Reske Sven N, Deisenhofer Sandra, Glatting Gerhard, Zlatopolskiy Boris D, Morgenroth Agnieszka, Vogg Andreas T J, Buck Andreas K, Friesen Claudia
Nuclear Medicine Clinic, Universität Ulm, Ulm, Germany.
J Nucl Med. 2007 Jun;48(6):1000-7. doi: 10.2967/jnumed.107.040337. Epub 2007 May 15.
Resistance to radiotherapy or chemotherapy is a common cause of treatment failure in high-risk leukemias. We evaluated whether selective nanoirradiation of DNA with Auger electrons emitted by 5-(123)I-iodo-4'-thio-2'-deoxyuridine ((123)I-ITdU) can induce cell kill and break resistance to doxorubicin, beta-, and gamma-irradiation in leukemia cells.
4'-thio-2'-deoxyuridine was radiolabeled with (123/131)I and purified by high-performance liquid chromatography. Cellular uptake, metabolic stability, DNA incorporation of (123)I-ITdU, and the effect of the thymidylate synthase (TS) inhibitor 5-fluoro-2'-deoxyuridine (FdUrd) were determined in HL60 leukemia cells. DNA damage was assessed with the comet assay and quantified by the olive tail moment. Apoptosis induction and irradiation-induced apoptosis inhibition by benzoylcarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD.fmk) were analyzed in leukemia cells using flow cytometry analysis.
The radiochemical purity of ITdU was 95%. Specific activities were 900 GBq/micromol for (123)I-ITdU and 200 GBq/micromol for (131)I-ITdU. An in vitro cell metabolism study of (123)I-ITdU with wild-type HL60 cells demonstrated an uptake of 1.5% of the initial activity/10(6) cells of (123)I-ITdU. Ninety percent of absorbed activity from (123)I-ITdU in HL60 cells was specifically incorporated into DNA. (123)I-ITdU caused extensive DNA damage (olive tail moment > 12) and induced more than 90% apoptosis in wild-type HL60 cells. The broad-spectrum inhibitor of caspases zVAD-fmk reduced (123)I-ITdU-induced apoptosis from more than 90% to less than 10%, demonstrating that caspases were central for (123)I-ITdU-induced cell death. Inhibition of TS with FdUrd increased DNA uptake of (123)I-ITdU 18-fold and the efficiency of cell kill about 20-fold. In addition, (123)I-ITdU induced comparable apoptotic cell death (>90%) in sensitive parental leukemia cells and in leukemia cells resistant to beta-irradiation, gamma-irradiation, or doxorubicin at activities of 1.2, 4.1, 12.4, and 41.3 MBq/mL after 72 h. This finding indicates that (123)I-ITdU breaks resistance to beta-irradiation, gamma-irradiation, and doxorubicin in leukemia cells.
(123)I-ITdU-mediated nanoirradiation of DNA efficiently induced apoptosis in sensitive and resistant leukemia cells against doxorubicin, beta-irradiation, and gamma-irradiation and may provide a novel treatment strategy for overcoming resistance to conventional radiotherapy or chemotherapy in leukemia. Cellular uptake and cell kill are highly amplified by inhibiting TS with FdUrd.
对放疗或化疗的耐药性是高危白血病治疗失败的常见原因。我们评估了用5-(123)I-碘-4'-硫代-2'-脱氧尿苷（(123)I-ITdU）发射的俄歇电子对DNA进行选择性纳米照射是否能诱导白血病细胞杀伤并打破对阿霉素、β射线和γ射线照射的耐药性。
4'-硫代-2'-脱氧尿苷用(123/131)I进行放射性标记，并通过高效液相色谱法纯化。在HL60白血病细胞中测定(123)I-ITdU的细胞摄取、代谢稳定性、DNA掺入以及胸苷酸合成酶（TS）抑制剂5-氟-2'-脱氧尿苷（FdUrd）的作用。用彗星试验评估DNA损伤，并用橄榄尾矩进行定量。使用流式细胞术分析在白血病细胞中分析苯甲酰羰基-Val-Ala-Asp-氟甲基酮（z-VAD.fmk）诱导的凋亡和照射诱导的凋亡抑制。
ITdU的放射化学纯度为95%。(123)I-ITdU的比活度为900 GBq/μmol，(131)I-ITdU的比活度为200 GBq/μmol。对野生型HL60细胞进行的(123)I-ITdU体外细胞代谢研究表明，(123)I-ITdU的摄取量为初始活性的1.5%/10(6)个细胞。HL60细胞中(123)I-ITdU吸收活性的90%特异性掺入DNA。(123)I-ITdU导致广泛的DNA损伤（橄榄尾矩>12），并在野生型HL60细胞中诱导超过90%的凋亡。半胱天冬酶广谱抑制剂zVAD-fmk将(123)I-ITdU诱导的凋亡从超过90%降低到不到10%，表明半胱天冬酶是(123)I-ITdU诱导细胞死亡的关键。用FdUrd抑制TS可使(123)I-ITdU的DNA摄取增加18倍，细胞杀伤效率提高约20倍。此外，在72小时后，当活性为1.2、4.1、12.4和41.3 MBq/mL时，(123)I-ITdU在敏感的亲本白血病细胞以及对β射线照射、γ射线照射或阿霉素耐药的白血病细胞中诱导了相当的凋亡细胞死亡（>90%）。这一发现表明(123)I-ITdU打破了白血病细胞对β射线照射、γ射线照射和阿霉素的耐药性。
(123)I-ITdU介导的DNA纳米照射有效地诱导了对阿霉素、β射线照射和γ射线照射敏感和耐药的白血病细胞凋亡，并可能为克服白血病对传统放疗或化疗的耐药性提供一种新的治疗策略。用FdUrd抑制TS可高度放大细胞摄取和细胞杀伤。
