EXPRESSION OF TISSUE NONSPECIFIC ALKALINE-PHOSPHATASE STIMULATES DIFFERENTIATED BEHAVIOR IN SPECIFIC TRANSFORMED-CELL POPULATIONS

Background: Tissue non-specific alkaline phosphatase (TN-AP) is a memb rane-bound glycoprotein enzyme which is characterized by its phosphohy drolytic activity. This enzyme is distributed virtually in all mammali an tissues during embryonic development (it can be demonstrated as ear ly as the 2-cell stage) where its expression is stage specific. The ex pression of TN-AP is frequently associated with cell differentiation a nd as such it has been used as a marker for this process. By employing a stable gene transfer and forced gene expression technique, previous findings suggested that TN-AP expression might influence cellular pro liferation and morphological differentiation. The focus of this study was to determine whether this was a cell-specific effect or not. Metho ds: The effects of TN-AP on various aspects of cellular activity were assessed by transferring and expressing the gene for this enzyme into three target populations; 1) CHO, 2) R1610, and 3) Rat-2. The paramete rs of cellular activity studied included cellular proliferation, cell cycle, cell migration, and tumorigenic potential (in the nude mouse). Cell cycle and cell proliferation analyses were accomplished, in part, through the use of fluorescence activated cell sorting (FAGS) as were determinations of cell-associated TN-AP activity and amount. Northern and southern blot analyses were used to estimate gene copy number and to evaluate gene expression respectively in transfected cell lines. R esults: Our data indicate that the TN-AP gene under control of various gene promoters was stably integrated into three fibroblast-like cell lines (CHO, R1610, and Rat-2). TN-AP activity and TN-AP protein levels were correlated to the strength of the various gene promoters, but no t to inserted gene copy numbers. The expression of the TN-AP gene in t hese three cell types further suggests cell-specific effects as demons trated by the following findings. The expression of TN-AP under contro l of a weak gene promoter in CHO and Rat-2 cells clearly decreased cel l proliferation and cell migration. However, the expression of TN-AP u nder control of either a weak or even a strong gene promoter in R1610 cells did not induce any changes in that cell line's behaviour (apart from expression of TN-AP). Such changes in CHO cells were also associa ted with a 2.2-fold increase in tubulin transcription as well as suppr essed tumorigenic potential. Conclusion: Taken together, these finding s suggest that the inhibitory effects of TN-AP expression on prolifera tion and cell migration are not non-specific and that high expression of TN-AP may induce changes in cell behaviour which may be consistent with or at least related to induction of terminal differentiation. (C) 1996 Wiley-Liss, Inc.