Changes in p53 expression can modify cell shape of ras-transformed fibroblasts and epitheliocytes

Abstract

p53 plays an important role in restriction of abnormal cell proliferation. Loss of this safeguard function induced by p53 mutations seems to be a key mechanism in oncogenesis. It cannot be excluded however, that in addition to elimination of p53-dependent checkpoints and/or apoptosis p53 mutations may cause additional effects that contribute to oncogenic transformation. In order to analyse the effects of wild-type (wt) and mutant p53 on expression of ras-induced morphological transformation we used the method of computer-assisted morphometry. The following parameters were determined: a) the area covered by the spread cells; b) dispersion and c) elongation of cell contours. The last two indices characterise cell shape. Elongation indicates the degree of bipolarity of cell contour and dispersion – the degree of its multipolarity. Transformation of Rat1 and mouse 10(3) fibroblasts by N-rasasp12 oncogene was accompanied by dramatic decrease of cell area and increase of dispersion and elongation. IAR-2 discoid epitheliocytes expressing exogenous ras oncogene transformed into polarised cells with decreased cell area. Fluorescent microscopic examination of actin cytoskeleton stained with rhodamine-phalloidin had shown that ras-induced transformation of IAR-2 cells is characterised by disappearance of circumferential actin bundle and straight fibers. Neither did we reveal actin stress-fibers in the ras-transformed Rat1 cells. Transduction of p53 cDNAs caused no significant changes in morphometric parameters of non-transformed parental Rat1, IAR-2 and 10(3) cells, but some of the p53 mutants modified cell shape of ras-transformed cells. p53-His273, unlike other tested p53 mutants (Tyr141, His194, Trp248), induced partial reversion of morphological transformation in both Rat1 fibroblasts and IAR-2 epitheliocytes. Its expression led to increase of average cell area, decrease of dispersion and elongation indices, and re-appearance of actin bundles. Exogenous wild-type p53 also caused some reversion of transformed phenotype of Rat/ras cells, but its effect was weaker than that of the p53-His273. In contrast, another p53 mutant p53-His175 was able to enhance ras-induced morphological transformation in p53-deficient murine 10(3) fibroblasts that is consistent with possible involvement of some gain of function activity of mutant p53 in modulation of cell shape. Possible pathways that might be responsible for p53-induced changes of cell morphology are discussed.