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Interaction of rhodamine 123 with mitochondria isolated from drug-sensitive and -resistant friend leukemia cells
qol. 127, No. 3,1985
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICA]IUNb
Pages 1039-1044
March 2 9 , 1 9 8 5
INTERACTION OF RHODAMINE 123 WITH MITOCHONDRIA ISOLATED FROM DRUGSENSITIVE AND -RESISTANT FRIEND LEUKEMIA CELLS Samir
Abou-Khalil 1., Wafa H. Abou-Khalil 1, L i l i a n Planas 2, Haim Tapiero 3, and Theodore J. Lampidis 2
Departments of ~ e d i c i n e and 2Oncology (Comprehensive Cancer Center), University of Miami School of Medicine, Miami, FL 33101 3D~partment de Pharmacologie Cellulaire, Institut de Cancgrologie et d'Immunog~n~tique (INSERM U-50) 94804 Villejuif, France Received February 19, 1985
Summary. Mitochondria isolated from Friend leukemia cell lines sensitive (FS) and resistant (FR) to rhodamine 123 (Rho123), showed respiratory control and ADP/O ratios indicative of well-coupled oxidative phosphorylation activity. When Rho123 was added to mitochondria from both cell lines, respiratory State 4 increased. The increase was higher in mitochondria isolated from resistant than from sensitive cells. Respiratory State 3 was slightly more inhibited by Rho123 in resistant than in sensitive cell mitochondria (98 and 82% inhibition, respectively). ~ i l e it is not clear how the uncoupling-like effects of Rho123 on State 4 contribute to cellular toxicity, our results indicate that differential cellular sensitivity to the drug does not correlate with inhibition of oxidative phosphorylation in mitochondria isolated from drug-sensitive and -resistant cells, o 1985 A c a d ~ i c Press, Inc.
The fluorescent mitochondrial probe rhodamine 123 (Rho123) has been shown to be selectively toxic to a number of tumorigenic (carcinoma) as compared to non-tumorigenic epithelial cell lines (1-3).
It was also shown that carcinoma
cells, when treated with Rho123, specifically accumulate and retain the drug in their mitochondria; whereas, in non-tumorigenic cells accumulation is less and their mitochondria lose the dye very quickly (4). Recently, it has been reported that Friend leukemia cells resistant to adriamycin are cross-resistant to Rho123 (5,6).
The accumulation and retention
of Rho123 in sensitive and resistant FLC parallel those found in carcinoma and non-tumorigenic cells.
These observations gave us an opportunity to study the
interaction of Rho123 with mitochondria from cells of the same origin with
* Author to whom correspondence should be addressed
1039
0006-291X/85 $1.50 Copyright © 1985 by Academtc Press, Inc. All rlghts of reproduction in any form reserved.
Vol. 127, No. 3, 1985
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
different sensitivities to the drug as opposed to mitochondria isolated from tumorigenic and non-tumorigenic epithelial cells of separate lineage (1-3). Through use of this cell system we were able to address the following question:
Is the cellular selective toxicity of Rho123 due to inherent dif-
ferences at the mitochondrial level?
To answer that question we have isolated
well-coupled mitochondria from drug-sensitive and -resistant Friend leukemia cells and determined the interaction of Rho123 with mitochondrial respiratory States 4 and 3.
The results indicate that mitochondrial oxidative phosphoryl-
ation does not appear to be involved in controlling differential sensitivity to the drug at the cellular level. MATERIALS AND METHODS pr@~aration of Cells. Rhodamine sensitive and rhodamine resistant Friend leukemia cells as described previously (6) were cultured in Eagle's Minimal Essential medium (GIBCO, Grand Island, N.Y.) supplemented with 10% fetal calf serum using 150 cm 2 tissue culture flasks incubated at 37°C under humidified 5% CO 2 atmosphere. Cells were harvested by centrifugation (1200 rpm for i0 min) when they reached a concentration of 2.5 to 3 x 106 cells/ml. Following centrifugation, i x 109 cells were washed in 30 ml of grinding medium consisting of 250 mM sucrose, 2 mM EDTA, and 1 mg/ml of bovine serum albumin, adjusted to pH 7.4 with KOH. Washing was followed by centrifugation and resuspension of the pelleted cells in the grinding medium to a concentation of 75 x 106 cells/ ml which were used for the preparation of mitochondria. Isolation of Mitochondria from Cells. Cells were fractionated by sonication at ice-cold temperature with the probe of a Fisher Sonic Dismembrator set at 60% output. A six-second sonication was repeated twice with a oneminute interval, and resulted in 90 to 95% disrupted cells. The sonicated suspension was immediately centrifuged at 800 x g for 12 minutes and the supernatant filtered through three layers of cheesecloth then centrifuged at 8500 x g for 14 minutes to obtain the mitochondrial pellet. This pellet was washed in the grinding medium (i x 109 cells/3 ml), centrifuged at 8500 x g for 14 minutes and resuspended in the grinding medium to give a final concentration of 35 to 40 mg of mitochondrial protein/ml. All procedures of isolation were conducted at 4°C. Protein Determination. Mitochondrial protein was determined by the Biuret nethod (7) using bovine serum albumin (fraction V) as a standard. Measurement of Oxidativ e Phosphorylation. Oxidative phosphorylation was ~etermined by oxypolarography as previously described (8). The reactions were :arried out in a 2-ml medium consisting of 150 mM sucrose, 25 mM glycylglycine, $0 mM KCI, and 1 mg/ml of bovine serum albumin, adjusted to pH 7.4 with KOH. ~etabolic States 4 and 3 were determined according to (9). RESULTS AND DISCUSSION Oxidative P h o s p h o r x l a t i o n o f Mitochondria Isolated from Cells in Culture. igures IA and 2A depict representative oxypolarographic tracings of isolated itochondria from Friend sensitive (FS) and Friend resistant (FR) leukemia ~lls, respectively.
These tracings are characteristic of well-coupled oxi-
Vol. 127, No. 3, 1985
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FS.Mito (5)Jg/ml)FS.Mito -~I-.-ADP ~ADP
FR.Mito (5.pg/ml)FR.Mito
Rh
Rh
---~-ADP ~x~,Dp
®
® Figure I.
Representative tracings of oxygen consumption by mitochondria of sensitive Friend leukemic cells (FS Mito) as measured by oxypolarography in the absence (A) or the presence (B) of rhodamine 123. Mitochondria (i mg protein), isolated from cells cultured as described under Materials and Methods, were incubated at 30°C in a 2-ml reaction medium consisting of 150 mM sucrose, 40 mM KCI, 25 mM glycylglycine, and I mg/ml of bovine serum albumin (pH 7.4); with i0 mM succinate, i ~g/mg protein of rotenone, and 5 mM phosphate added to the medium. ADP (180 nmol), 2,4-dinitrophenol or DNP (0.i mM), and rhodamine 123 or Rh (5 pg/ml) were added as indicated by arrows. The numbers along the traces give the initial rates of oxygen consumption expressed in nanogram-atoms oxygen/ min/mg mitoehondrial protein.
Figure 2.
Representative tracings of oxygen consumption by mitochondria of resistant Friend leukemic cells (FR Mito) in the absence (A) or the presence (B) of rhodamine 123 (Rh). Experimental conditions were as in Fig. I.
dative phosphorylation activity as evidenced by the following parameters:
a.
When oxidizing succinate, mitochondria from both cells gave an ADP/O ratio of 1.9, close to the theoretical value of 2.0; b. their respiratory control ratios ranged from 5 to 7; and c. the addition of 2,4-dinitrophenol consistently resulted in uncoupling.
The addition of 5 ~g/ml of Rho123 to mitochondria
from each cell type for two minutes altered respiratory State 3 and State 4 in both (Figs. IB & 2B).
Increasing the incubation time with the drug up to i0
minutes gave similar results (not shown).
Moreover, uncoupling by dinitro-
phenol of both mitochondria treated with the drug was not significantly affected (Figs. IB & 2B). Effects of Different Concentrations of Rhodamine 123 on Mitochondria.
The
addition of the drug at a concentration range varying between 0.5 to I0 pg/ml
1041
Vol. 127, No. 3, 1985
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A
T
200
,so
i ~ I/
i
N toe ~
i
i
B
I J I
Figure 3.
i
k
, , 3 5 RHODAMINE
FR Mito
[ IO
123 (yg/rnl)
Rhodamine 123 c o n c e n t r a t i o n dependent e f f e c t s on r e s p i r a t o r y S t a t e 4(A) and State 3(B) o f m i t o c h o n d r i a i s o l a t e d from b o t h s e n s i t i v e (FS) and resistant (FR) Friend leukemia cells. Values are means of three to six assays ~ S.E. computed under the conditions of Figures 1 and 2. Due to the different effect of rhodamine 123 on State ~, the percent of control of respiratory State 3 was calculated by comparing State 3 relative rate (respiratory rate of recorded State 3 - State 4) to that of the normal control.
to mitochondria from both FS and FR cells produced the following effects: i.
At 3 Ng/ml of drug, respiratory State 4 in mitochondria isolated
from FS cells was slightly increased and higher doses had no further effect. This slight uncoupling-like activity observed with FS mitochondria was similar to that observed with rat liver mitochondria (unpublished data and Refs. i0 and ii); however, such activity was more pronounced with FR mitochondria which reached a 2-fold increase at I0 ~g/ml of the drug (Fig. 3A) indicating that those organelles were more sensitive to the uncoupling-like effect of Rho123. 2.
Respiratory State 3 of mitochondria from both FS and FR cells was
~radually inhibited by increasing concentrations of Rho123.
Greater than 80%
nhibition was reached in both FS and FR mitochondria with i0 ~g/ml of the rug (Fig. 3B).
The apparent difference in the degree of inhibition of State 3
etween mitochondria from both cell types as shown in Fig. 3B does not parallel
1042
Vol. 127, No. 3, 1985
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS5
Table i. Comparative effects of rhodamine 123 and rhodamine 116 on mitochondria isolated from sensitive (FS) and resistant (FR) Friend leukemia cells FS Mitochondria
FR Mitochondria
Percent of Control
Addition (i0 ~g/ml)
State 4
Rhodamine 123
125 ~ 3
Rhodamine 116
95 ~ 6
State 3
18 ~
7
i00 ~ 12
Percent of control was determined as in Figure 3. two to six assays ± S.E.
State 4
State 3
211 ~ 17
0
103 ~
3
90
Values represent means of
the difference in Rho123 selective toxicity as observed at the cellular level (1-6).
Rather, it argues against the involvement of mitochondria in that se-
lectivity.
In fact our data support the recent findings that Rho123-sensitive
cells accumulate significantly more drug (5,6) than Rho123-resistant FLC and that this may be the basis for the selective toxicity. Comparative Effects of Rhodamine 123 and Rhodamine 116.
Table i shows
that in contrast to Rho123, the use of the dye rhodamine 116, which is an uncharged analogue, did not result in any effect on either State 4 or State 3 in both mitochondria.
This appears to be in agreement with the idea that the
positive charge of Rho123 is necessary for its translocation in respiring mitochondria (12), while
the neutral dye seems to remain ineffective outside
the mitochondria. In conclusion, well-coupled mitochondria were isolated from two Friend leukemia cell lines grown in culture.
Although these cell lines were character-
ized by differential sensitivity to the mitochondrial probe Rho123, the interaction of this drug with mitochondrial oxidative phosphorylation showed no correlation between cellular and mitochonrial sensitivity.
Unexpectedly,
this
activity was slightly more affected in mitochondria from resistant cells as compared to those from sensitive cells.
While this is a strong indication that
differential cellular sensitivity does not depend on differences residing at
1043
Vol. 127, No. 3, 1985
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the level of mitochondrial oxidative phosphorylation several other mitochondrial activities, including the observed uncoupling-like effect by Rho123, might be involved and remain to be explored. ACKNOWLEDGMENTS This work was supported in part by NIH Grant CA37109 to T.J.L. REFERENCES I. 2. 3. 4.
5. 6. 7. 8. 9. 10. 11. 12.
Lampidis, T.J., Bernal, S.D., Summerhayes, I.C., and Chen, L.B. (1982) Ann, N.Y. Acad. Sci. 397, 299-301. Bernal, S.D., Lampidis, T.J., Summerhayes, I.C., and Chen, L.B. (1982) Science 218, 1117-1119. Lampidis, T.J., Bernal, S.D., Summerhayes, I.C., and Chen, L.B. (1983) Cancer Res. 43, 716-720. Summerhayes, I.C., Lampidis, T.J., Bernal, D.S., Nadakavukaren, J.J., Nadakavukaren, K.K., Shepherd, E.L., and Chen, L.B. (1982) Proc. Natl. Acad. Sci. U.S.A., 79, 5292-5296. Lampidis, T.J., Munck, J.N., Bennoun, M., and Tapiero, H. (1984) Proc. Am. Assoc. Cancer Res. 25, 340. Tapiero, H., Munck, J.N., Fourcade, A., and Lampidis, T.J., (1984) Cancer Res. 44, 5544-5549. Allan, G., Gornall, A.G., Bardawill, C.J., and David, M.M. (1949) J. Biol. Chem. 177, 751-766. Abou-Khalil, S., Abou-Khalil, W.H., and Yunis, A.A. (1981) Arch. Biochem. Biophys. 209, 460-464. Chance, B., and Williams, G.R. (1956) Adv. Enzymol. 17, 65-134. Lampidis, T.J., Salet, C., Moreno, G., and Chen, L.B. (1984) Agents and Actions, 14, 3-10. Modica-Napolitano, J.S., Weiss, M.J., Chen, L.B. and Aprille, J.R. (1983) Biochem. Biophys. Res. Commun. 118, 717-723. Johnson, L.V., Walsh, M.L., Bockus, B.J., and Chen, L.B. (1981) J. Cell Biol. 88, 526-535.
I044
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICA]IUNb
Pages 1039-1044
March 2 9 , 1 9 8 5
INTERACTION OF RHODAMINE 123 WITH MITOCHONDRIA ISOLATED FROM DRUGSENSITIVE AND -RESISTANT FRIEND LEUKEMIA CELLS Samir
Abou-Khalil 1., Wafa H. Abou-Khalil 1, L i l i a n Planas 2, Haim Tapiero 3, and Theodore J. Lampidis 2
Departments of ~ e d i c i n e and 2Oncology (Comprehensive Cancer Center), University of Miami School of Medicine, Miami, FL 33101 3D~partment de Pharmacologie Cellulaire, Institut de Cancgrologie et d'Immunog~n~tique (INSERM U-50) 94804 Villejuif, France Received February 19, 1985
Summary. Mitochondria isolated from Friend leukemia cell lines sensitive (FS) and resistant (FR) to rhodamine 123 (Rho123), showed respiratory control and ADP/O ratios indicative of well-coupled oxidative phosphorylation activity. When Rho123 was added to mitochondria from both cell lines, respiratory State 4 increased. The increase was higher in mitochondria isolated from resistant than from sensitive cells. Respiratory State 3 was slightly more inhibited by Rho123 in resistant than in sensitive cell mitochondria (98 and 82% inhibition, respectively). ~ i l e it is not clear how the uncoupling-like effects of Rho123 on State 4 contribute to cellular toxicity, our results indicate that differential cellular sensitivity to the drug does not correlate with inhibition of oxidative phosphorylation in mitochondria isolated from drug-sensitive and -resistant cells, o 1985 A c a d ~ i c Press, Inc.
The fluorescent mitochondrial probe rhodamine 123 (Rho123) has been shown to be selectively toxic to a number of tumorigenic (carcinoma) as compared to non-tumorigenic epithelial cell lines (1-3).
It was also shown that carcinoma
cells, when treated with Rho123, specifically accumulate and retain the drug in their mitochondria; whereas, in non-tumorigenic cells accumulation is less and their mitochondria lose the dye very quickly (4). Recently, it has been reported that Friend leukemia cells resistant to adriamycin are cross-resistant to Rho123 (5,6).
The accumulation and retention
of Rho123 in sensitive and resistant FLC parallel those found in carcinoma and non-tumorigenic cells.
These observations gave us an opportunity to study the
interaction of Rho123 with mitochondria from cells of the same origin with
* Author to whom correspondence should be addressed
1039
0006-291X/85 $1.50 Copyright © 1985 by Academtc Press, Inc. All rlghts of reproduction in any form reserved.
Vol. 127, No. 3, 1985
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
different sensitivities to the drug as opposed to mitochondria isolated from tumorigenic and non-tumorigenic epithelial cells of separate lineage (1-3). Through use of this cell system we were able to address the following question:
Is the cellular selective toxicity of Rho123 due to inherent dif-
ferences at the mitochondrial level?
To answer that question we have isolated
well-coupled mitochondria from drug-sensitive and -resistant Friend leukemia cells and determined the interaction of Rho123 with mitochondrial respiratory States 4 and 3.
The results indicate that mitochondrial oxidative phosphoryl-
ation does not appear to be involved in controlling differential sensitivity to the drug at the cellular level. MATERIALS AND METHODS pr@~aration of Cells. Rhodamine sensitive and rhodamine resistant Friend leukemia cells as described previously (6) were cultured in Eagle's Minimal Essential medium (GIBCO, Grand Island, N.Y.) supplemented with 10% fetal calf serum using 150 cm 2 tissue culture flasks incubated at 37°C under humidified 5% CO 2 atmosphere. Cells were harvested by centrifugation (1200 rpm for i0 min) when they reached a concentration of 2.5 to 3 x 106 cells/ml. Following centrifugation, i x 109 cells were washed in 30 ml of grinding medium consisting of 250 mM sucrose, 2 mM EDTA, and 1 mg/ml of bovine serum albumin, adjusted to pH 7.4 with KOH. Washing was followed by centrifugation and resuspension of the pelleted cells in the grinding medium to a concentation of 75 x 106 cells/ ml which were used for the preparation of mitochondria. Isolation of Mitochondria from Cells. Cells were fractionated by sonication at ice-cold temperature with the probe of a Fisher Sonic Dismembrator set at 60% output. A six-second sonication was repeated twice with a oneminute interval, and resulted in 90 to 95% disrupted cells. The sonicated suspension was immediately centrifuged at 800 x g for 12 minutes and the supernatant filtered through three layers of cheesecloth then centrifuged at 8500 x g for 14 minutes to obtain the mitochondrial pellet. This pellet was washed in the grinding medium (i x 109 cells/3 ml), centrifuged at 8500 x g for 14 minutes and resuspended in the grinding medium to give a final concentration of 35 to 40 mg of mitochondrial protein/ml. All procedures of isolation were conducted at 4°C. Protein Determination. Mitochondrial protein was determined by the Biuret nethod (7) using bovine serum albumin (fraction V) as a standard. Measurement of Oxidativ e Phosphorylation. Oxidative phosphorylation was ~etermined by oxypolarography as previously described (8). The reactions were :arried out in a 2-ml medium consisting of 150 mM sucrose, 25 mM glycylglycine, $0 mM KCI, and 1 mg/ml of bovine serum albumin, adjusted to pH 7.4 with KOH. ~etabolic States 4 and 3 were determined according to (9). RESULTS AND DISCUSSION Oxidative P h o s p h o r x l a t i o n o f Mitochondria Isolated from Cells in Culture. igures IA and 2A depict representative oxypolarographic tracings of isolated itochondria from Friend sensitive (FS) and Friend resistant (FR) leukemia ~lls, respectively.
These tracings are characteristic of well-coupled oxi-
Vol. 127, No. 3, 1985
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FS.Mito (5)Jg/ml)FS.Mito -~I-.-ADP ~ADP
FR.Mito (5.pg/ml)FR.Mito
Rh
Rh
---~-ADP ~x~,Dp
®
® Figure I.
Representative tracings of oxygen consumption by mitochondria of sensitive Friend leukemic cells (FS Mito) as measured by oxypolarography in the absence (A) or the presence (B) of rhodamine 123. Mitochondria (i mg protein), isolated from cells cultured as described under Materials and Methods, were incubated at 30°C in a 2-ml reaction medium consisting of 150 mM sucrose, 40 mM KCI, 25 mM glycylglycine, and I mg/ml of bovine serum albumin (pH 7.4); with i0 mM succinate, i ~g/mg protein of rotenone, and 5 mM phosphate added to the medium. ADP (180 nmol), 2,4-dinitrophenol or DNP (0.i mM), and rhodamine 123 or Rh (5 pg/ml) were added as indicated by arrows. The numbers along the traces give the initial rates of oxygen consumption expressed in nanogram-atoms oxygen/ min/mg mitoehondrial protein.
Figure 2.
Representative tracings of oxygen consumption by mitochondria of resistant Friend leukemic cells (FR Mito) in the absence (A) or the presence (B) of rhodamine 123 (Rh). Experimental conditions were as in Fig. I.
dative phosphorylation activity as evidenced by the following parameters:
a.
When oxidizing succinate, mitochondria from both cells gave an ADP/O ratio of 1.9, close to the theoretical value of 2.0; b. their respiratory control ratios ranged from 5 to 7; and c. the addition of 2,4-dinitrophenol consistently resulted in uncoupling.
The addition of 5 ~g/ml of Rho123 to mitochondria
from each cell type for two minutes altered respiratory State 3 and State 4 in both (Figs. IB & 2B).
Increasing the incubation time with the drug up to i0
minutes gave similar results (not shown).
Moreover, uncoupling by dinitro-
phenol of both mitochondria treated with the drug was not significantly affected (Figs. IB & 2B). Effects of Different Concentrations of Rhodamine 123 on Mitochondria.
The
addition of the drug at a concentration range varying between 0.5 to I0 pg/ml
1041
Vol. 127, No. 3, 1985
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A
T
200
,so
i ~ I/
i
N toe ~
i
i
B
I J I
Figure 3.
i
k
, , 3 5 RHODAMINE
FR Mito
[ IO
123 (yg/rnl)
Rhodamine 123 c o n c e n t r a t i o n dependent e f f e c t s on r e s p i r a t o r y S t a t e 4(A) and State 3(B) o f m i t o c h o n d r i a i s o l a t e d from b o t h s e n s i t i v e (FS) and resistant (FR) Friend leukemia cells. Values are means of three to six assays ~ S.E. computed under the conditions of Figures 1 and 2. Due to the different effect of rhodamine 123 on State ~, the percent of control of respiratory State 3 was calculated by comparing State 3 relative rate (respiratory rate of recorded State 3 - State 4) to that of the normal control.
to mitochondria from both FS and FR cells produced the following effects: i.
At 3 Ng/ml of drug, respiratory State 4 in mitochondria isolated
from FS cells was slightly increased and higher doses had no further effect. This slight uncoupling-like activity observed with FS mitochondria was similar to that observed with rat liver mitochondria (unpublished data and Refs. i0 and ii); however, such activity was more pronounced with FR mitochondria which reached a 2-fold increase at I0 ~g/ml of the drug (Fig. 3A) indicating that those organelles were more sensitive to the uncoupling-like effect of Rho123. 2.
Respiratory State 3 of mitochondria from both FS and FR cells was
~radually inhibited by increasing concentrations of Rho123.
Greater than 80%
nhibition was reached in both FS and FR mitochondria with i0 ~g/ml of the rug (Fig. 3B).
The apparent difference in the degree of inhibition of State 3
etween mitochondria from both cell types as shown in Fig. 3B does not parallel
1042
Vol. 127, No. 3, 1985
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS5
Table i. Comparative effects of rhodamine 123 and rhodamine 116 on mitochondria isolated from sensitive (FS) and resistant (FR) Friend leukemia cells FS Mitochondria
FR Mitochondria
Percent of Control
Addition (i0 ~g/ml)
State 4
Rhodamine 123
125 ~ 3
Rhodamine 116
95 ~ 6
State 3
18 ~
7
i00 ~ 12
Percent of control was determined as in Figure 3. two to six assays ± S.E.
State 4
State 3
211 ~ 17
0
103 ~
3
90
Values represent means of
the difference in Rho123 selective toxicity as observed at the cellular level (1-6).
Rather, it argues against the involvement of mitochondria in that se-
lectivity.
In fact our data support the recent findings that Rho123-sensitive
cells accumulate significantly more drug (5,6) than Rho123-resistant FLC and that this may be the basis for the selective toxicity. Comparative Effects of Rhodamine 123 and Rhodamine 116.
Table i shows
that in contrast to Rho123, the use of the dye rhodamine 116, which is an uncharged analogue, did not result in any effect on either State 4 or State 3 in both mitochondria.
This appears to be in agreement with the idea that the
positive charge of Rho123 is necessary for its translocation in respiring mitochondria (12), while
the neutral dye seems to remain ineffective outside
the mitochondria. In conclusion, well-coupled mitochondria were isolated from two Friend leukemia cell lines grown in culture.
Although these cell lines were character-
ized by differential sensitivity to the mitochondrial probe Rho123, the interaction of this drug with mitochondrial oxidative phosphorylation showed no correlation between cellular and mitochonrial sensitivity.
Unexpectedly,
this
activity was slightly more affected in mitochondria from resistant cells as compared to those from sensitive cells.
While this is a strong indication that
differential cellular sensitivity does not depend on differences residing at
1043
Vol. 127, No. 3, 1985
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the level of mitochondrial oxidative phosphorylation several other mitochondrial activities, including the observed uncoupling-like effect by Rho123, might be involved and remain to be explored. ACKNOWLEDGMENTS This work was supported in part by NIH Grant CA37109 to T.J.L. REFERENCES I. 2. 3. 4.
5. 6. 7. 8. 9. 10. 11. 12.
Lampidis, T.J., Bernal, S.D., Summerhayes, I.C., and Chen, L.B. (1982) Ann, N.Y. Acad. Sci. 397, 299-301. Bernal, S.D., Lampidis, T.J., Summerhayes, I.C., and Chen, L.B. (1982) Science 218, 1117-1119. Lampidis, T.J., Bernal, S.D., Summerhayes, I.C., and Chen, L.B. (1983) Cancer Res. 43, 716-720. Summerhayes, I.C., Lampidis, T.J., Bernal, D.S., Nadakavukaren, J.J., Nadakavukaren, K.K., Shepherd, E.L., and Chen, L.B. (1982) Proc. Natl. Acad. Sci. U.S.A., 79, 5292-5296. Lampidis, T.J., Munck, J.N., Bennoun, M., and Tapiero, H. (1984) Proc. Am. Assoc. Cancer Res. 25, 340. Tapiero, H., Munck, J.N., Fourcade, A., and Lampidis, T.J., (1984) Cancer Res. 44, 5544-5549. Allan, G., Gornall, A.G., Bardawill, C.J., and David, M.M. (1949) J. Biol. Chem. 177, 751-766. Abou-Khalil, S., Abou-Khalil, W.H., and Yunis, A.A. (1981) Arch. Biochem. Biophys. 209, 460-464. Chance, B., and Williams, G.R. (1956) Adv. Enzymol. 17, 65-134. Lampidis, T.J., Salet, C., Moreno, G., and Chen, L.B. (1984) Agents and Actions, 14, 3-10. Modica-Napolitano, J.S., Weiss, M.J., Chen, L.B. and Aprille, J.R. (1983) Biochem. Biophys. Res. Commun. 118, 717-723. Johnson, L.V., Walsh, M.L., Bockus, B.J., and Chen, L.B. (1981) J. Cell Biol. 88, 526-535.
I044