RESUMO
Disorders of oxygen transport are commonly attributed to inadequate carrying capacity (anemia) but may also relate to inefficient gas exchange by red blood cells (RBCs), a process that is poorly characterized yet assumed to be rapid. Without direct measurements of gas exchange at the single-cell level, the barriers to O2 transport and their relationship with hematological disorders remain ill defined. We developed a method to track the flow of O2 in individual RBCs by combining ultrarapid solution switching (to manipulate gas tension) with single-cell O2 saturation fluorescence microscopy. O2 unloading from RBCs was considerably slower than previously estimated in acellular hemoglobin solutions, indicating the presence of diffusional barriers in intact cells. Rate-limiting diffusion across cytoplasm was demonstrated by osmotically induced changes to hemoglobin concentration (i.e., diffusive tortuosity) and cell size (i.e., diffusion pathlength) and by comparing wild-type cells with hemoglobin H (HbH) thalassemia (shorter pathlength and reduced tortuosity) and hereditary spherocytosis (HS; expanded pathlength). Analysis of the distribution of O2 unloading rates in HS RBCs identified a subpopulation of spherocytes with greatly impaired gas exchange. Tortuosity imposed by hemoglobin was verified by demonstrating restricted diffusivity of CO2, an acidic gas, from the dissipative spread of photolytically uncaged H+ ions across cytoplasm. Our findings indicate that cytoplasmic diffusion, determined by pathlength and tortuosity, is a major barrier to efficient gas handling by RBCs. Consequently, changes in RBC shape and hemoglobin concentration, which are common manifestations of hematological disorders, can have hitherto unrecognized and clinically significant implications on gas exchange.
Assuntos
Transporte Biológico/genética , Eritrócitos/metabolismo , Gases/sangue , Oxigênio/sangue , Adulto , Idoso , Dióxido de Carbono/sangue , Citoplasma/metabolismo , Feminino , Voluntários Saudáveis , Hemoglobinas/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Célula ÚnicaRESUMO
Cardiac contractile strength is recognised as being highly pH-sensitive, but less is known about the influence of pH on cardiac gene expression, which may become relevant in response to changes in myocardial metabolism or vascularization during development or disease. We sought evidence for pH-responsive cardiac genes, and a physiological context for this form of transcriptional regulation. pHLIP, a peptide-based reporter of acidity, revealed a non-uniform pH landscape in early-postnatal myocardium, dissipating in later life. pH-responsive differentially expressed genes (pH-DEGs) were identified by transcriptomics of neonatal cardiomyocytes cultured over a range of pH. Enrichment analysis indicated "striated muscle contraction" as a pH-responsive biological process. Label-free proteomics verified fifty-four pH-responsive gene-products, including contractile elements and the adaptor protein CRIP2. Using transcriptional assays, acidity was found to reduce p300/CBP acetylase activity and, its a functional readout, inhibit myocardin, a co-activator of cardiac gene expression. In cultured myocytes, acid-inhibition of p300/CBP reduced H3K27 acetylation, as demonstrated by chromatin immunoprecipitation. H3K27ac levels were more strongly reduced at promoters of acid-downregulated DEGs, implicating an epigenetic mechanism of pH-sensitive gene expression. By tandem cytoplasmic/nuclear pH imaging, the cardiac nucleus was found to exercise a degree of control over its pH through Na+/H+ exchangers at the nuclear envelope. Thus, we describe how extracellular pH signals gain access to the nucleus and regulate the expression of a subset of cardiac genes, notably those coding for contractile proteins and CRIP2. Acting as a proxy of a well-perfused myocardium, alkaline conditions are permissive for expressing genes related to the contractile apparatus.
Assuntos
Núcleo Celular , Miocárdio , Animais , Expressão Gênica , Mamíferos , Contração Miocárdica , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismoRESUMO
ATP fuels the removal of metabolic end-products, including H+ ions that profoundly modulate biological activities. Energetic resources in hypoxic tumor regions are constrained by low-yielding glycolysis, and any means of reducing the cost of acid extrusion, without compromising pH homeostasis, would therefore be advantageous for cancer cells. Some cancers express connexin channels that allow solute exchange between cells, and we propose that, via this route, normoxic cells supply hypoxic neighbors with acid-neutralizing HCO3- ions. This hypothesis was tested by imaging cytoplasmic pH in spheroidal tissue growths of connexin43-positive pancreatic cancer Colo357 cells during light-controlled H+ uncaging at the hypoxic core. Cytoplasmic acid retention at the core was halved in the presence of CO2/HCO3-, but this process requires a restorative HCO3- flux. The effect of CO2/HCO3- was ablated by connexin43 inhibition or knockdown. In connexin-decoupled spheroids, 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS), an inhibitor of HCO3- uptake, had no effect on cytoplasmic [H+] in the H+-uncaging region, indicating that DIDS-sensitive transport is not an adequate pH-regulatory strategy therein. With intact connexin-coupling, acid retention at the core increased upon DIDS treatment, indicating that HCO3- ions are taken up actively by peripheral cells and then transmitted passively to cells at the hypoxic core. Thus, the energetic burden of pH regulation is offloaded from hypoxic cells onto metabolically altruistic normoxic neighbors.-Dovmark, T. H., Hulikova, A., Niederer, S. A., Vaughan-Jones, R. D., Swietach, P. Normoxic cells remotely regulate the acid-base balance of cells at the hypoxic core of connexin-coupled tumor growths.
Assuntos
Equilíbrio Ácido-Base , Neoplasias/metabolismo , Hipóxia Tumoral/fisiologia , Trifosfato de Adenosina/metabolismo , Bicarbonatos/metabolismo , Linhagem Celular Tumoral , Conexina 43/antagonistas & inibidores , Conexina 43/genética , Conexina 43/metabolismo , Conexinas/metabolismo , Metabolismo Energético , Técnicas de Silenciamento de Genes , Glicólise , Humanos , Transporte de Íons , Modelos Biológicos , Neoplasias/patologia , Oxigênio/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Esferoides Celulares/metabolismo , Esferoides Celulares/patologiaRESUMO
Proliferation and invasion of cancer cells require favorable pH, yet potentially toxic quantities of acid are produced metabolically. Membrane-bound transporters extrude acid from cancer cells, but little is known about the mechanisms that handle acid once it is released into the poorly perfused extracellular space. Here, we studied acid handling by myofibroblasts (colon cancer-derived Hs675.T, intestinal InMyoFib, embryonic colon-derived CCD-112-CoN), skin fibroblasts (NHDF-Ad), and colorectal cancer (CRC) cells (HCT116, HT29) grown in monoculture or coculture. Expression of the acid-loading transporter anion exchanger 2 (AE2) (SLC4A2 product) was detected in myofibroblasts and fibroblasts, but not in CRC cells. Compared with CRC cells, Hs675.T and InMyoFib myofibroblasts had very high capacity to absorb extracellular acid. Acid uptake into CCD-112-CoN and NHDF-Ad cells was slower and comparable to levels in CRC cells, but increased alongside SLC4A2 expression under stimulation with transforming growth factor ß1 (TGFß1), a cytokine involved in cancer-stroma interplay. Myofibroblasts and fibroblasts are connected by gap junctions formed by proteins such as connexin-43, which allows the absorbed acid load to be transmitted across the stromal syncytium. To match the stimulatory effect on acid uptake, cell-to-cell coupling in NHDF-Ad and CCD-112-CoN cells was strengthened with TGFß1. In contrast, acid transmission was absent between CRC cells, even after treatment with TGFß1. Thus, stromal cells have the necessary molecular apparatus for assembling an acid-venting route that can improve the flow of metabolic acid through tumors. Importantly, the activities of stromal AE2 and connexin-43 do not place an energetic burden on cancer cells, allowing resources to be diverted for other activities.
Assuntos
Neoplasias Colorretais/metabolismo , Conexina 43/genética , Fator de Crescimento Transformador beta1/metabolismo , Ácidos/toxicidade , Proliferação de Células/efeitos dos fármacos , Antiportadores de Cloreto-Bicarbonato/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Conexina 43/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Humanos , Concentração de Íons de Hidrogênio , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
BACKGROUND: Tumour carbonic anhydrase IX (CAIX), a hypoxia-inducible tumour-associated cell surface enzyme, is thought to acidify the tumour microenvironment by hydrating CO2 to form protons and bicarbonate, but there is no definitive evidence for this in solid tumours in vivo. METHODS: We used 1H magnetic resonance spectroscopic imaging (MRSI) of the extracellular pH probe imidazolyl succinic acid (ISUCA) to measure and spatially map extracellular pH in HCT116 tumours transfected to express CAIX and empty vector controls in SCID mice. We also measured intracellular pH in situ with 31P MRS and measured lactate in freeze-clamped tumours. RESULTS: CAIX-expressing tumours had 0.15 pH-unit lower median extracellular pH than control tumours (pH 6.71 tumour vs pH 6.86 control, P = 0.01). Importantly, CAIX expression imposed an upper limit for tumour extracellular pH at 6.93. Despite the increased lactate concentration in CAIX-expressing tumours, 31P MRS showed no difference in intracellular pH, suggesting that CAIX acidifies only the tumour extracellular space. CONCLUSIONS: CAIX acidifies the tumour microenvironment, and also provides an extracellular pH control mechanism. We propose that CAIX thus acts as an extracellular pH-stat, maintaining an acidic tumour extracellular pH that is tolerated by cancer cells and favours invasion and metastasis.
Assuntos
Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Anidrase Carbônica IX/genética , Anidrase Carbônica IX/metabolismo , Neoplasias Colorretais/patologia , Ácido Láctico/análise , Animais , Hipóxia Celular , Proliferação de Células , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Células HCT116 , Humanos , Imidazóis/química , Camundongos , Transplante de Neoplasias , Espectroscopia de Prótons por Ressonância Magnética , Microambiente TumoralRESUMO
Biochemical signals acting on the nucleus can regulate gene expression. Despite the inherent affinity of nucleic acids and nuclear proteins (e.g. transcription factors) for protons, little is known about the mechanisms that regulate nuclear pH (pHnuc), and how these could be exploited to control gene expression. Here, we show that pHnuc dynamics can be imaged using the DNA-binding dye Hoechst 33342. Nuclear pores allow the passage of medium-sized molecules (calcein), but protons must first bind to mobile buffers in order to gain access to the nucleoplasm. Fixed buffering residing in the nucleus of permeabilized cells was estimated to be very weak on the basis of the large amplitude of pHnuc transients evoked by photolytic H(+)-uncaging or exposure to weak acids/bases. Consequently, the majority of nuclear pH buffering is sourced from the cytoplasm in the form of mobile buffers. Effective proton diffusion was faster in nucleoplasm than in cytoplasm, in agreement with the higher mobile-to-fixed buffering ratio in the nucleus. Cardiac myocyte pHnuc changed in response to maneuvers that alter nuclear Ca(2+) signals. Blocking Ca(2+) release from inositol-1,4,5-trisphosphate receptors stably alkalinized the nucleus. This Ca(2+)-pH interaction may arise from competitive binding to common chemical moieties. Competitive binding to mobile buffers may couple the efflux of Ca(2+)via nuclear pores with a counterflux of protons. This would generate a stable pH gradient between cytoplasm and nucleus that is sensitive to the state of nuclear Ca(2+) signaling. The unusual behavior of protons in the nucleus provides new mechanisms for regulating cardiac nuclear biology.
Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Núcleo Celular/metabolismo , Prótons , Algoritmos , Animais , Citoplasma/metabolismo , Difusão , Concentração de Íons de Hidrogênio , Modelos Químicos , RatosRESUMO
CO2 is produced abundantly by cardiac mitochondria. Thus an efficient means for its venting is required to support metabolism. Carbonic anhydrase (CA) enzymes, expressed at various sites in ventricular myocytes, may affect mitochondrial CO2 clearance by catalyzing CO2 hydration (to H(+) and HCO3(-)), thereby changing the gradient for CO2 venting. Using fluorescent dyes to measure changes in pH arising from the intracellular hydration of extracellularly supplied CO2, overall CA activity in the cytoplasm of isolated ventricular myocytes was found to be modest (2.7-fold above spontaneous kinetics). Experiments on ventricular mitochondria demonstrated negligible intramitochondrial CA activity. CA activity was also investigated in intact hearts by (13)C magnetic resonance spectroscopy from the rate of H(13)CO3(-) production from (13)CO2 released specifically from mitochondria by pyruvate dehydrogenase-mediated metabolism of hyperpolarized [1-(13)C]pyruvate. CA activity measured upon [1-(13)C]pyruvate infusion was fourfold higher than the cytoplasm-averaged value. A fluorescent CA ligand colocalized with a mitochondrial marker, indicating that mitochondria are near a CA-rich domain. Based on immunoreactivity, this domain comprises the nominally cytoplasmic CA isoform CAII and sarcoplasmic reticulum-associated CAXIV. Inhibition of extramitochondrial CA activity acidified the matrix (as determined by fluorescence measurements in permeabilized myocytes and isolated mitochondria), impaired cardiac energetics (indexed by the phosphocreatine-to-ATP ratio measured by (31)P magnetic resonance spectroscopy of perfused hearts), and reduced contractility (as measured from the pressure developed in perfused hearts). These data provide evidence for a functional domain of high CA activity around mitochondria to support CO2 venting, particularly during elevated and fluctuating respiratory activity. Aberrant distribution of CA activity therefore may reduce the heart's energetic efficiency.
Assuntos
Anidrases Carbônicas/metabolismo , Miócitos Cardíacos/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Dióxido de Carbono/metabolismo , Anidrase Carbônica II/metabolismo , Anidrase Carbônica IV/metabolismo , Compartimento Celular , Citoplasma/metabolismo , Metabolismo Energético , Corantes Fluorescentes , Concentração de Íons de Hidrogênio , Cinética , Masculino , Mitocôndrias Cardíacas/metabolismo , Ratos , Ratos WistarRESUMO
Carbonic anhydrase (CA) enzymes catalyze the chemical equilibration among CO2, HCO3(-) and H(+). Intracellular CA (CAi) isoforms are present in certain types of cancer, and growing evidence suggests that low levels correlate with disease severity. However, their physiological role remains unclear. Cancer cell CAi activity, measured as cytoplasmic CO2 hydration rate (kf), ranged from high in colorectal HCT116 (â¼2 s(-1)), bladder RT112 and colorectal HT29, moderate in fibrosarcoma HT1080 to negligible (i.e. spontaneous kf = 0.18 s(-1)) in cervical HeLa and breast MDA-MB-468 cells. CAi activity in cells correlated with CAII immunoreactivity and enzymatic activity in membrane-free lysates, suggesting that soluble CAII is an important intracellular isoform. CAi catalysis was not obligatory for supporting acid extrusion by H(+) efflux or HCO3(-) influx, nor for maintaining intracellular pH (pHi) uniformity. However, in the absence of CAi activity, acid loading from a highly alkaline pHi was rate-limited by HCO3(-) supply from spontaneous CO2 hydration. In solid tumors, time-dependence of blood flow can result in fluctuations of CO2 partial pressure (pCO2) that disturb cytoplasmic CO2-HCO3(-)-H(+) equilibrium. In cancer cells with high CAi activity, extracellular pCO2 fluctuations evoked faster and larger pHi oscillations. Functionally, these resulted in larger pH-dependent intracellular [Ca(2+)] oscillations and stronger inhibition of the mTORC1 pathway reported by S6 kinase phosphorylation. In contrast, the pHi of cells with low CAi activity was less responsive to pCO2 fluctuations. Such low pass filtering would "buffer" cancer cell pHi from non-steady-state extracellular pCO2. Thus, CAi activity determines the coupling between pCO2 (a function of tumor perfusion) and pHi (a potent modulator of cancer cell physiology).
Assuntos
Bicarbonatos/metabolismo , Dióxido de Carbono/metabolismo , Anidrases Carbônicas/metabolismo , Transdução de Sinais , Bicarbonatos/química , Dióxido de Carbono/química , Anidrases Carbônicas/química , Citoplasma/metabolismo , Citoplasma/patologia , Células HCT116 , Humanos , Concentração de Íons de Hidrogênio , Neoplasias/patologia , Pressão Parcial , PrótonsRESUMO
The degree to which cell membranes are barriers to CO2 transport remains controversial. Proteins, such as aquaporins and Rh complex, have been proposed to facilitate CO2 transport, implying that the nonchannel component of membranes must have greatly reduced CO2 permeability. To determine whether membrane CO2 permeation is rate limiting for gas transport, the spread of CO2 across multicellular tissue growths (spheroids) was measured using intracellular pH as a spatial readout. Colorectal HCT116 cells have basal water and NH3 permeability, indicating the functional absence of aquaporins and gas channels. However, CO2 diffusivity in HCT116 spheroids was only 24 ± 4% lower than in pure water, which can be accounted for fully by volume exclusion due to proteins. Diffusivity was unaffected by blockers of aquaporins and Rh complex (Hg(2+), p-chloromercuribenzoic acid, and 4,4'-diisothiocyano-2,2'-stilbene-disulfonic acid) but decreased under hypertonic conditions (by addition of 300 mOsm mannitol), which increases intracellular protein crowding. Similar CO2 diffusivity was measured in spheroids of T47D breast cells (basal water permeability) and NHDF-Ad fibroblasts (aquaporin-facilitated water permeability). In contrast, diffusivity of NH3, a smaller but less lipophilic gas, was considerably slower than in pure water, as expected from rate-limiting membrane permeation. In conclusion, membranes, even in the functional absence of proposed gas channels, do not restrict CO2 venting from tissue growths.-Hulikova, A., Swietach, P. Rapid CO2 permeation across biological membranes: implications for CO2 venting from tissue.
Assuntos
Transporte Biológico/fisiologia , Dióxido de Carbono/metabolismo , Permeabilidade da Membrana Celular/fisiologia , Membrana Celular/metabolismo , Amônia/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Células HCT116 , Humanos , Água/metabolismoRESUMO
Before molecular pathways in cancer were known to a depth that could predict targets, drug development relied on phenotypic screening, where the effectiveness of candidate chemicals is judged from functional readouts without considering the mechanisms of action. The unraveling of tumor-specific pathways has brought targets for molecularly driven drug discovery, but precedents in the field have shown that awareness of pathways does not necessarily predict therapeutic efficacy, and many cancers still lack druggable targets. Phenotypic screening therefore retains a niche in drug development where a targeted approach is not informative. We analyze the unique advantages of phenotypic screens, and how technological advances have improved their discovery power. Notable advances include the use of larger biological panels and refined protocols that address the disease-relevance and increase data content with imaging and omic approaches.
Assuntos
Antineoplásicos , Descoberta de Drogas , Neoplasias , Fenótipo , Humanos , Descoberta de Drogas/métodos , Neoplasias/tratamento farmacológico , Antineoplásicos/farmacologia , Animais , Ensaios de Seleção de Medicamentos Antitumorais/métodosRESUMO
Carbonic anhydrase IX (CA IX) is a hypoxia-induced cell surface enzyme expressed in solid tumors, and functionally involved in acidification of extracellular pH and destabilization of intercellular contacts. Since both extracellular acidosis and reduced cell adhesion facilitate invasion and metastasis, we investigated the role of CA IX in cell migration, which promotes the metastatic cascade. As demonstrated here, ectopically expressed CA IX increases scattering, wound healing and transwell migration of MDCK cells, while an inactive CA IX variant lacking the catalytic domain (ΔCA) fails to do so. Correspondingly, hypoxic HeLa cells exhibit diminished migration upon inactivation of the endogenous CA IX either by forced expression of the dominant-negative ΔCA variant or by treatment with CA inhibitor, implying that the catalytic activity is indispensable for the CA IX function. Interestingly, CA IX improves cell migration both in the absence and presence of hepatocyte growth factor (HGF), an established inducer of epithelial-mesenchymal transition. On the other hand, HGF up-regulates CA IX transcription and triggers CA IX protein accumulation at the leading edge of lamellipodia. In these membrane regions CA IX co-localizes with sodium bicarbonate co-transporter (NBCe1) and anion exchanger 2 (AE2) that are both components of the migration apparatus and form bicarbonate transport metabolon with CA IX. Moreover, CA IX physically interacts with AE2 and NBCe1 in situ, as shown here for the first time. Thus, our findings suggest that CA IX actively contributes to cell migration via its ability to facilitate ion transport and pH control at protruding fronts of moving cells.
Assuntos
Proteínas de Transporte de Ânions/metabolismo , Antígenos de Neoplasias/biossíntese , Antiporters/metabolismo , Anidrases Carbônicas/biossíntese , Movimento Celular/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Pseudópodes/metabolismo , Simportadores de Sódio-Bicarbonato/metabolismo , Animais , Proteínas de Transporte de Ânions/genética , Antígenos de Neoplasias/genética , Antiporters/genética , Bicarbonatos/metabolismo , Anidrase Carbônica IX , Anidrases Carbônicas/genética , Hipóxia Celular/fisiologia , Células HeLa , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Transporte de Íons/fisiologia , Estrutura Terciária de Proteína , Pseudópodes/genética , Proteínas SLC4A , Simportadores de Sódio-Bicarbonato/genética , Regulação para Cima/fisiologiaRESUMO
Acid-extrusion by active transport is important in metabolically active cancer cells, where it removes excess intracellular acid and sets the intracellular resting pH. Hypoxia is a major trigger of adaptive responses in cancer, but its effect on acid-extrusion remains unclear. We studied pH-regulation under normoxia and hypoxia in eight cancer cell-lines (HCT116, RT112, MDA-MB-468, MCF10A, HT29, HT1080, MiaPaca2, HeLa) using the pH-sensitive fluorophore, cSNARF-1. Hypoxia responses were triggered by pre-incubation in low O(2) or with the 2-oxoglutarate-dependent dioxygenase inhibitor dimethyloxalylglycine (DMOG). By selective pharmacological inhibition or transport-substrate removal, acid-extrusion flux was dissected into components due to Na(+)/H(+) exchange (NHE) and Na(+)-dependent HCO(3)(-) transport. In half of the cell-lines (HCT116, RT112, MDA-MB-468, MCF10A), acid-extrusion on NHE was the dominant flux during an acid load, and in all of these, bar one (MDA-MB-468), NHE-flux was reduced following hypoxic incubation. Further studies in HCT116 cells showed that <4-h hypoxic incubation reduced NHE-flux reversibly with a time-constant of 1-2 h. This was not associated with a change in expression of NHE1, the principal NHE isoform. Following 48-h hypoxia, inhibition of NHE-flux persisted but became only slowly reversible and associated with reduced expression of the glycosylated form of NHE1. Acid-extrusion by Na(+)-dependent HCO(3)(-) transport was hypoxia-insensitive and comparable in all cell lines. This constitutive and stable element of pH-regulation was found to be important for setting and stabilizing resting pH at a mildly alkaline level (conducive for growth), irrespective of oxygenation status. In contrast, the more variable flux on NHE underlies cell-specific differences in their dynamic response to larger acid loads.
Assuntos
Hipóxia Celular/fisiologia , Neoplasias/metabolismo , Ácidos/metabolismo , Transporte Biológico , Proteínas de Transporte de Cátions/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Células HCT116 , Células HT29 , Células HeLa , Homeostase , Humanos , Concentração de Íons de Hidrogênio , Neoplasias/patologia , Oxigênio/metabolismo , Trocador 1 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/metabolismoRESUMO
Acidic environments reduce the intracellular pH (pHi) of most cells to levels that are sub-optimal for growth and cellular functions. Yet, cancers maintain an alkaline cytoplasm despite low extracellular pH (pHe). Raised pHi is thought to be beneficial for tumor progression and invasiveness. However, the transport mechanisms underpinning this adaptation have not been studied systematically. Here, we characterize the pHe-pHi relationship in 66 colorectal cancer cell lines and identify the acid-loading anion exchanger 2 (AE2, SLC4A2) as a regulator of resting pHi. Cells adapt to chronic extracellular acidosis by degrading AE2 protein, which raises pHi and reduces acid sensitivity of growth. Acidity inhibits mTOR signaling, which stimulates lysosomal function and AE2 degradation, a process reversed by bafilomycin A1. We identify AE2 degradation as a mechanism for maintaining a conducive pHi in tumors. As an adaptive mechanism, inhibiting lysosomal degradation of AE2 is a potential therapeutic target.
Assuntos
Antiportadores de Cloreto-Bicarbonato , Proteínas de Membrana Transportadoras , Neoplasias , Proteínas de Transporte de Ânions/metabolismo , Antiporters/metabolismo , Linhagem Celular , Antiportadores de Cloreto-Bicarbonato/química , Antiportadores de Cloreto-Bicarbonato/metabolismo , Citoplasma/metabolismo , Concentração de Íons de Hidrogênio , Neoplasias/metabolismo , HumanosRESUMO
Propiogenic substrates and gut bacteria produce propionate, a post-translational protein modifier. In this study, we used a mouse model of propionic acidaemia (PA) to study how disturbances to propionate metabolism result in histone modifications and changes to gene expression that affect cardiac function. Plasma propionate surrogates were raised in PA mice, but female hearts manifested more profound changes in acyl-CoAs, histone propionylation and acetylation, and transcription. These resulted in moderate diastolic dysfunction with raised diastolic Ca2+, expanded end-systolic ventricular volume and reduced stroke volume. Propionate was traced to histone H3 propionylation and caused increased acetylation genome-wide, including at promoters of Pde9a and Mme, genes related to contractile dysfunction through downscaled cGMP signaling. The less severe phenotype in male hearts correlated with ß-alanine buildup. Raising ß-alanine in cultured myocytes treated with propionate reduced propionyl-CoA levels, indicating a mechanistic relationship. Thus, we linked perturbed propionate metabolism to epigenetic changes that impact cardiac function.
RESUMO
Intracellular pH (pH(i)), a major modulator of cell function, is regulated by acid/base transport across membranes. Excess intracellular H(+) ions (e.g. produced by respiration) are extruded by transporters such as Na(+)/H(+) exchange, or neutralized by HCO(3)(-) taken up by carriers such as Na(+)-HCO(3)(-) cotransport. Using fluorescence pH(i) imaging, we show that cancer-derived cell lines (colorectal HCT116 and HT29, breast MDA-MB-468, pancreatic MiaPaca2, and cervical HeLa) extrude acid by H(+) efflux and HCO(3)(-) influx, largely sensitive to dimethylamiloride and 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS), respectively. The magnitude of HCO(3)(-) influx was comparable among the cell lines and may represent a constitutive element of tumor pH(i) regulation. In contrast, H(+) efflux varied considerably (MDA-MB-468 > HCT116 > HT29 > MiaPaca2 > HeLa). When HCO(3)(-) flux was pharmacologically inhibited, acid extrusion in multicellular HT29 and HCT116 spheroids (â¼10,000 cells) was highly non-uniform and produced low pH(i) at the core. With depth, acid extrusion became relatively more DIDS-sensitive because the low extracellular pH at the spheroid core inhibits H(+) flux more than HCO(3)(-) flux. HCO(3)(-) flux inhibition also decelerated HCT116 spheroid growth. In the absence of CO(2)/HCO(3)(-), acid extrusion by H(+) flux in HCT116 and MDA-MB-468 spheroids became highly non-uniform and inadequate at the core. This is because H(+) transporters require extracellular mobile pH buffers, such as CO(2)/HCO(3)(-), to overcome low H(+) ion mobility and chaperone H(+) ions away from cells. CO(2)/HCO(3)(-) exerts a dual effect: as substrate for membrane-bound HCO(3)(-) transporters and as a mobile buffer for facilitating extracellular diffusion of H(+) ions extruded from cells. These processes can be augmented by carbonic anhydrase activity. We conclude that CO(2)/HCO(3)(-) is important for maintaining uniformly alkaline pH(i) in small, non-vascularized tumor growths and may be important for cancer disease progression.
Assuntos
Bicarbonatos/metabolismo , Dióxido de Carbono/metabolismo , Neoplasias/patologia , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Bicarbonatos/química , Transporte Biológico , Soluções Tampão , Linhagem Celular Tumoral , Difusão , Progressão da Doença , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Íons , Microscopia Confocal/métodos , Prótons , Esferoides Celulares/patologiaRESUMO
Growth of cancer cells in vitro can be attenuated by genetically inactivating selected metabolic pathways. However, loss-of-function mutations in metabolic pathways are not negatively selected in human cancers, indicating that these genes are not essential in vivo. We hypothesize that spontaneous mutations in 'metabolic genes' will not necessarily produce functional defects because mutation-bearing cells may be rescued by metabolite exchange with neighboring wild-type cells via gap junctions. Using fluorescent substances to probe intercellular diffusion, we show that colorectal cancer (CRC) cells are coupled by gap junctions assembled from connexins, particularly Cx26. Cells with genetically inactivated components of pH regulation (SLC9A1), glycolysis (ALDOA), or mitochondrial respiration (NDUFS1) could be rescued through access to functional proteins in co-cultured wild-type cells. The effect of diffusive coupling was also observed in co-culture xenografts. Rescue was largely dependent on solute exchange via Cx26 channels, a uniformly and constitutively expressed isoform in CRCs. Due to diffusive coupling, the emergent phenotype is less heterogenous than its genotype, and thus an individual cell should not be considered as the unit under selection, at least for metabolite-handling processes. Our findings can explain why certain loss-of-function mutations in genes ascribed as 'essential' do not influence the growth of human cancers.
Assuntos
Conexinas , Junções Comunicantes , Conexina 26/genética , Conexina 26/metabolismo , Conexinas/genética , Conexinas/metabolismo , Junções Comunicantes/metabolismo , Humanos , Mutação , Fenótipo , Isoformas de Proteínas/metabolismoRESUMO
BACKGROUND: Mild hemolysis occurs physiologically in neonates, but more severe forms can lead to life-threatening anemia. Newborns in developing regions are particularly at-risk due to the higher incidence of triggers (protozoan infections, sepsis, certain genetic traits). In advanced healthcare facilities, hemolysis is monitored indirectly using resource-intensive methods that probe downstream ramifications. These approaches could potentially delay critical decisions in early-life care, and are not suitable for point-of-care testing. Rapid and cost-effective testing could be based on detecting red blood cell (RBC)-specific proteins, such as carbonic anhydrase I (CAI), in accessible fluids (e.g., urine). METHODS: Urine was collected from 26 full-term male neonates and analyzed for CAI using immunoassays (ELISA, western blot) and proteomics (mass spectrometry). The cohort included a range of hemolytic states, including admissions with infection, ABO incompatibility, and receiving phototherapy. Data were paired with hemoglobin, serum bilirubin (SBR), and C-reactive protein (CRP) measurements. RESULTS: Urine from a control cohort (CRP < 20 mg/L, SBR < 125µmol/L) had no detectable CAI, in line with results from healthy adults. CAI excretion was elevated in neonates with raised SBR (>125 µmol/L), including those qualifying for phototherapy. Newborns with low SBR (<125 µmol/L) but elevated CRP (>20 mg/L) produced urine with strong CAI immunoreactivity. Proteomics showed that CAI was the most abundant RBC-specific protein in CAI-immunopositive samples, and did not associate with other RBC-derived peptides, indicating an intravascular hemolytic source followed by CAI-selective excretion. CONCLUSIONS: CAI is a direct biomarker of intravascular hemolysis that can be measured routinely in urine using non-invasive methods under minimal-laboratory conditions.
Assuntos
Anidrase Carbônica I , Hemólise , Doenças do Recém-Nascido , Anidrase Carbônica I/análise , Anidrase Carbônica I/metabolismo , Hemoglobinas , Humanos , Lactente , Recém-Nascido , Doenças do Recém-Nascido/diagnóstico , MasculinoRESUMO
CA IX is a transmembrane carbonic anhydrase isoenzyme predominantly expressed in human tumors in response to hypoxia and functionally implicated in adaptation of tumor cells to hypoxic stress via control of pH and cell adhesion. Intense investigations of the human CA IX as a hypoxic marker and a therapeutic target have been facilitated by specific monoclonal antibodies. However, no such reagents existed for the mouse CA IX ortholog. We generated five new anti-mouse CA IX monoclonal antibodies AM1-4, AM4-3, AM27-4, AM34-7 and AM35-1 produced using CA IX-deficient mice. The antibodies are suitable for various immunodetection methods including immunoblotting and immunohistochemistry. Using these reagents we show that the mouse CA IX is expressed in three out of nine tested mouse cell lines, namely in L929, MEF and TSA and is regulated by hypoxia and cell density similarly to human CA IX. We also demonstrate that the mouse CA IX exhibits hypoxia-related expression pattern in multicellular spheroids and in tumor xenografts. Our results indicate the use of the mouse model as suitable for further studies of CA IX role in tumor development and for its pre-clinical investigations. The new monoclonal antibodies represent potent tools for accomplishment of these future studies.