The amount of a specific cellular protein (p53) is a correlate of differentiation in embryonal carcinoma cells

A specific cellular protein of molecular weight of 53–55,000 (p53) has been shown to be induced in all SV40 transformed cells. A similar protein has also been shown to be present in embryonal carcinoma cells and in midgestation murine embryo primary cells, which are not infected by SV40. In embryo cell primaries the amount of the protein was shown to decrease with the increase in the stage of embryo development. As differentiation or decrease in cell growth rate can account for this, and since the growth rate of embryo primary cells cannot be measured, we chose to investigate various embryonal carcinoma cells. We report that the p53 is present in a pluripotent embryonal carcinoma cell OTT6050, and in its differentiated parietal endoderm derivative, PYS-2 cells. The amount of p53 is higher in the undifferentiated EC stem cells than in the differentiated PYS-2 (parietal endoderm) cells. The amount of the protein decreases in F9 embryonal carcinoma cells induced to differentiate to a parietal endoderm cell type by treatment with retinoic acid, as it does following spontaneous differentiation of OTT6050 EC cells. To determine if a change in growth rate, rather than differentiation, might acount for the diminished levels of this protein, the amount ofp53 was measured in growing and in growth arrested cell populations. When the growth rate of F9 cells was reduced by treatment with 8-bromocyclic AMP there was no change in the amount of p53. The half life of the p53 was compared in the undifferentiated and the differentiated cell types to determine if a change in stability might account, in part, for the altered levels of this protein. The p53 is found to be most stable in the SV40 transformed established clonal cells. It is less stable in the fibroblast clonal cells which were not transformed by SV40. The results of these experiments indicate that a decrease in the amount of p53 primarily correlates with differentiation in the embryonal carcinoma cell lines studied and not with cell growth rate. Furthermore, the decrease appears to be related (in part) to the decreased stability of the p53.     

Reversible effects of sodium butyrate on the differentiation of F9 embryonal carcinoma cells

We have studied effects of sodium butyrate on embryonal carcinoma F9 cell differentiation. In the presence of sodium butyrate, F9 cells underwent rapid and drastic morphological changes and expressed marked increases in mRNA levels of various differentiation markers. When sodium butyrate was removed from the cultures, all the examined phenotypes of F9 cell differentiation rapidly reverted to the characteristics of undifferentiated stem cells. However, under the same conditions, when cycloheximide or actinomycin D was added to the cultures, such phenotypic reversion was not observed, but high mRNA levels of the differentiation markers as well as altered cell morphology were retained. These results indicated that the effects of sodium butyrate on induction of teratocarcinoma cell differentiation were reversible and that de novo syntheses of some mRNA(s) and protein(s) were necessary for the reversion of differentiated cells to stem cells.     

G1 block of the cell cycle during differentiation of F9 cells correlates with accumulation of inhibitors of the activity of cyclin-kinase complexes of proteins p21/Waf1 and p27/Kip

F9 teratocarcinoma cells have a very short duration of the cell cycle with a short G1-period typical for early embryonic cells. The cells are capable of differentiating towards parietal endoderm cells after the treatment with retinoic acid (RA) and dibutyryl-cAMP (db-cAMP). This leads to changes in the cell cycle; in particular, G1-period becomes longer, and then differentiated F9 cells leave the cycle to stay in G0-phase. It was previously reported that undifferentiated F9 cells undergo no G1 arrest of the cell cycle after DNA damage (Malashicheva et al., 2000). In the present work mechanisms of accumulation of G1-phase cells during differentiation induced by retinoic acid and db-cAMP were studied. Kinase activity of cyclin-Cdk complexes regulating the G1/S transition was analyzed. In differentiated F9 cells, the activity of cyclin-Cdk complexes, comprising Cdk4 and Cdk2 kinases and cyclins A and E, was significantly decreased. A decrease of Cdk4 kinase activity correlates with a drop of the cyclin D1 content. The amount of p21/Waf1 and p27/Kip inhibitors of the cyclin-kinase complexes increased in differentiated F9 cells. p21/Waf1 protein, which undergoes proteasomal degradation in undifferentiated F9 cells, was shown to be stable in their differentiated derivatives. Besides, in differentiated F9 cells p21/Waf1 and p27/Kip proteins can be detected with Cdk4/Cdk2-cyclin E complexes, in contrast to undifferentiated cells. Thus, we suggest that a G1/G0 block of the cell cycle taking place upon differentiation of F9 cells is likely to be caused by a decrease in cyclin-kinase activity due to stabilization and accumulation of p21/Waf1 and p27/Kip inhibitors and to their ability to associate with Cdk-cyclin complexes.     

All-trans-retinoic acid-mediated modulation of p53 during neural differentiation in murine embryonic stem cells

All-trans-retinoic acid (RA) plays an important physiological role in embryonic development and is teratogenic in large doses in almost all species. p53, a tumor suppressor gene encodes phosphoproteins, which regulate cellular proliferation, differentiation, and apoptosis. Temporal modulation of p53 by retinoic acid was investigated in murine embryonic stem cells during differentiation and apoptosis. Undifferentiated embryonic stem cells express a high level of p53 mRNA and protein followed by a decrease in p53 levels as differentiation proceeds. The addition of retinoic acid during 8–10 days of differentiation increased the levels of p53 mRNA and protein, accompanied by accelerated neural differentiation and apoptosis. Marked increase in apoptosis was observed at 10–20 h after retinoic acid treatment when compared with untreated controls. Retinoic acid-induced morphological differentiation resulted in predominantly neural-type cells. Maximum increase in p53 mRNA in retinoic acid-treated cells occurred on day 17, whereas maximum protein synthesis occurred on days 14–17, which coincided with increased neural differentiation and proliferation. Increased p53 levels did not induce p21 transactivation, interestingly a decrease in p21 was observed on day 17 on exposure to retinoic acid. The level of p53 declined by day 21 of differentiation. The results demonstrated that retinoic acid-mediated apoptosis preceded the changes in p53 expression, suggesting that p53 induction does not initiate retinoic acid-induced apoptosis during development. However, retinoic acid accelerated neural differentiation and increased the expression of p53 in proliferating neural cells, corroborated by decreased p21 levels, indicating the importance of cell type and stage specificity of p53 function. This revised version was published online in July 2006 with corrections to the Cover Date.     

p53 plays a regulatory role in differentiation and apoptosis of central nervous system-associated cells.

This study demonstrated the involvement of the tumor suppressor protein p53 in differentiation and programmed cell death of neurons and oligodendrocytes, two cell types that leave the mitotic cycle early in development and undergo massive-scale cell death as the nervous system matures. We found that primary cultures of rat oligodendrocytes and neurons, as well as of the neuronal PC12 pheochromocytoma cell line, constitutively express the p53 protein. At critical points in the maturation of these cells in vitro, the subcellular localization of p53 changes: during differentiation it appears mainly in the nucleus, whereas in mature differentiated cells it is present mainly in the cytoplasm. These subcellular changes were correlated with changes in levels of immunoprecipitated p53. Infection of cells with a recombinant retrovirus encoding a C-terminal p53 miniprotein (p53 DD), previously shown to act as a dominant negative inhibitor of endogenous wild-type p53 activity, inhibited the differentiation of oligodendrocytes and of PC12 cells and protected neurons from spontaneous apoptotic death. These findings suggest that p53, upon receiving appropriate signals, is recruited into the nucleus, where it plays a regulatory role in directing primary neurons', oligodendrocytes, and PC12 cells toward either differentiation or apoptosis in vitro.     

Two distinct mechanisms regulate the levels of a cellular tumor antigen, p53.

The steady-state levels of p53 protein and p53 mRNA in transformed and nontransformed cells were examined to elucidate the mechanisms controlling expression of p53. mRNA levels were determined by Northern blot hybridization analysis, employing a p53-specific cDNA clone (M. Oren and A.J. Levine, Proc. Natl. Acad. Sci. U.S.A. 80:56-59, 1983), and protein levels were determined by the Western blotting technique. Analysis of p53 mRNA revealed a single polyadenylated mRNA species migrating at ca. 18S. Levels of p53 mRNA in simian virus 40-transformed cell line (SVT2) and in an homologous nontransformed cell line (3T3) were equivalent, although the steady-state levels of p53 protein were 25- to 100-fold higher in the SVT2 cells than in the 3T3 cells. A study with a non-virus-transformed cell system revealed a different result. Embryonal carcinoma cells (F9) were found to have nearly 20-fold higher levels of p53 mRNA in comparison with differentiated benign progeny cells. In this system the difference in p53 mRNA levels corresponded to the difference in p53 protein levels. Pulse-chase experiments were performed to study the half-life of p53 protein in these four types of cells. The turnover of p53 protein occurred with biphasic kinetics. In addition, it was found that protein synthesis inhibitors placed in the medium during the chase period prevented the turnover of p53 protein in transformed cells, but not in nontransformed (3T3) cells. These results provide evidence that the regulation of p53 expression in cells can occur at the level of p53 mRNA abundancy or p53 protein stability depending upon the experimental system under study, and that a regulated degradation process controls the turnover of p53 protein.     

Regulation of p53 in embryonic stem cells

Despite an increasing interest in the role of the p53 tumour suppressor protein in embryonic stem cells, not much is known about its regulation in this cell type.We show that the relatively high amount of p53 protein correlates with a higher amount of p53 RNA in ES cells compared to differentiated cells. Moreover, p53 RNA is more stable in embryonic stem cells and the p53 protein is more often transcribed. This is at least partly due to decreased expression of miRNA-125a and 125b in embryonic stem cells. Despite its cytoplasmic localisation, p53 is degraded in 26S proteasomes in embryonic stem cells. This process is controlled by Mdm2, the deubiquitinating enzyme Hausp and Ubc13. In contrast, the E3 ligase PirH2 appears to be less important for the control of p53 in embryonic stem cells. During differentiation, p53 protein and RNA levels are decreased which corresponds to increased expression of miRNA-125a and miRNA-125b.     

A role for p21 (WAF1) in the cAMP-dependent differentiation of F9 teratocarcinoma cells into parietal endoderm

Combined treatment of teratocarcinoma F9 cells with retinoic acid and dibutyryl-cAMP induces the differentiation into cells with a phenotype resembling parietal endoderm. We show that the levels of cyclin-dependent kinase inhibitor p21/WAF1/Cip1 (p21) protein and mRNA are dramatically elevated at the end of this differentiation, concomitantly with the appearance of p21 in the immunoprecipitated CDK2-cyclin E complex. The induction of differentiation markers could not be achieved by expression of ectopic p21 alone and still required treatment with differentiation agents. Clones of F9 cells transfected with sense or antisense p21 cDNA constructs revealed, upon differentiation, upregulated levels of mRNA for thrombomodulin, a parietal endoderm-specific marker, or increased fraction of cells in sub-G1 phase of the cell cycle, respectively. Consistent with this observation, whereas p21 was strictly nuclear in undifferentiated cells, a large proportion of differentiated cells had p21 localized also in the cytoplasm, a site associated with the antiapoptotic function of p21. Furthermore, p21 activated the thrombomodulin promoter in transient reporter assays and the p21 mutant defective in binding to cyclin E was equally efficient in activation. The promoter activity in differentiated cells was reduced by cotransfection of p21-specific siRNA or antisense cDNA. Coexpression of p21 increased the activity of the GAL-p300(1-1303) fusion protein on the GAL sites-containing TM promoter. This implies that p21 might act through a derepression of the p300 N-terminal-residing repression domain, thereby enhancing the p300 coactivator function. As differentiation of F9 cells into parietal endoderm-like cells requires the cAMP signaling, the results together suggest that the cyclin-dependent kinase inhibitor p21 may promote specifically this pathway in F9 cells.     

Potential role of mycophenolate mofetil in the management of neuroblastoma patients

In human neuroblastoma cell lines (LAN5, SHEP and IMR32), mycophenolic acid (MPA) at concentrations (10(-7)-10(-6) M) readily attainable during immunosuppressive therapy with mycophenolate mofetil (Cellcept), induces guanine nucleotide depletion leading to cell cycle arrest and apoptosis through a p53 mediated pathway (up-regulation of p53, p21 and bax and down-regulation of bcl-2 and survivin). MPA-induced apoptosis is also associated to a marked decrease of p27 protein. In the same cell lines MPA, at lower concentrations (50 nM), corresponding to the plasma levels of the active free drug during Cellcept therapy, induces differentiation toward the neuronal phenotype by causing a partial chronic guanine nucleotide depletion. MPA-induced differentiation is not associated to p27 accumulation as occurs using retinoic acid. At a fixed concentration of MPA a higher percentage of apoptotic or differentiated cells is obtained when non dialysed serum substitutes for the dialysed one, due to the higher hypoxanthine concentration in the former (about 10 microM) leading to competition on HPRT-mediated salvage of guanine. At hypoxanthine or oxypurinol concentrations higher than 1 microM (up to 100 microM) no further enhancement of MPA effects was obtained, in agreement with the recently described safety of the allopurinol-mycophenolate mofetil combination in the treatment of hyperuricemia of kidney transplant recipients. The apoptotic effects of MPA do not appear to be significantly increased by the UDP-glucuronosyltransferase inhibitor niflumic acid.     

Silencing of Ribosomal Protein S9 Elicits a Multitude of Cellular Responses Inhibiting the Growth of Cancer Cells Subsequent to p53 Activation

Background

Disruption of the nucleolus often leads to activation of the p53 tumor suppressor pathway through inhibition of MDM2 that is mediated by a limited set of ribosomal proteins including RPL11 and RPL5. The effects of ribosomal protein loss in cultured mammalian cells have not been thoroughly investigated. Here we characterize the cellular stress response caused by depletion of ribosomal protein S9 (RPS9).

Methodology/Principal Findings

Depletion of RPS9 impaired production of 18S ribosomal RNA and induced p53 activity. It promoted p53-dependent morphological differentiation of U343MGa Cl2:6 glioma cells as evidenced by intensified expression of glial fibrillary acidic protein and profound changes in cell shape. U2OS osteosarcoma cells displayed a limited senescence response with increased expression of DNA damage response markers, whereas HeLa cervical carcinoma cells underwent cell death by apoptosis. Knockdown of RPL11 impaired p53-dependent phenotypes in the different RPS9 depleted cell cultures. Importantly, knockdown of RPS9 or RPL11 also markedly inhibited cell proliferation through p53-independent mechanisms. RPL11 binding to MDM2 was retained despite decreased levels of RPL11 protein following nucleolar stress. In these settings, RPL11 was critical for maintaining p53 protein stability but was not strictly required for p53 protein synthesis.

Conclusions

p53 plays an important role in the initial restriction of cell proliferation that occurs in response to decreased level of RPS9. Our results do not exclude the possibility that other nucleolar stress sensing molecules act upstream or in parallel to RPL11 to activate p53. Inhibiting the expression of certain ribosomal proteins, such as RPS9, could be one efficient way to reinitiate differentiation processes or to induce senescence or apoptosis in rapidly proliferating tumor cells.     

Potential Role of Mycophenolate Mofetil in the Management of Neuroblastoma Patients

In human neuroblastoma cell lines (LAN5, SHEP and IMR32), mycophenolic acid (MPA) at concentrations (10^? 7–10^? 6 M) readily attainable during immunosuppressive therapy with mycophenolate mofetil (Cellcept), induces guanine nucleotide depletion leading to cell cycle arrest and apoptosis through a p53 mediated pathway (up‐regulation of p53, p21 and bax and down‐regulation of bcl‐2 and survivin). MPA‐induced apoptosis is also associated to a marked decrease of p27 protein. In the same cell lines MPA, at lower concentrations (50 nM), corresponding to the plasma levels of the active free drug during Cellcept therapy, induces differentiation toward the neuronal phenotype by causing a partial chronic guanine nucleotide depletion. MPA‐induced differentiation is not associated to p27 accumulation as occurs using retinoic acid. At a fixed concentration of MPA a higher percentage of apoptotic or differentiated cells is obtained when non dialysed serum substitutes for the dialysed one, due to the higher hypoxanthine concentration in the former (about 10 µM) leading to competition on HPRT‐mediated salvage of guanine. At hypoxanthine or oxypurinol concentrations higher than 1 µM (up to 100 µM) no further enhancement of MPA effects was obtained, in agreement with the recently described safety of the allopurinol‐mycophenolate mofetil combination in the treatment of hyperuricemia of kidney transplant recipients. The apoptotic effects of MPA do not appear to be significantly increased by the UDP‐glucuronosyltransferase inhibitor niflumic acid.