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R E Durand

Publications and source records attributed to R E Durand.

At least 19 recordsLinked to original sources
PubMed1992

Detection of hypoxic cells in a murine tumor with the use of the comet assay.

BACKGROUND: Hypoxic cells within solid tumors are likely to limit tumor curability by radiation therapy and some chemotherapeutic agents. PURPOSE: To quantify a hypoxic fraction in solid tumors, we developed a method which measures radiation-induced DNA single-strand breaks in individual tumor cells and makes use of the fact that three times more strand breaks are produced in aerobic than in hypoxic cells. METHODS: Immediately after irradiation with doses of 4-20 Gy, SCCVII squamous cell carcinomas growing in C3H mice were removed and cooled, and a single-cell suspension was prepared. These cells were then embedded in agarose, lysed in an alkaline solution, subjected to electrophoresis, and stained with a fluorescent DNA-binding dye. The amount and migration distance of damaged DNA from individual cells were scored by using a fluorescence image processing system, where differentially radio-sensitive aerobic and hypoxic cell populations resulted in bimodal damage distributions. Curve-fitting routines provided quantitative estimates of the fraction of hypoxic cells. RESULTS: After the mice were exposed to 10-20 Gy, the SCCVII tumors (450-600 mg) were shown to have a hypoxic fraction of 18.5% +/- 10.6% (mean +/- SD for 11 tumors), which compares well with the value of 11.6% observed using the paired survival curve method. CONCLUSIONS: Our results indicate that this method, which requires only a few thousand cells, is a rapid and sensitive way to detect hypoxic cells in solid animal tumors. IMPLICATIONS: Estimating hypoxia in accessible human tumors undergoing radiotherapy may be possible if the sensitivity of the method can be improved to allow detection of hypoxic cells after a dose of 2 Gy.

Animals↗
PubMed1992

Factors influencing DNA migration from individual cells subjected to gel electrophoresis.

Alkaline and neutral gel electrophoresis of individual mammalian cells allows detection of DNA single- and double-strand breaks, respectively. For both the alkaline and the neutral assays, lysis conditions influence how much DNA migrates, and factors in addition to DNA size play a role in migration. In particular, the tight packing of DNA in individual nuclei appears to reduce the ability to detect double-strand breaks in all of the genome. Tangling of DNA molecules is probably also responsible for the presence of "wings" associated with each nucleus after application of pulsed-field gel electrophoresis; these wings were aligned in the directions of the pulsed field, not along the resultant vector of the fields as was expected. The choice of fluorescent staining methods (propidium iodide, Hoechst 33342, or antibodies against bromodeoxyuridine) did not influence sensitivity for detecting DNA damage.

Cell Line↗
PubMed1992

Evaluation of bioreductive drugs in multicell spheroids.

The therapeutic potential of a variety of bioreductive agents, including misonidazole, RSU-1069, NFVO, mitomycin C, porfiromycin, and SR-4233 was evaluated using Chinese hamster V79 multicell spheroids in vitro. Fluorescence-activated cell sorting techniques were used to selectively recover cells from various depths within the spheroids to measure the differential cytotoxicity in the cells near the hypoxic core of the spheroid relative to the well oxygenated peripheral cells. At the high cell density found in spheroids (as in tissues in vivo) the differential toxicity observed was typically much less than expected, based on data from single cell systems. In some cases, this was due to lack of sufficient hypoxia in the spheroids; in other cases, drug treatment itself produced reoxygenation through metabolic or toxic effects during treatment. An unexpected observation of considerable concern was rapid bioreduction of the more active agents; this sometimes occurred at rates that exceeded drug delivery, resulting in considerably less efficacy when large hypoxic fractions were present (e.g. mitomycin C, NFVO, and SR-4233). This suggests that induction of hypoxia prior to bioreductive agent therapy may not be the most productive approach. Though none of the agents showed "ideal" properties, porfiromycin was judged to give the best combination of differential toxicity, longevity in situ, and ability to reach the entire hypoxic cell subpopulation.

Animals↗
PubMed1991

Keynote address: the influence of microenvironmental factors on the activity of radiation and drugs.

The inherent radio- and chemosensitivity of tumor cells clearly affects their response to treatment. Accumulating evidence, however, suggests that the biochemical and physiological status of the cell during treatment is at least as important. In this review, a critique of the current evidence for, and extent of, microenvironmental heterogeneity in tumors is presented, emphasizing human tumor cells in situ. The expected consequences of those changes on cellular response to radiation and chemotherapy is then briefly reviewed. Finally, the continuing interest in developing new therapeutic strategies for which the tumor microenvironment is an asset (as opposed to a liability) is discussed in the context of the dynamic nature of tumors, and the complexity of adequately analyzing combination treatments.

Acid-Base Equilibrium↗
PubMed1991

The response of hypoxic cells in SCCVII murine tumors to treatment with cisplatin and x rays.

Possible mechanisms of enhancement of radiation effects by cisplatin, including radiosensitization of hypoxic cells, drug-induced tumor reoxygenation, and inhibition of repair of sublethal radiation damage, were examined in the murine SCCVII model. Combination radiation/drug treatments were most effective when drug exposure preceded irradiation of animals breathing a reduced oxygen atmosphere, indicating that the primary interaction between the modalities was a cisplatin-induced increase in the oxygenation status of the acutely hypoxic cells in those tumors. Delivering cisplatin prior to or immediately after the first of two 5 Gy fractions was more effective than combinations with a single x-ray exposure, suggesting that proper sequences of the combined modalities may augment natural reoxygenation processes.

Animals↗
PubMed1991

Etoposide sensitivity and topoisomerase II activity in Chinese hamster V79 monolayers and small spheroids.

Chinese hamster V79 cells grown in suspension culture as spheroids are more resistant than monolayers to killing and mutation by ionizing radiation. A change in DNA conformation appears to accompany this increase in radiation resistance. We were therefore interested in whether the activity of topoisomerase II, a nuclear enzyme involved in DNA conformational changes and possibly in DNA repair, might differ in monolayers and small spheroids. One-day-old spheroid cells were more resistant than monolayer cells to the toxic effects of etoposide, a topoisomerase II inhibitor. Fewer strand breaks were induced by etoposide in spheroid DNA than monolayer DNA, as measured by the DNA precipitation and alkali unwinding assays, although identical amounts of damage were produced in monolayers and spheroids by the topoisomerase I inhibitor camptothecin, and the cell cycle specific agent, 5-fluorouracil. There was no evidence of a subpopulation of spheroid cells which were more resistant to etoposide, and no change in the rate of incorporation or DNA chain elongation in spheroids compared to monolayers. Topoisomerase II activity in 1-day-old spheroids, measured by decatenation of trypanosome kinetoplast DNA, was reduced to 68% of the monolayer value; in 3-4-day-old spheroids the level was 32.5%. These results indicate that topoisomerase II activity and sensitivity to a topoisomerase II inhibitor are reduced in 1-day-old spheroid cells. We suggest that the decrease in the activity of this enzyme may be linked to the change in DNA conformation in spheroids and the decrease in their radiation sensitivity.

Animals↗
PubMed1991

The radiation response of asynchronous cells at low dose: evidence of substructure.

Flow cytometry and cell sorting techniques have been used together with repeated measurement in an attempt to define better the radiation survival response of asynchronously dividing Chinese hamster V79-171 cells under aerobic and hypoxic conditions. Although the first two decades of cell inactivation have been examined, particular attention has been given to the low-dose range of a few grays, as used in individual radiation therapy treatments. A single linear-quadratic dose-response function was consistently unable to fit both the low-dose and high-dose data satisfactorily, suggesting a two-component response. Separate fitting of the low-dose and high-dose portions of the response yielded alpha and beta values which differed significantly (P = 0.001 to 0.002). The data are consistent with the hypothesis that the observed substructure simply reflects the presence of subpopulations of sensitive (G1-, G2-phase) and resistant (late S-phase) cells, which are resolved in these measurements. These results may have significance for certain situations in radiation therapy and in biophysical modeling of the radiation response.

Aerobiosis↗
PubMed1990

Schedule dependence for cisplatin and etoposide multifraction treatments of spheroids.

Cisplatin and etoposide are both widely used as antineoplastic chemotherapeutic agents. Two approaches are generally adopted when an attempt is made to maximize the efficiency of these drugs: concurrent use (where synergism is expected) or sequential administrations (exploiting the antiproliferative effects of etoposide). To differentiate between these approaches in a quantitative manner, we exposed an in vitro tumor model (V79 multicell spheroids) to the drugs, using treatment regimens with a constant weekly dose intensity. Some treatment schedules suggested the development of drug resistance, but this resulted from a changing growth fraction in the spheroids rather than from selection for or induction of cellular resistance. Fewer administrations of larger doses were generally less satisfactory than multiple, smaller treatments. The most effective sequence was an alternating regimen, by which the cytoreductive effects of cisplatin resulted in recruitment of quiescent cells into active proliferation, enhancing in turn the efficiency of subsequent etoposide treatment.

Animals↗
PubMed1990

Detection of etoposide resistance by measuring DNA damage in individual Chinese hamster cells.

The comet assay, which measures DNA strand breakage in individual cells, was used to examine the relation between DNA damage, cell survival, and resistance to the topoisomerase II inhibitor etoposide (VP-16). Chinese hamster V79-171b cells and a VP-16-resistant subline (VPr) were exposed to VP-16 as monolayers or spheroids. The comet assay was comparable in sensitivity to the DNA precipitation and alkali unwinding assays for detecting DNA strand breaks induced by VP-16. However, unlike conventional DNA damage assays, the comet assay also indicated heterogeneity in cell response. For V79 multicell spheroids exposed to VP-16, the external cycling cells were 50 times more sensitive to killing and DNA damage than the internal noncycling cells; the comet assay indicated the fraction of cells resistant to the drug. VPr cells, which were 10 times more resistant to killing and DNA damage by VP-16 than the parent cell line, could also be identified in mixed populations with the use of this method. These results suggest that the comet assay could be useful in predicting tumor cell response to DNA-damaging agents.

Animals↗
PubMed1990

Slow penetration of anthracyclines into spheroids and tumors: a therapeutic advantage?

Despite clear evidence that the effective penetration of the anthracycline antibiotics into experimental tumors or multicell spheroids is poor, these drugs exhibit clinical activity against a variety of solid tumors. In an attempt to understand this apparent contradiction, we used the Chinese hamster V79 spheroid system and flow cytometry techniques for intra-spheroid pharmacological studies of doxorubicin and daunomycin. Our results indicate that the slow delivery of the anthracyclines to the inner cells of spheroids is due to the rapid binding of the drug by cells in the outer layers. After exposure, the anthracyclines are retained much more effectively when cells remain in intact spheroids than when the spheroids have been dispersed, resulting in considerably more cytotoxicity in situ. This result indicates a need for considerable caution in attempting to predict the anti-tumor efficacy of drugs by using either conventional cell-culture systems, spheroids that have been disaggregated immediately post-exposure, or excision assays of tumors from experimental animals. Furthermore, our results suggest the need for a critical evaluation of the significance of the multidrug resistance (MDR) phenotype for cells surrounded by other drug-containing cells as opposed to single cells in drug-free culture medium.

Animals↗
PubMed1990

Intermittent blood flow in the KHT sarcoma--flow cytometry studies using Hoechst 33342.

The administration of the fluorescent DNA stain, Hoechst 33342, to mice bearing the KHT sarcoma, combined with flow cytometry, can be used to select cells according to their proximity to functional vasculature. Different protocols of administration of Hoechst 33342 were used in order to differentiate between the presence of temporary and chronically hypoxic cells. The results show a large difference in radiosensitivity between cells close to, and distant from, functional vasculature. However, this pattern of radiosensitivity is observed only when the staining period with Hoechst 33342 is short and coincides with the period of irradiation. When the radiation treatment is temporally divorced from the staining period then the radiosensitivity and staining intensity are not related. This result can be interpreted as indicating that hypoxic cells exist within this tumour as a result of fluctuations in tumour blood flow.

Animals↗
PubMed1990

Cisplatin and CCNU synergism in spheroid cell subpopulations.

The cytotoxicity of two antineoplastic drugs, cisplatin and CCNU, was evaluated in Chinese hamster V79 multicell spheroids using the drugs as single agents or combinations. Cells obtained from different depths within spheroids 550-750 microns in diameter showed different sensitivities to the two agents; the external cells of the spheroids were more sensitive than the internal cells to cisplatin, whereas the internal cells were most effectively killed by CCNU. Combining the two agents produced the expected 'complementary' activity, and in addition, synergism was observed between the drugs at exposure levels practical for clinical use. For the combination treatments, both the net pattern of cell killing through the spheroid and the degree of interaction between the agents (quantified using the combination index method) were a function of the dose ratio of the two drugs, and of overall treatment intensity. BCNU produced patterns of cell killing similar to CCNU, but showed little interaction with cisplatin. Our results suggest significant clinical potential in using CCNU with cisplatin, particularly since CCNU-cisplatin combinations were synergistic even in the cell subpopulations most resistant to each drug as a single agent.

Animals↗
PubMed1990

Respiration-induced oxygen gradients in cultured mammalian cells.

The effect of cell respiration on the availability of intracellular oxygen was investigated by comparing the radiosensitivities of respiring and non-respiring cells over a range of oxygen tensions. Monolayers of Chinese hamster V79 fibroblasts on glass Petri dishes were irradiated at respiration-inhibiting (4 degrees C) and normal (37 degrees C) cell-culturing temperatures. Desired extracellular oxygen concentrations were achieved by aspirating the culture medium above the cells prior to irradiation, leaving a residual thin film which prevented drying of the cells while allowing rapid equilibration with the overlying gas. Measurement of clonogenic survival revealed that, at equivalent extracellular oxygen concentrations, the cells irradiated at 37 degrees C were less radiosensitive than those irradiated at 4 degrees C. The difference in respiring and non-respiring cell radiosensitivity was dependent on cell shape, and decreased when the cells attached to the Petri dish surface were allowed to assume a flatter configuration. These results imply that at low extracellular oxygen tensions the oxygenation of critical cellular radiation target(s) is dependent on respiration and diffusion distance, as would be expected if oxygen gradients induced by respiration exist within and immediately around actively metabolizing cells.

Animals↗
PubMed1990

Multicell spheroids as a model for cell kinetic studies.

Cells growing in tissue culture as three-dimensional, multicellular aggregates called 'spheroids' typically show a decreasing growth fraction and development of quiescent subpopulations as the spheroids enlarge. Kinetic studies in a number of spheroid systems have indicated that the primary reason for the tumour-like growth is a progressive decrease in growth fraction, with only a modest elongation of cell cycle time in larger spheroids. In this paper, the cellular growth kinetics for spheroids of V79 Chinese hamster lung cells are reviewed, and the regrowth kinetics of cells resuming growth after recovery from quiescent regions of the spheroids are described. Further, the role of regrowth/repopulation in determining the spheroid response to anti-tumour cytotoxics is explored, with particular emphasis on treatment with cisplatin and etoposide. By separating the effects of cytotoxicity and regrowth in the overall spheroid response to anti-neoplastic drugs, it is suggested that 'drug resistance' in tumours can be a kinetic as well as a genetic problem.

Animals↗
PubMed1990

Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells measured using the "comet" assay.

A method for measuring DNA damage to individual cells, based on the technique of microelectrophoresis, was described by Ostling and Johanson in 1984 (Biochem. Biophys. Res. Commun. 123, 291-298). Cells embedded in agarose are lysed, subjected briefly to an electric field, stained with a fluorescent DNA-binding stain, and viewed using a fluorescence microscope. Broken DNA migrates farther in the electric field, and the cell then resembles a "comet" with a brightly fluorescent head and a tail region which increases as damage increases. We have used video image analysis to define appropriate "features" of the comet as a measure of DNA damage, and have quantified damage and repair by ionizing radiation. The assay was optimized for lysing solution, lysing time, electrophoresis time, and propidium iodide concentration using Chinese hamster V79 cells. To assess heterogeneity of response of normal versus malignant cells, damage to both tumor cells and normal cells within mouse SCC-VII tumors was assessed. Tumor cells were separated from macrophages using a cell-sorting method based on differential binding of FITC-conjugated goat anti-mouse IgG. The "tail moment", the product of the amount of DNA in the tail and the mean distance of migration in the tail, was the most informative feature of the comet image. Tumor and normal cells showed significant heterogeneity in damage produced by ionizing radiation, although the average amount of damage increased linearly with dose (0-15 Gy) and suggested similar net radiosensitivities for the two cell types. Similarly, DNA repair rate was not significantly different for tumor and normal cells, and most of the cells had repaired the damage by 30 min following exposure to 15 Gy. The heterogeneity in response did not appear to be a result of differences in response through the cell cycle.

Animals↗