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1.
Genes (Basel) ; 10(4)2019 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-30987199

RESUMO

Fluorescent light (FL) has been utilized for ≈60 years and has become a common artificial light source under which animals, including humans, spend increasing amounts of time. Although the solar spectrum is quite dissimilar in both wavelengths and intensities, the genetic consequences of FL exposure have not been investigated. Herein, we present comparative RNA-Seq results that establish expression patterns within skin, brain, and liver for Danio rerio, Oryzias latipes, and the hairless mouse (Mus musculus) after exposure to FL. These animals represent diurnal and nocturnal lifestyles, and ≈450 million years of evolutionary divergence. In all three organisms, FL induced transcriptional changes of the acute phase response signaling pathway and modulated inflammation and innate immune responses. Our pathway and gene clustering analyses suggest cellular perception of oxidative stress is promoting induction of primary up-stream regulators IL1B and TNF. The skin and brain of the three animals as well as the liver of both fish models all exhibit increased inflammation and immune responses; however, the mouse liver suppressed the same pathways. Overall, the conserved nature of the genetic responses observed after FL exposure, among fishes and a mammal, suggest the presence of light responsive genetic circuitry deeply embedded in the vertebrate genome.

2.
FASEB J ; 33(6): 6778-6788, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30807703

RESUMO

Maintenance of human embryonic stem cells (hESCs) with stable genome is important for their future use in cell replacement therapy and disease modeling. Our understanding of the mechanisms maintaining genomic stability of hESC and our ability to modulate them is essential in preventing unwanted mutation accumulation during their in vitro cultivation. In this study, we show the DNA damage response mechanism in hESCs is composed of known, yet unlikely components. Clustered oxidative base damage is converted into DNA double-strand breaks (DSBs) by base excision repair (BER) and then quickly repaired by ligase (Lig)3-mediated end-joining (EJ). If there is further induction of clustered oxidative base damage by irradiation, then BER-mediated DSBs become essential in triggering the checkpoint response in hESCs. hESCs limit the mutagenic potential of Lig3-mediated EJ by DNA break end protection involving p53 binding protein 1 (53BP1), which results in fast and error-free microhomology-mediated repair and a low mutant frequency in hESCs. DSBs in hESCs are also repaired via homologous recombination (HR); however, DSB overload, together with massive end protection by 53BP1, triggers competition between error-free HR and mutagenic nonhomologous EJ.-Kohutova, A., Raska, J., Kruta, M., Seneklova, M., Barta, T., Fojtik, P., Jurakova, T., Walter, C. A., Hampl, A., Dvorak, P., Rotrekl, V. Ligase 3-mediated end-joining maintains genome stability of human embryonic stem cells.

3.
Mol Carcinog ; 2018 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-30365185

RESUMO

Hepatocellular carcinoma (HCC) remains a deadly cancer, underscoring the need for relevant preclinical models. Male C3HeB/FeJ mice model spontaneous HCC with some hepatocarcinogenesis susceptibility loci corresponding to syntenic regions of human chromosomes altered in HCC. We tested other properties of C3HeB/FeJ tumors for similarity to human HCC. C3HeB/FeJ tumors were grossly visible at 4 months of age, with prevalence and size increasing until about 11 months of age. Histologic features shared with human HCC include hepatosteatosis, tumor progression from dysplasia to poorly differentiated, vascular invasion, and trabecular, oncocytic, vacuolar, and clear cell variants. More tumor cells displayed cytoplasmic APE1 staining versus normal liver. Ultrasound effectively detected and monitored tumors, with 85.7% sensitivity. Over 5000 genes were differentially expressed based on the GSE62232 and GSE63898 human HCC datasets. Of these, 158 and 198 genes, respectively, were also differentially expressed in C3HeB/FeJ. Common cancer pathways, cell cycle, p53 signaling and other molecular aspects, were shared between human and mouse differentially expressed genes. We established eigengenes that distinguish HCC from normal liver in the C3HeB/FeJ model and a subset of human HCC. These features extend the relevance and improve the utility of the C3HeB/FeJ line for HCC studies.

4.
Stem Cell Res ; 19: 113-117, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28129601

RESUMO

Pluripotent cells have been reported to exhibit lower frequencies of point mutations and higher levels of DNA repair than differentiated cells. This predicts that pluripotent cells are less susceptible to mutagenic exposures than differentiated cells. To test this prediction, we used a lacI mutation-reporter transgene system to assess the frequency of point mutations in multiple lines of mouse pluripotent embryonic stem cells and induced pluripotent cells, as well as in multiple lines of differentiated fibroblast cells, before and after exposure to a moderate dose of the mutagen, methyl methanesulfonate. We also measured levels of key enzymes in the base excision repair (BER) pathway in each cell line before and after exposure to the mutagen. Our results confirm that pluripotent cells normally maintain lower frequencies of point mutations than differentiated cells, and show that differentiated cells exhibit a large increase in mutation frequency following a moderate mutagenic exposure, whereas pluripotent cells subjected to the same exposure show no increase in mutations. This result likely reflects the higher levels of BER proteins detectable in pluripotent cells prior to exposure and supports our thesis that maintenance of enhanced genetic integrity is a fundamental characteristic of pluripotent cells.


Assuntos
Células-Tronco/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Epigenômica , Metanossulfonato de Metila/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos
5.
Aging (Albany NY) ; 8(11): 2754-2776, 2016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-27852980

RESUMO

Breast cancer incidence increases during aging, yet the mechanism of age-associated mammary tumorigenesis is unclear. Mammary stem cells are believed to play an important role in breast tumorigenesis, but how their function changes with age is unknown. We compared mammary epithelial cells isolated from young and old mammary glands of different cohorts of C57BL6/J and BALB/c mice, and our findings revealed that old mammary glands were characterized by increased basal cell pool comprised of mostly CD49fhi cells, altered luminal-to-basal cell ratio, and irregular ductal morphology. More interestingly, basal stem cells in old mice were increased in frequency, but showed a functional decline of differentiation and increased neoplastic transformation potential. Gene signature enrichment analysis revealed a significant enrichment of a luminal cell gene expression signature in the basal stem cell-enriched population from old mice, suggesting some luminal cells were expressing basal markers. Immunofluorescence staining confirmed the presence of luminal cells with high CD49f expression in hyperplastic lesions implicating these cells as undergoing luminal to basal phenotypic changes during aging. Whole transcriptome analysis showed elevated immune and inflammatory responses in old basal stem cells and stromal cells, which may be the underlying cause for increased CD49fhi basal-like cells in aged glands.


Assuntos
Envelhecimento/patologia , Transformação Celular Neoplásica/patologia , Glândulas Mamárias Animais/patologia , Neoplasias Mamárias Animais/patologia , Células-Tronco/patologia , Fatores Etários , Envelhecimento/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Feminino , Perfilação da Expressão Gênica , Inflamação/metabolismo , Inflamação/patologia , Integrina alfa6/metabolismo , Glândulas Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Células-Tronco/metabolismo
6.
Cancer Prev Res (Phila) ; 9(3): 245-52, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26667451

RESUMO

Hepatocellular carcinoma is increasingly important in the United States as the incidence rate rose over the last 30 years. C3HeB/FeJ mice serve as a unique model to study hepatocellular carcinoma tumorigenesis because they mimic human hepatocellular carcinoma with delayed onset, male gender bias, approximately 50% incidence, and susceptibility to tumorigenesis is mediated through multiple genetic loci. Because a human O(6)-methylguanine-DNA methyltransferase (hMGMT) transgene reduces spontaneous tumorigenesis in this model, we hypothesized that hMGMT would also protect from methylation-induced hepatocarcinogenesis. To test this hypothesis, wild-type and hMGMT transgenic C3HeB/FeJ male mice were treated with two monofunctional alkylating agents: diethylnitrosamine (DEN; 0.025 µmol/g body weight) on day 12 of life with evaluation for glucose-6-phosphatase-deficient (G6PD) foci at 16, 24, and 32 weeks or N-methyl-N-nitrosurea (MNU; 25 mg MNU/kg body weight) once monthly for 7 months starting at 3 months of age with evaluation for liver tumors at 12 to 15 months of age. No difference in abundance or size of G6PD foci was measured with DEN treatment. In contrast, it was unexpectedly found that MNU reduces liver tumor prevalence in wild-type and hMGMT transgenic mice despite increased tumor prevalence in other tissues. hMGMT and MNU protections were additive, suggesting that MNU protects through a different mechanism, perhaps through the cytotoxic N7-alkylguanine and N3-alkyladenine lesions which have low mutagenic potential compared with O(6)-alkylguanine lesions. Together, these results suggest that targeting the repair of cytotoxic lesions may be a good preventative for patients at high risk of developing hepatocellular carcinoma.


Assuntos
Alquilantes/farmacologia , Carcinoma Hepatocelular/prevenção & controle , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Dietilnitrosamina/farmacologia , Neoplasias Hepáticas Experimentais/prevenção & controle , Metilnitrosoureia/farmacologia , Proteínas Supressoras de Tumor/genética , Animais , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Técnicas Imunoenzimáticas , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Transgênicos
7.
Mutat Res ; 779: 124-33, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26201249

RESUMO

Increased paternal age is associated with a greater risk of producing children with genetic disorders originating from de novo germline mutations. Mice mimic the human condition by displaying an age-associated increase in spontaneous mutant frequency in spermatogenic cells. The observed increase in mutant frequency appears to be associated with a decrease in the DNA repair protein, AP endonuclease 1 (APEX1) and Apex1 heterozygous mice display an accelerated paternal age effect as young adults. In this study, we directly tested if APEX1 over-expression in cell lines and transgenic mice could prevent increases in mutagenesis. Cell lines with ectopic expression of APEX1 had increased APEX1 activity and lower spontaneous and induced mutations in the lacI reporter gene relative to the control. Spermatogenic cells obtained from mice transgenic for human APEX1 displayed increased APEX1 activity, were protected from the age-dependent increase in spontaneous germline mutagenesis, and exhibited increased apoptosis in the spermatogonial cell population. These results directly indicate that increases in APEX1 level confer protection against the murine paternal age effect, thus highlighting the role of APEX1 in preserving reproductive health with increasing age and in protection against genotoxin-induced mutagenesis in somatic cells.


Assuntos
DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Mutagênese/genética , Idade Paterna , Espermatogênese/genética , Animais , Apoptose/genética , Reparo do DNA/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/biossíntese , Regulação da Expressão Gênica no Desenvolvimento , Mutação em Linhagem Germinativa , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Espermatozoides/metabolismo , Espermatozoides/patologia
8.
Stem Cell Res ; 13(3 Pt A): 508-19, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25451711

RESUMO

The Disposable Soma Theory holds that genetic integrity will be maintained at more pristine levels in germ cells than in somatic cells because of the unique role germ cells play in perpetuating the species. We tested the hypothesis that the same concept applies to pluripotent cells compared to differentiated cells. Analyses of transcriptome and cistrome databases, along with canonical pathway analysis and chromatin immunoprecipitation confirmed differential expression of DNA repair and cell death genes in embryonic stem cells and induced pluripotent stem cells relative to fibroblasts, and predicted extensive direct and indirect interactions between the pluripotency and genetic integrity gene networks in pluripotent cells. These data suggest that enhanced maintenance of genetic integrity is fundamentally linked to the epigenetic state of pluripotency at the genomic level. In addition, these findings demonstrate how a small number of key pluripotency factors can regulate large numbers of downstream genes in a pathway-specific manner.


Assuntos
Genômica , Modelos Biológicos , Animais , Apoptose/genética , Imunoprecipitação da Cromatina , Reparo do DNA/genética , Bases de Dados Genéticas , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Genoma , Humanos , Camundongos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transcriptoma
9.
J Vis Exp ; (84): e50752, 2014 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-24637843

RESUMO

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases. LacI transgenic mice carry a recoverable λ phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of ß-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation. The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin(-)IL7R(-)Sca-1(+)cKit(++)(LSK) cells and other subpopulations of the hematopoietic system.


Assuntos
Análise Mutacional de DNA/métodos , Células-Tronco Hematopoéticas/fisiologia , Animais , Bacteriófago lambda/genética , DNA/análise , DNA/genética , Vetores Genéticos/genética , Células-Tronco Hematopoéticas/química , Repressores Lac/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutagênese
10.
Cancer Prev Res (Phila) ; 7(4): 445-55, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24520039

RESUMO

Perinatal exposure to bisphenol A (BPA) has been shown to cause aberrant mammary gland morphogenesis and mammary neoplastic transformation. Yet, the underlying mechanism is poorly understood. We tested the hypothesis that mammary glands exposed to BPA during a susceptible window may lead to its susceptibility to tumorigenesis through a stem cell-mediated mechanism. We exposed 21-day-old Balb/c mice to BPA by gavage (25 µg/kg/d) during puberty for 3 weeks, and a subset of animals were further challenged with one oral dose (30 mg/kg) of 7,12-dimethylbenz(a)anthracene (DMBA) at 2 months of age. Primary mammary cells were isolated at 6 weeks, and 2 and 4 months of age for murine mammary stem cell (MaSC) quantification and function analysis. Pubertal exposure to the low-dose BPA increased lateral branches and hyperplasia in adult mammary glands and caused an acute increase of MaSC in 6-week-old glands and a delayed increase of luminal progenitors in 4-month-old adult gland. Most importantly, pubertal BPA exposure altered the function of MaSC from different age groups, causing early neoplastic lesions in their regenerated glands similar to those induced by DMBA exposure, which indicates that MaSCs are susceptible to BPA-induced transformation. Deep sequencing analysis on MaSC-enriched mammospheres identified a set of aberrantly expressed genes associated with early neoplastic lesions in patients with human breast cancer. Thus, our study for the first time shows that pubertal BPA exposure altered MaSC gene expression and function such that they induced early neoplastic transformation.


Assuntos
Compostos Benzidrílicos/toxicidade , Transformação Celular Neoplásica/patologia , Depuradores de Radicais Livres/toxicidade , Glândulas Mamárias Animais/patologia , Neoplasias Mamárias Animais/induzido quimicamente , Neoplasias Mamárias Animais/patologia , Fenóis/toxicidade , Células-Tronco/efeitos dos fármacos , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proliferação de Células , Transformação Celular Neoplásica/efeitos dos fármacos , Células Cultivadas , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Glândulas Mamárias Animais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/patologia
11.
PLoS One ; 9(1): e87439, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24489914

RESUMO

Children are vulnerable to environmental mutagens, and the developing germline could also be affected. However, little is known about whether exposure to environmental mutagens in childhood will result in increased germline mutations in subsequent adult life. In the present study, male transgenic lacI mice at different ages (7, 25 and 60 days old) were treated with a known environmental mutagen (benzo[a]pyrene, B[a]P) at different doses (0, 50, 200 or 300 mg/kg body weight). Mutant frequency was then determined in a meiotic cell type (pachytene spermatocyte), a post-meiotic cell type (round spermatid) and epididymal spermatozoa after at least one cycle of spermatogenesis. Our results show that 1) mice treated with B[a]P at 7 or 25 days old, both being pre-adult ages, had significantly increased mutant frequencies in all spermatogenic cell types tested when they were 60 days old; 2) spermatogenic cells from mice treated before puberty were more susceptible to B[a]P-associated mutagenesis compared to adult mice; and 3) unexpectedly, epididymal spermatozoa had the highest mutant frequency among the spermatogenic cell types tested. These data show that pre-adult exposure to B[a]P increases the male germline mutant frequency in young adulthood. The data demonstrate that exposure to environmental genotoxins at different life phases (e.g., pre-adult and adult) can have differential effects on reproductive health.


Assuntos
Benzo(a)pireno/toxicidade , Mutação em Linhagem Germinativa , Mutagênicos/toxicidade , Espermátides/efeitos dos fármacos , Espermatócitos/efeitos dos fármacos , Animais , Análise Mutacional de DNA , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutagênese , Maturidade Sexual
12.
PLoS One ; 8(5): e63436, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23704908

RESUMO

The role of transforming growth factor-beta (TGF-ß) signaling in hepatocarcinogenesis remains controversial. We aimed to reveal TGF-ß signaling status in human and murine tissues of hepatocellular carcinoma (HCC) and the mechanisms that mediate TGF-ß's role in regulating HCC malignancy. Here, TGF-ß pathway component expression and activation in human and murine HCC tissues were measured with quantitative RT-PCR and Western blotting assays. The role of TGF-ß receptor and Smad signaling in the growth and survival of several HCC cell lines was determined with several in vitro and in vivo approaches. We found that TGF-ß receptor II (TßRII) expression was downregulated in two different HCC patient cohorts. Consistently, Smad3 phosphorylation was also downregulated in HCC tissues in comparison to that in adjacent normal tissues. Interestingly, many HCC cell lines were sensitive to TGF-ß and growth-inhibited by exogenous TGF-ß. However, stable knockdown of TßRII inhibited cell growth on plastic and in soft agar, and induced apoptosis resulting in suppressed subcutaneous tumor growth and metastatic potential in vivo. Furthermore, knockdown of Smad4 also led to a significant inhibition of growth on plastic and in soft agar with concomitant increase of apoptosis, PTEN expression, and reduced nuclear accumulation of linker region-phosphorylated Smad3. Taken together, TGF-ß signaling pathway plays a dichotomous role in hepatocellular carcinogenesis. It appears to suppress HCC development, but is retained for HCC cell survival and malignancy. Furthermore, Smad4 can mediate both growth inhibitory activity induced by exogenous TGF-ß and the survival activity induced by autocrine TGF-ß revealing a delicate selection of the two opposing activities of TGF-ß during HCC evolution.


Assuntos
Carcinogênese/patologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Animais , Apoptose/efeitos dos fármacos , Carcinogênese/efeitos dos fármacos , Carcinogênese/metabolismo , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Metástase Neoplásica , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/farmacologia
13.
Exp Hematol ; 41(8): 665-74, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23643835

RESUMO

Myelodysplastic syndrome (MDS) is considered a hematopoietic stem cell disease that is characterized by abnormal hematopoietic differentiation and a high propensity to develop acute myeloid leukemia. It is mostly associated with advanced age, but also with prior cancer therapy and inherited syndromes related to abnormalities in DNA repair. Recent technologic advances have led to the identification of a myriad of frequently occurring genomic perturbations associated with MDS. These observations suggest that MDS and its progression to acute myeloid leukemia is a genomic instability disorder, resulting from a stepwise accumulation of genetic abnormalities. The notion is now emerging that the underlying mechanism of this disease could be a defect in one or more pathways that are involved in responding to or repairing damaged DNA. In this review, we discuss these pathways in relationship to a large number of studies performed with MDS patient samples and MDS mouse models. Moreover, in view of our current understanding of how DNA damage response and repair pathways are affected by age in hematopoietic stem cells, we also explore how this might relate to MDS development.


Assuntos
Dano ao DNA , Síndromes Mielodisplásicas/genética , Animais , Apoptose , Reparo do DNA , Progressão da Doença , Instabilidade Genômica , Hematopoese , Humanos , Camundongos , Síndromes Mielodisplásicas/patologia
14.
Stem Cell Res ; 10(3): 396-404, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23466563

RESUMO

Identification of murine mammary stem cells (MaSCs) has been attempted with various in vitro and in vivo assays. While, the in vivo repopulation assay remains as the most definitive assay for MaSC detection, it is expensive, time-consuming, and technically challenging. The in vitro mammosphere assay was considered unreliable because of major concerns about its clonal origin. In the current study, co-culture experiments with mammary cells from fluorescent protein transgenic mice and time-lapse video microscopy revealed that >90% mammospheres formed from sorted basal epithelial-enriched cells were of clonal origin in terms of stem cell. These basal-cell derived mammospheres were further distinguished morphologically in a 3-dimensional extracellular matrix culture and functionally in the in vivo repopulation assay. Transplant of single mammospheres or the resultant 3-dimensional solid structures into gland-free mammary fat pads yielded a 70% success rate of multilineage mammary gland reconstitution. Thus, this in vitro sphere formation and differentiation assay is a reliable alternative to the in vivo repopulation assay for the study of MaSCs.


Assuntos
Glândulas Mamárias Animais/citologia , Células-Tronco/citologia , Animais , Células Cultivadas , Técnicas de Cocultura , Colágeno/química , Combinação de Medicamentos , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Laminina/química , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Vídeo , Proteoglicanas/química , Células-Tronco/metabolismo , Células-Tronco/patologia , Imagem com Lapso de Tempo
15.
Biol Reprod ; 88(4): 108, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23515674

RESUMO

Birth rates for older fathers have increased 30% since 1980. When combined with the increased risk for genetic and multifactorial disorders in children conceived by older fathers, paternal age has become an important health issue for modern society. Laboratory research in this area has been minimal, perhaps because of significant experimental barriers, not the least of which is inadequate access to fresh, disease-free human testicular tissue. Regardless, progress has been made and intriguing models supported by experimental evidence have been proposed. The putative mechanisms range from reduced DNA repair activity, leading to increased mutagenesis, to positive selection of germ cells harboring specific disease-causing mutations. There remain many important venues for research in this increasingly relevant phenomenon that impacts future generations.


Assuntos
Doenças Genéticas Inatas/etiologia , Idade Paterna , Envelhecimento/genética , Causalidade , Barreiras de Comunicação , Pai , Doenças Genéticas Inatas/epidemiologia , Doenças Genéticas Inatas/genética , Humanos , Conhecimento , Masculino , Modelos Biológicos , Mutagênese/genética , Mutagênese/fisiologia , Fatores de Risco
16.
Biol Reprod ; 88(1): 6, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23153565

RESUMO

Genetically based diseases constitute a major human health burden, and de novo germline mutations represent a source of heritable genetic alterations that can cause such disorders in offspring. The availability of transgenic rodent systems with recoverable, mutation reporter genes has been used to assess the occurrence of spontaneous point mutations in germline cells. Previous studies using the lacI mutation reporter transgenic mouse system showed that the frequency of spontaneous mutations is significantly lower in advanced male germ cells than in somatic cell types from the same individuals. Here we used this same mutation reporter transgene system to show that female germ cells also display a mutation frequency that is lower than that in corresponding somatic cells and similar to that seen in male germ cells, indicating this is a common feature of germ cells in both sexes. In addition, we showed that statistically significant differences in mutation frequencies are evident between germ cells and somatic cells in both sexes as early as mid-fetal stages in the mouse. Finally, a comparison of the mutation frequency in a general population of early type A spermatogonia with that in a population enriched for Thy-1-positive spermatogonia suggests there is heterogeneity among the early spermatogonial population such that a subset of these cells are predestined to form true spermatogonial stem cells. Taken together, these results support the disposable soma theory, which posits that genetic integrity is normally maintained more stringently in the germ line than in the soma and suggests that this is achieved by minimizing the initial occurrence of mutations in early germline cells and their subsequent gametogenic progeny relative to that in somatic cells.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Células Germinativas/fisiologia , Animais , Feminino , Genes Reporter , Repressores Lac/genética , Repressores Lac/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Mutação , Oócitos/metabolismo , Células de Sertoli/metabolismo , Espermatogônias/metabolismo , Antígenos Thy-1/genética , Antígenos Thy-1/metabolismo
17.
Mol Carcinog ; 52(4): 275-85, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22213062

RESUMO

The prevalence of hepatocellular carcinoma (HCC) was diminished from 60% to 18% at 15 months of age in C3HeB/FeJ male transgenic mice expressing hMGMT in our previous studies. To directly test if the methyltransferase activity is required for diminished tumor prevalence, two separate lines of transgenic mice bearing an enzymatically inactive form of hMGMT were used. In these lines, cysteine 145 was substituted with alanine (C145A). Expression of the hMGMT C145A transgene in liver was demonstrated by Northern blots and Western blots. Immunohistochemistry revealed predominantly nuclear localization of the hMGMT C145A protein. hMGMT C145A transgenic mice were crossed with lacI transgenic mice to assess mutant frequencies in the presence of the mutant protein. Mutant frequencies were similar among livers of lacI × hMGMT C145A bi-transgenic mice and lacI × wild-type (WT) mice. DNA sequence analysis of recovered lacI mutants revealed similar mutation spectra for hMGMT C145A and WT mice. The prevalence of HCC was also similar for the two tested lines of hMGMT C145A mice, 45% and 48% prevalence with median tumor sizes of 11 and 8 mm, and WT mice, 40% prevalence and median tumor size of 10 mm. These results provide evidence that residue C145 in hMGMT is required to reduce the prevalence of HCC in C3HeB/FeJ mice transgenic for hMGMT.


Assuntos
Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Fígado/patologia , O(6)-Metilguanina-DNA Metiltransferase/genética , Substituição de Aminoácidos , Animais , Carcinoma Hepatocelular/enzimologia , Ativação Enzimática , Humanos , Fígado/enzimologia , Fígado/metabolismo , Neoplasias Hepáticas/enzimologia , Masculino , Camundongos , Camundongos Transgênicos , O(6)-Metilguanina-DNA Metiltransferase/análise , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Transgenes
18.
Diabetes ; 61(11): 2776-86, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22807031

RESUMO

The antidiabetic and antiatherosclerotic effects of adiponectin make it a desirable drug target for the treatment of metabolic and cardiovascular diseases. However, the adiponectin-based drug development approach turns out to be difficult due to extremely high serum levels of this adipokine. On the other hand, a significant correlation between adiponectin multimerization and its insulin-sensitizing effects has been demonstrated, suggesting a promising alternative therapeutic strategy. Here we show that transgenic mice overexpressing disulfide bond A oxidoreductase-like protein in fat (fDsbA-L) exhibited increased levels of total and the high-molecular-weight form of adiponectin compared with wild-type (WT) littermates. The fDsbA-L mice also displayed resistance to diet-induced obesity, insulin resistance, and hepatic steatosis compared with WT control mice. The protective effects of DsbA-L overexpression on diet-induced insulin resistance, but not increased body weight and fat cell size, were significantly decreased in adiponectin-deficient fDsbA-L mice (fDsbA-L/Ad(-/-)). In addition, the fDsbA-L/Ad(-/-) mice displayed greater activity and energy expenditure compared with adiponectin knockout mice under a high-fat diet. Taken together, our results demonstrate that DsbA-L protects mice from diet-induced obesity and insulin resistance through adiponectin-dependent and independent mechanisms. In addition, upregulation of DsbA-L could be an effective therapeutic approach for the treatment of obesity and its associated metabolic disorders.


Assuntos
Adiponectina/metabolismo , Tecido Adiposo/metabolismo , Dieta Hiperlipídica/efeitos adversos , Glutationa Transferase/metabolismo , Resistência à Insulina , Obesidade/metabolismo , Regulação para Cima , Adiponectina/antagonistas & inibidores , Adiponectina/química , Tecido Adiposo/patologia , Animais , Resistência à Doença , Metabolismo Energético , Fígado Gorduroso/etiologia , Fígado Gorduroso/prevenção & controle , Glutationa Transferase/antagonistas & inibidores , Glutationa Transferase/genética , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Terapia de Alvo Molecular , Peso Molecular , Hepatopatia Gordurosa não Alcoólica , Obesidade/tratamento farmacológico , Obesidade/patologia , Obesidade/fisiopatologia , Especificidade de Órgãos , Consumo de Oxigênio , Estabilidade Proteica
19.
Mutat Res ; 744(2): 135-9, 2012 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-22314132

RESUMO

Spontaneous mutant frequency in the male germline increases with age, thereby increasing the risk of siring offspring with genetic disorders. In the present study we investigated the effect of age on ionizing radiation-induced male germline mutagenesis. lacI transgenic mice were treated with ionizing radiation at 4-, 15- and 26-month-old, and mutant frequencies were determined for pachytene spermatocytes and round spermatids at 15 days or 49 days after ionizing radiation treatment. Cells collected 15 days after treatment were derivatives of irradiated differentiating spermatogenic cells while cells collected 49 days later were derivatives of spermatogonial stem cells. The results showed that (1) spontaneous mutant frequency increased in spermatogenic cells recovered from nonirradiated old mice (26-months-old), particularly in the round spermatids; (2) mutant frequencies were significantly increased in round spermatids obtained from middle-aged mice (15-months-old) and old age mice (26-months-old) at 15 and 49 days after irradiation compared to the sham-treated old mice; and (3) pachytene spermatocytes obtained from 15- or 26-month-old mice displayed a significantly increased mutant frequency at 15 days post irradiation. This study indicates that age modulates the mutagenic response to ionizing radiation in the male germline.


Assuntos
Envelhecimento , Taxa de Mutação , Radiação Ionizante , Espermatócitos/efeitos da radiação , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos
20.
Mol Reprod Dev ; 78(12): 906-19, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21919107

RESUMO

The prevalence of spontaneous mutations increases with age in the male germline; consequently, older men have an increased risk of siring children with genetic disease due to de novo mutations. The lacI transgenic mouse can be used to study paternal age effects, and in this system, the prevalence of de novo mutations increases in the male germline at old ages. Mutagenesis is linked with DNA repair capacity, and base excision repair (BER), which can ameliorate spontaneous DNA damage, decreases in nuclear extracts of spermatogenic cells from old mice. Mice heterozygous for a null allele of the Apex1 gene, which encodes apurinic/apyrimidinic endonuclease I (APEN), an essential BER enzyme, display an accelerated increase in spontaneous germline mutagenesis early in life. Here, the consequences of lifelong reduction of APEN on genetic instability in the male germline were examined, for the first time, at middle and old ages. Mutant frequency increased earlier in spermatogenic cells from Apex1(+/-) mice (by 6 months of age). Nuclear DNA damage increased with age in the spermatogenic lineage for both wild-type and Apex1(+/-) mice. By old age, mutant frequencies were similar for wild-type and APEN-deficient mice. Mitochondrial genome repair also depends on APEN, and novel analysis of mitochondrial DNA (mtDNA) damage revealed an increase in the Apex1(+/-) spermatogenic cells by middle age. Thus, Apex1 heterozygosity results in accelerated damage to mtDNA and spontaneous mutagenesis, consistent with an essential role for APEN in maintaining nuclear and mtDNA integrity in spermatogenic cells throughout life.


Assuntos
Dano ao DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , DNA/genética , Espermatogênese/genética , Espermatozoides/fisiologia , Fatores Etários , Animais , Apoptose , Núcleo Celular/genética , DNA/metabolismo , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Heterozigoto , Modelos Logísticos , Masculino , Camundongos , Camundongos Transgênicos , Mutagênese/genética , Espermatozoides/química
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