RNA Sequencing and Cell Models of Virus-Associated Cancer (Review).

The review summarizes findings from the studies based on the application of technologies for transcriptome analysis to modern cellular model systems of human papillomavirus-associated cancer (HPV) (cervical cancer, head and neck tumors). A diversity of three-dimensional cancer models, such as spheroids, organoids (organotypic cultures), explants, mouse xenografts, are addressed. Particular attention is paid to the use of patient-derived biomaterial for establishing short-term cultures of primary tumor cells, as well as generating multicomponent (heterocellular) systems that comprise, together with the tumor component, other elements of its microenvironment. A number of unique biological properties of HPV-induced neoplasia are discussed, which make generating cell models a unique task. The novel findings in the field of molecular mechanisms of the onset and progression of HPV-associated cancer achieved by using RNA sequencing are presented for each variant of the model systems. These findings are considered in regard to applied aspects of their use, in terms of the opportunities for preclinical testing of new drugs, personalized diagnostics and selection of individual, most effective treatment regimens. The issues of drug resistance development, molecular-cellular heterogeneity, epigenetic reprogramming, and the role of the stromal microenvironment are reviewed. The paper accentuates the problems related to the limitations of the applicability of a particular model system. The areas with a significant lagging behind in omics research of virus-associated cancer in comparison with other types of oncological pathology and possible causes of this lag are noted. The future prospects for the development of model systems of HPV-associated tumors in the field of high-tech tissue engineering, in particular, the use of bioprinting and microfluidic biochips, are also outlined. The combination of these techniques with the methods of whole genome profiling will significantly increase the translational potential of the described model cell systems.

Expression of DROSHA and DICER genes in peripheral blood leukocytes in lung sarcoidosis.

AIM: To study the expression level of the genes DROSHA and DICER in peripheral blood leukocytes (PBL) of patients with sarcoidosis of the lungs. MATERIALS AND METHODS: The study included 32 patients diagnosed with persistent lung sarcoidosis (mean age 41.56±1.27 years) and 36 healthy donors (control; mean age 42.79±1.95 years). The level of expression of messenger RNA (mRNA) of the genes DROSHA and DICER were determined in PBL of healthy donors and patients with sarcoidosis of the lung by polymerase chain reaction in real time. RESULTS: As a result of the conducted researches it is established that the level of drosha gene expression in PBL patients with sarcoidosis of lungs is significantly reduced in comparison with the control (p<0.01). We also found a significant decrease in the number of Dicker gene transcripts in the PBL of the study group of patients (p<0.01). CONCLUSION: According to the results of the conducted studies, a significant decrease in the number of DROSHA and DICER transcripts in PBL patients with the development of lung sarcoidosis has been found, which can contribute to the pathogenesis of this disease.

[Remodeling of angiogenesis and lymphangiogenesis in cervical cancer development].

Ability to stimulate angiogenesis/lymphangiogenesis is recognized as an inherent feature of cancer cells providing necessary conditions for their growth and dissemination. "Angiogenic switch" is one of the earliest consequences of malignant transformation that encompasses a great number of genes and triggers a complex set of signaling cascades in endothelial cells. The processes of tumor microvasculature development are closely connected to the steps of carcinogenesis (from benign lesions to invasive forms) and occur through multiple deviations from the norm. Analysis of expression of proangiogenic factors at successive steps of cervical cancer development (intraepithelial neoplasia, cancer in situ, microinvasive, and invasive cancer) enables to reconstruct the regulatory mechanisms of (lymph-)angiogenesis and to discriminate the most important components. This review presents detailed analysis of literature data on expression of the key regulators of angiogenesis in cervical intraepithelial neoplasia and cervical cancer. Their possible involvement in molecular mechanisms of neoplastic transformation of epithelial cells, as well as invasion and tumor metastasis is discussed. Correlation between expression of proangiogenic molecular factors and various clinicopathological parameters is considered, the potential of their use in molecular diagnostics and targeted therapy of cervical cancer is reviewed. Particular attention is paid to relatively poorly studied regulators of lymphangiogenesis and "non-VEGF dependent", or alternative, angiogenic pathways that constitute the prospect of future research in the field.

[Septic Shock: Innate Molecular Genetic Mechanisms of the Development of Generalized Inflammation].

The capacity for immune surveilance and protection against genetically alien agents is a basic property of multicellular organisms, and increasing significance in realizing this, capacity is assigned to mechanisms of innate immunity. The data accumulated to date show that many components of these mechanisms have a very wide spectrum of biological functions and play essential roles at different stages of ontogeny. An illustrative example is the signal system activated by tumor necrosis factor alpha (TNFα), which is responsible for the inflammation process. Analysis of its structural organization has shown that signaling mechanisms initiating inflammation largely overlap with mechanisms of programmed cell death. This is why hypersecretion of TNFα may lead to systemic inflammatory reation, or septic shok, and, hence, have a fatal outcome. Although studies on the TNFα-dependent mechanism have long history, many aspects of its regulation remain obscure. In particular, this concerns the nature of interspecific differences in the sensitivity of mammals to TNFα action and the ability of TNFα to activate oppositely directed cell programs depending on cell type or ambient conditions. The numerous data obtained in studies on different experimental systems need generalization and critical analysis. This review is an attempt at such an analysis. Its scope is concentrated on modern views on the divergence of TNFα-induced signal at the level of intracellular receptor-associated proteins. A description is given to potential "molecular triggers" responsible for switching between the main TNFα-dependent signaling pathways: inflammation, apoptosis, and necroptosis. The contribution of necroptosis (genetically programmed necrotic cell death) to the development of systemic inflammation and the lethal effect of TNFα are described. Consideration is also given to various lines of mice possessing natural resistance or sensitivity to TNFα, which hold much promise as models for deciphering the molecular genetic bases of the regulation of innate immune reactions and other TNFα-dependent processes.

[Functional redistribution of caspase activities in K562 cells, induced for differentiation and apoptosis by thiazophosphol derivatives].

Activity of caspases 3, 9, 6 has been investigated in erythroleukemia K562 cells under conditions of induction or inhibition of erythroid differentiation using cyclophosphane (N'-bis-(beta-chlorethyl)-N'-O-trimethyl ester of diamide of phosphoric acid, antitumour agent of oncologic practice) and two newsynthetic thiazophosphol derivatives--2-t-butylamino-4-thioxo-4-chloromethyl-1,3,4-thiazophosphol-2-in, 2-anilino-4-thioxo-4-chloromethyl-1,3,4-thiazophosphol-2-in as modulators of differentiation. The treatment of K562 cells with cyclophosphane and the t-butylamine thiazophosphol derivative was accompanied by the induction of erythroid differentiation, activation of caspase 3 and caspase 9, followed by subsequent induction of apoptosis. Treatment of cancer cells with the aniline thiazophosphol derivative led to a loss of erythroid differentiation in cells and increased expression of the monocyte lineage associated surface antigen, activation of caspases 3, 9, 6, and induction of apoptosis. In the latter case the level of activity of caspase 3 was lower than in the cells treated in the presence of other compounds. Possible functional redistribution of activity of cell caspases involved in the realization of different directions of differentiation and apoptosis in K562 cells is discussed.

[Activity of genetically programmed cell death of lymphocytes in rheumatoid arthritis].

AIM: To study activity of genetically programmed cell death (PCD) of lymphocytes in patients with rheumatoid arthritis (RA). MATERIAL AND METHODS: Lymphocytes from 30 RA patients including 14 patients with RA history up to 2 years, and 12 healthy donors were studied for activity of caspase 4, 6, 8 usingfluorescence and caspase substrates 30, 60, 120, 150 and 180 min after start of the reaction and number of 1-2-thread serration of DNA with fluorescence of two DNA-tropic stains--EtBr and 4,6-DAPY Correlation was also studied between lymphocyte activity and RA activity, x-ray stage, duration, level of TNF-alpha. The trend in PCD activity of lymphocytes in immunosuppressive therapy was analysed. RESULTS: The activity of lymphocytic caspases in RA patients was low compared to healthy donors. A negative correlation was found between RA activity and activity of caspase 8 and 6. In RA therapy with methotrexate, sulphasalasine, glucocorticosteroids the activity of caspases and number of 1-thread serrations of DNA were subnormal. CONCLUSION: RA is associated with lymphocyte PCD disorders which may be involved in RA pathogenesis, in formation of clones of potentially autoaggressive lymphocytes, in particular. This process is not normalized by current methods of RA treatment causing unsatisfactory outcomes of RA therapy.

[Cellular mechanisms of induction of apoptosis in human erythroleukaemic K562 cells line treated by quinoline-N-oxide derivatives].

We have studied the influence of 2-(4'-nitrostyryl)-quinoline-1-oxide (2-NSQO) and 4-(4'-nitrostyryl)-quinoline-1-oxide (4-NSQO) on modulation activity of microsomal NADPH-oxidoreductases, concentration of nicotinamide enzymes and induction of apoptosis in human erythroleukaemic K562 cells. It was shown, that activity of microsomal NADPH-cytochrome c-reductases in cancer cells was inhibited by 10 microM 4-NSQO (15%), and 10 microM 2-NSQO (50%). Treatment of cells with these reagents for two days, was accompanied by caspases-9 and -3 activation, rise of EtBr and DAPI fluorescence related to DNA binding and induction of apoptosis. Apoptosis was preceded by the decrease of concentration of nicotinamide enzymes. Thus 4-NSQO is a promising compound for further experimental trials as an anticancer drug with low toxic action on the tissues of organism.

[Phorbol 12-myristate 13-acetate inhibits apoptosis in erythroleukemia K562 cells induced by some nucleosides].

We studied the ability of phorbol 12-myristate 13-acetat to prevent erythroid differentiation and apoptosis in erythroleukemic K562 cells induced by cytidine, thymidine, and guanosine. The exposure of cancer cells to combinations of phorbol 12-myrsitate 13-acetate (100 nM) nucleosides for two days led to a loss of hemoglobin production (marker of erythroid differentiation) in cells and increased expression of monocyte-macrophage lineage associated surface antigen CD14. The treatment of K562 cells with nucleosides only was accompanied by the activation of caspase-3 and caspase-9, rather than caspase-6, increased fluorescence of ethidium bromide and DAPI upon binding to DNA, and apoptosis. Intracellular activation of caspase-6, inhibition of caspase-9, a markedly decreased activity of caspase-3 and of fluorescence of DNA-binding dyes, and inhibition of apoptosis were observed when the cells were treated with phorbol 12-myeristet 13-acetate combined with nucleosides.

[Erythroid differentiation of K562 cells resistant to 2-(4'-dimethylaminostyryl)quinoline 1-oxide or 4-nitroquinoline 1-oxide is significantly increased after thymidine treatment]. / Eritroidnaia differentsirovka kletok K562, rezistentnykh k 2-(4-dimetilaminostiril)khinolin-1-oksidu ili 4-nitrokhinolin-1-oksidu, znachitel'no usilivaetsia pri obrabotke timidinom.

We studied the effect of certain differentiation inducers-thymidine, sodium butyrate, and dimethyl sulfoxide--on the cells of parental line K562 and the derived sublines resistant to quinoline xenobiotics 2-(4-dimethylaminostyryl)quinoline 1-oxide or 4-nitroquinoline 1-oxide. The cells of the both derived sublines demonstrate cross-resistance to these xenobiotics, while the subline resistant to 2-(4-dimethylaminostyryl)quinoline 1-oxide is also resistant to ethidium bromide and colchicine. Treatment of cells of the sublines with thymidine but neither sodium butyrate nor dimethyl sulfoxide for 2 or 4 days considerably increases intracellular content of hemoglobin as compared to the parental cells K562. The revealed effect is due to increased hemoglobin content in the cells rather than to the increased proportion of hemoglobin-containing cells, which results from decreased proliferation rate observed in all cases.

[Chemically-induced differentiation of the tumor cell lines]. / Khimicheski indutsirovannaia differentsirovka kletok opukholevykh linii.

We considered the biological effects of some chemical compounds, that stimulate cell differentiation, on the cells of tumor lines. Induced changes were analyzed in the expression of several transcriptional factors involved in the regulation of cell proliferation and apoptosis. Based on the generalized information about differentiating, apoptogenic, and antiproliferative effects of chemical inducers of differentiation, a conclusion was drawn that they affect the sensitivity of differentiated cells to the lytic action of natural killer cells.

[Phorbol-12-myristate-13-acetate prevents the inhibitory effect of A23187 on erythroid differentiation of K562 cells induced by dimethylsulfoxide]. / Forbol-12-miristat-13-atsetat otmeniaet ingibiruiushchee deistvie A23187 na éritroidnuiu differentsirovku kletok K562, indutsirovannuiu dimetilsul'foksidom.

The differentiating effect of DMSO on K562 cells was studied against the background of pretreatment of the cells by the modulators of activities of protein kinase C and Ca signaling, phorbol 12-myristate 13-acetate, and ionophore A23187. The 2-hour pretreatment of K562 cells with A23187 (1 microM), rather than phorbol 12-myristate 13-acetate (0.1 microM), led to inhibition of the differentiating effect of DMSO (0.1%) during the subsequent four days of incubation. When the cells were pretreated jointly with A23187 and phorbol 12-myristate 13-acetate, the DMSO-induced erythroid differentiation was restored. Analysis of the DNA-degrading effect of the reagents used on K562 cells by fluorescent dyes under the same conditions suggests that this activity is induced in the presence of DMSO in the cells pretreated with a combination of phorbol 12-myristate 13-acetate and A23187 or without pretreatment.

[Comparative analysis of differentiating and apoptogenic effects of cytidine, thymidine, and guanosine on cells of human erythromyeloleukemic cell line K562]. / Sravnitel'nyi analiz differentsiruiushchego i apoptogennogo deistvii tsitidina, timidina i guanozina na kletki éritromieloleikoznoi linii cheloveka K562.

The antiproliferative, differentiating and apoptogenic effects of purinic (cytidine and thymidine) and pyrimidinic (guanosine) nucleosides on K562 cells were investigated. Induction of erythroid differentiation (haemoglobin synthesis) in cancer cells was observed after 2 and 4 days incubation with 2 mM thymidine and 1 mM guanosine. Increase of haemoglobin synthesis in K 562 cells incubated with 2 mM cytidine was detected at 4 day. Analysis of the DNA-degrading effect (induction of apoptosis) of the reagents used on K 562 cells by fluorescent dyes and modulation activity of caspase 9 and also caspases 3,7,10 is induced with thymidine and guanosine but not with cytidine.

[Combination of thymidine and dexamethasone increases the K562 tumor cells sensitivity to human leukocytes cytotoxicity]. / Obrabotka kletok K562 timidinom na fone deksametazona povyshaet chuvstvitel'nost' opulevykh kletok k liticheskomu deistviiu leikotsitov cheloveka.

The influence of the number of differentiating agents on sensitivity of human erythroleukemic cells K562 to human leukocyte-mediated non-MHC-restricted lysis was studied. It has been shown that a 4-day treatment of cells K562 by dexamethasone (1 microM) or phorbol-12-myristate-13-acetate (100 nM) leads to a significant decrease in sensitivity of the treated cells to non-specific lysis mediated by human leukocytes. On the contrary, the treatment of cells K562 by a combination of dexamethasone and thymidine (2 mM) leads to an increased sensitivity of the treated cancer cells to non-specific lysis mediated by the above effector cells, compared with the situation when these cells were treated by dexamethasone only. The treatment of cells K562 by a combination of thymidine and phorbol-12-myristate-13-acetate demonstrates a tendency (P < 0.1) to increase the sensitivity to non-specific lysis mediated by human leukocytes, as compared with the cases, when these cells were treated by phorbol ester only. It has been shown that the changes in K562 cell sensitivity to lytic action of leukocytes, under the chosen incubation time and doses of the used agents, well compare with the changes of erythroid differentiation of the cancer cells in the same conditions.

[Changes in the K562 cell sensitivity to nonspecific lysis by human and rat leukocytes under the influence of sodium butyrate, dimethyl sulfoxide and phorbol-12-myristate-13-acetate]. / Izmenenie chuvstvitel'nosti kletok K562 k nespetsificheskomu lizisu leikotsitami cheloveka i krysy pod vliianiem biturata natriia, dimetilsul'foksida i forbol-12-miristat-13-atsetata.

We studied the ability of inducers and inhibitors of erythroid differentiation of K562 leukemia cells, such as sodium butyrate, dimethyl sulfoxide, and phorbol-12-myristate-13-acetate, respectively, to modulate sensitivity of these cells to non-specific lysis (non-restricted with respect to antigens of the major histocompatibility complex) mediated by natural human or rat killer cells. Unfractionated leukocytes from human peripheral blood or rat splenocytes were used as sources of natural killers. The induction of erythroid differentiation by sodium butyrate was accompanied by a significant increase in cell sensitivity to lysis with human peripheral blood lymphocytes; incubation of K562 cells in the mixture of sodium butyrate and dimethyl sulfoxide did not change cell sensitivity to lysis by both types of effector cells. The inhibition of sodium butyrate-induced erythroid differentiation with high doses of phorbol-12-myristate-13-acetate (100 nM; incubation was in the presence of both these agents simultaneously) resulted in an increased cell sensitivity to lysis with rat splenocytes. Incubation of K562 cells in a mixture of sodium butyrate, dimethyl sulfoxide, and phorbol-12-myristate-13-acetate (100 nM) produced greater lysis by human leukocytes, as compared with incubation in the mixture of sodium butyrate and dimethyl sulfoxide.

[Interaction of tumor cell differentiation induced in vitro and sensitivity of them to nonspecific lysis by natural killer cells. Possible mechanisms]. / Vzaimosviaz' indutsirovannoi in vitro differentsirovki kletok opukholevykh linii i chuvstvitel'nosti ikh k nespetsificheskomu lizisu estestvennymi killernymi kletkami. Vozmozhnye mekhanizmy.

The literature data on the influence of a large group of cancer cell differentiation inducers on the modulation of their susceptibility to non-MHC-restricted lysis (non-specific cytotoxicity, NCT) by natural killer (NK) cells have been analysed. A possible association between the apoptogenic action of differentiation inducers and their ability to modulate the NCT of cell targets by NK cells is discussed.