Krisztián Bácsi

The Candidate Oncogene CYP24A1: A Potential Biomarker for Colorectal Tumorigenesis

The main autocrine/paracrine role of the active metabolite of vitamin D3, 1α,25-dihydroxyvitamin D3 (1,25-D3), is inhibition of cell growth and induction of cell differentiation and/or apoptosis. Synthesis and degradation of the secosteroid occurs not only in the kidney but also in normal tissue or malignant extrarenal tissues such as the colon. Because 25-hydroxyvitamin D3 24-hydroxylase (CYP24A1) is considered to be the main enzyme determining the biological half-life of 1,25-D3, we have examined expression of the CYP24A1 mRNA (by real-time RT-PCR) and protein (by immunohistochemistry) in normal human colon mucosa, colorectal adenomas, and adenocarcinomas in 111 patients. Although 76% of the normal and benign colonic tissue was either completely devoid of or expressed very low levels of CYP24A1, in the majority of the adenocarcinomas (69%), the enzyme was present at high concentrations. A parallel increased expression of the proliferation marker Ki-67 in the same samples suggests that overexpression of CYP24A1 reduced local 1,25-D3 availability, decreasing its antiproliferative effect.

Effects of the lactase 13910 C/T and calcium-sensor receptor A986S G/T gene polymorphisms on the incidence and recurrence of colorectal cancer in Hungarian population.

BackgroundEpidemiological studies suggested the chemopreventive role of higher calcium intake in colorectal carcinogenesis. We examined genetic polymorphisms that might influence calcium metabolism: lactase (LCT) gene 13910 C/T polymorphism causing lactose intolerance and calcium-sensing receptor (CaSR) gene A986S polymorphism as a responsible factor for the altered cellular calcium sensation.Methods538 Hungarian subjects were studied: 278 patients with colorectal cancer and 260 healthy controls. Median follow-up was 17 months. After genotyping, the relationship between LCT 13910 C/T and CaSR A986S polymorphisms as well as tumor incidence/progression was investigated.Resultsin patient with colorectal cancer, a significantly higher LCT CC frequency was associated with increased distant disease recurrence (OR = 4.04; 95% CI = 1.71–9.58; p = 0.006). The disease free survival calculated from distant recurrence was reduced for those with LCT CC genotype (log rank test p = 0.008). In case of CaSR A986S polymorphism, the homozygous SS genotype was more frequent in patients than in controls (OR = 4.01; 95% CI = 1.33–12.07; p = 0.014). The number of LCT C and CaSR S risk alleles were correlated with tumor incidence (p = 0.035). The CCSS genotype combination was found only in patients with CRC (p = 0.033).ConclusionLCT 13910 C/T and CaSR A986S polymorphisms may have an impact on the progression and/or incidence of CRC.

The protective role of bone morphogenetic protein-8 in the glucocorticoid-induced apoptosis on bone cells

One of the side effects associated with glucocorticoid therapy is glucocorticoid-induced bone loss. Glucocorticoids partly detain bone formation via the inhibition of osteoblastic function, however, the exact mechanism of this inhibition remains elusive. In this study, we examined the effect of dexamethasone, an active glucocorticoid analogue, on cell viability and expression of bone remodelling-related genes in primary mouse calvarial and cloned MC3T3-E1 osteoblasts. Using sensitive biochemical assays, we demonstrated the apoptotic effect of dexamethasone on osteoblastic cells. Then, utilizing Taqman probe-based quantitative RT-PCR technology, gene expression profiles of 111 bone metabolism-related genes were determined. As a result of dexamethasone treatment we have detected significant apoptotic cell death, and six genes, including Smad3, type-2 collagen α-1, type-9 collagen α-1, matrix metalloproteinase-2, bone morphogenetic protein-4 and bone morphogenetic protein-8 showed (BMP-8) significant changes in their expression on a time- and concentration-dependent manner. BMP-8, (a novel player in bone-metabolism) exhibited a two orders of magnitude elevation in its mRNA level and highly elevated protein concentration by Western blot in response to dexamethasone treatment. The knockdown of BMP-8 by RNA interference significantly increased dexamethasone-induced cell death, confirming a protective role for BMP-8 in the glucocorticoid-induced apoptosis of osteoblasts. Our results suggest that BMP-8 might be an essential player in bone metabolism, especially in response to glucocorticoids.

CYP3A7*1C Polymorphism, Serum Dehydroepiandrosterone Sulfate Level, and Bone Mineral Density in Postmenopausal Women

The CYP3A7 enzyme metabolizes some steroid hormones, including dehydroepiandrosterone sulfate (DHEAS). The age-related decline of serum DHEAS levels is believed to contribute to osteoporosis. Previously, the CYP3A7*1C polymorphism has been shown to cause a persistent high CYP3A7 enzyme activity, resulting in lower levels of DHEAS in men. We hypothesized that the CYP3A7*1C polymorphism might contribute to bone loss through decreased levels of serum DHEAS in postmenopausal women. Postmenopausal women (n = 319) were divided into two subgroups: 217 with osteoporosis and 102 healthy controls. Genotyping, serum DHEAS measurement, and osteodensitometry of the lumbar spine and femoral neck were carried out in all subjects. Homozygous CYP3A7*1C carriers had significantly lower BMD at the lumbar spine compared to wild types (T score −3.27 ± 1.02 in CYP3A7*1C homozygous mutants vs. −1.35 ± 1.53 in wild types, P = 0.041). This association remained significant after adjustment for menopausal age, serum DHEAS level, alcohol consumption, steroid intake, smoking habits, and previous fractures. No association was found between genotypes and serum DHEAS levels in the total study population or in the subgroups. Serum DHEAS levels correlated positively with bone mineral density at the lumbar spine (r = 0.59, P = 0.042) after correction for age. Our data suggest that the CYP3A7 polymorphism might have an influence on bone mass at the lumbar spine independently of serum DHEAS concentrations.

Effect of Vertebroplasty Filler Materials on Viability and Gene Expression of Human Nucleus Pulposus Cells

Consequences of intradiscal cement leakage—often occurring after vertebral cement augmentation for the treatment of vertebral compression fractures—are still unknown. In this study, we have investigated the influences of vertebroplasty filler materials (polymethylmethacrylate‐, calcium phosphate‐ and calcium sulfate‐based bone cement) on isolated nucleus pulposus cells. Cell viability of cultured human nucleus pulposus cells were measured after treatment with vertebroplasty filler materials. Gene expression profile of selected genes was determined with quantitative real‐time PCR. The widely used polymethylmethacrylate and calcium phosphate cement significantly decreased cell number in a dose‐ and time‐dependent manner while calcium sulfate cement affected cell viability less. Expression of genes involved in matrix metabolism of nucleus pulposus—aggrecan, collagens, small proteoglycans—as well as important transcription factors have also significantly changed due to treatment (e.g., 2.5‐fold decrease in aggrecan expression was determined in cultures due to polymethylmethacrylate treatment). Our results suggest that vertebroplasty filler materials—depending on the type of applied material—can accelerate the degeneration of nucleus pulposus cells resulting in a less flexible disc in case of intradiscal cement leakage. This process may increase the risk of a subsequent new vertebral fracture, the main complication of vertebral augmentation.

Importance of dehydroepiandrosterone and dehydroepiandrosterone sulfate in different diseases

A dehidroepiandroszteron es szulfatalt szarmazeka androgen es osztrogen hormonok előanyaga, a gonadok szexualszteroid termelesehez jarul hozza. A dehidroepiandroszteron-szulfat csak deszulfatalodasa utan kepes reszt venni a folyamatban. A harmadik dekadtol kezdve a szerumszintjuk fokozatosan csokken, idősebb korban a maximalis ertek csupan 10–20%-a. Az alacsonyabb koncentracio kulonboző korallapotokhoz vezethet, mint a csontritkulas, a lipidmetabolizmus romlasa, sziv-errendszeri betegsegek, 2-es tipusu cukorbetegseg. Autoimmun betegsegben, szexualis diszfunkciok eseten szinten alacsonyabb szintet talaltak. Az intrakrinologia az endokrinologia specialis aga, jelentese, hogy a hormonszintezis es hatas ugyanazon szovetben, szekrecio nelkul jon letre. A magasabb helyi androgen- es/vagy osztrogenkoncentracio a hirsutismus, acne, seborrhea, az emlő es prosztatadaganatok patomechanizmusaban jelentős tenyező lehet. A dehidroepiandroszteron-potlas kedvező hatasat lattak posztmenopauzas csontritkulasban, lupus eryt...

Effect of menopause on gene expression pattern in bone tissue of nonosteoporotic women

Objective: Menopausal changes influence the growth, differentiation, and metabolism of bone tissue. Hormonal deficiency at the time of menopause results in marked increases in bone resorption and formation, leading to rapid bone loss. The aim of our investigation was to determine genes characterized by significantly changed mRNA expression rates in postmenopausal versus premenopausal nonosteoporotic bone tissue and to describe the interrelationships among these genes using multivariate data analysis. Methods: Ten bone tissue samples from postmenopausal nonosteoporotic women and seven bone tissue samples from premenopausal nonosteoporotic women were examined. The expression differences of 118 selected genes were analyzed in a TaqMan probe-based quantitative reverse transcriptase-polymerase chain reaction system. Results: The Mann-Whitney U test indicated significant differences in the expression of 29 genes of postmenopausal and premenopausal nonosteoporotic women. Twenty-eight genes, including extracellular matrix molecules and digesting enzymes, genes belonging to the transforming growth factor-&bgr;/bone morphogenic protein pathway, transcription factors, growth factors, and other candidate genes, were significantly up-regulated in postmenopausal women compared with premenopausal women. Only one gene (ENO1) showed down-regulation after menopause. Based on the multiple mRNA expression profiles of 118 genes, postmenopausal and premenopausal states could be differentiated by enhanced postmenopausal gene expression levels using principal components analysis. Canonical variates analysis demonstrated that postmenopausal and premenopausal nonosteoporotic bone tissues can be distinguished by expression analysis of genes controlled via estrogen receptor-&agr; and genes coding for extracellular matrix molecules. Conclusions: The menopausal state of bone tissue has been unambiguously defined by its complex gene transcription pattern. Significant differences observed in the gene expression profiles of estrogen-deficient human bone tissue provide further insight into the process of postmenopausal changes of bone metabolism.

Transcriptional profiling of immune system-related genes in postmenopausal osteoporotic versus non-osteoporotic human bone tissue ☆

The functional interaction between the immune system and bone metabolism has been established at both molecular and cellular levels. We have used non-parametric and multidimensional expression pattern analyses to determine significantly changed mRNA profile of immune system-associated genes in postmenopausal osteoporotic (OP) vs. non-osteoporotic (NOP) bone tissue. Seven bone tissue samples from OP patients and ten bone tissue samples from NOP women were examined in our study. The transcription differences of selected 44 genes were analyzed in Taqman probe-based quantitative real-time RT-PCR system. Mann-Whitney test indicated significantly down-regulated transcription activity of 3 genes (FCGR2A, NFKB1 and SCARA3) in OP bone tissue which have prominent role in (antibody) clearance, phagocytosis, pathogen recognition and inflammatory response. According to the canonical variates analysis results, the groups of postmenopausal OP and NOP women are separable by genes coding for cytokines, co-stimulators and cell surface receptors affected in innate immunity which have high discriminatory power. Based on the complex gene expression patterns in human bone cells, we could distinguish OP and NOP states from an immunological aspect. Our data may provide further insights into the changes of the intersystem crosstalk between the immune and skeletal systems, as well as into the local immune response in the altered microenvironment of OP bone.

Postmenopausal expression changes of immune system-related genes in human bone tissue.

IntroductionThe molecular and cellular interactions between the immune system and bone tissue have been established. Sex hormone deficiency after menopause has multifunctional role by influencing growth, differentiation, and metabolism of the skeletal and the immune system.DiscussionWe have used nonparametric and multidimensional expression pattern analyses to determine significantly changed mRNA profile of immune system-associated genes in postmenopausal (POST) and premenopausal (PRE) nonosteoporotic bone. Ten bone tissue samples from POST patients and six bone tissue samples from PRE women were examined in our study. The transcription differences of the selected 50 genes were analyzed in TaqMan probe-based quantitative real-time reverse transcriptase polymerase chain reaction system. Mann–Whitney test indicated significantly downregulated transcription activity of three genes (CD14, HLA-A/MHCI, ITGAM/CD11b) and upregulated expression of six genes (C3, CD86/B7-2, IL-10, IL-6, TGFB3, TNFSF11/RANKL) in postmenopausal bone. According to the canonical variate analysis results, the groups of POST and PRE women are separable by genes coding for cytokines, costimulator molecules, and cell surface receptors involved in antigen presentation and T cell stimulation processes which have high discriminatory power. Based on a complex gene expression pattern analysis of human bone tissue, we could distinguish POST and PRE states from an immunological aspect. Our data might provide further insight into the changes of the intersystem crosstalk between immune and skeletal homeostasis, as well as local immune response in the altered microenvironment of postmenopausal bone.

Gene expression patterns in the bone tissue of women with fibrous dysplasia.

Fibrous dysplasia is an isolated skeletal disorder caused by a somatic activating mutation of GNAS gene with abnormal unmineralized matrix overproduction and extensive undifferentiated bone cell accumulation in the fibro‐osseous lesions. The aim of our investigation was to identify genes that are differently expressed in fibrous versus non‐fibrous human bone and to describe the relationships between these genes using multivariate data analysis. Six bone tissue samples from female patients with fibrous dysplastia (FD) and seven bone tissue samples from women without FD (non‐FD) were examined. The expression differences of selected 118 genes were analyzed by the TaqMan probe‐based quantitative real‐time RT‐PCR system. The Mann–Whitney U‐test indicated marked differences in the expression of 22 genes between FD and non‐FD individuals. Nine genes were upregulated in FD women compared to non‐FD ones and 18 genes showed a downregulated pattern. These altered genes code for minor collagen molecules, extracellular matrix digesting enzymes, transcription factors, adhesion molecules, growth factors, pro‐inflammatory cytokines, and lipid metabolism‐affected substrates. Canonical variates analysis demonstrated that FD and non‐FD bone tissues can be distinguished by the multiple expression profile analysis of numerous genes controlled via a G‐protein coupled pathway and BMP cascade as well as genes coding for extracellular matrix composing molecules. The remarkable changed gene expression profile observed in the fibrous dysplastic human bone tissue may provide further insight into the pathogenetic process of fibrous degeneration of bone.