Expression of cyclin D1 and p53 and its correlation with proliferative activity in the spectrum of esophageal carcinomas induced after duodenal content reflux and 2,6-dimethylnitrosomorpholine administration in rats

Manuel Pera3, Pedro L Fernandez1, Miguel Pera, Antonio Palacín1, Antonio Cardesa1, Clemens Dasenbrock2, Thomas Tillman2 and Ulrich Mohr2

Service of Gastrointestinal Surgery and
1 Department of Pathology, Institute of Digestive Diseases, Hospital Clínic, IDIBAPS, University of Barcelona Medical School, Villarroel 170, 08036 Barcelona, Spain and
2 Institute of Experimental Pathology, Hannover Medical School, Hannover, Germany


    Abstract
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Alterations in expression of the p53 and cyclin D1 genes have been implicated in the development of esophageal carcinomas in both humans and animal models. We hypothesize that altered expression of cyclin D1 and p53 may be involved in the sequential development of esophageal carcinomas with glandular differentiation induced by the carcinogen, 2,6-dimethylnitrosomorpholine (DMNM) in rats with duodenal content reflux esophagitis. In the present study Sprague–Dawley rats were given DMNM 15 days after performing an esophago-jejunostomy in order to induce chronic duodenal content reflux esophagitis. Expression and localization of p53, cyclin D1 and Ki-67 were examined by immunohistochemical analyses. Twenty of 24 animals developed different types of esophageal carcinomas, including pure squamous carcinoma, adenosquamous carcinoma and pure adenocarcinoma. Undifferentiated basaloid areas were frequently observed in these tumors. Cyclin D1 overexpression was observed in hyperplastic lesions and increased through dysplasia and in undifferentiated areas of infiltrating carcinoma. Cyclin D1 expression coincided with increased Ki-67 expression and decreased along with cell differentiation. The p53 immunohistochemical pattern was parallel to that of cyclin D1, although the percentage of positive cells was usually smaller in all lesions and increased p53 expression started at the dysplastic stage. These findings suggest that overexpression of cyclin D1 may be an early event in DMNM-induced rat esophageal tumorigenesis, causing increased proliferation of esophageal stem cells. Abnormal p53 expression may then be required to promote the development of neoplastic transformation from dysplastic epithelium through invasive phenotype, being more evident in cancer cells with squamous differentiation.

Abbreviations: DMNM, 2,6-dimethylnitrosomorpholine; NMBA, N-nitrosomethylbenzylamine; PBS, phosphate-buffered saline; SCC, squamous cell carcinoma.


    Introduction
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Although the most frequent histological type of esophageal cancer is squamous cell carcinoma, the incidence of adenocarcinoma of the esophagus has risen rapidly in the USA and Western Europe (1). The causes for the recent emergence of esophageal adenocarcinoma are not well understood. There are reasons to suspect that gastroesophageal reflux plays an important part in the development of this type of cancer (2). Most of these adenocarcinomas arise in Barrett's esophagus, in which columnar cell metaplasia replaces the native squamous cell epithelium lining the distal esophagus (3). This premalignant lesion develops as a consequence of chronic reflux of acid and bile secretions into the esophagus. There is increasing evidence supporting a progression from metaplasia, to dysplasia and, finally, to adenocarcinoma (4).

Experimental models in laboratory rodents provide good tools for understanding the developmental mechanisms underlying carcinogenesis. Previous studies have shown that duodenal content reflux exerts a co-carcinogenic effect on 2,6-dimethylnitrosomorpholine (DMNM)-induced rat esophageal tumorigenesis (5). Pancreatobiliary secretions seem to stimulate expansion of the proliferative compartment of the squamous epithelium and it may explain in part the mechanism by which duodenal content reflux promotes esophageal carcinogenesis in experimental animals (6). In addition to the development of squamous cell carcinomas (SCCs), components of this mixed reflux (bile and pancreatic secretions) promote histopathological changes culminating in the development of both foci of glandular metaplasia and carcinomas with glandular differentiation (adenocarcinomas and adenosquamous carcinomas) (79). We have previously suggested that these latter types of tumors arise from multipotential stem cells of the basal layer of the squamous epithelium under the effect of duodenal content reflux secretions (7). All these animal models have provided some insights into the role of different components of duodenal content reflux on the development of Barrett's esophagus and esophageal adenocarcinoma in humans. Abnormal expression of many genes have been implicated in the neoplastic pathway leading to development of SCCs and its preneoplastic lesions such as papillomas and dysplasia in the rat model (10,11). Overexpression of cyclin D1 and its association with increased cell cycle proliferation have been described in the early stages of murine (1113) and human esophageal carcinogenesis (14,15). p53 mutations and mutational Ha-ras activation have been documented in rat esophageal papillomas (10,16). On the other hand, there is a lack of information regarding the molecular events which may ultimately predispose to the formation of esophageal adenocarcinomas in the rat model.

The purpose of the present study was to determine the possible role of two cell cycle-related genes (p53 and cyclin D1) and their correlation with the proliferative activity measured by Ki-67 in the spectrum of esophageal carcinomas induced by DMNM in rats with duodenal content reflux esophagitis.


    Material and methods
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Animals and treatment
Thirty 8-week-old Sprague–Dawley rats (Charles River, Germany), consisting of 15 males and 15 females weighing ~200 g at study start, were used in this study. Animals were housed two per cage in an environmentally controlled room maintained at 21 ± 2°C and 60 ± 15% relative humidity with a 12 h light/dark cycle. They were fed a pelleted diet (Altromin 1324; Altromin International, Germany) ad libitum and tap water. After a 1 week acclimatization period all animals underwent esophago-jejunostomy with gastric preservation, as previously described by our group, in order to produce chronic duodenal content reflux esophagitis (7). The surgical procedure was performed under ketamine (100 mg/kg body wt) and xylazine (4 mg/kg body wt) anesthesia. To induce esophageal neoplasms, DMNM provided by Professor U.Mohr (purity >98%) dissolved in olive oil was injected s.c. once weekly for life, at doses of 1/50 LD50. The doses were 468 mg/kg body wt (3.25 mmol) for males and 386 mg/kg body wt (2.26 mmol) for females. Carcinogen exposure began 15 days after performing esophago-jejunostomy. The animals were observed daily to assess their general health and any moribund animals were killed. Animals were scheduled to be killed at the beginning of the 32nd week after esophago-jejunostomy but were killed earlier when in a moribund condition. The study was approved by the Animal Care and Use Committee of the local authority at the Bezirksregierung, Hannover, Germany. All animals received humane care in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publication 85-93, revised 1985).

Tissue sample preparation
Animals were killed with an overdose of CO2 and subsequent exanguination. The esophagus was removed in toto and the lumen longitudinally opened by sectioning through the dorsal aspect of the esophageal wall. With the mucosal surface upward, the edges of the specimen were fixed to a cork plate with pins for macroscopic examination. Specimens were fixed in 10% buffered formalin. The esophagus was divided into three segments: proximal, middle and distal. The latter included the esophago-jejunal anastomosis. Step sections of the esophagus at 3 mm intervals were prepared in a longitudinal direction from each segment. These sections were embedded in paraffin, cut into 5 µm sections and stained with hematoxylin and eosin or with hematoxylin and eosin and Alcian blue for microscopic examination. Microscopically, esophageal lesions were classified as papillary hyperplasia, pure SCC, adenosquamous carcinoma and adenocarcinoma with two variants, pure glandular and signet-ring cell type.

Immunohistochemical staining
Paraffin-embedded tissue sections (5 µm) were deparaffinized and rehydrated. High temperature antigen retrieval was performed in 10 mM sodium citrate, pH 6.0, in a pressure cooker for 2 min. The slides remained in this solution for 20 min to cool. After a phosphate-buffered saline (PBS) rinse, tissue sections were treated with 0.3% hydrogen peroxide in methanol for 30 min to block endogenous peroxidase and then washed in PBS. Primary antibody incubations were done at room temperature for 60 min, followed by the EnVision Kit (HRP–mouse DAB+, K4007 or HRP–rabbit DAB+, K4003; Dako) procedure as visualization system. Primary antibodies used were as follows: rabbit anti-p53 (NCL-p53-CM5p at a dilution of 1:500; Novocastra Laboratories), rabbit anti-Ki67 (NCL-Ki67p at a dilution of 1:800; Novocastra Laboratories) and mouse anti-cyclin D1 (R124) (sc 6281 at a dilution of 1:40; Santa Cruz Biotechnology). The peroxidase reaction was developed with the use of diaminobenzidine. Finally, sections were counterstained with GIL I hematoxylin for 2 min. Cells were considered positive for p53, cyclin D1 and Ki-67 when evident nuclear staining could be identified. An estimation of the immunohistochemical expression of the above markers was made by counting at least 200 cells in random high power fields.


    Results
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Six of the 30 rats that had been operated on were killed during the early post-operative period and they were excluded from the study. Five animals were killed in a moribund condition within 3 months after esophago-jejunostomy. The remaining animals survived more than 6 months. Of these, 12 rats reached the scheduled end time point and were killed 32 weeks after performing esophago-jejunostomy.

Twenty of 24 animals developed esophageal carcinomas (Table IGo). The earliest tumor was observed in a rat killed 12 weeks after esophago-jejunostomy. All animals surviving beyond that time period developed carcinomas. The tumors were regularly distributed throughout the middle and distal third of the esophagus, although nine animals also developed tumors in the upper third of the esophagus. A summary of the neoplastic lesions is provided (Table IGo).


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Table I
 
Histopathological examination
Papillary hyperplasia was observed in most cases, consisting of thickening of the squamous epithelium with a variable degree of papillary outgrowth (Figure 1AGo).



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Fig. 1. (A) Lower power view of squamous papillary hyperplasia showing papillary projections and hyperkeratosis. H&E, x40. (B) Ki-67 expression in the two to three basal cell layers of papillary hyperplasia. Immunoperoxidase, x200. (C) Very faint and focal p53 expression in scattered basal cells of an area of papillary hyperplasia. Immunoperoxidase, x200. (D) Cyclin D1 immunohistochemical staining in an area of papillary hyperplasia. Note cyclin D1 nuclear staining in the basal and suprabasal layers of the squamous epithelium. Immunoperoxidase, x200.

 
Pure SCC was observed in 14 animals, mostly involving the lower two thirds of the esophagus and with an endophytic pattern of growth. These lesions consisted of infiltrating nests of squamous epithelium with nuclear atypia and frequent keratin formation. Squamous dysplasia accompanied the infiltrating lesions in some cases, whereas in others the squamous infiltrating cells seemed to emerge from undifferentiated basaloid cell nests (Figure 2AGo). Adenosquamous carcinoma was found in 19 animals, involving the upper third in seven cases. This type of neoplasia consisted of an admixture of the features of both adenocarcinomas and SCC, frequently in the form of small nests of squamous cells surrounding either mucus-filled lumina or small glands delineated by either cylindrical or signet-ring cells. Adenosquamous carcinomas were frequently observed in close relationship to pure SCCs. On occasions adenosquamous carcinoma nests seemed to emerge from basaloid-type neoplastic cells (Figure 3AGo). When coincidental squamous, adenosquamous and adenocarcinoma lesions were found in close proximity, a tendency for the latter to be in deeper areas was evident. Pure adenocarcinomas were observed in 11 animals. None of them were detected in the upper third of the esophagus. Microscopically, most adenocarcinomas consisted of proliferation of signet-ring cells which either individually infiltrated the esophageal wall or formed glandular structures with a variable component of cylindrical cells (Figure 4AGo). On occasions both signet-ring cells and neoplastic glandular structures were observed arising from undifferentiated basaloid neoplastic cells.



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Fig. 2. (A) Pure squamous cell carcinoma showing infiltrating nests of squamous epithelium with nuclear atypia and keratin formation. H&E, x200. (B) Ki-67 expression is more frequent in areas of undifferentiated basaloid SCC (bottom). Immunoperoxidase, x200. (C) p53 immunohistochemical staining. Note increased p53 expression in an area of undifferentiated basaloid cell phenotype (left). Immunoperoxidase, x200. (D) Cyclin D1 immunohistochemical staining in an area of SCC. Note intense and diffuse expression, mainly in areas of undifferentiated basaloid phenotype. Immunoperoxidase, x200.

 


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Fig. 3. (A) High power view of adenosquamous carcinoma. Gland formations are seen (arrows). H&E, x200. (B) Adenosquamous carcinoma showing Ki-67 expression limited to the area with undifferentiated basaloid phenotype. No expression is observed in the glandular compartment (top). Immunoperoxidase, x200. (C) Adenosquamous carcinoma showing frank p53 expression mostly limited to an area with squamous differentiation (bottom). Immunoperoxidase, x200. (D) Cyclin D1 immunohistochemical staining in adenosquamous carcinoma. Cyclin D1 nuclear staining in the undifferentiated basaloid areas (lower half), although some gland cells also express this marker. Immunoperoxidase, x200.

 


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Fig. 4. (A) Pure adenocarcinoma showing signet-ring cells infiltrating the esophageal wall. H&E, x200. (B) Pure esophageal adenocarcinoma showing no expression of Ki-67. Immunoperoxidase, x200. (C) A few scattered cells (arrow) showing positive staining for p53 in an area of adenocarcinoma. Immunoperoxidase, x200. (D) Low expression of cyclin D1 in an area of pure adenocarcinoma. Positive cells are mostly stromal fibroblast. Immunoperoxidase, x400.

 
Immunohistochemical expression of Ki-67, p53 and cyclin D1 expression of Ki-67
Normal esophageal epithelium, generally observed in the most proximal part of the upper third of the esophagus, showed expression of Ki-67 always restricted to the basal layer (Figure 5AGo). Areas with papillary hyperplasia consistently showed stronger expression of Ki-67 in the basal layer than that of adjacent normal squamous epithelium. An expansion of the proliferative compartment was usually seen with positive Ki-67 staining in the suprabasal layer (Figure 1BGo). Focal areas of squamous dysplasia, generally adjacent to squamous cell carcinomas, showed more intense and widespread positivity to Ki-67 involving most cell layers in comparison with the surrounding epithelium (Figure 5BGo). Differences in the degree of Ki-67 expression in SCCs with and without areas of undifferentiated component were observed. Well-differentiated areas of SCC usually showed Ki-67 positivity in <30% of the cells. In contrast, nearly 50% of the cells within the undifferentiated basaloid component stained positively for Ki-67 (Figure 2BGo). The analysis of Ki-67 expression in adenosquamous carcinomas was made both in areas with features of SCC and areas with glandular differentiation. The number of positively stained cells in areas with squamous differentiation was always higher (~30%) whereas the percentage decreased to <10% in those areas with adenocarcinoma features (Figure 3BGo). As in SCC, areas with undifferentiated basaloid phenotype showed even higher expression of Ki-67, reaching 50% of the cells. Pure esophageal adenocarcinomas, especially those cases with a predominant signet-ring cell pattern, were usually negative for Ki-67 staining except for a few cases showing scattered positive basal cells in some tubular-like glandular structures (Figure 4BGo).



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Fig. 5. (A) Ki-67 expression in the basal cell layer of normal epithelium. Immunoperoxidase, x200. (B) Dysplastic squamous epithelium showing intense and diffuse Ki-67 expression throughout the cell layers. Immunoperoxidase, x200. (C) Area of squamous dysplasia with intense p53 immunopositivity (top). Immunoperoxidase, x200. (D) Cyclin D1 is strongly and diffusely expressed by squamous dysplasia. Immunoperoxidase, x200.

 
Expression of p53
p53 expression was negative in areas of normal squamous epithelium. Only weak expression of p53 was found focally in basal cells of some areas of papillary hyperplasia (Figure 1CGo). Areas of squamous dysplasia were usually strongly positive for p53 (Figure 5CGo). Pure SCCs showed an average expression of p53 in 10% of the cells in well-differentiated superficial areas. However, p53 staining was observed in 40–70% of cells in those tumor compartments with basaloid cell phenotype (Figure 2CGo). Adenosquamous carcinoma only showed p53 expression, sometimes reaching 40–50% of cells, in those areas with squamous differentiation, thus coinciding with Ki-67 expression (Figure 3CGo). Pure adenocarcinomas were mostly negative, although in one case <10% positive cells were found.

Expression of cyclin D1
Normal squamous epithelium only expressed cyclin D1 in scattered basal cells, whereas an increased number of positive basal cells together with an increased number of positive cell layers were observed in papillary hyperplasia (Figure 1DGo). Cyclin D1 was strongly expressed by stromal fibroblasts in all types of lesions. Areas of squamous dysplasia showed cyclin D1 expression in many cells (Figure 5DGo) and this also occurred in SCC, predominantly in undifferentiated basaloid areas (Figure 2DGo). In adenosquamous cell carcinomas cyclin D1 expression was mainly observed in the squamous component and poorly differentiated basaloid areas (Figure 3DGo). Pure adenocarcinoma had cyclin D1 expression ranging from 0–10% of cells (Figure 4DGo).


    Discussion
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
It has been hypothesized that neoplastic progression in humans develops as a consequence of an acquired genetic instability and the subsequent evolution of clonal cell populations with accumulated genetic errors. In esophageal cancer, cell cycle alterations due to abnormalities involving tumor suppressor genes like p53, retinoblastoma (Rb) and cyclins have been reported (1719). Most experimental studies on murine chemical carcinogenesis of the esophagus have focused on the molecular mechanisms involved in the development of squamous cell carcinomas and their precursor lesions (papilloma and dysplasia). In our present study the combination of duodenal content reflux into the esophagus and DMNM administration resulted in the development of a spectrum of esophageal lesions such as papillary hyperplasia, dysplasia and carcinomas, many of the latter with areas of glandular differentiation. Our immunohistochemical analysis strongly suggests that cyclin D1 and p53 are very likely implicated in the development and progression of these esophageal lesions.

We have previously shown that either a combined reflux of bile and pancreatic secretions or a reflux of pancreatic secretions alone into the esophagus is able to stimulate a state of epithelial cell hyperproliferation (6) and, consistent with this, papillary hyperplasia in our current study showed increased cell proliferation with expansion of the proliferative compartment towards the suprabasal layers. Areas of squamous dysplasia were sometimes found in the epithelium adjacent to carcinomas and it is likely that in many cases dysplastic areas were overgrown by large carcinomas. Expression of Ki-67 was frequently diffuse throughout all the layers of dysplasia, indicating an active proliferative state. Due to the spectrum of differentiation of the carcinomas originating in our model of esophageal carcinogenesis we analyzed the expression of immunohistochemical markers in the different cell phenotypes separately. In many cases the superficial tumor areas directly beneath the surface epithelium consisted of irregular infiltrating nests of undifferentiated cells with a basaloid appearance. Ki-67 was strongly expressed by this phenotype, suggesting that it could be the most important proliferative compartment of esophageal carcinomas in our model. Indeed, Ki-67 expression decreased when either the squamous or glandular phenotype were acquired. When comparing glandular and squamous differentiation, the former was even less proliferative and areas of pure adenocarcinoma had the lowest degree of Ki-67 staining. This finding suggests that glandular differentiation is even more committed to a specific function (secretion) than squamous cells, which still retain some proliferative capability.

It seems that a critical genetic alteration in esophageal SCC in humans is the activation of cyclin D1 through gene amplification (19). There have been only a few reports of cyclin D1 expression in animal models of esophageal carcinogenesis (11,20). None of them have used a model combining carcinogenic treatment with chronic injury to the esophageal epithelium. Contrary to normal squamous epithelium, which only expresses cyclin D1 in scattered basal cells, in our study cyclin D1 overexpression was clearly observed in papillary squamous hyperplasia and also in areas of squamous dysplasia. Cyclin D1 was also strongly expressed in areas of undifferentiated basaloid carcinomas, but its expression was minimal in those areas with glandular differentiation. Two previous studies have evaluated cyclin D1 expression and its correlation with proliferative activity in a N-nitrosomethylbenzylamine (NMBA)-induced rat esophageal cancer model (11,20). In one report the amount of cyclin D1 was found to be negligible in normal rat esophageal epithelium, with a minimal increase in the expression levels in hyperplastic and dysplastic lesions (11). In contrast, esophageal papillomas and carcinomas each showed overexpression of cyclin D1 compared with hyperplastic and dysplastic lesions. The combination of carcinogen administration and chronic esophageal mucosal injury due to duodenal content reflux in our study might explain a higher degree of cellular proliferation and cyclin D1 expression in areas of papillary hyperplasia and dysplasia compared with the levels of cyclin D1 expression observed by Youssef et al. (11) in the same types of lesions. Both these studies found a high level of correlation between expression of cyclin D1 and proliferating cell nuclear antigen in papillomas. In our study there was a good correlation between proliferation assessed by Ki-67 staining and cyclin D1 in areas of papillary hyperplasia, dysplasia and undifferentiated basaloid areas of carcinoma. It is noteworthy that a correlation between expression of cyclin D1 and proliferation has also been observed in esophageal SCC in humans (21). The findings of this study suggest that cyclin D1 overexpression is an early event and may be important for the development of invasive carcinomas after lesions such as hyperplasia and dysplasia. This is in line with a previous study in humans, where cyclin D1 expression was found in early stages of SCC development, with no variation in the degree of staining between superficial and advanced lesions (22). In our study the amount of cyclin D1 was considered negligible in areas of glandular differentiation within adenosquamous carcinoma or pure adenocarcinoma, which suggests, as for Ki-67, a close relationship with cell phenotype. The coincidental expression of Ki-67 and cyclin D1 in dysplasia as well as in undifferentiated basaloid areas of carcinoma strongly support a proliferation-promoting effect of up-regulated cyclin D1. Indeed, transgenic mice with cyclin D1 overexpression in the squamous epithelium of the esophagus show increased cell proliferation and the development of a dysplastic phenotype (13).

It is likely, in this setting of increased cell proliferation, that other genetic alterations are acquired. Little is known about the role of p53 mutational inactivation in rodent esophageal tumors. p53 mutations have been detected in 30% of rat esophageal papillomas induced by NMBA (10). However, to our knowledge there is no conclusive data about the role of p53 inactivation in preinvasive lesions induced in rat esophageal carcinogenic models. In our present study papillary hyperplasia did not show abnormal p53 staining. Dysplastic areas always showed p53 expression, but the greater p53 staining was observed in undifferentiated basaloid areas of invasive carcinomas, reaching 40–70% of the cells. Pure SCC showed a lesser degree of p53 staining and little or no p53 expression was observed in areas with glandular differentiation. Therefore, p53 staining was greater in those areas of carcinoma where we also observed high cell proliferative activity. It is possible that the greater degree of cell proliferation observed in areas of dysplasia and undifferentiated basaloid carcinoma might be responsible for reactive expression of wild-type p53. Alternatively, p53 expression may represent subsequent genomic p53 alterations (mutations), although a non-causal relationship is also feasible. Indeed, p53 expression might also be related to differentiation rather than proliferation, being minimal when cells reach a terminal phenotype. Future analysis of the p53 tumor suppressor gene by direct sequence analysis will reveal the mechanisms leading to immunohistochemical p53 expression: gene mutation or wild-type protein overexpression. Although cyclin D1 overexpression may be an initiating event, mutant p53 may be required to promote the development of a dysplastic and SCC phenotype. A recent study in transgenic mice suggested that a combination of cyclin D1 overexpression and p53 inactivation plays a critical role in induction and progression of carcinogenesis in the oral–esophageal squamous epithelium (23).

A frequent finding in our experiment was the presence of undifferentiated basaloid areas in many carcinomas. The absence of submucosal glands in the rat esophagus suggests that carcinomas with glandular differentiation may arise from initiated stem cells of the basal layer of the squamous epithelium, a fact supported by the frequent finding of tumor nests with glandular differentiation adjacent to undifferentiated basaloid areas (Figure 3AGo). Destruction of the epithelial tissue architecture under the chronic effect of the biliopancreatic secretions may allow the proliferating stem cell population to be exposed to factors contained in the reflux material. Such proliferating cell populations may be particularly susceptible to accumulation of multiple genetic alterations and clonal expansion of mutant cells. This provides a larger cellular target for subsequent mutations and the eventual accumulation of sufficient genetic alterations within an individual cell or group of cells for progression from a benign proliferative lesion to full-blown malignancy. A minority of initiated cells will eventually proliferate to form a focus, with the potential for double differentiation, glandular and squamous, from the basal layer of the squamous epithelium (24). These foci will undergo further genetic changes which lead to the formation of carcinomas with areas of SCC and other areas of adenosquamous carcinoma.

On the basis of our observations we hypothesize that during tumor development in our rat model of esophageal carcinogenesis, one of the earliest events is cyclin D1 up-regulation, which may have an important role in initial proliferation of the squamous epithelium. Although we cannot exclude that such overexpression may be related to gene amplification, as stated in humans (25,26), the irritative effect of duodenal content reflux could induce cyclin D1 up-regulation by transcriptional induction or post-translational mechanisms (27). When undifferentiated neoplastic cells appear the proliferation-promoting role of cyclin D1 could be at its maximum, afterwards reverting to lower levels when differentiation (squamous or glandular) occurs. Contrary to Ki-67 and cyclin D1, which already seem up-regulated in hyperplastic lesions, abnormal p53 expression appears at the dysplastic stage and is also more intense in undifferentiated basaloid areas, suggesting that such abnormalities are acquired later in neoplastic transformation. p53 and cyclin D1 could then run in parallel throughout phenotypic evolution towards maximum differentiation (Figure 6Go).



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Fig. 6. Immunohistochemical pattern of cyclin D1, p53 and Ki-67 through the progression from papillary hyperplasia to different types of carcinoma.

 
In summary, we believe that the cyclin D1 and p53 cell cycle modulators could have an important role in murine esophageal carcinogenesis since cyclin D1 overexpression is an initial event at the hyperplastic stage, followed by that of p53 in dysplasia. An intervening phase of undifferentiated basaloid carcinoma with maximal cyclin D1 and p53 overexpression precedes the down-regulation of both markers towards differentiation. Future studies using this model might help to better understand some aspects of the histogenesis and process of malignant transformation of esophageal carcinomas with both glandular and squamous differentiation in humans.


    Notes
 
3 To whom correspondence should be addressed Email: mpera{at}medicina.ub.es Back


    Acknowledgments
 
We wish to thank Dr Antonio Martinez for his collaboration in Figures 1–5GoGoGoGoGo and Mrs Margarita Maynar and Elena Gonzalvo for their technical support. This work was supported by grant 97/1221 from the Fondo de Investigacion Sanitaria, Ministry of Health, Spain and HCICYT-SAF 97/0096.


    References
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 

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Received June 8, 2000; revised October 3, 2000; accepted October 5, 2000.