Sequestration of doxorubicin in vesicles in a multidrug-resistant cell line (LZ-100).

A multidrug-resistant Chinese hamster cell line, LZ-8, was subcultured in increasing levels of doxorubicin (DOX) until capable of growth in 100 micrograms/mL DOX. This new derivative, designated LZ-100, is the most DOX-resistant line in the LZ series, based on a comparison of Ki-1 values from cell survival studies. This increased level of drug resistance in LZ-100 cells did not result from (i) higher levels of P-glycoprotein (P-gp) in the plasma membrane compared with LZ-8 cells, since this protein constitutes approximately 20% of the total plasma membrane protein in both cell lines, or (ii) more efficient drug pumping by the same amount of P-gp, since efflux of rhodamine 123 and DOX was comparable in the two cell lines. However, an altered drug distribution was observed in LZ-100 cells compared with wild-type V79 cells; in LZ-100 cells DOX was largely excluded from the nucleus and was sequestered in vesicles in the cytoplasm. The number of vesicles per cell seen after DOX exposure corresponded with the level of drug resistance achieved by the LZ cell lines studied. DOX concentration-response experiments revealed that vesicle formation exhibited a biphasic relationship, with an initial rapid increase followed by a plateau where no further increase was observed. Time-course studies in LZ-100 cells revealed that the maximum number of DOX-containing vesicles per cell occurred 3-4 hr following initiation of DOX treatment. Radiation exposure (10 Gy) immediately preceding DOX treatment decreased the number of vesicles formed in LZ-100 cells by more than one-half and altered the subcellular distribution of DOX from an almost exclusively cytoplasmic to a homogeneous nuclear/cytoplasmic distribution. This redistribution was not a result of radiation inhibition of P-gp efflux. The inhibitory effect of radiation on vesicle formation increased with increasing radiation dose up to 10 Gy. Drug-containing vesicles were also observed in LZ-100 cells following exposure to mitoxantrone or daunorubicin (to which LZ-100 cells are also resistant), but fewer vesicles were observed than with DOX. These studies demonstrate that the drug sequestration phenomenon (i) occurs in cells exhibiting widely different levels of drug resistance, (ii) correlates with the level of drug resistance in LZ cell lines, (iii) occurs rapidly following exposure to DOX, mitoxantrone, or daunorubicin, and (iv) can be inhibited by irradiation.(ABSTRACT TRUNCATED AT 400 WORDS)

An enhanced ability for transforming adriamycin into a noncytotoxic form in a multidrug-resistant cell line (LZ-8).

Multidrug-resistant LZ-8 cells are 9000-fold more resistant to Adriamycin (ADRM) exposure than wild-type V79 cells. To understand more about the mechanisms producing such high level resistance, we tested whether LZ-8 cells inactivate ADRM toxicity to a greater extent than wild-type V79 cells. ADRM was recovered from (1) culture media of wild-type V79 and ADRM-resistant LZ-8 cells; (2) V79 and LZ-8 cells; and (3) LZ-8 cell plasma membrane, and the cytotoxicity was determined by treating V79 cells for 1 hr with a known concentration of the recovered ADRM. ADRM obtained from LZ-8 cells or its culture medium exhibited less cytotoxicity than that recovered from V79 cells or its culture medium. ADRM extracted from LZ-8 cell plasma membrane was noncytotoxic. HPLC analysis revealed that the extracted ADRM was structurally changed compared to stock ADRM. The retention time in the column was 7 min for stock ADRM, and 23 min for the recovered ADRM. Thus, LZ-8 cells have an increased ability to transform ADRM into a noncytotoxic form compared to wild-type V79 cells. This transformation involves structural conversion into a previously unidentified ADRM metabolite. The greatly increased survival of LZ-8 cells compared to V79 cells after ADRM treatment is due to at least two mechanisms: (1) an enhanced ability to inactivate the cytotoxicity of ADRM, and (2) increased drug efflux resulting from the amplification and overexpression of the pgp 1 gene in these cells. Our results suggest the possibility that P-glycoprotein participates in drug binding/inactivation in addition to serving as a drug efflux pump.

Interaction between radiation and drug damage in mammalian cells. VI. Radiation and doxorubicin age-response function of doxorubicin-sensitive and -resistant Chinese hamster cells.

A comparative study of the radiation and/or doxorubicin (DOX) survival response for synchronous populations of Chinese hamster V79 cells and two DOX-resistant variants (77A and LZ-8) was performed. The greatest cellular radiation sensitivity was observed in mitosis, while the greatest resistance was observed during late S phase for the three cell lines. The variation in radiation response throughout the cell cycle was expressed as a change in the width of the shoulder of the survival curves (Dq) with little change in D0. This suggests that each phase of the cell cycle has a different capacity for accumulation of radiation injury. The radiation age-response function for the three cell lines revealed that 77A and LZ-8 cells were more radiosensitive than V79 cells throughout the cell cycle. Exposure of synchronous populations to DOX (1.84 microM for V79, 9.21 microM for 77A, and 921 microM for LZ-8) for 1 h as a function of cell cycle phase revealed that V79, 77A, and LZ-8 cells exhibited the greatest sensitivity to DOX in mitosis and the most resistance to DOX during S phase, as indicated by the differences in the slope of the initial component of the survival curve. Levels of P-glyco-protein (P-gp) are probably not a factor contributing to DOX age-response function since P-gp levels remain constant throughout the cell cycle in all three cell lines. Synchronous populations of V79, 77A, and LZ-8 cells sequentially treated with DOX and radiation at various cell cycle phases were also analyzed. The results showed that the interaction between radiation and DOX damage resulted in a reduced cellular capacity for the accumulation of radiation damage throughout the cell cycle, as indicated by a decrease in the width of the shoulder of the survival curve. Overall, both DOX-sensitive V79 cells and DOX-resistant 77A and LZ-8 cells exhibited (1) a similar age-response function for radiation or DOX, and (2) no differences in the effects of DOX on radiation-induced damage throughout the cell cycle. These results indicate that acquired resistance to DOX associated with increased levels of P-gp in the cell membrane did not appear to affect the age-response function for radiation or DOX, and the nature of the interaction between damage caused by radiation and DOX was also not affected.