Meinhard Hahn

Increased expression of disintegrin-metalloproteinases ADAM-15 and ADAM-9 following upregulation of integrins α5β1 and αvβ3 in atherosclerosis

Regulation of αvβ3 and α5β1 integrin function plays a crucial role in atherosclerosis. Possible regulators of integrin–matrix interactions are integrin‐binding ADAMs (proteins with a disintegrin‐ and metalloproteinase‐domain), like ADAM‐15 and ADAM‐9. Molecular interactions between ADAM‐15, α5β1, and αvβ3 have been demonstrated. ADAM‐9 and ADAM‐15 were found to be interdependently regulated. This study, therefore, investigated whether the upregulation of integrins α5β1 and αvβ3 was correlated with the expression of integrin‐binding ADAMs in atherosclerotic processes. Human arterial and venous vascular smooth muscle cells (VSMCs) were incubated with PDGF over different time intervals up to a 3‐day culture period. mRNA concentrations, quantified by real‐time RT‐PCR and normalized to PBGD, of integrins αvβ3 and α5β1 were strongly increased after a 12‐h PDGF‐incubation in arterial and venous VSMC. ADAM‐15 and ADAM‐9 mRNA production showed a corresponding increase following integrin upregulation after a 24‐h incubation period. Western blot anaylsis revealed an increased protein expression of integrins and ADAMs in PDGF‐stimulated VSMC. Additionally, mRNA concentrations of atherosclerotic and normal human specimens were quantified by real‐time RT‐PCR. mRNA of ADAMs and integrins was significantly increased in atherosclerotic arteries compared to normal arteries. Immunohistochemistry of these specimens showed an increased expression and codistribution of both ADAMs and integrins in atherosclerosis. In conclusion, upregulation of ADAM‐15 and ADAM‐9 in atherosclerosis appears to follow an increase in α5β1 and αvβ3 integrins. Since α5β1 and αvβ3 are known to promote smooth muscle cell migration and proliferation, upregulation of ADAM‐15 and ADAM‐9 could balance integrin–matrix interactions and cell migration, thus modulating neointima progression. J. Cell. Biochem. 89: 808–823, 2003.

Validation of an algorithm for automatic quantification of nucleic acid copy numbers by real-time polymerase chain reaction.

Real-time quantitative polymerase chain reaction (PCR) with on-line fluorescence detection has become an important technique not only for determination of the absolute or relative copy number of nucleic acids but also for mutation detection, which is usually done by measuring melting curves. Optimum assay conditions have been established for a variety of targets and experimental setups, but only limited attention has been directed to data evaluation and validation of the results. In this work, algorithms for the processing of real-time PCR data are evaluated for several target sequences (p53, IGF-1, PAI-1, Factor VIIc) and compared to the results obtained by standard procedures. The algorithms are implemented in software called SoFAR, which allows fully automatic analysis of real-time PCR data obtained with a Roche LightCycler instrument. The software yields results with considerably increased precision and accuracy of quantifications. This is achieved mainly by the correction of amplification-independent signal trends and a robust fit of the exponential phase of the signal curves. The melting curve data are corrected for signal changes not due to the melting process and are smoothed by fitting cubic splines. Therefore, sensitivity, resolution, and accuracy of melting curve analyses are improved.

Estimating the relapse stage in chronic myeloid leukaemia patients after allogeneic stem cell transplantation by the amount of BCR-ABL fusion transcripts detected using a new real-time polymerase chain reaction method

We have used a new single‐step real‐time reverse transcription polymerase chain reaction (RT‐PCR) method to quantify BCR‐ABL transcripts, thereby estimating the relapse stage in chronic myeloid leukaemia patients after allogeneic transplants. In 402 samples from 172 patients, BCR‐ABL expression was determined and normalized, using the GAPDH housekeeping gene product as an endogenous reference. In our real‐time RT‐PCR assay, serial dilutions of RNA of the K562 cell line remained positive down to 7·5u2003pg. The median normalized BCR‐ABL amount differed significantly (Pu2003<u20030·001) between the various disease stages and was 0·06% (range 0·001–1·55%), 3·2% (range 1·4–5·6%) and 21·5% (range 6·8 −827%) in 17 patients with a molecular relapse, in eight patients with a cytogenetic relapse and in 10 patients with a haematological relapse respectively.

Influence of fluorophor dye labels on the migration behavior of polymerase chain reaction--amplified short tandem repeats during denaturing capillary electrophoresis.

The determination of the length of polymerase chain reaction (PCR)‐amplified short tandem repeats (STRs) by denaturing capillary electrophoresis (CE) is a standard procedure for purposes of genotyping. We show that dye‐specific mobility anomalies exist for 5′‐fluorophor‐labelled single‐stranded DNA (ssDNA) fragments in CE using the performance‐optimized polymer 4 (POP4) buffer sieving matrix, containing the entangled poly(N,N‐dimethylacrylamide) polymer, urea, and 2‐pyrrolidinone. The dye‐specific retardation effects relative to coseparated Gene Scan‐500 [TAMRA] standard fragments can lead to wrong genotyping, even for allele‐specific fragments of pentanucleotide STRs, when comparing the relative calculated sizes of identical fragments, labelled with rhodamine (ROX, TAMRA) or fluorescein dyes (FAM, 6‐FAM, HEX, JOE, NED, TET): The size of fluorescein dye‐labelled fragments of appr. 100 b in length appears to be smaller by up to 6.5 b. This effect becomes more dramatic with decreasing size: a 6‐FAM‐labelled 24‐mer oligonucleotide appeared to be smaller by 11u2009b. In contrast, in classical urea/polyacrylamide slab‐gel electrophoresis only a small dye‐specific retardation of identical fragments is observed. The dye‐specific effects are superimposed by weaker size and sequence‐dependent anomalies of fragment mobility. Therefore, in denaturing CE the coseparation of a defined allele ladder labelled with the same dye as the unknown sample fragments remains the method of choice for accurate genotyping.

Polymorphism of the pentanucleotide repeat d(AAAAT) within intron 1 of the human tumor suppressor gene p53 (17p13.1).

DyeDeoxy terminator cycle sequencing of allele-specific polymerase chain reaction products has shown that there is a highly polymorphic d(AAAAT) pentanucleotide repeat within the first intron of the human p53 gene. This provides a genetic marker for tumor suppressor p53 gene alterations.

Quantification of Retrotransposon XIR-2.5 Copy Number in Genomes of Poeciliidae Species

Recently quantitative PCR techniques are standard methods used for the precise quantification of low copy DNA target sequences [1], e.g. single or few DNA target molecules (viruses, bacteria) in biological samples or single copy genes in large genomes. In contrast, the precise quantification of high copy number elements in DNA genomes, e.g. transposons or retrotransposons, by quantitative PCR is not well represented in literature. For these targets so far conventional quantification techniques were used which are either time consuming and need large amounts of high molecular genomic DNA (Southern blots) [2] or expensive technical equipment (fluorescence in situ hybridization) [3], result in data of limited precision (c0t-analysis) [4] or have only a low dynamic range (in situ hybridization of radioactively labeled probes to interphase or metaphase chromosomes) [5].

Detection of p53 Allele Deletions in Human Cancer by Quantification of Genomic Copy Number

In most cases cancer is a genetic disease, characterized by a multiple step process [1], which involves the inactivation of tumor suppressor genes (e.g., by deletion of alleles) and the activation of oncogenes (e.g., by amplification of alleles). In healthy cells these misregulated genes are important regulators of cellular growth and cellular division, DNA repair, and apoptosis. p53 is the most prominent and most important tumor suppressor gene which becomes inactivated in nearly all cancer types, roughly 50% of all human tumors being affected by p53 inactivation [2]. The deletion of an allele or the inactivation by point mutations are the most prominent mechanisms for loss of function of p53. Tumors containing inactivated p53 are highly aggressive and often resistant to chemo- as well as radiotherapy. As the functional status of p53 has many clinical and therapeutical implications [3, 4, 5], rapid and sensitive tests for the presence or absence of functional p53 genes are needed. Several techniques have been described for the detection of p53 allele deletions: (a) analysis of polymorphic DNA markers (RFLPs, minisatellites, microsatellites) for loss of heterozygosity [6, 7]; (b) Southern blotting techniques [2]; (c) fluorescence in situ hybridizations [5, 8]; (d) comparative genomic hybridization [8, 9]; and (e) quantitative competitive PCR [10] which all have several disadvantages, e.g., that they require large amounts of high molecular DNA (b); that they involve cell culture techniques and fluorescence microscopy (c, d); that they depend on the presence of heterozygous allelotypes of DNA markers, application of radioactivity, or fluorescent labels plus DNA sequencers (a); that they require internal control templates (e); and that they only detect allele imbalance but in most cases do not allow discrimination between deletions and amplifications (a).

Quantitation of P53 Tumor Suppressor Gene Copy Number in Tumor Dna Samples by Competitive PCR in an Elisa-Format

During the last two decades molecular tumor biology has demonstrated that most tumors are the result of multi-step mutation processes (e.g. colorectal and lung carcinoma1,2, renal tumors3 and tumors of the skin4): several mutations must accumulate in the cellular genome and modify the functions of oncogenes and/or inactivate tumor suppressor genes, which control essential cellular processes such as cell division cycle or apoptosis, before a tumor develops. The tumor suppressor gene p53 is the gene most often affected by mutations in a large number of diverse, frequently occuring human tumors5. As typical for tumor suppressor genes, one copy of the p53 gene is often inactivated in tumor cells by point mutations, while the second one is deleted. Several clinical studies showed that the inactivation of p53 in tumors is accompanied by poor prognosis for the patients and tumor resistance against various types of chemotherapy or radiation therapy6. From a clinical point, therefore, a need for efficient procedures for the routine detection of p53 mutations exists. At present, several techniques can be used to detect deletions or losses of p53 alleles, e.g. (i) loss of heterozygosity (LOH) analysis of intragenic microsatellite polymorphisms7,8, (ii) fluorescence in-situ hybridization (FISH)6 or (iii) comparative genomic hybridization3. These techniques possess several disadvantages: for example in (i) the analysis of a patient is only informative in the case of a heterozygous allelotype of the polymorphism, in (ii) and (iii) one needs special, expensive equipment and great experience.

Quantitative PCR with Internal Standardization and OLA-ELISA Product Analysis for the p53 Tumor Suppressor Gene

Over the last nine years, several quantitative polymerase chain reaction (QPCR) techniques have been developed, and these are now frequently used for the quantification of DNA template copy numbers. However, only few of these PCR techniques are suitable for the precise and absolute quantification of the template copy number (1,2). For this purpose, we describe here a quantitative PCR strategy that uses a known amount of an internal standard DNA that is amplified in competition with the sample template, using one common PCR primer pair and identical primer binding sites for both templates (2-4). In the literature, several variants of internal control sequences were used for the purpose of standardization, e.g., (i) homologous gene sequences of closely related species; differing in few bp, slightly in length and/or absence or presence of restriction sites (5,6), (ii) sample DNA derived sequences that are shortened by a deletion (7) or (iii) lengthened by an insertion (8); (iv) competitor fragments that contain more (9); or (v) less (10) extended heterologous sequence strings; or (vi) differ only by one or two bp, thereby replacing a sample specific restriction site by unique one specific to the internal control DNA (1,11,12). But only the last type of the internal control templates that differ in a negligible manner from the sample DNA sequenc is suitable for precise quantifications, as could be shown by theoretical considerations (2) as well as experimentally (1). Even in the case of a homologous internal control sequence of identical length as the sample sequence and differing by less than 5% in sequence, the two templates are not amplified with the same efficiency and therefore do not fulfill the criteria of ideal competition (2), as could be shown (6).