Manganese superoxide dismutase protects the proliferative capacity of confluent normal human fibroblasts

We tested the hypothesis that manganese superoxide dismutase (MnSOD), an antioxidant enzyme, regulates the proliferative potential of confluent human fibroblasts. Normal human skin (AG01522) and lung (WI38, CCL-75) fibroblasts kept in confluence (>95% G(0)/G(1)) showed a significant decrease in their capacity to re-enter the proliferation cycle after 40-60 days. The inhibition of re-entry was accompanied with the age-dependent increase of p16 protein levels in the confluent culture. Adenoviral mediated overexpression of MnSOD during confluent growth suppressed p16, enhanced p21 protein accumulation, and protected fibroblasts against the loss of proliferation potential. Increases in p21 protein levels in MnSOD overexpressing confluent fibroblasts were independent of p53 protein levels. p53 protein levels did not change in control, replication-defective adenovirus containing an insertless vector (AdBgl II), or AdMnSOD-infected confluent cells cultured for 20 and 60 days. In addition, MnSOD-induced protection of the proliferation capacity of confluent fibroblasts was independent of their telomerase activity. However, telomerase-transformed fibroblasts showed increased MnSOD expression in confluent growth, maintaining their capacity to re-enter the proliferation cycle. Although inactivation of the retinoblastoma protein in cells subcultured from the 60-day confluent control, AdBgl II-, and AdMnSOD-infected fibroblasts was identical, only MnSOD-overexpressing cells showed a higher percentage of S-phase. These results support the hypothesis that a redox-sensitive checkpoint regulated the progression of fibroblasts from G(0)/G(1) to S-phase.     

MnSOD activity protects mitochondrial morphology of quiescent fibroblasts from age associated abnormalities

Previously, we have shown manganese superoxide dismutase (MnSOD) activity protects quiescent human normal skin fibroblasts (NHFs) from age associated loss in proliferative capacity. The loss in proliferative capacity of aged vs. young quiescent cells is often characterized as the chronological life span, which is clearly distinct from replicative senescence. We investigate the hypothesis that MnSOD activity protects the mitochondrial morphology from age associated damage and preserves the chronological life span of quiescent fibroblasts. Aged quiescent NHFs exhibited abnormalities in mitochondrial morphology including abnormal cristae formation and increased number of vacuoles. These results correlate with the levels of cellular reactive oxygen species (ROS) and mitochondrial morphology in MnSOD homozygous and heterozygous knockout mouse embryonic fibroblasts. The abnormalities in mitochondrial morphology in aged quiescent NHFs cultured in presence of 21% oxygen concentration were more severe than NHFs cultured in 4% oxygen environment. The alteration in mitochondrial morphology was associated with a significant increase in cell population doubling: 54 h in 21% compared to 44 h in 4% oxygen environment. Overexpression of MnSOD decreased ROS levels, and preserved mitochondrial morphology in aged quiescent NHFs. These results demonstrate that MnSOD activity protects mitochondrial morphology and preserves the proliferative capacities of quiescent NHFs from age associated loss.     

TRPC1: Getting physical in space

We tested a hypothesis that activation of growth-promoting pathways is required for cellular senescence. In the presence of serum, induction of p21 caused senescence, characterized by beta-Galactosidase staining, cell hypertrophy, increased levels of cyclin D1 and active TOR (target of rapamycin, also known as mTOR). Serum starvation and rapamycin inhibited TOR and prevented the expression of some senescent markers, despite high levels of p21 and cell cycle arrest. In the presence of serum, p21-arrested cells irreversibly lost proliferative potential. In contrast, when cells were arrested by p21 in the absence of serum, they retained the capacity to resume proliferation upon termination of p21 induction. In normal human cells such as WI38 fibroblasts and retinal pigment epithelial (RPE) cells, serum starvation caused quiescence, which was associated with low levels of cyclin D1, inactive TOR and slim-cell morphology. In contrast, cellular senescence with high levels of TOR activity was induced by doxorubicin (DOX), a DNA damaging agent, in the presence of serum. Inhibition of TOR partially prevented senescent phenotype caused by DOX. Thus growth stimulation coupled with cell cycle arrest leads to senescence, whereas quiescence (a condition with inactive TOR) prevents senescence.     

Enhanced expression of mitochondrial superoxide dismutase leads to prolonged in vivo cell cycle progression and up-regulation of mitochondrial thioredoxin

Mn superoxide dismutase (MnSOD) is an important mitochondrial antioxidant enzyme, and elevated MnSOD levels have been shown to reduce tumor growth in part by suppressing cell proliferation. Studies with fibroblasts have shown that increased MnSOD expression prolongs cell cycle transition time in G1/S and favors entrance into the quiescent state. To determine if the same effect occurs during tissue regeneration in vivo, we used a transgenic mouse system with liver-specific MnSOD expression and a partial hepatectomy paradigm to induce synchronized in vivo cell proliferation during liver regeneration. We show in this experimental system that a 2.6-fold increase in MnSOD activity leads to delayed entry into S phase, as measured by reduction in bromodeoxyuridine (BrdU) incorporation and decreased expression of proliferative cell nuclear antigen (PCNA). Thus, compared to control mice with baseline MnSOD levels, transgenic mice with increased MnSOD expression in the liver have 23% fewer BrdU-positive cells and a marked attenuation of PCNA expression. The increase in MnSOD activity also leads to an increase in the mitochondrial form of thioredoxin (thioredoxin 2), but not in several other peroxidases examined, suggesting the importance of thioredoxin 2 in maintaining redox balance in mitochondria with elevated levels of MnSOD.     

Late ROS accumulation and radiosensitivity in SOD1-overexpressing human glioma cells

This study investigates the hypothesis that CuZn superoxide dismutase (SOD1) overexpression confers radioresistance to human glioma cells by regulating the late accumulation of reactive oxygen species (ROS) and the G(2)/M-checkpoint pathway. U118-9 human glioma cells (wild type, neo vector control, and stably overexpressing SOD1) were irradiated (0-10 Gy) and assayed for cell survival, cellular ROS levels, cell-cycle-phase distributions, and cyclin B1 expression. SOD1-overexpressing cells were radioresistant compared to wild-type (wt) and neo vector control (neo) cells. Irradiated wt and neo cells showed a significant increase (approximately twofold) in DHE fluorescence beginning at 2 days postirradiation, which remained elevated at 8 days postirradiation. Interestingly, the late accumulation of ROS was suppressed in irradiated SOD1-overexpressing cells. The increase in ROS levels was followed by a decrease in cell growth and viability and an increase in the percentage of cells with sub-G(1) DNA content. SOD1 overexpression enhanced radiation-induced G(2) accumulation within 24 h postirradiation, which was accompanied by a decrease in cyclin B1 mRNA and protein levels. These results support the hypothesis that long after radiation exposure a "metabolic redox response" regulates radiosensitivity of human glioma cells.     

Cyclin-dependent kinase inhibitor p27(Kip1) is required for mouse mammary gland morphogenesis and function

We have studied the role of the cyclin-dependent kinase (Cdk) inhibitor p27(Kip1) in postnatal mammary gland morphogenesis. Based on its ability to negatively regulate cyclin/Cdk function, loss of p27 may result in unrestrained cellular proliferation. However, recent evidence about the stabilizing effect of p27 on cyclin D1-Cdk4 complexes suggests that p27 deficiency might recapitulate the hypoplastic mammary phenotype of cyclin D1-deficient animals. These hypotheses were investigated in postnatal p27-deficient (p27(-/-)), hemizygous (p27(+/)-), or wild-type (p27(+/+)) mammary glands. Mammary glands from p27(+/)- mice displayed increased ductal branching and proliferation with delayed postlactational involution. In contrast, p27(-/-) mammary glands or wild-type mammary fat pads reconstituted with p27(-/-) epithelium produced the opposite phenotype: hypoplasia, low proliferation, decreased ductal branching, impaired lobuloalveolar differentiation, and inability to lactate. The association of cyclin D1 with Cdk4, the kinase activity of Cdk4 against pRb in vitro, the nuclear localization of cyclin D1, and the stability of cyclin D1 were all severely impaired in p27(-/-) mammary epithelial cells compared with p27(+/+) and p27(+/-) mammary epithelial cells. Therefore, p27 is required for mammary gland development in a dose-dependent fashion and positively regulates cyclin D-Cdk4 function in the mammary gland.     

Sirt3-mediated deacetylation of evolutionarily conserved lysine 122 regulates MnSOD activity in response to stress

Genetic deletion of the mitochondrial deacetylase sirtuin-3 (Sirt3) results in increased mitochondrial superoxide, a tumor-permissive environment, and mammary tumor development. MnSOD contains a nutrient- and ionizing radiation (IR)-dependent reversible acetyl-lysine that is hyperacetylated in Sirt3?/? livers at 3 months of age. Livers of Sirt3?/? mice exhibit decreased MnSOD activity, but not immunoreactive protein, relative to wild-type livers. Reintroduction of wild-type but not deacetylation null Sirt3 into Sirt3?/? MEFs deacetylated lysine and restored MnSOD activity. Site-directed mutagenesis of MnSOD lysine 122 to an arginine, mimicking deacetylation (lenti-MnSOD(K122-R)), increased MnSOD activity when expressed in MnSOD?/? MEFs, suggesting acetylation directly regulates function. Furthermore, infection of Sirt3?/? MEFs with lenti-MnSOD(K122-R) inhibited in vitro immortalization by an oncogene (Ras), inhibited IR-induced genomic instability, and decreased mitochondrial superoxide. Finally, IR was unable to induce MnSOD deacetylation or activity in Sirt3?/? livers, and these irradiated livers displayed significant IR-induced cell damage and microvacuolization in their hepatocytes.     

RNA干扰Ｃyclin D1基因表达对瘢痕疙瘩成纤维细胞增殖和G1期调控的影响

为研究siRNA干扰瘢痕疙瘩成纤维细胞cyclin D1基因表达,对瘢痕疙瘩成纤维细胞的增殖、细胞周期和G1期调控的影响,构建了靶向cyclin D1的siRNA表达质粒.利用LipofecmmineTM2000转染体外培养的瘢痕疙瘩成纤维细胞,应用荧光定量PCR、RT-PCR检测cyclin D1 mRNA的干扰效果,应用MTT法、流式细胞仪检测细胞增殖和细胞周期的变化,应用免疫组织化学染色检测成纤维细胞中cyclin D1、CDK4、P16、pRb蛋白表达的影响.主要结果如F:a.靶向cyclin D1的特异性siRNA序列可以高效地抑制成纤维细胞cyclin D1基因表达,对照组与实验组在mRNA水平其表达抑制率分别为63.68%和92.83%(P<0.01);b.可以显著抑制瘢痕疙瘩成纤维细胞的增殖,改变细胞周期分布,G0/G1期细胞比例显著高于各对照组(P<0.05),细胞分裂被阻滞;c.免疫组化染色发现,转染72 h后,过表达的cyclin D1、CDK4和pRb蛋白,在瘢痕疙瘩成纤维细胞中均出现了不同程度的表达下调,而低表达的P16则呈上调表现.由上述结果可见,构建的靶向cyclin D1的RNAi表达质粒,可有效地抑制瘢痕疙瘩成纤维细胞cyclin D1基因表达,通过改变Gl期相关周期蛋白的水平,影响G1/S期的进程,显著地抑制成纤维细胞的增殖.     

Iron chelation study in a normal human hepatocyte cell line suggests that tumor necrosis factor receptor-associated protein 1 (TRAP1) regulates production of reactive oxygen species

Iron is an essential component of many proteins, and has crucial roles in the proper functioning of proteins involved in cellular respiration, proliferation, and differentiation. It has been recently reported that the deferoxamine (DFO), an iron chelator, induces mitochondrial dysfunction, characterized by an attenuation of oxidative phosphorylation, as well as senescence-like cellular morphology. However, the effects of DFO on mitochondrial heat shock proteins (HSPs) remain poorly understood. In this study, we examined the effect of DFO on tumor necrosis factor receptor-associated protein 1 (TRAP1), a representative mitochondrial HSP, in a normal human hepatocyte cell line, Chang cells. DFO specifically decreased TRAP1 levels, increasing reactive oxygen species (ROS) and caveolin-1 (Cav-1), a marker protein of senescence. To examine whether these effects of DFO are reversed, we established TRAP1-overexpressing Chang cells. DFO treatment to TRAP1-overexpressing cells resulted in decreases in levels of ROS, Cav-1, glutathione peroxidase (GPX), and manganese superoxide dismutase (MnSOD) levels as well as senescence-associated beta-galactosidase (SA beta-gal) activity. These results suggest that TRAP1 might play a role in protecting mitochondria against damaging stimuli via decrease of ROS generation.     

Cyclin D1 inhibits peroxisome proliferator-activated receptor gamma-mediated adipogenesis through histone deacetylase recruitment

The cyclin D1 gene encodes the labile serum-inducible regulatory subunit of a holoenzyme that phosphorylates and inactivates the retinoblastoma protein. Overexpression of cyclin D1 promotes cellular proliferation and normal physiological levels of cyclin D1 function to inhibit adipocyte differentiation in vivo. We have previously shown that cyclin D1 inhibits peroxisome proliferator-activated receptor (PPAR)gamma-dependent activity through a cyclin-dependent kinase- and retinoblastoma protein-binding-independent mechanism. In this study, we determined the molecular mechanism by which cyclin D1 regulated PPARgamma function. Herein, murine embryonic fibroblast (MEF) differentiation by PPARgamma ligand was associated with a reduction in histone deacetylase (HDAC1) activity. Cyclin D1-/- MEFs showed an increased propensity to undergo differentiation into adipocytes. Genetic deletion of cyclin D1 reduced HDAC1 activity. Reconstitution of cyclin D1 into the cyclin D1-/- MEFs increased HDAC1 activity and blocked PPARgamma-mediated adipogenesis. PPARgamma activity was enhanced in cyclin D1-/- cells. Reintroduction of cyclin D1 inhibited basal and ligand-induced PPARgamma activity and enhanced HDAC repression of PPARgamma activity. Cyclin D1 bound HDAC in vivo and preferentially physically associated with HDAC1, HDAC2, HDAC3, and HDAC5. Chromatin immunoprecipitation assay demonstrated that cyclin D1 enhanced recruitment of HDAC1 and HDAC3 and histone methyltransferase SUV39H1 to the PPAR response element of the lipoprotein lipase promoter and decreased acetylation of total histone H3 and histone H3 lysine 9. Collectively, these studies suggest an important role of cyclin D1 in regulation of PPARgamma-mediated adipocyte differentiation through recruitment of HDACs to regulate PPAR response element local chromatin structure and PPARgamma function.     

Induction of apoptosis by adenovirus-mediated manganese superoxide dismutase overexpression in SV-40-transformed human fibroblasts

Although mitochondrial reactive oxygen species (ROS) have been implicated both as an initiator and as an effector of apoptosis, the exact role of mitochondrial ROS has been difficult to establish due to the lack of an appropriate experimental system where ROS could be specifically generated from mitochondria and subsequent effects on cells analyzed. In this study, a manganese superoxide dismutase (MnSOD) activity-mediated apoptosis model was established and characterized. It was shown that despite early increases in the steady-state levels of ROS upon MnSOD overexpression, cellular oxidative damage was decreased significantly at later time points. Alterations in levels of peroxiredoxin (Prxn1) protein preceded the onset of apoptosis after MnSOD overexpression. A time course study demonstrated that increases in MnSOD activity prior to the onset of apoptosis correlated with alterations in the levels of nitration of tyrosine residue(s) of MnSOD protein. A direct correlation between MnSOD activity and the degree of apoptosis was demonstrated using a mutant MnSOD with decreased activity. The current study supports a causative role of mitochondrial ROS leading to the onset of apoptosis. The MnSOD activity-mediated apoptosis model described here could be further utilized to study mitochondrial apoptotic pathways.     

Overexpression of Manganese Superoxide Dismutase Promotes the Survival of Prostate Cancer Cells Exposed to Hyperthermia

It has been hypothesized that exposure of cells to hyperthermia results in an increased flux of reactive oxygen species (ROS), primarily superoxide anion radicals, and that increasing antioxidant enzyme levels will result in protection of cells from the toxicity of these ROS. In this study, the prostate cancer cell line, PC-3, and its manganese superoxide dismutase (MnSOD)-overexpressing clones were subjected to hyperthermia (43°C, 1?h). Increased expression of MnSOD increased the mitochondrial membrane potential (MMP). Hyperthermic exposure of PC-3 cells resulted in increased ROS production, as determined by aconitase inactivation, lipid peroxidation, and H₂O₂ formation with a reduction in cell survival. In contrast, PC-3 cells overexpressing MnSOD had less ROS production, less lipid peroxidation, and greater cell survival compared to PC-3 Wt cells. Since MnSOD removes superoxide, these results suggest that superoxide free radical or its reaction products are responsible for part of the cytotoxicity associated with hyperthermia and that MnSOD can reduce cellular injury and thereby enhance heat tolerance.     

PACSIN 2 represses cellular migration through direct association with cyclin D1 but not its alternate splice form cyclin D1b

Cyclin D1 overexpression is a common feature of many human malignancies. Genomic deletion analysis has demonstrated a key role for cyclin D1 in cellular proliferation, angiogenesis and cellular migration. To investigate the mechanisms contributing to cyclin D1 functions, we purified cyclin D1a-associated complexes by affinity chromatography and identified the PACSIN 2 (protein kinase C and casein kinase substrate in neurons 2) protein by mass spectrometry. The PACSIN 2, but not the related PACSIN 1 and 3, directly bound wild-type cyclin D1 (cyclin D1a) at the carboxyl terminus and failed to bind cyclin D1b, the alternative splicing variant of cyclin D1. PACSIN 2 knockdown induced cellular migration and reduced cell spreading in LNCaP cells expressing cyclin D1a. In cyclin D1^−/− mouse embryonic fibroblasts (MEFs), cyclin D1a, but not cyclin D1b, reduced the cell spreading to a polarized morphology. siPACSIN 2 had no effect on cellular migration of cyclin D1^−/− MEFs. Cyclin D1a restored the migratory ability of cyclin D1^−/− MEFs, which was further enhanced by knocking down PACSIN 2 with siRNA. The cyclin D1-associated protein, PACSIN 2, regulates cell spreading and migration, which are dependent on cyclin D1 expression.Key words: PACSIN 2, cyclin D1, polymorphism, cellular migration, cell spreading, cancer     

Inhibition of cell growth in NIH/3T3 fibroblasts by overexpression of manganese superoxide dismutase: Mechanistic studies

NIH/3T3 mouse fibroblasts were transfected with the cDNA for manganese superoxide dismutase (MnSOD), and two clones overexpressing MnSOD activity were subsequently characterized by comparison with parental and control plasmid-transfected cells. One clone with a 1.8-fold increase in MnSOD activity had a 1.5-fold increase in glutathione peroxidase (GPX) activity (increased GPX-adapted clone), while a second clone with a 3-fold increase in MnSOD activity had a 2-fold decrease in copper, zinc superoxide dismutase (CuZnSOD) activity (decreased CuZnSOD-adapted clone). Increased reactive oxygen species (ROS) levels compared with parental or control plasmid-transfected cells were observed in nonsynchronous cells in the increased GPX-adapted clone, but not in the decreased CuZnSOD-adapted clone. The two MnSOD-overexpressing clones showed different sensitivities to agents that generate oxidative stress. Flow cytometry analysis of the cell cycle showed altered cell cycle progression in both MnSOD-overexpressing clones. During logarithmic growth, both MnSOD-overexpressing clones showed increased mitochondrial membrane potential compared with parental and control plasmid-transfected cells. Both MnSOD-overexpressing clones showed a decrease in mitochondrial mass at the postconfluent phase of growth, suggesting that mitochondrial mass may be regulated by MnSOD and/or ROS levels. Our results indicate that adaptation of fibroblasts to overexpression of MnSOD can involve more than one mechanism, with the resultant cell phenotype dependent on the adaptation mechanism utilized by the cell. J. Cell. Physiol. 175:359–369, 1998. © 1998 Wiley-Liss, Inc.