Despite the widespread use of silver nanoparticles (AgNPs) in different fields and the amount of ... more Despite the widespread use of silver nanoparticles (AgNPs) in different fields and the amount of investigations available, to date, there are many contradictory results on their potential toxicity. In the present study, extensively characterized 20-nm AgNPs were investigated using optimized protocols and standardized methods to test several toxicological endpoints in different cell lines. The agglomeration/aggregation state of AgNPs in culture media was measured by dynamic light scattering (DLS). DNA and chromosomal damage on BEAS-2B and RAW 264.7 cells were evaluated
8-Oxoguanine, a common mutagenic DNA lesion, generates G:CNT:A transversions via mispairing with ... more 8-Oxoguanine, a common mutagenic DNA lesion, generates G:CNT:A transversions via mispairing with adenine during DNA replication. When operating normally, the MUTYH DNA glycosylase prevents 8-oxoguanine-related mutagenesis by excising the incorporated adenine. Biallelic MUTYH mutations impair this enzymatic function and are associated with colorectal cancer (CRC) in MUTYH-Associated Polyposis (MAP) syndrome. Here, we perform whole-exome sequencing that reveals a modest mutator phenotype in MAP CRCs compared to sporadic CRC stem cell lines or bulk tumours. The excess G:CN T:A transversion mutations in MAP CRCs exhibits a novel mutational signature, termed Signature 36, with a strong sequence dependence. The MUTYH mutational signature reflecting persistent 8-oxoG:A mismatches occurs frequently in the APC, KRAS, PIK3CA, FAT4, TP53, FAT1, AMER1, KDM6A, SMAD4 and SMAD2 genes that are associated with CRC. The occurrence of Signature 36 in other types of human cancer indicates that DNA 8-oxoguanine-related mutations might contribute to the development of cancer in other organs.
The production of reactive oxygen species (ROS) in mammalian cells is tightly regulated because o... more The production of reactive oxygen species (ROS) in mammalian cells is tightly regulated because of their potential to damage macromolecules, including DNA. To investigate possible links between high ROS levels, oxidative DNA damage, and genomic instability in mammalian cells, we established a novel model of chronic oxidative stress by coexpressing the NADPH oxidase human (h) NOX1 gene together with its cofactors NOXO1 and NOXA1. Transfectants of mismatch repair (MMR)-proficient HeLa cells or MMR-defective Msh2(-/-) mouse embryo fibroblasts overexpressing the hNOX1 complex displayed increased intracellular ROS levels. In one HeLa clone in which ROS were particularly elevated, reactive nitrogen species were also increased and nitrated proteins were identified with an anti-3-nitrotyrosine antibody. Overexpression of the hNOX1 complex increased the steady-state levels of DNA 8-oxo-7,8-dihydroguanine and caused a threefold increase in the HPRT mutation rate in HeLa cells. In contrast, additional oxidatively generated damage did not affect the constitutive mutator phenotype of the Msh2(-/-) fibroblasts. Because no significant changes in the expression of several DNA repair enzymes for oxidative DNA damage were identified, we suggest that chronic oxidative stress can saturate the cell's DNA repair capacity and cause significant genomic instability.
Investigations on T cell receptor δ chain (TcR δ) gene expression in freshly isolated normal larg... more Investigations on T cell receptor δ chain (TcR δ) gene expression in freshly isolated normal large granular lymphocytes, natural killer (NK) cell clones and NK bulk cultures revealed six Tδ transcripts (3.5, 3.1, 2.2, 2.0, 1.5 and 1.3 kb) which varied in number and relative abundance in the different samples. None of the six known Tδ variable regions was expressed and the Tδ locus was retained in its germ-line configuration. The origin and significance of these NK-associated Tδ transcripts are discussed.
The T cell receptor (TcR) β chain is encoded by a 1.3-kb mRNA which contains variable (V), divers... more The T cell receptor (TcR) β chain is encoded by a 1.3-kb mRNA which contains variable (V), diversity (D), joining (J) and constant (C) regions, A shorter 1.0-kb transcript with C but no V sequences is found in thymocytes, α/β and γ/δ T lymphocytes and natural killer cells. The origin, clonal variability and function of this transcript is unknown. We isolated cDNA clones representative of the 1.0-kb mRNA from cDNA libraries of either polyclonal or clonal natural killer and γ/δ lymphocytes. Ten different types of cDNA clones belonging to three separate groups were identified: (a) “J-C clones” consisting of one of five Jβ regions and the corresponding Cβ1 or Cβ2 regions; (b) “D-J-C clones” composed of Dβ1/or Dβ2/Jβ rearranged sequences and Cβ1 or Cβ2 sequences; (c) “5′C clones” made up of Cβ1 or Cβ2 regions preceded by the corresponding genomic 5′ flanking region. The presence of recombination signal sequences in J-C and D-J-C clones suggests that the first derive from mRNA transcribed from promoters located in the 5′ J region and the second from mRNA transcribed from promoters situated in the 5′ D region. Nucleotide sequence analysis demonstrated that only three of the ten types of clones isolated had the potential to code a short cytoplasmic Tβ protein with a variable D-Jβ N terminus. These findings have implications for the mechanisms of Tβ germ-line transcription and the function of the Tβ transcripts.
Despite the widespread use of silver nanoparticles (AgNPs) in different fields and the amount of ... more Despite the widespread use of silver nanoparticles (AgNPs) in different fields and the amount of investigations available, to date, there are many contradictory results on their potential toxicity. In the present study, extensively characterized 20-nm AgNPs were investigated using optimized protocols and standardized methods to test several toxicological endpoints in different cell lines. The agglomeration/aggregation state of AgNPs in culture media was measured by dynamic light scattering (DLS). DNA and chromosomal damage on BEAS-2B and RAW 264.7 cells were evaluated
8-Oxoguanine, a common mutagenic DNA lesion, generates G:CNT:A transversions via mispairing with ... more 8-Oxoguanine, a common mutagenic DNA lesion, generates G:CNT:A transversions via mispairing with adenine during DNA replication. When operating normally, the MUTYH DNA glycosylase prevents 8-oxoguanine-related mutagenesis by excising the incorporated adenine. Biallelic MUTYH mutations impair this enzymatic function and are associated with colorectal cancer (CRC) in MUTYH-Associated Polyposis (MAP) syndrome. Here, we perform whole-exome sequencing that reveals a modest mutator phenotype in MAP CRCs compared to sporadic CRC stem cell lines or bulk tumours. The excess G:CN T:A transversion mutations in MAP CRCs exhibits a novel mutational signature, termed Signature 36, with a strong sequence dependence. The MUTYH mutational signature reflecting persistent 8-oxoG:A mismatches occurs frequently in the APC, KRAS, PIK3CA, FAT4, TP53, FAT1, AMER1, KDM6A, SMAD4 and SMAD2 genes that are associated with CRC. The occurrence of Signature 36 in other types of human cancer indicates that DNA 8-oxoguanine-related mutations might contribute to the development of cancer in other organs.
The production of reactive oxygen species (ROS) in mammalian cells is tightly regulated because o... more The production of reactive oxygen species (ROS) in mammalian cells is tightly regulated because of their potential to damage macromolecules, including DNA. To investigate possible links between high ROS levels, oxidative DNA damage, and genomic instability in mammalian cells, we established a novel model of chronic oxidative stress by coexpressing the NADPH oxidase human (h) NOX1 gene together with its cofactors NOXO1 and NOXA1. Transfectants of mismatch repair (MMR)-proficient HeLa cells or MMR-defective Msh2(-/-) mouse embryo fibroblasts overexpressing the hNOX1 complex displayed increased intracellular ROS levels. In one HeLa clone in which ROS were particularly elevated, reactive nitrogen species were also increased and nitrated proteins were identified with an anti-3-nitrotyrosine antibody. Overexpression of the hNOX1 complex increased the steady-state levels of DNA 8-oxo-7,8-dihydroguanine and caused a threefold increase in the HPRT mutation rate in HeLa cells. In contrast, additional oxidatively generated damage did not affect the constitutive mutator phenotype of the Msh2(-/-) fibroblasts. Because no significant changes in the expression of several DNA repair enzymes for oxidative DNA damage were identified, we suggest that chronic oxidative stress can saturate the cell's DNA repair capacity and cause significant genomic instability.
Investigations on T cell receptor δ chain (TcR δ) gene expression in freshly isolated normal larg... more Investigations on T cell receptor δ chain (TcR δ) gene expression in freshly isolated normal large granular lymphocytes, natural killer (NK) cell clones and NK bulk cultures revealed six Tδ transcripts (3.5, 3.1, 2.2, 2.0, 1.5 and 1.3 kb) which varied in number and relative abundance in the different samples. None of the six known Tδ variable regions was expressed and the Tδ locus was retained in its germ-line configuration. The origin and significance of these NK-associated Tδ transcripts are discussed.
The T cell receptor (TcR) β chain is encoded by a 1.3-kb mRNA which contains variable (V), divers... more The T cell receptor (TcR) β chain is encoded by a 1.3-kb mRNA which contains variable (V), diversity (D), joining (J) and constant (C) regions, A shorter 1.0-kb transcript with C but no V sequences is found in thymocytes, α/β and γ/δ T lymphocytes and natural killer cells. The origin, clonal variability and function of this transcript is unknown. We isolated cDNA clones representative of the 1.0-kb mRNA from cDNA libraries of either polyclonal or clonal natural killer and γ/δ lymphocytes. Ten different types of cDNA clones belonging to three separate groups were identified: (a) “J-C clones” consisting of one of five Jβ regions and the corresponding Cβ1 or Cβ2 regions; (b) “D-J-C clones” composed of Dβ1/or Dβ2/Jβ rearranged sequences and Cβ1 or Cβ2 sequences; (c) “5′C clones” made up of Cβ1 or Cβ2 regions preceded by the corresponding genomic 5′ flanking region. The presence of recombination signal sequences in J-C and D-J-C clones suggests that the first derive from mRNA transcribed from promoters located in the 5′ J region and the second from mRNA transcribed from promoters situated in the 5′ D region. Nucleotide sequence analysis demonstrated that only three of the ten types of clones isolated had the potential to code a short cytoplasmic Tβ protein with a variable D-Jβ N terminus. These findings have implications for the mechanisms of Tβ germ-line transcription and the function of the Tβ transcripts.
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Papers by Ettore Meccia