Single and Coincident Intragenic Mutations Attributable to Gene Conversion in a Human Cell Line

Genetics ◽  
1997 ◽  
Vol 146 (4) ◽  
pp. 1429-1439
Author(s):  
Cynthia R Giver ◽  
Andrew J Grosovsky
Keyword(s):  
Cell Line ◽  
Gene Conversion ◽  
Point Mutations ◽  
Double Exchange ◽  
Detailed Examination ◽  
Human Cell Line ◽  
Human Lymphoblastoid Cell ◽  
Dna Base ◽  
Dna Strand ◽  
Conversion Tract

Two polymorphic sites are located within the heterozygous TK1 locus in the human lymphoblastoid cell line TK6: an inactivating frameshift in exon 4 of the nonfunctional allele and a phenotypically silent frameshift in exon 7 of the functional allele. Through the use of these intragenic polymorphisms and microsatellite markers that flank TK1, we demonstrate that partial gene conversion accounts for 3/75 (0.04) spontaneous and 9/163 (0.06) X-ray-induced TK1– mutants, thus comprising a significant component of forward mutations at this locus. In all cases, the conversion tract is <1 cM, rendering double exchange a remote alternate explanation for these results. Sequence analysis of full length TK1 cDNA provides rigorous exclusion of deletion events as a mechanism for generation of these allelotypes. Detailed examination of allelotypes in TK1– mutants identified two mechanisms for the generation of coincident sequence alterations that sometimes accompanied gene conversions. Mutations within the conversion tract were attributed to either error-prone gap filling synthesis during recombinational repair or mismatch repair within a heteroduplex region following branch migration. These findings suggest that a proportion of point mutations may not be targeted to sites of DNA base damage, but rather may arise as secondary consequences from the repair of DNA strand breaks.

The effect of anoxia on anthracycline-induced DNA damage in the RPMI-6410 human lymphoblastoid cell line

1982 ◽  
Vol 60 (9) ◽  
pp. 873-876 ◽  
Author(s):  
L. Brox ◽  
B. Gowans ◽  
R. To ◽  
A. Belch
Keyword(s):  
Cell Line ◽  
Lymphoblastoid Cell Line ◽  
Dna Strand Breaks ◽  
Primary Role ◽  
Alkaline Elution ◽  
Strand Breaks ◽  
Human Lymphoblastoid Cell ◽  
Strand Breakage ◽  
Human Lymphoblastoid Cell Line ◽  
Dna Strand

The alkaline elution procedure developed by Kohn and co-workers was used with the RPMI-6410 cultured human lymphoblastoid cell line to examine the hypothesis that anthracycline-induced DNA strand scission is mediated by oxygen- or superoxide-derived free radicals. Hypoxia was induced by gassing with nitrogen containing 5% carbon dioxide and less than 4 ppm oxygen. Alkaline elution studies showed hypoxia was induced, as the oxygen enhancement ratios for DNA strand breaks was 2.4 and 2.6 for the 250 R ± oxygen and the 500 R ± oxygen (1 R = 2.58 × 10−4 C/kg) experiments, respectively. The pattern of adriamycin-induced DNA strand breaks and cross-linking was not affected by hypoxia with 1-h adriamycin exposures between 0.05 and 1.0 μg/mL. Similarly, 1-h exposures of N-trifluoroacetyladriamycin-14-valerate at 3 or 10 μg/mL gave essentially identical alkaline elution profiles in the presence or absence of oxygen. These results do not support the hypothesis that oxygen-derived radicals play a primary role in anthracycline-induced DNA strand breakage.

scholarly journals Multiple-endpoint in vitro carcinogenicity test in human cell line TK6 distinguishes carcinogens from non-carcinogens and highlights mechanisms of action

2020 ◽  
Author(s):  
Katherine E. Chapman ◽  
Eleanor C. Wilde ◽  
Fiona M. Chapman ◽  
Jatin R. Verma ◽  
Ume-Kulsoom Shah ◽  
...  
Keyword(s):  
Cell Line ◽  
Human Cell Line ◽  
Test System ◽  
In Vitro Tests ◽  
Correct Identification ◽  
In Vitro Test ◽  
Human Lymphoblastoid Cell ◽  
Carcinogenic Potential

Abstract Current in vitro genotoxicity tests can produce misleading positive results, indicating an inability to effectively predict a compound’s subsequent carcinogenic potential in vivo. Such oversensitivity can incur unnecessary in vivo tests to further investigate positive in vitro results, supporting the need to improve in vitro tests to better inform risk assessment. It is increasingly acknowledged that more informative in vitro tests using multiple endpoints may support the correct identification of carcinogenic potential. The present study, therefore, employed a holistic, multiple-endpoint approach using low doses of selected carcinogens and non-carcinogens (0.001–770 µM) to assess whether these chemicals caused perturbations in molecular and cellular endpoints relating to the Hallmarks of Cancer. Endpoints included micronucleus induction, alterations in gene expression, cell cycle dynamics, cell morphology and bioenergetics in the human lymphoblastoid cell line TK6. Carcinogens ochratoxin A and oestradiol produced greater Integrated Signature of Carcinogenicity scores for the combined endpoints than the “misleading” in vitro positive compounds, quercetin, 2,4-dichlorophenol and quinacrine dihydrochloride and toxic non-carcinogens, caffeine, cycloheximide and phenformin HCl. This study provides compelling evidence that carcinogens can successfully be distinguished from non-carcinogens using a holistic in vitro test system. Avoidance of misleading in vitro outcomes could lead to the reduction and replacement of animals in carcinogenicity testing.

scholarly journals Elevated Levels of DNA Strand Breaks Induced by a Base Analog in the Human Cell Line with the P32T ITPA Variant

2010 ◽  
Vol 2010 ◽  
pp. 1-11 ◽  
Author(s):  
Irina S.-R. Waisertreiger ◽  
Miriam R. Menezes ◽  
James Randazzo ◽  
Youri I. Pavlov
Keyword(s):  
Cell Line ◽  
Human Cell Line ◽  
Dna Breaks ◽  
Nucleotide Biosynthesis ◽  
Purine Base ◽  
Strand Breaks ◽  
Inosine Triphosphate Pyrophosphatase ◽  
Base Analog ◽  
Deoxynucleoside Triphosphate ◽  
Dna Strand

Base analogs are powerful antimetabolites and dangerous mutagens generated endogenously by oxidative stress, inflammation, and aberrant nucleotide biosynthesis. Human inosine triphosphate pyrophosphatase (ITPA) hydrolyzes triphosphates of noncanonical purine bases (i.e., ITP, dITP, XTP, dXTP, or their mimic: 6-hydroxyaminopurine (HAP) deoxynucleoside triphosphate) and thus regulates nucleotide pools and protects cells from DNA damage. We demonstrate that the model purine base analog HAP induces DNA breaks in human cells and leads to elevation of levels of ITPA. A human polymorphic allele of theITPA, 94C->A encodes for the enzyme with a P32T amino-acid change and leads to accumulation of nonhydrolyzed ITP. The polymorphism has been associated with adverse reaction to purine base-analog drugs. The level of both spontaneous and HAP-induced DNA breaks is elevated in the cell line with the ITPA P32T variant. The results suggested that human ITPA plays a pivotal role in the protection of DNA from noncanonical purine base analogs.

Multiple dispersed spontaneous mutations: a novel pathway of mutation in a malignant human cell line

1991 ◽  
Vol 11 (6) ◽  
pp. 3163-3170
Author(s):  
J Harwood ◽  
A Tachibana ◽  
M Meuth
Keyword(s):  
Cell Line ◽  
Tumor Cells ◽  
Excision Repair ◽  
Point Mutations ◽  
Mutant Gene ◽  
Human Cell Line ◽  
Dna Lesions ◽  
Spontaneous Mutations ◽  
Base Pairs ◽  
Complex Genome

We analyzed the nature of spontaneous mutations at the autosomal locus coding for adenine phosphoribosyltransferase in the human colorectal carcinoma cell line SW620 to establish whether distinctive mutational pathways exist that might underlie the more complex genome rearrangements arising in tumor cells. Point mutations occur at a low rate in aprt hemizygotes derived from SW620, largely as a result of base substitutions at G.C base pairs to yield transversions and transitions. However, a novel pathway is evident in the form of multiple dispersed mutations in which two errors, separated by as much as 1,800 bp, fall in the same mutant gene. Such mutations could be the result of error-prone DNA synthesis occurring during normal replication or during long-patch excision-repair of spontaneously arising DNA lesions. This process could also contribute to the chromosomal instability evident in these tumor cells.

scholarly journals The human myeloma cell line LP-1: a versatile model in which to study early plasma-cell differentiation and c-myc activation

Blood ◽  
1989 ◽  
Vol 73 (4) ◽  
pp. 1020-1027
Author(s):  
L Pegoraro ◽  
F Malavasi ◽  
G Bellone ◽  
M Massaia ◽  
M Boccadoro ◽  
...  
Keyword(s):  
Cell Line ◽  
Plasma Cells ◽  
Point Mutations ◽  
Myeloma Cell ◽  
Human Cell Line ◽  
B Cell Maturation ◽  
Potential Partner ◽  
Human Myeloma Cell Line ◽  
Myc Gene ◽  
Conventional Cytogenetics

The characteristics of a human cell line (LP-1) derived from the peripheral blood of a patient with IgG-lambda myeloma in leukemic transformation are described. The cells resemble immature plasma cells in that they exhibit a membrane phenotype that is intermediate between late B lymphocytes and plasma cells, even though they secrete IgG- lambda chains. Treatment of LP-1 cells with 12–0 tetradecanoylphorbol- 13-acetate (TPA) or pokeweek mitogen (PWM) induces the appearance of surface markers and ultrastructural features typical of mature plasma cells but does not affect their proliferative activity. Molecular analysis of the cell line showed an increased expression of the c-myc protooncogene and the presence of abnormally sized transcripts. Conventional cytogenetics and pulsed-field gel electrophoresis showed no structural rearrangements of the c-myc gene, suggesting that the abnormal c-myc expression may be due to point mutations or small deletions within the gene. The LP-1 cell line is a useful model in which to study the process of B-cell maturation; such study may lead to the uncovering of unusual mechanisms of c-myc activation. Furthermore, the LP-1 cell is a potential partner in the generation of human hybridomas.

scholarly journals Multiple dispersed spontaneous mutations: a novel pathway of mutation in a malignant human cell line.

1991 ◽  
Vol 11 (6) ◽  
pp. 3163-3170 ◽  
Author(s):  
J Harwood ◽  
A Tachibana ◽  
M Meuth
Keyword(s):  
Cell Line ◽  
Tumor Cells ◽  
Excision Repair ◽  
Point Mutations ◽  
Mutant Gene ◽  
Human Cell Line ◽  
Dna Lesions ◽  
Spontaneous Mutations ◽  
Base Pairs ◽  
Complex Genome

We analyzed the nature of spontaneous mutations at the autosomal locus coding for adenine phosphoribosyltransferase in the human colorectal carcinoma cell line SW620 to establish whether distinctive mutational pathways exist that might underlie the more complex genome rearrangements arising in tumor cells. Point mutations occur at a low rate in aprt hemizygotes derived from SW620, largely as a result of base substitutions at G.C base pairs to yield transversions and transitions. However, a novel pathway is evident in the form of multiple dispersed mutations in which two errors, separated by as much as 1,800 bp, fall in the same mutant gene. Such mutations could be the result of error-prone DNA synthesis occurring during normal replication or during long-patch excision-repair of spontaneously arising DNA lesions. This process could also contribute to the chromosomal instability evident in these tumor cells.

scholarly journals The human myeloma cell line LP-1: a versatile model in which to study early plasma-cell differentiation and c-myc activation

Blood ◽  
1989 ◽  
Vol 73 (4) ◽  
pp. 1020-1027 ◽  
Author(s):  
L Pegoraro ◽  
F Malavasi ◽  
G Bellone ◽  
M Massaia ◽  
M Boccadoro ◽  
...  
Keyword(s):  
Cell Line ◽  
Plasma Cells ◽  
Point Mutations ◽  
Myeloma Cell ◽  
Human Cell Line ◽  
B Cell Maturation ◽  
Potential Partner ◽  
Human Myeloma Cell Line ◽  
Myc Gene ◽  
Conventional Cytogenetics

Abstract The characteristics of a human cell line (LP-1) derived from the peripheral blood of a patient with IgG-lambda myeloma in leukemic transformation are described. The cells resemble immature plasma cells in that they exhibit a membrane phenotype that is intermediate between late B lymphocytes and plasma cells, even though they secrete IgG- lambda chains. Treatment of LP-1 cells with 12–0 tetradecanoylphorbol- 13-acetate (TPA) or pokeweek mitogen (PWM) induces the appearance of surface markers and ultrastructural features typical of mature plasma cells but does not affect their proliferative activity. Molecular analysis of the cell line showed an increased expression of the c-myc protooncogene and the presence of abnormally sized transcripts. Conventional cytogenetics and pulsed-field gel electrophoresis showed no structural rearrangements of the c-myc gene, suggesting that the abnormal c-myc expression may be due to point mutations or small deletions within the gene. The LP-1 cell line is a useful model in which to study the process of B-cell maturation; such study may lead to the uncovering of unusual mechanisms of c-myc activation. Furthermore, the LP-1 cell is a potential partner in the generation of human hybridomas.

The Comet Assay with MCL-5 Cells as an Indicator of Genotoxic Treatment with Chemicals and Cigarette Smoke Condensate

2002 ◽  
Vol 30 (3) ◽  
pp. 331-339 ◽  
Author(s):  
Lucie Wolz ◽  
Günter Krause ◽  
Gerhard Scherer
Keyword(s):  
Cell Proliferation ◽  
Cell Line ◽  
Cigarette Smoke ◽  
Metabolic Activation ◽  
Strand Break ◽  
Mutation Induction ◽  
Cigarette Smoke Condensate ◽  
Human Lymphoblastoid Cell ◽  
Scge Assay ◽  
Dna Strand

The metabolically competent human lymphoblastoid cell line MCL-5 was treated with a panel of mutagens to assess the induction of DNA damage. Treatment effects were observed by monitoring cell proliferation and by single-cell gel electrophoresis (SCGE). The direct-acting mutagens benzo[ a]pyrene-7,8-diol 9, 10-epoxide (BPDE) and 1-methyl-3-nitro-1-nitrosoguanidine (MNNG), as well as pro-mutagens requiring metabolic activation, i.e. benzo[a]pyrene (BaP), 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyri-dine (PhIP), 4- N-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and cigarette-smoke condensate (CSC), were assayed by SCGE. Assay schemes were adapted for the MCL-5 cell line and for low levels of strand break induction, by inclusion of the DNA synthesis inhibitors cytosine arabinoside and hydroxyurea, and by extending the electrophoresis time. For all mutagens tested, dose-dependent increases of median and average tail moment values among 50 nucleoids per slide were observed. The determining factors for selecting the treatment doses for mutation-induction experiments were the solubility of BaP and PhIP in the exposure medium, and the cytotoxicity exhibited by BPDE, MNNG and CSC. Induction of DNA strand breaks was obtained at mutagen concentrations permitting sufficient cell proliferation, except in the case of MNNG.

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