Insights into Cadmium-Induced Carcinogenesis through an In Vitro Study Using C3H10T1/2Cl8 Cells: The Multifaceted Role of Mitochondria

In this paper, we report the metabolic characterization of two foci, F1 and F3, obtained at the end of Cell Transformation Assay (CTA), performed by treating C3H10T1/2Cl8 mouse embryo fibroblasts with 1 μM CdCl2 for 24 h. The elucidation of the cadmium action mechanism can be useful both to improve the in vitro CTA and to yield insights into carcinogenesis. The metabolism of the two foci was investigated through Seahorse and enzyme activity assays; mitochondria were studied in confocal microscopy and reactive oxygen species were detected by flow cytometry. The results showed that F1 focus has higher glycolytic and TCA fluxes compared to F3 focus, and a more negative mitochondrial membrane potential, so that most ATP synthesis is performed through oxidative phosphorylation. Confocal microscopy showed mitochondria crowded in the perinuclear region. On the other hand, F3 focus showed lower metabolic rates, with ATP mainly produced by glycolysis and damaged mitochondria. Overall, our results showed that cadmium treatment induced lasting metabolic alterations in both foci. Triggered by the loss of the Pasteur effect in F1 focus and by mitochondrial impairment in F3 focus, these alterations lead to a loss of coordination among glycolysis, TCA and oxidative phosphorylation, which leads to malignant transformation.

Two ISREs cooperatively regulate ISG expression in West Nile virus infected IFNAR -/- MEFs.

We previously identified a subset of interferon stimulated genes (ISGs) upregulated by West Nile virus (WNV) infection in wildtype mouse embryo fibroblasts (MEFs) after viral proteins had inhibited type 1 interferon (IFN)-mediated JAK-STAT signaling and also in WNV-infected RIG-I -/- , MDA5 -/- , STAT1 -/- , STAT2 -/- , IFNAR -/- , IRF3 -/- , IRF7 -/- , and IRF3/7 -/- MEFs. In this study, ISG upregulation by WNV infection in IFNAR -/- MEFs was confirmed by RNA-seq. ISG upregulation by WNV infection was inhibited in RIG-I -/- /MDA5 -/- MEFs. ISGs were upregulated in IRF1 -/- and IRF5 -/- MEFs but only minimally upregulated in IRF3/5/7 -/- MEFs, suggesting redundant IRF involvement. We previously showed that a single proximal interferon stimulated response element (ISRE) in the Oas1a and Oas1b promoters bound the ISGF3 complex after type 1 IFN treatment. In this study, we used wild-type and mutant promoter luciferase reporter constructs to identify critical regions in the Oas1b and Ifit1 promoters for gene activation in infected IFNAR -/- MEFs. Two ISREs were required in both promoters. Mutation of these ISREs in an Ifit1 promoter DNA probe reduced in vitro complex formation with infected nuclear extracts. An NF-κB inhibitor decreased Ifit1 promoter activity in cells and in vitro complex formation. IRF3 and p50 promoter binding were detected by ChIP for upregulated ISGs with two proximal ISREs. The data indicate that ISREs function cooperatively to upregulate the expression of some ISGs when type 1 IFN-signaling is absent with the binding complex consisting of IRF3, 5, and/or 7 and an NF-κB component(s) as well as other as yet unknown factors. AUTHOR SUMMARY Type 1 IFN signaling in mammalian cells induces formation of the ISGF3 transcription factor complex, which binds to interferon stimulated response elements (ISREs) in the promoters of interferon stimulated genes (ISGs) in the cell nucleus. Flavivirus proteins counteract type 1 IFN signaling by preventing either the formation or nuclear localization of ISGF3. A subset of ISRE-regulated ISGs was still induced in West Nile virus (WNV)-infected mouse embryo fibroblasts (MEFs) indicating that cells have an alternative mechanism for activating these ISGs. In this study, cellular components involved in this ISG upregulation mechanism were identified using gene-knockout MEFs and ChIP and critical promoter regions for gene activation were mapped using reporter assays. The data indicate cooperative function between two ISREs and required binding of IRF3, 5, and/or 7 and an NF-κB component(s). Moreover, type 1 IFN signaling-independent ISG activation requires different additional promoter activation regions than type 1 IFN-dependent activation.

Keap1 binds cytokine promoters upon virus infection and moderates their induction by recruiting NFκB p50 and G9a-GLP

Innate immunity requires a balance of positive and negative regulators of cytokine transcription. Keap1 deletion in mice alters innate immunity and inflammation. We investigated the influence of Keap1 on cytokine gene induction by Sendai virus infection in mouse embryo fibroblasts (MEFs). Keap1 bound to the Ifnb1, Tnf and Il6 promoters upon virus infection, and moderated viral induction of their transcription. Keap1 was required for viral induction of NFκB p50 and G9a-GLP lysine methyltransferase binding to these genes. Keap1 formed BiFC complexes with NFκB p50 that were localized to the nuclei in a subset of cells. Nrf2 counteracted viral induction of Keap1 binding to the promoters, and the effects of Keap1 on NFκB p50 and on G9a-GLP recruitment. Lysine methyltransferase inhibitors enhanced viral induction of transcription of the genes that were bound by Keap1 only in MEFs with intact Keap1, and not in Keap1-/- MEFs. They also enhanced NFκB p50 and NFκB p65 recruitment to these genes only in MEFs with intact Keap1, whereas they inhibited G9a-GLP recruitment. The reciprocal effects of Keap1 and of G9a-GLP lysine methyltransferase activity on chromatin binding by each other constitute a feedback circuit that moderates viral induction of cytokine transcription.SummaryVirus infection induces Keap1 binding to cytokine promoters, which recruits NFκB p50 and G9a-GLP and moderates their transcription.

Apoptotic stress induces Bax-dependent, caspase-independent redistribution of LINC complex nesprins

The canonical function of Bcl-2 family proteins is to regulate mitochondrial membrane integrity. In response to apoptotic signals the multi-domain pro-apoptotic proteins Bax and Bak are activated and perforate the mitochondrial outer membrane by a mechanism which is inhibited by their interaction with pro-survival members of the family. However, other studies have shown that Bax and Bak may have additional, non-canonical functions, which include stress-induced nuclear envelope rupture and discharge of nuclear proteins into the cytosol. We show here that the apoptotic stimuli cisplatin and staurosporine induce a Bax/Bak-dependent degradation and subcellular redistribution of nesprin-1 and nesprin-2 but not nesprin-3, of the linker of nucleoskeleton and cytoskeleton (LINC) complex. The degradation and redistribution were caspase-independent and did not occur in Bax/Bak double knockout (DKO) mouse embryo fibroblasts (MEFs). Re-expression of Bax in Bax/Bak DKO MEFs restored stress-induced redistribution of nesprin-2 by a mechanism which requires Bax membrane localization and integrity of the α helices 5/6, and the Bcl-2 homology 3 (BH3) domain. We found that nesprin-2 interacts with Bax in close proximity to perinuclear mitochondria in mouse and human cells. This interaction requires the mitochondrial targeting and N-terminal region but not the BH3 domain of Bax. Our results identify nesprin-2 as a Bax binding partner and also a new function of Bax in impairing the integrity of the LINC complex.

Mutagenicity of acrylamide and glycidamide in human TP53 knock-in (Hupki) mouse embryo fibroblasts

Acrylamide is a suspected human carcinogen formed during high-temperature cooking of starch-rich foods. It is metabolised by cytochrome P450 2E1 to its reactive metabolite glycidamide, which forms pre-mutagenic DNA adducts. Using the human TP53 knock-in (Hupki) mouse embryo fibroblasts (HUFs) immortalisation assay (HIMA), acrylamide- and glycidamide-induced mutagenesis was studied in the tumour suppressor gene TP53. Selected immortalised HUF clones were also subjected to next-generation sequencing to determine mutations across the whole genome. The TP53-mutant frequency after glycidamide exposure (1.1 mM for 24 h, n = 198) was 9% compared with 0% in cultures treated with acrylamide [1.5 (n = 24) or 3 mM (n = 6) for 48 h] and untreated vehicle (water) controls (n = 36). Most glycidamide-induced mutations occurred at adenines with A > T/T > A and A > G/T > C mutations being the most common types. Mutations induced by glycidamide occurred at specific TP53 codons that have also been found to be mutated in human tumours (i.e., breast, ovary, colorectal, and lung) previously associated with acrylamide exposure. The spectrum of TP53 mutations was further reflected by the mutations detected by whole-genome sequencing (WGS) and a distinct WGS mutational signature was found in HUF clones treated with glycidamide that was again characterised by A > G/T > C and A > T/T > A mutations. The WGS mutational signature showed similarities with COSMIC mutational signatures SBS3 and 25 previously found in human tumours (e.g., breast and ovary), while the adenine component was similar to COSMIC SBS4 found mostly in smokers’ lung cancer. In contrast, in acrylamide-treated HUF clones, only culture-related background WGS mutational signatures were observed. In summary, the results of the present study suggest that glycidamide may be involved in the development of breast, ovarian, and lung cancer.

β -Cypermethrin promotes the adipogenesis of 3T3-L1 cells via inducing autophagy and shaping an adipogenesis-friendly microenvironment

The toxicity of synthetic pyrethroids has garnered attention, and studies have revealed that pyrethroids promote fat accumulation and lead to obesity in mice. Nevertheless, the effect of β-cypermethrin (β-CYP) on adipogenesis and its underlying mechanism remains largely unknown. In this study, mouse embryo fibroblasts 3T3-L1 cells were exposed to β-CYP, and the cell viability, intracellular reactive oxygen species (ROS) level, autophagy, and adipogenesis were assessed to investigate the roles of oxidative stress and autophagy in the toxic effects of β-CYP on adipogenesis. The results demonstrated that treatment with 100 μΜ β-CYP elevated the ROS level, decreased mitochondrion membrane potential, stimulated autophagy, and enhanced the adipogenesis induced by the mixture of insulin, dexamethasone, and 3-isobutyl-1-methylxanthine. However, co-treatment with N-acetyl-L-cysteine partially blocked the abovementioned effects of β-CYP in 3T3-L1 cells. In addition, co-treatment with rapamycin, an autophagy agonist, enhanced the inductive effect of β-CYP on adipogenesis, whereas co-treatment with 3-methyladenine blocked the enhancement of adipogenesis caused by β-CYP. Moreover, β-CYP also altered the microenvironment of 3T3-L1 cells to an adipogenesis-friendly one by reducing the extracellular expression of miR-34a, suggesting that the culture media of β-CYP-treated 3T3-L1 cells could shift macrophages to M2 type. Taken together, the data obtained in the present study demonstrated that β-CYP promoted adipogenesis via oxidative stress-mediated autophagy disturbance, and it caused macrophage M2 polarization via the alteration of miR-34a level in the microenvironment. The study demonstrated the adipogenesis-promoting effect of β-CYP and unveiled the potential mechanism.