J. Jpn. Soc. Clin. Cytol. 1997; 36 (4): 349 `357. Key words: HL-60 cells TUNEL assay Apoptosis Cell smear Cell block Cytopathology A study for TUNEL a

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1 J. Jpn. Soc. Clin. Cytol. 1997; 36 (4): 349 `357. Key words: HL-60 cells TUNEL assay Apoptosis Cell smear Cell block Cytopathology A study for TUNEL assay as a method of detecting apoptotic cells Takako TAKEUCHI1), C. T., C. M. I. A. C., Akiko FUJIII), C. T., I. A. C., Yuka MIMURA4), M.T., Fumiko HIRAOKA2), C.T., I. A. C., Yuko HIRATA1), C.T., J. S. C., Miyuki KAMM1), C. T., I. A. C., Mie HARADA1), C.T., I. A. C., Hiroaki TAKEUCHI3), C. T., C. M. I. A. C., Tomoyuki MURAKAMI4), M. D. Department of Pathology, Shimonosekishi-Ishikai 1) Hospital 2)Department of Laboratory Medicine, 3)Department of Microbiology, 4)Department of Pathology, Yamaguchi University School of Medicine

3 Figure 1 Flow chart of sample preparations and TUNEL staining. = indicates the steps whose conditions were examined. "Equilibration buffer", TdT reaction solution composed of "TdT Enzyme" and "Reaction Buffer", "Stop/Wash Buffer" and "Auti -Digoxygenin-Peroxidase" solution were the agents included in ApopTagTM kit. Table 1 TUNEL-positive percentages on smears of apoptosis-induced HL-60 cells nder the protocol-directed TdT reaction a,b: A quite small number of cells remained on the slides of 24 hours and 1 week fixation with 10% formalin, and the positive ratios were 24.0% and 28.7%, respectively.

4 Table 2 TUNEL-positive percentages on smears of apoptosis-induced HL-60 cells under various TdT reactions without proteinase K treatment TdT Concentrations/Incubation Times a:<5%; and weak stainability b: n.d.; not done difficult to count exact number of positive-cells, because of extremely few cells Table 3 TUNEL-positive percentages on paraffin embedded sections of apoptosisinduced HL-60 cells under the protocol-directed TdT reaction without proteinase K treatment Time in Fixative *Proteinase K treatment:(-) *TdT enzyme activity: at the concentration indicated in the protocol of Ap optagtm for 1 hour Table 4 TUNEL-positive percentages on paraffin embedded sections of apoptosis-induced HL-60 cells under various TdT reactions with proteinase K treatment TdT Concentration/Incubation Time a:>; One experiment showed the number indicated at the right of">", while the other two experiments did more than 80% in the tripricated study. b:<; difficult to count exact number of positive cells, because of extremely few and low stainability : n. d.; not done

5 Table 5 TUNEL staining of 25 clinical samples (17 cases) according to the optimal conditions Normal mucosae were obtained from the same surgical materials including cancers listed immediately above them, respectively, on the table

7 Summary Since 1992, in situ TdT-mediated digoxygenin (biotin)- dutp nick end labeling (TUNEL) assay has become prevalent as a method of detecting apoptotic cells. However, our first trial with such specimens using a commercially available TUNEL kit and its protocol was disappointing, because of the considerable number of false-positive and false-negative cells. Therefore, we realized that this assay requires more studies about sample preparation and staining conditions to obtain higher specificity and sensitivity, especially in its application to cytologic smears and paraffin-embedded sec- In the present study, as a definite control of apoptotic tions. and non-apoptotic cells, human leukemia HL-60 cells treated with 0.15,u M campthotecin for 3 h were used. This treatment is known to induce apoptosis in approximately 30% of HL-60 cells. Smears and paraffin-embedded sections of those cells were prepared before and after fixation, respectively, under several conditions. Then, they were stained with agents included in a TUNEL assay kit (ApopTagTM, Oncor) at various concentrations of TdT enzyme and after various incubation times, with or without pretreatment with proteinase K. We regarded conditions resulting in 30% positive as optimal. We obtained good conditions for TUNEL assay for smears and paraffin-embedded sections of HL-60 cells. In addition, this method under those conditions fairly stained 25 surgically excised specimens prepared as touch smears and paraffin - embedded tissue sections. Notably, we also found that TUNEL staining is strongly influenced by fixation, proteinase K treatment, and TdT activity. Therefore, one should be very careful in evaluating results of TdT staining of materials whose preparatory conditions are unknown. 1) Kerr, J. F. R., Wyllie, A. H.,Currie, A. R. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 1972; 26: 239 `257. 2) Wyllie, A. H. Cell death: a new classification of separating apoptosis from necrosis. Cell death in biology and pathology. Edited by Bowen, I.D. and Lockshin, R. A. London, Cahpman and Hall Press: Lon- don, 1981; 9 `34. 3) Searle, J., Kerr, J. F. R., Bishop, C. J. Necrosis and apoptosis: distinct modes of cell death with funda-

8 mentally different significance. Pathol. Annu. 1982, 1981; 17: ) Kerr, J.F.R., Winterford, C.M., Harmom, B.V. Apoptosis : its significance in cancer and cancer therapy. Cancer 1994; 78: 2013 ` ) Fisher, D.E. Apoptosis in cancer therapy: crossing the threshold Cell 1994; 78: 539 `542. 6) Gorczyca, W., Bruno, S., Darzynkiewicz, R.J., Gong, J., Darzynkiewicz, Z. DNA strand breaks occurring during apoptosis: their early in situ detection by terminal deoxynucleotidyl transeferase and nick translation assays and prevention by serine protease inhibitors. Int. J. Oncol. 1992; 1: 639 `648. 7) Gavrieli, Y., Sherman, Y., Benn-Sasson, S.A. Identification of programmed cell death in situ via specific labeling of nuclear fragmentation. J. Cell Biol. 1992; 119: 493 `501. 8) Gorczyca, W., Gong, J. Ardrelt, B., Traganos, F., Darzynkiewicz, Z. The cell cycle related differences in susceptibility of HL-60 cells to apoptosis induced by various antitumor agents. Cencer Res. 1993; 53: 3186 ` ) Darzynkiewicz, Z., Bruno, S., Bino, G.D., Gorczyca, W., Hotz, M.A., Lassota, P. et al. Feature of apoptotic cells measured by flow cytometry. Cytometry 1992; 13: ) Murakami, T., Li, X., Gong, J., Bhatia, U., Traganos, F., Darzynkiewicz, Z. Inducation of apoptosis by 5- azacytidine: drug concentration-dependent differences in cell cycle specificity. Cancer Res. 1995; 55: 3093 ` ) Yasuda, M., Umemura, S., Osamura, R., Kenjo, T., Tsutsumi, Y. Apoptotic cells in the human endometrium and placental villi : pitfalls in applying the TUNEL method. Archives of Histology and Cytology 1995; 58: 185 `190.

第36巻第4号,1997年 357 a Photo b 1 Apoptosis-induced HL-60 cells by camptothecin : smear preparations stained by Papanicolaou (1-a) and TUNEL at the optimal condition (1-b).

9 第36巻第4号,1997年 357 a Photo b 1 Apoptosis-induced HL-60 cells by camptothecin : smear preparations stained by Papanicolaou (1-a) and TUNEL at the optimal condition (1-b). Cell death of HL-60 cells induced by 0.15 p CAM for 3 hours is exclusively apoptosis and its proportion is approximately 30%. Morphologically "apoptotic" cells, therefore, can be regarded as typical apoptotic cells who have received DNA strand breaks in nucleosomal units. A condition of TdT incubation examined in this study revealed a reasonable result of staining in both of percentage (Table 2) and morphology (1-b) (objective ; x 40). b a Photo 2 A case of colon cancer : a touch smear stained by Papanicolaou (2-a), a touch smear (2-b) and a paraffin embedded section (2-c) stained by TUNEL under the optimal conditions, respectively. Almost all of the pyknotic cells and nuclear materials which are morphologically suggested as apoptotic cells and apoptotic bodies are strongly stained by TUNEL, though some remains unstained c (objective a Photo 3 ; x 40). b Clinical materials stained with a TUNEL Staining Kit. The pictures show TUNEL staining for paraffin embedded tissue sections of normal gall bladder mucosa (3-a) and colon cancer (3-b) stained with ApopTagTM according to its protocol. False positive and false negative staining are strongly suggested from those pictures, respectively (objective ; x 40).

Visual Evaluation of Polka-dot Patterns Yoojin LEE and Nobuko NARUSE * Granduate School of Bunka Women's University, and * Faculty of Fashion Science,

Visual Evaluation of Polka-dot Patterns Yoojin LEE and Nobuko NARUSE * Granduate School of Bunka Women's University, and * Faculty of Fashion Science, Bunka Women's University, Shibuya-ku, Tokyo 151-8523