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It has been established previously that up to 40% of mouse CD34(+) hematopoietic stem cells are capable of internalizing exogenous dsDNA fragments both in vivo and ex vivo. Importantly, when mice are treated with a combination of cyclophosphamide and dsDNA, the repair of interstrand crosslinks in hematopoietic progenitors is attenuated, and their pluripotency is altered. Here we show for the first time that among various actively proliferating mammalian cell populations there are subpopulations capable of internalizing dsDNA fragments. In the context of cancer, such dsDNA-internalizing cell subpopulations display cancer stem cell-like phenotype. Furthermore, using Krebs-2 ascites cells as a model, we found that upon combined treatment with cyclophosphamide and dsDNA, engrafted material loses its tumor-initiating properties which we attribute to the elimination of tumor-initiating stem cell subpopulation or loss of its tumorigenic potential.

Introduction: Karanahan, a cancer treatment technology aimed at eradicating tumor-initiating stem cells, has already proven effective in 7 tumor models. Karanahan comprises the following procedures: (1) collecting surgical specimens, (2) determining the duration of the DNA repair process in tumor cells exposed to a cross-linking cytostatic agent, and (3) determining the time point, when cells, including tumor-initiating stem cells, are synchronized in the certain phase of the cell cycle after triple exposure to the cytostatic, becoming vulnerable for the terminal treatment, which is supposed to completely eliminate the rest of survived tumor-initiating stem cells. Determining these basic tumor properties allows to design the schedule for the administration of a cross-linking cytostatic and a complex composite DNA preparation. Being conducted in accordance with the schedule designed, Karanahan results in the large-scale apoptosis of tumor cells with elimination of tumor-initiating stem cells. Methods: Breast tumor specimens were obtained from patients, and basic tumor properties essential for conducting Karanahan therapy were determined. Results: We report the first use of Karanahan in patients diagnosed with breast cancer. Technical details of handling surgical specimens for determining the essential Karanahan parameters (tumor volume, cell number, cell proliferation status, etc) have been worked out. The terminally ill patient, who was undergoing palliative treatment and whose tumor specimen matched the required criteria, received a complete course of Karanahan. Conclusions: The results of the treatment conducted indicate that Karanahan technology has a therapeutic potency and can be used as a breast cancer treatment option. [ABSTRACT FROM AUTHOR]

Electron-microscopic analysis of the ultrastructure of the Krebs-2 carcinoma ascites cells in the first 90 min immediately after their exposure to fragmented double-stranded DNA has been performed. Morphological attributes of the treated cancer cells indicate the induction in these cells of destructive processes of presumably apoptotic type. The predominance of dystrophic-destructive changes in cells after the addition of DNA is supposed to be a consequence of the disturbance in metabolic processes caused by the experimental action. [ABSTRACT FROM AUTHOR]

Background: Extracellular double-stranded DNA participates in various processes in an organism. Here we report the suppressive effects of fragmented human double-stranded DNA along or in combination with cyclophosphamide on solid and ascites grafts of mouse Krebs-2 tumor cells and DNA preparation on human breast adenocarcinoma cell line MCF-7. Methods: Apoptosis and necrosis were assayed by electrophoretic analysis (DNA nucleosomal fragmentation) and by measurements of LDH levels in ascitic fluid, respectively. DNA internalization into MCF-7 was analyzed by flow cytometry and fluorescence microscopy. Results: Direct cytotoxic activity of double-stranded DNA (along or in combination with cyclophosphamide) on a solid transplant was demonstrated. This resulted in delayed solid tumor proliferation and partial tumor lysis due to necrosis of the tumor and adjacent tissues. In the case of ascites form of tumor, extensive apoptosis and secondary necrosis were observed. Similarly, MCF-7 cells showed induction of massive apoptosis (up to 45%) as a result of treatments with double-stranded DNA preparation. Conclusions: Double-stranded DNA (along or in combination with cyclophosphamide) induces massive apoptosis of Krebs-2 ascite cells and MCF-7 cell line (DNA only). In treated mice it reduces the integrity of gut wall cells and contributes to the development of systemic inflammatory reaction. [ABSTRACT FROM AUTHOR]

Abstract: We previously reported that fragments of exogenous double-stranded DNA can be internalized by mouse bone marrow cells without any transfection. Our present analysis shows that only 2% of bone marrow cells take up the fragments of extracellular exogenous DNA. Of these, ~45% of the cells correspond to CD34+ hematopoietic stem cells. Taking into account that CD34+ stem cells constituted 2.5% of the total cell population in the bone marrow samples analyzed, these data indicate that as much as 40% of CD34+ cells readily internalize fragments of extracellular exogenous DNA. This suggests that internalization of fragmented dsDNA is a general feature of poorly differentiated cells, in particular CD34+ bone marrow cells. When linearized plasmid DNA was used as a source of exogenous DNA, we observed that exonucleolytic processing and ligation of double-stranded DNA termini occurred in the bone marrow cells that had this DNA internalized. We also recovered “hybrid” plasmids that encompass kanamycin-resistance gene from the exogenous plasmid DNA and the fragments of plasmids from host enterobacteria, which is suggestive of recombination events taking place upon DNA internalization. CD34+ cells make up the distinctive bone marrow cell population that internalizes extracellular DNA. Cell cycle analysis of CD34+ cells treated with cyclophosphamide only or in combination with dsDNA, suggests that these cells have distinct biologic responses to these treatments. Namely, whereas upon cyclophosphamide treatment bone marrow stem cells become arrested at S–G2 phases, combined cyclophosphamide+dsDNA treatment leads to cell cycle progression without any delay. This indicates that when the genome is undergoing repair of interstrand crosslinks, injection of fragmented exogenous dsDNA results in immediate reconstitution of genome integrity. We observe that cyclophosphamide-only or a combined cyclophosphamide+dsDNA treatment of cells lead to two distinct waves of apoptosis in CD34+ progenitors. We also show that cyclophosphamide and cyclophosphamide+dsDNA injections promote division of CD34+ cells at distinct time periods. [Copyright &y& Elsevier]

Abstract: Morbidity and mortality in mice were observed upon administration of exogenous DNA following their pre-treatment with a cytostatic agent cyclophosphamide. Upon intraperitoneal injections, the fragments of exogenous DNA reached bone marrow cells. These cells were also found to internalize up to 1800kb of exogenous DNA ex vivo. The 18–24h time frame represents a final stage in the repair of DNA double-strand breaks, so when exogenous DNA was administered within this critical period of time, pathological changes were observed in many target organs. Namely, bone marrow cells underwent a sustained increase in apoptosis. Copy number of B1 and B2 DNA repeats in bone marrow cells remained unchanged, whereas in the control group of animals their levels were significantly decreased. Finally, the bone marrow cells of moribund animals completely lacked lymphoid progenitors, yet the CD34+ hematopoietic stem cell counts were normal. Histopathology analysis suggested that mice died due to accidental involution of lymphoid organs combined with a systemic inflammatory process induced by massive administration of exogenous DNA and depletion of lymphoid lineage. [Copyright &y& Elsevier]