Arylamine N-acetyltransferase 1 expression in breast cancer cell lines: a potential marker in estrogen receptor-positive tumors.

The prognosis for patients with estrogen receptor (ER)-positive breast cancer has improved significantly with the prescription of selective ER modulators (SERMs) for ER-positive breast cancer treatment. However, only a proportion of ER-positive tumors respond to SERMs, and resistance to hormonal therapies is still a major problem. Detailed analysis of published microarray studies revealed a positive correlation between overexpression of the drug metabolizing enzyme arylamine N-acetyltransferase type 1 (NAT1) and ER positivity, and increasing evidence supports a biological role for NAT1 in breast cancer progression. We have tested a range of ER-positive and ER-negative breast cancer cell lines for NAT1 enzyme activity, and monitored promoter and polyadenylation site usage. Amongst ER-positive lines, NAT1 activities ranged from 202 +/- 28 nmol/min/mg cellular protein (ZR-75-1) to 1.8 +/- 0.4 nmol/min/mg cellular protein (MCF-7). The highest levels of NAT1 activity could not be attributed to increased NAT1 gene copy number; however, we did detect differences in NAT1 promoter and polyadenylation site usage amongst the breast tumor-derived lines. Thus, whilst all cell lines tested accumulated transcripts derived from the proximal promoter, the line expressing NAT1 most highly additionally initiated transcripts initiating at a more distal, "tissue"-specific promoter. These data pave the way for investigating NAT1 transcripts as candidate prognostic markers in ER-positive breast cancer.

Differentiation of Campylobacter coli, Campylobacter jejuni, Campylobacter lari, and Campylobacter upsaliensis by a multiplex PCR developed from the nucleotide sequence of the lipid A gene lpxA.

We describe a multiplex PCR assay to identify and discriminate between isolates of Campylobacter coli, Campylobacter jejuni, Campylobacter lari, and Campylobacter upsaliensis. The C. jejuni isolate F38011 lpxA gene, encoding a UDP-N-acetylglucosamine acyltransferase, was identified by sequence analysis of an expression plasmid that restored wild-type lipopolysaccharide levels in Escherichia coli strain SM105 [lpxA(Ts)]. With oligonucleotide primers developed to the C. jejuni lpxA gene, nearly full-length lpxA amplicons were amplified from an additional 11 isolates of C. jejuni, 20 isolates of C. coli, 16 isolates of C. lari, and five isolates of C. upsaliensis. The nucleotide sequence of each amplicon was determined, and sequence alignment revealed a high level of species discrimination. Oligonucleotide primers were constructed to exploit species differences, and a multiplex PCR assay was developed to positively identify isolates of C. coli, C. jejuni, C. lari, and C. upsaliensis. We characterized an additional set of 41 thermotolerant isolates by partial nucleotide sequence analysis to further demonstrate the uniqueness of each species-specific region. The multiplex PCR assay was validated with 105 genetically defined isolates of C. coli, C. jejuni, C. lari, and C. upsaliensis, 34 strains representing 12 additional Campylobacter species, and 24 strains representing 19 non-Campylobacter species. Application of the multiplex PCR method to whole-cell lysates obtained from 108 clinical and environmental thermotolerant Campylobacter isolates resulted in 100% correlation with biochemical typing methods.

Activator protein-2gamma (AP-2gamma) expression is specifically induced by oestrogens through binding of the oestrogen receptor to a canonical element within the 5'-untranslated region.

The activator protein 2 (AP-2) transcription factors are essential proteins for oestrogenic repression of the ERBB2 proto-oncogene in breast cancer cells. In the present study, we have examined the possible oestrogenic regulation of AP-2 genes themselves in breast-tumour-derived lines. As early as 1 h after oestrogen treatment, AP-2gamma mRNA was markedly increased, whereas AP-2alpha was down-regulated, but with slower kinetics, and AP-2beta was not affected at all. Addition of anti-oestrogens ablated these effects. Modulation of the protein levels corresponded to changes in the transcript levels, thus suggesting that in oestrogen-treated cells, an inversion of the balance between AP-2alpha and AP-2gamma isoforms occurs. The 5'-untranslated region (5'-UTR) of the human AP-2gamma gene contains one consensus and one degenerate oestrogen-responsive element (ERE). Reporter constructs carrying the AP-2gamma promoter and the 5'-UTR were up-regulated by oestrogens in transient transfection assays. Deletion of the most conserved (but not of the degenerate) ERE from reporter constructs abrogated the oestrogenic response, although both ERE-containing segments were footprinted in DNaseI protection assays. In vitro binding assays demonstrated the ability of oestrogen receptor alpha (ERalpha) to bind to this site, and chromatin immunoprecipitation analysis of the endogenous gene showed that ERalpha occupies this region in response to oestrogens. We conclude that AP-2gamma is a primary oestrogen-responsive gene and suggest that AP-2 proteins may mediate some oestrogenic responses.

Characterization of the human activator protein-2gamma (AP-2gamma) gene: control of expression by Sp1/Sp3 in breast tumour cells.

The activator protein-2 (AP-2) family of DNA-binding transcription factors are developmentally regulated and also play a role in human neoplasia. In particular, the AP-2gamma protein has been shown to be overexpressed in a high percentage of breast tumours. In the present study, we report the complete sequence determination of the human TFAP2C gene encoding the AP-2gamma transcription factor plus the mapping of the transcription start site used in breast tumour-derived cells. The 5'-end of the gene lies within a CpG island and transcription is initiated at a single site within a classical initiator motif. We have gone on to investigate why some breast tumour-derived cell lines readily express AP-2gamma, whereas others do not, and show that the proximal promoter (+191 to -312) is differentially active in the two cell phenotypes. DNase footprinting led to the identification of three Sp1/Sp3-binding sites within this region, two of which are absolutely required both for promoter function and cell-type-specific activity. By Western blotting a panel of expressing and non-expressing breast tumour lines we show that the latter have higher levels of Sp3. Furthermore, increasing Sp3 levels in AP-2gamma-expressing cells led to the repression of AP-2gamma promoter activity, particularly when Sp3 inhibitory function was maximized through sumoylation. We propose that differences in the level and activity of Sp3 between breast tumour lines can determine the expression level of their AP-2gamma gene.

Physical and functional interactions among AP-2 transcription factors, p300/CREB-binding protein, and CITED2.

The transcriptional co-activators and histone acetyltransferases p300/CREB-binding protein (CBP) interact with CITED2, a transcription factor AP-2 (TFAP2) co-activator. p300/CBP, CITED2, and TFAP2A are essential for normal neural tube and cardiac development. Here we show that p300 and CBP co-activate TFAP2A in the presence of CITED2. TFAP2A transcriptional activity was modestly impaired in p300(+/-) and CBP(+/-) mouse embryonic fibroblasts; this was rescued by ectopic expression of p300/CBP. p300, TFAP2A, and endogenous CITED2 could be co-immunoprecipitated from transfected U2-OS cells indicating that they can interact physically in vivo. CITED2 interacted with the dimerization domain of TFAP2C, which is highly conserved in TFAP2A/B. In mammalian two-hybrid experiments, full-length p300 and TFAP2A interacted only when CITED2 was co-transfected. N-terminal residues of TFAP2A, containing the transactivation domain, are both necessary and sufficient for interaction with p300, and this interaction was independent of CITED2. Consistent with this, N-terminal residues of TFAP2A were required for p300- and CITED2-dependent co-activation. A histone acetyltransferase-deficient p300 mutant (D1399Y) did not co-activate TFAP2A and did not affect the expression or cellular localization of TFAP2A or CITED2. In mammalian two-hybrid experiments p300D1399Y failed to interact with TFAP2A, explaining, at least in part, its failure to function as a co-activator. Our results suggest a model wherein interactions among TFAP2A, CITED2, and p300/CBP are necessary for TFAP2A-mediated transcriptional activation and for normal neural tube and cardiac development.

FGFR4 overexpression in pancreatic cancer is mediated by an intronic enhancer activated by HNF1alpha.

Fibroblast growth factor receptor 4 (FGFR4) is expressed in 50-70% of pancreatic carcinomas (PC) and a similar proportion of derived cell lines. Here we determine the sites of FGFR4 transcriptional initiation which show a pattern characteristic of genes with GC-rich, TATA-less promoters. We have examined the chromatin structure around the FGFR4 gene in a panel of expressing and non-expressing PC lines using the DNase I hypersensitive site assay. One region of hypersensitivity, located largely within intron 1, was found to be greatly extended in expressing cells. Subsequent functional analyses using reporter assays demonstrated that this region was able to act as a cell-specific enhancer, only showing significant activity in PC lines expressing endogenous FGFR4. Transcription factors able to bind to the enhancer were investigated using footprinting and mobility shift assays and two binding sites for Sp1 proteins and two sites able to bind hepatic nuclear factor 1 (HNF1) proteins were identified. Further reporter assays using constructs mutated in each binding site demonstrated that HNF1 binding was essential for enhancer activity in expressing cells, an observation that correlated with the increased abundance of HNF1alpha in these same cells as measured by Western blotting. Finally we show that exogenous expression of HNF1 factors in an FGFR4 non-expressing line led to an induction of enhancer activity in reporter assays and also activated expression of the endogenous gene. We conclude that HNF1alpha is a major determinant of FGFR4 expression in PC.