S. S. Bogachev

Extracellular genomic DNA protects mice against radiation and chemical mutagens

BackgroundHigh doses of ionizing irradiation and chemical mutagens induce random mutations and chromosome aberrations in cells of affected organisms and cause acute symptoms, delayed increased risk of cancer and accelerated aging. The mechanism of disease development remains unclear and no treatment exists for consequences of the mutagenic damage.HypothesisWe have proposed recently that extracellular genomic DNA from tissue fluids of a healthy organism, innate receptor-mediated nuclear delivery of this DNA, and its homologous recombination with cellular genomic sequences might function concertedly as a natural proofreading mechanism for somatic cell genomes. Here we hypothesize that cells dying from irradiation or chemical mutagens release heavily damaged DNA fragments that propagate mutations and chromosome aberrations to DNA-recipient cells via this mechanism, inducing cell death and release of their mutated DNA again into the bloodstream. The repeated release of the mutated DNA followed by its incorporation into cellular genomes would spread mutational damage in the affected organism, thus making this DNA the etiologic agent of either radiation sickness or post-mutagen exposure syndrome. The hypothesis opens a possibility to inhibit and treat the disease via administration of non-mutated genomic DNA fragments that would compete with the circulating mutant DNA fragments, entering cells in greater numbers, leading to replacement of mutant segments in cellular genomes.Results and ConclusionsInjection of fragmented mouse DNA, but not human DNA, into lethally irradiated mice dramatically increased their survival. Similarly, the mouse DNA was more potent than human and salmon DNA in accelerating recovery of the normal leukocyte level in mice treated with the chemical mutagen cyclophosphamide. The species specificity of the DNA therapy suggests that the genomic sequences are the agent producing the effects.

Qualitative and quantitative characteristics of the extracellular DNA delivered to the nucleus of a living cell

BackgroundThe blood plasma and other intertissue fluids usually contain a certain amount of DNA, getting there due to a natural cell death in the organism. Cells of this organism can capture the extracellular DNA, whereupon it is delivered to various cell compartments. It is hypothesized that the extracellular DNA is involved in the transfer of genetic information and its fixation in the genome of recipient cell.ResultsThe existence of an active flow of extracellular DNA into the cell is demonstrated using human breast adenocarcinoma (MCF-7) cells as a recipient culture. The qualitative state of the DNA fragments delivered to the main cell compartments (cytoplasm and interchromosomal fraction) was assessed. The extracellular DNA delivered to the cell is characterized quantitatively.ConclusionIt is demonstrated that the extracellular DNA fragments in several minutes reach the nuclear space, where they are processed so that their linear size increases from about 500 bp to 10,000 bp. The amount of free extracellular DNA fragments simultaneously present in the nuclear space may reach up to 2% of the haploid genome. Using individual DNA fragments with a known molecular weight and sequence as an extracellular DNA, it is found that these fragments degrade instantly in the culture liquid in the absence of a competitor DNA and are delivered into the cell as degradants. When adding a sufficient amount of competitor DNA, the initial undegraded molecules of the DNA fragments with the known molecular weight and sequence are detectable both in the cytoplasm and nuclear space only at the zero point of experiments. The labeled precursor α-dNTP*, added to culture medium, was undetectable inside the cell in all the experiments.

A MULTI-SPACECRAFT VIEW OF A GIANT FILAMENT ERUPTION DURING 2009 SEPTEMBER 26/27

We analyze multi-spacecraft observations of a giant filament eruption that occurred during 2009 September 26 and 27. The filament eruption was associated with a relatively slow coronal mass ejection. The filament consisted of a large and a small part, and both parts erupted nearly simultaneously. Here we focus on the eruption associated with the larger part of the filament. The STEREO satellites were separated by about 117° during this event, so we additionally used SoHO/EIT and CORONAS/TESIS observations as a third eye (Earth view) to aid our measurements. We measure the plane-of-sky trajectory of the filament as seen from STEREO-A and TESIS viewpoints. Using a simple trigonometric relation, we then use these measurements to estimate the true direction of propagation of the filament which allows us to derive the true R/R ☉-time profile of the filament apex. Furthermore, we develop a new tomographic method that can potentially provide a more robust three-dimensional (3D) reconstruction by exploiting multiple simultaneous views. We apply this method also to investigate the 3D evolution of the top part of filament. We expect this method to be useful when SDO and STEREO observations are combined. We then analyze the kinematics of the eruptive filament during its rapid acceleration phase by fitting different functional forms to the height-time data derived from the two methods. We find that for both methods an exponential function fits the rise profile of the filament slightly better than parabolic or cubic functions. Finally, we confront these results with the predictions of theoretical eruption models.

Natural human gene correction by small extracellular genomic DNA fragments.

Classical gene targeting employs natural homologous recombination for a gene correction using a specially designed and artificially Delivered DNA construct but the method is very inefficient. On the other hand, small DNA fragments in the form of tiny chromatin-like articles naturally present in blood plasma can spontaneously penetrate into human cells and cell nuclei. We hypothesized that these natural DNA nanoparticles with recombinagenic free ends might be effective agents for gene replacement therapy. We demonstrate that a mixture of small fragments of total human chromatin from non-mutant cells added to a culture medium without transfection agents efficiently repaired a 47 base pair deletion in the CASP3 gene in 30% of treated human MCF7 breast cancer cells, as shown by restoration of caspase-3 apoptotic function and CASP3 DNA and mRNA structure. Such an innate gene replacement mechanism might function naturally in an organism using its own apoptotic DNA fragments. This mechanism might enable human cancer cell phenotype normalization in the presence of excess normal Cells.

Internalization of Exogenous DNA into Internal Compartments of Murine Bone Marrow Cells

Injections of exogenous DNA combined with a cytostatic agent cyclophosphamide (CP) cause illness and death in experimental mice. This phenomenon is referred to as delayed death. It has been found that fragments of exogenous DNA reach the bone marrow and enter the bone marrow cells (BMCs) 1–5 min after injections. Fragments of exogenous DNA are captured from culture medium by BMCs generated ex vivo. After joint incubation with BMCs of mice, the fragments of exogenous DNA are internalized into internal compartments in a nondegraded form. Up to 1800 kb of nucleic acid material can be present in each cell of BMCs generated ex vivo and treated with fragments of exogenous DNA. The fragments of exogenous DNA internalized in BMCs generated ex vivo of both intact and CP pretreated mice become circularized. In the case of intact mice, the fragments of exogenous DNA can form high-molecular weight structures in vivo. It is suggested that the exogenous fragments localized in BMC nuclei integrate into chromosome(s) of the recipient mouse genome when treated with CP and exogenous DNA.

Eradication of Krebs-2 primary ascites via a single-injection regimen of cyclophosphamide and double-stranded DNA

Previously, we reported on the development of a therapeutic regimen allowing eradication of primary murine Krebs-2 ascites transplants. This protocol involved multiple injections of dsDNA preparations administered during the NER and HR phases of repair of interstrand DNA cross-links induced by prior cyclophosphamide treatments. Mice treated under this protocol frequently developed secondary ascites, which indicated that some tumor-inducing cancer stem cells could survive the treatment and caused relapse. Further, we observed that animals receiving multiple dsDNA injections developed pronounced systemic inflammatory response. This prompted us to develop a more straightforward treatment regimen based on the synergistic activity of cyclophosphamide and dsDNA preparations, which would allow complete eradication of established primary Krebs-2 ascites and also be less toxic for the treated animals. This protocol relies on a precisely timed single injection of dsDNA during the NER/HR transition period of each repair cycle. Under this protocol, 8-day remission of Krebs-2 engrafted mice was achieved, which was similar to the В наших предыдущих работах был разработан режим элиминации первичного асцита на модели мышиной опухоли Кребс-2. В основе режима лежат многократные инъекции препаратов двуцепочечной ДНК, приходящиеся как на фазу NER, так и на фазу гомологической рекомбинации процесса репарации межце почечных сшивок после обработки животных циклофосфаном. Развивающийся вторичный асцит у обработанных таким образом животных свидетельствовал о том, что при такой обработке стволовые инициирующие раковые клетки не полностью элиминируются и впоследствии дают рецидив заболевания. Характерный симптомокомплекс системной воспалительной реакции предполагал токсическое воздействие многократных инъекций препарата ДНК. На основании этих фактов был разработан технологически простой режим синергичного действия цитостатика циклофосфана и препарата двуцепочечной ДНК, позволяющий полностью элиминировать первичный развитый асцит опухоли Кребс-2, со снижением уровня токсического воздействия на экспериментальных животных. В отличие от обсуждавшегося ранее режима, в настоящей разработке многократные инъекции препаратов двуцепочечной ДНК сведены к одной инъекции в точку демаркации фаз репарации NER и гомологической рекомбинации в каждом репаративном цикле. В результате такой обработки также удалось достичь 8-суточной ремиссии асцита Кребс-2 у всех животных в группе, что было показано при режиме многократных инъекций препаратов двуцепочечной ДНК. Средняя продолжительность жизни экспериментальных животных при однократных инъекциях увеличилась, что свидетельствует об уменьшении токсического воздействия терапии на животных.

Pathological changes in mice treated with cyclophosphamide and exogenous DNA

The synergistic action of the cytostatic drug cyclophosphamide (CP) and fragmented exogenous DNA causes illness and death in mice (Dolgova et al., 2011–2013). The observed “delayed death” effect was most clearly pronounced when the DNA preparation was administered 18 to 30 h after CP treatment. This time span was termed the “death window.”It was found that injections of exogenous DNA result in a sustained increase in bone-marrow cell (BMC) apoptosis, which occurs throughout the time of DNA administration (18–30 h). Exogenous DNA, both allogeneic and belonging to various taxa, induces BMC apoptosis. Plasmid DNA has the greatest effect on apoptosis induction. The analysis of reduction and restoration of the BMC subpopulations as the mice progressed to death revealed a virtually complete loss of the 12–20-μm fraction of the cell population (about 3–4% vs. 35–40% in the control), which corresponds to the maximum leukopenia on day 3 after CP treatment. However, the relative number of CD34+ hematopoietic stem cells (HSCs) from day 15 and until the end of the observation constituted 1.2–1.4%, which corresponds to the wild-type range. Comparison of BMC smears from the sternal bone marrow of the CP and CP + DNA groups of mice indicated that the BMC populations isolated from CP + DNA animals lack young committed lymphopoiesis progenitor cells. Moreover, the affected mice had immature blast cell types in their blood, which was never observed in healthy or CP-treated mice. Pathological and morphological analyses show that starting from posttreatment day 9, mice that received CP + DNA preparations displayed pronounced morphological changes in their lungs, liver, pancreas, central and peripheral immune system organs, and brain. Most of the pathological changes observed are consistent with a severe inflammatory response. This suggestion was proven by structural equivalents of functional involution of lymphoid organs, such as the thymus, spleen, and lymph nodes.We speculate that the death of treated animals resulted from multiple organ dysfunctions caused by accidental involution of lymphoid organs and the systemic inflammatory response syndrome, both associated with injections of fragmented exogenous DNA into experimental animals within the “death window,” which corresponds to the final step in the repair of the majority of CP-induced double-strand breaks.

Changes in the symptoms of hypothalamic diabetes insipidus after treatment with homologous exogenous DNA.

Hypothalamic diabetes insipidus is caused by defects in the synthesis and secretion of vasopressin. This hormone is synthesized in hypothalamic neurons; it is the main regulator of the water and electrolyte homeostasis of the internal medium [1, 2]. Morphological and genetic abnormalities in the hypothalamus– neurohypophysis system affecting the functioning of the vasopressin gene are accompanied by substantial changes in the pattern of water consumption and removal. Patients with diabetes insipidus drink an order of magnitude more water and form respectively larger amounts of hypoosmotic urine [3, 4]. Some immunoreactive vasopressin is found in the thymus, testis, ovary, and other organs whose specialized tissues synthesize steroid hormones or cytokines [5–8]. However, the expression of the vasopressin gene outside the hypothalamic nuclei is low and, in general, has no effect on the disease symptoms. Modulating effects of DNA preparations on the patterns of some physiological processes were reported recently [9, 10]. To study the possibility of the therapeutic correction of the symptoms of diabetes insipidus, we studied the effect of long-term treatment with exogenous homologous DNA.