The MDM2 oncoprotein has transforming potential that can be activated by overexpression, and it represents a critical regulator of the p53 tumor suppressor protein. To identify other factors with a potential role in influencing the expression and/or function of MDM2, we utilized a yeast two-hybrid screening protocol. Here we report that MDM2 physically interacts with a structurally related protein termed MDMX. The results obtained in these studies provide evidence that C-terminal RING finger domains, contained within both of these proteins, play an important role in mediating the association between MDM2 and MDMX. The interaction of these proteins interferes with MDM2 degradation, leading to an increase in the steady-state levels of MDM2. MDMX also inhibits MDM2-mediated p53 degradation, with subsequent accumulation of p53. Taken together, these data indicate that MDMX has the potential to regulate the expression and function of the MDM2 oncoprotein.An accumulating number of observations have implicated aberrant expression of the MDM2 oncogene in the pathogenesis of human neoplasias. This mammalian gene has transforming potential that can be activated by overexpression (1, 2). Originally identified as a gene amplified and overexpressed in a spontaneously transformed mouse 3T3 cell line (3), MDM2 is now known to be amplified in a variety of human tumors, particularly soft tissue sarcomas (4 -7). Additionally, there are several cases reported of tumor cells having an elevated expression of MDM2 that results from mechanisms other than gene amplification, including enhanced translation of MDM2 transcripts (8 -10).The MDM2 gene encodes a key negative regulator of the p53 tumor suppressor protein, and the role of MDM2 overexpression in cell transformation has been attributed, at least in part, to its disruption of the biological activities of p53 (11,12). MDM2 tightly associates with the N-terminal region of the p53 protein, inhibiting the trans-activation and G 1 growth arrest functions of p53 (13-16). Moreover, binding of MDM2 targets p53 for rapid degradation via the ubiquitin-proteasome pathway (17,18). In recent reports, evidence has been obtained suggesting that MDM2 can function as an E3 1 ubiquitin ligase and is responsible for targeting both itself, and p53, for degradation (19,20). Interestingly, the MDM2 gene itself is a transcriptional target of p53. When activated as a transcription factor, p53 binds to a promoter region within the first intron of the MDM2 gene and up-regulates its expression (21-24). Thus, there is evidence for an autoregulatory feedback loop involving the expression and function of MDM2 and p53 (23). Although the best characterized activities of MDM2 concern its functional interactions with p53, MDM2 also associates with other proteins. Some of these include E2F1 (25), pRb (26), and p300 (27). Such interactions could contribute to the transforming potential of MDM2 or may be concerned with modulating MDM2 function. Recently, a link between MDM2 and yet another tumor suppressor protein, ...
The p53 tumor suppressor protein can function as an activator and a repressor of gene transcription. Currently, the mechanism of transcriptional repression by p53 is poorly understood. To aid in clarifying this mechanism, we carried out studies designed to identify specific target genes that are down-regulated following p53 induction. Among the negative p53-response genes revealed by our screening protocols are those encoding stathmin (Op18), a tubulin-associated protein implicated in cell signaling pathways, and an FK506/rapamycin-binding protein, FKBP25. Stathmin and FKBP25 exhibit decreased expression in both human and murine immortalized and transformed cell lines following induction of wild-type p53 by several stimuli that result in DNA damage. Candidate p53-repressed genes such as these provide the necessary markers to delineate the mechanism and biological consequences of transcriptional repression mediated by p53.
Abstract:The regulation of 5-HT2A receptor expression by an antisense oligodeoxynucleotide, complementary to the coding region of rat 5-HT2A receptor mRNA, was examined in a cortically derived cell line and in rat brain. Treatment of A1A1 variant cells, which express the 5-HT2A receptor coupled to the stimulation of phosphatidylinositol (P1) hydrolysis, with antisense oligodeoxynucleotide decreased the maximal stimulation of P1 hydrolysis by the partial agonist quipazine and the number of 5-HT2A receptor sites as measured by the binding of 2~[125l]ĩ odolysergic acid diethylamide. Treatment of cells with random, sense, or mismatch oligodeoxynucleotide did not alter the stimulation of P1 hydrolysis by quipazine or 5-HT2A receptor number. Intracerebroventricular infusion of antisense, but not mismatch, oligodeoxynucleotide for 8 days resulted in a significant increase in cortical 5-HT2A receptor density and an increase in headshake behavior induced by the 5-HT2 receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane. The density of cortical 5-HT2A receptors was not altered by administration of antisense oligodeoxynucleotide for 1, 2, or 4 days. We hypothesize that in brain this antisense oligodeoxynucleotide relieved some form of translational suppression, resulting in an increase in 5-HT2A receptor expression. Key Words: 5-HT2A receptors-A1A1 variant cellsPhosphoinositide hydrolysis-Headshake behavior-Intracerebroventricular infusion. J. Neurochem. 71, 1457Neurochem. 71, -1463Neurochem. 71, (1998.Pharmacological, functional, as well as molecular criteria have contributed to the characterization of many subtypes of receptor for the neurotransmitter serotonin (5-hydroxytryptamine; 5-HT). The 5-HT2A receptor is found in high density in cortex, where it is enriched on pyramidal neurons Jakab and Goldman-Rakic, 1998), and in intermediate density in hypothalamus, caudate putamen, and nucleus accumbens (see Hoyer et al., 1994). 5-HT2A receptors have been implicated in a wide variety of behavioral and physiological processes as well as in the mechanism of action of psychotropic drugs. The 5-HT2A receptor appears to be the site of action of hallucinogenic compounds and has high affinity for atypical neuroleptics and many antidepressant drugs (Glennon, 1990;Leysen et al., 1993;Frazer, 1997).We have studied the regulation of expression of the 5-HT2A receptor by an antisense oligodeoxynucleotide (oligo) using a cortically derived cell line, A1A1 variant (AtAtv) cells, and in brain following intracerebroventricular infusion. A~A~v cells express 5-HT2A receptors coupled to the stimulation of phosphatidylinositol (P1) hydrolysis (Berg et al., 1994a). An antisense oligo designed to hybridize to the coding region of 5-HT2A receptor mRNA was used in these studies. MATERIALS AND METHODS OligosOligos, corresponding to coding region +57 to +74 (Julius et al., 1990) of rat 5-HT2A receptor cDNA (accession no. M30705), were used (5'to 3'): antisense, GGGCCA-TCACCTAATTGC; sense, GCAATTAGGTGATGGCCC; random, CCTCGTAATGCAGACTCG; mi...
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