Human IAPP-induced pancreatic ß cell toxicity and its regulation by autophagy.

Pancreatic islets in patients with type 2 diabetes mellitus (T2DM) are characterized by loss of ß cells and formation of amyloid deposits derived from islet amyloid polypeptide (IAPP). Here we demonstrated that treatment of INS-1 cells with human IAPP (hIAPP) enhances cell death, inhibits cytoproliferation, and increases autophagosome formation. Furthermore, inhibition of autophagy increased the vulnerability of ß cells to the cytotoxic effects of hIAPP. Based on these in vitro findings, we examined the pathogenic role of hIAPP and its relation to autophagy in hIAPP-knockin mice. In animals fed a standard diet, hIAPP had no toxic effects on ß cell function; however, hIAPP-knockin mice did not exhibit a high-fat-diet-induced compensatory increase in ß cell mass, which was due to limited ß cell proliferation and enhanced ß cell apoptosis. Importantly, expression of hIAPP in mice with a ß cell-specific autophagy defect resulted in substantial deterioration of glucose tolerance and dispersed cytoplasmic expression of p62-associated toxic oligomers, which were otherwise sequestrated within p62-positive inclusions. Together, our results indicate that increased insulin resistance in combination with reduced autophagy may enhance the toxic potential of hIAPP and enhance ß cell dysfunction and progression of T2DM.

A simple method for gene phasing using mate pair sequencing.

BACKGROUND: Recessive genes cause disease when both copies are affected by mutant loci. Resolving the cis/trans relationship of variations has been an important problem both for researchers, and increasingly, clinicians. Of particular concern are patients who have two heterozygous disease-causing mutations and could be diagnosed as affected (one mutation on each allele) or as phenotypically normal (both mutations on the same allele). Several methods are currently used to phase genes, however due to cost, complexity and/or low sensitivity they are not suitable for clinical purposes. METHODS: Long-range amplification was used to select and enrich the target gene (CYP21A2) followed by modified mate-pair sequencing. Fragments that mapped coincidently to two heterozygous sites were identified and used for statistical analysis. RESULTS: Probabilities for cis/trans relationships between heterozygous positions were calculated along with 99% confidence intervals over the entire length of our 10 kb amplicons. The quality of phasing was closely related to the depth of coverage and the number of erroneous reads. Most of the error was found to have been introduced by recombination in the PCR reaction. CONCLUSIONS: We have developed a simple method utilizing massively parallel sequencing that is capable of resolving two alleles containing multiple heterozygous positions. This method stands out among other phasing tools because it provides quantitative results allowing confident haplotype calls.

RNA sequencing identifies multiple fusion transcripts, differentially expressed genes, and reduced expression of immune function genes in BRAF (V600E) mutant vs BRAF wild-type papillary thyroid carcinoma.

CONTEXT: The BRAF V600E mutation (BRAF-MUT) confers an aggressive phenotype in papillary thyroid carcinoma, but unidentified additional genomic abnormalities may be required for full phenotypic expression. OBJECTIVE: RNA sequencing (RNA-Seq) was performed to identify genes differentially expressed between BRAF-MUT and BRAF wild-type (BRAF-WT) tumors and to correlate changes to patient clinical status. DESIGN: BRAF-MUT and BRAF-WT tumors were identified in patients with T1N0 and T2-3N1 tumors evaluated in a referral medical center. Gene expression levels were determined (RNA-Seq) and fusion transcripts were detected. Multiplexed capture/detection and digital counting of mRNA transcripts (nCounter, NanoString Technologies) validated RNA-Seq data for immune system-related genes. PATIENTS: BRAF-MUT patients included nine women, three men; nine were TNM stage I and three were stage III. Three (25%) had tumor infiltrating lymphocytes. BRAF-WT included five women, three men; all were stage I, and five (62.5%) had tumor infiltrating lymphocytes. RESULTS: RNA-Seq identified 560 of 13 085 genes differentially expressed between BRAF-MUT and BRAF-WT tumors. Approximately 10% of these genes were related to MetaCore immune function pathways; 51 were underexpressed in BRAF-MUT tumors, whereas 4 (HLAG, CXCL14, TIMP1, IL1RAP) were overexpressed. The four most differentially overexpressed immune genes in BRAF-WT tumors (IL1B; CCL19; CCL21; CXCR4) correlated with lymphocyte infiltration. nCounter confirmed the RNA-Seq expression level data. Eleven different high-confidence fusion transcripts were detected (four interchromosomal; seven intrachromosomal) in 13 of 20 tumors. All in-frame fusions were validated by RT-PCR. CONCLUSION: BRAF-MUT papillary thyroid cancers have reduced expression of immune/inflammatory response genes compared with BRAF-WT tumors and correlate with lymphocyte infiltration. In contrast, HLA-G and CXCL14 are overexpressed in BRAF-MUT tumors. Sixty-five percent of tumors had between one and three fusion transcripts. Functional studies will be required to determine the potential role of these newly identified genomic abnormalities in contributing to the aggressiveness of BRAF-MUT and BRAF-WT tumors.

Development of a multidisciplinary, multicampus subspecialty practice in endocrine cancers.

PURPOSE: Relative to more abundant neoplasms, endocrine cancers have been historically neglected, yet their incidence is increasing. We therefore sought to build interest in endocrine cancers, improve physician experience, and develop innovative approaches to treating patients with these neoplasms. METHODS: Between 2005 and 2010, we developed a multidisciplinary Endocrine Malignancies Disease Oriented Group involving all three Mayo Clinic campuses (Rochester, MN; Jacksonville, FL; and Scottsdale, AZ). In response to higher demand at the Rochester campus, we sought to develop a Subspecialty Tumor Group and an Endocrine Malignancies Tumor Clinic within the Division of Medical Oncology. RESULTS: The intended groups were successfully formed. We experienced difficulty in integration of the Mayo Scottsdale campus resulting from local uncertainty as to whether patient volumes would be sufficient to sustain the effort at that campus and difficulty in developing enthusiasm among clinicians otherwise engaged in a busy clinical practice. But these obstacles were ultimately overcome. In addition, with respect to the newly formed medical oncology subspecialty endocrine malignancies group, appointment volumes quadrupled within the first year and increased seven times within two years. The number of active therapeutic endocrine malignancies clinical trials also increased from one in 2005 to five in 2009, with all three Mayo campuses participating. CONCLUSION: The development of subspecialty tumor groups for uncommon malignancies represents an effective approach to building experience, increasing patient volumes and referrals, and fostering development of increased therapeutic options and clinical trials for patients afflicted with otherwise historically neglected cancers.

Development of a multidisciplinary, multicampus subspecialty practice in endocrine cancers.

OBJECTIVES: Relative to more abundant neoplasms, endocrine cancers have been historically neglected, yet their incidence is increasing. We therefore sought to build interest in endocrine cancers, improve physician experience, and develop innovative approaches to treating patients with these neoplasms. METHODS: Between 2005 and 2010, we developed a multidisciplinary Endocrine Malignancies Disease Oriented Group involving all 3 Mayo Clinic campuses (Rochester, Minnesota; Jacksonville, Florida; and Scottsdale, Arizona). In response to higher demand at the Rochester campus, we sought to develop a Subspecialty Tumor Group and an Endocrine Malignancies Tumor Clinic within the Division of Medical Oncology. RESULTS: The intended groups were successfully formed. We experienced difficulty in integration of the Mayo Scottsdale campus resulting from local uncertainty as to whether patient volumes would be sufficient to sustain the effort at that campus and difficulty in developing enthusiasm among clinicians otherwise engaged in a busy clinical practice. But these obstacles were ultimately overcome. In addition, with respect to the newly formed medical oncology subspecialty endocrine malignancies group, appointment volumes quadrupled within the first year and increased 7 times within 2 years. The number of active therapeutic endocrine malignancies clinical trials also increased from 1 in 2005 to 5 in 2009, with all 3 Mayo campuses participating. CONCLUSIONS: The development of subspecialty tumor groups for uncommon malignancies represents an effective approach to building experience, increasing patient volumes and referrals, and fostering development of increased therapeutic options and clinical trials for patients afflicted with otherwise historically neglected cancers.

Expression of wild-type and mutant S20G hIAPP in physiologic knock-in mouse models fails to induce islet amyloid formation, but induces mild glucose intolerance.

UNLABELLED: Aims/Introduction: Human islet polypeptide S20G mutation (hIAPP(S20G)) is associated with earlier onset type 2 diabetes and increased amyloidogenicity and cytotoxicity in vitro vs wild-type hIAPP (hIAPP(WT)), suggesting that amyloidogenesis may be pathogenic for type 2 diabetes. We compared the contributions of hIAPP(S20G) and hIAPP(WT) toward intra islet amyloid formation and development of type 2 diabetes in a unique physiologic knock-in mouse model. MATERIALS AND METHODS: We replaced the mouse IAPP gene (M allele) with hIAPP(WT) (W allele) and hIAPP(S20G) (G allele) via homologous recombination and backbred transgenic mice against C57Bl/6 strain 5 generations to minimize genetic variation. Mice (3 month old) were maintained on control (CD) or high fat diet (HFD) for 15 months and studied at 3 month intervals by oral glucose tolerance testing (OGTT) and pancreas histology to assess glucose homeostastis, amyloidogeneisis, islet mass, ß cell replication, and apoptosis. RESULTS: IAPP blood levels were indistinguishable in all mice. WW and GW mice maintained on both diets lacked intraislet amyloid at all ages. On both diets relative to MM controls WW and GW mice exhibit glucose intolerance (P < 0.008) with no differences in insulin secretion. However, GW mice secreted significantly more insulin (P < 0.03 that WW mice on both diets throughout the study. By 12 months on the high fat diet all mice increased their ß cell mass about 3-fold and were indistinguishable. CONCLUSIONS: Physiologic expression of hIAPP(WT) and hIAPP(S20G) in C57Bl/6 mice produces mild glucose intolerance with inappropriately normal insulin secretion that is independent of intraislet amyloid formation. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2011.00166.x, 2011).

The Putative PAX8/PPARγ Fusion Oncoprotein Exhibits Partial Tumor Suppressor Activity through Up-Regulation of Micro-RNA-122 and Dominant-Negative PPARγ Activity.

In vitro studies have demonstrated that the PAX8/PPARγ fusion protein (PPFP), which occurs frequently in follicular thyroid carcinomas (FTC), exhibits oncogenic activity. However, paradoxically, a meta-analysis of extant tumor outcome studies indicates that 68% of FTC-expressing PPFP are minimally invasive compared to only 32% of those lacking PPFP (χ(2) = 6.86, P = 0.008), suggesting that PPFP favorably impacts FTC outcomes. In studies designed to distinguish benign thyroid neoplasms from thyroid carcinomas, the previously identified tumor suppressor miR-122, a major liver micro-RNA (miR) that is decreased in hepatocellular carcinoma, was increased 8.9-fold (P < 0.05) in all FTC versus normal, 9.2-fold in FTC versus FA (P < 0.05), and 16.8-fold (P < 0.001) in FTC + PPFP versus FTC - PPFP. Constitutive expression of PPFP in the FTC-derived cell line WRO (WRO-PPFP) caused a 5-fold increase of miR-122 expression (P < 0.05) and a striking 5.1-fold reduction (P < 0.0001) in tumor progression compared to WRO-vector cells in a mouse xenograft model. Constitutive expression of either miR-122 or a dominant-negative PPARγ mutant in WRO cells was less effective than PPFP at inhibiting xenograft tumor progression (1.8-fold [P < 0.001] and 1.7-fold [P < 0.03], respectively). PPFP-induced up-regulation of miR-122 expression was independent of its known dominant-negative PPARγ activity. Up-regulation of miR-122 negatively regulates ADAM-17, a known downstream target, in thyroid cells, suggesting an antiangiogenic mechanism in thyroid carcinoma. This latter inference is directly supported by reduced CD-31 expression in WRO xenografts expressing PPFP, miR-122, and DN-PPARγ. We conclude that, in addition to its apparent oncogenic potential in vitro, PPFP exhibits paradoxical tumor suppressor activity in vivo, mediated by multiple mechanisms including up-regulation of miR-122 and dominant-negative inhibition of PPARγ activity.

Unique cellular and mitochondrial defects mediate FK506-induced islet ß-cell dysfunction.

OBJECTIVE: To determine biological mechanisms involved in posttransplantation diabetes mellitus caused by the immunosuppressant tacrolimus (FK506). METHODS: INS-1 cells and isolated rat islets were incubated with vehicle or FK506 and harvested at 24-hr intervals. Cells were assessed for viability, apoptosis, proliferation, cell insulin secretion, and content. Gene expression studies by microarray analysis, quantitative polymerase chain reaction, and motifADE analysis of the microarray data identified potential FK506-mediated pathways and regulatory motifs. Mitochondrial functions, including cell respiration, mitochondrial content, and bioenergetics were assessed. RESULTS: Cell replication, viability, insulin secretion, oxygen consumption, and mitochondrial content were decreased (P<0.05) 1.2-, 1.27-, 1.77-, 1.32-, and 1.43-fold, respectively, after 48-hr FK506 treatment. Differences increased with time. FK506 (50 ng/mL) and cyclosporine A (800 ng/mL) had comparable effects. FK506 significantly decreased mitochondrial content and mitochondrial bioenergetics and showed a trend toward decreased oxygen consumption in isolated islets. Cell apoptosis and proliferation, mitochondrial DNA copy number, and ATP:ADP ratios were not significantly affected. Pathway analysis of microarray data showed FK506 modification of pathways involving ATP metabolism, membrane trafficking, and cytoskeleton remodeling. PGC1-α mRNA was down-regulated by FK506. MotifADE identified nuclear factor of activated T-cells, an important mediator of ß-cell survival and function, as a potential factor mediating both up- and down-regulation of gene expression. CONCLUSIONS: At pharmacologically relevant concentrations, FK506 decreases insulin secretion and reduces mitochondrial density and function without changing apoptosis rates, suggesting that posttransplantation diabetes induced by FK506 may be mediated by its effects on mitochondrial function.

Autophagy protects against human islet amyloid polypeptide-associated apoptosis.

UNLABELLED: Aims/Introduction: Islets in type 2 diabetes are characterized by deposition of islet amyloid polypeptide (IAPP) as well as ß-cell dysfunction. The unique amyloidogenic character of human (h)IAPP is associated with cytotoxicity. Autophagy is a ubiquitous system of cellular recycling that contributes to cell survival. Thus, we examined whether autophagy could ameliorate hIAPP-associated cytotoxicity. MATERIALS AND METHODS: First, we used a COS-1 cell model, lacking endogenous IAPP that might affect cytotoxicity related to exogenous hIAPP. Next, we used the mouse ß-cell line, MIN-6 cells. Both cells were transfected with hIAPP or rat (r)IAPP expression constructs, or transfected with bicistronic vectors expressing green fluorescent protein (GFP) and either hIAPP or rIAPP for flow cytometry analysis. Cell viability and apoptosis markers were studied in relation to chemical or genetic modulation of autophagy. RESULTS: The viability of cells expressing hIAPP was significantly decreased as compared with those expressing rIAPP and the cleavage of pro-caspase-3 was elevated in hIAPP-transfected cells. The formation of autophagosomes and the conversion of microtubule-associated protein light chain 3B I to II were elevated in hIAPP-expressing cells. The viability of hIAPP-expressing cells was increased after treatment with rapamycin, an inducer of autophagy, and decreased after treatment with 3-methyladenine, an inhibitor of autophagy. In MIN-6 cells, annexin positive cells were increased by 3-methyladenine and decreased by rapamycin using flow cytometry. Knocking down of the autophagy protein 5 gene decreased hIAPP-transfected cell viability. CONCLUSIONS: Autophagy is co-localized with hIAPP expression and it plays a protective role in hIAPP-associated apoptosis. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2010.00065.x, 2010).

Distinct genetic changes characterise multifocality and diverse histological subtypes in papillary thyroid carcinoma.

AIMS: This study was undertaken to investigate the genetic factors underlying the development of multifocality and phenotypic diversity in multifocal papillary thyroid carcinoma (mPTC). METHODS: Loss of heterozygosity (LOH) and BRAF(V600E) mutation status were analysed in a total of 55 individual tumour foci from 18 cases of mPTC. The genetic findings and morphology of tumour foci were then compared. RESULTS: Multifocal PTC LOH rates were higher than observed previously in solitary PTC. Different patterns of LOH and BRAF(V600E) positivity separated follicular variant tumours and tumour foci from other PTC histological subtypes. In five cases, genetic alterations were detected in morphologically normal thyroid epithelium. CONCLUSIONS: These findings support the concept that multifocal PTCs develop through clonal selection from a field of pre-neoplastic cells, with morphotype differentiation correlating with specific tumour-genetic alterations. The relatively high genetic disarray in multifocal PTC may underlie their ability to spread throughout the thyroid gland.

Evaluation of the PAX8/PPARG translocation in follicular thyroid cancer with a 4-color reverse-transcription PCR assay and automated high-resolution fragment analysis.

BACKGROUND: Molecular testing of thyroid malignancies, in combination with cytologic and histologic examination, is becoming increasingly attractive as a tool for refining traditional morphologic diagnosis. The molecular changes associated with follicular thyroid carcinoma (FTC) are point mutations in RAS oncogenes or the presence of PAX8/PPARG (paired box 8/peroxisome proliferator-activated receptor gamma) rearrangement. METHODS: We developed and validated a clinical assay for the detection of PAX8/PPARG rearrangements that uses a 4-color reverse-transcription PCR (RT-PCR) assay and high-resolution fragment analysis. RESULTS: The RT-PCR assay is applicable for detecting the various described fusion transcripts of PAX8/PPARG in formalin-fixed, paraffin-embedded thyroid tissue and in fine-needle aspirate biopsy washes from thyroid nodules. The analytical sensitivity of the assay is 1 abnormal cell in a background of 100-10 000 translocation-negative cells. A comparison of the RT-PCR assay with dual-fusion fluorescence in situ hybridization showed an overall concordance of 95%. With this assay, we obtained a prevalence for the PAX8/PPARG rearrangement in FTC of 62% (13 of 21 cases), compared with a 5% prevalence (3 of 55) for other follicular cell-derived neoplasms. CONCLUSIONS: The introduction of this assay into clinical practice could provide useful information for the diagnosis and possibly for the prognosis and treatment of thyroid cancer in the future.

The role of the PAX8/PPARgamma fusion oncogene in the pathogenesis of follicular thyroid cancer.

When identified at early stages, most well-differentiated thyroid cancers are readily treated and yield excellent outcomes. Follicular thyroid cancer (FTC) however, when diagnosed at a late stage, may be very resistant to treatment, and exhibits 10-year survival rates less than 40%. Despite substantial progress in recent years, we still have limited understanding of the molecular and biological interrelationships between the various subtypes of benign and malignant follicular thyroid neoplasms. In contrast to the wealth of information available regarding papillary thyroid carcinoma (PTC), the triggering mechanisms of FTC development and the major underlying genetic alterations leading to follicular thyroid carcinogenesis remain obscure. Recent studies have focused on a chromosomal translocation, t(2;3) (q13;p25), fusing PAX8, a transcription factor that is essential for normal thyroid gland development, with the peroxisome proliferator-activated receptor gamma (PPARgamma), a member of the steroid/thyroid nuclear receptor family. This chromatin rearrangement results in the expression of a PAX8/PPARgamma fusion protein, designated PPFP, whose incidence is relatively common in FTC and may represent an initiating event in the genesis of FTC. Here we review progress on the studies of PPFP that assess its involvement in FTC tumorigenesis.

The Role of the PAX8/PPARgamma Fusion Oncogene in Thyroid Cancer.

Thyroid cancer is uncommon and exhibits relatively low mortality rates. However, a subset of patients experience inexorable growth, metastatic spread, and mortality. Unfortunately, for these patients, there have been few significant advances in treatment during the last 50 years. While substantial advances have been made in recent years about the molecular genetic events underlying papillary thyroid cancer, the more aggressive follicular thyroid cancer remains poorly understood. The recent discovery of the PAX8/PPARgamma translocation in follicular thyroid carcinoma has promoted progress in the role of PPARgamma as a tumor suppressor and potential therapeutic target. The PAX8/PPARgamma fusion gene appears to be an oncogene. It is most often expressed in follicular carcinomas and exerts a dominant-negative effect on wild-type PPARgamma, and stimulates transcription of PAX8-responsive promoters. PPARgamma agonists have shown promising results in vitro, although very few studies have been conducted to assess the clinical impact of these agents.

Opposing roles for p16Ink4a and p19Arf in senescence and ageing caused by BubR1 insufficiency.

Expression of p16(Ink4a) and p19(Arf) increases with age in both rodent and human tissues. However, whether these tumour suppressors are effectors of ageing remains unclear, mainly because knockout mice lacking p16(Ink4a) or p19(Arf) die early of tumours. Here, we show that skeletal muscle and fat, two tissues that develop early ageing-associated phenotypes in response to BubR1 insufficiency, have high levels of p16(Ink4a) and p19(Arf). Inactivation of p16(Ink4a) in BubR1-insufficient mice attenuates both cellular senescence and premature ageing in these tissues. Conversely, p19(Arf) inactivation exacerbates senescence and ageing in BubR1 mutant mice. Thus, we identify BubR1 insufficiency as a trigger for activation of the Cdkn2a locus in certain mouse tissues, and demonstrate that p16(Ink4a) is an effector and p19(Arf) an attenuator of senescence and ageing in these tissues.

The paired box-8/peroxisome proliferator-activated receptor-gamma oncogene in thyroid tumorigenesis.

The American Cancer Society estimates 30,180 new cases of thyroid cancer in the United States in 2006. Of all thyroid cancers, 15-20% are follicular thyroid carcinoma (FTC), making this the second most common thyroid malignancy (after papillary carcinoma). A proportion of FTC has been found to be associated with a chromosomal translocation, t (2, 3)(q13;p25), which fuses the thyroid-specific transcription factor paired box-8 with the peroxisome proliferator-activated receptor-gamma nuclear receptor, a ubiquitously expressed transcription factor. This fusion event causes expression of a paired box-8/peroxisome proliferator-activated receptor-gamma fusion protein (PPFP). PPFP is detected in approximately 30% of FTC. In this report we review data on the role of PPFP in FTC, its mechanism of oncogenesis, and PPFP targeting as a strategy in thyroid cancer treatment.

The human growth hormone gene contains a silencer embedded within an Alu repeat in the 3'-flanking region.

Alu family sequences are middle repetitive short interspersed elements (SINEs) dispersed throughout vertebrate genomes that can modulate gene transcription. The human (h) GH locus contains 44 complete and four partial Alu elements. An Sx Alu repeat lies in close proximity to the hGH-1 and hGH-2 genes in the 3'-flanking region. Deletion of the Sx Alu repeat in reporter constructs containing hGH-1 3'-flanking sequences increased reporter activity in transfected pituitary GC cells, suggesting this region contained a repressor element. Analysis of multiple deletion fragments from the 3'-flanking region of the hGH-1 gene revealed a strong orientation- and position-independent silencing activity mapping between nucleotides 2158 and 2572 encompassing the Sx Alu repeat. Refined mapping revealed that the silencer was a complex element comprising four discrete entities, including a core repressor domain (CRD), an antisilencer domain (ASE) that contains elements mediating the orientation-independent silencer activity, and two domains flanking the CRD/ASE that modulate silencer activity in a CRD-dependent manner. The upstream modulator domain is also required for orientation-independent silencer function. EMSA with DNA fragments representing all of the silencer domains yielded a complex pattern of DNA-protein interactions indicating that numerous GC cell nuclear proteins bind specifically to the CRD, ASE, and modulator domains. The silencer is GH promoter dependent and, in turn, its presence decreases the rate of promoter-associated histone acetylation resulting in a significant decrease of RNA polymerase II recruitment to the promoter. The silencer may provide for complex regulatory control of hGH gene expression in pituitary cells.

hMLH1 promoter methylation and silencing in primary endometrial cancers are associated with specific alterations in MBDs occupancy and histone modifications.

OBJECTIVE: To investigate the relationship between hMLH1 promoter methylation and changes in chromatin composition. To study how the occupancy of methyl CpG binding domain proteins (MBDs) and histone acetylation/methylation in hMLH1 promoter may participate in hMLH1 silencing. METHODS: 64 endometrial cancer samples were screened for hMLH1 mRNA expression. hMLH1 promoter methylation status was confirmed by methylation-specific PCR in cancers with high and low levels of hMLH1 expression. Chromatin immunoprecipitation was performed to compare the MBD occupancy and histone modifications between the methylated/silenced and unmethylated/active hMLH1 genes in multiple primary endometrial cancers. RESULTS: We demonstrated that MeCP2, MBD1 and MBD2, but not MBD3 and MBD4, specifically bind to methylated hMLH1 promoters. Hyperacetylated histones H3 and H4 were found to be associated with the unmethylated and transcriptionally active hMLH1 promoters. While H3 lysine-4 methylation was present in unmethylated hMLH1 promoters, H3 lysine-9 methylation was found exclusively in methylated promoters. Western blot analysis showed that similar global levels of MBDs and histones were present in the two cancer groups with high and low hMLH1 expression. CONCLUSIONS: A distinct combination of MBDs and histone modification is associated with the silencing of the hMLH1 gene. The changes in hMLH1 chromatin composition are closely related to methylation status of hMLH1 promoters. These changes are not accounted by the global expression levels of MBDs and histones in endometrial cancers.

Epigenetic-mediated upregulation of progesterone receptor B gene in endometrial cancer cell lines.

OBJECTIVES: To determine if epigenetic interference can restore progesterone receptor-B (PR-B) expression in PR-B negative endometrial adenocarcinoma cell lines, and to characterize the kinetics of PR-B induction mediated by DNA methyltransferase and histone deacetylase inhibitors. METHODS: The PR-B negative endometrioid cancer cell lines KLE and HEC-1B were used as study models. PR-B mRNA and protein expression levels were measured using real-time PCR and Western blot analysis, respectively. DNA methylation levels of the PR-B promoter were determined by methylation-specific PCR. Dose-response correlations and the duration of response to aza-deoxycytidine (ADC) and trichostatin A (TSA) were characterized. Cell responses to prolonged and repeated drug treatment were also examined. RESULTS: Relatively low concentrations of ADC and TSA over a 24-h period induced PR-B expression. Furthermore, ADC and TSA acted synergistically to reactivate PR-B expression. Depending on the cell line used, PR-B mRNA was induced 10-110 fold. This elevated PR-B expression continued for 48 h after drug withdrawal. Sustained upregulation of PR-B mRNA and protein was observed during prolonged and repeated drug treatment. CONCLUSION: The epigenetically silenced PR-B gene remains sensitive to changes in DNA demethylation and histone acetylation in uterine adenocarcinoma cell lines. Treatment with ADC and/or TSA results in a robust and sustainable PR-B upregulation. These small molecule epigenetic modifying agents may be used to sensitize poorly differentiated, PR-B negative endometrial cancers to progestational therapy.

Histone deacetylase inhibitors decrease DNA methyltransferase-3B messenger RNA stability and down-regulate de novo DNA methyltransferase activity in human endometrial cells.

It is well known that the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) acts synergistically with the DNA methyltransferase (DNMT) inhibitor 5-aza-2'-deoxycytidine (ADC) to reactivate DNA methylation-silenced genes. Moreover, in several studies, TSA was capable of inducing DNA demethylation even in the absence of ADC. Here we describe a mechanism by which HDAC inhibitors affect DNA methylation through their regulation on DNMT3B, a methyltransferase responsible for de novo DNA methylation. Using quantitative real-time PCR and Western blot analysis, we show that TSA down-regulates DNMT3B mRNA and protein expression in human endometrial cancer cells. This decrease in DNMT3B mRNA results in a significant reduction in de novo methylation activities. Further experiments indicated that TSA decreases DNMT3B mRNA stability and reduces its half-life from approximately 4 to 2.5 hours. We established that protein synthesis is required for posttranscriptional regulation, suggesting the involvement of an RNase and/or key mRNA stabilization factor(s) controlling the DNMT3B mRNA stability. Therefore, TSA may not only modify histone acetylation, but also potentially alter DNA methylation. Since the HDAC inhibitors are frequently used in epigenetic studies and are considered to be promising anticancer drugs, these new findings will have implications in both laboratory and clinical settings.

Opposite alterations of DNA methyltransferase gene expression in endometrioid and serous endometrial cancers.

OBJECTIVE: To examine the DNA methyltransferase (DNMT) mRNA and protein levels in endometrioid and serous cancers and to study the relationship between DNA methyltransferase expression and endometrial cancer development. METHODS: Normal endometrium, Grade I and Grade III endometrioid carcinoma tissues and cell lines, as well as serous cancer tissues, were analyzed for DNMT expression. Real-time PCR and Western blot techniques were employed to measure the mRNA and protein levels of the four DNA methyltransferases, DNMT1, DNMT2, DNMT3A, and DNMT3B. Immunohistochemistry was performed to detect alterations in DNMT nuclear localization and spatial organization patterns. RESULTS: While DNMT2 and DNMT3A expression appear to be normal, two- to fourfold increase in DNMT1 and DNMT3B were found in both Grade I and Grade III endometrioid cancers. In addition, the poorly differentiated cell lines expressed relatively higher DNMT levels than well-differentiated cells. In contrast to endometrioid carcinomas, serous cancers expressed substantially lower levels of DNMT1 and DNMT3B than normal controls, with four- and twofold reduction observed in DNMT1 and DNMT3B mRNA levels, respectively. Western blot analysis confirmed opposite expression patterns of DNMT1 and DNMT3B protein in endometrioid and serous cancers. Immunohistochemistry showed normal nuclear localization of DNMT1 and DNMT3B in Type I and Type II cancer specimens as well as cell cultures. CONCLUSION: Two opposite DNMT expression patterns were identified in endometrioid and serous cancers. The concerted upregulation in maintenance and de novo DNA methyltransferases in endometrioid carcinomas is consistent with a tendency for gene-specific hypermethylation observed in this histologic subtype, and may be implicated in tumor suppressor silencing. In contrast, the downregulation of maintenance and de novo DNA methyltransferases in serous cancers suggests that these tumors may contain hypomethylated genomic DNA, which has been associated with a higher mutation rate and is consistent with the known pathogenesis of serous-specific phenotypes. Taken together, the data suggest that divergent DNA methylation pathways may be implicated in the development of Type I and Type II endometrial cancers.