1,721,054 research outputs found
Trichostatin A preferentially reverses the upregulation of gene expression levels induced by gain of chromosome 7 in colorectal cancer cell lines
Get PDFEpithelial cancers are defined by a tumor-specific distribution of chromosomal aneuploidies that are maintained when cells metastasize and are conserved in cell lines derived from primary tumors. Correlations between genomic copy number and gene expression have been observed for different tumors including, colorectal (CRC), breast and pancreatic cancer. These ploidy-driven transcriptional deregulations are characterized by low-level expression changes of most genes on the affected chromosomes. The emergence of these aberrations at an early stage of tumorigenesis and the strong selection for the maintenance of these aneuploidies suggests that aneuploidy-dependent transcriptional deregulations might contribute to cellular transformation and maintenance of the malignant phenotype. The histone deacetylase inhibitor (HDACi) Trichostatin A (TSA) has anticancer effects and is well known to lead to large scale gene expression changes. Here we assessed if TSA could disrupt the aneuploidy-driven gene expression in the aneuploid colon cancer cell line SW480 and the artificially generated aneuploid cell line DLD-1 + 7. We found that TSA increases transcriptional activity throughout the genome, yet inhibits aneuploidy-induced gene expression changes on chromosome 7. Among the TSA affected genes on chromosome 7 we identified potential CRC oncogenes. These experiments represent the first attempt to explain how histone acetylation affects aneuploidy-driven gene expression changes. This article is protected by copyright. All rights reserved
Fetal Estetrol (E4) Inhibits Growth Hormone (GH) Signal Transduction in Breast Cancer Cell Lines
No full textObjective: Inhibition of GH signaling has been associated with increased longevity and cancer resistance. As the fetal estrogen estetrol was also linked to longevity and cancer resistance, we hypothesized that these effects of estetrol are mediated by inhibition of GH activity. Design: The CNMm-6TR mammary cell line, stably transfected with doxycycline (DOX)-inducible GHR (CNMm-cGHR cells), HEK293 cell line transfected with rGHR and the human T47D cell line were used to assess effects of estetrol on GH signal transduction. Proliferation was measured using the colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay (MTT). Spi-luciferase reporter activity, responsive to phosphorylated Stat5, was used to assess GH signaling. GHR mRNA expression was normalized to a reference gene. Results: Treatment of CNMm-cGHR cells with GH resulted in decreased proliferation rates. No effect was seen by the co-inhibition with estetrol. Using the spi-luc reporter activity, it was shown that GH activates the GHR/Jak2/ Stat5 pathway. Estetrol partially inhibited this activation. In the HEK-rGHR cells, estetrol was able to inhibit the GH induced activation of the spi-luc reporter, but only in the presence of an ERα expression plasmid. Effects of estetrol on the expression of GHR mRNA was assessed in T47D cells, a cell-line expressing ER, PR and GHR under natural promoters. After incubation with estetrol, a mean 4-fold inhibition of GHR mRNA expression was observed. Conclusion: Using different cell systems, it was shown that estetrol is able to inhibit GH signaling, a process dependent on the presence of the Estrogen Receptor (ER). Estetrol may therefore be used to inhibit GH-induced effects on breast cancer proliferation
Treating canine Cushing's syndrome: Current options and future prospects
Get PDFNaturally occurring hypercortisolism, also known as Cushing's syndrome, is a common endocrine disorder in dogs that can be caused by an adenocorticotrophic hormone (ACTH)-producing pituitary adenoma (pituitary-dependent hypercortisolism, PDH; 80–85% of cases), or by an adrenocortical tumor (ACT; 15–20% of cases). To determine the optimal treatment strategy, differentiating between these two main causes is essential. Good treatment options are surgical removal of the causal tumor, i.e. hypophysectomy for PDH and adrenalectomy for an ACT, or radiotherapy in cases with PDH. Because these options are not without risks, not widely available and not suitable for every patient, pharmacotherapy is often used. In cases with PDH, the steroidogenesis inhibitor trilostane is most often used. In cases with an ACT, either trilostane or the adrenocorticolytic drug mitotane can be used. Although mostly effective, both treatments have disadvantages. This review discusses the current treatment options for canine hypercortisolism, and considers their mechanism of action, efficacy, adverse effects, and effect on survival. In addition, developments in both adrenal-targeting and pituitary-targeting drugs that have the potential to become future treatment options are discussed, as a more selective and preferably also tumor-targeted approach could have many advantages for both PDH and ACTs
Steroidogenic factor-1 inverse agonists as a treatment option for canine hypercortisolism: in vitro study
Get PDFHypercortisolism is one of the most commonly diagnosed endocrinopathies in dogs, and new targeted medical treatment options are desirable. Steroidogenic factor-1 (SF-1), an orphan nuclear hormone receptor, is a key regulator of adrenal steroidogenesis, development, and growth. In pituitary-dependent hypercortisolism (PDH), high plasma ACTH concentrations increase the transcriptional activity of SF-1. In adrenal-dependent hypercortisolism, SF-1 expression is significantly greater in dogs with recurrence after adrenalectomy than in those without recurrence. Inhibition of SF-1 could therefore be an interesting treatment option in canine spontaneous hypercortisolism. We determined the effects of 3 SF-1 inverse agonists, compounds IsoQ A, #31, and #32, on cortisol production, on the messenger RNA (mRNA) expression of steroidogenic enzymes and SFs, and on cell viability, in primary adrenocortical cell cultures of 8 normal adrenal glands and of 3 cortisol-secreting adrenocortical tumors (ATs). To mimic PDH, the normal adrenocortical cell cultures were stimulated with ACTH. The results show that only compound #31 inhibited cortisol production and SF-1 target gene expression in non–ACTH-stimulated and ACTH-stimulated normal adrenocortical cells but did not affect cell viability. In the AT cell cultures, the effects of #31 on cortisol production and target gene expression were variable, possibly caused by a difference in the SF-1 mRNA expressions of the primary tumors. In conclusion, inhibition of SF-1 activity shows much promise as a future treatment for canine hypercortisolism
Melanocortin 2 receptor antagonists in canine pituitary-dependent hypercortisolism: in vitro studies
Get PDFCanine hypercortisolism is most often caused by an ACTH-secreting pituitary adenoma (pituitary-dependent hypercortisolism; PDH). An interesting target for a selective medical treatment of PDH would be the receptor for ACTH: the melanocortin 2 receptor (MC2R). In this study we investigated whether two peptide compounds, BIM-22776 (#776) and BIM-22A299 (#299), are effective MC2R antagonists in vitro. Their effects on cortisol production and mRNA expression of steroidogenic enzymes, MC2R and melanocortin 2 receptor accessory protein (MRAP) were evaluated in primary adrenocortical cell cultures (n = 8) of normal canine adrenal glands. Cortisol production stimulated by 50 nM ACTH was dose-dependently inhibited by #299 (inhibition 90.7 ± 2.3% at 5 μM) and by #776 (inhibition 38.0 ± 5.2% at 5 μM). The ACTH-stimulated mRNA expression of steroidogenic enzymes, MC2R and MRAP was significantly inhibited by both compounds, but most potently by #299. These results indicate that canine primary cell culture is a valuable in vitro system to test MC2R antagonists, and that these compounds, but especially #299, are effective MC2R antagonists in vitro. To determine its efficacy in vivo, further studies are warranted. Antagonism of the MC2R is a promising potential treatment approach in canine PDH
New Insights in the Functional Zonation of the Canine Adrenal Cortex
No full textBackground: Current understanding of adrenal steroidogenesis is that the production of aldosterone or cortisol depends on the expression of aldosterone synthase (CYP11B2) and 11b-hydroxylase cytochrome P450 (CYP11B1), respectively. However, this has never been studied in dogs, and in some species, a single CYP11B catalyzes both cortisol and aldosterone formation. Analysis of the canine genome provides data of a single CYP11B gene which is called CYP11B2, and a large sequence gap exists near the so-called CYP11B2 gene. Objectives: To investigate the zonal expression of steroidogenic enzymes in the canine adrenal cortex and to determine whether dogs have 1 or multiple CYP11B genes. Animals: Normal adrenal glands from 10 healthy dogs. Methods: Zona fasciculata (zF) and zona glomerulosa (zG) tissue was isolated by laser microdissection. The mRNA expression of steroidogenic enzymes and their major regulators was studied with RT-qPCR. Southern blot was performed to determine whether the sequence gap contains a CYP11B gene copy. Immunohistochemistry (IHC) was performed for 17a-hydroxylase/17,20-lyase (CYP17). Results: Equal expression (P = .62) of the so-called CYP11B2 gene was found in the zG and zF. Southern blot revealed a single gene. CYP17 expression (P = .05) was significantly higher in the zF compared with the zG, which was confirmed with IHC. Conclusions and Clinical Importance: We conclude that there is only 1 CYP11B gene in canine adrenals. The zone-specific production of aldosterone and cortisol is probably due to zone-specific CYP17 expression, which makes it an attractive target for selective inhibition of cortisol synthesis without affecting mineralocorticoid production in the zG
Red blood cell phosphate concentration and osmotic resistance during dietary phosphate depletion in dairy cows
No full textBACKGROUND: Hypophosphatemia in early lactating dairy cows has been implicated as primary cause for postparturient hemoglobinuria in cattle. Decreased availability of phosphorus has been proposed to reduce adenosine triphosphate synthesis of erythrocytes and thereby reduce osmotic resistance of these cells. HYPOTHESIS/OBJECTIVES: To study the effect of phosphorus depletion on the phosphate concentration ([Pi]) in plasma and erythrocytes and the osmotic resistance of erythrocytes and to determine the association between plasma [Pi] and erythrocyte [Pi]. ANIMALS: Ten healthy midlactating dairy cows in their 3rd to 5th lactation. METHODS: Prospective study. Dietary phosphorus depletion for 5 weeks followed by phosphorus supplementation. Plasma and erythrocyte [Pi] and erythrocyte osmotic resistance were measured. Four cows underwent continuous dextrose infusion at the end of phosphate depletion to exacerbate hypophosphatemia. RESULTS: Dietary P depletion resulted in a marked decline of the plasma [Pi] from 4.1 ± 1.0 mg/dL to a nadir of 1.7 ± 0,5 mg/dL, but did not alter erythrocyte [Pi] or osmotic resistance. Similarly, dextrose infusion induced a decline of the plasma [Pi] from 2.4 ± 0.5 mg/dL to 1.5 ± 0.5 mg/dL, but had no effect on erythrocyte [Pi] or osmotic resistance. CONCLUSIONS AND CLINICAL IMPORTANCE: In cattle, marked hypophosphatemia induced by dietary P depletion was neither associated with a decline in erythrocyte [Pi] nor with decreased osmotic resistance of erythrocytes. Phosphorus depletion alone is therefore unlikely to cause intravascular hemolysis and the plasma [Pi] is an unreliable index for the intracellular [Pi] of erythrocytes
The Evolution of Single Cell-derived Colorectal Cancer Cell Lines is Dominated by the Continued Selection of Tumor Specific Genomic Imbalances, Despite Random Chromosomal Instability
Get PDFIntratumor heterogeneity is a major challenge in cancer treatment. To decipher patterns of chromosomal heterogeneity, we analyzed six colorectal cancer cell lines by multiplex interphase FISH (miFISH). The mismatch repair deficient cell lines DLD-1 and HCT116 had the most stable copy numbers, whereas aneuploid cell lines (HT-29, SW480, SW620 and H508) displayed a higher degree of instability. We subsequently assessed the clonal evolution of single cells in two CRC cell lines, SW480 and HT-29, which both have aneuploid karyotypes but different degrees of chromosomal instability. The clonal compositions of the single cell-derived daughter lines, as assessed by miFISH, differed for HT-29 and SW480. Daughters of HT-29 were stable, clonal, with little heterogeneity. Daughters of SW480 were more heterogeneous, with the single cell-derived daughter lines separating into two distinct populations with different ploidy (hyper-diploid and near-triploid), morphology, gene expression and tumorigenicity. To better understand the evolutionary trajectory for the two SW480 populations, we constructed phylogenetic trees which showed ongoing instability in the daughter lines. When analyzing the evolutionary development over time, most single cell-derived daughter lines maintained their major clonal pattern, with the exception of one daughter line that showed a switch involving a loss of APC. Our meticulous analysis of the clonal evolution and composition of these colorectal cancer models shows that all chromosomes are subject to segregation errors, however, specific net genomic imbalances are maintained. Karyotype evolution is driven by the necessity to arrive at and maintain a specific plateau of chromosomal copy numbers as the drivers of carcinogenesis
ASXL3 is a Novel Pluripotency Factor in Human Respiratory Epithelial Cells and a Potential Therapeutic Target in Small Cell Lung Cancer
No full textIn this study, we generated induced pluripotent stem cells (iPSC) from normal human small airway epithelial cells (SAEC) to investigate epigenetic mechanisms of stemness and pluripotency in lung cancers. We documented key hallmarks of reprogramming in lung iPSC (Lu-iPSC) that coincided with modulation of more than 15,000 genes relative to parental SAEC. Of particular novelty, we identified the PRC2-associated protein, ASXL3 which was markedly upregulated in Lu-iPSC and small cell lung cancer (SCLC) lines and clinical specimens. ASXL3 overexpression correlated with increased genomic copy number in SCLC lines. ASXL3 silencing inhibited proliferation, clonogenicity and teratoma formation by Lu-iPSC, and diminished clonogenicity and malignant growth of SCLC cells in-vivo. Collectively, our studies validate the utility of the Lu-iPSC model for elucidating epigenetic mechanisms contributing to pulmonary carcinogenesis, and highlight ASXL3 as a novel candidate target for SCLC therapy
Treatment of multicentric or cranial mediastinal high-grade T-cell lymphoma in dogs with a first-line CCNU-L(-chlorambucil)-CHOP protocol
No full textThis retrospective study determined disease free survival (DFS) and progression free survival (PFS) in chemo-naïve dogs with multicentric or cranial mediastinal high-grade T-cell lymphoma, treated with a first-line CCNU-L(-chlorambucil)-CHOP protocol. Of thirteen dogs with multicentric lymphoma, 92.3% achieved a complete remission (CR), and the median DFS and PFS was 317 and 256 days, respectively. Three dogs had cranial mediastinal lymphoma, and achieved a CR with a median DFS and PFS of 978 and 1007 days, respectively. The oneand two-year DFS/PFS probability estimate for dogs with multicentric lymphoma was 0.50/0.46 and 0.42/0.38, respectively, for dogs with cranial mediastinal lymphoma 0.67/0.67. Neutropenia and thrombocytopenia were reported in 52.9% and 50% of the dogs, respectively, while 56.3% experienced grade 1-4 nephrotoxicity, hypothesized to be lomustine-induced. It was concluded that, compared to historical data, the currently described first-line CCNU-L(-chlorambucil)-CHOP protocol could benefit the survival of dogs with multicentric or cranial mediastinal highgrade T-cell lymphoma
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