum,242 calcium concentrations,149,243 anoxia and lactate,244 viral transformation,245 and serum stimulation.91,246 Preliminary immunolocalization data indicate that some of the HAS and hyaluronidases colocalize (A. Spicer et al., pers. commun.). All of this evidence supports, albeit indirect and tentative, the existence of the hyaluronasome structure. 

This subsection outlines the nMDS analysis of the microarray data on the gene transcriptional response of cell cycle-synchronized human fibroblasts to serum [21]. The dissimilarities are calculated as in the previous subsection. The data have been extensively analyzed with the aid of cluster analyses [21]. In contrast to the cluster analysis, for this example, nMDS clearly captures the time-dependence of the gene expression levels as if time correlation functions are explicitly analyzed (Fig. 26). 

FIGURE 18.2 Example of bridging from the gene expression data set of Iyer et al. (93) on the response of fibroblasts to serum. The qualitatively dissimilar patterns in (A) and (F) can be connected by intermediate patterns present in the same data set. 

Fig. IB. The effect of SAB on the repair kinetics of potentially lethal damage in normal human fibroblasts, Ewing s sarcoma, and human lung adenocarcinoma. No inhibitor (open circles) 8SAB (closed circles). Data points are means 1 standard deviation. Both normal fibroblast and human tumor cell cultures were grown and maintained in Eagle s MEM supplemented with 10% (VA ) fetal bovine serum, streptomycin and penicillin, at S7°C in a humidified chamber of 95% air and 5% CO2. Stock cultures in exponential growth were detached by trypsinization and appropriate cell numbers plated in 25 cm flasks and incubated to permit cell attachment. Inhibitors of poly(ADP-ribose) synthetase (SAB or BZ) were added to the appropriate cultures 2 hr prior to irradiation in air at room temperature. Following irradiation, the cells were exposed to the inhibitors for 24 hr, washed, and replenished with fresh medium. Refeeding was performed every S days and after 10-14 days, cells were fixed and the colonies stained with Giemsa. Only colonies of 50 cells or more were scored as survivors. All experiments were performed in triplicate and the survival data expressed as the mean S.E. of three separate survival experiments.

The XPS analysis of fibroblasts, epithelial cells, and smooth muscle cells washed in the absence or in the presence of serum proteins showed that they adsorb hardly any serum proteins in contrast with many materials mentioned in this section and in the section Data Interpretation and Evaluation To our knowledge, no recent studies of the surface of mammalian cells by XPS were reported. 

Fig. 9. The effect of P5C on PP-ribose-P levels in quiescent fibroblasts. Data from unpublished work in progress, Serum-starved quiescent human fibroblasts incubated in Eagle s MEM were treated with P5C (0.5 mM), 10% dialyzed fetal bovine serum, or both. After varying durations of treatment, the medium was removed and the monolayer was incubated for 45 minutes in Krebs-Ringer phosphate buffer with 5.5 mM glucose. Cells were then extracted with Tris-EDTA (Tris, 10 mM, pH 7.4 EDTA, 1 mM) heated to 100°C. PP-ribose-P levels in incubated control (O), P5C-treated ( ), serum-treated (A), and serum plus P5C-treated (A) cells are shown.

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