Frequent LOH3 is a hallmark for the presence of tumor suppressor genes on the affected chromosome regions. LOH on chromosome

Loss ofheterozygosity (LOH) on chromosome 18q21 is found frequently in various human cancers. Three candidate tumor suppressor genes, DCC (de leted in colorectal carcinomas), DM24 (deleted in pancreatic carcinomas, locus 4), and MADR2/JV18-1(MAD-relatedgene2), have been doned and Identified from this chromosome region. We have reported recently that LOH on chromosome 18q is observed frequently in neuroblastoma. Alterations of DCC are involved in many human tumors. DPC4 and MADR2/JVJ8-1 are recently demonstrated to be altered in pancreatic and colorectal cancers, respectively.To confirm if inactivation of the DCC, DPC4, and MADR2/ JV18-1 genes is involved in the pathogenesis of neuroblastoma and to clarify the mechanism of inactivation,we analyzed the expression of DCC, DPC4, and MADR2/JVI8-1 in neuroblastoma cell lines and primary tumors by reverse transcription-PCR and investigated the mutations in the coding regions of these genes by PCR/reverse transcription-PCR single-strand con formation polymorphism. We found that 12 of 25 (48%) cell lines and 14 of 32 (44%) primary tumors, including 3 with lSq LOH, had absent or reduced expression of DCC mRNA. Expression was more likely to be reduced in advanced (67%) than in early stage neuroblastomas (24%) (P 0.036), suggesting that inactivation of the DCC gene plays an important role in the progressionof neuroblastoma.Altered expressionoIDPC4 was found in six (24%) cell lines and six (19%) tumors. MADR2/JV18-1expression was re duced or absent only In four (16%) cell lines and three (9%) tumors. Muta dons ofthe DCC genes were examined in 25 of29 exons in neuroblastoma cell lines,andthoseexonsinwhichmutationswerefoundwerefurtherexamined in prlmal7 tumors. We found mi&sensemutations oIAAC (Asn)to AGC (Ser) at DCCcodesi 176 in one cell line and ACC (Thr) to ATC (lIe) at codon 1105 in one cell line and tumor, respectively; polymorphlsmsof CGA (ArgJ to GGA (Gly) at codon 201 and lTf (Phe) to UG (Leu) at codon 951 in most ofthe cell linesand tamors and a silentmutationofGAG (Glu)to GAA (Glu) at codon 118 in four cell lines and five primarytumors. We did not identify any mutations in theDPC4 and MADR2/JVI8-1 genes in neuroblastoma. Our results suggested that mutations of the DCC gene may be involved in the pathogenesis of neuroblastomas but failed to account for the relatively high frequency of the altered expression, implying that other mechanisms are responsible for the inactivation of the DCC gene in neuroblastoma. Low frequency of reduced or absent mRNA expression and lack of mutations in DPC4 and MADR2/JV18-1 genessuggested a limited role for these two genes in neuroblastoma.