Various factors are known to regulate cell growth and differentiation in epithelial-mesenchymal interactions. Keratinocyte growth factor (KGF), an epithelial-specific cytokine produced by dermal fibroblasts and other mesenchymal cells, appears to affect growth, migration, and differentiation in epithelial-mesenchymal interactions. We have previously shown that human embryonic skin fibroblasts induce anchorage-independent growth of HPV16 DNA-immortalized human uterine exocervical epithelial cells (HCE16/3 cell line) in cocultures of HCE16/3 cells and fibroblasts. Here we report that KGF may be a major factor influencing growth and behavior of HCE16/3 cells in the coculture system. KGF stimulated both DNA synthesis and proliferation of normal human cervical epithelial (HCE) cells and HCE16/3 cells and the increase was stronger in HCE16/3 cells than in HCE cells. SiHa cells, a cervical carcinoma cell line with integrated HPV16 DNA, did not respond to the KGF mitogen signal. KGF receptor (KGFR) studies suggested that the different responses to the KGF mitogen signal may be correlated with KGFR. In addition, KGF alone was able to induce anchorage-independent growth of HCE16/3 cells, suggesting a potential role for KGF in the transformation process of epithelial cells. However, the transcription of HPV16 early genes was suppressed by KGF in the immortalized HCE16/3 cells, and this appeared to be due to transcriptional repression rather than a posttranscriptional process according to nuclear run-on analysis. In contrast, viral gene expression was not affected by KGF in SiHa cells. Our results suggest that KGF is a bifunctional growth factor in the HPV-immortalized cells, a positive regulator of cell growth and negative regulator of HPV16 early gene expression.

Neurons were grown on plastic surfaces that were untreated, or treated with polylysine, laminin, or L1 and their growth cones were detached from their culture surface by applying known forces with calibrated glass needles. This detachment force was taken as a measure of the force of adhesion of the growth cone. We find that on all surfaces, lamellipodial growth cones require significantly greater detachment force than filopodial growth cones, but this differences is, in general, due to the greater area of lamellipodial growth cones compared to filopodial growth cones. That is, the stress (force/unit area) required for detachment was similar for growth cones of lamellipodial and filopodial morphology on all surfaces, with the exception of lamellipodial growth cones on L1-treated surfaces, which had a significantly lower stress of detachment than on other surfaces. Surprisingly, the forces required for detachment (760-3,340 mudynes) were three to 15 times greater than the typical resting axonal tension, the force exerted by advancing growth cones, or the forces of retraction previously measured by essentially the same method. Nor did we observe significant differences in detachment force among growth cones of similar morphology on different culture surfaces, with the exception of lamellipodial growth cones on L1-treated surfaces. These data argue against the differential adhesion mechanism for growth cone guidance preferences in culture. Our micromanipulations revealed that the most mechanically resistant regions of growth cone attachment were confined to quite small regions typically located at the ends of filopodia and lamellipodia. Detached growth cones remained connected to the substratum at these regions by highly elastic retraction fibers. The closeness of contact of growth cones to the substratum as revealed by interference reflection microscopy (IRM) did not correlate with our mechanical measurements of adhesion, suggesting that IRM cannot be used as a reliable estimator of growth cone adhesion.

We have examined the relationship between tension, an intrinsic stimulator of axonal elongation, and the culture substrate, an extrinsic regulator of axonal elongation. Chick sensory neurons were cultured on three substrata: (a) plain tissue culture plastic; (b) plastic treated with collagen type IV; and (c) plastic treated with laminin. Calibrated glass needles were used to increase the tension loads on growing neurites. We found that growth cones on all substrata failed to detach when subjected to two to threefold and in some cases 5-10-fold greater tensions than their self-imposed rest tension. We conclude that adhesion to the substrate does not limit the tension exerted by growth cones. These data argue against a "tug-of-war" model for substrate-mediated guidance of growth cones. Neurite elongation was experimentally induced by towing neurites with a force-calibrated glass needle. On all substrata, towed elongation rate was proportional to applied tension above a threshold tension. The proportionality between elongation rate and tension can be regarded as the growth sensitivity of the neurite to tension, i.e., its growth rate per unit tension. On this basis, towed growth on all substrata can be described by the simple linear equation: elongation rate = sensitivity x (applied tension - tension threshold) The numerical values of tension thresholds and neurite sensitivities varied widely among different neurites. On all substrata, thresholds varied from near zero to greater than 200 mudynes, with some tendency for thresholds to cluster between 100 and 150 mudynes. Similarly, the tension sensitivity of neurites varied between 0.5 and 5.0 microns/h/mudyne. The lack of significant differences among sensitivity or threshold values on the various substrata suggest to use that the substratum does not affect the internal "set points" of the neurite for its response to tension. The growth cone of chick sensory neurons is known to pull on its neurite. The simplest cytomechanical model would assume that both growth cone-mediated elongation and towed growth are identical as far as tension input and elongation rate are concerned. We used the equation above and mean values for thresholds and sensitivity from towing experiments to predict the mean growth cone-mediated elongation rate based on mean rest tensions. These predictions are consistent with the observed mean values.