ABCB1 Amplicon Contains Cyclic AMP Response Element-Driven TRIP6 Gene in Taxane-Resistant MCF-7 Breast Cancer Sublines.

A limited number of studies are devoted to regulating TRIP6 expression in cancer. Hence, we aimed to unveil the regulation of TRIP6 expression in MCF-7 breast cancer cells (with high TRIP6 expression) and taxane-resistant MCF-7 sublines (manifesting even higher TRIP6 expression). We found that TRIP6 transcription is regulated primarily by the cyclic AMP response element (CRE) in hypomethylated proximal promoters in both taxane-sensitive and taxane-resistant MCF-7 cells. Furthermore, in taxane-resistant MCF-7 sublines, TRIP6 co-amplification with the neighboring ABCB1 gene, as witnessed by fluorescence in situ hybridization (FISH), led to TRIP6 overexpression. Ultimately, we found high TRIP6 mRNA levels in progesterone receptor-positive breast cancer and samples resected from premenopausal women.

Sertoli Cells Possess Immunomodulatory Properties and the Ability of Mitochondrial Transfer Similar to Mesenchymal Stromal Cells.

It is becoming increasingly evident that selecting an optimal source of mesenchymal stromal cells (MSCs) is crucial for the successful outcome of MSC-based therapies. During the search for cells with potent regenerative properties, Sertoli cells (SCs) have been proven to modulate immune response in both in vitro and in vivo models. Based on morphological properties and expression of surface markers, it has been suggested that SCs could be a kind of MSCs, however, this hypothesis has not been fully confirmed. Therefore, we compared several parameters of MSCs and SCs, with the aim to evaluate the therapeutic potential of SCs in regenerative medicine. We showed that SCs successfully underwent osteogenic, chondrogenic and adipogenic differentiation and determined the expression profile of canonical MSC markers on the SC surface. Besides, SCs rescued T helper (Th) cells from undergoing apoptosis, promoted the anti-inflammatory phenotype of these cells, but did not regulate Th cell proliferation. MSCs impaired the Th17-mediated response; on the other hand, SCs suppressed the inflammatory polarisation in general. SCs induced M2 macrophage polarisation more effectively than MSCs. For the first time, we demonstrated here the ability of SCs to transfer mitochondria to immune cells. Our results indicate that SCs are a type of MSCs and modulate the reactivity of the immune system. Therefore, we suggest that SCs are promising candidates for application in regenerative medicine due to their anti-inflammatory and protective effects, especially in the therapies for diseases associated with testicular tissue inflammation.

The interconnection between cytokeratin and cell membrane-bound ß-catenin in Sertoli cells derived from juvenile Xenopus tropicalis testes.

Sertoli cells (SCs) play a central role in the determination of male sex during embryogenesis and spermatogenesis in adulthood. Failure in SC development is responsible for male sterility and testicular cancer. Before the onset of puberty, SCs are immature and differ considerably from mature cells in post-pubertal individuals regarding their morphology and biochemical activity. The major intermediate filament (IF) in mature SCs is vimentin, anchoring germ cells to the seminiferous epithelium. The collapse of vimentin has resulted in the disintegration of seminiferous epithelium and subsequent germ cell apoptosis. However, another IF, cytokeratin (CK) is observed only transiently in immature SCs in many species. Nevertheless, its function in SC differentiation is poorly understood. We examined the interconnection between CK and cell junctions using membrane ß-catenin as a marker during testicular development in the Xenopus tropicalis model. Immunohistochemistry on juvenile (5 months old) testes revealed co-expression of CK, membrane ß-catenin and E-cadherin. Adult (3-year-old males) samples confirmed only E-cadherin expression; CK and ß-catenin were lost. To study the interconnection between CK and ß-catenin-based cell junctions, the culture of immature SCs (here called XtiSCs) was employed. Suppression of CK by acrylamide in XtiSCs led to breakdown of membrane-bound ß-catenin but not F-actin and ß-tubulin or cell-adhesion proteins (focal adhesion kinase and integrin ß1). In contrast to the obvious dependence of membrane ß-catenin on CK stability, the detachment of ß-catenin from the plasma membrane via uncoupling of cadherins by Ca2+ chelator EGTA had no effect on CK integrity. Interestingly, CHIR99021, a GSK3 inhibitor, also suppressed the CK network, resulting in the inhibition of XtiSCs cell-to-cell contacts and testicular development in juvenile frogs. This study suggests a novel role of CK in the retention of ß-catenin-based junctions in immature SCs, and thus provides structural support for seminiferous tubule formation and germ cell development.

Epithelial-Mesenchymal Transition Promotes the Differentiation Potential of Xenopus tropicalis Immature Sertoli Cells.

Epithelial-mesenchymal transition (EMT) is a fundamental process in embryonic development by which sessile epithelial cells are converted into migratory mesenchymal cells. Our laboratory has been successful in the establishment of Xenopus tropicalis immature Sertoli cells (XtiSCs) with the restricted differentiation potential. The aim of this study is the determination of factors responsible for EMT activation in XtiSCs and stemness window acquisition where cells possess the broadest differentiation potential. For this purpose, we tested three potent EMT inducers-GSK-3 inhibitor (CHIR99021), FGF2, and/or TGF-ß1 ligand. XtiSCs underwent full EMT after 3-day treatment with CHIR99021 and partial EMT with FGF2 but not with TGF-ß1. The morphological change of CHIR-treated XtiSCs to the typical spindle-like cell shape was associated with the upregulation of mesenchymal markers and the downregulation of epithelial markers. Moreover, only CHIR-treated XtiSCs were able to differentiate into chondrocytes in vitro and cardiomyocytes in vivo. Interestingly, EMT-shifted cells could migrate towards cancer cells (HeLa) in vitro and to the injury site in vivo. The results provide a better understanding of signaling pathways underlying the generation of testis-derived stem cells.

Silurana Chromosomal Evolution: A New Piece to the Puzzle.

The African clawed frogs of the subgenus Silurana comprise both diploid and tetraploid species. The root of the polyploidization event leading to the extant Xenopus calcaratus, X. mellotropicalis, and X. epitropicalis is not fully understood so far. In X. mellotropicalis, we previously proposed 2 evolutionary scenarios encompassing complete (scenario A) or incomplete (scenario B) translocation of a heterochromatic block from chromosome 9 to 2 in a diploid ancestor. To resolve this puzzle, we performed FISH coupled with tyramide signal amplification (FISH-TSA) using 5 X. tropicalis and X. mellotropicalis single copy gene probes (gyg2, cept1, fn1, ndufs1, and sf3b1) reflecting borders of the heterochromatic blocks in X. tropicalis chromosome 9 (XTR 9) and X. mellotropicalis chromosome 9b (XME 9b) and XME 2a. cDNA sequencing recognized both homoeologous genes in X. mellotropicalis. Comparison of gene physical mapping between X. tropicalis and X. mellotropicalis clearly confirmed complete rather than incomplete translocation t(9;2) of the heterochromatic block in the diploid predecessor and thus favored scenario A regarding the formation of an ancestral allotetraploid karyotype.

Chromosome divergence during evolution of the tetraploid clawed frogs, Xenopus mellotropicalis and Xenopus epitropicalis as revealed by Zoo-FISH.

Whole genome duplication (WGD) generates new species and genomic redundancy. In African clawed frogs of the genus Xenopus, this phenomenon has been especially important in that (i) all but one extant species are polyploid and (ii) whole genome sequences of some species provide an evidence for genomic rearrangements prior to or after WGD. Within Xenopus in the subgenus Silurana, at least one allotetraploidization event gave rise to three extant tetraploid (2n = 4x = 40) species-Xenopus mellotropicalis, X. epitropicalis, and X. calcaratus-but it is not yet clear the degree to which these tetraploid genomes experienced rearrangements prior to or after allotetraploidization. To explore genome evolution during diversification of these species, we performed cytogenetic analyses of X. mellotropicalis, including assessment of the localization of nucleolar organizer region, chromosome banding, and determination of the p/q arm ratios for each chromosome pair. We compared these data to a previously characterized karyotype of X. epitropicalis. Morphometric, C-banding and Zoo-FISH data support a previously hypothesized common allotetraploid predecessor of these species. Zoo-FISH with whole chromosome painting (WCP) probes derived from the closely related diploid species X. tropicalis confirmed the existence of ten chromosomal quartets in X. mellotropicalis somatic cells, as expected by its ploidy level and tetraploid ancestry. The p/q arm ratio of chromosome 2a was found to be substantially different between X. mellotropicalis (0.81) and X. epitropicalis (0.67), but no substantial difference between these two species was detected in this ratio for the homoeologous chromosome pair 2b, or for other chromosome pairs. Additionally, we identified variation between these two species in the locations of a heterochromatic block on chromosome pair 2a. These results are consistent with a dynamic history of genomic rearrangements before and/or after genome duplication, a surprising finding given the otherwise relatively conserved genomic structure of most frogs.

A large pseudoautosomal region on the sex chromosomes of the frog Silurana tropicalis.

Sex chromosome divergence has been documented across phylogenetically diverse species, with amphibians typically having cytologically nondiverged ("homomorphic") sex chromosomes. With an aim of further characterizing sex chromosome divergence of an amphibian, we used "RAD-tags" and Sanger sequencing to examine sex specificity and heterozygosity in the Western clawed frog Silurana tropicalis (also known as Xenopus tropicalis). Our findings based on approximately 20 million genotype calls and approximately 200 polymerase chain reaction-amplified regions across multiple male and female genomes failed to identify a substantially sized genomic region with genotypic hallmarks of sex chromosome divergence, including in regions known to be tightly linked to the sex-determining region. We also found that expression and molecular evolution of genes linked to the sex-determining region did not differ substantially from genes in other parts of the genome. This suggests that the pseudoautosomal region, where recombination occurs, comprises a large portion of the sex chromosomes of S. tropicalis. These results may in part explain why African clawed frogs have such a high incidence of polyploidization, shed light on why amphibians have a high rate of sex chromosome turnover, and raise questions about why homomorphic sex chromosomes are so prevalent in amphibians.