Existence of a small subset of cancer cells referred to as tumor initiating stem cells (TISCs) largely responsible for tumor progression and resistance to chemotherapeutic cytostatic drugs reperesent an important recent paradigm shift. The present work is the first report in the series of papers from our group where we describe the development of anticancer therapy based on the selective targeting of TISCs. Here were characterize a cytoreductive activity of cyclophosphamide (CP), double-stranded DNA (dsDNA) and combinations thereof against the TISC population present in mouse Krebs-2 ascites. We evaluated engraftment potential of Krebs-2 cancer cells treated in ascites-bearing mice in vivo, followed by re-engraftment to congenic recipient mice in a form of a solid graft. These data indicate that with our approach TISCs can be completely eliminated even from a well-established ascites. We demonstrate that dsDNA-internalizing and CD34-positive cells are more sensitive to the synergistic effects of CP and dsDNA. When Krebs-2 ascites are treated with human DNA 1-12 hours post CP injection, this results in either elimination of cells that internalize TAMRA-labeled DNA (TISCs) or alters their phenotype, which is accompanied with the loss of surface expression of CD34. Next, we show that the timepoint 18 hrs post CP treatment is critical to the ongoing repair process in that it divides the repair into two phases: nucleotide excision repair + dsDNA break repair and homologous recombination. Importantly, both of these phases can be conveniently used for targeting the tumorigenic potential of the graft. In the context of monotherapy, CP is most effective against ascites grafts when administered as serial injections. To achieve maximum efficiency, the timing of consecutive injections must match the time when cancer cells found at G2/M during the first injection enter G1/S and/or the time of active repair via homologous recombination.

We performed a multicenter, double-blind, placebo-controlled, phase II clinical trial of human dsDNA-based preparation Panagen in a tablet form. In total, 80 female patients with stage II-IV breast cancer were recruited.

We report on the results of a phase II clinical trial of Panagen (tablet form of fragmented human DNA preparation) in breast cancer patients (placebo group n = 23, Panagen n = 57). Panagen was administered as an adjuvant leukoprotective agent in FAC and AC chemotherapy regimens. Pre-clinical studies clearly indicate that Panagen acts by activating dendritic cells and induces the development of adaptive anticancer immune response.

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 results of the multiple-injection treatment schedule. We observed an increase in the average life span of Krebs-2- transplanted mice on a single-injection regimen, which was consistent with reduced toxicity of such treatment.

Cumulative evidence obtained in this series of studies has guided the logic behind the development of a novel composite dsDNA-based preparation whose therapeutic application according to the specific regimen completely cures the mice engrafted with otherwise lethal Krebs-2 ascites. The likely mechanism involves elimination of TAMRA+ tumor-inducing stem cells (TISCs) from Krebs-2 tumors. We performed quantitative analysis of TISC dynamics in Krebs-2 ascites following treatment with the cytostatic drug cyclophosphamide (CP) and untreated control cells. In intact ascites, TISC percentage oscillates around a certain value. Following CP treatment and massive apoptosis of committed cancer cell subpopulation, we observed relative increase in TISC percentage, which is consistent with reduced susceptibility of TISCs to CP. Nonetheless, this treatment apparently synchronizes TISCs in a cell cycle phase when they become sensitive to further drug treatments. We describe the regimen of synergistic DNA + CP activity against Krebs-2 ascites. This protocol results in a complete cure of 50 % of Krebs-2 engrafted mice and involves three metronomic injections of CP exactly at the timepoints when repair cycles are about to finish combined with dsDNA injections 18 hours following each CP injection. The “final shot” uses CP + DNA treatment, which targets the surviving yet highly synchronized and therefore treatmentsensitive cells. The first three CP/DNA injections appear to arrest Krebs-2 cells in late S-G2-M phase and result in their simultaneous progression into G1-S phase. The timing of the “final shot” is crucial for the successful treatment, which eradicates tumorigenic cell subpopulation from Krebs-2 ascites. Additionally, we quantified the changes in several biochemical, cellular and morphopathological parameters in mice throughout different treatment stages.

In the present paper, we report on the series of experiments where multiple regimens of CP and dsDNA injections were tested for targeting the ascites form of murine Krebs-2 cancer in situ. We show that combining CP with cross-linked human and salmon dsDNA results in a synergistic toxicity for ascites-bearing mice, an observation supported by the histopathology analysis of organs and tissues of experimental animals. In contrast, using a composite mixture of native and cross-linked human and salmon DNA after CP injections leads to a significant increase in average lifespan of the treated mice. Further, we demonstrate that repeated rounds of CP+dsDNA injections result in dramatic anticancer effect. The timing of injections is chosen so that they target the cells that were insensitive to the previous treatments as they were in the G2/M phase. 3-4 rounds of injections are needed to eliminate the subpopulation of tumor-initiating cancer stem cells. Our experiments identified the regimen when complete resorption of the primary Krebs-2 ascites occurs in all of the treated animals, followed by a remarkable remission period lasting 7-9 days. Yet, this regimen does not prevent secondary site metastases (either solid or ascites form) from developing, which is likely caused by the migration of ascites cells into adjacent tissues or by incomplete eradication of cancer stem cells. To address these and other questions, we expanded the study and performed histopathology analysis, which indicated that secondary metastases is not the only cause of death. In fact, many animals displayed unfolding systemic inflammatory reaction which was culminated by multiple organ failure. Thus, we developed the concept for treating ascites form of Krebs-2 cancer, which allows elimination of the primary ascites.

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.

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.

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.

Abstract. There are some specific mechanisms in cell that detect the invasion into the cell of any foreign molecules, so-called pathogen-associated molecular patterns (PAMPs). The recognition of these PAMPs is realized by Toll-like receptors or is due to the cytosolic sensors action. In recent years much attention is directed to the investigation of foreign nucleic acids sensing and detection in cell. Nowadays some sensors of doublestranded DNA, such as STING, DAI, members of NOD-like protein family, RIG-like helicases, proteins of HIN-200 family are identified. Some of sensors interact with double-stranded DNA directly. The others are characterized as mediators of signal transduction from unknown DNA-sensor in the series of following molecular events. Induction of nuclear transcription factors NF-κB and IRF3/IRF7 to produce IFNβ or processing of proinflammatory cytokines IL-1β and IL-18 by means of inflammasome formation is a result of activation of cascade events induced by cytosolic DNA. In the review we combine information concerning signaling pathways characterized sensing of intracellular double-stranded DNA that results in the development of the cellular immune response.

Mice were observed to get sick and die upon administration of exogenous DNA in a specific period of time following their pretreatment with the cytostatic cyclophosphamide (CP) (Dolgova et al., 2011). It was established that exogenous DNA reaches internal compartments of bone marrow cells (BMCs) where it is processed (Dolgova et al., 2012a). Thus, BMCs appear to be the primary targets for the synergic action of these preparations (Dolgova et al., 2012b).In the present study, we show that the copy number for mouse interspersed genomic repeats decreases in the genome of mouse mononuclear cells as a result of interstrand cross-link (ICL) repair after pre-treatment with cytostatic CP. This phenomenon occurs within the time span from 18 to 24 h following CP injection, which corresponds to the final step in the repair of the majority of double-strand breaks (DSBs), as predominant intermediates in ICL repair. Injections of exogenous DNA in CP-pretreated mice preserve the copy number of interspersed repeats at the original level. Our results suggest that the fragments of exogenous DNA participate in ICL-induced DSB repair, thereby compromising the repair process.

The synergic 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 hours after CP treatment. This time span is designated as «death window». It was found that injections of exogenous DNA result in sustained increase in bone marrow cell (BMC) apoptosis, which occurs throughout the time of DNA administration (18–30 hours). Exogenous DNA, both allogeneic and belonging to various taxa induces BMCs apoptosis. Plasmid DNA has the greatest effect on apoptosis induction. The analysis of reduction and restoration of BMC subpopulations as the mice progressed to death revealed a virtually complete loss of the 12–20-mkm 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 amount of CD34+ hematopoietic stem cells (HSCs) from day 15 and till 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 indicates 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 severe inflammatory response. This suggestion is proven by structural equivalents of functional involution of lymphoid organs, such as 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.

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.”

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, similar to 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. (C) 2013 Elsevier B.V. All rights reserved.

We investigated the influence of Panagen DNA preparations on laboratory animals and IFN-induced human dendritic cells, as well as analyzed the data from a phase II clinical trial in the therapy of breast cancer. It was shown that this treatment resulted in increased number of CD8+/perforin+ T cells in peripheral lymphoid organs of experimental animals, in mixed lymphocyte culture population and in peripheral blood of breast cancer patients. Moreover, we demonstrated that when Panagen DNA preparations are used in combination with the standard FAC-based breast cancer therapies, non-specific immune response activity remains at the same levels as observed prior to therapy, whereas in FAC-placebo patients, non-specific immunity is greatly diminished.

Hemostimulating activity of Panagen substance representing the nucleoprotein complex extracted from the human placenta was studied using the model of cyclophosphan-induced myelosuppression in mice. Panagen was found to have hemostimulating properties comparable with activity of Derinat drug.

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 1800 kb of exogenous DNA ex vivo. The 18-24 h 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.

Exogenous allogenic DNA as nucleosome-free fragments reaches main cellular compartments (cytoplasm, nucleus) of human dendritic cells and deposits in the nuclear interchromosomal space without visibly changing in linear size. The presence of such allogenic fragmented DNA in medium in which human dendritic cells are cultured produces an enhancement of their allostimulatory activity. This enhancement is comparable to that produced by the standard maturation stimulus lipopolysaccharide Escherichia coli.

BACKGROUND:When cyclophosphamide and preparations of fragmented exogenous genomic double stranded DNA were administered in sequence, the regressive effect on the tumor was synergic: this combined treatment had a more pronounced effect than cyclophosphamide alone. Our further studies demonstrated that exogenous DNA stimulated the maturation and specific activities of dendritic cells. This suggests that cyclophosphamide, combined with DNA, leads to an immune response to the tumors that were grafted into the subjects post treatment.METHODS:Three-month old CBA/Lac mice were used in the experiments. The mice were injected with cyclosphamide (200 mkg per 1 kg body weight) and genomic DNA (of human, mouse or salmon sperm origin). The DNA was administered intraperitoneally or subcutaneously. After 23 to 60 days, one million tumor cells were intramuscularly grafted into the mice. In the final experiment, the mice were pre-immunized by subcutaneous injections of 20 million repeatedly thawed and frozen tumor cells. Changes in tumor growth were determined by multiplying the three perpendicular diameters (measured by caliper). Students' t-tests were used to determine the difference between tumor growth and average survival rate between the mouse groups and the controls.RESULTS:An analysis of varying treatments with cyclophosphamide and exogenous DNA, followed by tumor grafting, provided evidence that this combined treatment had an immunizing effect. This inhibitory effect in mice was analyzed in an experiment with the classical immunization of a tumor homogenate. The strongest inhibitory action on a transplanted graft was created through the following steps: cyclophosphamide at 200 mg/kg of body weight administered as a pretreatment; 6 mg fragmented exogenous DNA administered over the course of 3 days; tumor homogenate grafted 10 days following the final DNA injection.CONCLUSION:Fragmented exogenous DNA injected with cyclophosphamide inhibits the growth of tumors that are grafted to mice after this combined treatment.

Our study showed that protamine (80% w/w to DNA) effectively protected its molecules from degradation by native nucleases of the mammalian blood serum. Exogenous DNA bound to protamine effectively stimulated restoration of cyclophosphamide-induced leukopoiesis in mice. It is suggested that the phenomenon was due to repair processes taking place in hemopoietic stem cells damaged by a cross-linking cytostatic drug such as cyclophosphamide. DNA dosage may be reduced and the original DNA fragment size maintained by DNA binding to protamine. As a result, it might involve longer DNA fragments into repair processes of homologous recombination and eventually increase the cell's chances of getting rid of extensive damage.