ISSN 00954527, Cytology and Genetics, 2015, Vol. 49, No. 2, pp. 113–117. © Allerton Press, Inc., 2015. Original Ukrainian Text © S.V. Vernygorodskyi, L.V. Degtiariova, O.I. Iatsyna, Ya.B. Blume, A.I. Yemets, 2015, published in Tsitologiya i Genetika, 2015, Vol. 49, No. 2, pp. 42–47.
Role of Transcription Factors in Transdifferentiation of the Gastric Mucosa S. V. Vernygorodskyia, L. V. Degtiariovab, O. I. Iatsynac, Ya. B. Blumed, and A. I. Yemetsd a
National Pirogov Memorial Medical University, ul. Pirogova 56, Vinnytsya, 21018 Ukraine email:
[email protected] bBogomolets National Medical University, bul’v. Shevchenka 13, Kyiv, 01601 Ukraine c National Cancer Institute, ul. Lomonosova 33/43, Kyiv, 03022 Ukraine dInstitute of Food Biotechnology and Genomics, National Academy of Sciences of Ukraine, ul. Osypovskogo 2A, Kyiv, 04123 Ukraine Received April 14, 2014
Abstract—The analysis of intestinal differentiation transcription factor CDX2 in the gastric mucosa biopsies has been carried out. It was established that CDX2 by its own promoter activation pathway can obtain intes tinal phenotype for gastric mucosa cells. The loss of CDX2 expression in the nuclei of metaplastic epithelium may serve as a predictor of gastric mucosa malignization. Keywords: transcription factors, transdifferentiation, gastric mucosa DOI: 10.3103/S0095452715020115
INTRODUCTION Recently, the phenomenon of reprogramming the nucleus of a mature somatic cell has been intensively studied, because there is a prospect to obtain “patient specific” pluripotential cells that are similar to embry onic stem ones. While realizing this phenomenon under the influence of some factors, the activization of early embryogenesis genes and inhibiting of genes responsible for differentiation and specialization occur in the nucleus of a somatic cell. In case that the complete reprogramming specific genetic and epige netic information is lost, a cell acquires properties of pluripotential one. The complete reprogramming of a nucleus of a terminal differentiated somatic cell is proved during the experiment. This happens when the nucleus is transferred to a denucleated unfertilized ovule [1] and when mature specific cell conjugates with an embryonic stem one [2, 3]. In October 2012, John Gurdon and Shinya Yamanaka became Nobel laureates in medicine or physiology for the study of reprogramming of cells [4], but the investigation of mechanism and factors regulating realization of this biological phenomenon has not yet been finished. Up to the present, it was considered that differenti ated cells can emerge from embryonic or stem cells. But now we know that mature cells of one phenotype can transform into completely differentiated cells of another by means of transdifferentiation [5]. Dediffer entiation and cellular partition are normally signifi cant intermediate processes in the cell development, but they do not always take place. Transdifferentiation
associated with isolated change within the program of gene expression, is a direct link prototype between these two cell types. Presently, we have not found how specialization of intestinal endoderm occurs. We consider that intesti nal endoderm differentiates locally on the early stages and specialization is determined when dealing with surrounding mesenchyme tissue. We also assume that the expression of homeobox genes (Hox) specifies dif ferent organs according to the anterior–posterior axis of the body. These genes encode proteins regulating transcription and determining body structures and their position in anterior–posterior dimension. Acting in accordance with the genetic program, they initiate or suppress transcription of some genes under influ ence of interior factors and cause changes of cell struc ture and differentiation and organogenesis [6]. CDX1 and CDX2 are caudad linked homeobox transcription factors with selective localization in the nuclei of epithelial cells of human small and large intestine mucosa. But they are absent in the unchanged (normal) gastric mucosa. In the healthy intestinal mucosa, CDX2 is expressed mostly in differ entiated enterocytes of crypta and villi; CDX1 is expressed mostly in undifferentiated cells of prolifera tive crypta compartment [7]. However, as many stud ies proved, its aberrant expression in the upper parts of gastricintestinal tract can be an important pathogenic stage of intestinal metaplasia of gastric mucosa. Thus, they demonstrated that CDX2 activates the expression of intestinal mucin gene MUC2 in gastric cells induc
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ing intestinal transdifferentiation both in the gastric metaplasia sections and on particular gastric cancer kinds [8]. The studies conducted in on Cdx2trans genic mice [6, 7] and human gastrobiopsies [9, 10] also testify to the close pathogenic link of metaplasia and system of tissue genetic differentiation. The aim of our work is to define the role of tran scription factor CDX2 in origin of intestinal metapla sia of gastric mucosa, to substantiate its diagnostic and prognostic value, and to examine the prospects of CDX2 usage as immunohistochemical marker of some types of intestinal metaplasia. MATERIALS AND METHODS During 6 years, we examined 336 patients which were sent to endoscopic departments and consulting rooms of Vinnytsya region to specify their clinic diag nosis. There were 192 (57%) men and 144 women (43%). The main groups consisted of 68 patients with chronic atrophic gastritis with intestinal metaplasia because of the prevalent association of the latter with this disease. The comparative group included 30 patients with chronic atrophic gastritis without intestinal metapla sia. Average age of patients that were examined in the course of disease was 52.96 ± 1.13, and average disease duration as from the date of intestinal metaplasia diag nosis was 2.6 ± 0.63 years. In the process of fibroesophagogastroduodenos copy and chromoendoscopy with 0.5% water solution of methylene blue, we did plural biopsies (two bioptates from body and antral stomach and one from section of stomach angle) according to the require ments of modified Sydney system of chronic gastritis diagnostics and stained sections of gastric mucosa with the further study of bioptates. Biopsy material was noticed in 10% neutral formalin and after generally accepted processing paraffin blocks were made which were used for creating a microscopic section 5–7 μm thick. To determine metaplastic changes of gastric mucosa we used the following methods: general histo logical (staining with hematoxylin and eosin and according to Van Gieson) and histochemical (staining with glandular diamine according to Spicer, orcein together with alcian blue, aldehydefuchsin according to Gomori, and alcian blue at pH 1.0 and 2.5 in con junction with PASreaction). The persistence of Helicobacter pylori in gastric mucosa was determined with the use of a rapid urease test; cytologically according to Pappenheim and his tologically with Giemsa stain with the use of toluidine blue. Immunohistochemical studies were conducted in paraffin sections using the streptavidinbiotin visu alization method (Dako, Denmark). Antigen demask ing was carried out in citrate buffer at pH 6.0. As initial antibodies we used mouse and rabbit monoclonal anti bodies. Cell nuclei were counterstained with Mayer hematoxylin during 15–60 s.
Gene expression of transcription factor of intesti nal differentiation CDX2 was estimated with the use of mouse monoclonal antigens to nucleus antigen CDX2 and clone DAKCDX2 (Dako, Denmark). Mucin profile was determined using antibodies MUC5AC, MUC2, and MUC6 (clones CLH2, Ccp58 and CLH5) (Novocastra, United Kingdom). In prepara tions at 400fold microscope magnification, we deter mined the intestinal differentiation index (nucleus mark CDX2) in five accidentally selected fields of vision (≤500 cells) as a portion in percentage of posi tively stained nuclei of epithelial cells of gastric mucosa in three compartments (I—surface and foveolar epithelium; II—isthmus zone; III—gland base, middle and lower third of glands to basal sections). To estimate mucin expression (MUC5AC, MUC2, MUC6) in gas tric mucosa in the same sections, we used semiquantita tive estimation scale of staining intensity: 0 (absent)— absence of positive reaction in cells; 1 (weak)—to 30% of cells which reacted positively; 2 (moderate)—31– 60%; 3 (strong)—60% and more of stained cells [11]. RESULTS AND DISCUSSION We did not observe CDX2 expression during the immunohistochemical analysis of cases with morpho logically unchanged gastric mucosa and in patients with chronic nonatrophic gastritis and chronic atro phic gastritis without intestinal metaplasia. We took gastrobiptates from antral stomach section and body from 68 patients with chronic atrophic gastritis with metaplasia and found sections of replacement of spe cific gastric glands cells with metaplastic epithelium (intestinal and pyloric metaplasia). The complete intestinal metaplasia nidi of patients with chronic atrophic gastritis with intestinal metapla sia were characterized by the high level of intestinal factor expression of CDX2 transcription with nuclei of goblet cells and columnar epithelial cells with brush border (Fig. 1). During the histochemical analysis of goblet cells, we found that acid sialomucin predomi nated, and intestinal mucin MUC2 predominated during the immunohistochemical. We also detected the absence of neutral glycoproteins, acid sialo and sulfomucins and MUC5AC in columnar epithelial cells. Patients with complete intestinal metaplasia and H. pylory infection had the higher level of CDX2 expression in comparison with H. pylorinegative group and was 0.89 ± 0.01 (p < 0.001) (table). We man aged to detect moderate immunohistochemical reac tion on the presence of intestinal CDX2 differentia tion factor in nuclei of surface and foveolar epithelial cells of gastric mucosa and neck mucous cells on the early stage of intestinal metaplasia development before goblet cells have emerged. We can testify that CDX2 expression is typical for differentiated surface epithe lial cells and generative compartment cells. It indicates direct transdifferentiation of gastric epithelial cells CYTOLOGY AND GENETICS
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75% of patients did not have it (Fig. 2). At the same time, in sections of intestinal metaplasia with dysplas tically changed epithelium, we observed CDX2 expression disappearing. When we compared the study results concerning patients with chronic atrophic gastritis from H. pylori negative and H. pyloripositive groups, we found that focally expressed positive immunohistochemical reac tion dominated among infected patients. Disappear ing of CDX2marking of epithelial cell nuclei of gas tric mucosa in the sections attached to cancerous growth is a typical phenomenon for 98% of tests. At the same time, CDX2 expression was absent in cases of complete and incomplete intestinal metaplasia and was not correlated with the helicobacteriosis pres ence/absence (Fig 3.). In the group of patients with gastric cancer, there were only two people with moderately differentiated adenocarcinoma and weak CDX2 expression, but it was absent in the attached sections with intestinal metaplasia. But we did not identify CDX2 marking for 96% of patients with lowdifferentiated adenocarci noma and gastric signet ring cells. It is necessary to admit that patients with long intestinal metaplasia (more than 3–4 years) and mostly incomplete intesti nal metaplasia CDX2marking of affected sections was negative when infection H. pylori exists or not. The presence of CDX2 in epithelial cell nuclei of gastric mucosa proves the formation of socalled gas trointestinal phenotype of epithelial cells. It is typical that between CDX2positive goblet exocrinocytes synthesizing intestinal mucin MUC2 and gastric mucin MUC5AC there are cylindrical CDX2positive and CDX2negative epithelial cells with MUC5AC expression and acid (sialo, sulfomucins) and neutral mucins that can be detected by routine multicoloured histochemical methods. Immunohistochemical detection of positive expression of transcription factor CDX2 in surface epithelial cell nuclei and neck mucous cells of meta plasia gastric mucosa before goblet cells can prove that
Fig. 1. Marked expression of transcription factor of intes tinal differentiation of CDX2 in the nuclei of foveolar gas tric epithelial cells and nuclei of goblet and columnar epi thelial cells with brush border. Immunohistochemical marking CDX2. Magnification by ×300.
and involvement of stem cells in the process of differ entiation. After eradication of infection H. pylori, the number of gastric epithelial cells with CDX2positive nuclei decreased. This testifies to the possible warning of nucleus reprogramming of gastric mucosa epithelium if bacteria are eradicated in proper time. We are also to assert that CDX2 expression in nuclei of goblet cells in sections of complete intestinal metaplasia is not affected by infection eradication. This indicates attachment of intestinal phenotype of metaplasia epi thelium. Incomplete intestinal metaplasia is charac terized by weaker expression of CDX2 transcription factor concerning complete intestinal metaplasia, but
CDX2 expression of patients with chronic atrophic gastritis with intestinal metaplasia (M ± m) Expression level in groups of patients Nosology H. pylori (+)
H. pylori (–)
–
–
Complete intestinal metaplasia
0.89 ± 0.01
0.43 ± 0.040*
Incomplete intestinal metaplasia
0.24 ± 0.06**
0.16 ± 0.016**
Norm Chronic atrophic gastritis
(+) positive gastritis; (–) negative gastritis. CIM is complete intestinal metaplasia; IIM is incomplete intestinal metaplasia. * p < 0.001 to H. pylori(+); ** p < 0.001 to CIM. CYTOLOGY AND GENETICS
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Fig. 2. Negative CDX2 marking in the centers of incom plete intestinal metaplasia. Immunohistochemical mark ing CDX2. Magnification by ×200.
Fig. 3. Negative CDX2 marking in the centers of complete intestinal metaplasia and adenocarcinoma. Immunohis tochemical marking CDX2. Magnification by ×100.
the cellular phenotype changes into a gastric one and serve as an early marker of emergence of intestinal metaplasia. Vivid CDX2 expression in goblet cells and columnar epithelial cells of complete intestinal meta plasia sections indicates the ending of metaplasia pro cess and attachment of intestinal phenotype of epithe lial cells.
suppression of Sox2 expression and activization of transcription factor of intestinal CDX2 differentiation in gastric epithelial cells, with inhibition of gene MUC5AC an induction of gene MUC2.
When CDX2 expression increases, intestinal meta plasia gets complete and it indicates that gastric mucin (MUC5ACpositive) expression disappears in colum nar cells, and their brush borders emerges. Also, according to the data of Dimmler et al. [12], gastric factor of Shh. differentiation expression disappear. At the same time while CDX2 expression decreases, phe notype of glandular stomach epithelium is mixed (incomplete intestinal metaplasia). Except secretion of neutral glycoprotein, it produces sulfomucins in columnar epithelial cells. Such type of intestinal metaplasia (type III, incomplete large intestinal) was found more often in case of long (more 2–4 years) atrophic changes of gastric mucosa in patients with H. pylori and it was the most typical, according to our findings, for chronic atrophic pangastritis. It is also necessary to distinguish gastrointestinal phenotype of intestinal metaplasia that originated from persistence of helicobacteriosis. Separation of gastrointestinal phenotype of intestinal metaplasia is rather significant during further treatment and prog nosis for intestinal metaplasia. We found that disorder of regulation of family of transcription factors Sox2, Cdx2, Pdx1, Sonic hedge hog, and Oct1 are important for embryonic tissue development and in transdifferentiation of fundal and pyloric exocrinocytes into intestinal epithelium [13, 14]. One of the factors directly influencing the transcription factor function is H. pylori. The function Sox2 is regulated by interleukin (IL4) through activ ization of transcription factor STAT6 (Signal Trans ducer and Activator of Transcription) in gastric epi thelium. This regulation can be suppressed directly by H. pylori. Under the influence of H. pylori, we can see
The loss of CDX2 marking in the sections of intes tinal metaplasia (complete and incomplete) can be an unfavorable prognostic peculiarity of malignization, since these changes indicate disorders of cell differen tiation and tendencies to oncotransformation [15]. Considering the literature data, the absence of CDX2 marking in 96% of patients with stomach cancer con firm antioncogenic properties of this transcription fac tor. Similar CDX2 properties were observed in col orectal adenocarcinomas [16]. Thus, marking of tran scription factor CDX2 can be widely used for early intestinal metaplasia diagnostics and gastric mucosa oncotransformation. Our suppositions concerning antioncogenic properties of this transcription factor need further studying. Regarding intestinal metaplasia, the cellularbio logical peculiarities of epithelial cells depend on not only its type but also background process character in gastric mucosa. Hence, the differentiated approach based on the usage of molecularbiological markers to intestinal metaplasia phenomenon is not only of sci entific interest but also is a fundamental basis for the development of methodical approaches of prognosis and treatment of patients with intestinal metaplasia of gastric mucosa. Thus, the results of molecularbiological studies show that CDX2 can attach intestinal phenotype to cells by means of activization of its own promoter, but it contradicts the concept of metaplasia return. That is why the further studies of this phenomenon have to explain whether molecular mechanisms of emergence of intestinal metaplasia are identical during different pathological processes. CYTOLOGY AND GENETICS
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CONCLUSIONS Intestinal metaplasia of gastric mucosa is a hetero geneous state. Metaplasia changes of gastric epithe lium into an intestinal one can originate from different cell populations. The first possible one is called trans differentiation and it is a direct transition from differ entiated cells in the absence of cell proliferation. Alternative way is metaplasia, which can arise as a result of transformation of “stem” or “pluripotent cells” or cells that are able to restore themselves in an unlimited and prolong way. Intestinal metaplasia of gastric mucosa is irrevers ible while intestinal phenotype of epithelial cells (completely formed goblet cells) are attaching. According to our data, the intestinal metaplasia return is possible in case of reprogramming of gastric epithe lial cell nuclei until complete cell differentiation has finished. CDX2 is a starting transcription factor of nuclei reprogramming by intestinal type and can be used for early intestinal metaplasia diagnostics. Maximal CDX2 expression occurs when complete intestinal metapla sia is combined with the loss of MUC5AC expression by columnar epithelial cells and MUC2 production by goblet exocrinocytes and testifies to attachment of intestinal epithelium phenotype. Expression of intestinal factor of CDX2 transcrip tion with products of intestinal mucin MUC2 and gas tric mucin MUC5AC by goblet cells and columnar epi thelial cells is a formation marker of gastrointestinal phenotype of gastric mucosa epithelium—incomplete intestinal metaplasia. Negative CDX2 marking in intestinal epithelium nuclei can be an early marker of their malignization, and it is proved by marking absence in goblet cell nuclei and absorbing cells in the dysplasia gastric mucosa areas and sections attached to cancerous growth. This phenomenon is typical for 98% of patients. REFERENCES 1. Wilmut, I., Schnieke, A.E., McWhir, J., et al., Viable offspring derived from fetal and adult mammalian cells, Nature, 1997, vol. 385, pp. 810–813. 2. Tada, M., Takahama, Y., Abe, K., et al., Nuclear repro gramming of somatic cells by in vitro hybridization with ES cells, Curr. Biol., 2001, vol. 11, pp. 1553–1558. 3. Cowan, C.A., Atienza, J., Melton, D.A., and Eggan, K., Nuclear reprogramming of somatic cells after fusion with human embryonic stem cells, Science, 2005, vol. 309, pp. 1369–1373.
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Translated by N. Berestetska
