Biomedical subjects

Petr Pancoska

Publications and source records attributed to Petr Pancoska.

6 recordsLinked to original sources

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↗

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

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↗

PubMed2003

DNA sequence context and multiplex hybridization reactions: melting studies of heteromorphic duplex DNA complexes.

Heteromorphic hybrid duplex DNA complexes are duplex states, other than perfectly matched duplexes, that can form when single strands comprising several different perfectly matched duplexes are simultaneously present in solution. Such cross-hybridization "side reactions" are of particular nuisance in multiplex reaction schemes, where many strands are designed to hybridize in parallel fashion with only their corresponding perfect complement strand. Relative to the perfect match duplexes, the sequence dependent features of these heteromorphic duplex states and their thermodynamic stability are an important consideration for multiplex hybridization reaction design. We have measured absorbance versus temperature melting curves and performed differential scanning calorimetry measurements on various mixtures of eight different 24 base single strands. When perfect complementary pairs of strands are mixed in single reactions, four perfectly matched duplexes form. When mixtures of strands that are not perfectly matched are prepared and analyzed, melting transitions for cross-hybridization are observed along with significant hyperchromicity changes. This is indicative of a melting hybrid, heteromorphic duplex states formed from two nonperfectly matched strands. In addition, when both the perfectly matched and noncomplementary strands are mixed together (in multiplex hybridization reactions) at molar ratios of 1:1, 3:1, and 1:3, evidence of perfect duplex and heteromorphic duplex complexes is found in all cases. A new analytical tool for considering heterogeneous, duplex complexes in multiplex hybridization mixtures is presented and employed to interpret the acquired melting data.

Base Pair Mismatch↗

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↗