Epigenetic silencing of endothelin-3 in colorectal cancer.

Endothelins are expressed in a variety of human tissue and are involved in the processes as proliferation, migration and differentiation. The signal transduction pathway is a result of the endothelin-1-3 (ET1-3) binding to their receptors (ETAR, ETBR). ET-3 is a new candidate tumour suppressor gene, which is often downregulated or silenced in human cancer.The aim of the study was to examine DNA methylation of ET-3 genes in colorectal cancer (CRC) tissue samples in relation to the clinical stage (CS) of cancer. The paper is a continuation of our previously published results, which showed a four-fold transcriptional silencing of the ET-3 gene in the samples of colorectal cancer in comparison to normal tissues.A total of 66 paired CRC and normal (surgical margin) tissue samples were used in the study. The tumour tissues were collected from CRC patients in CS I-IV according the 7th edition of UICC TNM Classification of Malignant Tumours (CS I, n = 8; CS II, n = 20; CS III, n = 27; CS IV, n = 11). Assessment of epigenetic silencing of the ET-3 encoding gene was performed in three steps. The silencing of the ET-3 encoding gene was a result from methylation of the promoter sequence using methylation-specific PCR (MS-PCR). Analyses were performed using primers complementary for a CpG island in the first exon of the gene encoding ET-3. An epigenetic silence through methylation of 7.5% (5/66) in comparison to control was observed, including 10% of CS II (2/20), 7% of CS III (2/27) and 9% of CS IV (1/11). The controls and the samples of tumour in CS I showed no epigenetic silencing via methylation. In conclusion, epigenetic silencing of ET-3 in CRC could play a role in the progression than in the induction process. EDN3 would be a future target for epigenetic therapy in colorectal cancer, but further clinical studies are needed.

BCL2 and BAX mRNA concentration profile in fibrillary astrocytoma.

A high level of the BCL2 protein and the lack of apoptosis promoting protein BAX are beginning to be treated as markers of cellular resistance to anti-neoplastic drugs. The object of the study were specimens from stereotactic biopsy of Astrocytoma fibrillare in the central brain area, inaccessible to conventional surgery. The cytological preparations have been evaluated with histopathological and immunohistochemical methods in order to determine the origin of the tumour and assess cell proliferation activity. The molecular analysis conducted in order to determine the sensitivity of the tumour to radio- or chemotherapy included the determination of the number of mRNA BCL2 alpha and beta molecules and of BAX in 1 microg total RNA obtained from microscope slides. A higher expression of BAX than of BCL2-alpha is a prognosis for a positive result of chemo- or radiotherapy. A trace number of mRNA BCL2-beta molecules and a smaller number of mRNA BCL2-alpha molecules than mRNA BAX is a good prognosis for therapy.

Concentration profile of mRNA of kinine B1 and B2 receptors in the evaluation of colon pathologies.

Colon pathologies involve the activation of a number of inflammation mediators: cytokines, prostanoids and kinines. The expression patterns of the genes associated with their activation may provide a very sensitive marker of the physiological condition of the cells. This paper demonstrates certain statistically significant differences (p = 0.008) between the expression patterns of kinine B1 and B2 receptor encoding genes for colitis ulcerosa and a control group. The ratio of kinine B1/B2 concentrations changes significantly, on the average from 1.3 for a tissue assessed as healthy to 6.6 for colitis ulcerosa.

Expression of the histone H3 gene in the evaluation of cellular proliferation in colitis ulcerosa.

A comparison of the number of mRNA molecules of histone H3 in 1 microg total RNA extracted from colon sections sampled during colonoscopy was used to evaluate cellular proliferation activity in the rectum and sigmoid, in normal tissue and in colitis ulcerosa. Samples with similar intensity of the disease were selected for the study. Statistically significant differences between both groups of rectal sections were found in the expression of histone H3 encoding genes. The statistically significant result (p = 0.0485) indicates a more active division of cells in the healthy rectum, with no statistically significant differences in the sigmoid (p=0.9575).

[The assessment of expression of kininogen and bradykinin receptors genes in different vulvar pathologies by QPCR (TaqMan)]. / Ocena ekspresji genów kodujacych kininogen oraz receptory kinin w róznych stanach patologii sromu metoda QPCR (TaqMan).

OBJECTIVES: In different vulvar pathologies inflammatory process and pain are often observed. In these processes, kinins, released from kininogen, play an important role. Their effects are mediated by at least two types of bradykinin receptors--B1 and B2. B1 receptor appears in certain pathological states, B2 is widely distributed in normal tissues. The expression of genes coding kininogen, B1 and B2 receptors can be a very sensitive marker of tissue pathology. DESIGN: In the present study, the analysis of expression of genes coding kininogen, B1 and B2 was performed. The relation between the analysed genes expression and the pathology stage was analysed. MATERIALS AND METHODS: The specimens from condylomata accuminata, vulvar cancer and surgical margin were analysed. The number of DNA and mRNA copies of beta-actine, kininogen, B1 and B2 were examined basing on Q-PCR standard curves for beta-actine by use of Perkin Elmer-kit and the sequence detector ABI PRISM 7700-Taq Man application. RESULTS: In condylomata accuminata the high expression of mRNA of kininogen, B1 and B2 was found, while in vulvar cancer tissue, the expression of analysed genes was low. In the tissue from the tumour center, the lowest kinin genes expression was stated. CONCLUSIONS: The absence of kininogen and B2 mRNA expression characterised vulvar cancer tissue. The profile of expression of kininogen and its receptor genes can be a useful marker in the assessment of vulvar cancer surgical margin.

[Profiles of expression of genes coding kininogen and kinin receptors as a marker of tissue pathology in cervical cancer coexisting with HPV infection]. / Ekspresja genów receptorów dla kinin i kininogenu w raku szyjki macicy wspólistniejacym z infekcja HPV.

Kinins are peptides involved in inflammatory processes, vascular permeability, proliferation and mitogenesis of tumor cells. The majority of kinins actions are mediated through an interaction with cell surface bradykinin receptors BR1 and BR2. Kinins precursor is kininogen (kng). The changes in proteins are initiated by changes in the expression of genes coding these proteins, thus can be a valuable diagnostic markers of malignant processes including cervical carcinoma. The paper presents an analysis of kininogen-kinins receptor genes expression in women treated surgically because of a carcinoma of uterine cervix. Among the studied women in 5 cases previously brachyHDR therapy was applied. In all studied cases HPV 18 infection and in 2 cases a co-infection HPV 16/18 by use of Consensus Primers MY09, MY 11 and type specific primers for HPV 16, 18 was ascertained. In RNA extracts the number of the mRNA copies for kiningen, BR1 and BR2 was assessed using QRT-PCR Taq Man. The higher expression of BR1 than BR2 was marked in the tissue with cancer cells. In the patients after brachytherapy higher expression of BR2 than BR1 mRNA was found. The higher BR1 expression was also shown in iliac lymph nodes in patients with active neoplastic process, opposite to the patients after brachytherapy in whom higher BR2 expression was ascertained. The lack of expression of kng mRNA was found only in 3 specimens. The high expression of kinin receptors especially BR1 in infiltrating carcinoma margin can be a marker of pathology intensity: proliferative potential of neoplasms cells or chronic inflammatory state in the presence of invasive carcinoma.