Enhanced growth of colorectal aberrant crypt foci in fasted/refed rats involves changes in TGFß1 and p21CIP expressions
Giovanna Caderni1,
Maria-Giulia Perrelli2,
Fabio Cecchini2 and
Luciana Tessitore3,4
1 Dipartimento di Farmacologia Preclinica e Clinica, Università degli Studi di Firenze, Firenze, Italy,
2 Dipartimento di Scienze Cliniche e Biologiche, Ospedale `S. Luigi Gonzaga', Orbassano, Torino, Italy and
3 Dipartimento di Scienze Chimiche Alimentari Farmaceutiche e Farmacologiche, Università del Piemonte Orientale `Amedeo Avogadro', Vercelli, Italy
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Abstract
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We previously demonstrated that fasting/refeeding enhances the initiation phase of liver and colorectal carcinogenesis in rats. The present study was undertaken to establish whether cycles of fasting/refeeding carried out during the promotion phase of carcinogenesis may also affect the formation of aberrant crypt foci (ACF), preneoplastic lesions induced in the colon by azoxymethane (AOM). We were also interested in studying whether this effect might be mediated by changes in the proliferation, apoptosis or expression of TGFß1 and p21CIP genes in the colon. 44 male Fisher 344 rats were given a single dose of AOM (20 mg/kg s.c.) and one week later, they were exposed to 5 cycles of 4 days fasting followed by 710 days of refeeding (refed rats); controls were regularly fed; the rats were killed 2, 8 or 30 days after the last cycle of fasting. Fasting/refeeding caused a dramatic increase in crypt multiplicity when compared with regularly fed rats (AC/ACF was 4.30 ± 1.3 in refed and 2.38 ± 0.4 in regularly fed rats, P < 0.005 means ± SD), while no significant changes were observed in the number of ACF/colon. In the two experimental groups, cell proliferation was higher in ACF than in the surrounding mucosa, but proliferative indexes were higher and the apoptotic index lower in ACF of refed rats compared with regularly fed rats. TGFß1 expression was higher in the ACF of refed rats than in those of fully fed controls while p21CIP was less expressed in refed rats than in controls. These results suggest that fasting/refeeding is a risk factor for colon cancer and must be taken into account for cancer prevention in humans.
Abbreviations: ACF, aberrant crypt foci; AOM, azoxymethane; TdT, terminal deoxynucleotidyl transferase
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Introduction
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The influence of dietary habits on colorectal cancer is supported by a variety of epidemiological and experimental studies (1,2). In particular, a consistent association has been found between cancer and caloric intake: overfeeding in animals and overeating in humans being associated with a high incidence, while chronic caloric restriction is associated with a lower risk of cancer in many organs including the colon and rectum (2,3).
However, experimental studies have also demonstrated that short periods of hypocaloric diets or complete fasting followed by ad libitum refeeding enhance liver and mammary carcinogenesis (47). In agreement with these findings, we recently demonstrated that fasting/refeeding during the initiation phase with the carcinogen AOM increases colorectal carcinogenesis in rats (8,9). Fasting/refeeding has also been shown to increase cell proliferation in the colorectal epithelium (8,10), and high cell proliferation may increase carcinogenesis by expanding existing preneoplastic lesions.
Given these considerations and the fact that many individuals in Western countries where the incidence of colorectal cancer is high, engage unbalanced dietary habits with periods of dieting followed by binging or overeating, we wanted to study whether cycles of fasting/refeeding during the promotion phase of colorectal carcinogenesis would increase the number or growth of aberrant crypt foci (ACF) induced in the colon by azoxymethane (AOM). ACF are considered to be preneoplastic lesions in the development of colon cancer (1113), and although it has been demonstrated that not all ACF progress to cancer (14,15), they are widely used as endpoint in short-term carcinogenesis studies to determine the chemopreventive or promoting effects of drugs or dietary factors (16,17).
We were also interested in studying whether cycles of fasting and refeeding might induce changes in cell proliferation, apoptosis and the expression of TGFß1 and p21CIP (genes involved in cell cycle regulation) in ACF and in the normal surrounding mucosa.
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Materials and methods
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Animals and treatments
Forty-four 2-month-old male F344 rats, weighing about 150 g, were used (Charles River, Como, Italy). All animals were housed in plastic cages (34 in each cage) in a holding room and acclimatised to a 12:12 h lightdark cycle, controlled temperature (21 ± 1°C) and humidity (7080%) according to the European Union Regulations on the Care and Use of Laboratory Animals (18). After their arrival from the supplier, animals were quarantined for 1 week, during which they were fed a standard AIN-76 diet (Piccioni, Brescia, Italy). Rats were then given a single s.c. dose of 20 mg/kg of AOM (Sigma, Milan, Italy) and randomly allocated to one of the two experimental groups: one group was fed AIN-76 ad libitum; the other was fed the same diet but with five cycles of fasting for 4 days, followed by 710 days of refeeding (Figure 1
). Both groups had free access to water for all the duration of the experiment.

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Fig. 1. Experimental design: The total number of animals examined was 44. Control and refed rats were given a single dose of AOM (20 mg/kg). Control rats were regularly fed. Refed rats were exposed to 5 cycles of 4 days fasting (FAST), followed by regular feeding. At different times after AOM (arrows) rats were killed (10 rats/group after 64 days, 6 rats/group after 72 days and 6 rats/group after 92 days).
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To study the proliferative activity in the colorectal mucosa, rats were killed by carbon-dioxide asphyxia at various intervals after treatment with AOM (64 days (10 rats) and 70 and 92 days (6 rats each)). The rats were injected i.p. with a single dose of 500 mCi/kg of [3H]-thymidine (NEN, Boston, MA) 1 h before they were killed.
Determination of number and crypt multiplicity of foci
The colon and rectum were removed immediately after death, flushed with 0.9% cold NaCl solution and weighed. They were slit open longitudinally and divided into three segments: proximal, median and distal. Each segment was fixed in 10% formaldehyde solution between two filter papers in a Petri dish and stained with methylene blue as described by Bird (19), to evaluate the number of ACF and crypt multiplicity. ACF were classified as small (1-2-3-4 crypts/ACF), medium (5-6-7 crypts/ACF) or large (8 or more crypts/ACF).
Growth kinetic analysis
Growth parameters and gene expression were evaluated in histological sections of ACF and the surrounding mucosa. ACF were identified under the microscope in the colon stained with methylene blue and marked with permanent ink (Rotring, Germany). A small colon specimen (~2 mm2) including the marked ACF was then cut with a lancet, embedded in paraffin and cut into sections (4 µm) to be mounted on slides. The samples were stained with H&E for the determination of the mitotic index, or processed by the TUNEL method for the apoptotic index or processed for autoradiography for analysis of the labelling index. Each section was then observed under the microscope to identify the ACF, which was recognized as having a large crypt diameter often irregularly shaped. For each rat we scored at least 5 ACF which were chosen to have full longitudinal crypt sections (i.e. from the base to the bottom of the crypt ) to allow the determination of the proliferation indexes and apoptosis in at least 10 aberrant crypts. In the normal mucosa, at least 10 crypts/segment of the colorectum were used to determine each mitotic and labelling indexes while apoptotic index was determined in 20 crypts/rat.
Apoptosis was detected by the TUNEL method using the `In situ cell death detection fluorescein' kit (Boerhinger-Mannheim, Germany) which is an enzymatic system to label in situ apoptosis-induced DNA strand breaks. In this system, the terminal deoxynucleotidyl transferase (TdT) catalyzes the polymerization of fluorescein-dUTP nucleotides to free 3'-OH DNA ends in situ. Fluorescein labels incorporated in nucleotide polymers are detected and quantified by fluorescence microscopy (Leitz, Germany). This tailing reaction using TdT is described as the TUNEL (TdT-mediated dUTP nick end labelling) technique. The number of fluorescent cells, that is the number of cells with fragmented DNA, was determined for each crypt.
For autoradiography, sections of colorectal tissue were coated with NTB-2 Kodak emulsion, dried, immersed in liquid-scintillation fluid and sealed in a dark box at 80°C for 2 to 3 weeks. Slides were then developed and counterstained with H&E.
For each rat, we counted the number of cells on both sides of the well orientated crypts. A crypt is `well orientated' when the nuclei are visible from the base of the crypt to the luminal surface. We recorded the number and the position (from the bottom to the top of the crypt) of mitotic, apoptotic and labelled cells along the crypt column. For each rat, labelling, mitotic and apoptotic indexes (%) were calculated as the number of labelled, mitotic and apoptotic cells, respectively, counted in all the crypt sections of the same rat/number of cells in all the crypt sections of the same rat x100. The crypt column was divided into three equal parts (upper, medial and lower) to calculate the distribution of labelled, mitotic and apoptotic indexes along the crypt.
Immunohistochemistry
To measure the expression of TGFß1 and p21CIP proteins we cut tissue sections 3 µm thick. The sections were immersed in 0.3% H2O2 for 10 min to block endogenous peroxidase activity, incubated with sodium citrate buffer (Menarini, Florence, Italy) for 10 min in a microwave oven and incubated with goat serum (Dako, Milan, Italy) for 20 min. They were then reacted with primary mouse monoclonal antibody (Santa Cruz, CA, USA) diluted 1:50 for TGFß1 and 1:100 for p21CIP in TBS (Tris buffer saline) for 2 h. The sections were then incubated with secondary antibody conjugated to the avidine-biotin-peroxidase complex (Dako, Milan, Italy) for 1 h. Sections were stained with 3,3'-diaminobenzidine (DAB) (Boehringer-Mannheim, Milan, Italy) and counterstained with 1% hematoxylin to score `positive' cells which were identified as those with a dark brown colour.
For each rat TGFß1 and p21CIP expressions were calculated as the number of all the `positive cells' (those expressing the protein) counted in the crypts of the same rat/number of cells in all the crypt sections of the same rat x100. As described for the proliferative and apoptotic indexes, for each rat we scored at least 5 ACF which were chosen to have full longitudinal crypt sections to allow the determination of TGFß1 and p21CIP expressions in at least 10 aberrant crypts. In the normal mucosa, at least 10 normal crypts/rat were used to determine each parameter.
Statistical analysis
Data are expressed as mean ± SD; differences between groups were analyzed using Student's t-test (n = number of rats/group). A difference was considered statistically significant when the P-value was 0.05 or below.
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Results
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Fasting/refeeding enhances the growth of ACF
Body weights and food intake during the experiment are presented in Figure 2
and Figure 3
, respectively. Starting after the first cycle of fasting/refeeding (10 days after AOM) and throughout the course of all the experiment, body weight was lower (P
0.0001) in refed rats than in fully fed controls (Figure 2
). As expected, mean food intake in refed rats was higher (P < 0.001) after each cycle of fasting when compared with regularly fed rats (Figure 3
), but the total food intake of both groups was the same.

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Fig. 2. Body weight of fully fed controls (black points) and refed rats (white points) during the experimental period. Data are expressed as mean ± SD (n = 6). Starting after the first cycle of fasting/refeeding (10 days after AOM) and throughout the experiment, body weight was lower (P 0.0001) in refed rats than in fully fed controls.
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Fig. 3. Food intake in controls (black points) and refed rats (white points) during the experimental period. Data are expressed as mean ± SD (n = 6). After the five cycles of fasting and at the 92nd day, mean food intake in refed rats was higher (P < 0.001), compared with regularly fed rats.
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The results relative to ACF (Table I
) show that the number of ACF/colon at various times after AOM administration was similar in the two experimental groups. On the contrary, when comparing the two groups 70 and 92 days after AOM (8 and 30 days after the last cycle of fast, respectively) we found that fasting/refeeding caused a remarkable increase (P < 0.005) in crypt multiplicity when compared with regularly fed rats.
Fasting/refeeding increases mitotic and labelling indices and reduces the apoptotic index in the ACF of refed rats
Since loss of the concerted regulation of cell replication and active cell death is essential for non-cancerous tissue, we measured kinetic growth parameters and apoptosis in the ACF and in the surrounding mucosa of both refed and control rats (Table II
). As expected, in both groups the mitotic index was higher in ACF compared with the surrounding normal mucosa. Moreover, we found that both mitotic and labelling indexes were higher in the ACF of refed rats than in fully fed controls. The results also showed that the apoptotic index was significantly lower in the ACF of refed rats than in those of controls (Table II
). The determination of the distribution of the proliferative activity and apoptosis along the crypts did not show any significant difference between groups (data not shown).
p21CIPexpression was lower while TGFß1 expression was higher in ACF of refed rats
Given the variations in the proliferative and cell death kinetic parameters, we also investigated whether two of the main genes involved in the regulation of cell cycle of the colorectal epithelium, TGFß1 and p21CIP, could mediate the influence of fasting/refeeding on ACF growth. Levels of p21CIP and TGFß1 proteins were examined by immunohistochemistry in the putative preneoplastic lesions, ACF, and in the surrounding normal epithelium (Table III
). TGFß1 expression was found to be lower in ACF in comparison with the surrounding mucosa in both experimental groups, while TGFß1 expression was higher in the ACF of refed rats than in those of fully fed controls. We also found that p21CIP expression was higher in the ACF than in the normal mucosa and that in both ACF and normal mucosa, p21CIP was less expressed in refed rats than in controls; in particular, 64 days after AOM, none of the rats in the refed group expressed p21CIP protein in the normal mucosa. The multiplicity of the ACF examined for protein expression ranged from a minimum of 3 AC to a maximum of 6 AC/focus. No apparent differences in protein expression were found among ACF of different multiplicity (data not shown).
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Discussion
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The results of this study indicate that cycles of fasting/refeeding during the promotion phase of colorectal carcinogenesis enhance the growth of AOM-induced ACF, increase the proliferative activity and reduce cell death by apoptosis in ACF and in normal mucosa. The results also indicate that cycles of fasting/refeeding affect the expression of TGFß1 and p21CIP genes in both ACF and surrounding mucosa and that the expression of these two genes is altered in ACF as compared with normal mucosa.
Cyclic food restriction has been reported to abolish protection against mammary (5) and liver (4) carcinogenesis and more recently we showed that fasting/refeeding stimulated carcinogenesis in rat mammary glands (20) and liver (7,21,22). Contrary to these reports, other authors have shown that food restriction protect from carcinogenesis by enhancing apoptosis in preneoplastic cells (23), a finding in apparent discrepancy with the present data in which the refeeding period, after prolonged fasting, stimulates the ACF growth rate. It is possible that this stimulation might be due, in our experimental model, to nutritional factors or to the fact that fasting may have selected more aggressive ACF. Accordingly Corpet et al. (24) reported that food restriction decreased the proliferative activity in normal crypts but not in aberrant crypts induced in rats by AOM.
We also demonstrated that rats administered AOM after 4 days of fasting, developed larger ACF (8) and had a higher incidence of colorectal cancer (9). The data presented here clearly indicate that exposure of rats to various cycles of fasting/refeeding after AOM initiation, enhances crypt multiplicity of ACF without affecting the number of ACF. It is worth noting that crypt multiplicity has been proposed as the better predictor of colon cancer outcome (9,12,13). Therefore the present data demonstrate that not only the carcinogenesis initiation phase but also the promotion phase is affected by fasting/refeeding cycles. This effect was particularly striking at 92 days after AOM, when ACF multiplicity in the fasting/refeeding group was almost two times higher than in regularly fed rats. It is worth noting that such ample variation in crypt multiplicity has been reported in the literature only after treatment with potent chemopreventive agents such as polyethylene glycol which is able in rats to halve the multiplicity of AOM-induced ACF when compared with controls (16).
The mechanism underlying this effect seems to involve an increase in the proliferative activity in the mucosa together with a lower apoptotic index in the refed rats, thus favouring the growth of the ACF. The enhanced growth of ACF in refed rats may be explained, at least in part, by alteration in colon cell turnover observed during the refeeding which causes a wave of proliferation associated with a slowdown of apoptotis in ACF. Refeeding after fasting has been demonstrated to increase cell proliferation in colorectal mucosa in rodents (8,10).
In our study the variations in kinetic indices and apoptosis are accompanied by changes in the expression of cell cycle related genes, such as TGFß1 and p21CIP. TGFß1 is the main growth inhibitory factor in normal epithelium and p21CIP is the prototype of the CIP/KIP family of cyclin-dependent kinase inhibitor (CKI) well related to TGFß1 and known to mediate the effect of dietary factors on colorectal mucosa (2530).
Recently, it was reported that overexpression of either TGFß1 or R-TGFß1 by transfection into colon cancer cells inhibits cell growth and partially reverses the malignant phenotype (31,32). The expression of TGFß1 was lower in ACF than in surrounding mucosa. It may explain that ACF cells proliferate more than normal cells. Accordingly, administration of TGFß1 to rats treated with dimethylhydrazine reduces the number of aberrant crypts as well as tumor formation in the colon (33).
The results of our study also indicate that TGFß1 expression is increased in both ACF and the surrounding mucosa of the refed rats, a phenomenon difficult to explain. A similar increase in the expression of TGFß1 has been reported in hepatocytes of fasted rats (34). One could speculate that the cycles of starvation and refeeding decrease the sensitivity of colonocytes to TGFß1 with a consequent overexpression of TGFß1 in an attempt to restore normal proliferation. Accordingly, TGFß1 sensitivity has been reported to decrease during the process of colorectal carcinogenesis (35,36).
In refed rats, resistance to the action of TGFß1 may confer to the colorectal cells of ACF a growth advantage towards perifocal cells. On the other hand, TGFß1 may be available for inducing fibrogenesis (36), angiogenesis and immunosuppression (37) essential for tumor promotion.
Since TGFß1 has been reported to arrest the cell cycle at the late G1 phase by favouring accumulation of p21CIP(3840), we also determined the expression of this potential oncosuppressor gene which controls cell proliferation through the inhibition of cyclin-dependent kinase 2 (41,42). Indeed, the p21CIP gene was particularly interesting since loss of its function has been noted to be involved in the development of colorectal cancer (43). Consistent with these reports, we found that p21CIP expression was lower in the ACF and normal colorectal mucosa of refed rats when compared with that of fully fed animals. On the other hand, we also found that, irrespective of experimental group, p21CIP expression was higher in ACF than in normal mucosa, an apparently paradoxical finding similar to that reported by us in ACF and normal mucosa of resveratrol-treated and control rats (30).
In conclusion, in the present study we demonstrate that cycles of fasting/refeeding during the promotion phase of AOM-induced carcinogenesis enhance the growth of ACF, increase the proliferation activity and decrease apoptosis in preneoplastic lesions. Since these conditions have been associated with increased carcinogenesis (44,45), these results suggest that unbalanced dietary habits characterized by periods of fasting and binge eating similarly to those represented in this paper may represent a risk factor for colon cancer, even in humans. The identification of such risk factors which can and must be avoided may represent one of the best strategies for cancer prevention in humans.
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Notes
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4 To whom correspondence should be addressed at: Dipartimento di Scienze Cliniche e Biologiche, Università di Torino, Ospedale San Luigi Gonzaga, Regione Gonzole 10-10043 Orbassano (TO), Italy Email: tessitor{at}molinette.unito.it 
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Acknowledgments
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This study was supported by grants from Murst, Rome, Italy (40 and 60%).
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References
|
---|
-
Rogers,A.E., Zeisel,S.H. and Groopmann,J. (1993) Diet and cancerogenesis. Carcinogenesis, 14, 22052217.[Abstract]
-
World Cancer Research Fund and American Institute for Cancer Research. (1997) Food Nutrition and the Prevention of Cancer: A Global Perspective. BANTA Press, Menasha, WI, USA.
-
Miller,A.B., Berrino,F., Hill,M., Pietinen,P., Riboli,E. and Wahrendorf,J. (1994) Diet in the aetiology of cancer: a review. Eur. J. Cancer, 30A, 207220.
-
Lagopoulos,L., Sunahara,G.I., Wurzner,H., Dombrowsky,I. and Stalder,R. (1991) The effect of alternating dietary restriction and ad libitum feeding of mice on the development of diethylnitrosamine-induced liver tumors and its correlation to insulinemia. Carcinogenesis, 12, 311315.[Abstract]
-
Tagliaferro,A.R., Ronan,A.M., Meeker,L.D., Thompson,H.J. and Scott,A.L. (1996) Cyclic food restriction alters substrate utilization and abolishes protection from mammary carcinogenesis in female rats. J. Nutr., 126, 13981405.[ISI][Medline]
-
Muzio,G., Marengo,B., Salvo,R., Semeraro,A., Canuto,R.A. and Tessitore,L. (1999) Liver cancer is induced by a subnecrogenic dose of DENA when associated with fasting/refeeding: role of glutathione-transferase and lipid peroxidation. Free. Rad. Biol. Med., 26, 13141320.[ISI][Medline]
-
Tessitore,L. (2000) Apoptosis and cell proliferation are involved in the initiation of liver carcinogenesis by a subnecrogenic dose of diethylnitrosamine in refed rats. J. Nutr., 130, 104110.[Abstract/Free Full Text]
-
Premoselli,F., Sesca,E., Binasco,V., Caderni,G. and Tessitore,L. (1998) Fasting/refeeding before initiation enhances the growth of aberrant crypt foci induced by azoxymethane in rat colon and rectum. Int. J. Cancer, 77, 286294.[ISI][Medline]
-
Caderni,G., Bollito,E. and Tessitore,L. (1999) Colon cancer is induced by a single low dose of azoxymethane in fasted/refed rats. Nutr. Cancer, 35, 137142.[ISI][Medline]
-
Goodlad,R.A. and Wright,N.A. (1984) The effect of starvation and refeeding on intestinal cell proliferation in the mouse. Virchow Arch. (Cell Pathol.), 45, 6375.
-
McLellan,E.A. and Bird,R.P. (1988) Aberrant crypts: potential preneoplastic lesions in the murine colon. Cancer Res., 48, 61876192.[Abstract]
-
McLellan,E.A., Medline,A. and Bird,R.P. (1991) Sequential analyses of the growth and morphological characteristics of aberrant crypt foci: putative preneoplastic lesions. Cancer Res., 51, 52705274.[Abstract]
-
Pretlow,T.P., O'Riordan,M.A., Somich,G.A., Amini,S.B. and Pretlow,T.G. (1992) Aberrant crypts correlate with tumor incidence in F344 rats treated with azoxymethane and phytate. Carcinogenesis, 13, 15091512.[Abstract]
-
Yamada,Y., Yoshimi,N., Hirose,Y., Kawabata,K., Matsunaga,K., Shimizu,M., Hara,A. and Mori,H. (2000) Frequent beta-catenin mutations and accumulations of the protein in the putative preneoplastic lesions lacking macroscopic Aberrant Crypt Foci appearance, in rat colon carcinogenesis. Cancer Res., 60, 33233327.[Abstract/Free Full Text]
-
Yamada Y., Yoshimi,N., Hirose,Y., Matsunaga,K., Katayama,M., Sakata,K., Shimizu,M., Kuno, T and Mori,H. (2001) Sequential analysis of morphological and biological properties of beta-catenin-accumulated crypts, provable premalignant lesions independent of Aberrant Crypt Foci in rat colon carcinogenesis. Cancer Res., 61, 18741878.[Abstract/Free Full Text]
-
Corpet,D.E. and Parnaud,G. (1999) Polyethylene glycol, a potent suppressor of azoxymethane-induced colonic aberrant crypt foci in rats. Carcinogenesis, 20, 915918.[Abstract/Free Full Text]
-
Wargovich,M.J., Jimenez,A., Mckee,K., Steele,V.E. Velasco,M., Woods,J., Price,R., Gray,K. and Keloff,G.J. (2000)Efficacy of potential chemopreventive agents on rat colon aberrant crypt formation and progression. Carcinogenesis, 21, 11491155.[Abstract/Free Full Text]
-
European Community (1986) European Community Regulations on the Care and Use of Laboratory Animals, Law 86/609/EC.
-
Bird,R.P. (1987) Observation and quantification of aberrant crypts in the murine colon treated with a colon carcinogen: preliminary findings. Cancer Lett., 37, 147151[ISI][Medline]
-
Tessitore,L., Chiara,M., Sesca,E., Premoselli,F. and Binasco,V. (1997) Fasting during promotion, but not during initiation, enhances the growth of methylnitrosurea-induced mammary tumors. Carcinogenesis, 18, 16791681.[Abstract]
-
Tessitore,L. (1998) Hepatocellular carcinoma is induced by a subnecrogenic dose of diethylnitrosamine in previously fasted/refed rats. Nutr. Cancer, 32, 4954.[ISI][Medline]
-
Tessitore,L., Tomasi,C. and Greco,M. (1999) Fasting-induced apoptosis in rat liver is blocked by cycloheximide. Eur. J. Cell. Biol., 78, 573579.[ISI][Medline]
-
Grasl-Kraupp,B., Bursch,W., Ruttkay-Nedecky,B., Wagner,A., Lauer,B. and Shulte-Hermann,R. (1994) Food restriction eliminates preneoplastic cells through apoptosis and antagonizes carcinogenesis in rat liver. Proc. Natl Acad. Sci. USA, 91, 99959999.[Abstract/Free Full Text]
-
Corpet,D.E., Taché,S., Peiffer,G. (1997) Colon tumor promotion: is it a selection process? Effects of cholate, phytate and food restriction in rats on proliferation and apoptosis in normal and aberrant crypts. Cancer Lett., 114, 135138.[ISI][Medline]
-
Shin,S.I., Freedman,V.II., Risser,R. and Pollack,R. (1975) Tumorigenicity of virus-transformed cells in nude mice is correlated specifically with anchorage independent growth in vitro. Proc. Natl Acad. Sci. USA, 72, 44354439.[Abstract]
-
O'Neill,C., Jordan,P. and Ireland,G. (1986) Evidence for two distinct mechanisms in freshly explanted and 3T3 Swiss mouse fibroblasts. Cell, 44, 489496.[ISI][Medline]
-
Roberts,A.B. and Sporn,M.B. (1993) Physiological actions and clinical applications of transforming growth factor beta (TGFß). Growth Factors, 8, 19.[ISI][Medline]
-
Singhvi,R., Kumar,A., Lopets,G.P., Stephnopoulos,G.N., Wang,D.L., Whitesides,G.M. and Ingber,D.E. (1994) Engineering cell shape and function. Science, 264, 696698.[ISI][Medline]
-
Kletsas,D., Stathakos,D., Sorrentino,V. and Philipson,L. (1995) The growth-inhibitory block of TGFß is located close to the G1/ S border in the cell cycle. Exp. Cell. Res., 217, 477483.[ISI][Medline]
-
Tessitore,L., Davit,A., Sarotto,I. and Caderni,G. (2000) Resveratrol depresses the growth of colorectal aberrant crypt foci by affecting bax and p21cip expression. Carcinogenesis, 21, 16191622.[Abstract/Free Full Text]
-
MacKey,S.L., Auffenberg,T., Tannahill,C.L., Ksontini,R., Josephs,M.D., Nowak,M., Moldawer,L.L. and Copeland,E.M. (1998) Transfection of the type I (TGF-beta receptor into colon cancer cells increases receptor expression, inhibits cell growth and reduces the malignant phenotype. Ann. Surg., 227, 781789.[ISI][Medline]
-
Ye,S.C., Li,W., Liang,J., Zborowska,E., Venkateswarlu,S., Gong,J., Brattain,M.G. and Willson,J.K. (1999) Contextual effects of transforming growth factor beta on the tumorigenicity of human colon carcinoma cells. Cancer Res., 59, 47254731.[Abstract/Free Full Text]
-
Mikhailowsky,R., Shpitz,P., Polak-Charcon,S., Kost,Y., Segal,C., Fich,A. and Lamprecht,S.A. (1998) Controlled release of TGF-beta1 impedes rat colon carcinogenesis in vivo. Int. J. Cancer, 78, 618623.[ISI][Medline]
-
Shulte-Hermann,R., Bursh,W., Grasl-Kraupp,B., Huber,W. and Parzefall,W. (1994) In Cockburn,A. and Smith,L. (eds) Non Genotoxic Carcinogenesis. Springer Verlag, Berlin, 1, pp. 9120.
-
Reiss,M. (1997) Transforming growth factor-beta and cancer: a lovehate relationship? Oncol. Res., 9, 447457.[ISI][Medline]
-
Amendt,C., Schirmacher,P., Weber,H. and Blessing,M. (1998) Expression of a dominant negative type II TGF-beta receptor in mouse skin results in an increase in carcinoma incidence and an acceleration of carcinoma development. Oncogene, 17, 2534.[ISI][Medline]
-
Cui,W., Fowlis,D.J., Bryson,S., Duffie,E., Ireland,H., Balmain,A. and Akhurst,R.J. (1996) TGF-beta 1 inhibits the formation of benign skin tumours, but enhances progression to invasive spindle carcinomas in transgenic mice. Cell, 86, 531542.[ISI][Medline]
-
Gold,L.I. (1999) The role for transforming growth factor-beta (TGF-ß) in human cancer. Crit. Rev. Oncog., 10, 303360.[ISI][Medline]
-
Datto,M.B., Li,Y., Panus,J.F., Howe,D.J., Siong,Y. and Wang,X.F. (1995a) Transforming growth factor beta induces the cyclin-dependent kinase inhibitor p21 through a p53-independent mechanism. Proc. Natl Acad. Sci. USA, 92, 55455549.[Abstract]
-
Datto,M.B., Yu,Y. and Wang,X.F. (1995b) Functional analysis of the transforming growth factor beta responsive elements in the WAF1/Cip1/p21 promoter. J. Biol. Chem., 270, 2862328628.[Abstract/Free Full Text]
-
Li,C.Y., Suardet,L. and Little,J.B. (1995) Potential role of Waf1/Cip1/p21 as a mediator of TGF-beta cytoinhibitory effect. J. Biol. Chem., 270, 49714974.[Abstract/Free Full Text]
-
Estanyol,J.M., Jaumot,M., Casanovas,O., Rodriguez Vilarrupla,A., Agell,N. and Bach,S.O. (1999) The protein SET regulates the inhibitory effect of p21 (Cip1) on cyclin E-cyclin-dependent kinase 2 activity. J. Biol. Chem., 274, 3316133165.[Abstract/Free Full Text]
-
Polyak,K., Hamilton,S.R., Vogelstein,B. and Kinzler,K.W. (1996) Early alteration of cell-cycle-regulated gene expression in colorectal neoplasia. Am. J. Pathol., 149, 381387.[Abstract]
-
Newmark,H.L., Lipkin,M. and Maheshwari,N. (1990) Colonic hyperplasia and hyperproliferation induced by a nutritional stress diet with four components of western style diet. J. Natl Cancer Inst., 82, 491496.[Abstract]
-
Samaha,H.S., Kelloff,G.J., Steele,V., Rao,C.V. and Reddy,B.S. (1997) Modulation of apoptosis by sulindac, curcumin, phenylethyl-3-methylcaffeate and 6-phenylhexyl isothiocyanate: Apoptotic index as a biomarker in colon cancer chemoprevention and promotion. Cancer Res., 57, 13011305.[Abstract]
Received May 25, 2001;
revised October 23, 2001;
accepted October 24, 2001.