N-myc modulates expression of p73 in neuroblastoma

Abstract

The human p73 gene is a homolog of p53, which has been localized to chromosome 1p36 in a region that is frequently deleted in neuroblastoma. Transfection of the p73 gene into neuroblastoma cells that lack detectable p73 protein has been shown to result in growth suppression and to induce neuronal differentiation. In this study, we have identified by means of restriction landmark genome scanning (RLGS) a genomic fragment that was frequently reduced in intensity in neuroblastomas. The cloned fragment contained exon 1 of p73 as well as intronic and promoter sequences. We investigated the genomic and expression status of p73 and N-myc in 34 neuroblastoma tumors and 12 neuroblastoma cell lines. Approximately a third of neuroblastomas in our series exhibited deletion of p73. Most tumors analyzed exhibited reduced expression of p73, as determined by quantitative RT-PCR, in the absence of detectable p73 gene deletion. The reduced expression of p73 correlated with overexpression of N-myc in a statistically significant manner. The N-myc gene was transfected into two neuroblastoma cell lines that lacked N-myc amplification to determine its effect on p73 RNA levels. p73 was detectable at low level by RT-PCR in untransfected SK-N-AS cells and became undetectable following N-myc transfection, whereas in SH-EP1 cells, p73 levels were substantially reduced following transfection but remained detectable. Our data suggest that the N-myc gene modulates expression of p73, allowing neuroblastoma cells to escape the growth suppressing properties of p73.

Figure 1

Close-up sections of RLGS patterns from a neuroblastoma tumor (right) and normal DNA from the same subject (left). The black arrow points to reduced intensity of the p73 genomic fragment in the tumor relative to normal DNA. The white arrow points to an amplified fragment derived from the N-myc amplicon in the tumor [17].

Figure 2

Close-up sections of RLGS patterns from normal whole genomic DNA (left) and from the same DNA after spiking with cloned p73 genomic DNA (right), demonstrating increased intensity of the p73 genomic fragment.

Figure 3

Southern blot analysis of p73 in tumors and cell lines. The filters were also hybridized with a probe for a gene (GAC1) also located on chromosome 1. The adjusted ratio of intensity of the p73 band relative to the GAC1 is indicated. The designation NBT refers to tumors. Other designations refer to cell lines and four normal DNA samples.

Figure 4

RT-PCR analysis of p73 and N-myc expression in neuroblastoma tumors and cell lines. Ubiquitin RT-PCR products were used as control. A fetal adrenal and a fetal brain are included for reference, and the band intensity of their RT-PCR products for p73, N-myc, and ubiquitin were used as controls.

Figure 5

(A) N-Myc expression by Western analysis in SH-EP1 and SK-N-AS transfectants. The KCNR neuroblastoma cell line with amplified N-myc was used as a positive control. Equivalent protein loading is confirmed by GAPDH detection. (B) Reduced intensity of p73 RT-PCR products in SK-N-AS and SH-EP1 transfectants expressing N-Myc.