Molecular oxygen is one of the most important variables in modern cell culture systems. Fluctuations in its concentration can affect cell growth, differentiation, signaling, and free radical ...production. In order to maintain culture viability, experimental validity, and reproducibility, it is imperative that oxygen levels be consistently maintained within physiological “normoxic” limits. Use of the term normoxia, however, is not consistent among scientists who experiment in cell culture. It is typically used to describe the atmospheric conditions of a standard incubator, not the true microenvironment to which the cells are exposed. This error may lead to the situation where cells grown in a standard “normoxic” oxygen concentration may actually be experiencing a wide range of conditions ranging from hyperoxia to near-anoxic conditions at the cellular level. This apparent paradox is created by oxygen's sluggish rate of diffusion through aqueous medium, and the generally underappreciated effects that cell density, media volume, and barometric pressure can have on pericellular oxygen concentration in a cell culture system. This review aims to provide an overview of this phenomenon we have termed “consumptive oxygen depletion” (COD), and includes a basic review of the physics, potential consequences, and alternative culture methods currently available to help circumvent this largely unrecognized problem.
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•Oxygen (O2) is an often overlooked variable in cell and tissue culture systems.•We discuss differences between in vivo and in vitro O2 diffusion.•Alternative culturing technology to enhance O2 delivery are highlighted.•Guidelines to improve cell culture quality and minimize O2 fluctuations are proposed.
Epigenetic modifications represent mechanisms by which cells may effectively translate multiple signaling inputs into phenotypic outputs. Recent research is revealing that redox metabolism is an ...increasingly important determinant of epigenetic control that may have significant ramifications in both human health and disease. Numerous characterized epigenetic marks, including histone methylation, acetylation, and ADP-ribosylation, as well as DNA methylation, have direct linkages to central metabolism through critical redox intermediates such as NAD(+), S-adenosyl methionine, and 2-oxoglutarate. Fluctuations in these intermediates caused by both normal and pathologic stimuli may thus have direct effects on epigenetic signaling that lead to measurable changes in gene expression. In this comprehensive review, we present surveys of both metabolism-sensitive epigenetic enzymes and the metabolic processes that may play a role in their regulation. To close, we provide a series of clinically relevant illustrations of the communication between metabolism and epigenetics in the pathogenesis of cardiovascular disease, Alzheimer disease, cancer, and environmental toxicity. We anticipate that the regulatory mechanisms described herein will play an increasingly large role in our understanding of human health and disease as epigenetics research progresses.
Calcium uptake through the mitochondrial Ca2+ uniporter (MCU) is thought to be essential in regulating cellular signaling events, energy status, and survival. Functional dissection of the uniporter ...is now possible through the recent identification of the genes encoding for MCU protein complex subunits. Cancer cells exhibit many aspects of mitochondrial dysfunction associated with altered mitochondrial Ca2+ levels including resistance to apoptosis, increased reactive oxygen species production and decreased oxidative metabolism. We used a publically available database to determine that breast cancer patient outcomes negatively correlated with increased MCU Ca2+ conducting pore subunit expression and decreased MICU1 regulatory subunit expression. We hypothesized breast cancer cells may therefore be sensitive to MCU channel manipulation. We used the widely studied MDA-MB-231 breast cancer cell line to investigate whether disruption or increased activation of mitochondrial Ca2+ uptake with specific siRNAs and adenoviral overexpression constructs would sensitize these cells to therapy-related stress. MDA-MB-231 cells were found to contain functional MCU channels that readily respond to cellular stimulation and elicit robust AMPK phosphorylation responses to nutrient withdrawal. Surprisingly, knockdown of MCU or MICU1 did not affect reactive oxygen species production or cause significant effects on clonogenic cell survival of MDA-MB-231 cells exposed to irradiation, chemotherapeutic agents, or nutrient deprivation. Overexpression of wild type or a dominant negative mutant MCU did not affect basal cloning efficiency or ceramide-induced cell killing. In contrast, non-cancerous breast epithelial HMEC cells showed reduced survival after MCU or MICU1 knockdown. These results support the conclusion that MDA-MB-231 breast cancer cells do not rely on MCU or MICU1 activity for survival in contrast to previous findings in cells derived from cervical, colon, and prostate cancers and suggest that not all carcinomas will be sensitive to therapies targeting mitochondrial Ca2+ uptake mechanisms.
Expression of the antioxidant enzyme EcSOD in normal human mammary epithelial cells was not recognized until recently. Although expression of EcSOD was not detectable in non-malignant human mammary ...epithelial cells (HMEC) cultured in conventional two-dimensional (2D) culture conditions, EcSOD protein expression was observed in normal human breast tissues, suggesting that the 2D-cultured condition induces a repressive status of EcSOD gene expression in HMEC. With the use of laminin-enriched extracellular matrix (lrECM), we were able to detect expression of EcSOD when HMEC formed polarized acinar structures in a 3D-culture condition. Repression of the EcSOD-gene expression was again seen when the HMEC acini were sub-cultured as a monolayer, implying that lrECM-induced acinar morphogenesis is essential in EcSOD-gene activation. We have further shown the involvement of DNA methylation in regulating EcSOD expression in HMEC under these cell culture conditions. EcSOD mRNA expression was strongly induced in the 2D-cultured HMEC after treatment with a DNA methyltransferase inhibitor. In addition, epigenetic analyses showed a decrease in the degree of CpG methylation in the EcSOD promoter in the 3D versus 2D-cultured HMEC. More importantly, >80% of clinical mammary adenocarcinoma samples showed significantly decreased EcSOD mRNA and protein expression levels compared with normal mammary tissues and there is an inverse correlation between the expression levels of EcSOD and the clinical stages of breast cancer. Combined bisulfite restriction analysis analysis of some of the tumors also revealed an association of DNA methylation with the loss of EcSOD expression in vivo. Furthermore, overexpression of EcSOD inhibited breast cancer metastasis in both the experimental lung metastasis model and the syngeneic mouse model. This study suggests that epigenetic silencing of EcSOD may contribute to mammary tumorigenesis and that restoring the extracellular superoxide scavenging activity could be an effective strategy for breast cancer treatment.
Macrophages, including alveolar macrophages, are primary phagocytic cells of the innate immune system. Many studies of macrophages and inflammation have been done in mouse models, in which inducible ...NO synthase (NOS2) and NO are important components of the inflammatory response. Human macrophages, in contrast to mouse macrophages, express little detectable NOS2 and generate little NO in response to potent inflammatory stimuli. The human NOS2 gene is highly methylated around the NOS2 transcription start site. In contrast, mouse macrophages contain unmethylated cytosine-phosphate-guanine (CpG) dinucleotides proximal to the NOS2 transcription start site. Further analysis of chromatin accessibility and histone modifications demonstrated a closed conformation at the human NOS2 locus and an open conformation at the murine NOS2 locus. In examining the potential for CpG demethylation at the NOS2 locus, we found that the human NOS2 gene was resistant to the effects of demethylation agents both in vitro and in vivo. Our data demonstrate that epigenetic modifications in human macrophages are associated with CpG methylation, chromatin compaction, and histone modifications that effectively silence the NOS2 gene. Taken together, our findings suggest there are significant and underappreciated differences in how murine and human macrophages respond to inflammatory stimuli.
Alpha-tocopheryl succinate (alpha-TOS) is a selective inducer of apoptosis in cancer cells, which involves the accumulation of reactive oxygen species (ROS). The molecular target of alpha-TOS has not ...been identified. Here, we show that alpha-TOS inhibits succinate dehydrogenase (SDH) activity of complex II (CII) by interacting with the proximal and distal ubiquinone (UbQ)-binding site (Q(P) and Q(D), respectively). This is based on biochemical analyses and molecular modelling, revealing similar or stronger interaction energy of alpha-TOS compared to that of UbQ for the Q(P) and Q(D) sites, respectively. CybL-mutant cells with dysfunctional CII failed to accumulate ROS and underwent apoptosis in the presence of alpha-TOS. Similar resistance was observed when CybL was knocked down with siRNA. Reconstitution of functional CII rendered CybL-mutant cells susceptible to alpha-TOS. We propose that alpha-TOS displaces UbQ in CII causing electrons generated by SDH to recombine with molecular oxygen to yield ROS. Our data highlight CII, a known tumour suppressor, as a novel target for cancer therapy.
Porphyromonas gingivalis (P. gingivalis) is regarded as a keystone pathogen in destructive periodontal diseases. It expresses a variety of virulence factors, amongst them fimbriae that are involved ...in colonization, invasion, establishment and persistence of the bacteria inside the host cells. The fimbriae also were demonstrated to affect the host immune-response mechanisms. The major fimbriae are able to bind specifically to different host cells, amongst them peripheral blood monocytes. The interaction of these cells with fimbriae induces release of cytokines such as interleukin-1 (IL-1), IL-6, and tumor necrosis factor-α (TNF-α). The aim of this study was to generate recombinant major FimA protein from P. gingivalis W83 fimbriae and to prove its biological activity. FimA of P. gingivalis W83 was amplified from chromosomal DNA, cloned in a vector and transferred into Listeria innocua. (L. innocua).The expressed protein was harvested and purified using FPLC via a His trap HP column. The identity and purity was demonstrated by gel-electrophoresis and mass-spectrometry. The biological activity was assessed by stimulation of human oral epithelial cells and peripheral blood monocytes with the protein and afterwards cytokines in the supernatants were quantified by enzyme linked immunosorbent assay (ELISA) and cytometric bead array. Recombinant FimA could successfully be generated and purified. Gel-electrophoresis and mass-spectrometry confirmed that the detected sequences are identical with FimA. Stimulation of human monocytes induced the release of high concentrations of IL-1β, IL-6, IL-10 and TNF-α by these cells. In conclusion, a recombinant FimA protein was established and its biological activity was proven. This protein may serve as a promising agent for further investigation of its role in periodontitis and possible new therapeutic approaches.
•Recombinant FimA of P. gingivalis W83 was successfully cloned and prepared and the protein was positively identified as FimA.•The recombinant protein was biologically active by stimulating human monocytes to secrete pro-inflammatory cytokines.•It may serve as promising agent to further investigate FimA in periodontitis and for possible future therapeutic approaches.
Molecular subtypes of breast cancer are characterized by distinct patterns of gene expression that are predictive of outcome and response to therapy. The luminal breast cancer subtypes are defined by ...the expression of estrogen receptor-alpha (ERα)-associated genes, many of which are directly responsive to the transcription factor activator protein 2C (TFAP2C). TFAP2C participates in a gene regulatory network controlling cell growth and differentiation during ectodermal development and regulating ESR1/ERα and other luminal cell-associated genes in breast cancer. TFAP2C has been established as a prognostic factor in human breast cancer, however, its role in the establishment and maintenance of the luminal cell phenotype during carcinogenesis and mammary gland development have remained elusive. Herein, we demonstrate a critical role for TFAP2C in maintaining the luminal phenotype in human breast cancer and in influencing the luminal cell phenotype during normal mammary development. Knockdown of TFAP2C in luminal breast carcinoma cells induced epithelial-mesenchymal transition with morphological and phenotypic changes characterized by a loss of luminal-associated gene expression and a concomitant gain of basal-associated gene expression. Conditional knockout of the mouse homolog of TFAP2C, Tcfap2c, in mouse mammary epithelium driven by MMTV-Cre promoted aberrant growth of the mammary tree leading to a reduction in the CD24(hi)/CD49f(mid) luminal cell population and concomitant gain of the CD24(mid)/CD49f(hi) basal cell population at maturity. Our results establish TFAP2C as a key transcriptional regulator for maintaining the luminal phenotype in human breast carcinoma. Furthermore, Tcfap2c influences development of the luminal cell type during mammary development. The data suggest that TFAP2C has an important role in regulated luminal-specific genes and may be a viable therapeutic target in breast cancer.
Aging is a natural process involving complex interplay between environment, metabolism, and genes. Sirtuin genes and their downstream targets have been associated with lifespan in numerous organisms ...from nematodes to humans. Several target proteins of the sirtuin genes are key sensors and/or effectors of oxidative stress pathways including FOXO3, SOD3, and AKT1. To examine the relationship between single nucleotide polymorphisms (SNP) at candidate genes in these pathways and human lifespan, we performed a molecular epidemiologic study of an elderly cohort (≥65 years old.). Using age at death as a continuous outcome variable and assuming a co-dominant genetic model within the framework of multi-variable linear regression analysis, the genotype-specific adjusted mean age at death was estimated for individual SNP genotypes while controlling for age-related risk factors including smoking, body mass index, alcohol consumption and co-morbidity. Significant associations were detected between human lifespan and SNPs in genes SIRT3, SIRT5, SIRT6, FOXO3 and SOD3. Individuals with either the CC or CT genotype at rs107251 within SIRT6 displayed >5-year mean survival advantages compared to the TT genotype (5.5 and 5.9 years, respectively; q-value = 0.012). Other SNPs revealed genotype-specific mean survival advantages ranging from 0.5 to 1.6 years. Gender also modified the effect of SNPs in SIRT3, SIRT5 and AKT1 on lifespan. Our novel findings highlight the impact of sirtuins and sirtuin-related genotypes on lifespan, the importance of evaluating gender and the advantage of using age as a continuous variable in analyses to report mean age at death.
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