Is the mammalian cell plasma membrane a barrier to oxygen transport?

Oxygen transport in the Chinese hamster ovary (CHO) plasma membrane has been studied by observing the collision of molecular oxygen with nitroxide radical spin labels placed in the lipid bilayer portion of the membrane at various distances from the membrane surface using the long-pulse saturation-recovery electron spin resonance (ESR) technique. The collision rate was estimated for 5-, 12-, and 16-doxylstearic acids from spin-lattice relaxation times (T1) measured in the presence and absence of molecular oxygen. Profiles of the local oxygen transport parameters across the membrane were obtained showing that the oxygen diffusion-concentration product is lower than in water for all locations at 37 degrees C. From oxygen transport parameter profiles, the membrane oxygen permeability coefficients were estimated according to the procedure developed earlier by Subczynski et al. (Subczynski, W. K., J. S. Hyde, and A. Kusumi. 1989. Proceedings of the National Academy of Sciences, USA. 86:4474-4478). At 37 degrees C, the oxygen permeability coefficient for the plasma membrane was found to be 42 cm/s, about two times lower than for a water layer of the same thickness as the membrane. The oxygen concentration difference across the CHO plasma membrane at physiological conditions is in the nanomolar range. It is concluded that oxygen permeation across the cell plasma membrane cannot be a rate-limiting step for cellular respiration. Correlations of the form PM = cKs between membrane permeabilities PM of small nonelectrolyte solutes of mol wt less than 50, including oxygen, and their partition coefficients K into hexadecane and olive oil are reported. Hexadecane: c = 26 cm/s, s = 0.95; olive oil: c = 23 cm/s, s = 1.56. These values of c and s differ from those reported in the literature for solutes of 50 less than mol wt less than 300 (Walter, A., and J. Gutknecht. 1986. Journal of Membrane Biology. 90:207-217). It is concluded that oxygen permeability through membranes can be reliably predicted from measurement of partition coefficients.

Radiation induction of drug resistance in RIF-1: correlation of tumor and cell culture results.

The RIF-1 tumor line contains cells that are resistant to various anti-neoplastic drugs, including 5-fluorouracil (5FU), methotrexate (MTX), adriamycin (ADR), and etoposide (VP16). The frequency of these drug-resistant cells is increased after irradiation. The frequency of drug-resistant cells and the magnitude of radiation-induced drug resistance are different in cell culture than in tumors. The dose-response and expression time relationships for radiation induction of drug resistance observed in RIF-1 tumors are unusual. We hypothesize that at high radiation doses in vivo, we are selecting for cells that are both drug resistant and radiation resistant due to microenvironmental factors, whereas at low radiation doses in vivo and all radiation doses in vitro, we are observing true mutants. These studies indicate that there can be significant differences in drug-resistance frequencies between tumors and their cell lines of origin, and that radiation induction of drug resistance depends significantly on whether the induction is done in tumors or in cell culture. These results imply that theories about the induction of drug resistance that are based on cell culture studies may be inapplicable to the induction of drug resistance in tumors.

DNA content in nasopharyngeal carcinoma.

DNA analysis by flow cytometry was performed on tissue blocks from 41 patients with nasopharyngeal carcinoma. The histologic slides were reviewed by a pathologist and blindly classified according to the World Health Organization classification. The paraffin-embedded blocks were processed to obtain individual nuclei, which were then stained with propidium iodide. The nuclei were analyzed on a flow cytometer. Excluding 10 uninterpretable histograms, the remainder were interpreted blindly and classified as diploid or aneuploid. The Cox proportional hazards survival model was used to analyze stage, histology, radiation dose, and ploidy. We observed more diploids (23 of 31; 74%) than aneuploids (eight of 31; 26%). The 2-year survival rate of diploids was 55%, compared with 25% of aneuploids (P less than .05). We conclude that ploidy status is an independent prognostic factor in nasopharyngeal carcinoma.

Flow cytometric evaluation of chemosensitive and chemoresistant head and neck tumors.

For patients with head and neck squamous carcinoma, a clinical response to induction chemotherapy has correlated with a survival advantage. Similarly, patients with diploid tumors have displayed a survival advantage when compared with patients with aneuploid tumors. This study examined DNA content in 33 patients who had undergone induction chemotherapy as part of two clinical protocols to determine if there was a correlation between the patients with diploid tumors and the patients with a clinical response to chemotherapy. Although patients with stage III tumors had a longer disease-free survival than stage IV patients (p less than 0.0002), the addition of DNA content information did not improve the ability to predict response. Specifically, there was no correlation between DNA content and the response to chemotherapy. In addition, for this group of patients, a diploid DNA content was not correlated with a survival advantage. We conclude that DNA content information did not add significantly to the prediction of clinical outcome in these patients who received induction chemotherapy.

Drug resistance following irradiation of RIF-1 tumors: influence of the interval between irradiation and drug treatment.

RIF-1 tumors contain a small number of cells (1 to 100 per 10(6) cells) that are resistant to 5-fluorouracil, methotrexate, or adriamycin. The frequency of drug-resistant cells among individual untreated tumors is highly variable. Radiation, delivered in vivo at doses of 3 to 12 Gy, increases the frequency of methotrexate- and 5-fluorouracil-resistant cells, but not the frequency of adriamycin-resistant cells. The magnitude of induction of 5-fluorouracil and methotrexate resistance shows a complex dependence on the radiation dose and on the interval between irradiation and assessment of drug resistance. For a dose of 3 Gy, induced 5-fluorouracil and methotrexate resistance is seen only after an interval of 5 to 7 days, whereas for a dose of 12 Gy, high levels of induced resistance are observed 1 to 3 days after irradiation. The maximum absolute risk for induction of resistance is 4 per 10(4) cells per Gy for methotrexate, and 3 per 10(6) cells per Gy for 5-fluorouracil. These results indicate that tumor hypoxia may play a role in the increased levels of drug resistance seen after irradiation, and that both genetic and environmental factors may influence radiation-induction of drug resistance. These studies provide essential data for models of the development of tumor drug resistance, and imply that some of the drug resistance seen when chemotherapy follows radiotherapy may be caused by radiation-induced drug resistance.

Radiation induction of drug resistance in RIF-1 tumors and tumor cells.

The RIF-1 tumor cell line contains a small number of cells (1-20 per 10(6) cells) that are resistant to various single antineoplastic drugs, including 5-fluorouracil (5FU), methotrexate (MTX), and adriamycin (ADR). For 5FU the frequency of drug resistance is lower for tumor-derived cells than for cells from cell culture; for MTX the reverse is true, and for ADR there is no difference. In vitro irradiation at 5 Gy significantly increased the frequency of drug-resistant cells for 5FU, MTX, and ADR. In vivo irradiation at 3 Gy significantly increased the frequency of drug-resistant cells for 5FU and MTX, but not for ADR. The absolute risk for in vitro induction of MTX, 5FU, and ADR resistance, and for in vivo induction of 5FU resistance, was 1-3 per 10(6) cells per Gy; but the absolute risk for in vivo induction of MTX resistance was 54 per 10(6) cells per Gy. The frequency of drug-resistant cells among individual untreated tumors was highly variable; among individual irradiated tumors the frequency of drug-resistant cells was significantly less variable. These studies provide supporting data for models of the development of tumor drug resistance, and imply that some of the drug resistance seen when chemotherapy follows radiotherapy may be due to radiation-induced drug resistance.

Mechanisms of membrane damage for CHO cells heated in suspension.

Chinese hamster ovary cells were heated for 20 min at 45.5 degrees C in different conditions, and quantitative determinations of cellular membrane blebbing were performed for cells maintained at 25 degrees C and 37 degrees C after hyperthermia. The percentage of cells with blebs following heating was dependent upon the composition of the medium during heating and the posthyperthermia temperature after heating. The total extent of bleb formation after heating was independent of the calcium-ion concentration in the medium during heating; however, differences in the kinetics of bleb disappearance after heating point to the importance of Ca2+ concentration in the expression of heat damage. Without hyperthermia, blebs were formed on the cell-surface membrane with agents which block sulfhydryl groups or release calcium from cellular stores. The cells were protected from bleb formation when cells were incubated with glutathione before addition of sulfhydryl-blocking agents or heat treatment. Oligomycin did not prevent the formation of blebs, suggesting that this phenomenon is not energy-dependent. Only a small percentage of cells were covered with blebs when they were heated in saline solution. When cells were incubated with dbcAMP before heat, blebs did not appear at 25 degrees C. A possible interpretation for these observations is presented.

An evaluation of paramagnetic broadening agents for spin probe studies of intact mammalian cells.

Six transition metal ion complexes have been examined for their effects on the cell survival as well as their effectiveness in inducing the broadening of the electron spin resonance (ESR) spectra of nitroxide spin probes. These paramagnetic species are Ni(EDTA), Ni(DTPA), potassium tris(oxalato) chromate (chromium oxalate), K3Fe(CN)6, Cu(DTPA), and NiCl2. At 100 mM concentration, the typical concentration used in cell studies to broaden the extracellular nitroxide ESR signal, only Ni(EDTA) and Ni(DTPA) are found to be non-toxic to Chinese hamster ovary cells. The relative cytotoxicities of the six metal ion complexes are Cu(DTPA) greater than K3Fe(CN)6 greater than NiCl2 greater than chromium oxalate greater than Ni(DTPA) greater than Ni(EDTA). Thus, potassium ferricyanide and NiCl2, two most commonly used paramagnetic broadening agents, are relatively toxic to the cell. In contrast, among the six paramagnetic species tested here, chromium oxalate appears to be the most effective agent at non-toxic concentrations in inducing the broadening of the ESR spectra of both cationic and neutral nitroxide spin probes. By considering both their cytotoxicity and their effectiveness in causing line broadening of the nitroxide ESR spectra, chromium oxalate is a good paramagnetic broadening agent for spin probe studies of intact mammalian cells.

Cytotoxicity of commonly used nitroxide radical spin probes.

Since nitroxide radical spin probes are used frequently to test biophysical properties of cells, their use should be restricted to conditions that do not perturb normal cell growth and viability. Eight commonly used nitroxide radical spin probes have been tested for their effects on the survival of CHO cells. These include water-soluble spin probes Tempol, Tempamine, CTPO, CTPC and 4-maleimido-Tempo, and lipid soluble spin probes 5-Doxyl-, 12-Doxyl-, and 16-Doxylstearates. With the exception of 4-maleimido-Tempo, none of the water soluble spin labels inhibited cell survival at concentrations as high as 1 mM. At concentrations of 75 microM and higher, 4-maleimido-Tempo inhibited cell survival in a dose dependent manner. At concentrations commonly used for spin labeling of cells (30-50 microM) none of the lipid soluble spin probes tested was cytotoxic. At 100 microM only 5-Doxylstearate inhibited cell survival, whereas 12-Doxylstearate and 16-Doxylstearate had no effect.

Changes in bleb formation following hyperthermia treatment of Chinese hamster ovary cells.

Chinese hamster ovary cells in suspension cultures were heated for various times at 41.5, 43.5, and 45.5 degrees C, and quantitative determinations of microblebbing and macroblebbing of the cell membrane were performed for cells maintained at 4, 25, and 37 degrees C after hyperthermia. The percentage of cells with blebs following heating at 45.5 degrees C was dependent upon the duration of heating with increases from 40% for 5 min to 90% for 30 min. Cells exposed to lower temperatures exhibited less blebbing which was not quantifiable. The changes in bleb formation following 45.5 degrees C were dependent upon the posthyperthermia temperature: a slight decrease of macroblebbing at 25 degrees C, a decrease to 50% by 2 h at 37 degrees C, and a sharp decrease of macroblebbing to less than 10% by 1 h at 4 degrees C. Microblebbing increased slightly at 37 degrees C. When cells were transferred rapidly from the 4 degrees C posthyperthermia incubation to 37 degrees C, the bleb formation percentages returned rapidly to the higher levels which existed before posthyperthermia incubation at the lower temperatures. Gamma irradiation of 20 and 50 Gy produced only a small increase in microblebbing at longer periods (5 to 6 h) but no increase in macroblebbing. The survival of cells heated for 20 min at 45.5 degrees C was decreased 40% for suspension cells maintained at 4 degrees C for 2 to 3 h before incubation at 37 degrees C for colony formation compared to cells immediately incubated at 37 degrees C after heating. The survival of cells maintained at 25 degrees C after heating was not altered in comparison.

Effect of melanin on radiation response of CHO cells.

The effect of the presence of melanin on the response of mammalian cells to ionizing radiation was investigated in a model system utilizing the ability of Chinese hamster ovary cells to incorporate melanin by endocytosis. Cells were incubated in monolayer cultures from 2 to 20 hours with melanin prepared from 'beef eye' or synthesized by air oxidation of 3,4-dihydroxyphenylalanine. For asynchronous cultures, the survival curve parameters for cells incubated with both types of melanin were indistinguishable from those of the same cells without added melanin. The radiation response to fractionated doses of 6 Gy separated by various periods did not indicate any effect of melanin on the extent or kinetics of repair of sublethal damage. Likewise, the repair of potentially lethal damage in plateau phase cultures was unaffected by the presence of melanin. Thus the explanation for the clinical radiation resistance of melanomas in the absence of a direct radiation effect might more likely be found in consideration of other factors such as the role of melanin in oxygen consumption or in differentiation.

Effect of hypoxia on recovery from damage induced by heat and radiation in plateau-phase CHO cells.

The effect of hypoxia on the induction of and recovery from damage by radiation alone and in combination with heat has been investigated using plateau-phase Chinese hamster ovary (CHO) cells. Postirradiation hypoxia reduced the potentially lethal damage recovery (PLDR) in cells irradiated under an euoxic state and completely eliminated PLDR in cells irradiated under hypoxia. Cells which were maintained under hypoxia during both irradiation and a 4-hr recovery period and then incubated for a further period of 4 hr under euoxic conditions showed PLDR, suggesting that the inhibition of PLDR by hypoxia is reversible. Oligomycin, an inhibitor of energy metabolism, completely eliminated PLDR when present at a concentration of 1 microM during the postirradiation period. Pre- or postirradiation heat treatment at 42.5 degrees C for 30 min appreciably sensitized the cells to the induction of lethality. Thermal enhancement ratio (TER) was 1.7 for cells irradiated and heat treated under hypoxic conditions. The same heat treatment reduced the oxygen enhancement ratio (OER) associated with gamma radiation from 3.1 to 2.5. Cells subjected to this postirradiation heat treatment showed a small extent of PLDR, whereas the pre-heat-treated cells showed as much recovery as non-heat-treated cells. When hypoxic conditions prevailed during the post-treatment incubation period, PLDR was reduced in preheated cells and completely eliminated in postheated cells. The kinetics of interaction between heat and radiation damage were studied by introducing a time gap of 4 hr between the treatments. Cells maintained under euoxic conditions between the treatments showed an appreciable decrease in interaction, suggesting recovery from damage induced by the first treatment. Hypoxic conditions intervening the two treatments largely inhibited the loss of sensitization. Analysis of the results suggests that cells fail to recover from sublethal heat damage when held for 4 hr under hypoxic conditions. Cells held under hypoxic conditions partly recover from the radiation damage which subsequently interacts with sublethal heat damage, resulting in cell lethality.

Membrane fluidity of Chinese hamster ovary cells on plasma fibronectin-coated microcarriers. A spin label study.

Purified plasma fibronectin promotes the spreading of Chinese hamster ovary (CHO) cells on microcarriers in a serum-free medium. The promotion of 50% of cell spreading on microcarriers requires about 3.4 X 10(8) fibronectin molecules per bead. CHO cells spreading on plasma fibronectin-coated microcarriers had a more rigid cell membrane compared to CHO cells in suspension as determined by using 5-doxylstearate spin label. No detectable differences in the electron spin resonance (ESR) spectra of 12-doxylstearate spin-labeled CHO cells on plasma fibronectin-coated microcarriers and in suspension were observed, suggesting that the effects of plasma fibronectin on membrane fluidity are restricted to the polar head group region of the cell surface membrane. In addition, no significant differences in membrane fluidity and cell spreading were found between CHO cells spreading on plasma fibronectin-coated cytodex 1 (without denatured collagen) and cytodex 3 (with denatured collagen) microcarriers, indicating that a surface layer of denatured collagen is not required for plasma fibronectin to promote cell spreading and to rigidify the cell surface membrane.

Melanin endocytosis by cultured mammalian cells. A model for melanin in a cellular environment.

The incorporation of natural eumelanin from bovine eyes and synthetic 3,4-dihydroxy-phenylalanine (dopa) melanin into Chinese hamster ovary (CHO) cells is reported. The process is linear for at least 8 h. Electron microscopy showed phagocytosis of melanin, either as a single granule or in groups of granules, into cell lysosomes with subsequent degradation of the granule. The general features of the ingestion and degradation processes mimic those of the incorporation of melanosomes into keratinocytes. CHO cells with ingested melanin in general revealed properties very similar to those of the pigment-free CHO cell: cell division, oxygen consumption and plating efficiency were not greatly altered by moderate concentrations of pigment. This suggests that the CHO cell system may be useful for the study of pigment in a cellular environment; pigment-free CHO cells are well characterized and can serve as a good control. Preliminary applications are reported: demonstrations of (1) incorporation of metal ions (Al3+) into CHO cells using melanin as a carrier; (2) the ability of melanin to enhance the rate of oxygen consumption during photo-irradiation of the cells.

Inhibition of repair of premutational lesions in plateau phase Chinese hamster cells exposed to gamma radiation.

Induction and repair of forward mutations to 8-azaguanine resistance were studied in gamma irradiated, plateau phase Chinese hamster ovary cells. Mutation induction increased with dose with a relatively low induction for doses below 4 Gy and a steep increase thereafter. A close correlation between the ability of radiation to induce both lethality and mutations in plateau phase cells was evident. Recovery from potentially lethal damage resulted in a significant decrease in mutation frequency suggesting the possible involvement of an error free repair pathway. Mutation response at the end of recovery period was approximately linear with a slope of 2 X 10(-5) mutants per viable cells per Gy. This difference as compared to the immediate plating response supports the involvement of two types of damage in the induction of mutations: the nonrepairable, single hit component and a repairable component resulting from the interaction of lesions. Post-irradiation nonlethal hyperthermic treatment (42.5 degrees C; 30 min) sensitized the cells to killing as seen by the thermal enhancement ratio of 1.37. Interaction of hyperthermia, however, did not alter the mutation frequency obtained on immediate plating. Both post-irradiation hyperthermia and incubation at 4 degrees C inhibited most of the recovery from potentially lethal damage and also the repair of premutational lesions. These treatments resulted in a mutation frequency decrease of only 10-15% as compared to 50% seen in cells which actively repaired potentially lethal damage. The temperature dependence for the repair of premutational lesions suggests that the process is mediated by metabolically active steps.

Modification of mutation frequency in plateau phase Chinese hamster ovary cells exposed to gamma radiation during recovery from potentially lethal damage.

Induction of forward mutations leading to 8-azaguanine resistance was studied in plateau phase Chinese hamster ovary cells exposed to gamma-radiation. Survival and mutation frequency were assessed immediately after irradiation or after 6 hours of post-irradiation recovery at 37 degrees C. Cells exposed to doses above 4Gy showed appreciable increase in the survival level as a result of recovery from potentially lethal damage. The mutation frequency response curve for cells plated immediately after irradiation displayed an average induction rate of 5 X 4 X 10(-5) per viable cell per Gy in the dose range 4 to 10 Gy. Cells subjected to post-irradiation recovery showed a mutation frequency decrease of 30 to 50 per cent. The kinetics of repair of mutational lesions following exposure to 10 Gy gamma-radiation indicated a sharp decline in mutation frequency during the first hour of post-irradiation recovery and a slow decline thereafter. Analysis of these data suggests that the mutation frequency is significantly lower among the fraction of cells which survive by recovering from potentially lethal damage. The involvement of an error-free pathway that contributes to the repair of an appreciable part of the damage during the post-irradiation recovery period is discussed.

ESR studies of O2 uptake by Chinese hamster ovary cells during the cell cycle.

Magnetic interactions between dissolved oxygen and nitroxide radical spin probes lead to broadening of the ESR lines. We have used a closed-chamber method based on this property to determine the maximum rate of O2 uptake per cell (Vmax per cell) in cultured mammalian cells. A suitable spin probe and a cell suspension are mixed in an aerated medium, and the rate of disappearance of dissolved O2 is measured. The effects of temperature, pH, and microwave power on the determination of dissolved oxygen in solution were studied. For asynchronous Chinese hamster ovary cells, oxygen uptake is 3.8 X 10(7) oxygen molecules per cell per sec and appears to be enzymatically limited at oxygen concentrations greater than 10 microM. About 5-10 X 10(5) cells were used for each measurement, making it possible to study mitotically synchronized cells. Using this method, we have found that Vmax per unit cell volume changes during the cell cycle of Chinese hamster ovary cells from a minimum in mitosis to maxima in both G1 and late S phases. Advantages and limitations of spin probes for studying the O2 uptake of intact cells are discussed.