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1.
Endokrynol Pol ; 68(6): 631-641, 2017.
Article in English | MEDLINE | ID: mdl-29022645

ABSTRACT

INTRODUCTION: TRIP11 is a multifunctional protein localizing either to Golgi apparatus, acting as a golgin, or in the nucleus, acting as coactivator of transcription mediated by thyroid hormone receptor (THR) and hypoxia induced factor (HIF). Triiodothyronine (T3) regulates nuclear localization of TRIP11 by inducing its phosphorylation. The exact mechanism of this regulation unknown. The expressions of THR and HIF are disturbed in various cancers, including renal cell cancer (RCC). In this study we aimed to analyze: 1) the mechanism of T3-dependent subcellular localization of TRIP11; 2) the significance of TRIP11 and T3 signaling pathway in RCC progression. MATERIAL AND METHODS: TRIP11 subcellular localization was analyzed using immunocytochemistry in RCC-derived cell line treated with T3, T3-agarose and PI3K inhibitor, wortmannin. The expressions of TRIP11 and genes involved in T3 signaling and hypoxia were investigated using qPRC in 36 pairs of RCC tumor-control samples, followed by validation/survival analysis in an independent cohort of >450 renal cancer patients. RESULTS: Wortmannin disrupted T3-dependent nuclear transport of TRIP11. T3-agarose did not change TRIP11 localization, precluding extracellular T3-mediated mechanism. The expressions of TRIP11, HIF-1ß, THRA, THRB, FURIN, VEGFA, and GLUT1 were disturbed in renal cancer. Expressions of TRIP11 and HIF-1ß correlated with tumor grades. Decreased expressions of TRIP11, THRA, and THRB correlated with poor survival of RCC patients. CONCLUSIONS: 1) T3 induces nuclear TRIP11 localization via PI3K-dependent mechanism; 2) disturbed expression of T3 signaling pathway genes correlates with RCC progression. The specific mechanisms by which altered T3 signaling may contribute to RCC progression require further investigation.


Subject(s)
Carcinoma, Renal Cell/metabolism , Disease Progression , Nuclear Proteins/metabolism , Signal Transduction , Triiodothyronine/metabolism , Aryl Hydrocarbon Receptor Nuclear Translocator/genetics , Aryl Hydrocarbon Receptor Nuclear Translocator/metabolism , Carcinoma, Renal Cell/mortality , Cell Line, Tumor , Cytoskeletal Proteins , Gene Expression Regulation, Neoplastic , Humans , Nuclear Proteins/genetics , Phosphatidylinositol 3-Kinases/metabolism
2.
Mol Cell Endocrinol ; 442: 58-67, 2017 02 15.
Article in English | MEDLINE | ID: mdl-27940296

ABSTRACT

Type 1 iodothyronine deiodinase (DIO1) regulates peripheral metabolism of thyroid hormones that control cellular proliferation, differentiation and metabolism. The significance of DIO1 in cancer is unknown. In this study we hypothesized that diminished expression of DIO1, observed in renal cancer, contributes to the carcinogenic process in the kidney. Here, we demonstrate that ectopic expression of DIO1 in renal cancer cells changes the expression of genes controlling cell cycle, including cyclin E1 and E2F5, and results in inhibition of proliferation. The expression of genes encoding collagens (COL1A1, COL4A2, COL5A1), integrins (ITGA4, ITGA5, ITGB3) and transforming growth factor-ß-induced (TGFBI) is significantly altered in renal cancer cells with induced expression of DIO1. Finally, we show that overexpression of DIO1 inhibits migration of renal cancer cells. In conclusion, we demonstrate for the first time that loss of DIO1 contributes to renal carcinogenesis and that its induced expression protects cells against cancerous proliferation and migration.


Subject(s)
Cell Movement/physiology , Cell Proliferation/physiology , Iodide Peroxidase/metabolism , Kidney Neoplasms/metabolism , Cell Cycle/physiology , Cell Line, Tumor , Collagen/metabolism , Cyclin E/metabolism , E2F5 Transcription Factor/metabolism , Humans , Kidney/metabolism , Thyroid Hormones/metabolism , Transforming Growth Factors/metabolism
3.
PLoS One ; 11(5): e0155359, 2016.
Article in English | MEDLINE | ID: mdl-27171412

ABSTRACT

BACKGROUND: Translational control is a mechanism of protein synthesis regulation emerging as an important target for new therapeutics. Naturally occurring microRNAs and synthetic small inhibitory RNAs (siRNAs) are the most recognized regulatory molecules acting via RNA interference. Surprisingly, recent studies have shown that interfering RNAs may also activate gene transcription via the newly discovered phenomenon of small RNA-induced gene activation (RNAa). Thus far, the small activating RNAs (saRNAs) have only been demonstrated as promoter-specific transcriptional activators. FINDINGS: We demonstrate that oligonucleotide-based trans-acting factors can also specifically enhance gene expression at the level of protein translation by acting at sequence-specific targets within the messenger RNA 5'-untranslated region (5'UTR). We designed a set of short synthetic oligonucleotides (dGoligos), specifically targeting alternatively spliced 5'UTRs in transcripts expressed from the THRB and CDKN2A suppressor genes. The in vitro translation efficiency of reporter constructs containing alternative TRß1 5'UTRs was increased by up to more than 55-fold following exposure to specific dGoligos. Moreover, we found that the most folded 5'UTR has higher translational regulatory potential when compared to the weakly folded TRß1 variant. This suggests such a strategy may be especially applied to enhance translation from relatively inactive transcripts containing long 5'UTRs of complex structure. SIGNIFICANCE: This report represents the first method for gene-specific translation enhancement using selective trans-acting factors designed to target specific 5'UTR cis-acting elements. This simple strategy may be developed further to complement other available methods for gene expression regulation including gene silencing. The dGoligo-mediated translation-enhancing approach has the potential to be transferred to increase the translation efficiency of any suitable target gene and may have future application in gene therapy strategies to enhance expression of proteins including tumor suppressors.


Subject(s)
5' Untranslated Regions/genetics , Genes, Tumor Suppressor , Genetic Techniques , Protein Biosynthesis/genetics , Base Sequence , Cell Line, Tumor , Cyclin-Dependent Kinase Inhibitor p16/metabolism , Gene Expression Regulation , Humans , Nucleic Acid Conformation , Receptors, Thyroid Hormone/genetics , Regulatory Sequences, Nucleic Acid/genetics , Thermodynamics
4.
Postepy Biochem ; 60(1): 39-54, 2014.
Article in Polish | MEDLINE | ID: mdl-25033541

ABSTRACT

Translation initiation is a key rate-limiting step in cellular protein synthesis. A cap-dependent initiation is the most effective mechanism of the translation. However, some physiological (mitosis) and pathological (oxidative stress) processes may switch the classic mechanism to an alternative one that is regulated by an mRNA element such as IRES, uORF, IRE, CPE, DICE, AURE or CITE. A recently discovered mechanism of RNA hypoxia response element (rHRE)-dependent translation initiation, may change the view of oxygen-regulated translation and give a new insight into unexplained biochemical processes. Hypoxia is one of the better-known factors that may trigger an alternative mechanism of the translation initiation. Temporal events of oxygen deficiency within tissues and organs may activate processes such as angiogenesis, myogenesis, regeneration, wound healing, and may promote an adaptive response in cardiovascular and neurodegenerative diseases. On the other hand, growth of solid tumors may be accompanied by cyclic hypoxia, allowing for synthesis of proteins required for further progression of cancer cells. This paper provides a review of current knowledge on translational control in the context of alternative models of translation initiation.


Subject(s)
Models, Biological , Protein Biosynthesis/physiology , RNA/genetics , Response Elements , Animals , Frameshifting, Ribosomal/physiology , Humans , Hypoxia/metabolism , MicroRNAs/genetics , Neoplasms/metabolism , Peptide Chain Initiation, Translational , Protein Biosynthesis/genetics , RNA/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism
5.
PLoS One ; 9(5): e97624, 2014.
Article in English | MEDLINE | ID: mdl-24849932

ABSTRACT

Thyroid hormone receptor beta (THRB) gene is commonly deregulated in cancers and, as strengthened by animal models, postulated to play a tumor-suppressive role. Our previous studies revealed downregulation of THRB in clear cell renal cell carcinoma (ccRCC), but the culpable mechanisms have not been fully elucidated. Since epigenetic regulation is a common mechanism influencing the expression of tumor suppressors, we hypothesized that downregulation of THRB in renal cancer results from epigenetic aberrances, including CpG methylation and microRNA-dependent silencing. Our study revealed that ccRCC tumors exhibited a 56% decrease in THRB and a 37% increase in DNA methyltransferase 1 (DNMT1) expression when compared with paired non-neoplastic control samples. However, THRB CpG methylation analysis performed using BSP, SNaPshot and MSP-PCR consistently revealed no changes in methylation patterns between matched tumor and control samples. In silico analysis resulted in identification of four microRNAs (miR-155, miR-425, miR-592, and miR-599) as potentially targeting THRB transcript. Luciferase assay showed direct binding of miR-155 and miR-425 to 3'UTR of THRB, and subsequent in vivo analyses revealed that transfection of UOK171 cell line with synthetic miR-155 or miR-425 resulted in decreased expression of endogenous TRHB by 22% and 64%, respectively. Finally, real-time PCR analysis showed significant upregulation of miR-155 (354%) and miR-425 (162%) in ccRCC when compared with matched controls. Moreover, microRNA levels were negatively correlated with the amount of THRB transcript in tissue samples. We conclude that CpG methylation is not the major mechanism contributing to decreased THRB expression in ccRCC. In contrast, THRB is targeted by microRNAs miR-155 and miR-425, whose increased expression may be responsible for downregulation of THRB in ccRCC tumors.


Subject(s)
Carcinoma, Renal Cell/genetics , Epigenesis, Genetic , Kidney Neoplasms/genetics , Thyroid Hormone Receptors beta/genetics , 3' Untranslated Regions/genetics , Carcinoma, Renal Cell/pathology , Cell Line, Tumor , DNA (Cytosine-5-)-Methyltransferase 1 , DNA (Cytosine-5-)-Methyltransferases/genetics , DNA Methylation , Gene Expression Regulation, Neoplastic/genetics , Humans , Kidney Neoplasms/pathology , MicroRNAs/genetics
6.
PLoS One ; 6(9): e24541, 2011.
Article in English | MEDLINE | ID: mdl-21912701

ABSTRACT

Type 1 iodothyronine deiodinase (DIO1) catalyses the conversion of prohormone thyroxine to the active thyroid hormone 3,3',5-triiodothyronine (T3), important regulator of cell proliferation and differentiation. DIO1 expression is reduced in the most common type of kidney neoplasia, clear cell Renal Cell Carcinoma (ccRCC). MicroRNAs are small, non-coding RNAs that regulate gene expression at posttranscriptional levels. The aim of this study was to analyze the potential regulation of DIO1 expression by microRNAs in ccRCC. Bioinformatic analysis revealed that 3'UTR of the human DIO1 gene transcript contains miR-224 and miR-383 target sites, which are conserved across mammalian species. Semi-quantitative real-time PCR was used to analyze the expression of miR-224 and miR-383 in 32 samples of ccRCC tumors (T) and in 32 matched control (C) samples. We observed statistically significant (p = 0.0002) more than four fold increase in miR-224 expression and nearly two fold increase in miR-383 expression in samples T compared to samples C. Tumor specific changes in expression of miR-224 negatively correlated with changes in DIO1 expression and intracellular T3 concentration. Transfection of HeLa cell line with miR-224 and miR-383 suppressed the activity of a luciferase reporter containing the 3'UTR of DIO1. This was abolished when constructs mutated at the miR-224 and miR-383 target sites were used instead, indicating that miR-224 and miR-383 directly bind to DIO1 3'UTR. Finally, induced expression of miR-224 in Caki-2 cells resulted in significant (p<0.01) reduction of DIO1 mRNA. This study provides a novel miRNA-mediated regulatory mechanism of DIO1 expression in ccRCC.


Subject(s)
3' Untranslated Regions/genetics , Carcinoma, Renal Cell/complications , Hypothyroidism/complications , Hypothyroidism/genetics , Iodide Peroxidase/genetics , Kidney Neoplasms/complications , MicroRNAs/genetics , Carcinoma, Renal Cell/genetics , Carcinoma, Renal Cell/pathology , Gene Expression Regulation, Neoplastic/genetics , HeLa Cells , Humans , Hypothyroidism/metabolism , Kidney Neoplasms/genetics , Kidney Neoplasms/pathology , MicroRNAs/metabolism , Triiodothyronine, Reverse/metabolism
7.
Endokrynol Pol ; 62(2): 160-70, 2011.
Article in English | MEDLINE | ID: mdl-21528479

ABSTRACT

Alternative splicing of pre-mRNA is a process in which noncoding regions of primary transcript are removed and coding regions are joined in different manners to produce mRNA molecules of different sequences. Alternative splicing affects nearly all human genes and is a key source of diversity of proteins coded by a relatively small number of genes. Since alternative splicing is of crucial importance for the proper functioning of cells, including those involved in hormonal signalling, aberrations of alternative splicing can lead to disruption of cellular mechanisms and in consequence result in serious endocrine pathologies. Disturbances of alternative splicing include mutations of consensus splice regulatory sites and improprieties in the action of splicing factors, the proteins involved in regulating the process. In consequence of disturbed alternative splicing, improperly spliced mRNA and protein isoforms can be produced which can lead to disruption of function of their wild type counterparts. This review aims to discuss the role of alternative splicing in pathologies of the endocrine system and gives examples that highlight the importance of this process in the proper functioning of hormones, hormone receptors and other factors involved in hormonal regulation. The examples given include endocrine-related tumours (pituitary tumours, cancers of the thyroid, prostate, ovary and breast, and insulinoma), isolated growth hormone deficiency, and Frasier syndrome. Non-endocrine pathologies in which aberrant alternative splicing of transcripts of genes involved in hormonal signalling have been detected are also described. Finally, we discuss future perspectives on the possible usage of alternative splicing in diagnostics and therapy.


Subject(s)
Alternative Splicing/genetics , Endocrine Gland Neoplasms/genetics , Endocrine Gland Neoplasms/pathology , Frasier Syndrome/genetics , Human Growth Hormone/deficiency , Human Growth Hormone/genetics , Humans
8.
J Clin Endocrinol Metab ; 96(3): E546-53, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21159845

ABSTRACT

CONTEXT: Loss of the thyroid hormone receptor is common in tumors. In mouse models, a truncated THRB gene leads to thyroid cancer. Previously, we observed up-regulation of the expression of eight microRNAs (miRs) in papillary thyroid carcinoma (PTC) tumors. OBJECTIVE: Our objective was to determine whether THRB might be inhibited by miRs up-regulated in PTC. DESIGN: The potential binding of miR to the 3'-untranslated region of THRB was analyzed in silico. Direct inhibition by miRs binding to the cloned 3'-untranslated region of THRB was evaluated using luciferase assays. Inhibition of endogenous THRB and its target genes (DIO1 and APP) was examined in cell lines transfected by pre-miRs. The impact on thyroid hormone response element (TRE) was evaluated in promoter assays. Correlations between the expression of THRB and miRs was evaluated in 13 PTC tumor/normal tissue pairs. RESULTS: THRB contains binding sites for the top seven miRs up-regulated in PTC (P = 0.0000002). Direct interaction with THRB was shown for miR-21 and miR-146a. We observed lower levels of THRB transcripts in cell lines transfected with miR-21, -146a, and -221 (down-regulation of 37-48%; P < 0.0001), but not with miR-181a. THRB protein was suppressed down to 10-28% by each of four miRs. Concomitant expression of DIO1 and APP was affected (down-regulation of 32-66%, P < 0.0034 and up-regulation of 48-57%, P < 0.0002, respectively). All four miRs affected TRE activity in promoter assays. Down-regulation of luciferase occurred after transfection with pTRE-TK-Luc construct and each of four miRs. The analysis of tumor/normal tissue pairs revealed down-regulation of THRB in 11 of 13 pairs (1.3- to 9.1-fold), and up-regulation of miR-21, -146a, -181a, and -221 in almost all pairs. CONCLUSIONS: MiRs up-regulated in PTC tumors directly inhibit the expression of THRB, an important tumor suppressor gene.


Subject(s)
Carcinoma, Papillary/genetics , MicroRNAs/genetics , Thyroid Hormone Receptors beta/genetics , 3' Untranslated Regions/genetics , Amyloid beta-Protein Precursor/genetics , Apoptosis/genetics , Blotting, Western , Carcinoma , Carcinoma, Papillary/pathology , Cell Line, Tumor , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/genetics , Gene Expression Regulation, Neoplastic/physiology , Genes, Reporter/genetics , Humans , Luciferases/genetics , Polymorphism, Single Nucleotide , Reverse Transcriptase Polymerase Chain Reaction , Thyroid Cancer, Papillary , Thyroid Gland/pathology , Thyroid Neoplasms/genetics , Thyroid Neoplasms/pathology , Transcription, Genetic , Triiodothyronine/physiology
9.
Postepy Biochem ; 57(3): 257-65, 2011.
Article in Polish | MEDLINE | ID: mdl-22235651

ABSTRACT

Splicing of pre-mRNA is one of the post-transcriptional modifications in which introns are removed from primary transcript and exons are joined. pre-mRNA splicing reactions are catalyzed by dynamic complex called spliceosome. Exons can be joined on different manners in a process known as alternative splicing, that provide production of multiple mRNA and protein isoforms from relatively low number of genes. Alternative splicing is regulated by cis elements localized within exons or introns and trans-acting factors including spliceosome components and members of the SR and hnRNP protein family that exert antagonistic effects on splicing. Aberrant pre-mRNA splicing may be caused by mutations in cis elements and altered expression of splicing factors. This review describes disturbances in splicing of genes controlling proliferation and metastasis that can lead to tumoral transformation. Also, potential applications of abnormally spliced transcripts that may potentially serve as diagnostic biomarkers of cancer or targets in anticancer therapy are discussed.


Subject(s)
Alternative Splicing , Neoplasms/genetics , RNA Precursors/genetics , Animals , Cell Transformation, Neoplastic/genetics , Humans , Neoplasm Metastasis/genetics , Neoplasms/diagnosis , Neoplasms/pathology , Neoplasms/therapy , RNA, Messenger/genetics
10.
PLoS One ; 5(10): e13690, 2010 Oct 27.
Article in English | MEDLINE | ID: mdl-21082031

ABSTRACT

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is the most common type of renal cancer. One of the processes disturbed in this cancer type is alternative splicing, although phenomena underlying these disturbances remain unknown. Alternative splicing consists of selective removal of introns and joining of residual exons of the primary transcript, to produce mRNA molecules of different sequence. Splicing aberrations may lead to tumoral transformation due to synthesis of impaired splice variants with oncogenic potential. In this paper we hypothesized that disturbed alternative splicing in ccRCC may result from improper expression of splicing factors, mediators of splicing reactions. METHODOLOGY/PRINCIPAL FINDINGS: Using real-time PCR and Western-blot analysis we analyzed expression of seven splicing factors belonging to SR proteins family (SF2/ASF, SC35, SRp20, SRp75, SRp40, SRp55 and 9G8), and one non-SR factor, hnRNP A1 (heterogeneous nuclear ribonucleoprotein A1) in 38 pairs of tumor-control ccRCC samples. Moreover, we analyzed splicing patterns of five genes involved in carcinogenesis and partially regulated by analyzed splicing factors: RON, CEACAM1, Rac1, Caspase-9, and GLI1. CONCLUSIONS/SIGNIFICANCE: We found that the mRNA expression of splicing factors was disturbed in tumors when compared to paired controls, similarly as levels of SF2/ASF and hnRNP A1 proteins. The correlation coefficients between expression levels of specific splicing factors were increased in tumor samples. Moreover, alternative splicing of five analyzed genes was also disturbed in ccRCC samples and splicing pattern of two of them, Caspase-9 and CEACAM1 correlated with expression of SF2/ASF in tumors. We conclude that disturbed expression of splicing factors in ccRCC may possibly lead to impaired alternative splicing of genes regulating tumor growth and this way contribute to the process of carcinogenesis.


Subject(s)
Alternative Splicing , Apoptosis/physiology , Carcinoma, Renal Cell/genetics , Genes, Tumor Suppressor , Kidney Neoplasms/genetics , Oncogenes , Blotting, Western , Carcinoma, Renal Cell/pathology , Humans , Kidney Neoplasms/pathology , Polymerase Chain Reaction
11.
Biochim Biophys Acta ; 1802(11): 995-1005, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20691260

ABSTRACT

Thyroid hormone receptor ß1 (TRß1) is a hormone-dependent transcription factor activated by 3,5,3'-l-triiodothyronine (T3). TRß1 functions as a tumor suppressor and disturbances of the THRB gene are frequent findings in cancer. Translational control mediated by untranslated regions (UTRs) regulates cell proliferation, metabolism and responses to cellular stress, processes that are involved in carcinogenesis. We hypothesized that reduced TRß1 expression in clear cell renal cell cancer (ccRCC) results from regulatory effects of TRß1 5' and 3'UTRs on protein translation. We determined TRß1 expression and alternative splicing of TRß1 5' and 3'UTRs in ccRCC and control tissue together with expression of the type 1 deiodinase enzyme (coded by DIO1, a TRß1 target gene). Tissue concentrations of T3 (which are generated in part by D1) and expression of miRNA-204 (an mRNA inhibitor for which a putative interaction site was identified in the TRß1 3'UTR) were also determined. TRß1 mRNA and protein levels were reduced by 70% and 91% in ccRCC and accompanied by absent D1 protein, a 58% reduction in tissue T3 concentration and 2-fold increase in miRNA-204. Structural analysis of TRß1 UTR variants indicated that reduced TRß1 expression may be maintained in ccRCC by posttranscriptional mechanisms involving 5'UTRs and miRNA-204. The tumor suppressor activity of TRß1 indicates that reduced TRß1 expression and tissue hypothyroidism in ccRCC tumors is likely to be involved in the process of carcinogenesis or in maintaining a proliferative advantage to malignant cells.


Subject(s)
Carcinoma, Renal Cell/genetics , Kidney Neoplasms/genetics , Thyroid Hormone Receptors beta/genetics , Untranslated Regions/genetics , 5' Untranslated Regions/genetics , Alternative Splicing , Base Sequence , Blotting, Western , Carcinoma, Renal Cell/metabolism , Carcinoma, Renal Cell/pathology , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Humans , Iodide Peroxidase/genetics , Iodide Peroxidase/metabolism , Kidney/metabolism , Kidney/pathology , Kidney Neoplasms/metabolism , Kidney Neoplasms/pathology , MicroRNAs/genetics , MicroRNAs/metabolism , Models, Genetic , Molecular Sequence Data , Protein Biosynthesis , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Triiodothyronine/metabolism
14.
Thyroid ; 19(10): 1105-13, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19534619

ABSTRACT

BACKGROUND: Alternative splicing, one of the sources of protein diversity, is often disturbed in cancer. Type 1 iodothyronine deiodinase (DIO1) catalyzes deiodination of thyroxine generating triiodothyronine, an important regulator of cell proliferation and differentiation. The expression of DIO1 is disturbed in different types of cancer. The aim of the study was to analyze the alternative splicing of DIO1 and its possible disturbance in renal cancer. METHODS: Using real-time PCR, we analyzed 19 tissue samples (T) of renal cancer and 19 matched control samples (C) of the opposite pole of the kidney, not infiltrated by tumor, and 6 control samples (N) (nonneoplastic kidney abnormalities). RESULTS: Cloning of DIO1 mRNA isoforms revealed 11 different transcripts, among them 7 new splice variants, not previously reported. The expression of all variants of DIO1 was dramatically (>90%) and significantly (p < or = 0.0003) lowered in samples T compared to control samples C. The ratio of mRNA isoforms encoding DIO1 protein variants possessing or lacking the active center was lowered in samples T compared with control samples C, suggesting disturbed alternative splicing of DIO1. The expression of mRNA of splicing factors SF2/ASF (splicing factor-2/alternative-splicing factor) and hnRNPA1 (heterogeneous ribonucleoprotein A1), regulating 5'-splice site selection, was significantly but not proportionally lowered in samples T compared to samples C. The mRNA ratio of splicing factors SF2/ASF and hnRNPA1 correlated with the ratio of mRNA isoforms encoding DIO1 protein variants possessing or lacking the active center in controls C but not in samples T. CONCLUSIONS: Our results show that the expression and alternative splicing of DIO1 mRNA is disturbed in renal cancer, possibly due to changes in expression of splicing factors SF2/ASF and hnRNPA1.


Subject(s)
Gene Expression Regulation, Enzymologic/genetics , Iodide Peroxidase/genetics , Kidney Neoplasms/genetics , Protein Isoforms/genetics , Cloning, Molecular , DNA Primers , Databases, Genetic , Humans , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction
15.
Thyroid Res ; 1(1): 5, 2008 Oct 13.
Article in English | MEDLINE | ID: mdl-19014670

ABSTRACT

BACKGROUND: Triiodothyronine regulates proliferation acting as stimulator or inhibitor. E2F4 and E2F5 in complexes with pocket proteins p107 or p130 stop cells in G1, repressing transcription of genes important for cell cycle progression. p107 and p130 inhibits activity of cyclin/cdk2 complexes. Expression of all those proteins could be regulated by triiodothyronine. In clear cell renal cell carcinoma many disturbances in T3 signaling pathway was described, in that type of cancer also expression of some key G1 to S phase progression regulators was shown. METHODS: We investigated role of T3 and its receptors in regulation of proliferation of HK2, Caki-2, Caki-1 cell lines (cell counting, cytometric analysis of DNA content) and expression of thyroid hormone receptors, E2F4, E2F5, p107 and p130 (western blot and semi-quantitative real time PCR). Statistical analysis was performed using one-way ANOVA. RESULTS AND CONCLUSION: We show that T3 inhibits proliferation of HK2, and stimulates it in Caki lines. Those differences are result of disturbed expression of TR causing improper regulation of E2F4, E2F5, p107 and p130 in cancer cells.

16.
Thyroid ; 17(11): 1039-48, 2007 Nov.
Article in English | MEDLINE | ID: mdl-17910524

ABSTRACT

We show here that the promoter of E2F1 gene, encoding one of the key regulators of cell proliferation, is overly active in the presence of low amounts of triiodothyronine (T3) and in the presence of mutant thyroid hormone receptor. We also show that T3-thyroid hormone receptor pathway of regulation of molecular processes is disturbed in clear cell renal cell carcinoma (ccRCC) on several levels, including overexpression of thyroid hormone receptors and the disturbance of their binding to DNA and to the hormone. In comparison to the cancer-free kidneys and peritumoral respective control tissues, E2F1 mRNA and protein levels are significantly increased in cancer tissues. A significant correlation between E2F1 mRNA and protein levels has been found in both control types and ccRCCs. No correlation was observed between the amount of E2F1 mRNA and the amount of thyroid hormone receptors or their DNA or T3 binding activity, suggesting that the function of thyroid hormone receptors could be markedly disturbed in both tumor and peritumoral cells. In summary, we show that ccRCC is characterized by the overexpression of E2F1, which is likely a result of a deregulated control of T3-dependent molecular processes.


Subject(s)
Carcinoma, Renal Cell/metabolism , E2F1 Transcription Factor/biosynthesis , Receptors, Thyroid Hormone/physiology , Adult , Aged , Aged, 80 and over , DNA/metabolism , Female , Gene Expression Regulation, Neoplastic/physiology , Humans , Male , Middle Aged , Promoter Regions, Genetic/physiology , RNA, Messenger/metabolism , Thyroid Hormone Receptors alpha/biosynthesis , Thyroid Hormone Receptors beta/biosynthesis , Triiodothyronine/metabolism
17.
Acta Biochim Pol ; 54(3): 595-602, 2007.
Article in English | MEDLINE | ID: mdl-17726548

ABSTRACT

The expression of cyclin E gene (CCNE) in relation to the expression of its major regulatory protein, E2F1, was examined in clear cell renal cell carcinomas (ccRCC). We show that the overexpression of E2F1 is accompanied by the significant increase of the mean amounts of cyclin E mRNA, as well as of total cyclin E protein and its low molecular weight forms in cancer tissues as compared to peritumoral controls. A significant increase of the mean amount of total cyclin E was found in peritumoral tissues compared to cancer-free kidneys, suggesting that cancer cells might secrete factors having a profound influence on the metabolism of neighbouring tissues. A significant, positive correlations between E2F1 protein and total cyclin E mRNA, as well as between E2F1 protein and full length cyclin E protein were found in cancer-free kidneys and in peritumoral tissues, but not in ccRCCs. The overexpression of cyclin E positively correlated with the decreasing degree of tumor differentiation, implicating a role for cyclin E in the promotion of tumorigenesis.


Subject(s)
Carcinoma, Renal Cell/genetics , Cyclin E/genetics , Liver Neoplasms/genetics , Oncogene Proteins/genetics , Blotting, Western , Carcinoma, Renal Cell/metabolism , Cyclin E/metabolism , E2F1 Transcription Factor/genetics , E2F1 Transcription Factor/metabolism , Electrophoretic Mobility Shift Assay , Humans , Kidney/metabolism , Kidney/pathology , Liver Neoplasms/metabolism , Oncogene Proteins/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction
18.
Acta Biochim Pol ; 53(4): 641-50, 2006.
Article in English | MEDLINE | ID: mdl-17115080

ABSTRACT

In the present work, we have reviewed data showing that triiodothyronine and its nuclear receptors modify expression of different genes/proteins involved in cell cycle control beginning from growth factors (such as EGF and TGF-beta), to cell surface receptors (EGFR), as well as proteins acting at the cell membrane (Ras), various transcription factors (c-Fos, c-Myc, E2F1), cyclins, Cip/Kip family of cdk2 inhibitors, and p53 inhibitor Mdm2 (Table 1). We have shown how TRs are also able to modify the fate of a cell, thanks to their ability to form complexes with other transcription factors such as p53 - a key regulator of apoptosis and proliferation. Available data show that the function of thyroid hormones and of their receptors on cell proliferation is not homogenous. In fact, it strongly depends on the cell type, its developmental state (progenitor or differentiated), its patho-physiological state (normal or tumor cell), and the so-called 'cellular context'. Therefore, it is not possible to uniformly recommend T3 treatment or T3 depletion to stop or initiate proliferation of all cell types. Instead, a very individual and careful action should be considered.


Subject(s)
Cell Proliferation , Receptors, Thyroid Hormone/physiology , Thyroid Hormones/physiology , Animals , Gene Expression Regulation/physiology , Humans , Triiodothyronine/physiology
19.
Postepy Biochem ; 51(3): 288-96, 2005.
Article in Polish | MEDLINE | ID: mdl-16381173

ABSTRACT

The thyroid hormone biosynthesis is dependent on the level of the expression and the correct action of the basic proteins involved in this process: TSH receptor (TSH-R), sodium-iodine symporter (NIS), thyroglobulin (Tg), thyroid peroxidase (TPO) and iodothyronine deodinases type 1 (D1) and type 2 (D2). Transcription of the genes coding these proteins is regulated by TSH via cAMP cascade. Despite the thyroid-specific transcription factors are necessary for the transcription regulation of TSH-R, NIS, TG, TPO, DIO1 and DIO2 genes. Three thyroid transcription factors Titf1/Nkx2-1, Foxel and Pax8 seem to be essential for thyroid development, growth and function. Disturbances of the expression of these transcription factors and their co-operation could be responsible for the congenital thyroid gland disorders as well as in thyroid cancerogenesis.


Subject(s)
Thyroid Hormones/biosynthesis , Transcription Factors/metabolism , Animals , Humans , Nuclear Proteins/metabolism , Thyroid Diseases/genetics , Thyroid Diseases/physiopathology , Thyroid Gland/growth & development , Thyroid Nuclear Factor 1
20.
Thyroid ; 15(10): 1137-46, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16279847

ABSTRACT

Type 1 and type 2 iodothyronine 5' deiodinases (D1 and D2, respectively) catalyze the conversion of thyroxine (T(4)) to triiodothyronine (T(3)). Similar to other genes crucial for T(3) generation, D1 and D2 expression might be disturbed in papillary thyroid cancer (PTC) possible as a result of impairments in thyroid transcription factors Titf1/Nkx2-1 and Pax-8. The aim of the study was to investigate changes in the expression of D1 and D2 in PTC compared to changes in the expression of Titf1/Nkx2-1 and Pax-8. Although D1 and D2 activities were decreased in tumor samples (PTC) compared to control C samples (tissues from a nontumorous part of the gland), the differences were not statistically significant. Contrary to that, their mRNA levels were significantly decreased in PTC samples compared to C samples (p = 0.017 and p = 0.012, respectively). Interestingly there was clear discrepancy between enzymatic activity and mRNA level of both deiodinases. There was a statistically significant correlation between D1 and Pax-8 (r = 0.464, p = 0.039), D2 and Pax-8 (r = 0.461, p = 0.041), D2 and Titf1/Nkx2-1 mRNA levels (r = 0.526, p = 0.017). Our results show that changes in D1 and D2 expression in PTC, including the discrepancy between deiodinases activity and mRNA level, might possibly related to impaired Titf1/Nkx2-1 and Pax-8 action.


Subject(s)
Carcinoma, Papillary/metabolism , Iodide Peroxidase/biosynthesis , Nuclear Proteins/biosynthesis , Paired Box Transcription Factors/biosynthesis , Thyroid Neoplasms/metabolism , Transcription Factors/biosynthesis , Adult , Female , Gene Expression Regulation, Neoplastic , Humans , Male , Middle Aged , PAX8 Transcription Factor , RNA, Messenger/metabolism , Thyroid Gland/enzymology , Thyroid Gland/metabolism , Thyroid Nuclear Factor 1 , Iodothyronine Deiodinase Type II
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