Platelet-derived growth factor (PDGF) receptor-alpha activates c-Jun NH2-terminal kinase-1 and antagonizes PDGF receptor-beta -induced phenotypic transformation

Platelet-derived growth factor (PDGF) is a potent mitogen for mesenchymal cells. The PDGF B-chain (c-sis proto-oncogene) homodimer (PDGF BB) and v-sis, its viral counterpart, activate both alpha- and beta-receptor subunits (alpha-PDGFR and beta-PDGFR) and mediate anchorage-independent growth in NIH3T3 cells. In contrast, the PDGF A chain homodimer (PDGF AA) activates alpha-PDGFR only and fails to induce phenotypic transformation. In the present study, we investigated alpha- and beta-PDGFR specific signaling pathways that are responsible for the differences between the transforming ability of PDGF AA and BB. To study PDGF BB activation of beta-PDGFR, we established NIH3T3 clones in which alpha-PDGFR signaling is inhibited by a dominant-negative alpha-PDGFR, or an antisense construct of alpha-PDGFR. Here, we demonstrate that beta-PDGFR activation alone is sufficient for PDGF BB-mediated anchorage-independent cell growth. More importantly, inhibition of alpha-PDGFR signaling enhanced PDGF BB-mediated phenotypic transformation, suggesting that alpha-PDGFR antagonizes beta-PDGFR-induced transformation. While both alpha- and beta-receptors effectively activate ERKs, alpha-PDGFR, but not beta-PDGFR, activates stress-activated protein kinase-1/c-Jun NH(2)-terminal kinase-1 (JNK-1). Inhibition of JNK-1 activity using a dominant-negative JNK-1 mutant markedly enhanced PDGF BB-mediated anchorage-independent cell growth, demonstrating an antagonistic role for JNK-1 in PDGF-induced transformation. Consistently, overexpression of wild-type JNK-1 reduced PDGF BB-mediated transformation. Taken together, the present study showed that alpha- and beta-PDGFRs differentially regulate Ras-mitogen-activated protein kinase pathways critical for regulation of cell transformation, and transformation suppressing activity of alpha-PDGFR involves JNK-1 activation.     

Both platelet-derived growth factor receptor (PDGFR)-alpha and PDGFR-beta promote murine fibroblast cell migration

Cell motility plays a critical role for many physiological and pathological processes including wound healing, fibrosis, angiogenesis, and tumor metastasis. Platelet-derived growth factor (PDGF) is among the most potent stimuli for mesenchymal cell migration. The PDGF B-chain homodimer PDGF BB activates both alpha- and beta-receptor subunits (alpha-PDGFR and beta-PDGFR), and promotes cell migration in many cell types including fibroblasts and smooth muscle cells. PDGF-A chain homodimer PDGF AA activates alpha-PDGFR only, and its role for cell migration is still debatable. PDGF BB, but not PDGF AA, induces smooth muscle cell migration. Interestingly, alpha-PDGFR was shown to antagonize beta-PDGFR-induced smooth muscle cell migration. In the present study, we investigated the role of alpha-PDGFR and beta-PDGFR in PDGF-mediated cell migration of murine fibroblasts (NIH 3T3). Unlike smooth muscle cells, both PDGF AA and PDGF BB promoted NIH 3T3 cell migration. The effect of PDGF BB activation of beta-PDGFR alone for cell migration was examined using previously established NIH 3T3 clones in which alpha-PDGFR signaling is inhibited by a dominant-negative alpha-PDGFR, or an antisense construct of alpha-PDGFR. PDGF BB activation of beta-PDGFR alone was sufficient to induce cell migration, but the efficiency was significantly lower compared to PDGF activation of both receptors. These results showed that both alpha- and beta-PDGFRs promote fibroblast cell migration and their effects are additive. Taken together, we propose that cell-type specific alpha-PDGFR signaling is critical for regulation of mesenchymal cell migration in response to PDGF isoform, whereas beta-PDGFR mainly promotes cell migration.     

The p21(WAF1/CIP1) promoter is methylated in Rat-1 cells: stable restoration of p53-dependent p21(WAF1/CIP1) expression after transfection of a genomic clone containing the p21(WAF1/CIP1) gene

Rat-1 cells are used in many studies on transformation, cell cycle, and apoptosis. Whereas UV treatment of Rat-1 cells results in apoptosis, X-ray treatment does not induce either apoptosis or a cell cycle block. X-ray treatment of Rat-1 cells results in both an increase of p53 protein and expression of the p53-inducible gene MDM2 but not the protein or mRNA of the p53-inducible p21(WAF1/CIP1) gene, which in other cells plays an important role in p53-mediated cell cycle block. The lack of p21(WAF1/CIP1) expression appears to be the result of hypermethylation of the p21(WAF1/CIP1) promoter region, as p21(WAF1/CIP1) protein expression could be induced by growth of Rat-1 cells in the presence of 5-aza-2-deoxycytidine. Furthermore, sequence analysis of bisulfite-treated DNA demonstrated extensive methylation of cytosine residues in CpG dinucleotides in a CpG-rich island in the promoter region of the p21(WAF1/CIP1) gene. Stable X-ray-induced p53-dependent p21(WAF1/CIP1) expression and cell cycle block were restored to a Rat-1 clone after transfection with a P1 artificial chromosome (PAC) DNA clone containing a rat genomic copy of the p21(WAF1/CIP1) gene. The absence of expression of the p21(WAF1/CIP1) gene may contribute to the suitability of Rat-1 cells for transformation, cell cycle, and apoptosis studies.     

The lack of G1/S arrest of teratocarcinoma F9 cells is determinated by degradation of cyclin-dependent kinases inhibitor p21waf1/cip1

We have studied the ability of F9 teratocarcinoma cells to arrest in G1/S and G2/M checkpoints following gamma-irradiation. Wild-type p53 protein is rapidly accumulated in F9 cells after gamma-irradiation, however this is not followed by G1/S arrest; there is just a reversible delay of the cell cycle in G2/M. In order to elucidate the reasons of the lack of G1/S arrest in F9 cells we investigated the levels of regulatory cell cycle proteins: G1-cyclins, cyclin dependent kinases and kinase inhibitor p21WAF1/CIP1. We have shown that in spite of p53-dependent activation of p21WAF1/CIP1 promoter, p21WAF1/CIP1 protein is not revealed by different polyclonal and monoclonal antibodies, either by immunoblotting or by immunofluorescent staining. However, when cells are treated with specific proteasome inhibitor lactacystin, p21WAF1/CIP1 protein is revealed. We therefore suggest that p21WAF1/CIP1 protein is subjected to proteasome degradation in F9 cells and probably the lack of G1/S arrest after gamma-irradiation is due to this degradation. Thus, it is the combination of functionally active p53 with low level expression of p21WAF1/CIP1 that causes a short delay of the cell cycle progression in G2/M, rather than the G1-arrest after gamma-irradiation of F9 cells.     

Mechanisms of differential activation of target gene promoters by p53 hinge domain mutants with impaired apoptotic function

Suppression of tumor cell growth by p53 results from the activation of both apoptosis and cell cycle arrest functions that have been shown to be separable activities of p53. We report here that some mutants in the p53 hinge domain, a short linker between the DNA binding and tetramerization domains, differentially activated the promoters of p53 target genes and possessed an impaired apoptotic function. Our results indicate that the hinge domain may play an important role in differentially regulating p53 cell cycle arrest and apoptotic functions. However, the mechanisms by which p53 hinge domain mutants differentially activate its target genes, e.g. p21(WAF1/CIP1) and Bax, remain unknown. To investigate the possible mechanisms, recombinant p21(WAF1/CIP1) and Bax promoters were constructed, resulting in rearrangement of the existing p53 binding sites within a given promoter or actually swapping p53 binding sites between the two promoters. Our results suggest that multiple mechanisms of differential transactivation occur, depending on the molecular nature of the relevant hinge domain mutant, such as the possibility that dual separate DNA binding sites in the p21(WAF1/CIP1) promoter are responsible for the selective transactivation activity of p53 hinge domain mutant del300-327, which has a large deletion in the hinge domain. Lack of ideal p53 binding sites in the Bax promoter results in less potent activation than that seen with the p21(WAF1/CIP1) promoter when it is transactivated by hinge domain point mutant mutR306P or short deletion mutant del300-308 proteins. How the single mutation or the short deletion affect the conformation of p53 and consequently the transactivation of the Bax promoter will require further investigation of the relevant p53 protein: DNA-binding domain by NMR and x-ray crystallographic techniques.     

Adriamycin (ADM) induced apoptosis in Transitional Cell Cancer (TCC) cell lines accompanied by p21 WAF1/CIP1 induction

Genotoxic stimuli, including anticancer drugs, induce apoptosis in cancer cells through increase of p53, p21WAF1/CIP1 , at least in part. Bcl-2 and Bax modify this pathway or directly regulated by p53. Here we studied Adriamycin (ADM)-induced apoptosis in four human bladder cancer cell lines in respect of p53, p21WAF1/CIP1 and Bcl-2 family proteins. After ADM, treatment bladder cancer cells underwent dose-dependent cell death with typical morphologic features of apoptosis. Among four cell lines RT4 with wt p53, low ratio of Bcl-2 to Bax and induction of p21WAF1/CIP1 after ADM treatment, was the most sensitive to induction of apoptosis. Thus, p53, p21WAF1/CIP1 , Bcl-2 and Bax status might determine susceptibility of bladder cancer cells to ADM induced apoptosis.     

Alterations in TP53, cyclin D2, c-Myc, p21WAF1/CIP1 and p27KIP1 expression associated with progression in B-CLL

B-cell chronic lymphocytic leukaemia (B-CLL) originates from B lymphocytes that may differ in the activation level, maturation state or cellular subgroups in peripheral blood. Tumour progression in CLL B cells seems to result in gradual accumulation of the clone of resting B lymphocytes in the early phases (G0/G1) of the cell cycle. The G1 phase is impaired in B-CLL. We investigated the gene expression of five key cell cycle regulators: TP 53, c-Myc, cyclin D2, p21WAF1/CIP1 and p27KIP1, which primarily regulate the G1 phase of the cell cycle, or S-phase entry and ultimately control the proliferation and cell growth as well as their role in B-CLL progression. The study was conducted in peripheral blood CLL lymphocytes of 40 previously untreated patients. Statistical analysis of correlations of TP53, cyclin D2, c-Myc, p21WAF1/CIP1 and p27KIP1 expressions in B-CLL patients with different Rai stages demonstrated that the progression of disease was accompanied by increases in p53, cyclin D2 and c-Myc mRNA expression. The expression of p27KIP1 was nearly statistically significant whereas that of p21 WAF1/CIP1 showed no such correlation. Moreover, high expression levels of TP53 and c-Myc genes were found to be closely associated with more aggressive forms of the disease requiring earlier therapy.     

DNA损伤生物学反应中ATM对p21~(WAF1/CIP1)蛋白的直接磷酸化

毛细血管扩张性共济失调症突变蛋白 (mutatedinataxiatelangiectasia ,ATM)是直接感受DNA双链断裂损伤并起始诸多DNA损伤信号反应通路的主开关分子 .已有研究发现 ,DNA损伤生物学反应中 ,ATM可通过磷酸化活化p5 3,继而转录活化细胞周期检查点蛋白p2 1WAF1 CIP1的表达 ,而对于ATM是否直接参与p2 1WAF1 CIP1的早期活化迄今尚无实验证明 .通过免疫共沉淀反应 ,检测到细胞电离辐射 (ionizingradiation ,IR)反应早期ATM与p2 1WAF1 CIP1蛋白存在相互作用 .将p2 1WAF1 CIP1蛋白编码基因全长克隆入原核表达载体pGEX4T 2 ,经诱导表达及亲和层析纯化获取GST p2 1融合蛋白作为磷酸化底物 .体外磷酸化实验检测证明 ,IR活化的ATM具磷酸化p2 1WAF1 CIP1蛋白的功能 ,并且此磷酸化功能可被PI3K家族特异性抑制剂Wortmannin所抑制 .结果揭示了IR后ATM可通过直接磷酸化p2 1WAF1 CIP1蛋白 ,在IR致DNA损伤生物学反应早期调控p2 1WAF1 CIP1蛋白的快速活化过程     

Binding sequence-dependent regulation of the human proliferating cell nuclear antigen promoter by p53

Exposure of a lung epithelial cell line to ionizing radiation (IR) arrests cell cycle progression through 48 h post-exposure. Coincidentally, IR differentially activates expression of the cell cycle inhibitor, p21/WAF1, and the DNA replication protein, proliferating cell nuclear antigen (PCNA). p21/WAF1 mRNA levels remain elevated through 48 h post-exposure to IR, while PCNA mRNA levels increase transiently at early times. Since p21/WAF1 inhibits DNA replication by directly binding PCNA, the relative levels of the two proteins can determine cell cycle progression. The PCNA p53-binding site displayed reduced p53 binding affinity in vitro relative to the distal p21/WAF1 p53-binding site. Substitution of the p21/WAF1 site for the resident p53-binding site in the PCNA promoter altered the responses to increasing amounts of p53 or IR in transient expression assays. The p21/WAF1 p53-binding site sustained activation of the chimeric PCNA promoter under conditions (high p53 levels or high dose IR) that the PCNA p53-binding site did not. Binding site-specific regulation by wild-type p53 was not observed with mutant p53 harboring a serine to alanine change at amino acid 46. Limited activation of the PCNA promoter by p53 and its modified forms would restrict the amount of PCNA made available for DNA repair.     

The immunosuppressive agent tacrolimus induces p21WAF/CIP1WAF1/CIP1 via TGF-beta secretion

Tacrolimus (Tac) is more immunosuppressive drug compared to cyclosporine (CsA). Our previous studies have demonstrated that CsA induces the expression of p21WAF/CIP1 expression. In this study we explored if like CsA, Tac also induces expression of p21WAF/CIP1. We also determined if induction of p21WAF/CIP1 by Tac is dependent on TGF-beta. Using RT-PCR and Western blot analysis, we studied the induction of p21WAF/CIP1 mRNA and protein in human T cells and A-549 cells (human lung adenocarcinoma cells) by Tac. The stimulation of p21WAF/CIP1 promoter activity was studied by luciferase assay using p21WAF/CIP1-luc, chimeric plasmid DNA containing a p21WAF/CIP1 promoter segment and luciferase reporter gene. Using anti-TGF-beta antibody, we studied if induction of p21WAF/CIP1 by tacrolimus is dependent on TGF-beta. The results demonstrate that Tac induced p21WAF/CIP1 mRNA and protein expression as well as stimulated its promoter activity in T cells and A-549 cells. The induction of p21WAF/CIP1 expression by tacrolimus was dependent on TGF-beta since a neutralizing anti-TGF-beta antibody inhibited induction of p21WAF/CIP1in A-549 cells. These data support the hypothesis that cyclin inhibitor p21WAF/CIP1 might represent a unified mediator of the anti-proliferative effects of Tac and other immunosuppressive agents. Strategies involving p21WAF/CIP1 induction should be considered a viable alternative strategy to achieve immunosuppression possibly with reduced toxicity associated with current immunosuppression.