The cyclin-dependent kinase inhibitor p21 (WAF1) is required for survival of differentiating neuroblastoma cells.

We are employing recent advances in the understanding of the cell cycle to study the inverse relationship between proliferation and neuronal differentiation. Nerve growth factor and aphidicolin, an inhibitor of DNA polymerases, synergistically induce neuronal differentiation of SH-SY5Y neuroblastoma cells and the expression of p21WAF1, an inhibitor of cyclin-dependent kinases. The differentiated cells continue to express p21WAF1, even after removal of aphidicolin from the culture medium. The p21WAF1 protein coimmunoprecipitates with cyclin E and inhibits cyclin E-associated protein kinase activity. Each of three antisense oligonucleotides complementary to p21WAF1 mRNA partially blocks expression of p21WAF1 and promotes programmed cell death. These data indicate that p21WAF1 expression is required for survival of these differentiating neuroblastoma cells. Thus, the problem of neuronal differentiation can now be understood in the context of negative regulators of the cell cycle.     

The expression and activity of D-type cyclins in F9 embryonal carcinoma cells: modulation of growth by RXR-selective retinoids

The growth rate of malignant F9 embryonal carcinoma cells slows considerably following all-trans-retinoic acid-induced differentiation into benign parietal endoderm. To determine the mechanism of this process, we examined the expression of cyclins D1, D2, and D3 and the activity of their associated kinases. Cyclin D1 and D3 mRNA levels decreased during complete differentiation induced by all-trans-retinoic acid and dibutyryl cAMP, while the levels of cyclin D2 and the cyclin-dependent kinase (Cdk) inhibitor p27 mRNAs increased. Ultimately, terminally differentiated cells possessed 50% of the Cdk4-associated kinase activity observed in undifferentiated cells. Since numerous genes are differentially regulated during parietal endoderm differentiation, it is difficult to determine whether retinoic acid affects cell cycle gene expression directly or if these changes are caused by differentiation. We found that the retinoid X receptor (RXR)-selective agonists LG100153 and LG100268 significantly inhibited F9 cell growth without causing overt terminal differentiation as assessed by anchorage-independent growth and differentiation-associated gene expression. As seen in cells induced to differentiate by the RAR agonist all-trans-retinoic acid, RXR activation led to an increase in the number of cells in G1 phase. RXR agonists also sharply induced the levels of the Cdk regulatory subunits, cyclin D2 and D3. However, Cdk4-dependent kinase activity was reduced by RXR-selective retinoid treatment. These observations suggest that some retinoids can directly inhibit proliferation and regulate Cdk4-dependent kinase activity without inducing terminal differentiation.     

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.     

Expression of fibroblast growth factor by F9 teratocarcinoma cells as a function of differentiation

F9 teratocarcinoma stem cells treated with retinoic acid (RA) and dibutyryl cAMP (but2 cAMP) differentiate into embryonic parietal endoderm. Using heparin-affinity chromatography, endothelial cell proliferation assays, immunoprecipitation, and Western analysis with antibodies specific for acidic and basic fibroblast growth factors (FGFs), we detected biologically active FGF in F9 cells only after differentiation. A bovine basic FGF cDNA probe hybridized to 2.2-kb mRNAs in both F9 stem and parietal endoderm cells and to a 3.8-kb mRNA in F9 stem cells. A genomic DNA probe for acidic FGF hybridized to a 5.8-6.0-kb mRNA in both F9 stem and parietal endoderm cells, and to a 6.0-6.3-kb mRNA only in parietal endoderm cells. Although these FGF mRNAs were present in the stem cells, we could find no evidence that F9 stem cells synthesized FGFs, whereas differentiated F9 cells synthesized both acidic and basic FGF-like proteins. We conclude that biologically active factors with properties characteristic of acidic and basic FGF are expressed by F9 parietal endoderm cells after differentiation. Differentiating embryonic parietal endoderm thus may serve as a source of FGF molecules in the developing blastocyst, where these factors appear to play a central role in subsequent embryogenesis.     

p21(cip1) mRNA is controlled by endogenous transforming growth factor-beta1 in quiescent human hematopoietic stem/progenitor cells

Transforming growth factor-beta1 (TGF-beta1) has been described as an efficient growth inhibitor that maintains the CD34(+) hematopoietic progenitor cells in quiescence. The concept of high proliferative potential-quiescent cells or HPP-Q cells has been introduced as a working model to study the effect of TGF-beta1 in maintaining the reversible quiescence of the more primitive hematopoietic stem cell compartment. HPP-Q cells are primitive quiescent stem/progenitor cells on which TGF-beta1 has downmodulated the cytokine receptors. These cells can be released from quiescence by neutralization of autocrine or endogenous TGF-beta1 with a TGF-beta1 blocking antibody or a TGF-beta1 antisense oligonucleotide. In nonhematopoietic systems, TGF-beta1 cooperates with the cyclin-dependent kinase inhibitor, p21(cip1), to induce cell cycle arrest. We therefore analyzed whether endogenous TGF-beta1 controls the expression of the p21(cip1) in the CD34(+) undifferentiated cells using a sensitive in situ hybridization method. We observed that addition of anti-TGF-beta1 is followed by a rapid decrease in the level of p21(cip1) mRNA whereas TGF-beta1 enhances p21(cip1) mRNA expression concurrently with an inhibitory effect on progenitor cell proliferation. These results suggest the involvement of p21(cip1) in the cell cycle control of early human hematopoietic quiescent stem/progenitors and not only in the differentiation of more mature myeloid cells as previously described. The modulation of p21(cip1) observed in response to TGF-beta1 allows us to further precise the working model of high proliferative potential-quiescent cells.     

p21 and retinoblastoma protein control the absence of DNA replication in terminally differentiated muscle cells

During differentiation, skeletal muscle cells withdraw from the cell cycle and fuse into multinucleated myotubes. Unlike quiescent cells, however, these cells cannot be induced to reenter S phase by means of growth factor stimulation. The studies reported here document that both the retinoblastoma protein (Rb) and the cyclin-dependent kinase (cdk) inhibitor p21 contribute to this unresponsiveness. We show that the inactivation of Rb and p21 through the binding of the adenovirus E1A protein leads to the induction of DNA replication in differentiated muscle cells. Moreover, inactivation of p21 by E1A results in the restoration of cyclin E-cdk2 activity, a kinase made nonfunctional by the binding of p21 and whose protein levels in differentiated muscle cells is relatively low in amount. We also show that restoration of kinase activity leads to the phosphorylation of Rb but that this in itself is not sufficient for allowing differentiated muscle cells to reenter the cell cycle. All the results obtained are consistent with the fact that Rb is functioning downstream of p21 and that the activities of these two proteins may be linked in sustaining the postmitotic state.     

Calcitonin receptor-mediated growth suppression of HEK-293 cells is accompanied by induction of p21WAF1/CIP1 and G2/M arrest.

We investigated the mechanisms by which calcitonin (CT) suppresses cellular proliferation, using HEK-293 cells stably transfected with either the rat C1a CT receptor (CTR) or the insert-negative form of the human CTR. CT treatment of clonal cell lines expressing either receptor type, but not untransfected HEK-293 cells, strongly suppressed cell growth in a concentration-dependent manner. The reduction in cell growth with CT treatment could not be attributed to cellular necrosis or apoptotic cell death, the latter assessed by both DNA fragmentation analysis and caspase 3 (CPP-32) assay. Growth inhibition was associated with an accumulation of cells in the G2 phase of the cell cycle. CT treatment of the human and rat CTR-expressing cell lines resulted in a rapid and sustained induction of mRNA encoding the cyclin-dependent kinase inhibitor, p21WAF1/CIP1, increased levels of which were maintained at least 48 h after initiation of treatment. Western blot analysis showed a rapid corresponding increase in p21WAF1/CIP1 protein, whereas protein levels of another member of the cyclin-dependent kinase inhibitor family, p27kip1, were unchanged. In parallel with the induction of p21, CT treatment reduced levels of p53 mRNA and protein. CT treatment resulted in a specific cell cycle block in G2, which was associated with inhibition of Cdc2/cyclin B kinase activity as measured by histone H1 phosphorylation. There was no evidence for p21 association with this complex despite the inhibition of Cdc2 activity. Evidence that p21 induction was causative of cell growth suppression was obtained from p21 antisense oligonucleotide experiments. Treatment with a p21 antisense oligonucleotide blocked induction of p21 expression and significantly reduced the CT-mediated growth inhibition. These observations suggest that p21 is required for the G2 arrest in response to CT, but argue against a direct role of p21 in the inhibition of Cdc2 activity. These studies suggest a novel regulation of cell cycle progression by CT and will provide a basis for detailed examination of the molecular mechanisms involved.     

Reversible interconversion between primitive endoderm- and parietal endoderm-like F9 cells demonstrated by mRNAs expression

The differentiation of retinoic acid-treated F9 cells (primitive endoderm-like F9 cells) into parietal endoderm-like F9 cells induced by dibutyryl cAMP was studied as a culture model of the morphogenesis of early mouse embryo. For this purpose, 6 cDNA clones coding for mRNAs specifically expressed in parietal endoderm-like F9 cells were selected. Northern hybridization of RNA extracted from variously treated F9 cells to nick-translated plasmid DNA of these clones demonstrated the reversible expression of many mRNAs depending on the presence of dibutyryl cAMP in the culture medium. This result suggested that the differentiated state of parietal endoderm, which is formed from primitive endoderm at a position adjacent to the trophectoderm in mouse embryo, can be reversed if the local signal is removed. One of the selected clones, pLAM, hybridized to an mRNA of 6.3 kb and selected mRNA producing a laminin B subunit in an in vitro translation system. This clone has an inserted sequence of 3.1 kb. Among the restriction sites in this sequence, six were consistent with those in a 1.7 kb inserted sequence of pPE 49 and pPE 386, which were isolated by Barlow et al. as laminin B1 clones. An XbaI site found in both pPE 49 and pPE 386 was, however, not found at the corresponding position of pLAM. Dot hybridization of RNA with pLAM showed that expression of laminin B in F9 cells is stimulated more than 100-fold during differentiation of F9 stem cells into parietal endoderm-like F9 cells.     

p21(Cip-1/SDI-1/WAF-1) expression via the mitogen-activated protein kinase signaling pathway in insulin-induced chondrogenic differentiation of ATDC5 cells

The embryonal carcinoma-derived cell line, ATDC5, differentiates into chondrocytes in response to insulin or insulin-like growth factor-I stimulation. In this study, we investigated the roles of mitogen-activated protein (MAP) kinases in insulin-induced chondrogenic differentiation of ATDC5 cells. Insulin-induced accumulation of glycosaminoglycan and expression of chondrogenic differentiation markers, type II collagen, type X collagen, and aggrecan mRNA were inhibited by the MEK1/2 inhibitor (U0126) and the p38 MAP kinase inhibitor (SB203580). Conversely, the JNK inhibitor (SP600125) enhanced the synthesis of glycosaminoglycan and expression of chondrogenic differentiation markers. Insulin-induced phosphorylation of ERK1/2 and JNK but not that of p38 MAP kinase. We have previously clarified that the induction of the cyclin-dependent kinase inhibitor, p21(Cip-1/SDI-1/WAF-1), is essential for chondrogenic differentiation of ATDC5 cells. To assess the relationship between the induction of p21 and MAP kinase activity, we investigated the effect of these inhibitors on insulin-induced p21 expression in ATDC5 cells. Insulin-induced accumulation of p21 mRNA and protein was inhibited by the addition of U0126 and SB203580. In contrast, SP600125 enhanced it. Inhibitory effects of U0126 or stimulatory effects of SP600125 on insulin-induced chondrogenic differentiation were observed when these inhibitors exist in the early phase of differentiation, suggesting that MEK/ERK and JNK act on early phase differentiation. SB202580, however, is necessary not only for early phase but also for late phase differentiation, indicating that p38 MAP kinase stimulates differentiation by acting during the entire period of cultivation. These results for the first time demonstrate that up-regulation of p21 expression by ERK1/2 and p38 MAP kinase is required for chondrogenesis, and that JNK acts as a suppressor of chondrogenesis by down-regulating p21 expression.