Neuroblastoma cells with overexpressed MYCN retain their capacity to undergo neuronal differentiation.

Amplification of MYCN in neuroblastoma strongly correlates to unfavorable outcome, but little is known of how the high MYCN expression translates into an aggressive tumor phenotype. More aggressive neuroblastomas are generally immature and overexpression of exogenous MYCN in cultured neuroblastoma cells and other neuronal cell types has been reported to inhibit induced differentiation, suggesting a link between high MYCN expression and an immature phenotype. However, we show here that MYCN is expressed in human neuroblasts of sympathetic chain ganglia at fetal week 8.5, a developmental stage at which these neuroblasts express a number of sympathetic neuronal differentiation marker genes. Analyses of 28 neuroblastoma tumor specimens and 27 cell lines for the expression of MYCN and a panel of neuronal differentiation marker genes did not reveal any correlation between MYCN and marker gene expression levels. Finally, we tested five separate differentiation protocols and show that MYCN overexpressing neuroblastoma cells with a neuronal phenotype, derived from the non-MYCN-amplified human neuroblastoma cell line SK-N-SH, retain their capacity to differentiate despite constitutive MYCN overexpression. Our results show that high MYCN expression and sympathetic differentiation are compatible, and indirectly our findings lend support to previously published MYCN neuroblastoma tumor data, which suggest that in single MYCN copy neuroblastomas there is no direct correlation between a high cellular MYCN protein content and aggressive tumor cell behavior.

Expression of trkB in human neuroblastoma in relation to MYCN expression and retinoic acid treatment.

Expression of full-length trkB can be found in some highly malignant neuroblastoma tumors with an amplified MYCN gene. This contrasts sympathetic neuroblasts, from which neuroblastomas are thought to arise, which neither express trkB nor are dependent on the p145(trkB) ligands, brain-derived neurotrophic factor (BDNF) or neurotrophin-4/5, for their normal development. In this study we show that trkB was expressed in two out of five neuroblastoma tumors with amplified MYCN, while no trkB expression was observed when the MYCN gene was overexpressed in a non-MYCN-amplified neuroblastoma cell line. This shows that MYCN overexpression per se is not sufficient to induce trkB expression. trkB expression and BDNF responsiveness in neuroblastoma cells can be induced by all-trans-retinoic acid (RA). When SH-SY5Y cells were stimulated with a combination of RA and BDNF, norepinephrine and tyrosine hydroxylase levels were unaltered, showing that the cells did not change toward a more catecholaminergic sympathetic phenotype. However, expression of growth-associated protein 43, indicative of a neuronal phenotype, was elevated. Vesicular acetylcholine transporter, choline acetyl transferase, and neuropeptide tyrosine mRNA levels also increased in RA-BDNF-treated cells, which could suggest that these cells develop into a sympathetic cholinergic phenotype. In addition, treatment with RA-induced expression of the platelet-derived growth factor receptor-alpha. As previously shown for BDNF, platelet-derived growth factor stimulated growth of the RA-treated cells, findings that could have clinical relevance. If these receptors mediate a mitogenic signal in vivo also, this might limit the effect of RA treatment on neuroblastoma patients.

Differential experimental micrometastasis to lung, liver, and bone with lacZ-tagged CWR22R prostate carcinoma cells.

LacZ-tagged human prostate carcinoma CWR22Rv1 cells metastasize spontaneously to lung, liver, and bone from subcutaneous primary tumors in athymic nude mice; these organs are 'natural' targets of metastasis for the human disease. To evaluate the mechanism(s) of metastasis to these organs, an experimental metastasis model was used by taking advantage of the ultrasensitive detection of lacZ. Within I h after tail vein injection, micrometastases were forming in lung, liver, bone, kidney, and brain with very different quantitative levels. The kinetics of loss of unstable micrometastases and retention of stable ones were also very different in these organs. After injecting suspensions of single cells, both whole-organ and serial-section staining for lacZ revealed considerable heterogeneity in cell number of individual lung micrometastases while micrometastases in liver contained only I or 2 cells. The size of individual bone micrometastases also suggested only 1 or 2 cells. Tumor cells could also be detected in the small blood vessels of the lung within minutes after injection. These studies indicate that lacZ-tagged CWR22Rv1 cells after tissue culturing contain subsets of cells capable of establishing transient micrometastases in lung, liver, and bone after direct injection into the animal's circulation. Moreover, the quantitative and qualitative properties of the micrometastases in the three organs differ significantly, suggesting different mechanisms for stabilization and fates of micrometastases in these organs.