Tumor cell proliferation is considered to be a useful prognostic indicator of tumor aggressiveness and tumor response to therapy, but in vitro measurement of individual proliferation is complex and tedious work. PET imaging provides a noninvasive approach to measure tumor growth rate in situ. Early approaches have used 18F-FDG or methionine to monitor proliferation status. These 2 tracers detect changes in glucose and amino acid metabolism, respectively, and therefore provide only an indirect measure of proliferation status. More recent studies have focused on DNA synthesis itself as a marker of cell proliferation. Cell lines and tissues with a high proliferation rate require high rates of DNA synthesis. [11C]Thymidine was the first radiotracer for noninvasive imaging of tumor proliferation. The short half-life of 11C and rapid metabolism of [11C]thymidine in vivo make the radiotracer less suitable for routine use. Halogenated thymidine analogs such as 5-iodo-2-deoxyuridine (IUdR) can be successfully used as cell proliferation markers for in vitro studies because these compounds are rapidly incorporated into newly synthesized DNA. IUdR has been evaluated as a potential in vivo tracer in nuclear medicine, but the image quality and the calculation of proliferation rates are impaired by its rapid in vivo degradation. Hence, the thymidine analog 3'-deoxy-3'-18F-fluorothymidine (FLT) was recently introduced as a stable proliferation marker with a suitable nuclide half-life and stable in vivo. [18F]FLT is phosphorylated to 3-fluorothymidine monophosphate by thymidine kinase 1 and reflects thymidine kinase 1 activity in proliferating cell. [18F]FLT PET is feasible in clincal use and well correlates with cellular proliferation. Choline is a precursor for the biosynthesis of phospholipids (in particular, phosphatidylcholine), which is the essential component of all eukaryotic cell membranes and [11C]choline, which is a new marker for cellular proliferation.
Cell Line ; Cell Proliferation* ; Choline ; DNA ; Eukaryotic Cells ; Fluorodeoxyglucose F18 ; Glucose ; Half-Life ; Idoxuridine ; Membranes ; Metabolism ; Methionine ; Nuclear Medicine ; Phospholipids ; Thymidine ; Thymidine Kinase

Recently it is known that nitric oxide(NO) generated by an activatedmacrophage plays an important role in tumoricidal or bactericidal activity. Thisstudy was done to know the effects of NO produced by the activated macrophages onthe growth of the murine bladder tumor cell line (MBT-2). For the activation ofmacrophages, RAW 264.7 cell line ( macrophage-derived cell line) and peritonealmacrophages from Balb/c mouse were treated with interferon-r (INF-r),lipopolysaccharide ( LPS) or INF-r plus LPS and for the evaluation of growthinhibition of MBT -2 cell line, tritiated thymidine (3[H] -thymidine)incorporation was measured after coculturing of MBT-2 cell line with macrophageswhich had been activated to produce NO. The results are as follows : l.Peritoneal macrophages from Balb/c mouse could be activated to produce NO onlywhen they are stimulated by the combination of INF-r and LPS (35+/-1.0uM/L). Theycould produce a slight increase amount of NO when they had been stimulated byINF-r (11+/-2.5uM/ L) or LPS (14+/-3.5uM/L) compared to control macrophages(8+/-2.5uM/L). The induction of NO production by INF-r plus LPS could be abrogatedby the use of NG-monomethyl-L- arginine (NGMMA), a competitive inhibitor of NOsynthase (5+/-1.5 M/L). 2. RAW 264.7 cell lines could be activated to produce NOby the treatment of INF-r, LPS, and INF-r plus LPS (40+/-2.5uM/L, 37+/-3.0uM/Land 51+/-2.6uM/L respectively). When they are treated with NGMMA, they produced nomore NO even though they had been stimulated with INF-r plus LPS (14+/-4.0uM/L),and they could produce small .amount of NO(19+/-1.0uM/L) in the absence of thestimulation. 3. The incorporation of 3[H] -thymidine of the MBT-2 and peritonealmacrophages was reduced when the cells had been treated with INF-r plus LPScompared to the control (14,519+/-1,087cpm, 20,716+/-1,474cpm respectively).There was no reduction in the incorporation of 3[H] -thymidine when the cellshad been treated with INF-r or LPS alone. The incorporation of 3[H]-thymidineincreased when the cells had been treated with INF-r plus LPS in the presence ofNGMMA(19,622+/-1,341cpm). 4. The incorporation of 3[H] -thymidine of the MBT-2 and RAW 264.7 cell lines were reduced when the cells had been treated with INF-r, LPS and INF-r plus LPS (19.068+/-144cpm, 15,070+/-122cpm and 7,543+/-85cpm respectively) compared to control( 20,708+/-142cpm). When the cells had been pretreated with NGMMA, the incorporation of 3[H]- thymidine recovered to same degree (12,605+/-108cpm) compared to the cells stimulated with INF-r plus LPS. The above correlation of the NO production from macrophages which had been stimulated by INF-r and/or LPS and the inhibition of growth of the tumor cells suggests that NO produced by stimulated macrophages might be the responsible molecule in the defense system of the body against tumors.
Animals ; Arginine ; Cell Line* ; Macrophages* ; Macrophages, Peritoneal ; Metabolism* ; Mice ; Nitric Oxide ; Nitrogen* ; Thymidine ; Urinary Bladder Neoplasms

Thymidine is a commercially useful precursor for production of antiviral compounds such as stavudine and azidothymidine. Biosynthesis of thymidine by Escherichia coli BL21 (DE3) was studied using metabolic engineering methods. The deoA, tdk and udp of the salvage pathway were disrupted from E. coli BL21 to construct BS03 that produced 21.6 mg thymidine per liter. Additional deletion of pgi and pyrL increased the supply of thymidine precursors and the resulting strain BS05 produced 90.5 mg thymidine/L. At last, ushA, thyA, dut, ndk, nrdA and nrdB of thymidine biosynthetic pathway were overexpressed, and the resulting strain BS08 produced 272 mg thymidine/L. In fed-batch fermentation, BS08 accumulated 1248.8 mg thymidine/L. Metabolically engineered strain E. coli has potential applications for thymidine production.
Biosynthetic Pathways ; Escherichia coli ; genetics ; metabolism ; Fermentation ; Industrial Microbiology ; Metabolic Engineering ; Thymidine ; biosynthesis

OBJECTIVESTo study the change of ability to transform from 5'-deoxy-fluorouracil monophosphate (5'-DFUR) to fluorouracil (5-FU) in human colon cancer cell lines SW480 and LOVO which transfected with thymidine phosphorylase (TP) gene. And to discuss the anti-cancer activity of 5'-DFUR to SW480 and LOVO cells.

METHODSTP cDNA were transfected into human colorectal cancer cell lines SW480 and LOVO with the lentiviral vector, pLenti6.3_MCS_IRES2-EGFP. The transfection efficiency was analyzed by flow cytometer, the mRNA expression of TP was detected by RT-PCR, and the TP protein expression was detected by Western blot, and the volumes of 5-FU converted from 5'-DFUR both in 2 cells and medium were detected by high performance liquid chromatography (HPLC). The 50% inhibitory concentration (IC50) of 5'-DFUR on these 2 colon cancer cell lines both wild type and TP-transfected cells were evaluated by MTT assay.

RESULTSThe colorectal cancer cell lines SW480 and LOVO transfected with human TP cDNA were monitored 5 generations, and the transfections efficiency rate wea about 95%. Compared with wild type cell SW480 and LOVO, the RQ values of mRNA expression of SW480-TP and LOVO-TP were (695 ± 171) folds (t = -7.00, P = 0.002) and (282 ± 87) folds (t = -5.61, P = 0.030), respectively. Also TP protein expression in SW480-TP and LOVO-TP were higher than their parent cells shown by Western blot. The volume of 5-FU converted from 5'-DFUR in the medium cultured SW480-TP and LOVO-TP were increased compared with their parent cells, respectively (t = 19.406-66.921, P < 0.01), whereas few of 5-FU was detected both in wild, and TP-transfected cells. After transfected with TP cDNA, the IC50 of 5'-DFUR on SW480-TP and LOVO-TP were (587 ± 17) µmol/L and (1088 ± 89) µmol/L respectively, and there were significantly less than their parent cells (t = -32.59 and -8.52, P < 0.01).

CONCLUSIONSThe stabilized transfections of SW480 and LOVO with higher TP expression could be built with lentiviral vector. Transfected TP cDNA into SW480 and LOVO, could improve the expression both of TP mRNA and TP protein, increase the volume of 5-FU converted from 5'-DFUR in medium, and result in an enhancement of anticancer effect on these 2 cells.

Cell Line, Tumor ; Colonic Neoplasms ; pathology ; Floxuridine ; metabolism ; Fluorouracil ; metabolism ; Humans ; Thymidine Phosphorylase ; genetics ; Transcription, Genetic ; Transfection

OBJECTIVETo determine the contents of thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) in pancreatic cancer to provide a basis for the clinical use of capecitabine in pancreatic cancer patients.

METHODSThe contents of TP and DPD in pancreatic cancer and adjacent normal tissues from 20 patients were determined by ELISA and the TP to DPD ratios in the cancer and adjacent normal tissue were compared.

RESULTSTP content was 5- to 283-fold higher in tumor tissue (mean 74-fold) than in the adjacent normal tissue (P < 0.01). DPD in the cancer tissue increased significantly. So did the TP to DPD ratio, when compared to that in normal pancreatic tissue (P < 0.01).

CONCLUSIONThe increased TP to DPD ratio in pancreatic cancer suggests that capecitabine could be activated by the cancer, these capable of selectively kill the tumor cells.

Dihydrouracil Dehydrogenase (NADP) ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Humans ; Pancreas ; enzymology ; Pancreatectomy ; Pancreatic Neoplasms ; enzymology ; surgery ; Thymidine Phosphorylase ; metabolism

OBJECTIVETo investigate the mRNA expression of platelet-derived endothelial cell growth factor (PD-ECGF) in superficial bladder cancer and its significance.

METHODSPD-ECGF mRNA expressions were determined by RT-PCR in 28 cases of superficial bladder cancers and 6 cases of normal bladder mucosa. The relation between PD-ECGF mRNA expression and tumor invasion to lamina propria or recurrence after transurethral resection was also analyzed.

RESULTSSome degree of PD-ECGF mRNA expression was present in all the samples. The PD-ECGF mRNA level was 3.1-fold higher in pT(1) tumors than in normal bladder mucosa (t = 2.13, P < 0.05) and 2.2-fold higher in pT(1) tumors than in pT(a) tumors (t = 2.66, P < 0.05); G(3) tumors expressed 3.3-fold higher PD-ECGF mRNA than normal bladder mucosa (t = 2.44, P < 0.05) and 2.5-fold higher than G(1 - 2) tumors (t = 3.36, P < 0.01). Eleven cases recurred during the mean follow-up period of 18 months. Three-fold higher PD-ECGF mRNA expression was showed in cases who recurred after transurethral resection than that in cases who did not recur (t = 4.49, P < 0.01). The specificity and sensitivity of predicting tumor recurrence were 82.4% and 81.8% respectively using 0.095 as a cutoff value of PD-ECGF mRNA level in this group of superficial bladder cancer.

CONCLUSIONPD-ECGF mRNA expression correlates with tumor dedifferentiation and plays an important role in the early invasion in superficial bladder cancer. To analyze the PD-ECGF mRNA level contributes to the evaluations of tumor differentiation and invasion to lamina propria as well as recurrence prediction in superficial bladder cancer.

Carcinoma, Transitional Cell ; metabolism ; pathology ; Follow-Up Studies ; Humans ; RNA, Messenger ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Thymidine Phosphorylase ; genetics ; metabolism ; Urinary Bladder Neoplasms ; metabolism ; pathology

OBJECTIVETo explore the relation between the expressions of PD-ECGF and VEGF and the evolution of capillary hemangioma, so as to provide theoretical basis for treatment.

METHODSFourty cases with capillary hemangioma, proved by pathologic method, were randomly selected and divided into proliferative (n=22) and involuted groups (n=18), according to the Mulliken standard. 8 specimens from 8 children with prepuce operation were used as control group. All the specimens were fixed, embedded and underwent HE staining. The expression of PD-ECGF, VEGF and CD34 in endothelial cells were detected by immunohistochemistry. The microvessel-density (MVD) was also calculated. The results were analyzed by SPSS12.0.

RESULTSThe positive expression rates of PD-ECGF and VEGF were 95.45% (21/22) and 86.36% (19/22) in proliferative hemangioma, 77.78% (14/18) and 66.67% (12/ 18) in involuted hemangioma, 37.50% (3/8) and 37.50% (3/8) in normal skin. MVD in proliferative and involuted hemangioma and normal skin was 93.68 +/- 20.56, 51.94 +/- 20.73 and 17.50 +/- 5.30, respectively. There was a significant difference in PD-ECGF expression and MVD between the proliferative and involuted groups, or between the hemangioma and control groups (P < 0.05). The VEGF was significantly different between the proliferative and involuted groups, or between the proliferative and control groups (P < 0.05), but not between the involuted and control groups (P > 0.05). The expression of VEGF, PD-ECGD and MVD showed a positive relationship.

CONCLUSIONSPD-ECGF and VEGF have a synergetic effect in the proliferation of micro-vessels. PD-ECGF may enhance the activity of thymidine phosphorylase. They play an important role in the proliferation and involution of hemangioma.

Child, Preschool ; Female ; Hemangioma, Capillary ; metabolism ; pathology ; Humans ; Infant ; Male ; Neoplastic Syndromes, Hereditary ; metabolism ; pathology ; Thymidine Phosphorylase ; metabolism ; Vascular Endothelial Growth Factor A ; metabolism

PURPOSE: Several radioisotope-labeled thymidine derivatives such as [11C]thymidine was developed to demonstrate cell proliferation in tumor. But it is difficult to track metabolism with [11C]thymidine due to rapid in vivo degradation and its short physical half-life. 3'-[18F]fluoro-3'-deoxythymidine ([18F]FLT) was reported to have the longer half life of fluorine-18 and the lack of metabolic degradation in vivo. Here, we described the synthesis of the 3'-[18F]fluoro-3'-deoxythymidine ([18F]FLT) and compared with [18F]FET and [18F]FDG in cultured 9L cell and obtained the biodistribution and PET image in 9L tumor bearing rats. MATERIAL AND METHOD: For the synthesis of [18F]FLT, 3-N-tert-butoxycarbonyl-(5'-O-(4,4'-dimethoxytriphenylmethyl)-2'-deoxy-3'-O-(4-nitrobenzenesulfonyl)-beta-D-threopentofuranosyl)thymine was used as a FLT precursor, on which the tert-butyloxycarbonyl group was introduced to protect N3-position and nitrobenzenesulfonyl group. Radiolabeling of nosyl substitued precursor with 18F was performed in acetonitrile at 120 degrees C and deproteced with 0.5 N HCl. The cell uptake was measured in cultured 9L glioma cell. The biodistribution was evaluated in 9L tumor bearing rats after intravenous injection at 10 min, 30 min, 60 min and 120 min and obtained PET image 60 minutes after injection. RESULTS: The radiochemical yield was about 20-30% and radiochemical purity was more than 95% after HPLC purification. Cellular uptake of [18F]FLT was increased as time elapsed. At 120 min post-injection, the ratios of tumor/blood, tumor/muscle and tumor/brain were 1.61+/-0.34, 1.70+/-0.30 and 9.33+/-2.22, respectively. The 9L tumor was well visualized at 60 min post injection in PET image. CONCLUSION: The uptake of [18F]FLT in tumor was higher than in normal brain and PET image of [18F]FLT was acceptable. These results suggest the possibility of [18F]FLT as an imaging agent for brain tumor.
Animals ; Brain ; Brain Neoplasms ; Cell Proliferation ; Chromatography, High Pressure Liquid ; Glioma* ; Half-Life ; Injections, Intravenous ; Metabolism ; Rats* ; Thymidine

OBJECTIVETo detect the effect of interferon-α2a(IFN-α2a) on thymidine phosphorylase(TP) mRNA expression levels and the anticancer activity of 5-fluorouracil(5-FU) and 5'-deoxy-fluorouridine(5'-DFUR) in human colon carcinoma cell lines LOVO and SW480.

METHODSTwo human colon cancer cell lines LOVO and SW480 were cultured and treated with IFN-α2a at a series of dosage, and fluorescence quantitative PCR was carried out to detect the TP mRNA expression levels in these 2 cell lines. Then MTT assay and software Templet were used to determine the change of 50% inhibition concentration of 5-FU or 5'-DFUR combined with IFN-α2a on the two cell lines.

RESULTSThe TP mRNA expressions were up-regulated significantly by IFN-α2a at the doses of 500 U/ml and 5000 U/ml in LOVO(P<0.01). Compared with untreated cells(IFN-α2a 0 U/ml), no significance was found for TP mRNA expression levels in LOVO and SW480 treated by IFN-α2a at the dose of 50 U/ml (P>0.05). There was no significant difference for TP mRNA expression in SW480 between the dose of 0 U/ml and 500 U/ml of IFN-α2a(P>0.05), while a significant increace was detected at the dose of 5000 U/ml (P<0.01). No significant difference was found for the IC50 values after treatment of 5-FU combined with IFN-α2a (20 U/ml) on LOVO and SW480 compared with 5-FU alone, while the IC50 values after treatment of 5'-DFUR combined with IFN-α2a decreased significantly compared with 5'-DFUR alone(P<0.05).

CONCLUSIONThere is no direct inhibition effect of IFN-α2a on LOVO and SW480 in vitro, while it can up-regulate TP mRNA expression levels both in LOVO and SW480, and enhance the anticancer effect of 5'-DFUR on these 2 cell lines.

Cell Line, Tumor ; Colonic Neoplasms ; enzymology ; pathology ; Floxuridine ; pharmacology ; Humans ; Interferon-alpha ; pharmacology ; Recombinant Proteins ; pharmacology ; Thymidine Phosphorylase ; metabolism

OBJECTIVE: To investigate the mechanism of nucleolin (NCL) involved in lymphoma proliferation by regulating thymidine kinase 1 (TK1). METHODS: Twenty-three patients with diffuse large B-cell lymphoma (DLBCL) were selected and divided into initial treatment group (14 cases) and relapsed/refractory group (9 cases). Serum TK1 and C23 protein in peripheral blood mononuclear cells were detected. Cell models of CA46-NCL-KD (CA46-NCL-knockdown) and CA46-NCL-KNC (CA46-NCL-knockdown negative control) were established by lentivirus vector mediated transfection in Burkitt lymphoma cell line CA46. The half maximal inhibitory concentration (IC₅₀) of CA46-NCL-KD, CA46-NCL-KNC, and CA46 to adriamycin were detected by cell proliferation assay (MTS). The expression of NCL mRNA and protein in CA46-NCL-KD and CA46-NCL-KNC cells were dectected by Q-PCR and Western blot, respectively. The cell cycle of CA46-NCL-KD, CA46-NCL-KNC, and CA46 cells were detected by flow cytometry. The expression of TK1 protein in CA46-NCL-KD and CA46-NCL-KNC cells was detected by an enhanced chemiluminescence (ECL) dot blot assay. RESULTS: The level of serum TK1 in the initial treatment group was 0.43(0-30-1.01) pmol/L, which was lower than 10.56(2.19-14.99) pmol/L in the relapsed/refractory group (P<0-01), and the relative expression level of NCL protein in peripheral blood was also significantly lower. The IC₅₀ of CA46-C23-KD cells to adriamycin was (0.147±0.02) μg/ml, which was significantly lower than (0.301±0.04) μg/ml of CA46-C23-KNC cells and (0.338±0.05) μg/ml of CA46 cells (P<0.05). Compared with CA46-NCL-KNC cells, the expression of NCL mRNA and protein, TK1 protein decreased in CA46-NCL-KD cells, and the proportion of S phase and G₂/M phase also decreased, while G0/G1 phase increased in cell cycle. CONCLUSION The increased expression of NCL in DLBCL and CA46 cells indicates low sensitivity to drug. NCL may participate in regulation of lymphoma proliferation by affecting TK1 expression, thereby affecting the drug sensitivity.
Humans ; Leukocytes, Mononuclear/metabolism* ; Apoptosis ; Cell Line, Tumor ; Lymphoma ; Thymidine Kinase/pharmacology* ; Doxorubicin/pharmacology* ; Cell Division ; RNA, Messenger/genetics*