Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 7 de 7
Filter
Add more filters










Database
Language
Publication year range
1.
bioRxiv ; 2024 Jun 05.
Article in English | MEDLINE | ID: mdl-38895302

ABSTRACT

Background: Tanning bed users have a significantly increased risk of melanoma, but it remains unclear how indoor tanning drives melanomagenesis. Tanning bed radiation is often thought of as a substitute for natural UV radiation despite differences in the maximum doses, UV content, body sites exposed, and patterns of melanoma that arise. Methods: To better understand the epidemiologic trends and etiology of melanoma associated with tanning bed use, we described the patterns of melanoma in patients with quantifiable tanning bed usage and performed exome sequencing of 182 melanocytes from normal skin of a subset of these patients. Results: Tanning bed users were more likely than non-users to have melanoma on body sites with low cumulative levels of sun damage and were more likely to have multiple melanomas. The melanocytes in normal appearing skin from tanning bed users had higher mutation burdens, a higher proportion of melanocytes with pathogenic mutations, and distinct mutational signatures. These differences were most prominent over body sites that experience comparatively less exposure to natural sunlight. Conclusions: We conclude that tanning bed radiation induces melanoma by increasing the mutation burden of melanocytes and by mutagenizing a broader field of melanocytes than are typically exposed to natural sunlight. The unique signatures of mutations in skin cells of tanning users may be attributable to the distinct spectra of radiation emitted from solariums.

2.
Genome Biol ; 24(1): 273, 2023 Nov 30.
Article in English | MEDLINE | ID: mdl-38037084

ABSTRACT

Spatial transcriptomic technologies, such as the Visium platform, measure gene expression in different regions of tissues. Here, we describe new software, STmut, to visualize somatic point mutations, allelic imbalance, and copy number alterations in Visium data. STmut is tested on fresh-frozen Visium data, formalin-fixed paraffin-embedded (FFPE) Visium data, and tumors with and without matching DNA sequencing data. Copy number is inferred on all conditions, but the chemistry of the FFPE platform does not permit analyses of single nucleotide variants. Taken together, we propose solutions to add the genetic dimension to spatial transcriptomic data and describe the limitations of different datatypes.


Subject(s)
Formaldehyde , Neoplasms , Humans , Transcriptome , Paraffin Embedding , Neoplasms/genetics , Gene Expression Profiling , High-Throughput Nucleotide Sequencing
3.
Pigment Cell Melanoma Res ; 34(5): 905-917, 2021 09.
Article in English | MEDLINE | ID: mdl-33544968

ABSTRACT

Melanocyte stem cells (McSCs) are key components of the hair follicle (HF) stem cell system that regenerate differentiated melanocytes during successive HF cycles. To facilitate continued research on melanocyte development and differentiation and McSCs, we backcrossed inducible Dct-H2BGFP mice into the C57BL/6J background (B6-Dct-H2BGFP). We compared the expression pattern of B6-Dct-H2BGFP to that of Dct-H2BGFP mice on a mixed genetic background reported previously. To characterize B6-Dct-H2BGFP mice, we confirmed not only the expression of GFP in all melanocyte lineage cells, but also doxycycline regulation of GFP expression. Furthermore, ex vivo culture of the McSC subsets isolated by fluorescence-activated cell sorting (FACS) showed the propensity of bulge/CD34+ McSCs to differentiate with expression of non-melanocytic, neural crest lineage markers including glia (Gfap and CNPase, 73 ± 1% and 77 ± 2%, respectively), neurons (Tuj1 26 ± 5%), and smooth muscle (α-Sma, 31 ± 9%). In contrast, CD34-/secondary hair germ (SHG) McSCs differentiated into pigmented melanocytes, with higher expression of melanogenic markers Tyr (71 ± 1%), Tyrp1 (68 ± 4%), and Mitf (75 ± 7%). These results establish the utility of B6-Dct-H2BGFP bitransgenic mice for future in vivo studies of melanocytes requiring a defined genetic background.


Subject(s)
Cell Differentiation , Gene Expression Regulation , Melanocytes/metabolism , Membrane Glycoproteins/biosynthesis , Microphthalmia-Associated Transcription Factor/biosynthesis , Models, Biological , Oxidoreductases/biosynthesis , Stem Cells/metabolism , Animals , Membrane Glycoproteins/genetics , Mice , Mice, Transgenic , Microphthalmia-Associated Transcription Factor/genetics , Oxidoreductases/genetics
4.
PLoS Genet ; 15(4): e1008034, 2019 04.
Article in English | MEDLINE | ID: mdl-31017901

ABSTRACT

Melanocyte stem cells (McSCs) are the undifferentiated melanocytic cells of the mammalian hair follicle (HF) responsible for recurrent generation of a large number of differentiated melanocytes during each HF cycle. HF McSCs reside in both the CD34+ bulge/lower permanent portion (LPP) and the CD34- secondary hair germ (SHG) regions of the HF during telogen. Using Dct-H2BGFP mice, we separate bulge/LPP and SHG McSCs using FACS with GFP and anti-CD34 to show that these two subsets of McSCs are functionally distinct. Genome-wide expression profiling results support the distinct nature of these populations, with CD34- McSCs exhibiting higher expression of melanocyte differentiation genes and with CD34+ McSCs demonstrating a profile more consistent with a neural crest stem cell. In culture and in vivo, CD34- McSCs regenerate pigmentation more efficiently whereas CD34+ McSCs selectively exhibit the ability to myelinate neurons. CD34+ McSCs, and their counterparts in human skin, may be useful for myelinating neurons in vivo, leading to new therapeutic opportunities for demyelinating diseases and traumatic nerve injury.


Subject(s)
Antigens, CD34/metabolism , Melanocytes/immunology , Melanocytes/physiology , Stem Cells/immunology , Stem Cells/physiology , Animals , Cell Differentiation , Cells, Cultured , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Hair Color/physiology , Hair Follicle/cytology , Hair Follicle/physiology , Melanocytes/classification , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Mice, Nude , Mice, Transgenic , Myelin Basic Protein/deficiency , Myelin Basic Protein/genetics , Neural Crest/cytology , Neural Crest/immunology , Neural Crest/physiology , Pigmentation/physiology , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Regeneration/physiology , Stem Cells/classification
5.
Gene Expr Patterns ; 27: 76-84, 2018 01.
Article in English | MEDLINE | ID: mdl-29061525

ABSTRACT

Melanocytes are neural crest-derived cells that are responsible for mammalian hair follicle (HF) pigmentation. The Dct-LacZ transgenic mouse is extensively used to study melanocyte biology but lacks conditionally-inducible labelling and fluorescent labelling, enabling specific, viable isolation of melanocytes using fluorescence-activated cell sorting (FACS). Here, we have generated a Tet-off bitransgenic mouse model, Dct-H2BGFP, containing Dct-tTA and TRE-H2BGFP transgenes. Characterization of Dct-H2BGFP mice confirmed a pattern of Dct-H2BGFP expression in melanoblasts, melanocyte stem cells (McSCs), and terminally differentiated melanocytes similar to the expression pattern of previously published mouse models Dct-LacZ and iDct-GFP. GFP expression is regulated by doxycycline. GFP is shown to co-localize with melanocyte label-retaining cells (LRCs) identified through BrdU retention. The GFP-expressing cells identified in vivo in the bulge and the secondary hair germ of telogen HFs of Dct-H2BGFP mice express the melanocyte and melanocyte stem cell markers Dct and Kit. Using Dct-H2BGFP mice, we separated GFP-expressing cells from the telogen HF based on FACS and showed that GFP-expressing cells express high levels of Kit and Dct, and lower levels of HF epithelial keratin genes. We also show that GFP-expressing cells express high levels of the melanocyte differentiation genes Tyr, Tyrp1, and Pmel17, further substantiating their identity within the melanocyte lineage. Thus, Dct-H2BGFP mice are not only useful for the in vivo identification of melanocytic cells, but also for isolating them viably and studying their molecular and biological properties.


Subject(s)
Embryonic Stem Cells/cytology , Gene Expression Regulation, Developmental/drug effects , Hair Follicle/cytology , Melanocytes/cytology , Neural Crest/cytology , Tetracycline/pharmacology , Animals , Cell Lineage , Embryonic Stem Cells/drug effects , Embryonic Stem Cells/metabolism , Female , Flow Cytometry/methods , Hair Follicle/drug effects , Hair Follicle/metabolism , Lac Operon , Male , Melanocytes/drug effects , Melanocytes/metabolism , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Models, Animal , Neural Crest/drug effects , Neural Crest/metabolism , Oxidoreductases/genetics , Oxidoreductases/metabolism , Pigmentation , Promoter Regions, Genetic , Protein Synthesis Inhibitors/pharmacology , gp100 Melanoma Antigen/genetics , gp100 Melanoma Antigen/metabolism
6.
J Biol Chem ; 292(34): 14122-14133, 2017 08 25.
Article in English | MEDLINE | ID: mdl-28679534

ABSTRACT

Hsp70 is a protein chaperone that prevents protein aggregation and aids protein folding by binding to hydrophobic peptide domains through a reversible mechanism directed by an ATPase cycle. However, Hsp70 also binds U-rich RNA including some AU-rich elements (AREs) that regulate the decay kinetics of select mRNAs and has recently been shown to bind and stabilize some ARE-containing transcripts in cells. Previous studies indicated that both the ATP- and peptide-binding domains of Hsp70 contributed to the stability of Hsp70-RNA complexes and that ATP might inhibit RNA recruitment. This suggested the possibility that RNA binding by Hsp70 might mimic features of its peptide-directed chaperone activities. Here, using purified, cofactor-free preparations of recombinant human Hsp70 and quantitative biochemical approaches, we found that high-affinity RNA binding requires at least 30 nucleotides of RNA sequence but is independent of Hsp70's nucleotide-bound status, ATPase activity, or peptide-binding roles. Furthermore, although both the ATP- and peptide-binding domains of Hsp70 could form complexes with an ARE sequence from VEGFA mRNA in vitro, only the peptide-binding domain could recover cellular VEGFA mRNA in ribonucleoprotein immunoprecipitations. Finally, Hsp70-directed stabilization of VEGFA mRNA in cells was mediated exclusively by the protein's peptide-binding domain. Together, these findings indicate that the RNA-binding and mRNA-stabilizing functions of Hsp70 are independent of its protein chaperone cycle but also provide potential mechanical explanations for several well-established and recently discovered cytoprotective and RNA-based Hsp70 functions.


Subject(s)
HSP70 Heat-Shock Proteins/metabolism , RNA, Messenger/metabolism , Ribonucleoproteins/metabolism , Vascular Endothelial Growth Factor A/metabolism , AU Rich Elements , Allosteric Regulation , Binding Sites , HSP70 Heat-Shock Proteins/chemistry , HSP70 Heat-Shock Proteins/genetics , HeLa Cells , Humans , Immunoprecipitation , Kinetics , Mutation , Oligopeptides/genetics , Oligopeptides/metabolism , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Interaction Domains and Motifs , RNA/antagonists & inhibitors , RNA/metabolism , RNA Interference , RNA Stability , RNA, Messenger/chemistry , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Ribonucleoproteins/chemistry , Vascular Endothelial Growth Factor A/chemistry , Vascular Endothelial Growth Factor A/genetics
7.
Mol Cell Biol ; 33(1): 71-84, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23109422

ABSTRACT

The AU-rich elements (AREs) encoded within many mRNA 3' untranslated regions (3'UTRs) are targets for factors that control transcript longevity and translational efficiency. Hsp70, best known as a protein chaperone with well-defined peptide-refolding properties, is known to interact with ARE-like RNA substrates in vitro. Here, we show that cofactor-free preparations of Hsp70 form direct, high-affinity complexes with ARE substrates based on specific recognition of U-rich sequences by both the ATP- and peptide-binding domains. Suppressing Hsp70 in HeLa cells destabilized an ARE reporter mRNA, indicating a novel ARE-directed mRNA-stabilizing role for this protein. Hsp70 also bound and stabilized endogenous ARE-containing mRNAs encoding vascular endothelial growth factor (VEGF) and Cox-2, which involved a mechanism that was unaffected by an inhibitor of its protein chaperone function. Hsp70 recognition and stabilization of VEGF mRNA was mediated by an ARE-like sequence in the proximal 3'UTR. Finally, stabilization of VEGF mRNA coincided with the accumulation of Hsp70 protein in HL60 promyelocytic leukemia cells recovering from acute thermal stress. We propose that the binding and stabilization of selected ARE-containing mRNAs may contribute to the cytoprotective effects of Hsp70 following cellular stress but may also provide a novel mechanism linking constitutively elevated Hsp70 expression to the development of aggressive neoplastic phenotypes.


Subject(s)
AU Rich Elements , HSP70 Heat-Shock Proteins/metabolism , RNA, Messenger/metabolism , 3' Untranslated Regions , Binding Sites , Cyclooxygenase 2/genetics , Cyclooxygenase 2/metabolism , Gene Expression Regulation , HL-60 Cells , HSP70 Heat-Shock Proteins/genetics , HeLa Cells , Humans , Molecular Chaperones/metabolism , Protein Structure, Tertiary , RNA Processing, Post-Transcriptional , RNA Stability , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolism
SELECTION OF CITATIONS
SEARCH DETAIL
...