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Some multidrug resistant cell lines over-express the gene encoding the multidrug-resistance-associated protein (MRP). In all cell lines reported thus far, over-expression is associated with gene amplification. We have studied the predominant mechanisms of MRP over-expression in 4 human lung-cancer cell lines that cover a range of drug-resistance levels, and we have analyzed the MRP amplicon. In the SW-1573-derived, weakly resistant cell line 30.3M, MRP mRNA is elevated 3-fold in the absence of gene amplification. Run-on analysis shows that the increased MRP gene expression in this cell line is due to transcriptional activation. In the highly resistant GLC4/ADR and COR-L23/R cells, MRP gene amplification predominates, whereas in the moderately resistant MOR/R cells, gene amplification is combined with a mechanism resulting in an additional increase in the level of MRP mRNA. Fluorescence in situ hybridization shows that, in the GLC4/ADR cells, amplified MRP sequences are present both in double minute chromosomes (DM) and in homogeneously staining regions (HSR). By pulsed-field gel electrophoresis we show that the MRP-containing DM are 1 Mb in length. Chromosome-16-specific repetitive sequences adjacent to the MRP gene are also present in the DM and HSR, compatible with the involvement of these sequences in recombination events underlying MRP gene amplification. Our results show that low levels of drug resistance may arise by transcriptional activation of the MRP gene, whereas at high levels of drug resistance amplification of the MRP gene predominates, possibly facilitated by the presence of recombination-prone sequences.

The aim of this study was to determine whether significant inter-individual differences exist between skin fibroblast strains obtained from radiotherapy patients in both radiation-induced differentiation and collagen production in vitro, for use as potential parameters for a predictive assay for fibrosis following radiotherapy in patients. Morphological cell differentiation was determined 7 days after irradiation in seven early-passage primary human fibroblast cell strains and correlated with cell survival. Collagen production was measured in two cell strains by flow cytometry and incorporation of 3H-proline. There was a wide variation in the extent of radiation-induced differentiation for the seven cell strains, each showing a dose-related increase. The correlation between induced differentiation and cell survival was poor (r = 0.64) but statistically significant (p < 0.01). Collagen synthesis increased 7 days after irradiation for one cell strain (HF-48), as measured by incorporation of 3H-proline, but not in radiation sensitive AT-1 cells. The collagen I content of the two cell strains was assessed by flow cytometry but no significant differences were observed between the strains tested or with increasing dose. In conclusion, marked variations in radiation-induced fibroblast differentiation were observed between patients, this being an important criterion for a predictive assay.

Several methods were applied to determine the viability of hybridoma cells in suspension. These methods include dye inclusion and exclusion assays such as the classical trypan blue exclusion assay, the propidium iodide (PI) exclusion assay and the fluorescein diacetate (FDA) inclusion assay. Furthermore, the relation was studied between release of lactate dehydrogenase (LDH) by hybridoma cells and their viability. Also the ATP content of the cells and cellular heterogeneity as measured with a flow cytometer were determined in relation to cellular viability. The dye inclusion and exclusion assays using trypan blue, FDA, PI were shown to be useful methods to determine cellular viability. With the FDA and PI methods it was possible to obtain additional information about cells which are in a transition state between viable and non-viable. The viability according to the scatter properties of the cells appears to reflect the overall condition of the cells, although interpretation of the results is difficult. Measurement of LDH release in the culture fluid or the cytoplasmic ATP content could not be used as parameters for cell viability.