GM00637 Cells Research Articles

Mitochondrial receptor NIX meidates mitophagy and protects HeLa cells in against Cdinduced toxicity.

Heavy metals exposure are coupled with cellular damages. Our previous studies have demonstrated induction of ROS dependent mitochondrial damages and decreased cell viability in response to heavy metals exposure. Damaged mitochondria are selectively removed by PINK1/Parkin pathway in response to heavy metal exposures. Moreover, there exist several other mitophagy pathways, which are activated under various conditions and may crosstalk with each other. In present study we used HeLa cells which have mutated non‐functional Parkin, thus unable to conduct Parkin‐mediated mitophagy, to evaluate the role of other mitophagy pathways induced by Cd. Our results showed that Cd exposure could induce mitophagy in HeLa cells, lacking Parkin, and GM00637 cells, having intact Parkin, which was evaluated by translocation of mitochondria to lysosomes and decreased mitochondrial markers. In GM00637 cells, the mitophagy process was mediated by PINK1/Parkin pathway as confirmed by noticing Parkin translocation to mitochondria. However, in HeLa cells, no Parkin was detected. Interestingly, NIX expression was significantly increased in HeLa cells exceptionally. Moreover knocking down of NIX in HeLa cells resulted in decreased cell viability and inhibition of mitophagy process in HeLa cells. Finally, Cd exposure significantly increased NIX phosphorylation at serine‐81. These finding suggest a compensatory role of NIX in heavy metals mediated mitophagy when Parkin is not present.

NIX compensates lost role of parkin in cd-induced mitophagy in HeLa cells through phosphorylation

Our previous study demonstrated that cadmium (Cd) is an effective inducer of mitophagy, which is mainly mediated by PINK1/Parkin pathway. However, the role of other mitophagy pathways in Cd-induced mitophagy remains elusive. The present study employed HeLa cells, lacking fully functional Parkin, as a cell model to study Parkin-independent mitophagy pathway induced by Cd. Our results showed that BCL2/adenovirus E1B 19 kDa protein-interacting protein 3-like (Bnip3L/NIX), an outer mitochondrial membrane mitophagy receptor, could provide an alternate pathway for Cd-induced mitophagy in HeLa cells. Specifically, 10 μM Cd for 12 h induced mitophagy in GM00637 and HeLa cells which was assessed by mitochondrial fusion to lysosomes and decreased expression of mitochondrial markers such as COX-IV and HSP60. Notably, in GM00637 cells, Cd-induced mitophagy was predominantly mediated by PINK1/Parkin pathway as evinced by translocation of Parkin to mitochondria. Interestingly, in HeLa cells, significant increase in NIX expression was occurred and mitophagy was induced under Cd exposure, suggesting NIX compensates lost role of Parkin in Cd-induced mitophagy in HeLa cells. These results were verified by knocking down NIX using siRNA in HeLa cells, which lead to abolished mitophagy process. Moreover, NIX phosphorylation at serine-81 significantly increased in cells treated with Cd implying that phosphorylation of NIX plays an important role in NIX-mediated mitophagy. These findings reveal a novel mechanism of Cd toxicity and suggest a compensatory role of NIX in Cd-induced mitophagy.

Targeted Quantitative Proteomics Revealed Arsenite-induced Proteasomal Degradation of RhoB in Fibroblast Cells.

Arsenic is a toxicant widely present in the environment. Previous epidemiological and animal studies support that arsenic exposure is associated with elevated incidences of lung and skin cancers. Therefore, it is important to understand the molecular mechanisms through which arsenite initiates malignant transformation of lung and skin tissues. Ras superfamily of small GTPases assumes a crucial role in many cellular processes including transcription, protein synthesis, and trafficking. In addition, small GTPase signaling is known to be altered in many types of cancer. By employing a multiple-reaction monitoring (MRM)-based targeted proteomic method, we found that the protein level of RhoB was substantially decreased in IMR90 human lung fibroblast cells upon a 12-h exposure to 5 μM NaAsO2. In addition, the protein level of ectopically expressed RhoB was found to decline in a dose-dependent manner upon arsenite exposure in HEK293T, HeLa, and GM00637 cells as well as that of endogenous RhoB protein in IMR90 cells. Moreover, the arsenite-elicited down-regulation of RhoB was found to arise from enhanced proteasomal degradation. Taken together, we demonstrated, for the first time, that exposure to arsenite could attenuate the protein expression of RhoB through proteasomal degradation.

The toxicity and the acaricidal mechanism against Psoroptes cuniculi of the methanol extract of Adonis coerulea Maxim

ScopeAdonis coerulea Maxim. is a perennial herbaceous plant that grows in scrub, grassy slope areas, and as traditional medicine it has been used to treat animal acariasis for thousands of years. In this paper, we aimed to study the acute toxicity and cytotoxicity of the methanol extract of A. coerulea (MEAC) in vivo and in vitro for supporting the clinic uses. The acaricidal activity and the mechanism of action against Psoroptes cuniculi were investigated. ResultsThe results showed that isoorientin, luteolin and apigenin were the primary compounds in MEAC. The toxicity test showed that median lethal dose (LD50) and the 50% inhibitory concentration (IC50) of MEAC were estimated to be more than 5000mg/kg in mice in vivo and more than 50mg/ml against RAW 264.7 and GM00637 cells in the 3-(4, 5-dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide (MTT) test. After culturing with MEAC, the activities of superoxide dismutase (SOD), catalase (CAT), malonyldialdehyde (MDA), glutathione-S-transferase (GST), acetylcholinesterase (AChE) and Na+-K+-ATPase of mites were evaluated. Compared with the control group, SOD activity of MEAC-treated group of mites was inhibited, and CAT activity was activated at the preliminary phase but was gradually inhibited over the period of incubation. MDA content reached a peak at 6h and then gradually decreased. However, GST activity in the mites was activated in a dose- and time-dependent manner. AChE and Na+-K+-ATPase activities related to neural conduction, vital functions and the transmembrane ion gradient of the mites were inhibited. ConclusionMEAC is safe in the given doses in both the in vitro and the in vivo tests, can be applied in the clinic and it had good acaricidal activity. The extension of the incubation time in the mites led to dynamic disequilibrium between the production and clearing of superoxide anions, a disruption of the energy metabolism and the transmembrane ion gradient, and the inhibition of motor function. These factors may have resulted in mite death.

ATM mediates spermidine-induced mitophagy via PINK1 and Parkin regulation in human fibroblasts.

The ATM (ataxia telangiectasia mutated) protein has recently been proposed to play critical roles in the response to mitochondrial dysfunction by initiating mitophagy. Here, we have used ATM-proficient GM00637 cells and ATM-deficient GM05849 cells to investigate the mitophagic effect of spermidine and to elucidate the role of ATM in spermdine-induced mitophagy. Our results indicate that spermidine induces mitophagy by eliciting mitochondrial depolarization, which triggers the formation of mitophagosomes and mitolysosomes, thereby promoting the accumulation of PINK1 and translocation of Parkin to damaged mitochondria, finally leading to the decreased mitochondrial mass in GM00637 cells. However, in GM05849 cells or GM00637 cells pretreated with the ATM kinase inhibitor KU55933, the expression of full-length PINK1 and the translocation of Parkin are blocked, and the colocalization of Parkin with either LC3 or PINK1 is disrupted. These results suggest that ATM drives the initiation of the mitophagic cascade. Our study demonstrates that spermidine induces mitophagy through ATM-dependent activation of the PINK1/Parkin pathway. These findings underscore the importance of a mitophagy regulatory network of ATM and PINK1/Parkin and elucidate a novel mechanism by which ATM influences spermidine-induced mitophagy.

Sulfotransferase-1A1-dependent bioactivation of aristolochic acid I and N-hydroxyaristolactam I in human cells.

Aristolochic acids (AA) are implicated in the development of chronic renal disease and upper urinary tract carcinoma in humans. Using in vitro approaches, we demonstrated that N-hydroxyaristolactams, metabolites derived from partial nitroreduction of AA, require sulfotransferase (SULT)-catalyzed conjugation with a sulfonyl group to form aristolactam-DNA adducts. Following up on this observation, bioactivation of AA-I and N-hydroxyaristolactam I (AL-I-NOH) was studied in human kidney (HK-2) and skin fibroblast (GM00637) cell lines. Pentachlorophenol, a known SULT inhibitor, significantly reduced cell death and aristolactam-DNA adduct levels in HK-2 cells following exposure to AA-I and AL-I-NOH, suggesting a role for Phase II metabolism in AA activation. A gene knockdown, siRNA approach was employed to establish the involvement of selected SULTs and nitroreductases in AA-I bioactivation. Silencing of SULT1A1 and PAPSS2 led to a significant decrease in aristolactam-DNA levels in both cell lines following exposure to AA-I, indicating the critical role for sulfonation in the activation of AA-I in vivo Since HK-2 cells proved relatively resistant to knockdown with siRNAs, gene silencing of xanthine oxidoreductase, cytochrome P450 oxidoreductase and NADPH:quinone oxidoreductase was conducted in GM00637 cells, showing a significant increase, decrease and no effect on aristolactam-DNA levels, respectively. In GM00637 cells exposed to AL-I-NOH, suppressing the SULT pathway led to a significant decrease in aristolactam-DNA formation, mirroring data obtained for AA-I. We conclude from these studies that SULT1A1 is involved in the bioactivation of AA-I through the sulfonation of AL-I-NOH, contributing significantly to the toxicities of AA observed in vivo.

Abstract 5097: APE1/Ref-1 promotes cell adhesion and migration in cervical cancer cells

Abstract Apurinic and apyrimidinic endonuclease 1 (APE1)/redox effector factor-1 (Ref-1) is known to be involved in DNA base excision repair pathway and redox-dependent regulation of a number of transcription factors, such as AP1, p53, and Pax-8. It is expressed in numerous human cancers such as pancreatic cancer, prostate cancer, breast cancer, cervical cancer, and colon cancer. Our previous studies suggested APE1/Ref-1 can promote cancer progression through its involvement in cell proliferation, metastasis and angiogenesis; however, the molecular mechanism by which APE1/Ref-1 functions in these processes has not been characterized. In this study, we are the first to report that APE1/Ref-1 activates cell adhesion-related proteins to trigger cell migration. To investigate whether APE1/Ref-1 is involved in cell adhesion in normal fibroblasts, an adhesion assay was performed using GM00637 human fibroblast cell lines stably overexpressing APE1/Ref-1. Cell adhesion to vitronectin, a known ligand of the adhesion molecules integrin ανβ3 and integrin ανβ5, was significantly increased in APE1/Ref-1-overexpressing GM00637 cells compared to control fibroblast cells. FACS analysis showed APE1/Ref-1 induces the expression and activation of adhesion molecules including integrin αν and β3 in APE/Ref-1-overexpressing GM00637 cells. To evaluate whether APE1/Ref-1-mediated regulation of integrin ανβ3 is involved in cell migration in cervical cancer cells, a migration assay was performed using APE1/Ref-1-specific siRNA and an integrin ανβ3-specific antibody. Cell migration was significantly decreased in the absence of APE1/Ref-1 expression in cervical cancer cells, which was comparable with inhibition of integrin ανβ3. Consistent with these results, APE1/Ref-1 increased the activation and expression of integrin ανβ3 in both cervical cancer cells and cervical cancer patient tissue. Combined, these studies suggest that APE/Ref-1 can promote cell adhesion and migration through integrin ανβ3 signaling and thereby this signaling mechanism could contribute to cervical cancer progression. Citation Format: Mihwa Kim, Ho Jin You, Dae Joon Kim. APE1/Ref-1 promotes cell adhesion and migration in cervical cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5097. doi:10.1158/1538-7445.AM2015-5097

BLM is required for faithful chromosome segregation and its localization defines a class of ultrafine anaphase bridges

Mutations in BLM cause Bloom's syndrome, a disorder associated with cancer predisposition and chromosomal instability. We investigated whether BLM plays a role in ensuring the faithful chromosome segregation in human cells. We show that BLM-defective cells display a higher frequency of anaphase bridges and lagging chromatin than do isogenic corrected derivatives that eptopically express the BLM protein. In normal cells undergoing mitosis, BLM protein localizes to anaphase bridges, where it colocalizes with its cellular partners, topoisomerase IIIalpha and hRMI1 (BLAP75). Using BLM staining as a marker, we have identified a class of ultrafine DNA bridges in anaphase that are surprisingly prevalent in the anaphase population of normal human cells. These so-called BLM-DNA bridges, which also stain for the PICH protein, frequently link centromeric loci, and are present at an elevated frequency in cells lacking BLM. On the basis of these results, we propose that sister-chromatid disjunction is often incomplete in human cells even after the onset of anaphase. We present a model for the action of BLM in ensuring complete sister chromatid decatenation in anaphase.

Mutation Spectra Induced by α-Acetoxytamoxifen−DNA Adducts in Human DNA Repair Proficient and Deficient (Xeroderma Pigmentosum Complementation Group A) Cells

Tamoxifen, a breast cancer drug, has recently been approved for the chemoprevention of this disease. However, tamoxifen causes hepatic carcinomas in rats through a genotoxic mechanism and increases the risk of endometrial tumors in women. DNA adducts have been detected at low levels in human endometrium, and there is much interest in determining whether DNA damage plays a role in tamoxifen-induced endometrial carcinogenesis. This study investigates the mutagenicity of tamoxifen DNA adducts formed by alpha-acetoxytamoxifen, a reactive ester producing the major DNA adduct formed in livers of tamoxifen-treated rats. pSP189 plasmid DNA containing the supF gene was treated with alpha-acetoxytamoxifen and adduct levels (0.5-8.0 adducts per plasmid) determined by (32)P-postlabeling. Adducted plasmids were transfected into nucleotide excision repair proficient (GM00637) or deficient (GM04429, XPA) human fibroblasts. After replication, plasmids were recovered and screened in indicator bacteria. Relative mutation frequencies increased with the adduct level, with 1.3-3.6-fold higher numbers of mutations in the XP cells compared to the GM00637 cells, indicating that NER plays a significant role in the removal of these particular tamoxifen DNA adducts. The majority of sequence alterations (91-96%) occurred at GC base pairs, as did mutation hotspots, although the type and position of mutations was cell-specific. In both cell lines, as the adduct level increased, the proportion of GC --> AT transitions decreased and GC --> TA transversions, mutations known to arise from the major tamoxifen adducts, increased. Given the high mutagenicity of dG-N(2)-tamoxifen adducts, if not excised, they may potentially contribute to the initiation of endometrial cancer in women.

Metallothionein-III Prevents γ-Ray-induced 8-Oxoguanine Accumulation in Normal and hOGG1-depleted Cells

Metallothioneins (MT) play an important biological role in preventing oxidative damage to cells. We have previously demonstrated that the efficiency of the protective effect of MT-III against the DNA degradation from oxidative damage was much higher than that of MT-I/II. As an extension of the latter investigation, this study aimed to assess the ability of MT-III to suppress 8-oxoguanine (8-oxoG), which is one of the major base lesions formed after an oxidative attack to DNA and the mutant frequency of the HPRT gene in human fibroblast GM00637 cells upon exposure to gamma-rays. We found that human MT-III expression decreased the level of 8-oxoG and mutation frequency in the gamma-irradiated cells. Using an 8-oxoguanine DNA glycosylase (OGG1)-specific siRNAs, we also found that MT-III expression resulted in the suppression of the gamma-radiation-induced 8-oxoG accumulation and mutation in the OGG1-depleted cells. Moreover, the down-regulation of MT in human neuroblastoma SKNSH cells induced by MT-specific siRNA led to a significant increase in the 8-oxoG level, after exposure to gamma-irradiation. These results suggest that under the conditions of gamma-ray oxidative stress, MT-III prevents the gamma-radiation-induced 8-oxoG accumulation and mutation in normal and hOGG1-depleted cells, and this suppression might, at least in part, contribute to the anticarcinogenic and neuroprotective role of MT-III.

Enhanced expression of Mcm proteins in cancer cells derived from uterine cervix.

Minichromosome maintenance proteins (Mcm) 2-7 play essential roles in eukaryotic DNA replication. Several reports have indicated the usefulness of Mcm proteins as markers of cancer cells in histopathological diagnosis. However, their mode of expression and pathophysiological significance in cancer cells remain to be clarified. We compared the level of expression of Mcm proteins among human HeLa uterine cervical carcinoma cells, SV40-transformed human fibroblast GM00637 cells and normal human fibroblast WI-38 cells. All the proteins examined were detected in HeLa and GM cells at 6-10 times the level found in WI-38 cells on average. This increase was observed both in total cellular proteins and in the chromatin-bound fraction. Consistently, Mcm2 mRNA was enriched in HeLa cells to approximately four times the level in WI-38 cells, and the synthesis of Mcm4, 6 and 7 proteins was accelerated in HeLa cells. Immunohistochemical studies of surgical materials from human uterine cervix showed that Mcm3 and 4 are ubiquitously expressed in cancer cells. Further, the positive rate and level of Mcm3 and 4 expression appeared to be higher in cancer cells than in normal proliferating cells of the uterine cervix and dysplastic cells, suggesting that they can be useful markers to distinguish these cells.

Proteasome-dependent downregulation of p21(Waf1/Cip1) induced by reactive oxygen species.

After hydrogen peroxide (H(2)O(2)) treatment, the p21 (p21(Waf1/Cip1)) protein level in GM00637 fibroblast cells was rapidly decreased, reaching its nadir around 3 h. However, it rebounded within 5 hours to a level higher than that before treatment. Fluorescence microscopic analyses revealed that nuclear p21 was downregulated during the initial oxidative stress. H(2)O(2)-induced downregulation of p21 protein was accompanied by a gradual increase in p21 mRNA levels. Other inducers of genotoxic stress, such as treatment with adriamycin, a DNA damage compound, did not cause a significant decrease in p21 protein levels. Pretreatment of GM00637 cells with the proteasome inhibitors, lactacystin or MG132, completely blocked H(2)O(2)-induced p21 downregulation, suggesting that H(2)O(2) treatment accelerated p21 degradation. Conversely, cotreatment of cells with a protein synthesis inhibitor, cycloheximide, and H(2)O(2) drastically shortened the half-life of p21. Moreover, p21 mRNA levels were not downregulated by treatment with proteasome or protein synthesis inhibitors. Taken together, our studies indicate that oxidative stress induces rapid, but reversible, downregulation of functional p21 by accelerating its protein turnover.

Overexpression of human metallothionein-III prevents hydrogen peroxide-induced oxidative stress in human fibroblasts

Metallothioneins (MT) are ubiquitous low molecular weight metal binding proteins that may act as antioxidants. In the present study, the cloned human MT-III coding region was permanently transfected into GM00637 cells in order to investigate the antioxidative effects of this brain-specific MT isoform. GM00637/MT-III cells overexpressed MT-III mRNA versus pcDNA3 plasmid-transfected control cells (GM00637). When challenged with H 2O 2, the GM00637/MT-III cells displayed significantly more resistance than the GM00637 cells, as determined by cell cytotoxicity, lactate dehydrogenase leakage, and lipid peroxidation. In addition, the GM00637/MT-III cells were highly protected from the H 2O 2-induced production of reactive oxygen species and DNA damage. These results directly support the antioxidative role of MT-III, and demonstrate MT-III can scavenge free oxygen radicals and protect cells from oxidative stress.

Plk3 functionally links DNA damage to cell cycle arrest and apoptosis at least in part via the p53 pathway.

Polo-like kinase 3 (Plk3, previously termed Prk) contributes to regulation of M phase of the cell cycle (Ouyang, B., Pan, H., Lu, L., Li, J., Stambrook, P., Li, B., and Dai, W. (1997) J. Biol. Chem. 272, 28646-28651). Plk3 physically interacts with Cdc25C and phosphorylates this protein phosphatase predominantly on serine 216 (Ouyang, B., Li, W., Pan, H., Meadows, J., Hoffmann, I., and Dai, W. (1999) Oncogene 18, 6029-6036), suggesting that the role of Plk3 in mitosis is mediated, at least in part, through direct regulation of Cdc25C. Here we show that ectopic expression of a kinase-active Plk3 (Plk3-A) induced apoptosis. In response to DNA damage, the kinase activity of Plk3 was rapidly increased in an ATM-dependent manner, whereas that of Plk1 was markedly inhibited. Recombinant Plk3 phosphorylated in vitro a glutathione S-transferase fusion protein containing p53, but not glutathione S-transferase alone. Recombinant Plk1 also phosphorylated p53 but on residues that differed from those targeted by Plk3. Co-immunoprecipitation and pull-down assays demonstrated that Plk3 physically interacted with p53 and that this interaction was enhanced upon DNA damage. In vitro kinase assays followed by immunoblotting showed that serine 20 of p53 was a target of Plk3. Furthermore, expression of a kinase-defective Plk3 mutant (Plk3(K52R)) resulted in significant reduction of p53 phosphorylation on serine 20, which was correlated with a decrease in the expression of p21 and with a concomitant increase in cell proliferation. These results strongly suggest that Plk3 functionally links DNA damage to cell cycle arrest and apoptosis via the p53 pathway.

Reactive Oxygen Species-induced Phosphorylation of p53 on Serine 20 Is Mediated in Part by Polo-like Kinase-3

Upon exposure of cells to hydrogen peroxide (H(2)O(2)) phosphorylation of p53 was rapidly induced in human fibroblast GM00637, and this phosphorylation occurred on serine 9, serine 15, serine 20, but not on serine 392. In addition, H(2)O(2)-induced phosphorylation of p53 was followed by induction of p21, suggesting functional activation of p53. Induction of phosphorylation of p53 on multiple serine residues by H(2)O(2) was caffeine-sensitive and blocked in ATM(-/-) cells. Polo-like kinase-3 (Plk3) activity was also activated upon H(2)O(2) treatment, and this activation was ATM-dependent. Recombinant His(6)-Plk3 phosphorylated glutathione S-transferase (GST)-p53 fusion protein but not GST alone. When phoshorylated in vitro by His(6)-Plk3, but not by the kinase-defective mutant His6-Plk3(K52R), GST-p53 was recognized by an antibody specifically to serine 20-phosphorylated p53, indicating that serine 20 is an in vitro target of Plk3. Also serine 20-phosphorylated p53 was coimmunoprecipitated with Plk3 in cells treated with H(2)O(2). Furthermore, although H(2)O(2) strongly induced serine 15 phosphorylation of p53, it failed to induce serine 20 phosphorylation in Plk3-dificient Daudi cells. Ectopic expression of a Plk3 dominant negative mutant, Plk3(K52R), in GM00637 cells suppressed H(2)O(2)-induced serine 20 phosphorylation. Taken together, our studies strongly suggest that the oxidative stress-induced activation of p53 is at least in part mediated by Plk3.