A Systematic Protocol for the Characterization of Hsp90 Modulators

This is the author's accepted manuscript. Made available by the permission of the publisher.Several Hsp90 modulators have been identified including the N-terminal ligand geldanamycin (GDA), the C-terminal ligand novobiocin (NB), and the co-chaperone disruptor celastrol. Other Hsp90 modulators elicit a mechanism of action that remains unknown. For example, the natural product gedunin and the synthetic anti-spermatogenic agent H2-gamendazole, recently identified Hsp90 modulators, manifest biological activity through undefined mechanisms. Herein, we report a series of biochemical techniques used to classify such modulators into identifiable categories. Such studies provided evidence that gedunin and H2-gamendazole both modulate Hsp90 via a mechanism similar to celastrol, and unlike NB or GDA

Role of In vivo reflectance confocal microscopy in determining stability in vitiligo: A preliminary study

Background: Vitiligo is an acquired pigmentary disorder. In vivo reflectance confocal microscopy (RCM) reproducible imaging technique has already been reported to be useful in the diagnosis of other skin diseases. Objective: To define RCM features of vitiligo on different clinical stages. Materials and Methods: A total of 125 patients with a clinical diagnosis of vitiligo were included in this study. After informed consent, lesional skins of those vitiligo patients were characterized by using RCM. Five patients with inflammatory cell infiltration observed at the edge of skin lesions and another 5 patients without inflammatory cell infiltration were selected. Biopsies were performed at same sites of the RCM examination areas for histological and immune-histological analysis. Results: In the active stage of vitiligo, the RCM examination revealed that the bright dermal papillary rings presented at the dermoepidermal junction level in normal skin lost their integrity or totally disappeared, border between vitiligo lesion and normal skin became unclear, and highly refractile cells that referred to infiltrated inflammatory cells could be seen within the papillary dermis at the edge of the lesions. In the stable stage of vitiligo, the RCM showed a complete loss of melanin in lesional skin and a clear border between lesional and normal skin. Conclusion: A simple clinical examination with RCM may reliably and efficiently allow evaluation of the stability status of vitiligo lesions

Regulation of RUNX1/AML1 during the G2/M transition

The acute myeloid leukemia 1 (AML1, RUNX1) transcription factor is a key regulator of hematopoietic differentiation both in embryonic stem cells and mature hematopoietic progenitors. The RUNX1 protein is thought to play a role in the control of progression through the cell cycle. We have shown that post-transcriptional regulation of RUNX1 activity occurs, in part, through phosphorylation. To investigate whether transit through the cell cycle is associated with changes in the phosphorylation of RUNX1, we have derived phospho-specific antibodies against three of the five major phosphorylation sites in the transcriptional activation domain of RUNX1, S276, S303 and S462. Using these antibodies we demonstrate that treatment of Jurkat T-cells with nocodazole, a G2/M blocking compound, causes an increase in phosphorylation of these three amino acids. By elutriating the Jurkat cells, we are able to demonstrate that these amino acids are normally phosphorylated at the G2/M phase of the cell cycle. Using in vivo inhibitors and in vitro assays this phosphorylation appears to be dependent on Cdk1. We find that RUNX1 degradation occurs at the G2/M–G1 transition and is regulated by both Cdc20 and phosphoryation, suggesting that the anaphase promoting complex plays a role in modifying the level of this protein. Regulation of the extent of phosphorylation of RUNX1 may play a role in controlling the degradation of the protein, implying that additional E3 ligases may also be involved

Interferon-γ Induces Senescence in Normal Human Melanocytes

<div><p>Background</p><p>Interferon-γ (IFN-γ) plays an important role in the proceedings of vitiligo through recruiting lymphocytes to the lesional skin. However, the potential effects of IFN-γ on skin melanocytes and the subsequent contribution to the vitiligo pathogenesis are still unclear.</p><p>Objective</p><p>To investigate the effects of IFN-γ on viability and cellular functions of melanocytes.</p><p>Methods</p><p>Primary human melanocytes were treated with IFN-γ. Cell viability, apoptosis, cell cycle melanin content and intracellular reactive oxygen species (ROS) level were measured. mRNA expression was examined by real-time PCR. The release of interleukin 6 (IL-6) and heat shock protein 70 (HSP-70) was monitored by ELISA. β-galactosidase staining was utilized to evaluate melanocyte senescence.</p><p>Results</p><p>Persistent IFN-γ treatment induced viability loss, apoptosis, cell cycle arrest and senescence in melanocytes. Melanocyte senescence was characterized as the changes in pigmentation and morphology, as well as the increase of β-galactosidase activity. Increase of p21^Cip1/Waf1 protein was evident in melanocytes after IFN-γ treatment. IFN-γ induction of senescence was attenuated by siRNAs against p21, Janus kinase 2 (JAK2) or signal transducer and activator of transcription 1 (STAT1), but not by JAK1 siRNA nor by p53 inhibitor pifithrin-α. IFN-γ treatment increased the accumulation of intracellular ROS in melanocytes, while ROS scavenger N-acetyl cysteine (NAC) effectively inhibited IFN-γ induced p21 expression and melanocyte senescence. IL-6 and HSP-70 release was significantly induced by IFN-γ treatment, which was largely inhibited by NAC. The increase of IL-6 and HSP-70 release could also be observed in senescent melanocytes.</p><p>Conclusion</p><p>IFN-γ can induce senescence in melanocytes and consequently enhance their immuno-competency, leading to a vitiligo-prone milieu.</p></div

IFN-γ decreased viability of melanocytes, caused apoptosis and cell cycle arrest.

<p>Primary normal human Melanocytes were treated with various concentrations of IFN-γ (0, 100 or 1000 U/ml) for 72 h. Cell viability was then examined by MTS assay (A). Apoptosis was analyzed by flow cytometry after cells were stained with PI and Annexin V-FITC (B). (C) Cell cycle distribution of melanocytes was measured 24 h post IFN-γ treatment. Results are presented as mean ± SD from at least three independent melanocyte cultures. *P<0.05, **P<0.01, Student's t-test compared with controls.</p

JAK2 and STAT1 activities are necessary for IFN-γ caused melanocyte senescence.

<p>Melanocytes were transfected with JAK1, JAK2, STAT1 siRNAs or scrambled control siRNA (Ctrl). After 48 h, cells were treated with or without 100 U/ml IFN-γ for additional 7 days. (A) Cell viability of melancytes was measured by MTS assay. (B) Protein level of p21 was evaluated by Western blot. β-actin was probed as the loading control. (C) Percentages of SA-β-gal-positive cells were determined based on microscopic analysis.</p

Effects of IFN-γ on melanogenesis in normal melanocytes.

<p>(A) Melanocytes were treated with various concentrations of IFN-γ (0, 100 or 1000 U/ml) for 3 or 7days before melanin content was measured. The melanin content was normalized on the basis of protein concentration. (b–g) Total RNA was extracted from melanocytes treated with or without IFN-γ for 24 hours. Real-time PCR was then performed to evaluate the relative mRNA levels of (B) tyrosinase (TYR), (C) tyrosinase-related protein 1 (TYRP1), (D) Melan-A, (E) melanocyte protein 17 (PMEL17), (F) microphthalmia-associated transcription factor (MITF), and (G) dopachrome tautomerase (DCT). The values shown represent the mean ± SD of three independent melanocyte cultures. *P<0.05 and **P<0.01.</p

Involvement of Reactive Oxygen Species (ROS) in the IFN-γ induced senescence.

<p>(A) Melancytes were stimulated with indicated concentration of IFN-γ for 24 h. Generated ROS was detected with flow cytometer after labelled with the ROS sensor DCFH-DA. (B,C) Melanocytes were treated with or without 100 U/ml IFN-γ for 7 days in the presence of vehicle or 1 mM NAC. (B) Protein level of p21 was evaluated by Western blot. β-actin was probed as the loading control. (C) Percentages of SA-β-gal-positive cells were determined based on microscopic analysis. CON represents the cell culture without IFN-γ.</p