Cancer Research and Treatment

Jong Gu Rha

3 Articles

Detection of p16(INK4A) in the Mixed Cell Populations of Normal Peripheral Blood Mononuclear Cells and Cervical Cancer Cell Lines: Ji Young Kwon, Yoon Sung Jo, Ye Hoon Choi, Jong Gyu Chang, Ki Sung Ryu, Jong Gu Rha, Ku Taek Han; Cancer Res Treat. 2003;35(3):254-260. Published online June 30, 2003; DOI: https://doi.org/10.4143/crt.2003.35.3.254

PURPOSE
Human papilloma viruses (HPVs) play a central role in the pathogenesis of neoplastic lesions of the uterine cervix. The viral oncoprotein HPV E6 degrades the p53 protein, and the HPV E7 protein inactivates pRB and increases the expression of the CDK inhibitor, p16(INK4A). We investigated the usefulness of p16(INK4A) as a biologic marker for the cervical dysplastic and neoplastic cells.
MATERIALS AND METHODS
We examined the expression of p16(INK4A) and cytokeratin in a mixed population of normal peripheral blood mononuclear cells (PBMC) and the cervical cancer cell lines (HeLa, SiHa, and CasKi) using flow cytometry. RESULTS: The DNA indices of the HeLa, SiHa and CasKi cell lines were 1.89, 1.53 and 1.75, respectively, indicating that these cells are aneuploid cells. Furthermore, the positive rate of p16(INK4A) expression was 86.7% for the HeLa mixed population, 85.6% for the SiHa mixed population, and 92.2% for the CasKi mixed population. According to the FL3A vs FL3W histogram, electrical gating of the HeLa, SiHa and CasKi mixed populations showed the expression levels of both cytokeratin and p16(INK4A) to be identical, at 86.6%, 84.8% and 85.0%, respectively. These findings revealed that almost all cells selected through electrical gating were cervical cancer cells originating from the epithelium and which expressed cytokeratin and p16(INK4A). On the other hand, when each mixed population was electrically gated for normal PBMC, we found that the PBMCs expressed neither cytokeratin nor p16(INK4A).
CONCLUSION
Using flow cytometry, we observed the enhanced expression of p16(INK4A) in cervical cancer cell lines. These RESULTS suggest the usefulness of p16(INK4A) for the selective detection of cervical dysplastic and cancer cells in the liquid-based samples, which are taken from the cervices and contaminated with blood and stromal cells.

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2-Methoxyestradiol: A Hormonal Metabolite Modulates Stimulated T-Cells Function and proliferation
J.G.Y. Luc, R. Paulin, J.Y. Zhao, D.H. Freed, E.D. Michelakis, J. Nagendran
Transplantation Proceedings.2015; 47(6): 2057. CrossRef

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Multiparametric Flow Cytometry in Breast Cancer Cell Line (MCF-7) Stained with Fluorescein Isothiocyanate, Phycoerythrin, and Propidium Iodide: Ku Taek Han, Ki Sung Ryu, Sang Ha Han, Kweon In, Ji Min Song, Jang Heup Kim, Jong Kun Lee, Jong Gu Rha, Soo Pyung Kim, Hun Young Lee; J Korean Cancer Assoc. 1999;31(6):1129-1139.

Abstract PDF: 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.

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Inhibitory Effect of Transforming Growth Fasctor-β1 on Cellular Proliferation and c-myc Expression in Choriocarcinoma Cell Line: Sa Jin Kim, Jin Woo Kim, Jong Gu Rha, Guisera Lee, Seung Kyu Song, Sung Eun Namkoong; J Korean Cancer Assoc. 1995;27(6):961-968.

Abstract PDF: Modulation of cellular proliferation and c-myc oncogene expression after treatment of human choriocarcinoma cell line(BeWo) with trsnsorming growth factor-￥a1(TGF-￥a1) have been investigated by MTT [3-(4,5-dimethylthiaxol-2-yl)-2, 5-diphenyltetrazolium bromide] assay and Northern blotting analysis, respectively. TGF-￥a1 inhibited BeWo cell growth in a dose dependent manner at concentrations of 0.00110 ng/mL. When BeWo cells were exposed to 10 ng/ml of TGF-￥a1,c-myc was repressed after 4hr and completely aboiished at 24hr of stimulation. These results show that TGF-￥a1 may play an antiproliferative effect on BeWo cell growth via c-myc gene product. In vitro analysis of cellular and molecular alterations in BeWo cells in this study may aid in elucidating the mechanism of action of TGF-￥a1 on human choriocarcinoma tissues in vivo.

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