Kk. Millis et al., FORMATION, INTRACELLULAR-DISTRIBUTION AND EFFLUX OF GLUTATHIONE-BIMANE CONJUGATES IN DRUG-SENSITIVE AND DRUG-RESISTANT MCF-7 CELLS, Cancer chemotherapy and pharmacology, 40(2), 1997, pp. 101-111
The rate of reaction of monochlorobimane with glutathione (GSH) was me
asured in native human mammary MCF-7 adenocarcinoma cells (MCF-7wt) an
d sublines displaying resistance to 4-hydroperoxycyclophosphamide (MCF
-7hc) and adriamycin (MCF-7adr) prior to examination by epifluorescenc
e and confocal microscopy. After a 60-min incubation period at 37 degr
ees C, essentially all GSH was conjugated in the MCF-7wt and MCF-7adr
cell lines whereas only 80% of the GSH was conjugated in the MCF-7hc l
ine. All three lines displayed significant export of the conjugate fro
m the cell during this period, with the MCF-7adr line displaying the m
ost rapid efflux with 85% of the conjugate exported within 60 min. Epi
fluorescence microscopy detected an approximately 20% increase in inte
grated fluorescence intensity in the nuclear region in all three lines
. Confocal microscopy however, indicated that most of the cells examin
ed showed a homogeneous fluorescence distribution. The cells grown in
monolayers were found to be thicker in the nuclear region suggesting t
hat the observed increase in fluorescence intensity in the nuclear reg
ion in the images from epifluorescence microscopy was probably derived
from fluorescence from an out-of-focus plane. Cells depleted of GSH w
ith buthionine sulfoximine followed by treatment with mBCl showed sign
ificant fluorescence intensity resulting from nonspecific binding of t
his probe. These studies illustrate the need for measuring the rate of
GSH conjugate export and for determining probe specificity, and empha
sizes the need for using confocal techniques for the quantitative eval
uation of the distribution of intracellular fluorescence.