Transforming growth factor-β1 (TGF-β1) is a potent inhibitor of cellular growth and proliferation by G1 phase arrest or apoptosis. We investigated the association of TGF-β1 with the anti-proliferative effect of upstream stimulatory factor (USF) in Fischer rat thyroid cell line (FRTL-5) cells. [Methyl-3H] thymidine uptake was measured after treatment of FRTL-5 cells with TGF-β1 to identify its anti-proliferative effect. USF-1 and USF-2 proteins were in vitro translated, and an electrophoretic mobility shift assay was performed to identify the interaction between USF and the TGF-β1 promoter. FRTL-5 cells were transfected with USF cDNA, and then the expression of TGF-β1 was examined with Northern and Western blotting. The cell cycle-regulating proteins associated with TGF-β1 were also measured. TGF-β1 significantly inhibited [methyl-3H] thymidine uptake in FRTL-5 cells. Two specific binding sites for USF were found in the TGF-β1 promoter: -1,846~-1,841 (CACATG) and -621~-616 (CATGTG). Overexpression of USF increased both the mRNA levels and protein levels of TGF-β1. However, the expression of cyclin D1, CDK4, cyclin E, and CDK2, and the phosphorylation of retinoblastoma protein remained unchanged. Overexpression of USF in FRTL-5 cells increased the expression of TGF-β10 through specific binding to TGF-β1 promoter. However, the USF-induced expression of TGF-β1 did not cause G1 arrest.
Upstream Stimulatory Factor; Transforming Growth Factor beta1; FRTL-5 Cells
Brain and skin metastasis from urothelial carcinoma of the bladder is rare. There have been few case reports of the clinical course of patients with metastatic urothelial carcinoma of the brain and skin. In the present case, a 60-year-old man had undergone radical cystectomy with an ileal conduit owing to urothelial carcinoma (T1N0M0). The patient developed dizziness 9 years later and a solitary brain tumor was discovered in his left cerebellar hemisphere. The tumor was totally resected and the mass was verified to be metastatic urothelial carcinoma. One year after the metastasectomy of the brain lesion, multiple erythematous nodular lesions developed on his abdominal skin. The skin lesions were excised and verified to be metastatic urothelial carcinoma. This report describes this case of urothelial carcinoma of the bladder that metastasized to the brain and abdominal skin.
Neoplasm metastasis; Skin; Urinary bladder neoplasms
Tetrahydrobiopterin (BH4) deficiency is a genetic disorder associated with a variety of metabolic syndromes such as phenylketonuria (PKU). In this article, the signaling pathway by which BH4 deficiency inactivates mTORC1 leading to the activation of the autophagic pathway was studied utilizing BH4-deficient Spr-/- mice generated by the knockout of the gene encoding sepiapterin reductase (SR) catalyzing BH4 synthesis. We found that mTORC1 signaling was inactivated and autophagic pathway was activated in tissues from Spr-/- mice. This study demonstrates that tyrosine deficiency causes mTORC1 inactivation and subsequent activation of autophagic pathway in Spr-/- mice. Therapeutic tyrosine diet completely rescued dwarfism and mTORC1 inhibition but inactivated autophagic pathway in Spr-/- mice. Tyrosine-dependent inactivation of mTORC1 was further supported by mTORC1 inactivation in Pahenu2 mouse model lacking phenylalanine hydroxylase (Pah). NIH3T3 cells grown under the condition of tyrosine restriction exhibited autophagy induction. However, mTORC1 activation by RhebQ64L, a positive regulator of mTORC1, inactivated autophagic pathway in NIH3T3 cells under tyrosine-deficient conditions. In addition, this study first documents mTORC1 inactivation and autophagy induction in PKU patients with BH4 deficiency.
tetrahydrobiopterin; autophagy; mTORC1; tyrosine; phenylalanine; phenylketonuria; Akt; AMPK
Congenital adrenal hyperplasia (CAH) is characterized by decreased adrenal hormone production due to enzymatic defects and subsequent rise of adrenocorticotrophic hormone that stimulates the adrenal cortex to become hyperplastic, and sometimes tumorous. As the pathophysiology is basically a defect in the biosynthesis of cortisol, one may not consider CAH in patients with hypercortisolism. We report a case of a 41-yr-old man with a 4 cm-sized left adrenal tumorous lesion mimicking Cushing's syndrome who was diagnosed with CAH. He had central obesity and acanthosis nigricans involving the axillae together with elevated 24-hr urine cortisol level, supporting the diagnosis of Cushing's syndrome. However, the 24-hr urine cortisol was suppressed by 95% with the low dose dexamethasone suppression test. CAH was suspected based on the history of precocious puberty, short stature and a profound suppression of cortisol production by dexamethasone. CAH was confirmed by a remarkably increased level of serum 17-hydroxyprogesterone level. Gene mutation analysis revealed a compound heterozygote mutation of CYP21A2 (I173N and R357W).
Congenital Adrenal Hyperplasia; 21-Hydroxylase Deficiency; Hypercortisolism
RhoB, a member of the Rho subfamily of small GTPases, mediates diverse cellular functions, including cytoskeletal organization, cell transformation and vesicle trafficking. The thymus undergoes progressive decline in its structure and function after puberty. We found that RhoB was expressed in thymic medullary epithelium. To investigate a role of RhoB in the regulation of thymic epithelial organization or thymocyte development, we analyzed the thymi of RhoB-deficient mice. RhoB-deficient mice were found to display earlier thymic atrophy. RhoB deficiency showed significant reductions in thymus weight and cellularity, beginning as early as 5 weeks of age. The enhanced expression of TGF-β receptor type II (TGFβRII) in thymic medullary epithelium was observed in RhoB-null mice. In addition, the expression of fibronectin, which is shown to be regulated by TGF-β signaling, was accordingly increased in the mutant thymic medulla. Since there is no age-related change of RhoB expression in the thymus, it is unlikely that RhoB in thymic epithelium directly contributes to age-related thymic involution. Nevertheless, our findings strongly support a physiological role of RhoB in regulation of thymus development and maintenance through the inhibition of TGF-β signaling in thymic medullary epithelium.
atrophy; RhoB; thymus; TGF-β
Low quality clinical trials have a possibility to have errors in the process of deriving the results and therefore distort the study. Quality assessment of clinical trial is necessary in order to prevent any clinical application erroneous results is important. Randomized controlled trial (RCT) is a design for evaluate the effectiveness of medical procedure. This study was conducted by extracting the RCTs from the original articles published in the Journal of Korean Medical Science (JKMS) from 1986 to 2011 and conducting a qualitative analysis using three types of analysis tools: Jadad scale, van Tulder scale and Cochrane Collaboration risk of bias Tool. To compare the quality of articles of JKMS, quality analysis of the RCTs published in Yonsei Medical Journal (YMJ) and Korean Journal of Internal Medicine was also conducted. In the JKMS, YMJ and Korean Journal of Internal Medicine, the quantitative increase of RCT presented over time was observed but no qualitative improvement of RCT was observed over time. From the results of this study, it is required for the researchers to plan for and perform higher quality studies.
Randomized Controlled Trial; Journal Article
Parathyroid neoplasms; Parathyroid cancer, adult; Hypercalcemia
Papillary thyroid carcinoma (PTC) is the most common malignant thyroid tumor, and 36-69% of PTC cases are caused by mutations in the BRAF gene. The substitution of a valine for a glutamic acid (V600E) comprises up to 95-100% of BRAF mutations; therefore, most diagnostic methods, including allele-specific PCR and real-time PCR, are designed to detect this mutation. Nevertheless, other mutations can also comprise the genetic background of PTC. Recently, a novel and sensitive technique called mutant enrichment with 3'-modified oligonucleotides (MEMO) PCR has been introduced. When we applied allelespecific PCR and MEMO-PCR for the detection of the BRAF V600E mutation, we found an unusual 3' bp deletion mutation (c.1799_1801delTGA) only when using MEMO-PCR. This deletion results in the introduction of a glutamic acid into the B-Raf activation segment (p.V600_K601delinsE), leading to an elevated basal kinase activity of BRAF. This is the first report of a rare 3 bp BRAF deletion in a PTC patient that could not be detected by allele-specific PCR.
Papillary thyroid carcinoma; BRAF; Deletion; Mutation; Mutant enrichment with 3'-modified oligonucleotides (MEMO) PCR; Korean
The acceptance rate for journal publication of the abstracts presented at the annual Korean Urological Association (KUA) meeting, the time to publication, and the effect of abstract characteristics on the publication pattern were analyzed and compared with data for abstracts from other major urological meetings.
Materials and Methods
A total of 1,005 abstracts listed in the abstract books of the 2006 (58th) and 2007 (59th) annual KUA meetings were analyzed, and their subsequent publication as listed in PubMed or KoreaMed between August 2006 and August 2011 was evaluated.
A total of 41.59% of abstracts were published as full-length reports. Abstracts on sexual dysfunction, neurourology, prostate cancer, basic research, and benign prostatic hyperplasia showed the highest publication rates (54%, 52.27%, 48%, 47.56%, and 45%, respectively). It took 19.01±12.83 months on average for abstracts to be published in a journal, whereas it took 25.24±14.64 months and 17.51±11.89 months for publication in foreign and Korean journals, respectively (p<0.001).
Approximately 40% of studies presented as abstracts at the KUA meeting are subsequently published as full-length articles. The KJU is the most targeted journal. The mean time to publication is 1.5 years, and publication seems to be influenced by the study subject.
Abstracts; Journal article; Peer review, research
A single surgeon skilled in conventional laparoscopic surgery used laparoendoscopic single-site surgery (LESS) to treat benign urological diseases. This study reports our surgical results and introduces a simple technique with tips based on our experience.
Materials and Methods
LESS surgery was performed on 116 patients by use of a homemade single-port device composed of an Alexis wound retractor and a powder-free surgical glove. Cases were 44 varicocelectomies (including 8 bilateral cases), 38 renal cyst marsupializations (including 3 bilateral cases), 26 ureterolithotomies (with 1 concomitant ureterolithotomy and contralateral renal cyst marsupialization), 4 prostatic enucleations, and 4 bladder rupture repairs. The mean patient age was 44.43±16.46 years (range, 11 to 76 years), and the male-to-female ratio was 87:29.
In one ureterolithotomy case, LESS was converted to conventional laparoscopic surgery. The mean operative time was 87.03±45.03 minutes, the estimated blood loss was 61.90 ml (range, 0 to 2,000 ml), and the mean hospital stay was 3.03±2.12 days. Two patients underwent single-port transvesical enucleation of the prostate (STEP) requiring patient-controlled anesthesia. No patients developed major complications, and all patients were satisfied, with 75.86% expressing a high degree of satisfaction.
We report successful treatment outcomes for LESS in 116 cases of benign urological disease. Our findings suggest that LESS can replace conventional laparoscopy.
Laparoscopy; Surgical procedures, minimally invasive; Urology
The aim of this study was to assess the prevalence of diabetes and to study the effects of excess growth hormone (GH) on insulin sensitivity and β-cell function in Korean acromegalic patients. One hundred and eighty-four acromegalic patients were analyzed to assess the prevalence of diabetes, and 52 naïve acromegalic patients were enrolled in order to analyze insulin sensitivity and insulin secretion. Patients underwent a 75 g oral glucose tolerance test with measurements of GH, glucose, insulin, and C-peptide levels. The insulin sensitivity index and β-cell function index were calculated and compared according to glucose status. Changes in the insulin sensitivity index and β-cell function index were evaluated one to two months after surgery. Of the 184 patients, 17.4% were in the normal glucose tolerance (NGT) group, 45.1% were in the pre-diabetic group and 37.5% were in the diabetic group. The insulin sensitivity index (ISI0,120) was significantly higher and the HOMA-IR was lower in the NGT compared to the diabetic group (P = 0.001 and P = 0.037, respectively). The ISI0,120 and disposition index were significantly improved after tumor resection. Our findings suggest that both insulin sensitivity and β-cell function are improved by tumor resection in acromegalic patients.
Insulin Resistance; Acromegaly
We analyzed the prescriptions of alpha-blockers and phosphodiesterase 5 inhibitors (PDE5Is) in the urology department as well as in other departments of the general hospital.
We investigated the frequency of prescription of alpha-blockers and PDE5Is from 3 general hospitals from January 1, 2007 to December 31, 2009. For alpha-blockers, data were collected from patients to whom alpha-blockers were prescribed from among patients recorded as having benign prostatic hyperplasia according to the 5th Korean Standard Classification of Diseases. For PDE5Is, data were collected from patients to whom PDE5Is were prescribed by the urology department and by other departments. Alpha-blockers were classified into tamsulosin, alfuzosin, doxazosin, and terazosin, whereas PDE5Is were classified into sildenafil, tadalafil, vardenafil, udenafil, and mirodenafil.
Alpha-blockers were prescribed to 11,436 patients in total over 3 years, and the total frequency of prescriptions was 68,565. Among other departments, the nephrology department had the highest frequency of prescription of 3,225 (4.7%), followed by the cardiology (3,101, 4.5%), neurology (2,576, 3.8%), endocrinology (2,400, 3.5%), pulmonology (1,102, 1.6%), and family medicine (915, 1.3%) departments in order. PDE5Is were prescribed to 2,854 patients in total over 3 years, and the total frequency of prescriptions was 10,558. The prescription frequency from the urology department was 4,900 (46.4%). Among other departments, the endocrinology department showed the highest prescription frequency of 3,488 (33.0%), followed by the neurology (542, 5.1%), cardiology (467, 4.4%), and family medicine (407, 3.9%) departments in order.
A high percentage of prescriptions of alpha-blockers and PDE5Is were from other departments. For more specialized medical care by urologists is required in the treatment of lower urinary tract symptoms and erectile dysfunction.
Adrenergic alpha-1 Receptor Antagonists; Phosphodiesterase 5 Inhibitors; Prescriptions
Transurethral resection of the prostate is the most common surgery for benign prostatic hyperplasia. However, it doesn't work best for men with very large prostate and bladder stones. Herein we report our initial experience with concomitant laparoendoscopic single-site surgery and finger-assisted single-port transvesical enucleation of the prostate for the treatment of the condition.
Urinary bladder calculi; Prostatic hyperplasia; Laparoscopy; Minimally invasive surgical procedures
Because low-quality trials may lead to erroneous conclusions, quality assessments are necessary. Thus, in this study, we scrutinized randomized controlled trials (RCTs) published in the Korean Journal of Urology (KJU) to assess their quantity and quality.
Materials and Methods
Upon extracting RCTs from all articles published in the KJU from 1991 to 2010, assessments were made on the basis of the Jadad scale and the adequacy of allocation concealment. The selections and assessments were performed independently by two researchers, and adjustment of the differences was done by a third-party researcher. In addition, the factors that may affect quality were analyzed.
A total of 3,516 original articles were searched and 28 RCTs were extracted. In the 1990s, RCTs constituted only 0.27% of the total original articles, but in the 2000s, RCTs constituted 1.34%. The mean total Jadad score increased from 1.6 points in the 1990s to 1.65 points in the 2000s. However, the percentage of "good quality" trials also increased from 20% to 30.43%. As for adequate allocation concealment, one study was observed in the 2000s. The aspect most lacking was appropriate dropout and double-blinding. Studies with medical interventions or funded or examined by institutional review boards tended to receive higher quality assessments.
Although RCTs consistently increased in both quantity and quality, in future studies, researchers should continue to strive toward achieving adequate allocation concealment and appropriate double-blinding. In addition, researchers must become more interested in receiving external funding and undergoing examination by institutional review boards.
Korea; Prospective studies; Random allocation; Urology
The identification of genome-wide cis-regulatory modules (CRMs) and characterization of their associated epigenetic features are fundamental steps toward the understanding of gene regulatory networks. Although integrative analysis of available genome-wide information can provide new biological insights, the lack of novel methodologies has become a major bottleneck. Here, we present a comprehensive analysis tool called combinatorial CRM decoder (CCD), which utilizes the publicly available information to identify and characterize genome-wide CRMs in a species of interest. CCD first defines a set of the epigenetic features which is significantly associated with a set of known CRMs as a code called ‘trace code’, and subsequently uses the trace code to pinpoint putative CRMs throughout the genome. Using 61 genome-wide data sets obtained from 17 independent mouse studies, CCD successfully catalogued ∼12 600 CRMs (five distinct classes) including polycomb repressive complex 2 target sites as well as imprinting control regions. Interestingly, we discovered that ∼4% of the identified CRMs belong to at least two different classes named ‘multi-functional CRM’, suggesting their functional importance for regulating spatiotemporal gene expression. From these examples, we show that CCD can be applied to any potential genome-wide datasets and therefore will shed light on unveiling genome-wide CRMs in various species.
Graves' disease (GD) is caused by thyroid-stimulating hormone receptor (TSHR) and thyroid-stimulating immunoglobulin (TSI). We used a recently introduced, technically enhanced TSI bioassay to assess its diagnostic value and determine the cut-off in patients in high iodine intake area.
In a cross-sectional setting, we collected serum from 67 patients with untreated GD, 130 with GD under treatment, 22 with GD in remission, 42 with Hashimoto's thyroiditis, 12 with subacute thyroiditis, 20 with postpartum thyroiditis, and 93 euthyroid controls. TSI was measured using the Thyretain™ bioassay, which is based on Chinese hamster ovary cells transfected with chimeric TSHR (Mc4). TSI levels are reported as a specimen-to-reference ratio percentage (SRR%).
The TSI levels in patients with GD (either treated or not) were significantly higher than those of the remaining patients (p < 0.05). The new bioassay showed a sensitivity of 97.0% and a specificity of 95.9% with a cut-off value of 123.0 SRR% for GD. A weak correlation was found between TSI and thyrotropin-binding inhibiting immunoglobulin (TBII) (rs = 0.259, p = 0.03), but no correlation was found between TSI and tri-iodothyronine or free thyroxine.
The Mc4-CHO bioassay showed comparable diagnostic value for GD with the conventional TBII assay. We propose a cut-off of 123.0 SRR% in areas where iodine intake is high.
Graves disease; Immunoglobulins, thyroid-stimulating; Biological assay; Receptors, thyrotropin; Thyrotropin-binding inhibitory immunoglobulin
Long-term survivors of childhood cancer appear to have an increased risk for the metabolic syndrome, subsequent type 2 diabetes and cardiovascular disease in adulthood compared to healthy children. The purpose of this study was to investigate the frequency of the metabolic syndrome and associated factors in childhood cancer survivors at a single center in Korea.
We performed a retrospective review of medical records of 98 childhood cancer survivors who were diagnosed and completed anticancer treatment at Samsung Medical Center, Seoul, Korea between Jan. 1996 and Dec. 2007. Parameters of metabolic syndrome were evaluated between Jan. 2008 and Dec. 2009. Clinical and biochemical findings including body fat percentage were analyzed.
A total of 19 (19.4%) patients had the metabolic syndrome. The median body fat percentage was 31.5%. The body mass index and waist circumference were positively correlated with the cranial irradiation dose (r=0.38, P<0.001 and r=0.44, P<0.00, respectively). Sixty-one (62.2%) patients had at least one abnormal lipid value. The triglyceride showed significant positive correlation with the body fat percentage (r=0.26, P=0.03). The high density lipoprotein cholesterol showed significant negative correlation with the percent body fat (r=-0.26, P=0.03).
Childhood cancer survivors should have thorough metabolic evaluation including measurement of body fat percentage even if they are not obese. A better understanding of the determinants of the metabolic syndrome during adolescence might provide preventive interventions for improving health outcomes in adulthood.
Cancer survivor; Metabolic syndrome; Body composition; Fat percentage
The use of graft materials in bladder mucosa has been examined in animal models, but debate exists over which materials are effective. Intestine has been used as a substitute in augmentation cystoplasty for patients with neuropathic bladder, but serious adverse effects of the operation have occurred in some instances. We report a case of a successful repair of an enterovesical fistula by use of bovine pericardium. The patient has remained well for 2.5 years. We suggest that bovine pericardium may be a suitable option as a bladder substitute.
Pericardium; Radiation injuries; Urinary bladder fistula
Most foreign bodies in the lower genitourinary tract are self-inserted via the urethra as the result of exotic impulses, psychometric problems, sexual curiosity, or sexual practice while intoxicated. Diagnosis of these foreign bodies can be done by clinical history, physical examination, and image studies of the patient. The treatment of foreign bodies is determined by their size, location, shape, and mobility. In most cases, minimally invasive procedures such as endoscopic removal are recommended to prevent bladder and urethral injuries. In some cases, however, surgical treatment should be done if the foreign bodies cannot be removed by the endoscopic procedure or further injuries are expected as a result of the endoscopic procedures. Herein we present 2 cases of self-inserted lower genitourinary foreign bodies with a brief review of the literature.
Foreign bodies; Urethra; Urinary bladder
Resistance to thyroid hormone (RTH) is an autosomal dominant hereditary disorder that is difficult to diagnose because of its rarity and variable clinical features. The magnitude of RTH is caused by mutations in the thyroid hormone receptor beta (TRβ) gene. We recently treated a 38-yr-old woman with RTH who had incidental papillary thyroid carcinoma. She presented with goiter and displayed elevated thyroid hormone levels with an unsuppressed TSH. She was determined to harbor a missense mutation of M310T in exon 9 of the TRβ gene, and diagnosed with generalized RTH. This mutation has not yet been reported in Korea. RTH is very rare and easily overlooked, but should be considered in patients who present with goiter and elevated thyroid hormone levels with an unsuppressed TSH. The association between thyroid cancer and RTH needs further study.
Thyroid Hormone Resistance Syndrom; Thyroid Hormone Receptor; Thyroid Hormone Receptors beta; Mutation; Thyroid Neoplasms
The existence of an association between lung function and metabolic syndrome (MetS) has been debated in cases involving non-obese subjects. To address this debate, we performed a cross-sectional study to investigate the association between lung function and MetS in both obese and non-obese populations.
The present study consisted of a total of 1,951 Korean male subjects. In this study group, we investigated relationships between lung function and MetS risk factors such as fasting serum glucose, systolic blood pressure (SBP), insulin resistance index, waist circumference (WC), and hemoglobin A1C level.
Forced vital capacity (FVC) values were significantly lower in the MetS group compared with those of the non-MetS group. In both non-obese (body mass index [BMI] < 25 kg/m2) and obese subjects (BMI ≥ 25 kg/m2), fasting serum glucose, hemoglobin A1C level, insulin resistance index, SBP, WC, and the prevalences of diabetes and MetS were significantly higher in subjects in the lowest FVC quartile compared with those in the highest FVC quartile. Odds ratios for the presence of MetS risk factors, after adjusting for age and height, ranged from 1.21 to 1.39 (P < 0.01) for a one standard deviation decrease in FVC.
The results of our study suggest that decreased vital capacity in Korean adult male subjects is associated with MetS, irrespective of obesity.
FEV1; FVC; Insulin resistance; Metabolic syndrome
Hypoparathyroidism is an abnormality of calcium metabolism characterized by low serum levels of parathyroid hormone in spite of hypocalcemia. The causes of hypoparathyroidism are numerous. Activating mutations in the calcium-sensing receptor (CaSR) gene are well-known causes of familial isolated hypoparathyroidism, also known as autosomal dominant hypocalcemia (ADH). Here we describe members of a Korean family with a heterozygous Pro221Leu mutation causing ADH. This case is the first report in Korea.
Hypoparathyroidism; Hypocalcemia; Receptors, Calcium-Sensing
This study was undertaken to evaluate the incidence and risk factors associated with relative adrenal insufficiency (RAI) in Korean critically-ill patients. All patients who were admitted to the Medical Intensive Care Unit (MICU) of Samsung Medical Center between January 1, 2006 and April 30, 2007 were prospectively evaluated using a short corticotropin stimulation test on the day of admission. RAI was defined as an increase in the serum cortisol level of <9 µg/dL from the baseline after administration of 250 µg of corticotropin. In all, 123 patients were recruited and overall the incidence of RAI was 44% (54/123). The presence of septic shock (P=0.001), the Simplified Acute Physiology Score (SAPS) II (P=0.003), the Sequential Organ Failure Assessment (SOFA) score (P=0.001), the mean heart rate (P=0.040), lactate levels (P=0.001), arterial pH (P=0.047), treatment with vasopressors at ICU admission (P=0.004), and the 28-day mortality (P=0.041) were significantly different between patients with and without RAI. The multivariate analysis showed that the SOFA score was an independent predictor of RAI in critically-ill patients (odd ratio=1.235, P=0.032). Our data suggest that RAI is frequently found in Korean critically-ill patients and that a high SOFA score is an independent predictor of RAI in these patients.
Adrenal Insufficiency; SOFA Score; Critical Illness
Cisplatin and carboplatin are the primary first-line therapies for the treatment of ovarian cancer. However, resistance to these platinum-based drugs occurs in the large majority of initially responsive tumors, resulting in fully chemoresistant, fatal disease. Although the precise mechanism(s) underlying the development of platinum resistance in late-stage ovarian cancer patients currently remains unknown, CpG-island (CGI) methylation, a phenomenon strongly associated with aberrant gene silencing and ovarian tumorigenesis, may contribute to this devastating condition.
To model the onset of drug resistance, and investigate DNA methylation and gene expression alterations associated with platinum resistance, we treated clonally derived, drug-sensitive A2780 epithelial ovarian cancer cells with increasing concentrations of cisplatin. After several cycles of drug selection, the isogenic drug-sensitive and -resistant pairs were subjected to global CGI methylation and mRNA expression microarray analyses. To identify chemoresistance-associated, biological pathways likely impacted by DNA methylation, promoter CGI methylation and mRNA expression profiles were integrated and subjected to pathway enrichment analysis.
Promoter CGI methylation revealed a positive association (Spearman correlation of 0.99) between the total number of hypermethylated CGIs and GI50 values (i.e., increased drug resistance) following successive cisplatin treatment cycles. In accord with that result, chemoresistance was reversible by DNA methylation inhibitors. Pathway enrichment analysis revealed hypermethylation-mediated repression of cell adhesion and tight junction pathways and hypomethylation-mediated activation of the cell growth-promoting pathways PI3K/Akt, TGF-beta, and cell cycle progression, which may contribute to the onset of chemoresistance in ovarian cancer cells.
Selective epigenetic disruption of distinct biological pathways was observed during development of platinum resistance in ovarian cancer. Integrated analysis of DNA methylation and gene expression may allow for the identification of new therapeutic targets and/or biomarkers prognostic of disease response. Finally, our results suggest that epigenetic therapies may facilitate the prevention or reversal of transcriptional repression responsible for chemoresistance and the restoration of sensitivity to platinum-based chemotherapeutics.
We have developed a new bioinformatics approach called ECMFinder (Evolutionary Conserved Motif Finder). This program searches for a given DNA motif within the entire genome of one species and uses the gene association information of a potential transcription factor-binding site (TFBS) to screen the homologous regions of a second and third species. If multiple species have this potential TFBS in homologous positions, this program recognizes the identified TFBS as an evolutionary conserved motif (ECM). This program outputs a list of ECMs, which can be uploaded as a Custom Track in the UCSC genome browser and can be visualized along with other available data. The feasibility of this approach was tested by searching the genomes of three mammals (human, mouse and cow) with the DNA-binding motifs of YY1 and CTCF. This program successfully identified many clustered YY1- and CTCF-binding sites that are conserved among these species but were previously undetected. In particular, this program identified CTCF-binding sites that are located close to the Dlk1, Magel2 and Cdkn1c imprinted genes. Individual ChIP experiments confirmed the in vivo binding of the YY1 and CTCF proteins to most of these newly discovered binding sites, demonstrating the feasibility and usefulness of ECMFinder.