Louie Naumovski

ASPP proteins specifically stimulate the apoptotic function of p53.

We identified a family of proteins termed ASPP. ASPP1 is a protein homologous to 53BP2, the C-terminal half of ASPP2. ASPP proteins interact with p53 and specifically enhance p53-induced apoptosis but not cell cycle arrest. Inhibition of endogenous ASPP function suppresses the apoptotic function of endogenous p53 in response to apoptotic stimuli. ASPP enhance the DNA binding and transactivation function of p53 on the promoters of proapoptotic genes in vivo. Two tumor-derived p53 mutants with reduced apoptotic function were defective in cooperating with ASPP in apoptosis induction. The expression of ASPP is frequently downregulated in human breast carcinomas expressing wild-type p53 but not mutant p53. Therefore, ASPP regulate the tumor suppression function of p53 in vivo.

The p53-binding protein 53BP2 also interacts with Bc12 and impedes cell cycle progression at G2/M.

Using the yeast two-hybrid system, we have isolated a cDNA (designated BBP, for Bcl2-binding protein) for a protein (Bbp) that interacts with Bcl2. Bbp is identical to 53BP2, a partial clone of which was previously isolated in a two-hybrid screen for proteins that interact with p53. In this study, we show that specific interactions of Bbp/53BP2 with either Bcl2 or p53 require its ankyrin repeats and SH3 domain. These interactions can be reproduced in vitro with bacterially expressed fusion proteins, and competition experiments indicate that Bcl2 prevents p53 from binding to Bbp/53BP2. BBP/53BP2 mRNA is abundant in most cell lines examined, but the protein cannot be stably expressed in a variety of cell types by transfection. In transiently transfected cells, Bbp partially colocalizes with Bcl2 in the cytoplasm and results in an increased number of cells at G2/M, possibly accounting for the inability to obtain stable transfectants expressing the protein. These results demonstrate that a single protein can interact with either Bcl2 or p53 both in yeast cells and in vitro. The in vivo significance of these interactions and their potential consequences for cell cycle progression and cell death remain to be determined.

Vascular Cell Apoptosis Cell Type–Specific Modulation by Transforming Growth Factor-β1 in Endothelial Cells Versus Smooth Muscle Cells

BACKGROUND It is postulated that vascular lesion formation and remodeling involves a balance between vascular cell death and cell proliferation. Transforming growth factor-beta1 (TGF-beta1) is a pleiotropic factor expressed within vascular cells that regulates cell growth in a tissue-specific manner. This study tested the hypothesis that the control of vascular cell apoptosis involves cell type-specific regulation by TGF-beta1. METHODS AND RESULTS In response to serum withdrawal, cultured endothelial cells and vascular smooth muscle cells exhibited apoptosis as evidenced by DNA laddering and quantitated by analysis of nuclear chromatin morphology. Addition of TGF-beta1 to endothelial cells in serum-free media further potentiated the induction of apoptosis in a dose-dependent fashion. Moreover, TGF-beta1 promoted endothelial cell death despite the presence of 10% serum. However, endothelial cells plated on collagen I were resistant to TGF-beta1-induced apoptosis. This antiapoptotic influence of the matrix was mimicked by integrin activation with anti-beta1 antibodies and associated with increased expression of the antiapoptotic factor bcl-2. In accord with the hypothesis that the modulation of antiapoptotic gene expression may mediate the effects of TGF-beta1 and beta1 integrins on cell fate, we observed that endothelial cells with constitutive upregulation of bcl-2 remained viable despite exposure to TGF-beta1 in serum-free conditions. In contrast to the proapoptotic effect of TGF-beta1 in endothelial cells, addition of TGF-beta1 to vascular smooth muscle cells in serum-free media inhibited apoptosis. CONCLUSIONS These findings suggest that the effect of cytokines such as TGF-beta1 on cell fate is contextual and is modulated by cell-matrix interactions in a cell type-specific manner.

Groups of p53 target genes involved in specific p53 downstream effects cluster into different classes of DNA binding sites.

The tumor suppressor protein p53, once activated, can cause either cell cycle arrest or apoptosis through transactivation of target genes with p53 DNA binding sites (DBS). To investigate the role of p53 DBS in the regulation of this profound, yet poorly understood decision of life versus death, we systematically studied all known and potential p53 DBS. We analysed the DBS separated from surrounding promoter regions in yeast and mammalian assays with and without DNA damage. p53 efficiently utilized the DBS of MDM2 and of genes connected to cell cycle arrest, DNA repair and the death receptor pathway of apoptosis. However, p53 was unable to utilize two-thirds of the isolated DBS, a subset that included almost all DBS of apoptosis-related genes. Neither ASPP2, a p53-interacting protein reported to specifically stimulate p53 transcriptional activity on apoptosis-related promoters, nor DNA damage resulted in p53 utilization of isolated DBS of apoptosis-related genes. Thus, a major regulation of p53 activity occurs at the level of p53 DBS themselves by posing additional requirements for the successful utilization of apoptosis-related DBS.

ENDOTHELIAL CELL APOPTOSIS IN CAPILLARY NETWORK REMODELING

We hypothesized that the regulation of apoptosis is an important determinant of capillary network structure. Using human umbilical vein endothelial cells (HUVEC) in in vitro model systems of capillary tube formation, we initially documented that apoptosis is a prominent feature of network formation. Perturbations of integrin‐matrix signaling by the administration of either colchicine or an anti‐αvβ3 antibody resulted in the dissolution of the tubular network in association with increased apoptosis. The activation of the αvβ3 integrin induced increased expression of the anti‐apoptotic gene bcl‐2 and conferred resistance to the proapoptotic effect of TGF‐β1. In contrast to the stable networks formed by HUVEC, bovine aortic endothelial cells (BAEC) exhibited a more dynamic process of network formation and spontaneous involution. The inhibition of BAEC apoptosis by stable transfection of bcl‐2 prevented the involution of the network. We hypothesized that TGF‐β1 present within the model system mediated network involution by inducing BAEC death. Indeed, blockade of TGF‐β1 with neutralizing antibodies reduced BAEC apoptosis and preserved the network structure. As observed with HUVEC networks, stable BAEC networks formed during blockade of TGF‐β1 were also dependent on the survival‐promoting effects of matrix‐integrin interactions. This study suggests that capillary network structure is determined by the balance of proapoptotic vs. anti‐apoptotic signals mediated by the engagement of cytokine and integrin receptors within the milieu. J. Cell. Physiol. 178:359–370, 1999.

Motexafin Gadolinium and Zinc Induce Oxidative Stress Responses and Apoptosis in B-Cell Lymphoma Lines

There is an emerging appreciation of the importance of zinc in regulating cancer cell growth and proliferation. Recently, we showed that the anticancer agent motexafin gadolinium (MGd) disrupted zinc metabolism in A549 lung cancer cells, leading, in the presence of exogenous zinc, to cell death. Here, we report the effect of MGd and exogenous zinc on intracellular levels of free zinc, oxidative stress, proliferation, and cell death in exponential phase human B-cell lymphoma and other hematologic cell lines. We find that increased levels of oxidative stress and intracellular free zinc precede and correlate with cell cycle arrest and apoptosis. To better understand the molecular basis of these cellular responses, gene expression profiling analyses were conducted on Ramos cell cultures treated with MGd and/or zinc acetate. Cultures treated with MGd or zinc acetate alone elicited transcriptional responses characterized by induction of metal response element-binding transcription factor-1 (MTF-1)-regulated and hypoxia-inducible transcription factor-1 (HIF-1)-regulated genes. Cultures cotreated with MGd and zinc acetate displayed further increases in the levels of MTF-1- and HIF-1-regulated transcripts as well as additional transcripts regulated by NF-E2-related transcription factor 2. These data provide insights into the molecular changes that accompany the disruption of intracellular zinc homeostasis and support a role for MGd in treatment of B-cell hematologic malignancies.

Interferon-inducible Myc/STAT-interacting protein Nmi associates with IFP 35 into a high molecular mass complex and inhibits proteasome-mediated degradation of IFP 35.

Nmi is an interferon (IFN)-inducible protein homologous to IFN-inducible protein IFP 35. The homology consists of a novel Nmi/IFP 35 domain (NID) of 90–92 amino acids that is repeated in tandem in each protein and mediates Nmi-Nmi protein interactions and subcellular localization. In a yeast two-hybrid screen with a fragment of Nmi protein containing both NIDs, we identified an interaction between Nmi and IFP 35. Deletion derivatives of the proteins indicate that both NIDs are required for the interaction between Nmi and IFP 35. In mammalian cells, Nmi and IFP 35 co-immunoprecipitate and co-localize in large cytoplasmic speckles. Nmi and IFP 35 proteins associate into a high molecular mass complex of 300–400 kDa as determined by native gel electrophoresis and gel filtration. The association of Nmi and IFP 35 into a complex can be demonstrated in multiple cell lines and is not dependent on treatment with IFN. Short term and long term cultures of transfected HEK293 cells suggest that Nmi and IFP 35 proteins stabilize each other through complex formation. IFP 35 appears to be more labile because Nmi was stable in the absence of IFP 35, whereas IFP 35 was degraded in the absence of Nmi. A deletion analysis revealed that Nmi must interact with IFP 35 to prevent its degradation and that the amino terminus of Nmi is required, but not sufficient, for this function. Inhibition of the proteasome, but not other proteases, led to increased levels of IFP 35. Thus, we have shown that Nmi and IFP 35 associate into a protein complex, that IFP 35 is degraded in a proteasome-mediated process, and that a novel function of Nmi is to prevent IFP 35 degradation. The stabilization of IFP 35 by Nmi may serve to amplify the physiologic effects of IFNs.

Apoptosis-stimulating protein of p53 (ASPP2) heterozygous mice are tumor-prone and have attenuated cellular damage–response thresholds

The expression of ASPP2 (53BP2L), a proapoptotic member of a family of p53-binding proteins, is frequently suppressed in many human cancers. Accumulating evidence suggests that ASPP2 inhibits tumor growth; however, the mechanisms by which ASPP2 suppresses tumor formation remain to be clarified. To study this, we targeted the ASPP2 allele in a mouse by replacing exons 10–17 with a neoR gene. ASPP2−/− mice were not viable because of an early embryonic lethal event. Although ASPP2+/− mice appeared developmentally normal, they displayed an increased incidence of a variety of spontaneous tumors as they aged. Moreover, γ-irradiated 6-week-old ASPP2+/− mice developed an increased incidence of high-grade T cell lymphomas of thymic origin compared with ASPP2+/+ mice. Primary thymocytes derived from ASPP2+/− mice exhibited an attenuated apoptotic response to γ-irradiation compared with ASPP2+/+ thymocytes. Additionally, ASPP2+/− primary mouse embryonic fibroblasts demonstrated a defective G0/G1 cell cycle checkpoint after γ-irradiation. Our results demonstrate that ASPP2 is a haploinsufficient tumor suppressor and, importantly, open new avenues for investigation into the mechanisms by which disruption of ASPP2 pathways could play a role in tumorigenesis and response to therapy.

Proapoptotic p53-Interacting Protein 53BP2 Is Induced by UV Irradiation but Suppressed by p53

ABSTRACT p53 is an important mediator of the cellular stress response with roles in cell cycle control, DNA repair, and apoptosis. 53BP2, a p53-interacting protein, enhances p53 transactivation, impedes cell cycle progression, and promotes apoptosis through unknown mechanisms. We now demonstrate that endogenous 53BP2 levels increase following UV irradiation induced DNA damage in a p53-independent manner. In contrast, we found that the presence of a wild-type (but not mutant) p53 gene suppressed 53BP2 steady-state levels in cell lines with defined p53 genotypes. Likewise, expression of a tetracycline-regulated wild-type p53 cDNA in p53-null fibroblasts caused a reduction in 53BP2 protein levels. However, 53BP2 levels were not reduced if the tetracycline-regulated p53 cDNA was expressed after UV damage in these cells. This suggests that UV damage activates cellular factors that can relieve the p53-mediated suppression of 53BP2 protein. To address the physiologic significance of 53BP2 induction, we utilized stable cell lines with a ponasterone A-regulated 53BP2 cDNA. Conditional expression of 53BP2 cDNA lowered the apoptotic threshold and decreased clonogenic survival following UV irradiation. Conversely, attenuation of endogenous 53BP2 induction with an antisense oligonucleotide resulted in enhanced clonogenic survival following UV irradiation. These results demonstrate that 53BP2 is a DNA damage-inducible protein that promotes DNA damage-induced apoptosis. Furthermore, 53BP2 expression is highly regulated and involves both p53-dependent and p53-independent mechanisms. Our data provide new insight into 53BP2 function and open new avenues for investigation into the cellular response to genotoxic stress.

Disrupted differentiation and oncogenic transformation of lymphoid progenitors in E2A-HLF transgenic mice.

ABSTRACT The hepatic leukemia factor (HLF) gene codes for a basic region-leucine zipper (bZIP) protein that is disrupted by chromosomal translocations in a subset of pediatric acute lymphoblastic leukemias. HLF undergoes fusions with the E2Agene, resulting in chimeric E2a-Hlf proteins containing the E2a transactivation domains and the Hlf bZIP DNA binding and dimerization motifs. To investigate the in vivo role of this chimeric bZIP protein in oncogenic transformation, its expression was directed to the lymphoid compartments of transgenic mice. Within the thymus, E2a-Hlf induced profound hypoplasia, premature involution, and progressive accumulation of a T-lineage precursor population arrested at an early stage of maturation. In the spleen, mature T cells were present but in reduced numbers, and they lacked expression of the transgene, suggesting further that E2a-Hlf expression was incompatible with T-cell differentiation. In contrast, mature splenic B cells expressed E2a-Hlf but at lower levels and without apparent adverse or beneficial effects on their survival. Approximately 60% of E2A-HLF mice developed lymphoid malignancies with a mean latency of 10 months. Tumors were monoclonal, consistent with a requirement for secondary genetic events, and displayed phenotypes of either mid-thymocytes or, rarely, B-cell progenitors. We conclude that E2a-Hlf disrupts the differentiation of T-lymphoid progenitors in vivo, leading to profound postnatal thymic depletion and rendering B- and T-cell progenitors susceptible to malignant transformation.