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Changes in p53 expression can modify cell shape of ras-transformed fibroblasts and epitheliocytes
N Gloushankova1, V Ossovskaya, J Vasiliev
1Institute of Cancerogenesis, Cancer Research Center, Russian Academy of Sciences, Moscow.
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.
Insights
Mutant p53 proteins can influence cancer development beyond inhibiting cell death. Specific p53 mutations, like p53-His273, can partially reverse ras-induced cell transformation, while others may enhance it.
Area of Science:
- Cell Biology
- Molecular Biology
- Oncology
Background:
- The tumor suppressor protein p53 is crucial for preventing abnormal cell proliferation.
- Loss of p53 function through mutations is a key event in oncogenesis.
- p53 mutations might contribute to cancer through mechanisms beyond checkpoint control and apoptosis.
Purpose of the Study:
- To investigate the effects of wild-type (wt) and mutant p53 on ras-induced morphological transformation using computer-assisted morphometry.
- To analyze changes in cell area, shape (dispersion and elongation), and actin cytoskeleton organization.
Main Methods:
- Computer-assisted morphometry to quantify cell area, dispersion, and elongation.
- Fluorescent microscopy with rhodamine-phalloidin to visualize the actin cytoskeleton.
- Expression of N-ras oncogene and various p53 cDNAs (wt and mutants) in Rat1 fibroblasts, IAR-2 epitheliocytes, and p53-deficient murine 10(3) fibroblasts.
Main Results:
- Ras-induced transformation led to decreased cell area and increased shape irregularity (dispersion, elongation) in fibroblasts and epitheliocytes.
- Ras transformation disrupted actin cytoskeleton organization, characterized by loss of actin bundles and stress fibers.
- p53-His273 mutant partially reversed ras-induced morphological transformation, restoring cell area and actin organization.
- Wild-type p53 showed a weaker reversion effect compared to p53-His273.
- p53-His175 mutant enhanced ras-induced transformation in p53-deficient cells, suggesting a gain-of-function activity.
Conclusions:
- Mutant p53 proteins can modulate cell morphology and influence oncogenic transformation, potentially through gain-of-function mechanisms.
- Specific p53 mutants, like p53-His273, exhibit tumor-suppressive activity by reverting transformed phenotypes.
- The study highlights the complex role of p53 mutations in cancer beyond their canonical functions.