67Gallium (67Ga) is a radionuclide which accumulates in hematological malignancies and is used for diagnostic imaging. We investigated in this in vitro study the cell cycle dependency of cellular uptake and cytotoxicity of 67Ga. Cell cycle synchronization of cells was achieved by counterflow centrifugal elutriation and the use of cytostatic drugs. The human lymphoma cell lines U-937 and U-715 were used and in elutriation experiments we also used the leukemic cell line HL-60. The transferrin receptor (CD71) expression, 67Ga uptake and cell proliferation inhibition were the parameters measured. We also studied cytotoxicity in various schedules for combination of 67Ga and drugs and the residual proliferative capacity was measured. The CD71 expression in the three cell lines increased from 106-177% on S phase cells and from 118-233% on G2M cells, as compared to the G0/G1 cell fraction. The 67Ga uptake varied from 108-127% for S cells and 128-139% for G2M cells. The drugs chosen induced cell cycle phase accumulation in S and/or G2M phase during preincubation. 67Ga preincubation induced accumulation in the G2M phase. Almost all combinations of 67Ga and drugs resulted in a non-interactive effect, except for methotrexate which resulted in an antagonistic effect. No preferential effect of any of the incubation schemes was seen. CD71 expression and 67Ga uptake were increased in S and G2M cells. Combination of 67Ga with drugs which arrest cells in these cell cycle phases did not result in a change in cytotoxicity. However, these results implicate that 67Ga and the cytostatic drugs tested except for methotrexate might be used together or sequentially in therapy.
Summary Gallium-67 is a radionuclide that accumulates in haematological malignancies and is used for diagnostic purposes. Uptake of 67Ga into the cell occurs via the transferrin receptor, which is differentially expressed during the various cell cycle phases. With the aim of selectively increasing 67Ga uptake, we studied whether the transferrin receptor (TfR) expression could be modulated in the U937 and U715 lymphoma cell lines by cytostatic drugs inducing cell cycle phase accumulation. We tested clinically relevant drugs such as 1-f-D-arabinofuranosylcytosine (Ara-C), hydroxyurea and methotrexate. Cytotoxicity was determined by testing the clonogenic capacity of the lymphoma cell lines. All three drugs induced an increase in S-phase content, TfR expression and 67Ga uptake in U937 and U715 single cells. The combinations of drugs and 67Ga resulted in an additive effect on the clonogenic capacity. In U937 spheroids, cultured by the fibrin clot technique, we found an accumulation in the S-phase too as well as an increase of the transferrin receptor expression after Ara-C preincubation. As in single cells 67Ga uptake was increased without synergistic effects on the clonogenic capacity. In conclusion, priming with drugs induces increased transferrin receptor expression and 67Ga uptake. Inhibition of clonogenic capacity was additive rather than synergistic.
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