EXPRESSION AND MUTATIONAL ANALYSIS OF THE DCC, DPC4, AND MADR2 JV18-1GENES IN NEUROBLASTOMA/

Loss of heterozygosity (LOH) on chromosome 18q21 is found frequently i n various human cancers. Three candidate tumor suppressor genes, DCC ( deleted in colorectal carcinomas), DPC4 (deleted in pancreatic carcino mas, locus 4), and MADR2/JV18-1 (MAD-related gene 2), have been cloned and identified from this chromosome region. We hare reported recently that LOH on chromosome 18q is observed frequently in neuroblastoma. A lterations of DCC are involved in many human tumors. DPC4 and MADR2/JV 18-1 are recently demonstrated to be altered in pancreatic and colorec tal cancers, respectively. To confirm if inactivation of the DCC, DPC4 , and MADR2/ JV18-1 genes is involved in the pathogenesis of neuroblas toma and to clarify the mechanism of inactivation, we analyzed the exp ression of DCC, DPC4, and MADR2/JV18-1 in neuroblastoma cell lines and primary tumors qv reverse transcription-PCR and investigated the muta tions in the coding regions of these genes by PCR/reverse transcriptio n-PCR single-strand conformation polymorphism. We found that 12 of 25 (48%) cell lines and 14 of 32 (44%) primary tumors, including 3 with 1 8q LOH, had absent or reduced expression of DCC mRNA. Expression was m ore likely to be reduced in advanced (67%) than in early-stage neurobl astomas (24%) (P = 0.036), suggesting that inactivation of the DCC gen e plays an important role in the progression of neuroblastoma. Altered expression of DPC4 was found in six (24%) cell lines and six (19%) tu mors. MADR2/JV18-1 expression nas reduced or absent only in four (16%) cell lines and three (9%) tumors. Mutations of the DCC genes were exa mined in 25 of 29 exons in neuroblastoma cell lines, and those exons i n which mutations were found were further examined in primary tumors, We found missense mutations of AAC (Asn) to AGC (Ser) at DCC codon 176 in one cell line and ACC (Thr) to ATC (Ile) at codon 1105 in one cell line and tumor, respectively; polymorphisms of CGA (Arg) to GGA (Gly) at codon 201 and TTT (Phe) to TTG (Leu) at codon 951 in most of the c ell lines and tumors; and a silent mutation of GAG (Glu) to GAA (Glu) al codon 118 in four cell lines and five primary tumors. We did not id entify any mutations in the DPC I and MADR2/JV18-1 genes in neuroblast oma. Our results suggested that mutations of the DCC gene may be invol ved in the pathogenesis of neuroblastomas but failed to account for th e relatively high frequency of the altered expression, implying that o ther mechanisms are responsible for the inactivation of the DCC gene i n neuroblastoma. Low frequency of reduced or absent mRNA expression an d lack of mutations in DPC4 and MADR2/JV18-1 genes suggested a Limited role for these two genes in neuroblastoma.