Cancer Research and Treatment

Original 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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Relationship between Expressions of Tumor - Associated Antigen MAGE-3 and p53 Proteins during Cell Cycle by Bivariate Analysis of Flow Cytometry: Hee Kyoung Chang, Deok Jun Kim, Kang Dae Lee, Hwan Jung Roh, G Spagnoli; J Korean Cancer Assoc. 1999;31(4):784-792.

Abstract PDF: PURPOSE
MAGE (melanoma antigen gene) is a tumor associated antigen, presented by HLA class I molecules, which is recognized by cytotoxic T lymphocytes. The expression of MAGE proteins are confined to malignant tumor tissues, except for the normal testis and placental tissues. Therefore, MAGE may be a potential target for immunotherapy of malignant tumors. However, biological aspects associated with the cell cycle are not yet described.
MATERIALS AND METHODS
The material used for this study was a novel human squamous cell carcinoma cell line (PNUH-12) from the hypopharynx, which had one point mutation of 78th base, C to G, in exon 7 of p53 gene. To understand the role of MAGE in relation to cell cycle and its relationship with p53 as the Gl checkpoint regulator, the expressions of MAGE-3 protein and mvtant p53 (Mtp53) were accessed by flow cytometry and immunohistochemistry. Double stains for MAGE-3/Mtp53 was analyzed with bivariate flow cytometry. DNA histograms using MAGE-3/PI (DNA) and Mtp53/PI (DNA) were also analyzed.
RESULTS
The expression rate of MAGE-3 and Mtp53 were 83% and 85%, respectively. MAGE-3 was expressed in cytoplasm, while M:p53 were expressed in the nuclei of the tumor cells on the immunohistochemical sections. With bivariate analyses, coexpression rate of MAGE-3/Mtp53 was 0.96, and MAGE-3 and Mtp53 constantly showed high levels throughout the cell cycle except Go.
CONCLUSIONS
These results mean that (I) MAGE-3 might have yet unknown relationship with mutant p53, (2) expressions of MAGE-3 and Mtp53 are not dependent on the cell cycle in PNUH-12 hypopharyngeal squamous carcinoma cell line, and suggest that MAGE-3 might have a role as important as p53 during the development of malignant tumors.

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Correlation between Proliferative Index by DNA Flow Cytometry and Histological Features in Stomach Cancer: Hyoung Kug Kim, Myeong Soo Lee, Hong Yong Kim, Se Hwan Han, Seok Yong Rhu, Hong Joo Kim, Young Doug Kim; J Korean Cancer Assoc. 1998;30(6):1078-1088.

Abstract PDF: PURPOSE
Stomach cancer is the most prevalent malignancy in Korea. The survival rate in advanced stage disease has stayed in less than 50%. One of the possible explanation for dismal outcome of stomach cancer is various biologic behavior of cancer cells of heterogeneous clones. Introduction of flow cytometric analysis has provided objective information of cancer cell kinetics, and it could help us in deciding the appropriate adjuvant therapy. The prospective study was undertaken to evaluate the clinical implication of DNA ploidy and each proliferative fraction by DNA flowcytometry. The other aim of the study was to evaluate which one is the most valuable index for proliferative activity of cancer cells.
MATERIALS AND METHODS
One hundred and fifty-four patients who underwent gastric resection for primary stomach cancer were included in this study. Male to female ratio was 2.1: 1, and mean age was 58.2 years (range: 26-81). Resected cancer tissues were immediately transported to the flow cytometry laboratory, and analyses for DNA content and cell cycle distribution were carried out by FACScan. The results of flow cytometric analysis were studied in correlation with clinical and histologic parameters; depth of invasion, lymph node metastasis, distant metastasis, stage, Laurens classification, histologic types and grade.
RESULTS
The frequency of aneuploid cancer was 40.3% (62 cases). The mean value of GO/Gl fraction was 75.9% and that of S-phase was 16.0%. Decrease of GO/Gl correlates with lymph node metastasis (p 0.015) and stage (p-0.046). Aneuploid cancer exhibited significant decrease of GO/Gl fraction. However, there was no significant conelation between decreased GO/Gl and depth of invasion, distant metastasis, Laurens classi- fication, differentiation of the cancer cells. Patients with metastasis to the lymph node or distant organs had increased S-phase fraction (p-0.032). High S-phase fraction also correlates with advanced stage (p-0.011) and ploidy of the oancer cells (p=0.001). When the ploidy of the tumor was analysed with clinical variables, aneuploid pattern was increased in cancer cells with intestinal type according to Laurens classificatian (p=0.042), Diploid cancer had significantly lower level of S-phase fraction than aneuploid cancer (p 0.001).
CONCLUSION
Ploidy and growth fraction of the stomach cancer reflected the extent of disease in different aspects. However, there was no single parameter which reflected the extent of disease and degree of malignant potential. Furthermore, there is a possibility that S-phase & action alone is not an accurate parameter for the proliferative activity of stomach cancer cells. In conclusion, flow cytometric analyses is a valuable study providing us more precise information about biologic properties of cancer cells. However, further evaluation with longer follow-up period is imperative because the ultimate value as an prognostic factors can be estimated in respective of clinical outcomes.

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Flow Cytometric Analysis of BRCA1 Protein in Sporadic Breast Cancer: Seung Moo Lee, Kyung Soon Sons, Hee Dae Lee; J Korean Cancer Assoc. 1998;30(4):701-710.

Abstract PDF: PURPOSE
To study the subcellular localization with flow-cytometry and to evaluate their prognostic values.
MATERIALS AND METHODS
The breast tissues were obtained from 28 patients with breast cancer and 6 patients with benign mass. The expression of BRCA1 protein was analyzed with the flow cytometry(Coulter Epics-XL, Coulter Corps, FL, USA) using the monoclonal antibody(BRCA1(Ab-1), Calbiochem, MA, USA) before and after nuclear and cytoplasmic permeabilization in association with DNA ploidy analysis. Several BRCA1 protein indices were derived including 95 percentile channel fluorescence(95% CF) and mean channel fluorescence(MCF) and percentage of BRCA 1 positive cell population arbitarily defined as those above 0.12 channel fluorescence.
RESULTS
Cytoplasmic 95% CF were higher in breast cancer(n=28, 0.65+/-0.26) than in benign mass(n=6, 0.40+/-0.13, p=0.0211). Cytoplasmic BRCAl positive cell percentages were significantly higher in malignant tissues(24.0+/-10.3) than in benign mass(43.4+/-15.2, p=0.0059). Cytoplasmic BRCA1 positive cell percentages were significantly different according to the stages(stage I vs II, 32.6+/-9.8 vs 48.3+/-18.8, p=0.048, stage I vs stage III, 32.6+/-9.8 vs 47.0+/-10.9, p=0.010). The BRCA1 protein indices were not significantly correlated with histologic grades and DNA indices(aneuploidy, S phase and proliferation fractions).
CONCLUSIONS
Flowcytometric assay offers an alternative approach to evaluating BRCA1 protein status of breast cancer tissue and detection of cytoplasmic BRCA1 protein by this method may help to understand the role of BRCA1 in breast cancer cell biology. The further study on cytoplasmic or nuclear BRCA1 protein in association with clinical therapeutic response or prognosis seems to be warranted.

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A Study on the Tropism of Epstein-Barr Virus: Choon Hae Chung, Mi Ja Lee, Ho Jong Jeon; J Korean Cancer Assoc. 1997;29(6):954-964.

Abstract PDF: PURPOSE
The Epstein-Barr Virus (EBV) is associated with a variety of human lymphocytic and epithelial malignancies. EBV is thought to display exclusive tropism for B lymphocytes, follicular dendritic cells, and pharyngeal epithelia via specific receptors (C3d receptors, CR2, CD21). Recent evidence, however, challenged this belief. We designed this experiment to determine the incidence of EBV receptor in various malignant tumor cell lines and normal lymphocyte subsets.
MATERIALS AND METHODS
We have examined the incidence of EBV receptor, CD21 on the 10 healthy adult peripheral blood (PB), 10 umbilical cord blood (CB), 4 immortalized lymphoblastoid B cells by EBV infection (CSUP-1, CSUP-2, CSUP-3, CSUP-4), 3 EBV-positive B cell lymphoma cell lines (Jiyoye, IM-9, PTLC-1), 1 EBV-negative B cell lymphoma cell line (JeKo-1), 3 T cell lymphoma and leukemia cell lines (CCRF-CEM, H9, CEM-CM3), one histiocytic lymphoma cell line (U-937) and 5 gastric cancer cell lines (KATO III, AGS, SNU-1, SNU-5, and SNU-16). EBV receptor, C3d receptor was identified by flow cytometry (FACSCalibur) using FITC-conjugated or PE-conjugated CD21 monoclonal antibody. Also we investigated the expression of CD3, CD5, CD7, CD19, CD20, IgM, IgG, Ig and Ig by using FITC-conjugated or PE-conjugated monoclonal antibody, on above cell lines.
RESULTS
The expressions of CD21 molecule were 10.99 3.84% and 9.22 5.39% in adult PB lymphocytes and CB lymphocytes, respectively. The anti-human CD21 antibody was positive for CD19-positive or CD20-positive B lymphocytes. The CD3-positive or CD7-positive T lymphocytes were negative for anti-human CD21 antibody in PB and CB. But, CD21 antibody was weakly positive for CD5-positive lymphocytes. EBV-positive cell lines expressed variable ranges from 0.9% to 5.2% for CD21 antigen, while EBV-negative lymphoma cell line, JeKo-1 expressed 5.5%. All T lymphoma and leukemia cell lines and gastric cancer cell lines did not express CD21 antigen. But U-937 expressed 14.4% for CD21 antigen.
CONCLUSION
These results suggested that the CD21 antigen was expressed in CD20 or CD19-positive mature B cells, CD5-dim positive lymphocytes, some EBV-positive and negative B cell lymphoma cell lines, and a histiocytic lymphoma cell line. Further evaluation on the nature of CD5-dim positive cells, which was expressing CD21 molecule, is revealed, especially in reference to EBV association in some peculiar subtypes of peripheral T cell lymphoma.

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Assessment of Cell Proliferation in Primary and Recurrent Colorectal Cancers - Expression of Transforming Growth Factor - α and Prolifer: Jin Sil Seong, Sun Hee Sung, Jung Woon Lee, Hyun Soo Shin, Charn Il Park, Oh Hun Kwon; J Korean Cancer Assoc. 1995;27(2):223-230.

Abstract PDF: Cell proliferation potential has been found to be a significant biological parameter correlated with the clinical outcome. This study was ta investigate the cell proliferation potential in primary and recurrent colorectal tumor tissues. Using paraffin-embedded tissues from the paired primary and recurrent tumors of l0 patients, a simple hematoxylineosin stain was done and immunohistochemical stains for trans- forming growth factor-a(TGF-a) and proliferating cell nuclear antigen(PCNA) were performed through a labeled streptavidine biotin method. DNA contents and S-phase fraction(SPF) of the cells were assessed by flowcytometric DNA analysis. The degree of differentiation was poorer in the recurrent tumors than in primary tumors. In 4 primary tumors with mixed adenocacinoma and mucinous adenocarcinoma, only the mucinous adenocarcinoma companent was shown in the recurrent tumors. There was no difference in TGF-a expression between the primary and the recurrent tumors however, PCNA was overexpressed in the recurrent tumors comparing to the primary tumors. Flow cytometric DNA analysis was successful in 7 paired cases. There was change of the ploidy from the diploidy to the aneuploidy in 4 cases. SPF showed remarkable increase in the recurrent tumors comparing to the primary tumors. These results show high proliferative potential of the recurrent colorectal tumors, which can be measured using PCNA expression and SPF as biomarkers. Based on the results of this study, an effort to establish more refined method to predict recurrence should be pursued.

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Usefulness of MIB1 Expression as a Prognostic Factor in Transitional Cell Carcinoma of Urinary Bladder: Han Kyeum Kim, Young Sik Kim, In Sun Kim; J Korean Cancer Assoc. 1995;27(3):459-468.

Abstract PDF: Tumor proliferation is inversely associated with survival in patients with transitional cell carcinoma of urinary bladder. Ki67 and MIB1 monoclonal antibodies directed against different epitopes of the same proliferation-related antigen. Whereas Ki67 works only on frozen section, MIBl may be used also on fixed sections. In order to determine whether MIB1 is clinically a useful proliferation marker as well as prognostic one or not, MIB1 expresaion, in. bladder cancers were compared with conventional prognostic parameters such: as Ash histolbgic grade, and proliferation index including S-phase fraction calculated by flow cytometry. The expression of MIBI was assessed immunohistochemically in 36 cases of transitional cell carcinoma with anti-MIBl monoclonal antibody which can recognize a formai'in resistent epitope of Ki67 in formalin-fixed, paraffin wax embedded sections after incubation fn a microwave oven. Then, image analysis was employed for quantitation of immunostained MIBl expression. MIBI expression rates were highly correlated with Ash histologic grade. And, two pa- rameters revealed a good correlatian in linear regression analysis(Y = 4.16X - 3.01). Also, prolife- ration index and S-phase fraction calculated by flow cytometry were highly correlated with MIB1 expression(r=0.92, r=0.87, respectively) Therefore, MIB1 immunostaining is considered as a useful prognostic marker in transitional cell carcinoma of urinary bladder and a reason- able substitute for the Ki67 monoclonal antibody. In addition, the advantages of MIB1 immunostaining on paraffin sections include the feasibility of retrospective studies and of obtaining clear morphologic specimens that are optimal for use with computer-assisted image analysis systems.

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Cell Kinetics Studies of Human Epithelial Cancers with Bromodeoxyuridine Labeling: Jin Sil Seong, Jung Woon Lee, Eun Ji Chung, Yung Tae Kim, Jae Wook Kim, Nam Kyu Kim, Sung Joon Hong, Eun Chan Choi, Won Sang Lee, Oh Hun Kwon, Gwi Eon Kim; J Korean Cancer Assoc. 1995;27(5):783-790.

Abstract PDF: Cell kinetic parameters of labeling index (LI), duration of S-phase (Ts), and potential doubling time (Tpot) were analyzed following infusion of bromodeoxyuridine (BUdR) in 33 patients with various epithelial cancers. Twelve uterine cervical cancers, 9 rectal cancers, 7 head and neck caneers, and 5 bladder cancers were included. Biopsies were taken about 4-6 h after 200 mg/m(2) BUdR infusion and the samples were anaiyzed with bivariate DNA /BUdR flow cytometry. The distribution of cell kinetic parameters for the 33 epithelial cancers showed a large range of values for each parameter. The median LI, Ts, and Tpot were 4.5%, 10.8 h, and 242.3 h, respectively. Eight among 33 patients (24.2%) showed aneuploidy. In aneuploid tumors the distribution of LI, Ts, and Tpot was in relatively small range. Aneupliod tumors appeared to show higher LI and shorter Tpot than those in diploid tumors. In diploid tumors, the poesibility of normal cell contamination could not be ruled out. The results of this study would be a basis for future trial to predict which ones would show tumor clonogen repopulation during radiotherapy so that benefit from altered fractionated radiotherapy.

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Flow Cytometric DNA Analysis of Breast Carcinoma: Kwang Sun Suh, Chin Sun Bae; J Korean Cancer Assoc. 1996;28(2):253-261.

Abstract PDF: Nuclear DNA content in 35 cases of breast carcinomas was determined by flow cytometry using paraffin-embedded archival tissue, in order to investigate the correlation between DNA ploidy and pathologic parameters, such as histologic type and degree of differentiation. DNA aneuploidy was found in 18 cases(51.4%). DNA ploidy was correlated with histologic type, tumor grade, and degree of tumar necrosis(P<0.005), but not with patient age, tumor size, or axillary nodal status(P>0.1). There was a tendency for high grade diploid tumors to have a higher S-phase fraction than low grade tumors, although it was statistica11y insignificant. Four patients out of 18 with aneuploid tumors died of breast cancer 10 months to 2 years after operation. However, it was impossible to analyse clinical and pathologic parameters to predict biologic behavior because of the small number of cases and inadequate follow-up data.

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