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Two colon cancer cell lines, HT-29 (human) and DHD/K12/TRb (rat), were grown as monolayer cultures to various confluence degrees. The cytotoxic efficacies of doxorubicin and 4'-deoxydoxorubicin, evaluated by a survival assay, and the nuclear drug concentrations, measured by microspectrofluorometry, were shown to progressively decrease with the augmentation of confluence. This confluence dependent resistance (CDR) to anthracyclines was demonstrated independent of the multidrug resistance drug efflux mechanism. The cellular uptake of three compounds (sodium [51Cr]chromate, D-[14C]alanine, L-[14C]glucose) known to passively diffuse across the cell membrane as anthracyclines do was also reduced in confluent cells. After trypsin cell detachment, the kinetics of reversion of the sodium [51Cr]chromate uptake decrease and that of CDR were similar. Therefore, CDR may be attributed to a reduction of anthracycline cell intake due to a general alteration of passive diffusion across the cell membrane. However, CDR is only partly explained by this phenomenon since a reduced sensitivity of confluent cells was observed compared with nonconfluent cells for a similar amount of drug in their nuclei. CDR could explain the high resistance to anthracyclines of some solid tumors, such as colon tumors, in which cancer cells are tightly aggregated.

Intranuclear drug concentration in cells treated with doxorubicin (DXR) or with 4'-deoxy-4'-iododoxorubicin (IDX) was measured by means of a quantitative microspectrofluorometric technique recently developed by us. Resolution of free and bound drug contributions in fluorescence emission spectra, as collected from a microvolume of single living cell nuclei, provided concentration data with about 10% indetermination. Uptake of DXR and IDX into the nucleus of K562 cells and DXR-resistant K562/DXR cells could then be studied with a sensitive, nondestructive technique. Growth inhibitory concentrations of K562 and K562/DXR cells, when measured with respect to drug content in the medium, differed by a factor of 25 in the case of DXR and by a factor of three in the case of IDX. By contrast, intranuclear drug concentrations measured at corresponding growth inhibitory concentrations are found to be nearly constant, i.e., independent of cellular-resistant phenotype and of anthracycline structure. This result supports an identical mechanism of action for the two drugs, most probably targeted to the nucleus, and ascribes to intracellular transport the different potency of the two drugs in the two cell lines.