Abstract
A novel human gene (PLU-1) has been identified which shows a highly restricted expression in normal adult tissues but which is consistently expressed in breast cancers. A fragment of the PLU-1 cDNA was identified by differentially screening a fetal brain library with cDNAs prepared from ce-1 cells (a human mammary epithelial cell line overexpressing c-ErbB2) treated or untreated with the antibody 4D5, which inhibits c-ErbB2 phosphorylation. Clones covering the full cDNA sequence of 6.4 kilobases were isolated from a breast cancer cDNA library. Although expression of PLU-1 in ce-1 cells is regulated by signaling from c-ErbB2, the gene is expressed in all the breast cancer cell lines examined, in cells cultured from primary breast cancers, and in the invasive and in situ components of primary breast cancers. Translation of the open reading frame predicts a protein of 1544 amino acids, which contains three PHD/LAP motifs, a specific DNA-binding domain found in a Drosophila protein (dri) and novel domains showing extensive homology with other human and non human gene products. Transient transfection of cell lines with MYC-tagged PLU-1 showed the protein to be localized in the nucleus and associated with discrete foci. The presence of the dri motif and PHD/LAP fingers together with the clear nuclear localization and consistent expression in breast cancers, suggest a role for PLU-1 in regulating gene expression in breast cancers.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s)AJ132440
Isolation of the PLU-1 Gene—The MTSV1–7 cell line was derived by immortalization of luminal epithelial cells cultured from human milk [2], and the ce-1 cell line was developed by transfection of MTSV1–7 with c-ErbB2 cDNA [3]
We have described here the initial characterization of a novel human gene, PLU-1, which shows restricted expression in normal adult tissues, but which is consistently expressed in primary breast cancers and in breast cancer cells lines
Summary
Cell Lines—MTSV1–7, ce-1, T47D, and ZR75 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FCS (Life Technologies, Inc.) and 0.3 g/ml glutamine. The cDNA library from the human breast carcinoma cell line ZR75 was oligo(dT)-primed, and cDNA sequences were cloned into the uni-ZAP XR vector (Stratagene) with a XhoI site at the 3Ј end and EcoRI site at the 5Ј end [17]. Construction of a Tagged PLU-1 Mammalian Expression Vector— Based on the analysis of the restriction enzymes in the sequence and the amino acid coding sequence, the mammalian expression vector pcDNA 3.1 (Ϫ)/MYC-His A (Invitrogen) with a C-terminal MYC-His tag driven by the cytomegalovirus promotor was selected for constructing the tagged gene. The recombinant clones were sequenced over the joins and PCR regions and the final construct with a 4.781-kb insert is referred as plu1-ORF/MYC-His A
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