Nonquiescent Cells Research Articles

Continuous maintenance of transformed fibroblasts under reduced serum conditions: utility as a model system for investigating growth factor-specific effects in nonquiescent cells.

We report the continuous growth maintenance of untransformed and chemically transformed fibroblasts (AKR-2B, AKR-MCA cells) in low concentrations of serum (0.1% FBS). The cell lines established (AKR-0.1F, MCA-0.1F) proliferated at rates comparable to cells maintained under high serum conditions (10% FBS). Complete removal of serum from the cells did not induce quiescence. The MCA-0.1F cells were more similar to the untransformed AKR-2B fibroblasts in their morphology, saturation density, inability to form colonies under anchorage-independent conditions, steady-state level of c-myc expression, and kinetics of induction of c-myc in response to specific growth factors. This report demonstrates the utility of this cell line as a nonquiescent model system for investigating growth factor-specific effects in serum-free, cycling cells. Addition of transforming growth factor-beta (TGF-beta) (5 ng/ml) to proliferating MCA-0.1F cells, in the absence of any serum, induced a multilayered growth pattern at confluency, similar to that of AKR-MCA cells maintained in 10% FBS. Other growth factors tested did not elicit this effect. The induction of this growth pattern by TGF-beta was associated with a sustained induction of the c-myc proto-oncogene at confluency, but not with a restoration of anchorage-independent growth. The data suggest that TGF-beta may play a role in the up-regulation of c-myc at confluency previously described for AKR-MCA cells maintained in 10% serum.

Fluorescence ratio imaging microscopy: temporal and spatial measurements of cytoplasmic pH.

Fluorescence ratio imaging microscopy (Tanasugarn, L., P. McNeil, G. Reynolds, and D. L. Taylor, 1984, J. Cell Biol., 98:717-724) has been used to measure the spatial variations in cytoplasmic pH of individual quiescent and nonquiescent Swiss 3T3 cells. Fundamental issues of ratio imaging that permit precise and accurate temporal and spatial measurements have been addressed including: excitation light levels, lamp operation, intracellular probe concentrations, methods of threshold selection, photobleaching, and spatial signal-to-noise ratio measurements. Subcellular measurements can be measured accurately (less than 3% coefficient of variation) in an area of 3.65 microns 2 with the present imaging system. Quiescent Swiss 3T3 cells have a measured cytoplasmic pH of 7.09 (0.01 SEM), whereas nonquiescent cells have a pH of 7.35 (0.01 SEM) in the presence of bicarbonate buffer. A unimodal distribution of mean cytoplasmic pH in both quiescent and nonquiescent cells was identified from populations of cells measured on a cell by cell basis. Therefore, unlike earlier studies based on cell population averages, it can be stated that cells in each population exhibit a narrow range of cytoplasmic pH. However, the mean cytoplasmic pH can change based on the physiological state of the cells. In addition, there appears to be little, if any, spatial variation in cytoplasmic pH in either quiescent or nonquiescent Swiss 3T3 cells. The pH within the nucleus was always the same as the surrounding cytoplasm. These values will serve as a reference point for investigating the role of temporal and spatial variations in cytoplasmic pH in a variety of cellular processes including growth control and cell movement.

Multiple hormones stimulate the production of somatomedin by cultured human fibroblasts.

Although immunoreactive somatomedin (IR-SM) secretion by cultured human fibroblasts has been reported, the hormonal control of IR-SM production is not well defined. This study concerns the effects of several hormones and growth factors on IR-SM production by cultured human fibroblasts. Platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) had concentration-dependent stimulatory effects on IR-SM production when added to confluent quiescent cultures. When PDGF, FGF, macrophage growth factor (MGF), or epidermal growth factor (EGF) was added transiently for 5 h and the cells subsequently incubated in SM-C deficient, platelet-poor plasma (SM-C-deficient PPP), persistent stimulation of IR-SM production occurred. In contrast, transient exposure to other known stimuli of IR-SM production, such as human GH, T4, and insulin, resulted in no stimulation. Continuous exposure of quiescent, high density fibroblast cultures to EGF, insulin, hydrocortisone, or T4 incubated in the presence of serum-free medium alone resulted in no significant stimulation. During simultaneous incubation with PDGF and SM-C-deficient PPP, however, hydrocortisone, T4, EGF, and insulin produced concentration-dependent increases in IR-SM production. After transient exposure to PDGF, only hydrocortisone and T4 were stimulatory in the presence of SM-C-deficient PPP. We conclude that competence factors, such as PDGF and FGF, are potent stimuli of IR-SM production in the presence of serum-free medium, and that this hormonal signal can be remembered by the cell even after withdrawal of the growth factor. In contrast, progression factors, such as hydrocortisone, T4, and insulin, are effective only in nonquiescent cells. EGF and insulin require simultaneous incubation with PDGF. The PDGF-stimulated cell, therefore, responded differently to hormonal stimuli of IR-SM production than did the quiescent cell.

Variations in growth and cell cycle time of arterial smooth muscle cells cultured at low density.

The population kinetics of cultured rat arterial smooth muscle cells replated at various low densities were studied by direct counting and observation of the cells. Population doubling time decreases with increasing initial density of the culture. These variations in population doubling times depend on both the variation in the percentage of quiescent cells and on the variation of the mean cell cycle time of non-quiescent cells.