The New General Biological Property of Stem-like Tumor Cells (Part II: Surface Molecules, Which Belongs to Distinctive Groups with Particular Functions, Form a Unique Pattern Characteristic of a Certain Type of Tumor Stem-like Cells).

An ability of poorly differentiated cells of different genesis, including tumor stem-like cells (TSCs), to internalize extracellular double-stranded DNA (dsDNA) fragments was revealed in our studies. Using the models of Krebs-2 murine ascites carcinoma and EBV-induced human B-cell lymphoma culture, we demonstrated that dsDNA internalization into the cell consists of several mechanistically distinct phases. The primary contact with cell membrane factors is determined by electrostatic interactions. Firm contacts with cell envelope proteins are then formed, followed by internalization into the cell of the complex formed between the factor and the dsDNA probe bound to it. The key binding sites were found to be the heparin-binding domains, which are constituents of various cell surface proteins of TSCs-either the C1q domain, the collagen-binding domain, or domains of positively charged amino acids. These results imply that the interaction between extracellular dsDNA fragments and the cell, as well as their internalization, took place with the involvement of glycocalyx components (proteoglycans/glycoproteins (PGs/GPs) and glycosylphosphatidylinositol-anchored proteins (GPI-APs)) and the system of scavenger receptors (SRs), which are characteristic of TSCs and form functional clusters of cell surface proteins in TSCs. The key provisions of the concept characterizing the principle of organization of the "group-specific" cell surface factors of TSCs of various geneses were formulated. These factors belong to three protein clusters: GPs/PGs, GIP-APs, and SRs. For TSCs of different tumors, these clusters were found to be represented by different members with homotypic functions corresponding to the general function of the cluster to which they belong.

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.

[Novel Glycyrrhetinic Acid Derivative Soloxolone Methyl Inhibits the Inflammatory Response and Tumor Growth in vivo].

Due to wide spreading of inflammatory disease and imperfection of available anti-inflammatory drugs, mainly associated with their serious side effects, searching for new anti-inflammatory agents is a pressing problem. Natural triterpenoids and their synthetic analogs are a promising source of new drugs. In this study, we have investigated the anti-inflammatory and antitumor effects in vivo of the glycyrrhetinic acid derivative soloxolone methyl (SM), or methyl 2-cyano-3,12-dioxo-18ßH-olean-9(ll),l(2)-dien-30-oate. SM was shown to efficiently suppress the development of edema in a mouse model of carrageenan- or histamine-induced acute inflammation. SM also inhibited the tumor growth and reduced the tumor cell count in the ascitic fluid in mice bearing Krebs-2 carcinoma, the development of which is accompanied by an inflammatory process in the surrounding tissues.

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.

A strategy to eradicate well-developed Krebs-2 ascites in mice.

We describe the strategy, which allows curing experimental mice engrafted with Krebs-2 ascites. The strategy is based on the facts that i) Krebs-2 tumor-initiating stem cells (TISCs) are naturally capable of internalizing fragments of extracellular double-stranded DNA (dsDNA); ii) upon delivery into TISCs, these dsDNA fragments interfere with the on-going DNA repair process so that TISCs either die or lose their tumorigenic potential. The following 3-step regimen of therapeutic procedures leading to eradication of Krebs-2 ascites is considered. Firstly, three timed injections of cyclophosphamide (CP) exactly matching the interstrand cross-link (ICL) repair phases that lead to synchronization of ascites cells in late S/G2/M. Secondly, additional treatment of ascites 18 hours post each CP injection (at NER/HR transition timepoint) with a composite dsDNA-based preparation interfering with the NER and HR repair pathways, so that tumorigenic properties of ascites cells are compromised. Thirdly, final treatment of mice with a combination of CP and dsDNA injections as ascites cells undergo apoptotic destruction, and the surviving TAMRA+ TISCs arrested in late S/G2/M phases massively enter into G1/S, when they regain sensitivity to CP+dsDNA treatment. Thus, this regimen assures that no viable cells, particularly Krebs-2 TISCs, remain.

Identification of cancer stem cells and a strategy for their elimination.

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.

Switching between control and phytohaemagglutinin-containing diets affects growth of Krebs II ascites cells and produces differences in the levels of putrescine, spermidine and spermine.

Almost twice as many ascites tumour cells were recovered from mice pre-fed for 3 days on a lactalbumin (La)-based control diet, injected with Krebs II ascites cells and then maintained on the same diet for a further 8 days, when compared with mice fed on a phytohaemagglutinin-containing (PHA) diet for the whole period. A dietary switch on the day of injection of tumour cells produced an intermediate effect; mice switched to the La diet after pre-feeding on PHA for 3 days developed somewhat more tumour cells than when the opposite dietary switch was performed. The polyamine content in the tumour cells was lowest in the mice fed on La, and highest in mice fed PHA for the duration of the experiment, respectively. Since large amounts of extraneous polyamines are required in order to sustain tumour growth, and the hyperplastic growth of the gut which occurs in response to injesting the lectin is a polyamine-dependent process, it is evident that the two growth signals compete with one another for important nutrients/growth factors, including polyamines.

A diet containing the lectin phytohaemagglutinin (PHA) slows down the proliferation of Krebs II cell tumours in mice.

Mice injected intraperitoneally with Krebs II cells and then fed on a diet containing the lectin phytohaemagglutinin (PHA) developed ascites tumours more slowly than mice fed on a control diet. After an 8-day period following injection the number of cells recovered from mice maintained on the PHA diet was half that from those fed the control diet. A switch of diet from control to PHA on day 4 after injection resulted in a large decrease in number of tumour cells recovered. Mice injected s.c. also developed tumours at later times when fed on the PHA diet. A quantitative of ribosomes in polysome-containing fractions showed no major differences in protein synthesis in control mice and those fed the PHA diet.

[Effects of exogenous poly(A)+ mRNA on antigenic properties and growth of Krebs-2 tumor cells]. / Vliianie ékzogennoi poli(A)+ mRNK na antigennye svoistva i rost kletok opukholi Krebs-2.

The treatment of Krebs-2 ascitic tumor cells (TC) with total RNA from the liver of Wistar rats (2 mg/ml) altered their antigenicity. As a result, the growth of treated TC in contrast with untreated TC was inhibited when transplanted i. m. to mice preimmunized with rat liver homogenate. Investigations of poly(A+)mRNA, rRNA and tRNA isolated from the same tissue established that the alteration of antigenicity is due to mRNA (8-24 micrograms/ml). In the cytotoxicity assay with antisera against rat Wistar antigens, the expression of rat antigens in TC treated mRNA was observed in the next cell generations.

Treatment of mice bearing a Krebs ascitic tumor by means of a protocol based on radioactive copper (64Cu). III. Efficiency of the anticancer treatment according to the stage of tumor development.

The treatment described previously (Anticancer Res 9:947-954,1989) was efficient when applied on day 1 or 6 after the injection of 5 x 10(5) Krebs ascitic cells in mice. Under our experimental conditions, all the untreated mice died within 12 to 25 days. The experiments described show that the treatment developed, is efficient when applied on day 11 but not on day 16. To understand the difference in the treatment efficiency between these two days, we tested the 64Cu incorporation inside the ascitic cells. It was observed that 64Cu incorporation exists on day 11 but no longer on day 16. On day 16, the inefficiency of the treatment must be correlated with the non-incorporation of 64Cu inside the ascitic cells. As the tumor growth is also arrested on day 16, an irreversible stage is reached. A model was developed to explain the results obtained. In this model, cancer develops in 3 successive stages. In the first stage, the cellular functioning is under the control of the malignant tumor cells and the number of cells with the "cancer functioning" is increasing with time, but decreasing after removal of the malignant tumor; in the second stage, tumor and cancer both develop independently, meaning that the number of cells with the "cancer functioning" will continue to increase after the removal of the malignant tumor; in the third stage, each cell of the organism has the "cancer functioning" and the characteristics of malignancy will by retroaction.

[Antiproliferative action of swine leukocyte interferon and of an inhibitor of interferon action]. / Antiproliferativnoe deistvie svinogo leikotsitarnogo interferona i ingibitora deistviia interferona.

The results of the study of the antiproliferative effect of swine leukocyte interferon and of an inhibitor of interferon effect in experimental mice with transplanted Krebs-2 ascitic carcinoma cells are presented. The interferon inhibitor exerted antiproliferative effect similar to that of native swine interferon. Combined use of swine interferon and interferon inhibitor did not lead to summation of the antiproliferative effect of these preparations.

Effects of growth rate and methotrexate on folate polyglutamates and folylpolyglutamate hydrolase activity in Krebs ascites cells.

The activity of folylpolyglutamate hydrolase was measured throughout intraperitoneal growth of Krebs ascites cells in mice and after exposure to methotrexate. Hydrolase activity was lowest during the log phase of growth. Methotrexate administered intraperitoneally during log growth caused a dose- and time-dependent increase in hydrolase activity. Modest changes were observed in endogenous folate polyglutamate chain length distributions throughout growth and upon exposure to methotrexate, but these changes could not be correlated with hydrolase activity.

Ascites tumor invasion of mouse peritoneum studied by high-voltage electron microscope stereoscopy.

Interaction of Krebs-2 and Ehrlich tetraploid cells with NYLR/Nya mouse peritoneum mesothelium and penetration of basal lamina and elastic reticulum were studied. Invasion of abdominal viscera was rare. Invading cells had a shrunken nucleus and cytoplasm like the "dark cells" of hyperplastic epithelia. High-voltage electron microscope stereoscopy showed that invasive cells pass through small holes in the elastic reticulum by adherence to the reticulum and by constriction of the cells. High voltage electron microscopy stereoscopy of collagen fibers near tumor cells indicated that fragmentation and loss of collagen is minimal. Rapid progression by ascites transfer appears to produce anchorage-independent cells adapted to ascites fluid growth, but new selection steps must be adopted to concentrate strongly invasive subpopulations.

Comparison of the rate of aminoacylation of tRNA isolated from NMRI mouse liver with tRNA isolated from Krebs II ascites or mouse plasmacytoma cells.

A comparison of the initial rates of aminoacylation of tRNAs isolated from different sources for 17 amino acids was performed. tRNA was isolated from NMRI mouse liver (tRNA L) and from Krebs II ascites tumors (tRNA Asc), and aminoacyl-tRNA synthetases were prepared from the latter cells. The aminoacylation of tRNA Asc was 31-88% slower than the charging of tRNA L. In similar studies, tRNA from a mouse plasmacytoma tumor (tRNA Mt) and from suspension cultured cells of the same cell line (tRNA M) were compared to tRNA L in the aminoacylation reaction catalysed by synthetases isolated from tumor or suspension cultured cells. About half of the tRNAs (Mt or M) for the 17 amino acids tested differed in charging rate when compared to tRNA L, but the differences were not as great as those observed in the experiments where tRNA Asc and tRNA L were compared.

Comparison of phosphorylation of ribosomal proteins from HeLa and Krebs II ascites-tumour cells by cyclic AMP-dependent and cyclic GMP-dependent protein kinases.

Phosphorylation of eukaryotic ribosomal proteins in vitro by essentially homogeneous preparations of cyclic AMP-dependent protein kinase catalytic subunit and cyclic GMP-dependent protein kinase was compared. Each protein kinase was added at a concentration of 30nM. Ribosomal proteins were identified by two-dimensional gel electrophoresis. Almost identical results were obtained when ribosomal subunits from HeLa or ascites-tumour cells were used. About 50-60% of the total radioactive phosphate incorporated into small-subunit ribosomal proteins by either kinase was associated with protein S6. In 90 min between 0.7 and 1.0 mol of phosphate/mol of protein S6 was incorporated by the catalytic subunit of cyclic AMP-dependent protein kinase. Of the other proteins, S3 and S7 from the small subunit and proteins L6, L18, L19 and L35 from the large subunit were predominantly phosphorylated by the cyclic AMP-dependent enzyme. Between 0.1 and 0.2 mol of phosphate was incorporated/mol of these phosphorylated proteins. With the exception of protein S7, the same proteins were also major substrates for the cyclic GMP-dependent protein kinase. Time courses of the phosphorylation of individual proteins from the small and large ribosomal subunits in the presence of either protein kinase suggested four types of phosphorylation reactions: (1) proteins S2, S10 and L5 were preferably phosphorylated by the cyclic GMP-dependent protein kinase; (2) proteins S3 and L6 were phosphorylated at very similar rates by either kinase; (3) proteins S7 and L29 were almost exclusively phosphorylated by the cyclic AMP-dependent protein kinase; (4) protein S6 and most of the other proteins were phosphorylated about two or three times faster by the cyclic AMP-dependent than by the cyclic GMP-dependent enzyme.

Inhibiting effect of vitamins C and B12 on the mitotic activity of ascites tumors.

The mitotic activity of the transplantable mouse tumors, Sarcoma 37, Krebs-2, and Ehrlich carcinomas, in the ascites form, were inhibited after treatment with a mixture of vitamins C and B12 with no apparent toxic side effects. These vitamins when administered alone, at the same dosage, did not seem to have any apparent effect on mitosis or the morphology of the cells studied. Microscopic examination of the stained ascites fluid taken from the mice treated with the vitamin mixture showed few tumor cells, and these in various stages of disintegration. Also, an increase in lymphocytes, monocytes and neutrophils were noticed; however, later in the experiment, no tumor cells could be found and monocytes and macrophages were abundant.