Subcellular localization of meta-tetra(hydroxyphenyl)chlorin (mTHPC) in HT29 human colon adenocarcinoma cells has been studied by means of fluorescence microscopy. The observed diffuse intracellular distribution of mTHPC fluorescence outside the nucleus indicates general staining of cellular organelles. No changes in dye fluorescence pattern are evident during and immediately after cell illumination. Alternatively, the changes in mTHPC fluorescence pattern are observed upon subsequent cell incubation, and are characterized by the appearance of distinct bright fluorescence zones. Reaching a maximum 1 h after illumination, modifications of the fluorescence pattern then gradually disappear in parallel with the formation of plasma membrane blebs, suggesting that cell necrotic lysis is taking place. Photosensitized damage to mitochondria and the Golgi apparatus has been studied using fluorescent probes 1 h after irradiation, the stage of extensive cytoplasm vacuolization, and reveal alterations of these organelles. Changes in the mTHPC fluorescence pattern and mTHPC photocytotoxicity, as measured by the MTT test 24 h after illumination, are inhibited by sodium azide, a singlet oxygen quencher.

The multidrug resistance (MDR) modulating activity of SDZ‐PSC833 (PSC), a non‐immunosuppressive cyclosporine analogue, was investigated and compared with cyclosporin A (CSA) in bone marrow clinical specimens from 45 patients with acute myeloid leukaemia (AML) taken at diagnosis, using double‐labelling flow cytometry with simultaneous determination of P‐glycoprotein (PGP) expression and intracellular daunorubicin fluorescence (IDF). On the basis of pre‐clinical results in multidrug‐resistant K562 leukaemic cells, concentrations leading to iso‐effective complete restoration of IDF were used: 5 and 10 μmol/l, respectively for PSC and CSA. In the clinical specimens, PGP expression was correlated with a significant decrease in IDF. PSC was found to be significantly more potent than CSA since it was found to induce a significant increase in IDF in a higher number of cases and to a higher extent than CSA. PGP‐unrelated activity of PSC was also observed in specimens expressing no PGP but exhibiting low IDF, thus probably expressing alternative resistance mechanisms. The results confirm the potency of PSC as MDR‐modulating agent in clinical AML specimens whose resistance pattern differed from that of highly resistant cell models and suggest that the activity of PSC is not limited to P‐glycoprotein inhibition.

A tamoxifen-resistant cell line (MCF7TAM) was established from tamoxifen-sensitive MCF-7 human breast cancer cells expressing estrogen receptors. Though the resistant cell line grows in the presence of tamoxifen, estrogen receptors continue to be expressed at similar levels as in the parental cell line. However, estrogen receptors appeared to be altered in the resistant cell line since important discrepancies are observed between results obtained with ligand binding assays and immunoenzymatic assays, tending to show modifications of estrogen receptor ligand binding capacity. The intracellular distribution of tamoxifen in sensitive and resistant cell lines was investigated using fluorescence of eosin–tamoxifen ionic association. Fluorescence emission spectra of eosin, tamoxifen and eosin–tamoxifen complex (λex = 480 nm) were analyzed and the maximal fluorescence intensity found for the complex (λem = 540 nm) was four times higher than that of eosin alone, while tamoxifen alone did not emit any fluorescence in this spectral range. In MCF-7 cells, tamoxifen was found to be mainly located surrounding the nucleus, although nuclear fluorescence intensity was significantly lower. No highly fluorescent granules were observed in the resistant cell lines as opposed to sensitive cells. Improvement of this fluorescence microscopy methodology could appear of interest, taking into account the complexity of tamoxifen resistance molecular pathways.

Video epifluorescence microscopy and image analysis are used for studying anthracycline resistance in breast cancer cells. In order to perform a semi-quantitative image analysis, several deconvolution algorithms are tested and validated on model beads. The most performant algorithm is applied to fluorescent biological specimens. We show that deconvolution makes image segmentation easier. Semi-quantitative measurements on resulting images are correlated with results obtained by cytometry.

A tamoxifen resistant cell line (MCF7(TAM)) was established from tamoxifen sensitive MCF-7 human adenocarcinoma cells expressing estrogen receptors. The resistant cell line was found to express estrogen receptors to similar level as the parent cell line but the receptors were found to be altered, having lost their ability to bind estradiol or tamoxifen. The fluorescence of eosin-tamoxifen ionic association was used to investigate intracellular location of tamoxifen in both sensitive and resistant cell lines. Fluorescence emission spectra of eosin, tamoxifen and eosin-tamoxifen complex (lambda exc=480mn) were analysed and showed that maximal fluorescence intensity of the complex (lambda em=540 nm) was four times higher than that of eosin alone while tamoxifen alone did not emit any fluorescence in this spectral range. In MCF-7 cells, tamoxifen was found to be diffusively located in the cytoplasm and nuclear fluorescence intensity was significantly lower. No difference was observed in fluorescence intensity or location in tamoxifen resistant cells, although it has been previously correlated with clinical responsiveness. Improvement of this fluorescence microscopy methodology appears necessary to provide accurate results taking into account the complexicity of tamoxifen resistance molecular pathways.