HPD overexpression predicts poor prognosis in breast cancer.

BACKGROUND: The enzyme, 4-hydroxyphenylpyruvate dioxygenase (HPD), is critical to tyrosine metabolism; its deficiency can cause tyrosinemia. However, its precise contribution to tumorigenesis is unclear. Here, we investigated the correlation between HPD expression and prognosis in patients with breast cancer. METHODS: 145 breast cancer specimens were selected to analyze HPD protein expression by immunohistochemistry and evaluate its relationship to patients' clinicopathological features. HPD localization was confirmed in MCF-7 and MDA-MB-231 breast cancer cells, using immunofluorescence staining. The expression of HPD protein was detected in breast cancer and cancer-adjacent normal tissues using Western blot analysis. Survival rates were calculated by the Kaplan-Meier method. RESULTS: We found that HPD protein was mainly located in the cytoplasm/nucleoli/perinucleus in breast cancer cells, as shown by immunofluorescence staining in MCF-7 and MDA-MB-231 cells, and immunohistochemistry in breast cancer and adjacent normal tissues (HPD protein expression-breast cancer: 46.9% [68/145], ductal carcinoma in situ [DCIS]: 22.6% [12/53], and normal tissues: only 4.8% [2/42]). Similarly, the Western blot results further confirmed the increased expression of HPD in breast cancer compared with cancer-adjacent normal tissues (P < 0.05). HPD expression level was positively correlated with histological grade and clinical stage, and inversely correlated with 10-year overall survival (OS) rates, in patients with breast cancer. Among patients with breast cancer, those with high HPD expression had worse OS rates than those with low HPD expression. Additionally, when patients were subgrouped by disease stage or grade, those with high HPD expression had worse OS rates than those with low HPD expression for each respective stage or grade. CONCLUSIONS: Our findings indicate that HPD may be a useful prognostic predictor, and a potential therapeutic target for patients with breast cancer.

HpdR is a transcriptional activator of Sinorhizobium meliloti hpdA, which encodes a herbicide-targeted 4-hydroxyphenylpyruvate dioxygenase.

Sinorhizobium meliloti hpdA, which encodes the herbicide target 4-hydroxyphenylpyruvate dioxygenase, is positively regulated by HpdR. Gel mobility shift and DNase I footprinting analyses revealed that HpdR binds to a region that spans two conserved direct-repeat sequences within the hpdR-hpdA intergenic space. HpdR-dependent hpdA transcription occurs in the presence of 4-hydroxyphenylpyruvate, tyrosine, and phenylalanine, as well as during starvation.

Human 4-hydroxyphenylpyruvate dioxygenase gene (HPD).

Overlapping DNA fragments spanning approximately 21 kb of genomic DNA and encompassing the human 4-hydroxyphenylpyruvate dioxygenase gene (HPD) have been cloned by screening a human leukocyte genomic library and by PCR amplification of human fibroblastic DNA. A continuous gene sequence of 20,890 nucleotides was established, including 1957 bp of the 5'-flanking region. The 4-hydroxyphenylpyruvate dioxygenase gene is composed of 14 exons interrupted by 13 introns, all exhibiting conventional vertebrate splicing. Computer analysis of the DNA sequence revealed 12 complete repetitive Alu elements, 1 in the 5'-flanking region and 11 in the intervening segments of the gene. The transcriptional initiation site was mapped to a position 35 nt upstream of the translational start point. The computer analysis also identified several potential transcription regulatory elements, including one CRE site, two AP-2 sites, and two Sp1 sites, in the sequence upstream of the transcription initiation site. Functional analysis of promoter activity by transient transfection of chloramphenicolacetyl transferase reporter plasmids revealed a possible involvement of cyclic adenosine monophosphate in the regulation of transcription. The highest level of expression of 4-hydroxyphenylpyruvate dioxygenase was found in human liver tissue as demonstrated by Northern blot analysis.

Isolation of cDNA clones for genes showing enhanced expression in barley leaves during dark-induced senescence as well as during senescence under field conditions.

Senescence of barley (Hordeum vulgare L. cv. Carina) primary foliage leaves was induced by transfer of the plants into darkness for 2 d. Under these conditions senescence was characterized by a light-reversible decline in the efficiency of photosystem II, and in chlorophyll and protein contents. To isolate senescence-associated genes a differential display of cDNA fragments amplified from reversely transcribed RNA was employed. By this method, gene expression in leaves of control plants collected at the onset of the dark period was compared with gene expression in senescing leaves collected at the end of the extended dark period. The expression of the genes represented by various differentially displayed cDNA fragments was examined by Northern blot hybridizations with RNA derived from primary foliage leaves before and after induction of senescence by darkness. In order to test whether these genes with enhanced expression during dark-induced senescence also show enhanced expression during natural senescence, Northern blot hybridizations were carried out with RNA samples prepared from flag leaves of barley plants during maturation and senescence under field conditions. Five of the cDNA fragments representing transcripts associated with dark-induced senescence, as well as with natural senescence, were selected as probes for screening a cDNA library from senescent flag leaves. With one probe a larger cDNA including a complete open reading frame with homology to the sequence of a known proteinase inhibitor was found. Another cDNA isolated by this means showed high sequence similarity with a gene coding for a 4-hydroxyphenylpyruvate dioxygenase. The other three larger cDNA clones isolated by this procedure so far do not show significant homologies with known sequences.

The C-terminal of rat 4-hydroxyphenylpyruvate dioxygenase is indispensable for enzyme activity.

We have cloned and overexpressed rat 4-hydroxyphenylpyruvate dioxygenase (4HPPD) in Escherichia coli. The soluble, active recombinant enzyme was shown to contain both 4HPPD and alpha-ketoisocaproate dioxygenase (alpha KICD) activity. However, upon truncation of the 14 amino acids at the C-terminus by site-directed mutagenesis, the resulting mutant enzyme (rat F antigen) exhibited complete loss of 4HPPD and alpha KICD activities. This finding suggests that the C-terminal extension domain plays an essential role in the catalytic activity of the enzyme.

A nonsense mutation in the 4-hydroxyphenylpyruvic acid dioxygenase gene (Hpd) causes skipping of the constitutive exon and hypertyrosinemia in mouse strain III.

4-Hydroxyphenylpyruvic acid dioxygenase (HPD; EC 1.13.11.27) is an important enzyme in tyrosine catabolism in most organisms. Decreased activity of 4-hydroxyphenylpyruvic acid dioxygenase in the liver of mouse strain III is associated with tyrosinemia. We report a nucleotide substitution that generates a termination codon in exon 7 of the 4-hydroxyphenylpyruvic acid dioxygenase gene in III mice. This mutation is associated with partial exon skipping, and most of the mRNA lacks sequences corresponding to exon 7. The partial exon skipping apparently is the result of a nonsense mutation in the exon. Mouse strain III is a model for human tyrosinemia type 3 (McKusick 276710), and this strain together with recently established models for tyrosinemia type 1 will facilitate studies of hereditary tyrosinemias.

Structure of the human 4-hydroxyphenylpyruvic acid dioxygenase gene (HPD).

4-Hydroxyphenylpyruvic acid dioxygenase (HPD) is an important enzyme in tyrosine catabolism in most organisms. The activity of this enzyme is expressed mainly in the liver and developmentally regulated in mammals, and a genetic deficiency in this enzyme in humans and mice leads to hereditary tyrosinemia type 3. Using human HPD cDNA as a probe, a chromosomal gene related to HPD was isolated from human gene libraries. The human HPD gene is over 30 kb long and is split into 14 exons. The extract size and boundaries of exon blocks were determined, and all of the splice donor and acceptor sites conformed to the GT/AG rule. Analysis of the 5' flanking sequence of the gene suggests that expression of the gene is regulated by hepatocyte-specific and liver-enriched transcription factors, as well as by hormones. These features of the 5' flanking region of the gene are similar to those of other genes that are specifically expressed in hepatocytes and that are developmentally regulated.