Dihydrophenazine-derived oligomers from industrial waste as sustainable superior cathode materials for rechargeable lithium-ion batteries.

Organic materials with the 5,10-dihydrophenazine motif are superior cathode materials for lithium-ion batteries. However, the difficult accessibility and low capacity of such cathodes materials are obstacles to their practical applications. Herein, two novel oligomers, termed poly(5-methyl-10-(2-methacryloxypropyl)-5,10-dihydrophenazine) (PMPPZ) and poly(5-methyl-10-(2-methacryloxyethyl)-5,10-dihydrophenazine) (PMEPZ), were effectively synthesized from an industrial waste phenazine. Both oligomers were exploited successfully as excellent cathode materials for sustainable lithium-ion batteries. PMPPZ and PMEPZ exhibited good electrochemical stability and high initial discharge specific capacities of 88 mA h g-1 and 152 mA h g-1, respectively. Furthermore, upon in situ composition with MWCNTs, a composite material, named PMEPZ-MWCNTs, was achieved with enhanced stability and superior specific discharge capacity with the active-site utilization rate of up to 99%. PMEPZ-MWCNTs delivers high initial discharge capacity of up to 303 mA h g-1 and even 252 mA h g-1 after 300 cycles. Both oligomers exhibit double-electron transfer mechanisms. This work affords an alternative approach to utilizing phenazine as a useful material, circumventing the emission of vast environment harmful gases.

The impact of RNA standardization and heterogeneous gene expression on the results of cDNA array of human breast carcinoma.

cDNA microarray is an established technique. However, difficulties such as handling tissue samples under RNase-free conditions, the heterogeneous tumor composition, i.e. non-malignant versus malignant cells and different pathologic types of malignant cells, and lack of appropriate reference may limit the potentially benefit of this method in clinical use. In this study, we examined how standardization of gene expression to total mg RNA or mg tissue and tumor heterogeneity affect the final results. We found that the gene expression of human breast tumors was approximately 9 times higher in malignant tissue as compared to the non-malignant tissue when expressed per total mg RNA, but approximately 40 times higher when expressed per mg tissue. Genes that were expected to act as housekeeping genes (PUC18, RPL and beta-actin) varied between different parts of the tumor and also between non-malignant and malignant tissues, excluding them as reference genes. We also found that the gene expression differed in various parts of the breast tumor, probably due to a mixture of different types of cells, i.e. non-malignant and malignant cells. To find out if the variations in the gene expression were due to cell heterogeneity we used microdissection to collect malignant cells separately. We found that the gene expression was markedly different in the isolated malignant cells as compared to the gene expression of the bulk tumor tissue. Thus, to be able to evaluate results from cDNA array gene expression experiments it is, to our opinion, necessary to work with pure tumor cell populations, until solid information is available on the impact of stromal component. Housekeeping genes should be handling with care and mg tissue may be preferred instead of microg RNA for standardization.

In vivo biological activity of the histone deacetylase inhibitor LAQ824 is detectable with 3'-deoxy-3'-[18F]fluorothymidine positron emission tomography.

Histone deacetylase inhibitors (HDACI) are emerging as growth inhibitory compounds that modulate gene expression and inhibit tumor cell proliferation. We assessed whether 3'-deoxy-3'-[(18)F]fluorothymidine-positron emission tomography ([18F]FLT-PET) could be used to noninvasively measure the biological activity of a novel HDACI LAQ824 in vivo. We initially showed that thymidine kinase 1 (TK1; EC2.7.1.21), the enzyme responsible for [18F]FLT retention in cells, was regulated by LAQ824 in a drug concentration-dependent manner in vitro. In HCT116 colon carcinoma xenograft-bearing mice, LAQ824 significantly decreased tumor [18F]FLT uptake in a dose-dependent manner. At day 4 of treatment, [18F]FLT tumor-to-heart ratios at 60 minutes (NUV60) were 2.16 +/- 0.15, 1.86 +/- 0.13, and 1.45 +/- 0.20 in vehicle, and 5 and 25 mg/kg LAQ824 treatment groups, respectively (P < or = 0.05). LAQ825 at 5 mg/kg also significantly reduced both TK1 levels and [18F]FLT uptake at day 10 but not at day 2 (P < or = 0.05). [18F]FLT NUV60 correlated significantly with cellular proliferation (r = 0.68; P = 0.0019) and was associated with drug-induced histone H4 hyperacetylation. Of interest to [18F]FLT-PET imaging, both TK1 mRNA copy numbers and protein levels decreased in the order vehicle >5 mg/kg LAQ824 > 25 mg/kg LAQ824, providing a rationale for the use of [18F]FLT-PET in this setting. We also observed increases in Rb hypophosphorylation and p21 levels, factors that could have contributed to the alteration in TK1 transcription in vivo. In conclusion, we have shown the utility of [18F]FLT-PET for monitoring the biological activity of the HDACI, LAQ824. Drug-induced changes in tumor [18F]FLT uptake were due, at least in part, to reductions in TK1 transcription and translation.

Thymidine kinase 1: a proliferation marker for determining prognosis and monitoring the surgical outcome of primary bladder carcinoma patients.

Reliable markers for monitoring bladder tumor therapy are needed to evaluate treatment effectiveness. Thymidine kinase 1 (TK1) is an enzyme involved in DNA synthesis and therefore proliferation-dependent. Serum concentration of TK1 (STK1) correlates with malignancy in various types of cancer, thus reflecting treatment results. This study explores for the first time the use of STK1 concentration, both as a prognostic marker and to monitor the outcome of bladder carcinoma surgery. STK1 in 56 bladder carcinoma patients was measured pre-operatively, and post-operatively at 1 week and 1, 3, and 6 months, using an immune ECL dot blot assay. An anti-TK1 chicken IgY antibody was used to determine STK1 concentrations. Mean pre-operative STK1 of bladder carcinoma patients was significantly higher than that of healthy individuals, with no overlap of individual values. STK1 concentrations increased significantly with tumor stage (I-III) and T-values (T1-T2), but not tumor grade (G1-G4). STK1 gradually declined, being 66% lower after 1 week. STK1 reached the level of healthy controls at 1 month and remained there for at least 6 months, post-operatively until this study ended. Since STK1 concentration correlates with tumor stage, degree of invasion and metastasis, and monitors the surgical outcome, it can be a reliable index to diagnose and determine prognosis in post-operative bladder carcinoma.

Thymidine kinase 1 in serum predicts increased risk of distant or loco-regional recurrence following surgery in patients with early breast cancer.

BACKGROUND: The prognostic value of the concentration of serum thymidine kinase 1 (S-TK1) with regard to recurrence in low risk breast cancer patients, 3 months after surgery was evaluated. PATIENTS AND METHODS: The concentration of S-TK1 in serum was determined in 120 breast cancer patients at the time of surgery and in 67 patients 3 months after surgery, by anti-TK1 chicken IgY antibody, using a dot-blot immuno-assay. The S-TK1 concentration was compared with the serological activity of thymidine kinase (STK) and of carbohydrate antigen (CA 15-3). RESULTS: A statistically significant trend (unadjusted) was found for recurrence (distant or loco-regional) in patients with a higher S-TK1 concentration, as compared with patients with a lower S-TK1 concentration. A multivariate analysis gave the same results. The hazard rate ratio for developing distant and/or loco-regional recurrence in patients with a higher S-TK1 concentration was about six to seven times higher than in patients with a lower S-TK1 concentration. CONCLUSION: Our results indicate that the S-TK1 concentration is higher in patients developing distant and/or loco-regional recurrence 3 months post-surgery.

Time-dependent RNA degradation affecting cDNA array quality in spontaneous canine tumours sampled using standard surgical procedures.

Heterogeneous gene expression in tumours and the degradation of RNA when sampling under non-RNAse-free conditions may limit the potential benefit of cDNA array studies. This study examines changes in the integrity of RNA by means of RNA gel electrophoresis at various post-operative intervals on canine mammary tumours (n=10) and malignant lymphoma (n=1). The tumours were cut into pieces (3-5 mm diameter, approximately 50 mg) and kept in tubes without RNAse-free buffer at room temperature. No special precautions were taken to avoid the influences of Rnase; rather, normal surgical procedures were used. We found that total RNA of the mammary tumours started to degrade within 30 min of the operation, and the rate of degradation increased up to 4 h, which was the last time point included in this study. RNA in the lymphoma tumours degraded more rapidly, and was completely degraded at 30 min post-operation. The degradation of mRNA in the mammary tumours, as studied by human cDNA arrays, was heterogeneous, i.e. some mRNA degraded completely, some only partially. This indicates that the mRNA degradation rate varied depending on the type of mRNA. However, since we found that gene expression differs depending on the part of the mammary tumour examined, one cannot exclude that the variation in the mRNA degradation rate may simply reflect heterogeneous gene expression within the tumour. We conclude that RNA integrity is unaffected immediately after sampling under non-RNAse-free conditions; however, the tumour sample should be preserved under RNAse-free conditions within 15 min to avoid RNA degradation. This is a much shorter time interval than previously reported in other similar studies; however, these studies generally treated normal tissue, under which 3-5 h non-RNAse-free conditions have been found not to affect RNA quality.

Concentration of thymidine kinase 1 in serum (S-TK1) is a more sensitive proliferation marker in human solid tumors than its activity.

Activity of thymidine kinase 1 in serum (STK) is a useful marker for leukaemia and lymphoma, but not for solid tumors. We investigate thymidine kinase 1 concentration in serum (S-TK1) as a potential tumor marker. S-TK1 concentration and STK activity levels were determined in 9 human malignant diseases (breast, gastric, rectal, colorectal, lung, brain cancer, hepatoma, lymphoma, leukaemia) and in benign and non-cancerous diseases, representing 850 preoperative cases. Healthy volunteers (n=43) were used as positive controls. S-TK1 concentration was determined by ECL dot blot assay and STK activity levels by an RIA assay. S-TK1 concentrations and STK activity levels in preoperative malignant patients were significantly higher than in healthy individuals, in patients with benign tumors and in those with non-cancerous diseases. Significant correlations between concentration and activity level were only found in healthy individuals, in patients with benign tumors, and in some patients with malignancies, i.e. leukaemia, and breast and gastric cancers. About 90-95 percent of the malignant patients showed S-TK1 concentrations above those of the healthy controls. The corresponding value for STK activity was about 75 percent. When sera from malignant patients were diluted with sera from healthy individuals, S-TK1 concentrations and STK activity levels decreased more than expected. This indicates the presence of a compound (or compounds) in the serum of healthy individuals that destabilises S-TK1. We conclude that S-TK1 concentration is a more sensitive tumor marker in solid malignancies than is STK activity.

Early detection of tumor response to chemotherapy by 3'-deoxy-3'-[18F]fluorothymidine positron emission tomography: the effect of cisplatin on a fibrosarcoma tumor model in vivo.

We have assessed the potential of [18F]fluorothymidine positron emission tomography ([18F]FLT-PET) to measure early cytostasis and cytotoxicity induced by cisplatin treatment of radiation-induced fibrosarcoma 1 (RIF-1) tumor-bearing mice. Cisplatin-mediated arrest of tumor cell growth and induction of tumor shrinkage at 24 and 48 hours, respectively, were detectable by [18F]FLT-PET. At 24 and 48 hours, the normalized uptake at 60 minutes (tumor/liver radioactivity ratio at 60 minutes after radiotracer injection; NUV60) for [18F]FLT was 0.76 +/- 0.08 (P = 0.03) and 0.51 +/- 0.08 (P = 0.03), respectively, compared with controls (1.02 +/- 0.12). The decrease in [18F]FLT uptake at 24 hours was associated with a decrease in cell proliferation assessed immunohistochemically (a decrease in proliferating cell nuclear antigen labeling index, LI(PCNA), from 14.0 +/- 2.0% to 6.2 +/- 1.0%; P = 0.001), despite the lack of a change in tumor size. There were G1-S and G2-M phase arrests after cisplatin treatment, as determined by cell cycle analysis. For the quantitative measurement of tumor cell proliferation, [18F]FLT-PET was found to be superior to [18F]fluorodeoxyglucose-PET (NUV60 versus LIPCNA: r = 0.89, P = 0.001 and r = 0.55, P = 0.06, respectively). At the biochemical level, we found that the changes in [18F]FLT and [18F]fluorodeoxyglucose uptake were due to changes in levels of thymidine kinase 1 protein, hexokinase, and ATP. This work supports the further development of [18F]FLT-PET as a generic pharmacodynamic readout for early quantitative imaging of drug-induced changes in cell proliferation in vivo.

Expression of cell proliferating genes in patients with non-small cell lung cancer by immunohistochemistry and cDNA profiling.

Thymidine kinase 1 (TK1) is a key enzyme involved in the synthesis of DNA precursors and thus, cell proliferation-dependent. Antibodies against TK1 have provided attractive tools for cancer diagnosis. Expression of TK1 in 158 non-small cell lung cancer (NSCLC) patients with 59 adenocarcinoma (AC) and 99 squamous cell carcinoma (SCC) was determined by anti-TK1 monoclonal antibody (mAb) 1E3 (AC, n=50; SCC, n=70). Parallel tumor sections were stained for Ki-67 (MIB-1), and TK1 expression was also investigated with anti-TK1 chicken IgY Ab (AC, n=9; SCC, n=29; normal lung tissues, n=10). In one AC and one SCC patient, gene profiling was done by cDNA array. Using the mAb 1E3, a significantly higher TK1 labeling index (LI) of AC patients was found (68%) compared to the LI of Ki-67 (36%). This difference was due to a significantly higher TK1 LI of tumor stage II and grade 2. Although no difference in the LI of TK1 and Ki-67 of SCC patients was found (54 vs. 53%), significantly higher TK1 LI of SCC patients of tumor grade 1 was found. Using the anti-TK1 IgY Ab, a higher TK1 LI of AC patients (78%) and SCC patients (66%) was found compared to staining with mAb 1E3 (68 vs. 54%), but it was not significantly different. Samples stained only for TK1 represented mostly tumor stages I and II and grades 1 and 2 of both AC and SCC. AC patients whose samples stained only for Ki-67 were found to be in stage I and grade 1. cDNA profiling showed that the expression of BRCA1, cyclin B1 and cdc2p34 was higher in AC compared to SCC, while the expression of IGFBP-3 and EGFR was higher in SCC. TK1 is apparently a more reliable marker in AC patients than Ki-67. However, a combination of the two markers may help identify patients of different stages and grades more efficiently, and cyclin/kinase complexes and growth factors/receptors may be useful markers in distinguishing AC from SCC.

The uptake of 3'-deoxy-3'-[18F]fluorothymidine into L5178Y tumours in vivo is dependent on thymidine kinase 1 protein levels.

PURPOSE: The aim of this study was to investigate the role of thymidine kinase 1 (TK1) protein in 3'-deoxy-3'-[18F]fluorothymidine ([18F]FLT) positron emission tomography (PET) studies. METHODS: We investigated the in vivo kinetics of [18F]FLT in TK1+/- and TK1-/- L5178Y mouse lymphoma tumours that express different levels of TK1 protein. RESULTS: [18F]FLT-derived radioactivity, measured by a dedicated small animal PET scanner, increased within the tumours over 60 min. The area under the normalised tumour time-activity curve were significantly higher for the TK1+/- compared with the -/- variant (0.89+/-0.02 vs 0.79+/-0.03 MBq ml(-1) min, P=0.043; n=5 for each tumour type). Ex vivo gamma counting of tissues excised at 60 min p.i. (n=8) also revealed significantly higher tumour [18F]FLT uptake for the TK1+/- variant (6.2+/-0.6 vs 4.6+/-0.4%ID g(-1), P=0.018). The observed differences between the cell lines with respect to [18F]FLT uptake were in keeping with a 48% higher TK1 protein in the TK1+/- tumours versus the -/- variant (P=0.043). On average, there were no differences in ATP levels between the two tumour variants (P=1.00). A positive correlation between [18F]FLT accumulation and TK1 protein levels (r=0.68, P=0.046) was seen. Normalisation of the data for ATP content further improved the correlation (r=0.86, P=0.003). CONCLUSION: This study shows that in vivo [18F]FLT kinetics depend on TK1 protein expression. ATP may be important in realising this effect. Thus, [18F]FLT-PET has the potential to yield specific information on tumour proliferation in diagnostic imaging and therapy monitoring.

Cytosolic thymidine kinase is a specific histopathologic tumour marker for breast carcinomas.

Thymidine kinase 1 (TK1), an enzyme involved in the synthesis of precursors for DNA, and thus proliferation dependent, has been suggested as a good tumour marker. We have recently developed poly/monoclonal antibodies against TK1, which proved useful for diagnostics in both serum and immunohistochemistry of cancer patients. The anti-TK1 monoclonal antibodies (mAbs) 1D11 and 1E3 were characterized by Western blot, immunoprecipitation and flow cytometry. TK1 mAbs and Ki-67 mAb were then used for immunohistochemistry staining of tumour sections from 54 patients with ductal infiltrated breast carcinoma. Results showed the relative number of patients with positively stained tumours for TK1 (mAb 1D11) and for Ki-67 (mAb MIB-1) were 47 and 41%, respectively, significantly related (p=0.007). Combination of TK1 mAbs 1D11 and 1E3 increased this number to 56%, due to detection of a significantly higher number of patients with grade 2 tumours. Patients with stage II and grade 2 tumours showed significantly higher TK1 staining when compared to stage I and grade 1. Ki-67 staining was significantly higher in stage III and grade 3. The tumours only stained for TK1 represented higher stages and grades, while tumours staining only for Ki-67 were of lower stages and grades. Combining TK1 and Ki-67 increased the number of patients with positively stained tumours to 69%. In conclusion, TK1 is a reliable marker for identification of patients with grade 2 tumours. The highest number of patients with positively stained tumours were obtained when both TK1 and Ki-67 markers were used.

Genes related to growth regulation, DNA repair and apoptosis in an oestrogen receptor-negative (MDA-231) versus an oestrogen receptor-positive (MCF-7) breast tumour cell line.

The molecular mechanism(s) behind the development of endocrine resistance in breast cancer remains controversial. Here, we compare the capability of oestrogen receptor (ER)-negative cells (MDA-231) versus ER-positive tamoxifen-sensitive cells (MCF-7) to handle DNA repair, transmit signals from damaged DNA, initiate cell death via apoptosis, and then to control transmitted signals from the cell cycle and to synthesize growth factors and receptors. Genes related to these events were studied by cDNA micro-array. Normal human breast cells (H2F) and human lymphoblastoid tumour cells (CEM) were used as controls. Of the 18 genes investigated, 10 genes showed differences in their expression between the cell types. The ER-negative cells showed higher expressions of BRCA1, BRCA2, cdc2, cyclin B1, cyclin D1, cyclin E, IGFBP-3, TGF-alpha, TGF beta 2 and a lower expression of TGF beta R1. No differences in the expressions of bax, bcl-2, p53, p21 and GADD45 were found between the two cell lines. We found that the ER-negative cells were characterized by: (1) a stimulated expression of growth factors and cell cycle regulation compounds, (2) improved DNA repair capacity, but (3) no change in DNA damage signals and apoptotic pathways. Improved DNA repair capacity of ER-negative cells would have a growth advantage over ER-positive tumours when receiving antitumour therapy.

3'-deoxy-3'-[18F]fluorothymidine as a new marker for monitoring tumor response to antiproliferative therapy in vivo with positron emission tomography.

3'-Deoxy-3'-[(18)F]fluorothymidine ([(18)F]FLT) has been proposed as a new marker for imaging tumor proliferation by positron emission tomography (PET). The uptake of [(18)F]FLT is regulated by cytosolic S-phase-specific thymidine kinase 1 (TK1). In this article, we have investigated the use of [(18)F]FLT to monitor the response of tumors to antiproliferative treatment in vivo. C3H/Hej mice bearing the radiation-induced fibrosarcoma 1 tumor were treated with 5-fluorouracil (5-FU; 165 mg/kg i.p.). Changes in tumor volume and biodistribution of [(18)F]FLT and 2-[(18)F]fluoro-2-deoxy-D-glucose ([(18)F]FDG) were measured in three groups of mice (n = 8-12/group): (a) untreated controls; (b) 24 h after 5-FU; and (c) 48 h after 5-FU. In addition, dynamic [(18)F]FLT-PET imaging was performed on a small animal scanner for 60 min. The metabolism of [(18)F]FLT in tumor, plasma, liver, and urine was determined chromatographically. Proliferation was determined by staining histological sections for proliferating cell nuclear antigen (PCNA). Tumor levels of TK1 protein and cofactor (ATP) were determined by Western blotting and bioluminescence, respectively. Tumor [(18)F]FLT uptake decreased after 5-FU treatment (47.8 +/- 7.0 and 27.1 +/- 3.7% for groups b and c, respectively, compared with group a; P < 0.001). The drug-induced reduction in tumor [(18)F]FLT uptake was significantly more pronounced than that of [(18)F]FDG. The PET image data confirmed lower tumor [(18)F]FLT retention in group c compared with group a, despite a trend toward higher radiotracer delivery for group c. Other than phosphorylation in tumors, [(18)F]FLT was found to be metabolically stable in vivo. The decrease in tumor [(18)F]FLT uptake correlated with the PCNA-labeling index (r = 0.71, P = 0.031) and tumor volume changes after 5-FU treatment (r = 0.58, P = 0.001). In this model system, the decrease in [(18)F]FLT uptake could be explained by changes in catalytic activity but not translation of TK1 protein. Compared with group a, TK1 levels were lower in group b (78.2 +/- 5.2%) but higher in group c (141.3 +/- 9.1%, P < 0.001). In contrast, a stepwise decrease in ATP levels was observed from group a to b to c (P < 0.001). In conclusion, we have demonstrated the ability to measure tumor response to antiproliferative treatment with [(18)F]FLT and PET. In our model system, the radiotracer uptake was correlated with PCNA-labeling index. The decrease in [(18)F]FLT uptake after 5-FU was more pronounced than that of [(18)F]FDG. [(18)F]FLT is, therefore, a promising marker for monitoring antiproliferative drug activity in oncology that warrants additional testing.

Production and characterisation of a novel chicken IgY antibody raised against C-terminal peptide from human thymidine kinase 1.

Egg yolk is a good source of highly specific antibodies against mammalian antigens because of the phylogenetic distance between birds and mammals. Chicken egg yolk immunoglobulins (IgY) were generated to a synthetic 31-amino acid peptide from the C-terminal of human HeLa thymidine kinase 1 (TK1) enzyme. The anti-TK1 IgY antibody was purified using affinity chromatography against the 31-amino acid peptide. The purified antibody inhibited the catalytic activity of the TK1 enzyme in the CEM TK1(+) cells and recognized the 25-kDa subunit and tetrameric form of TK1, which has a pI value of 8.3. No immunoreaction was observed in CEM TK1(-) cells. Western blot of the serum TK1 (S-TK1) also showed that only a single band was found in the serum of patients with malignancies. No band was seen in healthy serum. Furthermore, dot blots and enhanced chemiluminescence (ECL) detection of S-TK1 performed on sera of preoperative patients with gastric cancer (GC) (n=31) and healthy controls (n=62) showed that the levels of S-TK1 in the sera of cancer patients were significantly different (P<0.01). Using ECL dot blots, 0.1 pg of TK1 in 3 microl sera could be detected. Immunohistostaining of tissues in the 11 advanced-stage cancer patients (four breast carcinomas, three hepatocarcinomas and four thyroid carcinomas) indicated that a strong staining of TK1 enzyme was found in the cytoplasm of malignant cells. No staining or weak staining was seen in normal tissues. We suggest that screening for TK1 using anti-TK1 IgY may be potentially useful for serological and immunohistochemical detection of TK1 as an early prognosis and for monitoring patients undergoing treatment.

A comparative study: immunohistochemical detection of cytosolic thymidine kinase and proliferating cell nuclear antigen in breast cancer.

To explore the expression of cytosolic thymidine kinase 1 (TK1) as a cell proliferative marker in human breast cancers, immunohistochemistry was used to detect the expression of TK1 in 52 malignant breast lesions, 20 benign breast lesions, and 16 normal breast tissues. The results were compared to the expression of proliferating cell nuclear antigen (PCNA) in the same specimens. The TK1-labelling index (TK1-LI) and PCNA-labeling index (PCNA-LI) were significantly higher in malignant lesions than in nonmalignant lesions (p < 0.0001 and p < 0.0013, respectively). The TK1-LI (78.9%) in malignant lesions was higher compared to PCNA-LI (64.5%). No significant difference was found for TK1-LI and PCNA-LI between benign lesions and normal tissues. Concerning the tumor stages and the tumor grades, TK1-LI showed a significant correlation with the increased tumor stages (p = 0.023) and tumor grades (p = 0.009). However, PCNA-LI was neither significantly different in tumor stages (p = 0.062) nor in tumor grades (p = 0.073). We conclude that TK1 might be a more accurate marker than PCNA for estimation of cell proliferation and malignant potentials in breast carcinomas.

Involvement of a substrate cycle between thymidine and thymidylate in the regulation of DNA precursor pool in ehrlich ascites tumour.

In Ehrlich ascites tumour (EAT) cells the main route for dTTP required for DNA synthesis is closely related to thymidylate synthesis activity via the de novo pathway. However, more than 10-time of thymidylate (dTMP) is synthesised by cytosolic thymidine kinase (TK1) via the salvage pathway than needed for DNA synthesis in this cells. Therefore, this study focus to determine if a substrate cycle exists between thymidine (dTdR) and dTMP in the EAT cells. Results show that the ratio of K'eq/Q for the TK1 reaction is 264.2 and for the thymidylate 5'-phosphatase (dTMPase) reaction is 110.9 in the exponentially growing cells, respectively. Since the apparent ratios of K'eq/Q for both reactions are different from equality (ñ1) by two orders, it appears as a non-equilibrium reaction. This indicates that when TK1 and dTMPase are simultaneously active in the exponentially growing cells, a substrate cycle results. The regulation of the excess of non-essential products of dTdR/dTMP for DNA synthesis is involved in a substrate cycle for maintaining a balanced nucleotide pool, hence ensuring a balanced supply of the DNA precursor in the exponentially growing cells. As the tumours continue to grow, cells reached the stationary phase. The ratio of K'eq/Q for TK1 reaction is 7.7 and for the dTMPase reaction is 81.1, showing less than the equilibrium of two orders of magnitude. In this case, dTMPase could not act with TK1 together to form a pair of reaction, leading to an elevated concentration of intracellular dTMP and a dramatically excretion of dTdR into the ascites fluid.