Aflatoxin-transformed C3H/10T1/2 cells overexpress protein kinase C and have an altered response to phorbol ester treatments.

Abstract

The aflatoxin B1-transformed C3H/10T1/2 (10T1/2) cell line 7SA has disordered growth in culture and is tumorigenic in syngeneic mice. Chronic exposure (14 days) of 10T1/2 and 7SA cells to phorbol 12,13-dibutyrate (PDBu) increased the saturation density of 10T1/2 cells but dramatically slowed the entry of 7SA cells into the log phase of growth without affecting their final saturation density. Similar PDBu treatment of low-density cultures dramatically decreased the size of 7SA colonies. Both cell lines bound [3H]PDBu in a specific and saturable manner. Analysis of this binding yielded linear Scatchard plots for both cell lines with distinctly different Kd values (10.7 nM for 10T1/2 versus 54.5 nM for 7SA). The total amount of [3H]PDBu bound was 2-fold greater in the 7SA cells versus the 10T1/2 cell line. Both cell lines released arachidonic acid following a 2-h exposure to PDBu; however, the magnitude of the response of the 7SA cells was only one-half that of the 10T1/2 cells. Western blot analysis of protein kinase C (PKC) using specific anti-PKC antibodies revealed a greater total amount of PKC alpha in the 7SA cells relative to an equal number of 10T1/2 cells. No immunoreactive PKC alpha was found in either cell line 16 h after exposure to 600 nM PDBu; however, PKC alpha returned to control levels in both cell lines 24 h after removal of the phorbol ester. These results suggest that an overexpression of PKC alpha may play a role in the altered biological properties of aflatoxin-transformed 10T1/2 cells.