13th balkan biochemical biophysical days & meeting on metabolic disorders’ programme & abstracts

ORAL PRESENTATION 4

EXPRESSION OF ESTROGEN RECEPTOR β IN BREAST CARCINOMAS

To determine whether estrogen receptor beta (ERβ) expression is associated with estrogen receptor α (ERα) and progesterone receptor (PR) status we compared its expression with levels of ERα and PR. Thirty two samples of breast carcinomas were examined for ERα, ERβ and PR. Levels of ERα and PR were measured by conventional biochemical techniques. Expression of ERβ mRNA was determined by RT-PCR. Briefly, RNA from tumor samples was isolated according the method of Chomczynski and Sacchi (1987); total RNA was reverse transcribed with oligo dT primer; PCR was performed using the specific primers positioned in exons 4 and 6. PCR (254 bp product) were analyzed on polyacrilamide gel electrophoresis. In 9 carcinomas ERβ mRNA was not detected, and 23 carcinomas (71,8%) were positive for ERβ expression. Level of expression was quantified densitometricaly and relatively ranged from 1 to 3. Results conform positive correlation between ERα and PR status. Analysis of correlation between ERα and ERβ expression as wel as ERβ expression and PR shows:

 absence of any correlation between ERα and ERβ;

 statistical significant (hi-square test, p=0,03) inverse correlation between ERβ and PR status.

Therefore, expression of ERα and ERβ are independent one from the other, but increased expression of ERβ is connected with decreasing of ER functionalities represented through decreased PR expression.

OCTOBER 15 , 2003 - WEDNESDAY

HALL A

LECTURE 1

THE ROLES OF CYP1A AND CYP2E IN CHEMICAL CARCINOGENESIS

It has been established that approximately 80% of human cancers are attributable to environmental agents and about 70% of the human cancers are caused by chemical compounds. The majority of the organic carcinogens is not reactive as such but must undergo enzymatic reactions to form electrophilic species. The first step in biotransformation is usually the oxidative step, catalyzed by microsomal cytochrome P450 (CYP) dependent monooxygenases. Upto now more than 2000 P450 genes are sequenced and 265 P450 families have been identified and 18 of them are found in humans. Although other P450s and other mechanisms may activate the carcinogens, CYP1A enzymes are the most significant and 90% of the known carcinogens are metabolically activated by CYP1A to the proximate and ultimate carcinogens.

Persistent organic pollutions such as polyaromatic hydrocarbons (PAHs), dioxins, polychlorinated biphenyls (PCBs) specifically induce CYP1A1 through Ah receptor mediated mechanism. At the same time, CYP1A1 mostly converts PAH- and PCB-type precarcinogens to their carcinogenic epoxides or other oxygenated metabolites. These epoxides, in turn bind DNA covalently forming DNA-adducts which may cause cancer in the years to come. Thus induction of CYP1A1 is used to measure both exposure and resulting toxic carcinogenic effects of these types of organic pollutants. (Arınç, E., Şen, A., Bozcaarmutlu, A.: Pure & Appl. Chem. 72, 985-994, 2000). Greater CYP1A1 induction may result in high levels of activated carcinogens, and consequently to higher degree of persistent DNA-adduct formation or to enhanced oxidative DNA damage.

Good correlation between smoking and lung cancer is established. Individuals with a high inducible phenotype CYP1A1 show high risk of lung cancer. Gene-environment interactions are more pronounced at lower levels of cigarette exposure in which the susceptibility to lung cancer increased in the case of patients with either CYP1A1 mutated alleles and GSTM1 null gene.

On the other hand, CYP1A2 activates many arylamines and amides present in food and cigarette smoking. CYP1A2 exhibits polymorphisms and inducibility. Aflatoxin B1 (AFB1) must be activated to a reactive epoxide prior to exerting carcinogenic effects. CYP1A2 is the predominant human CYP enzyme involved in the activation of AFB1. In the presence of hepatitis B virus (HBV), a relative risk for liver cancer upon exposure of AFB1 increased 30-fold.

CYP2E1 is also involved in chemical carcinogenesis, both in the metabolic activation of small molecular weight organic chemicals and in the generation of ROS. N-Nitrosodimethylamine (NDMA) is a procarcinogen that is activated by CYP2E1 dependent NDMA N-demethylase. NDMA is known to be carcinogenic in liver, kidney and lung. Pyridine, constituent of tobacco and tobacco smoke, has tumor-promoting and teratogenic activities. Following in vitro pyridine treatment of rabbits, NDMA N-demethylase activity of both liver and lung is stimulated by 6.9- and 5.2-fold, respectively indicating that pyridine increases the metabolic activation of NDMA and in turn, may potentiate formation of liver and lung cancers significantly (Arınç, E., Adalı, O., Gençker-Özkan, A. M.: Arch. Toxicol., 34, 329-334, 2000). In addition, experiments carried out in our laboratory demonstrated that in the experimentally induced diabetic rabbits, both CYP2E1 and NDMA N-demethylase activity have been increased about two-fold in kidney and liver of rabbits. Therefore, it is expected that the incidence of tumor formation due to exposure to NDMA will be more pronounced in diabetic state because of CYP2E1 induction.

LECTURE 2

Z. Lygerou

For cells to maintain genome integrity, S-phase must alternate with mitosis at every cell cycle. Licensing of DNA for replication takes place only after completion of mitosis and ensures once per cell cycle replication.

Cdt1, a central licensing factor conserved across evolution, is tightly controlled in human cells, so that it is present only in the G1 phase, when licensing is legitimate. Over-expression of Cdt1 leads to genomic instability. We have shown that ubiquitin dependent proteolysis ensures that Cdt1 is destroyed as soon as S-phase starts. Cdt1 is undetectable in cells accumulating cyclin A, suggesting that phosphorylation by cdk2/cdc2 – cyclin A complexes targets Cdt1 for degradation. Geminin, a molecular inhibitor of Cdt1, accumulates in cells which do not contain Cdt1, suggesting that Geminin’s inhibitory function over Cdt1 might be redundant for the majority of the mammalian cell cycle. When primary cells exit the cell cycle to G0, both Cdt1 and Geminin protein and mRNA levels are decreased. In contrast, cancer cells over-express both these proteins.

In order to study the localization, dynamics and interactions of licensing factors in the living cell, we constructed fusions of Cdt1 and Geminin to Green Fluorescent Protein (GFP), Cyan Fluorescent Protein (CFP) and Yellow Fluorescent Protein (YFP) and characterized their behavior after transfection into mammalian cultured cells. Time-lapse microscopy and fluorescence lifetime imaging microscopy (FLIM) allows us to identify the location and timing of protein-protein and protein-DNA interactions as cells progress through the cell cycle.

the effect of InsulIn level on steroId receptors

Steroid receptors are key molecules in steroid hormone action. It has been proposed that a nonsteroidal hormone such as insulin may exert an influence on steroid receptors and alter the cell responses to steroid hormone action. Diabetes mellitus is very frequent metabolic disorder that also has implications on steroid hormone action. The main goal of the presented study was to elucidate the effects of insulin treatment and insulin deficiency on steroid receptors.

The experiments were performed on 3-months-old male Wistar rats. Intact and streptozotocin-pretreated rats (60mg/kg, 7 days before hormone treatment) were injected with insulin (200g/kg) or hormonal solvent 3h before sacrifice. Protein contents of estrogen (ER), progesterone (PR), androgen (AR) and glucocorticoid receptors (GR) in liver were analyzed by immunoblotting method using appropriate specific antibodies.

The obtained results point out that streptozotocin did not cause significant changes in protein content of any kind of analyzed steroid receptors. Insulin injection significantly decreased the AR protein content in intact rats, while there were no changes in protein content of other three analyzed steroid receptors in rat liver. Insulin given to insulin-deficient animals did not cause changes in steroid receptor content compare to intact rats. However, in comparison to diabetic rats there was the significant decrease in PR content, whereas protein contents of other steroid receptors were unchanged.

The presented data indicate that insulin treatment and insulin deficient state did not alter the protein content of ER and GR in rat liver. The levels of PR and AR were not influenced by streptozotocin treatment, while, depend on endogenous level of insulin, hormone treatment decreased their content.

The research was supported by grant from Ministry of Science, Technology and Development, Republic of Serbia: ”Insulin and steroid receptors as mediators of hormone actions and their cross-talk under physiological and nonphysiological conditions”. (Grant number 1999).

ORAL PRESENTATION 2

Rıza Köksal ÖZGÜL¹, Hakan DURUKAN², Ayşe TURAN³, Cihan ÖNER¹, Ay ÖĞÜŞ¹, Debora B. FARBER⁴

Stargardt disease (STGD), the most prevalent autosomal recessively inherited macular dystrophy is characterized by severe reduction of central visual acuity and leading to partial or complete blindness due to photoreceptor degeneration. The responsible gene for STGD encodes a ATP-binding cassette transporter, ABCA4 which has been shown to be involved in retinoid transport in the retina. Molecular characterization of the ABCA4 gene led to the identification of many mutations in Stargardt disease, cone-rod dystrophy (CRD), autosomal recessive retinitis pigmentosa (arRP) and age- related macular degeneration (AMD). By the screening of the ABCA4 gene in various types of inherited retinal degenerations, more than two hundred disease-associated mutations reported in different populations.

In this study, all fifty exons and flanking intronic sequences of the ABCA4 gene were screened by Single Strand Conformation Polymorphism (SSCP) in a cohort of 40 Turkish patients with STGD and arRP. After SSCP analysis, DNA fragments showing different migration patterns were sequenced.

Our results revealed the presence of three novel mutations (C54G, T829M, IVS19-6C>A), two mutations previously reported (R212C, IVS28+4C>T) and several polymorphic changes in the ABCA4 gene among Turkish patients affected with Stargardt and arRP.

This is the first report on the mutation profile of the ABCA4 gene in Turkish patients. Further studies will be helpful in understanding of complex genotype- phenotype relationship in ABCA4 gene in our population.

ORAL PRESENTATION 3

Meltem MÜFTÜOĞLU¹, Özlem DALMIZRAK¹, Bülent ELİBOL², Ayşe ERCAN¹, Gülnihal KULAKSIZ¹, Hamdi ÖĞÜŞ¹, Turgay DALKARA² and Nazmi ÖZER¹