Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.5.1.61 (porphobilinogen deaminase)
 637 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In recent years, large discrepancies were described in the success rate of the tyrosinase reverse transcription polymerase chain reaction (RT-PCR) for detecting melanoma cells in the peripheral blood of melanoma patients. We present a quality control study in which we analysed the reproducibility of detection of tyrosinase and MART-1 transcripts in 106 blood samples from 68 melanoma patients (mainly stages III and IV). With this study, we aimed to improve insight in the reproducibility of a RT-PCR for the detection of (minimal) amounts of circulating melanoma cells. We performed two reverse transcriptions on each mRNA sample and performed tyrosinase and MART-1 nested PCRs in duplicate per cDNA sample. Thus, four tyrosinase and four MART-1 measurements were performed per blood sample. In our study, the majority of blood samples was negative for tyrosinase (80%) or MART-1 (66%). Only four samples were positive in all four determinations for tyrosinase and seven for MART-1. Variable results (1-3 times positive results) were obtained for tyrosinase and MART-1 in 16% and 27% respectively. MART-1 PCR had a better performance than tyrosinase PCR. Sensitivity increased when both markers were used. We reasoned that the low number of melanoma marker PCR-positive blood samples can be explained by differences in mRNA quality. By using real-time quantitative PCR, we found that this was not the case: amplification of porphobilinogen deaminase (PBGD), a low copy household gene, was not different in blood samples in which a melanoma marker was not detected from groups in which this marker was detected more or less consistently (1-4 times). When applying real-time quantitative PCR for tyrosinase and MART-1, we found that a low amount of SK-MEL-28 cell equivalents was present in the blood of melanoma patients, with a higher number of equivalents in the group with a consistently positive result. We conclude that low reproducibility of a repeated assay for the detection of circulating melanoma cells is not caused by differences in mRNA quality between the samples, but due to low numbers of amplifiable target mRNA molecules in the mRNA sample. Use of more than one marker and repetition of the assay will increase the probability of finding positive PCR results.
Br J Cancer 1999 May
PMID:Reproducibility of detection of tyrosinase and MART-1 transcripts in the peripheral blood of melanoma patients: a quality control study using real-time quantitative RT-PCR. 1036 Jun 70

Recently, considerable interest has been given to photodynamic therapy of cancer using delta-aminolaevulinic acid to induce protoporphyrin IX as the cell photosensitizer. One advantage of this modality is that protoporphyrin IX is cleared from tissue within 24 h after delta-aminolaevulinic acid administration. This could allow for multiple treatment regimens because of little concern regarding the accumulation of the photosensitizer in normal tissues. However, the haem biosynthetic pathway would have to be fully functional after the first course of therapy to allow for subsequent treatments. Photosensitization of cultured R3230AC rat mammary adenocarcinoma cells with delta-aminolaevulinic acid-induced protoporphyrin IX resulted in the inhibition of porphobilinogen deaminase, an enzyme in the haem biosynthetic pathway, and a concomitant decrease in protoporphyrin IX levels. Cultured R3230AC cells exposed to 0.5 mM delta-aminolaevulinic acid for 27 h accumulated 6.07 x 10(-16) mol of protoporphyrin IX per cell and had a porphobilinogen deaminase activity of 0.046 fmol uroporphyrin per 30 min per cell. Cells cultured under the same incubation conditions but exposed to 30 mJ cm(-2) irradiation after a 3-h incubation with delta-aminolaevulinic acid showed a significant reduction in protoporphyrin IX, 2.28 x 10(-16) mol per cell, and an 80% reduction in porphobilinogen deaminase activity to 0.0088 fmol uroporphyrin per 30 min per cell. Similar effects were evident in irradiated cells incubated with delta-aminolaevulinic acid immediately after, or following a 24 h interval, post-irradiation. There was little gain in efficacy from a second treatment regimen applied within 24 h of the initial treatment, probably a result of initial metabolic damage leading to reduced levels of protoporphyrin IX. These findings suggest that a correlation may exist between the delta-aminolaevulinic acid induction of porphobilinogen deaminase activity and the increase in intracellular protoporphyrin IX accumulation.
Br J Cancer 1999 Jun
PMID:Delta-aminolaevulinic acid-induced photodynamic therapy inhibits protoporphyrin IX biosynthesis and reduces subsequent treatment efficacy in vitro. 1036 7

Protoporphyrin IX, induced by the exogenous addition of delta-aminolevulinic acid, reaches different levels in different tumor cells. Because many of the steps in heme biosynthesis, of which protoporphyrin IX is penultimate, are located in the mitochondria, we surmised that the mitochondrial content of cells may relate to the amount of protoporphyrin IX synthesized in response to excess delta-aminolevulinic acid. We observed that accumulation of MitoTracker, a fluorescent mitochondrial probe, delta-aminolevulinic acid-induced protoporphyrin IX levels, and porphobilinogen deaminase activity all presented with the same cell-line-dependent rank order among the four different neoplastic cells. This rank order, however, differed for cytochrome c oxidase activity, the final enzyme in mitochondrial electron transport, and for accumulation of radioactive label from [(14)C]delta-aminolevulinic acid. The data demonstrate that enzymes involved in heme biosynthesis, in general, display a rank order associated with mitochondrial content. These data imply that such parameters may have value as prognosticators of cells to produce delta-aminolevulinic acid-induced protoporphyrin IX, a photosensitizer for photodynamic therapy of cancer.
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PMID:Relationship of delta-aminolevulinic acid-induced protoporphyrin IX levels to mitochondrial content in neoplastic cells in vitro. 1055 64

5-Aminolaevulinic acid (ALA)-induced porphyrin biosynthesis, which is used for ALA-based photodynamic therapy (ALA-PDT), was studied in tissues of 10 patients with Barrett's oesophagus (BE) and adenocarcinoma of the oesophagus (AC) undergoing oesophagectomy at a mean time interval of 6.7 h after the ingestion of ALA (60 mg kg(-1)). In BE, AC, squamous epithelium (SQ) and gastric cardia, the activities of the haem biosynthetic enzymes porphobilinogen deaminase (PBG-D) and ferrochelatase (FC) and the PDT power index--the ratio between PBG-D and FC in BE and AC in comparison with SQ--were determined before ALA ingestion. Following ALA administration, ALA, porphobilinogen, uroporphyrin I and PPIX were determined in tissues and plasma. The PDT power index did not predict the level of intracellular accumulation of PPIX found at 6.7 h. In BE, there was no selectivity of PPIX accumulation compared to SQ, whereas in half of patients with AC selectivity was found. Higher haem biosynthetic enzyme activities (i.e. PBG-D) and lower PPIX precursor concentrations were found in BE and AC compared to SQ. It is therefore possible that PPIX levels will peak at earlier time intervals in BE and AC compared to SQ.
Br J Cancer 2000 Aug
PMID:Porphyrin biosynthesis in human Barrett's oesophagus and adenocarcinoma after ingestion of 5-aminolaevulinic acid. 1094 4

Understanding the regulation and control of heme/porphyrin biosynthesis is critical for the optimization of the delta-aminolevulinic-acid (ALA)-mediated photodynamic therapy of cancer, in which endogenously produced protoporphyrin IX (PPIX) is the photosensitizer. The human breast cancer cell line MCF-7, the rat mammary adenocarcinoma cell line R3230AC, the mouse mammary tumor cell line EMT-6 and the human mesothelioma cell line H-MESO-1 were used to study ALA-induced PPIX levels and their relationship to delta-aminolevulinic acid dehydratase (ALA-D) activity in vitro. Incubation of these cell lines with 0.5 mM ALA for 3 h resulted in a significant increase in PPIX accumulation, compared with control cells, but there was no significant change in ALA-D activity. Exposure of cells incubated with ALA to 30 mJ/cm2 of fluorescent light, a dose that would cause a 50% reduction in cell proliferation, did not significantly alter the activity of ALA-D. Increasing the activity of porphobilinogen deaminase (PBGD), the enzyme immediately subsequent to ALA-D, by four- to seven-fold via transfection of cells with PBGD complementary DNA did not alter the activity of ALA-D. However, incubation of cells with various concentrations of succinyl acetone, a potent inhibitor of ALA-D, caused a concomitant decline in both PPIX accumulation and ALA-D activity. These data imply that when cells are exposed to exogenous ALA, ALA-D is an important early-control step in heme/porphyrin biosynthesis and that regulation of PPIX synthesis by this dehydratase may impact the effectiveness of ALA-mediated photosensitization.
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PMID:Is delta-aminolevulinic acid dehydratase rate limiting in heme biosynthesis following exposure of cells to delta-aminolevulinic acid? 1128 Oct 29

Recently, considerable interest has been directed to red-fluorescence photodiagnosis of brain and other tumours during surgery using the protoporphyrin IX natural precursor, 5-aminolaevulinic acid. In the present study we focused on the role of the rate-limiting enzyme porphobilinogen deaminase in glioma C6 cell activity, differentiation and sub-cellular distribution. Over-expression of the human housekeeping porphobilinogen deaminase in the glioma cells, using the housekeeping-porphobilinogen deaminase plasmid, induced a G1 cell cycle attenuation accompanied by increases in enzyme activity and c6 differentiation toward astrocytes. Visualisation of subcellular localisation of the porphobilinogen deaminase using the independent techniques of fluorescence immuno-staining with specific anti-human porphobilinogen deaminase antibodies and cellular expression of porphobilinogen deaminase fused to green fluorescent protein, revealed (unexpectedly) a major fraction of porphobilinogen deaminase in the nucleus and only a minor fraction in the cytoplasm. Both C and N terminals of porphobilinogen deaminase fused to green fluorescent protein revealed a major fraction of the newly synthesized fused porphobilinogen deaminase in the nucleus. Furthermore, newborn rat brain cells grown in a primary culture showed the same localisation pattern of porphobilinogen deaminase in the nuclei. Stimulation of C6 glioma cell differentiation by butyrate induced a marked decrease in porphobilinogen deaminase both in the nucleus and in the cytoplasm as determined by Western blotting and fluorescence immuno-localisation. These findings suggest a possible dual role for housekeeping porphobilinogen deaminase in fast dividing glioma cells, one related to the porphyrin synthesis pathway and another coupled to nuclear function, which might be linked to tumorigenesis.
Br J Cancer 2002 Mar 18
PMID:Nuclear distribution of porphobilinogen deaminase (PBGD) in glioma cells: a regulatory role in cancer transformation? 1195 37

Quantitation of target mRNAs using the reverse-transcription polymerase chain reaction found a widespread field of application in diverse biomedical diagnostic assays. However, the problem of varying sample quality has to be solved by correcting target molecule amounts through detection of an endogenous control template. The choice of an appropriate reference gene is still object of debate as pseudogene co-amplification and expression level variations may limit the usefulness of some currently used reference reactions. We compared quantitative expression levels of the commonly used endogenous reference genes beta-actin (beta-actin), beta-2-microglobulin (beta2-MG) and porphobilinogen deaminase (PBDG) using the TaqMan chemistry. With these assays we investigated the respective expression patterns in K562 cells and leucocytes of normal individuals as well as of malignoma patients. In K562 cells 1544+246 beta-actin, 65+30 beta2-MG and 22+/-8 PBDG copies/cell were detected. In normal leucocytes 491+/-97 beta-actin, 40+/-17 beta2-MG and <1 PBDG copies/cell were quantified. Leucocytes of various malignancies exhibited 84+/-51 beta-actin, 106+/-8 beta2-MG and <1 PBDG copies/cell. We conclude that beta2-MG is the most suitable reference gene tested as its variation between different sample origins and within distinct cell types was acceptable low.
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PMID:Quantitative analysis of beta-actin, beta-2-microglobulin and porphobilinogen deaminase mRNA and their comparison as control transcripts for RT-PCR. 1200 44

One strategy to predict clinical outcome in patients with acute myeloid leukemia (AML) is detection of minimal residual disease (MRD) after achievement of hematologic complete remission (CR). We established a real-time RT-PCR assay by use of TaqMan technology for the identification of MRD by quantification of the most frequent fusion transcripts resulting from t(9;11)(p22;q23). To achieve comparable PCR efficiencies between the different PCR assays, primers were chosen to obtain amplicons of nearly identical lengths. MLL/AF9 copy numbers were normalized to the housekeeping gene porphobilinogen deaminase (PBGD). The sensitivity of the assay, as determined at the cellular level, was comparable to that of qualitative single-round RT-PCR. Samples from eight patients with t(9;11)-positive AML were analyzed. At diagnosis and relapse, normalized copy numbers were positive and ranged from 490 to 5,558. Samples from two of seven patients collected at the time of CR became negative, whereas five cases still had positive normalized copy numbers with values between 5 and 5,286. The implications of MRD detection by MLL/AF9 fusion transcript quantification for the clinical management of t(9;11)-positive AML have to be determined in further studies.
Genes Chromosomes Cancer 2003 Nov
PMID:Development of a real-time RT-PCR assay for the quantification of the most frequent MLL/AF9 fusion types resulting from translocation t(9;11)(p22;q23) in acute myeloid leukemia. 1450 4

Protoporphyrin IX (PpIX) synthesis by malignant cells is clinically exploited for photodiagnosis and photodynamic therapy following administration of 5-aminolevulinic acid (ALA). The expression and activity of the housekeeping porphobilinogen deaminase (PBGD) was correlated to PpIX synthesis in differentiating B16 melanoma cells. Differentiation was stimulated by two inducers, butyrate and hexamethylene bisacetamide (HMBA), both of which promote the formation of typical melanosomes and melanin, as well as morphological changeover. A marked decrease in total PBGD activity and PpIX synthesis was observed following stimulation by butyrate, while HMBA induced an opposite effect. In contrast, ferrochelatase levels remained unchanged. Photodynamic inactivation of the cells undergoing differentiation was largely dependent on the PpIX accumulation, which was modulated by the two inducers butyrate and HMBA. Fluorescence immunostaining with anti-PBGD antibodies revealed a major PBGD fraction in the nucleus and a minor fraction in the cytosol. This nuclear localisation pattern was confirmed by expression of PBGD fused to green fluorescence protein. We suggest that efficient photodynamic therapy of cancer facilitated by ALA administration can be enhanced using combined therapeutic modalities.
Br J Cancer 2004 May 04
PMID:Differentiation-dependent photodynamic therapy regulated by porphobilinogen deaminase in B16 melanoma. 1515 May 93

Telomeres cap chromosome ends and are pivotal for DNA stability. Deregulation of the telomere stabilising enzyme telomerase in malignancy has implications in diagnosis, prognosis and therapeutics of cancer. Quantification of the expression of the telomerase catalytic subunit, hTERT, using the LightCycler TeloTAGGG hTERT Quantification kit is not optimal for analysis of chronic myeloid leukemia (CML) samples. The internal control, porphobilinogen deaminase (PBGD) is amplified in a separate tube to hTERT and has an unstable genomic localisation of 11q23. Our laboratory thus developed a real-time reverse transcriptase polymerase chain reaction which co-amplifies hTERT and either mitochondrial single-stranded DNA binding protein 1 (ssBP1) or ubiquitin C (UBC).
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PMID:Real-time quantitative RT-PCR for human telomere elongation reverse transcriptase in chronic myeloid leukemia. 1523 74


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