p21(WAF¹/C¹P¹) deficiency induces mitochondrial dysfunction in HCT116 colon cancer cells.

p21(WAF1/CIP1) is a critical regulator of cell cycle progression. However, the role of p21 in mitochondrial function remains poorly understood. In this study, we examined the effect of p21 deficiency on mitochondrial function in HCT116 human colon cancer cells. We found that there was a significant increase in the mitochondrial mass of p21(-/-) HCT116 cells, as measured by 10-N-nonyl-acridine orange staining, as well as an increase in the mitochondrial DNA content. In contrast, p53(-/-) cells had a mitochondrial mass comparable to that of wild-type HCT116 cells. In addition, the expression levels of the mitochondrial biogenesis regulators PGC-1α and TFAM and AMPK activity were also elevated in p21(-/-) cells, indicating that p21 deficiency induces the rate of mitochondrial biogenesis through the AMPK-PGC-1α axis. However, the increase in mitochondrial biogenesis in p21(-/-) cells did not accompany an increase in the cellular steady-state level of ATP. Furthermore, p21(-/-) cells exhibited significant proliferation impairment in galactose medium, suggesting that p21 deficiency induces a defect in the mitochondrial respiratory chain in HCT116 cells. Taken together, our results suggest that the loss of p21 results in an aberrant increase in the mitochondrial mass and in mitochondrial dysfunction in HCT116 cells, indicating that p21 is required to maintain proper mitochondrial mass and respiratory function.

Glucose deprivation is associated with Chk1 degradation through the ubiquitin-proteasome pathway and effective checkpoint response to replication blocks.

Chk1 plays a key role in the DNA replication checkpoint and in preserving genomic integrity. Previous studies have shown that reduced Chk1 function leads to defects in the checkpoint response and is closely associated with tumorigenesis. Here, we report that glucose deprivation caused the degradation of Chk1 protein without perturbing cell cycle progression. The induction of Chk1 degradation in response to glucose deprivation was observed in various cancer cell lines and in normal human fibroblasts. Therefore, it appears to be a universal phenomenon in mammalian cells. A specific proteasome inhibitor blocked glucose deprivation-induced Chk1 degradation. Ubiquitination of Chk1 was detected, indicating that the proteasome-ubiquitin pathway mediates Chk1 degradation upon glucose deprivation. Mechanistic studies have demonstrated that ATR-dependent phosphorylation of Chk1 at the Ser317 and Ser345 sites is not required, suggesting that the molecular mechanism for Chk1 degradation upon glucose deprivation is distinct from genotoxic stress-induced degradation. Under conditions of glucose deprivation, the cells manifested a defective checkpoint response to replication stress, camptothecin or hydroxyurea. The forced expression of Myc-Chk1 partially rescued the defective response to the replication block upon glucose deprivation. Taken together, our results indicate that glucose deprivation induces ubiquitin-mediated Chk1 degradation and defective checkpoint responses, implying its potential role in genomic instability and tumor development.

Melatonin suppresses doxorubicin-induced premature senescence of A549 lung cancer cells by ameliorating mitochondrial dysfunction.

Melatonin is an indolamine that is synthesized in the pineal gland and shows a wide range of physiological functions. Although the anti-aging properties of melatonin have been reported in a senescence-accelerated mouse model, whether melatonin modulates cellular senescence has not been determined. In this study, we examined the effect of melatonin on anticancer drug-induced cellular premature senescence. We found that the doxorubicin (DOX)-induced senescence of A549 human lung cancer cells and IMR90 normal lung cells was substantially inhibited by cotreatment with melatonin in a dose-dependent manner. Mechanistically, the DOX-induced G2/M phase cell cycle arrest and the decrease in cyclinB and cdc2 expression were not affected by melatonin. However, the DOX-induced increase in intracellular levels of ROS, which is necessary for premature senescence, was completely abolished upon melatonin cotreatment. In addition, the reduction in mitochondrial membrane potential that occurs upon DOX treatment was inhibited by melatonin. An aberrant increase in mitochondrial respiration was also significantly suppressed by melatonin, indicating that melatonin ameliorates the mitochondrial dysfunction induced by DOX treatment. The treatment of A549 cells with luzindole, a potent inhibitor of melatonin receptors, failed to prevent the effects of melatonin treatment on mitochondrial functions and premature senescence in cells also treated with DOX; this suggests that melatonin suppresses DOX-induced senescence in a melatonin receptor-independent manner. Together, these results reveal that melatonin has an inhibitory effect of melatonin on premature senescence at the cellular level and that melatonin protects A549 cells from DOX-induced senescence. Thus, melatonin might have the therapeutic potential to prevent the side effects of anticancer drug therapy.

Nek6 suppresses the premature senescence of human cancer cells induced by camptothecin and doxorubicin treatment.

Cellular senescence plays an important role in tumor suppression. The mitotic kinase Nek6 has recently been shown to be overexpressed in various cancers and has been implicated in tumorigenesis. Previously, we reported that the down-regulation of Nek6 expression was required for p53-induced senescence. In this study, we examined the effect of Nek6 overexpression on the premature senescence of cancer cells induced by the anticancer drugs camptothecin (CPT) and doxorubicin (DOX). We found that CPT- and DOX-induced morphology changes and increases in senescence-associated ß-galactosidase staining were significantly inhibited in EJ human bladder cancer cells and H1299 human lung cancer cells overexpressing HA-Nek6. DOX-induced G2/M cell cycle arrest and the reduction in cyclin B and cdc2 levels after DOX treatment were significantly reduced by Nek6 overexpression. In addition, an increase in the intracellular levels of ROS in response to DOX was also inhibited in cells overexpressing Nek6. These results suggest that the increased expression of Nek6 renders cancer cells resistant to premature senescence, and targeting Nek6 could be an efficient strategy for cancer treatment.

Isolation and functional analysis of the human glioblastoma-specific promoter region of the human GD3 synthase (hST8Sia I) gene.

We identified the promoter region of the human GD3 synthase (hST8Sia I) gene to elucidate the mechanism underlying the regulation of hST8Sia I expression in human glioblastoma cells. The 5'-rapid amplification of cDNA end using mRNA prepared from U-87MG cells revealed the presence of transcription start site of hST8Sia I gene, and the 5'-terminal analysis of its product showed that transcription started from 648 nucleotides upstream of the translational initiation site. Functional analysis of the 5'-flanking region of the hST8Sia I gene by transient expression method revealed that the region from -638 to -498 is important for transcriptional activity of the hST8Sia I gene in U-87MG and T98G cells. This region lacks apparent TATA and CAAT boxes, but contains putative binding sites for transcription factors AREB6 and Elk-1. Site-directed mutagenesis and transient transfection assays demonstrated that both AREB6 and Elk-1 elements in this region were required for the promoter activity in U-87MG and T98G cells. These results indicated that both AREB6 and Elk-1 might play an essential role in the transcriptional activity of hST8Sia I gene essential for GD3 synthesis in human glioblastoma cells.

Photodynamic Approach for Teratoma-Free Pluripotent Stem Cell Therapy Using CDy1 and Visible Light.

Pluripotent stem cells (PSC) are promising resources for regeneration therapy, but teratoma formation is one of the critical problems for safe clinical application. After differentiation, the precise detection and subsequent elimination of undifferentiated PSC is essential for teratoma-free stem cell therapy, but a practical procedure is yet to be developed. CDy1, a PSC specific fluorescent probe, was investigated for the generation of reactive oxygen species (ROS) and demonstrated to induce selective death of PSC upon visible light irradiation. Importantly, the CDy1 and/or light irradiation did not negatively affect differentiated endothelial cells. The photodynamic treatment of PSC with CDy1 and visible light irradiation confirmed the inhibition of teratoma formation in mice, and suggests a promising new approach to safe PSC-based cell therapy.

The inhibition of Nek6 function sensitizes human cancer cells to premature senescence upon serum reduction or anticancer drug treatment.

The induction of premature senescence in cancer cells was proposed as an effective cancer treatment strategy. In this paper, we show that the inhibition of Nek6 expression by Nek6 siRNA-mediated knockdown or the overexpression of a dominant negative form of Nek6 (Nek6KM) induced premature senescence as well as cell death under reduced serum conditions in multiple cancer cell lines, including both p53 wild-type and p53 mutant/null backgrounds. Moreover, cancer cells expressing Nek6KM exhibited significantly increased premature senescence upon treatment with the anticancer drugs doxorubicin (DOX) and camptothecin (CPT). Significantly, the overexpression of Nek6KM also inhibited tumor growth and promoted premature senescence in vivo in a xenograft mouse model. Taken together, our results further confirm that Nek6 plays an important role in the premature senescence of cancer cells, suggesting that Nek6 may be a potential therapeutic target for human cancers.

Loss of Med1/TRAP220 promotes the invasion and metastasis of human non-small-cell lung cancer cells by modulating the expression of metastasis-related genes.

Med1/TRAP220 is an essential component of the TRAP/Mediator complex. In this study, we present a novel function of Med1 in human non-small-cell lung cancer (NSCLC) progression. We found that the loss of Med1 expression was strongly associated with increased rates of invasion and metastasis in NSCLC patients. Consistent with lung cancer patient data, the knockdown of Med1 in NSCLC cell lines led to an increase in cell migration and invasion. Med1-depleted cells displayed an increase in metastasis in a xenograft tumor model and in an in vivo metastasis assay. Moreover, a microarray analysis revealed that the mRNA levels of the metastasis-related genes uPAR, ID2, ID4, PTP4A1, PKP3, TGM2, PLD1, TIMP2, RGS2, and HOXA4 were altered upon Med1 knockdown. Collectively, these results suggest that the loss of Med1 increases the invasive potential of human NSCLC cells by modulating the expression of metastasis-related genes.

Transcriptional activation of human GM3 synthase (hST3Gal V) gene by valproic acid in ARPE-19 human retinal pigment epithelial cells.

The present study demonstrated that valproic acid (VPA) transcriptionally regulates human GM3 synthase (hST3Gal V), which catalyzes ganglioside GM3 biosynthesis in ARPE-19 human retinal pigment epithelial cells. For this, we characterized the promoter region of the hST3Gal V gene. Functional analysis of the 5'-flanking region of the hST3Gal V gene revealed that the -177 to -83 region functions as the VPA-inducible promoter and that the CREB/ATF binding site at -143 is crucial for VPA-induced expression of hST3Gal V in ARPE-19 cells. In addition, the transcriptional activity of hST3Gal V induced by VPA in ARPE-19 cells was inhibited by SP600125, a c-Jun N-terminal kinase (JNK) inhibitor. In summary, our results identified the core promoter region in the hST3Gal V promoter and for the first time demonstrated that ATF2 binding to the CREB/ATF binding site at -143 is essential for transcriptional activation of hST3Gal V in VPA-induced ARPE-19 cells.

Nek6 overexpression antagonizes p53-induced senescence in human cancer cells.

Nek6 is an NIMA-related kinase that plays a critical role in mitotic cell cycle progression. Recent studies have shown that Nek6 is upregulated in various human cancers, but the function of Nek6 in tumorigenesis is largely unknown. Here, we examined the role of Nek6 in cellular senescence. Our data revealed that Nek6 expression is decreased both in both the replicative senescence of human normal fibroblasts and premature senescence induced by p53 expression in EJ human bladder cancer cells and H1299 human lung cancer cells. Interestingly, the enforced expression of Nek6 in EJ and H1299 cells completely suppresses p53-induced senescence, whereas the expression of kinase-dead Nek6 did not affect p53-induced senescence. Mechanistic studies revealed that cell cycle arrest in the G1 and G2/M phases, as well as the reduction of cyclin B and cdc2 protein level upon p53 expression were significantly reduced by Nek6 overexpression. In addition, p53-induced increases in intracellular levels of ROS were also inhibited in cells overexpressing Nek6. These results suggest that the downregulation of Nek6 expression is required for the onset of p53-induced cellular senescence and imply a possible role of Nek6 in tumorigenesis.