4192

MDK

MK|NEGF2;midkine (neurite growth-promoting factor 2);MDK;midkine (neurite growth-promoting factor 2)

ARAP|amphiregulin-associated protein|midgestation and kidney protein|midkine|neurite outgrowth-promoting factor 2|neurite outgrowth-promoting protein

11p11.2

protein-coding

It is important to develop a system to express therapeutic genes in tumor cells with sufficient selectivity for cancer gene therapy. Midkine (MK) is a newly identified heparin-binding growth factor that is transiently expressed in the early stages of retinoic acid-induced differentiation of embryonal carcinoma cells. It has been reported that many human malignant tumors express high levelsof MK mRNA or protein. However, no MK expression is detected in human or mouse liver. These interesting features of MK led us to examine the MK promoter as a candidate for tumor-specific gene expression. We thus developed new recombinant adenoviral (Ad) vectors containing either luciferase reporter gene (AdMKLuc) or herpes simplex thymidine kinase gene (AdMKTK) under the control of the human MK promoter. AdMKLuc achieved relatively high activity in Wilms' tumor (G-401) and neuroblastoma (SK-N-SH) cell lines. In addition, AdMKTK induced marked cell death in response to ganciclovir (GCV) in these same lines. Conversely, very low activity of the MK promoter was observed in mouse liver in vivo compared with the cytomegalovirus promoter. Importantly, AdMKTK + GCV did not induce liver toxicity, whereas substantial toxicity was seen with AdCMVTK + GCV treatment. Onthe basis of these findings, we conclude that the MK promoter is a candidate tumor-specific promoter for Wilms' tumor or neuroblastoma.

It is important to develop a system to express therapeutic genes in tumor cells with sufficient selectivity for cancer gene therapy. Midkine (MK) is a newly identified heparin-binding growth factor that is transiently expressed in the early stages of retinoic acid-induced differentiation of embryonal carcinoma cells. It has been reported that many human malignant tumors express high levelsof MK mRNA or protein. However, no MK expression is detected in human or mouse liver. These interesting features of MK led us to examine the MK promoter as a candidate for tumor-specific gene expression. We thus developed new recombinant adenoviral (Ad) vectors containing either luciferase reporter gene (AdMKLuc) or herpes simplex thymidine kinase gene (AdMKTK) under the control of the human MK promoter. AdMKLuc achieved relatively high activity in Wilms' tumor (G-401) and neuroblastoma (SK-N-SH) cell lines. In addition, AdMKTK induced marked cell death in response to ganciclovir (GCV) in these same lines. Conversely, very low activity of the MK promoter was observed in mouse liver in vivo compared with the cytomegalovirus promoter. Importantly, AdMKTK + GCV did not induce liver toxicity, whereas substantial toxicity was seen with AdCMVTK + GCV treatment. Onthe basis of these findings, we conclude that the MK promoter is a candidate tumor-specific promoter for Wilms' tumor or neuroblastoma.

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood and arises from cells of the developing sympathoadrenergic lineage. Activating mutations in the gene encoding the ALK tyrosine kinase receptor predispose for NB. Here, we focus on the normal function of Alk signaling in the control of sympathetic neuron proliferation, as well as on the effects of mutant ALK. Forced expression of wild-type ALK and NB-related constitutively active ALK mutants in cultures of proliferating immature sympathetic neurons results in a strong proliferation increase, whereas Alk knockdown and pharmacological inhibition of Alk activity decrease proliferation. Alk activation upregulates NMyc and trkB and maintains Alk expression by an autoregulatory mechanism involving Hand2. The Alk-ligand Midkine (Mk) is expressed in immature sympathetic neurons and in vivoinhibition of Alk signaling by virus-mediated shRNA knockdown of Alk and Mk leads to strongly reduced sympathetic neuron proliferation. Taken together, these results demonstrate that the extent and timing of sympathetic neurogenesis is controlled by Mk/Alk signaling. The predisposition for NB caused by activating ALK mutations may thus be explained by aberrations of normal neurogenesis, i.e. elevated and sustained Alk signaling and increased NMyc expression.

Embryonal kidney cell tumors develop in rats given the alkylating agent N-nitroso-N'-methylurea as neonates. These tumors resemble the childhood Wilms tumors in their histopathology. Deletions and mutations in the Wilms tumor suppressor gene, WT1, are present in up to 6% of childhood nephroblastomas. To investigate the role of WT1 in rat kidney tumorigenesis, we studied the genetic alterations in WT1 and its target genes. Point mutations were found in WT1 cDNA in 7 of 18 kidney tumors. Mesenchymal tumors contained G-->A transition mutations in codons 128, 364, and 372, typical of the methylating action of N-nitroso-N'-methylurea on DNA. Each of the four nephroblastomas contained the same T-->A mutation at codon 111 of WT1, reflective of transversion mutagenesis by N-nitroso-N'-methylurea in vivo. Like Wilms tumors, mRNA levels of WT1, IGF2,Pax-2, and MK genes were higher than newborn kidney in the majority of the tumors. The histopathology of the rat kidney tumors and the genetic alterations are reminiscent of those observed in Wilms tumors, establishing this as a relevant model system for the human disease.