A deficiency in Mdm2 binding protein inhibits Myc-induced B-cell proliferation and lymphomagenesis.

Mdm2 binding protein (MTBP) has been implicated in cell-cycle arrest and the Mdm2/p53 tumor suppressor pathway through its interaction with Mdm2. To determine the function of MTBP in tumorigenesis and its potential role in the Mdm2/p53 pathway, we crossed Mtbp-deficient mice to Emu-myc transgenic mice, in which overexpression of the oncogene c-Myc induces B-cell lymphomas primarily through inactivation of the Mdm2/p53 pathway. We report that Myc-induced B-cell lymphoma development in Mtbp heterozygous mice was profoundly delayed. Surprisingly, reduced levels of Mtbp did not lead to an increase in B-cell apoptosis or affect Mdm2. Instead, an Mtbp deficiency inhibited Myc-induced proliferation and the upregulation of Myc target genes necessary for cell growth. Consistent with a role in proliferation, Mtbp expression was induced by Myc and other factors that promote cell-cycle progression and was elevated in lymphomas from humans and mice. Therefore, Mtbp functioned independent of Mdm2 and was a limiting factor for the proliferative and transforming functions of Myc. Thus, Mtbp is a previously unrecognized regulator of Myc-induced tumorigenesis.

MDM2 interacts with the C-terminus of the catalytic subunit of DNA polymerase epsilon.

MDM2 is induced by p53 in response to cellular insults such as DNA damage and can have effects upon the cell cycle that are independent or downstream of p53. We used a yeast two-hybrid screen to identify proteins that bind to MDM2 and which therefore might be involved in these effects. We found that MDM2 can bind to the C-terminus of the catalytic subunit of DNA polymerase epsilon (DNA pol epsilon), to a region that is known to be essential in yeast. In an in vitro system we confirmed that MDM2 could bind to the homologous regions of both mouse and human DNA pol epsilon and to full-length human DNA pol epsilon. DNA pol epsilon co-immunoprecipitated with MDM2 from transfected H1299 cells and also from a HeLa cell nuclear extract. We show here that the DNA pol epsilon-interacting domain of MDM2 is located between amino acids 50 and 166. Our studies provide evidence that MDM2 interacts with a region of DNA pol epsilon that plays a critical role in the function of DNA pol epsilon.

A novel cellular protein (MTBP) binds to MDM2 and induces a G1 arrest that is suppressed by MDM2.

The MDM2 protein, through its interaction with p53, plays an important role in the regulation of the G(1) checkpoint of the cell cycle. In addition to binding to and inhibiting the transcriptional activation function of the p53 protein, MDM2 binds, inter alia, to RB and the E2F-1.DP-1 complex and in so doing may promote progression of cells into S phase. Mice transgenic for Mdm2 possess cells that have cell cycle regulation defects and develop an altered tumor profile independent of their p53 status. MDM2 also blocks the growth inhibitory effects of transforming growth factor-beta1 in a p53-independent manner. We show here that a novel growth regulatory molecule is also the target of MDM2-mediated inhibition. Using a yeast two-hybrid screen, we have identified a gene that encodes a novel cellular protein (MTBP) that binds to MDM2. MTBP can induce G(1) arrest, which in turn can be blocked by MDM2. Our results suggest the existence of another growth control pathway that may be regulated, at least in part, by MDM2.

Identification of a tumor-derived p53 mutant with novel transactivating selectivity.

MDM2 is a p53-responsive molecule that when overexpressed, can alter growth control pathways via p53-dependent and independent mechanisms. We have identified a mutant p53 containing line that expresses high levels of transcripts that are regulated by the p53-responsive promoter of the MDM2 gene. Analysis of cloned product obtained from these tumor cells revealed that they harbor a mutant p53 protein (possessing an Arg to Gln substitution at codon 213) that is a potent transactivator of MDM2 expression. Consistent with this activity, the R213Q mutant was found to have the ability to interact with DNA sequences located within the MDM2 promoter. In contrast to previously described tumor-derived p53 mutants which retain MDM2 transactivation function and possess partial growth suppressive activity, the R213Q mutant is severely compromised in its ability to induce p53-regulated transcripts that encode for proteins involved in cell-cycle arrest and apoptosis. The R213Q mutant can also be expressed at high levels in stably transfected cells and cells that harbor this mutant possess elevated levels of MDM2 protein. The R213Q mutant was also found to be able to up-regulate MDM2 during a genotoxic stress response. R213Q is the first described tumor-derived p53 mutant that is deficient at up-regulating both cell cycle arrest and apoptotic factors, but is highly proficient at inducing the growth-promoting molecule MDM2.

Tumour epidermal growth factor receptor, erbB-2 and cathepsin D in node-negative invasive breast cancer: their impact on the selection of patients for systemic adjuvant therapy.

OBJECTIVE: To determine the feasibility and the economic impact of tumour EGFR, erbB-2 and cathepsin-D measurements in women with node-negative breast cancer. DESIGN: Consecutive tumour samples received at a regional steroid receptor laboratory from patients with node-negative breast cancer were evaluated with commercially available kits to determine EGFR, erbB-2 and cathepsin-D levels. SETTING: All node-negative patients whose tumours were submitted to the steroid receptor laboratory from November 1992 to March 1994 were included (n = 142). A control group of concurrent node-negative breast cancer patients from the London Regional Cancer Centre (LRCC) database were also evaluated to determine the representativeness of our sample. MAIN OUTCOME MEASURE: To determine the proportion of patients who were positive for the 3 newer prognostic factors relative to their risk of relapse. RESULTS: We found 75 positive values in 69 patients (48.6%). We demonstrated that each factor identified a different high-risk subgroup. Epidermal growth factor receptor (EGFR) positivity (> 10 fmol/mg protein) was found in 16.3% of patients, with 19.9% of patients positive for erbB-2 (> 250 units/mg protein) and 17.3% positive for cathepsin D (> 70 pmol/mg protein). Between 10% and 23.2% more node-negative patients currently seen in a regional cancer centre could be offered systemic adjuvant chemotherapy based on a single positive new factor. CONCLUSIONS: These tumour evaluations are straightforward using material already available in a regional steroid receptor laboratory or on tumour tissue available to pathologists. The economic impact is minimal; the 1995 cost of performing all 3 evaluations is Can$425-616 (US$304-440) per patient treated depending on the number of assays per run. Prospective clinical trials incorporating tumour EGFR, erbB-2 and cathepsin D are feasible and economically viable.

The pathway regulating MDM2 protein degradation can be altered in human leukemic cells.

The MDM2 protein regulates the functional activity of the p53 tumor suppressor through direct physical association. Signals that control MDM2 expression are poorly understood but are likely to play an important role in the regulation of p53 activity. We show here that the half-life of MDM2 protein is shorter in proliferating than in quiescent peripheral blood mononuclear cells. We also demonstrate that MDM2 protein half-life is extended in some, but not all, p53 mutant human leukemic cell lines. In at least one of these p53 mutant lines, increased MDM2 protein stability is associated with higher amounts of MDM2 protein. Moreover, we demonstrate that MDM2 protein accumulates to a much greater extent in proteasome inhibitor-treated cells containing unstable MDM2 than in cells possessing stable MDM2. These results demonstrate that MDM2 expression is regulated by events that control the stability of the protein and suggest that the normal regulation of MDM2 turnover can be altered in tumor cell lines.

mdm2-P2 transcript levels predict the functional activity of the p53 tumor suppressor in primary leukemic cells.

The mdm2-P2 promoter is a transcriptional target of the p53 tumor suppressor. The aim of this study was to determine if there is an association between the level of these transcripts and the status of the p53 gene in human leukemic cells. A correlation between mdm2-P2 transcript levels and p53 gene status was observed in all cell lines examined. Primary malignant cells from 10 leukemic patients were also analysed for both mdm2-P2 levels and p53 gene status. All five patients with detectable mdm2-P2 transcripts possessed wild-type p53 alleles. However, only two of five patients with undetectable mdm2-P2 transcripts possessed mutant p53. mdm2-P2 levels were also determined in primary leukemic cells from 14 additional cases both before and after in vitro exposure to cisplatin. The p53 gene was found to be wild-type in all cases where mdm2-P2 levels were induced by cisplatin. There were four cases where no, or only modest, increases in mdm2-P2 levels were detected after cisplatin exposure. Two of these patients were found to harbor mutant p53 while one other possessed leukemic cells with elevated levels of mdm2 protein. These results show that induction of mdm2-P2 transcripts can be used to predict the presence of transcriptionally active p53 in primary leukemic cells.

The mdm2 proto-oncogene.

The mouse double minute 2 (mdm2) proto-oncogene was originally discovered as one of three genes that was amplified in a tumorigenic cell line derived from non-transformed Balb/c cells. Consistent with the expression pattern of mdm2 in these cells, it was later shown that the transforming potential of the mdm2 proto-oncogene can be activated by experimental overexpression. Overexpression of mdm2 protein been detected in a number of diverse human malignancies, indicating that this oncogene plays a key role in human carcinogenesis. One mechanism by which mdm2 overexpression may lead to uncontrolled cellular proliferation is through its ability to physically associate with the p53 tumor suppressor and block p53's growth suppressive functions. Forced overexpression of mdm2 has been shown to block the transactivation, cell cycle arrest and apoptotic functions of p53. The mdm2 gene has also been shown to be a transcriptional target of p53 and the induction of p53 transcriptional activity leads to increases in mdm2 RNA and protein levels. Thus, it appears that an auto-regulatory feedback loop exists between these two proteins which keeps the growth suppressive functions of p53 in check during normal cell cycling. However, this block is thought to be overcome during certain cellular insults, including DNA damage, so that p53 can regulate the expression of genes involved in cell cycle arrest and/or apoptosis. Genetic lesions leading to elevated levels of mdm2 likely impair the ability of p53 to orchestrate the expression of genes controlling cell cycle progression during cellular insults. This may lead to the propagation of genetic errors, genomic instability and ultimately to an increase in the rate of tumor cell evolution. There is also recent evidence which suggests that mdm2 may play roles in p53-independent pathways regulating cellular proliferation. mdm2 has recently been shown to interact with the retinoblastoma tumor suppressor protein p(Rb), and the E2F-1 and DP1 transcription factors. These, and other clinical, cellular and biochemical studies relating to the mdm2 oncogene are reviewed here. In addition, a proposed role for mdm2 in pathways controlling cell cycle response to cellular perturbations is presented.

Altered expression of p53 and mdm-2 proteins at diagnosis is associated with early treatment failure in childhood acute lymphoblastic leukemia.

PURPOSE: To determine whether potential alteration in p53 function through p53 gene mutation or mdm-2 overexpression correlates with early treatment failure in childhood acute lymphoblastic leukemia (ALL). PATIENTS AND METHODS: Diagnostic marrow samples from 34 children were analyzed for p53 gene alterations by western blot and SSCP/DNA sequence analysis and for mdm-2 overexpression by western blot analysis. These samples were derived from two groups of children with ALL: 17 good outcome patients who are in long-term continuous complete remission and 17 poor outcome patients who did not achieve a complete remission or relapsed within 6 months of achieving remission. RESULTS: Two children within the poor outcome group were found to have p53 gene mutations. Furthermore, five poor outcome patients were shown to have greater than 10-fold overexpression of mdm-2 protein compared with the mean level of mdm-2 protein measured in the good outcome group. Aberrant p53 protein expression was found in only one good outcome patient, whereas no good outcome children were found to have elevated levels (> 10-fold) of mdm-2 protein. CONCLUSION: We show for the first time that potential alteration in p53 function in childhood ALL is more common (P = .036) in cases of early treatment failure than in children who remain in long-term continuous remission.

Expression of 14-3-3 gamma in injured arteries and growth factor- and cytokine-stimulated human vascular smooth muscle cells.

One of the most critical cellular events in disease states such as vascular restenosis is the cytokine-induced activation of vascular smooth muscle cells (VSMCs) resulting in intimal thickening. Identification of the molecular mechanisms of VSMC activation is crucial in understanding the initiation of vascular restenosis. In this report, we show that one 14-3-3 protein family isoform, gamma, is transcriptionally up-regulated in rat carotid arteries after balloon angioplasty. 14-3-3 gamma protein induction localizes to both the media and neointima in such injured vessels. Because it has been shown that some members of the 14-3-3 family may play an important role in cellular proliferation by binding to and activating the protein kinase Raf-1 and VSMCs constitute the major cellular component of the restenotic lesion, we investigated the expression of this message in serum- and cytokine-stimulated human VSMCs. Both serum and selected cytokines induce 14-3-3 gamma mRNA and protein, the magnitude of which correlates with the degree of cellular stimulation. 14-3-3 gamma mRNA, however, does not increase when other cell types are stimulated with specific growth factors. Human tissue distribution of 14-3-3 gamma mRNA indicates that in contrast to other 14-3-3 proteins, the gamma isoform is highly expressed in VSMCs and skeletal and heart muscle, suggesting an important role for the gamma isoform in muscle tissue as well. These results indicate that 14-3-3 gamma expression increases in response to vessel damage and proliferative signals and may implicate a role for the gamma isoform of 14-3-3 in VSMC activation and metabolism.

Analysis of p53 and mdm-2 expression in 18 patients with Sézary syndrome.

Sézary syndrome is a leukaemic form of cutaneous T-cell lymphoma which presents with multiple cytogenetic abnormalities and responds poorly to chemotherapy. Because of the importance of the p53 tumour suppressor in maintaining genomic stability and in sensitizing transformed cells to DNA damaging agents, we looked for alternations which may affect p53 functions in 18 patients with Sézary syndrome. Cytogenetic analysis suggested frequent p53 gene inactivation since 6/18 patients had loss of one copy of 17p. However, single-strand conformational polymorphism (SSCP) revealed that p53 gene mutations are relatively rare, occurring in only two of 18 Sézary patients. Neither of these two patients was missing a copy of 17p. Possible abnormalities of p53 pathway function through mdm-2 over-expression were also investigated. Although all 18 patients had normal levels of mdm-2 RNA 4/18 over-expressed mdm-2 protein. One patient with advanced disease and the highest percentage of malignant cells overexpressed mdm-2 protein and possessed a nonsense p53 gene mutation. The five patients with abnormalities of p53 or mdm-2 were found to have significantly highest absolute lymphocyte counts and higher absolute numbers of Sézary cells (P=0-021 and 0.027 respectively). In summary, molecular alternations of 17p and potential p53 pathway abnormalities are a common event in Sézary syndrome and appear to be associated with more advanced disease.

High incidence of potential p53 inactivation in poor outcome childhood acute lymphoblastic leukemia at diagnosis.

Previous studies have indicated that p53 gene mutations were an uncommon event in acute lymphoblastic leukemia (ALL) in children. In one series of 330 patients, p53 mutations were seen in fewer than 3%. We analyzed bone marrow mononuclear cells derived from 10 children with ALL at diagnosis who subsequently failed to achieve a complete remission or who developed relapse within 6 months of attaining complete remission for p53 gene mutations and mdm-2 overexpression. We found that three children had p53 gene mutations, and four overexpressed mdm-2. Also, experiments comparing relative levels of mdm-2 RNA and protein in these patients demonstrated that mdm-2 overexpression can occur at the transcriptional and posttranscriptional level in primary leukemic cells. Although we were unable to link Waf-1 RNA expression with p53 status in childhood ALL, our data show potential p53 inactivation by multiple mechanisms in a large percentage of these patients and demonstrate that these alterations can be detected at diagnosis. Inactivation of the p53 pathway may, therefore, be important in children with ALL who fail to respond to treatment and may be useful for the early identification of children requiring alternative therapies.

Enhanced translation: a novel mechanism of mdm2 oncogene overexpression identified in human tumor cells.

The cellular mdm2 gene, which has potential transforming activity that can be activated by overexpression, is amplified in a significant percentage of human sarcomas and in other mammalian tumors. Proteins encoded by the mdm2 gene can bind to, and inhibit the function of, the protein product of the p53 tumor suppressor gene. As reported here, we have identified human choriocarcinoma cell lines that express high levels of mdm2 proteins as well as the p53 protein. Several lines of evidence demonstrate that the p53 in these tumor cells has a wild-type nucleotide sequence, although the protein exhibits an extended half-life. Further, the more than 100-fold overexpression of mdm2 proteins in these cells cannot be explained by gene amplification, elevated RNA expression, or altered protein stability; rather our data indicate that elevated mdm2 protein levels in these choriocarcinoma cell lines result from enhanced translation. This mechanism has not previously been implicated in the regulation of mdm2 gene expression, and it represents a novel means by which the potential transforming activity of the mdm2 oncogene could be activated.

Physical and functional interaction between wild-type p53 and mdm2 proteins.

The mdm2 oncogene, which is often amplified in mammalian tumors, produces a number of transcripts that encode distinct protein forms. Previous studies demonstrating that overexpression of the mdm2 gene can activate its transforming potential, and can inhibit the transcriptional activation function of p53, prompted us to begin to explore possible functional differences among the various mdm2 products. Utilizing a transient transfection assay, we have evaluated four naturally occurring murine mdm2 forms for their ability to inhibit p53-mediated transcriptional activation of reporter genes regulated by p53 response elements. Three of these mdm2 forms were found to physically associate with the wild-type p53 protein and to possess the ability to inhibit its transactivation function. A fourth form failed to exhibit either of these functions. This last mdm2 form lacks the N-terminal protein domain that is present in the other three splice forms examined, pointing to this region as one that is critical for complex formation with the p53 protein. Identifying such differences among mdm2 proteins provides important clues for dissecting their functional domains, and emphasizes that defining the individual properties of these products will be critical in elucidating the overall growth control function of the mdm2 gene.

Interleukin-1 alpha mediates an alternative pathway for the antiproliferative action of poly(I.C) on human endothelial cells.

The antiproliferative effect of double-stranded RNA (dsRNA) on human tumor and normal cells has been well established. However, the genes involved in the dsRNA-induced antiproliferative response and the molecular mechanisms by which this occurs remain less well defined. We have studied the ability of synthetic dsRNA, polyinosinic:polycytidylic acid (poly(I.C)) to modify human umbilical vein endothelial cell (HUVEC) growth and report that poly(I.C) induces a dose-dependent inhibition of HUVEC proliferation in vitro. In addition, the mRNA levels for the cytokines interleukin-1 alpha (IL-1 alpha) and interferon-beta 1 are induced in poly(I.C)-treated cells. Moreover, the growth inhibitory effects of poly(I.C) are relieved when cells are grown in the presence of an IL-1 alpha antisense oligonucleotide to the human IL-1 alpha transcript. Thus, the effects of poly(I.C) appear to be mediated, in part, through the function of IL-1 alpha, suggesting an alternative pathway for dsRNA-mediated inhibition of human endothelial cell growth.

Cellular and enzymatic activities of a synthetic heteropolymer double-stranded RNA of defined size.

We have synthesized a novel heteropolymer double-stranded RNA (dsRNA) molecule of defined length and strandedness (dsRNA309) and evaluated its ability to induce cytokine gene expression, activate dsRNA-dependent enzymes, and inhibit both tumor cell growth and virus replication. Unlike the conventionally studied synthetic homopolymer dsRNAs, polyinosinic acid:polycytidylic acid (poly(I-C)) and its mismatched analogue polyinosinic:polycytidylic, uridylic acid (poly(I-C12,U), dsRNA309 possessed restricted biological activity. dsRNA309 was unable to inhibit tumor cell growth or efficiently induce cytokine (i.e. interferon-beta and interleukin-1 alpha) gene expression. However, dsRNA309 was able to inhibit virus replication and activate dsRNA-dependent intracellular enzymes, 2'-5' oligoadenylate synthetase (2'-5' A synthetase) and the dsRNA-activated inhibitor kinase in in vitro assay systems. Overall, dsRNA309 provided a means for examining the mechanisms governing the dsRNA-regulated antiviral and antiproliferative responses, and studies with dsRNA309 demonstrated that the ability of a synthetic dsRNA to activate dsRNA-dependent intracellular enzymes does not necessarily predict the same gene inducing capacity.

RNA abundance measured by a lysate RNase protection assay.

We describe a sensitive ribonuclease protection assay that we have used to measure the amount of interferon-beta RNA directly in lysates of human cells. Cell lysates were prepared in concentrated guanidine thiocyanate. Molecular hybridization with RNA probes was then performed directly in crude cell lysate, and native RNase-resistant duplexes were characterized by polyacrylamide gel electrophoresis. Comparison of interferon-beta RNA abundance by quantitative solution hybridization and lysate RNase protection showed that lysate RNase protection was highly quantitative. A high degree of reproducibility of the method was determined with a glyceraldehyde-3-phosphate dehydrogenase "housekeeping" gene probe. Sensitivity of lysate RNase protection was determined using both induced interferon-beta RNA and synthetic human endogenous reverse transcriptase RNA as target. The lysate RNase protection method was able to measure as few as 10(4)-10(5) RNA molecules.