Replication in mammalian cells recapitulates the locus-specific differences in somatic instability of genomic GAA triplet-repeats

Friedreich ataxia is caused by an expanded (GAA·TTC)(n) sequence in intron 1 of the FXN gene. Small pool PCR analysis showed that pure (GAA·TTC)(44+) sequences at the FXN locus are unstable in somatic cells in vivo, displaying both expansions and contractions. On searching the entire human and mouse genomes we identified three other genomic loci with pure (GAA·TTC)(44+) sequences. Alleles at these loci showed mutation loads of <1% compared with 6.3–30% for FXN alleles of similar length, indicating that somatic instability in vivo is regulated by locus-specific factors. Since distance between the origin of replication and the (CTG·CAG)(n) sequence modulates repeat instability in mammalian cells, we tested if this could also recapitulate the locus-specific differences for genomic (GAA·TTC)(n) sequences. Repeat instability was evaluated following replication of a (GAA·TTC)(115) sequence in transfected COS1 cells under the control of the SV40 origin of replication located at one of five different distances from the repeat. Indeed, depending on the location of the SV40 origin relative to the (GAA·TTC)(n) sequence, we noted either no instability, predominant expansion or both expansion and contraction. These data suggest that mammalian DNA replication is a possible mechanism underlying locus-specific differences in instability of GAA triplet-repeat sequences

Whispering-gallery mode resonators for highly unidirectional laser action

Optical microcavities can be designed to take advantage of total internal reflection, which results in resonators supporting whispering-gallery modes (WGMs) with a high-quality factor (Q factor). One of the crucial problems of these devices for practical applications such as designing microcavity lasers, however, is that their emission is nondirectional due to their radial symmetry, in addition to their inefficient power output coupling. Here we report the design of elliptical resonators with a wavelength-size notch at the boundary, which support in-plane highly unidirectional laser emission from WGMs. The notch acts as a small scatterer such that the Q factor of the WGMs is still very high. Using midinfrared (λ ∼ 10 μm) injection quantum cascade lasers as a model system, an in-plane beam divergence as small as 6 deg with a peak optical power of ∼5 mW at room temperature has been demonstrated. The beam divergence is insensitive to the pumping current and to the notch geometry, demonstrating the robustness of this resonator design. The latter is scalable to the visible and the near infrared, thus opening the door to very low-threshold, highly unidirectional microcavity diode lasers.Physic

Role of transcript and interplay between transcription and replication in triplet-repeat instability in mammalian cells

Triplet-repeat expansions cause several inherited human diseases. Expanded triplet-repeats are unstable in somatic cells, and tissue-specific somatic instability contributes to disease pathogenesis. In mammalian cells instability of triplet-repeats is dependent on the location of the origin of replication relative to the repeat tract, supporting the ‘fork-shift’ model of repeat instability. Disease-causing triplet-repeats are transcribed, but how this influences instability remains unclear. We examined instability of the expanded (GAA•TTC)n sequence in mammalian cells by analyzing individual replication events directed by the SV40 origin from five different locations, in the presence and absence of doxycycline-induced transcription. Depending on the location of the SV40 origin, either no instability was observed, instability was caused by replication with no further increase due to transcription, or instability required transcription. Whereas contractions accounted for most of the observed instability, one construct showed expansions upon induction of transcription. These expansions disappeared when transcript stability was reduced via removal or mutation of a spliceable intron. These results reveal a complex interrelationship of transcription and replication in the etiology of repeat instability. While both processes may not be sufficient for the initiation of instability, transcription and/or transcript stability seem to further modulate the fork-shift model of triplet-repeat instability

DNA methylation status of REIC/Dkk-3 gene in human malignancies

The REIC (reduced expression in immortalized cells)/Dkk-3 is down-regulated in various cancers and considered to be a tumor suppressor gene. REIC/Dkk-3 mRNA has two isoforms (type-a,b). REIC type-a mRNA has shown to be a major transcript in various cancer cells, and its promoter activity was much stronger than that of type-b. In this study, we examined the methylation status of REIC/Dkk-3 type-a in a broad range of human malignancies. We examined REIC/Dkk-3 type-a methylation in breast cancers, non-small-cell lung cancers, gastric cancers, colorectal cancers, and malignant pleural mesotheliomas using a quantitative combined bisulfite restriction analysis assay and bisulfate sequencing. REIC/Dkk-3 type-a and type-b expression was examined using reverse transcriptional PCR. The relationships between the methylation and clinicopathological factors were analyzed. The rate of REIC/Dkk-3 type-a methylation ranged from 26.2 to 50.0% in the various primary tumors that were examined. REIC/Dkk-3 type-a methylation in breast cancer cells was significantly heavier than that in the other cell lines that we tested. REIC/Dkk-3 type-a methylation was inversely correlated with REIC/Dkk-3 type-a expression. There was a correlation between REIC/Dkk-3 type-a and type-b mRNA expression. REIC/Dkk-3 type-a expression was restored in MDA-MB-231 cells using 5-aza-2'-deoxycytidine treatment. We found that estrogen receptor-positive breast cancers were significantly more common among the methylated group than among the non-methylated group. REIC/Dkk-3 type-a methylation was frequently detected in a broad range of cancers and appeared to play a key role in silencing REIC/Dkk-3 type-a expression in these malignancies

Goettingen Minipigs (GMP): Comparison of Two Different Models for Inducing Diabetes

Purpose: Preclinical experiments on large animals are indispensable for evaluating the effectiveness of diabetes therapies. Miniature swine are well suited for such studies due to their physiological and pathophysiological responses. Methods: We compare two methods for inducing diabetes in Goettingen minipigs (GMP), in five with the beta cell toxin streptozotocin (STZ) and in five other GMP by total pancreatectomy (PE). Glucose homeostasis was assessed with the intravenous glucose-tolerance test (IVGTT) and continual monitoring of interstitial glucose levels. At conclusion of the observation period, the pancreata were examined histologically. Three non-diabetic GMP served as control group. Results: The IVGTT revealed markedly diabetic profiles in both GMP groups. STZ-GMP were found to harbor residual C-peptides and scattered insulin-positive cells in the pancreas. PE-GMP survived the total pancreatectomy only with intensive postoperative care. Conclusions: Although both methods reliably induced diabetes in GMP, the PE-GMP clearly had more health problems and required a greater expenditure of time and resources. The PE-GMP model, however, was better at eliminating endogenous insulin and C-peptide than the STZ-GMP model

Wnt signalling in human breast cancer: expression of the putative Wnt inhibitor Dickkopf-3 (DKK3) is frequently suppressed by promoter hypermethylation in mammary tumours

INTRODUCTION: Expression of the putative Wnt signalling inhibitor Dickkopf-3 (DKK3) is frequently lost in human cancer tissues because of aberrant 5'-cytosine methylation within the DKK3 gene promoter. Since other Wnt signalling inhibitors have been reported to be targets of epigenetic inactivation in human breast cancer, we questioned if DKK3 expression is also epigenetically silenced during breast carcinogenesis and therefore might contribute to oncogenic Wnt signalling commonly found in this disease. METHODS: DKK3 mRNA expression and DKK3 promoter methylation were determined by RT-PCR, realtime PCR and methylation-specific PCR in breast cell lines (n = 9), normal breast tissues (n = 19) and primary breast carcinomas (n = 150), respectively. In vitro DNA demethylation was performed by incubating breast cell lines with 5-aza-2'-deoxycytidine and trichostatin A. DKK3 protein expression was analysed by immunohistochemistry in breast carcinomas (n = 16) and normal breast tissues (n = 8). Methylation data were statistically correlated with clinical patient characteristics. All statistical evaluations were performed with SPSS 14.0 software. RESULTS: DKK3 mRNA was downregulated in 71% (five of seven) of breast cancer cell lines and in 68% of primary breast carcinomas (27 of 40) compared with benign cell lines and normal breast tissues, respectively. A DNA demethylating treatment of breast cell lines resulted in strong induction of DKK3 mRNA expression. In tumourous breast tissues, DKK3 mRNA downregulation was significantly associated with DKK3 promoter methylation (p < 0.001). Of the breast carcinomas, 61% (92 of 150) revealed a methylated DKK3 promoter, whereas 39% (58 of 150) retained an unmethylated promoter. Loss of DKK3 expression in association with DKK3 promoter methylation (p = 0.001) was also confirmed at the protein level (p < 0.001). In bivariate analysis, DKK3 promoter methylation was not associated with investigated clinicopathological parameters except patient age (p = 0.007). CONCLUSIONS: DKK3 mRNA expression and consequently DKK3 protein expression become frequently downregulated during human breast cancer development due to aberrant methylation of the DKK3 promoter. Since DKK3 is thought to negatively regulate oncogenic Wnt signalling, DKK3 may be a potential tumour suppressor gene in normal breast tissue

Movement Behavior of High-Heeled Walking: How Does the Nervous System Control the Ankle Joint during an Unstable Walking Condition?

The human locomotor system is flexible and enables humans to move without falling even under less than optimal conditions. Walking with high-heeled shoes constitutes an unstable condition and here we ask how the nervous system controls the ankle joint in this situation? We investigated the movement behavior of high-heeled and barefooted walking in eleven female subjects. The movement variability was quantified by calculation of approximate entropy (ApEn) in the ankle joint angle and the standard deviation (SD) of the stride time intervals. Electromyography (EMG) of the soleus (SO) and tibialis anterior (TA) muscles and the soleus Hoffmann (H-) reflex were measured at 4.0 km/h on a motor driven treadmill to reveal the underlying motor strategies in each walking condition. The ApEn of the ankle joint angle was significantly higher (p<0.01) during high-heeled (0.38±0.08) than during barefooted walking (0.28±0.07). During high-heeled walking, coactivation between the SO and TA muscles increased towards heel strike and the H-reflex was significantly increased in terminal swing by 40% (p<0.01). These observations show that high-heeled walking is characterized by a more complex and less predictable pattern than barefooted walking. Increased coactivation about the ankle joint together with increased excitability of the SO H-reflex in terminal swing phase indicates that the motor strategy was changed during high-heeled walking. Although, the participants were young, healthy and accustomed to high-heeled walking the results demonstrate that that walking on high-heels needs to be controlled differently from barefooted walking. We suggest that the higher variability reflects an adjusted neural strategy of the nervous system to control the ankle joint during high-heeled walking

EWS/ETS Regulates the Expression of the Dickkopf Family in Ewing Family Tumor Cells

BACKGROUND: The Dickkopf (DKK) family comprises a set of proteins that function as regulators of Wnt/beta-catenin signaling and has a crucial role in development. Recent studies have revealed the involvement of this family in tumorigenesis, however their role in tumorigenesis is still remained unclear. METHODOLOGY/PRINCIPAL FINDINGS: We found increased expression of DKK2 but decreased expression of DKK1 in Ewing family tumor (EFT) cells. We showed that EFT-specific EWS/ETS fusion proteins enhance the DKK2 promoter activity, but not DKK1 promoter activity, via ets binding sites (EBSs) in the 5' upstream region. EWS/ETS-mediated transactivation of the promoter was suppressed by the deletion and mutation of EBSs located upstream of the DKK2 gene. Interestingly, the inducible expression of EWS/ETS resulted in the strong induction of DKK2 expression and inhibition of DKK1 expression in human primary mesenchymal progenitor cells that are thought to be a candidate of cell origin of EFT. In addition, using an EFT cell line SK-ES1 cells, we also demonstrated that the expression of DKK1 and DKK2 is mutually exclusive, and the ectopic expression of DKK1, but not DKK2, resulted in the suppression of tumor growth in immuno-deficient mice. CONCLUSIONS/SIGNIFICANCE: Our results suggested that DKK2 could not functionally substitute for DKK1 tumor-suppressive effect in EFT. Given the mutually exclusive expression of DKK1 and DKK2, EWS/ETS regulates the transcription of the DKK family, and the EWS/ETS-mediated DKK2 up-regulation could affect the tumorigenicity of EFT in an indirect manner

Bypass Mechanisms of the Androgen Receptor Pathway in Therapy-Resistant Prostate Cancer Cell Models

Background: Prostate cancer is initially dependent on androgens for survival and growth, making hormonal therapy the cornerstone treatment for late-stage tumors. However, despite initial remission, the cancer will inevitably recur. The present study was designed to investigate how androgen-dependent prostate cancer cells eventually survive and resume growth under androgen-deprived and antiandrogen supplemented conditions. As model system, we used the androgen-responsive PC346C cell line and its therapy-resistant sublines: PC346DCC, PC346Flu1 and PC346Flu2. Methodology/Principal Findings: Microarray technology was used to analyze differences in gene expression between the androgen-responsive and therapy-resistant PC346 cell lines. Microarray analysis revealed 487 transcripts differentiallyexpressed between the androgen-responsive and the therapy-resistant cell lines. Most of these genes were common to all three therapy-resistant sublines and only a minority (,5%) was androgen-regulated. Pathway analysis revealed enrichment in functions involving cellular movement, cell growth and cell death, as well as association with cancer and reproductive system disease. PC346DCC expressed residual levels of androgen receptor (AR) and showed significant down-regulation of androgen-regulated genes (p-value = 10 27). Up-regulation of VAV3 and TWIST1 oncogenes and repression of the DKK3 tumor-suppressor was observed in PC346DCC, suggesting a potential AR bypass mechanism. Subsequent validation of these three genes in patient samples confirmed that expression was deregulated during prostate cancer progression