Enhancement of the Antitumor and Antimetastatic Effect of Topotecan and Normalization of Blood Counts in Mice with Lewis Carcinoma by Tdp1 Inhibitors-New Usnic Acid Derivatives.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is an important DNA repair enzyme and one of the causes of tumor resistance to topoisomerase 1 inhibitors such as topotecan. Inhibitors of this Tdp1 in combination with topotecan may improve the effectiveness of therapy. In this work, we synthesized usnic acid derivatives, which are hybrids of its known derivatives: tumor sensitizers to topotecan. New compounds inhibit Tdp1 in the micromolar and submicromolar concentration range; some of them enhance the effect of topotecan on the metabolic activity of cells of various lines according to the MTT test. One of the new compounds (compound 7) not only sensitizes Krebs-2 and Lewis carcinomas of mice to the action of topotecan, but also normalizes the state of the peripheral blood of mice, which is disturbed in the presence of a tumor. Thus, the synthesized substances may be the prototype of a new class of additional therapy for cancer.

Impact of Double-Stranded RNA Internalization on Hematopoietic Progenitors and Krebs-2 Cells and Mechanism.

It is well-established that double-stranded RNA (dsRNA) exhibits noticeable radioprotective and radiotherapeutic effects. The experiments conducted in this study directly demonstrated that dsRNA was delivered into the cell in its native form and that it induced hematopoietic progenitor proliferation. The 68 bp synthetic dsRNA labeled with 6-carboxyfluorescein (FAM) was internalized into mouse hematopoietic progenitors, c-Kit+ (a marker of long-term hematopoietic stem cells) cells and CD34+ (a marker of short-term hematopoietic stem cells and multipotent progenitors) cells. Treating bone marrow cells with dsRNA stimulated the growth of colonies, mainly cells of the granulocyte-macrophage lineage. A total of 0.8% of Krebs-2 cells internalized FAM-dsRNA and were simultaneously CD34+ cells. dsRNA in its native state was delivered into the cell, where it was present without any signs of processing. dsRNA binding to a cell was independent of cell charge. dsRNA internalization was related to the receptor-mediated process that requires energy from ATP. Synthetic dsRNA did not degrade in the bloodstream for at least 2 h. Hematopoietic precursors that had captured dsRNA reinfused into the bloodstream and populated the bone marrow and spleen. This study, for the first time, directly proved that synthetic dsRNA is internalized into a eukaryotic cell via a natural mechanism.

The Lipophilic Purine Nucleoside-Tdp1 Inhibitor-Enhances DNA Damage Induced by Topotecan In Vitro and Potentiates the Antitumor Effect of Topotecan In Vivo.

The use of cancer chemotherapy sensitizers is a promising approach to induce the effect of clinically used anticancer treatments. One of the interesting targets is Tyrosyl-DNA Phosphodiesterase 1 (Tdp1), a DNA-repair enzyme, that may prevent the action of clinical Topoisomerase 1 (Top1) inhibitors, such as topotecan (Tpc). Tdp1 eliminates covalent Top1-DNA (Top1c) complexes that appear under the action of topotecan and determines the cytotoxic effect of this drug. We hypothesize that Tdp1 inhibition would sensitize cells towards the effect of Tpc. Herein, we report the synthesis and study of lipophilic derivatives of purine nucleosides that efficiently suppress Tdp1 activity, with IC50 values in the 0.3-22.0 µM range. We also showed that this compound class can enhance DNA damage induced by topotecan in vitro by Comet assay on human cell lines HeLa and potentiate the antitumor effect of topotecan in vivo on a mice ascitic Krebs-2 carcinoma model. Thereby, this type of compound may be useful to develop drugs, that sensitize the effect of topotecan and reduce the required dose and, as a result, side effects.

Promising New Inhibitors of Tyrosyl-DNA Phosphodiesterase I (Tdp 1) Combining 4-Arylcoumarin and Monoterpenoid Moieties as Components of Complex Antitumor Therapy.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is an important DNA repair enzyme in humans, and a current and promising inhibition target for the development of new chemosensitizing agents due to its ability to remove DNA damage caused by topoisomerase 1 (Top1) poisons such as topotecan and irinotecan. Herein, we report our work on the synthesis and characterization of new Tdp1 inhibitors that combine the arylcoumarin (neoflavonoid) and monoterpenoid moieties. Our results showed that they are potent Tdp1 inhibitors with IC50 values in the submicromolar range. In vivo experiments with mice revealed that compound 3ba (IC50 0.62 µM) induced a significant increase in the antitumor effect of topotecan on the Krebs-2 ascites tumor model. Our results further strengthen the argument that Tdp1 is a druggable target with the potential to be developed into a clinically-potent adjunct therapy in conjunction with Top1 poisons.

Five-year disease-free survival among stage II-IV breast cancer patients receiving FAC and AC chemotherapy in phase II clinical trials of Panagen.

BACKGROUND: 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. METHODS: We analyzed 5-year disease-free survival of patients recruited into the trial. RESULTS: Five-year disease-free survival in the placebo group was 40 % (n = 15), compared with the Panagen arm - 53 % (n = 51). Among stage III patients, disease-free survival was 25 and 52 % for placebo (n = 8) and Panagen (n = 25) groups, respectively. Disease-free survival of patients with IIIB + C stage was as follows: placebo (n = 6)-17 % vs Panagen (n = 18)-50 %. CONCLUSIONS: Disease-free survival rate (17 %) of patients with IIIB + C stage breast cancer receiving standard of care therapy is within the global range. Patients who additionally received Panagen demonstrate a significantly improved disease-free survival rate of 50 %. This confirms anticancer activity of Panagen. TRIAL REGISTRATION: ClinicalTrials.gov NCT02115984 from 04/07/2014.

Combination of cyclophosphamide and double-stranded DNA demonstrates synergistic toxicity against established xenografts.

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.

Results of multicenter double-blind placebo-controlled phase II clinical trial of Panagen preparation to evaluate its leukostimulatory activity and formation of the adaptive immune response in patients with stage II-IV breast cancer.

BACKGROUND: 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. METHODS: Patients received three consecutive FAC (5-fluorouracil, doxorubicin and cyclophosphamide) or AC (doxorubicin and cyclophosphamide) adjuvant chemotherapies (3 weeks per course) and 6 tablets of 5 mg Panagen or placebo daily (one tablet every 2-3 hours, 30 mg/day) for 18 days during each chemotherapy course. Statistical analysis was performed using Statistica 6.0 software, and non-parametric analyses, namely Wilcoxon-Mann-Whitney and paired Wilcoxon tests. To describe the results, the following parameters were used: number of observations (n), median, interquartile range, and minimum-maximum range. RESULTS: Panagen displayed pronounced leukostimulatory and leukoprotective effects when combined with chemotherapy. In an ancillary protocol, anticancer effects of a tablet form of Panagen were analyzed. We show that Panagen helps maintain the pre-therapeutic activity level of innate antitumor immunity and induces formation of a peripheral pool of cytotoxic CD8+ perforin + T-cells. Our 3-year follow-up analysis demonstrates that 24% of patients who received Panagen relapsed or died after the therapy, as compared to 45% in the placebo cohort. CONCLUSIONS: The data collected in this trial set Panagen as a multi-faceted "all-in-one" medicine that is capable of simultaneously sustaining hematopoiesis, sparing the innate immune cells from adverse effects of three consecutive rounds of chemotherapy and boosting individual adaptive immunity. Its unique feature is that it is delivered via gastrointestinal tract and acts through the lymphoid system of intestinal mucosa. Taken together, maintenance of the initial levels of innate immunity, development of adaptive cytotoxic immune response and significantly reduced incidence of relapses 3 years after the therapy argue for the anticancer activity of Panagen. TRIAL REGISTRATION: ClinicalTrials.gov NCT02115984 from 04/07/2014.

Immunotherapy of hepatocellular carcinoma with small double-stranded RNA.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with limited therapeutic options. Since HCC has been shown to be immunogenic, immunotherapy is considered a promising therapeutic approach. Small interfering RNAs (siRNAs), depending on their structure and sequence, can trigger the innate immune system, which can potentially enhance the adaptive anticancer immune response in the tumor-bearing subjects. Immunostimulatory properties of nucleic acids can be applied to develop adjuvants for HCC treatment. METHODS: The transplantable HCC G-29 tumor in male CBA/LacSto (CBA) mice was used to study the effects of immunostimulatory RNA on tumor growth. Tumor size, metastases area in different organs of mice and mouse survival rate were analyzed. Furthermore the mouse serum IFN-α levels were measured using ELISA. RESULTS: In the present study, we found that a 19-bp RNA duplex (ImmunoStimulattory RNA or isRNA) with 3-nt overhangs at the 3'-ends of specific sequence displays immunostimulatory, antitumor, and antimetastatic activities in mice bearing HCC G-29. Our results demonstrate that isRNA strongly increases the level of interferon-α (IFN-α) by up to 25-fold relative to the level in mice injected with Lipofectamine alone (Mock), and to a lesser extent increases the level of proinflammatory cytokine interleukin-6 (IL-6) (by up to 5.5-fold relative to the Mock level), in mice blood serum. We showed that isRNA reliably (P < 0.05) inhibits primary tumor growth in mice compared to the mock group. Furthermore, injections of isRNA significantly enhanced necrotic processes in the center of the primary tumor, and decreased by twofold the width of the undifferentiated peripheral zone and the number of mitotic cells in this zone. The results showed that isRNA efficiently reduces the area of metastases in the liver, kidneys, and heart of CBA/LacSto mice with HCC. CONCLUSIONS: The obtained results clearly demonstrate immunostimulatory and antimetastatic properties of the isRNAs in mice with HCC. Consequently, this short double-stranded RNA can be considered as a potential adjuvant for the therapy of HCC.

Ligand-directed acid-sensitive amidophosphate 5-trifluoromethyl-2'-deoxyuridine conjugate as a potential theranostic agent.

Herein, we report a novel strategy to engineer an acid-sensitive anticancer theranostic agent using a vector-drug ensemble. The ensemble was synthesized by directly conjugating the linoleic acid (LA)-modified branched polyethyleneimine with a chemotherapeutic drug trifluorothymidine. Linoleic acid residues were grafted onto 25 kDa polyethyleneimine (PEI) by treating PEI with linoleic acid chloroanhydride. 5-Trifluoromethyl-2'-deoxyuridine (trifluorothymidine, TFT) was introduced into LA-PEI conjugate by phosphorylating the conjugate with amidophosphate of trifluorothymidine 5'-monophosphate (pTFT), which had been activated by its conversion into the N,N-dimethylaminopyridine derivative. The extent of mononucleotide analog incorporation in the polymer was regulated by the ratio of pTFT to the polymer during the synthesis. Samples containing 20-70 TFT residues per PEI molecule were obtained. The cytotoxicity of PEI-LA-pTFT conjugates decreased with increasing nucleotide content, as examined using the MTT method. Due to the presence of fluorine atoms, TFT-based conjugates could be detected directly in the animals by (19)F magnetic resonance imaging. In addition, the presence of the amidophosphate group in PEI-LA-pTFT conjugates allowed their detection by in vivo(31)P NMR spectroscopy. Indeed, the (31)P NMR signal of a phosphoramide (δ ~ 12 ppm) was observed in the mouse muscle tissue treated with PEI-LA-pTFT conjugate along with the signals from endogenous phosphorus-containing compounds. At the same time, the use of PEI-LA-pTFT conjugate for chemotherapeutic drug delivery is limited due to the low release of pTFT from the carrier. To enhance the release of the drug from the conjugate in the endosomes, PEI-LA polymer was coupled with urocanic acid (UA), which bears imidazole ring and thus can form an acid-labile P-N bond with pTFT. The PEI-LA-UA-pTFT conjugate containing 30 residues of UA and 40 residues of pTFT was tested against the murine Krebs-II ascites carcinoma, grown as an ascetic tumor. The intraperitoneal injection of the conjugates resulted in prolongation of the animals' life and to the complete disappearance of the tumor after three injections.

Effects of human exogenous DNA on production of perforin-containing CD8+ cytotoxic lymphocytes in laboratory setting and clinical practice.

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.

Triterpenoid saponins from the roots of Acanthophyllum gypsophiloides Regel.

Two new triterpenoid saponins 1 and 2 were isolated from the methanol extract of the roots of Acanthophyllum gypsophiloides Regel. These saponins have quillaic acid or gypsogenin moieties as an aglycon, and both bear similar sets of two oligosaccharide chains, which are 3-O-linked to the triterpenoid part trisaccharide α-L-Arap-(1→3)-[α-D-Galp-(1→2)]-ß-D-GlcpA and pentasaccharide ß-D-Xylp-(1→3)-ß-D-Xylp-(1→3)-α-L-Rhap-(1→2)-[ß-D-Quip-(1→4)]-ß-D-Fucp connected through an ester linkage to C-28. The structures of the obtained saponins were elucidated by a combination of mass spectrometry and 2D NMR spectroscopy. A study of acute toxicity, hemolytic, anti-inflammatory, immunoadjuvant and antifungal activity was carried out. Both saponins 1 and 2 were shown to exhibit immunoadjuvant properties within the vaccine composition with keyhole limpet hemocyanin-based immunogen. The availability of saponins 1 and 2 as individual pure compounds from the extract of the roots of A. gypsophiloides makes it a prospective source of immunoactive agents.

Experimental Comparison of the In Vivo Efficacy of Two Novel Anticancer Therapies.

BACKGROUND/AIM: We compared the therapeutic efficacy of two recently developed experimental anticancer technologies: 1) in situ vaccination based on local immunotherapy with CpG oligonucleotides and anti-OX40 antibodies to activate antitumor immune response and 2) "Karanahan" technology [from the Sanskrit karana ('source') + han ('to kill')] based on the combined injection of cyclophosphamide and double-stranded DNA to eradicate cancer stem cells. MATERIALS AND METHODS: The anticancer approaches were compared on three types of mouse malignant tumors with different grades of immunogenicity: weakly immunogenic carcinoma Krebs-2, moderately immunogenic Lewis carcinoma, and highly immunogenic A20 В-cellular lymphoma. RESULTS: Our results indicated that in situ vaccination was the most effective against the highly immunogenic tumor А20. In addition, "Karanahan" demonstrated high efficiency in all types of tumors, regardless of their immunogenicity or size. CONCLUSION: "Karanahan" therapy showed higher efficacy relative to in situ vaccination with CpG oligonucleotides and anti-OX40 antibodies.

Effect of double-stranded DNA on maturation of dendritic cells in vitro.

A preparation of human genomic fragmented double-stranded DNA (dsDNA) was used as maturation stimulus in cultures of human dendritic cells (DCs) generated in compliance with the interferon protocol. Culturing of the DCs in medium with 5µg/ml of the DNA preparation was associated with a decrease in the relative proportion of CD14 + cells and an increase in that of CD83 + cells. These changes are markers of DC maturation. The efficiency with which the DNA preparation was able to elicit DC maturation was commensurate with that of lypopolysaccharide from bacterial cell, the standard inducer of DC maturation. Generated ex vivo, matured in the presence of the human DNA preparation, pulsed with tumor antigens mouse DCs were used as a vaccine in biological tests for its antitumor activity. The experimental results demonstrate that reinfusion of mature pulsed with tumor antigens DCs cause a statistically significant suppression of tumor graft growth.

Exogenous allogenic fragmented double-stranded DNA is internalized into human dendritic cells and enhances their allostimulatory activity.

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.

A strategy of tumor treatment in mice with doxorubicin-cyclophosphamide combination based on dendritic cell activation by human double-stranded DNA preparation.

BACKGROUND: Immunization of mice with tumor homogenate after combined treatment with cyclophosphamide (CP) and double-stranded DNA (dsDNA) preparation is effective at inhibition of growth of tumor challenged after the treatment. It was assumed that this inhibition might be due to activation of the antigen-presenting cells. The purpose was to develop improved antitumor strategy using mice. We studied the combined action of cytostatics doxorubicin (Dox) plus CP with subsequent dsDNA preparation on tumor growth. METHODS: Three-month old CBA/Lac mice were used in the experiments. Mice were injected with CP and human dsDNA preparation. The percentage of mature dendritic cells (DCs) was estimated by staining of mononuclear cells isolated from spleen and bone marrow 3, 6, and 9 days later with monoclonal antibodies CD34, CD80, and CD86. In the next set of experiments, mice were given intramuscularly injections of 1-3 × 105 tumor cells. Four days later, they were injected intravenously with 6-6.7 mg/kg Dox and intraperitoneally with 100-200 mg/kg CP; 200 mkg human DNA was injected intraperitoneally after CP administration. Differences in tumor size between groups were analyzed for statistical significance by Student's t-test. The MTT-test was done to determine the cytotoxic index of mouse leucocytes from treated groups. RESULTS: The conducted experiments showed that combined treatment with CP and dsDNA preparation produce an increase in the total amount of mature DCs in vivo. Treatment of tumor bearers with preparation of fragmented dsDNA on the background of pretreatment with Dox plus CP demonstrated a strong suppression of tumor growth in two models. RLS, a weakly immunogenic, resistant to alkalyting cytostatics tumor, grew 3.4-fold slower when compared with the control (p < 0.001). In experiment with Krebs-2 tumor, only 2 of the 10 mice in the Dox+CP+DNA group had a palpable tumor on day 16. The cytotoxic index of leucocytes was 86.5% in the Dox+CP+DNA group, but it was 0% in the Dox+CP group. CONCLUSIONS: Thus, the set of experiments we performed showed that exogenous dsDNA, when administered on the background of pretreatment with Dox plus CP, has an antitumor effect possibly due to DC activation.

Characterization of biological peculiarities of the radioprotective activity of double-stranded RNA isolated from Saccharomyces сerevisiae.

THE PURPOSE OF THE ARTICLE: Protection from ionizing radiation is the most important component in the curing malignant neoplasms, servicing atomic reactors, and resolving the situations associated with uncontrolled radioactive pollutions. In this regard, discovering new effective radioprotectors as well as novel principles of protecting living organisms from high-dose radiation is the most important factor, determining the new approaches in medical and technical usage of radiation. MATERIALS AND METHODS: Experimental animals were irradiated on the γ-emitter (Cs137) with a dose of 9.4 Gy. Radioprotective properties of several agents (total RNA, single-stranded RNA, double-stranded RNA and B-190) were estimated by the survival/death rates of experimental animals within 30-90 d. Pathomorphological examination of internal organs end electron microscope assay was done on days 9-12 after irradiation. Cloning and other molecular procedures were performed accordingly to commonly accepted protocols. For assessment of the internalization of labeled nucleic acid, bone marrow cells were incubated with double-stranded RNA labeled with 6-FAM fluorescent dye. Cells with internalized double-stranded RNA were assayed using Axio Imager M1 microscope. In the other experiment, bone marrow cells after incubation with double-stranded RNA were stained with Cy5-labeled anti-CD34 antibodies and assayed using Axioskop 2 microscope. RESULTS: In this study, several biological features of the radioprotective action of double-stranded RNA are characterized. It was shown that 160 µg of the double-stranded RNA per mouse protect experimental animals from the absolutely lethal dose of γ-radiation of 9.4 Gy. In different experiments, 80-100% of irradiated animals survive and live until their natural death. Radioprotective properties of double-stranded RNA were found to be independent on its sequence, but strictly dependent on its double-stranded form. Moreover, double-stranded RNA must have 'open' ends of the molecule to exert its radioprotective activity. CONCLUSIONS: Experiments indicate that radioprotective effect of double-stranded RNA is tightly bound to its internalization into hematopoietic stem cells, which further repopulate the spleen parenchyma of irradiated mice. Actively proliferating progenitors form the splenic colonies, which further serve as the basis for restoration of hematopoiesis and immune function and determine the survival of animals received the lethal dose of radiation.

Combined therapy with cyclophosphamide and DNA preparation inhibits the tumor growth in mice.

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.

Efficacy of a new cancer treatment strategy based on eradication of tumor-initiating stem cells in a mouse model of Krebs-2 solid adenocarcinoma.

Krebs-2 solid carcinoma was cured using a new "3+1" strategy for eradication of Krebs-2 tumor-initiating stem cells. This strategy was based on synchronization of these cells in a treatment-sensitive phase of the cell cycle. The synchronization mechanism, subsequent destruction of Krebs-2 tumor-initiating stem cells, and cure of mice from a solid graft were found to depend on the temporal profile of the interstrand cross-link repair cycle. Also, the temporal profile of the Krebs-2 interstrand repair cycle was found to have a pronounced seasonal cyclicity at the place of experiments (Novosibirsk, Russia). As a result, the therapeutic effect that is based on application of the described strategy, originally developed for the "winter repair cycle" (November-April), is completely eliminated in the summer period (June-September). We conclude that оne of the possible and the likeliest reasons for our failure to observe the therapeutic effects was the seasonal cyclicity in the duration of the interstrand repair cycle, the parameter that is central to our strategy.

Gene expression profiling of tumor-initiating stem cells from mouse Krebs-2 carcinoma using a novel marker of poorly differentiated cells.

Using the ability of poorly differentiated cells to natively internalize fragments of extracellular double-stranded DNA as a marker, we isolated a tumorigenic subpopulation present in Krebs-2 ascites that demonstrated the features of tumor-inducing cancer stem cells. Having combined TAMRA-labeled DNA probe and the power of RNA-seq technology, we identified a set of 168 genes specifically expressed in TAMRA-positive cells (tumor-initiating stem cells), these genes remaining silent in TAMRA-negative cancer cells. TAMRA+ cells displayed gene expression signatures characteristic of both stem cells and cancer cells. The observed expression differences between TAMRA+ and TAMRA- cells were validated by Real Time PCR. The results obtained corroborated the biological data that TAMRA+ murine Krebs-2 tumor cells are tumor-initiating stem cells. The approach developed can be applied to profile any poorly differentiated cell types that are capable of immanent internalization of double-stranded DNA.