RESUMO
UNLABELLED: We report the first, to our knowledge, findings describing the relationships between both static and dynamic analysis parameters of 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) PET and the expression of the proliferation marker Ki-67, and the protein expression and enzymatic activity of thymidine kinase-1 (TK1) in surgically resected lung lesions. METHODS: Static and dynamic analyses (4 rate constants and 2 compartments) of (18)F-FLT PET images were performed in a cohort of 25 prospectively accrued, clinically suspected lung cancer patients before surgical resection (1 lesion was found to be benign after surgery). The maximal and overall averaged expression of Ki-67 and TK1 were determined by semiquantitative analysis of immunohistochemical staining. TK1 enzymatic activity was determined by in vitro assay of extracts prepared from flash-frozen samples of the same tumors. RESULTS: Static (18)F-FLT uptake (partial-volume-corrected maximum-pixel standardized uptake value from 60- to 90-min summed dynamic data) was significantly correlated with the overall (ρ = 0.57, P = 0.006) and maximal (ρ = 0.69, P < 0.001) immunohistochemical expressions of Ki-67 and TK1 (overall expression: ρ = 0.65, P = 0.001; maximal expression: ρ = 0.68, P < 0.001) but not with TK1 enzymatic activity (ρ = 0.34, P = 0.146). TK1 activity was significantly correlated with TK1 protein expression only when immunohistochemistry was scored for maximal expression (ρ = 0.52, P = 0.029). Dynamic analysis of (18)F-FLT PET revealed correlations between the flux constant (K(FLT)) and both overall (ρ = 0.53, P = 0.014) and maximal (ρ = 0.50, P = 0.020) TK1 protein expression. K(FLT) was also associated with both overall (ρ = 0.59, P = 0.005) and maximal (ρ = 0.63, P = 0.002) Ki-67 expression. We observed no significant correlations between TK1 enzyme activity and K(FLT). In addition, no significant relationships were found between TK1 expression, TK1 activity, or Ki-67 expression and any of the compartmental rate constants. CONCLUSION: The absence of observable correlations of the imaging parameters with TK1 activity suggests that (18)F-FLT uptake and retention within cells may be complicated by a variety of still undetermined factors in addition to TK1 enzymatic activity.
Assuntos
Fluordesoxiglucose F18/farmacologia , Neoplasias Pulmonares/diagnóstico por imagem , Tomografia por Emissão de Pósitrons/métodos , Timidina Quinase/biossíntese , Ciclo Celular , Proliferação de Células , Estudos de Coortes , Feminino , Humanos , Imuno-Histoquímica/métodos , Antígeno Ki-67/biossíntese , Cinética , Neoplasias Pulmonares/patologia , Masculino , Prognóstico , Fatores de TempoRESUMO
An investigation was conducted to determine if the 5-fluoro analog of TPI (5-chloro-6-[(2-iminopyrrolidin-1-yl)methyl]uracil), a potent inhibitor of human thymidine phosphorylase (TP), has an IC(50) in a range that might allow to use it labeled for imaging of TP expression in vivo. The previously unreported fluoro analog, TPI(F), was prepared and tested against TPI and TPI(Br) using an inhibition assay of [H-3]thymidine cleavage. An assay, performed in the presence of 0.4 mg/ml of human TP, yielded IC(50) values of 2.5 nM, 2.7 nM, and 9.0 nM for TPI, TPI(Br), and TPI(F), respectively. The results indicate that further studies to develop (18)F-labeled TPI(F) as a potential radiopharmaceutical for PET imaging of TP expression in vivo are warranted.
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Pirrolidinas/síntese química , Pirrolidinas/farmacologia , Timidina Fosforilase/antagonistas & inibidores , Uracila/análogos & derivados , Uracila/síntese química , Uracila/farmacologia , Catálise/efeitos dos fármacos , Inibidores Enzimáticos/química , Humanos , Concentração Inibidora 50 , Pirrolidinas/química , Timidina/química , Timidina/metabolismo , Uracila/químicaRESUMO
BACKGROUND: We previously identified a number of genes which were methylated significantly more frequently in the tumor compared to the non-cancerous lung tissues from non-small cell lung cancer (NSCLC) patients. Detection of methylation profiles of genes in NSCLC could provide insight into differential pathways to malignancy and lead to strategies for better treatment of individuals with NSCLC. METHODS: We determined the DNA methylation status of 27 genes using quantitative MethyLight assays in lung tumor samples from 117 clinically well-characterized NSCLC patients. RESULTS: Hypermethylation was detected in one of more of the genes in 106 (91%) of 117 cases and was detected at high levels (percentage methylation reference (PMR)> or =4%) in 79% of NSCLC cases. Methylation of APC, CCND2, KCNH5 and, RUNX was significantly more frequent in adenocarcinomas compared to squamous cell carcinomas (SCC), while methylation of CDKN2A was more common in SCC. Hypermethylation of KCNH5, KCNH8, and RARB was more frequent in females compared to males. Hypermethylation of APC and CCND2 was inversely associated with proliferation score assessed by Ki-67 level. CONCLUSIONS: Our findings of differential gene hypermethylation frequencies in tumor tissues from patients with adenocarcinoma or squamous cell cancers and in females compared to males suggests that further investigation is warranted in order to more fully understand the potential disparate pathways and/or risk factors for NSCLC associated with histologic type and gender.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Metilação de DNA , DNA/análise , Neoplasias Pulmonares/genética , Fatores Sexuais , Idoso , Biópsia , Carcinoma Pulmonar de Células não Pequenas/epidemiologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/fisiopatologia , Diferenciação Celular/genética , Proliferação de Células , Ciclina D2/genética , Feminino , Genes APC , Humanos , Neoplasias Pulmonares/epidemiologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/fisiopatologia , Masculino , Pessoa de Meia-Idade , Receptores do Ácido Retinoico/genética , Fatores de Risco , FumarRESUMO
The thymidine salvage pathway enzymes thymidine kinase 1 (TK1) and thymidine phosphorylase (TP) compete for thymidine as a substrate and catalyze opposing synthetic and catabolic reactions that have been implicated in the control of proliferation and angiogenesis, respectively. We investigated the relationship between the expression of TK1 and TP as they relate to proliferation (Ki-67 labeling index) and angiogenesis (Chalkley count of CD31-stained blood vessels) in a series of 110 non-small-cell lung cancer (NSCLC) tumors from patients prospectively enrolled in an imaging trial. TK1 and TP exhibited similar patterns of immunohistochemical distribution, in that each was found in both the nucleus and the cytoplasm of tumor cells. Each enzyme exhibited a significant positive correlation between its levels of nuclear and cytoplasmic expression. A significant positive correlation between TK1 expression and the Ki-67 labeling index (r = 0.53, p<0.001) was observed. TP was significantly positively correlated with Chalkley scoring of CD31 staining in high vs low Chalkley scoring samples (mean TP staining of 115.8 vs 79.9 scoring units, p<0.001), respectively. We did not observe a substantial inverse correlation between the TP and TK1 expression levels in the nuclear compartment (r = -0.17, p=0.08). Tumor size was not found to be associated with TK1, TP, Ki-67, or Chalkley score. These findings provide additional evidence for the role of thymidine metabolism in the complex interaction of proliferation and angiogenesis in NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/irrigação sanguínea , Carcinoma Pulmonar de Células não Pequenas/patologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/patologia , Timidina Quinase/metabolismo , Timidina Fosforilase/metabolismo , Idoso , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Núcleo Celular/enzimologia , Proliferação de Células , Ensaios Clínicos como Assunto , Citoplasma/enzimologia , Feminino , Humanos , Imuno-Histoquímica , Antígeno Ki-67/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Pessoa de Meia-Idade , Neovascularização Patológica , Coloração e Rotulagem , Carga TumoralRESUMO
INTRODUCTION: The activity of the pyrimidine salvage pathway enzyme thymidine kinase 1 (TK1) is tightly cell cycle regulated and has been investigated as a prognostic indicator of cancer in a variety of tissues. However, using the in vitro assay of TK1 to rank order a series of unique tumor samples by their TK1 activity can be problematic due to the complex nature of TK1 enzyme substrate kinetics. We present a refined TK1 in vitro assay and method of analysis which address these problems. METHODS: Extracts were prepared of the resected lung lesions from eight patients and assayed for TK1 activity using an in vitro assay modified to account for nonlinearities in extract protein concentration. A separate extract of exponentially growing A549 human lung carcinoma cells was used as a cross-assay control. RESULTS: In extracts prepared from eight frozen samples of resected human lung lesions, TK1 activity (mean=0.0070+/-0.0077 pmol [(3)H]-TMP/microg protein/minute) was 2 orders of magnitude below that of exponentially growing A549 human lung carcinoma cells (mean=0.1572+/-0.0218 pmol [(3)H]-TMP/microg protein/minute; n=9). TK1 activity was nonlinear with respect to extract protein concentration in both groups, with A549 cell extracts exhibiting evidence of positive cooperativity which could not be explained by the presence of detergents in the cell lysis buffer. Lung tumor extracts demonstrated evidence of negative cooperativity. CONCLUSIONS: The modified TK1 assay takes into account these nonlinearities by averaging the results of several complete time-course curves measured over a range of extract protein concentrations. An extract prepared from exponentially growing A549 cells is included in each assay for use as a cross-assay control. We demonstrate that these modifications allow for the accurate rank ordering of TK1 activity in solid tumors.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/enzimologia , Neoplasias Pulmonares/enzimologia , Timidina Quinase/análise , Células Cultivadas , Humanos , Proteínas de Neoplasias/metabolismo , Compostos Radiofarmacêuticos , TimidinaRESUMO
Two human cell lines (A549 and U937) with cytosolic thymidine phosphorylase (TP) activity were used to evaluate the potential of 5'-deoxy-5'-[F-18]fluorothymidine ([F-18]DFT) as a tracer of intracellular TP expression. Cellular metabolism of DFT led to the production of 5-[F-18]fluoro-2,5-dideoxy-D-ribose-1alpha-phosphate ([F-18]FddR-1P), in analogy to the metabolism of thymidine, which produces 2-deoxy-D-ribose-1alpha-phosphate (dR-1P). A549 cells showed the highest production rate of FddR-1P. After A549 cells were exposed to [F-18]DFT for 40 min, the relative intracellular concentration of [F-18]FddR-1P was more than sevenfold higher in cells than its precursor in the incubating medium. For the same amount of time, a twofold concentration was seen in U937 cells. However, uptake ratios did not rank with the corresponding TP activities found in cell extracts [TP activity ratio (U937:A549)=1.6] that were independently determined with a labeled thymidine/thymine cleavage assay. The discrepancy of TP activity ratios was traced to the instability of FddR-1P in cells. This was evident from the fact that cells accumulated radioactivity by producing FddR-1P, but activity also effluxed from cells over 1 h when the medium was subsequently made tracer free. The dominant labeled molecule released by cells was characterized as a neutral and lipophilic molecule, which was presumed to be a deoxynucleoside. Our results indicate that [F-18]DFT would not be effective for imaging TP expression because its initial metabolite undergoes further conversion to a diffusible secondary metabolite, allowing activity loss from cells.
Assuntos
Didesoxinucleosídeos/farmacocinética , Neoplasias/diagnóstico por imagem , Neoplasias/metabolismo , Timidina Fosforilase/metabolismo , Linhagem Celular Tumoral , Didesoxinucleosídeos/química , Ativação Enzimática , Humanos , Taxa de Depuração Metabólica , Cintilografia , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Células U937RESUMO
UNLABELLED: The thymidine analog (18)F-3'-deoxy-3' -fluorothymidine (FLT) is being used clinically for PET imaging of tumor proliferation. Appropriate use of this tracer requires validating the mechanisms by which it accumulates in dividing cells. We tested the accuracy with which FLT uptake predicted the activity of cytosolic thymidine kinase-1 (TK(1)), an enzyme that is upregulated before and during DNA synthesis. METHODS: Cultured A549 human lung carcinoma cells were manipulated to a range of proliferation rates from actively dividing to growth arrested. Uptake of radiolabeled FLT was compared with cell cycle activity, which was expressed as the percentage of cells in S phase, and with activity of cytosolic TK(1). We also compared uptake of FLT and deoxyglucose. We genetically manipulated A549 cells by transfecting them with human papillomavirus type 16 E6 (designated A549-E6) to abrogate function of the tumor suppressor gene, p53. Although radiation typically inhibits progression of mammalian cells through the cell cycle, abrogation of p53 function eliminates this inhibition. We then compared FLT uptake with the percentage of cells in S phase and TK(1) activity in irradiated A549-E6 cells and in irradiated control cells having normal p53 function and the expected radiation-induced growth delay. RESULTS: A549 cells with only 3%-5% cells in S phase took up little FLT and had low levels of TK(1) activity. When cells were stimulated to grow by being placed into fresh medium, we observed a strong correlation between increased FLT uptake and increased TK(1) activity. As expected, FLT uptake varied much more as a function of growth than did uptake of deoxyglucose. Nonproliferating A549 cells did not enter the cell cycle if they were irradiated before being placed into fresh medium, and they did not accumulate FLT or show elevated TK(1) activity. In contrast, radiation did not inhibit the cell cycle progression of A549-E6 cells. When subcultured, they began to grow and showed increased uptake of FLT commensurate with greater TK(1) activity. CONCLUSION: In cultured A549 cells FLT uptake is positively correlated with cell growth and TK(1) activity. Inhibition of cell cycle progression prevents FLT uptake and increased TK(1) activity. These results suggest that FLT images reflect TK(1) activity and the percentage of cells in S phase.