Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0019204 (hepatocellular carcinoma)
 71,386 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cultured rat hepatoma cells, H4-II-E-C3, are known to possess a phenylananine hydroxylating system which is sufficient to enable them to grow in tyrosine-depleted medium. Using standard procedures of auxotroph enrichment with this cell line, we have isolated tyrosine auxotrophs for the first time. We report in this paper the class of auxotrophs with (a) reduced (15-64% of wild type) or (b) absent activity of phenylalanine hydroxylase, an enzymic component of the phenylalanine hydroxylating system. This class of auxotroph presumably contains either lower (a) [or zero (b)] levels of normal phenylalanine hydroxylase protein than wild type, or mutant phenylalanine hydroxylase protein with lowered (or zero) activity. The two subgroups of auxotrophs (a) and (b) differ from each other in their revertibility and their growth behavior in the tyrosine-free medium. Over a 12-month period of testing, the auxotrophs have been highly stable with respect to their phenylalanine hydroxylase activity and growth phenotype in tyrosine-free medium. Such auxotrophs should facilitate genetic and biochemical study of the genes controlling the phenylalanine hydroxylation system and the study of phenylketonuria.
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PMID:Genetics of the mammalian phenylalanine hydroxylase system: I. Isolation of phenylalanine hydroxylase-deficient tyrosine auxotrophs from rat hepatoma cells. 19 52

Mutations in the human phenylalanine hydroxylase gene associated with two prevalent mutant alleles have been identified and shown to be in linkage disequilibrium with the corresponding mutant restriction fragment length polymorphism haplotypes. These results suggest the possibility of carrier detection in the population without a prior family history of phenylketonuria (PKU). Furthermore, recombinant retroviruses containing the full-length human phenylalanine hydroxylase cDNA have been constructed and used to transduce functional enzymatic activity into cultured hepatoma cells. Together with the recent success in retroviral infection of primary mouse hepatocytes, it will be possible to use the mouse model to investigate somatic gene therapy for PKU.
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PMID:Molecular basis of phenylketonuria and recombinant DNA strategies for its therapy. 289 2

P-chlorophenylalanine is an irreversible inhibitor of rat phenylalanine hydroxylase in vivo and in rat hepatoma cells and is frequently administered to rodents to create an animal model for phenylketonuria. We investigated the effect of p-chlorophenylalanine on production of human phenylalanine hydroxylase in human hepatoma cells and cells transformed with the recombinant human phenylalanine hydroxylase gene. P-chlorophenylalanine inhibited production of the human enzyme in human hepatoma cells and transformed mouse hepatoma cells but had no effect on the production of the enzyme in transformed NIH3T3 cells or in E. coli. Thus, phenylalanine hydroxylase inhibition does not result from a simple interaction between the drug and enzyme.
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PMID:P-chlorophenylalanine does not inhibit production of recombinant human phenylalanine hydroxylase in NIH3T3 cells or E. coli. 294 45

Phenylketonuria (PKU) is caused by deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH). A full-length human PAH cDNA sequence has been inserted into pzip-neoSV(X), which is a retroviral vector containing the bacterial neo gene. The recombinant has been transfected into psi 2 cells, which provide synthesis of the retroviral capsid. Recombinant virus was detected in the culture medium of the transfected psi 2 cells, which is capable of transmitting the human PAH gene into mouse NIH 3T3 cells by infection leading to stable incorporation of the recombinant provirus. Infected cells express PAH mRNA, immunoreactive PAH protein, and exhibit pterin-dependent phenylalanine hydroxylase activity. The recombinant virus is also capable of infecting a mouse hepatoma cell line that does not normally synthesize PAH. PAH activity is present in the cellular extracts and the entire hydroxylation system is reconstituted in the hepatoma cells infected with the recombinant viruses. Thus, recombinant viruses containing human PAH cDNA provide a means for introducing functional PAH into mammalian cells of hepatic origin and can potentially be introduced into whole animals as a model for somatic gene therapy for PKU.
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PMID:Retroviral-mediated gene transfer of human phenylalanine hydroxylase into NIH 3T3 and hepatoma cells. 345 78

Cells deficient in phenylalanine hydroxylase (PAH) are tyrosine auxotrophs and will not survive in tyrosine-free media. PAH activity can be constituted in cultured cells by infection with recombinant retroviruses carrying a human PAH cDNA. Mouse hepatoma cells transformed with recombinant PAH will grow in tyrosine-free media since these cells constitutively synthesize the cofactor tetrahydrobiopterin which is essential for PAH activity. NIH3T3 cells transformed with the PAH cDNA express the PAH apoenzyme, but this enzyme is inactive in vivo since these cells do not synthesize biopterin. We describe a method of selection for PAH in the fibroblast-like NIH3T3 cells involving tyrosine-free media supplemented with biopterin, reducing agents, and antioxidants. Cells transformed with the recombinant PAH gene exhibit PAH activity in culture and will grow in the biopterin-supplemented tyrosine-free media. Metabolic selection for PAH activity provides a new selectable marker for gene transfer experiments. This method is shown to be useful in the production of high titers of recombinant retroviruses carrying PAH and provides a model for experiments in somatic gene therapy of phenylketonuria.
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PMID:Selection for phenylalanine hydroxylase activity in cells transformed with recombinant retroviruses. 347 Sep 52

Mammalian phenylalanine hydroxylase (PAH) catalyses the conversion of L-phenylalanine to L-tyrosine in the presence of dioxygen and tetrahydrobiopterin; it is a highly regulated enzyme. Little is known about the rates of synthesis and degradation of PAH in vivo. The enzyme has been reported to have a half-life of approx. 2 days in rat liver and 7-8 h in rat hepatoma cells, but the mechanism of its degradation is not known. In the present study it is shown that the tetrameric form of the recombinant wild-type human enzyme is a substrate for the ubiquitin-conjugating enzyme system in the cytosolic fraction of rat testis. Our findings support the conclusion that multi-/poly-ubiquitination of human PAH plays a key role in the turnover of this cytosolic liver enzyme and provides a mechanism for the increased turnover observed for a number of recombinant mutant forms of the enzyme related to the metabolic disorder phenylketonuria, when expressed in eukaryotic cells.
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PMID:Recombinant human phenylalanine hydroxylase is a substrate for the ubiquitin-conjugating enzyme system. 892 Oct 3

An A-->T substitution in cDNA nucleotide 1197 (c.1197A/T) of the human phenylalanine hydroxylase (PAH) gene has been regarded as a silent mutation, because both the wild-type (GUA) and the mutant (GUU) alleles encode a valine residue at codon 399 (V399 V). The nucleotide c.1197 is located at the 3'-end of exon 11at position -3 of the exon-intron junction. To explore whether the substitution exerts any effects on the processing of the PAH mRNA, illegitimate PAH transcripts from lymphoblast cultures of a phenylketonuria (PKU) patient heterozygous for c.1197A/T were analyzed by the polymerase chain reaction following reverse-transcription (RT-PCR). mRNAs with an exon 11 deletion were revealed. Furthermore, by using an R408 W mutation in the paternal allele as a marker, sequence analysis of the RT-PCR products indicates that virtually all PAH transcripts from the maternal allele with the c. 1197A/T substitution do not contain exon 11. To address whether this substitution is the main determinant for exon skipping, PAH minigenes with or without the substitution were constructed and transfected to a human hepatoma cell line. Analysis of the transcription products by S1 nuclease mapping clearly indicated that such exon 11 skipping was directly associated with the c.1197A/T substitution. Thus, this study demonstrates that the c.1197A/T substitution in the PAH gene is not just a neutral polymorphism but a mutation that induces post-transcriptional skipping of exon 11 leading to a PKU phenotype.
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PMID:A silent mutation induces exon skipping in the phenylalanine hydroxylase gene in phenylketonuria. 1121 2

Tetrahydrobiopterin (BH4)-responsive phenylalanine hydroxylase (PAH) deficiency is a recently recognized variant of phenylketonuria, with a probable multifactorial molecular basis. In this study we have investigated the effect of BH4 on PAH gene expression in human hepatoma. Our results show that increased BH4 levels result in an enhancement of PAH activity and PAH protein, due to longer turnover rates, while PAH mRNA levels remain unchanged. This was confirmed for mutant PAH proteins (A309V, V388M and Y414C) associated to in vivo BH4 responsiveness, validating previous studies. We can conclude that there is no effect of the cofactor on PAH gene transcription, probably being the chemical chaperone effect of BH4 stabilizing mutant PAH proteins the major underlying mechanism of the response.
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PMID:Analysis of the effect of tetrahydrobiopterin on PAH gene expression in hepatoma cells. 1650 82

Phenylketonuria (PKU), the most frequent disorder of amino acid metabolism, is caused by mutations in human phenylalanine hydroxylase gene (PAH), leading to deficient enzyme activity. Previously reported but uncharacterized PAH gene mutation, p.S231F (c.692C > T), was detected in Serbian patients with classical PKU. We analyzed p.S231F PAH protein in prokaryotic (Escherichia coli) and eukaryotic expression system (hepatoma cells). In both systems the mutant enzyme was unstable. Residual enzyme activity in vitro was approximately 1%. Mutation p.S231F PAH was not activated by pre-incubation with phenylalanine substrate. We found no GroEL/GroES chaperone effect and slightly positive effect of the (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH(4)) on the stabilization of the protein structure. Our findings were in accordance with severe patients' phenotypes. In conclusion, p.S231F should be classified as a functionally null PAH gene mutation as it drastically reduces stability and activity of the PAH enzyme in vitro.
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PMID:The Missense p.S231F phenylalanine hydroxylase gene mutation causes complete loss of enzymatic activity in vitro. 1962 56

Treatment with tetrahydrobiopterin (BH4) is the latest therapeutic option approved for patients with phenylketonuria (PKU)-one of the most frequent inborn metabolic diseases. PKU or phenylalanine hydroxylase (PAH) deficiency is caused by mutations in the PAH gene. Given that some PAH mutations are responsive to BH4 treatment while others are non-responsive, for every novel mutation that is discovered it is essential to confirm its pathogenic effect and to assess its responsiveness to a BH4 treatment in vitro, before the drug is administered to patients. We found a c.676C>A (p.Gln226Lys) mutation in the PAH gene in two unrelated patients with PKU. The corresponding aberrant protein has never been functionally characterized in vitro and its response to BH4 treatment is unknown. Computational analyses proposed that glutamine at position 226 is an important, evolutionary conserved amino acid while the substitution with lysine probably disturbs tertiary protein structure and impacts posttranslational PAH modifications. Using hepatoma cellular model, we demonstrated that the amount of mutant p.Gln226Lys PAH detected by Western blot was only 1.2% in comparison to wild-type PAH. The addition of sepiapterin, intracellular precursor of BH4, did not increase PAH protein yield thus marking p.Gln226Lys as BH4-non-responsive mutation. Therefore, computational, experimental, and clinical data were all in accordance showing that p.Gln226Lys is a severe pathogenic PAH mutation. Its non-responsiveness to BH4 treatment in hepatoma cellular model should be considered when deciding treatment options for PKU patients carrying this mutation. Consequently, our study will facilitate clinical genetic practice, particularly genotype-based stratification of PKU treatment.
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PMID:Functional Characterization of Novel Phenylalanine Hydroxylase p.Gln226Lys Mutation Revealed Its Non-responsiveness to Tetrahydrobiopterin Treatment in Hepatoma Cellular Model. 2965 78


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