Recently, it was shown that MYCN amplified cells spontaneously expulse extrachromosomally amplified gene copies by micronuclei formation. Furthermore, it was shown that these cells lose their malignant phenotype and start to age. We tested whether it is possible to encourage neuroblastoma tumor cells to enter the senescence pathway by low concentrations of the micronuclei-inducing drug hydroxyurea (HU). We studied the effect of HU on 12 neuroblastoma cell lines with extra- or intrachromosomally amplified MYCN copies and without amplification. Two extrachromosomally amplified neuroblastoma cell lines (with double minutes) were investigated in detail. Already after 3 weeks of HU treatment, the BrdU uptake dropped to 25% of the starting cells. After 4 weeks, enlarged and flattened cells (F-cells) and increased granularity in the majority of cells were observed. A drastic reduction of the MYCN copy number-down to one copy per cell-associated with CD44 and MHCI upregulation in up to 100% of the HU treated neuroblastoma cells was found after 5-8 weeks. Telomere length was reduced to half the length within 8 weeks of HU treatment, and telomerase activity was not detectable at this time, while being strongly expressed at the beginning. All these features and the expression of senescence-associated-beta-galactosidase (SA-beta-GAL) in up to 100% of the cells support the hypothesis that these cells entered the senescence pathway. Thus, low-dose HU is a potent senescence elicitor for tumor cells with gene amplification, possibly representing an attractive additional strategy for treatment of this subset of tumors.

MYCN amplification is associated with poor prognosis in neuroblastoma disease. To improve our understanding of the influence of the MYCN amplicon and its corresponding expression, we investigated the 2p expression pattern of MYCN amplified (n ‫؍‬ 13) and nonamplified (n ‫؍‬ 4) cell lines and corresponding primary tumors (n ‫؍‬ 3) using the comparative expressed sequence hybridization technique. All but one MYCN amplified cell line displayed overexpression at 2p. Expression peaks were observed frequently at 2pter and less frequently at 2p24 (MYCN locus), 2p23.3-23.2, and/or 2p23.1. Importantly, cell lines and two corresponding primary tumors displayed expression peaks at similar loci. No significant 2p24 expression level was observed for those cell lines displaying a low amplification rate (n ‫؍‬ 3) by comparative genomic hybridization. Only the cell lines with an enhanced peak at 2p23.2-23.3 displayed coamplification of the ALK gene (2p23.2), reported to be associated with unfavorable prognosis. Finally, two of four cell lines without MYCN amplification, both derived from patients with poor outcome, also showed an expression peak at 2p23.2. These data indicate that, besides MYCN, other genes proximal and distal to MYCN are highly expressed in neuroblastoma. The prognostic significance of expression peaks at 2p23.2-23.3, independent of MYCN and ALK status, remains to be investigated.

We investigated induction of chromosome aberrations (CA) in human lymphocytes when exposed to 150 MeV and spread out Bragg peak (SOBP) proton beams, and 199 MeV/u carbon beam which are currently widely used for cancer treatment and simultaneously are important components of cosmic radiation. For a comparison, the boron ions of much lower energy 22 MeV/u and a 60 Co γ rays were used. Dose–effect curves as well as the distributions of CA were studied using Poisson and Neyman type A statistics. Systematics of experimentally determined parameters, their dependence on applied doses and irradiation quality are presented.

Abstract. Chromosome aberrations in human lymphocytes exposed to different doses of particle radiation: 150 MeV and spread out Bragg peak proton beams, 22 MeV/u boron beam and 199 V/u carbon beam were studied. For comparison, an experiment with 60 Co γ-rays was also performed. We investigated distributions of aberration frequency and the shape of dose-response curves for the total aberration yield as well as for exchange and non-exchange aberrations, separately. Applying the linear-quadratic model, we could derive a relation between the fitted parameters and the ion track radius which could explain experimentally observed curvature of the dose-response curves. The results compared with physical expectations clearly show that the biological effects of cell repair are much more important than the ion track overlapping.