Increased ENT2 Expression and Its Association With Altered Purine Metabolism in Different Stages of Colorectal Cancer Cell Lines

Background Colorectal cancer (CRC) is one of the most prevalent malignant cancers worldwide. Although the purine metabolism pathway is known to be vital for cancer cells survival mechanism, not much is known on ENT2 role in CRC development and its association with purine metabolites. Hence this study is aimed to determine the level of hypoxanthine phosphoribosyl transferase (HPRT), hypoxanthine, uric acid (UA), and the activity of xanthine oxidase (XO) and relate the findings with the ENT2 expression level in different CRC stages. Methods and results Normal colon cell line; CCD-841CoN and a panel of human CRC cell lines; SW480, HCT15 and HCT116, representing different CRC stages; Dukes’ B, C, and D respectively, have been used to measure HPRT, hypoxanthine/xanthine, UA levels and the activity of XO by biochemical assays. The level of ENT2 gene expression was also performed by qRT-PCR. The levels of HPRT, hypoxanthine were significantly higher (P< 0.05), while XO and UA were lower (P< 0.05) in all CRC stages as compared to the normal colon cells. Furthermore, ENT2 expression was found to be increased in all CRC stages. Despite having the highest level of HPRT and hypoxanthine, ENT2 level is lower in Dukes' D when compared to Dukes' B and C. Conclusion The rate of salvage pathway is increased in CRC development as indicated by the elevated levels of HPRT and hypoxanthine in different CRC stages. Increase ENT2 expression implies its importance in assisting hypoxanthine uptake. This step is vital in order to increase DNA synthesis via hypoxanthine recycling. Thus, ENT2 may be a potential marker in therapeutic development.

Detailed Study of HPRT1 Gross Deletions Found in 10 Italian Lesch-Nyhan Families

Background: Lesch-Nyhan disease (LND) is an X-linked rare pathology involving the purine nucleotides salvage pathway. Its incidence is estimated in 1:350.000 born. The condition is due to mutations in the HPRT1 (hypoxanthine phosphoribosyl transferase 1) gene of which in our cohort 28% (10/35) are large deletions. In order to better assess the nature of the observed deletions in our LND population we analyzed 10 families carrying large deletions in the HPRT1 gene region and studied the underlying pathogenic mechanisms. Methods: We performed PCR based localization of the break points and sequenced the gap-junction fragments. Bioinformatics analysis was performed through several web tools on the 5’ and 3’ break points to determine the factors involved in the deletion mechanism. Results: We precisely mapped 10 unique large deletions involving the HPRT1 gene region that span from 300 bp to 64 kbp. No common breakpoints were found and each deletion appears to be family specific. Conclusions: The deletions in the HPRT1 gene area are consistent with the Micro homology-Mediated Break-Induced Replication (MMBIR) mechanism. There are strong links with Alu-s and no recurrent break points with all of the observed deletions being unique. The relatively large amount of deletions in the HPRT1 region is peculiar and linked with the almost absolute lack of polymorphic sites in the HPRT1 gene making it a very interesting region for further studies.

Metabolome analysis of masseter muscle in senescence-accelerated mouse-prone 8 (SAMP8)

Frailty is a vulnerable state that marks the transition to long-term care for the elders. Recently, the relationship between frailty and oral function has been attracting attention. By clarifying the specific metabolic changes in the masseter muscle, we aimed to contribute to maintenance of masticatory function. The purpose of this study is to clarify the changes in masseter muscle of senescence-accelerated mouse-prone 8 (SAMP8) mice metabolites and metabolic pathways due to aging. Capillary electrophoresis-mass spectrometry metabolome analysis was performed on the masseter muscle of 12-week-old, 40-week-old, and 55-week-old mice. Expression analysis was performed by reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence for the metabolome pathways extracted by metabolome analysis that considered to be related to aging. Nineteen metabolites had a significant difference in absolute quantitative values and were considered to affect the first principal component by factor loading. The extracted metabolic pathways were glycolysis, polyamine metabolome pathway, and purine metabolome pathway. RT-PCR was performed on the extracted metabolome pathways. Expression of the spermidine synthase and hypoxanthine phosphoribosyl transferase genes with significant differences by RT-PCR was confirmed by immunofluorescence. The metabolic pathways considered to be related to aging in masseter muscle were glycolysis, polyamine metabolic pathway, and purine metabolic pathway.

Overexpression of HPRT1 is associated with poor prognosis in head and neck squamous cell carcinoma

Hypoxanthine phosphoribosyl transferase (HPRT1), as a salvage pathway enzyme, plays a crucial role in modulating the cell cycle and has been reported to be overexpressed in multiple cancers. Nevertheless, the relationship between the HPRT1 and Head and Neck Squamous Cell Carcinomas (HNSCC) has not been investigated so far. We first evaluated the expression of HPRT1 at transcriptomic and proteomic levels in tumor and healthy control tissues and its clinical value using The Cancer Genome Atlas (TCGA), Human Protein Atlas, Kaplan-Meier Plotter databases, GSE107591, and quantitative real-time PCR analysis. Then, we employed the COSMIC and cBioPortal databases to assess the mutations of the HPRT1 gene and their association with survival outcomes of patients with HNSCC. Finally, we performed the functional enrichment analysis for HPRT1 co-expressed genes in HNSCC utilizing the Enrichr database. The mRNA and protein expressions of HPRT1 were significantly elevated in HNSCC compared with normal tissues. Besides, the upregulation of HPRT1 expression was correlated with age, sex, pathological stage, and histological grades of HNSCC patients. Moreover, the increased expression of HPRT1 in cancer tissues exhibited a strong capacity for being a promising biomarker for the diagnosis and prognosis of patients with HNSCC. The co-expressed genes of HPRT1 were mainly enriched in several cancer-related processes such as DNA replication and cell cycle. The present study demonstrated that the overexpression of HPRT1 is significantly correlated with the progression of HNSCC and may serve as a useful biomarker for the early detection and risk stratification of patients with HNSCC.

Hypoxanthine Phosphoribosyl Transferase 1 Is Upregulated, Predicts Clinical Outcome and Controls Gene Expression in Breast Cancer

Hypoxanthine phosphoribosyl transferase 1 (HPRT1) is traditionally believed to be a housekeeping gene; however, recent reports suggest that it is upregulated in several cancers and is associated with clinical outcomes. HPRT1 is located on chromosome X and encodes the HPRT enzyme, which functions in recycling nucleotides to supply for DNA and RNA synthesis in actively dividing cells. Here, we used transcriptomic analyses to interrogate its expression across all known cancer types and elucidated its role in regulating gene expression in breast cancer. We observed elevated HPRT1 RNA levels in malignant tissues when compared to normal controls, indicating its potential as a diagnostic and prognostic marker. Further, in breast cancer, the subtype-specific analysis showed that its expression was highest in basal and triple-negative breast cancer, and HPRT1 knockdown in breast cancer cells suggested that HPRT1 positively regulates genes related to cancer pathways. Collectively, our results essentially highlight the importance of and change the way in which HPRT1’s function is studied in biology, warranting careful examination of its role in cancer.

Development of a high-throughput screening system for identification of novel reagents regulating DNA damage in human dermal fibroblasts

Ultraviolet (UV) radiation is a major inducer of skin aging and accumulated exposure to UV radiation increases DNA damage in skin cells, including dermal fibroblasts. In the present study, we developed a novel DNA repair regulating material discovery (DREAM) system for the high-throughput screening and identification of putative materials regulating DNA repair in skin cells. First, we established a modified lentivirus expressing the luciferase and hypoxanthine phosphoribosyl transferase (HPRT) genes. Then, human dermal fibroblast WS-1 cells were infected with the modified lentivirus and selected with puromycin to establish cells that stably expressed luciferase and HPRT (DREAM-F cells). The first step in the DREAM protocol was a 96-well-based screening procedure, involving the analysis of cell viability and luciferase activity after pretreatment of DREAM-F cells with reagents of interest and post-treatment with UVB radiation, and vice versa. In the second step, we validated certain effective reagents identified in the first step by analyzing the cell cycle, evaluating cell death, and performing HPRT-DNA sequencing in DREAM-F cells treated with these reagents and UVB. This DREAM system is scalable and forms a time-saving high-throughput screening system for identifying novel anti-photoaging reagents regulating DNA damage in dermal fibroblasts.