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Wolmer-Solberg et al., reported that six human neuroblastoma cell lines and the vast majority of clinical neuroblastoma samples contained HCMV DNA and expressed HCMV proteins. We could not replicate the data and therefore remain skeptical towards the prevalence of HCMV DNA in neuroblastomas.

Established tumour cell lines are ubiquitous tools in research, but their representativity is often debated. One possible caveat is that many cell lines are derived from cells with genomic instability, potentially leading to genotype changes in vitro. We applied SNP-array analysis to an established tumour cell line (WiT49). Even though WiT49 exhibited chromosome segregation errors in 30% of cell divisions, only a single chromosome segment exhibited a shift in copy number after 20 population doublings in culture. In contrast, sub-populations derived from single cells expanded for an equal number of population doublings showed on average 5.8 and 8.9 altered segments compared to the original culture and to each other, respectively. Most copy number variants differentiating these single cell clones corresponded to pre-existing variations in the original culture. Furthermore, no sub-clonal variation was detected in any of the populations derived from single cells. This indicates that genetic bottlenecks resulting from population reduction poses a higher threat to genetic representativity than prolonged culture per se, even in cell lines with a high rate of genomic instability. Genetic bottlenecks should therefore be considered a potential caveat in all studies involving sub-cloning, transfection and other conditions leading to a temporary reduction in cell number.

One extra chromosome copy (i.e., trisomy) is the most common type of chromosome aberration in cancer cells. The mechanisms behind the generation of trisomies in tumor cells are largely unknown, although it has been suggested that dysfunction of the spindle assembly checkpoint (SAC) leads to an accumulation of trisomies through failure to correctly segregate sister chromatids in successive cell divisions. By using Wilms tumor as a model for cancers with trisomies, we now show that trisomic cells can form even in the presence of a functional SAC through tripolar cell divisions in which sister chromatid separation proceeds in a regular fashion, but cytokinesis failure nevertheless leads to an asymmetrical segregation of chromosomes into two daughter cells. A model for the generation of trisomies by such asymmetrical cell division accurately predicted several features of clones having extra chromosomes in vivo, including the ratio between trisomies and tetrasomies and the observation that different trisomies found in the same tumor occupy identical proportions of cells and colocalize in tumor tissue. Our findings provide an experimentally validated model explaining how multiple trisomies can occur in tumor cells that still maintain accurate sister chromatid separation at metaphase-anaphase transition and thereby physiologically satisfy the SAC.

Neuroblastoma is a childhood tumour derived from cells of the sympathetic nervous system, which are arrested at low differentiation stages. Low differentiation stage and high tumour stage correlate to poor outcome. In earlier studies we have found that hypoxia induces dedifferentiation of neuroblastoma cells, which implies that hypoxia evokes a more aggressive phenotype. Here we employ microarray analysis to investigate the hypoxic effects, in neuroblastoma cells, on the expression of a larger set of genes. Genes involved in survival and treatment resistance were upregulated, which support the concept of an aggressive hypoxic phenotype. The microarray results further strengthened the concept of hypoxia-induced dedifferentiation of neuroblastoma cells. Hypoxia-treated neuroblastoma cells were reoxygenated to determine the persistence of the hypoxic phenotype. Based on neuronal- and neural crest marker gene expression analysis, we conclude that the hypoxic phenotype persisted for at least 24 h upon reoxygenation. Hence, there was no selection for dedifferentiated cells; instead the hypoxic phenotype appears adaptable and dynamically reversible. Major transcription factors that act in response to hypoxia are the hypoxia-inducible factors HIF-1a and HIF-2a. Intriguingly, HIF-2a, but not HIF-1a, was detected adjacent to blood vessels in neuroblastoma specimen, indicating a role for HIF-2a at more physiological oxygen levels. Analysis of HIF-protein levels in neuroblastoma cells exposed to hypoxia (1% O2) or 5% O2 (mimicking a more physiological oxygen level) revealed that HIF-1a is primarily and only transiently induced at 1% O2. HIF-2a, on the other hand, is induced at both 1 and 5% O2 and its protein levels increase over time. In a microarray analysis we extracted a set of genes with regulation patterns similar to that of the HIF-protein patterns seen in neuroblastoma cells grown at either 1 or 5% O2. We propose that the differences in HIF target gene utilization are d