P1 and P2 purinergic receptor signal transduction in rat type II pneumocytes

Extracellular ATP is a potent agonist of surfactant phosphatidylcholine (PC) exocytosis from type II pneumocytes in culture. We studied P1 and P2 receptor signal transduction in type II pneumocytes. The EC50 for ATP on PC exocytosis was 10(-6) M, whereas the EC50 for ADP, AMP, adenosine, and the nonmetabolizable ATP analogue alpha,beta-methylene ATP was 10(-4) M. The rank order of agonists for PC exocytosis was ATP greater than ADP greater than AMP greater than adenosine greater than alpha,beta-methylene ATP. The rank order of agonists for phosphatidylinositol (PI) hydrolysis was ATP greater than ADP, whereas AMP, adenosine, and alpha,beta-methylene ATP did not stimulate PI hydrolysis. ATP (10(-4) M) caused a 15-fold increase in adenosine 3',5'-cyclic monophosphate (cAMP) production, and the nonmetabolizable adenosine analogue 5'-N-ethylcarboxyamidoadenosine (10(-6) M) increased cAMP production threefold. The effects of both these agonists on cAMP production were completely inhibited by the adenosine antagonist 8-phenyltheophylline (10(-5) M). The effects of ATP (10(-4) M) on PC exocytosis were inhibited 38% by 10(-5) M 8-phenyltheophylline. Thus, ATP regulates PC exocytosis by activating P2 receptors, which stimulate PI hydrolysis to inositol phosphate, as well as by activating P1 receptors, which stimulate cAMP production. Interactions between the P1 and P2 pathways may explain the high potency of extracellular ATP as an agonist of PC exocytosis.     

Substrate specificity of T4 RNA-ligase. The role of phosphate nucleotide residues in the formation of a covalent AMP-RNA-ligase complex]

The inhibiting effect of adenosine, AMP, ADP, ATP, gamma-thio ATP (I), beta,gamma-imine ATP (II), beta,gamma-methylene ATP (III), P1,P3-di(adenosine-5') triphosphate (IV), P1,P4-di(adenosine-5') tetraphosphate (V) and adenosine 5'-tetraphosphate (VI) on the first step of the T4 RNA ligase reaction was studied. All the compounds tested, with the exception of adenosine, appeared to be competitive inhibitors of the first step of the enzymatic reaction. The inhibition constants (Ki) for the ATP analogs were determined. The data obtained suggest that the efficiency of inhibition depends on the number of phosphate groups and on the structure of ATP analogs. All the compounds under study (I-VI), except for AMP and ADP, form covalent AMP-RNA ligase complexes.     

Growth inhibition of transformed mouse fibroblasts by adenine nucleotides occurs via generation of extracellular adenosine

The growth of transformed mouse fibroblasts (3T6 cells) in medium containing 5% fetal bovine serum was inhibited after treatment with concentrations greater than 50 microM ATP, ADP, or AMP. Adenosine, the common catabolite of the nucleotides, had no effect on cell growth at concentrations below 1 mM. However, the following results indicate that the toxicity of ATP, ADP, and AMP is mediated by serum- and cell-associated hydrolysis of the nucleotides to adenosine. 1) ADP and AMP, but not ATP, were toxic to 3T6 cells grown in serum-free medium or medium in which phosphohydrolase activity of serum was inactivated. Under these conditions, the cells exhibited cell-associated ADPase and 5'-nucleotidase activity, but little ecto-ATPase activity. 2) Inhibition of adenosine transport in 3T6 cells by dipyridamole or S-(p-nitrobenzyl)-6-thioinosine prevented the toxicity of ATP in serum-containing medium and of ADP and AMP in serum-free medium. 3) A 16-24-h exposure to 125 microM AMP or ATP was needed to inhibit cell growth under conditions where serum- and cell-associated hydrolysis of the nucleotides generated adenosine in the medium continuously over the same time period. In contrast, 125 microM adenosine was completely degraded to inosine and hypoxanthine within 8-10 h. Furthermore, multiple doses of adenosine added to the cells at regular intervals over a 16-h period were significantly more toxic than an equivalent amount of adenosine added in one dose. Treatment of 3T6 cells with AMP elevated intracellular ATP and ADP levels and reduced intracellular UTP levels, effects which were inhibited by extracellular uridine. Uridine also prevented growth inhibition by ATP, ADP, and AMP. These and other results indicate that serum- and cell-associated hydrolysis of adenine nucleotides to adenosine suppresses growth by adenosine-dependent pyrimidine starvation.     

Airway effects of purine nucleosides and nucleotides and release with bronchial provocation in asthma

Adenosine, AMP, and ADP all caused similar concentration-related bronchoconstriction when inhaled by patients with asthma, whereas the adenosine hydrolysis product inosine had no effect. Geometric mean provocation concentrations of adenosine AMP and ADP causing a 20% fall in forced expiratory volume in 1 s (PCf20) were 2.34, 4.27, and 2.19 mumol/ml and 40% fall in specific airway conductance (PCs40) 3.16, 5.01, and 2.0 mumol/ml. Bronchoconstriction was rapid in onset, reaching a maximum 2-5 min after a single inhalation of AMP. In 31 asthmatic subjects a positive correlation was established between airway responsiveness to histamine, as an index of non-specific responsiveness, and airway reactivity to adenosine (PCf20, r = 0.60; PCs40, r = 0.64; P less than 0.01). Following bronchial provocation with allergen in nine subjects, plasma levels of adenosine increased from a mean base line of 5.4 +/- 0.9 to 9.6 +/- 2.0 ng/ml at 15 min (P less than 0.01) in parallel with a fall in forced expiratory volume in 1 s. With methacholine provocation bronchoconstriction reached maximum 2-5 min postchallenge being followed by, but not accompanied by, significant increases in plasma levels of adenosine. These data suggest that adenosine is a specific bronchoconstrictor that may contribute to airflow obstruction in asthma.     

Effects of chlorogenic acid,caffeine and coffee on components of the purinergic system of streptozotocin-induced diabetic rats

We evaluated the effect of chlorogenic acid (CGA), caffeine (CA) and coffee (CF) on components of the purinergic system from the cerebral cortex and platelets of streptozotocin-induced diabetic rats. Animals were divided into eight groups: control animals treated with (I) water (WT), (II) CGA (5 mg/kg), (III) CA (15 mg/kg) and (IV) CF (0.5 g/kg), and diabetic animals treated with (V) WT, (VI) CGA (5 mg/kg), (VII) CA (15 mg/kg) and (VIII) CF (0.5 g/kg). Our results showed an increase (173%) in adenosine monophosphate (AMP) hydrolysis in the cerebral cortex of diabetic rats. In addition, CF treatment increased adenosine diphosphate (ADP) and AMP hydrolysis in group VIII synaptosomes. Platelets showed an increase in ectonucleotidase activity in group V, and all treatments reduced the increase in adenosine triphosphate and ADP hydrolysis. Furthermore, there was an increase in platelet aggregation of 72% in the diabetic rats, and CGA and CF treatment reduced platelet aggregation by nearly 60% when compared to diabetic rats. In this context, we can suggest that CGA and CF treatment should be considered a therapeutic and scientific target to be investigated in diseases associated with hyperglycemia.     

Adenine Nucleotide Hydrolysis in Patients with Aseptic and Bacterial Meningitis

The meningitis is a disease with high mortality rates capable to cause neurologic sequelae. The adenosine (the final product of ATP hydrolysis by ectonucleotidases), have a recognized neuroprotective actions in the central nervous system (CNS) in pathological conditions. The aim of the present study was evaluate the adenine nucleotides hydrolysis for to verify one possible role of ATP, ADP and AMP hydrolysis in inflammatory process such as meningitis. The hydrolysis was verified in cerebrospinal fluid (CSF) from human patients with aseptic and bacterial meningitis. Our results showed that the ATP hydrolysis was reduced 12.28% (P < 0.05) in bacterial meningitis and 22% (P < 0.05) in aseptic meningitis. ADP and AMP hydrolysis increased 79.13% (P < 0.05) and 26.37% (P < 0.05) in bacterial meningitis, respectively, and 57.39% (P < 0.05) and 42.64% (P < 0.05) in aseptic meningitis, respectively. This may be an important protective mechanism in order to increase adenosine production.     

Breast cancer in the eastern province of Saudi Arabia

In the Kingdom of Saudi Arabia (KSA), hospital and population based statistics have shown that breast cancer has the highest crude frequency rate among Saudi women. The scarcity of reports about the disease in the KSA has been the impetus to this analysis about breast cancer in the eastem province of KSA. Data on female patients with invasive breast carcinoma seen at King Fahd Hospital of the University in the eastern province of KSA, were retrospectively reviewed. The analysis intended to examine the pattern of the disease and the outcome for patients. Between 1985 and 1995, 292 patients were identified. Their median age±SD (standard deviation) was 42±10.5 years. Most patients were younger than 50 years (78%) and were predominantly premenopausals (79%). Only 25 (9%) of patients had stage I cancer, whilst 130 (44%), 90 (30%), and 47 (16%) had stage II, III, and IV, respectively. Among patients with known axillary nodal status (242 patients), only 37% were node-negative whilst 32% and 31% had 1–3, and ≥4 positive nodes, respectively. Adjuvant chemotherapy and tamoxifen were commonly offered; nonetheless, other adjuvant modalities were rarely utilised. The median follow-up ±SD of all patients was 62.3±8.9 months: 152 patients (52%) were alive with no evidence of disease, 25 (9%) were alive with evidence of disease, and 115 (39%) were dead from breast cancer or its related complications. The median survival of the entire group was not obtained, but the 10-year projected survival was 55%. For stage I and II patients, 118 (76%) were alive with a projected 10-year actuarial survival of 64%. On the other hand, only 51 (57%) of patients with stage III disease were alive with a median survival of 41.5 months (95% Confidence interval (CI), 18.9 to 51.3). Patients with stage IV disease demonstrated a poor outcome with a median survival of 23.5 (95%, CI 12.2 to 31.4). Multivariate analyses were performed to explore the influence of independent variables on overall survival (OS) for patients with non-metastatic disease. Besides the expected adverse effect of disease progression, the favourable influence of adjuvant chemotherapy and tamoxifen prevailed. The amount of benefit gained from tamoxifen, however, was small. Similar analyses were undertaken to determine the influence of independent variables on progression-free survival (PFS). These analyses ascertained the adverse effects of advanced stage and the favourable impact of adjuvant chemotherapy. Breast cancer in the KSA has features that are distinctive from those of industrialised countries. Survival data, however, were comparable. The favourable influence of adjuvant chemotherapy was evident on both OS and PFS. Adjuvant tamoxifen, however, had little effect. Due to its infrequent use, the role of other adjuvant modalities could not be asserted.     

Role of membrane-bound 5''-nucleotidase in nucleotide uptake by the moderate halophile Vibrio costicola

Intact cells of Vibrio costicola hydrolyzed ATP, ADP, and AMP. The membrane-bound 5'-nucleotidase (C. Bengis-Garber and D. J. Kushner, J. Bacteriol. 146:24-32, 1981) was solely responsible for these activities, as shown by experiments with anti-5'-nucleotidase serum and with the ATP analog, adenosine 5'-(beta gamma-imido)-diphosphate. Fresh cell suspensions rapidly accumulated 8-14C-labeled adenine 5'-nucleotides and adenosine. The uptake of ATP, ADP, and AMP (but not the adenosine uptake) was inhibited by adenosine 5'-(beta gamma-imido)-diphosphate similarly to the inhibition of the 5'-nucleotidase. Furthermore, the uptake of nucleotides had Mg2+ requirements similar to those of the 5'-nucleotidase. The uptake of ATP was competitively inhibited by unlabeled adenosine and vice versa; inhibition of the adenosine uptake by ATP occurred only in the presence of Mg2+. These experiments indicated that nucleotides were dephosphorylated to adenosine before uptake. The hydrolysis of [alpha-32P]ATP as well as the uptake of free adenosine followed Michaelis-Menten kinetics. The kinetics of uptake of ATP, ADP, and AMP also each appeared to be a saturable carrier-mediated transport. The kinetic properties of the uptake of ATP were compared with those of the ATP hydrolysis and the uptake of adenosine. It was concluded that the adenosine moiety of ATP was taken up via a specific adenosine transport system after dephosphorylation by the 5'-nucleotidase.     

In vitro effect of homocysteine on nucleotide hydrolysis by blood serum from adult rats

During the past few years, elevated blood levels of homocysteine (Hcy) have been linked to increased risk of premature coronary artery disease, stroke and thromboembolism. These processes can be also related to the ratio adenine nucleotide/adenosine, since extracellularly these nucleotides are associated with modulation of processes such as platelet aggregation, vasodilatation and coronary flow. Furthermore, there are some studies that suggest a relationship between Hcy and plasma adenosine concentrations. The sequential hydrolysis of ATP to adenosine by soluble nucleotidases constitutes one of the systems for rapid inactivation of circulating adenine nucleotides. Thus, the main objective of this study was to evaluate if Hcy can participate in the modulation of the extracellular adenine nucleotide hydrolysis by rat blood serum. Our results showed that Hcy, at final concentrations of 5.0 mM, inhibits in vitro ATP, ADP and AMP hydrolysis by 26, 21 and 16%, respectively. Also Hcy, at final concentrations of 8.0mM, inhibited the in vitro hydrolysis of ATP, ADP and AMP by 46, 44 and 44%, respectively. Kinetic analysis showed that the inhibitions of the three adenine nucleotide hydrolyses in the presence of Hcy, by serum of adult rats, is of the uncompetitive type. The IC50 calculated from the results obtained were 6.52+/-1.75 mM (n = 4), 5.18 +/- 0.64 mM (n = 3) and 5.16 +/- 1.22 mM (n = 3) for ATP, ADP and AMP hydrolysis, respectively.     

曲妥珠单抗辅助或新辅助治疗HER2 阳性转移性乳腺癌的临床结果

目的:比较人表皮生长因子受体2过表达(HER2+)的患者既往接受以曲妥珠单抗为基础辅助或新辅助治疗后再次接受针曲妥珠单抗治疗的临床结果。方法:共247例(I-III期170例,IV期77例)HER2+转移性乳腺癌患者,其中首次接受曲妥珠单抗治疗患者211例(I-III期134例,IV期77例),再次接受曲妥珠单抗治疗患者36例(I-III期)。使用Cox比例风险回归和logistic回归分析首次或提前接受曲妥珠单抗治疗的患者的预后的临床结果,生存评估使用Kaplan-Meier法。结果:I-III期HER2+转移性乳腺癌患者中,未预先接受曲妥珠单抗治疗组的中位总生存期为36个月和预先接受曲妥珠单抗治疗组为28个月(危害比[HR],1.45;95%CI,1.05-2.02[P=0.012]);I-IV期HER2+转移性乳腺癌患者中,未预先使用曲妥珠单抗组的中位总生存期为37个月,客观缓解率58%;临床获益率77%;预先使用曲妥珠单抗组为25个月,客观缓解率28%;临床获益率37%;调整后的比值比为客观缓解率0.37(9%CI,0.18-0.77;P=0.010)和临床获益率0.28(95%CI,0.14-0.59;P=0.014)。单因素分析没有提前接受曲妥珠单抗治疗组中位总生存率较长(P=0.012)。多因素分析发现总生存率没有显著差异(P=0.19)。结论:当曲妥珠单抗用于转移性疾病,没有提前接受曲妥珠单抗治疗的HER2+乳腺癌患者临床结果优于提前接受曲妥珠单抗治疗的患者。     

'Arimidex' (anastrozole) versus tamoxifen as adjuvant therapy in postmenopausal women with early breast cancer--efficacy overview

ATAC, a randomized, double-blind trial, compared tamoxifen (20 mg) with anastrozole (‘Arimidex’) (1 mg) alone, and the combination of anastrozole plus tamoxifen (combination), as adjuvant endocrine treatment for postmenopausal patients with early breast cancer. Patients with operable invasive breast cancer following completion of primary therapy, who were candidates to receive adjuvant endocrine therapy, were eligible for this study. Primary endpoints were disease-free survival (DFS) and tolerability. Other endpoints included time to recurrence (TTR: censoring non-breast cancer deaths before recurrence) and the incidence of contralateral breast cancer. A total of 9366 patients were included in this study (N=3125, 3116 and 3125 for anastrozole, tamoxifen and the combination, respectively). Median duration of therapy was 30.7 months and median follow-up was 33.3 months. The total numbers of events were 317, 379 and 383 for anastrozole, tamoxifen and the combination, respectively. DFS was significantly improved in the overall population for anastrozole versus tamoxifen (hazard ratio (HR)=0.81, 95% confidence interval (CI) (0.71–0.96), P=0.013). Anastrozole showed improved TTR compared with tamoxifen (HR=0.79, CI (0.67–0.94), P=0.008), which improved even further in the ER+ and/or PR+ subgroup (HR=0.73, CI (0.59–0.90), P=0.003). The incidences of hot flushes, thromboembolic events, ischaemic cerebrovascular events, vaginal bleeding/discharge and endometrial cancer were significantly reduced with anastrozole compared with tamoxifen (P<0.03 for all). Musculoskeletal disorders and fractures were significantly reduced in patients receiving tamoxifen compared with those on anastrozole (P<0.03 for both). No increase in hip fractures was seen for anastrozole versus tamoxifen (11 versus 13, respectively). Combination treatment was equivalent to tamoxifen in terms of both efficacy and tolerability. Anastrozole showed superior efficacy to tamoxifen for DFS, TTR and contralateral breast cancer. Early findings show anastrozole to be an effective and well-tolerated endocrine option for the treatment of postmenopausal patients with early breast cancer. For the first time a choice now exists for adjuvant endocrine treatment for postmenopausal women with hormone responsive tumours. Longer follow-up will further define the benefit/risk of anastrozole adjuvant therapy.     

Involvement of inositol 1,4,5-trisphosphate and calcium in the action of adenine nucleotides on aortic endothelial cells

ADP and ATP, in the 1-100 microM range of concentrations, increased the formation of inositol phosphates in bovine aortic endothelial cells. The accumulation of inositol trisphosphate in response to adenine nucleotides was rapid (maximum at 15 s) and transient. This material was identified as the biologically active isomer inositol 1,4,5-trisphosphate on the basis of its retention time by high-performance liquid chromatography on an anion-exchange resin. AMP and adenosine have no effect on inositol phosphates. The action of ATP and ADP was mimicked with an equal potency and activity by their phosphorothioate analogs, ATP gamma S and ADP beta S, and with a lower potency by adenosine 5'-(beta,gamma-imido)triphosphate, whereas adenosine 5'-(alpha,beta-methylene)triphosphate, was inactive. In the same range of concentrations, ADP and ATP induced an efflux of 45Ca2+ from prelabeled bovine aortic endothelial cells and increased the fluorescence emission by cells loaded with quin-2. Here, too, AMP and adenosine were completely inactive. The outflow of 45Ca2+ induced by ADP was partially maintained in a calcium-free medium. These data suggest that in aortic endothelial cells, P2-purinergic receptors, of the P2Y subtype, are coupled to the hydrolysis of phosphatidylinositol bisphosphate by a phospholipase C. It is likely that the release of prostacyclin and endothelium-derived relaxing factor in response to ADP and ATP is a consequence of this initial event.     

Substrate binding modes of purine and pyrimidine nucleotides to human ecto-5′-nucleotidase (CD73) and inhibition by their bisphosphonic acid derivatives

Human ecto-5-nucleotidase (CD73) is involved in purinergic signalling, which influences a diverse range of biological processes. CD73 hydrolyses AMP and is the major control point for the levels of extracellular adenosine. Inhibitors of CD73 thus block the immunosuppressive action of adenosine, a promising approach for cancer immunotherapy. Interestingly, ADP and ATP are competitive inhibitors of CD73, with the most potent small-molecule inhibitors to date being non-hydrolysable ADP analogues. While AMP is the major substrate of the enzyme, CD73 has been reported to hydrolyse other 5′-nucleoside monophosphates. Based on a fragment screening campaign at the BESSY II synchrotron, we present the binding modes of various deoxyribo- and ribonucleoside monophosphates and of four additional fragments binding to the nucleoside binding site of the open form of the enzyme. Kinetic analysis of monophosphate hydrolysis shows that ribonucleotide substrates are favoured over their deoxyribose equivalents with AMP being the best substrate. We characterised the initial step of AMP hydrolysis, the binding mode of AMP to the open conformation of CD73 and compared that to other monophosphate substrates. In addition, the inhibitory activity of various bisphosphonic acid derivatives of nucleoside diphosphates was determined. Although AMPCP remains the most potent inhibitor, replacement of the adenine base with other purines or with pyrimidines increases the K_i value only between twofold and sixfold. On the other hand, these nucleobases offer new opportunities to attach substituents for improved pharmacological properties.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11302-021-09802-w.     

A non-functional retinoblastoma tumor suppressor (RB) pathway in premenopausal breast cancer is associated with resistance to tamoxifen

The retinoblastoma tumor suppressor (RB) is important for retaining cell cycle control and loss of RB function is commonly observed in various malignancies. Experimental and animal studies have shown that RB knockdown in ER+ (estrogen receptor) cell lines and xenografts leads to resistance to tamoxifen, indicating that RB-inactivation could be linked to impaired response to specific cancer treatments. To address this issue, we utilized a unique randomized trial including 500 premenopausal breast cancer patients receiving either two years of adjuvant tamoxifen treatment or no treatment after surgery, and defined the tamoxifen response in RB-subgroups. Non-functional RB tumors were defined by lack of concordance between RB-phosphorylation and proliferation, in comparison to RB-functional tumors displaying comparable RB-phosphorylation and proliferation. In the ER+ tumors harboring a functional RB pathway (N=204), patients benefited from adjuvant tamoxifen with fewer breast cancer recurrences (HR=0.53, 95% CI 0.34-0.81, P=0.003). In the small subgroup of ER+ and RB non-functional tumors there was no benefit of tamoxifen (HR=2.28, 95% CI 0.51-10.3, P=0.28). In a multivariate analysis, the interaction between status of the RB pathway and treatment was significant (P=0.010), validating that despite being a small subgroup of ER+ breast cancer, RB functional status appears to be linked to response to tamoxifen treatment. These findings are in line with earlier experimental data altogether suggesting that analyses of RB status in breast cancer have the potential to be one among other future predictive factors that needs to be analyzed in order to successfully identify patients that will benefit from tamoxifen treatment.     

α-Tocopherol regulates ectonucleotidase activities in synaptosomes from rats fed a high-fat diet

α-Tocopherol (α-Toc) is involved in various physiologic processes, which present antioxidant and neuroprotective properties. High-fat diets have an important role in neurodegenerative diseases and neurological disturbances. This study aimed to investigate the effects of treatment with α-Toc and the consumption of high-fat diets on ectonucleotidase activities in synaptosomes of cerebral cortex, hippocampus and striatum of rats. Animals were divided into four different groups, which received standard diet (control), high-fat saturated diet (HF), α-Toc and high-fat saturated diet plus α-Toc (α-Toc + HF). High-fat saturated diet was administered ad libitum and α-Toc by gavage using a dose of 50 mg·kg(-1). After 3 months of treatment, animals were submitted to euthanasia, and cerebral cortex, hippocampus and striatum were collected for biochemical assays. Results showed that adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP) hydrolysis in the cerebral cortex, hippocampus and striatum were decreased in HF in comparison to the other groups (P < 0·05). When rats that received HF were treated with α-Toc, the activity of the ectonucleotidases was similar to the control. ATP, ADP and AMP hydrolysis in the cerebral cortex, hippocampus and striatum were increased in the α-Toc group when compared with the other groups (P < 0·05). These findings demonstrated that the HF alters the purinergic signaling in the nervous system and that the treatment with α-Toc was capable of modulating the adenine nucleotide hydrolysis in this experimental condition.     

Mechanisms of hydrolysis of adenosine 5'-triphosphate, adenosine 5'-diphosphate, and inorganic pyrophosphate in aqueous perchloric acid

The acid-catalyzed hydrolysis of adenosine 5'-triphosphate (ATP) has been found to give rise both to adenosine 5'-diphosphate (ADP) and inorganic phosphate and to adenosine 5'-phosphate (AMP) and inorganic pyrophosphate. Kinetic and isotope studies on the mechanism of hydrolysis of ATP therefore depend on a knowledge of the mechanism of hydrolysis of the polyphosphate products, ADP and inorganic pyrophosphate. The latter reactions have been studied over the acidity range 1--5 M perchloric acid at 25 degrees C while the more complex problem of the hydrolysis of ATP has been followed at a single acidity (3 M perchloric acid). The positions of bond fission have been determined for both ATP and ADP.     

Kinetic characterization of ATP diphosphohydrolase and 5'-nucleotidase activities in cells cultured from submandibular salivary glands of rats

The participation of ecto-ATP diphosphohydrolase (CD39; ecto-NTPDase) and ecto-5'-nucleotidase (CD73) activities in the nucleotide hydrolysis by salivary gland cells from rats was evaluated. We investigated the biochemical characteristics of these ectoenzymes in cells cultured from submandibular salivary glands of rats. The V(max) for the hydrolysis of ATP, ADP and AMP were 2275+/-153 (mean+/-SEM, n = 4), 941+/-96 (mean+/-SEM, n = 5) and 175+/-5 (mean+/-SEM, n = 5) nmol Pi liberated per min per mg of protein, respectively. The K(m) values for ATP, ADP and AMP were 224+/-8 microM (mean+/-SEM, n = 4), 163+/-15 microM (mean+/-SEM, n = 5) and 117+/-5 microM (mean+/-SEM, n = 5), respectively. The competition plot showed that ATP and ADP were hydrolyzed at the same active site on the enzyme. It may be postulated that the physiological role for this ecto-enzyme cascade is to terminate the action of the co-transmitter ATP, generating adenosine.     

Ano1/TMEM16A Overexpression Is Associated with Good Prognosis in PR-Positive or HER2-Negative Breast Cancer Patients following Tamoxifen Treatment

The calcium-activated chloride channel Ano1 (TMEM16A) is overexpressed in many tumors. Although Ano1 overexpression is found in breast cancer due to 11q13 amplification, it remains unclear whether signaling pathways are involved in Ano1 overexpression during breast cancer tumorigenesis in vivo. Estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) have been known to contribute to breast cancer progression. It is unclear whether Ano1 is associated with clinical outcomes in breast cancer patients with different ER, PR and HER2 status. In the present study, we investigated the Ano1 expression in 431 patients with invasive ductal breast carcinoma and 46 patients with fibroadenoma, using immunohistochemistry, and analyzed the association between Ano1 expression and clinical characteristics and outcomes of breast cancer patients with different ER, PR, and HER2 status. Ano1 was overexpressed in breast cancer compared with fibroadenoma. Ano1 was significantly more associated with breast cancer with the lower clinical stage (stage I or II), or triple-negative status. Mostly importantly, Ano1 overexpression was associated with good prognosis in patients with the PR-positive or HER2-negative status, and in patients following tamoxifen treatment. Multivariate Cox regression analysis showed that Ano1 overexpression was a prognostic factor for longer overall survival in PR-positive or HER2-negative patients, and a predictive factor for longer overall survival in patients following tamoxifen treatment. Our findings suggest that Ano1 may be a potential marker for good prognosis in PR-positive or HER2-negative patients following tamoxifen treatment. The PR and HER2 status defines a subtype of breast cancer in which Ano1 overexpression is associated with good prognosis following tamoxifen treatment.     

Nucleotide interconversions in microtubule protein preparations, a significant complication for accurate measurement of GTP hydrolysis in the presence of adenosine 5'-(beta, gamma-imidotriphosphate)

Pursuing the observation of Carlier and Pantaloni [Carlier, M.-F., & Pantaloni, D. (1982) Biochemistry 21, 1215-1224] that adenosine 5'-(beta, gamma-imidotriphosphate) (pNHppA) strongly inhibited tubulin-independent phosphatases in microtubule protein preparations, we observed with a number of commercial preparations of pNHppA that a major proportion of the terminal phosphate of [gamma-32P]GTP added to microtubule protein preparations was rapidly converted into ATP. Initially postulating degradation of pNHppA to AMP followed by stepwise conversion of AMP to ATP, we isolated two nucleoside monophosphate kinase activities from microtubule protein capable of generating ATP from AMP + GTP. The amounts of these enzymes in microtubule protein preparations, however, are probably too low to account for rapid ATP formation. Instead, ATP formation most likely is caused by nucleoside diphosphate kinase acting on ADP contaminating commercial pNHppA preparations. Such ADP contamination was demonstrated by high-performance liquid chromatography, with the amount of ATP formed with different pNHppA preparations proportional to the amount of ADP contamination. Repurification of commercial pNHppA until it was free of contaminating ADP also resulted in the elimination of ATP formation. The repurified pNHppA potently inhibited GTP hydrolysis in microtubule protein preparations. In addition, especially when supplemented with equimolar Mg2+, the repurified pNHppA strongly inhibited GTP hydrolysis and microtubule assembly in reaction mixtures containing purified tubulin and heat-treated microtubule-associated proteins (which contain negligible amounts of tubulin-independent phosphatase activity). We conclude that studies of microtubule-dependent GTP hydrolysis which make use of pNHppA must be interpreted with extreme caution.