Determination of radiosensitivity in established and primary squamous cell carcinoma cultures using the micronucleus assay.

In this study, the cytokinesis-block micronucleus assay (CBMN) was used to measure radiosensitivity in three established cell lines (SCC-61, V175 and V134) and 10 primary cell cultures of squamous cell carcinoma (SCC) of the head and neck. Assessment involved optimisation of the assay to determine cytochalasin-B (CB) concentration and sampling time postirradiation. A much closer correlation between dose-response data measured in the clonogenic and micronucleus assays was found when the micronucleus assay was performed under standardised conditions for each cell line (2 micrograms/ml CB: 48 h postirradiation) instead of predetermined optimised assay conditions. This indicates that, for these SCC cell lines, the CBMN assay may be able to predict in vitro radiosensitivity. To be of clinical use in predicting radiosensitivity, the CBMN assay also needs to be evaluated with primary cell cultures. In this study, no relationship between micronucleus frequency at 2 or 6 Gy and patient clinical outcome 12 months following surgery and radiotherapy was seen. Similarly, no association between patient outcome and tumour stage, nodal stage and histology was observed. These CBMN assay data from the primary cell cultures are presently inconclusive as a measure of patient tumour radiosensitivity.

The regulation of propionate oxidation in Prototheca zopfii.

1. Whole cell suspensions of Prototheca zopfii grown on propionate oxidize propionate, acrylate, malonic semialdehyde and acetate immediately, whereas acetate-grown cells only oxidize acrylate or propionate rapidly after a lag of 20-30min. This adaptation to propionate is slowed down by 8-azaguanine or p-fluorophenylalanine, and is not influenced by adding an ammonium salt or an amino acid mixture. 2. The adaptation involves induction of the enzymes of beta-oxidation of propionate. 3. A small proportion (5-8%) of the activities of propionyl-CoA dehydrogenase, beta-hydroxypropionate dehydrogenase and malonic semialdehyde dehydrogenase are consistently associated with mitochondria isolated from propionate-grown cells. 4. Such mitochondria will oxidize propionyl-CoA, beta-hydroxypropionate and malonic semialdehyde, and the respiration rates with these substrates in the presence of inorganic phosphate are ADP-dependent. 5. Mitochondria from acetate-grown cells do not contain detectable activities of the enzymes of propionate oxidation.

Propionate assimilation in the flagellate Polytomella caeca. An inducible mitochondrial enzyme system.

1. The assimilation of propionate by Polytomella caeca involves the beta-oxidation of this fatty acid. 2. Propionate-grown cells immediately oxidize propionate, beta-hydroxypropionate, malonic semialdehyde and acetate; acetate-grown cells oxidize propionate rapidly only after a lag of 2hr., and this adaptation of resting cells to propionate involves the formation of the enzymes of beta-oxidation. 3. The beta-hydroxypropionate dehydrogenase and malonic semialdehyde dehydrogenase activities of both propionate-grown and propionate-adapted cells are partly located in mitochondrial fractions. 4. Mitochondria isolated from propionate-grown cells, and also those from acetate-grown cells fully adapted to propionate, oxidize succinate, alpha-oxoglutarate, beta-hydroxypropionate and malonic semialdehyde; oxidation of these substrates is tightly coupled to the phosphorylation of ADP. 5. Mitochondria from acetate-grown cells exhibit ADP-dependent oxidation of succinate and alpha-oxoglutarate, but do not oxidize beta-hydroxypropionate or malonic semialdehyde. Mitochondria isolated from acetate-grown cells adapted to propionate for 5hr. slowly oxidize beta-hydroxypropionate and malonic semialdehyde, but no tightly coupled phosphorylation is detectable. 6. Two of the inducible enzymes of propionate oxidation are located within the NAD-impermeable barrier and appear to be membrane-bound. 7. The formation of the inducible enzymes is inhibited by cycloheximide and actinomycin D, but not by chloramphenicol.

The micronucleus assay: an evaluation of its use in determining radiosensitivity in vitro.

The cytokinesis-block micronucleus assay was used to measure radiosensitivity in vitro in a panel of seven cell lines. Six of these cell lines were used to study the major parameters of this assay. We observed varying sensitivities following cytochalasin-B exposure. Treatment with 1 microgram/ml cytochalasin-B for 24 h reduced cell survival in four of the six cell lines by > 60%. Cytochalasin-B concentration and post-irradiation culture time were both found to influence cell-response. In three cell lines (V39, V134 and HX142), a decrease in cytochalasin-B concentration (2-0.5 microgram/ml) resulted in an increase in the frequency of radiation-induced micronuclei per binucleate cell. In other cell lines, either the opposite (V7M, CHO-K1) or no effect (WiDr) was seen. A linear dose-response was observed between induced damage expressed as the frequency of micronuclei and radiation dose in all but one melanoma (V39) cell line. Evidence for radiation-induced division-delay, with the maximum frequency of binucleation in irradiated cultures occurring 24-48 h after that of controls, was only seen in two cell lines. Of particular note, and in contrast to some other published reports, was the lack of a general correlation between cell-response measured in the clonogenic and the cytokinesis-block micronucleus assays. Consideration of lethal lesions, determined from the clonogenic dose-response curve, with respect to micronucleus frequency showed a complex relationship, with one micronucleus per binucleate cell corresponding to a wide range of lethal lesions depending on the cell line.(ABSTRACT TRUNCATED AT 250 WORDS)