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Biomedical subjects

R E Meyn

Publications and source records attributed to R E Meyn.

At least 109 records · Page 6Linked to original sources
PubMed1984

Cell synchrony techniques. I. A comparison of methods.

Selected cell synchrony techniques, as applied to asynchronous populations of Chinese hamster ovary (CHO) cells, have been compared. Aliquots from the same culture of exponentially growing cells were synchronized using mitotic selection, mitotic selection and hydroxyurea block, centrifugal elutriation, or an EPICS V cell sorter. Sorting of cells was achieved after staining cells with Hoechst 33258. After synchronization by the various methods the relative distribution of cells in G1, S, or G2 + M phases of the cell cycle was determined by flow cytometry. Fractions of synchronized cells obtained from each method were replated and allowed to progress through a second cell cycle. Mitotic selection gave rise to relatively pure and unperturbed early G1 phase cells. While cell synchrony rapidly dispersed with time, cells progressed through the cell cycle in 12 hr. Sorting with the EPICS V on the modal G1 peak yielded a relatively pure but heterogeneous G1 population (i.e. early to late G1). Again, synchrony dispersed with time, but cell-cycle progression required 14 hr. With centrifugal elutriation, several different cell populations synchronized throughout the cell cycle could be rapidly obtained with a purity comparable to mitotic selection and cell sorting. It was concluded that, either alone or in combination with blocking agents such as hydroxyurea, elutriation and mitotic selection were both excellent methods for synchronizing CHO cells. Cell sorting exhibited limitations in sample size and time required for synchronizing CHO cells. Its major advantage would be its ability to isolate cell populations unique with respect to selected cellular parameters.

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PubMed1984

Cell synchrony techniques. II. Analysis of cell progression data.

CHO cells which have been sorted by mitotic detachment, centrifugal elutriation and fluorescence activated cell sorting have been followed for up to 14 hr by flow cytometry to examine their progression characteristics. Mathematical modelling techniques were used to provide quantitative estimates of the cell-cycle parameters. Mitotic detachment gives an 11.2-hr cycle time with mean transit times TG1, TS and TG2M equal to 3.2, 5.6 and 2.4 respectively. Cells prepared by central elutriation in an early G1 state have a 14-hr cycle time with TG1, TS and TG2M of 5.7, 6.0 and 2.3 hr. Populations prepared by centrifugal elutriation enriched in early S and late S and G2M have transit times of 2.7, 5.9 and 1.6 hr and 4.9, 6.7 and 2.1 hr with cycle times of 11.2 and 13.2 hr respectively. Cell sorting for a G1 population gives transit times of 9.8, 8.0 and 3.6 for an overall 21.4-hr cycle time.

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PubMed1984

6-Thioguanine-induced DNA damage as a determinant of cytotoxicity in cultured Chinese hamster ovary cells.

The mechanism of action of 6-thioguanine (TG) has been examined in cultured Chinese hamster ovary cells by direct measurement of the incorporation of the compound into DNA and by analysis of the resulting DNA damage. The predominant lesions as monitored by alkaline elution were DNA strand breaks. Very few, if any, interstrand or DNA-protein cross-links could be definitively observed. The cytotoxicity of TG as measured by colony-forming ability appeared closely related with its incorporation into DNA and the DNA strand scission events. As TG concentrations were increased, cytotoxicity, DNA incorporation, and strand scission reached a plateau; this result is consistent with earlier reports that TG produces a reversible block of DNA synthesis. Strand breaks appeared to be related to the incorporation of TG into DNA, since the addition of 1 microM cycloheximide during a 24-hr treatment with 3 microM TG prevented the cytotoxicity, prevented incorporation of TG into DNA, and eliminated the strand breaks. Alkali-labile sites were detected in the DNA of TG-treated cells by alkaline elution at pH 12.8, suggesting that depurination of TG residues by a glycosylase mechanism may occur. It is also postulated that TG residues are recognizable by the long-patch repair system, since UV-sensitive cells deficient for long-patch repair were more sensitive to TG than were wildtype cells. Furthermore, caffeine (1 mM) was shown to enhance the lethality of TG (3 microM), as monitored by colony formation, without altering levels of TG incorporation into DNA or the strand scission as measured immediately after treatment. This result, coupled with the known delayed cytotoxic response of TG, suggests that gaps may occur in newly synthesized DNA opposite TG residues and that the repair of these gaps by a postreplication repair mechanism is inhibited by caffeine.

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PubMed1984

Neocarzinostatin-mediated DNA damage and repair in wild-type and repair-deficient Chinese hamster ovary cells.

The formation and repair of neocarzinostatin (NCS)-mediated DNA damage were examined in two strains of Chinese hamster ovary cells. The response in strain EM9, a mutant line selected for its sensitivity to ethyl methanesulfonate and shown to have a defect in the repair of X-ray-induced DNA breaks, was compared with that observed in the parental strain (AA8). The DNA strand breaks and their subsequent rejoining were measured using the method of elution of DNA from filters under either alkaline (for single-strand breaks), or nondenaturing conditions (for double-strand breaks). Colony survival assays showed that the mutant was more sensitive to the action of NCS than was the parental strain by a factor of approximately 1.5. Elution analyses showed that the DNA from both strains was damaged by NCS; the mutant displayed more damage than the parent under the same treatment conditions. Single-strand breaks were produced with a frequency of about 10 to 15 times the frequency of double-strand breaks. Both strains were able to rejoin both single-strand breaks and double-strand breaks induced by NCS treatment. The strand break data suggest that the difference in NCS-mediated cytotoxicity between EM9 and AA8 cells may be directly related to the enhanced production of DNA strand breaks in EM9. However, the fact that much higher doses of NCS were required in the DNA studies compared to the colony survival assays implies that either a small number of DNA breaks occur in a critical region of the genome, or that lesions other than DNA strand breaks are partly responsible for the observed cytotoxicity.

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PubMed1984

The efficiency of DNA strand-break repair in two fibrosarcoma tumors and in normal tissues of mice irradiated in vivo with X rays.

We have used alkaline elution to study the repair of X-ray-induced DNA strand breaks in vivo in two fibrosarcoma tumors and in several normal mouse tissues after whole-body irradiation of mice with 10-12.5 Gy of X rays. Both tumors were found to repair damage significantly faster and to a greater extent than any of the normal tissues, so that by 2 hr after irradiation the level of damage in both tumors was indistinguishable from unirradiated control values. Of the normal tissues studied, liver repaired the fastest. The kinetics for the other normal tissues were essentially the same, showing an appreciable level (7-16%) of unrepaired lesions still evident after 2 hr. Even as late as 12 hr there was a significant amount of residual damage in some tissues, with testes and spleen showing the greatest level (ca. 15%). The repair kinetics for each tissue were not appropriately described by a sum of two exponentials. In contrast, previously reported data for many homogeneous mammalian cell systems in vitro and for some tissues in vivo have shown biphasic repair kinetics. This difference may be related to heterogeneity of both cell type and environment within the tissue populations used in the investigation. The faster repair of DNA strand breaks by tumor cells relative to cells from normal tissues was not readily explainable in terms of such radiobiological parameters as overall tissue oxygenation or sulfhydryl content. Rather, it appears that the degree of differentiation of the cells within the tissue population may be a major determinant of repair proficiency. Based on a model incorporating a competition between repair and fixation of sublethal lesions, these data are consistent with the idea that tumor cells may have a repair, and hence survival, advantage over normal cells in response to ionizing radiation.

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PubMed1984

Enhancement of the DNA cross-linking activity of nitrogen mustard by misonidazole and diethyl maleate in a mouse fibrosarcoma tumor in vivo.

We have used the technique of alkaline elution to determine the amount of nitrogen mustard (HN2)-induced DNA cross-linking in a murine fibrosarcoma tumor in vivo. Mice were either treated with HN2 directly or were pretreated with misonidazole (MISO) or diethyl maleate prior to injection with HN2. Two types of HN2-induced DNA lesions were detected, namely, proteinase K-sensitive and -resistant cross-links. Pretreatment with MISO did not appear to affect the ratio of the two types of lesion. In mice treated with HN2 alone, the amount of cross-linking reached a high level by 0.5 hr postinjection, after which these lesions were repaired, 62% of cross-links being removed between 0.5 hr and 6 hr postinjection. Pretreatment of mice with MISO resulted in substantial alterations in both the magnitude and time course of cross-linking during the first few hr after injection of HN2. Both MISO and diethyl maleate enhanced the number of cross-links formed at 0.5 hr postinjection. Furthermore, in MISO-pretreated mice, only 18% of the cross-links present at 0.5 hr had been removed by 6 hr postinjection. This early enhancement is possibly related to glutathione depletion resulting in reduced intracellular inactivation of HN2. Since repair processes were determined not to be saturated at the level of lesions under study, these data suggest that, in addition to the initial glutathione depletion resulting in an increased burden of damage, MISO may also inhibit DNA repair processes, possibly via a hypoxia-dependent interaction between MISO reduction products and DNA or repair enzymes. Assuming that DNA cross-linking is related to the cytotoxicity of HN2, these effects may account for the MISO enhancement of HN2 toxicity toward various biological systems which have been reported previously. It appears that chemosensitization may result from a variety of factors, with the relative importance of each factor depending on the particular drug being used.

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PubMed1983

Relationship between cytotoxicity and DNA damage in mammalian cells treated with anthracenedione derivatives.

The effects of two anthracenedione derivatives on in vitro cell survival and DNA of Chinese hamster ovary (CHO) cells were investigated. The two drugs studied were 1,4-dihydroxy-5,8-bis-(2-[2-hydroxyethyl)amino)ethylamino)-9,10-anthracenedione (DHAQ, NSC No. 279836) and 1,4-bis-(2-[2-hydroxyethyl)-amino)ethylamino)-9,10-anthracenedione (HAQ, NSC No. 287513). DHAQ was 100-fold more potent in reducing cell survival than HAQ. DNA strand breaks were assayed by alkaline elution. DHAQ (10 ng/ml) caused more strand breakage than 1000 ng/ml HAQ. This difference correlates well with their differences in ability to kill cells.

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PubMed1983

Enhancement of the DNA cross-linking activity of melphalan by misonidazole in vivo.

The technique of alkaline elution has been adapted for the study of drug-induced DNA cross-link formation in vivo. Pretreatment with misonidazole (MISO) enhances the number of cross-links formed in a fibrosarcoma and in the spleen and gut of mice for periods up to 48 h following a single injection of melphalan (MEL). The tumour was sensitized by a greater factor (2.05) than either of the normal tissues (enhancement factor 1.4-1.5). This enhancement did not appear to be related to inhibition of the repair of actual cross-links. Rather, the effect was explicable in terms of one of two alternative models. Firstly, MISO pretreatment could result in a greater amount of binding of MEL to DNA at early times after injection. This may be the result of altered pharmacokinetics of MEL, or of enhanced intracellular uptake of MEL due to MISO pretreatment. Secondly, MISO may exert its affect by inhibition of the repair of cross-links or monoadducts at early times post-injection, which would not be observed in this study. The possible involvement of glutathione depletion in chemosensitization by MISO was investigated by comparison with the effect of diethyl maleate (DEM), a known thiol-depleting reagent. Glutathione depletion, while perhaps being important, could not account for all of the effects observed.

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PubMed1983

Hyperthermic potentiation of unrejoined DNA strand breaks following irradiation.

Previous reports have suggested that the potentiation of cellular radiation sensitivity by hyperthermia may be due to its inhibition of the repair of single-strand breaks in DNA. Such inhibition could result in increased numbers of unrejoined breaks at long times following irradiation, lesions that are presumed to be lethal to the cell. As a test of this hypothesis, the amounts of residual strand-break damage in cells following combined hyperthermia and ionizing radiation were measured. The results show that hyperthermia does significantly enhance the relative number of unrejoined strand breaks as measured by the technique of alkaline elution and that the degree of enhancement is dependent on both the temperature and duration of the hyperthermia treatment. For example, compared to unheated cells, the proportion of unrejoined breaks measured 8 hr after irradiation was increased by a factor of 1.5 in cells that were treated for 30 min at 43 degrees C, by a factor of 6 for cells treated for 30 min at 45 degrees C, and by a factor of 4 for cells treated at 43 degrees C for 2 hr. In experiments in which the sequence of heat and irradiation were varied, a high degree of correlation was observed between the resulting level of cell killing and the relative numbers of unrejoined strand breaks. The greatest effects on both of these parameters were observed in those protocols in which the irradiation was delivered either during, just before, or just after the heat treatment.

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PubMed1983

Variation in normal and tumor tissue sensitivity of mice to ionizing radiation-induced DNA strand breaks in vivo.

The efficiency of DNA strand break formation in normal and tumor tissues of mice was measured using the technique of alkaline elution coupled with a microfluorometric determination of DNA. This methodology allowed measurement of the DNA strand breaks produced in tissues irradiated in vivo with doses of radiation comparable to those used in radiotherapy (i.e., 1.0 gray) without the necessity for the cells to be dividing and incorporating radioactive precursors to label the DNA. The results showed that substantial differences existed among various tissues in terms of the amount of DNA strand break damage produced for a given dose of radiation. Of the normal tissues, the most breaks were produced in bone marrow and the least were produced in gut. Furthermore, strand break production was relatively inefficient in the tumor compared to the normal tissues. The efficiency of DNA strand break formation measured in the cells from the tissues irradiated in vitro was much more uniform and considerably greater than that measured in vivo, suggesting that the normal tissues in the animal may be radiobiologically hypoxic.

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PubMed1982

Defective removal of DNA cross-links in a repair-deficient mutant of Chinese hamster cells.

To further understand the relationships between DNA damage, DNA repair, and cellular end points such as survival and mutation, the repair capacity of a DNA repair-deficient mutant (strain UV-20) of Chinese hamster ovary cells was characterized in response to DNA cross-linking agents. This mutant, previously shown to be hypersensitive to killing by both ultraviolet light and the cross-linking agent mitomycin C, was also found to be extremely sensitive to cis-diamminedichloroplatinum, another DNA cross-linking agent. The efficiency of DNA cross-link removal after treatment with mitomycin C or cis-diamminedichloroplatinum was measured using the technique of alkaline elution and compared in wild-type Chinese hamster ovary cells and strain UV-20. Wild-type cells removed 80 or 95% of the cross-links within 24 hr after treatment with cis-diamminedichloroplatinum or mitomycin C, respectively. In contrast, UV-20 cells, which were equally as susceptible to cross-link damage as were wild-type cells, removed only a small proportion of the cross-links made by either agent. These results emphasize the importance of DNA repair processes in modulating the cytotoxic effects of chemicals that produce DNA cross-link damage and suggest that cross-link repair in Chinese hamster ovary cells is controlled by a pathway that also repairs damage from ultraviolet radiation.

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PubMed1981

Recovery of Chinese hamster cells from mercuric chloride exposure.

The effects of chronic treatments with HgCl2 on cell survival, DNA replication, and cell progression in cultured Chinese hamster ovary cells were investigated. The ability of these cells to recover from the effects was also characterized. Exposure of cells to 4 X 10(-5) M HgCl2 for 30 min killed about 50% of the cells, and this proportion did not increase with continued exposure up to 24 h. The rate of DNA replication was reduced to 28% of the control rate in the presence of HgCl2. However, when the cells were returned to medium without HgCl2, the rate of DNA replication recovered to 88% of control after 3 h of exposure and 55% of control after 8 h of exposure. The cell doubling time was increased from a control value of 16 h to 31 h in the presence of HgCl2. When the exposed cells were returned to medium without HgCl2, the doubling time returned to 16 h. The rate of progression of cells from G1 phase to S phase was greatly reduced in the presence of HgCl2, and no recovery was observed in this case when the cells were transferred to normal medium. These findings suggested a correlation between ability to recover and the cytotoxic effects of HgCl2.

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PubMed1980

Cycle-dependent anticancer drug cytotoxicity in mammalian cells synchronized by centrifugal elutriation.

The cycle-dependent cytotoxicity of seven chemotherapy agents was compared with the use of subpopulations of Chinese hamster ovary cells separated into the various phases of the cell cycle by centrifugal elutriation. The proportion of cells killed by either beta-cytosine arabinoside or hydroxyurea agreed well with the proportion of S-phase cells in the treated subpopulations. Cell-cycle survival patterns were also determined for bleomycin (BLM), adriamycin (ADR), cis-diamminedichloroplatinum (II) (cis-DDP), Asaley, and Yoshi 864. Three of the agents (cis-DDP, Asaley, and Yoshi 864) had similar cell-cycle survival patterns in that they all preferentially killed cells in the G1 phase. In contrast, BLM preferentially killed cells in G2+M phase, whereas ADR was most cytotoxic to cells in middle to late S-phase. This investigation demonstrated that separation of cultured mammalian cells into the various cell-cycle phases by centrifugal elutriation allows the rapid determination of the phase-dependent cytotoxic effects exerted by chemotherapy agents.

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PubMed1980

Thermal enhancement of DNA damage in mammalian cells treated with cis-diamminedichloroplatinum(II).

The cytotoxic effects of cis-diamminedichloroplatinum(II) (cis-DDP) were shown to be strongly potentiated by hyperthermia. The molecular mechanisms responsible for this potentiation were investigated by assaying the degree of DNA cross-linking produced under the different drug treatment conditions by the technique of alkaline elution. The results showed that the cells treated with the drug at 43 degrees had a greater amount of DNA cross-linking immediately after treatment than did cells treated with the drug at 37 degrees, indicating a possible thermal enhancement of drug uptake by the cells. Whereas the hyperthermia potentiated the cytotoxicity of cis-DDP by a factor of nearly 10, the degree of DNA cross-linking was only enhanced by a factor of 6.5, suggesting that while a large portion of the enhanced cytotoxicity may be attributed to the increased cross-linking other factors may also play some role. The possible influence of hyperthermia on the repair of the DNA damage induced by cis-DDP was investigated; however, no significant differnence in the rate of disappearance of cross-links between cells treated at 37 or 43 degrees was observed. Advantageous combination of chemotherapy with cis-DDP and hyperthemia for the treatment of cancer is implied by these results.

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