Ronald M. Humphrey

THE FATE OF PYRIMIDINE DIMERS IN THE DNA OF ULTRAVIOLET‐IRRADIATED CHINESE HAMSTER CELLS

Abstract—As an aid to understanding the relationship between dimer repair and cellular recovery, we have studied dimer removal and replication of dimer‐containing DNA in Chinese hamster ovary (CHO) cells irradiated with ultraviolet light (254 nm). These investigations demonstrated that (1) dimers are not excised as polynucleotides of less than 500,000 mol. wt, (2) fractionation of the ultraviolet dose does not enhance dimer excision, (3) dimer‐containing DNA is replicated in ultraviolet‐irradiated CHO cells, and (4) the dimers are conserved in the replicated DNA. These findings support the proposed mechanism of bypass of photoproducts during DNA replication in mammalian cells.

Deoxyribonucleic Acid Synthesis in Ultraviolet-Light-Irradiated Chinese Hamster Cells

Two mammalian cell lines, Chinese hamster ovary (CHO) which can recover colony-forming ability between fractionated doses of ultraviolet light (UV), and Chinese hamster B-14FAF28 which cannot recover, were tested for the ability to bypass UV-induced photoproducts in DNA during postirradiation DNA synthesis. The molecular weight distributions of newly synthesized DNA in UV-irradiated populations of both cell lines showed evidence for photoproduct bypass. Hence, the bypass mechanism does not correlate with recovery after UV.

Evaluation of S phase synchronization by analysis of DNA replication in 5-bromodeoxyuridine☆

Abstract The S phase kinetics have been evaluated in cells synchronized with either thymidine or hydroxyurea by direct analysis of the proportion of DNA semi-conservatively replicated as a function of time after release from the inhibitor. The proportion of DNA replicated was determined by growing the cells in medium containing 5-bromodeoxyuridine (BUdR) and subsequently measuring the amount of DNA that acquired increased buoyant density in CsCl gradients. The results confirm previous reports that substantial DNA synthesis occurs during TdR treatment. In contrast, HU provided a population of cells very nearly at the G 1-S interphase since 95 % of the DNA replicated synchronously after its removal. It is proposed that by measuring the rate and maximum extent of DNA replication with BUdR during S phase one can evaluate different synchrony methods for use in experiments designed to study aspects of semiconservative DNA replication.

COMPARISONS OF TRITIATED THYMIDINE, TRITIATED WATER, AND COBALT-60 GAMMA RAYS IN INDUCING CHROMOSOMAL ABERRATIONS.

Reports have indicated that ,-radiation from tritium-labeled thymidine (H3TdR) incorporated into deoxyribonucleic acid (DNA) inhibits the survival and growth of tissue culture cells and ascites tumor cells (1-4); produces cytological effects in chick embryos (5); causes an increase in polyploid cells in the rat liver (6); damages spermatogonia (4, 7, 8); causes chromosome aberrations in plants (9-11), leukocytes (12), and regenerating rat liver cells (13); induces tumors in mice (14); and produces both lethality and mutations in mice (15) and bacteria (16-18). Except for the bacterial work, most of these studies are not quantitative in terlms of the amount of intranuclear incorporation measured either as the number of tritium disintegrations in the cell or as the dose in rads required to cause the effects observed. The object of the present study is (1) to determine the relationship between the number of visible chromosomal aberrations and the number of tritium disintegrations in the DNA, and (2) to compare the radiation dose delivered from H3TdR required to produce a certain amount of chromosomal damage with the radiation dose from chronic fAand y-radiation required to produce the same amount of damage.

The response of Chinese hamster ovary cells to fast-neutron radiotherapy beams. II. Sublethal and potentially lethal damage recovery capabilities.

The recovery capabilities of mammalian cells in culture were investigated after irradiation with fast neutrons. Two different neutron beams (mean neutron energy 7 and 21 MeV) produced at the Texas A & M Variable Energy Cyclotron were used in these studies. The ability of fast-neutron-irradiated cells to recover from sublethal damage was tested in fractionated dose experiments. No recovery could be detected after irradiation with total doses of fast neutrons up to 300 rad. After a total dose of 500 rad, a recovery factor of about 1.5 was observed, which was considerably less than that observed when the cells were irradiated with60 Co γ rays to a comparable survival level. The recovery capabilities of cells were not significantly different for the two neutron beams. The ability of fast-neutron-irradiated cells to recover from potentially lethal damage was tested by irradiating cells in the plateau phase of growth and plating the cells either immediately after the irradiation or 8 hr later. No difference in ...

The Response of Chinese Hamster Ovary Cells to Fast Neutron Radiotherapy Beams: III. Variation in Relative Biological Effectiveness with Position in the Cell Cycle

The response of synchronized Chinese hamster cells to fast neutrons was investigated. Two different neutron beams (mean energies 7 and 21 MeV) produced at the Texas A & M University Variable Energy Cyclotron were used in these studies. The results showed that the variation in sensitivity of cells to neutrons as a function of position in the cell cycle was substantially reduced compared to the variation observed with60 Co γ rays. The degree of this reduction was similar for the two neutron beams studied. A detailed analysis of single-dose survival curves generated at radiation-sensitive and radiation-resistant positions in the cell cycle demonstrated that the relative biological effectiveness (RBE) of the sensitive moiety was much smaller than that of the resistant cells. For example, the RBE at the clinically relevant neutron dose of 75 rad was 4.7 for the mid- to late-S phase cells, 2.2 for the mitotic cells, and 1.7 for the G1/ S cells. Moreover, the

Effects of ultraviolet irradiation on the rate and sequence of DNA replication in synchronized Chinese hamster cells.

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Provisional assignment of TPI, GPI, and PEPD to Chinese hamster autosomes 8 and 9: a cytogenetic basis for functional haploidy of an autosomal linkage group in CHO cells

of the sensitive moiety was do...

Transformation of UV-hypersensitive Chinese hamster ovary cell mutants with UV-irradiated plasmids.

The effects of ultraviolet light (UV) irradiation on the rate of DNA replication in synchronized Chinese hamster ovary (CHO) cells were investigated. A technique for measuring semiconservative DNA replication was employed that involved growing the cells in medium containing 5-bromodeoxyuridine and subsequently determining the amount of DNA that acquired hybrid buoyant density in CsCl density gradients. One of the advantages of this technique was that it allowed a characterization of the extent of DNA replication as well as rate after irradiation. It was found that while there was a dose-dependent reduction in the rate of DNA replication following UV-irradiation, doses of up to 10 J/m2 (which produce many dimers per replication) did not prevent the ultimate replication of the entire genome. Hence, we conclude that dimers cannot be absolute blocks to DNA replication. In order to account for the total genome replication observed, a mechanism must exist that allows genome replication between dimers. The degree of reduction in the rate of replication by UV was the same whether the cells were irradiated at the G1-S boundary or 1 h into S-phase. Previous work had shown that cells in early S-phase are considerably more sensitive to UV than cells at the G1-S boundary. Experiments specifically designed to test for reiterative replication showed that UV does not induce a second round of DNA replication within the same S-phase.

The effects of ionizing radiation on the kinetics of DNA replication in synchronized Chinese hamster ovary cells.

Concordant segregation analysis of Chinese hamster (Cricetulus griseus) isozymes and chromosomes segregating from interspecific somatic cell hybrids made with mouse C11D cells revealed the locations of GPI and PEPD on chromosome 9 and TPI on chromosome 8 in both euploid Chinese hamster and CHO cells. The patterns of electrophoretically detectable shift mutants of these loci in CHO cells were consistent with the observed presence of two normally banded chromosome 8s and monosomy for chromosome 9. These findings and the isolation of three independent, null PEPD mutants in only 527 ethyl methansulfonate-exposed clones indicate that the high frequency of recovery of recessive drug resistant mutants in CHO cells may be due not only to haploidy caused by deletions and monosomy but also by great sensitivity of certain loci to particular mutagens.