Search PubMed⌕ Search

Biomedical subjects

W F Morgan

Publications and source records attributed to W F Morgan.

At least 73 records · Page 4Linked to original sources
PubMed1990

Chromosome aberration induction in Chinese hamster ovary cells by restriction enzymes with different methylation sensitivity.

The isoschizomer pair MspI and HpaII were used to investigate whether the putative specificity of restriction endonucleases would be maintained when they were introduced into mammalian cells. Although both enzymes recognize the sequence CCGG, HpaII will cut only if the internal cytosine is unmethylated, whereas MspI will cut regardless of the methylation status. Cleavage results in a cohesive-end DNA double-strand break, which can lead to the formation of chromosome aberrations. Since mammalian DNA is heavily methylated, one would expect MspI to be much more effective than HpaII at inducing chromosome aberrations in Chinese hamster ovary cells. In fact, during G1, MspI induced a greater than 90-fold higher number of aberrations than did HpaII. Cell cycle studies indicated that during early S there was a 30-fold increase in HpaII-induced aberrations. This increase may be due to increased accessibility of replicating hypomethylated DNA. Cells that were treated with the demethylating agent 5-aza-2'-deoxycytidine (AzdC) displayed only a moderate increase in HpaII-induced aberrations during G1. This observation, together with the results of restriction enzyme analysis of genomic DNA, indicated that demethylation was incomplete. The effects of AzdC on the induction of aberrations by MspI suggested that AzdC increases chromatin accessibility. Our results were consistent with the expected specificity of MspI and HpaII. Thus, it appears that restriction endonucleases can play a useful role in determining the biological consequences of DNA double-strand breaks.

Animals↗
PubMed1989

Increased chromosomal radiosensitivity of a Chinese hamster ovary cell line that inducibly expresses the Eco RI restriction endonuclease.

We have transfected a Chinese hamster ovary cell line (CHO 6) with a plasmid that inducibly expresses the Eco RI restriction endonuclease gene in the presence of cadmium sulfate (CdSO4). Expression of Eco RI results in DNA double-strand breaks, which can lead to chromosome aberrations. The new line, designated CHO 10, also has a low level of constitutive expression of Eco RI in the absence of CdSO4 without any cytogenetic effect. This suggested that these cells may be efficient at repairing low levels of DNA double-strand breaks. To test this, both cell lines were exposed to ionizing radiation, and aberration yields were analyzed with or without induction of Eco RI. CHO 10 cells showed increased radiosensitivity after G1 irradiation, but after G2 exposure, only doses greater than or equal to 0.4 Gy caused more damage in CHO 10 cells. We conclude that CHO 10 cells can tolerate constitutive expression of Eco RI, but that when the cells are subjected to additional stress, in this case ionizing radiation, they become very sensitive to DNA double-strand breaks.

Animals↗
PubMed1989

Quiescent human lymphocytes do not contain DNA strand breaks detectable by alkaline elution.

On the basis of qualitative assays, quiescent lymphocytes have previously been reported to have numerous DNA strand breaks, which are thought to be repaired after mitogenic stimulation by a process associated with poly(ADP-ribosyl)ation. Using alkaline elution, a very sensitive assay for quantifying DNA single-strand breakage, we found no evidence for a high frequency of DNA strand breaks in unstimulated human peripheral blood lymphocytes. No differences in elution profiles were observed between unstimulated lymphocytes and lymphocytes 4 or 48 h after addition of the mitogen phytohemagglutinin (PHA). Furthermore, addition of 3-aminobenzamide (3AB), an inhibitor of poly(ADP-ribose) synthetase, or aphidicolin, an inhibitor of DNA polymerase alpha, did not increase the amount of DNA eluting from the filter after PHA stimulation. In contrast to reported studies of mouse splenic lymphocytes, we found that human lymphocytes were able to replicate and divide in the presence of the ADP-ribosylation inhibitor. Human lymphocytes were also capable of proliferating in nicotinamide-free medium, with or without 3AB, indicating that ADP-ribosylation is not a requirement for lymphocyte differentiation. We therefore consider it unlikely that peripheral human lymphocytes contain significant numbers of strand breaks that play any role in their stimulation or differentiation in response to PHA.

Aphidicolin↗
PubMed1989

Measurement of sister chromatid exchanges and their relationship to DNA damage, repair and cell killing.

We have concentrated on how SCE is measured, how it correlates with other measures of cellular damage, and how SCE can be used as a tool to study cell cycle kinetics. We have not discussed the biological significance of SCEs, i.e. why they occur spontaneously, and what role, if any, in cellular preservation they play. Similarly, our discussion of the mechanisms of formation of SCE has been deliberately brief. These are important considerations, but the answers to them are still largely only speculative. What is clear is that SCE is an extremely interesting biological phenomenon from a variety of points of view, and even though the mechanisms underlying SCE are not yet well understood, measurement of these events has produced much useful and provocative information.

Animals↗
PubMed1989

Restriction endonucleases do not induce sister-chromatid exchanges in Chinese hamster ovary cells.

Bacterial restriction enzymes offer the unique opportunity to determine the biological and cytogenetic consequences of DNA double-strand breakage. To examine the role of various types of breaks in sister-chromatid exchange (SCE) formation, we used restriction enzymes with different recognition sequences and different cutting frequencies to generate DNA double-strand breaks in Chinese hamster ovary (CHO) cells. The restriction enzymes were introduced by electroporation into exponentially growing cells during the second replication cycle in bromodeoxyuridine, and SCEs were analyzed at mitosis. Contrary to results reported by others, we found no increase in SCE frequency in cells exposed to restriction enzymes despite the presence of numerous cells with chromatid aberrations. These data suggest that DNA double-strand breaks do not lead to SCE formation.

Animals↗
PubMed1988

Nuclear fragmentation and premature chromosome condensation induced by heat shock in S-phase Chinese hamster ovary cells.

Beginning in early S phase, synchronized Chinese hamster ovary cells were heated for 12 h at 41.5 degrees C, during which time the cells progressed through S, G2, and into mitosis. Upon return to 37 degrees C, some of the interphase cells developed a fragmented nuclear morphology, and the fraction of cells exhibiting this effect reached 40% after entering the first mitosis. About 50% of the mitotic cells accumulated with Colcemid at the second mitosis exhibited premature chromosome condensation (metaphase-associated PCC) in an amount equal to approximately one to two chromosomes per diploid cell. There was also a low frequency of spontaneous S and G2 PCC (0.5 to 1.5% in controls); this distinctly different type of PCC was observed in heated interphase cells at a frequency of 2.0 to 7.0% before the first wave of entry into mitosis. Heated S-phase cells exhibited a chromosome break frequency of about 0.9 breaks per cell when analyzed at the first mitosis. This value agrees well with the observed surviving fraction of 40%, if we assume that one break per cell is a lethal event, and that chromosome damage has a Poisson frequency distribution in the cell population; however, this correlation may not be valid for tumor cell populations. Between the first and second mitoses, an increase in hyperthermia-induced aneuploidy was observed which was apparently due to unequal division, since mitotic cells containing PCC were excluded from the chromosome number frequency analyses. Although this induction of aneuploidy was observed independently of PCC, we conclude that abnormal division is necessary for both effects, and both PCC and unequal division result in an increase in aneuploidy. Previous studies using transmission electron microscopy demonstrated a precocious reformation of the nuclear membrane around metaphase chromosomes in Chinese hamster ovary cells heated at this temperature (R. A. Coss et al., Cancer Res., 39: 1911-1918, 1979). We suggest that such heat-induced changes in nuclear envelope formation lead to nuclear fragmentation and ultimately result in metaphase-associated PCC.

Animals↗
PubMed1988

Mutagenesis.

Explore the source record for details and available documents.

Benzamides↗
PubMed1988

Inducible expression and cytogenetic effects of the EcoRI restriction endonuclease in Chinese hamster ovary cells.

The cytogenetic endpoints sister chromatid exchange (SCE) and chromosome aberrations are widely used as indicators of DNA damage induced by mutagenic carcinogens. Chromosome aberrations appear to result directly from DNA double-strand breaks, but the lesion(s) giving rise to SCE formation remains unknown. Most compounds that induce SCEs induce a spectrum of lesions in DNA. To investigate the role of double-strand breakage in SCE formation, we constructed a plasmid that gives rise to one specific lesion, a staggered-end ("cohesive") DNA double-strand break. This plasmid, designated pMENs, contains a selectable marker, neo, which is a bacterial gene for neomycin resistance, and the coding sequence for the bacterial restriction endonuclease EcoRI attached to the mouse metallothionein gene promoter. EcoRI recognizes G decreases AATTC sequences in DNA and makes DNA double-strand breaks with four nucleotides overhanging as staggered ends. Cells transfected with pMENS were resistant to the antibiotic G418 and contained an integrated copy of the EcoRI gene, detectable by DNA filter hybridization. The addition of the heavy metal CdSO4 resulted in the intracellular production of EcoRI, as measured by an anti-EcoRI antibody. Cytogenetic analysis after the addition of CdSO4 indicated a dramatic increase in the frequency of chromosome aberrations but very little effect on SCE frequency. Although there was some intercellular heterogeneity, these results confirm that DNA double-strand breaks do result in chromosome aberrations but that these breaks are not sufficient to give rise to SCE formation.

Animals↗
PubMed1987

Cell cycle-dependent potentiation of X-ray-induced chromosomal aberrations by 3-aminobenzamide.

The poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide had dramatically different effects on X-ray-induced cytogenetic damage in human lymphocytes depending on the stage of the cell cycle in which cells were irradiated. 3-Aminobenzamide (0.08-3.00 mM) potentiated the frequency of chromosomal aberrations when lymphocytes were irradiated in G1, S, or late G2. No effect was observed, however, when lymphocytes were irradiated in G0 or at the S/G2 boundary 6 h before termination of culture. These results indicate that poly(ADP-ribose) polymerase may be involved in chromosomal repair of radiation damage only during specific stages of the cell cycle.

Benzamides↗
PubMed1987

The role of acentric chromosome fragments in gene amplification.

We assessed the role of acentric chromosome fragments in gene amplification by using cell fusion techniques to introduce the fragmented chromosomes of a donor Chinese hamster ovary (CHO) cell line that contained the dihydrofolate reductase (dhfr) gene(s) into a CHO cell line deficient for dhfr. Chromosome fragments were successfully integrated into cells at a frequency of approximately 3%. Methotrexate-resistant variants arose much more frequently in two cell lines derived from these successful cell fusions than in wild-type CHO cells. The hybrid cell lines also amplified their dhfr genes more readily than did the CHO cell line used as dhfr donor.

Animals↗
PubMed1987

3-Aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase, is a stimulator, not an inhibitor, of DNA repair.

An inhibitor of poly(ADP-ribose) synthesis, 3-aminobenzamide (3AB), at low concentrations (0.01-0.1 mM) was found to reduce strand-break frequencies and increase repair replication in human lymphoid cells damaged by methyl methanesulfonate. A concentration of 0.1 mM 3AB was adequate to produce a maximum effect on strand-break frequencies and repair replication. This evidence, together with our previous measurements, demonstrates that 3AB cannot be regarded as an inhibitor of DNA repair; rather, it actually accelerates the ligation of DNA repair patches. Previous considerations of 3AB as a repair inhibitor may have derived from the use of excessive concentrations above 1 mM that may have stimulated additional damage and from the use of ethyl alcohol as a solvent for 3AB. Interpretations of the role of single-strand breaks and poly(ADP-ribose) in DNA repair, differentiation, and gene activity may need reevaluation because they have frequently been based on an erroneous notion of 3AB as a repair inhibitor, when its mode of action is, in fact, more complex.

Benzamides↗
PubMed1987

Induced sister chromatid exchange frequency is not increased in homogeneously staining regions that contain amplified genes.

Gene amplification is a process by which cells become resistant to selective agents by increasing gene copy number and overproducing specific enzymes. The molecular mechanism by which gene amplification occurs is unknown, but unequal sister chromatid exchange (SCE) has been suggested as one possibility. Unequal SCE results in one chromatid containing an extra copy of a selected gene that is deleted in the sister chromatid. Two predictions of the unequal SCE model are that agents that increase SCE frequency would increase gene amplification, and that SCE would be more prevalent in arrays of amplified units. We examined SCE frequency in the Chinese hamster ovary cell line MK42, which contains amplified dihydrofolate reductase genes that are stably integrated as a homogeneously staining region in chromosome #2. Under treatment conditions known to increase amplification of the dihydrofolate reductase gene, a number of agents did increase SCE frequency in MK42 cells. The frequency of SCE in the amplified region of chromosome #2, however, was no different from that expected if SCE were induced randomly as a function of chromosome length. These data, therefore, do not support the prediction of unequal SCE as a model for gene amplification. Our experiments, however, do not test the idea that unequal SCE at normal frequencies may lead to gene amplification, nor do they rule out the possibility of transient periods early in amplification, during which the homogeneously staining region may be a "hot spot" for SCE.

Animals↗
PubMed1987

Effect of exogenous thymidine on sister-chromatid exchange frequency in Chinese hamster ovary cells with bromodeoxyuridine- and chlorodeoxyuridine-substituted chromosomes.

There are conflicting reports on the effect of exogenous thymidine (dThd) on the frequency of sister-chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells. Thymidine has been reported either to increase or to have no effect on SCE frequency under similar experimental conditions. To resolve this controversy, we have carried out a series of experiments to examine the effect of dThd on CHO cells cultured with 5-bromodeoxyuridine (BrdUrd). In addition, we have examined the effect of dThd on CHO cells cultured with 5-chlorodeoxyuridine (CldUrd), a much more potent inducer of SCEs than BrdUrd. The addition of 100 microM dThd to the culture medium caused a consistent decrease in the yield of SCEs in cells grown in BrdUrd for two cell cycles. The decrease was even greater when cells were grown in dThd and CldUrd. Analysis of twin and single SCEs indicated that dThd must be present during the first cell cycle to reduce the frequency of SCEs. Because excess dThd is thought to have an effect when DNA replicates on a template substituted with a halogenated nucleoside, dThd at concentrations from 100 microM to 9 mM was added to cultures for the second cell cycle after a first cell cycle in BrdUrd. In this experiment, the presence of dThd increased SCE frequency in a dose-dependent manner. The results suggest that if dThd competes with halogenated nucleosides and thus decreases their incorporation into DNA, SCEs are suppressed in the subsequent cell cycle, whereas if excess dThd creates a dNTP pool imbalance, SCEs can be increased.

Animals↗
PubMed1987

Differences in the stimulation of repair replication by 3-aminobenzamide in lymphoblastoid cells damaged by methylmethanesulfonate or ultraviolet light.

Human lymphoblastoid cells damaged by u.v. light accumulated DNA breaks in the presence of cytosine arabinoside and hydroxyurea at a frequency similar to that of cells damaged by methylmethanesulfonate. 3-Aminobenzamide (1 mM) reduced the net strand-break frequency detected after either kind of damage. Repair replication, however, was stimulated only in methylmethanesulfonate-damaged cells. This stimulation is therefore not related directly to the DNA strand-break frequencies and concomitant poly(ADP-ribose) synthesis, but depends on some other cellular response specific to alkylating agents.

Benzamides↗
PubMed1987

Unique DNA repair properties of a xeroderma pigmentosum revertant.

A group A xeroderma pigmentosum revertant with normal sensitivity was created by chemical mutagenesis. It repaired (6-4) photoproducts normally but not pyrimidine dimers and had near normal levels of repair replication, sister chromatid exchange, and mutagenesis from UV light. The rate of UV-induced mutation in a shuttle vector, however, was as high as the rate in the parental xeroderma pigmentosum cell line.

4-Nitroquinoline-1-oxide↗
PubMed1986

Potentiation of sister chromatid exchange by 3-aminobenzamide is not modulated by topoisomerases or proteases.

Poly(ADP-ribose) is synthesized in response to DNA strand breaks and covalently modifies numerous intracellular proteins. We have proposed that this modification regulates, i.e., inhibits, the activity of these enzymes, e.g., topoisomerases and proteases, which could otherwise cause additional DNA damage or alterations in chromatin structure. Inhibition of poly(ADP-ribose) polymerase by 3-amino-benzamide (3AB) in cells exposed to DNA-damaging agents would, according to this proposal, eliminate the regulatory role of ADP-ribosylation. When Chinese hamster ovary cells are cultured with methyl methanesulfonate (MMS) and 3AB, a synergistic increase in sister chromatid exchange frequency is observed. We investigated the regulatory role of poly(ADP-ribose) polymerase to see if topoisomerases or proteases are involved in this synergistic increase. Cells were exposed to MMS or the intercalating agent 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), 3AB, and either the topoisomerase inhibitor novobiocin or the protease inhibitor antipain. Neither novobiocin nor antipain affected the synergistic response of MMS and 3AB or the additive response of m-AMSA and 3AB. These results suggest that topoisomerases or proteases do not account for the effect of 3AB on sister chromatid exchange frequency after DNA damage.

Aminoacridines↗