Inducing respiratory complex I impairment elicits an increase in PGC1α in ovarian cancer.

Anticancer strategies aimed at inhibiting Complex I of the mitochondrial respiratory chain are increasingly being attempted in solid tumors, as functional oxidative phosphorylation is vital for cancer cells. Using ovarian cancer as a model, we show that a compensatory response to an energy crisis induced by Complex I genetic ablation or pharmacological inhibition is an increase in the mitochondrial biogenesis master regulator PGC1α, a pleiotropic coactivator of transcription regulating diverse biological processes within the cell. We associate this compensatory response to the increase in PGC1α target gene expression, setting the basis for the comprehension of the molecular pathways triggered by Complex I inhibition that may need attention as drawbacks before these approaches are implemented in ovarian cancer care.

Respiratory Complex I dysfunction in cancer: from a maze of cellular adaptive responses to potential therapeutic strategies.

Mitochondria act as key organelles in cellular bioenergetics and biosynthetic processes producing signals that regulate different molecular networks for proliferation and cell death. This ability is also preserved in pathologic contexts such as tumorigenesis, during which bioenergetic changes and metabolic reprogramming confer flexibility favoring cancer cell survival in a hostile microenvironment. Although different studies epitomize mitochondrial dysfunction as a protumorigenic hit, genetic ablation or pharmacological inhibition of respiratory complex I causing a severe impairment is associated with a low-proliferative phenotype. In this scenario, it must be considered that despite the initial delay in growth, cancer cells may become able to resume proliferation exploiting molecular mechanisms to overcome growth arrest. Here, we highlight the current knowledge on molecular responses activated by complex I-defective cancer cells to bypass physiological control systems and to re-adapt their fitness during microenvironment changes. Such adaptive mechanisms could reveal possible novel molecular players in synthetic lethality with complex I impairment, thus providing new synergistic strategies for mitochondrial-based anticancer therapy.

The Release of Inflammatory Mediators from Acid-Stimulated Mesenchymal Stromal Cells Favours Tumour Invasiveness and Metastasis in Osteosarcoma.

Osteosarcoma is the most frequent primary malignant bone tumour with an impressive tendency to metastasise. Highly proliferative tumour cells release a remarkable amount of protons into the extracellular space that activates the NF-kB inflammatory pathway in adjacent stromal cells. In this study, we further validated the correlation between tumour glycolysis/acidosis and its role in metastases. In patients, at diagnosis, we found high circulating levels of inflammatory mediators (IL6, IL8 and miR-136-5p-containing extracellular vesicles). IL6 serum levels significantly correlated with disease-free survival and 18F-FDG PET/CT uptake, an indirect measurement of tumour glycolysis and, hence, of acidosis. In vivo subcutaneous and orthotopic models, co-injected with mesenchymal stromal (MSC) and osteosarcoma cells, formed an acidic tumour microenvironment (mean pH 6.86, as assessed by in vivo MRI-CEST pH imaging). In these xenografts, we enlightened the expression of both IL6 and the NF-kB complex subunit in stromal cells infiltrating the tumour acidic area. The co-injection with MSC also significantly increased lung metastases. Finally, by using 3D microfluidic models, we directly showed the promotion of osteosarcoma invasiveness by acidosis via IL6 and MSC. In conclusion, osteosarcoma-associated MSC react to intratumoural acidosis by triggering an inflammatory response that, in turn, promotes tumour invasiveness at the primary site toward metastasis development.

Current methodologies to detect circulating tumor cells: a focus on ovarian cancer.

Identification of circulating tumor cells (CTC) in liquid biopsies opens a window of opportunities for the optimization of clinical management of oncologic patients. In ovarian cancer (OC), which involves atypical routes of metastatic spread, CTC analyses may also offer novel insights about the mechanisms behind malignant progression of the disease. However, current methodologies struggle to precisely define CTC number in the peripheral blood of OC patients, and the isolation of viable cells for further characterization is still challenging. The biggest limitation is the lack of methodological standardization for OC CTC detection, preventing comprehensive definition of their clinical potential required for the transfer to practice. Here we describe and compare methods for CTC analysis that have been implemented for OC thus far, discussing pros, cons and improvements needed. We identify biophysical separation approaches as optimal for CTC enrichment. On the other hand, the identification of specific tumor antigens or gene transcripts, despite displaying drawbacks related to tumor heterogeneity, still remains the best approach for OC CTC detection.

Sarcoma treatment in the era of molecular medicine.

Sarcomas are heterogeneous and clinically challenging soft tissue and bone cancers. Although constituting only 1% of all human malignancies, sarcomas represent the second most common type of solid tumors in children and adolescents and comprise an important group of secondary malignancies. More than 100 histological subtypes have been characterized to date, and many more are being discovered due to molecular profiling. Owing to their mostly aggressive biological behavior, relative rarity, and occurrence at virtually every anatomical site, many sarcoma subtypes are in particular difficult-to-treat categories. Current multimodal treatment concepts combine surgery, polychemotherapy (with/without local hyperthermia), irradiation, immunotherapy, and/or targeted therapeutics. Recent scientific advancements have enabled a more precise molecular characterization of sarcoma subtypes and revealed novel therapeutic targets and prognostic/predictive biomarkers. This review aims at providing a comprehensive overview of the latest advances in the molecular biology of sarcomas and their effects on clinical oncology; it is meant for a broad readership ranging from novices to experts in the field of sarcoma.

Benign albeit glycolytic: MCT4 expression and lactate release in giant cell tumour of bone.

Giant cell tumour of bone (GCTB) is a histologically benign, locally aggressive skeletal lesion with an unpredictable propensity to relapse after surgery and a rare metastatic potential. The microscopic picture of GCTB shows different cell types, including multinucleated giant cells, mononuclear cells of the macrophage-monocyte lineage, and spindle cells. The histogenesis of GCTB is still debated, and morphologic, radiographic or molecular features are not predictive of the clinical course. Characterization of the unexplored cell metabolism of GCTB offers significant clues for the understanding of this elusive pathologic entity. In this study we aimed to characterize GCTB energetic metabolism, with a particular focus on lactate release and the expression of monocarboxylate transporters, to lie down a novel path for understanding the pathophysiology of this tumour. We measured the expression of glycolytic markers (GAPDH, PKM2, MCT4, GLUT1, HK1, LDHA, lactate release) in 25 tissue samples of GCTB by immunostaining and by mRNA and ELISA analyses. We also evaluated MCT1 and MCT4 expression and oxidative markers (JC1 staining and Bec index) in tumour-derived spindle cell cultures and CD14+ monocytic cells. Finally, we quantified the intratumoural and circulating levels of lactate in a series of 17 subjects with GCTB. In sharp contrast to the benign histological features of GCTB, we found a high expression of glycolytic markers, with particular reference to MCT4. Unexpectedly, this was mainly confined to the giant cell, not proliferating cell component. Accordingly, GCTB patients showed higher levels of blood lactate as compared to healthy subjects. In conclusion, taken together, our data indicate that GCTB is characterized by a highly glycolytic metabolism of its giant cell component, opening new perspectives on the pathogenesis, the natural history, and the treatment of this lesion.

Acid microenvironment promotes cell survival of human bone sarcoma through the activation of cIAP proteins and NF-κB pathway.

Extracellular acidification is a very common cause of stress in tumor microenvironment and of Darwinian pressure. In acid areas of the tumor, most cancer cells are-albeit slowly proliferating-more resistant to cell death than those in well-perfused regions. Tumor acidosis can directly regulate the expression of pro-survival proteins since a low extracellular pH activates the caspase-dependent cell death machinery. This mechanism has never been explored in bone sarcomas. We cultured osteosarcoma and Ewing sarcoma cells under low pH (pH 6.5), and we performed deep-sequencing and protein analysis. Both in in vitro and in vivo models, acidification activity enhanced tumor cells survival. However, we did not observe any change in ERK1 phosphorylation. On the contrary, both at the mRNA and protein level, we found a significant induction of TRAF adaptor proteins and of cIAP proteins (BIRC2 and/or BIRC3). As a consequence, the downstream nuclear transcription factor kappa B (NF-κB) survival pathway was increased. Furthermore, the treatment with the cIAP inhibitor LCL161 reverted the protection from apoptosis under low pH. In vitro results were confirmed both in Ewing sarcoma xenograft and in osteosarcoma patients, since the analysis of tumor tissues demonstrated that the levels of expression of TRAF1 or NF-κB1 significantly correlate with the level of expression of the vacuolar ATPase (V-ATPase), the most important proton pump in eukaryotes. Moreover, in the tissue sections of xenograft model, the nuclear translocation of RelB, a key subunit of the NF-κB transcriptional complex, localized in the tumor region that also corresponded to the acid microenvironment associated with the highest levels of expression of LAMP2 and V-ATPase, in the internal area of the tumor, as revealed by immunohistochemistry. Our data confirm that tumor acid microenvironment activates a stress-regulated switch to promote cell survival of bone sarcoma, and support the hypothesis that this mechanism is mediated by the recruitment of TRAF/cIAP complexes. Altogether, these results suggest that TRAF/cIAP can be considered as a target for anti-cancer therapies.

Pre-clinical Models for Studying the Interaction Between Mesenchymal Stromal Cells and Cancer Cells and the Induction of Stemness.

Mesenchymal stromal cells (MSC) have essential functions in building and supporting the tumour microenvironment, providing metastatic niches, and maintaining cancer hallmarks, and it is increasingly evident that the study of the role of MSC in cancer is crucial for paving the way to clinical opportunities for novel anti-cancer therapies. To date, the vast majority of preclinical models that have been used for studying the effect of reactive MSC on cancer growth, metastasis, and response to therapy has been mainly based on in vitro flat biology, including the co-culturing with cell compartmentalization or with cell-to-cell contact, and on in vivo cancer models with different routes of MSC inoculation. More complex in vitro 3D models based on spheroid structures that are formed by intermingled MSC and tumour cells are also capturing the interest in cancer research. These are innovative culture systems tailored on the specific tumour type and that can be combined with a synthetic extracellular matrix, or included in in silico technologies, to more properly mimic the in vivo biological, spatial, biochemical, and biophysical features of tumour tissues. In this review, we summarized the most popular and currently available preclinical models for evaluating the role of MSC in cancer and their specific suitability, for example, in assaying the MSC-driven induction of epithelial-to-mesenchymal transition or of stem-like traits in cancer cells. Finally, we enlightened the need to carefully consider those parameters that might unintentionally strongly affect the secretome in MSC-cancer interplay and introduce confounding variables for the interpretation of results.

Cause and effect of microenvironmental acidosis on bone metastases.

Skeletal involvement is a frequent and troublesome complication in advanced cancers. In the process of tumor cells homing to the skeleton to form bone metastases (BM), different mechanisms allow tumor cells to interact with cells of the bone microenvironment and seed in the bone tissue. Among these, tumor acidosis has been directly associated with tumor invasion and aggressiveness in several types of cancer although it has been less explored in the context of BM. In bone, the association of local acidosis and cancer invasiveness is even more important for tumor expansion since the extracellular matrix is formed by both organic and hard inorganic matrices and bone cells are used to sense protons and adapt or react to a low pH to maintain tissue homeostasis. In the BM microenvironment, increased concentration of protons may derive not only from glycolytic tumor cells but also from tumor-induced osteoclasts, the bone-resorbing cells, and may influence the progression or symptoms of BM in many different ways, by directly enhancing cancer cell motility and aggressiveness, or by modulating the functions of bone cells versus a pro-tumorigenic phenotype, or by inducing bone pain. In this review, we will describe and discuss the cause of acidosis in BM, its role in BM microenvironment, and which are the final effectors that may be targeted to treat metastatic patients.

Validation of Suitable Housekeeping Genes for the Normalization of mRNA Expression for Studying Tumor Acidosis.

Similar to other types of cancer, acidification of tumor microenvironment is an important feature of osteosarcoma, and a major source of cellular stress that triggers cancer aggressiveness, drug resistance, and progression. Among the different effects of low extracellular pH on tumor cells, we have recently found that short-term exposure to acidosis strongly affects gene expression. This alteration might also occur for the most commonly used housekeeping genes (HKG), thereby causing erroneous interpretation of RT-qPCR data. On this basis, by using osteosarcoma cells cultured at different pH values, we aimed to identify the ideal HKG to be considered in studies on tumor-associated acidosis. We verified the stability of 15 commonly used HKG through five algorithms (NormFinder, geNorm, BestKeeper, ΔCT, coefficient of variation) and found that no universal HKG is suitable, since at least four HKG are necessary for proper normalization. Furthermore, according to the acceptable range of values, YWHAZ, GAPDH, GUSB, and 18S rRNA were the most stable reference genes at different pH. Our results will be helpful for future investigations focusing on the effect of altered microenvironment on cancer behavior, particularly on the effectiveness of anticancer therapies in acid conditions.

Prominent role of RAB39A-RXRB axis in cancer development and stemness.

In this study, we found that RAB39A, a member of the RAS oncogene family, was selectively expressed in cancer cells of different histotypes, by analyzing gene expression in human osteosarcoma cells and the cancer stem cells (CSCs) and by comparing them with normal cells through global transcriptomics and principal component analyses. We further validated RAB39A as a therapeutic target, by silencing its expression. The silencing impaired cancer stemness and spherogenic ability in vitro, as well as tumorigenesis in vivo. RNA-seq analyses in the silenced spheres suggested that RAB39A is associated downstream with RXRB and KLF4. Notably, RXRB expression was inhibited in RAB39A-silenced CSCs. Induced overexpression of RXRB in RAB39A-silenced cells restored spherogenic ability and tumorigenesis, confirming RXRB as a major effector of RAB39A. Quantitative RT-PCR analysis of ∼400 human cancer tissues showed that RAB39A was highly expressed in sarcomas and in malignancies of lymphoid, adrenal and testicular tissues. Our data provide the rationale for targeting of the RAB39A-RXRB axis as a therapy for aggressive cancers.

MDA-MB-231 breast cancer cells fuel osteoclast metabolism and activity: A new rationale for the pathogenesis of osteolytic bone metastases.

Recent progress in dissecting the molecular paracrine circuits of cancer and stromal cells in bone metastases (BM) are offering new options to improve current merely palliative approach. The study of tumor-stroma metabolic interplay may further ameliorate this scenario. In this context, we demonstrated that highly glycolytic MDA-MB-231 cancer cells, that form osteolytic BM in vivo, release a large amount of lactate at a significantly higher level than MCF7 cells. Thus, we speculated that lactate released from carcinoma cells is uptaken and metabolically used by osteoclasts, the key players of osteolysis associated with BM. First, we demonstrated that the release of lactate at the bone site is mediated by monocarboxylate transporter 4 (MCT4), as revealed by immunostaining and MCT4 localization at the plasma membrane of tumor cells in mouse model of BM and in human tissue sections of BM. Then, we showed that in vitro lactate is uptaken by osteoclasts to be used as a fuel for the oxidative metabolism of osteoclasts, ultimately enhancing Type I collagen resorption. The passive transport of lactate into osteoclasts was mediated by MCT1: MCT1 expression is significantly upregulated during osteoclast differentiation and Type I collagen resorption is significantly impaired when osteoclasts are treated with 7-(N-benzyl-N-methylamino)-2-oxo-2H-chromene-3-carboxylic acid, an MCT-1 inhibitor. Together, these data demonstrate that lactate released by glycolytic breast carcinoma cells in the bone microenvironment promotes the formation of osteolytic lesions, and provide the rationale for further studies on the use of MCT1 targeting as a novel therapeutic approach in advanced cancer patients with BM.

Intratumoral acidosis fosters cancer-induced bone pain through the activation of the mesenchymal tumor-associated stroma in bone metastasis from breast carcinoma.

Cancer-induced bone pain (CIBP) is common in patients with bone metastases (BM), significantly impairing quality of life. The current treatments for CIBP are limited since they are often ineffective. Local acidosis derived from glycolytic carcinoma and tumor-induced osteolysis is only barely explored cause of pain. We found that breast carcinoma cells that prefer bone as a metastatic site have very high extracellular proton efflux and expression of pumps/ion transporters associated with acid-base balance (MCT4, CA9, and V-ATPase). Further, the impairment of intratumoral acidification via V-ATPase targeting in xenografts with BM significantly reduced CIBP, as measured by incapacitance test. We hypothesize that in addition to the direct acid-induced stimulation of nociceptors in the bone, a novel mechanism mediated by the acid-induced and tumor-associated mesenchymal stroma might ultimately lead to nociceptor sensitization and hyperalgesia. Consistent with this, short-term exposure of cancer-associated fibroblasts, mesenchymal stem cells, and osteoblasts to pH 6.8 promotes the expression of inflammatory and nociceptive mediators (NGF, BDNF, IL6, IL8, IL1b and CCL5). This is also consistent with a significant correlation between breakthrough pain, measured by pain questionnaire, and combined high serum levels of BDNF and IL6 in patients with BM, and also by immunofluorescence staining showing IL8 expression that was more in mesenchymal stromal cells rather than in tumors cells, and close to LAMP-2 positive acidifying carcinoma cells in BM tissue sections. In summary, intratumoral acidification in BM might promote CIBP also by activating the tumor-associated stroma, offering a new target for palliative treatments in advanced cancer.

Energy metabolism in osteoclast formation and activity.

Osteoclastogenesis and osteolysis are energy-consuming processes supported by high metabolic activities. In human osteoclasts derived from the fusion of monocytic precursors, we found a substantial increase in the number of mitochondria with differentiation. In mature osteoclasts, mitochondria were also increased in size, rich of cristae and arranged in a complex tubular network. When compared with immature cells, fully differentiated osteoclasts showed higher levels of enzymes of the electron transport chain, a higher mitochondrial oxygen consumption rate and a lower glycolytic efficiency, as evaluated by extracellular flux analysis and by the quantification of metabolites in the culture supernatant. Thus, oxidative phosphorylation appeared the main bioenergetic source for osteoclast formation. Conversely, we found that bone resorption mainly relied on glycolysis. In fact, osteoclast fuelling with galactose, forcing cells to depend on Oxidative Phosphorylation by reducing the rate of glycolysis, significantly impaired Type I collagen degradation, whereas non-cytotoxic doses of rotenone, an inhibitor of the mitochondrial complex I, enhanced osteoclast activity. Furthermore, we found that the enzymes associated to the glycolytic pathway are localised close to the actin ring of polarised osteoclasts, where energy-demanding activities associated with bone degradation take place. In conclusion, we demonstrate that the energy required for osteoclast differentiation mainly derives from mitochondrial oxidative metabolism, whereas the peripheral cellular activities associated with bone matrix degradation are supported by glycolysis. A better understanding of human osteoclast energy metabolism holds the potential for future therapeutic interventions aimed to target osteoclast activity in different pathological conditions of bone.

Altered pH gradient at the plasma membrane of osteosarcoma cells is a key mechanism of drug resistance.

Current therapy of osteosarcoma (OS), the most common primary bone malignancy, is based on a combination of surgery and chemotherapy. Multidrug resistance mediated by P-glycoprotein (P-gp) overexpression has been previously associated with treatment failure and progression of OS, although other mechanisms may also play a role. We considered the typical acidic extracellular pH (pHe) of sarcomas, and found that doxorubicin (DXR) cytotoxicity is reduced in P-gp negative OS cells cultured at pHe 6.5 compared to standard 7.4. Short-time (24-48 hours) exposure to low pHe significantly increased the number and acidity of lysosomes, and the combination of DXR with omeprazole, a proton pump inhibitor targeting lysosomal acidity, significantly enhanced DXR cytotoxicity. In OS xenografts, the combination treatment of DXR and omeprazole significantly reduced tumor volume and body weight loss. The impaired toxicity of DXR at low pHe was not associated with increased autophagy or lysosomal acidification, but rather, as shown by SNARF staining, with a reversal of the pH gradient at the plasma membrane (ΔpHcm), eventually leading to a reduced DXR intracellular accumulation. Finally, the reversal of ΔpHcm in OS cells promoted resistance not only to DXR, but also to cisplatin and methotrexate, and, to a lesser extent, to vincristine. Altogether, our findings show that, in OS cells, short-term acidosis induces resistance to different chemotherapeutic drugs by a reversal of ΔpHcm, suggesting that buffer therapies or regimens including proton pump inhibitors in combination to low concentrations of conventional anticancer agents may offer novel solutions to overcome drug resistance.

Identification and Validation of Housekeeping Genes for Gene Expression Analysis of Cancer Stem Cells.

The characterization of cancer stem cell (CSC) subpopulation, through the comparison of the gene expression signature in respect to the native cancer cells, is particularly important for the identification of novel and more effective anticancer strategies. However, CSC have peculiar characteristics in terms of adhesion, growth, and metabolism that possibly implies a different modulation of the expression of the most commonly used housekeeping genes (HKG), like b-actin (ACTB). Although it is crucial to identify which are the most stable HKG genes to normalize the data derived from quantitative Real-Time PCR analysis to obtain robust and consistent results, an exhaustive validation of reference genes in CSC is still missing. Here, we isolated CSC spheres from different musculoskeletal sarcomas and carcinomas as a model to investigate on the stability of the mRNA expression of 15 commonly used HKG, in respect to the native cells. The selected genes were analysed for the variation coefficient and compared using the popular algorithms NormFinder and geNorm to evaluate stability ranking. As a result, we found that: 1) Tata Binding Protein (TBP), Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ), Peptidylprolyl isomerase A (PPIA), and Hydroxymethylbilane synthase (HMBS) are the most stable HKG for the comparison between CSC and native cells; 2) at least four reference genes should be considered for robust results; 3) the use of ACTB should not be recommended, 4) specific HKG should be considered for studies that are focused only on a specific tumor type, like sarcoma or carcinoma. Our results should be taken in consideration for all the studies of gene expression analysis of CSC, and will substantially contribute for future investigations aimed to identify novel anticancer therapy based on CSC targeting.

Modulation of TGFbeta 2 levels by lamin A in U2-OS osteoblast-like cells: understanding the osteolytic process triggered by altered lamins.

Transforming growth factor beta (TGFbeta) plays an essential role in bone homeostasis and deregulation of TGFbeta occurs in bone pathologies. Patients affected by Mandibuloacral Dysplasia (MADA), a progeroid disease linked to LMNA mutations, suffer from an osteolytic process. Our previous work showed that MADA osteoblasts secrete excess amount of TGFbeta 2, which in turn elicits differentiation of human blood precursors into osteoclasts. Here, we sought to determine how altered lamin A affects TGFbeta signaling. Our results show that wild-type lamin A negatively modulates TGFbeta 2 levels in osteoblast-like U2-OS cells, while the R527H mutated prelamin A as well as farnesylated prelamin A do not, ultimately leading to increased secretion of TGFbeta 2. TGFbeta 2 in turn, triggers the Akt/mTOR pathway and upregulates osteoprotegerin and cathepsin K. TGFbeta 2 neutralization rescues Akt/mTOR activation and the downstream transcriptional effects, an effect also obtained by statins or RAD001 treatment. Our results unravel an unexpected role of lamin A in TGFbeta 2 regulation and indicate rapamycin analogs and neutralizing antibodies to TGFbeta 2 as new potential therapeutic tools for MADA.

V-ATPase is a candidate therapeutic target for Ewing sarcoma.

Suppression of oxidative phosphorylation combined with enhanced aerobic glycolysis and the resulting increased generation of protons are common features of several types of cancer. An efficient mechanism to escape cell death resulting from intracellular acidification is proton pump activation. In Ewing sarcoma (ES), although the tumor-associated chimeric gene EWS-FLI1 is known to induce the accumulation of hypoxia-induced transcription factor HIF-1α, derangements in metabolic pathways have been neglected so far as candidate pathogenetic mechanisms. In this paper, we observed that ES cells simultaneously activate mitochondrial respiration and high levels of glycolysis. Moreover, although the most effective detoxification mechanism of proton intracellular storage is lysosomal compartmentalization, ES cells show a poorly represented lysosomal compartment, but a high sensitivity to the anti-lysosomal agent bafilomycin A1, targeting the V-ATPase proton pump. We therefore investigated the role of V-ATPase in the acidification activity of ES cells. ES cells with the highest GAPDH and V-ATPase expression also showed the highest acidification rate. Moreover, the localization of V-ATPase was both on the vacuolar and the plasma membrane of all ES cell lines. The acidic extracellular pH that we reproduced in vitro promoted high invasion ability and clonogenic efficiency. Finally, targeting V-ATPase with siRNA and omeprazole treatments, we obtained a significant selective reduction of tumor cell number. In summary, glycolytic activity and activation of V-ATPase are crucial mechanisms of survival of ES cells and can be considered as promising selective targets for the treatment of this tumor.

Continuous enzyme-coupled assay of phosphate- or pyrophosphate-releasing enzymes.

A coupled enzyme assay able to monitor the kinetics of reactions catalyzed by phosphate- or pyrophosphate-releasing enzymes is presented here. The assay is based on the concerted action of inorganic pyrophosphatase (PPase), purine nucleoside phosphorylase (PNPase), and xanthine oxidase (XOD). In the presence of phosphate, PNPase catalyzes the phosphorolysis of inosine, generating hypoxanthine, which is oxidized to uric acid by XOD. The uric acid accordingly formed can be spectrophotometrically monitored at 293 nm, taking advantage of a molar extinction coefficient which is independent of pH between 6 and 9. The coupled assay was tested using DNA polymerases as a model system. The activity of Klenow enzyme was quantitatively determined, and it was found in agreement with the corresponding activity determined by traditional methods. Moreover, the continuous coupled assay was used to determine Km and Vmax of Klenow enzyme, yielding values in good agreement with previous observations. Finally, the coupled assay was also used to determine the activity of partially purified DNA polymerases, revealing its potential use to monitor purification of phosphate- or pyrophosphate-releasing enzymes.