SHORT COMMUNICATIONS
mRNA Expression of Topoisomerase II in Human Tumors and Normal Tissues Ryungsa KIM1, Naoki HIRABAYASHI1, Masahiko NISHIYAMA 1, Takashi YORISHIMA1, Tetsuya TOGEa and Kosuke OKADA2 ABSTRACT: T h e cellular levels o f topoisomerase II expression were compared between 10 fresh h u m a n tumors and normal tissues to predict the selective anticancer effect o f its inhibitors such as adriamycin and VP-16. Topoisomerase II expression was observed in 9 o f the 10 tumor tissues (90.0 per cent), 3 of which showed extremely high levels, whereas only 5 o f the normal tissues (50.0 per cent) expressed any cellular topoisomerase II and the levels were not higher than those seen in the cancer cells. Six o f the 9 positive tumors showed a higher level o f topoisomerase II expression than the normal tissues, while the other 3 showed the same level. It can be interpreted from these results that topoisomerase II inhibitors could be effective in cancer patients due to the greater level of this enzyme in tumor cells than in normal tissues. Thus, it is suggested that a comparative analysis o f topoisomerase II expression between tumors and normal tissues may be useful for predicting the selective cytotoxicity of topoisomerase II inhibitors in clinical practice. KEY WORDS: topoisomerase II level, topoisomerase II inhibitor, h u m a n tumors and normal tissues
R e c e n t l y , much attention has b e e n focused on mammalian DNA topoisomerase II as a target of various anticancer agents. 1-~ It has b e e n clearly demonstrated that adriamycin or etoposide (VP-16) induces reversible protein linked DNA damage and inhibits the DNA rejoining reaction? -~ Moreover, the level a n d / o r activity of this enzyme is closely related with cell proliferation 6 and thus, the enzymatic fluctuation in Cells strongly influ-
1The Department of Surgery, Research Institute for Nuclear Medicine and Biology, Hiroshima University and 2the Division of Blood Transfusion, Hiroshima University Hospital, Hiroshima,Japan Reprint requests to: Ryungsa Kim, MD, The Department of Surgery, Research Institute for Nuclear Medicine and Biology, Hiroshima University, 1-2-3, Kasumi Minami-ku, Hiroshima 734,Japan
ences the effect of topoisomerase II anticancer agents. However, few reports have shown the difference in topoisomerase II levels between h u m a n tumor cells and normal cells. In this paper, w e a n a l y z e the cellular levels o f topoisomerase II expression in 10 tumors and the adjacent normal tissues. T h e levels o f topoisomerase II expression were analyzed by the RNA dot blot. For the isolation of total RNA, ten fresh h u m a n tumors and each of the respective adjacent normal tissues were frozen immediately after surgery and stored at --70~ until the analysis. Total cellular RNA was isolated by the guanidinium isothiocyanate method.' Two pg of each RNA preparation was denatured in 10 per cent formaldehyde and applied to nitrocellulose filters for dot blot hybridi, zation2 A s2P-labelled t o p o i s o m e r a s e H
JAPANESEJOURNALOF SURGERY,VOL.21, No. 5 pp. 587-589, 1991
Kim et al.
588
Jpn. J. Surg. September 1991
Table 1. Comparison of Topoisomerase II Expression Levels between Human Tumors and Normal Tissues Topo I I Expression Tumor Normal
Patient No.
Tumor Type
1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
Gastric ca. Gastric ca. Gastric ca. Gastric ca. Gastric ca. Rectal ca. Liver meta. (Rectum) Hepatocellular ca. Lung ca. Renal meta. (Pancreas)
Actin Topo II expression Ieve|/~
Fig. 1. Typical results oftopoisomerase II expression. Total cellular RNA (2 /+g) from tumors and normal tissues was spotted onto nitrocellulose filters. The filters were hybridized to the topoisomerase II probe and then rehybridized to the/3-actin as an internal control. Upper rows indicate those hybridized to the topoisomerase II probe and lower rows, those hybridized to the 13-actin probe. T; tumor, N; normal. p r o b e , 9 kindly p r o v i d e d by Dr. L.F. Liu, J o h n s Hopldns University, USA, and a /3actin p r o b e as an internal control were made by the olligolabelling m e t h o d ? ~ T h e filters were prehybridized at 42~ in 50 per cent formamide, 5 X Denhardts reagent, 5 X SSC (NaC1/sodium citrate, p H 7.0) a n d 200 ktg denatured salmon sperm DNA/ml. Hybridization was p e r f o r m e d overnight in the same solution containing 32P-labelled probes. After hybridization, the filters were washed in 2 X SSC with 0.1 p e r cent SDS (sodium dodecyl sulfate) followed by a final wash in 0.1 X SSC a n d 0.1 per cent SDS at 65~ Autoradiograms p e r f o r m e d at --70~ for 1-2 days a n d
-+ + + ~ + ~+ ntt+
--+ -+ + -+ + --
the intensity o f autoradiograms quantified by densitometric analysis. T e n tumors, comprising 5 gastric, 1 rectal, 1 hepatocellular, 1 lung, 1 metastatic liver and 1 metastatic renal cancer, as well as the adjacent n o r m a l tissues, were studied. Topoisomerase II expression was observed in 9 o f the 10 t u m o r tissues (90.0 p e r cent), 3 o f which showed extremely high levels. O n the other hand, topoisomerase II expression was observed in o n l y 5 o f the adjacent normal tissues (50.0 p e r cent). Six o f the 9 topoisomerase I I detected tumors showed higher levels of expression t h a n the adjacent normal tissues, while similar expression was observed in the other 3 tumors. T h e s e results indicate that at least in some tumors the levels o f topoisomerase II are higher than those in normal tissues. Since the levels and regulation o f this enzyme in proliferating t u m o r cells differ f r o m those in quiescent or n o r m a l cells, 11,12 a c o m p a r a t i v e study o f topoisomerase I I expression should be perf o r m e d for effective use o f the inhibitors. T h e topoisomerase I I in tumor cells may be regulated through the RNA transcription or protein degradation. H,13 A l t h o u g h f u r t h e r clinical a n d genetic studies on topoisomerase II expression in t u m o r cells will be required, our study, which indicates the higher level o f topoisomerase II expression in t u m o r cells, may provide useful information about the clinical effects
Volume 21 Number 5
Topoisomerase H expression
of topoisomerase II inhibitors. ( R e c e i v e d f o r p u b l i c a t i o n o n O c t . 3, 1990)
8.
REFERENCES 9. 1. Lock RB, Ross WE. DNA topoisomerases in cancer therapy. Anti Cancer Drug Design 1987; 2: 151164. 2. Drlica K, Franco RJ. Inhibitors of DNA topoisomerases. Biochemistry 1988; 27: 2253-2259. 3. Liu LF. DNA topoisomerase poisons as antitumor drugs. Annu Rev Biochem 1989; 58: 351-357. 4. Hsiang Y-H, Hertzberg R, Hecht S, Liu LF. Camptothecin induced protein-linked DNA breaks via mammalian DNA topoisomerase I. J Biol Chem 1985; 260: 14873-14878. 5. Tewey KM, Chen GL, Nelson EM, Liu LF. Intercalative antitumor drugs interfere with the breakageunion reaction of mammalian DNA topoisomerase II.J Biol Chem 1984; 259: 9182-9187. 6. Sullivan DM, Glisson BS, Hodges PK, SmallwoodKentro S, Ross WE. Proliferation dependence of topoisomerase II-mediated drug action. Biochemistry 1986; 25: 2248-2256. 7. Maniatis T, Fritsch EF, Sambrook J. Molecular
10.
11.
12.
13.
589
cloning: a laboratory mannual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 1982. Thomas PS. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci 1980; 77: 5201-5205. Tsai-Pflugfelder M, Liu LF, Liu AA, Tewey KM, Whang-PengJ, Knutsen T, Huebner K, Croce CM, Wang JC. Cloning and sequencing of cDNA encoding human DNA topoisomerase II and localization of the gene to chromosome region 17q21-22. Proc Natl Acad Sci 1988; 85: 7177-7181. Feiberg AP, Vogelstein B. A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 1983; 132: 6-13. Hsiang Y-H, Wu H-Y, Liu LF. Proliferation-dependent regulation of DNA topoisomerase II in cultured human cells. Cancer Res 1988; 48: 32303235. HsiangY-H, Wu H-Y, Liu LF. Topoisomerase: novel therapeutic targets in cancer chemotherapy. Biochem Parmacot 1988; 17: 1801-1802. Heck MMS, Hittelman WN, Earnshaw WC. Differential expression of DNA topoisomerase I and II during the eukaryotic cell cycle. Proc Natl Acad Sci 1988; 85: 1086-1090.
mRNA Expression of Topoisomerase II in Human Tumors and Normal Tissues Ryungsa KIM1, Naoki HIRABAYASHI1, Masahiko NISHIYAMA 1, Takashi YORISHIMA1, Tetsuya TOGEa and Kosuke OKADA2 ABSTRACT: T h e cellular levels o f topoisomerase II expression were compared between 10 fresh h u m a n tumors and normal tissues to predict the selective anticancer effect o f its inhibitors such as adriamycin and VP-16. Topoisomerase II expression was observed in 9 o f the 10 tumor tissues (90.0 per cent), 3 of which showed extremely high levels, whereas only 5 o f the normal tissues (50.0 per cent) expressed any cellular topoisomerase II and the levels were not higher than those seen in the cancer cells. Six o f the 9 positive tumors showed a higher level o f topoisomerase II expression than the normal tissues, while the other 3 showed the same level. It can be interpreted from these results that topoisomerase II inhibitors could be effective in cancer patients due to the greater level of this enzyme in tumor cells than in normal tissues. Thus, it is suggested that a comparative analysis o f topoisomerase II expression between tumors and normal tissues may be useful for predicting the selective cytotoxicity of topoisomerase II inhibitors in clinical practice. KEY WORDS: topoisomerase II level, topoisomerase II inhibitor, h u m a n tumors and normal tissues
R e c e n t l y , much attention has b e e n focused on mammalian DNA topoisomerase II as a target of various anticancer agents. 1-~ It has b e e n clearly demonstrated that adriamycin or etoposide (VP-16) induces reversible protein linked DNA damage and inhibits the DNA rejoining reaction? -~ Moreover, the level a n d / o r activity of this enzyme is closely related with cell proliferation 6 and thus, the enzymatic fluctuation in Cells strongly influ-
1The Department of Surgery, Research Institute for Nuclear Medicine and Biology, Hiroshima University and 2the Division of Blood Transfusion, Hiroshima University Hospital, Hiroshima,Japan Reprint requests to: Ryungsa Kim, MD, The Department of Surgery, Research Institute for Nuclear Medicine and Biology, Hiroshima University, 1-2-3, Kasumi Minami-ku, Hiroshima 734,Japan
ences the effect of topoisomerase II anticancer agents. However, few reports have shown the difference in topoisomerase II levels between h u m a n tumor cells and normal cells. In this paper, w e a n a l y z e the cellular levels o f topoisomerase II expression in 10 tumors and the adjacent normal tissues. T h e levels o f topoisomerase II expression were analyzed by the RNA dot blot. For the isolation of total RNA, ten fresh h u m a n tumors and each of the respective adjacent normal tissues were frozen immediately after surgery and stored at --70~ until the analysis. Total cellular RNA was isolated by the guanidinium isothiocyanate method.' Two pg of each RNA preparation was denatured in 10 per cent formaldehyde and applied to nitrocellulose filters for dot blot hybridi, zation2 A s2P-labelled t o p o i s o m e r a s e H
JAPANESEJOURNALOF SURGERY,VOL.21, No. 5 pp. 587-589, 1991
Kim et al.
588
Jpn. J. Surg. September 1991
Table 1. Comparison of Topoisomerase II Expression Levels between Human Tumors and Normal Tissues Topo I I Expression Tumor Normal
Patient No.
Tumor Type
1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
Gastric ca. Gastric ca. Gastric ca. Gastric ca. Gastric ca. Rectal ca. Liver meta. (Rectum) Hepatocellular ca. Lung ca. Renal meta. (Pancreas)
Actin Topo II expression Ieve|/~
Fig. 1. Typical results oftopoisomerase II expression. Total cellular RNA (2 /+g) from tumors and normal tissues was spotted onto nitrocellulose filters. The filters were hybridized to the topoisomerase II probe and then rehybridized to the/3-actin as an internal control. Upper rows indicate those hybridized to the topoisomerase II probe and lower rows, those hybridized to the 13-actin probe. T; tumor, N; normal. p r o b e , 9 kindly p r o v i d e d by Dr. L.F. Liu, J o h n s Hopldns University, USA, and a /3actin p r o b e as an internal control were made by the olligolabelling m e t h o d ? ~ T h e filters were prehybridized at 42~ in 50 per cent formamide, 5 X Denhardts reagent, 5 X SSC (NaC1/sodium citrate, p H 7.0) a n d 200 ktg denatured salmon sperm DNA/ml. Hybridization was p e r f o r m e d overnight in the same solution containing 32P-labelled probes. After hybridization, the filters were washed in 2 X SSC with 0.1 p e r cent SDS (sodium dodecyl sulfate) followed by a final wash in 0.1 X SSC a n d 0.1 per cent SDS at 65~ Autoradiograms p e r f o r m e d at --70~ for 1-2 days a n d
-+ + + ~ + ~+ ntt+
--+ -+ + -+ + --
the intensity o f autoradiograms quantified by densitometric analysis. T e n tumors, comprising 5 gastric, 1 rectal, 1 hepatocellular, 1 lung, 1 metastatic liver and 1 metastatic renal cancer, as well as the adjacent n o r m a l tissues, were studied. Topoisomerase II expression was observed in 9 o f the 10 t u m o r tissues (90.0 p e r cent), 3 o f which showed extremely high levels. O n the other hand, topoisomerase II expression was observed in o n l y 5 o f the adjacent normal tissues (50.0 p e r cent). Six o f the 9 topoisomerase I I detected tumors showed higher levels of expression t h a n the adjacent normal tissues, while similar expression was observed in the other 3 tumors. T h e s e results indicate that at least in some tumors the levels o f topoisomerase II are higher than those in normal tissues. Since the levels and regulation o f this enzyme in proliferating t u m o r cells differ f r o m those in quiescent or n o r m a l cells, 11,12 a c o m p a r a t i v e study o f topoisomerase I I expression should be perf o r m e d for effective use o f the inhibitors. T h e topoisomerase I I in tumor cells may be regulated through the RNA transcription or protein degradation. H,13 A l t h o u g h f u r t h e r clinical a n d genetic studies on topoisomerase II expression in t u m o r cells will be required, our study, which indicates the higher level o f topoisomerase II expression in t u m o r cells, may provide useful information about the clinical effects
Volume 21 Number 5
Topoisomerase H expression
of topoisomerase II inhibitors. ( R e c e i v e d f o r p u b l i c a t i o n o n O c t . 3, 1990)
8.
REFERENCES 9. 1. Lock RB, Ross WE. DNA topoisomerases in cancer therapy. Anti Cancer Drug Design 1987; 2: 151164. 2. Drlica K, Franco RJ. Inhibitors of DNA topoisomerases. Biochemistry 1988; 27: 2253-2259. 3. Liu LF. DNA topoisomerase poisons as antitumor drugs. Annu Rev Biochem 1989; 58: 351-357. 4. Hsiang Y-H, Hertzberg R, Hecht S, Liu LF. Camptothecin induced protein-linked DNA breaks via mammalian DNA topoisomerase I. J Biol Chem 1985; 260: 14873-14878. 5. Tewey KM, Chen GL, Nelson EM, Liu LF. Intercalative antitumor drugs interfere with the breakageunion reaction of mammalian DNA topoisomerase II.J Biol Chem 1984; 259: 9182-9187. 6. Sullivan DM, Glisson BS, Hodges PK, SmallwoodKentro S, Ross WE. Proliferation dependence of topoisomerase II-mediated drug action. Biochemistry 1986; 25: 2248-2256. 7. Maniatis T, Fritsch EF, Sambrook J. Molecular
10.
11.
12.
13.
589
cloning: a laboratory mannual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 1982. Thomas PS. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci 1980; 77: 5201-5205. Tsai-Pflugfelder M, Liu LF, Liu AA, Tewey KM, Whang-PengJ, Knutsen T, Huebner K, Croce CM, Wang JC. Cloning and sequencing of cDNA encoding human DNA topoisomerase II and localization of the gene to chromosome region 17q21-22. Proc Natl Acad Sci 1988; 85: 7177-7181. Feiberg AP, Vogelstein B. A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 1983; 132: 6-13. Hsiang Y-H, Wu H-Y, Liu LF. Proliferation-dependent regulation of DNA topoisomerase II in cultured human cells. Cancer Res 1988; 48: 32303235. HsiangY-H, Wu H-Y, Liu LF. Topoisomerase: novel therapeutic targets in cancer chemotherapy. Biochem Parmacot 1988; 17: 1801-1802. Heck MMS, Hittelman WN, Earnshaw WC. Differential expression of DNA topoisomerase I and II during the eukaryotic cell cycle. Proc Natl Acad Sci 1988; 85: 1086-1090.
