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Downregulation of Telomerase Maintenance-Related ACD Expression in Patients Undergoing Immunosuppresive Therapy Following Kidney Transplantation

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Downregulation of Telomerase Maintenance-Related ACD Expression in Patients Undergoing Immunosuppresive Therapy Following Kidney Transplantation 2224 EXPERIMENTAL AND THERAPEUTIC MEDICINE 10: 2224-2230, 2015 Downregulation of telomerase maintenance-related ACD expression in patients undergoing immunosuppresive therapy following kidney transplantation AGNIESZKA WITKOWSKA1, BARBARA STRZALKA-MROZIK2, ALEKSANDER OWCZAREK3, JOANNA GOLA2, URSZULA MAZUREK2, WLADYSLAW GRZESZCZAK1 and JANUSZ GUMPRECHT1 1Department of Internal Medicine, Diabetology and Nephrology, Medical University of Silesia, 41-800 Zabrze, Silesia; 2Department of Molecular Biology; 3Division of Statistics, Medical University of Silesia, 41-200 Sosnowiec, Silesia, Poland Received August 30, 2014; Accepted July 23, 2015 DOI: 10.3892/etm.2015.2785 Abstract. Chronic administration of immunosuppressants gene transcription, and thus TPP1 protein expression, may has been associated with long-term consequences, including enhance the capacity for cell immortalization, despite normal a higher risk of neoplasm development. The processes regu- levels of other key telomere maintenance factors, in patients lating telomere function exert a major influence on human undergoing immunosuppressive therapy. Furthermore, the cancer biology. The present study aimed to assess the effect of results indicate that TPP1 has potential for use as an early immunosuppressive therapy on the expression of genes asso- clinical marker and/or therapeutic target for cancer in patients ciated with telomere maintenance and protection in patients following organ transplantation. following renal transplantation. A total of 51 patients that had undergone kidney transplantation and 54 healthy controls Introduction were enrolled in the study. The 51 transplant patients received a three-drug immunosuppressive regimen consisting of cyclo- Solid organ transplant recipients are at an elevated risk of cancer sporine A, prednisone and mycophenolate mofetil. In stage 1 development. Compared with the general population, cancers of the study, the expression profiles of 123 transcripts, which develop more rapidly, occur earlier and metastasize more represented 70 genes, were assessed in peripheral mononuclear widely in this group of patients (1). Following kidney trans- blood cells using an oligonucleotide microarray technique in plantation the risk of cancer development increases 2-4-fold; 8 transplant recipients and 4 healthy control subjects. Among thus, following cardiovascular disease, kidney transplantation the analyzed transcripts, the expression levels of 4 differed is a major cause of morbidity (2). Certain types of cancer are significantly between the studied groups; however, only the particularly over-represented among the transplant patient ACD (adrenocortical dysplasia homolog) gene, encoding the population; for example, marked increases in incidence telomere-binding protein POT1-interacting protein 1 (TPP1), have been observed in oncovirus-related tumors, such as was sufficiently specific for telomere homeostasis. The expres- Kaposi's sarcoma, skin cancer and lymphoma, which are sion of ACD was downregulated in transplant recipients associated with a >20-fold increase in risk. In addition, the (fold change, 2.11; P=0.006). In stage 2 of the study, reverse rate of kidney malignancies is increased 15-fold in transplant transcription-quantitative polymerase chain reaction analysis patients compared with the general population (3). Common of ACD, DKC1 and hTERT mRNA was conducted for all malignancies, such as lung, ovarian, colon or gastric cancer, transplant patients and control subjects. The results confirmed have an incidence that is ~2-fold higher, while the incidence of the downregulation of the ACD gene in patients that had leukemia, liver, gynecological, bladder and testicular tumors received immunosuppressive therapy (P=0.002). The results increases ~5-fold following renal transplantation (3). The of the present study indicate that the downregulation of ACD increased risk of carcinogenesis observed in patients following renal transplantation is the result of conventional risk factors (genetic, immune or environmental), in addition to risk factors specific to transplant recipients (primarily immunosuppressive therapy and, in certain cases, oncogenic viruses) (1); however, Correspondence to: Dr Agnieszka Witkowska, Department of immunosuppressive therapy appears to be the major factor Internal Medicine, Diabetology and Nephrology, Medical University of Silesia, 3 Maja 13/15, 41-800 Zabrze, Silesia, Poland responsible for the increased cancer incidence following trans- E-mail: [email protected] plantation (4). The development of cancer is a multistage process Key words: telomerase, TPP1, ACD gene, immunosupression, involving numerous mutations and/or chromosomal aberra- kidney transplantation tions; therefore, cancer is regarded as a disease of genomic instability (5). The majority of the genetic aberrations that are characteristic of cancer can be initiated by telomere dysfunc- WITKOWSKA et al: ACD DOWNREGULATION IN PATIENTS UNDERGOING IMMUNOSUPPRESIVE THERAPY 2225 tion (6,7). Telomeres are nucleoprotein structures that protect In stage 2 of the study, reverse transcription-quantitative the ends of eukaryotic chromosomes. The formation of a polymerase chain reaction (RT-qPCR) analysis of the selected telosome, which is a DNA loop structure with a number of telomere maintenance factors was performed in all patients in associated proteins, including the six core factors telomeric the study and control groups. repeat-binding factor 1 (TRF1), TRF2, telomeric repeat- binding factor 2-interacting protein 1, TERF1-interacting Subjects. A total of 51 kidney transplantation patients and nuclear factor 2, protection of telomeres protein 1 (POT1) 54 healthy control subjects were enrolled in the study. and POT1-interacting protein 1 (TPP1), protects the very Kidney transplant recipients (35 men and 16 women; age, end of a telomere against a DNA break, preventing chromo- 48.7±6.8 years), who had undergone renal transplantation somal end-to-end fusions, misrepair and degradation (6,8-11). surgery an average of 9.6 years previously, were treated with a Telomere shortening promotes genome instability, and three-drug homogenic immunosuppressive regimen, consisting shortened telomeres have been reported to be common and of cyclosporine A (CsA), prednisone and mycophenolate prevalent early genetic alterations in cancer initiation (12,13). mofetil (MMF) or mycophenolate sodium. Doses were deter- Furthermore, the stability of telomere length determines mined based on the patients weight, and all patients underwent immortalization, which is the obligatory step of cancer devel- serum drug concentration monitoring. Patients with an acute opment (14). In immortal cells, a balance is maintained between disease were excluded (C-reactive protein-negative). Fifty-four the loss of telomeric DNA due to degradation or incomplete medication-free healthy subjects (11 men and 43 women) were replication and telomere elongation, which is performed by a selected for the control group. A total of 7 euglycemic trans- DNA polymerase known as telomerase (15). Human telom- plant recipients (5 men and 2 women; age, 46.5±8.6 years) and erase is composed of telomerase RNA (hTR) and a catalytic 4 healthy controls (2 men and 2 women; age, 59.0±4.5 years) subunit, telomerase reverse transcriptase (hTERT) (16,17). were selected for microarray assay analysis in stage 1 of the Telomerase is believed to be strongly repressed in normal study. human somatic tissues, but reactivated in 85-90% of human cancer tissues (18,19); however, a previous study has indicated Tissue samples. Venous blood samples were collected and that, even in the presence of proficient telomerase activity and stored in tubes containing EDTA, and a 7.5-ml sample from normal telomere length, telomere-associated proteins have an each patient was centrifuged using Ficoll-Conray density important role in cancer (9). gradient centrifugation (specific gravity, 1.077) for 30 min at As telomere dysfunction has been implicated in leukemia 350 x g (Immuno-Biological Laboratories Co., Ltd., Gunma, and cancer development (9-11,20), the various factors involved Japan), immediately after blood collection. A 10-ml sample in telomere maintenance may emerge as potential cancer was collected from patients selected for microarray assay. markers and/or therapeutic targets. The identification of novel cancer markers is particularly important for patients receiving RNA extraction. Total RNA was extracted from the PBMCs immunosuppressive therapy, as these patients are at an using TRIzol® reagent (Invitrogen Life Technologies, Carlsbad, increased risk of cancer and require more frequent diagnostic CA, USA) and then treated with DNase I (MBI Fermentas; screening for the early identification of cancer. The aim of the Thermo Fisher Scientific, Vilnius, Lithuania) according to the present study, therefore, was to assess the effect of immuno- manufacturer's instructions. The quality of the extracted RNA suppressive therapy on the expression of genes associated with was tested electrophoretically using ethidium bromide-stained telomere maintenance and protection in patients following 0.8% agarose gel. The results were analyzed and recorded renal transplantation. using an electrophoresis documentation system (Fisher Biotec, Perth, WA, Australia) and LabImage-1D software, version 2.7.2 Materials and methods (Kapelan Bio-Imaging GmbH, Leipzig, Germany). The total RNA concentration was measured spectrophotometrically Study design. In order to establish
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