Department of Gynaecologic Oncology, Radiumhemmet, Institute of Oncology and Pathology, Karolinska University Hospital and Institute, Stockholm, Sweden. caroline.lundgren@karolinska.se
The objective of this study was to explore the protein expression pattern in normal endometrial mucosa (n = 5) and endometrial carcinoma (n = 15) of low (diploid) and high (aneuploid) malignancy potential by two-dimensional gel electrophoresis (2-DE). The specimens were evaluated for histopathologic subtype, stage and grade in relation to DNA ploidy. A match-set consisting of five samples from normal endometrium, eight diploid and seven aneuploid tumours was created. All the diploid and three of the aneuploid tumours were of endometrioid subtype, while the remaining four were of uterine seropapillary type. There were 192 protein spots differentiating diploid tumours from normal endometrium and 238 protein spots were separating aneuploid tumours from normal endometrium (p
The expression of the 17 beta-hydroxysteroid dehydrogenase (17-HSD) gene in a series of human breast cancer cell lines was studied by Northern blot hybridization with a cDNA probe and by a time-resolved immunofluorometric assay using polyclonal antibodies against the enzyme protein. The 17-HSD enzyme protein concentration was measured in the 800 x g cell extract. A high concentration was measured in the BT-20 cell line, corresponding to one-fourth of the average concentration in placental tissue. Western blot analysis indicated that the antigen corresponded to a single Mr 35,000 band. In 2 other cell lines (MDA-MB-361 and T-47D), the 17-HSD protein concentration was much lower, but still measurable, whereas in the remaining 5 cell lines (HBL-100, MCF-7, MDA-MB-231, MDA-MB-468, and ZR-75-1) it was below the detection limit of the assay. Treatment of the cells for 5 days with the synthetic progestin, ORG2058, resulted in an increase of the 17-HSD protein concentration only in the T-47D cell line. By Northern blot analysis, a low level of 2.3-kilobase mRNA transcripts was detected in all 8 cell lines. In addition, a 1.3-kilobase 17-HSD mRNA was present in the samples from the 3 cell lines containing measurable amounts of 17-HSD protein in the cell extract, and the band intensities were proportional to the amount of protein measured with the immunofluorometric assay. Only in the T-47D cell line did progestin treatment correspond to an increased amount of the 17-HSD 1.3-kilobase mRNA. These results suggest that the 1.3-kilobase mRNA for 17-HSD is the form most closely associated with protein expression and is also the only form responding to the progestin induction of the 17-HSD gene.
17 beta-Hydroxysteroid dehydrogenases (17HSDs) regulate the biological activity of sex steroid hormones in a variety of tissues by catalyzing the interconversions between highly active steroid hormones, e.g. estradiol and testosterone, and corresponding less active hormones, estrone and androstenedione. Epidemiological and endocrine evidence indicates that estrogens play a role in the etiology of breast cancer, while androgens are involved in mechanisms controlling the growth of normal and malignant prostatic cells. Using LNCaP prostate cancer cell lines, we have developed a cell model to study the progression of prostate cancer. In the model LNCaP cells are transformed in culture condition into more aggressive cells. Our data suggest that substantial changes in androgen and estrogen metabolism occur in the cells, leading to increased production of active estrogens during the process. In breast cancer, the reductive 17HSD type 1 activity is predominant in malignant cells, while the oxidative 17HSD type 2 mainly seems to be present in non-malignant breast epithelial cells. Deprivation of an estrogen response by using specific 17HSD type 1 inhibitors is a tempting approach in treating estrogen-dependent breast cancer. Our recent studies demonstrate that in addition to sex hormone target tissues, estrogens may be important in the development of cancer in some other tissues previously not considered to be estrogen target tissues, such as the gastrointestinal tract.
17Beta-hydroxysteroid dehydrogenase type 2: independent prognostic significance and evidence of estrogen protection in female patients with colon cancer.
Biocenter Oulu, Research Center for Molecular Endocrinology, WHO Collaborating Centre for Research on Reproductive Health, P.O. Box 5000, University of Oulu, FIN-90014 Oulu, Finland.
The mRNA expression of 17beta-hydroxysteroid dehydrogenase (17HSD) types 1 and 2 enzymes catalyzing opposite reaction of estrogen metabolism was investigated in colon cancer. Further, the significance of the 17HSD type 2 enzyme as a possible marker of colorectal cancer (CRC) prognosis was studied. In the normal mucosa, 17HSD type 2 mRNA was predominantly expressed in the surface epithelium and in the upper parts of the crypts. In the lamina propria expression was seen in endothelial cells and mononuclear phagocytes. In colorectal tumors, 17HSD type 2 expression was in most cases downregulated. Female patients had significantly more cancers with high 17HSD type 2 mRNA expression (n=11/35; 31%) than male patients (n=3/39; 8%) (P=0.02). We observed a significant impact of 17HSD type 2 mRNA expression on survival in female patients with distal colorectal cancer (n=24), with an overall cumulative 5-year survival rate of 54% in those with low 17HSD type 2 mRNA expression. None of the female patients with high 17HSD type 2 mRNA expression survived (n=11; P=0.0068; log rank 7.32). In male patients, no significant association with survival was observed. Our data provide evidence suggesting that low 17HSD type 2 mRNA expression is an independent marker of favorable prognosis in females with distal colorectal cancer, supporting the presence of gender- and location-related differences in the pathogenesis of colon cancer.
50bp deletion in the promoter for superoxide dismutase 1 (SOD1) reduces SOD1 expression in vitro and may correlate with increased age of onset of sporadic amyotrophic lateral sclerosis.
The objective was to test the hypothesis that a described association between homozygosity for a 50bp deletion in the SOD1 promoter 1684bp upstream of the SOD1 ATG and an increased age of onset in SALS can be replicated in additional SALS and control sample sets from other populations. Our second objective was to examine whether this deletion attenuates expression of the SOD1 gene. Genomic DNA from more than 1200 SALS cases from Ireland, Scotland, Quebec and the USA was genotyped for the 50bp SOD1 promoter deletion. Reporter gene expression analysis, electrophoretic mobility shift assays and chromatin immunoprecipitation studies were utilized to examine the functional effects of the deletion. The genetic association for homozygosity for the promoter deletion with an increased age of symptom onset was confirmed overall in this further study (p=0.032), although it was only statistically significant in the Irish subset, and remained highly significant in the combined set of all cohorts (p=0.001). Functional studies demonstrated that this polymorphism reduces the activity of the SOD1 promoter by approximately 50%. In addition we revealed that the transcription factor SP1 binds within the 50bp deletion region in vitro and in vivo. Our findings suggest the hypothesis that this deletion reduces expression of the SOD1 gene and that levels of the SOD1 protein may modify the phenotype of SALS within selected populations.
Since beta2-adrenergic receptors are important regulators of blood pressure, genetic variation in this receptor could explain risk of elevated blood pressure in selected individuals. We tested the hypothesis that Gly16Arg, Gln27Glu, and Thr164Ile in the beta2-adrenergic receptor gene associated with elevated blood pressure.
We genotyped 9185 individuals from the adult Danish general population.
Allele frequencies of 16Arg, 27Glu, and 164Ile were 0.38, 0.44, and 0.01, respectively. Among women never treated with antihypertensive medication those heterozygous for Thr164Ile versus non-carriers had increased diastolic blood pressure (P=0.02). Women heterozygous for Thr164Ile versus non-carriers had an odds ratio for elevated blood pressure of 1.93 (95% CI: 1.30-2.86). Finally, women double heterozygous for Thr164Ile and Gln27Glu or Gly16Arg versus non-carriers at all 3 loci had an odds ratio for elevated blood pressure of 2.49 (1.28-4.85) or 3.19 (1.46-6.97). In men, blood pressure was not influenced by this genetic variation.
In women Thr164Ile heterozygosity is associated with increased diastolic blood pressure, and represent a risk factor for elevated blood pressure in women in the general population. This was most pronounced in those women also heterozygous for Gln27Glu or Gly16Arg.
To evaluate C to T substitution at nucleotide 677 of N(5), N(10)-methylenetetrahydrofolate reductase gene in women with prior preeclamptic or normotensive pregnancies.
Methylenetetrahydrofolate reductase genotypes were determined in 113 Finnish women with preeclamptic first pregnancies and 103 controls with one or more normotensive pregnancies, using polymerase chain reaction and restriction enzyme analysis. Preeclampsia was defined as severe in 100 women who fulfilled one or more of the subsequent criteria: systolic blood pressure (BP) at least 160 mmHg, diastolic BP at least 110 mmHg, or proteinuria at least 2 g per 24-hour urine collection.
There were no significant differences in prevalences of the methylenetetrahydrofolate reductase genotypes (CC, CT, and TT) between groups (57%, 40%, and 3% in the preeclamptic group and 54%, 39%, and 7%, respectively, in controls). The frequency of the T677 allele was 0.23 in the preeclamptic group and 0.26 in the control group (difference 0.03; 95% confidence interval -0.08, 0.14; P =.51). Our sample had 60% power to detect a difference of the allele frequencies similar to that (0.12) reported previously. The result was similar when analysis was restricted to patients with severe preeclampsia (T677 allele frequency 0.22).
A carrier status for the T677 allele of the methylenetetrahydrofolate reductase gene does not predispose to preeclampsia, at least in the Finnish population.
A genetic basis for otitis media is established, however, the role of rare variants in disease etiology is largely unknown. Previously a duplication variant within A2ML1 was identified as a significant risk factor for otitis media in an indigenous Filipino population and in US children. In this report exome and Sanger sequencing was performed using DNA samples from the indigenous Filipino population, Filipino cochlear implantees, US probands, Finnish, and Pakistani families with otitis media. Sixteen novel, damaging A2ML1 variants identified in otitis media patients were rare or low-frequency in population-matched controls. In the indigenous population, both gingivitis and A2ML1 variants including the known duplication variant and the novel splice variant c.4061?+?1?G>C were independently associated with otitis media. Sequencing of salivary RNA samples from indigenous Filipinos demonstrated lower A2ML1 expression according to the carriage of A2ML1 variants. Sequencing of additional salivary RNA samples from US patients with otitis media revealed differentially expressed genes that are highly correlated with A2ML1 expression levels. In particular, RND3 is upregulated in both A2ML1 variant carriers and high-A2ML1 expressors. These findings support a role for A2ML1 in keratinocyte differentiation within the middle ear as part of otitis media pathology and the potential application of ROCK inhibition in otitis media.
Aberrant expression of E-cadherin and beta-catenin in association with transforming growth factor-beta1 in urinary bladder lesions in humans after the Chernobyl accident.
This study examines the molecular pathways of cell-cell communication in chronic inflammatory processes associated with long-term low-dose urinary bladder exposure to ionizing radiation in people without major disease living more than 19 years in radio-contaminated areas of Ukraine after the Chernobyl accident. Patterns of components of the E-cadherin/beta-catenin complex, and transforming growth factor-beta1 (TGF-beta1) and inducible nitric oxide synthase (iNOS) expression were immunohistochemically evaluated in urinary bladder biopsies from 52 males with benign prostate hyperplasia and 8 females with chronic cystitis (group 1). For comparison, 25 males and 6 females living in non-contaminated areas of Ukraine were also investigated (group 2). Fourteen patients with primary urothelial carcinomas, which were operated on before the Chernobyl accident, were included as a carcinoma group. Chronic proliferative atypical cystitis ('Chernobyl cystitis') was observed in group 1 patients. Foci of dysplasia and carcinoma in situ were found in 51 (85%) and 34 (57%) of the 60 cases, respectively. Chronic cystitis with areas of dysplasia was detected in only 4 (13%) cases of 31 group 2 patients. Statistically significant differences in immunohistochemical scores for TGF-beta1 in the urothelium and lamina propria, iNOS in the urothelium and both beta-catenin and E-cadherin in the cytoplasm were observed between groups 1 and 2 with marked expression in group 1. Furthermore, TGF-beta1 overexpression and alteration in E-cadherin/beta-catenin complexes in bladder urothelium might play a crucial role in urinary bladder carcinogenesis in humans exposed to long-term low-dose ionizing radiation.
Mesial temporal lobe epilepsy (MTLE) is one of the most common types of the intractable epilepsies and is most often associated with hippocampal sclerosis (HS), which is characterized by pronounced loss of hippocampal pyramidal neurons. microRNAs (miRNAs) have been shown to be dysregulated in epilepsy and neurodegenerative diseases, and we hypothesized that miRNAs could be involved in the pathogenesis of MTLE and HS.
miRNA expression was quantified in hippocampal specimens from human patients using miRNA microarray and quantitative real-time polymerase chain reaction RT-PCR, and by RNA-seq on fetal brain specimens from domestic pigs. In situ hybridization was used to show the spatial distribution of miRNAs in the human hippocampus. The potential effect of miRNAs on targets genes was investigated using the dual luciferase reporter gene assay.
miRNA expression profiling showed that 25 miRNAs were up-regulated and 5 were down-regulated in hippocampus biopsies of MTLE/HS patients compared to controls. We showed that miR-204 and miR-218 were significantly down-regulated in MTLE and HS, and both were expressed in neurons in all subfields of normal hippocampus. Moreover, miR-204 and miR-218 showed strong changes in expression during fetal development of the hippocampus in pigs, and we identified four target genes, involved in axonal guidance and synaptic plasticity, ROBO1, GRM1, SLC1A2, and GNAI2, as bona fide targets of miR-218. GRM1 was also shown to be a direct target of miR-204.
miR-204 and miR-218 are developmentally regulated in the hippocampus and may contribute to the molecular mechanisms underlying the pathogenesis of MTLE and HS.
