ABSTRACT
PURPOSE: Multiparametric flow cytometry is a powerful tool for analyzing the phenotypic, cell kinetic and ploidy heterogeneity of tumor cell populations. But there are major problems such as inaccurate results by the contribution of non-neoplastic cell contamination and the substantial spectral overlap of PI (propidium iodide) into PE (phycoery- thrin) fluorescent emissions on a standard flow cytometer. Recent studies suggested that the emission spectral overlap from PI into PE could be sufficiently compensated electrically and the cytokeratin, a marker for epithelial tumor cells, are successfully used in conjunction with DNA specific dye so as to obtain DNA profiles selectively for cytokeratin-positive tumor cells. The aim of this study was to investigate the feasibility that multiparametric analysis in heterogeneous cell populations of cell lines like solid tumors, which were stained triply with PE, fluorescein isothiocyanate FITC, and PI, can be done without any influences by the contaminated normal diploid cell populations and without spectral overlap between fluorochromes on a standard flow cytometer. MATERIALS AND METHODS: MCF-7 cell lines and heterogeneous cell populations mixed with MCF-7 cells and human peripheral blood lymphocytes were fixed with 1% paraformal- dehyde and permeabilized with 100% methanol. Cytokeratin was labeled with PE and some proliferat!on-associated markers were labeled with FITC, which were followed by DNA staining by PI. These triply stained cells were measured on a standard FACScan flow cytometer equipped with 488 nm single laser and those acquired data were analyzed with WinList 3.0 and ModFit LT software programs on personal computor. RESULTS: Coefficient of variation (CV) of GoG1> peak of MCF-7 cells alone was 4.3. GoG1, S, and G2M phase fractions were 44.9%, 45.9%, and 9.2% respectively. FITC, PE and PI fluorochromes could be detected without any interference between them. CVs of GoG1 peak of PBL and MCF-7 cells in those heterogeneous population were 2.3 and 4.2 respectively. The DNA index of MCF-7 cells was 1.7. MCF-7 cells expressed the cyto- keratin, PCNA, p53, c-erbB/2 and c-myc antigen and in contrast, PBL did not express cytokeratin. The cell cycle phase fractions and oncoprotein expressions could be detected separately in diploid PBL and aneuploid MCF-7 cells in the mixed cell population without any influences by each other. CONCLUSION: These results suggested that the cellular antigen expressions of the malignant cells can be analyzed selectively without influences of fluorescent signals from nonneo- plastic cells. The neoplastic tumor subpopulations are clearly identified on the basis of both ploidy status and antigen expressions. The positive cytokeratin expressions indicate that they were derived from the epithelium, providing objective evidence of the tissue of origin and more precise analysis of DNA contents, ploidy, and oncogene expressions selectively with possible correlation between them. Thus, this method offers new possibilities for multiparameter flow cytometric analysis in the heterogeneous solid tumor cell populations.
Subject(s)
Humans , Aneuploidy , Breast Neoplasms , Breast , Cell Cycle , Cell Line , Diploidy , DNA , Epithelium , Flow Cytometry , Fluorescein , Fluorescein-5-isothiocyanate , Fluorescent Dyes , Keratins , Lymphocytes , MCF-7 Cells , Methanol , Oncogenes , Phycoerythrin , Plastics , Ploidies , Population Characteristics , Proliferating Cell Nuclear Antigen , PropidiumABSTRACT
BACKGROUND: The basic treatment of malignant tumors is surgery, radiotherapy, chemotherapy. Even though, the object of these treatments is to kill cancer cells, they have limitations. So, in future studies of treatment of cancer, we should look into increasing human immune response using gene therapy in order to induce damage to tumor cells. OBJECTIVE: The cell growth inhibitory effect of cervical cancer cells was investigated by direct transfection using liposome(pRcCMVp53/lipofectin). and by indirect transfection using Adenovirus(AdCMVp53). METHODS: The cervical cancer cell lines we used in this study were HPV16 positive, having inhibitory gene, wild p53 gene, CaSki, SiHa, HPV18 positive HeLa, HeLaS3 and HPV negative C33A, HT3, LacZ gene was used as the marker gene for the transfection efficacy. Direct transfection was done by using lipofectin (pRcCMVp53/lipofectin) and indirect transfection was done by using virus, AdCMVp53. The effect of tumor cell growth inhibition was measured by cell counting assay. RESULT: Inhibition of growth of cervical cancer cells in cell counts of direct transfection was CaSki(88.5%), SiHa(59.1%), HeLa(86.0%), HeLaS3(78.0%), C33A(91.3%) and HT3(74.0%). Inhibition of growth of cervical cancer cells in cell counts of indirect transfection was CaSki(97.4%), SiHa(91.6%), HeLa(95.8%), HeLaS3(99.7%), C33A(97.3%) and HT3(87.4%). CONCLUSION: The inhibition of cell growth of cervical cancer cells by direct and indirect transfection was significantly reduced, and showed little differences depending on the type of cells. These results will have a great meaning in treating cervical cancer patients using gene therapy by direct or indirect transfection
Subject(s)
Humans , Adenoviridae , Cell Count , Cell Line , Drug Therapy , Genes, p53 , Genetic Therapy , Lac Operon , Plasmids , Radiotherapy , Transfection , Uterine Cervical NeoplasmsABSTRACT
OBJECTIVE: Human papillomavirus (HPV) has been identified in the majority of invasive cervical cancer patient and has been found to contribute in a significant way to the genesis of human cervical cancer. HPV has two transforming genes that encode the oncoproteins E6 and E7, E6 can form complexes with p53 and promote p53 degradation, E7 inhibit retinoblastoma protein (RB). The p53 protein is as a phosphoprotein which co-immunoprecipitated with the SV40 T-Antigen. The wild type p53 protein is capable of suppressing the tumorigenic phenotype and regulating cell cycle. Adeno-associated virus(AAV) is a linear single stranded DNA parvovirus which is dependent upon cotransfection by a second unrelated virus to undergo productive infection. It has been well documented that AAV DNA integrates into cellular DNA as one to several tandem copies joined to cellular DNA through the termini. In order to introduce wild type p53 through AAV virus into a cervical cancer patient for gene therapy, we had constructed recombinant p53 adeno associated virus plasmid (pAAVCMVp53). METHODS: pAAVCMVp53 was created new AAV-vector system, pRc/CMVp53 including p53 cDNA and AAV-derivative vector, pASPA-AAV-CMV-polyA were made to HindIII/blunt fragments. Eluated 1.8 kb fragment of wild type p53 cDNA was ligated to pAAV-CMV-polyA, 4.9 kb fragment deprived hASPA cDNA. RESULT: Recombinant AAVCMVp53 was constructed by using pRc/CMVp53 andpASPA-AAV-CMV-polyA. This pAAVCMVp53 was confirmed by various restriction enzyme-digestions and Southern-blotting. This new vector system will be studied on expression, stability in cervical cancer cell lines and animals. CONCLUSION: This system will be one of the useful vector system for cervical cancer gene therapy.
Subject(s)
Animals , Humans , Antigens, Viral, Tumor , Cell Cycle , Cell Line , Clone Cells , DNA , DNA, Complementary , DNA, Single-Stranded , Genetic Therapy , Oncogene Proteins , Oncogenes , Parvovirus , Phenotype , Plasmids , Retinoblastoma Protein , Satellite Viruses , Uterine Cervical NeoplasmsABSTRACT
We compared the therapeutic effects of concomitant chemoradiotherapy (CRT) using cisplatin to single radiotherapy (RT) in uterine cervical cancer. 34 cases of non-operable uterine cervical cancer were reviewed retrospectively from Mar, 1993 to May, 1996 in St. Mary' s Hospital. The patients were randomly selected to compare the effects of both methods. 22 patients were included in CRT group and 12 patients in RT group. The results were as follows: 1. The decrease of tumor size was not significant (2.17 cm in CRT and 1.95 cm in RT) (p=0.61), but the number of responders of CRT group was larger than that of RT group significantly (p0.05) 3. The overall survival rate showed no difference between two groups (p>0.05). The disease-free survivals for 38 months were 17.02% in CRT and 11.36% in RT, but it was not significant (p>0.05). In conclusion, concomitant chemoradiotherapy showed better rate of response, but size of tumor decrease and tumor markers showed no difference. CRT might improve the overall survival and disease-free survival, although it was not significant in this study. The clinical significance of CRT remains to be determined in large randomized clinical trial.
Subject(s)
Humans , Chemoradiotherapy , Cisplatin , Disease-Free Survival , Radiotherapy , Retrospective Studies , Survival Rate , Biomarkers, Tumor , Uterine Cervical NeoplasmsABSTRACT
The incidence of malignant change of ovarian mature teratoma is 1~2%. The majority is squamous cell cancer, the others was adenocarcinoma. Neuroepithelial tissue was frequently detected in mature cystic teratoma, but their malignant change was extremely rare. Only, two cases of neuroblastoma of ovarian teratoma were reported in the world. We report one case of neuroblastoma arising in ovarian mature teratoma with a brief review. Our case is the third reported one in the world.
Subject(s)
Female , Adenocarcinoma , Incidence , Neoplasms, Squamous Cell , Neuroblastoma , Ovary , TeratomaABSTRACT
Flow cytometry, a useful tool for measuring DNA content and cell differentiation as expressed by cell surface markers, is utilized to measure multiple antigens, especially surface antigen, intracellular oncoprotein, and DNA content, simultaneously. For this simultaneous detection, several methods off ixation and permeabilization have been used with limited values. In this study, 20 ng/ml of lysolecithin in 1% paraformaldehyde solution was utilized for fixation and permeabilization of cultured promyelocytic leukemic cells(HL 60). The cells were first stained with phycoerythrin (PE)-conjugated monoclonal antibody to the cell surface My 7 antigen and then were fixed and permeabilized with 20 ng/ml of lysolecithin in 1% partormaldehyde solution. After incubation, the fixed and permeabilized cells were stained with monoclonal antibody to intracellular c-myc antigen, which were followed by fluorescein isothiocyanate (FITC)-conjugated secondary antibody. The c-myc stained cells were finally stained for DNA content with 7-amino-actinomycin D(7-AAD). This procedure permits excellent staining for intracellular oncoproteins and preservation of surface antigens with relatively low cofficients of variation (CV) for the G0G1 peak of the DNA histograms and suggests that the sequential staining procedure of surface antigen, intracellular antigen, and DNA content will be extended for the study of correlations with cellular differentiation, expression of oncoproteins, and cell cycle analysis in the cells which are obtained from human malignant diseases using a 488 nm single laser flow cytometry.
Subject(s)
Humans , Antigens, Surface , Cell Cycle , Cell Differentiation , DNA , Flow Cytometry , Fluorescein , Oncogene Proteins , PhycoerythrinABSTRACT
Transforming growth factor-beta1(TGF-beta1) is known to be a potent growth inhibitor for many cell types, including most epithelial cells. In skin keratinocytes, TGF-beta1 has been shown to inhibit growth and to rapidly reduce c-myc expression. However, the molecular mechanism of TGF-beta1 action on cell growth of cervical carcinoma has not yet been elucidated. We thus assessed the effect of TGF-beta1 on the growth of cervical carcinoma cell lines. Two cervical squamous carcinoma cell lines were incubated with varying concentration of TGF-beta 1, and growth inhibition was evaluated with tetrazolium-based colorimetric assay. After culturing in concentrations of 0.1~10 ng/ml in both cell lines. Northern blot analysis revealed c-myc mRNA expression was suppressed by 10 ng/ml of TGF-beta 1 following 6-hour of treatment in both cell lines. These results suggest that TGF-beta1 inhibits the growth of cervical carcinoma cells by suppressing c-myc oncogene expression.
Subject(s)
Blotting, Northern , Carcinoma, Squamous Cell , Cell Line , Epithelial Cells , Genes, myc , Keratinocytes , Oncogenes , RNA, Messenger , Skin , Transforming Growth Factor beta , Transforming Growth Factor beta1 , Uterine Cervical NeoplasmsABSTRACT
A retrogpective review of hematologic rnonitoring involving aggressive chemotherapy was careiyd out ta assese whetber there ia a predictable relatiorship between the white blood ce11 count end the platelet count as a refleetion of bone marrow toxicity and when maximum myeloauppression occur during a treatment program. This data revealed that the white blood cell and granulocyte levels are closely related and that myeloeuppression can oceur during any course of CAP(cyclophosphamide, adriamycin, and cisplatin), VBP(vinblastine, bleomycin, and cisplatin) chemotherspy in gynecological cancer. Thus, for these treatment regimena in gynecoldgical malignancies, the white blood cell and granulocyte count is sufficient for momtoing toxicity and adjusting future courses of chemotherapy. There are no bone merrow depresaions by the treatment regirnens for the gestational trophoblastic disease.
Subject(s)
Humans , Bleomycin , Bone Marrow , Doxorubicin , Drug Therapy , Equidae , Gestational Trophoblastic Disease , Granulocytes , Leukocytes , Platelet CountABSTRACT
No abstract available.