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67Ga and 59Fe uptake in human melanoma cells
lnrrrnrrr~~nol Jurrrnul a/ Nuclear Medicine end Biolay): Pergamon Press Lid 1979 Printed m Great Britain
Vol. 6. pp. 23 to 28
67Ga and 59Fe Uptake in Human Melanoma Cells* U. K. TERNER, H. WONG. A. A. NOUJAIM,? and J. R. HILL
B. C. LENTLE
Department of Nuclear Medicine, Cross Cancer Institute, Division of Bionucleonics. Radiopharmacy, Faculty of Pharmacy and Pharmaceutical Sciences.liniversity of Alberta and Department of Pathology, University of Alberta Hospital. Edmonton. Alberta. Canada (Receiwd 30 Srpternhvr 1978)
Human melanoma cells were incubated with 67Ga-transferrin and 59Fe-transferrin in the presence of free unlabeled iron as well as 59Fe and 67Ga and increasing PO:- ion concentration. It was observed that factors which influence the uptake of 59Fe by melanoma cells have a similar effect on the uptake of 67Ga by the tumor cells. Furthermore, it was noted that when melanoma cells were incubated with either ‘251-transferrin-59Feor ““I-transferrin-h7Ga the lz51 activity associated with the cells was similar in both cases *and remained constant over the various incubation times. However, the rate of uptake of ““Fe and “Ga by the tumor cells differed. The uptake of 59Fe was very rapid over the first Ifh. After that time the rate of uptake was reduced. while in the case of gallium the uptake was linear over the time period tested. This would suggest that 1’51-transferrin-59Fe has a higher affinity for the cell surface binding site than does ‘251-transferrin-h7Ga. It may also further suggest that for s9Fe the rate of transferrin receptor complexation is close to. or exceeds.the rate-limiting step which is responsible for transporting the 59Fe across the melanoma cell membrane. This does not appear to be the case with gallium.
INTRODUCTION GALLIUM-67 as the citrate has long been used
as a diagnostic agent in oncology. W h ile the exact mechanism of tumor
localization
is still
a subject of divergent opinions it would appear that metal binding and storage proteins such as transferrin, (1,2) lactoferrin’3’ and ferritint4’ do play a role in the transport and localization of “Ga. Bearing this in m ind we undertook to examine the similarities
and differences that
exist in the uptake of 67Ga and 59Fe by cultured melanoma cells in the presence of various “modifier” agents. These findings are the sub-
ject of this paper. METHODS AND MATERIALS A human melanoma tissue culture cell line was used in this investigation. This cell line has been established in the Cross Cancer Institute and is found to be gallium-avid. The tissue culture was maintained in RPMI-1640 growth medium to which was added 15% fetal calf serum, 100 units/ml penicillin, 100 pg/ml streptomycin, 0.29 mg/ml glutamine and 1 m M sodium pyruvate (a11obtained from Gibco.
Grand Island, N.Y.). Cell suspensions were obtained by washing the monolayer of cells in the incubstmn flask with versene butTer (Gibco) followed by a very brief treatment with 0.257; trypsin. The cells wzre then washed twice with lOm1 volume of LeiboLitz (L-15) medium and the final cell suspension consisted of L-15 medium containing 10” cells/ml. The viability of the cells was determined by their ability to exclude 0.17; trypan blue and usually the viability exceeded 98%. The cell suspension (10” cells) was then incubated at 37’C in a normal atmosphere, with approximately 0.1 /Xi b7Ga-citrate, “Ga-transferrin. ‘“Fetransferrin. ’“1-transferrin-” ‘Ga or I2 “I-transfcrrin-59Fe. At the end of each incubation period. cell viability was again determined and the cells were separated from the supernatant by centrifugation. The cells were washed twice with 0.99!,NaCl solution and the radioactivity of both the cell pellets and combined supernatants determined separately. A minimum of three replicates was used in each experimerit and each experiment was repeated two or three times. Labrliny of transferrin (4 59Fe-transferrin and b7Ga-transferrin.
Human apotransferrin (obtained from Sigma Biochemicals) dissolved in 0.9% NaCl solution 2 mg/ml was incubated with either h7Ga-citrate(NEN) or “FeCI,(NEN) at a pH adjusted to 7.4 with 0.1 N *This work was supported by a Research Grant NaOH solution. The incubate was allowed to stand overnight at room temperature. Separation of free from the National Cancer Institute of Canada. t To whom reprint requests should be addressed. s9Fe or “Ga from that bound to transferrin wao 23
achieved by means of a Sephadex G-50 column (Pharmacia) with 0.9”,, NaCl solution used as eluant. (b) Pwpwrrfiorl oj ‘“l-r~urz.~~~r~in-59Fe and 12’1twnsfiJrl-irv6’Gu Human apotransferrin (Sigma) was labeled with “‘1 by the ICI method. essentially as described by RHF.“) The ‘151-transferrin in 0.05 M HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid) 0.1 M KCI, pH 7.4 was then incubated with 59Fe-NTA (nitrilotriacetate) or “Ga-NTA. FeNTA was prepared by the addition of SO-loo&Ii of a ‘9FeCIJ solution in HCI to a 3 M excess of NTA solution. This mixture was allowed to stand for i h, and to this was added 2-5 mg “‘1-transferrin in 0.05 M HEPES 0.1 M KC1 pH 7.4. The final pH was adjusted to 7.4 with 0.1 M NaHCO,. The incubation mixture was allowed to stand overnight at room temperature (21 C). The “free” “‘Fe was separated from the “Fe bound to transferrin by means of a Sephadex G-50 (Pharmacia) 0.8 cm x 30cm column equilibrated with 0.1 M KCI. 0.05 M HEPES pH 7.4. The column was eluted with a similar solvent system. “Ga-NTA was prepared by acidifying “Ga-citrate (0.2-0.5 mCi) NEN with an equal volume of 8 N HCI to convert “‘Ga-citrate to “GaCl,. The mixture was applied to a 0.7 x 2.5 cm Dowex 1 x 4 ion exchange resin previously equilibrated Wh 8 N HCI. The column was eluted with 2-3 ml 8 N HCI to remove the free citrate followed by 0.1 N HCI which eluted the “Ga as chloride. The “GaCl, solution was first incubated for ) h with a three-fold molar excess of NTA, and the pH was then adjusted slowly with NaOH to pH 2.5-3.0. A 0.1 M solution of NaHCO, (IOC-200 ~1) was then added raising the pH to 5-6. To the reaction mixture was then added 2-5mg of human ‘““I-apotransferrin dissolved in l-l.5 ml of 0.1 M KCI, 0.05 M HEPES pH 7.4. The final pH of the incubate was adjusted to 7.4 with 0.1 M NaHCO, solution. After incubating overnight the reaction mixture was separated as described above. Final protein concentrations were determined by U.V. analysis (280 nm). The activities associated with the cell pellet and supernatant was determined by means of a Searle Model-l 195 Gamma Ray Spectrometer. Correction for crossover counts (s9Fe in I “1 channel or “Ga in “‘1 channel) was affected for each sample. in cases of dual-labeled samples.
RESULTS
AND
FIG. 1. Effect of increasing iron concentrations (0.010. 0.015, 0.030, 0.050, 0.090 and 0.18 pg/ml) on “Ga uptake by melanoma cells incubated with “Ga-transferrin. Each point is a mean & 1 SD. of three determinations after 3 h incubations.
transferrin to the melanoma cell membrane transport system. Next, the role of phosphate ions on the uptake of both “Ga and 59Fe was examined by incubating melanoma cells with “Ga-transferrin or 59Fe-transferrin. It has been shown (‘) that PO:- can remove Fe from transferrin and thus make it unavailable for transport across a cell membrane. Figure 2 clearly indicates that as the concentration of PO:- is increased in the incubation medium containing melanoma cells in the presence of either “‘Gatransferrin or s9Fe-transferrin there is a parallel decrease in uptake of both ions by the cultured tumor cells. This decrease in uptake
DISCUSSION
The influence of increasing the concentrations of iron concentration and its affect on the uptake of (“Ga by cells when incubated with 67Ga-transferrin is shown in Fig. 1. The melanoma cells were incubated with increasing concentrations of non-radioactive FeCl,. From Fig. 1 it can be clearly seen that Fe has an inhibitory effect on the uptake of 6JGa by melanoma cells incubated with “Ga-transferrin complex. This could be interpreted as competition of Fe for the same binding sites as those occupied by “Ga on the transferrin molecule. The displaced “Ga would then no longer be available for active transport by
pg Phosphote in Incubate (Iml)
Fig. 2. ElTect of increasing phosphate concentrations (60, 90, 120 and 180 pg/ml) in the incubation medium on the uptake of 67Ga(A) or 59Fe(o) by melanoma cells incubated with either “Ga-fransferrin or 59Fetransferrin respectively. The uptake of 59Fe(0) from 59FeCl, was also determined. Each point is the mean _+
1
S.D.
of
three
determinations
incubations.
after
3h
TABLE
1. Transferrin
Material “FeCI, (0.08 nCi)* “Fe-transferrint (0.02 PCi) 5yFe-transferrin + free ‘9Fe (0.08 LtCi) “‘Fe-transferrin + free 59Fe (0.16 nCi)
as carrier for “9Fe uptake in melanoma cells Activity in cell fraction”
0o increase above expected value --I__.
311 * 135 997 * 128 1616 a 135
24”,,
1928 k 51
23” 0
I’Mean * I S.D. of three determinations. * Uptake calculated as 0.23 1 0.20°/,/10h viable cells. t Amount of transferrin added was 0.024 mg/l ml of incubate containing live cells.
of both “Fe and “Ga would suggest that a similar phenomenon occurs with 67Ga-transferrin as is the case with 59Fe-transferrin. Since it has been suggested that transferrin is a carrier for both 59Fe and 67Ga, a series of experiments were conducted to define the role of transferrin in the active transport process.The findings for both 5QFeand 67Ga are illustrated in Tables 1 and 2 respectively. In the case of s9Fe uptake by melanoma cells the amount of 59Fe taken up by the cells from either 59FeCl, or ‘“Fe-transferrin was less than when similar concentrations were added simultaneously. In fact a 24:/, increase in uptake of radio-iron by the melanoma cells was observed, in excess of that taken up when FeC13 0.08 FCi, (0.024 mg) was added singly to the cell incubate. A strikingly similar effect was observed when “Ga-citrate and “Ga-transferrin were added in combination to the incubate (Table 2). The uptake again exceeded that determined for 67Ga-transferrin or “Ga-citrate alone.
IOh
Further experiments were carried out using specifically labeled 1Z51-transferrin-h7Ga and 1’51-transferrin-59Fe. These were designed to measure both the relative rate at which the ‘“Fe as well as thle (“Ga is taken up by the melanoma cells in the incubate, while also trying to determine the fate of the carrier protein. the 1251-labeled transferrin. For this study various concentrations of both lZ51-transferrin-59Fe and 1’51-transferrin-67Ga were incubated over varying time intervals. The results are summarized in Fig. 3 and Table 3. From the data given here it is apparent that both 59Fe and h7Ga are taken up quite rapidly by the melanoma cells. However, the rate of uptake of each isotope differs. The rate of uptake of 59Fe is very rapid for the first i # h with the uptake being 3.5% per 10’ live cells, but after that time period the rate of uptake seems to level off, so that at 4.5 h the uptake was 4.07: per IO6 cells when 0.018 mg ’“‘I-transferrin -59F~ewas used. By comparison, in the case of gallium the rate of uptake in-
2. The uptake of 67Ga by melanoma cells incubated with a constant concentration of “Ga-transferrin, plus increasing quantities of “Ga-citrate
TABLE
Material b7Ga-citrate (0.03 i&i) h7Ga-tran~ferrin (0.022 ptci) “‘Ga-transferrin + free 67Ga (0.03 PCi) h7Ga-transferrin + free 67Ga (0.06pCi) “Ga-transferrin + free 67Ga (0.12nCi)
Activity in cell fraction”
O0 increase above expected value
210 k 56 380 -t 20 645 5 65
+ 9.3”,,
1048 k 80
$- 3 1.O”;,
1465 2 150
$ 20. I”,,
‘Each value is the mean f 1 SD. of three determinations. Concentration of transferrin used-0045 mgin 1 ml incubate containing 1Oh live melanoma cells. Incubation time was 3 h.
26
U. K. Trrnrr.
H. Wang, A. il. Not/;aim. B. C. Lrnrle and J. R. Hill
FIG. 3. Uptake of 59Fe(o)and 123(o) in melanoma cells incubatedwith ‘251-transferrin-s9Fe. Also shown
is the uptake of 67Ga(A)and “‘I(A) in cells incubated with ’251-transferrin-h7Ga. Each point representsthe mean of six determinations.
creases steadily over the 4.5 h incubation period with an uptake of 3.3% when the same amount of ’251-transferrin-67Ga was added to lo6 cells in the 1 m l incubate. It is also interesting to note that while the uptake of 59Fe from ‘251-transferrin-59Fe is affected by the relative concentration of ’251-transferrin-59Fe added to the incubation medium, this does not appear to be the case with ‘251-transferrin-67Ga. In the case of gallium there appears to be no difference in the relative rate of uptake (expressed as a percentage of the total activity added) at the concentrations used in this experiment (Table 3). Thus both 59Fe and 67Ga appear to enter the melanoma cells using transferrin as a carrier molecule. Furthermore, factors which influence the uptake of one ion also affect the uptake of the other and in a similar manner. The observation that the addition of increasing concentrations of FeCl, (non radioactive) to the incubation medium containing 67Gatransferrin and melanoma cells cause a marked reduction of 67Ga uptake by the tumor cells suggests that iron competes for the same active transport site as the transferrin molecule. This implies that iron displaces the 67Ga from the active transport site on the transferrin, hence making it unavailable for active uptake by the melanoma cells. While HILL et ~1.“~’ found that the addition of very low concentrations of Fe to a leukocyte culture system actually enhanced the uptake of 67Ga from the incubation medium this may be explained by the fact that in these experiments all concentrations of Fe were low enough to permit occupation of protein binding sites not occupied by Ga. This could conceivably increase the affinity of cer-
tain carrier proteins, especially transferrin, for the membrane receptor sites.”‘.I 3, However, HILL rr (I/.“~’ also observed that at higher concentrations of Fe, the uptake of Ga by the culture cells was reduced. The fact that iron has a much higher binding constant with respect to the specific binding sites than other metal ions has been demonstrated by a number of workers.“.‘.“,’ 5.16)The reduction in uptake of 67Ga by human melanoma tissue culture cells in the presence of iron is consistent with the findings of HARRIS and SEPHTON’~) who used mouse myeloma cells as a model. The observation of others that phosphate ions affect dissociation of Fe from transferrinc6’ and Ga from transferrin’“! hence making these ions unavailable for transport across the cell membrane, is supported by our findings (Fig. 2). As the phosphate concentration increased in the incubation medium a parallel inhibition of uptake of both “Ga and “Fe by melanoma cells was observed. The reduction in uptake by cells incubated with either “Ga-transferrin or 59Fe-transferrin reflects the dissociation of 67Ga or 99Fe from transferrin. This would therefore make it unavailable for transport into the cell, which indeed was observed (Fig. 2). These observations further emphasize the active transport role played by transferrin and would suggest that positive diffusion of (“Ga across the membrane as suggested by Iro et al. (11) does play only a m inor if not insignificant role in the total uptake of gallium by tumors. GRAMS et ~1.‘~’observed a similar inhibitory effect by phosphate ions on the uptake of 67Ga by cultured leukemic cells. In the potentiation experiments of both 67Ga and 59Fe (Tables 1 and 2), the addition of 67Ga-citrate and 59FeC1, to their respective incubation medium resulted in a higher uptake than was expected from the active transport by labeled transferrin or passive diffusion. This would suggest that transferrin which has donated an ion to the cell membrane, or monometal labeled transferrin, could easily bind free 67Ga or 59Fe and transport it back to the cell membrane. Furthermore it has been shown” *.l 3*1‘.18) that transferrin which has both specific binding sites occupied has a much higher binding affinity than mono-metalic transferrin for the cell surface receptors, which would ultimately lead to the active transport of iron or gallium across the cell membrane. This could explain the accelerated increase in uptake of both 59Fe and 67Ga by the melanoma cells. This concept is further substantiated by the double-labeled transferrin studies using either ’251-transferrin-67Ga or ’251-transferrin-59Fe (Fig. 3. Table 3). The 1251 associated with the
67Ga and 59Fe uptake
in human mekunomu crl,‘s
cell fraction remained constant when expressed as a percentage of the total label in the incubate. This would suggest that in both cases the binding of labeled transferrin to the melanoma cell membrane is a function of the concentration of transferrin in the medium and would appear to be linear in the range of concentrations tested. However, the differences in the rate of uptake of 59Fe when compared to “Ga from the transferrin may signify that 59Fe labeled transferrin has a higher affinity for the tumor cell membrane than does “Ga-transferrin. This observation agrees with that of others.‘8.19’Moreover at higher concentrations the rate of uptake is reduced. suggesting that there may be a rate limiting step in the transport of 59Fe across the membrane. This does not appear to be the case in the uptake of “Ga from 1’51-transferrin-h7Ga (Fig. 3. Table 3). The relatively linear uptake of gallium by the tumor cells irrespective of the concentration used over the various time periods may in fact reflect a lower affinity for the transferrin membrane binding site in the case of galliumlabeled transferrin as compared to iron-transferrin. This lower affinity may in fact be below the rate limiting step of the membrane transport protein responsible for carrying the metal ion into the cell milieu. Also, if the theory of differing affinities for the membrane transferrin receptor is correct, then Ga-transferrin would only be able to dislodge newly formed apotransferrin from the surface binding sites of the melanoma cells at a rate slower than that of Fe-transferrin. While the factors which influence the uptake of iron from s9Fe-transferrin by melanoma cells have a similar effect on the uptake of gallium from “Ga-transferrin, the differences in the rate of uptake of each of the metal ions
can be explained by differing affinities which s9Fe-transferrin and 67Ga-transferrin have for the melanoma membrane binding site.
REFERENCES I. 2. 3. 4. 5. 6.
7. 8. 9. IO. 1 I. I 2. 13. 14. 15. 16. 17. 18. 19.
CLALSEN J.. ED~LINC; C. and FOGH J. Canw Rex 34, 193 I (I 974). GUNASIXERA S. W.. KING; L. J. and LAVENDER P. J. Clinicrr chirrl. Acru 39. 401 (1972). HOFFER P. B.. HUBERTY J. and KHAYAIN-BASHI H. J. nucl. Mrd. 18. 713 (1977). HEGGE F. N.. MAHLER D. J. and LARSEN S. M. .I. Jd. Med. 18. 937 (1977). REIF A. E. J. n~rcl. Mrd. 9. I48 (1968). SHAIX A. L.. OYAMA J.. REINHART R. W. and MILLER J. R. Proc. Sot. op. Bid. Add. 87. 443 (1954). BATES G. W., BILL~PS C. and SAI.TMAN P. J. hiol. Chcrn. 242. 2810 (1967). HARRIS A. W. and SEPHTON R. G. Car~ccr RKS. 37, 3634 (I 977). GRAMS R. A.. Wtw J. and GLI~)
Vol. 6. pp. 23 to 28
67Ga and 59Fe Uptake in Human Melanoma Cells* U. K. TERNER, H. WONG. A. A. NOUJAIM,? and J. R. HILL
B. C. LENTLE
Department of Nuclear Medicine, Cross Cancer Institute, Division of Bionucleonics. Radiopharmacy, Faculty of Pharmacy and Pharmaceutical Sciences.liniversity of Alberta and Department of Pathology, University of Alberta Hospital. Edmonton. Alberta. Canada (Receiwd 30 Srpternhvr 1978)
Human melanoma cells were incubated with 67Ga-transferrin and 59Fe-transferrin in the presence of free unlabeled iron as well as 59Fe and 67Ga and increasing PO:- ion concentration. It was observed that factors which influence the uptake of 59Fe by melanoma cells have a similar effect on the uptake of 67Ga by the tumor cells. Furthermore, it was noted that when melanoma cells were incubated with either ‘251-transferrin-59Feor ““I-transferrin-h7Ga the lz51 activity associated with the cells was similar in both cases *and remained constant over the various incubation times. However, the rate of uptake of ““Fe and “Ga by the tumor cells differed. The uptake of 59Fe was very rapid over the first Ifh. After that time the rate of uptake was reduced. while in the case of gallium the uptake was linear over the time period tested. This would suggest that 1’51-transferrin-59Fe has a higher affinity for the cell surface binding site than does ‘251-transferrin-h7Ga. It may also further suggest that for s9Fe the rate of transferrin receptor complexation is close to. or exceeds.the rate-limiting step which is responsible for transporting the 59Fe across the melanoma cell membrane. This does not appear to be the case with gallium.
INTRODUCTION GALLIUM-67 as the citrate has long been used
as a diagnostic agent in oncology. W h ile the exact mechanism of tumor
localization
is still
a subject of divergent opinions it would appear that metal binding and storage proteins such as transferrin, (1,2) lactoferrin’3’ and ferritint4’ do play a role in the transport and localization of “Ga. Bearing this in m ind we undertook to examine the similarities
and differences that
exist in the uptake of 67Ga and 59Fe by cultured melanoma cells in the presence of various “modifier” agents. These findings are the sub-
ject of this paper. METHODS AND MATERIALS A human melanoma tissue culture cell line was used in this investigation. This cell line has been established in the Cross Cancer Institute and is found to be gallium-avid. The tissue culture was maintained in RPMI-1640 growth medium to which was added 15% fetal calf serum, 100 units/ml penicillin, 100 pg/ml streptomycin, 0.29 mg/ml glutamine and 1 m M sodium pyruvate (a11obtained from Gibco.
Grand Island, N.Y.). Cell suspensions were obtained by washing the monolayer of cells in the incubstmn flask with versene butTer (Gibco) followed by a very brief treatment with 0.257; trypsin. The cells wzre then washed twice with lOm1 volume of LeiboLitz (L-15) medium and the final cell suspension consisted of L-15 medium containing 10” cells/ml. The viability of the cells was determined by their ability to exclude 0.17; trypan blue and usually the viability exceeded 98%. The cell suspension (10” cells) was then incubated at 37’C in a normal atmosphere, with approximately 0.1 /Xi b7Ga-citrate, “Ga-transferrin. ‘“Fetransferrin. ’“1-transferrin-” ‘Ga or I2 “I-transfcrrin-59Fe. At the end of each incubation period. cell viability was again determined and the cells were separated from the supernatant by centrifugation. The cells were washed twice with 0.99!,NaCl solution and the radioactivity of both the cell pellets and combined supernatants determined separately. A minimum of three replicates was used in each experimerit and each experiment was repeated two or three times. Labrliny of transferrin (4 59Fe-transferrin and b7Ga-transferrin.
Human apotransferrin (obtained from Sigma Biochemicals) dissolved in 0.9% NaCl solution 2 mg/ml was incubated with either h7Ga-citrate(NEN) or “FeCI,(NEN) at a pH adjusted to 7.4 with 0.1 N *This work was supported by a Research Grant NaOH solution. The incubate was allowed to stand overnight at room temperature. Separation of free from the National Cancer Institute of Canada. t To whom reprint requests should be addressed. s9Fe or “Ga from that bound to transferrin wao 23
achieved by means of a Sephadex G-50 column (Pharmacia) with 0.9”,, NaCl solution used as eluant. (b) Pwpwrrfiorl oj ‘“l-r~urz.~~~r~in-59Fe and 12’1twnsfiJrl-irv6’Gu Human apotransferrin (Sigma) was labeled with “‘1 by the ICI method. essentially as described by RHF.“) The ‘151-transferrin in 0.05 M HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid) 0.1 M KCI, pH 7.4 was then incubated with 59Fe-NTA (nitrilotriacetate) or “Ga-NTA. FeNTA was prepared by the addition of SO-loo&Ii of a ‘9FeCIJ solution in HCI to a 3 M excess of NTA solution. This mixture was allowed to stand for i h, and to this was added 2-5 mg “‘1-transferrin in 0.05 M HEPES 0.1 M KC1 pH 7.4. The final pH was adjusted to 7.4 with 0.1 M NaHCO,. The incubation mixture was allowed to stand overnight at room temperature (21 C). The “free” “‘Fe was separated from the “Fe bound to transferrin by means of a Sephadex G-50 (Pharmacia) 0.8 cm x 30cm column equilibrated with 0.1 M KCI. 0.05 M HEPES pH 7.4. The column was eluted with a similar solvent system. “Ga-NTA was prepared by acidifying “Ga-citrate (0.2-0.5 mCi) NEN with an equal volume of 8 N HCI to convert “‘Ga-citrate to “GaCl,. The mixture was applied to a 0.7 x 2.5 cm Dowex 1 x 4 ion exchange resin previously equilibrated Wh 8 N HCI. The column was eluted with 2-3 ml 8 N HCI to remove the free citrate followed by 0.1 N HCI which eluted the “Ga as chloride. The “GaCl, solution was first incubated for ) h with a three-fold molar excess of NTA, and the pH was then adjusted slowly with NaOH to pH 2.5-3.0. A 0.1 M solution of NaHCO, (IOC-200 ~1) was then added raising the pH to 5-6. To the reaction mixture was then added 2-5mg of human ‘““I-apotransferrin dissolved in l-l.5 ml of 0.1 M KCI, 0.05 M HEPES pH 7.4. The final pH of the incubate was adjusted to 7.4 with 0.1 M NaHCO, solution. After incubating overnight the reaction mixture was separated as described above. Final protein concentrations were determined by U.V. analysis (280 nm). The activities associated with the cell pellet and supernatant was determined by means of a Searle Model-l 195 Gamma Ray Spectrometer. Correction for crossover counts (s9Fe in I “1 channel or “Ga in “‘1 channel) was affected for each sample. in cases of dual-labeled samples.
RESULTS
AND
FIG. 1. Effect of increasing iron concentrations (0.010. 0.015, 0.030, 0.050, 0.090 and 0.18 pg/ml) on “Ga uptake by melanoma cells incubated with “Ga-transferrin. Each point is a mean & 1 SD. of three determinations after 3 h incubations.
transferrin to the melanoma cell membrane transport system. Next, the role of phosphate ions on the uptake of both “Ga and 59Fe was examined by incubating melanoma cells with “Ga-transferrin or 59Fe-transferrin. It has been shown (‘) that PO:- can remove Fe from transferrin and thus make it unavailable for transport across a cell membrane. Figure 2 clearly indicates that as the concentration of PO:- is increased in the incubation medium containing melanoma cells in the presence of either “‘Gatransferrin or s9Fe-transferrin there is a parallel decrease in uptake of both ions by the cultured tumor cells. This decrease in uptake
DISCUSSION
The influence of increasing the concentrations of iron concentration and its affect on the uptake of (“Ga by cells when incubated with 67Ga-transferrin is shown in Fig. 1. The melanoma cells were incubated with increasing concentrations of non-radioactive FeCl,. From Fig. 1 it can be clearly seen that Fe has an inhibitory effect on the uptake of 6JGa by melanoma cells incubated with “Ga-transferrin complex. This could be interpreted as competition of Fe for the same binding sites as those occupied by “Ga on the transferrin molecule. The displaced “Ga would then no longer be available for active transport by
pg Phosphote in Incubate (Iml)
Fig. 2. ElTect of increasing phosphate concentrations (60, 90, 120 and 180 pg/ml) in the incubation medium on the uptake of 67Ga(A) or 59Fe(o) by melanoma cells incubated with either “Ga-fransferrin or 59Fetransferrin respectively. The uptake of 59Fe(0) from 59FeCl, was also determined. Each point is the mean _+
1
S.D.
of
three
determinations
incubations.
after
3h
TABLE
1. Transferrin
Material “FeCI, (0.08 nCi)* “Fe-transferrint (0.02 PCi) 5yFe-transferrin + free ‘9Fe (0.08 LtCi) “‘Fe-transferrin + free 59Fe (0.16 nCi)
as carrier for “9Fe uptake in melanoma cells Activity in cell fraction”
0o increase above expected value --I__.
311 * 135 997 * 128 1616 a 135
24”,,
1928 k 51
23” 0
I’Mean * I S.D. of three determinations. * Uptake calculated as 0.23 1 0.20°/,/10h viable cells. t Amount of transferrin added was 0.024 mg/l ml of incubate containing live cells.
of both “Fe and “Ga would suggest that a similar phenomenon occurs with 67Ga-transferrin as is the case with 59Fe-transferrin. Since it has been suggested that transferrin is a carrier for both 59Fe and 67Ga, a series of experiments were conducted to define the role of transferrin in the active transport process.The findings for both 5QFeand 67Ga are illustrated in Tables 1 and 2 respectively. In the case of s9Fe uptake by melanoma cells the amount of 59Fe taken up by the cells from either 59FeCl, or ‘“Fe-transferrin was less than when similar concentrations were added simultaneously. In fact a 24:/, increase in uptake of radio-iron by the melanoma cells was observed, in excess of that taken up when FeC13 0.08 FCi, (0.024 mg) was added singly to the cell incubate. A strikingly similar effect was observed when “Ga-citrate and “Ga-transferrin were added in combination to the incubate (Table 2). The uptake again exceeded that determined for 67Ga-transferrin or “Ga-citrate alone.
IOh
Further experiments were carried out using specifically labeled 1Z51-transferrin-h7Ga and 1’51-transferrin-59Fe. These were designed to measure both the relative rate at which the ‘“Fe as well as thle (“Ga is taken up by the melanoma cells in the incubate, while also trying to determine the fate of the carrier protein. the 1251-labeled transferrin. For this study various concentrations of both lZ51-transferrin-59Fe and 1’51-transferrin-67Ga were incubated over varying time intervals. The results are summarized in Fig. 3 and Table 3. From the data given here it is apparent that both 59Fe and h7Ga are taken up quite rapidly by the melanoma cells. However, the rate of uptake of each isotope differs. The rate of uptake of 59Fe is very rapid for the first i # h with the uptake being 3.5% per 10’ live cells, but after that time period the rate of uptake seems to level off, so that at 4.5 h the uptake was 4.07: per IO6 cells when 0.018 mg ’“‘I-transferrin -59F~ewas used. By comparison, in the case of gallium the rate of uptake in-
2. The uptake of 67Ga by melanoma cells incubated with a constant concentration of “Ga-transferrin, plus increasing quantities of “Ga-citrate
TABLE
Material b7Ga-citrate (0.03 i&i) h7Ga-tran~ferrin (0.022 ptci) “‘Ga-transferrin + free 67Ga (0.03 PCi) h7Ga-transferrin + free 67Ga (0.06pCi) “Ga-transferrin + free 67Ga (0.12nCi)
Activity in cell fraction”
O0 increase above expected value
210 k 56 380 -t 20 645 5 65
+ 9.3”,,
1048 k 80
$- 3 1.O”;,
1465 2 150
$ 20. I”,,
‘Each value is the mean f 1 SD. of three determinations. Concentration of transferrin used-0045 mgin 1 ml incubate containing 1Oh live melanoma cells. Incubation time was 3 h.
26
U. K. Trrnrr.
H. Wang, A. il. Not/;aim. B. C. Lrnrle and J. R. Hill
FIG. 3. Uptake of 59Fe(o)and 123(o) in melanoma cells incubatedwith ‘251-transferrin-s9Fe. Also shown
is the uptake of 67Ga(A)and “‘I(A) in cells incubated with ’251-transferrin-h7Ga. Each point representsthe mean of six determinations.
creases steadily over the 4.5 h incubation period with an uptake of 3.3% when the same amount of ’251-transferrin-67Ga was added to lo6 cells in the 1 m l incubate. It is also interesting to note that while the uptake of 59Fe from ‘251-transferrin-59Fe is affected by the relative concentration of ’251-transferrin-59Fe added to the incubation medium, this does not appear to be the case with ‘251-transferrin-67Ga. In the case of gallium there appears to be no difference in the relative rate of uptake (expressed as a percentage of the total activity added) at the concentrations used in this experiment (Table 3). Thus both 59Fe and 67Ga appear to enter the melanoma cells using transferrin as a carrier molecule. Furthermore, factors which influence the uptake of one ion also affect the uptake of the other and in a similar manner. The observation that the addition of increasing concentrations of FeCl, (non radioactive) to the incubation medium containing 67Gatransferrin and melanoma cells cause a marked reduction of 67Ga uptake by the tumor cells suggests that iron competes for the same active transport site as the transferrin molecule. This implies that iron displaces the 67Ga from the active transport site on the transferrin, hence making it unavailable for active uptake by the melanoma cells. While HILL et ~1.“~’ found that the addition of very low concentrations of Fe to a leukocyte culture system actually enhanced the uptake of 67Ga from the incubation medium this may be explained by the fact that in these experiments all concentrations of Fe were low enough to permit occupation of protein binding sites not occupied by Ga. This could conceivably increase the affinity of cer-
tain carrier proteins, especially transferrin, for the membrane receptor sites.”‘.I 3, However, HILL rr (I/.“~’ also observed that at higher concentrations of Fe, the uptake of Ga by the culture cells was reduced. The fact that iron has a much higher binding constant with respect to the specific binding sites than other metal ions has been demonstrated by a number of workers.“.‘.“,’ 5.16)The reduction in uptake of 67Ga by human melanoma tissue culture cells in the presence of iron is consistent with the findings of HARRIS and SEPHTON’~) who used mouse myeloma cells as a model. The observation of others that phosphate ions affect dissociation of Fe from transferrinc6’ and Ga from transferrin’“! hence making these ions unavailable for transport across the cell membrane, is supported by our findings (Fig. 2). As the phosphate concentration increased in the incubation medium a parallel inhibition of uptake of both “Ga and “Fe by melanoma cells was observed. The reduction in uptake by cells incubated with either “Ga-transferrin or 59Fe-transferrin reflects the dissociation of 67Ga or 99Fe from transferrin. This would therefore make it unavailable for transport into the cell, which indeed was observed (Fig. 2). These observations further emphasize the active transport role played by transferrin and would suggest that positive diffusion of (“Ga across the membrane as suggested by Iro et al. (11) does play only a m inor if not insignificant role in the total uptake of gallium by tumors. GRAMS et ~1.‘~’observed a similar inhibitory effect by phosphate ions on the uptake of 67Ga by cultured leukemic cells. In the potentiation experiments of both 67Ga and 59Fe (Tables 1 and 2), the addition of 67Ga-citrate and 59FeC1, to their respective incubation medium resulted in a higher uptake than was expected from the active transport by labeled transferrin or passive diffusion. This would suggest that transferrin which has donated an ion to the cell membrane, or monometal labeled transferrin, could easily bind free 67Ga or 59Fe and transport it back to the cell membrane. Furthermore it has been shown” *.l 3*1‘.18) that transferrin which has both specific binding sites occupied has a much higher binding affinity than mono-metalic transferrin for the cell surface receptors, which would ultimately lead to the active transport of iron or gallium across the cell membrane. This could explain the accelerated increase in uptake of both 59Fe and 67Ga by the melanoma cells. This concept is further substantiated by the double-labeled transferrin studies using either ’251-transferrin-67Ga or ’251-transferrin-59Fe (Fig. 3. Table 3). The 1251 associated with the
67Ga and 59Fe uptake
in human mekunomu crl,‘s
cell fraction remained constant when expressed as a percentage of the total label in the incubate. This would suggest that in both cases the binding of labeled transferrin to the melanoma cell membrane is a function of the concentration of transferrin in the medium and would appear to be linear in the range of concentrations tested. However, the differences in the rate of uptake of 59Fe when compared to “Ga from the transferrin may signify that 59Fe labeled transferrin has a higher affinity for the tumor cell membrane than does “Ga-transferrin. This observation agrees with that of others.‘8.19’Moreover at higher concentrations the rate of uptake is reduced. suggesting that there may be a rate limiting step in the transport of 59Fe across the membrane. This does not appear to be the case in the uptake of “Ga from 1’51-transferrin-h7Ga (Fig. 3. Table 3). The relatively linear uptake of gallium by the tumor cells irrespective of the concentration used over the various time periods may in fact reflect a lower affinity for the transferrin membrane binding site in the case of galliumlabeled transferrin as compared to iron-transferrin. This lower affinity may in fact be below the rate limiting step of the membrane transport protein responsible for carrying the metal ion into the cell milieu. Also, if the theory of differing affinities for the membrane transferrin receptor is correct, then Ga-transferrin would only be able to dislodge newly formed apotransferrin from the surface binding sites of the melanoma cells at a rate slower than that of Fe-transferrin. While the factors which influence the uptake of iron from s9Fe-transferrin by melanoma cells have a similar effect on the uptake of gallium from “Ga-transferrin, the differences in the rate of uptake of each of the metal ions
can be explained by differing affinities which s9Fe-transferrin and 67Ga-transferrin have for the melanoma membrane binding site.
REFERENCES I. 2. 3. 4. 5. 6.
7. 8. 9. IO. 1 I. I 2. 13. 14. 15. 16. 17. 18. 19.
CLALSEN J.. ED~LINC; C. and FOGH J. Canw Rex 34, 193 I (I 974). GUNASIXERA S. W.. KING; L. J. and LAVENDER P. J. Clinicrr chirrl. Acru 39. 401 (1972). HOFFER P. B.. HUBERTY J. and KHAYAIN-BASHI H. J. nucl. Mrd. 18. 713 (1977). HEGGE F. N.. MAHLER D. J. and LARSEN S. M. .I. Jd. Med. 18. 937 (1977). REIF A. E. J. n~rcl. Mrd. 9. I48 (1968). SHAIX A. L.. OYAMA J.. REINHART R. W. and MILLER J. R. Proc. Sot. op. Bid. Add. 87. 443 (1954). BATES G. W., BILL~PS C. and SAI.TMAN P. J. hiol. Chcrn. 242. 2810 (1967). HARRIS A. W. and SEPHTON R. G. Car~ccr RKS. 37, 3634 (I 977). GRAMS R. A.. Wtw J. and GLI~)