Biomedical subjects

Ute M Moll

Publications and source records attributed to Ute M Moll.

26 records · Page 2Linked to original sources

PubMed2003

deltaNp73 facilitates cell immortalization and cooperates with oncogenic Ras in cellular transformation in vivo.

TP73, despite significant homology to TP53, is not a classic tumor suppressor gene, since it exhibits upregulation of nonmutated products in human tumors and lacks a tumor phenotype in p73-deficient mice. We recently reported that an N-terminally truncated isoform, DeltaNp73, is upregulated in breast and gynecological cancers. We further showed that DeltaNp73 is a potent transdominant inhibitor of wild-type p53 and TAp73 in cultured human tumor cells by efficiently counteracting their target gene transactivations, apoptosis, and growth suppression functions (A. I. Zaika et al., J. Exp. Med. 6:765-780, 2002). Although these data strongly suggest oncogenic properties of DeltaNp73, this can only be directly shown in primary cells. We report here that DeltaNp73 confers resistance to spontaneous replicative senescence of primary mouse embryo fibroblasts (MEFs) and immortalizes MEFs at a 1,000-fold-higher frequency than occurs spontaneously. DeltaNp73 cooperates with cMyc and E1A in promoting primary cell proliferation and colony formation and compromises p53-dependent MEF apoptosis. Importantly, DeltaNp73 rescues Ras-induced senescence. Moreover, DeltaNp73 cooperates with oncogenic Ras in transforming primary fibroblasts in vitro and in inducing MEF-derived fibrosarcomas in vivo in nude mice. Wild-type p53 is likely a major target of DeltaNp73 inhibition in primary fibroblasts since deletion of p53 or its requisite upstream activator ARF abrogates the growth-promoting effect of DeltaNp73. Taken together, DeltaNp73 behaves as an oncogene that targets p53 that might explain why DeltaNp73 upregulation may be selected for during tumorigenesis of human cancers.

Animals↗

PubMed2003

Detection of mitochondrial localization of p53.

p53 is a master regulator of cell death pathways and has transcription-dependent and transcription- independent modes of action. Mitochondria are major signal transducers in apoptosis and are critical for p53-dependent cell death. Recently, we discovered that a fraction of stress-induced wild-type p53 protein rapidly translocates to mitochondria during p53-dependent apoptosis. Suborganellar localization by various methods shows that p53 predominantly localizes to the surface of mitochondria. Moreover, bypassing the nucleus by targeting p53 to mitochondria is sufficient to induce apoptosis in p53-null cells, without requiring further DNA damage. Here, we describe subcellular fractionation as a classic technique for detecting mitochondrial p53 in cell extracts. It consists of cell homogenization by hypo-osmotic swelling, removal of nuclear components by low-speed centrifugation, and mitochondrial isolation by a discontinuous sucrose density gradient. p53 and other mitochondrial proteins can then be detected by standard immunoblotting procedures. The quality of mitochondrial isolates can be verified for purity and intactness.

Animals↗

PubMed2003

Analysis of nuclear and cytoplasmic degradation of p53 in cells after stress.

Until recently, the accepted model held that p53 degradation occurs exclusively on cytoplasmic proteasomes and, hence, has an absolute requirement for nuclear export of p53 via the CRM1 pathway. However, proteasomes are abundant in both cytosol and nucleus. We recently analyzed HDM2-mediated degradation of endogenous p53 in the presence of various CRM1 blockers. We found that significant HDM2-mediated degradation takes place despite nuclear export blockade, indicating that endogenous p53 degradation occurs locally in the nucleus, in parallel to cytoplasmic degradation. Here, we describe how subcellular fractionation can be used to monitor nuclear and cytoplasmic degradation of endogenous wild-type p53 during the recovery phase after a stress stimulus. The fractions are then analyzed by immunoblotting in a time-dependent fashion. Vimentin and lamin A proteins are used to monitor the purity of the cytosolic and nuclear fractions, respectively, and to control for equal loading.

Animals↗

PubMed2003

Mutational analysis of p53 in human tumors: direct DNA sequencing and SSCP.

This chapter describes the techniques necessary to identify p53 genomic mutations in either frozen or paraffin-embedded tumor samples. DNA is extracted from the tumor samples and then used as a template to amplify the p53 coding sequences by polymerase chain reaction (PCR). The PCR products are characterized by agarose gel electrophoresis. Subsequently, mutations can be detected by direct DNA sequencing of the PCR products, followed by acrylamide gel electrophoresis and autoradiography. Alternatively, PCR products containing mutations can be identified by their aberrant mobility on denaturing acrylamide gels, using single-stranded conformation polymorphism (SSCP).

DNA Mutational Analysis↗

PubMed2003

Mutational analysis of p53 in human tumors: immunocytochemistry.

Mutations of the p53 gene are the most common genetic changes in human malignancies; therefore their detection is of practical importance. In contrast to wild-type p53 in resting normal cells, mutant p53 proteins are easily detectable by immunocytochemical methods due to their abnormally extended half-life. Several methods of immunocytochemistry can be used to analyze the presence and localization of p53 protein in cells or tissues. The most important is immunocytochemical p53 staining of sections from paraffin embedded tissues. This method is used as a relatively reliable surrogate marker for p53 mutations and has the advantage of being easy, fast, and suitable for mass screening of large archival tissue banks. Frozen sections can also be used to detect and localize the p53 proteins. p53 can also be detected in tissue culture cells. p53 can be detected in situ through a secondary antibody coupled to a fluorescent dye or an enzymatic activity that reacts with certain chromogens.

DNA Mutational Analysis↗

PubMed2003

The MDM2-p53 interaction.

Activation of the p53 protein protects the organism against the propagation of cells that carry damaged DNA with potentially oncogenic mutations. MDM2, a p53-specific E3 ubiquitin ligase, is the principal cellular antagonist of p53, acting to limit the p53 growth-suppressive function in unstressed cells. In unstressed cells, MDM2 constantly monoubiquitinates p53 and thus is the critical step in mediating its degradation by nuclear and cytoplasmic proteasomes. The interaction between p53 and MDM2 is conformation-based and is tightly regulated on multiple levels. Disruption of the p53-MDM2 complex by multiple routes is the pivotal event for p53 activation, leading to p53 induction and its biological response. Because the p53-MDM2 interaction is structurally and biologically well understood, the design of small lipophilic molecules that disrupt or prevent it has become an important target for cancer therapy.

Animals↗

PubMed2002

DeltaNp73, a dominant-negative inhibitor of wild-type p53 and TAp73, is up-regulated in human tumors.

p73 has significant homology to p53. However, tumor-associated up-regulation of p73 and genetic data from human tumors and p73-deficient mice exclude a classical Knudson-type tumor suppressor role. We report that the human TP73 gene generates an NH(2) terminally truncated isoform. DeltaNp73 derives from an alternative promoter in intron 3 and lacks the transactivation domain of full-length TAp73. DeltaNp73 is frequently overexpressed in a variety of human cancers, but not in normal tissues. DeltaNp73 acts as a potent transdominant inhibitor of wild-type p53 and transactivation-competent TAp73. DeltaNp73 efficiently counteracts transactivation function, apoptosis, and growth suppression mediated by wild-type p53 and TAp73, and confers drug resistance to wild-type p53 harboring tumor cells. Conversely, down-regulation of endogenous DeltaNp73 levels by antisense methods alleviates its suppressive action and enhances p53- and TAp73-mediated apoptosis. DeltaNp73 is complexed with wild-type p53, as demonstrated by coimmunoprecipitation from cultured cells and primary tumors. Thus, DeltaNp73 mediates a novel inactivation mechanism of p53 and TAp73 via a dominant-negative family network. Deregulated expression of DeltaNp73 can bestow oncogenic activity upon the TP73 gene by functionally inactivating the suppressor action of p53 and TAp73. This trait might be selected for in human cancers.

Animals↗

PubMed2002

Nuclear degradation of p53 occurs during down-regulation of the p53 response after DNA damage.

The principal regulator of p53 stability is HDM2, an E3 ligase that mediates p53 degradation via the ubiquitin-26S proteasome pathway. The current model holds that p53 degradation occurs exclusively on cytoplasmic proteasomes and hence has an absolute requirement for nuclear export of p53 via the CRM-1 pathway. However, proteasomes are abundant in both cytosol and nucleus, and no studies have been done to determine under what physiological circumstances p53 degradation might occur in the nucleus. We analyzed HDM2-mediated degradation of endogenous p53 in the presence of various nuclear export inhibitors of CRM-1, including leptomycin B (LMB), a noncompetitive, specific, and fast-acting inhibitor; and HTLV1-Rex protein, a potent competitive inhibitor. We found that significant HDM2-mediated p53 degradation took place in the presence of LMB or HTLV1-Rex, indicating that endogenous p53 degradation occurs locally in the nucleus, in parallel to cytoplasmic degradation. Moreover, p53 null cells that coexpressed export-defective mutants of p53 and HDM2 retained partial competence for p53 degradation. It is important that nuclear degradation of p53 occurred during the poststress recovery phase of a p53 response, after DNA damage ceased. We propose that the capability of local p53 degradation within the nucleus provides a tighter and faster control during the down-regulatory phase, when an active p53 program needs to be turned off quickly.

Active Transport, Cell Nucleus↗