A functional p53-responsive intronic promoter is contained within the human<i>mdm2</i>gene

Zauberman, Ayelet; Flusberg, Deborah; Haupt, Ygal; Barak, Yaacov; Oren, Moshe

doi:10.1093/nar/23.14.2584

Cited by 272 publications

(254 citation statements)

References 29 publications

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“…The MDM2 gene is very weakly stimulated by wt p53 and the low levels of the MDM2 protein p90 are produced mainly due to the basal activity of the P1 promoter (Zauberman et al, 1995a). (B) In a normal cell under stress (DNA damage, hypoxia, metabolic changes and immunological response), the MDM2 gene is expressed only when wt p53 has activated the targets that mediate its 'protective' action (e.g.…”

Section: Resultsmentioning

confidence: 99%

“…Electrophoretic mobility shift assay (EMSA) The p53RE of the MDM2 gene The p53RE of the MDM2 contains two p53 binding sites (underlined italic regions), separated by 18 nucleotides (Zauberman et al, 1995a). The sequence of this region is: 5'-TTGAGCT-GGTCAAGTTCAGACACGTTC-CGAAACTGCAGTAAAAG-GAGTTAAGTCCTGACTTTGTCT-CCAGCTC-3'.…”

Section: Mdm2 and P53 Western Blot Analysis -Mdm2 Immunoprecipitationmentioning

confidence: 99%

“…Reporter plasmid pGL2hmdm-HX-luc was constructed, as described previously (Zauberman et al, 1995a), by excising the HindIII-XhoI fragment from the amplified hMDM2 DNA, and subcloning it into pGL2-Basic (Promega). The representative p53 mutants, V157F, R209T (group A), V173E (group B) and R273H (group C), were generated by site-directed mutagenesis of the parental wtp53 vector pCB6p53wt.…”

Section: Controlsmentioning

confidence: 99%

“…The list of genes that possess these DNA sites is rapidly increasing and includes: waf-J/cip-J (El-Deiry et al, 1993), gadd45 (Kastan et al, 1992 and references therein), MDM2 (Zauberman et al, 1995a), bax (Miyashita and Reed, 1995), cyclin G (Zauberman et al, 1995b), insulin-like growth factor binding protein 3 (IGF-BP3) (Buckbinder et al, 1995), epidermal growth factor receptor (EGF-r) (Deb et al, 1994), transforming growth factor-a (TGF-a) (Shin et al, 1995), proliferating cell nuclear antigen (PCNA) (Morris et al, 1996), thrombospondin-J (Dameron et al, 1994), fas/APO-1 (Owen-Schaub et al, 1995), Rb (Osifchin et al, 1994), cyclin D , ras (Spandidos et al, 1995;Zhang et al, 1995) and p53 itself (Deffie et al, 1993).…”

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confidence: 99%

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Effects of p53 mutants derived from lung carcinomas on the p53-responsive element (p53RE) of the MDM2 gene

Gorgoulis

Zacharatos²,

Manolis³

et al. 1998

Br J Cancer

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Summary The present study represents a continuation of previous works in which we observed that lung carcinomas co-expressing MDM2 protein and p53 mutants (mt p53) exhibited more aggressive behaviour. In the above studies, we suggested a 'gain of function' mechanism of mt p53 proteins based on the fact that the MDM2 gene possesses a p53-responsive element (MDM2-p53RE). In this study, to prove our hypothesis, we selected 12 cases from a series of 51 bronchogenic carcinomas. In these 12 cases, we examined the ability of the expressed mt p53 to bind the MDM2-p53RE and correlated the findings with MDM2 expression. Furthermore, we constructed four of these p53 mutants and studied their transactivation properties by co-transfecting them with a reporter plasmid carrying MDM2-p53RE in the p53 null non-smallcell lung carcinoma cell line (NSCLC) H1299. We observed mutant p53 protein DNA-binding activity, which depended on the nature and the position of the amino acid substitution. The fact that the cases with DNA-binding activity were accompanied with MDM2 protein isoforms' overexpression is indicative of a 'gain of function' phenotype. This hypothesis was enforced by the findings of the transfection experiments, which revealed that certain p53 mutants enhanced the expression of the luciferase reporter gene either directly or indirectly via a dominant positive effect on the wild-type p53. In conclusion, this work is one first attempt to examine if the deregulation of the p53/MDM2 autoregulatory feedback loop is due to novel properties of certain p53 mutants in the specific environment of a subset of bronchogenic carcinomas.Keywords: p53 mutations; p53-responsive element; MDM2 gene; MDM2 isoforms; lung cancer The p53 oncosuppressor gene is mapped to chromosomal region 17pl3 and encodes a 393 amino acid (aa), 53-kDa nuclear phosphoprotein. The protein is divided into three main structural and functional domains. The first 42 amino acids at the N-terminus constitute the transactivation domain, the residues between amino acids 120 and 290 make up the sequence-specific DNA binding domain, whereas residues 310-393 at the C-terminus contain the nuclear localization signals, the tetramerization domain and the extreme carboxy-terminus that allosterically regulates p53 specific DNA binding. p53 protein is involved in vital aspects of the cell life, such as control of cell cycle checkpoints G, and G2, maintaining genomic integrity, DNA repair, replication, transcription, programmed cell death (apoptosis) and differentiation. Functional loss of p53, mostly via mutations, is considered the most common genetic lesion in human cancer (Greenblatt et al, 1994). Therefore, an understanding of the functions of p53 may help elucidate key steps in carcinogenesis. The cellular effects of p53 are mediated either by protein-protein interaction or by binding to DNA regulatory elements. In the first case, p53 interacts with factors of the replication, transcription and repair machinery (reviewed by:

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Section: Resultsmentioning

confidence: 99%

Section: Mdm2 and P53 Western Blot Analysis -Mdm2 Immunoprecipitationmentioning

confidence: 99%

Section: Controlsmentioning

confidence: 99%

mentioning

confidence: 99%

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Effects of p53 mutants derived from lung carcinomas on the p53-responsive element (p53RE) of the MDM2 gene

Gorgoulis

Zacharatos²,

Manolis³

et al. 1998

Br J Cancer

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“…The mouse gene contains at least 12 exons spread over about 25 kb of DNA (Montes de Oca Luna et al, 1996;Jones et al, 1996). Two promoters were characterized: the ®rst one is located upstream of the gene and is expressed constitutively whereas the second one is present in the ®rst intron and is controlled by p53 in both mouse and human through two adjacent p53 binding sites (Wu et al, 1993;Juven et al, 1993;Barak et al, 1994;Zauberman et al, 1995). Activation of Mdm2 expression by p53 provides the opportunity for feedback control of p53 activity that could play an important role in cell recovery after repair of damaged DNA.…”

Section: Mdm2 Gene Structure and Expressionmentioning

confidence: 99%

Mdm2: keeping p53 under control

1997

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The Mdm2 gene is overexpressed in several human tumors. The oncogenic potential of Mdm2 is partially explained by the inhibition of the activity of the tumor suppressor protein p53. Determination of the threedimensional structure of complexes between Mdm2 and the N-terminal p53 peptide provided a molecular basis for the inhibition of the transcriptional function of p53 by Mdm2. More dramatically, p53 is targeted by Mdm2 for rapid degradation. The Mdm2 gene itself is activated by p53, which gives the opportunity for feed-back control of p53 activity. Keeping p53 under control is most likely the major task of Mdm2 during early development. Recently, evidence was provided for an alternative, p53-independent function of Mdm2.

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A MOLECULAR AND IMMUNOHISTOCHEMICAL STUDY OF THE MDM2 PROTEIN ISOFORMS ANDp53 GENE PRODUCT IN BRONCHOGENIC CARCINOMA

et al. 1996

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Forty‐one bronchogenic carcinomas were investigated for expression of MDM2 protein isoforms and their relationship to p53 protein levels and p53 gene alterations using molecular and immunohistochemical techniques. The findings were correlated with the pathological features of the carcinomas. MDM2 protein was overexpressed in 26 cases (63 per cent). Western blot analysis with two monoclonal antibodies, 1B10 and IF2, revealed three MDM2 protein isoforms, p90, p57 and p76/74. p90 and p57 are capable of interacting with p53 protein, while p76/74 is not. Various patterns of MDM2 isoforms were seen. Although no correlation between the patterns and pathological features was observed, lymph node metastases were more frequent in the cases with MDM2 overexpression (P<0·005). In 3 out of 17 specimens of normal lung tissue examined, there was a low level of expression of p90. Molecular analysis revealed that MDM2 overexpression was a consequence of increased transcription rather than MDM2 gene amplification. p53 protein was overexpressed in 21 cases (51 per cent) and p53 gene alterations (mutations+allelic deletions) were detected in 23 patients (56 per cent). A high degree of concordance (76 per cent) between p53 mutations and p53 staining was noticed (P<10−5). p53 gene alterations were significantly associated with lymph node disease (P<0·01). MDM2 and p53 proteins were simultaneously detected in 21 cases (51 per cent), of which 17 (42 per cent) showed p53 and MDM2 overexpression. The latter group was positively correlated with p53 mutations (P<0·05). A strong correlation between MDM2/p53 co‐expression and lymph node metastases was observed (P<0·001). The findings suggest that MDM2 overexpression is a common event in bronchogenic carcinoma. The selective expression of some MDM2 isoforms in neoplastic tissue and not in the surrounding normal areas underscores the pathological role of the various MDM2 products. Finally, the coexistence of MDM2 protein(s) and p53 aberrations (mutations and/or overexpression) in a subset of lung carcinomas may be indicative of a ‘gain of function’ phenotype, with more aggressive characteristics.

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A functional p53-responsive intronic promoter is contained within the humanmdm2gene

Cited by 272 publications

References 29 publications

Effects of p53 mutants derived from lung carcinomas on the p53-responsive element (p53RE) of the MDM2 gene

Effects of p53 mutants derived from lung carcinomas on the p53-responsive element (p53RE) of the MDM2 gene

Mdm2: keeping p53 under control

A MOLECULAR AND IMMUNOHISTOCHEMICAL STUDY OF THE MDM2 PROTEIN ISOFORMS ANDp53 GENE PRODUCT IN BRONCHOGENIC CARCINOMA

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