Primary and metastatic brain cancer genomics and emerging biomarkers for immunomodulatory cancer treatment.

Recent studies with immunomodulatory agents targeting both cytotoxic T-lymphocyte protein 4 (CTLA4) and programmed cell death 1 (PD1)/programmed cell death ligand 1 (PDL1) have shown to be very effective in several cancers revealing an unexpected great activity in patients with both primary and metastatic brain tumors. Combining anti-CTLA4 and anti-PD1 agents as upfront systemic therapy has revealed to further increase the clinical benefit observed with single agent, even at cost of higher toxicity. Since the brain is an immunological specialized area it's crucial to establish the specific composition of the brain tumors' microenvironment in order to predict the potential activity of immunomodulatory agents. This review briefly summarizes the basis of the brain immunogenicity, providing the most updated clinical evidences in terms of immune-checkpoint inhibitors efficacy and toxicity in both primary and metastatic brain tumors with the final aim of defining potential biomarkers for immunomodulatory cancer treatment.

Production of an egg yolk antibody against Parietaria judaica 2 allergen.

Specific antibodies are essential tools for studying proteins as well as for diagnostic research in biomedicine. The egg yolk of immunized chicken is an inexpensive source of high-quality polyclonal antibodies. The 12-kDa Parietaria judaica 2 allergen was expressed as a fusion protein and was used to immunize Leghorn chickens. In this paper, we show, using 2-dimensional gel electrophoresis and immunoblotting, that chicken antibodies raised against a recombinant allergen can be used to recognize similar proteins from a pollen raw extract. Allergen identity was confirmed by nanoLC-nanospray-tandem mass spectrometry analysis. Our data demonstrate for the first time that a synergistic combination of molecular biology, 2-dimensional PAGE, and use of nonmammalian antibodies represents a powerful tool for reliable identification of allergens.

p21WAF1-derived peptides linked to an internalization peptide inhibit human cancer cell growth.

We tested the ability of synthetic peptides derived from p21(WAF1), fused to the internalization peptide sequence derived from Antennapedia, to inhibit the growth of cancer cells in two human ovarian cancer cell lines expressing wild-type p53 or not. Two fused peptides corresponding to p21(WAF1) regions 17-33 and 63-77 inhibited cell growth in both cell lines while the same peptides without the internalization sequence were inactive. The fused peptides prevented growth at concentrations which inhibited cyclin-dependent kinase 2 and cdc2 activity, thus demonstrating that the peptides act by mimicking the action of p21(WAF1) on kinases. This study illustrates the potential pharmacological use of small peptides fused with the Antennapedia internalization sequence in proliferative disorders. The approach may be extended to other diseases in which cell penetration of a peptide may be of therapeutic benefit. More stable drug-like molecules with better pharmacological properties could be designed based on the results obtained with peptides.

Labeling of rat neurons by anti-GluR3 IgG from patients with Rasmussen encephalitis.

The authors report the immunocytochemical localization in rat brain of affinity-purified anti-GluR3 (glutamate receptor) antibodies from two patients with Rasmussen encephalitis (RE) and from immunized rabbits. The distribution of immunolabeling was similar using antibodies from rabbits and patients with RE. No electrophysiologic responses were elicited from acutely dissociated kainate-responsive neurons isolated from rat brain when these antibodies were applied. These findings show that anti-GluR3 antibodies purified from patients with RE bind to specific regions of the CNS but do not act through an excitotoxic mechanism.

Calcium influx in rat thalamic relay neurons through voltage-dependent calcium channels is inhibited by enkephalin.

High and low voltage-activated, transient (HVA and LVA,T) Ca2+ currents are crucial in determining the characteristic thalamic firing pattern, during the oscillatory mode. The modulatory effects induced by D-ala2-D-leu5-enkephalin (DADLE) on voltage-dependent Ca2+ channels have been investigated on acutely dissociated neurons from rat ventro-basal (VB) thalamus, by means of whole cell patch-clamp technique. DADLE (400 nM) reduced HVA Ca2+ channel currents in 37 out of 44 cells tested (-53 +/- 5.3% to 0 mV test potential, n = 24,). In 50% of the cases DADLE induced an effect which was persistent at all the potentials tested, i.e. a voltage-independent one. In the remaining neurons, the inhibition partially or totally disappeared on the currents evoked at the highest potentials. DADLE was also able to inhibit LVA Ca2+ channels (-40% in five out of 12 cells). In conclusion, thalamic relay neurons present opioid receptors negatively coupled to both HVA and LVA Ca2+ channels. The presence of two inhibitory effects of DADLE on the total HVA Ca2+ channels has been observed, and they are distinguishable on the basis of their sensitivity to voltage. It is suggested that Ca2+ current modulation may play a role in the production and tuning of the rhythmic burst discharge in these neurons.

Valproate selectively reduces the persistent fraction of Na+ current in neocortical neurons.

The effect of valproate (VPA) on Na+ currents (INa), was studied by means of voltage clamp recordings using whole-cell patch clamp configuration in 21 acutely dissociated neocortical neurons. Concentrations of VPA up to 200 microM failed to induce any detectable decrease in fast INa (I(Naf)), but the persistent fraction (I(NaP)) was significantly reduced by low VPA concentrations (10-30 microM), corresponding to the lower values of the 'therapeutic' range in epileptic patients. Since it is known that I(NaP) critically regulates the firing properties of pyramidal neurons, it is suggested that the anticonvulsant effectiveness of VPA is mainly due to its effect on I(NaP).

Protein kinase C-dependent modulation of Na+ currents increases the excitability of rat neocortical pyramidal neurones.

The effect of the protein kinase C (PKC) activator 1-oleoyl-2-acetyl-sn-glycerol (OAG) on TTX-sensitive Na+ currents in neocortical pyramidal neurones was evaluated using voltage-clamp and intracellular current-clamp recordings. In pyramid-shaped dissociated neurones, the addition of OAG to the superfusing medium consistently led to a 30% reduction in the maximal peak amplitude of the transient sodium current (I(Na,T)) evoked from a holding potential of -70 mV. We attributed this inhibitory effect to a significant negative shift of the voltage dependence of steady-state channel inactivation (of approximately 14 mV). The inhibitory effect was completely prevented by hyperpolarising prepulses to potentials that were more negative than -80 mV. A small but significant leftward shift of INa,T activation was also observed, resulting in a slight increase of the currents evoked by test pulses at potentials more negative then -35 mV. In the presence of OAG, the activation of the persistent fraction of the Na+ current (INa,P) evoked by means of slow ramp depolarisations was consistently shifted in the negative direction by 3.9+/-0.5 mV, while the peak amplitude of the current was unaffected. In slice experiments, the OAG perfusion enhanced a subthreshold depolarising rectification affecting the membrane response to the injection of positive current pulses, and thus led the neurones to fire in response to significantly lower depolarising stimuli than those needed under control conditions. This effect was attributed to an OAG-induced enhancement of INa,P, since it was observed in the same range of potentials over which I(Na,P) activates and was completely abolished by TTX. The qualitative firing characteristics of both the intrinsically bursting and regular spiking neurones were unaffected when OAG was added to the physiological perfusing medium, but their firing frequency increased in response to slight suprathreshold depolarisations. The obtained results suggest that physiopathological events working through PKC activation can increase neuronal excitability by directly amplifying the I(Na,P)-dependent subthreshold depolarisation, and that this facilitating effect may override the expected reduction in neuronal excitability deriving from OAG-induced inhibition of the maximal INa, T peak amplitude.

Inhibition of transient and persistent Na+ current fractions by the new anticonvulsant topiramate.

The actions of the antiepileptic drug topiramate (TPM) on Na+ currents were assessed using whole-cell patch-clamp recordings in dissociated neocortical neurons and intracellular recordings in neocortical slices. Relatively low TPM concentrations (25-30 microM) slightly inhibited the persistent fraction of Na+ current in dissociated neurons and reduced the Na+-dependent long-lasting action potential shoulders, which can be evoked in layer V pyramidal neurons after Ca++ and K+ current blockade. Conversely, the same drug concentrations were ineffective in reducing the amplitude of the fast Na+-dependent action potentials evoked in slices or the peak of transient Na+ (INaf) current evoked in isolated neurons from a physiological holding potential. Consistent INaf inhibition became, however, evident only when the neuronal membrane was kept depolarized to enhance resting Na+ channel inactivation. TPM (100 microM) was ineffective on the voltage dependence of activation but induced a leftward shift of the steady-state INaf inactivation curve. The drug-induced inhibitory effect increased with the duration of membrane depolarization, and the recovery of INaf after long membrane depolarizations was slightly delayed in comparison with that observed under control conditions. The obtained evidence suggests that the anticonvulsant action of TPM may operate by stabilizing channel inactivation, which can be induced by depolarizing events similar to those occurring in chronic epileptic conditions. Concurrently, the slight but significant inhibition of the persistent fraction of the Na+ current, obtained with the application of relatively low TPM concentrations, may contribute toward its anticonvulsant effectiveness by modulating the near-threshold depolarizing events that are sustained by this small current fraction.

Characterization of cyclin B1 expression in human cancer cell lines by a new three-parameter BrdUrd/cyclin B1/DNA analysis.

Flow cytometric cyclin expression/DNA content analysis, now commonly used, provides useful information on the mechanisms regulating cell cycle progression. However, this biparametric analysis does not make a clear-cut distinction between G1 and S-early or between S-late and G2M phase cells. This paper proposes a new three-parameter flow cytometric method with which to determine cyclin B1 levels in single cells in different cell cycle phases by coupling bromodeoxyuridine (BrdUrd) immunodetection and DNA content. DNA denaturation by HCl did not alter the level of cyclin B1. Differences in cyclin B1 expression were observed in seven human cancer cell lines of different origin. The percentage of cyclin B1-positive cells and the cyclin B1 content per cell indicated different patterns. In some cases cyclin B1 accumulation preceded the G2M checkpoint, at which its content usually started to rise. Using available easily reproducible techniques, this flow cytometric approach gives details of intracellular variability in cyclin expression.

Characterization of the new immunosuppressive drug undecylprodigiosin in human lymphocytes: retinoblastoma protein, cyclin-dependent kinase-2, and cyclin-dependent kinase-4 as molecular targets.

Undecylprodigiosin (UP) is the first described member of a family of related compounds showing immunosuppressive activity. We have investigated the biological effect and mechanism of action of UP in human lymphocytes. We show that UP blocks the proliferation of purified peripheral human T and B lymphocytes with an IC50 of 3 to 8 ng/ml and following stimulation by all mitogens used, with no effect on cell death. At the concentrations active on fresh lymphocytes, UP has no significant effect on the proliferation of different leukemic cell lines. UP blocks T cell activation in mid to late G1 phase and before entry into S phase, as shown by analysis of the cell cycle and of the expression of c-myc, IL-2, transferrin receptor, and B-myb. UP inhibits only partially the expression of IL-2R, suggesting that the major target of UP is localized downstream from the interaction between IL-2 and its receptor. The expression of cell cycle genes was investigated. The phosphorylation of the retinoblastoma protein was completely blocked by UP, an event alone sufficient to explain the block of S phase entry and the inhibition of proliferation. The induction of cyclin D2 and the decrease in p27 were not inhibited by UP, whereas the induction of cyclin E, cyclin A, cyclin-dependent kinase-2, and cyclin-dependent kinase-4 was strongly inhibited, potentially explaining the inhibition of retinoblastoma protein phosphorylation. These data clearly show that the site of action of UP is different from that of both cyclosporin A and rapamycin, and that this new class of compounds may, therefore, be good candidates for combined therapy.

Mode of action of thiocoraline, a natural marine compound with anti-tumour activity.

Thiocoraline, a new anticancer agent derived from the marine actinomycete Micromonospora marina, was found to induce profound perturbations of the cell cycle. On both LoVo and SW620 human colon cancer cell lines, thiocoraline caused an arrest in G1 phase of the cell cycle and a decrease in the rate of S phase progression towards G2/M phases, as assessed by using bromodeoxyuridine/DNA biparametric flow cytometric analysis. Thiocoraline does not inhibit DNA-topoisomerase II enzymes in vitro, nor does it induce DNA breakage in cells exposed to effective drug concentrations. The cell cycle effects observed after exposure to thiocoraline appear related to the inhibition of DNA replication. By using a primer extension assay it was found that thiocoraline inhibited DNA elongation by DNA polymerase alpha at concentrations that inhibited cell cycle progression and clonogenicity. These studies indicate that the new anticancer drug thiocoraline probably acts by inhibiting DNA polymerase alpha activity.

Cell cycle perturbations and apoptosis induced by isohomohalichondrin B (IHB), a natural marine compound.

Isohomohalichondrin B (IHB), a novel marine compound with anti-tumoral activity, extracted from the Lissodendorix sponge, inhibits GTP binding to tubulin, preventing microtubule assembly. Cell cycle perturbations and apoptosis induced by IHB were investigated on selected human cancer cell lines by using flow cytometric and biochemical techniques. Monoparameter flow cytometric analysis showed that 1 h IHB exposure caused a delayed progression through S-phase, a dramatic block in G2M phase of the cell cycle and the appearance of tetraploid cell population in LoVo, LoVo/DX, MOLT-4 and K562 cells. At 24 h after IHB exposure, the majority of cells blocked in G2M were in prophase as assessed by morphological analysis and by the fact that they expressed high levels of cyclin A/cdc2 and cyclin B1/cdc2. At 48 h, all cells were tetraploid as assessed by biparameter cyclin A/DNA and cyclin B1/DNA content analysis. Apoptotic death was detected in both leukaemic MOLT-4 and K562 cells, which express wild-type and mutated p53 respectively, when the cells were blocked in mitotic prophase. In conclusion, IHB is a novel potent anti-tumour drug that causes delayed S-phase progression, mitotic block, tetraploidy and apoptosis in cancer cell lines.

Specificity of the HP1 chromo domain for the methylated N-terminus of histone H3.

Recent studies show that heterochromatin-associated protein-1 (HP1) recognizes a 'histone code' involving methylated Lys9 (methyl-K9) in histone H3. Using in situ immunofluorescence, we demonstrate that methyl-K9 H3 and HP1 co-localize to the heterochromatic regions of Drosophila polytene chromosomes. NMR spectra show that methyl-K9 binding of HP1 occurs via its chromo (chromosome organization modifier) domain. This interaction requires methyl-K9 to reside within the proper context of H3 sequence. NMR studies indicate that the methylated H3 tail binds in a groove of HP1 consisting of conserved residues. Using fluorescence anisotropy and isothermal titration calorimetry, we determined that this interaction occurs with a K(D) of approximately 100 microM, with the binding enthalpically driven. A V26M mutation in HP1, which disrupts its gene silencing function, severely destabilizes the H3-binding interface, and abolishes methyl-K9 H3 tail binding. Finally, we note that sequence diversity in chromo domains may lead to diverse functions in eukaryotic gene regulation. For example, the chromo domain of the yeast histone acetyltransferase Esa1 does not interact with methyl- K9 H3, but instead shows preference for unmodified H3 tail.