Monika Puzianowska-Kuznicka

Expression of thyroid hormone receptors is disturbed in human renal clear cell carcinoma

Human renal clear cell carcinoma (RCCC) accounts for up to 2% of human cancers. To find out if thyroid hormone (T3) and its receptors (TRs) play a role in tumorigenesis of RCCC, the expression of TRs was evaluated on mRNA and protein level. It was found that TRalpha (both alpha1 and alpha2) mRNA amount was significantly decreased in tumors while compared with healthy kidney tissue, and this decrease was deepest in G1 (well differentiated) RCCCs. In contrast, TRalpha1 protein was 1.6x overexpressed in tumors. TRbeta1 mRNA amount was overexpressed in 30% and significantly decreased in 70% of examined tumors. On the protein level, TRbeta1 amount was 1.7x lower in tumors than in healthy controls.

p53-dependent repression of the human MCL-1 gene encoding an anti-apoptotic member of the BCL-2 family: the role of Sp1 and of basic transcription factor binding sites in the MCL-1 promoter.

Abstract p53 regulates transcription of one anti-apoptotic and four pro-apoptotic members of the BCL-2 family, but nothing is known about the regulation of MCL-1, another anti-apoptotic member of this family, by p53. Confocal microscopic analysis of COS1, HEK 293 and HeLa cells transfected with a p53 expression plasmid demonstrated a decrease in the signal of endogenous MCL-1 compared to neighboring non-transfected cells. Transcription regulation assays showed that the 1826 bp human MCL-1 promoter fragment was repressed up to 30-fold by wild-type p53 in a dose-dependent manner. As shown by electrophoretic mobility shift assays, Sp1 binding to the sites located in the -295 to +16 MCL-1 promoter fragment was decreased in the presence of p53. However, the MCL-1 promoter devoid of all Sp1 binding sites was still repressed by p53, albeit 2-fold weaker than the wild-type promoter. Overexpression of Sp1 reduced p53-dependent repression of the MCL-1 promoter only up to 2.2-fold. Transcription regulation assays performed with MCL-1 promoter deletion mutants showed that most of the p53 inhibitory effect was mediated by the -41 to +16 bp promoter fragment containing binding sites only for TATA-binding protein and other basal transcription factors. We propose a novel, promoter-based mechanism by which p53 down-regulates expression of the anti-apoptotic MCL-1 protein.

The ER and ageing II: calcium homeostasis.

Increase in intracellular Ca(2+) concentration occurs by Ca(2+) influx through the plasma membrane and by Ca(2+) release from intracellular stores. The ER is the most important Ca(2+) store. Its stress, characterized by the impairment of Ca(2+) homeostasis and by the accumulation of misfolded proteins, can be induced by different factors. In turn, it induces defense mechanisms such as unfolded protein response, and when it is severe and prolonged, activation of the apoptotic pathway. Damage to the ER, impairment of its function, and a decreased level of its Ca(2+)-handling proteins might all play a role in physiological ageing by handicapping the ER stress response. Thus, healthy ageing is accompanied by subtle alterations of Ca(2+) homeostasis and signaling, including alterations in the ER Ca(2+) load and release. The expression and/or function of ryanodine receptors, IP3 receptors, and SERCA Ca(2+) pumps located in the ER membrane, and Ca(2+)-binding proteins within ER lumen all seem to be affected in aged cells. Data are presented on age-dependent, tissue-specific changes in ER-related Ca(2+) homeostasis in skeletal, cardiac and smooth muscles, as well as in the nervous and immune systems. Disturbances of Ca(2+) homeostasis and of signaling are potential targets for intervention in aged humans.

A novel homozygous p.Arg527Leu LMNA mutation in two unrelated Egyptian families causes overlapping mandibuloacral dysplasia and progeria syndrome.

Mandibuloacral dysplasia (MAD) is a rare disease resulting from a mutation of LMNA gene encoding lamins A and C. The most common mutation associated with this disease is a homozygous arginine 527 replacement by histidine. Three female patients originating from two unrelated families from Northeast Egypt were examined. Their growth was retarded; they had microcephaly, widened cranial sutures, prominent eyes and cheeks, micrognathia, dental crowding, hypoplastic mandible, acro-osteolysis of distal phalanges, and joint contractures. In addition, they presented some progeroid features, such as pinched nose, premature loss of teeth, loss of hair, scleroderma-like skin atrophy, spine rigidity, and waddling gait. The clinical presentation of the disease varied between the patient originating from Family 1 and patients from Family 2, suggesting that unknown, possibly epigenetic factors, modify the course of the disease. The first symptoms of the disease appeared at the age of 2.5 (a girl from Family 1), 5, and 3 years (girls from Family 2). All patients had the same, novel homozygous c.1580G>T LMNA mutation, resulting in the replacement of arginine 527 by leucine. Computational predictions of such substitution effects suggested that it might alter protein stability and increase the tendency for protein aggregation, and as a result, might influence its interaction with other proteins. In addition, restriction fragment-length polymorphism analysis performed in 178 unrelated individuals showed that up to 1.12% of inhabitants of Northeast Egypt might be heterozygous carriers of this mutation, suggesting the presence of a founder effect in this area.

Interleukins 6 and 15 Levels Are Higher in Subcutaneous Adipose Tissue, but Obesity Is Associated with Their Increased Content in Visceral Fat Depots

Excess adiposity is associated with chronic inflammation, which takes part in the development of obesity-related complications. The aim of this study was to establish whether subcutaneous (SAT) or visceral (VAT) adipose tissue plays a major role in synthesis of pro-inflammatory cytokines. Concentrations of interleukins (IL): 1β, 6, 8 and 15 were measured at the protein level by an ELISA-based method and on the mRNA level by real-time PCR in VAT and SAT samples obtained from 49 obese (BMI > 40 kg/m2) and 16 normal-weight (BMI 20–24.9 kg/m2) controls. IL-6 and IL-15 protein concentrations were higher in SAT than in VAT for both obese (p = 0.003 and p < 0.0001, respectively) and control individuals (p = 0.004 and p = 0.001, respectively), while for IL-1β this was observed only in obese subjects (p = 0.047). What characterized obese individuals was the higher expression of IL-6 and IL-15 at the protein level in VAT compared to normal-weight controls (p = 0.047 and p = 0.016, respectively). Additionally, obese individuals with metabolic syndrome had higher IL-1β levels in VAT than did obese individuals without this syndrome (p = 0.003). In conclusion, concentrations of some pro-inflammatory cytokines were higher in SAT than in VAT, but it was the increased pro-inflammatory activity of VAT that was associated with obesity and metabolic syndrome.

Cognitive Performance and Functional Status Are the Major Factors Predicting Survival of Centenarians in Poland

BACKGROUND Clinical and biochemical predictors of extreme longevity would be useful in geriatric practice but have still not been clearly defined. METHODS To identify the best nongenetic predictors of survival in centenarians, we examined 340 individuals aged 100+ years. A detailed questionnaire was completed, and comprehensive geriatric assessment and blood analyses were performed. Survival of study participants was checked annually over the period of 10 years. RESULTS In the univariate Cox proportional hazards model, a longer survival of centenarians was correlated with a higher adjusted Mini-Mental State Examination (MMSE(adj)) score (p < .000001), higher Activities of Daily Living (ADL) and adjusted Instrumental Activities of Daily Living (IADL(adj)) scores (p < .000001 and p = .00008, respectively), and younger age at the time of testing (p = .005). Blood pressure, lipid profile, and C-reactive protein and hemoglobin concentrations were not associated with survival. Multivariate analysis including age, sex, and the MMSE(adj) and ADL scores showed that both MMSE(adj) and ADL predicted survival (HR = 0.978 per each MMSE(adj) point, 95% CI: 0.964-0.993, p = .004; HR = 0.900 per each ADL point, 95% CI: 0.842-0.962, p = .002, respectively). In multivariate analysis with the ADL score substituted by the IADL(adj) score, the only predictor of survival was MMSE(adj) (HR = 0.973 per each MMSE(adj) point, 95% CI: 0.958-0.988, p = .0006). CONCLUSIONS Cognitive and functional performances are predictors of survival in centenarians.

Triiodothyronine utilizes phosphatidylinositol 3-kinase pathway to activate anti-apoptotic myeloid cell leukemia-1

Triiodothyronine (T(3)) regulates apoptosis in cells according to their developmental stage, cell type, and pathophysiological state. The molecular mechanisms of this regulation, however, have been largely unknown. In this work, we show that the expression of the myeloid cell leukemia-1 (MCL-1) protein, an anti-apoptotic member of B-cell lymphoma-2 (BCL-2) family, increases in thyroid hormone receptor-expressing human kidney-2 (HK2) cells upon 6-h incubation in 100 nM T(3); we also describe the molecular mechanisms leading to this phenomenon. Transcription regulation assays performed in human embryonic kidney (HEK) 293 cells show that 100 nM T(3) increases transcription from the MCL-1 promoter twofold in the presence of thyroid hormone receptor beta1, but not of its alpha1 isoform. However, this increase is not a result of direct activation via the thyroid hormone-response element, TRE-DR4, located at the -998 to -983 position in this promoter; furthermore, the presence of 9-cis-retinoic acid receptor is not required. The promoters activation is abolished in the presence of phosphatidylinositol 3-kinase (PI3-K) inhibitor, wortmannin. The -295 to -107 promoter fragment contains all sequences involved in T(3)-dependent activation of the MCL-1 promoter, and cAMP-responsive element located at the -262 to -255 position is a major mediator in this process. Therefore, MCL-1 expression is activated by T(3), which increases its promoter activity by a non-genomic mechanism using the PI3-K signal transduction pathway. We propose that this is another mechanism by which T(3) regulates apoptosis.

The —351A/G polymorphism of ESR1 is associated with risk of myocardial infarction but not with extreme longevity

BACKGROUND Women live longer than men. Some possible reasons for this advantage are the protection provided by high concentrations of 17β-estradiol (E2) during the premenopausal period and polymorphic variants of the estrogen receptors (ERs), which mediate various cardiovascular functions of E2. METHODS We tested whether the -351A/G and -397T/C polymorphisms of the ERα-encoding ESR1 were associated with extreme longevity. The genomic DNA of 148 centenarians (C), 414 young controls (Y), and 208 myocardial infarction patients (MI) was analyzed by RFLP-PCR. RESULTS Both polymorphisms were equally distributed in the Y, C, and in centenarians never diagnosed with MI (HC). In centenarians, none of these polymorphisms was associated with a particular lipid profile. The AA genotype of the -351A/G polymorphism was less frequent in the C, HC and Y groups than in MI patients (p=0.058, p=0.021, and p=0.004, respectively). In MI patients, the GG genotype of the -351A/G polymorphism was associated with significantly lower mean total cholesterol, LDL, and HDL levels compared to the AG (p=0.0194, p=0.0213, and p=0.0367, respectively) and AA genotypes (p=0.0014, p=0.0078, and p=0.0448, respectively). CONCLUSIONS The -351A/G ESR1 polymorphism might be associated with MI, but not with extreme longevity.

Vitamin D Receptor Binding to DNA Is Altered without the Change in Its Expression in Human Renal Clear Cell Cancer

Vitamin D co-regulates cell proliferation, differentiation and apoptosis, the processes that are disturbed in cancer tissues. It acts through the vitamin D nuclear receptor (VDR) that binds to DNA in the regulatory sequences of the target genes. As the kidney is one of the key organs for vitamin D metabolism and action, we analyzed VDR expression and its DNA binding activity in human renal clear cell cancer. 24 tumors, 24 controls that were excised from the opposite pole of the same kidney and 7 controls originating from kidneys without cancer were examined. Independently of tumor grading neither Northern blots nor immunoblotting demonstrated statistically significant differences of the mean VDR mRNA and protein amounts, respectively, in the cancer as compared to both control types. In contrast, the amount of VDR-DNA complexes was lower in 52.2% of the tumors in comparison to their corresponding controls. After normalization against VDR receptor protein amount in 34.8% of the tumors VDR-DNA binding was at least 3–4 times weaker than in the controls. However, the expression of vitamin D-dependent P21 gene on the mRNA level was not decreased in these cancers. It remains to be elucidated if altered VDR function due to its impaired binding to DNA contributes to the process of tumorigenesis, and what potential vitamin D-dependent mechanisms are involved in this process.

Association between vitamin D concentration and levels of sex hormones in an elderly Polish population with different genotypes of VDR polymorphisms (rs10735810, rs1544410, rs7975232, rs731236)

BACKGROUND Vitamin D co-regulates the synthesis of sex hormones in part by interaction with its nuclear receptor. The aim of this study was to determine whether there is an association of vitamin D concentration vs the level of sex hormones in elderly Polish individuals with different genotypes of the vitamin D receptor (VDR) gene. MATERIALS AND METHODS Rs10735810, rs1544410, rs7975232, and rs731236 polymorphisms of VDR, the serum sex hormone level, free estrogen index (FEI) and free androgen index (FAI) as well as vitamin D, were evaluated in 766 persons (362 women and 404 men) selected from 5695 Polish population, aged 65-90years from the PolSenior survey. RESULTS We observed that women with GG (rs731236), TT (rs7975232), BB (rs1544410) and FF (rs10735810) genotypes were characterized by a significant correlation between vitamin D vs testosterone concentration and FAI value. We found a significant correlation between testosterone level and FAI vs vitamin D concentration in men with heterozygote AG in the rs731236 polymorphism and in the GG (rs7975232), the BB (rs1544410), and the Ff (rs10735810) genotypes. CONCLUSION In elderly selected Polish population with different genotypes of VDR polymorphisms, a statistically significant relationship between vitamin D concentration vs testosterone level was observed.