Tohoku
J. Exp.
Flow Cancer
Med.,
1992,
168, 307-310
Cytometric Cell
DNA
Analysis
of Lung
Lines
SHINTO HIRO OKADA, SHUNSUKE KOBAYASHI,HIROHISA INABA, NOBUYUKISATO, TORU HASUMI,WATARUSHOJI, HIROYUKIYOSHIDAand SHIGEFUMIFUJIMURA Department of Surgery, The Research Institute for Tuberculosis and Cancer, Tohoku University, Sendai 980
OKADA,S., KOBAYASHI, S., INABA,H., SATO,N., HASUMI,T., SHOJI, W., YOSHIDA, H. and FUJIMURA,S. Flow Cytometric DNA Analysis of Lung Cancer Cell Lines. Tohoku J. Exp. Med., 1992, 168 (2), 307-310 Tumor DNA content (ploidy) was analyzed by use of flow cytometry (FCM) in 17 lung cancer cell lines which were subcultured in our laboratory. The study included 6 adenocarcinomas, 2 squamous cell carcinomas, l adenosquamous cell carcinoma, 5 large cell carcinomas, and 3 small cell carcinomas. Of the 17 lung carcinoma cell lines, 15 revealed aneuploid patterns with DNA index above 1.1, whereas one had diploid. The mean DNA index (DI) in adenocarcinoma, was 1.34+0.09, DI 1.6, in squamous cell carcinoma, DI 1.0 in adenosquamous cell carcinoma, DI 1.70±0.66 in large cell carcinoma, and DI 1.29 in small cell carcinoma. Of the 17 cell lines, three lines showed multiploid patterns with clinically poor prognosis and indicated heterogeneity. Flow cytometric DNA analysis using lung cancer cell lines could provide further basic study of lung cancer cells and give a useful information on the degree of the malignancy clinically, lung cancer ; in vitro ; flow cytometry ; DNA index
DNA content has been proven to be clinical interest in various tumors. Many studies on DNA analysis of fresh specimens have been reported (Klein et al 1982; Jacobsen et al. 1983; Friedlander et al. 1984; Strang et al. 1986). However, there are few studies on DNA analysis of human tumor cell lines, especially cell lines of lung cancer. The purpose of this study is to determine (1) whether cellular DNA content is a stable feature of the lung cancer cell lines and (2) whether there is a correlation between the DNA index and prognosis. Tumor DNA content (ploidy) was analyzed by use of flow cytometry (FCM) in 17 lung cancer cell lines. MATERIALS AND METHODS The study included 17 lung cancer lines of 6 adenocarcinomas, 2 squamous cell carcinomas, 1 adenosquamous cell carcinoma, 5 large cell carcinomas, and 3 small cell carcinomas which have been subcultured in our laboratory (Table 1). 10,000 cells were measured by FCM (EPICS 750) in each analysis. Prior to analysis, the cells were treated Address for reprints : 4-1 Seiryomachi, Aoba-ku, Sendai 980, Japan. 307
S. Okada
308 TABLE 1.
In
vitro biological
et al.
characteri sties
of lung
cancer
cell lines
RESULTS Of the lung carcinoma cell lines, 15 revealed aneuploid patterns with DNA index above 1.1, whereas one (AS, adenosquamous cell carcinoma) had diploid (Table 1, Figs. 1, 2, 3).
Fig. 1. lines.
DNA histogram
of lung cancer
cell
Fig. 2. lines.
DNA histogram
of lung cancer
cell
Flow
Cytometric
TABLE 2.
Fig. 3. lines.
DNA histogram
DNA Analysis
Relationship
of lung cancer
between
cell
of Lung DNA
index
Cancer and
Cell Lines clinical
Fig. 4. Relationship and prognosis.
309
stage
between
DNA
index
The mean DNA index (DI) in adenocarcinoma was 1.34±0.09, DI 1.6 in squamous cell' carcinoma, DI 1.0 in adenosquamous cell carcinoma, DI 1.70 + 0.66 in large cell carcinoma, and DI 1.29 in small cell carcinoma. The cell lines with DNA index above 2.0 included one squamous cell carcinoma (Sq2) and two large cell carcinomas (L1, L2), which prognosis were 7, 2, 2.5 months, respectively. Of the 17 cell lines, three lines (one cell line of small cell carcinoma Sl, and
310
S. Okada
et al.
two cell lines of large cell carcinoma Ll, L2) showed multiploid patterns clinically poor prognosis (25, 2, 2.5 months, respectively Table 2, Fig. 4).
with
DISCUSSION Since most human solid tumor specimen contain both benign and malignant portions, a DNA histogram represents the sum of these cell components, which reveals a distinct DNA content. The histogram may reveal one peak with a wide coefficient of variation (CV), a peak with skewed distribution pattern, or two or more separate peaks. In other words, it may be difficult to differentiate aneuploid tumor cells with a near diploid DNA content from benign diploid cells in some cases. DNA distribution histogram in cancer cell lines provides DNA content of only true cancer cells, which can be easy and simple to interpret DNA histogram. Three cell lines (one cell line of small cell carcinoma and two cell lines of large cell carcinoma) gave multiploidy patterns which indicated heterogeneity in cell populations. The fact that clinically small cell carcinoma and large cell carcinoma show heterogeneity supports our data. The present study implied that DNA ploidy pattern and DNA index in lung cancer cell lines could be prognostic factors in clinic. Flow cytometric DNA analysis using cell lines of lung cancer could provide further basic study of lung cancer cells and give a useful information on the degree of the malignancy clinically. Acknowledgments The authors thank Ms.Mayumi Chiba for her technical assistance on tissue culture. References 1) Friedlander, M., Hedley, D.W. & Taylor, J.W. (1984) Clinical and biological significance of aneuploidy in human tumors. J. Olin. Pathol., 37, 961-974. 2) Jakobsen, A., Kristensen, P.B. & Poulsen, H.K. (1983) Flow cytometric classification of biopsy specimens from cervical intraepithelial neoplasia. Cytometry,4, 166-169. 3) Klein, F., Herr, H., Sogani, P., Whitmore, W., Jr. & Melamed, M. (1982) Detection and follow-up of carcinoma of the urinary bladder by flow cytometry. Cancer, 50, 389-395. 4) Strang, P., Stendahl, U., Frankendal, B. & Lindgren, A. (1986) Flow cytometric DNA patterns in cervical carcinoma. Acta Radial. Oncol., 25, 249-254.
J. Exp.
Flow Cancer
Med.,
1992,
168, 307-310
Cytometric Cell
DNA
Analysis
of Lung
Lines
SHINTO HIRO OKADA, SHUNSUKE KOBAYASHI,HIROHISA INABA, NOBUYUKISATO, TORU HASUMI,WATARUSHOJI, HIROYUKIYOSHIDAand SHIGEFUMIFUJIMURA Department of Surgery, The Research Institute for Tuberculosis and Cancer, Tohoku University, Sendai 980
OKADA,S., KOBAYASHI, S., INABA,H., SATO,N., HASUMI,T., SHOJI, W., YOSHIDA, H. and FUJIMURA,S. Flow Cytometric DNA Analysis of Lung Cancer Cell Lines. Tohoku J. Exp. Med., 1992, 168 (2), 307-310 Tumor DNA content (ploidy) was analyzed by use of flow cytometry (FCM) in 17 lung cancer cell lines which were subcultured in our laboratory. The study included 6 adenocarcinomas, 2 squamous cell carcinomas, l adenosquamous cell carcinoma, 5 large cell carcinomas, and 3 small cell carcinomas. Of the 17 lung carcinoma cell lines, 15 revealed aneuploid patterns with DNA index above 1.1, whereas one had diploid. The mean DNA index (DI) in adenocarcinoma, was 1.34+0.09, DI 1.6, in squamous cell carcinoma, DI 1.0 in adenosquamous cell carcinoma, DI 1.70±0.66 in large cell carcinoma, and DI 1.29 in small cell carcinoma. Of the 17 cell lines, three lines showed multiploid patterns with clinically poor prognosis and indicated heterogeneity. Flow cytometric DNA analysis using lung cancer cell lines could provide further basic study of lung cancer cells and give a useful information on the degree of the malignancy clinically, lung cancer ; in vitro ; flow cytometry ; DNA index
DNA content has been proven to be clinical interest in various tumors. Many studies on DNA analysis of fresh specimens have been reported (Klein et al 1982; Jacobsen et al. 1983; Friedlander et al. 1984; Strang et al. 1986). However, there are few studies on DNA analysis of human tumor cell lines, especially cell lines of lung cancer. The purpose of this study is to determine (1) whether cellular DNA content is a stable feature of the lung cancer cell lines and (2) whether there is a correlation between the DNA index and prognosis. Tumor DNA content (ploidy) was analyzed by use of flow cytometry (FCM) in 17 lung cancer cell lines. MATERIALS AND METHODS The study included 17 lung cancer lines of 6 adenocarcinomas, 2 squamous cell carcinomas, 1 adenosquamous cell carcinoma, 5 large cell carcinomas, and 3 small cell carcinomas which have been subcultured in our laboratory (Table 1). 10,000 cells were measured by FCM (EPICS 750) in each analysis. Prior to analysis, the cells were treated Address for reprints : 4-1 Seiryomachi, Aoba-ku, Sendai 980, Japan. 307
S. Okada
308 TABLE 1.
In
vitro biological
et al.
characteri sties
of lung
cancer
cell lines
RESULTS Of the lung carcinoma cell lines, 15 revealed aneuploid patterns with DNA index above 1.1, whereas one (AS, adenosquamous cell carcinoma) had diploid (Table 1, Figs. 1, 2, 3).
Fig. 1. lines.
DNA histogram
of lung cancer
cell
Fig. 2. lines.
DNA histogram
of lung cancer
cell
Flow
Cytometric
TABLE 2.
Fig. 3. lines.
DNA histogram
DNA Analysis
Relationship
of lung cancer
between
cell
of Lung DNA
index
Cancer and
Cell Lines clinical
Fig. 4. Relationship and prognosis.
309
stage
between
DNA
index
The mean DNA index (DI) in adenocarcinoma was 1.34±0.09, DI 1.6 in squamous cell' carcinoma, DI 1.0 in adenosquamous cell carcinoma, DI 1.70 + 0.66 in large cell carcinoma, and DI 1.29 in small cell carcinoma. The cell lines with DNA index above 2.0 included one squamous cell carcinoma (Sq2) and two large cell carcinomas (L1, L2), which prognosis were 7, 2, 2.5 months, respectively. Of the 17 cell lines, three lines (one cell line of small cell carcinoma Sl, and
310
S. Okada
et al.
two cell lines of large cell carcinoma Ll, L2) showed multiploid patterns clinically poor prognosis (25, 2, 2.5 months, respectively Table 2, Fig. 4).
with
DISCUSSION Since most human solid tumor specimen contain both benign and malignant portions, a DNA histogram represents the sum of these cell components, which reveals a distinct DNA content. The histogram may reveal one peak with a wide coefficient of variation (CV), a peak with skewed distribution pattern, or two or more separate peaks. In other words, it may be difficult to differentiate aneuploid tumor cells with a near diploid DNA content from benign diploid cells in some cases. DNA distribution histogram in cancer cell lines provides DNA content of only true cancer cells, which can be easy and simple to interpret DNA histogram. Three cell lines (one cell line of small cell carcinoma and two cell lines of large cell carcinoma) gave multiploidy patterns which indicated heterogeneity in cell populations. The fact that clinically small cell carcinoma and large cell carcinoma show heterogeneity supports our data. The present study implied that DNA ploidy pattern and DNA index in lung cancer cell lines could be prognostic factors in clinic. Flow cytometric DNA analysis using cell lines of lung cancer could provide further basic study of lung cancer cells and give a useful information on the degree of the malignancy clinically. Acknowledgments The authors thank Ms.Mayumi Chiba for her technical assistance on tissue culture. References 1) Friedlander, M., Hedley, D.W. & Taylor, J.W. (1984) Clinical and biological significance of aneuploidy in human tumors. J. Olin. Pathol., 37, 961-974. 2) Jakobsen, A., Kristensen, P.B. & Poulsen, H.K. (1983) Flow cytometric classification of biopsy specimens from cervical intraepithelial neoplasia. Cytometry,4, 166-169. 3) Klein, F., Herr, H., Sogani, P., Whitmore, W., Jr. & Melamed, M. (1982) Detection and follow-up of carcinoma of the urinary bladder by flow cytometry. Cancer, 50, 389-395. 4) Strang, P., Stendahl, U., Frankendal, B. & Lindgren, A. (1986) Flow cytometric DNA patterns in cervical carcinoma. Acta Radial. Oncol., 25, 249-254.
