Identification and characterization of a biallelic deletion in chromosome band 18q21.1 of human breast cancer cell line HCC-1428

Representational difference analysis (RDA) was performed on the genome of human breast cancer cell line HCC-1428 to identify novel candidate tumor suppressor gene loci. A biallelic deletion in the HCC-1428 genome was identified at the 18q21.1 chromosome band. This deletion was mapped using novel and established 18q21.1 sequence tagged sites (STSs) and is 260 to 330 kilobases in size. The deletion was confirmed by Southern blot analysis. Sequence analysis of candidate single nucleotide polymorphisms (SNPs) of the genome of the paired normal cell line, HCC-1428 BL, confirmed that the deletion eliminates both constitutive alleles. This deletion is in between but does not alter the open reading frames (ORFs) of candidate tumor suppressor genes Smad4 and DCC.; Sequence analysis determined that the HCC-1428 cell line harbors a novel, in frame three amino acid deletion mutation of the Smad4 candidate tumor suppressor gene. The mutation, "p.Glu49_Lys51del," eliminates codons 49 through 51 (glutamate-lysine-lysine) of the Smad4 MH 1 domain and is the second documented ORF mutation of Smad4 in a human breast tumor. Pulse chase analysis of p.Glu49_Lys51del Smad4 vs. normal Smad4 demonstrates destabilization of the mutant protein. The relevance of these findings to the search for novel tumor suppressor genes on human chromosome arm 18q and to further elucidation of the biochemistry of the Smad4 protein is discussed.