Mutation Research: Fundamental And Molecular Mechanisms Of Mutagenesis, Apr 1, 1977
Three polycyclic hydrocarbons, benz(a)anthracene. 3-methylcholanthrene and 7,12-dImethylbenr(a)an... more Three polycyclic hydrocarbons, benz(a)anthracene. 3-methylcholanthrene and 7,12-dImethylbenr(a)anthracene, have been studied in a cellmediated mutagenesis system using B H K 21 cells to metabolize the hydrocarbons and V-79 cells as targets for detecting induced cytotoxicity and mutation. In large-scale experiments, the D N A of V-79 cells was analyzed by column chromatography to determine the nature and true extent of reaction of hydrocarbons with deoxyribonucleosides. Products with D N A formed by the two carcinogenic compounds were qualitatively very similar to those reported to occur in vivo and in primary cell cultures. Binding indices were calculated from the tritium content of DNA-hydrocarbon products, related to overall metabolism, for these two compounds together with benzo(a)pyrene and 'I-methylbenz(a)anthracene using data from a previous study. These values reflected differences in carcinogenic potency between the compounds. Induced mutation frequencies were related to the extent of D N A reaction with each compound. A t equivalent extents of D N A reaction with hydrocarbon products, levels of induced mutation were not significantly different.
Cell transformation assays (CTAs) have long been proposed as in vitro methods for the identificat... more Cell transformation assays (CTAs) have long been proposed as in vitro methods for the identification of potential chemical carcinogens. Despite showing good correlation with rodent bioassay data, concerns over the subjective nature of using morphological criteria for identifying transformed cells and a lack of understanding of the mechanistic basis of the assays has limited their acceptance for regulatory purposes. However, recent drivers to find alternative carcinogenicity assessment methodologies, such as the Seventh Amendment to the EU
Telomerase is required for the complete replication of chromosomal ends. In tumors, the human tel... more Telomerase is required for the complete replication of chromosomal ends. In tumors, the human telomerase reverse transcriptase subunit (hTERT) is up-regulated, thereby removing a critical barrier for unlimited cell proliferation. To understand more about hTERT regulation, we measured hTERT RNA levels by quantitative reverse transcription (RT)PCR. Telomerase-positive cell lines were found to contain between 0.2 and 6 molecules of spliced hTERT RNA per cell, whereas in telomerasenegative cells, the number of molecules was below the sensitivity of the assay (<0.004 molecules/cell). Intron-containing, immature hTERT RNA was observed only in nuclei of telomerase-positive cells, which suggests that hTERT RNA levels are transcriptionally regulated. Microcell transfer of a normal chromosome 3 into the human breast carcinoma cell line (21NT) abolishes telomerase activity and induces senescence. Endogenous hTERT transcripts were undetectable in the nuclei of 21NT-chromosome 3 hybrids, even...
Losses of heterozygosity involving chromosomes 9 and 10 are frequent events in the development an... more Losses of heterozygosity involving chromosomes 9 and 10 are frequent events in the development and progression of cutaneous malignant melanoma. To investigate whether specifically deleted chromosomal regions encode tumor suppressor genes (TSGs), we introduced normal chromosome 10 into the tumorigenic human metastatic melanoma cell line UACC-903 by microcell fusion. In addition, two chromosome 9 derivatives that were microdeleted in the region of the p16INK4A/p15INK4B locus were transferred to determine whether an additional melanoma TSG or TSGs reside on chromosome 9p, as indicated by previous melanoma allele loss studies. In comparison to parental cells, microcell hybrids generated with chromosomes 9 (microdeleted) and 10 displayed reduced anchorage-independent growth in soft agar and markedly reduced tumorigenicity in athymic (nu/nu) mice. These data define a TSG or TSGs that function independently of p15/p16 on chromosome 9 and provide evidence for a TSG (or TSGs) on chromosome 1...
Cardiovascular diseases are a dominant cause of death in the developed world and new ways to unde... more Cardiovascular diseases are a dominant cause of death in the developed world and new ways to understand the underlying cellular, molecular and functional mechanisms are needed. Novel opportunities to address this need have arisen from the ability to produce cardiomyocytes from human pluripotent stem cells (hPSCs). Here, we will discuss our work towards in vitro modelling of genetic diseases that affect the electrophysiology, structure or survival of heart cells. For example, modelling the potentially fatal condition of long QT syndrome using hiPSCderived cardiomyocytes shows that the electrophysiological and pharmacological responses observed in the in vitro models accurately replicate known patient outcomes. These models can also be used to evaluate the utility of genetic-based corrective therapies. Such observations go some way to validating hPSC models as a useful screening and development tool for drug and genetic therapy. Similarly, studies have shown these hPSCcardiomyocytes offer real potential in drug safety assessment, providing novel ways to address an issue that has placed a socioeconomic burden on the pharmaceutical for several decades. Finally, we will summarise our work towards automating hPSC culture and differentiation to provide high content screening opportunities for industry.
Mutation Research: Fundamental And Molecular Mechanisms Of Mutagenesis, Apr 1, 1977
Three polycyclic hydrocarbons, benz(a)anthracene. 3-methylcholanthrene and 7,12-dImethylbenr(a)an... more Three polycyclic hydrocarbons, benz(a)anthracene. 3-methylcholanthrene and 7,12-dImethylbenr(a)anthracene, have been studied in a cellmediated mutagenesis system using B H K 21 cells to metabolize the hydrocarbons and V-79 cells as targets for detecting induced cytotoxicity and mutation. In large-scale experiments, the D N A of V-79 cells was analyzed by column chromatography to determine the nature and true extent of reaction of hydrocarbons with deoxyribonucleosides. Products with D N A formed by the two carcinogenic compounds were qualitatively very similar to those reported to occur in vivo and in primary cell cultures. Binding indices were calculated from the tritium content of DNA-hydrocarbon products, related to overall metabolism, for these two compounds together with benzo(a)pyrene and 'I-methylbenz(a)anthracene using data from a previous study. These values reflected differences in carcinogenic potency between the compounds. Induced mutation frequencies were related to the extent of D N A reaction with each compound. A t equivalent extents of D N A reaction with hydrocarbon products, levels of induced mutation were not significantly different.
Cell transformation assays (CTAs) have long been proposed as in vitro methods for the identificat... more Cell transformation assays (CTAs) have long been proposed as in vitro methods for the identification of potential chemical carcinogens. Despite showing good correlation with rodent bioassay data, concerns over the subjective nature of using morphological criteria for identifying transformed cells and a lack of understanding of the mechanistic basis of the assays has limited their acceptance for regulatory purposes. However, recent drivers to find alternative carcinogenicity assessment methodologies, such as the Seventh Amendment to the EU
Telomerase is required for the complete replication of chromosomal ends. In tumors, the human tel... more Telomerase is required for the complete replication of chromosomal ends. In tumors, the human telomerase reverse transcriptase subunit (hTERT) is up-regulated, thereby removing a critical barrier for unlimited cell proliferation. To understand more about hTERT regulation, we measured hTERT RNA levels by quantitative reverse transcription (RT)PCR. Telomerase-positive cell lines were found to contain between 0.2 and 6 molecules of spliced hTERT RNA per cell, whereas in telomerasenegative cells, the number of molecules was below the sensitivity of the assay (<0.004 molecules/cell). Intron-containing, immature hTERT RNA was observed only in nuclei of telomerase-positive cells, which suggests that hTERT RNA levels are transcriptionally regulated. Microcell transfer of a normal chromosome 3 into the human breast carcinoma cell line (21NT) abolishes telomerase activity and induces senescence. Endogenous hTERT transcripts were undetectable in the nuclei of 21NT-chromosome 3 hybrids, even...
Losses of heterozygosity involving chromosomes 9 and 10 are frequent events in the development an... more Losses of heterozygosity involving chromosomes 9 and 10 are frequent events in the development and progression of cutaneous malignant melanoma. To investigate whether specifically deleted chromosomal regions encode tumor suppressor genes (TSGs), we introduced normal chromosome 10 into the tumorigenic human metastatic melanoma cell line UACC-903 by microcell fusion. In addition, two chromosome 9 derivatives that were microdeleted in the region of the p16INK4A/p15INK4B locus were transferred to determine whether an additional melanoma TSG or TSGs reside on chromosome 9p, as indicated by previous melanoma allele loss studies. In comparison to parental cells, microcell hybrids generated with chromosomes 9 (microdeleted) and 10 displayed reduced anchorage-independent growth in soft agar and markedly reduced tumorigenicity in athymic (nu/nu) mice. These data define a TSG or TSGs that function independently of p15/p16 on chromosome 9 and provide evidence for a TSG (or TSGs) on chromosome 1...
Cardiovascular diseases are a dominant cause of death in the developed world and new ways to unde... more Cardiovascular diseases are a dominant cause of death in the developed world and new ways to understand the underlying cellular, molecular and functional mechanisms are needed. Novel opportunities to address this need have arisen from the ability to produce cardiomyocytes from human pluripotent stem cells (hPSCs). Here, we will discuss our work towards in vitro modelling of genetic diseases that affect the electrophysiology, structure or survival of heart cells. For example, modelling the potentially fatal condition of long QT syndrome using hiPSCderived cardiomyocytes shows that the electrophysiological and pharmacological responses observed in the in vitro models accurately replicate known patient outcomes. These models can also be used to evaluate the utility of genetic-based corrective therapies. Such observations go some way to validating hPSC models as a useful screening and development tool for drug and genetic therapy. Similarly, studies have shown these hPSCcardiomyocytes offer real potential in drug safety assessment, providing novel ways to address an issue that has placed a socioeconomic burden on the pharmaceutical for several decades. Finally, we will summarise our work towards automating hPSC culture and differentiation to provide high content screening opportunities for industry.
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