Application of the isotope-dilution principle to the analysis of factors affecting the incorporation of (3H) uridine and (3H) cytidine into cultured lymphocytes. Evaluation of pools in serum and culture media.
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ABSTRACT:
SUBMITTER: Forsdyke DR
PROVIDER: S-EPMC1178176 | biostudies-other | 1971 Dec
REPOSITORIES: biostudies-other
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Project description:1. Rat thymus cells were incubated in homologous serum (10%) and medium 199. The effects of various quantities of thymidine or deoxycytidine (0-30mum) on the radioactive labelling of cells with the corresponding radioactive deoxynucleoside were examined. From plots of the reciprocal of the radioactivity incorporated against the total deoxynucleoside concentration (;isotope-dilution plots'), values were obtained for (a) the V(max.) of the rate-limiting step governing incorporation of the deoxynucleoside, and (b) the concentration of the pool of compounds competing with the radioactive deoxynucleoside at that rate-limiting step. From changes in these values under different experimental conditions inferences were drawn on the position and control of the rate-limiting step within intact cells. 2. Isotope-dilution plots for deoxycytidine were linear, whereas plots for thymidine were bimodal, indicating an abrupt increase in both the V(max.) and pool concentration at a critical thymidine concentration (approx. 5mum). The bimodality was removed by amethopterin. The V(max.) determined with deoxy[U-(14)C]cytidine was approximately equal to the sum of the V(max.) determined with deoxy[5-(3)H]cytidine and the V(max.) determined with [Me-(3)H]thymidine at thymidine concentrations above 5mum. 3. The thymidine competitor pool at thymidine concentrations above 5mum was approximately equal to the sum of the deoxycytidine competitor pool and the thymidine competitor pool at thymidine concentrations below 5mum. The pools were independent of cell concentration and dependent on serum concentration. 4. These results were explained on the following basis. Deoxycytidine in serum (16mum) is the major source of both cytosine and, by way of thymidylate synthetase, thymine, in the DNA of thymus cells. Serum deoxycytidine normally maintains a sufficient intracellular concentration of dTTP to inhibit partially the activity of thymidine kinase. When the dTTP concentration is lowered, either by decreasing the concentration of deoxycytidine or by inhibiting thymidylate synthetase, the activity of thymidine kinase increases. The activity of thymidine kinase may also be increased by concentrations of thymidine greater than 5mum, which overcome the inhibition of the enzyme by dTTP. At concentrations of thymidine below 5mum, thymidine kinase limits the rate of labelling with [Me-(3)H]thymidine and the radioactivity is diluted by a pool of unlabelled thymidine in serum (4mum). At thymidine concentrations greater than 5mum, the activity of DNA polymerase limits the rate of labelling and the radioactivity is diluted both by serum thymidine and, indirectly, by serum deoxycytidine.
| S-EPMC1166202 | biostudies-other
Project description:Uridine-cytidine kinase (UCK), including human UCK2, are a family of enzymes that generally phosphorylate both uridine and cytidine. However, UCK of Thermus thermophilus HB8 (ttCK) phosphorylates only cytidine. This cytidine-restricted activity is thought to depend on Tyr93, although the precise mechanism remains unresolved. Exhaustive mutagenesis of Tyr93 in ttCK revealed that the uridine phosphorylation activity was restored only by replacement of Tyr93 with His or Gln. Replacement of His117 in human UCK2, corresponding to residue Tyr93 in ttCK, by Tyr resulted in a loss of uridine phosphorylation activity. These findings indicated that uridine phosphorylation activity commonly depends on a single residue in the UCK family.
| S-EPMC5614712 | biostudies-literature
Project description:1. Rat lymph-node cells cultured in serum and medium 199 were activated to transform and proliferate by concanavalin A. Initial cell activation was assessed by measuring the enhanced radioactive labelling of cells with [(3)H]uridine produced by concanavalin A during the first 6h of culture. 2. In medium containing serum the degree of activation was dependent on the ratio of concanavalin A to non-diffusible serum macromolecules; however, cells could be activated to a normal extent in a medium containing only diffusible molecules. This indicates that certain serum macromolecules buffer cell receptor sites against reaction with concanavalin A. 3. At high concanavalin A concentrations labelling was depressed below that of control cultures without concanavalin A. This inhibition of labelling (i) occurred in calf serum, but not in homologous serum, (ii) was removed by pretreatment of serum with various complement inhibitors, and (iii) first appeared after 1h of culture following an initial phase of cell activation by concanavalin A. Cells pre-labelled with [(3)H]uridine slowly released the label into the culture medium; this rate of release was suddenly accelerated after 1h of culture with concanavalin A if complement was present. The results suggest that inhibition of labelling requires the sequential binding of concanavalin A and then complement to the cell surface. 4. Results from experiments in which calf serum was mixed in various proportions with calf serum which had been preheated to inactivate complement, suggest (i) a requirement for complement in stoicheiometric quantities dependent on the number of cells being inhibited, and (ii) that preheated serum can inactivate complement in unheated serum. 5. The proliferative response over 3 days of culture was assessed by measuring the enhanced labelling of cells with [(3)H]thymidine produced by concanavalin A. In preheated calf serum two types of inhibition were noted. (i) A progressive inhibition at high concanavalin A concentrations so that the optimum response was shifted to lower concanavalin A concentrations as the duration of culture was extended; it is suggested that this reflects the secretion of complement by cultured cells. (ii) An inhibition of the optimum response appearing late in the culture period at high cell concentrations; it is suggested that this is due to the exhaustion of medium nutrients in most actively growing cultures.
| S-EPMC1177655 | biostudies-other
Project description:5-Formyl-deoxyuridine (fdU) and 5-formyl-deoxycytidine (fdC) are formyl-containing nucleosides that are created by oxidative stress in differentiated cells. While fdU is almost exclusively an oxidative stress lesion formed from deoxythymidine (T), the situation for fdC is more complex. Next to formation as an oxidative lesion, it is particularly abundant in stem cells, where it is more frequently formed in an epigenetically important oxidation reaction performed by α-ketoglutarate dependent TET enzymes from 5-methyl-deoxycytidine (mdC). Recently, it was shown that genomic fdC and fdU can react with the ϵ-aminogroups of nucleosomal lysines to give Schiff base adducts that covalently link nucleosomes to genomic DNA. Here, we show that fdU features a significantly higher reactivity towards lysine side chains compared with fdC. This result shows that depending on the amounts of fdC and fdU, oxidative stress may have a bigger impact on nucleosome binding than epigenetics.
| S-EPMC8518870 | biostudies-literature
Project description:1. Partially purified pig blood lymphocytes were stimulated to transform in culture with phytohaemagglutinin. Initial cell activation was assessed by measuring the increase of uridine incorporation into RNA induced by phytohaemagglutinin. The phytohaemagglutinin/serum ratio for this effect was similar to that required for transformation; however, no inhibition at high phytohaemagglutinin/serum ratios was found during cell activation. 2. Without replenishment of medium the pool of competitors with added uridine for incorporation fell to zero during 2 days of culture. At certain critical pool concentrations uridine itself could stimulate the rate of uridine incorporation. 3. Most of the tritium from [5-(3)H]uridine added at the initiation of culture had been incorporated into RNA by the end of the second day of culture; the subsequent loss of radioactivity preceded a fall in the total RNA content of cultures. 4. RNA was qualitatively examined on sucrose density gradients. In the first 30min. after the addition of phytohaemagglutinin, increased labelling occurred predominantly between the 28s and 4s regions of the gradients. On the second day of culture with phytohaemagglutinin mainly RNA sedimenting beyond the 28s region of gradients was labelled in 30min.
| S-EPMC1198646 | biostudies-other
Project description:The rising interest in understanding the functions, regulation, and maintenance of the epitranscriptome calls for robust and accurate analytical methods for the identification and quantification of post-transcriptionally modified nucleosides in RNA. Monomethylations of cytidine and adenosine are common post-transcriptional modifications in RNA. Herein, we developed an LC-MS/MS/MS coupled with the stable isotope-dilution method for the sensitive and accurate quantifications of 5-methylcytidine (m(5)C), 2'-O-methylcytidine (Cm), N(6)-methyladenosine (m(6)A), and 2'-O-methyladenosine (Am) in RNA isolated from mammalian cells and tissues. Our results showed that the distributions of m(5)C, Cm and Am are tissue-specific. In addition, the 2'-O-methylated ribonucleosides (Cm and Am) are present at higher levels than the corresponding methylated nucleobase products (m(5)C and m(6)A) in total RNA isolated from mouse brain, pancreas, and spleen but not mouse heart. We also found that the levels of m(5)C, Cm, and Am are significantly lower (by 6.5-43-fold) in mRNA than in total RNA isolated from HEK293T cells, whereas the level of m(6)A was slightly higher (by 1.6-fold) in mRNA than in total RNA. The availability of this analytical method, in combination with genetic manipulation, may facilitate the future discovery of proteins involved in the maintenance and regulation of these RNA modifications.
| S-EPMC4711271 | biostudies-literature
Project description:Lung cancer has the highest morbidity and mortality among all cancers. Discovery of early diagnostic and prognostic biomarkers of lung cancer can greatly facilitate the survival rate and reduce its mortality. In our study, by analyzing Gene Expression Omnibus and Oncomine databases, we found a novel potential oncogene uridine-cytidine kinase 2 (UCK2), which was overexpressed in lung tumor tissues compared to adjacent nontumor tissues or normal lung. Then we confirmed this finding in clinical samples. Specifically, UCK2 was identified as highly expressed in stage IA lung cancer with a high diagnostic accuracy (area under the receiver operating characteristic curve > 0.9). We also found that high UCK2 expression was related to poorer clinicopathological features, such as higher T stage and N stage and higher probability of early recurrence. Furthermore, we found that patients with high UCK2 expression had poorer first progression survival and overall survival than patients with low UCK2 expression. Univariate and multivariate Cox regression analyses showed that UCK2 was an independent risk factor related with worse DFS and OS. By gene set enrichment analysis, tumor-associated biological processes and signaling pathways were enriched in the UCK2 overexpression group, which indicated that UCK2 might play a vital role in lung cancer. Furthermore, in cytology experiments, we found that knockdown of UCK2 could suppress the proliferation and migration of lung cancer cells. In conclusion, our study indicated that UCK2 might be a potential early diagnostic and prognostic biomarker for lung cancer.
| S-EPMC6726693 | biostudies-literature
Project description:PurposeUridine-cytidine kinase (UCK) 2 is a rate-limiting enzyme involved in the salvage pathway of pyrimidine-nucleotide biosynthesis. Recent studies have shown that UCK2 is overexpressed in many types of cancer and may play a crucial role in activating antitumor prodrugs in human cancer cells. In the current study, we evaluated the potential prognostic value of UCK2 in breast cancer.MethodsWe searched public databases to explore associations between UCK2 gene expression and clinical parameters in patients with breast cancer. Gene set enrichment analysis (GSEA) was performed to identify biological pathways associated with UCK2 gene expression levels. Survival analyses were performed using 10 independent large-scale breast cancer microarray datasets.ResultsWe found that UCK2 mRNA expression was elevated in breast cancer tissue compared with adjacent nontumorous tissue or breast tissue from healthy controls. High UCK2 levels were correlated with estrogen receptor negativity (p<0.001), advanced tumor grade (p<0.001), and poor tumor differentiation (p<0.001). GSEA revealed that UCK2-high breast cancers were enriched for gene sets associated with metastasis, progenitor-like phenotypes, and poor prognosis. Multivariable Cox proportional hazards regression analyses of microarray datasets verified that high UCK2 gene expression was associated with poor overall survival in a dose-response manner. The prognostic power of UCK2 was superior to that of TNM staging and comparable to that of multiple gene signatures.ConclusionThese findings suggest that UCK2 may be a promising prognostic biomarker for patients with breast cancer.
| S-EPMC5500396 | biostudies-literature
Project description:IntroductionUridine-cytidine kinase 2 (Uck2) has been demonstrated to activate antitumor prodrugs and regulate chemosensitivity in cancer cells. However, the expression and function of Uck2 in hepatocellular carcinoma (HCC) remain unknown. In this study, we were the first to systematically elucidate the role of Uck2 in HCC.Patients and methodsUck2 gene expression was queried between normal liver tissues and HCC in a web-based data mining platform (www.oncomine.org). Uck2 gene expression in tissue microarray was determined by immunohistochemical staining. The clinical and prognostic significance of Uck2 expression was statistically analyzed. Stable cell lines with increased Uck2 expression were established using lentivirus-based vectors, and RNAi technology was used to transiently downregulate Uck2 expression. Cell migration and invasion were assessed by using wound-healing and transwell assays, respectively. mRNA and protein expression levels were determined using quantitative real-time PCR and Western blotting, respectively.ResultsWe report the upregulation of Uck2 expression in HCC tissues. We explored the relationship between Uck2 levels and the clinicopathological features of HCC patients. High Uck2 notably correlated with early recurrence and poor prognosis in HCC patients. Uck2 expression in HCC cell lines regulated the cell migration and invasion capacities in vitro. The stable overexpression of Uck2 in Bel-7402 cells promoted their metastasis ability in vivo. Furthermore, the Uck2 upregulation increased the MMP2/9 expression and activated the Stat3 signaling pathway. In addition, WP1066, a Stat3 inhibitor, neutralized the effects of Uck2 on the MMP2/9 expression and the migration and invasion capacities of HCC cells.ConclusionOur data suggest that Uck2 promotes HCC cell migration and invasion via the Stat3 signaling pathway and might be a novel potential target in HCC therapy.
| S-EPMC6267774 | biostudies-literature
Project description:1. Shortly after the addition of phytohaemagglutinin to cultures, partially purified pig blood lymphocytes showed increased labelling of RNA by [5-(3)H]uridine and began to attach to the glass of the culture tubes. 2. In the presence of phytohaemagglutinin most cells became attached to glass during the first 5hr. of culture; those first attaching had a low mean RNA/DNA ratio. In the absence of phytohaemagglutinin cell attachment to glass was diminished. 3. During the first 5hr. of culture the rates of increase of radioactivity/unit of DNA of the attached and unattached populations in the presence of phytohaemagglutinin were of the same order and exceeded that of cultures without phytohaemagglutinin. 4. The increase of labelling in cultures to which phytohaemagglutinin was added after cells had sedimented to glass was greater than that occurring in cultures in which cells were resuspended before phytohaemagglutinin was added.
| S-EPMC1198807 | biostudies-other