Our long term goal is to understand how radiation and drugs interact in cells. To study this, a series of multidrug resistant Chinese hamster cell lines were produced by selection in increasing levels of ADRM, culminating in LZ-100 (grown in 100 mu-g/ml). LZ-100 cells exhibit three mechanisms of resistance: enhanced drug efflux via P-glycoprotein (P-gp), increased inactivation of ADRM cytoxicity by structural modification of the molecule, and sequestration of ADRM into vesicles. The hypothesis to be tested is that radiation exposure produces cell membrane effects that impair the sequestration of ADRM into vesicles by LZ-100 after ADRM treatment. The specific .
aims are to: 1) characterize the drug sequestration mechanism in LZ-100; 2) determine the effect of radiation on the drug sequestration mechanism; and 3) determine the relative importance of radiation induced membrane damage in affecting vesicle formation.
Aim 1 will be accomplished by utilizing electron microscopy to determine the origin of vesicles, determining the presence of P-gp in isolated vesicles using antibody staining and Western blotting, blocking P-gp externally on LZ cell membrane to determine if vesicle formation is inhibited and identifying vesicle contents using HPLC and searching for lysosomal enzymes.
Aim 2 will be accomplished by quantitating the effect of radiation on vesicle formation using light and electron microscopy, quantitating cell survival after radiation/ADRM treatment, and quantitating membrane turnover.
Aim 3 will be accomplished by comparing the ability of membrane damaging agents with DNA damaging agents to inhibit vesicle formation in LZ-100. If X-irradiation does effect the drug sequestration mechanism in LZ-100, this suggests that the cell membrane is important in the cellular response to radiation. In addition, these studies will provide important information concerning how the drug sequestration mechanism functions and its significance to multidrug resistance, the function of P-gp in vesicle formation/function, the effect of radiation on the drug sequestration mechanism, and will allow new, combined (drug and radiation) therapeutic strategies for human cancer to be formulated.
