A novel 1p36.2 located gene, APITD1, with tumour-suppressive properties and a putative p53-binding domain, shows low expression in neuroblastoma tumours

Abstract

Neuroblastoma is characterised by a lack of TP53 mutations and no other tumour suppressor gene consistently inactivated has yet been identified in this childhood cancer form. Characterisation of a new gene, denoted APITD1, in the neuroblastoma tumour suppressor candidate region in chromosome 1p36.22 reveals that APITD1 contains a predicted TFIID-31 domain, representing the TATA box-binding protein-associated factor, TAF(II)31, which is required for p53-mediated transcription activation. Two different transcripts of this gene were shown to be ubiquitously expressed, one of them with an elevated expression in foetal tissues. Primary neuroblastoma tumours of all different stages showed either very weak or no measurable APITD1 expression, contrary to the level of expression observed in neuroblastoma cell lines. A reduced pattern of expression was also observed in a set of various tumour types. APITD1 was functionally tested by adding APITD1 mRNA to neuroblastoma cells, leading to the cell growth to be reduced up to 90% compared to control cells, suggesting APITD1 to have a role in a cell death pathway. Furthermore, we determined the genomic organisation of APITD1. Automated genomic DNA sequencing of the coding region of the gene as well as the promoter sequence in 44 neuroblastoma tumours did not reveal any loss-of-function mutations, indicating that mutations in APITD1 is not a common abnormality of neuroblastoma tumours. We suggest that low expression of this gene might interfere with the ability for apoptosis through the p53 pathway.

Figure 1

(A) Map of the 1p36.2 neuroblastoma tumour suppressor gene candidate region. The location of the genes is based on data from the UCSC genome browser; April 2002 draft sequence. The homozygously deleted region on chromosome 1p in a neuroblastoma cell line, found by Ohira et al (2000), is indicated by a dark shaded box, while the neuroblastoma SRO as defined by our group (Martinsson et al, 1995) is shown as a light grey box. The region includes the genes UBE4B, KIF1B, PGD, APITD1, CORT, DFFA and PEX14. ICAT is located just distal of the region. APITD1 is marked with a black box. Arrows indicate the transcriptional direction of the genes. The scale in the top is the approximate distance from the 1p-terminal, in mega base pairs. (B) The genomic organisation of the APITD1 gene, with the two alternative first exons, is shown with intron sizes indicated in kb. The location of the primers used for amplification of transcripts A and B from cDNA for RT–PCR expression analysis is also illustrated.

Figure 2

The two alternative transcript versions of the APITD1 gene. Both transcripts share exons 2–5, but they differ in the starting exons and in the 3′UTR sequences. Exon 1A is located approximately 160 bp upstream of exon 1B in the genomic sequence. The translated sequence from the ORF in transcript A and the in-frame ORF in transcript B, which starts immediately prior to the putative TFIID-31 domain, is shown. The amino-acid sequence with significant similarity to the TFIID-31 domain is marked in bold face and italics. The last 622 bp of transcript A (chr1_29_927.b), which consists of exon 2 from the nearby located gene CORT and noncoding genomic sequence, is not shown.

Figure 3

Conserved amino acids in the translated APITD1 transcript A ORF. The first nine amino acids in the translated ORF did not align to the translated expressed sequences from other organisms in the TIGR database, and they are not shown. Percent identity with the human sequence is shown within brackets after the name of each organism. Residues which are identical or chemically similar to the human amino-acid sequence are highlighted in grey. The residues which are identical or similar among all aligned sequences are indicated under the alignment, and the TFIID-31 similar domain is framed.

Figure 4

Transcript size of the APITD1 gene. Northern blot analysis of total RNA isolated from the neuroblastoma cell lines SK-N-AS and SH-SY5Y. The blot was hybridised with a radio labelled probe. (A) APITD1. (B) ACTB internal loading control.

Figure 5

RT–PCR expression analysis of transcripts A and B in APITD1. Amplification of APITD1 transcript A and APITD1 transcript B in each sample is compared to the amplification of ACTB. (A) Fairly ubiquitous expression of both transcripts in a set of normal adult and foetal tissues (CLONTECH). Lanes 1–24, PCR products from the indicated tissues. (B) Reduced expression of APITD1 gene products in neuroblastoma tumours of different stages compared to neuroblastoma cell lines and adult and foetal normal tissues (OriGene). The outcome of the patients, the stage of neuroblastoma and the status of chromosome 1p is indicated above the upper panel; NED, no evidence of disease; DOD, dead of disease; 1, 2, 2a, 3, 4 and 4S, stages of neuroblastoma; −, negative for 1p deletion; +, positive for 1p deletion; ±, uncertain result (based on FISH and microsatellite analysis). Lanes 1–17, neuroblastoma tumours; lanes 18–26, neuroblastoma cell lines and normal tissues as indicted above the panel. (C) Reduced expression of APITD1 gene products in various tumours. Lanes 1–14, tumours of various types as indicated above the panel.

Figure 6

PCR amplification of APITD1 exon 4 in a human/rodent somatic cell hybrid mapping panel. Amplification products of the appropriate size are obtained in the chromosome 1 cell hybrid and in the human cell line control exclusively.

Figure 7

Comparative growth curves of cell lines after transfection of 1.5 μg GFP mRNA; 0.3 μg APITD1A mRNA+1.2 μg GFP mRNA or 1.5 μg APITD1A mRNA, respectively. (A) SK-N-AS (neuroblastoma). (B) SK-N-BE(2) (neuroblastoma). (C) K562 (lymphoblast). (D) 293 (transformed embryonal kidney).

Figure 8

Expression of GFP in transfected cells shows that the mRNA has been introduced into the cells. (A) K562 (lymphoblast). (B) SK-N-AS (neuroblastoma). (C) SK-N-BE (neuroblastoma).