A novel mouse model of rhabdomyosarcoma underscores the dichotomy of MDM2-ALT1 function in vivo.

Alternative splicing of the oncogene murine double minute 2 (MDM2) is induced in response to genotoxic stress. MDM2-ALT1, the major splice variant generated, is known to activate the p53 pathway and impede full-length MDM2's negative regulation of p53. Despite this perceptible tumor-suppressive role, MDM2-ALT1 is also associated with several cancers. Furthermore, expression of MDM2-ALT1 has been observed in aggressive metastatic disease in pediatric rhabdomyosarcoma (RMS), irrespective of histological subtype. Therefore, we generated a transgenic MDM2-ALT1 mouse model that would allow us to investigate the effects of this splice variant on the progression of tumorigenesis. Here we show that when MDM2-ALT1 is ubiquitously expressed in p53 null mice it leads to increased incidence of spindle cell sarcomas, including RMS. Our data provide evidence that constitutive MDM2-ALT1 expression is itself an oncogenic lesion that aggravates the tumorigenesis induced by p53 loss. On the contrary, when MDM2-ALT1 is expressed solely in B-cells in the presence of homozygous wild-type p53 it leads to significantly increased lymphomagenesis (56%) when compared with control mice (27%). However, this phenotype is observable only at later stages in life (⩾18 months). Moreover, flow cytometric analyses for B-cell markers revealed an MDM2-ALT1-associated decrease in the B-cell population of the spleens of these animals. Our data suggest that the B-cell loss is p53 dependent and is a response mounted to persistent MDM2-ALT1 expression in a wild-type p53 background. Overall, our findings highlight the importance of an MDM2 splice variant as a critical modifier of both p53-dependent and -independent tumorigenesis, underscoring the complexity of MDM2 posttranscriptional regulation in cancer. Furthermore, MDM2-ALT1-expressing p53 null mice represent a novel mouse model of fusion-negative RMS.

Behavioral effects and pharmacokinetics of gamma-hydroxybutyrate (GHB) precursors gamma-butyrolactone (GBL) and 1,4-butanediol (1,4-BD) in baboons.

RATIONALE: Gamma-butyrolactone (GBL) and 1,4-butanediol (1,4-BD) are prodrugs for gamma-hydroxybutyrate (GHB). Like GHB, GBL and 1,4-BD are drugs of abuse, but their behavioral effects may differ from GHB under some conditions. OBJECTIVES: The first study compared the behavioral effects of GBL (32-240 mg/kg) and 1,4-BD (32-240 mg/kg) with each other and to effects previously reported for GHB (32-420 mg/kg). A second study determined GHB pharmacokinetics following intragastric administration of GHB, GBL, and 1,4-BD. METHODS: Operant responding for food, observed behavioral effects, and a fine-motor task occurred at multiple time intervals after administration of drug or vehicle. In a separate pharmacokinetics study, blood samples were collected across multiple time points after administration of GHB, GBL, and 1,4-BD. RESULTS: Like GHB, GBL, and 1,4-BD impaired performance on the fine-motor task, but the onset of motor impairment differed across drugs. GBL and 1,4-BD dose dependently decreased the number of food pellets earned, but at lower doses than previously observed for GHB. Similar to GHB, both GBL and 1,4-BD produced sedation, muscle relaxation, gastrointestinal symptoms, and tremors/jerks. Administration of GBL and 1,4-BD produced higher maximum concentrations of GHB with shorter times to maximum concentrations of GHB in plasma when compared to GHB administration. CONCLUSIONS: GBL and 1,4-BD produced behavioral effects similar to those previously reported with GHB and the time course of effects were related to blood levels of GHB. Given their higher potency and faster onset of effects, the abuse liability of GBL and 1,4-BD may be greater than GHB.

An analysis of the utility of differential outcome procedures in drug discrimination research.

In differential outcomes procedures, the correlation of unique reinforcers with distinct discriminative stimuli can decrease the amount of time needed for response acquisition and improve terminal accuracy of responding. The drug discrimination assay is widely used to categorize psychoactive drugs as similar or dissimilar and to describe underlying neurochemical changes associated with drug administration. Because the drug discrimination assay relies heavily upon initial response acquisition and continuing terminal accuracy, a procedure successful at shortening acquisition time and improving terminal accuracy would be beneficial. The present studies examined differences in acquisition of drug stimulus control between rats exposed to differential outcome procedures and rats exposed to the outcomes, in a non-systematic way, in two experiments. The first experiment examined acquisition of control by (+/-)-3,4-methylenedioxymethamphetamine (MDMA), D-amphetamine and saline; the second examined control by MDMA, (+)-lysergic acid diethylamide (LSD) and saline. Neither initial acquisition nor terminal accuracy was influenced by differential outcomes in either experiment. Although the differential outcome effect has been demonstrated in many situations, it does not appear to be useful in the drug discrimination assay. Possible reasons for the lack of an observed effect are discussed.

Antagonism of the discriminative stimulus effects of (+)-7-OH-DPAT by remoxipride but not PNU-99194A.

The dopamine (DA) agonist 7-hydroxy-N,N-di-n-propyl-2-amino-tetralin (7-OH-DPAT) has been used extensively as a tool to investigate the role of DA D(3) receptors in the reinforcing and discriminative stimulus properties of psychostimulant drugs. The present study examined the relative importance of D(3) vs. D(2) receptor actions in the discriminative stimulus effects of (+)-7-OH-DPAT (0.03 mg/kg, sc) in 16 male Sprague-Dawley rats trained to discriminate this compound from saline in a two-lever, water-reinforced operant procedure under a FR 20 schedule. Stimulus generalization and antagonism tests were conducted with cocaine and with various selective D(2) and D(3) receptor ligands. In contrast to previous findings that (+)-7-OH-DPAT substitutes for cocaine, the present results demonstrated that cocaine does not produce stimulus generalization in animals trained to discriminate (+)-7-OH-DPAT. Although two D(3)-preferring agonists, PD-128907 and pramipexole, produced complete stimulus generalization to the training drug, two highly selective D(3) antagonists (PNU-99194A, PD 152255) failed to block the discriminative stimulus effects of (+)-7-OH-DPAT. However, the D(2) antagonist remoxipride (3.0 mg/kg) produced a rightward shift in the (+)-7-OH-DPAT dose-response curve. These findings suggest that D(2) receptors are critically involved in mediating the cue properties of (+)-7-OH-DPAT. However, alternative interpretations that PNU-99194A is not entirely D(3) receptor selective should also be considered.

A three-choice discrimination procedure dissociates the discriminative stimulus effects of d-amphetamine and (+/-)-MDMA in rats.

(+/-)-3,4-Methylenedioxymethamphetamine (MDMA) produces subjective effects in humans that are similar to, but distinguishable from, those of psychostimulants. Drug discrimination studies in nonhumans have yielded inconsistent results regarding the similarities between MDMA and the psychomotor stimulant d-amphetamine. This study successfully used a 3-choice operant procedure to establish MDMA and d-amphetamine as discriminative stimuli in rats. Cocaine produced complete substitution for d-amphetamine, and LSD produced dose-dependent increases in MDMA-appropriate responding with nearly complete substitution (78%) for MDMA. The hallucinogen 2,5-dimethoxy-4-bromoamphetamine only partially substituted for MDMA and severely disrupted response rate. Fenfluramine and both isomers of 3,4-methylenedioxyamphetamine (MDA) all produced complete substitution for MDMA. The serotonin-receptor antagonist pirenpirone only partially blocked MDMA discrimination.

The dopamine D3 receptor antagonist PNU-99194A fails to block (+)-7-OH-DPAT substitution for D-amphetamine or cocaine.

The present study examined the role of dopamine D3 receptor actions in the stimulus generalization produced by (+)-7-OH-DPAT in rats trained to discriminate either D-amphetamine or cocaine from saline. Twelve male Sprague-Dawley rats were trained to discriminate D-amphetamine (1.0 mg/kg) and 12 rats were trained to discriminate cocaine (5.0 mg/kg) from saline in a two-choice, water-reinforced operant procedure. Stimulus generalization tests were administered with the D3 receptor-preferring agonist, (+)-7-hydroxy-N, N-di-n-propyl-2-aminotetralin ((+)-7-OH-DPAT, 0.01-1.0 mg/kg) as well as the D3-preferring antagonist, 5,6-di-methoxy-2-(dipropylamino)indan-hydrochloride (PNU-99194A, 5-40 mg/kg). PNU-99194A (10-40 mg/kg) was also administered in combination with the training dose of D-amphetamine or cocaine to test for antagonism of each training drug cue. Finally, to assess the role of D3 receptor actions in the stimulus generalization produced by (+)-7-OH-DPAT (0.1 mg/kg), PNU-99194A (10, 20 mg/kg) was tested in combination with this compound in each training group. The results showed complete stimulus generalization with (+)-7-OH-DPAT in rats trained to discriminate D-amphetamine, although only partial stimulus generalization was observed with this compound in rats trained to discriminate cocaine. PNU-99194A produced partial substitution for both training drugs, and failed to block the discriminative stimulus effects of either D-amphetamine or cocaine. Moreover, this compound failed to block the stimulus generalization produced by (+)-7-OH-DPAT in rats trained to discriminate D-amphetamine. These results question the importance of D3 receptor actions in the discriminative stimulus effects of psychostimulants and their similarities to (+)-7-OH-DPAT.