A non-covalent interaction between small ubiquitin-like modifier-1 and Zac1 regulates Zac1 cellular functions

Zac1, a zinc-finger protein that regulates apoptosis and cell cycle arrest 1, such as p53, can induce cell-cycle arrest and apoptosis. The transactivation and coactivation functions of Zac1 may occur at non-promyelocytic leukemia nuclear body (PML-NB) sites in the presence of other PML-NB components, including ubiquitin-conjugating 9 (Ubc9). It is unclear whether post-translational modification of Zac1 by the small ubiquitin-like modifier SUMO plays a role in the coactivation functions of Zac1 for the regulation of the p21 gene. Mutagenesis experiments revealed that the two SUMO-binding lysine residues of Zac1, K237 and K424, repress the transactivation activity of Zac1. Studies using a SUMO-1 C-terminal di-glycine motif mutant that is deficient in the ability to form covalent bonds with lysines, SUMO-1 (GA), and a dominant-negative Ubc9 construct (C93S) indicated that SUMO-1 might regulate Zac1 transactivation and coactivation via a non-covalent interaction. Unlike the wild-type Zac1, which induced apoptosis, the Zac1 (K237/424R) double mutant had the ability to induce autophagy. The functional role of p21 remains to be investigated. SUMO-1 selectively suppressed the induction of the p21 gene and protein by wild-type Zac1 but not by the Zac1 (K237/424R) double mutant. Moreover, wild-type Ubc9 but not Ubc9 (C93S) further potentiated the suppression of SUMO-1 in all Zac1-induced p21 promoter activities. Our data reveal that p21 may be an important factor for the prevention of Zac1-induced apoptosis without affecting autophagosome formation. This work indicates that Zac1 functions are regulated, at least in part, via non-covalent interactions with SUMO-1 for the induction of p21, which is important for the modulation of apoptosis.     

Down-regulation of p21 contributes to apoptosis induced by HPV E6 in human mammary epithelial cells

Infection with human papillomaviruses (HPV) is strongly associated with the development of cervical cancer. The HPV E6 gene is essential for the oncogenic potential of HPV. E6 induces cell proliferation and apoptosis in cervical cancer precursor lesions and in cultured cells. Although induction of telomerase and inactivation of the tumor suppressor p53 play important roles for E6 to promote cell growth, the molecular basis of E6-induced apoptosis is poorly understood. While it is expected that inactivation of p53 by E6 should lead to a reduction in cellular apoptosis, numerous studies demonstrated that E6 could in fact sensitize cells to apoptosis. Understanding the mechanism of p53-independent apoptosis is of clinical significance. In the present study, we investigated the mechanism of apoptosis during E6-mediated immortalization of primary human mammary epithelial cell (HMEC). E6 by itself is sufficient to immortalize HMECs and is believed to do so at least in part by activation of telomerase. During the process of E6-mediated HMEC immortalization, an increased apoptosis was observed. Mutational analysis demonstrated that E6-induced apoptosis was distinct from its ability to promote cell proliferation, activate telomerase, or degrade p53. While the known pro-apoptotic E6 target proteins such as Bak or c-Myc did not appear to play an important role, down-regulation of the cyclin-dependent kinase inhibitor p21^Waf1/Cip1 (p21) by E6 correlated with its ability to induce apoptosis. Ectopic expression of p21 inhibited E6-induced apoptosis. Moreover, a p53 degradation defective E6 mutant was competent for p21 down-regulation and apoptosis induction. The anti-apoptotic function of p21 may not simply be the result of p21-induced growth arrest. These studies demonstrate an E6 activity to down-regulate p21 that is important for induction of apoptosis.     

Triptolide Induces Growth Inhibition and Apoptosis of Human Laryngocarcinoma Cells by Enhancing p53 Activities and Suppressing E6-Mediated p53 Degradation

Triptolide, an active compound extracted from Chinese herb Leigongteng (Tripterygium wilfordii Hook F.), shows a broad-spectrum of anticancer activity through its cytotoxicity. However, the efficacy of triptolide on laryngocarcinoma rarely been evaluated, and the mechanism by which triptolide-induced cellular apoptosis is still not well understood. In this study, we found that triptolide significantly inhibited the laryngocarcinoma HEp-2 cells proliferation, migration and survivability. Triptolide induces HEp-2 cell cycle arrest at the G1 phase and apoptosis through intrinsic and extrinsic pathways since both caspase-8 and -9 are activated. Moreover, triptolide enhances p53 expression by increasing its stability via down-regulation of E6 and E6AP. Increased p53 transactivates down-stream target genes to initiate apoptosis. In addition, we found that short time treatment with triptolide induced DNA damage, which was consistent with the increase in p53. Furthermore, the cytotoxicity of triptolide is decreased by p53 knockdown or use of caspases inhibitor. In conclusion, our results demonstrated that triptolide inhibits cell proliferation and induces apoptosis in laryngocarcinoma cells by enhancing p53 expression and activating p53 functions through induction of DNA damage and suppression of E6 mediated p53 degradation. These studies indicate that triptolide is a potential anti-laryngocarcinoma drug.     

Induction of endoreduplication by a JNK inhibitor SP600125 in human lung carcinoma A 549 cells

The effect of the pan c-Jun N-terminal kinase (JNK) inhibitor SP600125 on the proliferation of human lung carcinoma A549 cells has been evaluated. We have shown that SP600125 completely inhibited the proliferation of A549 cells, the cycle arrest being in G2/M phase. When cells were treated with SP600125 for >12h, a cell population with DNA content of 4n to 8n was detected. Moreover, the effect of SP600125 on the expression of cell cycle related proteins was an upregulation of p53 protein accompanied by an increase in its molecular mass. Prolonged SP600125 treatment downregulated p21, Bax and Mdm2 expression, but increased the level of the cellular p53-Mdm2 complex. Taken together, we show that SP600125 could induce G2/M cell cycle arrest and endoreduplication in a p21 independent manner, and that SP600125 could also post-translationally modify p53 to modify its function. Our data show that basic JNK activity plays an important role in the progression of the cell cycle at G2/M cell phase.     

Notch1-mediated signaling regulates proliferation of porcine satellite cells (PSCs)

Notch signaling is an evolutionarily conserved cell–cell communication mechanism involved in the regulation of cell proliferation, differentiation and fate decisions of mammalian cells. In the present study, we investigated the possible requirement for Notch signaling in the proliferation and differentiation of porcine satellite cells. We show that Notch1, 2 and 3 are expressed in cultured porcine satellite cells. Knock-down of NOTCH1, but not NOTCH2 and NOTCH3, decreases the proliferation of porcine satellite cells. In contrast, enhancement of NOTCH1 expression via treatment of porcine satellite cells with recombinant NF-κB increases the proliferation of porcine satellite cells. The alteration of porcine satellite cell proliferation is associated with significant changes in the expression of cell cycle related genes (cyclin B1, D1, D2, E1 and p21), myogenic regulatory factors (MyoD and myogenin) and the Notch effector Hes5. In addition, alteration of Notch1 expression in porcine satellite cells causes changes in the expression of GSK3β-3. Taken together, these findings suggest that of the four notch-related genes, Notch1is likely to be required for regulating the proliferation and therefore the maintenance of porcine satellite cells in vivo, and do so through activation of the Notch effector gene Hes5.     

Sepiapterin regulates cell proliferation and migration: its association with integrin α3β1 and p53 in human lung cancer cells

Tetrahydrobiopterin (BH₄) has been known to be an essential cofactor for nitric oxide synthase as well as the aromatic amino acid hydroxylases, which are involved in regulation of cellular fates including proliferation, migration and differentiation. In the present study, we report that sepiapterin, a stable form of BH₄ precursor, modulates proliferation and migration in human lung cancer cells. Sepiapterin induction of cell proliferation in p53 wild-type A549 cells, but not in p53-deficient H1299 cells, is accompanied by enhanced expression of cell cycle-related proteins such as cyclin-dependent kinase 4 (Cdk4), cyclin D and cyclin E, and reduced expression of Cdk inhibitor p21^WAF1/Cip1, demonstrating that sepiapterin-induced mitogenic responses might be associated with p53 expression status in lung cancer cells. In addition, sepiapterin enhances cell migration in A549 cells, but not H1299 cells. Finally, we show that sepiapterin induces A549 cell proliferation and migration through the activation of Akt and p70^S6K signaling pathways, as evidenced by using Akt and p70^S6K inhibitor. Collectively, these findings indicate that sepiapterin might play differential roles in regulation of cellular fates, depending on the status of p53 expression in lung cancer.     

Increased stability of P21(WAF1/CIP1) mRNA is required for ROS/ERK-dependent pancreatic adenocarcinoma cell growth inhibition by pyrrolidine dithiocarbamate

We present evidence that pyrrolidine dithiocarbamate (PDTC) inhibits growth of p53-negative pancreatic adenocarcinoma cell lines via cell cycle arrest in the S-phase, while it has no effect on primary fibroblast proliferation. Growth inhibition of cancer cells is dependent on ROS and ERK1/2 induction as indicated by a significantly reduced PDTC-associated growth inhibition by the free radical scavenger N-acetyl-L-cysteine (NAC) or the MEK/ERK1/2 inhibitor (PD98059). Moreover, ERK1/2 induction is dependent on ROS production as demonstrated by a complete removal of PDTC-mediated ERK1/2 phosphorylation by NAC. p21(WAF1/CIP1) activation has a central role in growth inhibition by PDTC, as revealed by P21(WAF1/CIP1) silencing experiments with antisense oligonucleotide, and occurs via increased mRNA stability largely mediated by ROS/ERK induction. Conversely, PDTC does not affect P21(WAF1/CIP1) gene expression in primary fibroblasts, although it is able to activate p53 and the p53-regulated antioxidant SESN2. These results suggest that the resistance of fibroblasts to the cytotoxic action of PDTC may be related to the up-regulation of p53-dependent antioxidant genes. Finally, in vivo studies on PaCa44 cells subcutaneously xenografted in nude mice show that treatment with 100 or 200 mg/kg PDTC reduces of 30% or 60% the tumour volume, respectively, and does not cause any apparent form of toxicity.     

Subcellular localization of Daxx determines its opposing functions in ischemic cell death

This study examined the role of Daxx in ischemic stress. Upon ischemic stress, nuclear export of Daxx to the cytoplasm was observed in primary myocytes as well as in various cell lines. Daxx silencing using siRNAs was detrimental in tethering PML-nuclear body (PML-NB) constituents together. Overexpression of Daxx (W621A) caused nuclear export of p53 independently of PML and promoted ischemic cell death via activation of JNK. Conversely, overexpression of Daxx (S667A) prevented dissociation of PML-NB constituents and protected cells from ischemic death. Collectively, our results demonstrate that the subcellular localization of Daxx determines its role in ischemic cell death.