Expression Of Hypoxanthine-guanine Phosphoribosyltransferase Research Articles

Autophagy and unfolded protein response induction: a crosstalk between street rabies virus and the host.

The endoplasmic reticulum (ER) response mechanism to cellular stress is mediated by the unfolded protein response/ER-associated degradation (UPR/ERAD) pathway. A viral infection can trigger ER stress and engage some transcription factors, depending on the host cell and virus type, activating or inhibiting autophagy. The relationship between ER response and autophagy in rabies has not been investigated yet. In the present study, the mouse brain was infected with street rabies virus (SRABV). Total RNA was extracted from the brains of animals, and cDNA was synthesized. Next, real-time PCR assay was performed using specific primers. The expression of hypoxanthine-guanine phosphoribosyltransferase (Hprt), CCAAT/enhancer binding protein homologous protein (CHOP), apoptosis signal-regulating kinase 1 (ASK1), activating transcription factor 6 (ATF6), and caspase 3 (CASP3) genes was also investigated. Based on the results, SRABV caused significant changes in the mRNA expression of ATF6, CHOP, and ASK1 genes in the brains of infected mice in the control group (group V). Treatment of infected cells with the pIRES-EGFP-Beclin-1 vector and rapamycin caused changes in nearly most of the parameters. However, alterations in CASP3 gene expression were only observed when the vector and the virus were simultaneously injected into the cells. Overall, protection and autophagy against cell death induced by SRABV infection can be achieved by activating the ER stress pathway, followed by a marked increase in the expression of ATF6, CHOP, ASK1, and CASP3 genes.

HPRT1 Deficiency Induces Alteration of Mitochondrial Energy Metabolism in the Brain

Alterations in function of hypoxanthine guanine phosphoribosyl transferase (HPRT), one of the major enzymes involved in purine nucleotide exchange, lead to overproduction of uric acid and produce various symptoms of Lesch-Nyhan syndrome (LNS). One of the hallmarks of LNS is maximal expression of HPRT in the central nervous system with the highest activity of this enzyme in the midbrain and basal ganglia. However, the nature of neurological symptoms has yet to be clarified in details. Here, we studied whether HPRT1 deficiency changes mitochondrial energy metabolism and redox balance in murine neurons from the cortex and midbrain. We found that HPRT1 deficiency inhibits complex I-dependent mitochondrial respiration resulting in increased levels of mitochondrial NADH, reduction of the mitochondrial membrane potential, and increased rate of reactive oxygen species (ROS) production in mitochondria and cytosol. However, increased ROS production did not induce oxidative stress and did not decrease the level of endogenous antioxidant glutathione (GSH). Thus, disruption of mitochondrial energy metabolism but not oxidative stress could play a role of potential trigger of brain pathology in LNS.

Abstract 1840: Isolation and evaluation of human nanobodies against hypoxanthine guanine phosphoribosyltransferase (HPRT) and their potential use for the detection and targeting of lung, breast and colon cancer cells

Abstract Since the clinical relevance of hypoxanthine guanine phosphoribosyltransferase (HPRT) as both a cancer biomarker and a potential tumor target is increasing, we isolated human nanobodies (Nbs) against HPRT and tested their potential to detect and target cytosolic and membrane associated HPRT in lung, breast and colon cancer cells. HPRT is a purine salvage pathway enzyme that catalyzes the conversion of hypoxanthine to inosine monophosphate and guanine to guanosine monophosphate. Until recently, HPRT was considered as a housekeeping gene and its clinical relevance was mainly limited to congenital central nervous disorders such as Lesch-Nyhan disease. However, recent gene expression analysis across all cancers in The Cancer Genome Atlas (TCGA) database and analysis of HPRT expression within cancer patients' tissues has revealed that the expression of HPRT is elevated in the majority of malignant tissues in comparison to normal tissues. Moreover, multiple studies have recently shown that HPRT may be associated with the cell membrane in lung, colon cancer and lymphoma cells, and not in normal cells. Thus, HPRT is a potential immunotherapeutic target for cancer. Using phage display technology we isolated 384 nanobody clones from the Christ human single domain antibody (dAb) library against human HPRT. Ten of the most sensitive Nbs were sequenced and evaluated to determine their sensitivity using dose response curves. Four parameter logistic regression analysis of the top 10 HPRT nanobodies showed R squared values ranging from 0.9388-0.9989 and a sigmoidal shape denoting a concentration-dependent antigen-ligand relationship. The anti-HPRT Nbs were able to detect minimum HPRT amounts ranging from 10-20 ng/ml. Validation of the anti-HPRT Nbs was conducted with a siRNA HPRT knockdown in A549 cells. A549 siRNA HPRT knockdown cell lysate showed at least a 5-fold reduction in the overall signal in phage ELISA compared to A549 wild type cell lysate (p<0.001) indicating the specificity of the Nbs to HPRT. In addition, flow cytometry analysis showed that the anti-HPRT Nbs were able to detect membrane associated HPRT on A549 (20%), NCI-H460 (23%), HT-29 (41%) , SW620 (46%) and MDA-MB-231 (32%) cancer cell lines. Anti-HPRT Nbs did not show significant binding to normal mononuclear cells compared to cancer cells (p<0.002). Anti-HPRT Nbs were successfully expressed and purified using the pET-scFv expression system. Human anti-HPRT Nbs may be used for the detection of both cytosolic and membrane associate HPRT. Future directions of this research will explore the uses of anti-HPRT-Nb-based therapies and their use in other applications such as chimeric antigen receptors (CARs) for cell adoptive therapies. Citation Format: Edwin J. Velazquez, Tyler B. Humpherys, Toni O. Mortimer, David M. Bellini, Kathryn R. Smith, Rachel M. Morris, Abigail Goodman, Kim L. O'Neill. Isolation and evaluation of human nanobodies against hypoxanthine guanine phosphoribosyltransferase (HPRT) and their potential use for the detection and targeting of lung, breast and colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1840.

Overexpression and surface localization of HPRT in prostate cancer provides a potential target for cancer specific antibody mediated cellular cytotoxicity

We chose to evaluate Hypoxanthine Guanine Phosphoribosyltransferase (HPRT) as a possible biomarker for prostate cancer due to its involvement in nucleotide synthesis and cell cycle progression. We utilized two prostate cancer cell lines (PC3 and DU145) along with patient tissue and knockdowns to evaluate overall HPRT expression. The surface localization of HPRT was determined utilizing flow cytometry, confocal microscopy, and scanning electron microscopy followed by ADCC to evaluate targeting potential. We found significant upregulation of HPRT within malignant samples with approximately 47% of patients had elevated levels of HPRT compared to normal controls. We also observed a significant association between HPRT and the plasma membrane of DU145 cells (p = 0.0004), but found no presence on PC3 cells (p = 0.14). This was confirmed with scanning electron microscopy and confocal microscopy. ADCC experiments were performed to determine whether HPRT could be used as a target antigen for selective cell-mediated killing. We found that DU145 cells treated with HPRT antibodies had a significantly higher incidence of cell death than both isotype treated samples and PC3 cells treated with the same concentrations of HPRT antibody. Finally, we determined that p53 had a significant impact on HPRT expression both internally and on the surface of cancer cells. These results suggest HPRT as a possible biomarker target for the treatment of patients with prostate cancer.

Evaluation of the upregulation and surface expression of hypoxanthine guanine phosphoribosyltransferase in acute lymphoblastic leukemia and Burkitt\u2019s B cell lymphoma

BackgroundThe aim of this study is to determine whether Hypoxanthine Guanine Phosphoribosyltransferase (HPRT) could be used as a biomarker for the diagnosis and treatment of B cell malignancies. With 4.3% of all new cancers diagnosed as Non-Hodgkin lymphoma, finding new biomarkers for the treatment of B cell cancers is an ongoing pursuit. HPRT is a nucleotide salvage pathway enzyme responsible for the synthesis of guanine and inosine throughout the cell cycle.MethodsRaji cells were used for this analysis due to their high HPRT internal expression. Internal expression was evaluated utilizing western blotting and RNA sequencing. Surface localization was analyzed using flow cytometry, confocal microscopy, and membrane biotinylation. To determine the source of HPRT surface expression, a CRISPR knockdown of HPRT was generated and confirmed using western blotting. To determine clinical significance, patient blood samples were collected and analyzed for HPRT surface localization.ResultsWe found surface localization of HPRT on both Raji cancer cells and in 77% of the malignant ALL samples analyzed and observed no significant expression in healthy cells. Surface expression was confirmed in Raji cells with confocal microscopy, where a direct overlap between HPRT specific antibodies and a membrane-specific dye was observed. HPRT was also detected in biotinylated membranes of Raji cells. Upon HPRT knockdown in Raji cells, we found a significant reduction in surface expression, which shows that the HPRT found on the surface originates from the cells themselves. Finally, we found that cells that had elevated levels of HPRT had a direct correlation to XRCC2, BRCA1, PIK3CA, MSH2, MSH6, WDYHV1, AK7, and BLMH expression and an inverse correlation to PRKD2, PTGS2, TCF7L2, CDH1, IL6R, MC1R, AMPD1, TLR6, and BAK1 expression. Of the 17 genes with significant correlation, 9 are involved in cellular proliferation and DNA synthesis, regulation, and repair.ConclusionsAs a surface biomarker that is found on malignant cells and not on healthy cells, HPRT could be used as a surface antigen for targeted immunotherapy. In addition, the gene correlations show that HPRT may have an additional role in regulation of cancer proliferation that has not been previously discovered.

Hypoxanthine Guanine Phosphoribosyltransferase expression is negatively correlated with immune activity through its regulation of purine synthesis.

The purpose of this study was to examine the effects of elevated Hypoxanthine Guanine Phosphoribosyltransferase (HPRT) on the immune response in the tumor microenvironment. HPRT expression was evaluated in cancer patients and correlated with cytokine expression, survival, and immune cell infiltration. An HPRT knockdown cell line was created to evaluate HPRT impact on purine expression and subsequent purine treatment was administered to immune cells to determine their influence on cell activation. HPRT expression was negatively correlated with the general expression of both pro-inflammatory and anti-inflammatory cytokines. Additionally, HPRT expression was also negatively correlated with the infiltration of immune cell subsets: B-cells, CD4 + T cells, macrophages, neutrophils, and dendritic cells (p < 0.001) and CD8 + T-cells (p < 0.01). When HPRT was knocked down in a Raji cell line, the levels of adenosine were reduced significantly compared to the wild type. When examining the level of Ca2+ influx of Raji compared to the HPRT Raji knockdown cell, there was a significant decrease in calcium influx in the knockdown cells when compared to the wild type cells. This demonstrates that HPRT had a significant impact on overall cell activation and the ability of the cells to properly influx calcium needed for their activation. We conclude that purine levels significantly reduce immune cell activation in cancer and the upregulation of HPRT in malignant tissue is a contributing factors to the immunosuppressive microenvironment.

Abstract 2369: HPRT overexpression may contribute to the immunosuppressive tumor microenvironment

Abstract The purpose of this study was to examine the possible effects that Hypoxanthine Guanine Phosphoribosyltransferase (HPRT) may have on the immune response in the tumor microenvironment. HPRT is a salvage pathway enzyme responsible for recycling guanosine throughout the cell cycle, and we have shown that it is significantly upregulated in numerous solid malignancies. As a necessary component of the S phase in the cell cycle, guanosine has also been regarded as a potential neuroprotectant in the central nervous system (CNS). We thus hypothesized that there may be a correlation between the observed upregulation of HPRT and the relative downregulation of the immune system mediated by the differential regulation of guanosine. Using data from The Cancer Genome Atlas (TCGA), we analyzed over 200 cytokine genes and found a negative correlation between the level of HPRT expression in cancer and the general expression level of both pro-inflammatory and anti-inflammatory cytokine expression. We further analyzed data showing the relative infiltration level of individual immune cell subsets in solid tumors compared with elevated HPRT levels. We found the most significant negative correlations between HPRT expression and immune cell infiltration for B-cells (p &amp;lt; 0.001), CD8+ T-cells (p &amp;lt; 0.01), CD4+ T-cells (p &amp;lt; 0.001), macrophages (p &amp;lt; 0.001), neutrophils (p &amp;lt; 0.001), and dendritic cells (p &amp;lt; 0.001) in lung squamous cell carcinoma. Other significant negative correlations were found in kidney renal clear cell carcinoma, prostate adenocarcinoma, and breast invasive carcinoma. To further analyze this reduction in infiltration, we examined the relationship between HPRT expression and both costimulatory and coinhibitory molecules and found a significant negative correlation. Because of these observed associations between HPRT levels and cytokine expression, we hypothesized that the increased guanosine production was the mechanism by which HPRT was affecting the immune system in the tumor microenvironment. To test this, we examined the level of Ca2+ influx using Fluo-4 assays of B-cell (Raji), T-cell (Jurkat), and monocyte (THP-1) cell lines when treated with guanosine as a measure of overall cell activation. We then compared this activation to untreated cells and cells treated with adenosine, a known anti-inflammatory agent. When treated with guanosine, a significant reduction in activation was found in THP-1s (p &amp;lt; 0.01) and Rajis (p &amp;lt; 0.01), but not in Jurkats. However, under hypoxic and hypoglycemic conditions similar to those commonly found within the tumor microenvironment, Jurkat activation was significantly reduced (p &amp;lt; 0.05) when treated with guanosine. From this data, we conclude that guanosine levels significantly reduce immune cell activation in the tumor microenvironment and that the upregulation of HPRT in malignant tissue may be one of the contributing factors of the immunosuppressive nature of the microenvironment. Citation Format: Michelle H. Townsend, Zachary D. Ewell, Claudia M. Tellez Freitas, Dallas J. Larsen, Eliza L. Bitter, Kelsey B. Bennion, Stephen R. Piccolo, K Scott Weber, Richard A. Robison, Kim L. O'Neill. HPRT overexpression may contribute to the immunosuppressive tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2369.

Abstract 264: Differential expression of HPRT in prostate cancer leads to investigation of its ADCC effects

Abstract As 1 in 41 American men will die of prostate cancer, this study proposes hypoxanthine- guanine phosphoribosyl transferase (HPRT) as another therapeutic target in the repertoire of prostate cancer immunotherapeutic targets. We evaluated the HPRT enzyme because of its role in the purine salvage pathway, nucleotide synthesis, and cell cycle progression. To evaluate the role of HPRT in prostate cancer, PC3 and DU145 prostate cancer cells were used because of their markedly different levels of HPRT expression. Flow cytometry and scanning electron microscopy showed the surface localization of HPRT while immunohistochemistry revealed HPRT upregulation within tissue. We consistently found a significant association between HPRT and the plasma membrane of DU145 cells, but found no HPRT presence on the surface of PC3 cells. Flow cytometry showed insignificant (p = 0.14) changes in fluorescence when PC3 cells were exposed to HPRT antibodies, while there was a significant increase in fluorescence on DU145 cells (p = 0.0004). To confirm this finding, we used gold conjugated antibodies to determine the distribution of HPRT across the membrane with an electron microscope. This analysis further supported the surface presence of HPRT on DU145 cells as the gold weight percentage of DU145 cells increased significantly when exposed to HPRT antibodies (p &amp;lt; 0.0001). In addition to its surface expression on DU145 cells, elevated levels of HPRT were found in 47% of prostate patient tissue samples compared to healthy controls (n = 25), while 53% of patients had no HPRT upregulation (n = 28). Additionally, RNA-seq data from The Cancer Genome Atlas (TCGA) was used to evaluate general HPRT levels in patients with prostate cancer (n = 502) and healthy individuals (n = 52). This data revealed a significant (p &amp;lt; 0.0001) increase in HPRT levels upon malignancy. While some patients’ levels were consistent with healthy control levels, there was a significant number of patients with increased protein expression upon cancer development. Because of the consistent and notable presence of HPRT on the surface of DU145 cells and in the tissue of prostate cancer patients, we began to investigate HPRT as a therapeutic target. Through MTS assays and live cell imaging, we assessed the potential of HPRT antibodies in an ADCC format. We observed increased DU145 cell death in wells treated with HPRT antibody when compared to wells treated with an isotype antibody and untreated wells (p &amp;lt; 0.01 and p &amp;lt; 0.0001, respectively). Furthermore, the amount of PC3 cell death in HPRT treated wells was insignificant when compared to isotype wells and untreated wells (p &amp;gt; 0.05). These results strongly indicate a unique relationship between prostate cancer cells and HPRT and supports the use of an HPRT antibody to harness the patient’s immune system in treating subsets of prostate cancer. Citation Format: Michelle H. Townsend, Kelsey A. Bennion, Zac E. Ence, Eliza E. Bitter, Abi M. Felsted, John E. Lattin, McKay D. Reese, Stephen R. Piccolo, Kim L. O'Neill. Differential expression of HPRT in prostate cancer leads to investigation of its ADCC effects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 264.

Abstract 562: HPRT surface localization on prostate cancer cells as a biomarker for immunotherapy

Abstract The purpose of this study is to evaluate the expression and possible upregulation of the salvage pathway enzyme hypoxanthine guanine phosphoribosyltransferase (HPRT) in prostate cancer cells to determine if it could serve as a biomarker for prostate cancer diagnosis and treatment. Prostate cancer is the second most lethal cancer in men, and an estimated 26,730 men will die from the disease in 2017. We chose to evaluate the HPRT enzyme due to its involvement in nucleotide synthesis and cell cycle progression. Two prostate cancer cell lines were used for this analysis (PC3 and DU145) along with malignant tissue from 35 patients with prostate carcinoma. The surface localization of HPRT was determined utilizing flow cytometry and scanning electron microscopy, while upregulation within tissue was assessed using immunohistochemistry. Additionally, RNA-seq data from TCGA was used to evaluate general HPRT upregulation in patients with prostate cancer (n = 502) when compared to healthy individuals (n = 52). Throughout our investigation, we found a significant association between HPRT and the plasma membrane of DU145 cells, but found no presence on PC3 cells. Flow cytometry showed insignificant (p = 0.14) changes in fluorescence when PC3 cells were exposed to HPRT antibodies, while there was a significant increase in fluorescence when DU145 cells were treated with fluorescent HPRT antibody (p = 0.0004). To determine the distribution of HPRT across the membrane and ensure the observed expression was not due to cytoplasmic HPRT, gold conjugated antibodies were used for analysis with an electron microscope. The distribution of the gold on the cell surface showed random HPRT binding across the membrane with no patterns of localization. This analysis aided in confirming HPRT surface presence as the gold weight % of DU145 cells increased significantly when exposed to HPRT antibodies (p &amp;lt; 0.0001). In addition to being presented on the surface of DU145 cells, tissue samples revealed variable HPRT expression as approximately 53% of prostate cancer patients evaluated had elevated levels of HPRT compared to normal controls, while 47% of patients had no upregulation. TCGA data revealed a significant (p = 1.53x10-4) increase in HPRT levels upon malignancy. While some patients had levels consistent with healthy controls, there was a significant number of patients with increased protein expression upon cancer development. The control of HPRT expression within these cancer cells has been linked preliminarily to p53 functionality. While PC3 cells are null for p53, DU145 cells have a gain of function (GOF) p53 mutation. GOF p53 mutations are known to influence salvage pathway enzyme expression and is an influential gene in HPRT expression in these cancer cells. These results strongly indicate a unique relationship between prostate cancer cells and HPRT and suggest HPRT as a possible biomarker for the detection and treatment of patients with prostate cancer. Citation Format: Michelle H. Townsend, Abigail M. Felsted, Taylor P. Cox, Zachary E. Ence, Stephen R. Piccolo, Richard A. Robison, Kim O'Neill. HPRT surface localization on prostate cancer cells as a biomarker for immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 562.

Elevated Expression of Hypoxanthine Guanine Phosphoribosyltransferase within Malignant Tissue

Hypoxanthine Guanine Phosphoribosyltransferase (HPRT) is a housekeeping enzyme involved in the purine synthesis of guanine and inosine in the salvage pathway. While other salvage pathway enzymes, such as TK1, have been established as biomarkers for both cancer cell proliferation and cancer development, little research been done to evaluate whether HPRT has the same potential as a cancer biomarker. We designed this study to determine if HPRT has value as an identifier of malignancy within the most common types of cancer. We utilized histological samples from lung, colon, prostate, and breast cancer with additional normal tissue to evaluate whether there was any elevation of HPRT within malignant samples. In addition, we also assessed general HPRT expression within patient’s samples from The Cancer Genome Atlas (TCGA) to confirm clinical relevance. We found that within a subset of patients, there was significant elevation of HPRT when compared to normal tissue controls. This elevation was seen in 33-55% of the malignant samples and appears to have no dependence on cancer stage. There were slight differences in staining patterns among all the organ types, but overall each organ displayed the same pattern of ‘HPRT high’ and ‘HPRT low’ populations within malignant samples. We found that in our TCGA samples, there was a similar elevation of HPRT that was significant when compared to normal controls. Overall, as an upregulated enzyme that does not directly correlate with stage, HPRT could become a valuable marker in the early diagnosis of a variety of solid tumors.

Genetic Modification of Mouse Bone Marrow by Lentiviral Vector-Mediated Delivery of Hypoxanthine-Guanine Phosphoribosyltransferase Short Hairpin RNA Confers Chemoprotection Against 6-Thioguanine Cytotoxicity

We have recently developed a novel and highly efficient strategy that exclusively uses the purine analog 6-thioguanine (6TG) for both pretransplantation conditioning and post-transplantation chemoselection of hypoxanthine-guanine phosphoribosyltransferase (HPRT)-deficient bone marrow (BM). In a mouse BM transplantation model, combined 6TG preconditioning and in vivo chemoselection consistently achieved >95% engraftment of HPRT-deficient donor BM and long-term reconstitution of histologically and immunophenotypically normal hematopoiesis in both primary and secondary recipients, without significant toxicity and in the absence of any other cytotoxic conditioning regimen. To translate this strategy for combined 6TG conditioning and chemoselection into a clinically feasible approach, it is necessary to develop methods for genetic modification of normal hematopoietic stem cells (HSC) to render them HPRT-deficient and thus 6TG-resistant. Here we investigated a strategy to reduce HPRT expression and thereby confer protection against 6TG myelotoxicity to primary murine BM cells by RNA interference (RNAi). Accordingly, we constructed and validated a lentiviral gene transfer vector expressing short-hairpin RNA (shRNA) that targets the murine HPRT gene. Our results showed that lentiviral vector-mediated delivery of HPRT-targeted shRNA could achieve effective and long-term reduction of HPRT expression. Furthermore, in both an established murine cell line as well as in primary murine BM cells, lentiviral transduction with HPRT-targeted shRNA was associated with enhanced resistance to 6TG cytotoxicity in vitro. Hence this represents a translationally feasible method to genetically engineer HSC for implementation of 6TG-mediated preconditioning and in vivo chemoselection.

HPRT-Deficiency Dysregulates cAMP-PKA Signaling and Phosphodiesterase 10A Expression: Mechanistic Insight and Potential Target for Lesch-Nyhan Disease?

Lesch-Nyhan Disease (LND) is the result of mutations in the X-linked gene encoding the purine metabolic enzyme, hypoxanthine guanine phosphoribosyl transferase (HPRT). LND gives rise to severe neurological anomalies including mental retardation, dystonia, chorea, pyramidal signs and a compulsive and aggressive behavior to self injure. The neurological phenotype in LND has been shown to reflect aberrant dopaminergic signaling in the basal ganglia, however there are little data correlating the defect in purine metabolism to the neural-related abnormalities. In the present studies, we find that HPRT-deficient neuronal cell lines have reduced CREB (cAMP response element-binding protein) expression and intracellular cyclic AMP (cAMP), which correlates with attenuated CREB-dependent transcriptional activity and a reduced phosphorylation of protein kinase A (PKA) substrates such as synapsin (p-syn I). Of interest, we found increased expression of phosphodiesterase 10A (PDE10A) in HPRT-deficient cell lines and that the PDE10 inhibitor papaverine and PDE10A siRNA restored cAMP/PKA signaling. Furthermore, reconstitution of HPRT expression in mutant cells partly increased cAMP signaling synapsin phosphorylation. In conclusion, our data show that HPRT-deficiency alters cAMP/PKA signaling pathway, which is in part due to the increased of PDE10A expression and activity. These findings suggest a mechanistic insight into the possible causes of LND and highlight PDE10A as a possible therapeutic target for this intractable neurological disease.

A Novel Method of Combined Preconditioning and In Vivo Chemoselection Using 6-Thioguanine Alone Results in Highly Efficient Reconstitution of Normal Hematopoiesis with HPRT-Deficient Bone Marrow

Introduction: Hematopoietic stem cell transplantation (HSCT) is a mainstay of treatment for hereditary disorders and lymphohematopoietic malignancies, and is the most commonly employed modality for ex vivo gene therapy. However, this procedure often entails myeloablative conditioning by irradiation and/or cytotoxic drugs which incur significant morbidity, and low engraftment rates frequently limit efficacy in the absence of a selective advantage that amplifies donor cells. Methods & Results: Here we tested a novel strategy for combined conditioning and chemoselection based on the fact that 6-thioguanine (6TG) myelotoxicity is dependent on its conversion to thioguanine nucleotide by hypoxanthine-guanine phosphoribosyltransferase (HPRT), and that HPRT-deficiency per se does not impair hematopoietic cell development or function. In a murine model of bone marrow (BM) transplantation, our initial results demonstrated that, at appropriate doses, 6TG induces selective myeloablation in HPRT-wild type (wt) recipients, without detectable adverse effects on extra-hematopoietic tissues. In contrast, HPRT-deficient donor mice were highly resistant to its cytotoxic effects, and transplantation of HPRT-deficient BM achieved rescue of HPRT-wt recipients from lethal hematopoietic failure. Using an optimized protocol, we tested 6TG as a single agent, both for myeloablative conditioning of 6TG-sensitive HPRT-wt recipients, and for in vivo chemoselection of 6TG-resistant HPRT-deficient donor BM after transplantation. Combined 6TG conditioning and chemoselection achieved highly efficient engraftment (˜95%) without significant toxicity and in the absence of any other cytotoxic conditioning regimen. Longterm reconstitution of histologically and immunophenotypically normal BM was achieved in primary and secondary recipients, indicating that 6TG selection amplified the self-renewing, pluripotent HSC population from HPRT-deficient donor BM. In current studies, we are testing lentiviral vector-mediated shRNA expression for knockdown of HPRT expression, in order to apply this combined 6TG conditioning and chemoselection procedure to transplantation of HPRT-wt donor-derived BM after genetic modification. Conclusion: Our results suggest that this protocol for highly efficient conditioning and in vivo chemoselection may be generally applicable as a strategy to improve HSCT engraftment efficiency and confer a selective advantage to genetically modified cells after ex vivo gene therapy.

Lesch–Nyhan Syndrome: mRNA expression of HPRT in patients with enzyme proven deficiency of HPRT and normal HPRT coding region of the DNA

Inherited mutation of the purine salvage enzyme, hypoxanthine guanine phosphoribosyltransferase (HPRT) gives rise to Lesch–Nyhan syndrome (LNS) or Lesch–Nyhan variants (LNV). We report a case of two LNS affected members of a family with deficiency of activity of HPRT in intact cultured fibroblasts in whom mutation could not be found in the HPRT coding sequence but there was markedly decreased HPRT expression of mRNA.

Knockdown of HPRT Enables Selection of Genetically Modified Human Hematopoietic Progenitor Cells.

AbstractAbstract 3772Genetic modification of autologous hematopoietic stem cells (HSC) has the potential for effective treatment of a wide variety of inherited blood disorders. However, HSC gene therapy has shown limited clinical efficacy (with notable exceptions), in part because of the small proportion of engrafted genetically corrected HSCs. The use of drug-resistance genes to enable selection for transduced HSCs has been explored, but with limited success. Previous studies from our laboratory have indicated that murine HSC can be selected with 6-Thioguanine (6TG), a relatively non-toxic drug used in the treatment of leukemias, after knocking down the expression of hypoxanthine-guanine phosphoribosyltransferase (HPRT), an enzyme that metabolizes 6TG to its active state. We sought to determine if these findings can be translated to human hematopoietic cells. In the present study, we transduced human myeloid (Molm13, MV4-11) and lymphoid cell lines (Reh) with lentiviral vectors expressing shRNA constructs targeting HPRT or a non-targeted control sequence (Ctrl). Two of the most promising constructs directed against HPRT (491 and 50) were studied in more detail to determine which is most effective. Cells were selected in puromycin and cell lysates analyzed for HPRT gene expression. Reverse-transcription, real-time PCR (RT qPCR) and western blotting demonstrated that construct 491 was most efficient in knocking down HPRT in human hematopoietic cell lines compared to construct 50 (and Ctrl). To determine whether knockdown of HPRT provided resistance to 6TG, cells were cultured in the absence or presence of different doses of 6TG and live cell concentrations were determined. While Ctrl transduced cells decreased in a dose dependent manner after 72h of 6TG treatment, cells transduced with constructs 491 and 50 were relatively resistant to 6TG. IC50 values for construct 491 were significantly higher (114μM for Molm13 and 46μM for Reh cell lines) than construct 50 (1μM for Molm13 and 10μM for Reh) in comparison to control transduced cells (0.4μM for Molm13 and 3.5μM for Reh). We assessed cell death in human hematopoietic cell lines by annexin V staining after exposure to 6TG at 48 and 72h. As expected, control transduced cells died of apoptosis upon 6TG treatment, while 491 and 50 transduced cells were resistant. Furthermore, 491 transduced cells were more resistant to apoptosis than 50 transduced cells. Based on these results, construct 491 was used to transduce human CD34+ progenitor cells isolated from umbilical cord blood along with control shRNA. Transduction efficiency varied from 25–35% as determined by %GFP expression by flow cytometry. Sorted GFP+ cells showed reduced expression of HPRT in 491 transduced cells compared to controls, as measured by RT qPCR. Similar to the effects in cell lines, in vitro proliferation of control transduced CD34+ cells diminished in response to increasing 6TG concentrations. There was an increase in the percentage of GFP+ cells in 6TG treated 491 transduced cells compared to untreated controls in a dose dependent fashion, indicating a selective advantage conferred to 491 transduced cells in the presence of 6TG. Importantly, 491 transduced cells continued to proliferate despite treatment with 6TG. Like 6TG, cisplatin requires mismatch repair (MMR) for cytotoxicity. To determine if HPRT knockdown had off-target effects impairing MMR, transduced cells were also treated with cisplatin. Both control and 491 transduced cells stopped proliferating in the presence of cisplatin indicating that MMR remained intact. These data indicate that human hematopoietic progenitor cells can be selected in vitro by knock-down of HPRT and treatment with 6TG. Xenografts of Ctrl and 491 transduced human CD34+ cord blood cells have been generated and are being treated with 6TG to determine if human cells can be selected with 6TG in vivo. Disclosures:Off Label Use: Off label use of 6-thioguanine will be suggested.

Increased CUG Triplet Repeat-binding Protein-1 Predisposes to Impaired Adipogenesis with Aging

Preadipocyte differentiation capacity declines between middle and old age. Expression of the adipogenic transcription factors, CCAAT/enhancer-binding protein (C/EBP) alpha and peroxisome proliferator-activated receptor gamma (PPARgamma), is lower in differentiating preadipocytes from old than young animals, although no age-related changes occur in C/EBPbeta mRNA, which is upstream of C/EBPalpha and PPARgamma. C/EBPbeta-liver-enriched inhibitory protein (C/EBPbeta-LIP), a truncated C/EBPbeta isoform that is a dominant inhibitor of differentiation, increases with aging in rat fat tissue and preadipocytes. CUG triplet repeat-binding protein-1 (CUGBP1) binds to C/EBPbeta mRNA, increasing C/EBPbeta-LIP translation. Abundance and nucleotide binding activity of CUGBP1 increased with aging in preadipocytes. CUGBP1 overexpression in preadipocytes from young animals increased C/EBPbeta-LIP and impaired adipogenesis. Decreasing CUGBP1 in preadipocytes from old rats by RNA interference reduced C/EBPbeta-LIP abundance and promoted adipogenesis. Tumor necrosis factor-alpha, levels of which are elevated in fat tissue with aging, increased CUGBP1 protein, CUGBP1 binding activity, and C/EBPbeta-LIP in preadipocytes from young rats. Thus, CUGBP1 contributes to regulation of adipogenesis in primary preadipocytes and is responsive to tumor necrosis factor-alpha. With aging, preadipocyte CUGBP1 abundance and activity increases, resulting in enhanced translation of the C/EBPbeta-LIP isoform, thereby blocking effects of adipogenic transcription factors, predisposing preadipocytes from old animals to resist adipogenesis. Altered translational processing, possibly related to changes in cytokine milieu and activation of stress responses, may contribute to changes in progenitor differentiation and tissue function with aging.

Proteomic analysis of Down's syndrome patients with gout

Background In this study, the expression of hypoxanthine-guanine phosphoribosyl transferase (HPRT) in Down's syndrome patients with gout (DS/G) was determined, and possible underlying mechanisms of gout were characterized using proteomic tools. Methods Serum was obtained from DS/G, healthy controls and gout patients (without DS), recruited from the rheumatology clinic. Baseline enzyme assays were recorded and RT-PCR used to identify HPRT gene expression. 2-D electrophoresis and mass spectrometry were utilized to determine a plausible explanation concerning the mechanisms leading to increased uric acid levels in DS patients. Results Two DS patients were diagnosed with gouty arthritis. Their HPRT enzyme activity was slightly lower than that of normal controls. HPRT expression was also slightly decreased in DS/G patients compared with controls. Serum protein profiles of these two DS/G patients revealed that haptoglobin α chain and apolipoprotein A1 (ApoA1) were both significantly down-regulated. Protein expression was validated by immunoblot. Conclusion Our results revealed that low levels of haptoglobin in the two DS/G patients were related to renal dysfunction may have affected uric acid excretion and caused gout. However, decreased ApoA1 revealed a positive correlation between defective lipid metabolism and gouty arthritis in DS/G patients.

Serglycin-deficient Cytotoxic T Lymphocytes Display Defective Secretory Granule Maturation and Granzyme B Storage

Cytotoxic T lymphocytes eliminate infected and tumor cells mainly by perforin/granzyme-induced apoptosis. Earlier studies suggested that serglycin-proteoglycans form macromolecular complexes with granzymes and perforin in the cytotoxic granule. Serglycin-proteoglycans may also be involved in the delivery of the cytolytic machinery into target cells. We have developed a serglycin-deficient mouse strain, and here we studied the importance of serglycin-proteoglycans for various aspects of cytotoxic T lymphocyte function. 35SO4(2-) radiolabeling of serglycin-deficient cells demonstrated a dramatic reduction of incorporated label as compared with wild type cells, indicating that serglycin is by far the dominating proteoglycan species produced by the cytotoxic T lymphocyte. Moreover, lack of serglycin resulted in impaired ability of cytotoxic T lymphocytes to produce secretory granule of high electron density, although granule of lower electron density were produced both in wild type and serglycin-deficient cells. The serglycin deficiency did not affect the mRNA expression for granzyme A, granzyme B, or perforin. However, the storage of granzyme B, but not granzyme A, Fas ligand, or perforin, was severely defective in serglycin-deficient cells. Serglycin-deficient cells did not display defects in late cytotoxicity toward target cell lines. Taken together, these results point to a key role for serglycin in the storage of granzyme B and for secretory granule maturation but argue against a major role for serglycin in the apoptosis mediated by cytotoxic T lymphocytes.

Real-time RT-PCR for the Detection of Beta-adrenoceptor Messenger RNAs in Small Human Endomyocardial Biopsies

Quantification of mRNAs from extremely small human samples remains a challenge. Requiring minimal amounts of tissue and no post-reaction manipulation, real-time reverse transcriptase-polymerase chain reaction (RT-PCR) is an attractive method to quantitatively assess the expression of rare mRNAs. We evaluated the applicability of the technique on RNA extracted from human endomyocardial biopsies and isolated cardiomyocytes, and compared the technique to the RT-competitive PCR approach. Primers and probes were designed to amplify the three subtypes of human β -adrenoceptors (β 1-,β 2- and β 3 AR), as well as reference genes such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Hypoxanthine-guanine phosphoribosyltransferase (HPRT), and the oncogene ABL by real-time RT-PCR. Specific primers and a deleted competitor were synthetized to compare the quantitation of the β 3 AR mRNA expression by RT-competitive PCR. We validated the technique on human cardiomyocytes either freshly isolated or selectively excised from fixed sections of human myocardium by Laser Capture Microdissection. The standard curves obtained for the cDNA's analysed showed mean slopes comprised between −3.3 and −3.7. Inter- and intra-assay variablility of gene quantitation was reflected by mean values of the variance coefficients of Ct of 4.84±1.13% and 2.73±0.39% or 3.32±1.03% and 2.21±0.24% (corresponding to percent variances of copy numbers of 83.07±12.72% and 34.45±9.03% or 47.40±8.59% and 23.83±3.16%) for human β 3 AR and GAPDH genes, respectively. The expression of GAPDH, HPRT and ABL mRNA was characterized by a very low dispersion of individual values across cardiac pathologies, suggesting that these genes may be used as reference genes in quantitative PCR studies. Finally, we applied the technique to detect rare mRNAs, such as β -AR mRNAs, from small human endomyocardial biopsies and even isolated cardiomyocytes. Real-time RT-PCR is appropriate to quantitate rare messenger RNAs, including in extremely small human tissue samples. This method appears very promising for futures studies of gene expression in several pathophysiological conditions, including heart failure.

Suppressed expression of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) in an irradiation-attenuated Plasmodium berghei XAT strain

Plasmodium berghei XAT (XAT) is a non-reversible, non-lethal type malaria parasite strain derived from the highly virulent lethal P. berghei NK65 (NK65) by X-irradiation. The difference in polypeptide expression between NK65 and XAT was examined in this study. Western blot patterns of the parasite polypeptides showed that a 30-kDa polypeptide was not detected in XAT. In the present paper, we focused the study on the difference in the expression of the 30-kDa polypeptide between XAT and NK65. Although several other significant differences were noted in the spots shown by two-dimensional gel electrophoresis, the 30-kDa polypeptide was isolated by means of preparative 2D-gel electrophoresis followed by HPLC, and N-terminal amino acid sequence of the polypeptide was eventually determined. Complementary DNA clones encoding the 30-kDa polypeptide were isolated and characterized. Full-length cDNA clones from XAT encoded a protein of 231 amino acid residues with a 693-bp open reading frame. The deduced amino acid sequence exhibited 67% identity with that for P. falciparum hypoxanthine-guanine phosphoribosyltransferase (HGPRT; EC 2.4.2.8), suggesting that this protein is P. berghei HGPRT. Northern blot analysis revealed that expression of HGPRT in XAT was only one-eighth of that in NK65. This finding indicates that HGPRT gene expression is markedly suppressed in XAT. The amino acid sequence of HGPRT from NK65 was identical to that from XAT. This finding showed that the amino acid sequence of XAT-HGPRT was not mutated and had not undergone deletion.