Chapter 3 Density Differential Responses of Normal Versus Transformed 10T½ Murine Embryonic Fibroblasts

Summary

In optimized bioassays, a balance of favorable cell-cell interactions, per-volume nutrient availability and per-volume waste accumulation is achieved. Both normal and transformed 10T½ murine embryonic fibroblasts displayed density-dependent generation time shifts, with optimal doubling efficiency observed at 1000 cells/milliliter/2.0 cm2 surface area. In normal 10T½ fibroblasts, TGF-β1 stimulated DNA synthesis as monitored by tritiated thymidine incorporation. In methylcholanthrene-transformed fibroblasts, however, TGF-β1 reduced an already-high basal level of DNA synthesis. These results suggest that even as transformed cells displayed an altered response to TGF-β1 when compared with normal fibroblasts, the mechanisms governing cell-cell interaction remained intact. The contribution of intercellular interactions to general population fitness based on generation time reflected the importance of considering population dynamics in designing and validating bioassays.

Introduction

In optimized bioassays, a balance of favorable cell-cell interactions, per-volume nutrient availability and per-volume waste accumulation is achieved. Both normal and transformed 10T½ murine embryonic fibroblasts displayed density-dependent generation time shifts, with optimal doubling efficiency observed at 1000 cells/milliliter/2.0 cm2 surface area. In normal 10T½ fibroblasts, TGF-β1 stimulated DNA synthesis as monitored by tritiated thymidine incorporation. In methylcholanthrene-transformed fibroblasts, however, TGF-β1 reduced an already-high basal level of DNA synthesis. These results suggest that even as transformed cells displayed an altered response to TGF-β1 when compared with normal fibroblasts, the mechanisms governing cell-cell interaction remained intact. The contribution of intercellular interactions to general population fitness based on generation time reflected the importance of considering population dynamics in designing and validating bioassays.

The advent of tissue culture has been critical in advancing studies of cellular physiology and in vitro biochemistry. Cell culture techniques eliminate the need for conducting many biological studies in animal model systems to preclude legal and ethical dilemmas inherent for in vivo models. In addition, in vitro techniques enabled control of environmental parameters. Sample homogeneity may therefore be achieved from the natural selection of fit cells (Freshney, 1994).

Optimizing and validating a cell culture system is critical for investigating pathways underlying biological processes, including carcinogenesis, toxicology, and proliferation. Bioassay validation requires rigorous maintenance of the cell culture protocol, including media selection, trypsinization method, cryostorage conditions, incubation parameters, and seeding density.

Normal cell populations require cell-cell contact. This requirement correlates with an observed optimal growth rate within the cellular population, arising from the secretion and reception of growth factors by neighboring cells via a paracrine or juxtacrine mechanism. In addition, autocrine-regulated feedback programs by individual cells influence the efficiency of paracrine and juxtacrine mechanisms between cells within the population.

Shifts in generation time of the total cell population reflect environmental suitability for growth of the individual cell. Consistent plating density and feeding schedule should therefore not be discounted and is, in fact, critical for maintaining fidelity and authenticity of cellular responses to extracellular signals in cell-based assays. Inconsistent culture program may render spurious conclusions. Changes in cell cycle kinetics under variable culturing methods may be due to shifts in generation time leading to an altered response rather than reflecting a true correlation to growth factor signaling.

The choice of cell line in which biological mechanisms are investigated is important in the rational design of bioassays. The C3H-10T½ cell line used in these studies were derived from selective cloning of the C3H Heston inbred mice. These murine embryonic fibroblasts were subjected to a modified 3T3-passaging method (Reznikoff et al, 1973a). To align with the cell passaging designation established for 3T3 fibroblasts, the cell culture designation of C3H-10T½ fibroblasts delineated a ten-day passaging program where 0.5 * 105 cells were seeded into 60-mm dishes during continuous culture (Reznikoff et al, 1973a).

C3H-10T½ murine fibroblasts are versatile in vitro cell systems and have been used as experimental models of normal and transformed pathways. The non-spontaneously transforming phenotype of C3H-10T½ fibroblasts is key in experimental reproducibility and signaling fidelity. Moreover, the ease of inducible transformation in this cell line allows diverse studies spanning chemical carcinogenesis and toxicology (Poole et al, 1983), hyperoxia-affected cellular physiology (Djurhuus et al, 1998), and ionizing radiation studies on cell cycle checkpoints (Crompton et al, 1998). Here, we examined the optimal seeding density for C3H-10T½ embryonic fibroblasts and a polycyclic hydrocarbon methylcholanthrene-transformant, MCA-10T½. In this model system, culture parameters including selection of media, incubation conditions, feeding schedule, and trypsinization method were kept constant. Only initial plating densities were varied. Changes in generation time were investigated among three plating densities to determine the optimal seeding density in bioassays utilizing this fibroblast model.

Materials and Methods

Cell Culture. Cells were cultured according to Materials and Methods, Chapter 2. MCA-10T½ fibroblasts were observed to have lost the property of contact inhibition of growth (Reznikoff et al, 1973b).

Calculation of Generation Time. Generation time was calculated according to Materials and Methods, Chapter 2.

Results and Discussion

The effects of differing seeding densities on the generation time of murine embryonic fibroblasts were examined. Normal or transformed fibroblasts were cultured from an initial density of 680, 1000, or 5000 cells per 2.0 cm2 growth area. Cells were collected every 24 hours and total cell number tabulated via Coulter particle-size counting. Generation time was calculated according to Materials and Methods.

Normal fibroblasts plated at 5000 per 2.0 cm2 exhibited a longer generation time when compared with cells plated at 1000 per 2.0 cm2. Figure 3-1. Thus, an increase in generation time correlated with a decrease in doubling efficiency. Methylcholanthrene-transformed fibroblasts also exhibited a similar albeit less-exaggerated density-dependent shift in generation time, Figure 3-2. In the transformed state, an apparent loss of sensitivity in density-dependent generation time-shift suggested a loss of requirement for cell-cell contact.

Phenotypic differences between normal and transformed 10T½ fibroblasts included morphology, sensitivity to contact inhibition, and ability to form overlapping cellular clusters or foci. Transformed cells also responded differently to extracellular signals when compared with normal cells. Given that in normal 10T½ fibroblasts, TGF-β1 was a potent mitogen, the effect of TGF-β1 on DNA synthesis in both normal and methylcholanthrene-transformed fibroblasts were examined. C3H- or MCA-10T½ fibroblasts were grown to confluence and serum-starved overnight before treating with TGF-β1 for 48 hours. Intracellular total thymidine pools and DNA synthesis were monitored with tritiated thymidine (3H-Thd). TGF-β1 stimulated thymidine uptake in both normal and transformed fibroblasts, although the basal level of thymidine uptake in transformed cells was higher than those observed in normal cells, Figure 3-3A. TGF-β1 stimulated DNA synthesis in normal fibroblasts, Figure 3-3B, whereas cells receiving no growth factor (control) exhibited low levels of thymidine incorporation. Transformed fibroblasts, on the other hand, maintained high basal DNA synthesis that was reduced under TGF-β1 treatment. The MCA-derivatives of 10T½ fibroblasts therefore responded differently to TGF-β1 than did the C3H counterparts.

Taken together, these results suggest that under chemical transformation where growth factor response for DNA synthesis was altered, density-dependence of population generation time remained intact.

Previous investigations in fibroblast transformations in the C3H cell line had suggested the importance of plating density on the final phenotypic outcome of the transformed cells. When benzopyrene-transformed fibroblasts were seeded at high density, foci formation was suppressed (Haber et al, 1977). Cell-cell communication may therefore play an important role in mediating transformation. Early transformation studies in C3H fibroblasts with methylcholanthrene (MCA) also demonstrated a seeding density-dependence on transformation frequency, where the number of malignantly transformed foci decreased with increasing cell density and decreasing volume of medium per cell (Reznikoff et al, 1973b). The location of foci formation also supported density dependence of transformation: foci were found to increase at the outer rims of the plate where cells were sparse, suggesting the inhibition of transformation by dense populations of normal cells towards the center of the culture environment (Reznikoff et al, 1973b).

Early investigations in population dynamics in 10T½ fibroblasts showed in transformed cells, certain wild-type growth control mechanisms remained intact and that as cell density increased under serum stimulation, intercellular communication at the membrane level may suppress the transformed phenotype (Bertram, 1977). Serum therefore had a suppressive effect on density-dependent transformation in MCA-10T½ fibroblasts. We demonstrated here that TGF-β1 potently stimulated DNA synthesis in the C3H-phenotype but reduced DNA synthesis in the transformed phenotype. Methylcholanthrene transformed fibroblasts may be secreting growth factors, including TGF-β1, which act in an autocrine fashion to stimulate growth in the basal state without exogenously added growth factors. A threshold in cellular crowding may create an opposing force to contain the local cell population. Foci formation would therefore be evident of an upset of this balance.

In mesenchymal cells such as C3H fibroblasts, TGF-β1 stimulated cell cycle progression while in epithelial cells, TGF-β1 potently inhibited cell cycle progression. Examination of cellular morphology during carcinogenesis showed TGF-β1-resistant epithelial cells as assuming a fibroblast-like morphology. Changes in intercellular communication may prove relevant to the enigmatic switch between TGF-β1 effects in cells of mesenchymal and epithelial origin. In dissecting the biochemical pathways underlying this switch in response, therefore, both alterations in signaling pathways as well as changes in spatial cellular assignment must be considered as contributing factors.

Co-culture experiments with endothelial cells and 10T½ fibroblasts demonstrated a specific involvement cell-cell contact in mediating mutual growth inhibition of endothelial cells and fibroblasts. Conditioned medium from contacting co-cultures also inhibited DNA synthesis of both cell types in a mechanism that was not governed by TGF-β1, PDGF-AA, or bFGF (Hirschi et al, 1999). Endothelial cells in this co-culture model recruited mesenchymal cells to differentiate to a pericyte lineage. Since endothelial cells comprise blood vessel structures, the basis of heterotypic cellular recruitment and induction of differentiation underlies vascular pathogenesis.

Obtaining reproducible and relevant results from in vitro bioassays demand careful maintenance of the cellular growth state between new cell passages so that observed responses to experimental manipulation would remain consistent. In designing and validating bioassays, in vitro parameters considered in experimental design must extend beyond characterizing responses to extracellular signals. In addition to establishing a consistent cell culturing protocol to maintain the characteristic phenotype for a cell line, mechanical population requirements such as cell seeding density should be optimized for the model system. Cells within the plated population must achieve balance between critical cell-cell contact and nutrient limitation or waste product accumulation inherent within the population. The crowdedness within a cell population determined in part the general fitness of the population based on the availability of shared nutrients, efficiency of gas exchange, and accumulation of toxic waste products.

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