Overexpression of Mdm2 in mice reveals a p53-independent role for Mdm2 in tumorigenesis.

The Mdm2 proto-oncogene is amplified to high copy numbers in human sarcomas and is overexpressed in a wide variety of other human cancers. Because Mdm2 protein forms a complex with the p53 tumor suppressor protein and down-regulates p53 function, the oncogenic potential of Mdm2 is presumed to be p53-dependent. To model these conditions in mice, we have used the entire Mdm2 gene, under transcriptional control of its native promoter region, as a transgene to create mice that overexpress Mdm2. The transgenic mice are predisposed to spontaneous tumor formation, and the incidence of sarcomas observed in the Mdm2-transgenic mice in the presence or absence of functional p53 demonstrates that, in addition to Mdm2-mediated inactivation of p53, there exists a p53-independent role for Mdm2 in tumorigenesis.

hSmad5 gene, a human hSmad family member: its full length cDNA, genomic structure, promoter region and mutation analysis in human tumors.

hSmad (mothers against decapentaplegic)-related proteins are important messengers within the Transforming Growth Factor-beta1 (TGF-beta1) superfamily signal transduction pathways. To further characterize a member of this family, we obtained a full length cDNA of the human hSmad5 (hSmad5) gene by rapid amplification of cDNA ends (RACE) and then determined the genomic structure of the gene. There are eight exons and two alternative transcripts; the shorter transcript lacks exon 2. We identified the hSmad5 promoter region from a human genomic YAC clone by obtaining the nucleotide sequence extending 1235 base pairs upstream of the 5' end of the cDNA. We found a CpG island consistent with a promoter region, and we demonstrated promoter activity in a 1232 bp fragment located upstream of the transcription initiation site. To investigate the frequency of somatic hSmad5 mutations in human cancers, we designed intron-based primers to examine coding regions by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. Neither homozygous deletions or point mutations were found in 40 primary gastric tumors and 51 cell lines derived from diverse types of human cancer including 20 cell lines resistant to the growth inhibitory effects of TGF-beta1. These results suggest that the hSmad5 gene is not commonly mutated and that other genetic alterations mediate the loss of TGF-beta1 responsiveness in human cancers.

Hepatitis B virus X protein and p53 tumor suppressor interactions in the modulation of apoptosis.

We have reported previously that the hepatitis B virus oncoprotein, HBx, can bind to the C terminus of p53 and inhibit several critical p53-mediated cellular processes, including DNA sequence-specific binding, transcriptional transactivation, and apoptosis. Recognizing the importance of p53-mediated apoptosis for maintaining homeostasis and preventing neoplastic transformation, here we further examine the physical interaction between HBx and p53 as well as the functional consequences of this association. In vitro binding studies indicate that the ayw and adr viral subtypes of HBx bind similar amounts of glutathione S-transferase-p53 with the distal C terminus of HBx (from residues 111 to 154) being critical for this interaction. Using a microinjection technique, we show that this same C-terminal region of HBx is necessary for sequestering p53 in the cytoplasm and abrogating p53-mediated apoptosis. The transcriptional transactivation domain of HBx also maps to its C terminus; however, a comparison of the ability of full-length and truncated HBx protein to abrogate p53-induced apoptosis versus transactivate simian virus 40- or human nitric oxide synthase-2 promoter-driven reporter constructs indicates that these two functional properties are distinct and thus may contribute to hepatocarcinogenesis differently. Collectively, our data indicate that the distal C-terminal domain of HBx, independent of its transactivation activity, complexes with p53 in the cytoplasm, partially preventing its nuclear entry and ability to induce apoptosis. These pathobiological effects of HBx may contribute to the early stages of hepatocellular carcinogenesis.

The tumorigenic potential and cell growth characteristics of p53-deficient cells are equivalent in the presence or absence of Mdm2.

The Mdm2 oncoprotein forms a complex with the p53 tumor suppressor protein and inhibits p53-mediated regulation of heterologous gene expression. Recently, Mdm2 has been found to bind several other proteins that function to regulate cell cycle progression, including the E2F-1/DP1 transcription factor complex and the retinoblastoma tumor-suppressor protein. To determine whether Mdm2 plays a role in cell cycle control or tumorigenesis that is distinct from its ability to modulate p53 function, we have examined and compared both the in vitro growth characteristics of p53-deficient and Mdm2/p53-deficient fibroblasts, and the rate and spectrum of tumor formation in p53-deficient and Mdm2/p53-deficient mice. We find no difference between p53-deficient fibroblasts and Mdm2/p53-deficient fibroblasts either in their rate of proliferation in culture or in their survival frequency when treated with various genotoxic agents. Cell cycle studies indicate no difference in the ability of the two cell populations to enter S phase when treated with DNA-damaging agents or nucleotide antimetabolites, and p53-deficient fibroblasts and Mdm2/p53-deficient fibroblasts exhibit the same rate of spontaneous immortalization following long-term passage in culture. Finally, p53-deficient mice and Mdm2/p53-deficient mice display the same incidence and spectrum of spontaneous tumor formation in vivo. These results demonstrate that deletion of Mdm2 has no additional effect on cell proliferation, cell cycle control, or tumorigenesis when p53 is absent.

Genomic organization of the mouse double minute 2 gene.

Transfection of the mouse double minute 2 (Mdm2) oncogene has been found to induce immortalization of primary cells and to transform cultured cells. Amplification and/or overexpression of human MDM2 has been documented in a large percentage of human cancers. Mouse and human Mdm2 cDNA have been cloned from transformed cells and the cDNA sequence of both genes have been reported previously. In this report, we present the gene structure of mouse Mdm2. Comparison of the coding sequences of the Mdm2 gene with the previously reported cDNA sequence and with Mdm2 sequences obtained from an Mdm2-bearing cosmid clone capable of inducing transformation revealed that the reported cDNA sequence was in error, and that Mdm2-induced transformation of cells does not require an activating mutation in Mdm2. Ligation-anchor PCR analysis of transcripts produced from the P1 and P2 promoters indicates that transcription initiates at sites upstream of those reported previously for both promoters.