mutational status of HER-2, C-MYC, H-RAS and TP53 genes and methylation status of P14 and P16 genes

Uvod: Pleomorfni adenom (PA) je spororastući benigni tumor koji se obično javlja u petoj deceniji života i mnogo se češće razvija kod žena. Najčešće pogađa velike pljuvačne žlezde, a čak u preko 70% slučajeva lokalizovan je u zaušnoj (parotidnoj) pljuvačnoj žlezdi. Uglavnom se uspešno leči hirurški, ali je ipak to jedini benigni tumor pljuvačnih žlezda koji pokazuje sklonost ka malignoj transformaciji u karcinom u pleomorfnom adenomu (CXPA). U prvim, citogenetičkim studijama, analizom kariotipa pokazano je u najvećem broju slučajeva prisustvo karakteristične hromozomske translokacije koja uključuje PLAG1 ili HMGA2 gene. Međutim, kod ovih, kao i kod tumora sa neizmenjenim kariotipom, u nastanak i progresiju tumora su uključeni i drugi mehanizmi. Narušena mitogena signalna kaskada, koja u normalnim fiziološkim uslovima započinje na receptorima za faktore rasta (HER-2 npr.), jedan je od ključnih mehanizama odgovornih za rast tumorske mase. Aberantni mitogeni signal može takođe biti generisan i endogeno kao rezultat mutacione aktivacije onko-proteina iz familije Ras. Jedan od mogućih mehanizama prenosa mitogenog signala realizuje se preko ciklin zavisnih kinaza, čiji je snažni regulator protein p16. Mitogeni signal prenosi se dalje u nukleus gde, između ostalih c-myc, stimuliše transkripciju gena uključenih u ćelijsku deobu. Ukoliko se u genomu dogode oštećenja, jedan od ključnih tumor supresorskih gena – TP53, zaustaviće ćelijski ciklus i/ili ćeliju uvesti u apoptozu. Jedan od pozitivnih regulatora p53 funkcije je p14, koji vezuje Mdm2, čime onemogućava formiranje Mdm2/p53 inhibitornog kompleksa. Na funkciju i stabilnost ovih proteina mogu da utiču različite mutacije, a na nivo ekspresije različiti epigenetički mehanizmi, u prvom redu metilacija promotora. Cilj: Cilj ove doktorske disertacije je ispitivanje povezanosti mutacija u genima HER-2, c-myc, H-ras i TP53, kao i metilacionog statusa promotora p16 i p14 gena sa nastankom, progresijom i malignom transformacijom pleomorfnih adenoma parotidnih pljuvačnih žlezda...Introduction: Pleomorphic adenoma (PA) is a slow-growing benign tumor that usually develops in the fourth or fifth decade of life, more often in women. It is mostly found in the major salivary glands, and over 70% are localized in the parotid salivary gland. Surgical removal is usually successful and the patient is cured. However it is the only benign salivary gland tumor with a tendency toward malignant transformation into carcinoma ex pleomorphic adenoma (CXPA). PAs have been extensively studied from a cytogenetic point of view and indeed the presence of specific chromosomal translocations involving PLAG1 or HMGA2 gene loci have been reported in many instances. There has been much less studies on other molecular events potentially involved in tumor development and progression. Disruption of the mitotic signaling cascade is one of the key mechanisms responsible for tumor growth. The aberrant signaling does not necessarily start from growth factors and their receptors. It may be generated endogenously, for instance from the mutated Ras protein. One possible transmission of the mitogen signal is through cyclin dependent kinases that are strongly regulated by the p16 protein. The signal is transmitted to the nucleus where c-myc, among others, stimulates the transcription of the cell cycle regulatory genes. Accumulation of DNA damage leads to cell cycle arrest and programmed cell death under the control of p53. p14 interactions with Mdm2 initiate p53-dependant cell cycle arrest and apoptosis. Lack of p14 results in higher levels of Mdm2 and p53 inhibition. Expression, function and stability of these cancer gene products may be considerably affected by mutations, and epimutations – primarly gene promoter methylation. Aim: The aim of this doctoral dissertation was to assess the frequency and thus the relevance of mutations in HER-2, c-myc, H-ras and TP53 genes, as well as methylation status of p16 and p14 gene promoters in the development, progression and malignant transformation of pleomorphic adenoma of parotid salivary glands..

Reduced light exposure mitigates streptozotocin-induced vascular changes and gliosis in diabetic retina by an anti-inflammatory effect and increased retinal cholesterol turnover

Diabetic retinopathy is not cured efficiently and changes of lifestyle measures may delay early retinal injury indiabetes. The aim of our study was to investigate the effects of reduced daily light exposure on retinal vascularchanges in streptozotocin (STZ)-induced model of DM with emphasis on inflammation, Aqp4 expression, visualcycle and cholesterol metabolism-related gene expression in rat retina and RPE. Male Wistar rats were dividedinto the following groups: 1. control; 2. diabetic group (DM) treated with streptozotocin (100 mg/kg); 3. groupexposed to light/dark cycle 6/18 h (6/18); 4. diabetic group exposed to light/dark cycle 6/18 h (DM+6/18).Retinal vascular abnormalities were estimated based on lectin staining, while the expression of genes involved inthe visual cycle, cholesterol metabolism, and inflammation was determined by qRT-PCR. Reduced light exposurealleviated vasculopathy, gliosis and the expression of IL-1 and TNF-α in the retina with increased perivascularAqp4 expression. The expression of genes involved in visual cycle and cholesterol metabolism was significantlyup-regulated in RPE in DM+6/18 vs. DM group. In the retina only the expression of APOE was significantlyhigher in DM+6/18 vs. DM group. Reduced light exposure mitigates vascular changes and gliosis in DM via itsanti-inflammatory effect, increased retinal cholesterol turnover and perivascular Aqp4 expression