-
Investigative Ophthalmology & Visual... Nov 2023The proliferative and neurogenic potential of retinal Müller glia after injury varies widely across species. To identify the endogenous mechanisms regulating the...
PURPOSE
The proliferative and neurogenic potential of retinal Müller glia after injury varies widely across species. To identify the endogenous mechanisms regulating the proliferative response of mammalian Müller glia, we comparatively analyzed the expression and function of nestin, an intermediate filament protein established as a neural stem cell marker, in the mouse and rat retinas after injury.
METHODS
Nestin expression in the retinas of C57BL/6 mice and Wistar rats after methyl methanesulfonate (MMS)-induced photoreceptor injury was examined by immunofluorescence and Western blotting. Adeno-associated virus (AAV)-delivered control and nestin short hairpin RNA (shRNA) were intravitreally injected to rats and Müller glia proliferation after MMS-induced injury was analyzed by BrdU incorporation and immunofluorescence. Photoreceptor removal and microglia/macrophage infiltration were also analyzed by immunofluorescence.
RESULTS
Rat Müller glia re-entered the cell cycle and robustly upregulated nestin after injury whereas Müller glia proliferation and nestin upregulation were not observed in mice. In vivo knockdown of nestin in the rat retinas inhibited Müller glia proliferation while transiently stimulating microglia/macrophage infiltration and phagocytic removal of dead photoreceptors.
CONCLUSIONS
Our findings suggest a critical role for nestin in the regulation of Müller glia proliferation after retinal injury and highlight the importance of cross species analysis to identify the molecular mechanisms regulating the injury responses of the mammalian retina.
Topics: Animals; Mice; Rats; Cell Proliferation; Eye Injuries; Methyl Methanesulfonate; Mice, Inbred C57BL; Nestin; Rats, Wistar; Neuroglia
PubMed: 37934159
DOI: 10.1167/iovs.64.14.8 -
Nucleic Acids Research Apr 2008N3-methyl-adenine (3MeA) is the major cytotoxic lesion formed in DNA by S(N)2 methylating agents. The lesion presumably blocks progression of cellular replicases because...
N3-methyl-adenine (3MeA) is the major cytotoxic lesion formed in DNA by S(N)2 methylating agents. The lesion presumably blocks progression of cellular replicases because the N3-methyl group hinders interactions between the polymerase and the minor groove of DNA. However, this hypothesis has yet to be rigorously proven, as 3MeA is intrinsically unstable and is converted to an abasic site, which itself is a blocking lesion. To circumvent these problems, we have chemically synthesized a 3-deaza analog of 3MeA (3dMeA) as a stable phosphoramidite and have incorporated the analog into synthetic oligonucleotides that have been used in vitro as templates for DNA replication. As expected, the 3dMeA lesion blocked both human DNA polymerases alpha and delta. In contrast, human polymerases eta, iota and kappa, as well as Saccharomyces cerevisiae poleta were able to bypass the lesion, albeit with varying efficiencies and accuracy. To confirm the physiological relevance of our findings, we show that in S. cerevisiae lacking Mag1-dependent 3MeA repair, poleta (Rad30) contributes to the survival of cells exposed to methyl methanesulfonate (MMS) and in the absence of Mag1, Rad30 and Rev3, human polymerases eta, iota and kappa are capable of restoring MMS-resistance to the normally MMS-sensitive strain.
Topics: Adenine; DNA Damage; DNA Glycosylases; DNA Replication; DNA-Directed DNA Polymerase; Gene Deletion; Humans; Kinetics; Methyl Methanesulfonate; Mutagens; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 18281311
DOI: 10.1093/nar/gkn058 -
Mutation Research. Genetic Toxicology... Sep 2019In accordance with the 3 Rs to reduce in vivo testing, more advanced in vitro models, moving from 2D monolayer to 3D cultures, should be developed for prediction of... (Comparative Study)
Comparative Study
In accordance with the 3 Rs to reduce in vivo testing, more advanced in vitro models, moving from 2D monolayer to 3D cultures, should be developed for prediction of human toxicity of industrial chemicals and environmental pollutants. In this study we compared cytotoxic and genotoxic responses induced by chemicals in 2D and 3D spheroidal cultures of the human liver cancer cell line HepG2. HepG2 spheroids were prepared by hanging drop technology. Both 3D spheroids and 2D monolayer cultures were exposed to different chemicals (colchicine, chlorpromazine hydrochloride or methyl methanesulfonate) for geno- and cytotoxicity studies. Cytotoxicity was investigated by alamarBlue assay, flow cytometry and confocal imaging. DNA damage was investigated by the comet assay with and without Fpg enzyme for detection of DNA strand breaks and oxidized or alkylated base lesions. The results from the cyto- and genotoxicity tests showed differences in sensitivity comparing the 2D and 3D HepG2 models. This study shows that human 3D spheroidal hepatocellular cultures can be successfully applied for genotoxicity testing by the comet assay and represent a promising advanced in vitro model for toxicity testing.
Topics: Cell Culture Techniques; Cell Survival; Chlorpromazine; Colchicine; Comet Assay; DNA Damage; DNA, Neoplasm; Guanine; Hep G2 Cells; Humans; Hydrogen Peroxide; Laboratory Proficiency Testing; Methyl Methanesulfonate; Sensitivity and Specificity; Spheroids, Cellular
PubMed: 31561895
DOI: 10.1016/j.mrgentox.2019.03.006 -
Genetics Oct 1984Six schemes were used to identify 80 independent recessive lethal deficiencies of linkage group (LG) II following X-ray treatment of the nematode Caenorhabditis elegans....
Six schemes were used to identify 80 independent recessive lethal deficiencies of linkage group (LG) II following X-ray treatment of the nematode Caenorhabditis elegans. Complementation tests between the deficiencies and ethyl methanesulfonate-induced recessive visible, lethal and sterile mutations and between different deficiencies were used to characterize the extents of the deficiencies. Deficiency endpoints thus helped to order 36 sites within a region representing about half of the loci on LG II and extending over about 5 map units. New mutations occurring in this region can be assigned to particular segments of the map by complementation tests against a small number of deficiencies; this facilitates the assignment of single-site mutations to particular genes, as we illustrate. Five sperm-defective and five oocyte-defective LG II sterile mutants were identified and mapped. Certain deficiency-by-deficiency complementation tests allowed us to suggest that the phenotypes of null mutations at two loci represented by visible alleles are wild type and that null mutations at a third locus confer a visible phenotype. A segment of LG II that is about 12 map units long and largely devoid of identified loci seems to be greatly favored for crossing over.
Topics: Animals; Caenorhabditis; Chromosome Deletion; Chromosome Mapping; Genes, Lethal; Genes, Recessive; Genetic Complementation Test; Genetic Linkage; Methyl Methanesulfonate
PubMed: 6500256
DOI: 10.1093/genetics/108.2.331 -
ELife Nov 2021Three-methyl cytosine (3meC) are toxic DNA lesions, blocking base pairing. Bacteria and humans express members of the AlkB enzymes family, which directly remove 3meC....
Three-methyl cytosine (3meC) are toxic DNA lesions, blocking base pairing. Bacteria and humans express members of the AlkB enzymes family, which directly remove 3meC. However, other organisms, including budding yeast, lack this class of enzymes. It remains an unanswered evolutionary question as to how yeast repairs 3meC, particularly in single-stranded DNA. The yeast Shu complex, a conserved homologous recombination factor, aids in preventing replication-associated mutagenesis from DNA base damaging agents such as methyl methanesulfonate (MMS). We found that MMS-treated Shu complex-deficient cells exhibit a genome-wide increase in A:T and G:C substitutions mutations. The G:C substitutions displayed transcriptional and replicational asymmetries consistent with mutations resulting from 3meC. Ectopic expression of a human AlkB homolog in Shu-deficient yeast rescues MMS-induced growth defects and increased mutagenesis. Thus, our work identifies a novel homologous recombination-based mechanism mediated by the Shu complex for coping with alkylation adducts.
Topics: Alkylation; Homologous Recombination; Methyl Methanesulfonate; Mutagenesis; Mutagens; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 34723799
DOI: 10.7554/eLife.68080 -
Stem Cell Research Mar 2017Pluripotent 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 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.
Topics: Animals; Cell Differentiation; Cell Line; DNA Damage; DNA Repair; Epigenomics; Methyl Methanesulfonate; Mice; Mice, Inbred C57BL; Mutagenesis; Stem Cells
PubMed: 28129601
DOI: 10.1016/j.scr.2016.12.029 -
The Journal of Toxicological Sciences Aug 2003Methyl methanesulfonate (MMS), a methylating agent, is known to be a genotoxicant in testis. The purpose of this study was to investigate roles of oxidative...
Methyl methanesulfonate (MMS), a methylating agent, is known to be a genotoxicant in testis. The purpose of this study was to investigate roles of oxidative stress-responsive proteins, heme oxygenase-1 (HO-1) and metallothionein-1/2 (MT-1/2), in genotoxicity of MMS. Cadmium, a potent genotoxicity inducer, induced HO-1 and MT-1/2 in rat livers and kidneys. Then we comparatively investigated MMS-induced HO-1 and MT-1/2 in rat livers, kidneys and testes. We found that a single administration of MMS (40 mg/kg) resulted in the induction of MT-1/2 mRNA in the liver, but not HO-1 mRNA, reaching maximum level at 6 hr and returning to the control levels by 24 hr. Interestingly, MMS induced both HO-1 and MT-1/2 mRNAs in the kidney. In contrast, MMS induced HO-1 mRNA, but not MT-1/2 mRNA in the testis. Since HO-1 and MT-1/2 have been recognized to respond to various oxidative stimuli, we further examined the inducing effect of MMS on these two proteins. MMS at dosages of 20 to 40 mg/kg for 2 consecutive weeks induced HO-1 mRNA (123 to 187% of the control) and protein (274 to 404% of the control) in rat testes. However, MT-1/2 mRNA was not induced by MMS administration, although a high level of expression was observed in comparison with the liver and kidney. These findings suggest that MMS induces HO-1 and/or MT-1/2 mRNA and its protein tissue-dependently, and the heme catabolites by HO-1 in the testis may contribute in some manner to its genotoxicity.
Topics: Animals; Blotting, Western; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Kidney; Liver; Male; Metallothionein; Methyl Methanesulfonate; Microsomes; Mutagens; RNA, Messenger; Rats; Rats, Sprague-Dawley; Testis
PubMed: 12974610
DOI: 10.2131/jts.28.181 -
Aging Jan 2021The naked mole rat (NMR), is the longest-living rodent species, and is extraordinarily resistant to cancer and aging-related diseases. The molecular basis for these...
The naked mole rat (NMR), is the longest-living rodent species, and is extraordinarily resistant to cancer and aging-related diseases. The molecular basis for these unique phenotypic traits of the NMR is under extensive research. However, the role of regulated cell death (RCD) in the longevity and the protection from cancer in the NMR is still largely unknown. RCD is a mechanism restricting the proliferation of damaged or premalignant cells, which counteracts aging and oncotransformation. In this study, DNA damage-induced cell death in NMR fibroblasts was investigated in comparison to RCD in fibroblasts from . The effects of methyl methanesulfonate, 5-fluorouracil, and etoposide in both cell types were examined using contemporary cell death analyses. Skin fibroblasts from were found to be more resistant to the action of DNA damaging agents compared to fibroblasts from . Strikingly, our results revealed that NMR cells also exhibit a limited apoptotic response and seem to undergo regulated necrosis. Taken together, this study provides new insights into the mechanisms of cell death in NMR expanding our understanding of longevity, and it paves the way towards the development of innovative therapeutic approaches.
Topics: Animals; Cells, Cultured; DNA Damage; Fibroblasts; Longevity; Methyl Methanesulfonate; Mice; Mole Rats; Regulated Cell Death
PubMed: 33510044
DOI: 10.18632/aging.202577 -
Acta Crystallographica. Section F,... Jun 2019Chagas disease, which is caused by Trypanosoma cruzi, affects more than six million people worldwide. Cruzain is the major cysteine protease involved in the survival of...
Chagas disease, which is caused by Trypanosoma cruzi, affects more than six million people worldwide. Cruzain is the major cysteine protease involved in the survival of this parasite. Here, the expression, purification and crystallization of this enzyme are reported. The cruzain crystals diffracted to 1.2 Å resolution, yielding two novel cruzain structures: apocruzain and cruzain bound to the reversible covalent inhibitor S-methyl thiomethanesulfonate. Mass-spectrometric experiments confirmed the presence of a methylthiol group attached to the catalytic cysteine. Comparison of these structures with previously published structures indicates the rigidity of the cruzain structure. These results provide further structural information about the enzyme and may help in new in silico studies to identify or optimize novel prototypes of cruzain inhibitors.
Topics: Apoproteins; Crystallography, X-Ray; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Drug Design; Methyl Methanesulfonate; Models, Molecular; Protein Conformation; Protozoan Proteins
PubMed: 31204688
DOI: 10.1107/S2053230X19006320 -
International Journal of Molecular... Jul 2021In order to tackle the study of DNA repair pathways, the physical and chemical agents creating DNA damage, the genotoxins, are frequently employed. Despite their...
In order to tackle the study of DNA repair pathways, the physical and chemical agents creating DNA damage, the genotoxins, are frequently employed. Despite their utility, their effects are rarely restricted to DNA, and therefore simultaneously harm other cell biomolecules. Methyl methanesulfonate (MMS) is an alkylating agent that acts on DNA by preferentially methylating guanine and adenine bases. It is broadly used both in basic genome stability research and as a model for mechanistic studies to understand how alkylating agents work, such as those used in chemotherapy. Nevertheless, MMS exerts additional actions, such as oxidation and acetylation of proteins. In this work, we introduce the important notion that MMS also triggers a lipid stress that stems from and affects the inner nuclear membrane. The inner nuclear membrane plays an essential role in virtually all genome stability maintenance pathways. Thus, we want to raise awareness that the relative contribution of lipid and genotoxic stresses when using MMS may be difficult to dissect and will matter in the conclusions drawn from those studies.
Topics: Antineoplastic Agents, Alkylating; DNA Damage; DNA Repair; Hep G2 Cells; Humans; Lipids; Methyl Methanesulfonate; Mutagens; Nuclear Envelope; Retinal Pigment Epithelium
PubMed: 34299079
DOI: 10.3390/ijms22147461