Radiodinated meta-iodobenzylguandine (MIBG) is increasingly used for the diagnosis and targeted radiotherapy of neuro-adrenergic tumors. We have investigated various conditions for specific tumor loading and prolonged retention of this radiopharmaceutical in poorly differentiated SK-N-SH neuroblastoma and highly differentiated PC-12 pheochromocytoma cells. At a constant value of drug concentration x incubation time, short incubations were superior to protracted incubations for maximal cell loading. This effect was most pronounced in the SH-N-SH neuroblastoma cells. In highly differentiated pheochromocytoma cells, the levels of MIBG storage remained high and unchanged during incubations up to 46 hr in label-free medium, while primitive SK-N-SH cells lost 40-50% of accumulated drug by diffusion. In PC-12 cells, susceptibility of stored MIBG to exocytotic release induced by acetylcholine or K+ was similar to that of natural norepinephrine (NE) and prevented by the Ca(++)-channel blockers verapamil and nifedipine. Conversely, granule-poor SK-N-SH cells were insensitive to exocytotic release of MIBG. Uptake and retention capacities were minimally impaired by an externally delivered radiation dose of 5 Gy to mimic the radiobiological effect of 131I-MIBG in tumors. In pre-irradiated cultures, drug uptake was even stimulated, probably due to enrichment in non-proliferating cells. An autoradiographic comparison of the (sub)cellular distributions of 3H-norepinephrine and 125I-MIBG showed that routine conditions of cell fixation and sample processing do not yield reliable results regarding localization of MIBG.

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In childhood acute lymphoblastic leukaemia there are large interpatient variations in levels of the apoptosis-regulating proteins Bax and Bcl-2, but the molecular basis for this variation is unknown. Point-mutations in bax have been reported in cell lines derived from haematological malignancies. Frameshift mutations, which result in reduced Bax levels, have also been found in colon cancer of the microsatellite mutator phenotype. Bcl-2 overexpression, or gain of function mutations in the open reading frame (ORF) or in the translational repressor, the upstream ORF (uORF) of bcl-2, might also be important in deregulating its function or expression. We have therefore analyzed 21 bone marrow aspirates from untreated childhood acute lymphoblastic leukaemia and 2 from myeloid leukaemia for mutations in bax and bcl-2. DNA sequence analysis of the ORFs of bax and bcl-2 and of the uORF of bcl-2 revealed no mutations, despite the large range in expression levels. Thus, mutations within the (u)ORFs of bax and bcl-2 that (in)activate or deregulate Bax and Bcl-2 are infrequent in primary childhood acute leukaemia and do not play a major role in regulation of the encoded proteins in this disease.

Background: The therapeutic use of [ 131 I]meta-iodobenzylguanidine ([ 131 I]MIBG) is often accompanied by hematological toxicity, mainly consisting of persistent and severe thrombocytopenia. While MIBG accumulates in neuroblastoma cells via selective uptake by the norepinephrine transporter (NET), the serotonin transporter (SERT) is responsible for cellular uptake of MIBG in platelets. In this study, we have investigated whether pharmacological intervention with selective serotonin reuptake inhibitors (SSRIs) may prevent radiotoxic MIBG uptake in platelets without affecting neuroblastoma tumor uptake. Methods: To determine the transport kinetics of SERT for [ 125 I]MIBG, HEK293 cells were transfected with SERT and uptake assays were conducted. Next, a panel of seven SSRIs was tested in vitro for their inhibitory potency on the uptake of [ 125 I]MIBG in isolated human platelets and in cultured neuroblastoma cells. We investigated in vivo the efficacy of the four best performing SSRIs on the accumulation of [ 125 I]MIBG in nude mice bearing subcutaneous neuroblastoma xenografts. In ex vivo experiments, the diluted plasma of mice treated with SSRIs was added to isolated human platelets to assess the effect on [ 125 I]MIBG uptake. Results: SERT performed as a low-affinity transporter of [ 125 I]MIBG in comparison with NET (K m = 9.7 μM and 0.49 μM, respectively). Paroxetine was the most potent uptake inhibitor of both serotonin (IC 50 = 0.6 nM) and MIBG (IC 50 = 0.2 nM) in platelets. Citalopram was the most selective SERT inhibitor of [ 125 I]MIBG uptake, with high SERT affinity in platelets (IC 50 = 7.8 nM) and low NET affinity in neuroblastoma cells (IC 50 = 11.940 nM). The in vivo tested SSRIs (citalopram, fluvoxamine, sertraline, and paroxetine) had no effect on [ 125 I]MIBG uptake levels in neuroblastoma xenografts. In contrast, treatment with desipramine, a NET selective inhibitor, resulted in profoundly decreased xenograft [ 125 I]MIBG levels (p < 0.0001). In ex vivo [ 125 I]MIBG uptake experiments, 100-and 34-fold diluted murine plasma of mice treated with citalopram added to isolated human platelets led to a decrease in MIBG uptake of 54-76%, respectively.