MDM2 and MDMX can interact differently with ARF and members of the p53 family.

Members of the p53 family of transcription factors have essential roles in tumor suppression and in development. MDM2 is an essential regulator of p53 that can inhibit the transcriptional activity of p53, shuttle p53 out of the nucleus, and target p53 for ubiquitination-mediated degradation. Little is known about the interaction and selectivity of different members of the p53 family (p53, p63, and p73) and the MDM2 family (MDM2 and MDMX). Here we show that the transcriptional activities of p53 and p73, but not that of p63, were inhibited by both MDM2 and MDMX. Consistent with these, we found that MDMX can physically interact with p53 and p73, but not with p63. Moreover, ectopically expressed MDM2 and MDMX could induce alterations in the subcellular localization of p73, but did not affect the subcellular localization of p53 and p63. Finally, we demonstrate that while ARF can interact with MDM2 and inhibit the regulation of p53 by MDM2, no interaction was found between ARF and MDMX. These data reveal that significant differences and selectivity exist between the regulation of different members of the p53 family by MDM2 and MDMX.

Cleavage of cyclin A at R70/R71 by the bacterial protease OmpT.

Previous work has shown that cyclin A can be cleaved at Arg-70/Arg-71 by a proteolytic activity present in an in vitro-coupled transcription/translation system by using rabbit reticulocyte lysate programmed by plasmid DNA encoding p27(KIP1), a cyclin-dependent kinase inhibitor, but not by plasmid DNAs encoding other cyclin-dependent kinases inhibitors. Here we report that cyclin A is also cleaved by translation product programmed by plasmid DNA encoding cyclin B. Several findings indicate that the cleavage activity in this assay is provided by the bacterial protease OmpT, which cofractionates with cyclin B and p27(KIP1) plasmid DNAs and is thus carried over into the coupled in vitro transcription/translation reactions. (i) Cleavage activity appeared even when transcription or translation of the cyclin B or p27(KIP1) was blocked. (ii) Activity resembling OmpT, a serine protease that cleaves between dibasic residues, routinely copurifies with p27(KIP1) and cyclin B plasmid DNAs. (iii) Both cyclin A cleavage activity and OmpT activity are heat stable, resistant to denaturation, and inhibited by Zn(2+), Cu(2+), or benzamidine. (iv) Cyclin A cleavage activity is detected when using lysates or DNAs prepared from Escherichia coli strains that contained OmpT but not with strains lacking OmpT. (v) Purified OmpT enzyme itself cleaves cyclin A at R70/R71. These data indicate that OmpT can be present in certain DNA preparations obtained by using standard plasmid purification protocols, and its presence can potentially affect the outcome and interpretation of studies carried out using in vitro-translated proteins.

On the concentrations of cyclins and cyclin-dependent kinases in extracts of cultured human cells.

Cyclins and cyclin-dependent kinases (CDKs) are key regulators of the human cell cycle. Here we have directly measured the concentrations of the G(1) and G(2) cyclins and their CDK partners in highly synchronized human cervical carcinoma cells (HeLa). To determine the exact concentrations of cyclins and CDKs in the cell extracts, we developed a relatively simple method that combined the use of (35)S-labeled standards produced in rabbit reticulocyte lysates and immunoblotting with specific antibodies. Using this approach, we formally demonstrated that CDC2 and CDK2 are in excess of their cyclin partners. We found that the concentrations of cyclin A2 and cyclin B1 (at their peak levels in the G(2) phase) were about 30-fold less than that of their partner CDC2. The peak levels of cyclin A2 and cyclin E1, at the G(2) phase and G(1) phase, respectively, were only about 8-fold less than that of their partner CDK2. These ratios are in good agreement with size fractionation analysis of the relative amount of monomeric and complexed forms of CDC2 and CDK2 in the cell. All the cyclin A2 and cyclin E1 are in complexes with CDC2 and CDK2, but there are some indications that a significant portion of cyclin B1 may not be in complex with CDC2. Furthermore, we also demonstrated that the concentration of the CDK inhibitor p21(CIP1/WAF1) induced after DNA damage is sufficient to overcome the cyclin-CDK2 complexes in MCF-7 cells. These direct quantitations formally confirmed the long-held presumption that CDKs are in excess of the cyclins in the cell. Moreover, similar approaches can be used to measure the concentration of any protein in cell-free extracts.

Degradation of cyclin A does not require its phosphorylation by CDC2 and cyclin-dependent kinase 2.

Many cyclins are degraded by the ubiquitination/proteasome pathways involving the anaphase-promoting complex and SCF complexes. These degradations are frequently dependent on phosphorylation by cyclin-dependent kinases (CDKs), providing a self-limiting mechanism for CDK activity. Here we present evidence from in vitro and in vivo assay systems that the degradation of human cyclin A can be inhibited by kinase-inactive mutants of CDK2 and CDC2. One obvious interpretation of these results is that like other cyclins, CDK-dependent phosphorylation of the cyclin A may be involved in cyclin A degradation. Our data indicated that CDK2 can phosphorylate cyclin A on Ser-154. Site-directed mutagenesis of Ser-154 abolished the phosphorylation by recombinant CDK2 in vitro and the majority of cyclin A phosphorylation in the cell. Activation of CDK2 and binding to SKP2 or p27(KIP1) were not affected by the phosphorylation of Ser-154. Surprising, in marked contrast to cyclin E, where phosphorylation of Thr-380 by CDK2 is required for proteolysis, degradation of cyclin A was not affected by Ser-154 phosphorylation. It is likely that the stabilization of cyclin A by the kinase-inactive CDKs was mainly due to a cell cycle effect. These data suggest an important difference between the regulation of cyclin A and cyclin E.

G1 versus G2 cell cycle arrest after adriamycin-induced damage in mouse Swiss3T3 cells.

Cell cycle arrest after different types of DNA damage can occur in either G1 phase or G2 phase of the cell cycle, involving the distinct mechanisms of p53/p21(Cip1/Waf1) induction, and phosphorylation of Cdc2, respectively. Treatment of asynchronously growing Swiss3T3 cells with the chemotherapeutic drug adriamycin induced a predominantly G2 cell cycle arrest. Here we investigate why Swiss3T3 cells were arrested in G2 phase and not in G1 phase after adriamycin-induced damage. We show that adriamycin was capable of inducing a G1 cell cycle arrest, both during the G0-G1 transition and during the G1 phase of the normal cell cycle. In G0 cells, adriamycin induced a prolonged cell cycle arrest. However, adriamycin caused only a transient cell cycle delay when added to cells at later time points during G0-G1 transition or at the G1 phase of normal cell cycle. The G1 arrest correlated with the induction of p53 and p21(Cip1/Waf1), and the exit from the arrest correlated with the decline of their expression. In contrast to the G1 arrest, adriamycin-induced G2 arrest was relatively tight and correlated with the Thr-14/Tyr-15 phosphorylation of cyclin B-Cdc2 complexes. The relative stringency of the G1 versus G2 cell cycle arrest may explain the predominance of G2 arrest after adriamycin treatment in mammalian cells.

MDM2 and MDMX inhibit the transcriptional activity of ectopically expressed SMAD proteins.

Transforming growth factor-beta (TGF-beta) inhibits cell proliferation in many cell types, and acquisition of TGF-beta resistance has been linked to tumorigenesis. One class of proteins that plays a key role in the TGF-beta signal transduction pathway is the SMAD protein family. MDM2, a key negative regulator of p53, has recently been shown to suppress TGF-beta-induced growth arrest in a p53-independent manner. Here we show that MDM2 and the structurally related protein MDMX can inhibit the transcriptional activity of ectopically expressed SMAD1, SMAD2, SMAD3, and SMAD4. Immunofluorescence staining indicated that ectopically expressed SMAD4 was present in both the cytoplasm and nucleus, and MDM2 and NIDMX were localized mainly to the nucleus and cytoplasm, respectively. When SMAD4 was coexpressed with either MDM2 or MDMX, nuclear accumulation of SMAD4 was strikingly inhibited. We have no evidence that SMAD4 binds directly to MDM2 or MDMX; hence, the inactivation and nuclear exclusion of SMAD4 by MDM2/MDMX may involve other indirect mechanisms.

MDM2 and MDMX bind and stabilize the p53-related protein p73.

The p53 gene encodes one of the most important tumor suppressors in human cells and undergoes frequent mutational inactivation in cancers. MDM2, a transcriptional target of p53, binds p53 and can both inhibit p53-mediated transcription [1] [2] and target p53 for proteasome-mediated proteolysis [3] [4]. A close relative of p53, p73, has recently been identified [5] [6]. Here, we report that, like p53, p73alpha and the alternative transcription product p73beta also bind MDM2. Interaction between MDM2 and p53 represents a key step in the regulation of p53, as MDM2 promotes the degradation of p53. In striking contrast to p53, the half-life of p73 was found to be increased by binding to MDM2. Like MDM2, the MDM2-related protein MDMX also bound p73 and stabilized the level of p73. Moreover, the growth suppression functions of p73 and the induction of endogenous p21, a major mediator of the p53-dependent growth arrest pathway, were enhanced in the presence of MDM2. These differences between the regulation of p53 and p73 by MDM2/MDMX may highlight a physiological difference in their action.

Differential responses of proliferating versus quiescent cells to adriamycin.

The relative sensitivity of proliferating and quiescent cells to DNA-damaging agents is a key factor for cancer chemotherapy. Here we undertook a reevaluation of the way that proliferating and quiescent cells differ in their responses and fate to adriamycin-induced damage. Distinct types of assays that measure membrane integrity, metabolic activity, cell size, DNA content, and the ability to proliferate were used to compare growing and quiescent Swiss3T3 fibroblasts after adriamycin treatment. We found that immediately after adriamycin treatment of growing cells, p53 and p21(Cip1/Waf1) were induced but the cells remained viable. In contrast, less p53 and p21(Cip1/Waf1) were induced in quiescent cells after adriamycin treatment, but the cells were more prone to immediate cell death, possibly involving apoptosis. Adriamycin induced a G2/M cell cycle arrest in growing cells and a concomitant increase in cell size. In contrast, adriamycin induced an increase in sub-G1 DNA content in quiescent cells and a decrease in cell size. In contrast to the short-term responses, adriamycin-treated quiescent cells have a better long-term survival and proliferation potential than adriamycin-treated growing cells in colony formation assays. These data suggest that proliferating and resting cells are remarkably different in their short-term and long-term responses to adriamycin.

Regulation of cyclin A-Cdk2 by SCF component Skp1 and F-box protein Skp2.

Cyclin A-Cdk2 complexes bind to Skp1 and Skp2 during S phase, but the function of Skp1 and Skp2 is unclear. Skp1, together with F-box proteins like Skp2, are part of ubiquitin-ligase E3 complexes that target many cell cycle regulators for ubiquitination-mediated proteolysis. In this study, we investigated the potential regulation of cyclin A-Cdk2 activity by Skp1 and Skp2. We found that Skp2 can inhibit the kinase activity of cyclin A-Cdk2 in vitro, both by direct inhibition of cyclin A-Cdk2 and by inhibition of the activation of Cdk2 by cyclin-dependent kinase (CDK)-activating kinase phosphorylation. Only the kinase activity of Cdk2, not of that of Cdc2 or Cdk5, is reduced by Skp2. Skp2 is phosphorylated by cyclin A-Cdk2 on residue Ser76, but nonphosphorylatable mutants of Skp2 can still inhibit the kinase activity of cyclin A-Cdk2 toward histone H1. The F box of Skp2 is required for binding to Skp1, and both the N-terminal and C-terminal regions of Skp2 are involved in binding to cyclin A-Cdk2. Furthermore, Skp2 and the CDK inhibitor p21(Cip1/WAF1) bind to cyclin A-Cdk2 in a mutually exclusive manner. Overexpression of Skp2, but not Skp1, in mammalian cells causes a G1/S cell cycle arrest.

Supplementation with vitamins C and E enhances cytokine production by peripheral blood mononuclear cells in healthy adults.

The effect of supplementation with vitamins C and E on cytokine production of healthy adult volunteers was studied in a single-blind trial. Ten subjects in each group received daily vitamin C (1 g ascorbic acid), vitamin E (400 mg dl-alpha-tocopheryl acetate), or vitamins C and E for 28 d. Plasma concentrations of alpha-tocopherol, ascorbate, and lipid peroxides as well as the production of cytokines by peripheral blood mononuclear cells (PBMCs) were measured before, during, and at the end of the supplementation and 1 wk later. PBMCs were cultured in the presence of absence of lipopolysaccharide for 24 h. The interleukin 1 (IL-1), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-alpha) in the culture supernates were assayed by enzyme-linked immunosorbent assay methods. Production of IL-1 beta and TNF-alpha in the group supplemented with vitamins C and E was significantly higher (P < 0.05) than that of the groups given vitamin E or vitamin C alone. The enhancing effect of supplementation with a combination of vitamins E and C coincided with peak plasma alpha-tocopherol and ascorbate concentrations and the lowest plasma lipid peroxide concentrations (P < 0.05) on day 14. In addition, an in vitro experiment with PBMCs showed that vitamins E and C reduced lipopolysaccharide-induced prostaglandin E2 production and enhanced TNF-alpha production. These results indicate that combined supplementation with vitamins C and E is more immunopotentiating than supplementation with either vitamin alone in healthy adults.