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Elena Giulotto - One of the best experts on this subject based on the ideXlab platform.
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Increased Gene Amplification in Immortal Rodent Cells Deficient for the DNA-dependent Protein Kinase Catalytic Subunit
Cancer Research, 2001Co-Authors: Chiara Mondello, Paola Rebuzzini, Manuela Dolzan, Scott Edmonson, Guillermo E Taccioli, Elena GiulottoAbstract:Gene amplification is one of the most frequent genome anomalies observed in tumor cells, whereas it has never been detected in cells of normal origin. A large body of evidence indicates that DNA double-strand breaks (DSBs) play a key role in initiating Gene amplification. In mammals, DSBs are mainly repaired through the nonhomologous end-joining pathway (NHEJ) that requires a functional DNA-dependent protein kinase catalytic subunit (DNA-PKcs). In rodent cell lines, N -(phosphonacetyl)-l-aspartate (PALA) resistance is considered a measure of Gene amplification because it is mainly attributable to amplification of the carbamyl- P -synthetase aspartate transcarbamylase dihydro-orotase ( CAD ) Gene. In this paper we show that the radiosensitive hamster cell line V3, which is defective in DSB repair because of a mutation in the DNA-PKcs Gene, displays also an increased frequency of Gene amplification. In these cells, we found that the amplification of the CAD Gene occurs with a frequency and a rate more than one order of magnitude higher than in control cell lines, although it relies on the same mechanisms. When the same analysis was performed in mouse embryo fibroblasts (MEFs) obtained from animals in which the DNA-PKcs Gene was ablated by homologous recombination, a higher frequency of amplification compared with the controls was found only after cellular immortalization. In primary DNA-PKcs −/− MEFs, PALA treatment induced a block in the cell cycle, and no PALA-resistant clones were found. Our results indicate that the lack of DNA-PKcs increases the probability that Gene amplification occurs in a Genetic background already permissive, like that of immortalized cells, although it is not sufficient to make normal cells able to amplify.
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Late onset of CAD Gene amplification in unamplified PALA resistant Chinese hamster mutants.
Cancer Letters, 2000Co-Authors: Elena Mucciolo, Chiara Mondello, Livia Bertoni, Elena GiulottoAbstract:In rodent cells, resistance to PALA (N-phosphonacetyl-L-aspartate) has always been found associated with amplification of the CAD Gene (carbamyl-P synthetase, aspartate transcarbamylase, dihydro-orotase). We describe two PALA resistant Chinese hamster mutant cell lines in which amplification of the CAD Gene was not present. The PALA resistant phenotype was stable when the cells were grown in non-selective medium. However, after prolonged growth in the presence of the same drug concentration used for selection, cells with increased CAD Gene copy number and higher levels of resistance overrode the original population. In these cell populations, a heteroGeneous organization of the CAD Genes was revealed by fluorescence in situ hybridization on mitotic chromosomes indicating that the additional copies of the Gene were Generated in several ways, such as non-disjunction and breakage-fusion-bridge cycles. The clastogenic effect of PALA, evidenced as chromosomal aberrations in the cells grown in the presence of the drug, could have favored the late onset of the amplified mutants. It is tempting to speculate that, during the expansion of tumor populations, different drug resistance mechanisms, including Gene amplification, could occur in succession and lead to the Generation of cells highly resistant to chemotherapeutic agents.
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Selection of N-(phosphonacetyl)-L-aspartate resistant Chinese hamster mutants in the presence of the uridine uptake inhibitor dipyridamole.
Anticancer research, 1995Co-Authors: L. Tessera, Livia Bertoni, Elena Mucciolo, Elena GiulottoAbstract:In mammalian cells selected in culture for resistance to PALA the CAD Gene is amplified and these cells are a widely used model system to study Gene amplification. Selection of resistant mutants is routinely performed in medium supplemented with dialyzed serum, because the cytotoxic effect of PALA is reversed by uridine, which is contained in serum. We have shown that in Chinese hamster cells dipyridamole reduced uridine uptake to less than 5% with limited effect on cell survival. Moreover, in medium supplemented with complete serum and 10 microM dipyridamole the toxicity of PALA was similar to that obtained in medium containing dialyzed serum. We then used 10 microM dipyridamole to inhibit uridine uptake during selection of PALA resistant colonies and found that both the frequency and the type of mutants were as those obtained in the presence of dialyzed serum. In particular, in the five mutants tested, the mechanism of resistance to PALA was amplification of the CAD Gene.
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localization of the chinese hamster CAD Gene reveals homology between human chromosome 2p and chinese hamster 7q
Genomics, 1993Co-Authors: Livia Bertoni, Carmen Attolini, Silvana Simi, Elena GiulottoAbstract:The trifunctional enzyme CAD catalyzes the first three steps of pyrimidine biosynthesis. By using fluorescence in situ hybridization the authors have localized the Chinese hamster CAD Gene on chromosome 7q11-q13 of diploid fibroblasts. Other Genes previously assigned to chromosome 7 include acid phosphatase-1, the M2 subunit of ribonucleotide reductase and ornithine decarboxylase. These Genes are also syntenic with CAD on human chromosome 2p. They have then mapped CAD on the pericentromeric region of two different rearranged chromosomes (Z8p and R2q) in a cell line derived from Chinese hamster ovary. The presence of CAD on Z8 and R2 indicates that they derive from rearrangements involving chromosome 7. 14 refs., 2 figs.
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Localization of the Chinese Hamster CAD Gene Reveals Homology between Human Chromosome 2p and Chinese Hamster 7q
Genomics, 1993Co-Authors: Livia Bertoni, Carmen Attolini, Silvana Simi, Elena GiulottoAbstract:The trifunctional enzyme CAD catalyzes the first three steps of pyrimidine biosynthesis. By using fluorescence in situ hybridization we have localized the Chinese hamster CAD Gene on chromosome 7q11-q13 of diploid fibroblasts. Other Genes previously assigned to chromosome 7 include acid phosphatase-1, the M2 subunit of ribonucleotide reductase and ornithine decarboxylase. These Genes are also syntenic with CAD on human chromosome 2p. We have then mapped CAD on the pericentromeric region of two different rearranged chromosomes (Z8p and R2q) in a cell line derived from Chinese hamster ovary. The presence of CAD on Z8 and R2 indicates that they derive from rearrangements involving chromosome 7.
Livia Bertoni - One of the best experts on this subject based on the ideXlab platform.
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Late onset of CAD Gene amplification in unamplified PALA resistant Chinese hamster mutants.
Cancer Letters, 2000Co-Authors: Elena Mucciolo, Chiara Mondello, Livia Bertoni, Elena GiulottoAbstract:In rodent cells, resistance to PALA (N-phosphonacetyl-L-aspartate) has always been found associated with amplification of the CAD Gene (carbamyl-P synthetase, aspartate transcarbamylase, dihydro-orotase). We describe two PALA resistant Chinese hamster mutant cell lines in which amplification of the CAD Gene was not present. The PALA resistant phenotype was stable when the cells were grown in non-selective medium. However, after prolonged growth in the presence of the same drug concentration used for selection, cells with increased CAD Gene copy number and higher levels of resistance overrode the original population. In these cell populations, a heteroGeneous organization of the CAD Genes was revealed by fluorescence in situ hybridization on mitotic chromosomes indicating that the additional copies of the Gene were Generated in several ways, such as non-disjunction and breakage-fusion-bridge cycles. The clastogenic effect of PALA, evidenced as chromosomal aberrations in the cells grown in the presence of the drug, could have favored the late onset of the amplified mutants. It is tempting to speculate that, during the expansion of tumor populations, different drug resistance mechanisms, including Gene amplification, could occur in succession and lead to the Generation of cells highly resistant to chemotherapeutic agents.
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Selection of N-(phosphonacetyl)-L-aspartate resistant Chinese hamster mutants in the presence of the uridine uptake inhibitor dipyridamole.
Anticancer research, 1995Co-Authors: L. Tessera, Livia Bertoni, Elena Mucciolo, Elena GiulottoAbstract:In mammalian cells selected in culture for resistance to PALA the CAD Gene is amplified and these cells are a widely used model system to study Gene amplification. Selection of resistant mutants is routinely performed in medium supplemented with dialyzed serum, because the cytotoxic effect of PALA is reversed by uridine, which is contained in serum. We have shown that in Chinese hamster cells dipyridamole reduced uridine uptake to less than 5% with limited effect on cell survival. Moreover, in medium supplemented with complete serum and 10 microM dipyridamole the toxicity of PALA was similar to that obtained in medium containing dialyzed serum. We then used 10 microM dipyridamole to inhibit uridine uptake during selection of PALA resistant colonies and found that both the frequency and the type of mutants were as those obtained in the presence of dialyzed serum. In particular, in the five mutants tested, the mechanism of resistance to PALA was amplification of the CAD Gene.
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localization of the chinese hamster CAD Gene reveals homology between human chromosome 2p and chinese hamster 7q
Genomics, 1993Co-Authors: Livia Bertoni, Carmen Attolini, Silvana Simi, Elena GiulottoAbstract:The trifunctional enzyme CAD catalyzes the first three steps of pyrimidine biosynthesis. By using fluorescence in situ hybridization the authors have localized the Chinese hamster CAD Gene on chromosome 7q11-q13 of diploid fibroblasts. Other Genes previously assigned to chromosome 7 include acid phosphatase-1, the M2 subunit of ribonucleotide reductase and ornithine decarboxylase. These Genes are also syntenic with CAD on human chromosome 2p. They have then mapped CAD on the pericentromeric region of two different rearranged chromosomes (Z8p and R2q) in a cell line derived from Chinese hamster ovary. The presence of CAD on Z8 and R2 indicates that they derive from rearrangements involving chromosome 7. 14 refs., 2 figs.
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Localization of the Chinese Hamster CAD Gene Reveals Homology between Human Chromosome 2p and Chinese Hamster 7q
Genomics, 1993Co-Authors: Livia Bertoni, Carmen Attolini, Silvana Simi, Elena GiulottoAbstract:The trifunctional enzyme CAD catalyzes the first three steps of pyrimidine biosynthesis. By using fluorescence in situ hybridization we have localized the Chinese hamster CAD Gene on chromosome 7q11-q13 of diploid fibroblasts. Other Genes previously assigned to chromosome 7 include acid phosphatase-1, the M2 subunit of ribonucleotide reductase and ornithine decarboxylase. These Genes are also syntenic with CAD on human chromosome 2p. We have then mapped CAD on the pericentromeric region of two different rearranged chromosomes (Z8p and R2q) in a cell line derived from Chinese hamster ovary. The presence of CAD on Z8 and R2 indicates that they derive from rearrangements involving chromosome 7.
Jeffrey N. Davidson - One of the best experts on this subject based on the ideXlab platform.
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the evolutionary history of the first three enzymes in pyrimidine biosynthesis
BioEssays, 1993Co-Authors: Jeffrey N. Davidson, Kuey C Chen, Robert S Jamison, Lisa A Musmanno, Christine B. KernAbstract:Some metabolic pathways are nearly ubiquitous among organisms: the Genes encoding the enzymes for such pathways must therefore be ancient and essential. De novo pyrimidine biosynthesis is an example of one such metabolic pathway. In animals a single protein called CAD carries the first three steps of this pathway. The same three enzymes in prokaryotes are associated with separate proteins. The CAD Gene appears to have evolved through a process of Gene duplication and DNA rearrangement, leading to an in-frame Gene fusion encoding a chimeric protein. A driving force for the creation of eukaryotic Genes encoding multienzymatic proteins such as CAD may be the advantage of coordinate expression of enzymes catalyzing steps in a biosynthetic pathway. The analogous structure in bacteria is the operon. Differences in the translational mechanisms of eukaryotes and prokaryotes may have dictated the different strategies used by organisms to evolve coordinately regulated Genes.
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Complete hamster CAD protein and the carbamylphosphate synthetase domain of CAD complement mammalian cell mutants defective in de novo pyrimidine biosynthesis
Somatic Cell and Molecular Genetics, 1992Co-Authors: Lisa A Musmanno, Robert S Jamison, Richard S. Barnett, Edward Buford, Jeffrey N. DavidsonAbstract:The mammalian CAD Gene codes for a 240-kDa multifunctional protein that catalyzes the first three steps of de novo pyrimidine biosynthesis. Previously, the longest cDNA construct available was missing approximately 500 bp of coding sequence at the 5′ end, thereby lacking the sequence to encode the entire carbamylphosphate synthetase (CPSase) domain. Here, a complete CAD hamster cDNA is constructed, placed into a mammalian expression vector, and transfected into hamster cells deficient in CAD. Transfectants show coordinately restored levels of all three enzyme activities and the presence of full-length CAD protein. A derivative construct of the CAD cDNA was Generated that should encode only the CPSase domain. When transfected into mammalian cells, a protein was synthesized that had significant CPSase activity both in vivo and in vitro. The two constructs Generated in this study will facilitate the study of CAD structure, function, and allosteric regulation.
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An unusual Alu repeat sequence within the CAD Gene.
Journal of Molecular Evolution, 1991Co-Authors: Jeffrey N. Davidson, Nada H. Khattar, Kuey-chu ChenAbstract:There are several hundred thousand members of the Alu repeat family in the human genome. Those Alu elements sequenced to date appear to fit into subfamilies. A novel Alu has been found in an intron of the human CAD Gene: it appears to be due to rearrangement between Alu repeats belonging to two different subfamilies. Further sequence data from this intron suggest that the Alu element may have rearranged prior to its entry into the CAD Gene. Such findings indicate that, in addition to single nucleotide substitutions and deletions, DNA rearrangements may be a factor in Generating the diversity of Alu repeats found in primate genomes.
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Organization and nucleotide sequence of the 3' end of the human CAD Gene.
DNA and Cell Biology, 1990Co-Authors: Jeffrey N. Davidson, Gadiparthi N. Rao, Lee Niswander, Carla Andreano, Celeste Tamer, Kuey-chu ChenAbstract:ABSTRACT Aspartate transcarbamylase (ATCase) is found as a monofunctional protein in prokaryotes and as a part of a multifunctional protein in fungi and animals. In mammals, this enzyme along with ...
Stephen Safe - One of the best experts on this subject based on the ideXlab platform.
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estrogen receptor sp1 complexes are required for induction of CAD Gene expression by 17β estradiol in breast cancer cells
Endocrinology, 2003Co-Authors: Shaheen Khan, Maen Abdelrahim, Ismael Samudio, Stephen SafeAbstract:The CAD Gene is trifunctional and expresses carbamoylphosphate synthetase/aspartate carbamyltransferase/dihydroorotase, which are required for pyrimidine biosynthesis. CAD Gene activities are induced in MCF-7 human breast cancer cells, and treatment of MCF-7 or ZR-75 cells with 10 nm 17β-estradiol (E2) resulted in a 3- to 5-fold increase in CAD mRNA levels in both cell lines. The mechanism of hormone-induced CAD Gene expression was further investigated using constructs containing the growth-responsive −90 to +115 (pCAD1) region of the CAD Gene promoter. E2 induced reporter Gene (luciferase) activity in MCF-7 and ZR-75 cells transfected with pCAD1, which contains three upstream GC-rich and two downstream E-box motifs. Deletion and mutation analysis of the CAD Gene promoter demonstrated that only the GC boxes that bind Sp1 protein were required for E2 responsiveness. Results of electrophoretic mobility shift and chromatin immunoprecipitation assays show that both Sp1 and estrogen receptor α interact with th...
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Estrogen Receptor/Sp1 Complexes Are Required for Induction of CAD Gene Expression by 17β-Estradiol in Breast Cancer Cells
Endocrinology, 2003Co-Authors: Shaheen Khan, Maen Abdelrahim, Ismael Samudio, Stephen SafeAbstract:The CAD Gene is trifunctional and expresses carbamoylphosphate synthetase/aspartate carbamyltransferase/dihydroorotase, which are required for pyrimidine biosynthesis. CAD Gene activities are induced in MCF-7 human breast cancer cells, and treatment of MCF-7 or ZR-75 cells with 10 nm 17β-estradiol (E2) resulted in a 3- to 5-fold increase in CAD mRNA levels in both cell lines. The mechanism of hormone-induced CAD Gene expression was further investigated using constructs containing the growth-responsive −90 to +115 (pCAD1) region of the CAD Gene promoter. E2 induced reporter Gene (luciferase) activity in MCF-7 and ZR-75 cells transfected with pCAD1, which contains three upstream GC-rich and two downstream E-box motifs. Deletion and mutation analysis of the CAD Gene promoter demonstrated that only the GC boxes that bind Sp1 protein were required for E2 responsiveness. Results of electrophoretic mobility shift and chromatin immunoprecipitation assays show that both Sp1 and estrogen receptor α interact with th...
Jua Munozblanco - One of the best experts on this subject based on the ideXlab platform.
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cloning expression and immunolocalization pattern of a cinnamyl alcohol dehydrogenase Gene from strawberry fragaria ananassa cv chandler
Journal of Experimental Botany, 2002Co-Authors: Rosario Lancoportales, Nieves Medinaescoba, Jua A Lopezraez, Jose A Gonzalezreyes, Jose M Villalba, Enriqueta Moyano, J L Caballero, Jua MunozblancoAbstract:: Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) catalyses the conversion of p-hydroxy-cinnamaldehydes to the corresponding alcohols and is considered a key enzyme in lignin biosynthesis. By a differential screening of a strawberry (Fragariax ananassa cv. Chandler) fruit specific subtractive cDNA library, a full-length clone corresponding to a CAD Gene was isolated (FxaCAD1). Northern blot and quantitative real time PCR studies indicated that the strawberry FxaCAD1 Gene is expressed in fruits, runners, leaves, and flowers but not in roots. In addition, the Gene presented a differential expression in fruits along the ripening process. Moreover, by screening of a strawberry genomic library a CAD Gene was isolated (FxaCAD2). Similar to that found in other CAD Genes from higher plants, this strawberry CAD Gene is structured in five exons and four introns. Southern blot analyses suggest that, probably, a small CAD Gene family exists in strawberry. RT-PCR studies indicated that only the FxaCAD1 Gene was expressed in all the fruit ripening stages and vegetative tissues analysed. The FxaCAD1 cDNA was expressed in E. coli cells and the corresponding protein was used to raise antibodies against the strawberry CAD polypeptide. The antibodies obtained were used for immunolocalization studies. The results showed that the CAD polypeptide was localized in lignifying cells of all the tissues examined (achenes, fruit receptacles, runners, leaves, pedicels, and flowers). Additionally, the cDNA was also expressed in yeast (Pichia pastoris) as an extracellular protein. The recombinant protein showed activity with the characteristic substrates of CAD enzymes from angiosperms, indicating that the Gene cloned corresponds to a CAD protein.
