Depressed affect and time perception.
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Adult↗
Biomedical subjects
B D Crawford
Publications and source records attributed to B D Crawford.
5-Azacytidine-induced conversion to cadmium resistance correlates with early S phase replication of inactive metallothionein genes in synchronized CHO cells.
Previous studies have shown both hypermethylation and late replication of DNA sequences to be associated with gene inactivity. To determine whether there is a causal relationship between patterns of DNA methylation and replication timing during S phase, we have examined the timing of replication of the inactive, hypermethylated metallothionein (MT) I and II genes in synchronized, cadmium-sensitive (Cds) CHO cells. The time of S-phase replication of the MT genes was ascertained by determining the period of S phase wherein cadmium-resistant (Cdr) cells could be induced with highest frequency by pulse treatment of synchronized Cds cells with the hypomethylating drug 5-azacytidine (5-aza-CR), and by analyzing Southern blots of density fractionated DNAs isolated from synchronized cells pulse-labeled with BrdU during different intervals after release from hydroxyurea blockade. Southern filter hybridization analyses demonstrated replication of both MTI and II gene sequences within the first half of S phase. Consistent with this result, phenotypic conversion of Cds to Cdr was maximal immediately after hydroxyurea release and decreased abruptly within three hours. The replication of inactive hypermethylated MT genes in early S phase argues that transcriptional inactivity and gene-specific hypermethylation are not sufficient conditions for late DNA replication.
Animals↗
Assignment of RAS proto-oncogenes in Chinese hamsters: implications for mammalian gene linkage conservation and neoplasia.
HRAS and KRAS are the cellular homologs of the oncogenic transforming genes found in the Harvey strain of murine sarcoma virus and the Kirsten murine sarcoma virus, respectively. Phyla as diverse as insects, birds, and mammals possess distinct HRAS and KRAS sequences, suggesting that these genes are essential to metazoa. In this report, we used a clone panel of Chinese hamster X mouse C11D somatic cell hybrids segregating hamster chromosomes to map those genes. Southern filter hybridization analyses of the hybrids revealed that hamster HRAS and KRAS gene sequences are on chromosomes 3 and 8, respectively. These gene assignments are consistent with the conservation of autosomal gene linkage groups observed among hamsters, humans, and mice and may provide insight into specific chromosomal alterations that have been observed during the spontaneous neoplastic transformation of Chinese hamster fibroblasts in vitro.
Animals↗
Oncogenes and linkage groups: conservation during mammalian chromosome evolution.
Proto-oncogenes, which represent the cellular progenitors of the transforming genes harbored by acute transforming oncogenic retroviruses, have been highly conserved during vertebrate evolution. In this report, we have assigned experimentally a subset of proto-oncogenes (SRC, ABL, FES, and FMS-all related to the SRC family) to Chinese hamster chromosomes by Southern filter hybridization analyses of DNAs isolated from both somatic cell hybrids and flow-sorted hamster chromosomes. These results demonstrate that several autosomal linkage groups containing proto-oncogenes originated prior to the radiation and speciation of mammals and have remained remarkably stable for nearly 80 million years.
Animals↗
Coordinate amplification of metallothionein I and II genes in cadmium-resistant Chinese hamster cells: implications for mechanisms regulating metallothionein gene expression.
We describe here the derivation, characterization, and use of clonal cadmium-resistant (Cdr) strains of the Chinese hamster cell line CHO which differ in their metallothionein (MT) induction capacity. By nondenaturing polyacrylamide gel electrophoresis, we showed that the stable Cdr phenotype is correlated with the augmented expression of both isometallothioneins (MTI and MTII). In cells resistant to concentrations of CdCl2 exceeding 20 microM, coordinate amplification of genes encoding both isometallothioneins was demonstrated by using cDNA MT-coding sequence probes and probes specific for 3'-noncoding regions of Chinese hamster MTI and MTII genes. Molecular and in situ hybridization analyses supported close linkage of Chinese hamster MTI and MTII genes, which we have mapped previously to Chinese hamster chromosome 3. This suggests the existence of a functionally related MT gene cluster in this species. Amplified Cdr variants expressing abundant MT and their corresponding Cds parental CHO cells should be useful for future studies directed toward elucidating the mechanisms that regulate expression of the isometallothioneins.
Animals↗
Construction and analysis of DNA sequence libraries from flow-sorted chromosomes: practical and theoretical considerations.
We describe the construction and analysis of recombinant DNA libraries representative of chromosomes 1 and 2 of Chinese hamster (Cricetulus griseus). Propidium-iodide stained chromosomes were purified by flow cytometric analysis and sorting, and EcoRI digests of purified DNA were cloned into the bacteriophage vector Charon 4A. These libraries contain DNA complementary to 63% and 69% of nick-translated DNA derived from flow-purified chromosomes 1 and 2, respectively. However, sequences complementary to only 24% and 35% of a total Chinese hamster genomic DNA tracer were hybridized in parallel renaturation experiments. The chromosome 2 library contained DNA sequences encoding dihydrofolate reductase (dhfr), a gene previously mapped to Chinese hamster chromosome 2. No sequences complementary to dhfr were found in the library constructed from chromosome 1 DNA. These analyses are discussed with regard to the current limitations and future strategies for the construction of chromosome-specific DNA sequence libraries of high purity and completeness.
Animals↗
Assignment of genes encoding metallothioneins I and II to Chinese hamster chromosome 3: evidence for the role of chromosome rearrangement in gene amplification.
Cadmium resistant (Cdr) variants with coordinately amplified metallothionein I and II (MTI and MTII) genes have been derived from both Chinese hamster ovary and near-euploid Chinese hamster cell lines. Cytogenetic analyses of Cdr variants consistently revealed breakage and rearrangement involving chromosome 3p. In situ hybridization with a Chinese hamster MT-encoding cDNA probe localized amplified MT gene sequences near the translocation breakpoint involving chromosome 3p. These observations suggested that both functionally related, isometallothionein loci are linked on Chinese hamster chromosome 3. Southern blot analyses of DNAs isolated from a panel of Chinese hamster X mouse somatic cell hybrids which segregate hamster chromosomes confirmed that both MTI and MTII are located on chromosome 3. We speculate that rearrangement of chromosome 3p could be causally involved with the amplification of MT genes in Cdr hamster cell lines.
Animals↗
Neoplastic conversion of preneoplastic Syrian hamster cells: rate estimation by fluctuation analysis.
Analysis of the role of gene mutations in the multistep process of neoplastic transformation requires that the discrete steps in carcinogenesis first be dissected. Toward this end, we have isolated and characterized preneoplastic Syrian hamster cells which exhibit in vitro a trait highly correlated with neoplastic conversion in vivo. Previous findings (J. C. Barrett, Cancer Res. 40:91-94, 1980) indicate that spontaneous neoplastic transformation of Syrian hamster cells occurs in at least two steps. An intermediate stage, characterized by an aneuploid established cell line which has a propensity to become neoplastic spontaneously upon further growth in vitro, has been described. These preneoplastic cells differ from diploid early-passage Syrian hamster cells in becoming capable of anchorage-independent growth in semisolid agar, as well as becoming neoplastic in vivo when attached to a solid substrate. Evidence presented here demonstrates that anchorage-independent conversion in vitro is a reliable marker for neoplastic conversion in this cell system. Fluctuation analyses, patterned after those described by Luria and Delbruck for microbial genetics, demonstrate that anchorage-independent variants are generated randomly from clonally derived preneoplastic cells at the rate of 10(-8) to 10(-7) variants per cell per generation. These results establish a multistep stochastic process for transformation in vitro and indicate that conversion to anchorage independence may be necessary for Syrian hamster cells to become tumorigenic. The possible role of gene mutation in this step during neoplastic progression is discussed.
Aneuploidy↗
Comparison between mutagenesis in normal and transformed Syrian hamster fibroblasts: difference in the temporal order of HPRT gene replication.
A highly tumorigenic subdiploid cell line, BP6T, derived in our laboratory from Syrian hamster embryo (SHE) cells, is amenable to studies of somatic mutation in vitro. Cellular and biochemical characterization of clonally derived BP6T cells resistant to 6-thioguanine (TGr) or ouabain (Quar) demonstrated these mutants to be similar qualitatively to mutants of SHE cells characterized previously (Barrett et al., 1978). BP6T TGr mutants resistant to 6-thioguanine are cross-resistant to 8-azaguanine, lack HPRT activity, exhibit a low frequency of reversion and arise spontaneously at a rate of approximately 5 X 10(-7) mutants per cell per generation. BP6T Ouar mutants were shown to be highly resistant to ouabain-mediated inhibition of 86Rb influx, indicating an alteration in the Na+/K+ ATPase. These studies on the BP6T cell line provide the experimental basis for a comparative study of the mutagenic responses of normal, diploid SHE cells versus those of related, but transformed aneuploid cells. Highly synchronized cultures of these 2 cells were mutagenized by pulse treatment with BrdU during different periods of S phase, followed immediately by near-UV irradiation. The induced mutation frequencies so obtained provided information about the temporal order of replication of genes encoding HPRT and Na+/K+ ATPase in both SHE and BP6T cells. The temporal pattern of replication of Na+/K+ ATPase gene loci is similar in both cell types, but the temporal order of replication of the HPRT gene is significantly different between SHE and BP6T cells (mid-late S phase, versus early S phase, resp.). This observed difference emphasizes the caution required in the study of mutagenesis and DNA replication using transformed, aneuploid cells under the assumption that the underlying mechanisms are the same for normal, diploid cells.
Animals↗
Correlation of in vitro growth properties and tumorigenicity of Syrian hamster cell lines.
Several in vitro phenotypic characteristics frequently associated with neoplastic cells were examined in a series of spontaneous and benzo(a)pyrene-induced Syrian hamster clonal cell lines which differed in their degree of tumorigenicity. Nonparametric statistical analysis demonstrated cloning efficiency in semisolid agar, enhanced fibrinolytic activity, decreased serum requirement for growth, decreased organization of intracellular actin, and increased cloning efficiency in liquid medium to be correlated with tumorigenicity. These correlations were not only qualitative but also quantitative. This suggests that the factors determining the degree of tumorigenicity of a cell can be cellular growth properties.
Actins↗
Butyric acid suppression of the in vitro neoplastic state of Syrian hamster cells.
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Animals↗
Regulation of requirements for anchorage-independent growth of Syrian hamster fibroblasts by somatic mutation.
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Adenine↗
Quantitation of fibrinolytic activity of Syrian hamster fibroblasts using 3H-labeled fibrinogen prepared by reductive alkylation.
Tritium-labeled fibrinogen with a specific activity of 2.0 X 10(7) cpm/mg was prepared by the method of reductive alkylation. The use of the 3H-fibrinogen as a substrate for detection of both intracellular and extracellular fibrinolytic activity derived from cultures of benzo(a)pyrene-transformed Syrian hamster cell lines was examined in cell-free assays, 3H-fibrinogen enabled reliable quantitation of the fibrinolytic activity associated with neoplastic cells. The elevated extracellular fibrinolytic activity in the transformed cell lines as compared to normal hamster embryo cultures was demonstrated with this substrate. The ease with which large quantities of 3H-fibrinogen of high specific activity and prolonged half-life can be prepared makes the use of this substrate an attractive alternative to 125I-labeled fibrinogen.
Alkylation↗
Temporal acquistion of enhanced fibrinolytic activity by syrian hamster embryo cells following treatment with benzo(a)pyrene.
Following treatment of Syrian hamster embryo cells with benzo(a)pyrene, the time required for the expression of enhanced fibrinolytic activity was examined. For this study, the fibrin-agarose overlay method was developed to distinguish the activity of normal and transformed colonies of hamster cells. Colonies possessing enhanced fibrinolytic activity were not observed one passage (2 weeks after treatment). Morphologically transformed colonies, which exhibited no enhanced fibrinolytic activity, were observed 8 days following treatment. In contrast to these two early changes, cells capable of growth in soft agar were observed much later (6 to 8 weeks after treatment). Untreated Syrian hamster embryo cells generally senesced and did not exhibit enhanced fibrinolytic activity. Approximately 1 of 10 untreated cultures escaped senescence and evolved as a continuous cell line; such cultures frequently exhibited enhanced fibrinolytic activity. These results suggest that the acquisition of enhanced fibrinolytic activity, while perhaps not a cause of neoplastic transformation, may reflect a loss of control of the normal function of the cellular genetic apparatus during the process of transformation.
Benzopyrenes↗