- Email: [email protected]
Expression of the cyclin D1 gene in rat colorectal aberrant crypt foci and tumors induced by azoxymethane
Cancer Letters 140 (1999) 99±104
Expression of the cyclin D1 gene in rat colorectal aberrant crypt foci and tumors induced by azoxymethane K. Otori a, b,*, K. Sugiyama a, S. Fukushima b, H. Esumi a a
Investigative Treatment Division, National Cancer Center, Research Institute East, 6-5-1 Kashiwanoha, Kashiwa, Chiba, 277 Japan b First Department of Pathology, Osaka City, University Medical School, 1-4-3, Asahi-machi, Abeno-ku, Osaka, 545-8585 Japan Received 13 January 1999; accepted 10 February 1999
Abstract Cyclin D1 is a cell cycle regulator which is overexpressed in a variety of human cancers. We examined overexpression of cyclin D1 in several stages of rat colorectal carcinogenesis induced by azoxymethane (AOM) treatment. The level of cyclin D1 in 13 aberrant crypt foci (ACF) (atypical hyperplasias), 22 colorectal tumors (14 non-invasive adenocarcinomas and eight invasive adenocarcinomas) was assessed by immunostaining using a polyclonal antibody. Cell proliferation of these samples was investigated by measurement of 5-bromo-2 0 -deoxyuridine-labeling index. Indices of cyclin D1-positive cells in adenocarcinomas and atypical hyperplasias were signi®cantly higher than that in normal crypts (P , 0:05). Moreover, cyclin D1positive rates in the two types of adenocarcinomas were signi®cantly higher than that in atypical hyperplasias (P , 0:05). Staining of nuclear cyclin D1 was very strong in almost all adenocarcinomas and four ACF. Comparisons of BrdU-positive indices in colorectal lesions showed similar results to the cyclin D1-positive indices. These results suggested that overexpession of cyclin D1 occurs early in the multistep carcinogenesis, and plays an important role in rat colorectal carcinogenesis. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cyclin D1; Aberrant crypt foci; Colorectal tumor; Colorectal carcinogenesis
1. Introduction Cyclin D1 is a protooncogene located on one of the most frequently ampli®ed chromosomal regions (11q13) in human carcinomas [1]. It is expressed in the G1 phase of the cell cycle, and is thought to regulate the G1±S transition point of the cell cycle by acting in conjunction with cyclin-dependent kinase cdk4 [2-4]. The overexpression of cyclin D1 has been reported in a variety of human cancers and chemical carcinogen-induced mouse tumors [5-14].
* Corresponding author. Tel.: 181-6-66453736; fax: 181-666463093.
However, the expression of cyclin D1 has not been examined enough in colorectal carcinogenesis. Since Bird reported the existence of aberrant crypts in the murine colon treated with a colon carcinogen [15], aberrant crypt foci (ACF) have become widely accepted to be the precursor or neoplastic lesions of colorectal tumors. ACF exhibit hyperplasia or dysplasia histopathologically, and harbor K-ras gene mutations, and increased expression of the oncogene c-fos in rats [16-18]. However, the expression of the cyclin D1 gene in rat ACF has not been reported. It is unclear whether overexpression of cyclin D1 occurs in the early stages of colorectal carcinogenesis. The present study was designed to examine colorectal ACF and tumors from AOM-treated rats for overex-
0304-3835/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(99)00058-0
100
K. Otori et al. / Cancer Letters 140 (1999) 99±104
pression of cyclin D1 in multistage colorectal carcinogenesis by immunohistochemistry.
each ACF and tumor was made by two pathologists (SF and KO).
2. Materials and methods
2.5. Immunohistochemical analyses
2.1. Chemicals
All specimens were assessed for cyclin D1 protein nuclear accumulation. Three micrometer paraf®nembedded tissue sections were deparaf®nized, heated in a hot water bath at 908C for 20 min in 6 M urea, and then stained using a 1:200 diluted rabbit polyclonal antibody to human cyclin D1 (UBI, Lake placid, NY) by the avidin±biotin±peroxidase complex method. The sections of human bladder carcinoma tissues, which express cyclin D1 protein, were used as a positive control. BrdU was immunohistochemically stained by the method of Tiwawech et al. [19] with some modi®cations in serial sections of colonic tissues obtained from rats treated i.p. with BrdU. Brie¯y, sections were deparaf®nized with three changes of xylene and hydrated using a graded series of alcohol. Endogenous peroxidase was blocked using 3% hydrogen peroxide (H2O2) at room temperature for 5 min, and then tissue sections were treated with 2N HCl at room temperature for 20 min and neutralized with Tris-buffered saline. Subsequently, they were incubated with 0.02% actinase E (Kaken, Tokyo, Japan) at 378C for 20 min. After incubation with normal horse serum at room temperature for 30 min, they were incubated overnight at 48C with mouse monoclonal antibody to BrdU (Dakopatts, Denmark) diluted 1:1000. The subsequent steps were the same as those for cyclin D1 staining. Vertical sections of aberrant and normal crypts were used for determination of the cyclin D1-positive and BrdU-labeling index. Representative cyclin D1-or BrdU-stained areas in the sections of ACF and tumors were selected. Indices of positive cells were determined by counting the number of positive cells among at least 1000 cells in these areas, and indicated as percentages.
Azoxymethane (AOM) and 5-bromo-2 0 -deoxyuridine (BrdU) were purchased from Sigma (St Louis, MO). 2.2. Animals and their maintenance A total of 24 male F344 rats, 5 weeks old, were purchased from Charles River Japan (Kanagawa, Japan), and quarantined for 1 week before the start of the experiment. Animals were housed three to a wire cage, in an air-conditioned room. The animals were allowed free access to water and food (CE-2; CLEA, Tokyo, Japan). The room temperature was controlled at 22 ^ 28C, and the relative humidity, at 55 ^ 10%. Fluorescent lighting provided a 12-h light/ dark cycle. 2.3. Treatment of carcinogen One group of 21 male F344 rats was treated s.c. with 15 mg/kg body weight of AOM three times at 3 day-intervals. A control group of three rats was treated with saline in the same way. Animals were sacri®ced under ether anesthesia at week 32 from the initial treatment of AOM. Twenty-one AOMtreated rats were injected i.p. with BrdU at a dose of 100 mg/kg body weight 1 h before each death. 2.4. Histological examination The colons were promptly removed, and 10% phosphate-buffered formalin solution was injected into the lumens. The colons were cut along the longitudinal median axis, then opened and ®xed ¯at between ®lter papers. After staining with methylene blue (0.2% in distilled water) for 30 s, ACF and tumors were identi®ed under a stereo microscope. All tumors and large ACF ( . 7 crypts/focus) were removed under a stereo microscope. These tissues were embedded in paraf®n. Serial vertical sections of the tissues were made for hematoxylin and eosin (H.E.) staining and immunohistochemical staining. The histological diagnosis of
2.6. Statistical analysis Statistical analysis of the data was performed using a computer-assisted Fisher's test with Stat View software for the Macintosh computer, version 4.0 (Abacus Concepts, Berkeley, CA). A value of P , 0:05 was considered statistically signi®cant.
K. Otori et al. / Cancer Letters 140 (1999) 99±104
101
Fig. 1. Serial vertical sections of ACF ((A1±3) original magni®cation £ 200) and a tumor) ((B1±3) original magni®cation £ 165) obtained from rat colon treated with AOM. (A1,B2) were hematoxylin and eosin stained sections. (A2,B2) were the sections stained with anticyclin D1 antibody. (A3,B3) were the sections stained with anti-BrdU antibody. Notes: ACF (A) and a tumor (B) were diagnosed as atypical hyperplasia and non-invasive adenocarcinoma, respectively. Nuclear cyclin D1-positive cells were observed in (A2,B2). Apparent staining of cytoplasmic cyclin D1 was seen in (B2), whereas the staining was weak in (A2). BrdU-positive cells were observed both in (A3,B3).
3. Results The colons of AOM-treated and control rats were evaluated for colorectal lesions at week 32 from the initial AOM-treatment by a stereo microscope. All of the AOM-treated rats had several ACF, and 13 of 21 AOM-treated rats harbored colorectal tumors ( . 0.5 mm in diameter). None of the control animals had any ACF and tumors.
We examined 35 sections of colorectal lesions, including all 22 colonic tumors and 13 large ACF (.7 crypts/focus), and ten sections of apparently normal mucosa by histological and immunological analyses. All 13 ACF were diagnosed as atypical hyperplasias. Fourteen of 22 colorectal tumors were diagnosed as non-invasive adenocarcinomas, and the other eight colorectal tumors as invasive adenocarcinomas. Nuclear or cytoplasmic cyclin D1 staining was
102
K. Otori et al. / Cancer Letters 140 (1999) 99±104
Fig. 2. Cyclin D1-positive indices in different colonic lesions.
observed in almost all the colorectal lesions and concurrently a few normal crypts simultaneously. Staining of nuclear cyclin D1 in both non-invasive and invasive adenocarcinomas was very strong (Fig. 1B2). Very weak staining of nuclear cyclin D1 was found in nine of 13 atypical hyperplasias and a few normal crypts. However, four of the 13 atypical hyperplasias (ACF) showed strong staining of nuclear cyclin D1 as in adenocarcinomas (Fig. 1A2). There were no apparent histological or cytological difference between nuclear cyclin D1-positive and negative ACF. Cyclin D1-positive indices in the two types of adenocarcinomas and in atypical hyperplasias were signi®cantly higher than that in normal crypts (P , 0:05; Fig. 2). Moreover, cyclin D1-positive rates in two types of adenocarcinomas were signi®cantly higher than that in the atypical hyperplasias (P , 0:05; Fig. 2). There was no signi®cant difference
Fig. 3. BrdU-positive indices in different colonic lesions.
in the cyclin D1-positive indices between non-invasive and invasive adenocarcinomas. We examined cell proliferation activity in the colorectal ACF and tumors. BrdU-positive indices of these lesions were similar to the cyclin D1-positive indices (Fig. 3). However, the distribution of BrdUpositive cells were different between ACF and adenocarcinomas. The positive cells in adenocarcinomas tended to be located in surface areas of epithelium, compared with atypical hyperplasias (Fig. 1A3,B3). No clear association between the distribution of BrdU- and cyclin D1-positive cells in the tissue sections was found. 4. Discussion We have evaluated the overexpression of cyclin D1 in rat colorectal lesions induced by AOM treatment by immunohistochemistry. The levels of expression of cyclin D1 in both non-invasive and invasive adenocarcinomas were signi®cantly higher than that in atypical hyperplasias (ACF). The cell proliferative activity (BrdU positive index) was also signi®cantly different between these lesions. Moreover, some atypical hyperplasias (ACF) showed strong positive staining for nuclear cyclin D1. These observations suggest that overexpression of cyclin D1 is an early event of, and plays an important role in colorectal tumorigenesis. The ampli®cation and overexpression of the cyclin D1 gene has been reported in many human tumors and preneoplastic lesions [20,21]. Hinds et al. [22] indicated that the cyclin D1 functioned like an oncogene and was a key factor in human tumor development by in vitro studies. Bartkova et al. [23] suggested that alterations of cyclin D1 expression are a common feature of malignancies in diverse histogenesis. We and other groups have reported that K-ras gene mutations were present in preneoplastic and neoplastic lesions in the colorecetum of human and rat [16,18,24]. Recently, it was shown that cyclin D1 overexpression was induced by activated ras in rat intestinal cells [25]. Overexpression of cyclin D1 induced malignant transformation in primary rat embryo ®broblasts in cooperation with activated ras [26]. These data indicate that overexpression of cyclin D1 may be caused by K-ras gene mutation, and may
K. Otori et al. / Cancer Letters 140 (1999) 99±104
then play a crucial role in rat and human colorectal tumorigenesis. Positive staining for nuclear cyclin D1 was seen in adenomatous polyps and adenocarcinomas, and some ACF could be diagnosed as carcinoma in situ in human colorectum [21,27]. In our current study, it was clear that some rat ACF exhibited strong staining for nuclear cyclin D1 as well as adenocarcinomas. These ACF might represent a very early stage of the progression of ACF into more advanced lesions. Wang et al. [14] suggested that overexpression of cyclin D1 and cyclin E occurred relatively early in N-nitrosomethylbenzylamine (NMBA)-induced rat esophageal tumorigenesis. It might be of interest to examine whether cyclin E is overexpressed in ACF and tumors of the rat colon in the same way as cyclin D1. Recently, the relationship between overexpression of cyclin D1 and the prognosis of human carcinomas was examined [11,28,29]. In esophageal carcinoma, cyclin D1 overexpression is suggested to be a useful marker of prognosis [29]. Similarly, signi®cant correlations were also observed in breast tumors [28]. The prognostic importance of cyclin D1 overexpression in human colorectal carcinoma might be worth evaluating in further studies.
Acknowledgements This work was supported in part by a Grant for the second term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health and Welfare, and a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan. KO was awarded a Research Resident Fellowship from the Foundation for Promotion of Cancer Research.
[4] [5]
[6]
[7]
[8]
[9]
[10] [11]
[12] [13] [14]
[15] [16]
References [1] T. Motokura, A. Arnold, Cyclins and oncogenesis, Biochim. Biophys. Acta 1155 (1993) 63±78. [2] H. Matsushima, M.F. Roussel, E.A. Ashmun, C. Sherr, Colony-stimulating factor 1 regulates novel cyclins during the Gi phase of the cell cycle, Cell 65 (1991) 701±713. [3] W. Jiang, S.M. Kahn, P. Zhou, Y.J. Zhang, A.M. Cacace, A.S. Infante, S. Doi, R.M. Santella, I.B. Weinstein, Overexpression of cyclin D1 in rat ®broblasts causes abnormalities in growth
[17]
[18]
103
control, cell cycle progression and gene expression, Oncogene 8 (1993) 3447±3457. T. Hunter, J. Pines, Cyclins and cancer II: cyclin D and CDK inhibitors come of age, Cell 79 (1994) 573±582. W. Jiang, Y.J. Zhang, S.M. Kahn, M.C. Hollstein, R.M. Santella, S.H. Lu, C.C. Harris, R. Montesano, I.B. Weinstein, Altered expression of the cyclin D1 and retinoblastoma gene in human esophageal cancer, Proc Natl. Acad. Sci. USA 90 (1993) 9026±9030. F.S. Leach, S.J. Elledge, C.J. Sherr, L.K.V. Willson, S. Markowitz, K.W. Kinzler, B. Vogelstein, Ampli®cation of cyclin gene in colorectal carcinomas, Cancer Res. 53 (1993) 1986±1989. J. Bartkova, J. Lukas, M. Atrauss, J. Bartek, The PRAD-1/ cyclin D1 oncogene product accumulates aberrantly in a subset of colorectal carcinoma, Int. J. Cancer 58 (1994) 568±573. T. Callender, A. El-Naggar, M.S. Lee, R. Frankenthaler, M.A. Luna, J.G. Batsakis, PRAD-1 (CCND1)/cyclin D1 oncogene ampli®cation in primary head and neck squamous call carcinoma, Cancer 74 (1994) 152±158. N. Nishida, Y. Fukuda, T. Komeda, R. Kita, T. Sando, M. Furukawa, M. Amenomori, I. Shibagaki, K. Nakao, M. Ikenaga, K. Ishizaki, Ampli®cation and overexpression of the cyclin D1 gene in aggressive human hepatocellular carcinoma, Cancer Res. 54 (1994) 3107±3110. S.Y. Zhang, J. Caamano, F. Cooper, X. Guo, A.J.P. KleinSzanto, Immunohistochemistry of cyclin D1 in human breast cancer, Am. J. Clin. Pathol. 102 (1994) 695±698. D.C. Bettcher, J. Heighway, P.S. Hasleton, H.J. Altermatt, W.D.J. Ryder, T. Cerny, N. Thatcher, Prognostic signi®cance of CCND1 (cyclin D1) overexpression in primary resected non-small-cell lung cancer, Br. J. Cancer 73 (1996) 294±300. W.C. Hung, C.Y. Chai, J.S. Huang, L.Y. Chuang, Expression of cyclin D1 and c-K-ras gene product in human epithelial ovarian tumors, Human Pathol. 27 (1996) 1324±1328. T. Nikaido, S.F. Li, T. Shiozawa, S. Fujii, Coabnormal expression of cyclin D1, p53 protein in human uterine endometrial carcinomas, Cancer 78 (1996) 1248±1253. Q.S. Wang, C.L.K. Sabourin, H. Wang, G.D. Stoner, Overexpression of cyclin D1 and cyclin E in N-nitrosomethylbenzylamine-induced rat esophageal tumorigenesis, Carcinogenesis 17 (1996) 1583±1588. R.P. Bird, Observation and quanti®cation of aberrant crypts in the murine colon treated with a colon carcinogen: preliminary ®ndings, Cancer Lett. 37 (1987) 147±151. S.A. Stopera, L.C. Murphy, R.P. Bird, Evidence for a ras gene mutation in azoxymethane-induced colonic aberrant crypts in Sprague±Dawley rats: earliest recognizable precursor lesions of experimental colon cancer, Carcinogenesis 13 (1992) 2081±2085. S.A. Stopera, R.P. Bird, Colonic aberrant crypt foci are associated with increased expression of c-fos: the possible role of modi®ed c-fos expression in preneoplastic lesions in colon cancer, Carcinogenesis 13 (1992) 573±578. N. Shivapurkar, Z. Tang, A. Ferreira, S. Nasim, C. Garett, O. Alabaster, Sequential analysis of K-ras mutations in aberrant
104
[19]
[20] [21]
[22] [23] [24]
K. Otori et al. / Cancer Letters 140 (1999) 99±104 crypt foci and colonic tumors by azoxymethane in Fisher-344 rats on high-risk diet, Carcinogenesis 15 (1994) 775±778. D. Tiwawech, R. Hasegawa, Y. Kurata, M. Tatematsu, M. Shibata, W. Thamavit, N. Ito, Dose-dependent effects of 2acetylamino¯uorene on hepatic foci development and cell proliferation in rats, Carcinogenesis 12 (1991) 985±990. T. Wang, R. Cardiff, L. Zukerberg, E. Lees, A. Arnold, E.V. Schmidt, Mammary hyperplasia and carcinoma in MMTVcyclin D1 transgenic mice, Nature 369 (1994) 669±671. N. Arber, H. Hibshoosh, S.F. Moss, T. Sutter, Y. Zhang, M. Begg, S. Wang, B. Weinstein, P.R. Holt, Increased expression of cyclin D1 is an early event in multistage colorectal carcinogenesis, Gastroenterology 110 (1996) 669±674. P.W. Hinds, S.F. Dowdy, E.N. Eaton, A. Arnold, R.A. Weinberg, Function of a human cyclin gene as an oncogene, Proc. Natl. Acad. Sci. USA 91 (1994) 709±713. J. Bartkova, J. Lukas, M. Atrauss, J. Bartek, Cyclin D1 oncoprotein aberrantly accumulates in malignancies of diverse histogenesis, Oncogene 10 (1995) 775±778. K. Otori, K. Suglyama, T. Hasebe, S. Fukushima, H. ESumi, Emergence of adenomatous aberrant crypt foci (ACF) from
[25] [26]
[27]
[28]
[29]
hyperplastic ACF with concomitant increase in cell proliferation, Cancer Res. 55 (1995) 4743±4746. J. Filmus, A.I. Robles, W. Shi, M.J. Wong, L.L. Colombo, C.J. Conti, Induction of cyclin D1 overexpression by activated ras, Oncogene 9 (1994) 3627±3633. H. Lovec, A. Sewing, F.C. Lucibello, R.M.T. MoÈroÈy, Oncogenic activity of cyclin D1 revealed through cooperation with Ha-ras: link between cell cycle control and malignant transformation, Oncogene 9 (1994) 323±326. A.K. Konstantakos, I.M. Siu, T.G. Pretlow, T.A. Stellato, T.P. Pretlow, Human aberrant crypt foci with carcinoma in situ from a patient with sporadic colon cancer, Gastroenterology 111 (1996) 772±777. G.G. Mcintosh, J.J. Anderson, I. Milton, M. Steward, A.H. Parr, M.D. Thomas, J.A. Henry, B. Angus, T.W.J. Lennard, C.H.W. Home, Determination of the prognostic value of cyclin D1 overexpression in breast cancer, Oncogene 11 (1995) 885±891. H. Naitoh, J. Shibata, A. Kawaguchi, M. Kodama, T. Hattori, Overexpression and localization of cyclin D1 mRNA and in esophageal cancer, Am. J. Pathol. 146 (1995) 1161±1169.
Expression of the cyclin D1 gene in rat colorectal aberrant crypt foci and tumors induced by azoxymethane K. Otori a, b,*, K. Sugiyama a, S. Fukushima b, H. Esumi a a
Investigative Treatment Division, National Cancer Center, Research Institute East, 6-5-1 Kashiwanoha, Kashiwa, Chiba, 277 Japan b First Department of Pathology, Osaka City, University Medical School, 1-4-3, Asahi-machi, Abeno-ku, Osaka, 545-8585 Japan Received 13 January 1999; accepted 10 February 1999
Abstract Cyclin D1 is a cell cycle regulator which is overexpressed in a variety of human cancers. We examined overexpression of cyclin D1 in several stages of rat colorectal carcinogenesis induced by azoxymethane (AOM) treatment. The level of cyclin D1 in 13 aberrant crypt foci (ACF) (atypical hyperplasias), 22 colorectal tumors (14 non-invasive adenocarcinomas and eight invasive adenocarcinomas) was assessed by immunostaining using a polyclonal antibody. Cell proliferation of these samples was investigated by measurement of 5-bromo-2 0 -deoxyuridine-labeling index. Indices of cyclin D1-positive cells in adenocarcinomas and atypical hyperplasias were signi®cantly higher than that in normal crypts (P , 0:05). Moreover, cyclin D1positive rates in the two types of adenocarcinomas were signi®cantly higher than that in atypical hyperplasias (P , 0:05). Staining of nuclear cyclin D1 was very strong in almost all adenocarcinomas and four ACF. Comparisons of BrdU-positive indices in colorectal lesions showed similar results to the cyclin D1-positive indices. These results suggested that overexpession of cyclin D1 occurs early in the multistep carcinogenesis, and plays an important role in rat colorectal carcinogenesis. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cyclin D1; Aberrant crypt foci; Colorectal tumor; Colorectal carcinogenesis
1. Introduction Cyclin D1 is a protooncogene located on one of the most frequently ampli®ed chromosomal regions (11q13) in human carcinomas [1]. It is expressed in the G1 phase of the cell cycle, and is thought to regulate the G1±S transition point of the cell cycle by acting in conjunction with cyclin-dependent kinase cdk4 [2-4]. The overexpression of cyclin D1 has been reported in a variety of human cancers and chemical carcinogen-induced mouse tumors [5-14].
* Corresponding author. Tel.: 181-6-66453736; fax: 181-666463093.
However, the expression of cyclin D1 has not been examined enough in colorectal carcinogenesis. Since Bird reported the existence of aberrant crypts in the murine colon treated with a colon carcinogen [15], aberrant crypt foci (ACF) have become widely accepted to be the precursor or neoplastic lesions of colorectal tumors. ACF exhibit hyperplasia or dysplasia histopathologically, and harbor K-ras gene mutations, and increased expression of the oncogene c-fos in rats [16-18]. However, the expression of the cyclin D1 gene in rat ACF has not been reported. It is unclear whether overexpression of cyclin D1 occurs in the early stages of colorectal carcinogenesis. The present study was designed to examine colorectal ACF and tumors from AOM-treated rats for overex-
0304-3835/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(99)00058-0
100
K. Otori et al. / Cancer Letters 140 (1999) 99±104
pression of cyclin D1 in multistage colorectal carcinogenesis by immunohistochemistry.
each ACF and tumor was made by two pathologists (SF and KO).
2. Materials and methods
2.5. Immunohistochemical analyses
2.1. Chemicals
All specimens were assessed for cyclin D1 protein nuclear accumulation. Three micrometer paraf®nembedded tissue sections were deparaf®nized, heated in a hot water bath at 908C for 20 min in 6 M urea, and then stained using a 1:200 diluted rabbit polyclonal antibody to human cyclin D1 (UBI, Lake placid, NY) by the avidin±biotin±peroxidase complex method. The sections of human bladder carcinoma tissues, which express cyclin D1 protein, were used as a positive control. BrdU was immunohistochemically stained by the method of Tiwawech et al. [19] with some modi®cations in serial sections of colonic tissues obtained from rats treated i.p. with BrdU. Brie¯y, sections were deparaf®nized with three changes of xylene and hydrated using a graded series of alcohol. Endogenous peroxidase was blocked using 3% hydrogen peroxide (H2O2) at room temperature for 5 min, and then tissue sections were treated with 2N HCl at room temperature for 20 min and neutralized with Tris-buffered saline. Subsequently, they were incubated with 0.02% actinase E (Kaken, Tokyo, Japan) at 378C for 20 min. After incubation with normal horse serum at room temperature for 30 min, they were incubated overnight at 48C with mouse monoclonal antibody to BrdU (Dakopatts, Denmark) diluted 1:1000. The subsequent steps were the same as those for cyclin D1 staining. Vertical sections of aberrant and normal crypts were used for determination of the cyclin D1-positive and BrdU-labeling index. Representative cyclin D1-or BrdU-stained areas in the sections of ACF and tumors were selected. Indices of positive cells were determined by counting the number of positive cells among at least 1000 cells in these areas, and indicated as percentages.
Azoxymethane (AOM) and 5-bromo-2 0 -deoxyuridine (BrdU) were purchased from Sigma (St Louis, MO). 2.2. Animals and their maintenance A total of 24 male F344 rats, 5 weeks old, were purchased from Charles River Japan (Kanagawa, Japan), and quarantined for 1 week before the start of the experiment. Animals were housed three to a wire cage, in an air-conditioned room. The animals were allowed free access to water and food (CE-2; CLEA, Tokyo, Japan). The room temperature was controlled at 22 ^ 28C, and the relative humidity, at 55 ^ 10%. Fluorescent lighting provided a 12-h light/ dark cycle. 2.3. Treatment of carcinogen One group of 21 male F344 rats was treated s.c. with 15 mg/kg body weight of AOM three times at 3 day-intervals. A control group of three rats was treated with saline in the same way. Animals were sacri®ced under ether anesthesia at week 32 from the initial treatment of AOM. Twenty-one AOMtreated rats were injected i.p. with BrdU at a dose of 100 mg/kg body weight 1 h before each death. 2.4. Histological examination The colons were promptly removed, and 10% phosphate-buffered formalin solution was injected into the lumens. The colons were cut along the longitudinal median axis, then opened and ®xed ¯at between ®lter papers. After staining with methylene blue (0.2% in distilled water) for 30 s, ACF and tumors were identi®ed under a stereo microscope. All tumors and large ACF ( . 7 crypts/focus) were removed under a stereo microscope. These tissues were embedded in paraf®n. Serial vertical sections of the tissues were made for hematoxylin and eosin (H.E.) staining and immunohistochemical staining. The histological diagnosis of
2.6. Statistical analysis Statistical analysis of the data was performed using a computer-assisted Fisher's test with Stat View software for the Macintosh computer, version 4.0 (Abacus Concepts, Berkeley, CA). A value of P , 0:05 was considered statistically signi®cant.
K. Otori et al. / Cancer Letters 140 (1999) 99±104
101
Fig. 1. Serial vertical sections of ACF ((A1±3) original magni®cation £ 200) and a tumor) ((B1±3) original magni®cation £ 165) obtained from rat colon treated with AOM. (A1,B2) were hematoxylin and eosin stained sections. (A2,B2) were the sections stained with anticyclin D1 antibody. (A3,B3) were the sections stained with anti-BrdU antibody. Notes: ACF (A) and a tumor (B) were diagnosed as atypical hyperplasia and non-invasive adenocarcinoma, respectively. Nuclear cyclin D1-positive cells were observed in (A2,B2). Apparent staining of cytoplasmic cyclin D1 was seen in (B2), whereas the staining was weak in (A2). BrdU-positive cells were observed both in (A3,B3).
3. Results The colons of AOM-treated and control rats were evaluated for colorectal lesions at week 32 from the initial AOM-treatment by a stereo microscope. All of the AOM-treated rats had several ACF, and 13 of 21 AOM-treated rats harbored colorectal tumors ( . 0.5 mm in diameter). None of the control animals had any ACF and tumors.
We examined 35 sections of colorectal lesions, including all 22 colonic tumors and 13 large ACF (.7 crypts/focus), and ten sections of apparently normal mucosa by histological and immunological analyses. All 13 ACF were diagnosed as atypical hyperplasias. Fourteen of 22 colorectal tumors were diagnosed as non-invasive adenocarcinomas, and the other eight colorectal tumors as invasive adenocarcinomas. Nuclear or cytoplasmic cyclin D1 staining was
102
K. Otori et al. / Cancer Letters 140 (1999) 99±104
Fig. 2. Cyclin D1-positive indices in different colonic lesions.
observed in almost all the colorectal lesions and concurrently a few normal crypts simultaneously. Staining of nuclear cyclin D1 in both non-invasive and invasive adenocarcinomas was very strong (Fig. 1B2). Very weak staining of nuclear cyclin D1 was found in nine of 13 atypical hyperplasias and a few normal crypts. However, four of the 13 atypical hyperplasias (ACF) showed strong staining of nuclear cyclin D1 as in adenocarcinomas (Fig. 1A2). There were no apparent histological or cytological difference between nuclear cyclin D1-positive and negative ACF. Cyclin D1-positive indices in the two types of adenocarcinomas and in atypical hyperplasias were signi®cantly higher than that in normal crypts (P , 0:05; Fig. 2). Moreover, cyclin D1-positive rates in two types of adenocarcinomas were signi®cantly higher than that in the atypical hyperplasias (P , 0:05; Fig. 2). There was no signi®cant difference
Fig. 3. BrdU-positive indices in different colonic lesions.
in the cyclin D1-positive indices between non-invasive and invasive adenocarcinomas. We examined cell proliferation activity in the colorectal ACF and tumors. BrdU-positive indices of these lesions were similar to the cyclin D1-positive indices (Fig. 3). However, the distribution of BrdUpositive cells were different between ACF and adenocarcinomas. The positive cells in adenocarcinomas tended to be located in surface areas of epithelium, compared with atypical hyperplasias (Fig. 1A3,B3). No clear association between the distribution of BrdU- and cyclin D1-positive cells in the tissue sections was found. 4. Discussion We have evaluated the overexpression of cyclin D1 in rat colorectal lesions induced by AOM treatment by immunohistochemistry. The levels of expression of cyclin D1 in both non-invasive and invasive adenocarcinomas were signi®cantly higher than that in atypical hyperplasias (ACF). The cell proliferative activity (BrdU positive index) was also signi®cantly different between these lesions. Moreover, some atypical hyperplasias (ACF) showed strong positive staining for nuclear cyclin D1. These observations suggest that overexpression of cyclin D1 is an early event of, and plays an important role in colorectal tumorigenesis. The ampli®cation and overexpression of the cyclin D1 gene has been reported in many human tumors and preneoplastic lesions [20,21]. Hinds et al. [22] indicated that the cyclin D1 functioned like an oncogene and was a key factor in human tumor development by in vitro studies. Bartkova et al. [23] suggested that alterations of cyclin D1 expression are a common feature of malignancies in diverse histogenesis. We and other groups have reported that K-ras gene mutations were present in preneoplastic and neoplastic lesions in the colorecetum of human and rat [16,18,24]. Recently, it was shown that cyclin D1 overexpression was induced by activated ras in rat intestinal cells [25]. Overexpression of cyclin D1 induced malignant transformation in primary rat embryo ®broblasts in cooperation with activated ras [26]. These data indicate that overexpression of cyclin D1 may be caused by K-ras gene mutation, and may
K. Otori et al. / Cancer Letters 140 (1999) 99±104
then play a crucial role in rat and human colorectal tumorigenesis. Positive staining for nuclear cyclin D1 was seen in adenomatous polyps and adenocarcinomas, and some ACF could be diagnosed as carcinoma in situ in human colorectum [21,27]. In our current study, it was clear that some rat ACF exhibited strong staining for nuclear cyclin D1 as well as adenocarcinomas. These ACF might represent a very early stage of the progression of ACF into more advanced lesions. Wang et al. [14] suggested that overexpression of cyclin D1 and cyclin E occurred relatively early in N-nitrosomethylbenzylamine (NMBA)-induced rat esophageal tumorigenesis. It might be of interest to examine whether cyclin E is overexpressed in ACF and tumors of the rat colon in the same way as cyclin D1. Recently, the relationship between overexpression of cyclin D1 and the prognosis of human carcinomas was examined [11,28,29]. In esophageal carcinoma, cyclin D1 overexpression is suggested to be a useful marker of prognosis [29]. Similarly, signi®cant correlations were also observed in breast tumors [28]. The prognostic importance of cyclin D1 overexpression in human colorectal carcinoma might be worth evaluating in further studies.
Acknowledgements This work was supported in part by a Grant for the second term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health and Welfare, and a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan. KO was awarded a Research Resident Fellowship from the Foundation for Promotion of Cancer Research.
[4] [5]
[6]
[7]
[8]
[9]
[10] [11]
[12] [13] [14]
[15] [16]
References [1] T. Motokura, A. Arnold, Cyclins and oncogenesis, Biochim. Biophys. Acta 1155 (1993) 63±78. [2] H. Matsushima, M.F. Roussel, E.A. Ashmun, C. Sherr, Colony-stimulating factor 1 regulates novel cyclins during the Gi phase of the cell cycle, Cell 65 (1991) 701±713. [3] W. Jiang, S.M. Kahn, P. Zhou, Y.J. Zhang, A.M. Cacace, A.S. Infante, S. Doi, R.M. Santella, I.B. Weinstein, Overexpression of cyclin D1 in rat ®broblasts causes abnormalities in growth
[17]
[18]
103
control, cell cycle progression and gene expression, Oncogene 8 (1993) 3447±3457. T. Hunter, J. Pines, Cyclins and cancer II: cyclin D and CDK inhibitors come of age, Cell 79 (1994) 573±582. W. Jiang, Y.J. Zhang, S.M. Kahn, M.C. Hollstein, R.M. Santella, S.H. Lu, C.C. Harris, R. Montesano, I.B. Weinstein, Altered expression of the cyclin D1 and retinoblastoma gene in human esophageal cancer, Proc Natl. Acad. Sci. USA 90 (1993) 9026±9030. F.S. Leach, S.J. Elledge, C.J. Sherr, L.K.V. Willson, S. Markowitz, K.W. Kinzler, B. Vogelstein, Ampli®cation of cyclin gene in colorectal carcinomas, Cancer Res. 53 (1993) 1986±1989. J. Bartkova, J. Lukas, M. Atrauss, J. Bartek, The PRAD-1/ cyclin D1 oncogene product accumulates aberrantly in a subset of colorectal carcinoma, Int. J. Cancer 58 (1994) 568±573. T. Callender, A. El-Naggar, M.S. Lee, R. Frankenthaler, M.A. Luna, J.G. Batsakis, PRAD-1 (CCND1)/cyclin D1 oncogene ampli®cation in primary head and neck squamous call carcinoma, Cancer 74 (1994) 152±158. N. Nishida, Y. Fukuda, T. Komeda, R. Kita, T. Sando, M. Furukawa, M. Amenomori, I. Shibagaki, K. Nakao, M. Ikenaga, K. Ishizaki, Ampli®cation and overexpression of the cyclin D1 gene in aggressive human hepatocellular carcinoma, Cancer Res. 54 (1994) 3107±3110. S.Y. Zhang, J. Caamano, F. Cooper, X. Guo, A.J.P. KleinSzanto, Immunohistochemistry of cyclin D1 in human breast cancer, Am. J. Clin. Pathol. 102 (1994) 695±698. D.C. Bettcher, J. Heighway, P.S. Hasleton, H.J. Altermatt, W.D.J. Ryder, T. Cerny, N. Thatcher, Prognostic signi®cance of CCND1 (cyclin D1) overexpression in primary resected non-small-cell lung cancer, Br. J. Cancer 73 (1996) 294±300. W.C. Hung, C.Y. Chai, J.S. Huang, L.Y. Chuang, Expression of cyclin D1 and c-K-ras gene product in human epithelial ovarian tumors, Human Pathol. 27 (1996) 1324±1328. T. Nikaido, S.F. Li, T. Shiozawa, S. Fujii, Coabnormal expression of cyclin D1, p53 protein in human uterine endometrial carcinomas, Cancer 78 (1996) 1248±1253. Q.S. Wang, C.L.K. Sabourin, H. Wang, G.D. Stoner, Overexpression of cyclin D1 and cyclin E in N-nitrosomethylbenzylamine-induced rat esophageal tumorigenesis, Carcinogenesis 17 (1996) 1583±1588. R.P. Bird, Observation and quanti®cation of aberrant crypts in the murine colon treated with a colon carcinogen: preliminary ®ndings, Cancer Lett. 37 (1987) 147±151. S.A. Stopera, L.C. Murphy, R.P. Bird, Evidence for a ras gene mutation in azoxymethane-induced colonic aberrant crypts in Sprague±Dawley rats: earliest recognizable precursor lesions of experimental colon cancer, Carcinogenesis 13 (1992) 2081±2085. S.A. Stopera, R.P. Bird, Colonic aberrant crypt foci are associated with increased expression of c-fos: the possible role of modi®ed c-fos expression in preneoplastic lesions in colon cancer, Carcinogenesis 13 (1992) 573±578. N. Shivapurkar, Z. Tang, A. Ferreira, S. Nasim, C. Garett, O. Alabaster, Sequential analysis of K-ras mutations in aberrant
104
[19]
[20] [21]
[22] [23] [24]
K. Otori et al. / Cancer Letters 140 (1999) 99±104 crypt foci and colonic tumors by azoxymethane in Fisher-344 rats on high-risk diet, Carcinogenesis 15 (1994) 775±778. D. Tiwawech, R. Hasegawa, Y. Kurata, M. Tatematsu, M. Shibata, W. Thamavit, N. Ito, Dose-dependent effects of 2acetylamino¯uorene on hepatic foci development and cell proliferation in rats, Carcinogenesis 12 (1991) 985±990. T. Wang, R. Cardiff, L. Zukerberg, E. Lees, A. Arnold, E.V. Schmidt, Mammary hyperplasia and carcinoma in MMTVcyclin D1 transgenic mice, Nature 369 (1994) 669±671. N. Arber, H. Hibshoosh, S.F. Moss, T. Sutter, Y. Zhang, M. Begg, S. Wang, B. Weinstein, P.R. Holt, Increased expression of cyclin D1 is an early event in multistage colorectal carcinogenesis, Gastroenterology 110 (1996) 669±674. P.W. Hinds, S.F. Dowdy, E.N. Eaton, A. Arnold, R.A. Weinberg, Function of a human cyclin gene as an oncogene, Proc. Natl. Acad. Sci. USA 91 (1994) 709±713. J. Bartkova, J. Lukas, M. Atrauss, J. Bartek, Cyclin D1 oncoprotein aberrantly accumulates in malignancies of diverse histogenesis, Oncogene 10 (1995) 775±778. K. Otori, K. Suglyama, T. Hasebe, S. Fukushima, H. ESumi, Emergence of adenomatous aberrant crypt foci (ACF) from
[25] [26]
[27]
[28]
[29]
hyperplastic ACF with concomitant increase in cell proliferation, Cancer Res. 55 (1995) 4743±4746. J. Filmus, A.I. Robles, W. Shi, M.J. Wong, L.L. Colombo, C.J. Conti, Induction of cyclin D1 overexpression by activated ras, Oncogene 9 (1994) 3627±3633. H. Lovec, A. Sewing, F.C. Lucibello, R.M.T. MoÈroÈy, Oncogenic activity of cyclin D1 revealed through cooperation with Ha-ras: link between cell cycle control and malignant transformation, Oncogene 9 (1994) 323±326. A.K. Konstantakos, I.M. Siu, T.G. Pretlow, T.A. Stellato, T.P. Pretlow, Human aberrant crypt foci with carcinoma in situ from a patient with sporadic colon cancer, Gastroenterology 111 (1996) 772±777. G.G. Mcintosh, J.J. Anderson, I. Milton, M. Steward, A.H. Parr, M.D. Thomas, J.A. Henry, B. Angus, T.W.J. Lennard, C.H.W. Home, Determination of the prognostic value of cyclin D1 overexpression in breast cancer, Oncogene 11 (1995) 885±891. H. Naitoh, J. Shibata, A. Kawaguchi, M. Kodama, T. Hattori, Overexpression and localization of cyclin D1 mRNA and in esophageal cancer, Am. J. Pathol. 146 (1995) 1161±1169.