Abstract
The proto-oncogene neu (HER2 or c-erbB2) is overexpressed with or without gene amplification in 20-30% of breast cancers. In patients, neu amplification or overexpression in breast and ovarian cancer correlates with poor prognosis and tumor resistance to chemotherapy. neu-induced transformation can be reversed by the suppression of neu gene transcription. To further understand how neu gene transcription is regulated and to identify a possible transcriptional repressor(s) of neu, we identified a negative regulatory element known previously to be located within a 1-kilobase (kb) DNA fragment of an unknown sequence, upstream of the proximal neu gene promoter. One of several DNA fragments subcloned from this region suppressed transcriptional activity of the proximal neu gene promoter. Sequencing of the 1-kb fragment confirmed the location of the repressor element to be between an AluI and a RsaI sites, around 1.4 kb upstream to the translation start site. Various deletions were introduced into the AluI-RsaI fragment and subcloned into both the native neu promoter and a heterologous thymidine kinase promoter. Subsequent transfections and reporter gene assays in cell lines of various tissues of origin confirmed and narrowed the repressor activity to a 120-base pair NlaIV-MslI fragment located between -1385 and -1266. Importantly, specific protein binding activity to this element could be detected with nuclear extracts isolated from these cell lines. In contrast, a 28-base pair MslI-RsaI fragment (-1265 to -1238), located immediately 3' of the putative repressor element, was found to form protein-DNA complexes with only nuclear extracts isolated from a colon carcinoma cell line. This specific protein binding activity correlated with a previously unknown transcriptional stimulatory activity only in this cell line.
Highlights
Neu was originally identified as the transforming gene in ethylnitrosourea-induced neuroblastomas in BDIX rats [1, 2]
As many as 20 –30% of human breast and ovarian cancers are found to exhibit HER2 gene amplification or overexpression (19, 20, and reviewed in Ref. 16), which correlates with reduced survival [19, 20]
Our discussion will be limited to several sites that were located within the minimized functional repressor element as noted in a later section
Summary
Enzymes and Reagents—Restriction enzymes and other DNA modifying enzymes including Klenow fragment, T4 polymerase, T4 ligase, and calf intestinal phosphatase were purchased from Life Technologies Inc., New England Biolabs (Mississauga, Ontario), Roche Molecular Biochemicals, or Amersham Pharmacia Biotech. Thin layer chromatography (TLC) plates were the products of Eastman Kodak Co. Cell culture media and reagents were obtained from Life Technologies Inc. Plasmids—The plasmid pBluescript(IIKS) (Stratagene, La Jolla, CA) was used for general subcloning purposes. The pNeuEcoRVCAT construct containing the proximal 500-bp neu gene promoter linked to the CAT gene has been previously described [36]. This construct was used as a reporter for analyzing the effects of various subcloned DNA fragments on the transcriptional activity of the native neu gene promoter. The TLC method of CAT assays was performed as described previously [36], except that the standard [14C]chloramphenicol was replaced with 1-[dichloroacetyl-114C]deoxychloramphenicol (Amersham Pharmacia Biotech) [40]. The gel was dried under vacuum in a gel dryer and exposed to a Kodak XAR-5 film at Ϫ80 °C
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