RESUMEN
Induced pluripotent stem cells (iPSCs) can in principle differentiate into any cell of the body, and have revolutionized biomedical research and regenerative medicine. Unlike their human counterparts, mouse iPSCs (miPSCs) are reported to silence transposable elements and prevent transposable element-mediated mutagenesis. Here we apply short-read or Oxford Nanopore Technologies long-read genome sequencing to 38 bulk miPSC lines reprogrammed from 10 parental cell types, and 18 single-cell miPSC clones. While single nucleotide variants and structural variants restricted to miPSCs are rare, we find 83 de novo transposable element insertions, including examples intronic to Brca1 and Dmd. LINE-1 retrotransposons are profoundly hypomethylated in miPSCs, beyond other transposable elements and the genome overall, and harbor alternative protein-coding gene promoters. We show that treatment with the LINE-1 inhibitor lamivudine does not hinder reprogramming and efficiently blocks endogenous retrotransposition, as detected by long-read genome sequencing. These experiments reveal the complete spectrum and potential significance of mutations acquired by miPSCs.
Asunto(s)
Células Madre Pluripotentes Inducidas , Humanos , Ratones , Animales , Retroelementos/genética , Elementos Transponibles de ADN/genética , Mutación , Elementos de Nucleótido Esparcido Largo/genéticaRESUMEN
LINE-1 (L1) retrotransposons are a source of insertional mutagenesis in tumor cells. However, the clinical significance of L1 mobilization during tumorigenesis remains unclear. Here, we applied retrotransposon capture sequencing (RC-seq) to multiple single-cell clones isolated from five ovarian cancer cell lines and HeLa cells and detected endogenous L1 retrotransposition in vitro. We then applied RC-seq to ovarian tumor and matched blood samples from 19 patients and identified 88 tumor-specific L1 insertions. In one tumor, an intronic de novo L1 insertion supplied a novel cis-enhancer to the putative chemoresistance gene STC1. Notably, the tumor subclone carrying the STC1 L1 mutation increased in prevalence after chemotherapy, further increasing STC1 expression. We also identified hypomethylated donor L1s responsible for new L1 insertions in tumors and cultivated cancer cells. These congruent in vitro and in vivo results highlight L1 insertional mutagenesis as a common component of ovarian tumorigenesis and cancer genome heterogeneity.
Asunto(s)
Evolución Molecular , Elementos de Nucleótido Esparcido Largo/genética , Neoplasias Ováricas/patología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Metilación de ADN , Resistencia a Antineoplásicos , Femenino , Regulación Neoplásica de la Expresión Génica , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Pérdida de Heterocigocidad/genética , Mutagénesis Insercional , Mutación , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genéticaRESUMEN
An increasing number of people are getting tattoos; however, many regret the decision and seek their removal. Lasers are currently the most commonly used method for tattoo removal; however, treatment can be lengthy, costly, and sometimes ineffective, especially for certain colors. Ingenol mebutate is a licensed topical treatment for actinic keratoses. Here, we demonstrate that two applications of 0.1% ingenol mebutate can efficiently and consistently remove 2-week-old tattoos from SKH/hr hairless mice. Treatment was associated with relocation of tattoo microspheres from the dermis into the posttreatment eschar. The skin lesion resolved about 20 days after treatment initiation, with some cicatrix formation evident. The implications for using ingenol mebutate for tattoo removal in humans are discussed.
RESUMEN
Enhancement of regulatory T cell (Treg cell) frequency and function is the goal of many therapeutic strategies aimed at treating type 1 diabetes (T1D). The interleukin-2 (IL-2) pathway, which has been strongly implicated in T1D susceptibility in both humans and mice, is a master regulator of Treg cell homeostasis and function. We investigated how IL-2 pathway defects impact Treg cells in T1D-susceptible nonobese diabetic (NOD) mice in comparison with protected C57BL/6 and NOD congenic mice. NOD Treg cells were reduced in frequency specifically in the lymph nodes and expressed lower levels of CD25 and CD39/CD73 immunosuppressive molecules. In the spleen and blood, Treg cell frequency was preserved through expansion of CD25(low), effector phenotype Treg cells. Reduced CD25 expression led to decreased IL-2 signaling in NOD Treg cells. In vivo, treatment with IL-2-anti-IL-2 antibody complexes led to effective upregulation of suppressive molecules on NOD Treg cells in the spleen and blood, but had reduced efficacy on lymph node Treg cells. In contrast, NOD CD8(+) and CD4(+) effector T cells were not impaired in their response to IL-2 therapy. We conclude that NOD Treg cells have an impaired responsiveness to IL-2 that reduces their ability to compete for a limited supply of IL-2.
Asunto(s)
Interleucina-2/metabolismo , Linfocitos T Reguladores/inmunología , Alelos , Animales , Antígenos CD/metabolismo , Movimiento Celular , Proliferación Celular , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Haplotipos/genética , Terapia de Inmunosupresión , Ganglios Linfáticos/metabolismo , Recuento de Linfocitos , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Páncreas/patología , Fenotipo , Factor de Transcripción STAT5/metabolismo , Transducción de Señal , Bazo/metabolismo , Regulación hacia ArribaRESUMEN
Ingenol mebutate has recently been approved by the Federal Drug Administration (USA) as a topical treatment for actinic keratoses. Herein, we describe the efficacy of ingenol mebutate for the topical treatment of squamous cell carcinoma (SCC) using a wild-type mouse model (SKH1) and the UV-induced mouse SCC cell line, T7. Daily treatment for 2 days with 0.25 % ingenol mebutate gel produced a cure rate of 70 %, with 0 % for placebo gel. Electron microscopy revealed swelling of cancer cell mitochondria within 1 h, with disruption of the inner mitochondrial membranes evident at 6 h post treatment. Primary necrosis of cancer cells was clearly evident by 24 h. Treatment was associated with local haemorrhage and a prodigious neutrophil infiltrate, with anti-T7 antibodies also detected. This is the first report of the successful treatment of SCC tumours with ingenol mebutate gel in wild-type mice, and supports the view that ingenol mebutate induces primary necrosis and activates the immune system.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma de Células Escamosas/tratamiento farmacológico , Diterpenos/farmacología , Neoplasias Cutáneas/tratamiento farmacológico , Administración Cutánea , Animales , Antineoplásicos/administración & dosificación , Carcinoma de Células Escamosas/inmunología , Carcinoma de Células Escamosas/ultraestructura , Diterpenos/administración & dosificación , Femenino , Geles , Masculino , Ratones , Ratones Pelados , Microscopía Electrónica , Mitocondrias/efectos de los fármacos , Mitocondrias/patología , Dilatación Mitocondrial/efectos de los fármacos , Necrosis , Infiltración Neutrófila/efectos de los fármacos , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/ultraestructura , Factores de TiempoRESUMEN
Skin cancer is the most prevalent cancer worldwide and is primarily caused by chronic UV exposure. Here, we describe the topical field-directed treatment of SKH1/hr mice with UVB-damaged skin with ingenol mebutate, a new topical drug shown to be effective for the treatment of actinic keratosis (AK). Application of 0.05% ingenol mebutate gel to photo-damaged skin resulted in a ≈70% reduction in the number of skin lesions that subsequently emerged compared with placebo treatment. Ingenol mebutate treatment also reduced the number of mutant p53 keratinocyte patches by ≈70%. The treatment resulted in epidermal cell death, acute inflammation, recruitment of neutrophils, hemorrhage, and eschar formation, all of which resolved over several weeks. Ingenol mebutate field-directed treatment might thus find utility in the removal of subclinical precancerous cells from UV-damaged skin. Field-directed treatment may be particularly suitable for patients who have AKs surrounded by UV-damaged skin.