Tumor Suppresser Gene p 53 Expression in Premalignant Lesions and Gastric Carcinoma-Prognostic Value

Th e aim of the study was to verify the presence of mutated tumor suppresser gene p in intestinal mucosa with histologically confi rmed premalignant lesions and gastric carcinoma, and assess its prognostic value. Th e paper presents prospective study that included  patients with gastric adeno-carcinoma of intestinal type that were treated at Gastroenterohepatology Clinic, and  patients with histologically confi rmed chronic atrophic H. pylori positive gastritis. In the mucosa biopsy samples, we analyzed presence, frequency and severity of infl ammatory-regenerative, metaplastic and dysplastic changes. We typed intestinal metaplasia immunohistochemically and confi rmed the presence of p onco-protein in antigen positive gastric carcinoma cells, and evaluated its prognostic value. Our results suggest that H. pylori acts as an initiator of infl ammatory processes in gastric mucosa, which are followed by emergence of precancerous lesions. p is expressed late in carcinogenesis () and as such, may be considered as an indicator of transformation of premalignant into malignant lesion.

Tumor Suppresser Gene p53 Expression in Premalignant Lesions and Gastric Carcinoma -Prognostic Value Zora Vukobrat-Bijedić¹*, Svjetlana Radović², Azra Husić-Selimović¹, Srđan Gornjaković¹ Introduction Molecular dissection of gastric carcinoma development revealed involvement of numerous genetic alterations ().Genetic instability of tumor suppresser genes inactivation and telomerase reactivation are responsible in the early stages of gastric carcinogenesis, while oncologene activation enhances growth factors and cytokine expression thus leading towards gastric carcinoma.Genetic instability stimulates gastric carcinogenesis in several ways ().Somatic mutations in microsatellite sequences caused by DNA replication errors may present the source of genetic instability.Chromosome instability is caused by the replication of errors in telomere reductions coupled with telomerase reactivation.Normally, in the absence of telomerase activity, telomeres are progressively reduced with each somatic cell division and aging.Th e reduction may function as a mitotic clock used by cells to calculate divisions leading towards replicative cell maturity.Telomerase activity strongly correlates with the progression of malignancy (,).Inactivation of multiple tumor suppresser genes caused by either mutation or deletion is the most frequently observed genetic abnormality in gastric carcinoma.Anomalies in p, APC, MCC, DCC genes were found in - tumors.Mutations in p are the most frequent genetic abnormalities in human carcinoma.p acts as a DNA linking factor and transcription regulator thus supporting genetic control over initiation of S phase of cell division.It also has an important role in apoptosis as well as fi nal cell differentiation.In  it was discovered as a cell protein, labeled viral oncoprotein, large tumor antigen (T-ag) in monkey cells transformed with SV -Simian virus.It was not identifi ed in normal cells, while its levels signifi cantly increase in transformed tumor cell lines ().Accumulation of mutations at p locus occurs in the loss of alleles -loss of heterozygocity (LOH).Th e loss was recorded in the later events and the progression into carcinoma.In well diff erentiated gastric carcinoma coupled with chronic atrophic gastritis, mutations most frequently occur in A-T pairs with high incidence of transmission.p also correlates with aneuploidia, depth of tumor invasion and uncertain clinical outcome ().

Subjects and Methods
Th e paper presents prospective study of the potential for gastric adenocarcinoma development, which included three viewpoints: clinical, patho-histological and microbiological.Th e study included  patients with gastric carcinoma that were treated at the Gastroenterohepatology Clinic.All the patients were subjected to endoscopic examination and biopsy was performed in antropyloric region, lesser curvature and corpus.Carcinoma was histologically verifi ed.We also analyzed sample of mucosa - cm removed from the border of tumor lesion.Biopsy samples were conserved in   buff ered neutral formalin, paraffi n embedded and sliced with microtome into  μm thick sections.Th e sections were stained with Heamatoxylin and Eosin (HE staining), immunohistochemically for p antibodies.Based on well-defi ned histological criteria we graded infl ammatory-regenerative changes as chronic superfi cial gastritis or chronic atrophic gastritis of the degree I, II and III.Th e degree of activity was defined either as active or dormant phase based on leukocyte infi ltration.Using the standing criteria for simple separation of inflammatory-regenerative from dysplastic changes we defi ned epithelial dysplasia as slight, moderately severe and severe.Classification into one of the groups was conducted based on the analysis of  criteria that are visually graded -.Following the assessment the slides were prepared for immunohistochemical analysis if IM and assigned into one of the three possible categories.Th e categorization was based on detailed analysis of mucines and morphological changes in epithelia.The analysis included degree of expansion and type of IM.Antibody monoclonal mouse anti human p protein (code No M, clone Do-, lot  by Dako) was used in p visualization.p was targeted in infl ammatory-regenerative changes in gastric carcinoma, various stages of dysplastic changes, in IM, and in carcinoma.Immunohistochemical status of p protein recognizes grade:  -no antigen positive cells,  -antigen present in less than   cells,  --  of antigen positive cells, and  --  positive cells.Intensity of p coloration was graded as:  -pale yellow,  -darker-yellow,  -brown.According to the location of p positive cells we marked that they take up /, / or / (the total) of the crypt length.Our intent was to determine correlation between the expression of the studied antigen and the degree of morphological change, and to explore prognostic value of p in pre-cancerous lesions in gastric mucosa as well as in gastric carcinoma.

Results
Immunohistochemical staining confi rmed presence of p tumor suppresser proteina in  subjects with gastric carcinoma.p was found in more than   cells in  subjects while in  subjects it was present in less than 

Discussion
Tumor suppresser gene p is located in short hand of chromosome  (p .).It codes for nuclear phosphoprotein of  kD, which acts in cell cycle control.Normal protein coded by this gene labeled wild-type supports normal cell phenotype.A part of its function pertains to the control of DNA damage and unplanned DNA synthesis, while the ultimate eff ect of p (initiation of apoptosis) would depend on cell type and the extent of the detected DNA damage ().Protein p acts as growth protein.It acts at G/S restriction point of cell cycle by controlling the damages on DNA.In untransformed cells p protein level is very low with half-life of - minutes.It appears that the levels of p mRNA expression correlate with cells proliferation degree.Just before S phase p mRNA level increases - times, before it reverts to its normal low level in between cell divisions.It reaches its peak in organogenesis.In apoptosis p levels are also quite elevated ().Intracellular levels of p increase - times as the cell cycle progresses in order to reach its maximum in the late G phase, just before S phase.Th us, the progression of cell cycle towards the succeeding S phase is blocked, and the time necessary for DNA repair provided.Hence, p protein modulates the genes' transcription process, monitors DNA synthesis and, when needed, induces apoptosis in G phase during the arrested cell growth (,).Mutations in p gene result from various envi-ronmental factors.When one allele is lost and the other sustains point mutations, the gene loses suppressers role which results in genetic instability in cells.In this way, tumor suppresser gene loses its role and, in certain cases gains ability to promote cell proliferation ().Th is ability extends life time of inadequately controlled cells and they may become susceptible to additional genetic alteration, which lead them into malignant transformation ().Semi-quantitative method of p antigen positive cells detection showed p presence in  () subjects with gastric carcinoma. out of  (,) subjects were classifi ed grade II, which means antigen presence in more than  cells.Coloration intensity was pale yellow and was found in cells along the total crypt length.In  out of  (,) subjects it was classifi ed grade I and was found in less than  cells.Coloration was pale yellow and was found in cells along the total crypt length.In the control group, antigen was positive in only one () subject.That patient was diagnosed with atrophic gastritis grade II in active phase associated with moderately severe epithelial dysplasia as well as intestinal metaplasia.More than  cells were antigen positive and the case was classified as grade II.Coloration was dark yellow (grade III) and was found in the cells along the total crypt length.Our research shows alteration of p in  subjects with intestinal type gastric carcinoma.Among the cases of pre-malignant lesions, we identifi ed p alteration in only one case of moderately severe dysplasia and chronic atrophic gastritis of grade II.Sasaki et al. states that p expression closely correlates with the progression of changes into carcinoma, and its levels are lower in early carcinogenesis and pre-cancerous lesions.Considering concurrence of the results we may accept this statement.Our results, which show p alteration in  patients with gastric carcinoma, concur with previous fi ndings.

Conclusion
Our results show expression of p protein as a late event in gastric carcinoma as well as chronic atrophic gastritis grade II in active phase with moderately severe dysplasia.Th is event is correlated with moderately severe dysplasia and gastric carcinoma and indicates progression of dysplasia as a pre-cancerous lesion into malignancy.Th erefore, p protein cannot be used as an initial screening marker.However, it may be useful in the assessment of moderately severe and severe epithelial dysplasia reaction and its evolvement from pre-malignant lesion into gastric carcinoma.
BASIC MEDICAL SCIENCES 2007; 7 (1): 7-10 ZORA VUKOBRATBIJEDIĆ ET AL.: TUMOR SUPPRESSER GENE p53 EXPRESSION IN PREMALIGNANT LESIONS AND GASTRIC CARCINOMA -PROGNOSTIC VALUEcells.In all subjects p was found in cells along the total crypt length.ONCO-PROTEIN P EXPRESSION In our control group of subjects diagnosed with H. pylori positive chronic atrophic gastritis p was detected in one subject in more than   cells taking up / of a crypt.