Characterization of a doxorubicin-resistant murine melanoma line: Studies on cross-resistance and its circumvention

Abstract

A B16 mouse melanoma cell line resistant to doxorubicin was obtained by continuous in vitro exposure to the drug. The ID50 for this line was 200 times higher than that for the parental cell line. The resistant cell line had some biological characteristics similar to those of the sensitive parental cell line, like saturation density and protein content. Differences were found in doubling time which was longer, cloning efficiency which was lower and DNA content which was higher in the resistant as compared to the parental line. Intracellular distribution of doxorubicin was also different having a nuclear-cytoplasmic ratio higher in sensitive than in resistant cells. Melanin content was an unstable feature in the sensitive cell line, whereas melanin was always present in resistant cells. Resistance to doxorubicin was maintained during 50 in vitro passages in the absence of the drug. Cross-resistance was found with vincristine and other anthracyclines, like daunorubicin and 4'-epi-doxorubicin but not with cis-platinum, and a new doxorubicin derivative, 4'-deoxy-4'-iodio-doxorubicin. The B16 line showed a lower resistance index to 4'-deoxy-doxorubicin and 4-demethoxy-daunorubicin (30 and 3 respectively), as compared to doxorubicin. Doxorubicin-resistance was partially circumvented by pretreatment of resistant cells with verapamil, a calcium chelating agent, and by trifluoperazine, a calmodulin-antagonist.

In vivo characterization of a doxorubicin resistant B16 melanoma cell line

“…The lower colonization potential of the resistant line is directly related to the cloning efficiency which has been found to be 10 times lower for the resistant line (Supino et al, 1986) and confirm similar findings on B16 melanoma subclones (Stackpole et al, 1985b). Such a correlation does not exist between the cloning efficiency and the tumourigenicity of these two lines as already reported for B16 melanoma cells in culture (Kreider & Schomayer, 1975 (Biedler et al, 1975;Biedler et al, 1983) Nowak et al, 1973).…”

“…B16V cells show a pattern similar to B16 cells while B16VDXR cells, as found in vitro (Supino et al, 1986), show besides a first and a second peak similar to those of sensitive cells, a third peak shifted towards higher values (hypertetraploid).…”

“…The fact that B16VDXR showed after in vivo growth the same higher DNA content compared to the sensitive line B16V found in vitro (Supino et al, 1986), indicates that no cell selection occurred during in vivo growth. Similarly no selection seems to have occurred in the sensitive line B16V obtained by growing the parental B16 melanoma in vitro.…”

“…As reported for other murine tumours (Schabel et al, 1983), no cross-resistance was found for this The main biological properties that characterize the B16VDXR line in vivo compared to the parent line B16V are the longer latency period with consequent longer survival time and the lower colonization capacity, all features which go along with lower malignancy as reported for other drugresistant tumours (Biedler et al, 1983). The longer latency of the B16VDXR line correlates with the in vitro longer doubling time (25 vs. 15h) (Supino et al, 1986) and this might be responsible for the different sensitivity to both DX and cis-DDP. In fact it has been reported that while rapidly growing tumours are more sensitive to DX than slowly growing tumours, the opposite is true for cis-DDP (Mattern et al, 1981).…”

“…Detailed description of the in vitro assays of levels of resistance has been reported (Supino et al, 1986). Briefly, cells were treated at cell seeding with different concentrations of DX.…”