CP27 affects viability,proliferation, attachment and gene expression in embryonic fibroblasts

CP27 is a gene that has been cloned from an E11 early embryonic library and has been suggested to mediate early organogenesis (Diekwisch et al., 1999, Gene 235, 19). We have hypothesized that CP27 exhibits its effects on organogenesis by affecting individual cell function. Based on the CP27 expression pattern we have selected the CP27 expressing embryonic fibroblast cell line BALB/c 3T3 to determine the effects of CP27 on cell function. CP27 loss of function strategies were performed by adding 5, 12.5 or 25 micro g/ml anti-CP27 antibody to cultured BALB/c 3T3 cells and comparing the results to controls in which identical concentrations of rabbit serum were added to the culture medium. Other controls included an antibody against another extracellular matrix protein amelogenin (negative control) and anti-CP27 antibodies directed against other areas of the CP27 molecule (positive control). Following cell culture, cell viability, apoptosis, cell proliferation, cell shape, cellular attachment and fibronectin matrix production were assayed using MTT colourimetric assay, BrdU staining, morphometry, immunostaining and western blot analysis. Block of CP27 function using an antibody strategy resulted in the following significant changes: (i) reduced viability, (ii) increased number of apoptotic cells, (iii) reduced proliferation, (iv) alterations in cell shape, (v) loss of attachment, and (vi) reduction in fibronectin matrix production. There was also a redistribution in fibronectin matrix organization demonstrated by immunohistochemistry. We conclude that CP27 plays an important role in the maintance of normal cell function and that CP27 block leads to significant changes in cellular behaviour.