Biomedical subjects

Ute M Moll

Publications and source records attributed to Ute M Moll.

At least 19 recordsLinked to original sources

PubMed2006

Akt-mediated cisplatin resistance in ovarian cancer: modulation of p53 action on caspase-dependent mitochondrial death pathway.

Akt is a determinant of cisplatin [cis-diammine-dichloroplatinum (CDDP)] resistance in ovarian cancer cells, and this may be related to the regulation of p53. Precisely how Akt facilitates CDDP resistance and interacts with p53 is unclear. Apoptotic stimuli induce second mitochondria-derived activator of caspase (Smac) release from mitochondria into the cytosol, where it attenuates inhibitor of apoptosis protein-mediated caspase inhibition. Whereas Smac release is regulated by p53 via the transactivation of proapoptotic Bcl-2 family members, it is unclear whether p53 also facilitates Smac release via its direct mitochondrial activity. Here we show that CDDP induces mitochondrial p53 accumulation, the mitochondrial release of Smac, cytochrome c, and HTR/Omi, and apoptosis in chemosensitive but not in resistant ovarian cancer cells. Smac release was p53 dependent and was required for CDDP-induced apoptosis. Mitochondrial p53 directly induced Smac release. Akt attenuated mitochondrial p53 accumulation and Smac/cytochrome c/Omi release and conferred resistance. Inhibition of Akt facilitated Smac release and sensitized chemoresistant cells to CDDP in a p53-dependent manner. These results suggest that Akt confers resistance, in part, by modulating the direction action of p53 on the caspase-dependent mitochondrial death pathway. Understanding the precise etiology of chemoresistance may improve treatment for ovarian cancer.

Amino Acid Sequence↗

PubMed2006

WT p53, but not tumor-derived mutants, bind to Bcl2 via the DNA binding domain and induce mitochondrial permeabilization.

The induction of apoptosis by p53 in response to cellular stress is its most conserved function and crucial for p53 tumor suppression. We recently reported that p53 directly induces oligomerization of the BH1,2,3 effector protein Bak, leading to outer mitochondrial membrane permeabilization (OMMP) with release of apoptotic activator proteins. One important mechanism by which p53 achieves OMMP is by forming an inhibitory complex with the anti-apoptotic BclXL protein. In contrast, the p53 complex with the Bcl2 homolog has not been interrogated. Here we have undertaken a detailed characterization of the p53-Bcl2 interaction using structural, biophysical, and mutational analyses. We have identified the p53 DNA binding domain as the binding interface for Bcl2 using solution NMR. The affinity of the p53-Bcl2 complex was determined by surface plasmon resonance analysis (BIAcore) to have a dominant component KD 535 +/- 24 nm. Moreover, in contrast to wild type p53, endogenous missense mutants of p53 are unable to form complexes with endogenous Bcl2 in human cancer cells. Functionally, these mutants are all completely or strongly compromised in mediating OMMP, as measured by cytochrome c release from isolated mitochondria. These data implicate p53-Bcl2 complexes in contributing to the direct mitochondrial p53 pathway of apoptosis and further support the notion that the DNA binding domain of p53 is a dual function domain, mediating both its transactivation function and its direct mitochondrial apoptotic function.

Binding Sites↗

PubMed2006

Patterns of p73 N-terminal isoform expression and p53 status have prognostic value in gynecological cancers.

The goal of this study was to determine whether patterns of expression profiles of p73 isoforms and of p53 mutational status are useful combinatorial biomarkers for predicting outcome in a gynecological cancer cohort. This is the first such study using matched tumor/normal tissue pairs from each patient. The median follow-up was over two years. The expression of all 5 N-terminal isoforms (TAp73, DeltaNp73, DeltaN'p73, Ex2p73 and Ex2/3p73) was measured by real-time RT-PCR and p53 status was analyzed by immunohistochemistry. TAp73, DeltaNp73 and DeltaN'p73 were significantly upregulated in tumors. Surprisingly, their range of overexpression was age-dependent, with the highest differences delta (tumor-normal) in the youngest age group. Correction of this age effect was important in further survival correlations. We used all 6 variables (five p73 isoform levels plus p53 status) as input into a principal component analysis with Varimax rotation (VrPCA) to filter out noise from non-disease related individual variability of p73 levels. Rationally selected and individually weighted principal components from each patient were then used to train a support vector machine (SVM) algorithm to predict clinical outcome. This SVM algorithm was able to predict correct outcome in 30 of the 35 patients. We use here a mathematical tool for pattern recognition that has been commonly used in e.g. microarray data mining and apply it for the first time in a prognostic model. We find that PCA/SVM is able to test a clinical hypothesis with robust statistics and show that p73 expression profiles and p53 status are useful prognostic biomarkers that differentiate patients with good vs. poor prognosis with gynecological cancers.

Age Factors↗

PubMed2005

Clinical relevance of dominant-negative p73 isoforms for responsiveness to chemotherapy and survival in ovarian cancer: evidence for a crucial p53-p73 cross-talk in vivo.

PURPOSE: We aimed to determine the clinical role of the p53 family members p53 and p73 in the responsiveness to platinum-based chemotherapy and survival in ovarian cancer, considering their cross-talk and the p53 polymorphism at codon 72. EXPERIMENTAL DESIGN: A detailed analysis of p53 and p73 in a series of 122 ovarian cancers was done. We used a functional yeast-based assay to determine the p53 mutational status. Red yeast colonies, indicating mutant p53, were subsequently sequenced to determine the specific p53 alteration. p53 mutations were divided into two groups according to their previous characterization in the literature: those that efficiently inhibit transcriptionally active TAp73 function and those that do not. A p53 polymorphism at codon 72 was determined in corresponding normal tissue or blood of ovarian cancer patients. Isoform-specific p73 expression analysis using real-time reverse transcription-PCR has previously been done in the majority of ovarian cancers included in this study. In a retrospective chart review, responsiveness to chemotherapy was assessed, and survival data with long follow-up times were collected. RESULTS: Eighty of 122 (65.6%) of ovarian cancers harbored p53 mutations. p53 mutational status was an important determinant of responsiveness to platinum-based chemotherapy in all patients with a residual tumor of <2 cm in diameter after initial surgery (wild-type versus mutant, P = 0.029). In addition, p53 mutational status was a strong prognosticator for recurrence-free and overall survival (P < 0.0001 and P = 0.003, respectively) in univariate analyses. High expression levels of dominant-negative p73 isoforms (DeltaNp73 and DeltaN'p73) significantly correlated with chemotherapeutic failure (P = 0.048) and with worse recurrence-free and overall survival in patients with p53 mutant cancers (P = 0.048 and P = 0.005, respectively). Eight p53 mutations, present in 19 cases, were found that efficiently inhibit TAp73 (i.e., 175H, 220C, 245S, 245D, 248W, 248Q, 266E, and 273H). Patients with p53 mutations that efficiently inhibit TAp73 function had a significantly shorter overall survival than patients with p53 mutations of unknown effect on TAp73 (P = 0.044). The p53 polymorphism at codon 72 had no influence on responsiveness to chemotherapy or survival. CONCLUSION: We provide the first clinical evidence that dominant-negative p73 isoforms contribute to drug resistance in vivo, underscoring the importance of a p53-p73 cross-talk. NH2-terminally truncated p73 isoforms were of significant clinical effect by providing an additional unfavorable factor for response to platinum-based chemotherapy and survival in p53 mutant ovarian cancers.

Adenocarcinoma, Mucinous↗

PubMed2005

Mitochondrially targeted p53 has tumor suppressor activities in vivo.

Complex proapoptotic functions are essential for the tumor suppressor activity of p53. We recently described a novel transcription-independent mechanism that involves a rapid proapoptotic action of p53 at the mitochondria and executes the shortest known circuitry of p53 death signaling. Here, we examine if this p53-dependent mitochondrial program could be exploited for tumor suppression in vivo. To test this, we engage Emu-Myc transgenic mice, a well-established model of p53-dependent lymphomagenesis. We show that exclusive delivery of p53 to the outer mitochondrial membrane confers a significant growth disadvantage on Emu-Myc-transformed B-cells of p53-deficient or alternate reading frame-deficient genotypes, resulting in efficient induction of apoptosis and impinged proliferation. Conversely, normal cells from thymus, spleen, and bone marrow showed poor infectivity with these viruses. This proof-of-principle experiment shows that exclusive reliance on the direct mitochondrial program exerts a significant tumor suppressor activity in vivo. Our in vivo data on the direct mitochondrial apoptotic p53 program lays the groundwork to further investigate its efficacy and safety and to address its possible therapeutic value in the future.

Amino Acid Sequence↗

PubMed2005

Transcription-independent pro-apoptotic functions of p53.

Induction of apoptosis is one of the central activities by which p53 exerts its tumor-suppressing function. Aside from its primary function as a transcription factor, it can promote apoptosis independent of transcription. Recent studies have started to define the mechanisms of non-transcriptional pro-apoptotic p53 activities operating within the intrinsic mitochondria-mediated pathway of apoptosis. So far, two different mechanisms have been described, each of which was assigned to a specific localization of the p53 protein, either in the cytosol or directly at the mitochondria. Although mechanistically different, both transcription-independent modes of apoptosis induction converge, as they both initiate permeabilization of the outer mitochondrial membrane via activation of the pro-apoptotic Bcl-2 family members Bax or Bak.

Apoptosis↗

PubMed2005

Viral and cellular oncogenes induce rapid mitochondrial translocation of p53 in primary epithelial and endothelial cells early in apoptosis.

In p53-dependent apoptosis in response to genotoxic and hypoxic stress, a fraction of induced wild-type p53 rapidly translocates to mitochondria, triggering a rapid first wave of mitochondrial membrane permeabilization and apoptosis that is later fortified by the transcriptional program of p53. However, whether this direct mitochondrial program also occurs upon oncogenic stress is unknown. In normal cells, oncogenic signals can induce a p53-dependent fail-safe mechanism to counter uncontrolled proliferation by engaging p53-dependent apoptosis. To address whether mitochondrial p53 contributes to oncogene-induced fail-safe apoptosis, p53 translocation was determined in primary human epithelial and endothelial cells overexpressing c-Myc, E1A or E2F1. Serum starvation of these cells, but not of control cells, triggered rapid p53 accumulation at mitochondria, accompanied by cytochrome c and SMAC release and followed by apoptosis. Our data establishes the contribution of the transcription-independent mitochondrial p53 pathway to apoptosis of primary cells in response to deregulated oncogenes.

Adenovirus E1A Proteins↗

PubMed2005

Stress-induced p53 runs a transcription-independent death program.

The transcription-independent p53-mediated apoptotic response has obtained a solid mechanistic basis in recent years. A fraction of stress-induced wild type p53 protein rapidly translocates to mitochondria in response to genotoxic, hypoxic, and oxidative stresses in established cell lines and primary cells, as well as in physiological and pathophysiologic stress responses in the animal. While the groundwork of mechanisms and kinetics of direct mitochondrial p53 activities is laid out, the quantitative contribution of this pathway to total p53-mediated apoptosis and tumor suppression in vivo remains to be elucidated. An update on these efforts is given here.

Animals↗

PubMed2005

Macrophage migration inhibitory factor MIF interferes with the Rb-E2F pathway.

Macrophage migration inhibitory factor (MIF) is implicated in the regulation of inflammation and cell growth. We previously showed that MIF is a potent modulator of p53- and E2F-dependent pathways that are activated in response to oncogenic signaling. Here, we characterize the functional link between MIF and E2F transcription factors. Our results demonstrate that MIF-deficient cells exhibit E2F-dependent growth alterations and reduced susceptibility to oncogenic transformation. The basis for this transformation resistance is a perturbed function of the C-terminal Rb binding region of E2F4. However, inactivation of Rb or substitution of the E2F4 C-terminal domain by the E2F1 C-terminal region rescues the transformation defect. Importantly, the involvement of E2F factors in DNA replication rather than in regulation of transcription determines their oncogenic properties in the context of MIF deficiency. A proinflammatory molecule interfering with tumor suppression and DNA replication provides a compelling molecular link for the association of chronic inflammation and tumorigenesis.

Animals↗

PubMed2004

Stress-induced p53 runs a direct mitochondrial death program: its role in physiologic and pathophysiologic stress responses in vivo.

It is now well established that a fraction of stress-induced wtp53 protein rapidly translocates to mitochondria in immortalized and transformed cells in culture. Mitochondrial p53 interacts with anti-apoptotic proteins of the Bcl 2 family at the outer mitochondrial membrane, resulting in membrane permeabilization, release of death effectors such as cytochrome C and subsequent rapid apoptosis. The significance and relevance of this direct mitochondrial p53 program to the overall p53-mediated stress response in vivo is underlined by a number of recent studies in animals and primary cells. They all support a role for this direct pathway in the physiologic and pathophysiologic response to genotoxic and hypoxic insults and occur precisely in those tissues where p53 plays a critical role in mediating apotpotis rather than cell cycle arrest.

Animals↗

PubMed2004

Rational design of DNA sequences for nanotechnology, microarrays and molecular computers using Eulerian graphs.

Nucleic acids are molecules of choice for both established and emerging nanoscale technologies. These technologies benefit from large functional densities of 'DNA processing elements' that can be readily manufactured. To achieve the desired functionality, polynucleotide sequences are currently designed by a process that involves tedious and laborious filtering of potential candidates against a series of requirements and parameters. Here, we present a complete novel methodology for the rapid rational design of large sets of DNA sequences. This method allows for the direct implementation of very complex and detailed requirements for the generated sequences, thus avoiding 'brute force' filtering. At the same time, these sequences have narrow distributions of melting temperatures. The molecular part of the design process can be done without computer assistance, using an efficient 'human engineering' approach by drawing a single blueprint graph that represents all generated sequences. Moreover, the method eliminates the necessity for extensive thermodynamic calculations. Melting temperature can be calculated only once (or not at all). In addition, the isostability of the sequences is independent of the selection of a particular set of thermodynamic parameters. Applications are presented for DNA sequence designs for microarrays, universal microarray zip sequences and electron transfer experiments.

Algorithms↗

PubMed2004

Transdominant DeltaTAp73 isoforms are frequently up-regulated in ovarian cancer. Evidence for their role as epigenetic p53 inhibitors in vivo.

Despite strong homology, the roles of TP53 and TP73 in tumorigenesis seem to be fundamentally different. In contrast to TP53, tumor-associated overexpression of TP73 in many different cancers, combined with virtual absence of inactivating mutations and lack of a cancer phenotype in the TP73 null mouse are inconsistent with a suppressor function but instead support an oncogenic function. The discovery of NH(2)-terminally truncated p73 isoforms, collectively called DeltaTAp73, is now the focus of intense interest because they act as potent transdominant inihibitors of wild-type p53 and transactivation-competent TAp73. Therefore, establishing deregulated DeltaTAp73 expression in tumors could be the crucial link to decipher which of the two opposing roles of this bipolar gene is the biologically relevant one. This study is the largest to date and encompasses 100 ovarian carcinomas with complete expression profile of all NH(2)-terminal isoforms, discriminating between TAp73 and DeltaTAp73 (DeltaNp73, DeltaN'p73, Ex2p73, and Ex2/3p73) by isoform-specific real-time reverse transcription-PCR. We find that the set of NH(2)-terminal p73 isoforms distinguishes ovarian cancer patients from healthy controls and thus is a molecular marker for this diagnosis. Ovarian cancers strongly and almost universally overexpress DeltaN'p73 compared with normal tissues (95% of cancers). About one-third of tumors also exhibit concomitant up-regulation of the antagonistic TAp73, whereas only a small subgroup of tumors overexpress DeltaNp73. Thus, deregulation of the E2F1-responsive P1 promoter, rather than the alternate P2 promoter, is mainly responsible for the production of transdominant p53/TAp73 antagonists in ovarian cancer. Tumor stage, grade, presence of metastases, p53 status, and residual disease after resection are significant prognostic markers for overall and recurrence-free survival. A trend is found for better overall survival in patients with low expression of DeltaN'p73/DeltaNp73, compared with patients with high expression. A strong correlation between deregulated DeltaTAp73 and p53 status exists. p53 wild-type cancers exhibit significantly higher deregulation of DeltaN'p73, DeltaNp73, and Ex2/3p73 than p53 mutant cancers. This data strongly supports the hypothesis that overexpression of transdominant p73 isoforms can function as epigenetic inhibitors of p53 in vivo, thereby alleviating selection pressure for p53 mutations in tumors.

Adult↗

PubMed2004

Efficient RNA interference depends on global context of the target sequence: quantitative analysis of silencing efficiency using Eulerian graph representation of siRNA.

Several aspects of gene silencing by small interfering RNA duplexes (siRNA) influence the efficiency of the silencing. They can be divided into two categories, one covering the cell-specific factors and the other covering molecular factors of the RNA interference (RNAi). A prerequisite for sequence-based siRNA design is that hybridization thermodynamics is the dominant factor. Our assumption is that cell-specific parameters (cell line, degradation, cross-hybridization, target conformation, etc.) can be pooled into an average cellular factor. Our hypothesis is that the molecular basis of the positional dependence of siRNA-induced gene silencing is the uniqueness of context of a corresponding target sequence segment relative to all other such segments along the attacked RNA. We encode this context into descriptors derived from Eulerian graph representation of siRNAs and show that the descriptor based upon the contextual similarity and predicted thermodynamic stability correlates with the experimentally observed silencing efficiency of human lamin A/C gene. We further show that information encoded in this regression function is generalizable and can be used as a predictor of siRNA efficiency in unrelated genes (CD54 and PTEN). In summary, our method represents an evolution of siRNA design from the currently used algorithms which are only qualitative in nature.

Base Sequence↗

PubMed2004

In vivo mitochondrial p53 translocation triggers a rapid first wave of cell death in response to DNA damage that can precede p53 target gene activation.

p53 promotes apoptosis in response to death stimuli by transactivation of target genes and by transcription-independent mechanisms. We recently showed that wild-type p53 rapidly translocates to mitochondria in response to multiple death stimuli in cultured cells. Mitochondrial p53 physically interacts with antiapoptotic Bcl proteins, induces Bak oligomerization, permeabilizes mitochondrial membranes, and rapidly induces cytochrome c release. Here we characterize the mitochondrial p53 response in vivo. Mice were subjected to gamma irradiation or intravenous etoposide administration, followed by cell fractionation and immunofluorescence studies of various organs. Mitochondrial p53 accumulation occurred in radiosensitive organs like thymus, spleen, testis, and brain but not in liver and kidney. Of note, mitochondrial p53 translocation was rapid (detectable at 30 min in thymus and spleen) and triggered an early wave of marked caspase 3 activation and apoptosis. This caspase 3-mediated apoptosis was entirely p53 dependent, as shown by p53 null mice, and preceded p53 target gene activation. The transcriptional p53 program had a longer lag phase than the rapid mitochondrial p53 program. In thymus, the earliest apoptotic target gene products PUMA, Noxa, and Bax appeared at 2, 4, and 8 h, respectively, while Bid, Killer/DR5, and p53DinP1 remained uninduced even after 20 h. Target gene induction then led to further increase in active caspase 3. Similar biphasic kinetics was seen in cultured human cells. Our results suggest that in sensitive organs mitochondrial p53 accumulation in vivo occurs soon after a death stimulus, triggering a rapid first wave of apoptosis that is transcription independent and may precede a second slower wave that is transcription dependent.

Animals↗

PubMed2004

p63 and p73: roles in development and tumor formation.

The tumor suppressor p53 is critically important in the cellular damage response and is the founding member of a family of proteins. All three genes regulate cell cycle and apoptosis after DNA damage. However, despite a remarkable structural and partly functional similarity among p53, p63, and p73, mouse knockout studies revealed an unexpected functional diversity among them. p63 and p73 knockouts exhibit severe developmental abnormalities but no increased cancer susceptibility, whereas this picture is reversed for p53 knockouts. Neither p63 nor p73 is the target of inactivating mutations in human cancers. Genomic organization is more complex in p63 and p73, largely the result of an alternative internal promoter generating NH2-terminally deleted dominant-negative proteins that engage in inhibitory circuits within the family. Deregulated dominant-negative p73 isoforms might play an active oncogenic role in some human cancers. Moreover, COOH-terminal extensions specific for p63 and p73 enable further unique protein-protein interactions with regulatory pathways involved in development, differentiation, proliferation, and damage response. Thus, p53 family proteins take on functions within a wide biological spectrum stretching from development (p63 and p73), DNA damage response via apoptosis and cell cycle arrest (p53, TAp63, and TAp73), chemosensitivity of tumors (p53 and TAp73), and immortalization and oncogenesis (DeltaNp73).

Amino Acid Sequence↗

PubMed2003

The Role of p63 and p73 in tumor formation and progression: coming of age toward clinical usefulness. Commentary re: F. Koga et al., Impaired p63 expression associates with poor prognosis and uroplakin III expression in invasive urothelial carcinoma of the bladder. Clin. Cancer Res., 9: 5501-5507, 2003, and P. Puig et al., p73 Expression in human normal and tumor tissues: loss of p73alpha expression is associated with tumor progression in bladder Cancer. Clin. Cancer Res., 9: 5642-5651, 2003.

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Animals↗

PubMed2003

p53 has a direct apoptogenic role at the mitochondria.

p53 induces apoptosis by target gene regulation and transcription-independent signaling. However, a mechanism for the latter was unknown. We recently reported that a fraction of induced p53 translocates to the mitochondria of apoptosing tumor cells. Targeting p53 to mitochondria is sufficient to launch apoptosis. Here, we provide evidence that p53 translocation to the mitochondria occurs in vivo in irradiated thymocytes. Further, we show that the p53 protein can directly induce permeabilization of the outer mitochondrial membrane by forming complexes with the protective BclXL and Bcl2 proteins, resulting in cytochrome c release. p53 binds to BclXL via its DNA binding domain. We probe the significance of mitochondrial p53 and show that tumor-derived transactivation-deficient mutants of p53 concomitantly lose the ability to interact with BclXL and promote cytochrome c release. This opens the possibility that mutations might represent "double-hits" by abrogating the transcriptional and mitochondrial apoptotic activity of p53.

Animals↗

PubMed2003

Nuclear and cytoplasmic degradation of endogenous p53 and HDM2 occurs during down-regulation of the p53 response after multiple types of DNA damage.

The principal regulator of p53 stability is HDM2, an E3 ligase mediating p53 degradation via the ubiquitin-26S proteasome pathway. Until recently, the accepted model held that p53 degradation occurs exclusively on cytoplasmic proteasomes, with an absolute requirement for nuclear export of p53 via the CRM1 pathway. However, 26S proteasomes are abundant in cytosol and nucleus. Using forced overexpression of HDM2 in mutant p53 tumor cells, we previously found that p53 degradation occurs in both the nucleus and the cytoplasm. p53 null cells coexpressing export-defective p53 and HDM2 retained partial competence for p53 degradation, challenging the obligatory export model. Because the ability of local nuclear destruction might add important control in switching off the p53 pathway, we now test this notion for physiological situations in untransfected cells and determine the significance of this regulation. Despite nuclear export blockade by leptomycin B and HTLV1-Rex protein, two potent CRM1 inhibitors, nuclear degradation of endogenous wild-type p53 and HDM2 occurs during down-regulation of the p53 response. This was seen in RKO and U2OS cells recovering from all major forms of DNA damage, including UV, gamma-IR, camptothecin, or cisplatinum. Moreover, significant nuclear degradation of endogenous p53 and HDM2 occurs in isolated nuclear fractions prepared from these recovering cells. Furthermore, nuclear proteasomes efficiently degrade ubiquitinated p53 in vitro. Our data indicate that in nonlethal outcomes of cellular stress, when DNA damage has been successfully repaired and the active p53 response needs to be down-regulated quickly to resume normal homeostasis, both nuclear and cytoplasmic proteasomes are recruited to efficiently degrade the elevated p53 and HDM2 protein levels. The physiological significance of local nuclear destruction lies in the fact that it adds tighter control and speed to switching the p53 pathway off.

Cell Nucleus↗