Thioredoxin-1 as a biological predictive marker for selecting diffuse large B-cell lymphoma patients for etoposide-containing treatment.

OBJECTIVE: In diffuse large B-cell lymphoma (DLBCL), there is an unmet medical need to select patients who would benefit from intensified frontline treatments such as adding etoposide to an R-CHOP regimen. METHODS: The present work included a retrospective clinical analysis of two patient cohorts and an in vitro study. Primary biopsy samples from DLBCL patients treated with an etoposide-containing high-dose regimen (n = 37) and etoposide-containing frontline treatment (n = 69, R-CHOEP) were studied using immunohistochemical thioredoxin-1 (Trx1) staining. Two DLBCL cell lines expressing Trx1 were cultured, and their expression was silenced using the small interfering RNA knockdown technique. Chemoresistance was tested with doxorubicin, etoposide, vincristine, prednisolone and carboplatin. RESULTS: Thioredoxin-1 knockdown sensitised DLBCL cells to doxorubicin (P < .0001) but decreased etoposide-induced cell death (P < .00001). In DLBCL patients who received etoposide-containing frontline treatment, low cytoplasmic Trx1 expression was associated with inferior 5-year overall survival (46% vs 76%, P = .026) and disease-specific survival (68% vs 90%, P = .026). CONCLUSIONS: Strong Trx1 expression appears to increase drug resistance to doxorubicin but sensitises cells to etoposide. This implies that Trx1 expression might be the first predictive biological marker to select the patients who might benefit from adding etoposide to R-CHOP immunochemotherapy.

Peroxiredoxin 6 Serum Levels and Risk of Neutropenic Infections in Diffuse Large B-cell Lymphoma.

BACKGROUND/AIM: Diffuse large B-cell lymphoma (DLBCL) is an aggressive malignancy where antioxidant enzyme peroxiredoxin 6 (Prx6) has previously been associated with adverse outcomes. Its systemic effects in DLBCL are unknown. MATERIALS AND METHODS: This study included 53 patients with DLBCL, five patients with primary central nervous system lymphoma (PCNSL) and 20 healthy controls. The expression of Prx6 was evaluated immunohistochemically in DLBCL tissue samples and compared to its expression in blood serum. RESULTS: Prx6 expression was the highest in healthy controls, followed by DLBCL patients and PCNSL patients. Febrile neutropenic infection after the first treatment course was associated with low pre-treatment Prx6 serum levels (<14 ng/ml) (p=0.025, OR=8.615, 95% confidence interval=1.032-71.933). Serum levels of Prx6 recovered after treatment (p=0.006). CONCLUSION: Patients with low Prx6 levels might be more prone to treatment-related adverse effects through elevated levels of oxidative stress.

ß-Adrenergic agonists mediate enhancement of ß1-adrenergic receptor N-terminal cleavage and stabilization in vivo and in vitro.

The ß(1)-adrenergic receptor (ß(1)AR) is the predominant ßAR in the heart and is the main target for ß-adrenergic antagonists, widely used in the treatment of cardiovascular diseases. Previously, we have shown that the human (h) ß(1)AR is cleaved in its N terminus by a metalloproteinase, both constitutively and in a receptor activation-dependent manner. In this study, we investigated the specific events involved in ß(1)AR regulation, focusing on the effects of long-term treatment with ß-adrenergic ligands on receptor processing in stably transfected human embryonic kidney 293(i) cells. The key findings were verified using the transiently transfected hß(1)AR and the endogenously expressed receptor in neonatal rat cardiomyocytes. By using flow cytometry and Western blotting, we demonstrated that isoproterenol, S-propranolol, CGP-12177 [4-[3-[(1,1-dimethylethyl)amino]2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one], pindolol, and timolol, which displayed agonistic properties toward the ß(1)AR in either the adenylyl cyclase or the mitogen-activated protein kinase signaling pathways, induced cleavage of the mature cell-surface receptor. In contrast, metoprolol, bisoprolol, and CGP-20712 [1-[2-((3-carbamoyl-4-hydroxy)phenoxy)ethylamino]-3-[4-(1-methyl-4-trifluoromethyl-2-imidazolyl)phenoxy]-2-propanol], which showed no agonistic activity, had only a marginal or no effect. Importantly, the agonists also stabilized intracellular receptor precursors, possibly via their pharmacological chaperone action, and they stabilized the receptor in vitro. The opposing effects on the two receptor forms thus led to an increase in the amount of cleaved receptor fragments at the plasma membrane. The results underscore the pluridimensionality of ß-adrenergic ligands and extend this property from receptor activation and signaling to the regulation of ß(1)AR levels. This phenomenon may contribute to the exceptional resistance of ß(1)ARs to downregulation and tendency toward upregulation following long-term ligand treatments.

Association of the beta-1 adrenergic receptor carboxyl terminal variants with left ventricular hypertrophy among diabetic and non-diabetic survivors of acute myocardial infarction.

BACKGROUND: The beta-1 adrenergic receptor (beta1AR) plays a fundamental role in the regulation of cardiovascular functions. It carries a nonsynonymous single nucleotide polymorphism in its carboxyl terminal tail (Arg389Gly), which has been shown to associate with various echocardiographic parameters linked to left ventricular hypertrophy (LVH). Diabetes mellitus (DM), on the other hand, represents a risk factor for LVH. We investigated the possible association between the Arg389Gly polymorphism and LVH among non-diabetic and diabetic acute myocardial infarction (AMI) survivors. METHODS: The study population consisted of 452 AMI survivors, 20.6% of whom had diagnosed DM. Left ventricular parameters were measured with two-dimensional guided M-mode echocardiography 2-7 days after AMI, and the Arg389Gly polymorphism was determined using a polymerase chain reaction-restriction fragment length polymorphism assay. RESULTS: The Arg389 homozygotes in the whole study population had a significantly increased left ventricular mass index (LVMI) when compared to the Gly389 carriers (either Gly389 homozygotes or Arg389/Gly389 heterozygotes) [62.7 vs. 58.4, respectively (p = 0.023)]. In particular, the Arg389 homozygotes displayed thicker diastolic interventricular septal (IVSd) measures when compared to the Gly389 carriers [13.2 vs. 12.3 mm, respectively (p = 0.004)]. When the euglycemic and diabetic patients were analyzed separately, the latter had significantly increased LVMI and diastolic left ventricular posterior wall (LVPWd) values compared to the euglycemic patients [LVMI = 69.1 vs. 58.8 (p = 0.001) and LVPWd = 14.2 vs. 12.3 mm (p < 0.001), respectively]. Furthermore, among the euglycemic patients, the Arg389 homozygotes displayed increased LVMI and IVSd values compared to the Gly389 carriers [LVMI = 60.6 vs. 56.3, respectively (p = 0.028) and IVSd = 13.1 vs. 12.0 mm, respectively (p = 0.001)]. There was no difference in the LVMI and IVSd values between the diabetic Arg389 homozygotes and Gly389 carriers. CONCLUSIONS: The data suggest an association between the beta1AR Arg389Gly polymorphism and LVH, particularly the septal hypertrophy. The Arg389 variant appears to confer a higher risk of developing LVH than the corresponding Gly389 variant among patients who have suffered AMI. This association cannot be considered to be universal, however, since it does not appear to exist among diabetic AMI survivors.

Human beta1-adrenergic receptor is subject to constitutive and regulated N-terminal cleavage.

The beta(1)-adrenergic receptor (beta(1)AR) is the predominant betaAR in the heart, mediating the catecholamine-stimulated increase in cardiac rate and force of contraction. Regulation of this important G protein-coupled receptor is nevertheless poorly understood. We describe here the biosynthetic profile of the human beta(1)AR and reveal novel features relevant to its regulation using an inducible heterologous expression system in HEK293(i) cells. Metabolic pulse-chase labeling and cell surface biotinylation assays showed that the synthesized receptors are efficiently and rapidly transported to the cell surface. The N terminus of the mature receptor is extensively modified by sialylated mucin-type O-glycosylation in addition to one N-glycan attached to Asn(15). Furthermore, the N terminus was found to be subject to limited proteolysis, resulting in two membrane-bound C-terminal fragments. N-terminal sequencing of the fragments identified two cleavage sites between Arg(31) and Leu(32) and Pro(52) and Leu(53), which were confirmed by cleavage site and truncation mutants. Metalloproteinase inhibitors were able to inhibit the cleavage, suggesting that it is mediated by a matrix metalloproteinase or a disintegrin and metalloproteinase (ADAM) family member. Most importantly, the N-terminal cleavage was found to occur not only in vitro but also in vivo. Receptor activation mediated by the betaAR agonist isoproterenol enhanced the cleavage in a concentration- and time-dependent manner, and it was also enhanced by direct stimulation of protein kinase C and adenylyl cyclase. Mutation of the Arg(31)-Leu(32) cleavage site stabilized the mature receptor. We hypothesize that the N-terminal cleavage represents a novel regulatory mechanism of cell surface beta(1)ARs.

The endoplasmic reticulum Ca2+-pump SERCA2b interacts with G protein-coupled receptors and enhances their expression at the cell surface.

Calcium (Ca(2+)) plays a pivotal role in both cellular signaling and protein synthesis. However, it is not well understood how calcium metabolism and synthesis of secreted and membrane-bound proteins are related. Here we demonstrate that the sarco(endo)plasmic reticulum Ca(2+) ATPase 2b (SERCA2b), which maintains high Ca(2+) concentration in the lumen of the endoplasmic reticulum, interacts specifically with the human delta opioid receptor during early steps of receptor biogenesis in human embryonic kidney 293 cells. The interaction involves newly synthesized incompletely folded receptor precursors, because the association between the delta opioid receptor and SERCA2b (i) was short-lived and took place soon after receptor translation, (ii) was not affected by misfolding of the receptor, and (iii) decreased if receptor folding was enhanced by opioid receptor pharmacological chaperone. The physical association with SERCA2b was found to be a universal feature among G protein-coupled receptors within family A and was shown to occur also between the endogenously expressed luteinizing hormone receptor and SERCA2b in rat ovaries. Importantly, active SERCA2b rather than undisturbed Ca(2+) homeostasis was found to be essential for delta opioid receptor biogenesis, as inhibition of its Ca(2+) pumping activity by thapsigargin reduced the interaction and impaired the efficiency of receptor maturation, two phenomena that were not affected by a Ca(2+) ionophore A23187. Nevertheless, inhibition of SERCA2b did not compromise the functionality of receptors that were able to mature. Thus, we propose that the association with SERCA2b is required for efficient folding and/or membrane integration of G protein-coupled receptors.

Inefficient maturation of the rat luteinizing hormone receptor. A putative way to regulate receptor numbers at the cell surface.

Increasing evidence suggests that the folding and maturation of monomeric proteins and assembly of multimeric protein complexes in the endoplasmic reticulum (ER) may be inefficient not only for mutants that carry changes in the primary structure but also for wild type proteins. In the present study, we demonstrate that the rat luteinizing hormone receptor, a G protein-coupled receptor, is one of these proteins that matures inefficiently and appears to be very prone to premature degradation. A substantial portion of the receptors in stably transfected human embryonic kidney 293 cells existed in immature form of M(r) 73,000, containing high mannose-type N-linked glycans. In metabolic pulse-chase studies, only approximately 20% of these receptor precursors were found to gain hormone binding ability and matured to a form of M(r) 90,000, containing bi- and multiantennary sialylated N-linked glycans. The rest had a propensity to form disulfide-bonded complexes with a M(r) 120,000 protein in the ER membrane and were eventually targeted for degradation in proteasomes. The number of membrane-bound receptor precursors increased when proteasomal degradation was inhibited, and no cytosolic receptor forms were detected, suggesting that retrotranslocation of the misfolded/incompletely folded receptors is tightly coupled to proteasomal function. Furthermore, a proteasomal blockade was found to increase the number of receptors that were capable of hormone binding. Thus, these results raise the interesting possibility that luteinizing hormone receptor expression at the cell surface may be controlled at the ER level by regulating the number of newly synthesized proteins that will mature and escape the ER quality control and premature degradation.