Lewis Carcinoma Cells Research Articles

МЕХАНИЗМЫ ПРОТИВООПУХОЛЕВОГО ДЕЙСТВИЯ МАЛЫХ ДОЗ РАДИАЦИИ, СВЯЗАННЫЕ С АКТИВАЦИЕЙ ЗАЩИТНЫХ СИСТЕМ КЛЕТКИ

Background: The effects of ionizing radiation (IR) involve a highly orchestrated series of events in cells, including DNA damage and repair, cell death, and changes in the level of proliferation associated with the stage of the cell cycle. A large number of existing studies in literature have examined the activity of genes and their regulators in mammalian cells in response to high doses of ionizing radiation. Although there are many studies, the research in effect of low doses of ionizing radiation remains limited. Though much progress has been made in understanding the basic principles of effects of low doses radiation on individual components of biological systems, less is known about how low doses affect target molecules and regulate the cellular networks (e.g., activation of the immune system, genes and their regulators in the phenomenon of hormesis, the formation of an adaptive response). These observations determined the purpose of the work: to investigate the activity of genes and non-coding RNAs (long non-coding RNAs and microRNAs) in various organs of mice with transplanted Lewis carcinoma after low doses radiation. Material and methods: 24 female mice C57Bl/6 were transplanted subcutaneously with Lewis carcinoma cells (105 cells in 0.2 ml of Hanks’ solution). Total 4-fold X-ray irradiation with an interval of 4 days at a dose of 0.075 Gy (0.85 Gy/min) was performed on the RUST M1 from 6 days after transplantation; the tumor size was measured daily. The mice were divided into the following groups: biocontrol, biocontrol+irradiation, tumor and tumor+irradiation. On the 19th day from the beginning of the experiment, the mice were euthanized. The expression profiles of mRNA genes, long non-coding RNAs and microRNAs controlling the response to radiation were determined in the bone marrow, thymus, spleen and tumor of mice. Results: Fractionated low doses irradiation of mice with transplanted Lewis carcinoma caused a growth decrease of implanted tumour cells compared to the similar group without irradiation. At the same time, there was an activation of oncosuppressors, and a decrease in the activity of oncogenes in the thymus and spleen of mice with tumor and irradiation. In the tumor group, without irradiation, the number of activated oncogenes prevailed over the number of inactivated ones. Conclusion: Thus, the low doses radiation exposure led to the activation of antitumor immunity in mice, which emerged in slowing tumor growth in animals and was represented in the induction of oncosuppressors and inhibition of oncogenes expression.

Exposition of Phosphatidylserine in Lewis Carcinoma Cells under the Action of Anphen Sodium and Hydrogen Peroxide

Exposition of Phosphatidylserine in Lewis Carcinoma Cells under the Action of Anphen Sodium and Hydrogen Peroxide

Apoptotic effect of the anphen sodium antioxidant in combination with H2O2 on Lewis carcinoma cells

It was shown by immunoblotting that the introduction of an antitumor antioxidant, sodium 2-carboxy-2-(N-acetylamino)-3-(3,5-di-tert-butyl-4-hydrophenyl)propanoate (anphen sodium), into a suspension of Lewis carcinoma cells induces a sharp decrease in the contents of the monomer and homodimer of the anti-apoptotic Bcl-2 protein. According to fluorescence analysis, the number of apoptotic cells sharply increased 1 h after exposure to anphen sodium; preliminary addition of 5 μmol L-1 of hydrogen peroxide increased the cell membrane permeability of anphen sodium and the number of apoptotic cells. The onset of apoptosis detected using fluorophores is comparable in time with the start of the decline of the Bcl-2 level. It was assumed that the mechanism of action of anphen sodium may include the interaction of the agent with the hydrophobic (BH3) domain of Bcl-2 family proteins.

Non-irradiated Bystander Fibroblasts Attenuate Damage to Irradiated Cancer Cells

Introduction and aim: Radiation-induced bystander effect is described as a different type of responses, displayed by non-irradiated neighbouring cells, induced by signals transmitted from irradiated cells. These responses in bystander cells include genetic damage (SCE, micronuclei, genomic instability), apoptosis induction and other non-necessarily detrimental effects. Bystander effect might bear some implications for coexisting normal cells non-targeted by cancer radiotherapy. However, it is possible, that bystander effect can act in opposite direction, and non-irradiated cells can in some way influence the response of targeted cells. Our experiments in vitro were aimed at evaluating this concept. Materials and methods: Lung Lewis carcinoma cells (LLC), growing in monolayer in 6-well plates, were irradiated with 2 or 4 Gy dose of X-rays (using a 6 MeV accelerator suited for therapeutic purposes). After irradiation, the cells were co-cultured with non-irradiated NIH3T3 mice fibroblasts, the latter growing (in monolayer) in special inserts. Such system allowed separation of the two kinds of cells, with medium freely circulating through the separation membrane (pore size 0.4 µm). Thus, species released by irradiated cells could be transmitted to non-irradiated neighbours and vice versa. Results and discussion: The bystander effects, caused by irradiated cancer cells, which were observed in non-targeted fibroblasts included dose-dependent elevation of micronucleus and apoptosis frequency indicating that irradiated cancer cells can induce damage in normal fibroblast cells. However, irradiated LLC cancer cells co-incubated with fibroblasts presented lower levels of this type of cytogenetic damage and apoptosis in comparison with LLC cells incubated after irradiation without fibroblasts on inserts or without inserts at all. Our current studies attempt to search for agents and signalling responsible for observed bilateral bystander effects. Supported by the Polish Ministry of Science and High Education, grant no N406 101/31/3870

DAP kinase links the control of apoptosis to metastasis.

DAP kinase is a new type of calcium/calmodulin-dependent enzyme that phosphorylates serine/threonine residues on proteins. Its structure contains ankyrin repeats and the 'death' domain, and it is associated with the cell cytoskeleton. The gene encoding DAP kinase was initially isolated as a positive mediator of apoptosis induced by interferon-gamma, by using a strategy of functional cloning. We have now tested whether this gene has tumour-suppressive activity. We found that lung carcinoma clones, characterized by their highly aggressive metastatic behaviour and originating from two independent murine lung tumours, did not express DAP kinase, in contrast to their low-metastatic counterparts. Restoration of DAP kinase to physiological levels in high-metastatic Lewis carcinoma cells suppressed their ability to form lung metastases after intravenous injection into syngeneic mice, and delayed local tumour growth in a foreign 'microenvironment' Conversely, in vivo selection of rare lung lesions following injection into syngeneic mice of low-metastatic Lewis carcinoma cells or of DAP kinase transfectants, was associated with loss of DAP kinase expression. In situ TUNEL staining of tumour sections revealed that DAP kinase expression from the transgene raised the incidence of apoptosis in vivo. DAP-kinase transfectants also showed increased sensitivity in vitro to apoptotic stimuli, of the sort encountered by metastasizing cells at different stages of malignancy. We propose that loss of DAP kinase expression provides a unique mechanism that links suppression of apoptosis to metastasis.

Lewis肺癌実験モデルにおけるLaserメス減量術後の影響

Background. Malignant tumor cells are believed to be shed into the bloodstream during conventional surgical resection of a primary tumor. In head and neck neoplasms, laser resection (Nd: YAG and CO2) is one treatment for resection of organ-confined primary tumors. To date, however, the evidence of malignant tumor cell dissemination during laser resection remains unclear. In this study, the author have examined the effect of laser reduction on the shedding of tumor cells into blood and micrometastasis formation in the lung using a marker gene-tagged mouse Lewis lung carcinoma model.Methods. 1×106 Lewis carcinoma cells transfected with hygromycin-resistant gene (Hyg) were injected subcutaneously into C57BL male mice, and tumor cells in blood and the lung were estimated after 10 minutes of laser reduction of primary tumors leaving 1/5 of them by polymerase chain reaction for Hyg as atarget gene. Macroscopic metastasis was estimated by counting visible parietal nodules in the lung after fixation in Bouin's solution.Results and conclusion. The detection rate of circulating tumor cells in blood was higher in the Nd: YAG (54.5%) and CO2 (60.0%) laser treatment group than in the control group (30.0%). Macroscopic metastasis in the lung were also increased in the Nd: YAG and CO2 laser treatment group, when compared with the control group. These results suggest that laser reduction of malignant tumor enhances tumor cell shedding into the bloodstream.

A 92-kDa human immunostimulatory protein.

We purified to apparent homogeneity a human urinary glycoprotein of 92 kDa (HGP.92) that, administered intravenously at 250 micrograms/kg, fully protected mice against a lethal inoculum of Listeria monocytogenes. Since HGP.92 protected scid mice, which lack B and T lymphocytes, this increased resistance to Listeria did not appear to be lymphocyte mediated. Furthermore, inflammatory macrophages incubated with 6 nM HGP.92 inhibited the growth of Lewis carcinoma cells in vitro. These two activities appeared to depend on an oligosaccharide moiety, as they were lost after N-Glycanase treatment of HGP.92. Thus, the biological activity of HGP.92 was in some way related to a glycan moiety.

Immunostimulatory human urinary protein.

We have previously extracted a protein from inflammatory mouse granuloma that fully protects normal mice against lethal Listeria monocytogenes infection. We now report that polyclonal antibodies directed against this protein react with a human urinary fraction that also provides full protection of normal mice against L. monocytogenes. Murine monoclonal antibodies completely inhibit the protective activity of the human urinary fraction. We have purified to apparent homogeneity a human glycoprotein of 43 kDa (HGP.43), pI = 3.2 +/- 0.2. HGP.43 injected i.v. at 250 micrograms/kg fully protected mice against a lethal inoculum of L. monocytogenes. Such resistance followed injection of HGP.43, 4 days before and, still significantly, 8 hr after Listeria infection. In scid mice lacking T and B cells, similar resistance to L. monocytogenes was observed. Inflammatory murine macrophages became cytostatic against Lewis carcinoma cells after incubation with 1.5 nM HGP-43.

Membrane characteristics and functional activity of phagocytic alveolar macrophages

The intravascular (iv) or intramuscular (im) inoculations of a suspension of Lewis carcinoma cells induced metastatic tumor nodules in the lungs of C57Bl/6 mice. The administration of curantil (dipyridamole) (500 mg per 20 g-mouse, per os) once a week beginning one day after iv tumor cells inoculation (2 X 10(5) cells) reduced 4-fold the number of tumor nodules. The administration of curantil one day after im tumor cells inoculation (2 X 10(6) cells) reduced 2-fold the number of tumor nodules. The combined treatment of curantil (4-, 10-days after inoculation) and well-known inducer of interferon poly I:poly C (50 mg per 20 g-mouse, 1-, 7-, 14-days after inoculation tumor cells) had no synergistic effect.

Correlation between circulating cancer cells and incidence of metastases.

Quantitative aspects of the behaviour of B16 melanoma and Lewis lung carcinoma cells during the post-intravasation stages of metastasis were examined in relation to their spontaneous metastatic potential. Cancer cells were isolated from the blood of mice bearing i.m. tumours throughout tumour growth using a novel discontinuous gradient centrifugation technique. Four times more Lewis carcinoma cells than B16 melanoma cells were shed into the circulation, although the numbers of cells shed from either tumour were orders of magnitude more than the numbers of spontaneous pulmonary metastases which developed. Greater numbers of Lewis carcinoma cells were also shed as clumps and with leukocytes attached. However, although similar numbers of radiolabelled melanoma and Lewis carcinoma cells were arrested in the lungs after i.v. injection, fewer carcinoma cells were retained there and 12 times fewer Lewis carcinoma nodules developed in the lungs following injection of non-radiolabelled cells. It appears that the low lung colonization potential of the Lewis lung carcinoma is compensated for during spontaneous metastases by the numbers of cells shed from primary tumours as single cells and as clumps.

Immunostimulatory mouse granuloma protein.

Earlier studies have shown that from subcutaneous talc-induced granuloma in mice, a fraction could be extracted that fully protected mice against Listeria monocytogenes. Using standard biochemical procedures--i.e., ammonium sulfate fractionation, preparative electrophoresis, gel filtration chromatography, isoelectric focusing, and preparative polyacrylamide gel electrophoresis--we have now purified an active factor to homogeneity. A single band was obtained in NaDodSO4/polyacrylamide gel with an apparent Mr of 55,000. It migrated with alpha 1-globulins and the isoelectric point was 5 +/- 0.1. The biological activity was destroyed with Pronase but not with trypsin and a monospecific polyclonal rabbit antiserum was obtained. The intravenous injection of 5 micrograms of this "mouse granuloma protein" fully protects mice against a lethal inoculum of L. monocytogenes. Moreover, after their incubation with 10 nM mouse granuloma protein, mouse peritoneal cells became cytostatic against Lewis carcinoma cells.

Inflammation et résistance antitumorale III. — effets précoces d'une réaction inflammatoire sur lévolution de la tumeur de lewis

Following the inflammatory reaction induced in mice by subcutaneous implantation into the dorsal area of talc embedded in a calcium phosphate gel, one day after the intradermal inoculation of Lewis carcinoma cells in the sternal area, the tumour growth was delayed. In contrast with a decreased mitotic index and a tumour cell injury, no differences were found between the controls and the treated animals concerning the average number of vessels or the number of leukocytes infiltrating the tumour.

Inflammation and host resistance against pathogens.

After a review of all factors released in an inflammatory focus which act on pathogens, a summary of recent findings concerning the influence of an inflammation on the remote control of mouse resistance against pathogens is given. In treated mice, the ability of spleen and liver macrophages to killListeria was found to be increased between 3 and 18 days following the induction of the inflammation. An increased resistance against the Lewis carcinoma was found, even when the mice were treated one day after the inoculation of malignant cells. In the treated mice, an increased cellular multiplication was observed in bone marrow, thymus, spleen, lymph nodes whereas no variations were observed in other tissues. The higher host resistance is correlated with an increased leucopoiesis and the activation of liver and spleen macrophages. The serum of treated animals is ablein vitro to activate peritoneal macrophages against Lewis carcinoma cells, to stimulate the multiplication of bone marrow cells and to agglutinate malignant cells. In order to explain the mechanisms of such an increased host resistance, investigations were undertaken to isolate and characterize possible active substances in the granuloma and in the serum from granuloma-bearing mice, and to check the possible activity of mediators of inflammatory reactions. Concerning the granuloma and the serum, it was possible, following electrofocusing, to extract and to isolate a fraction which is able to protect mice against a lethal inoculum ofListeria. Among the known mediators of acute inflammation, it was found not only that kinins are able to increase the spreading of macrophagesin vitro but that kinins or some of their degradation products are able,in vivo, to increase the resistance of mice againstListeria and the Lewis carcinoma. These results emphasize the importance of local non-specific inflammatory reactions on macrophages.