NW/. Med. i?io/. Vol. 17, No. 3. pp. 331-334. Inr. J. Radiar. Appl. Insrrum. Parr B Printed in Great Britain. All rights reserved
1990
Copyright
c
0883-2897/90 $3.00 + 0.00 1990 Pergamon Press plc
Effects of Iron-binding Proteins on In Wro Uptake of 67Ga-Citrate by Tumor Cells L. J. ANGHILERI,*
P. THOUVENOT, F. BRUNOTTE, M. C. CRONE ESCANYE and J. ROBERT
Nuclear Medicine Service, Faculty of Medicine, University of Nancy, Nancy, France (Received 12 February 1989; in revised.form 20 July 1989)
A comparative study of carrier-free 67Gacitrate uptake by Ehrlich ascites tumor cells in the presence of lactoferrin, transferrin and ferritin has demonstrated that lactoferrin considerably increases the uptake of “Ga, and that this increase seems to be determined by its iron-load. The other iron-binding proteins assayed have a null or negative effect. Their behavior in the presence of sodium citrate supports the concept of lactoferrin-binding by the cells as responsible for the uptake. The different behavior of 67Gaacitrate iron-binding protein complexes appears to support this hypothesis.
Introduction It has been suggested (Larson, 1978) that transferrin (TF) plays a strong role in the localization of “‘Gacitrate by tumors. Gallium binds to at least 3 iron-binding proteins (IBP): transferrin (TF), lactoferrin (LF) and ferritin (FT) (Hoffer, 1980). In spite of the considerable bibliography on TF and LF interaction with gallium (Terner et al., 1981; Weiner et al., 1981; Vallabhajosula et al., 1980; Hoffer et al., 1977) there is little information concerning the effects of iron saturation of iron-binding proteins on “Gacitrate uptake by tumor cells. The aim of this experimental work has been to investigate the effects of the iron-load of iron-binding proteins on the in tlitro uptake of 67Ga-citrate by Ehrlich carcinoma ascites cells.
Materials and Methods LF from human milk with low iron content, (il)LF, (0.8 bound Fe/mol, mol. wt 77,000) of 98% purity; iron-saturated LF from human milk, (is)LF, of 90% purity; human TF, holo form, iron-saturated, (is)TF, of 98% purity; and FT from horse spleen (type I), FT, were all from SIGMA Chemical Co., St Louis, Missouri, U.S.A., and used as supplied. AI1 the experiments were performed in duplicate series using different batches of carrier-free 6’Gaacitrate, specific activity 1 mCi/mL from
*Reprint requests should be addressed to: Dr Leopold J. Anghileri. Service de Medecine Nucliaire. 18 rue Lionnois. 54000 Nancy, France.
Mallinckrodt Diagnostica, Petten, The Netherlands. No other heavy metal ions were present in this solution. The experiments were performed with Ehrhch ascites cells maintained in the ascitic form by weekly inoculation into Swiss mice. Eight days after inoculation pools of cells from at least 5 animals were used for each experiment. Care was taken to minimize ascites contamination with blood. To study the effects of preincubation with IBP, 4-5 x 10’ tumor cells in 1 mL Tyrode medium were incubated at 37°C for 15 min with 0.2 mg IBP. After washing twice with cold Tyrode to eliminate unbound IBP the cells were resuspended in 1 mL Tyrode containing 0.5 PCi carrier-free 67Ga-citrate and incubated at 37°C for 30min. In a parallel series of incubations, the same number of cells from the same pool as used for the preincubation assay were incubated at 37’C for 30 min with 0.2 mg IBP plus 0.5 PCi carrier-free 67Ga-citrate. In both cases, after incubation with 67Ga-citrate, the cells were centrifuged, washed twice with Tyrode and counted for radioactivity. In all the experiments, cells incubated under the same experimental conditions but without IBP were used as control, and their 67Gaacitrate uptake referred as 100%. To evaluate the probable contribution of 67Ga bound to the IBP added to the cells, 0.2 mg of IBP in 0.5 mL Tyrode were incubated with 0.5 PCi carrier-free 6’Gaacitrate at 37°C for 30 min. Then, the radioactivity bound to IBP was separated by column chromatography on a Sephadex G-50 and counted. To assess the effects of citrate ion on 67Ga uptake by tumor cells in the presence of IBP, a similar experimental protocol was used, but 1 mM sodium
331
L. J. ANCHILERI et al.
332
citrate was present in the incubation medium containing 67Ga-citrate. The influence of IBP concentration on “‘Ga uptake by tumor cells was studied using the same experimental protocol but increasing the IBP concentration in the incubation medium from 2 to 2OOpg. In addition we have investigated the tumor cell uptake of 67Ga-citrate-IBP complexes that were prepared as follows: 10mg of IBP in 0.5 mL Tyrode containing 200 pCi of carrier-free 67Ga-citrate were incubated at 37°C for 2 h. After incubation the complexes were precipitated adding saturated (NH,), SO4 (pH 7.2) to 75% saturation. The insoluble fraction was washed twice with 75% saturation (NH,)*S04 (pH 7.2) solution, and after draining the excess of liquid, dissolved in 10 mL of saline. Radiochemical purity of these complexes was assayed by column chromatography on a Sephadex G-50. To evaluate the tumor cell uptake of these complexes, a series of tubes containing 5-5.5 x 10’ cells in 1 mL Tyrode were incubated at 37°C for 30min with 0.2 mg IBP as the complex 67Ga-citrate-IBP. After incubation the cells were processed as described.
Results The presence of (is)LF during preincubation or during incubation (in the same medium containing 67Ga-citrate) increases the uptake of the control cells by almost 700%. For all the other assayed IBP, with the exception of (iI)LF, the uptakes are equal or lower than that corresponding to control cells (Table 1). If we compare, under the same experimental conditions, 67Gaacitrate uptake by the cells in the presence of IBP with the 67Ga-citrate bound by IBP in the absence of cells, there is no correlation between the values. The “Ga bound by IBP (in the absence of cells) is: 7.9% for (is)LF, 3.0% for (is)TF, 752.0% for (il)LF, 58.5% for (if)TF, and 379.0% for FT, respectively, of the values corresponding to the tumor cell uptake in the
presence of the same amount of IBP (Table 1, column D). Citrate ion inhibition of 67Gacitrate uptake by tumor cells preincubated with IBP is fairly similar to the one corresponding to control cells (Table 1, column B, row b). For the control cells the inhibition value is -6O.O%, and only when (is)LF and (il)LF are incubated together with 67Ga-citrate plus 1 mM sodium citrate is a much lower inhibitory effect of citrate ion observed: +3.5% for (is)LF and - 13.4% for (il)LF. In the absence of cells, the binding of 67Ga-citrate by IBP, in the presence of 1 mM sodium citrate shows the same pattern of individual values (Table 1, column E) as that corresponding to binding in the absence of sodium citrate (Table 1, column D). The complex 67Ga-citrate-(is)TF is strongly bound by tumor cells (Fig. l), 5-10 times as much as the other IBP-complexes, but the yield of complex formation under identical experimental conditions is very low, 6% for (is)TF, 89% for (is)LF, 92% for (il)LF, 53% for (if)TF and 90% for FT. The ratio of cellular uptake of radioactivity from the complex to radioactivity taken up in the presence of 1 mM sodium citrate is about 1.O (0.9-l .2), while the same ratio for 67Gacitrate alone is more than double (2.1-3.1) (Table 2, column c). The concentration of IBP in the incubation medium is important for determining the extent of 67Gacitrate-binding by tumor cells in the presence of (is)LF and (il)LF. All the other IBP show very little difference in the concentration range 2-200~18 (Fig. 2). The effects of incubation time for 67Ga-citrate alone, or in the presence of (is)LF or (is)TF show an almost steady value after 30 min incubation (Fig. 3).
Discussion From these experimental results it is clear that (is)LF greatly increases 67Ga uptake while the effects
Table I. Effects of IBP on “Gaxitrate
uptake by Ehrlich ascites cells
“Ga uptake by Ehrlich tumor cells B
C
D
E
+3.5 - 50.3
1.9(7.9)(l)
4.2 (10.0) (2)
0. I (3.0)
0.5 (44.2)
(is)LF
(a) (b)
681.6 k 14.8. 699.2 5 70.8
1037.2 f 82.6’ 466.0 + 67.9
(is)TF
(a) (b)
85.4 k 4.1 86.8 + 10.6
32.5 f 4.0 34.7 k 9.6
(il)LF
I”bl
(if)TF
(a)
FT
(b)
“Ga bound to IBP in absence of cells
A
-64.1 -63.4
190.0 f 10.0 262.3 + 38.4
13.4 -53.4
29.0 (752.0)
16.2 (568.4)
50.9 k 12.5 81.2+4.4
38.4 + 5.4 28.5 f 6.8
56. I -65.1
1.7 (58.5)
3.5 (205.9)
70.4 f 8.2 9X.8 k 17.2
28.9 i 3.0 39.2 f 7.7
-62.8 -80.0
IO.1 (379.0)
6.4 (395. I)
67.0 + 3.5 158.0 + 4.5
A. Uptake in presence of IBP (as % of cells without IBP); B, uptake in presence of IBP + I mM sodium citrate (as % of cells without IBP); C. as % of uptake by cells + IBP in absence of I mM sodium citrate (for cells without IBP the value is -60.0); D, in citro binding by IBP in absence of cells (as % of to&l “Ga) and (I) as % of “Ga taken up by cells + IBP from the same dose of “Ga; E, in aim binding by IBP in absence of cells but in presence of 1mM sodium citrate (as % of total 67Ga) and (2) as % of 6’Ga taken up by cells + IBP from the Same dose of “Ga. (a). Single incubation; (b). preincubation with IBP. ‘Mean value i SD of five tubes.
Iron-binding protein and 67Ga+zitrate uptake
333
A : (is)LF B : (is)TF C : (II)LE D : IWTF E : FT
0 TIME
5,
20
10 tmln)
60
30
Fig. 1. In aitro uptake of 67Ga-citrate-IBP complexes (0) and of “‘Gaxitrate (A) by Ehrlich ascites tumor cells (4-S x 10’ cells): A, (is)LF complex; B, (is)TF complex; C, (iI)LF complex; D, (if)TF complex; and E, TF complex. The values correspond to the percentage of total dose and are the mean f SD of five incubation tubes.
of (il)LF are lower but still positive. On the contrary, (is)TF, (if)TF and FT have a negative effect on 67Ga uptake. The enhancement of 67Ga uptake by LF is in agreement with the observation of other authors (Hoffer et al., 1977) and the fact that (il)LF is less effective suggests that the iron-load of the LF might determine the order of 67Ga uptake. The null or negative effects of TF are in agreement with previous reports indicating that TF may play a role only in certain tumors by a TF-receptor mediated cellular uptake of “Ga (Larson er al., 1981), and with the in citw effect of animal plasma on 67Ga uptake (Anghileri er ul., 1988). The fact that (is)LF and (is)TF bind less “Gaacitrate than the (ii) or (if) forms in the absence of tumor cells respectively (Table 1, column D), while in the presence of tumor cells they show the opposite
effect (Table 1, column A, row a) raises the question of whether the iron carried over by IBP does determine 67Ga-citrate uptake.
Table
2. Yields
of *‘Ga-citrate-IBP
complex
by Ehrlich
“Ga
1990
Copyright
c
0883-2897/90 $3.00 + 0.00 1990 Pergamon Press plc
Effects of Iron-binding Proteins on In Wro Uptake of 67Ga-Citrate by Tumor Cells L. J. ANGHILERI,*
P. THOUVENOT, F. BRUNOTTE, M. C. CRONE ESCANYE and J. ROBERT
Nuclear Medicine Service, Faculty of Medicine, University of Nancy, Nancy, France (Received 12 February 1989; in revised.form 20 July 1989)
A comparative study of carrier-free 67Gacitrate uptake by Ehrlich ascites tumor cells in the presence of lactoferrin, transferrin and ferritin has demonstrated that lactoferrin considerably increases the uptake of “Ga, and that this increase seems to be determined by its iron-load. The other iron-binding proteins assayed have a null or negative effect. Their behavior in the presence of sodium citrate supports the concept of lactoferrin-binding by the cells as responsible for the uptake. The different behavior of 67Gaacitrate iron-binding protein complexes appears to support this hypothesis.
Introduction It has been suggested (Larson, 1978) that transferrin (TF) plays a strong role in the localization of “‘Gacitrate by tumors. Gallium binds to at least 3 iron-binding proteins (IBP): transferrin (TF), lactoferrin (LF) and ferritin (FT) (Hoffer, 1980). In spite of the considerable bibliography on TF and LF interaction with gallium (Terner et al., 1981; Weiner et al., 1981; Vallabhajosula et al., 1980; Hoffer et al., 1977) there is little information concerning the effects of iron saturation of iron-binding proteins on “Gacitrate uptake by tumor cells. The aim of this experimental work has been to investigate the effects of the iron-load of iron-binding proteins on the in tlitro uptake of 67Ga-citrate by Ehrlich carcinoma ascites cells.
Materials and Methods LF from human milk with low iron content, (il)LF, (0.8 bound Fe/mol, mol. wt 77,000) of 98% purity; iron-saturated LF from human milk, (is)LF, of 90% purity; human TF, holo form, iron-saturated, (is)TF, of 98% purity; and FT from horse spleen (type I), FT, were all from SIGMA Chemical Co., St Louis, Missouri, U.S.A., and used as supplied. AI1 the experiments were performed in duplicate series using different batches of carrier-free 6’Gaacitrate, specific activity 1 mCi/mL from
*Reprint requests should be addressed to: Dr Leopold J. Anghileri. Service de Medecine Nucliaire. 18 rue Lionnois. 54000 Nancy, France.
Mallinckrodt Diagnostica, Petten, The Netherlands. No other heavy metal ions were present in this solution. The experiments were performed with Ehrhch ascites cells maintained in the ascitic form by weekly inoculation into Swiss mice. Eight days after inoculation pools of cells from at least 5 animals were used for each experiment. Care was taken to minimize ascites contamination with blood. To study the effects of preincubation with IBP, 4-5 x 10’ tumor cells in 1 mL Tyrode medium were incubated at 37°C for 15 min with 0.2 mg IBP. After washing twice with cold Tyrode to eliminate unbound IBP the cells were resuspended in 1 mL Tyrode containing 0.5 PCi carrier-free 67Ga-citrate and incubated at 37°C for 30min. In a parallel series of incubations, the same number of cells from the same pool as used for the preincubation assay were incubated at 37’C for 30 min with 0.2 mg IBP plus 0.5 PCi carrier-free 67Ga-citrate. In both cases, after incubation with 67Ga-citrate, the cells were centrifuged, washed twice with Tyrode and counted for radioactivity. In all the experiments, cells incubated under the same experimental conditions but without IBP were used as control, and their 67Gaacitrate uptake referred as 100%. To evaluate the probable contribution of 67Ga bound to the IBP added to the cells, 0.2 mg of IBP in 0.5 mL Tyrode were incubated with 0.5 PCi carrier-free 6’Gaacitrate at 37°C for 30 min. Then, the radioactivity bound to IBP was separated by column chromatography on a Sephadex G-50 and counted. To assess the effects of citrate ion on 67Ga uptake by tumor cells in the presence of IBP, a similar experimental protocol was used, but 1 mM sodium
331
L. J. ANCHILERI et al.
332
citrate was present in the incubation medium containing 67Ga-citrate. The influence of IBP concentration on “‘Ga uptake by tumor cells was studied using the same experimental protocol but increasing the IBP concentration in the incubation medium from 2 to 2OOpg. In addition we have investigated the tumor cell uptake of 67Ga-citrate-IBP complexes that were prepared as follows: 10mg of IBP in 0.5 mL Tyrode containing 200 pCi of carrier-free 67Ga-citrate were incubated at 37°C for 2 h. After incubation the complexes were precipitated adding saturated (NH,), SO4 (pH 7.2) to 75% saturation. The insoluble fraction was washed twice with 75% saturation (NH,)*S04 (pH 7.2) solution, and after draining the excess of liquid, dissolved in 10 mL of saline. Radiochemical purity of these complexes was assayed by column chromatography on a Sephadex G-50. To evaluate the tumor cell uptake of these complexes, a series of tubes containing 5-5.5 x 10’ cells in 1 mL Tyrode were incubated at 37°C for 30min with 0.2 mg IBP as the complex 67Ga-citrate-IBP. After incubation the cells were processed as described.
Results The presence of (is)LF during preincubation or during incubation (in the same medium containing 67Ga-citrate) increases the uptake of the control cells by almost 700%. For all the other assayed IBP, with the exception of (iI)LF, the uptakes are equal or lower than that corresponding to control cells (Table 1). If we compare, under the same experimental conditions, 67Gaacitrate uptake by the cells in the presence of IBP with the 67Ga-citrate bound by IBP in the absence of cells, there is no correlation between the values. The “Ga bound by IBP (in the absence of cells) is: 7.9% for (is)LF, 3.0% for (is)TF, 752.0% for (il)LF, 58.5% for (if)TF, and 379.0% for FT, respectively, of the values corresponding to the tumor cell uptake in the
presence of the same amount of IBP (Table 1, column D). Citrate ion inhibition of 67Gacitrate uptake by tumor cells preincubated with IBP is fairly similar to the one corresponding to control cells (Table 1, column B, row b). For the control cells the inhibition value is -6O.O%, and only when (is)LF and (il)LF are incubated together with 67Ga-citrate plus 1 mM sodium citrate is a much lower inhibitory effect of citrate ion observed: +3.5% for (is)LF and - 13.4% for (il)LF. In the absence of cells, the binding of 67Ga-citrate by IBP, in the presence of 1 mM sodium citrate shows the same pattern of individual values (Table 1, column E) as that corresponding to binding in the absence of sodium citrate (Table 1, column D). The complex 67Ga-citrate-(is)TF is strongly bound by tumor cells (Fig. l), 5-10 times as much as the other IBP-complexes, but the yield of complex formation under identical experimental conditions is very low, 6% for (is)TF, 89% for (is)LF, 92% for (il)LF, 53% for (if)TF and 90% for FT. The ratio of cellular uptake of radioactivity from the complex to radioactivity taken up in the presence of 1 mM sodium citrate is about 1.O (0.9-l .2), while the same ratio for 67Gacitrate alone is more than double (2.1-3.1) (Table 2, column c). The concentration of IBP in the incubation medium is important for determining the extent of 67Gacitrate-binding by tumor cells in the presence of (is)LF and (il)LF. All the other IBP show very little difference in the concentration range 2-200~18 (Fig. 2). The effects of incubation time for 67Ga-citrate alone, or in the presence of (is)LF or (is)TF show an almost steady value after 30 min incubation (Fig. 3).
Discussion From these experimental results it is clear that (is)LF greatly increases 67Ga uptake while the effects
Table I. Effects of IBP on “Gaxitrate
uptake by Ehrlich ascites cells
“Ga uptake by Ehrlich tumor cells B
C
D
E
+3.5 - 50.3
1.9(7.9)(l)
4.2 (10.0) (2)
0. I (3.0)
0.5 (44.2)
(is)LF
(a) (b)
681.6 k 14.8. 699.2 5 70.8
1037.2 f 82.6’ 466.0 + 67.9
(is)TF
(a) (b)
85.4 k 4.1 86.8 + 10.6
32.5 f 4.0 34.7 k 9.6
(il)LF
I”bl
(if)TF
(a)
FT
(b)
“Ga bound to IBP in absence of cells
A
-64.1 -63.4
190.0 f 10.0 262.3 + 38.4
13.4 -53.4
29.0 (752.0)
16.2 (568.4)
50.9 k 12.5 81.2+4.4
38.4 + 5.4 28.5 f 6.8
56. I -65.1
1.7 (58.5)
3.5 (205.9)
70.4 f 8.2 9X.8 k 17.2
28.9 i 3.0 39.2 f 7.7
-62.8 -80.0
IO.1 (379.0)
6.4 (395. I)
67.0 + 3.5 158.0 + 4.5
A. Uptake in presence of IBP (as % of cells without IBP); B, uptake in presence of IBP + I mM sodium citrate (as % of cells without IBP); C. as % of uptake by cells + IBP in absence of I mM sodium citrate (for cells without IBP the value is -60.0); D, in citro binding by IBP in absence of cells (as % of to&l “Ga) and (I) as % of “Ga taken up by cells + IBP from the same dose of “Ga; E, in aim binding by IBP in absence of cells but in presence of 1mM sodium citrate (as % of total 67Ga) and (2) as % of 6’Ga taken up by cells + IBP from the Same dose of “Ga. (a). Single incubation; (b). preincubation with IBP. ‘Mean value i SD of five tubes.
Iron-binding protein and 67Ga+zitrate uptake
333
A : (is)LF B : (is)TF C : (II)LE D : IWTF E : FT
0 TIME
5,
20
10 tmln)
60
30
Fig. 1. In aitro uptake of 67Ga-citrate-IBP complexes (0) and of “‘Gaxitrate (A) by Ehrlich ascites tumor cells (4-S x 10’ cells): A, (is)LF complex; B, (is)TF complex; C, (iI)LF complex; D, (if)TF complex; and E, TF complex. The values correspond to the percentage of total dose and are the mean f SD of five incubation tubes.
of (il)LF are lower but still positive. On the contrary, (is)TF, (if)TF and FT have a negative effect on 67Ga uptake. The enhancement of 67Ga uptake by LF is in agreement with the observation of other authors (Hoffer et al., 1977) and the fact that (il)LF is less effective suggests that the iron-load of the LF might determine the order of 67Ga uptake. The null or negative effects of TF are in agreement with previous reports indicating that TF may play a role only in certain tumors by a TF-receptor mediated cellular uptake of “Ga (Larson er al., 1981), and with the in citw effect of animal plasma on 67Ga uptake (Anghileri er ul., 1988). The fact that (is)LF and (is)TF bind less “Gaacitrate than the (ii) or (if) forms in the absence of tumor cells respectively (Table 1, column D), while in the presence of tumor cells they show the opposite
effect (Table 1, column A, row a) raises the question of whether the iron carried over by IBP does determine 67Ga-citrate uptake.
Table
2. Yields
of *‘Ga-citrate-IBP
complex
by Ehrlich
“Ga
