Up-regulation of GADD45α expression by NSAIDs leads to apoptotic and necrotic colon cancer cell deaths

Growth arrest and DNA damage inducible 45 alpha (GADD45α) is a central player in mediating apoptosis induced by a variety of stress stimuli and genotoxic agents. Regular usage of nonselective nonsteroidal anti-inflammatory drugs (NSAIDs) such as indomethacin and sulindac is associated with reduced risk for various cancers, including colon cancer. The role of GADD45α in NSAID-induced colon cancer cell cytotoxicity is unknown. In this study, we report that indomethacin and sulindac sulfide treatments up-regulate GADD45α mRNA expression and protein levels in colon cancer HT-29, RKO and Caco-2 cells. This up-regulation of GADD45α is accompanied by necrotic cell death and apoptosis. Anti-sense suppression of GADD45α expression inhibited indomethacin and sulindac sulfide-induced necrotic cell death and apoptosis. These findings confirm a role for GADD45α in NSAID-induced cytotoxicity, a mechanism for the anti-neoplastic effect of NSAIDs in colon tumorigenesis and cancer growth.     

Ribosomal protein S7 regulates arsenite-induced GADD45α expression by attenuating MDM2-mediated GADD45α ubiquitination and degradation

The stress-responding protein, GADD45α, plays important roles in cell cycle checkpoint, DNA repair and apoptosis. In our recent study, we demonstrate that GADD45α undergoes a dynamic ubiquitination and degradation in vivo, which process can be blocked by the cytotoxic reagent, arsenite, resulting in GADD45α accumulation to activate JNKs cell death pathway, thereby revealing a novel mechanism for the cellular GADD45α functional regulation. But the factors involved in GADD45α stability modulations are unidentified. Here, we demonstrated that MDM2 was an E3 ubiquitin ligase for GADD45α. One of MDM2-binding partner, ribosomal protein S7, interacted with and stabilized GADD45α through preventing the ubiquitination and degradation of GADD45α mediated by MDM2. This novel function of S7 is unrelated to p53 but seems to depend on S7/MDM2 interaction, for the S7 mutant lacking MDM2-binding ability lost its function to stabilize GADD45α. Further investigations indicated that arsenite treatment enhanced S7–MDM2 interaction, resulting in attenuation of MDM2-dependent GADD45α ubiquitination and degradation, thereby leading to GADD45α-dependent cell death pathway activation. Silencing S7 expression suppressed GADD45α-dependent cytotoxicity induced by arsenite. Our findings thus identify a novel function of S7 in control of GADD45α stabilization under both basal and stress conditions and its significance in mediating arsenite-induced cellular stress.     

Selection of valid reference genes for expression studies of hepatic cell lines under IFN-α treatment

The proper selection of reference genes to normalize the quantitative real-time PCR (RT-qPCR) results under particular experimental conditions is crucial for validation of the gene quantification data. Herein, using SYBR green RT-qPCR, five reference genes (GAPDH, ACTB, HMBS, HPRT-1 and TBP) were evaluated to determine the most stable reference genes in hepatic cell lines (Huh-7 and HepG₂) under IFN-α treatment conditions. Analyses by geNorm program ranked GAPDH and HPRT-1 in Huh-7 and that of ACTB and HMBS in HepG₂ cells as the most stable reference genes under IFN-α treatment. While, same reference gene pairs were ranked by NormFinder program in Huh-7 cells, GAPDH was assessed as the most stable gene in HepG₂ group by this program, implying the importance of the employed algorithm in comparative interpretation of the data. Finally, cumulative analyses by one-way ANOVA, geNorm and NormFinder programs indicated that use of two reference genes (HMBS and GAPDH) in Huh-7 and three (HMBS, ACTB and GAPDH) in HepG₂ cells would greatly improve the normalization of the RT-qPCR data under IFN-α. Data presented in this paper will aid the selection of the most stable reference genes in RT-qPCR studies on evaluation of hepatic viral proteins and IFN pathway.     

Abnormal expression ofα-L-fucosidase in lymphoid cell lines of fucosidosis patients

Fucosidosis is an autosomal recessive lysosomal storage disease due to a deficiency of-L-fucosidase activity in tissues and body fluids. Exponentially growing lymphoid cell cultures from four fucosidosis patients had 2.7-fold to 15.6-fold less extracellular-L-fucosidase protein and 28.8-fold to 144.0-fold less intracellular-L-fucosidase protein with negligible catalytic activity, compared to the mean of 19 control cultures. The percentage of total-L-fucosidase protein released extracellularly by cultures from the four patients was 64 to 85%, compared to 35±9% for control cultures. Intracellular and extracellular enzyme forms in fucosidosis and control cell lines were glycoproteins containing polypeptide chains ofM _r=52,000. During a 1.5-hr pulse-label with³⁵S-methionine,-L-fucosidase was synthesized by control cells and two fucosidosis cell lines as an intracellular form withM _r=58,000. During a subsequent 21-hr chase with unlabeled methionine, mutant enzyme was almost entirely processed to an extracellular form withM _r=62,000. In contrast, only 25–30% of control enzyme was processed to an extracellular form (M _r=62,000), with the remainder retained intracellularly (M _r=60,000). In the other two fucosidosis cell lines,-L-fucosidase was synthesized as an intracellular form withM _r=56,000 that was processed to an extracellular form withM _r=60,000. In summary, the fucosidosis mutation(s) affected the catalytic activity, quantity, and extracellular release of-L-fucosidase as expressed by lymphoid cells.This work was funded by NIH Grants DK 32161 to R. A. DiCioccio and GM 28428 to J. K. Darby.     

The increased expression of DEC1 gene is related to HIF-1α protein in gastric cancer cell lines

Overexpression of differentiated embryo chondrocyte 1 (DEC1) has been reported to contribute to the cellular differentiation, proliferation, and apoptosis of various cancers. Our previous studies have shown that DEC1 was highly expressed in gastric cancer (GCa) tissues. However, there is no report about the expression of DEC1 in GCa cell lines until now. In this study, We evaluated the mRNA and protein expression of DEC1 and hypoxia-inducible factor 1α (HIF-1α) under normoxic and hypoxic conditions in six GCa cell lines: BGC-823, MGC80-3, MKN1, AGS, FU97 and SGC-7901. An HIF-1α protein inhibitor was used to analyze the association of DEC1 and HIF-1α expression. Under normoxia, the mRNA expression of both HIF-1α and DEC1 was moderate, whereas the protein expression of DEC1 was higher than that of HIF-1α. Hypoxia induced the mRNA expression of DEC1 and the protein expression of HIF-1α and DEC1 in a time-dependent manner but had no effect on the mRNA expression of HIF-1α. Furthermore, inhibition of HIF-1α protein expression resulted in a significant decrease in both the mRNA and protein expression of DEC1. Taken together, DEC1 expression is correlated with HIF-1α protein in GCa cell line, blockage of HIF-1α protein led to reduced DEC1 expression. The efficacy of inhibiting HIF-1α and DEC1 expression should be tested in clinical trials as possible treatment for GCa.     

High frequency of-β-hexosaminidase deficiency in lymphoblastoid cell lines

The activity of seven lysosomal enzymes was determined in 25 lymphoblastoid cell lines. These lines included normal controls transformed with Epstein-Barr virus, Burkitt's lymphomas and other lymphomas with or without EBV genome.Four lines were deficient in total β-hexosaminidase activity. The deficiency was as severe as that of the variant O (Sandhoff's disease) of clinical β-hexosaminidase deficiency. The electrophoretic pattern was also similar to that observed in Sandhoff's disease.The possible mechanisms explaining the high frequency of β-hexosaminidase deficiency in lymphoblastoid cell lines are discussed.     

Genetically engineered cell lines for α1-antitrypsin expression

Objectives

To establish genetically modified cell lines that can produce functional α1-antitrypsin (AAT), by CRISPR/Cas9-assisted homologous recombination.

Results

α1-Antitrypsin deficiency (AATD) is a monogenic heritable disease that often results in lungs and liver damage. Current augmentation therapy is expensive and in short of supply. To develop a safer and more effective therapeutic strategy for AATD, we integrated the AAT gene (SERPINA1, NG_008290.1) into the AAVS1 locus of human cell line HEK293T and assessed the safety and efficacy of CRISPR/Cas9 on producing potential therapeutic cell lines. Cell clones obtained had the AAT gene integrated at the AAVS1 locus and secreted approx. 0.04 g/l recombinant AAT into the medium. Moreover, the secreted AAT showed an inhibitory activity that is comparable to plasma AAT.

Conclusions

CRISPR/Cas9-mediated engineering of human cells is a promising alternative for generating isogenic cell lines with consistent AAT production. This work sheds new light on the generation of therapeutic liver stem cells for AATD.

     

Regulation of cellular Gsα levels and basal adenylyl cyclase activity by expression of the β2-adrenoceptor in neuroblastoma cell lines

Mouse neuroblastoma x rat glioma hybrid NG108-15 and mouse neuroblastoma x embryonic hamster brain NCB20 cells were transfected with a construct containing a human 2 adrenoceptor cDNA under the control of the actin promoter. Clones were selected on the basis of resistance to geneticin sulphate and those expressing a range of levels of the receptor expanded for further study. Membranes from a clone of NG108-15 cells expressing high levels of the receptor (N22) but not one expressing only low levels of the receptor (N17) exhibited a markedly elevated adenylyl cyclase activity when measured in the presence of Mg²⁺ compared to wild type cells. This was not due to elevated levels of the adenylyl cyclase catalytic moiety however as there was no difference in these membranes when the adenylyl cyclase activity was measured in the presence of Mn²⁺. The elevated basal activity was partially reversed by addition of the -adrenoceptor antagonist propranolol. Agonist activation of N22 but not N17 cells led to a large selective down-regulation of cellular G_s levels which was independent of the generation of cyclic AMP. Isoprenaline stimulation of adenylyl cyclase activity and of the specific high affinity binding of [³H] forskolin was achieved with substantially greater potency (some 30 fold) in N22 cell membranes than in N17. By contrast agonist activation of the endogenously expressed IP prostanoid receptor caused stimulation of adenylyl cyclase and stimulation of high affinity [³H] forskolin binding which was equipotent in each of N22, N17 and wild type NG108-15 cells. Agonist activation of the IP prostanoid receptor caused an equivalent degree of G_s down-regulation in each cell type. Expression of an epitope tagged variant of G_s in NG108-15 cells resulted in prostanoid agonist-induced down-regulation of this polypeptide in a manner indistinguishable from that of wild type G_s. Isolation of clones of NCB20 cells expressing high levels of the 2 adrenoceptor also resulted in a specific agonist-induced down-regulation of G_s.     

In search of expression bottlenecks in recombinant CHO cell lines—a case study

The efficient production of recombinant proteins such as antibodies typically involves the screening of an extravagant number of clones in order to finally select a stable and high-producing cell line. Thereby, the underlying principles of a powerful protein machinery, but also potential expression limitations, often remain poorly understood. To shed more light on this topic, we applied several different techniques to investigate a previously generated cell line (4B3-IgA), which expressed recombinant immunoglobulin A (IgA) with an unusually low specific productivity. Results were compared to the host cell line and to another recombinant CHO cell line (3D6-IgA) expressing another IgA that binds to an overlapping epitope. The low specific productivity of clone 4B3-IgA could not be explained by GCN or mRNA levels, but insufficiencies in protein maturation and/or secretion were determined. Despite the presence of free light chain polypeptides, they occasionally failed to associate with their heavy chain partners. Consequently, heavy chains were misassembled and accumulated to form intracellular aggregates, so-called Russell bodies. These protein deposits evoked the expression of increased amounts of ER-resident chaperones to combat the induced stress. Despite bottlenecks in protein processing, the cells’ quality checkpoints remained intact, and predominantly correctly processed IgA was exported into the culture medium. The results of our study demonstrated that recombinant protein expression was impaired by heavy chain aggregation despite the presence of a disposable light chain and revealed elevated chaperone formation in combination with limited antibody assembly. Our studies suggest that the primary amino acid sequence and consequently the resulting structure of an expressed protein need to be considered as a factor influencing a cell’s productivity.     

Therapeutic ROS targeting of GADD45γ in the induction of G2/M arrest in primary human colorectal cancer cell lines by cucurbitacin E

Cucurbitacin E (CuE) or α-elaterin is a natural compound previously shown to be an antifeedant as well as a potent chemopreventive agent against several types of cancer. The present study investigated the anticancer effects of CuE on colorectal cancer (CRC) using primary cell lines isolated from five CRC patients in Taiwan, Specifically, we explored the anti-proliferation and cell cycle G2/M arrest induced by CuE in CRC cells. MPM-2 flow cytometry tests show that CuE-treated cells accumulated in metaphase (CuE 2.5–7.5 μM). Results further indicate that CuE produced G₂/M arrest as well as the downregulation of CDC2 and cyclin B1 expression and dissociation. Both effects increased proportionally with the dose of CuE; however, the inhibition of proliferation, arrest of mitosis, production of reactive oxygen species (ROS), and loss of mitochondrial membrane potential (ΔΨm) were found to be dependent on the quantity of CuE used to treat the cancer cells. In addition, cell cycle arrest in treated cells coincided with the activation of the gene GADD45(α, β, γ). Incubation with CuE resulted in the binding of GADD45γ to CDC2, which suggests that the delay in CuE-induced mitosis is regulated by the overexpression of GADD45γ. Our findings suggest that, in addition to the known effects on cancer prevention, CuE may have antitumor activities in established CRC.     

Mechanism of HCV's resistance to IFN-α in cell culture involves expression of functional IFN-α receptor 1

The mechanisms underlying the Hepatitis C virus (HCV) resistance to interferon alpha (IFN-α) are not fully understood. We used IFN-α resistant HCV replicon cell lines and an infectious HCV cell culture system to elucidate the mechanisms of IFN-α resistance in cell culture. The IFN-α resistance mechanism of the replicon cells were addressed by a complementation study that utilized the full-length plasmid clones of IFN-α receptor 1 (IFNAR1), IFN-α receptor 2 (IFNAR2), Jak1, Tyk2, Stat1, Stat2 and the ISRE- luciferase reporter plasmid. We demonstrated that the expression of the full-length IFNAR1 clone alone restored the defective Jak-Stat signaling as well as Stat1, Stat2 and Stat3 phosphorylation, nuclear translocation and antiviral response against HCV in all IFN-α resistant cell lines (R-15, R-17 and R-24) used in this study. Moreover RT-PCR, Southern blotting and DNA sequence analysis revealed that the cells from both R-15 and R-24 series of IFN-α resistant cells have 58 amino acid deletions in the extracellular sub domain 1 (SD1) of IFNAR1. In addition, cells from the R-17 series have 50 amino acids deletion in the sub domain 4 (SD4) of IFNAR1 protein leading to impaired activation of Tyk2 kinase. Using an infectious HCV cell culture model we show here that viral replication in the infected Huh-7 cells is relatively resistant to exogenous IFN-α. HCV infection itself induces defective Jak-Stat signaling and impairs Stat1 and Stat2 phosphorylation by down regulation of the cell surface expression of IFNAR1 through the endoplasmic reticulum (ER) stress mechanisms. The results of this study suggest that expression of cell surface IFNAR1 is critical for the response of HCV to exogenous IFN-α.     

GADD45α inhibition of DNMT1 dependent DNA methylation during homology directed DNA repair

In this work, we examine regulation of DNA methyltransferase 1 (DNMT1) by the DNA damage inducible protein, GADD45α. We used a system to induce homologous recombination (HR) at a unique double-strand DNA break in a GFP reporter in mammalian cells. After HR, the repaired DNA is hypermethylated in recombinant clones showing low GFP expression (HR-L expressor class), while in high expressor recombinants (HR-H clones) previous methylation patterns are erased. GADD45α, which is transiently induced by double-strand breaks, binds to chromatin undergoing HR repair. Ectopic overexpression of GADD45α during repair increases the HR-H fraction of cells (hypomethylated repaired DNA), without altering the recombination frequency. Conversely, silencing of GADD45α increases methylation of the recombined segment and amplifies the HR-L expressor (hypermethylated) population. GADD45α specifically interacts with the catalytic site of DNMT1 and inhibits methylation activity in vitro. We propose that double-strand DNA damage and the resulting HR process involves precise, strand selected DNA methylation by DNMT1 that is regulated by GADD45α. Since GADD45α binds with high avidity to hemimethylated DNA intermediates, it may also provide a barrier to spreading of methylation during or after HR repair.     

Estrogen receptor β agonists affect growth and gene expression of human breast cancer cell lines

Expression of estrogen receptor β (ERβ) has been described to reduce growth of cancer cell lines derived from hormone-dependent tumors, like breast cancer. In this study we tested to what extent two ERβ agonists, androgen derivative 3β-Adiol and flavonoid Liquiritigenin, would affect growth and gene expression of different ERβ-positive human breast cancer cell lines. Under standard cell culture conditions, we observed 3β-Adiol to inhibit growth of MCF-7 cells in a dose-dependent manner, whereas growth of BT-474 and MCF-10A cells was suppressed by the maximum concentration (100 nM) only. When treated in serum-free medium, all cell lines except of MDA-MB-231 were responsive to 1 nM 3β-Adiol, and ZR75-1 cells exhibited a dose-dependent antiproliferative response. Providing putative mechanisms underlying the observed growth-inhibitory effect, expression of Ki-67 or cyclins A2 and B1 was downregulated after 3β-Adiol treatment in all responsive lines. In contrast, treatment with lower doses of Liquiritigenin did not affect growth. In MCF-7 cells, the highest dose of this flavonoid exerted proliferative effects accompanied by increased expression of cyclin B1, PR and PS2, indicating unspecific activation of ERα. In conclusion, the ERβ agonists tested exerted distinct concentration-dependent and cell line-specific effects on growth and gene expression. The observed inhibitory effects of 3β-Adiol on breast cancer cell growth encourage further studies on the potential of this and other ERβ agonists as targeted drugs for breast cancer therapy.     

C-terminal region of GADD34 regulates eIF2α dephosphorylation and cell proliferation in CHO-K1 cells

GADD34 is a member of a growth arrest and DNA damage (GADD)-inducible gene family. Here, we established a novel Chinese hamster ovary (CHO)-K1-derived cell line, CHO-K1-G34M, which carries a nonsense mutation (termed the Q525X mutation) in the GADD34 gene. The Q525X mutant protein lacks the C-terminal 66 amino acids required for GADD34 to bind to and activate protein phosphatase 1 (PP1). We investigated the effects of GADD34 with or without the Q525X mutation on the phosphorylation status of PP1 target proteins, including the α subunit of eukaryotic initiation factor 2 (eIF2α) and glycogen synthase kinase 3β (GSK3β). CHO-K1-G34M cells had higher levels of eIF2α phosphorylation compared to the control CHO-K1-normal cells both in the presence and absence of endoplasmic reticulum stress. Overexpression of the wild-type GADD34 protein in CHO-K1-normal cells largely reduced eIF2α phosphorylation, while overexpression of the Q525X mutant did not produce similar reductions. Meanwhile, neither wild type nor Q525X mutation of GADD34 affected the GSK3β phosphorylation status. GADD34 also did not affect the canonical Wnt signaling pathway downstream of GSK3β. Cell proliferation rates were higher, while expression levels of the cyclin-dependent kinase inhibitor p21 were lower in CHO-K1-G34M cells compared to the CHO-K1-normal cells. The GADD34 Q525X mutant had a reduced ability to inhibit cell proliferation and enhance p21 expression of the CHO-K1-normal cells compared to the wild-type GADD34 protein. These results suggest that the GADD34 protein C-terminal plays important roles in regulating not only eIF2α dephosphorylation but also cell proliferation in CHO-K1 cells.     

Patterns of constitutive and IFN-γ inducible expression of HLA class II molecules in human melanoma cell lines

Major histocompatibility complex (MHC) class II proteins (HLA-DR, HLA-DP and HLA-DQ) play a fundamental role in the regulation of the immune response. The level of expression of human leukocyte antigen (HLA) class II antigens is regulated by interferon-gamma (IFN-gamma) and depends on the status of class II trans-activator protein (CIITA), a co-activator of the MHC class II gene promoter. In this study, we measured levels of constitutive and IFN-gamma-induced expression of MHC class II molecules, analysed the expression of CIITA and investigated the association between MHC class II transactivator polymorphism and expression of different MHC class II molecules in a large panel of melanoma cell lines obtained from the European Searchable Tumour Cell Line Database. Many cell lines showed no constitutive expression of HLA-DP, HLA-DQ and HLA-DR and no IFN-gamma-induced increase in HLA class II surface expression. However, in some cases, IFN-gamma treatment led to enhanced surface expression of HLA-DP and HLA-DR. HLA-DQ was less frequently expressed under basal conditions and was less frequently induced by IFN-gamma. In these melanoma cell lines, constitutive surface expression of HLA-DR and HLA-DP was higher than that of HLA-DQ. In addition, high constitutive level of cell surface expression of HLA-DR was correlated with lower inducibility of this expression by IFN-gamma. Finally, substitution A-->G in the 5' flanking region of CIITA promoter type III was associated with higher expression of constitutive HLA-DR (p<0.005). This study yielded a panel of melanoma cell lines with different patterns of constitutive and IFN-gamma-induced expression of HLA class II that can be used in future studies of the mechanisms of regulation of HLA class II expression.