Larry E. Hopwood

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.

Electron spin resonance studies of changes in membrane fluidity of Chinese hamster ovary cells during the cell cycle.

Electron spin resonance (ESR) spin-label methods were used with 5-doxyl-stearic acid as a probe to investigate membrane fluidity of Chinese hamster ovary (CHO) cells during the cell cycle. A 35 GHz ESR technique was developed to study membrane fluidity of intact cells. This technique requires only about 1/6 the amount of cells compared to the conventional spin-label techniques. With this technique we observed a cyclic change of membrane fluidity during the cell cycle of CHO cells: cells in mitosis had the highest membrane fluidity, whereas cells in G1 and early S phases had the lowest membrane fluidity.

Cytofluorometric analysis of the DNA content in ovarian carcinoma and its relationship to patient survival

Forty‐one patients with epithelial malignancies of the ovary treated at the Medical College of Wisconsin Affiliated Hospitals from 1976 to 1984 had paraffin embedded tissue available for review. Of the 41 patients, 40 had adequate material to provide 50 micron sections that were evaluated with flow cytometry to determine DNA content. Tumor‐ and patient‐related parameters were then correlated with the results of flow cytometry. Overall survival at 5 years in these patients was 43%, and relapse‐free survival was 50%. Forty percent of the tumors were diploid, and 60% were aneuploid. Five‐year survival for diploid patients was 74% with a relapse‐free survival of 71%. Corresponding overall and relapse‐free survivals for aneuploid patients were 22% and 35%, respectively. Distribution of the patients by histology, stage, and grade was equal between the diploid and aneuploid groups. All patients were treated with whole abdominal plus concomitant pelvic boost irradiation; total doses to the whole abdomen ranged from 10 Gy to 46 Gy (1000 to 4600 cGy) with the median dose being 37.5 Gy. Approximately 40% of the patients received chemotherapy, which usually consisted of a single agent (Alkeran, Burroughs Welcome, Research Triangle Park, NC). This retrospective study of patients with ovarian cancer treated with radiation therapy suggests the possibility that determinations of DNA ploidy may be useful in selecting patients whose poor prognosis dictates that a more aggressive therapy be used.

ESR studies on membrane fluidity of Chinese hamster ovary cells grown on microcarriers and in suspension.

Abstract Electron spin resonance (ESR) spin label methods were used to study membrane fluidity of Chinese hamster ovary (CHO) cells grown on microcarriers and in suspension using 5-doxylstearic acid spin label as a probe. CHO cells grown on microcarriers had a more rigid cell membrane compared to CHO cells grown in suspension culture. CHO cells removed from the surface of the microcarriers by either trypsinization, EDTA treatment or osmotic shock had a membrane fluidity similar to that of CHO cells grown in suspension culture. Conversely, when the cells grown in suspension culture were attached and flattened on the surface of the microcarriers the fluidity decreased. Moreover, membrane fluidity of CHO cells grown on microcarriers changed as a function of the population density, whereas that of the cells in suspension did not. This implies that cell adhesion and/or cell-cell interactions influence the fluidity of 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.

Combined modality of 2-formylpyridine monothiosemicarbazonato copper(II) and radiation☆

Abstract The cupric complex of 2-formylpyridine monothiosemicarbazone, CuL + , is much more potent than the metal free ligand, solvated cupric sulfate, or ferric- 2-formylpyridine monothiosemicarbazone as determined by single-cell survival of CHO cells. CuL + is toxic throughout the cell cycle but most toxic at the G,/S interpbase where CuL + delays the onset of S-phase. Survival of synchronous and asynchronous CHO cells with CuL + and radiation suggest a near additive interaction when CuL + is present when DNA damage is repaired.

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.

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.

Mechanisms of membrane damage for CHO cells heated in suspension

SummaryChinese hamster ovary cells were heated for 20 min at 45.5°C in different conditions, and quantitative determinations of cellular membrane blebbing were performed for cells maintained at 25°C and 37°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°C. A possible interpretation for these observations is presented.

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.