Stoyan Chakarov

DNA Repair and Cell Differentiation—Does Getting Older Means Getting Wiser as Well?

ABSTRACT The cell DNA needs to be constantly repaired in order to preserve the genome integrity and to ensure the fidelity of transcription. Its been known that a marked difference exists between rates of certain types of repair of transcribed genes and non-transcribed DNA. Recently theres been accumulating evidence that state of terminal differentiation of the cell reflects on the NER profile, attenuating ubiquitous repair in all genome regions and focusing on transcription-dependent repair. This may be viewed not as a differentiation-associated defect but, rather, as a compromise with the integrity of the genome in its bulk for the sake of exclusive repair of regions where active transcription is under way.

An Experimental Model for Assessment of Global DNA Repair Capacity

ABSTRACT Cellular DNA is constantly subjected to a significant amount of damage resulting from extracellular as well as intracellular processes. There are various mechanisms to repair DNA damage among which nucleotide excision repair (NER) pathway has the highest degree of versatility for recognizing and repairing potentially hazardous DNA modifications. It is difficult, however, to measure the rate of DNA repair as the synthesis of DNA related to repair constitutes only a small fraction of the overall rate of DNA synthesis in the cell. The current paper presents an experimental setup that allows measuring the rate of repair DNA synthesis based on suppression of the replicative DNA synthesis in order to differentiate the repair-associated synthetic activity. The proposed methodology allows for assessment of repair rate of the DNA damage of any type and in any DNA region undergoing repair irrelevant of the structure of the region in question. It could serve as a basis for development of diagnostic methods for assessment of the capacity for global DNA repair.

DO WE NEED MORE HUMAN EMBRYONIC STEM CELL LINES

ABSTRACT The enormous potential of human embryonic stem cells is fueling continuous research aimed at establishment of new lines of these cells. Currently, research groups from 24 countries have reported derivation of over 1000 human embryonic stem cell lines. Because of the controversy surrounding the derivation of these cells from human embryos it is important to clarify whether the existing hESC lines are sufficient for basic research and future therapeutic applications. Here we briefly review some of the most important arguments justifying the need for continuing derivation of human embryonic stem cell lines.

Children of the Sun, Children of the Moon—A Mini-Panel for Assessment of Inter-Individual Variation Between the Capacity of Healthy Individuals to Repair Everyday Genotoxic Insults

ABSTRACT The DNA repair machinery of healthy human cells usually manages the consequences of the daily barrage of DNA damage for years and decades before any adverse effects related to genotoxic impact become manifest. There is significant variance, however, even between healthy individuals, in regard to their ability to detect and repair genotoxic damage. Some aspects of this variance exist throughout the life of the individual (genetic factors, such as polymorphisms in genes coding for products acting in the repair of DNA damage), while others (e.g. telomere length) may be the outcome of the genotype-phenotype interplay, modified by environmental factors. Numerous markers for assessment of capacity to combat genotoxic damage have been described so far, with only some of them having a value of their own under physiological and/or pathological conditions. In the present study we provide the results from the evaluation of a mini-panel (p53 P72R, XPCins83PAT, rate of telomere attrition) for assessing the capacity of healthy individuals to repair genotoxic damage, and outline the possible fields of application.

p53—Guardian Angel and Archangel

ABSTRACT p53 is a master regulator of the cell cycle, capable of assessing the severity and the scope of damage to the cellular DNA, integrating the signals from the cell under stress and delivering the final decision about the destiny of the cell—undertaking repair activities; entering replicative senescence; inducing cell death; resorting to translesion transactions or altering the metabolism or the expression pattern of the cell. Proper functioning of p53 and its related pathways is essential in multicellular eukaryotes, with failures in the DNA-binding and transactivation properties of p53 usually resulting in cancer. Recent research on some common polymorphic variants of p53 that exhibit differential properties in their ability to induce cell cycle arrest or apoptosis indicate that p53 is not only the ‘guardian angel’ of the genome, as is commonly believed, keeping its integrity in check and disposing of damaged cells, but it is as well the ‘archangel’ that is responsible for cutting down the lifespan of the organism by the mechanism of ageing. One function can hardly exist without the other, and it is very individual as to whether carriership of one polymorphic form or another would be beneficial in the particular case, considering the general status of the patient and the specificities of the pathogenesis of the disease or condition in question. Age of the patient seems to be a major determining factor, as the differential pattern in the properties of p53 seems to become more pronounced as age advances.

Current state of the opportunities for derivation of germ-like cells from pluripotent stem cells: are you a man, or a mouse?

The concept of pluripotency as a prerogative of cells of early mammal embryos and cultured embryonic stem cells (ESC) has been invalidated with the advent of induced pluripotent stem cells. Later, it became clear that the ability to generate all cell types of the adult organism is also a questionable aspect of pluripotency, as there are cell types, such as germ cells, which are difficult to produce from pluripotent stem cells. Recently it has been proposed that there are at least two different states of pluripotency; namely, the naïve, or ground state, and the primed state, which may differ radically in terms of timeline of existence, signalling mechanisms, cell properties, capacity for differentiation into different cell types, etc. Germ-like male and female rodent cells have been successfully produced in vitro from ESC and induced pluripotent stem cells. The attempts to derive primate primordial germ cells (PGC) and germ cells in vitro from pluripotent stem cells, however, still have a low success rate, especially with the female germline. The paper reviews the properties of rodent and primate ESC with regard to their capacity for differentiation in vitro to germ-like cells, outlining the possible caveats to derivation of PGC and germ cells from primate and human pluripotent cells.

Investigation of the role of MMP3 -1171insA polymorphism in cutaneous malignant melanoma – a preliminary study

Coetaneous malignant melanoma is the most aggressive cancer of the skin with a high rate of mortality worldwide. Degradation of basement membranes and extracellular matrix is an essential step in cancer invasion and metastasis. Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) play key roles in this step. MMP-3 also called stromelysin-1 was one of the first proteinases found to be associated with cancer. In the gene of MMP-3 (MMP3), an insertion/deletion of an A nucleotide at position -1171 in promoter region has been identified and shown to effect the expression activity of the gene. The present study was conducted to investigate the relation of MMP3 -1171insA polymorphism with skin malignant melanoma risk in a pilot case-control study of Bulgarian patients (n = 26) and unaffected controls (n = 172). The genotypes of controls and melanoma patients were in Hardy-Weinberg equilibrium. The results showed no statistically significant difference both in genotype and allele frequencies of MMP3 -1171insA polymorphism between melanoma patients and healthy controls either in crude analyses (p = 0.360 and 0.790, c2-test) or after adjustment for age and sex. The comparison of some clinical characteristics between the patients with different genotypes showed a trend for longer survival of patients with 6A/6A genotype compared to the carriers of 5A allele (5A/5A+5A/6A genotypes, p = 0.118, Log rank test). The results of our current preliminary study do not provide evidence for the role of the promoter polymorphism -1171insA in MMP3 as a risk factor for development of coetaneous melanoma, but suggest its implication in progression of the diseases.

Measuring Telomere Length—From Ends to Means

ABSTRACT Telomeres are ribonucleoproteid complexes capping the ends of linear genomes and protecting them from attrition and fusion. The rate of telomere shortening is related to cellular aging and a loss of telomere length is observed in a significant proportion of human diseases and conditions. Telomerase activity may serve as a useful marker for capacity for self-renewal and potential for differentiation in stem cells. The present paper reviews the present-day methods for measuring telomere length in cell populations and/or assessment of telomerase activity—Southern hybridisation, flow cytometry/Flow FISH, real-time PCR and some less popular methods such as TRAP analysis and STELA, assessing the advantages and weaknesses of each method and outlining the possible fields of application.

Rapid and Efficient Method for Production of T4 Endonuclease V by Heterologous Expression in E.Coli

ABSTRACT T4 endonuclease V is specific to cyclobutane-pyrimidine dimers produced by UV-irradiation. When applied topically in mammals, it induces strand breaks in the altered DNA, thus triggering the cellular DNA repair system. Application of T4 endo V-containing preparations may ameliorate sun-induced damage in healthy persons as well as in patients defective in DNA repair capacity. We propose a rapid and efficient method for production of T4 endonuclease V without the need for phage inoculation of bacteria, thus allowing for easier and cost-effective manufacturing of T4endo V of the purity required by the GMP standards for medicinal preparations for use in humans.

Isolation and Properties of Recombinant Human High Mobility Group I/Y Chromosomal Protein

ABSTRACTRecombinant human high mobility group chromosomal protein I/Y has been expressed in E. coli BL21(DE3) strain using the expression system of Studier et al. (1990). Cells were transformed with bacterial plasmid pET15bHMG-I/Y containing cDNA of the human HMG I/Y protein inserted in-frame with a sequence coding six cnosecutive histidines at the N-terminus of the protein (Thanos and Maniatis, 1992). The recombinant protein has been isolated from the total protein extract by selective binding of the 6xHis-tag on Ni2+-NTA resin (Qiagen) using metalo belate affinty chromatography. The protein was tested by SDS polyacrylamide gel electrophoresis and immunochemical detection with anti rat brain HMG I antibodies (IgG fraction). The immunoperoxidase test was performed on Western blots with 4-chloro-l-naphtol/H2O2 as a substrate and on dot blots with luminol as an enhanced hemiluminiscence emitting substrate (ECL—Amersham Life Sci.). By its electrophoretic and immunochemical reactions the obtained recombinant ...