OVEREXPRESSION OF PKC-EPSILON IN R6 FIBROBLASTS CAUSES INCREASED PRODUCTION OF ACTIVE TGF-BETA

In previous studies, our laboratory demonstrated that Rat 6 (R6) fibro blasts which stably overproduce high levels of PKC epsilon display abn ormalities in growth control that are characteristic of malignant tran sformation (Cacace et al., 1993, Oncogene, 8:2095-2104). The R6-PKC ep silon overproducing cell lines also exhibited a decreased growth facto r requirement. The present study demonstrates that conditioned medium (CM) from two individual clones, R6-PKC epsilon 10 and 30, stimulates DNA synthesis in control R6-C1 cells. Maximal DNA synthesis and morpho logic transformation was achieved in control cells when they were trea ted with medium from R6-PKC epsilon cells grown in the presence of TPA (TPA-CM). Size fractionation of the TPA-CM from PKC epsilon. 30 cells revealed that this activity is due to a factor(s) that has an apparen t molecular weight in the range of 10-30 kD and is heat and acid stabl e. This factor, like TGF beta 1, stimulated anchorage-independent grow th of NRK cells. Western blot analysis (under nonreducing conditions) of the TPA-CM from R6-PKC epsilon 30 and R6-PKC epsilon 10 cells revea led the presence of the 25 kD active forms of TGF beta 2 and 3. These active forms of TCF beta were not found in the CM of control R6 cells, or R6 cells that overexpress PKC alpha or PKC beta 1. The addition of a pan-specific TGF beta antibody to NRK cells treated with the 10-30 kD fraction of TPA-CM from PKC epsilon 30 cells blocked the ability of this material to stimulate thymidine incorporation. Taken together, t hese studies suggest that the oncogenic activity of PKC epsilon in R6 cells is due, at least in part, to its ability to induce production of the active forms of TGF beta 2 and 3. (C) 1998 Wiley-Liss, Inc.