Interleukin-3 dependent c-myc protein expression during the cell cycle of murine mast cells.

Aim of the study was to evaluate the relationship between the mitogenic stimulus interleukin-3 to normal murine mast cells and the cell cycle dependent expression of the nuclear c-myc protein. In order to do that on a cell by cell basis, we measured the nuclear c-myc protein simultaneously by flow cytometry, via specific monoclonal antibodies, and the DNA content via the intercalating dye propidium iodide. When cells were deprived from interleukin-3 (IL-3), proliferation was inhibited and the majority of cells arrested in early G1 (G1A, characterized by low c-myc content). Readdition of IL-3 resulted in a slow transition of cells from G1A to late G1 (G1B, at higher c-myc content) before DNA synthesis started. G1A cells with low c-myc content do not undertake DNA synthesis. Using a stathmokinetic methodology we confirmed that the G1A cells are early postmitotic G1 phase cells. The low content of c-myc within these cells appears a direct consequence of reduced c-myc levels during mitosis. Cumulatively, the data suggest that c-myc protein levels of murine mast cells fall at mitosis and that these levels must rise before cells can traverse the G1 phase. Our data are compatible with a model in which c-myc protein content of G1 phase cells has to reach a critical threshold before the cells can move further into the cell cycle.     

Effects of maternal marijuana and cocaine use on fetal growth

To investigate the effects on infants of the use of marijuana and cocaine during pregnancy and to compare the importance of urine assays with that of interviews in ascertaining drug use, we prospectively studied 1226 mothers, recruited from a general prenatal clinic, and their infants. On the basis of either interviews or urine assays conducted prenatally or post partum, 27 percent of the subjects had used marijuana during pregnancy and 18 percent had used cocaine. When only positive urine assays were considered, the corresponding values were 16 percent and 9 percent, respectively. When potentially confounding variables were controlled for in the analysis, the infants whose mothers had positive urine assays for marijuana, as compared with the infants whose mothers were negative according to both interviews and urine assays, had a 79-g decrease in birth weight (P = 0.04) and a 0.5-cm decrement in length (P = 0.02). Women who had positive assays for cocaine, as compared with nonusers, had infants with a 93-g decrease in birth weight (P = 0.07), a 0.7-cm decrement in length (P = 0.01), and a 0.43-cm-smaller head circumference (P = 0.01). To compare our findings with those of other investigators who did not use urine assays, we repeated the analyses, considering only self-reported use of marijuana (23 percent) and cocaine (13 percent). There were no significant associations between such use as determined by interviews alone and any of the measures of outcome. We conclude that the use of marijuana or cocaine during pregnancy is associated with impaired fetal growth and that measuring a biologic marker of such use is important to demonstrate the association.     

Role of potassium channels in the antinociception induced by agonists of alpha2-adrenoceptors

1. The effect of the administration of pertussis toxin (PTX) as well as modulators of different subtypes of K+ channels on the antinociception induced by clonidine and guanabenz was evaluated in the mouse hot plate test. 2. Pretreatment with pertussis toxin (0.25 microg per mouse i.c.v.) 7 days before the hot-plate test, prevented the antinociception induced by both clonidine (0.08-0.2 mg kg(-1), s.c.) and guanabenz (0.1-0.5 mg kg(-1), s.c.). 3. The administration of the K(ATP) channel openers minoxidil (10 microg per mouse, i.c.v.), pinacidil (25 microg per mouse, i.c.v.) and diazoxide (100 mg kg(-1), p.o.) potentiated the antinociception produced by clonidine and guanabenz whereas the K(ATP) channel blocker gliquidone (6 microg per mouse, i.c.v.) prevented the alpha2 adrenoceptor agonist-induced analgesia. 4. Pretreatment with an antisense oligonucleotide (aODN) to mKv1.1, a voltage-gated K+ channel, at the dose of 2.0 nmol per single i.c.v. injection, prevented the antinociception induced by both clonidine and guanabenz in comparison with degenerate oligonucleotide (dODN)-treated mice. 5. The administration of the Ca2+-gated K+ channel blocker apamin (0.5-2.0 ng per mouse, i.c.v.) never modified clonidine and guanabenz analgesia. 6. At the highest effective doses, none of the drugs used modified animals' gross behaviour nor impaired motor coordination, as revealed by the rota-rod test. 7. The present data demonstrate that both K(ATP) and mKv1.1 K+ channels represent an important step in the transduction mechanism underlying central antinociception induced by activation of alpha2 adrenoceptors.     

Comparative effects of L-methionine, S-adenosyl-L-methionine and 5'-methylthioadenosine on the growth of preneoplastic lesions and DNA methylation in rat liver during the early stages of hepatocarcinogenesis.

Male Wistar rats, initiated with diethylnitrosamine (DENA), were subjected to a selection treatment, according to the "resistant hepatocyte" model, followed or not followed by phenobarbital (PB). Rats received, for 3 weeks after selection, 4 i.m. doses (96 mmol/kg) of L-methionine, S-adenosyl-L-methionine (SAM), or 5'-methylthioadenosine (MTA), a SAM catabolite formed during polyamine synthesis or by spontaneous splitting of SAM at physiologic temperature and pH. They were then killed. In some rats, SAM and MTA treatments were started 20 weeks after initiation. The animals were killed 3 weeks later and persistent (neoplastic) nodules (PN) were collected. Some rat groups received 1/2 and 1/4 of the above SAM and MTA doses, or 1/8 of the above MTA dose. SAM and MTA, but not methionine, caused a dose-dependent decrease in number and surface area of gamma-glutamyltranspeptidase (GGT)-positive foci, and in labeling index (LI) of focal cells, coupled with remodeling. SAM and MTA liver contents, SAM/S-adenosylhomocysteine (SAH) ratio and overall methylation of liver DNA were low during the development of GGT-positive foci. SAM, but not methionine, caused a dose-dependent recovery of SAM content and DNA methylation, and a partial reconstitution of liver MTA pool. Exogenous MTA only induced the reconstitution of MTA pool, without affecting SAM level and DNA methylation. Recovery of SAM and MTA pool and DNA methylation was found in the rats subjected to SAM plus MTA, indicating the absence of inhibition of DNA methyltransferases in vivo by MTA. MTA also inhibited liver reparative growth in partially hepatectomized rats, without modifying SAM content and DNA methylation of regenerating liver (RL). A high activity of ornithine decarboxylase (ODC) was found in the liver, during the development of preneoplastic foci, and in PN. This activity was inhibited by SAM and MTA treatments. Although MTA was more effective than SAM, the decrease in ODC activity was coupled with a larger fall in DNA synthesis in SAM-treated than in MTA-treated rats. Thus the antipromotion effect of SAM could not merely depend on its (spontaneous) transformation into MTA. Although MTA production may play a role in the SAM antipromotion effect, other mechanisms could be involved. A role of DNA methylation in the inhibition of growth by SAM is suggested. MTA is a potential chemopreventive agent for liver carcinogenesis.     

Epidermal growth-factor receptor and erbb2 protein in colorectal tissue - comparison between cancer and normal mucosa

Epidermal growth factor receptor (EGFr) and p185neu protein were measured in 55 samples of carcinoma and 55 of normal colorectal mucosa from the same patient, using a ligand binding assay and an ELISA method respectively. The binding characteristics of EGFr were similar in cancer and normal tissue. The concentrations of both EGFr and p185 showed gaussian distribution and were not significantly different between normal and cancer tissue, although a trend toward higher levels of EGFr in normal mucosa was found. Moreover, no significative variations were found in the ratios between cancer and normal tissue after desaturation of the EGFr. No correlations were found between EGFr and p185 and the main clinopathological parameters.     

Dual IGF1R/IR inhibitors in combination with GD2-CAR T-cells display a potent anti-tumor activity in diffuse midline glioma H3K27M-mutant

BackgroundDiffuse midline gliomas (DMG) H3K27M-mutant, including diffuse intrinsic pontine glioma (DIPG), are pediatric brain tumors associated with grim prognosis. Although GD2-CAR T-cells demonstrated significant anti-tumor activity against DMG H3K27M-mutant in vivo, a multimodal approach may be needed to more effectively treat patients. We investigated GD2 expression in DMG/DIPG and other pediatric high-grade gliomas (pHGG) and sought to identify chemical compounds that would enhance GD2-CAR T-cell anti-tumor efficacy.MethodsImmunohistochemistry in tumor tissue samples and immunofluorescence in primary patient-derived cell lines were performed to study GD2 expression. We developed a high-throughput cell-based assay to screen 42 kinase inhibitors in combination with GD2-CAR T-cells. Cell viability, western blots, flow-cytometry, real time PCR experiments, DIPG 3D culture models, and orthotopic xenograft model were applied to investigate the effect of selected compounds on DIPG cell death and CAR T-cell function.ResultsGD2 was heterogeneously, but widely, expressed in the tissue tested, while its expression was homogeneous and restricted to DMG/DIPG H3K27M-mutant cell lines. We identified dual IGF1R/IR antagonists, BMS-754807 and linsitinib, able to inhibit tumor cell viability at concentrations that do not affect CAR T-cells. Linsitinib, but not BMS-754807, decreases activation/exhaustion of GD2-CAR T-cells and increases their central memory profile. The enhanced anti-tumor activity of linsitinib/GD2-CAR T-cell combination was confirmed in DIPG models in vitro, ex vivo, and in vivo.ConclusionOur study supports the development of IGF1R/IR inhibitors to be used in combination with GD2-CAR T-cells for treating patients affected by DMG/DIPG and, potentially, by pHGG.     

Ethanol-Induced Extracellular Signal Regulated Kinase: Role of Dopamine D1 Receptors

Background:  Addictive drugs activate extracellular signal regulated kinase (ERK) in brain regions critically involved in their affective and motivational properties. The aim of this study was to demonstrate the ethanol-induced activation of ERK in the nucleus accumbens (Acb) and in the extended amygdala [bed nucleus of the stria terminalis lateralis (BSTL) and central nucleus of the amygdala (CeA)] and to highlight the role of dopamine (DA) D₁ receptors in these effects.
Methods:  Ethanol (0.5, 1, and 2 g/kg) was administered by gavage and ERK phosphorylation was determined in the nucleus Acb (shell and core), BSTL, and CeA by immunohistochemistry. The DA D₁ receptor antagonist, SCH 39166 (SCH) (50 μg/kg), was administered 10 minutes before ethanol (1 g/kg).
Results:  Quantitative microscopic examination showed that ethanol, dose-dependently increased phospho-ERK immunoreactivity (optical and neuronal densities) in the shell and core of nucleus Acb, BSTL, and CeA. Pretreatment with SCH fully prevented the increases elicited by ethanol (1 g/kg) in all brain regions studied.
Conclusions:  The results of this study indicate that ethanol, similar to other addictive drugs, activates ERK in nucleus Acb and extended amygdala via a DA D₁ receptor-mediated mechanism. Overall, these results suggest that the D₁ receptors/ERK pathway may play a critical role in the motivational properties of ethanol.