MYC expression promotes the proliferation of neural progenitor cells in culture and in vivo

Abstract

Primitive neuroectodermal tumors (PNETs) are pediatric brain tumors that result from defects in signaling molecules governing the growth and differentiation of neural progenitor cells. We used the RCAS-TVA system to study the growth effects of three genetic alterations implicated in human PNETs on a subset of neural progenitor cells that express the intermediate filament protein, nestin. The genetic alterations tested were: 1) overexpression of the cellular oncoprotein, MYC; 2) activation of transcription factor, beta-catenin; and 3) haploinsufficiency of Ptc, the hedgehog receptor gene. The RCAS-TVA system uses an avian retroviral vector, RCAS, to target gene expression to specific cell types in transgenic mice. To express exogenous genes in neural progenitor cells, we used Ntv-a mice. In these mice, the Nestin gene promoter drives expression of TVA, the cell surface receptor for the virus. Ectopic expression of MYC, but not activated beta-catenin, promoted the proliferation of neural progenitor cells in culture and in the cerebral leptomeninges in vivo. These effects were equally penetrant in mice with Ptc+/- and Ptc+/+ genetic backgrounds. Although overexpression of MYC is not sufficient to cause intraparenchymal tumors, it may facilitate PNET formation by sustaining the growth of undifferentiated progenitor cells.

Figure 1

Expression of RCAS-MYC and RCAS-β-catenin in Ntv-a mouse brain cultures. (A) RCAS vector carrying exogenous gene (human c-MYC cDNA) 3′ of the retroviral env gene. Although RCAS vectors replicate only in avian cells, the exogenous gene is expressed in both avian and mammalian cells as a spliced message from the constitutive retroviral promoter, LTR. (B) Western blot showing expression of human MYC protein (67 kDa) in a mouse brain culture infected with RCAS-MYC retrovirus. No expression was detected in cells infected with control virus carrying LACZ (9E10 antibody, 1:100). (C) RT-PCR analysis of brain cultures from three different Ntv-a mice (lanes 1–3) infected with RCAS-MYC or RCAS-β-catenin and two uninfected cultures (lanes 4 and 5). Total RNA was used as template in RT-PCR reactions with oligonucleotide primers specific for either human MYC or CTNNB1 coding sequences within the integrated RCAS proviruses. The MYC product (838 bp) or CTNNB1 (819 bp) product was present in all infected cultures, but in none of the uninfected cultures. As a positive control, we used RNA from virus producer cell lines, DF1-MYC or DF1-CTNNB1 (lane 6). Parallel PCRs showed no amplification from residual traces of genomic DNA in the RNA samples (lanes 1–6).

Figure 2

In vitro growth stimulation of Ntv-a mouse brain cultures by RCAS-MYC. (A–C) Cell growth curves for Ntv-a mouse brain cultures infected with the indicated RCAS vectors. Cultures were derived from the following mice: (A) R4 with Ptc^+/- genetic background, (B) WT3 with Ptc^+/+ background, and (C) R7 (Ptc^+/-) and WT6 (Ptc^+/+). Data points represent averages of three cell counts with standard deviations less the 38% of the mean (highly significant on the log scale). (D–E) Phase contrast photomicrographs showing cell morphology of Ntv-a brain culture R2 (Ptc^+/-) infected with RCAS-MYC (D) and uninfected (E). Cells were photographed after comparable time periods in culture. Scale bar, 70 µm.

Figure 3

Histopathology of leptomeningeal cell aggregates induced by RCAS-MYC. (A) Cell aggregate abutting pial surface of the brain from Ntv-a/Ptc^+/- mouse (M35) injected with RCAS-MYC. An artery within the subarachnoid space is visible in the upper left. H&E. (B) Immunoperoxidase staining of Ntv-a/Ptc^+/- mouse brain section (M129) using antibody, 9E10 (1:50), specific for human MYC. Cells within the subarachnoid space show nuclear IR (arrows) and cytoplasmic IR (arrowhead). (C) Immunoperoxidase staining of Ntv-a/Ptc^+/+ mouse brain section (M108) showing MYC IR (9E10 antibody, 1:50). All cells in the aggregate show cytoplasmic staining. (D) Immunoperoxidase staining of the same mouse brain specimen as shown in panel C (M111), using antibody, 35-Z6 (1:50), specific for leukocyte transmembrane protein, CD45. Only a few scattered cells are CD45⁺ (arrows). (E) Nestin expression in Ntv-a brain culture infected with RCAS-MYC (401 antibody, 1:500). (F) Leptomeningeal cell aggregate in Ntv-a/Ptc^+/- mouse brain (M111) showing expression of nestin (401 antibody, 1:50). (G) Leptomeningeal cell aggregate in Ntv-a/Ptc^+/- mouse brain (M129) showing expression of notch1 (A6 antibody, 1:50). (H) Leptomeningeal cell aggregate in Ntv-a/Ptc^+/+ mouse brain (M108) showing expression of βIII tubulin (TuJ1 antibody, 1:400). Scale bar, 15 µm for panels A–E; 9 µm for panels F–H.