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Transfection of B16 Melanoma Cells with IL-3 Gene and Its in Vitro Proliferative Features

Chinese Journal of Cancer Biotherapy.1994;0(01):-.

IL-3 has effects on a wide variety of cell types, including immature cells of the immune cells, and mature immune cells such as granulocytes. In the purpose of studying the immunoregulatory function of IL-3 and its potential role in cancer therapy, we established a IL-3-secreting tumor model using gene transfection to deliver locally IL-3 to tumor site. First, we constructed IL-3 expression vector BMGNeo-mIL-3, then transfected it into B16 murine melanoma cells. By G418 resistant screening and limiting dilution, we obtained a transfectant that expressed high levels of IL-3 (806U / ml) . The IL-3 expression of the transfectant was confirmed by Northern blot analyses. Although the wild-type B16 cells and the B16-Neo cells transfected with BMGNeo did not express IL-3, the IL-3 expression in B16 cells had no obvious effect on the in vitro proliferation of the transfectant. These results showed we had successfully established a IL-3-secreting tumor cell clone which would enable us to further study its in vivo tumorigenicity and immune function.

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Application of MTT Colorimetric Assay in Research of Cytokine Activity and Cytotoxicity of Killer Cells

Zhigang TIAN ; Jianhua ZHANG

Chinese Journal of Cancer Biotherapy.1994;0(01):-.

We used MTT assay to test the cellular cytotoxicity ( NK, LAK, CTL, Macrophage), cytokine activities ( 1L-1, 1L-2, 1L-6, TNF), proliferation of lymphocytes and chemosensitivity of tumor cells, and compared it with radioactive isotope assay. The results showed that the MTT assay may be used to test the cellular cytotoxicity, cytokine ac-tivity, proliferation of lymphocytes and chemosensitivity of tumor cells. We think it is a simple, rapid, economic and safety method.

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Inhibitory Effect of Antibody Against Basic Fibroblast Growth Factor on Androgen or Glucocorticoid-induced Growth of Shionogi Carcinoma 115 Cells

Jian LU ; Jinshan ZHANG

Chinese Journal of Cancer Biotherapy.1994;0(01):-.

The effects of androgen and glucocorticoid on the proliferation of SC-3 mouse breast carcinoma cells and their mechanisms were investigated in a serum-free culture. Both physiological concentrations of testosterone ( T) and pharmacological concentrations of dexamethasone ( Dex) significantly increased the cell proliferation. The growth-stimulatory effects of two hormones were mediated through their own receptors.SC-3 cells stimulated by T and Dex were also found to secrete fibroblast growth factor (FGF) like factor into conditioned medium (CM) . The addition of anti-FGF antibody in the medium inhibited T, Dex and bFGF-induced proliferation of SC-3 cells. Furthermore, the presence of FGF receptors in SC-3 cells were demonstrated. These results indicated that androgen and glucocorticoid-in-duced enhancement of SC-3 cell growth is mediated through an induction of FGF-like growth factor which acts on SC-3 cells as an autocrine growth factor.

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Inducing Effects of Retinamide (RII) on Tumor Cells with Different Metastatic Ability

Jin GAO ; Liqun HAN ; Jian CAO

Chinese Journal of Cancer Biotherapy.1994;0(01):-.

Newly synthesized retinoic acid derivative-retinamide (RII) was employed as differentiation inducing agent. Cultured tumor cells of epithelial origin were exposed to 10-5mol/L RII for 5 passages. These cells included mouse fore stomach carcinoma (MFC, lung metastatic rate 85%), human nasopharyngeal carcinoma (CNE-2Z, 56%), and clonal variants from CNE-2Z, CNE2L2 (100%) and CNE2L4 (13%) . The results of serial observation were as follows. The growth, clone-forming ability, motility, invasiveness of these tumor cells were obviously inhibted. Their adhesion to fibronectin and laminin was increased. Morphological ultrastructure changes, mainly of surface structure, were also observed. These changes suggested that RII made metastatic cells less aggressive and showed a tendency toward differentiation. After exposured to RII, different changes of oncogene and antioncogene expression of these tumor cells were detected. For example, RE caused expression of nm23in MFC cells, but down regulated (decreased) its expression in CNE2L2 cells and similar changes of rasH, fos, nm23, Rb. Expression were observed for CNE-2Z cells and its clonal variants CNE2L2 CNE2ZL2. RII down regulated both these oncogene and antioncogenes in both CNE-2Z and CNE2L2, but up regulated all of them in CNE2L4. The results indicated that oncogene and antioncogene may play different roles in different tumor cells, the same factor (RII) may lead to different changes of gene-expression in different metastatic tumor cells. So the function of oncogene and antioncogene may be of relativity and may be influenced by multifactors. Our data were mostly from in vitro experiments. It could not be deduced completely to a level as a whole in vivo.

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Studies on the Mechanisms of the Apoptotic Cell Death of Target Cells Mediated by Human Monocytes

Haidong DONG ; Zhenchao QIAN

Chinese Journal of Cancer Biotherapy.1994;0(01):-.

The mechanisms of cytotoxicity mediated by pokeweed-mitogen (PWM)-activated human peripheral blood monocytes was investigated in this study. By DNA electrophoresis and propidium iodide ( Pl) -DNA staining flow cytometry, the study demonstrated that apoptotic cell death of target U937 cells and Raji cells was induced in lectin-depen-dent monocyte-mediated cytotoxicity ( LDMC) . The LDMC-mediated DNA fragmentation in U937 cells could be completly inhibited by anti-TNF-?monoclonal antibodies McAbs, instead of the addition of monosaccharide (N-acetyl glucosamine, Glc NAC) . In contrast, Glc NAC inhibited the DNA fragmentation in Raji cells induced by PWM-monocytes while the anti-TNF-a McAbs had no effect. By flow cytometry, we also demonstrated that PWM could bind with tumor cells as well as with monocytes, at the stimulation of the production of nitric oxide ( NO-) from monocytes. This NO-production was enhanced in the presence of target cells, but the enhacement was abolished by the treatment of GlcNAc.The presence of Lectin-like receptors on the surface of monocytes and tumor cells may bring the effector target cells together, thus facilitating the induction of apoptosis in target cells by triggering the production of cytolytic factors and the modification of target cell surface antigens.

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Anti-T Cell Immunotoxin for GVHD Prophylaxis in Allogeneic Bone Marrow Transplantation

Beifen SHEN ; Yan LI ; Xing CHEN

Chinese Journal of Cancer Biotherapy.1994;0(01):-.

Bone marrow transplantation was considered effective treatment for leukemias and other haematological diseases. Acute graft v-host disease (GVHD) is a major complication of allogeneic bone marrow transplantation. The T lymphocytes were specially depleted by anti-CD5, CD2, CD8, CD27 monoclonal antibody-ricin immunotoxins. Inhibition of the protein synthesis and T-cell functions in the target cells was observed in vitro. At 10 9mol / L of the immunotoxin, 3 logs (99.9%) of target cells were killed, but no cytotoxicity on nontarget cells. At the same concentration, the anti T cell immunotoxin had no any influence on the proliferating rate of CFU-GM and BFU E. None of the ten patients who received T cell depleted bone marrow developed grade III or IV acute GVHD.

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Discussion on the Establishment of Animal Tumor Models and Their Standards

Jin GAO

Chinese Journal of Cancer Biotherapy.1994;0(01):-.

Animal tumor models, as replicas of human tumors, are of important significance concerning the investigation of the mechanism of tumorigenesis, tumor progression, tumor prevention and therapy, when establishing animal tumor models, we should choose suitable species of animals and corresponding carcinogens. One species of animal differs greatly from others. The same carcinogen may induce different tumor in different species of animals. Thus it is most important that animals should be selected properly to obtain animal tumor models suitable for experiment. Animal tumor models comprise spontaneons tumor models, inducing tumor models and transplantable tumor models. This paper will focus on the transplantable animal tumor models. The sources of human tumors (transplanted to immunodeficiency animals) are mainly biopsy tissues, surgically resected tumor specimens and human tumor cell lines. The basic necessity to establish transplantable tumors includes collecting fresh, non-necrotic non-capsulated tumor tissue within 1-2 hours after resection, selecting host animal (including immunodeficiency animals) should be around 4 weeks old, and the most frequent innoculating site is dorsal subcutaneous. Successful establishment of transplantable tumor models should satisfy the following standards: 15 - 20 successive generations (at least 3 - 4 animals each generation); 100 % growth after transplantation; least self-extinction (not definitely zero); stable growth rate; similar life span of host (high reproductivity); low host response (suitable for in vivo growth in host); histological similarity with primary tumor.

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Cloning of cDNAs Encoding Extra-cellular Domain of Human IL-6, IL-10, IL-13 and SCF

Zhongbin LIU ; Zhigang TIAN ; Rui SUN

Chinese Journal of Cancer Biotherapy.1994;0(01):-.

Human IL-6,IL-10,IL-13 and SCF cDNAs encoding extra - cellular domain were isolated by reverse transcription polymerase reaction (RT-PCR). These cDNA fragments were inserted into expression vector PBV220 which contains PRP_(L), promoters and SD sequence. Then the E. coli DH5? were tronsformed with the recombinant plasmids. After screening, the clones carrying those recombinant plasmids were identified. SDS-PAEG and biological assay indicated that human IL-6, IL-10 and IL-13 were expressed in E.coli. The expression level of rhIL-6 was as high as 30% of the total bacterial protein.

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Experimental Tumor Immunotherapy with TNF Gens Transduced Cells

Zhihai QIN ; Thomas BLANKENSTEIN

Chinese Journal of Cancer Biotherapy.1994;0(01):-.

The tumor necrosis factor(TNF) secreted by gene modified tumor cells can lead to very effective tumor rejection. This effect of TNF on tumor growth is mediated mainly by the induction of an antitumor immune response. It requires a local and continuous presence of TNF at the tumor site. Tumor suppression induced by TNF is close dependent and a complete tumor eradication must not be accompanied by systemic toxic side effects. A complex pattern of tumor infiltrating cells has been observed in TNF producing tumors, consisting of macrophages, CD4+ and CD8~(+)T cells. For efficient tumor inhibition maccophages and CD8~(+)T cells are needed,whereas CD4~(+)T cells seem to be innocent bystander cells. TNF is also effective in T eell deficient mice, but in most cases for complete tumor elimination T ceils have to be present. Depending on the cell lines used or the levels of TNF secreted by transduced tumor cells, systemic toxicity of TNF has also been observed including cachexia or wasting of the experimental animals. In some cases, TNF gene transfected tumors did not show growth inhibition in vivo, but rather, their metastases were enhanced. Using TNF producing tumor cells as vaccine, no systemic protective immunity against a parental tumor cell challenge has been observed.

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Treatment of 50 Acute Leukemia Patients with Autologous Hematopoietic Stem Cell Transplantation

Haichuan LIU ; Shanqian YAO ; Fangding LOU

Chinese Journal of Cancer Biotherapy.1994;0(01):-.

We treated 50 acute leukemia patients with autologous hematopoietic stem cell transplantation. Among them, 10 patients were transplanted with unpurged autologous bone marrow (ABMT), 22 patients with purged autologous bone marrow(PABMT), 18 Patients with autologous blood hematopoietic stem cell (ABHSCT). The three years disease-free living incidences in the three groups were 75%,69.9% and 72.7%, respectively. The relapse rates were 36.6%, 27.2%, and 31.4%, respectively.A high three-year disease-free incidence and low relapse rate were found in the patients with less than two months between diagnosis and achieving complete remission (CR), transplantation after 6 months of achieving CR, conditioning treatment with chemotherapy and total body irradiation (TBI) and acute myeloid leukemia (AML). The results indicated that there are some correlations between the amount of infused mononuclear cells and the recovery of bone marrow hematopoiesis. No significant difference of therapeutic effiency was demonstrated among the three groups.

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