Valproic acid radiosensitizes anaplastic thyroid cells through a decrease of the DNA damage repair capacity.

BACKGROUND: Anaplastic thyroid cancer (ATC) represents a rare lethal human malignancy with poor prognosis. Multimodality treatment, including radiotherapy, is recommended to improve local control and survival. Valproic acid (VA) is a clinically available histone deacetylase inhibitor with a well-documented side effect profile. In this study, we aim to investigate the combined effect of VA with photon irradiation in vitro. METHODS: Anaplastic thyroid cancer cells (8505c) were used to investigate the radiosensitizing effect of VA. RESULTS: VA sensitized cells to photon irradiation. VA increased radiation-induced apoptosis and radiation-induced DNA damage measured by γH2AX foci induction. Furthermore, VA prolonged γH2AX foci disappearance over time in irradiated cells and decreased the radiation-induced levels of mRNA of key DNA damage repair proteins of the homologous recombination (HR) and the nonhomologous end joining (NHEJ) pathways. CONCLUSIONS: VA at a clinically safe dose enhance the radiosensitivity of 8505c cells through an increase in radiation-induced apoptosis and a disruption in the molecular mechanism of HR and NHEJ DNA damage repair pathways.

Induction of apoptosis in T lymphoma cells by long-term treatment with thyroxine involves PKCζ nitration by nitric oxide synthase.

Thyroid hormones are important regulators of cell physiology, inducing cell proliferation, differentiation or apoptosis, depending on the cell type. Thyroid hormones induce proliferation in short-term T lymphocyte cultures. In this study, we assessed the effect of long-term thyroxine (T4) treatment on the balance of proliferation and apoptosis and the intermediate participants in T lymphoma cells. Treatment with T4 affected this balance from the fifth day of culture, inhibiting proliferation in a time-dependent manner. This effect was associated with apoptosis induction, as characterized through nuclear morphological changes, DNA fragmentation, and Annexin V-FITC/Propidium Iodide co-staining. In addition, increased iNOS gene and protein levels, and enzyme activity were observed. The generation of reactive oxygen species, depolarization of the mitochondrial membrane, and a reduction in glutathione levels were also observed. The imbalance between oxidants and antioxidants species is typically associated with the nitration of proteins, including PKCζ, an isoenzyme essential for lymphoma cell division and survival. Consistently, evidence of PKCζ nitration via proteasome degradation was also observed in this study. Taken together, these results suggest that the long-term culture of T lymphoma cells with T4 induces apoptosis through the increased production of oxidative species resulting from both augmented iNOS activity and the loss of mitochondrial function. These species induce the nitration of proteins involved in cell viability, promoting proteasome degradation. Furthermore, we discuss the impact of these results on the modulation of T lymphoma growth and the thyroid status in vivo.

Possible involvement of stress hormones and hyperglycaemia in chronic mild stress-induced impairment of immune functions in diabetic mice.

Stress, an important aspect of modern life, has long been associated with an altered homeostatic state. Little is known about the effect of the life stress on the outcome of diabetes mellitus, especially related to the higher risk of infections. Here, we evaluate the effects of chronic mild stress (CMS) exposure on the evolution of type I diabetes induced by streptozotocin administration in BALB/c mice. Exposure of diabetic mice to CMS resulted in a significant reduction of survival and a sustained increase in blood glucose values. Concerning the immune response, chronic stress had a differential effect in mice with diabetes with respect to controls, showing a marked decrease in both T- and B-cell proliferation. No correlation was found between splenic catecholamine or circulating corticosterone levels and the proliferative response. However, a significant negative correlation was found between glucose levels and concanavalin A- and lipopolysaccharide-stimulated proliferative responses of T and B cells. A positive correlation between blood glucose and splenic catecholamine concentrations was found in diabetic mice but not in controls subjected to CMS. Hence, the present report shows that diabetic mice show a worse performance in immune function after stress exposure, pointing to the importance of considering life stress as a risk factor for patients with diabetes.

Chronic restraint stress impairs T-cell immunity and promotes tumor progression in mice.

Long-term exposure to stressful situations can affect the immune system. The T-cell response is an important component of anti-tumoral immunity. Hence, impairment of the immune function induced by a chronic stressor has been postulated to alter the immunosurveillance of tumors, thus leading to a worse neoplastic prognosis. Here, we show that chronic restraint stress affects T-cell mediated immunity in mice. This was evidenced by a decrease of mitogen-induced T-cell proliferation, a reduction in CD4(+)T lymphocyte number and a decrease of tumor necrosis factor-alpha (TNF-alpha) and Interferon-gamma (IFN-gamma) production in stressed mice. Additionally, mice subjected to chronic restraint stress displayed an enhancement of tumor growth in a syngeneic lymphoma model, i.e. an increase of tumor proliferation and a reduction of animal survival. Finally, stressed mice had a reduced specific cytotoxic response against these tumor cells. These results suggest that chronic exposure to stress promotes cancer establishment and subsequent progression, probably by depressing T-cell mediated immunity. The T-cell immunity impairment as well as the tumor progression enhancement emphasize the importance of the therapeutic management of stress to improve the prognosis of cancer patients.

Detection of Helicobacter and Campylobacter spp. from the aquatic environment of marine mammals.

The mechanism by which Helicobacter species are transmitted remains unclear. To examine the possible role of environmental transmission in marine mammals, we sought the presence of Helicobacter spp. and non-Helicobacter bacteria within the order Campylobacterales in water from the aquatic environment of marine mammals, and in fish otoliths regurgitated by dolphins. Water was collected from six pools, two inhabited by dolphins and four inhabited by seals. Regurgitated otoliths were collected from the bottom of dolphins' pools. Samples were evaluated by culture, PCR and DNA sequence analysis. Sequences from dolphins' water and from regurgitated otoliths clustered with 99.8-100% homology with sequences from gastric fluids, dental plaque and saliva from dolphins living in those pools, and with 99.5% homology with H. cetorum. Sequences from seals' water clustered with 99.5% homology with a sequence amplified from a Northern sea lion (AY203900). Control PCR on source water for the pools and from otoliths dissected from feeder fish were negative. The findings of Helicobacter spp. DNA in the aquatic environment suggests that contaminated water from regurgitated fish otoliths and perhaps other tissues may play a role in Helicobacter transmission among marine mammals.

Helicobacter spp. from gastric biopsies of stranded South American fur seals (Arctocephalus australis).

Gastrointestinal lesions with uncertain etiology have been widely described among pinnipeds. The aim of our study was to investigate the presence of Helicobacter spp. in the gastric mucosa of South American fur seals (Arctocephalusaustralis). Gastric biopsies from thirteen seals, stranded on the shores of the Southwestern Atlantic Ocean in Argentina, were evaluated for the presence of Helicobacter spp. by PCR and DNA sequence analysis. Six gastric biopsies were positive for Helicobacter spp. Pairwise sequence comparisons showed less than 95% identity to novel Helicobacter spp. described from pinnipeds from North America and Australia. However, phylogenetic analysis revealed that the South American fur seal sequences clustered with 99-100% homology with H. cetorum, a species isolated from dolphins and whales. The presence of H. cetorum in pinnipeds, if confirmed by its isolation from the gastric mucosa of these mammals, demonstrates the wide host range of this bacterium in the marine environment.

Impaired immune responses in streptozotocin-induced type I diabetes in mice. Involvement of high glucose.

Diabetes is widely believed to predispose to serious infections. However, the mechanisms linking diabetes and immunosuppression are not well defined. One potential mediator of the altered defence mechanisms is hyperglycaemia. It has been identified as the main factor contributing to the development of diseases associated with diabetes mellitus. In this study we analyse the immune response in diabetes and the direct effect of hyperglycaemia on T and B lymphocyte reactivity. Diabetes induced an early decrease in IgG levels in the secondary response. However, both primary responses against a T-cell-dependent or independent antigen were affected after 6 months of diabetes induction. T- and B- cell proliferation was only decreased at this time. To gain insight into the potential mechanisms involved, we evaluated the influence of hyperglycaemia over the immune response. Pre-incubation of lymph node and spleen cells in a high glucose (HG) containing medium led to a significant time- and dose-dependent decrease in T- and B-cell proliferation. This effect was associated with the presence of HG-derived supernatants. Still viable cells after HG exposition were able to improve their proliferative response when cultured with the mitogen in a fresh standard medium. HG diminished cell viability, increased apoptosis and induced oxidative stress in lymphocytes. These results indicate that HG concentrations can directly affect lymphoid cell growth. An increase in oxidative stress would be implicated in this deleterious effect. The possibility that prolonged exposure to pathologically HG concentrations would result in the immunosuppressive state observed in diabetes is also discussed.

The thyroid status reprograms T cell lymphoma growth and modulates immune cell frequencies.

UNLABELLED: In spite of considerable evidence on the regulation of immunity by thyroid hormones, the impact of the thyroid status in tumor immunity is poorly understood. Here, we evaluated the antitumor immune responses evoked in mice with different thyroid status (euthyroid, hyperthyroid, and hypothyroid) that developed solid tumors or metastases after inoculation of syngeneic T lymphoma cells. Hyperthyroid mice showed increased tumor growth along with increased expression of cell cycle regulators compared to hypothyroid and control tumor-bearing mice. However, hypothyroid mice showed a higher frequency of metastases than the other groups. Hyperthyroid mice bearing tumors displayed a lower number of tumor-infiltrating T lymphocytes, lower percentage of functional IFN-γ-producing CD8(+) T cells, and higher percentage of CD19(+) B cells than euthyroid tumor-bearing mice. However, no differences were found in the distribution of lymphocyte subpopulations in tumor-draining lymph nodes (TDLNs) or spleens among different experimental groups. Interestingly, hypothyroid TDLN showed an increased percentage of regulatory T (Treg) cells, while hyperthyroid mice displayed increased number and activity of splenic NK cells, which frequency declined in spleens from hypothyroid mice. Moreover, a decreased number of splenic myeloid-derived suppressor cells (MDSCs) were found in tumor-bearing hyperthyroid mice as compared to hypothyroid or euthyroid mice. Additionally, hyperthyroid mice showed increased cytotoxic activity, which declined in hypothyroid mice. Thus, low levels of intratumoral cytotoxic activity would favor tumor local growth in hyperthyroid mice, while regional and systemic antitumor response may contribute to tumor dissemination in hypothyroid animals. Our results highlight the importance of monitoring the thyroid status in patients with T cell lymphomas. KEY MESSAGES: T cell lymphoma phenotype is paradoxically influenced by thyroid status. Hyperthyroidism favors tumor growth and hypothyroidism rises tumor dissemination. Thyroid status affects the distribution of immune cell types in the tumor milieu. Thyroid status also modifies the nature of local and systemic immune responses.

Haloperidol effect on intracellular signals system coupled to alpha1-adrenergic receptor in rat cerebral frontal cortex.

The induction of intracellular signals coupled to alpha1-adrenoceptor by haloperidol, were studied in rat cerebral frontal cortex. The neuroleptic exerts a biphasic effect on nitric oxide synthase (NOS), inhibiting the enzymatic activity at low concentrations (10(-9) M), while higher concentrations (10(-5) M) increased it. Protein kinase C (PKC) and phosphoinositol turnover (PIs) were involved in these actions, as haloperidol induced PKC translocation at low concentrations, and increased PIs turnover at high concentrations. All the effects of haloperidol were blocked by the alpha-adrenoceptor antagonist prazosin and the phospholipase C (PLC) inhibitor NCDC. The possibility that a cross-talk between both enzymatic pathways depending on the neuroleptic concentration used in rat cerebral frontal cortex, is also discussed.

Fluoxetine action on murine T-lymphocyte proliferation: participation of PKC activation and calcium mobilisation.

The present study was undertaken to analyse the effect of fluoxetine upon murine T-lymphocyte proliferation. We found that fluoxetine exerted a dual effect, which depended on the degree of lymphocyte activation: at mitogenic concentration (2 microg/mL) of concavalin A (Con A), we observed an inhibitory effect on cellular proliferation, whereas, on submitogenic Con A concentration (1 microg/mL), fluoxetine stimulated the cellular response. Given these facts, we studied PKC activation and calcium mobilisation in both stimulatory and inhibitory effects of fluoxetine on T-cell proliferation. We observed that fluoxetine increased PKC translocation obtained with 1 microg/mL Con A concentration, whereas PKC was degraded when 2 microg/mL was used. This mechanism is thought to be mediated by calcium mobilisation. According to our results, fluoxetine seemed to modulate calcium influx, which, in turn, would influence PKC translocation, modulating the immune response.

Increased proliferative activity, loss of beta-adrenergic receptor function and class I major histocompatibility complex antigen surface expression in a modified lymphoma cell line.

The molecular interaction of class I major histocompatibility complex (MHC) antigens (Ag) and of beta-adrenergic receptors was previously demonstrated on lymphocytes. By long-term culturing with high concentration of foetal calf serum, the murine S49 lymphoma cell line was modified (S49m) giving phenotypic alterations in beta-adrenergic receptors and class I Ag expression. S49m cells displayed a reduced number of beta-adrenergic sites that were uncoupled to the adenylate cyclase system. These were unable to respond to beta agonist stimulation, despite the fact that direct activation of Gs could be achieved with aluminium tetrafluoride. Although S49m cells showed normal expression of the thy 1.2 Ag, they displayed no expression of class I Ag of the d haplotype. This was assessed by the evident lack of cytotoxic activity of specific monoclonal antibodies (Mo Ab) and of their binding. When performing IFI staining on permeabilized cells, we found positive staining with anti-class d Ab inside the cell. This loss of expression and activity of beta-adrenoceptors and the internalization of class I Ag were accompanied by a higher rate of proliferation in S49m cells. The possibility that the loss of both molecules would modify the biology of the cell is also discussed.

Role of nitric oxide in cardiac beta-adrenoceptor-inotropic response.

We examined some of the signalling events in the negative modulation of isoproterenol-induced stimulation of contractility in rat isolated atria. Isoproterenol-mediated positive inotropic response is accompanied by the stimulation of nitric oxide synthase (NOS) and an increase in the production of cyclic GMP (cGMP). Inhibition of NOS and guanylate cyclase increased the dose-response curve of isoproterenol on contractility. Inhibitors of calcium flux or calcium calmodulin, but not of protein kinase C, abrogated these mechanisms. The existence of a modulatory negative inotropic-cyclic GMP-mediated mechanism limiting the effect of beta-adrenergic stimulation in myocardium is discussed.

In vivo iron and zinc deficiency diminished T- and B-selective mitogen stimulation of murine lymphoid cells through protein kinase C-mediated mechanism.

Zinc and iron are crucial mineral components of human diet, because their deficiency leads to several disorders, including alterations of the immune function. It has been demonstrated, in both humans and rodents, that a diminished number of lymphoid cells and a loss of lymphocyte activity accompany deprivation of these essential minerals. The aim of this work was to analyze if iron and/or zinc imbalances regulate lymphocyte activity and the intracellular signals involved in the effect. Mice from the BALB/c strain were fed with iron- and/or zinc-deficient or mineral-supplemented diets, according to the American Institute of Nutrition Rodent Diets. Levels of iron and zinc were assessed in blood, liver, or bone samples. Selective mitogen stimulation of T- and B-lymphocytes were performed. We found a diminished proliferative response in T- and B-lymphocytes from zinc- and/or iron-deficient animals with respect to controls. These effects were related to decreased mitogen-induced translocation of protein kinase C (PKC) activity to cell membranes on both cell types from all animals fed with deficient diets. Our results demonstrate that iron and zinc deficiencies affect both T- and B-lymphocyte function by PKC-dependent mechanisms.

Colchicine blocks beta adrenoceptor and class I antigen-specific interactions.

Previously we have demonstrated a molecular relationship between H-2 class I antigens and beta adrenoceptors from cardiac tissue. Here we show this type of interaction taking place with beta adrenoceptors from splenic cells and their purified membranes and the participation of cytoskeletal proteins in the phenomenon. Alloimmune, as well as anti-class I but not anti-class II, antibodies were able to inhibit in a competitive manner the binding of (-)-[3H]dihydroalprenolol to splenic lymphocytes and their purified membranes, and to increase cyclic AMP levels in intact cells as a consequence of beta adrenoceptor activation. Furthermore, colchicine (a microtubule disrupting drug), but not cytochalasin B (a microfilament disrupting drug), was able to abrogate alloimmune antibody inhibition over the beta radioligand binding to its receptor on both intact splenocytes and their membranes. Alloantibody actions were significantly diminished by peripheral protein solubilization in purified spleen cell membranes. These data pointed indirectly to the participation of a colchicine binding protein in class I antigen hormone-receptor associations.

Selective cytoprotective effect of histamine on doxorubicin-induced hepatic and cardiac toxicity in animal models.

The aim of the present work was to evaluate the potential protective effect of histamine on Doxorubicin (Dox)-induced hepatic and cardiac toxicity in different rodent species and in a triple-negative breast tumor-bearing mice model. Male Sprague Dawley rats and Balb/c mice were divided into four groups: control (received saline), histamine (5 mg/kg for rats and 1 mg/kg for mice, daily subcutaneous injection starting 24 h before treatment with Dox), Dox (2 mg/kg, intraperitoneally injected three times a week for 2 weeks) and Dox+histamine (received both treatments). Tissue toxicity was evaluated by histopathological studies and oxidative stress and biochemical parameters. The combined effect of histamine and Dox was also investigated in vitro and in vivo in human MDA-MB-231 triple-negative breast cancer model. Heart and liver of Dox-treated animals displayed severe histological damage, loss of tissue weight, increased TBARS levels and DNA damage along with an augment in serum creatine kinase-myocardial band. Pretreatment with histamine prevented Dox-induced tissue events producing a significant preservation of the integrity of both rat and mouse myocardium and liver, through the reduction of Dox-induced oxidative stress and apoptosis. Histamine treatment preserved anti-tumor activity of Dox, exhibiting differential cytotoxicity and increasing the Dox-induced inhibition of breast tumor growth. Findings provide preclinical evidence indicating that histamine could be a promising candidate as a selective cytoprotective agent for the treatment of Dox-induced cardiac and hepatic toxicity, and encourage the translation to clinical practice.

Stimulation of phosphoinositide hydrolysis via class I antigen-specific recognition in murine cardiac tissue.

Induction of polyphosphoinositide hydrolysis in cardiac tissue by specific recognition of class I histocompatibility antigens was assayed. C3H (H-2k) mice auricles were labelled with myo-[3H]inositol precursor and inositol phosphate production in the presence or absence of anti-class I k products was measured. Anti-class I, but not anti-class II products specifically increased phosphoinositide turnover. This increment was partially blocked by muscarinic cholinergic and alpha-adrenergic blockers and even more so by the phospholipase C inhibitor NCDC. Alloantibodies specifically directed against class I antigens could then exert stimulation of phospholipase C-mediated phosphoinositide hydrolysis through the interaction with muscarinic cholinergic and/or alpha-adrenergic receptors. The induction of intracellular second messengers by class I antigens and hormone-receptor interactions is discussed.

Down-regulation of beta-adrenergic receptors induced by mitogen activation of intracellular signaling events in lymphocytes.

The expression of beta-adrenergic receptors on murine lymphocytes stimulated with concanavalin A was studied. A decrease in beta-adrenoceptor number on T lymphocytes and a diminished response to specific agonist stimulation at the peak of proliferation was found. The blockade of cell proliferation by tyrosine kinases or protein kinase C inhibitors reversed the decrease in beta-adrenoceptor number. PMA plus ionophore or interleukin-2 but not PMA alone were able to induce beta-adrenoceptor down-regulation accompanying cellular proliferation. These results showed that the intracellular signals triggered during lymphocyte activation are involved in beta-adrenoceptor down-regulation and it would represent the loss of a mechanism that exerts negative neuroimmune control of cellular proliferation.

Altered beta-adrenoceptor function associated to protein kinase C activation in hyperproliferative T lymphocytes.

beta-Adrenoceptor (betaAR) expression and function as well as its modulation via intracellular transduction signals, were analyzed on the T cell lymphoma BW5147. Independently to the kinetic of proliferation and relative to the number of receptors displayed in normal T lymphocytes, BW5147 cells displayed a decreased number of betaAR, uncoupled to adenylate cyclase, but coupled to protein kinase C stimulation. This last effect was impaired by a beta-antagonist and by blockers of the enzymatic pathways involved in T lymphocyte proliferation, inducing a recovery of betaAR sites. Down-regulation of betaAR would implicate the loss of a negative neuroimmune control mechanism for lymphocyte proliferation. The coupling of the remaining sites to a positive signal for cellular activation, would contribute to establish an hyperproliferative state.

Major histocompatibility complex modulation of beta-adrenoceptor function.

Reciprocal interaction between beta-adrenoceptor specific ligand occupancy and alloantibody binding to specific antigens of cardiac and smooth muscle tissues was observed. Interference of alloimmune antibody fixation to both cardiac and oviductal tract preparations by beta 1 or beta 2 selective blockers, respectively, was obtained by means of indirect immunofluorescence assays. Reciprocally, alloimmune IgG and monoclonal antibodies directed to class I H-2 antigens, behaving as beta-adrenoceptor agonists, modified the contractility of both tissues, increasing intracellular levels of cyclic AMP (cAMP). Additionally, alloantibodies were also capable of inhibiting specific beta-adrenoceptor radioligand binding to purified cardiac and smooth muscle membranes. These data suggested a modulation of beta-adrenoceptor function by antibodies directed against H-2 class I histocompatibility molecules, probably through molecular interactions between both structures.