ABSTRACT
PURPOSE: Cytokines and their corresponding cell surface receptors are involved in intercellular signalling pathways and in the radioresistance of normal and malignant cells. The aim was the characterization of the expression of intracellular cytokines, their receptors and apoptosis-associated markers under the influence of radiation. MATERIAL AND METHODS: Two Ewing tumours were characterized in vitro before and 4, 24 and 72 h after radiation with 5 and 10 Gy, and in vivo 4, 6 and 15 days after radiation with 5 and 30 Gy by five parameter flow cytometry. Direct fluorescence-conjugated antibodies directed against intracellular cytokines (interferon-gamma, tumour necrosis factor [TNF]-alpha, interleukin 1) and their receptors (CD119, CD120a, CD121a) were used. Annexin V and 7-amino-actinomycin D were used to identify radiation-induced apoptosis. RESULTS: Inter- and intra-individual heterogeneities were identified by the expression of cytokine receptors and the intracellular cytokine profile before radiation. Time- and dose-dependent up-regulation of the cytokines TNF-alpha and interleukin 1 were found in vitro. In vivo, an up-regulation of CD120a and CD121a was detectable on tumour cell subpopulations. For interferon-gamma and CD119, no changes were seen. CONCLUSIONS: The observed radiation-induced changes of cytokine and receptor profile are an indication for complex intercellular interactions in view of radioresistance-associated mechanisms between cell populations within one individual tumour. The observed heterogeneous response on radiation might have therapeutic implications for an individualized therapy based on combined radiation and cytokine modulation, defined by flow cytometric characterization of markers potentially informative for radioresistance.
Subject(s)
Cytokines/biosynthesis , Receptors, Cytokine/biosynthesis , Sarcoma, Ewing/metabolism , Animals , Annexin A5/pharmacology , Antigens, CD/biosynthesis , Apoptosis , CD11 Antigens/biosynthesis , Cell Division , Cell Line, Tumor , Cytokines/metabolism , DNA/metabolism , Dactinomycin/pharmacology , Dose-Response Relationship, Drug , Dose-Response Relationship, Radiation , Enzyme Inhibitors/pharmacology , Flow Cytometry , Fluorescent Dyes/pharmacology , Humans , Immunophenotyping , Interferon-gamma/metabolism , Interleukin-1/metabolism , Mice , Mice, Nude , Microscopy, Fluorescence , Neoplasm Transplantation , Radiation, Ionizing , Receptors, Interferon/biosynthesis , Receptors, Interleukin-1/biosynthesis , Receptors, Tumor Necrosis Factor/biosynthesis , Receptors, Tumor Necrosis Factor, Type I , Time Factors , Tumor Necrosis Factor-alpha/metabolism , Up-Regulation , Interferon gamma ReceptorABSTRACT
PURPOSE: Adhesion molecules, cytokines and their corresponding cell-surface receptors are involved in intercellular signalling pathways, radioresistance and metastasis-mediating mechanisms of malignant cells. The aim was the characterization of changes in the marker profile of Ewing tumour cell subpopulations under the influence of radiation. MATERIALS AND METHODS: Three Ewing tumours were characterized in vitro and in vivo in a xenograft model before and after radiation by five-parameter flow cytometry. Antibodies directed against cell surface and intracellular antigens, apoptosis-associated markers and the DNA dye 7-aminoactinomycin D were used. RESULTS: Tumour cell subpopulations were identified by expression of adhesion molecules and cytokine receptors, intracellular cytokines, apoptotic markers and DNA content. Heterogeneous changes of flow cytometric profile were identified on tumour cell subpopulations after radiation. CONCLUSIONS: The changed profile of tumour cells under radiation might be associated with biological changes of tumour subpopulations in view of radioresistance and metastatic potential and might be useful to identify intercellular regulation mechanisms and to define parameters being predictive for a response to therapy.
Subject(s)
Dactinomycin/analogs & derivatives , Sarcoma, Ewing/pathology , Animals , Annexin A5/metabolism , Apoptosis , CD56 Antigen/biosynthesis , Cell Adhesion , Cytokines/metabolism , DNA/metabolism , Dactinomycin/pharmacology , Flow Cytometry , Humans , Immunophenotyping , Intercellular Adhesion Molecule-1/biosynthesis , Mice , Mice, Nude , Neoplasm Transplantation , Time Factors , Tumor Cells, Cultured , Tumor Necrosis Factor-alpha/metabolism , Up-RegulationABSTRACT
BACKGROUND: Heterogeneity in human malignant tumors is a well-described phenomenon and of interest with regard to subpopulations with differences in clonality, metastatic potential, and response to therapy under different treatment regimes. The aim of this study was the simultaneous characterization of surface markers and DNA content of solid tumors to identify tumor cell subpopulations and to study the association between the expression of antigens and DNA content. METHODS: In the present study, six different malignant tumors grown as xenografts in nude mice were characterized by five-parameter flow cytometry. Immunophenotyping was performed using a variety of direct fluorescence-conjugated antibodies. In all cases, simultaneous detection of DNA content was done after staining with 7-aminoactinomycin D. RESULTS: Tumor cells were characterized by light scatter properties, antigen expression, and DNA content. Tumor cell heterogeneity, subpopulations, and DNA content-dependent antigen expression were identified. CONCLUSIONS: This method offers the possibility of characterizing solid tumors according to their immunophenotype and DNA content. The results obtained can be used to identify changes in immunophenotypic and DNA profiles of tumor cell populations before and after therapy and might be useful to define parameters predictive for response to therapy.