Thymidine Phosphorylase Enzyme Activity Research Articles

P05.1 Efficacy and tolerability of miglustat in patients with Niemann – Pick C disease

and dysarthria. She had left sided dystonia since 6 years of age. Because of her fatigue she was bound toWheel chair for 4 years. The parents were not consangineous. Her examination revealed that she was cachectic, had total ophthalmoplegia, pitosis, left facial weakness. Tonus was increased on the left extremities, and had rigidity on the left upper extremity, deep tendon reflexes were hypoactive. Muscle strength was 3/5 on lower extremities. Bilateral babinsky was positive. Previously made tests were as follows; brain MRI; showed hyperintensity on basal ganglions especially on putamen and globus pallidus. MRI spectroscopy showed constant lactate peak on thalamus. MELAS; MERRF; NARP mutations were normal. Muscle biopsy was normal, EEG; normal. With the neurological findings she was thought to have MNGIE: BAER revealed bilateral moderate degree sensory-neural deafness, electromyography revealed demyelinating neuropathy on lower extremities, serum lactate level was normal. Thymidine phosphorylase enzyme activity was normal. Since Thymidine phosphorylase was normal she was reassesed and for Coeliac disease anti-tissue transglutaminase IgA (t-TGIgA) and antigliadin IgA and IgG (AGA IgA and IgG) titres were studied and found to be high. Small intestine biopsy revealed gluten-sensitive enteropathy stage III and she was diagnosed as Coeliac disease. After gluten-free diet her neurological findings improved. We presented this case to show the broad spectrum of neurologic findings in Coeliac Disease.

Interferons upregulate thymidine phosphorylase expression via JAK-STAT-dependent transcriptional activation and mRNA stabilization in human glioblastoma cells

Overexpression of the angiogenic enzyme thymidine phosphorylase (TP) in tumor cells and/or infiltrating macrophages correlates with increased microvessel density and poor prognosis in various tumor types including glioma. The present study examined how the TP gene expression is regulated by different types of interferons (IFNs) in human T98G and A172 glioblastoma cells. Both type I (alpha, beta) and type II (gamma) IFNs upregulated TP mRNA and protein expression while inhibiting cell proliferation. IFN-induced TP mRNA accumulation was not inhibited by the protein synthesis inhibitor cycloheximide, but was strongly blocked by the transcription inhibitor actinomycin D, as well as by transcription factor decoy oligodeoxynucleotides containing the putative IFN response element or the gamma-activated sequence in the TP promoter. The Janus kinase (JAK) inhibitor AG-490 blocked both IFN-induced STAT1 (signal transducers and activators of transcription 1) phosphorylation and TP expression. All IFNs increased the stability of TP mRNA as well. In addition, IFN-evoked TP enzyme activity enhanced the cytotoxicity of 5-fluorouracil (5-FU). These findings indicate that TP expression may be upregulated by IFNs via the JAK-STAT signaling pathway and both transcriptional and posttranscriptional mechanisms. Combined treatment with IFN and 5-fluorouracil may be a useful therapeutic strategy for malignant gliomas.

Definitive diagnosis of mitochondrial neurogastrointestinal encephalomyopathy by biochemical assays.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is caused by mutations in the gene encoding thymidine phosphorylase (TP). The clinical manifestations of MNGIE are recognizable and homogeneous, but in the early stages, the disease is often misdiagnosed. This study assesses the reliability of biochemical assays to diagnose MNGIE. We studied 180 patients with clinical features suggestive of MNGIE, 14 asymptomatic TP mutation carriers, and 20 controls. TP enzyme activity in the buffy coat was determined by a fixed-time method, and the plasma nucleosides thymidine (dThd) and deoxyuridine (dUrd) were assessed by a gradient-elution reversed phase HPLC method. TP was sequenced through standard procedures in patients who met the clinical criteria for MNGIE. Twenty-five of the 180 patients fulfilled the clinical criteria for MNGIE and had homozygous or compound heterozygous TP mutations. All had drastically decreased TP activity [mean (SD), 10 (15) nmol thymine formed. h(-1). (mg protein)(-1) vs 634 (217) nmol thymine formed. h(-1). (mg protein)(-1) for the controls]. Relative to the control mean, TP activities were reduced to 35% in mutation carriers and 65% in MNGIE-like patients. All 25 MNGIE patients had detectable plasma dThd [8.6 (3.4) micromol/L] and dUrd [14.2 (4.4) micromol/L]. Controls, carriers, and MNGIE-like patients showed no detectable plasma dThd and dUrd. We propose a diagnostic algorithm based on the determination of plasma dThd and dUrd, TP activity in buffy coat, or both to make a definitive diagnosis of MNGIE. Increased concentrations of dThd (>3 micromol/L) and dUrd (>5 micromol/L) in plasma or a decrease in buffy coat TP activity to </=8% relative to controls is sufficient to diagnose MNGIE.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE): a disease of two genomes.

Mitochondrial encephalomyopathies are clinically and genetically heterogeneous because mitochondria are the products of 2 genomes: mitochondrial DNA (mtDNA) and nuclear DNA (nDNA). Among the mendelian-inherited mitochondrial diseases are defects of intergenomic communication, disorders due to nDNA mutations that cause depletion and multiple deletions of mtDNA. Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder of intergenomic communication and is defined clinically by 1) severe gastrointestinal dysmotility; 2) cachexia; 3) ptosis, ophthalmoparesis, or both; 4) peripheral neuropathy; and 5) leukoencephalopathy. Skeletal muscle biopsies of patients have revealed abnormalities of mtDNA and mitochondrial respiratory chain enzymes. The disease is caused by mutations in the thymidine phosphorylase (TP) gene. TP protein catalyzes phosphorolysis of thymidine to thymine and deoxyribose 1-phosphate. In MNGIE patients, TP enzyme activity is reduced drastically, and plasma thymidine and deoxyuridine are elevated dramatically. We have hypothesized that alterations of nucleoside metabolism cause an imbalanced mitochondrial nucleotide pool that leads to depletion and deletions of mtDNA. MNGIE is a recognizable clinical syndrome caused by mutations in TP. The diagnosis can be confirmed by measuring TP activity in buffy coat or plasma levels of thymidine and deoxyuridine. Reduction of circulating thymidine and deoxyuridine in MNGIE patients may be therapeutic.

Localization of Thymidine Phosphorylase Expression in Colorectal Carcinoma Tissues by in situ RT-PCR Assay

Abundant expression of thymidine phosphorylase (TP) mRNA and protein was observed in human carcinoma tissues relative to the corresponding normal tissues. However, the distribution of cells that express TP has not yet been determined. In this study, we examined enzyme activity of TP in 24 primary colorectal carcinoma tissues and the corresponding normal tissue. Using in situ reverse-transcription polymerase chain reaction (RT-PCR), a new methodology to determine the localization of genes of interest objectively and clearly, cell distribution was compared. The expression of TP mRNA is observed in normal infiltrating cells adjacent to colon carcinoma cells by in situ RT-PCR. Similar to the protein activity, the average TP mRNA expression (amplified products/×400 field) in cases of Dukes C and D is 76.8, while that of Dukes A and B is 42.5, being significantly different (p = 0.001). Moreover, the number of amplified products in colon carcinoma tissues showed a significant correlation with the enzyme activity evaluated by high-performance liquid chromatography (p < 0.01, R = 0.76). In conclusion, the expression of TP mRNA is localized in stromal tissues as determined by in situ RT-PCR and can reflect the level of TP enzyme activity.

Altered thymidine metabolism due to defects of thymidine phosphorylase.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive human disease due to mutations in the thymidine phosphorylase (TP) gene. TP enzyme catalyzes the reversible phosphorolysis of thymidine to thymine and 2-deoxy-D-ribose 1-phosphate. We present evidence that thymidine metabolism is altered in MNGIE. TP activities in buffy coats were reduced drastically in all 27 MNGIE patients compared with 19 controls. All MNGIE patients had much higher plasma levels of thymidine than normal individuals and asymptomatic TP mutation carriers. In two patients, the renal clearance of thymidine was approximately 20% that of creatinine, and because hemodialysis demonstrated that thymidine is ultrafiltratable, most of the filtered thymidine is likely to be reabsorbed by the kidney. In vitro, fibroblasts from controls catabolized thymidine in medium; by contrast, MNGIE fibroblasts released thymidine. In MNGIE, severe impairment of TP enzyme activity leads to increased plasma thymidine. In patients who are suspected of having MNGIE, determination of TP activity in buffy coats and thymidine levels in plasma are diagnostic. We hypothesize that excess thymidine alters mitochondrial nucleoside and nucleotide pools leading to impaired mitochondrial DNA replication, repair, or both. Therapies to reduce thymidine levels may be beneficial to MNGIE patients.

Thymidine phosphorylase: a two-face Janus in anticancer chemotherapy.

Several cytokines and growth factors modulate angiogenesis through a fine tuned paracrine or autocrine mode of action. Among them is plateled-derived endothelial cell growth factor (PD-ECGF), which is highly is expressed in tumors, and is angiogenic by stimulation of endothelial cell migration. Studies have shown that PD-ECGF is identical to the well known enzyme thymidine phosphorylase (TP), which is involved in thymidine metabolism and homeostasis. Interestingly, PD-ECGF plays an angiogenic role as a result of its TP enzyme activity. In light of these findings, PD-ECGF/TP should not be considered a true growth factor, and its PD-ECGF name is now actually a misnomer. Recently, TP activity was thought of as an interesting potential two-face target for controling tumor-dependent angiogenesis. In fact, on one hand, its high levels of expression in tumors compared to non-neoplastic regions, and its broad substrate specificity suggested that TP could be used as an enzymatic tool to locally activate anticancer nucleoside bases or base analogs. On the other hand, its enzyme-dependent angiogenic activity engendered the search for specific inhibitors to reduce TP-dependent angiogenesis. This review will describe TP, its activity, its possible mechanisms of action and its role in angiogenesis. Particular attention will be focused on the design and biological characterization of novel TP inhibitors which recently showed promising anticancer activity.

Thymidine phosphorylase activity required for tumor angiogenesis and growth

Thymidine phosphorylase (TP) is identical to platelet-derived endothelial cell growth factor (PD-ECGF), and has angiogenic activity. We examined the involvement of TP activity in tumor growth and angiogenesis. KB cells were transfected with wild-type or mutant (L148R) PD-ECGF cDNA, and two sublines with high TP activity, KB/wt4 and KB/wt6, and one subline with no TP activity, KB/L148R, were cloned, respectively. The doubling times of these subclones in vitro were similar to that of KB cells. However, the growth of KB/wt4 and KB/wt6 cells was significantly faster when xenografted into nude mice than that of control cells with no TP activity. The tumors with high TP activity (KB/wt4 and KB/wt6) had significantly more microvessels than those with no TP activity (KB/-, KB/CV and KB/L148R) (P<0.01). These results, taken together with previous reports, suggest that the TP enzyme activity itself is involved in angiogenesis and growth of the KB tumors.

Elevated Thymidine Phosphorylase Activity in Psoriatic Lesions Accounts for the Apparent Presence of an Epidermal “Growth Inhibitor,” But Is Not in Itself Growth Inhibitory

An apparent tissue-specific growth inhibitor, or chalone, obtained from psoriatic lesions was tentatively identified in the 100-kDa fraction based upon inhibition of DNA synthesis, as measured by [3H]-thymidine uptake by a squamous cell carcinoma cell line, SCC 38. This fraction, however, failed to inhibit SCC 38 cell growth when assessed directly in a neutral red uptake assay. Characterization of the inhibitor of [3H]-thymidine uptake revealed it to have biochemical properties identical to thymidine phosphorylase: 1) molecular weight close to 100 kDa, 2) isoelectric point of 4.2, and 3) thymidine phosphorylase enzyme activity. Thus, we conclude that its ability to inhibit [3H]-thymidine uptake was due to thymidine catabolism rather than inhibition of DNA synthesis or growth inhibition. Examination of thymidine phosphorylase activity in keratome biopsies from psoriatic and normal skin demonstrated a twentyfold increase in activity in psoriatic lesions relative to non-lesional or normal skin. This increase in metabolism of thymidine was due to thymidine phosphorylase rather than uridine phosphorylase activity. The correlation between increased thymidine phosphorylase activity and increased keratinocyte proliferation in vitro (cultured) and in vivo (psoriasis), suggests that this enzyme may play a critical role in providing the thymidine necessary for keratinocyte proliferation.