Breast cancer brain metastases - A 12 year review of treatment outcomes.

BACKGROUND: Breast cancer (BC) is the 2nd commonest cause of brain metastases (BM). This retrospective review investigates the applicability of prognostic scores and highlights different outcomes for patients with HER2 positive compared to triple negative (TN) subtypes. METHODS: Two hundred and seventy four patients received whole brain radiotherapy for BC BM (01/2000-12/2011). The primary objective was to determine factors influencing overall survival (OS). All information relevant to primary BC, disease recurrence, treatment, outcome and cause of death (either neurological (NP) or systemic progression (SP)) were collected. Univariate (UV) and multivariate (MV) Cox regression analysis were used. RESULTS: One hundred and forty four patients (53%) were ER positive, 104 (38%) HER2 positive and 57 (21%) TN. Median age at BM was 53 (27-81) years and median OS from BM diagnosis 7.3 (5.7-8.9) months. On MV analysis, Her2 status, RPA score, surgery, stereotactic radiotherapy, and absence of TN disease were independent prognostic factor for OS. NP was the cause of death in 69.2% of HER2 positive patients and 17.3% had SP. Of the TN patients, 29.8% had NP and 54.4% SP (p < 0.001). CONCLUSION: A consistent OS advantage is noted for HER2 positive BM cases and inclusion of BC subtype in the breast GPA score should improve the prognostic factors' sensitivity. The unique presentations, response to treatment and causes of death for HER2 positive patients means more aggressive focal therapy should be considered and studied in the context of clinical trials. For TN BM patients with poor performance status, best supportive care may be appropriate.

Multipotent neurotrophin antagonist targets brain-derived neurotrophic factor and nerve growth factor.

Brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) are members of the neurotrophin family that normally play a role in the development and maintenance of the nervous system. However, neurotrophin dysregulation has been implicated in several neurodegenerative diseases and psychiatric disorders including Alzheimer's disease, Parkinson's disease, neuropathic pain, depression, and substance abuse. Despite their central role in the nervous system, neurotrophins have proved to be an elusive pharmacological target. Here, we describe a novel multipotent neurotrophin antagonist, 3-[(5E)-4-oxo-5-[[5-(4-sulfamoylphenyl)-2-furyl]methylene]-2-thioxo-thiazolidin-3-yl]propanoic acid (Y1036). Y1036 binds BDNF (K(D) = 3.5 +/- 0.3 microM) and NGF (K(D) = 3.0 +/- 0.4 microM) preventing either BDNF or NGF from interacting with their obligate receptor(s). Y1036 prevents both BDNF- and NGF-mediated trk activation, downstream activation of the p44/42 mitogen-activated protein kinase pathway, and neurotrophin-mediated differentiation of dorsal-root ganglion sensory neurons. Identification of a BDNF- and NGF-specific antagonist is of considerable interest in the study and treatment of diseases where dysregulation of multiple neurotrophins has been implicated.

Sociodemographic and clinical characteristics of patients presenting with first-episode psychosis and concurrent substance misuse.

AIMS: To determine the incidence of concurrent substance misuse among individuals entering first-episode psychosis treatment, and examine whether patients with concurrent substance misuse differ on variables relevant to service planning. METHODS: Consecutive patients (n=376) were assessed using standardized instruments. RESULTS: Twenty-two per cent met abuse or dependence criteria for a single substance, and 8% met criteria for two substances. Most met criteria for marijuana or marijuana and alcohol. The majority of patients with a concurrent disorder were male, and on average they were 3.5 years younger at psychosis onset. Patients with a concurrent disorder experienced worse 'positive' and anxiety symptoms in the month before treatment. Premorbidly they had better social functioning, but worse academic functioning. CONCLUSIONS: There are important differences between patients entering first-episode psychosis treatment with and without a concurrent substance misuse. Early intervention efforts might be informed by our accumulating knowledge about the characteristics of patients who have both disorders.

The influence of radiotherapy on capsule formation and aesthetic outcome after immediate breast reconstruction using biodimensional anatomical expander implants.

BACKGROUND: Capsular contracture occurs more frequently when immediate breast reconstruction (IBR) is associated with radiotherapy (RT) in a post-mastectomy field. The aim of this study was to investigate the impact of RT on surgical outcome after IBR using a single implant type. METHODS: One hundred and thirty-six breast reconstructions were studied in 114 patients: 62 reconstructions were performed using submuscular implants alone and 74 had an implant-assisted latissimus dorsi myocutaneous flap using a McGhan 150 biodimensional permanent expander implant. Data were prospectively collected on capsule contracture, geometric measurements, photographic assessments and pain scores. The median follow-up was 4 (range, 2-5) years. RESULTS: The mean age of the 114 patients studied was 45 (range, 20-77) years. Forty-four reconstructed breasts received RT. Capsule formation was detected in 13/92 (14.1%) reconstructed breasts with no RT and in 17/44 (38.6%) reconstructed breasts with RT. On univariate analysis, RT was the only variable related to capsule formation (p<0.001). Significant differences in geometric measurements of symmetry were identified in patients with capsules compared with those without capsules. Photographic assessments were worse in the capsule group: mean photo score 8 (95% CI 8, 8.5) compared with the no capsule group 6.5 (95% CI 5, 7.5), p<0.001. Persistent pain two years or more after surgery was present in 8/30 patients with capsules and 1/106 with no capsule group, p<0.01. Capsule formation is three times more likely to occur after IBR in association with an RT field. However, as more than 60% of patients do not get capsules despite RT at four years, implant-assisted tissue expansion techniques using a biodimensional device is a viable breast reconstructive option in selected cases.

The binding of zinc and copper ions to nerve growth factor is differentially affected by pH: implications for cerebral acidosis.

It has recently been shown that transition metal cations Zn2+ and Cu2+ bind to histidine residues of nerve growth factor (NGF) and other neurotrophins (a family of proteins important for neuronal survival) leading to their inactivation. Experimental data and theoretical considerations indicate that transition metal cations may destabilize the ionic form of histidine residues within proteins, thereby decreasing their pK(a) values. Because the release of transition metal cations and acidification of the local environment represent important events associated with brain injury, the ability of Zn2+ and Cu2+ to bind to neurotrophins in acidic conditions may alter neuronal death following stroke or as a result of traumatic injury. To test the hypothesis that metal ion binding to neurotrophins is influenced by pH, the effects of Zn2+ and Cu2+ on NGF conformation, receptor binding and NGF tyrosine kinase (trkA) receptor signal transduction were examined under conditions mimicking cerebral acidosis (pH range 5.5-7.4). The inhibitory effect of Zn2+ on biological activities of NGF is lost under acidic conditions. Conversely, the binding of Cu2+ to NGF is relatively independent of pH changes within the studied range. These data demonstrate that Cu2+ has greater binding affinity to NGF than Zn2+ at reduced pH, consistent with the higher affinity of Cu2+ for histidine residues. These findings suggest that cerebral acidosis associated with stroke or traumatic brain injury could neutralize the Zn2+-mediated inactivation of NGF, whereas corresponding pH changes would have little or no influence on the inhibitory effects of Cu2+. The importance of His84 of NGF for transition metal cation binding is demonstrated, confirming the involvement of this residue in metal ion coordination.

Zinc inhibits p75NTR-mediated apoptosis in chick neural retina.

It has previously been documented that Zn2+ inhibits TrkA-mediated effects of NGF. To evaluate the ability of Zn2+ to attenuate the biological activities of NGF mediated by p75NTR, we characterized the effects of this transition metal cation on both binding and the pro-apoptotic properties of the NGF-p75NTR interaction. Binding of NGF to p75NTR displayed higher affinity in embryonic chick retinal cells than in PC12 cells. NGF induced apoptosis in dissociated cultures of chick neural retina. The addition of 100 microM Zn2+ inhibited binding and chemical cross-linking of 125I-NGF to p75NTR, and also attenuated apoptosis mediated by this ligand-receptor interaction. These studies lead to the conclusion that Zn2+ antagonizes NGF/p75NTR-mediated signaling, suggesting that the effect of this transition metal cation can be either pro- or anti-apoptotic depending on the cellular context.

Involvement of oxidative stress in ascorbate-induced proapoptotic death of PC12 cells.

Ascorbate is a reducing agent, but it is also known to oxidize cellular components under specific conditions. The mechanism of this oxidative action, however, is not well established. Ascorbate treatment increased lipid peroxide content in PC12 cells, but did not increase quantities of lipid peroxide when homogenates of PC12 cells were treated with ascorbate, suggesting that cellular integrity is required for ascorbate to generate lipid peroxidation. However, dehydroascorbate increased lipid peroxide production in both intact PC12 cells and the cell homogenates. These differential effects of ascorbate and dehydroascorbate on intact cells versus homogenates suggest that the dehydroascorbate in cytosol induces an oxidative stress. Ascorbate in culture medium is rapidly oxidized to dehydroascorbate, which is transported into cells by a glucose transporter (GLUT). The GLUT antagonists wortmannin and cytochalasin B, or a high concentration of glucose, blocked (14)C uptake (from ascorbate) in a time-dependent manner and suppressed lipid peroxide production in PC12 cells. These observations support the concept that ascorbate is oxidized to dehydroascorbate, which is transported into cells via GLUT. The dehydroascorbate induces oxidative stress. The oxidative stress triggered apoptosis according to ceramide production, caspase-3 activation, and TUNEL. We have concluded that ascorbate is taken up after oxidation to dehydroascorbate via a "dehydroascorbate transporter" (GLUT), and the dehydroascorbate generates an oxidative stress which triggers apoptosis. These studies have significant implications for conditions under which a high concentration of ascorbate in a tissue is released during a period of hypoxia (e.g., stroke) and taken up during a reperfusion period as dehydroascorbate. Inhibiting uptake of dehydroascorbate may offer novel therapeutic strategies to alleviate brain damage during a reperfusion period.

Oxidative stress induced by ascorbate causes neuronal damage in an in vitro system.

Of particular physiological interest, ascorbate, the ionized form of ascorbic acid, possesses strong reducing properties. However, it has been shown to induce oxidative stress and lead to apoptosis under certain experimental conditions. Ascorbate in the brain is released during hypoxia, including stroke, and is subsequently oxidized in plasma. The oxidized product (dehydroascorbate) is transported into neurons via a glucose transporter (GLUT) during a reperfusion period. The dehydroascorbate taken up by cells is reduced to ascorbate by both enzymatic and non-enzymatic processes, and the ascorbate is stored in cells. This reduction process causes an oxidative stress, due to coupling of redox reactions, which can induce cellular damage and trigger apoptosis. Ascorbate treatment decreased cellular glutathione (GSH) content, and increased the rates of lipid peroxide production in rat cortical slices. Wortmannin, a specific inhibitor of phosphatidylinositol (PI)-3-kinase (a key enzyme in GLUT translocation), prevented the ascorbate induced-decrease of GSH content, and suppressed ascorbate-induced lipid peroxide production. However, wortmannin was ineffective in reducing hydrogen peroxide (H(2)O(2))-induced oxidative stress. The oxidative stress caused ceramide accumulation, which was proportionally changed with lipid peroxides when the cortical slices were treated with ascorbate. These differential effects support the hypothesis that GLUT efficiently transports the dehydroascorbate into neurons, causing oxidative stress.

The common neurotrophin receptor p75NTR enhances the ability of PC12 cells to resist oxidative stress by a trkA-dependent mechanism.

Functional role(s) for the common neurotrophin receptor p75NTR in nerve growth factor (NGF) signaling have yet to be fully elucidated. Many studies have demonstrated that p75NTR can enhance nerve growth factor-induced survival mediated via the trkA receptor. In addition, newly identified pathways for p75NTR signaling have included distinct p75NTR-specific and trk-independent effects which generally appear to be pro-apoptotic. In the present study, we have examined the influence of p75NTR on NGF-mediated protective effects from hydrogen peroxide (H2O2)-induced apoptotic cell death of PC12 cells. Exposure of PC12 cells to H2O2 resulted in Caspase-3 activation and apoptosis. NGF protected PC12 cells against H2O2-mediated apoptosis in a dose-dependent manner and inhibited Caspase-3 activation. These effects of NGF required activation of both PI 3-kinase and MAP kinase signal pathways. When NGF binding to p75NTR was blocked by treating cells with either BDNF or PD90780, and where p75NTR expression was reduced by treating cells with antisense oligonucleotide to p75NTR, the protective effects of NGF were attenuated. Further, NGF had no effect on cell viability in PC12nn5 cells, which express only p75NTR. When trk-mediated signal transduction was blocked, leaving p75NTR signaling activated, PC12 cells were not more vulnerable to H2O2. These data suggest that p75NTR enhances the ability of PC12 cells to resist oxidative stress by a trkA-dependent mechanism, potentially by allosteric mechanisms. Further, potential trkA-independent and pro-apoptotic signaling of p75NTR does not contribute to apoptotic cell death of PC12 cells in a setting of oxidative insult.

The effects of ascorbic acid on dopamine-induced death of PC12 cells are dependent on exposure kinetics.

The role of ascorbic acid on dopamine (DA) oxidation-mediated cytotoxicity was studied using the PC12 cell line. DA cytotoxicity was slightly attenuated by ascorbic acid, whereas the cytotoxicity of 6-hydroxydopamine (6-OHDA), a DA oxidation product, was markedly potentiated. To elucidate the relationship between the ascorbic acid effect and the degree of DA oxidation, ascorbic acid was added in a time-dependent fashion after DA treatment. We found greater cell death the later ascorbic acid was applied. Treatment of cells with glutathione alleviated DA- and 6-OHDA-induced cell death, while L-buthionine sulfoximine potentiated DA and 6-OHDA cytotoxicity. Ascorbic acid combined with glutathione eliminated the toxicity of DA and 6-OHDA. These results suggest that the interaction between DA and ascorbic acid is dependent upon the degree of DA oxidation and glutathione availability.

Sublethal hypoxia up-regulates corticotropin releasing factor receptor type 1 in fetal hippocampal neurons.

We analysed the influence of oxygen-glucose deprivation (OGD) on the expression of corticotropin releasing factor (CRF) receptors (CRF-R1 and CRF-R2) in fetal hippocampal neurons in vitro. A 2 h exposure of neurons to OGD resulted in death of 18+/-2.8% cells at 24 h following exposure, which was considered sublethal hypoxia. Expression of both receptors was quantitated by competitive reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. Levels of mRNA for CRF-R1 were increased 3.2-fold compared to control neurons, while CRF-R2 mRNA levels remained unchanged. The increase of CRF-R1 mRNA levels was observed at 6 h and peaked at 24 h. CRF-R1 protein levels were also increased by 2.4-fold and 1.7-fold at 24 h and 48 h, respectively. These data suggest that the effects of CRF on neuronal survival are mediated in part through the induction and expression of CRF-R1 following a hypoxic/ischemic insult.

Specific inhibition of growth factor-stimulated extracellular signal-regulated kinase 1 and 2 activation in intact cells by electroporation of a growth factor receptor-binding protein 2-Src homology 2 binding peptide.

Activation of the Ras pathway is central to mitogenesis by a variety of growth factors such as the epidermal growth factor, platelet-derived growth factor, or hepatocyte growth factor. Ras activation requires the function of adaptors such as growth factor receptor-binding protein 2, which can bind either directly or indirectly through Src homology 2 domains to the activated receptor. To examine the role of the Src homology 2 domain of growth factor receptor-binding protein 2 in the mitogenic response triggered by these growth factors, we introduced a peptide (PVPE-phosphono-methylphenylalanine-INQS) that can selectively bind this domain into mouse, rat, or human cells growing on conductive indium-tin oxide-coated glass by in situ electroporation. Cells were subsequently stimulated with growth factors and assessed for activation of a downstream target, extracellular signal-regulated kinase (ERK) 1/2, by probing with antibodies specific for its activated form. Electrodes and slides were configured to provide nonelectroporated control cells side by side with the electroporated ones, both growing on the same type of indium-tin oxide-coated glass surface. The data demonstrate that the peptide can cause a dramatic inhibition of epidermal growth factor or platelet-derived growth factor-mediated ERK1/2 activation and DNA synthesis in vivo, compared with its control phenylalanine-containing counterpart. In contrast, the peptide had a very limited effect on hepatocyte growth factor-triggered ERK1/2 activation and DNA synthesis. These results demonstrate the potential of the in situ electroporation approach described here in the study of the coupling of activated receptor tyrosine kinases to the ERK1/2 cascade.

The interaction of neurotrophins with the p75NTR common neurotrophin receptor: a comprehensive molecular modeling study.

Neurotrophins are a family of proteins with pleiotropic effects mediated by two distinct receptor types, namely the Trk family, and the common neurotrophin receptor p75NTR. Binding of four mammalian neurotrophins, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5), to p75NTR is studied by molecular modeling based on X-ray structures of the neurotrophins and the extracellular domain of p55TNFR, a homologue of p75NTR. The model of neurotrophin/receptor interactions suggests that the receptor binding domains of neurotrophins (loops I and IV) are geometrically and electrostatically complementary to a putative binding site of p75NTR, formed by the second and part of the third cysteine-rich domains. Geometric match of neurotrophin/receptor binding domains in the complexes, as characterized by shape complementarity statistic Sc, is comparable to known protein/protein complexes. All charged residues within the loops I and IV of the neurotrophins, previously determined as being critical for p75NTR binding, directly participate in receptor binding in the framework of the model. Principal residues of the binding site of p75NTR include Asp47, Lys56, Asp75, Asp76, Asp88, and Glu89. The additional involvement of Arg80 and Glu53 is specific for NGF and BDNF, respectively, and Glu73 participates in binding with NT-3 and NT-4/5. Neurotrophins are likely to induce similar, but not identical, conformational changes within the p75NTR binding site.

Prooxidant effects of ascorbate in rat brain slices.

Ascorbate is a well-known reducing agent, but it can generate oxidative potential under appropriate condition. In rat cerebral cortex homogenate, 1 mM ascorbate decreased thiobarbituric acid-reactive substances (TBARS) content to 86% +/- 4% of control values, confirming that ascorbate is a reducing agent. However, ascorbate increased TBARS, in a dose-related manner, in slices prepared from cerebral cortex. Ferrous ion (Fe2+) had little effect on ascorbate-induced lipid oxidation in cortical slices, and EDTA did not have an influence on the ascorbate-induced oxidative action. Conversely, ascorbate plus Fe2+ elevated TBARS content to more than threefold over ascorbate alone in tissue homogenates. In summary, ascorbate is a reducing agent in the brain tissue homogenate but has an oxidizing effect in brain slices. A hypothesis is proposed to explain the oxidative effects of ascorbate in cortical slices, wherein extracellular ascorbate is oxidized to dehydroascorbate, which is rapidly carried into the cells via a glucose transporter (GLUT). The dehydroascorbate in cytosol is then reduced back to ascorbate, and, during the reduction process, cellular components are oxidized.

Characterization of antiallodynic actions of ALE-0540, a novel nerve growth factor receptor antagonist, in the rat.

There is growing evidence that nerve growth factor (NGF) may function as a mediator of persistent pain states. We have identified a novel nonpeptidic molecule, ALE-0540, that inhibits the binding of NGF to tyrosine kinase (Trk) A or both p75 and TrkA (IC50 5.88 +/- 1. 87 microM, 3.72 +/- 1.3 microM, respectively), as well as signal transduction and biological responses mediated by TrkA receptors. ALE-0540 was tested in models of neuropathic pain and thermally-induced inflammatory pain, using two routes of administration, a systemic i.p. and a spinal intrathecal (i.th.) route. Morphine was also tested for comparison in the antiallodynia model using mechanical stimuli. We show that either i.p. or i.th. administration of ALE-0540 in rats produced antiallodynia in the L5/L6 ligation model of neuropathic pain. The calculated A50 values (and 95% confidence intervals) for ALE-0540 administered i.p. and i. th. were 38 (17.5-83) mg/kg and 34.6 (17.3-69.4) microgram, respectively. ALE-0540 given i.th., at doses of 30 and 60 microgram, also blocked tactile allodynia in the thermal sensitization model. Although morphine displayed greater potency [A50 value of 7.1 (5.6-8. 8) mg/kg] than ALE-0540 in anti-allodynic effect when given i.p. to L5/L6-ligated rats, it was not active when administered i.th. These data suggest that a blockade of NGF bioactivity using a NGF receptor antagonist is capable of blocking neuropathic and inflammatory pain and further support the hypothesis that NGF is involved in signaling pathways associated with these pain states. ALE-0540 represents a nonpeptidic small molecule which can be used to examine mechanisms leading to the development of agents for the treatment of pain.

Estimation of PC12 cell numbers with acid phosphatase assay and mitochondrial dehydrogenase assay: dopamine interferes with assay based on tetrazolium.

Both the acid-phosphatase and mitochondrial dehydrogenase assay have been used to quantify cell numbers. The commonly used acid-phosphatase assay uses p-nitrophenyl phosphate as a substrate, while the mitochondrial dehydrogenase assay is based on the conversion of tetrazolium to formazan. Our experimental results showed that the former assay was more sensitive in detecting small numbers of PC12 cells (200-10,000 cells/well), whereas the latter was useful for larger numbers of cells (2000-40,000 cells/well). The number of PC12 cells decreased after dopamine treatment, according to the acid-phosphatase assay and by direct cell counts under a light microscope. However, the optical densities measured by the mitochondrial dehydrogenase assay increased after dopamine treatment. We tried to clarify discrepancies between the two assays, since dopamine is an important neurotransmitter and both assays are commonly used to estimate cell numbers. To elucidate the interference between dopamine and tetrazolium salt, cell-free control experiments were performed. Dopamine and other catecholamines (adrenaline and noradrenaline) reacted with tetrazolium and, thus, produced a false positive reaction in the assay. We therefore conclude that the tetrazolium assay is not a suitable method for evaluating the number of catecholamine-treated cells, while the acid-phosphatase assay is reliable and sensitive.

Zinc and copper inhibit nerve growth factor-mediated protection from oxidative stress-induced apoptosis.

We have previously provided evidence that two transition metal cations, Zn2+ and Cu2+, can alter the conformation of nerve growth factor (NGF), rendering it unable to bind to its receptors or to activate signal transduction pathways. In the present study, we have assessed the influence of Zn2+ and Cu2+ on NGF-mediated protection from an oxidative insult. Exposure of rat pheochromocytoma (PC12) cells to hydrogen peroxide resulted in an increase in cell death via apoptosis, which was inhibited by NGF. Zn2+ and Cu2+, when added to cultures at a concentration of 100 microM, prevented NGF-mediated survival-promoting effects. Neither of these ions had an effect on basal cell viability (in the absence of NGF) after an oxidative insult. These results demonstrate that Zn2+ and Cu2+ can selectively inhibit NGF-mediated resistance to an oxidative stress, and have significant implications for neuronal function under both physiological and pathological (e.g. cerebral ischemia) conditions.

Induction of cell death by radiotherapy.

Ionising radiation remains one of the most effective tools in the therapy of cancer. It combines the properties of an extremely efficient DNA-damaging agent with a high degree of spatial specificity. Nonetheless, there remain considerable differences in the outcome for treatment of tumours of differing histological type treated by radiotherapy. Tumours arising from lymphoid or germ cells are significantly more radiocurable than most solid tumours of epithelial origin. The molecular mechanisms underlying such differences in cellular radiosensitivity are the subject of current research. When normal mammalian cells are subjected to stress signals--e.g. radiation, chemotherapeutic drugs, oxygen deficiency--a range of gene products involved in the sensing and signalling of such stresses are activated. The response of eukaryotic cells to ionising radiation includes activation of DNA repair pathways and cell cycle checkpoints, with subsequent full 'biological' recovery or cell death. Radiation induces two different modes of cell death termed mitotic or clonogenic cell death, and apoptosis. Until recent years, there was surprisingly little mechanistic understanding of the events following induction of physical damage by radiation and biological outcome for the cell. There have been recent major advances in our understanding of the signal transduction pathways involved in determining the fate of cells after irradiation.

Glutathione protects PC12 cells from ascorbate- and dopamine-induced apoptosis.

The role of reduced glutathione (GSH) on ascorbate- and dopamine-induced apoptosis in PC12 cells was investigated. Ascorbate is a potent reducing agent and is thus expected to protect against dopamine-induced apoptosis. However, we found that both ascorbate and dopamine killed PC12 cells and ascorbate enhanced dopamine-induced toxicity. The EC50 of cell toxicity induced by ascorbate, dopamine and dopamine plus 0.1 mM ascorbate during 24-h treatment were 0.93+/-0.15 mM, 0.18+/-0.05 mM and 0.13+/-0.04 mM, respectively. When the medium contained 10 mM GSH, the EC50 increased approximately three- and sevenfold for ascorbate and dopamine, respectively. With increased treatment duration, no further toxic effects of ascorbate or dopamine were observed. The GSH synthesis inhibitor, DL-buthionine-(S,R)-sulfoximine (BSO), induced cell toxicity and potentiated the toxic effects of ascorbate and dopamine, suggesting that endogenous GSH participates in protecting against basal oxidative stress. We conclude that both ascorbate and dopamine induce apoptosis in PC12 cells and further that GSH protects them from apoptosis. This study indicates that the toxic effects of ascorbate are potentially due to an oxidative mechanism, similar to that induced by dopamine.

Reciprocal modulation of TrkA and p75NTR affinity states is mediated by direct receptor interactions.

Equilibrium binding of 125I-nerve growth factor (125I-NGF) to cells coexpressing the tyrosine kinase receptor A (TrkA) and common neurotrophin receptor (p75NTR), cells coexpressing both receptors where p75NTR is occupied, and cells expressing only p75NTR, revealed reciprocal modulation of receptor affinity states. Analysis of receptor affinity states in PC12 cells, PC12 cells in the presence of brain-derived neurotrophic factor (BDNF), and PC12nnr5 cells suggested that liganded and unliganded p75NTR induce a higher affinity state within TrkA, while TrkA induces a lower affinity state within p75NTR. These data are consistent with receptor allosterism, and prompted a search for TrkA/p75NTR complexes in the absence of NGF. Chemical crosslinking studies revealed high molecular weight receptor complexes that specifically bound 125I-NGF, and were immunoprecipitated by antibodies to both receptors. The heteroreceptor complex of TrkA and p75NTR alters conformation and/or dissociates in the presence of NGF, as indicated by the ability of low concentrations of NGF to prevent heteroreceptor crosslinking. These data suggest a new model of receptor interaction, whereby structural changes within a heteroreceptor complex are induced by ligand binding.