Thymidine Kinase 1 mRNA Research Articles

DNA nanosensor based on bipedal 3D DNA walker-driven proximal catalytic hairpin assembly for sensitive and fast TK1 mRNA detection.

Hyperproliferative diseases are the first step for tumor formation; thymidine kinase 1 (TK1) mRNA is closely related to cell proliferation. Therefore, the risk of malignant proliferation can be identified by sensitively detecting the variance in TK1 mRNA concentration, which can be used for tumor auxiliary diagnosis and monitoring tumor treatment. Owing to the low abundance and instability of TK1 mRNA in real samples, the development of a sensitive and fast mRNA detection method is necessary. A DNA nanosensor that can be used for detecting TK1 mRNA based on bipedal 3D DNA walker-driven proximal catalytic hairpin assembly (P-CHA) was developed. P-CHA hairpins were hybridized to a linker DNA strand coupled with magnetic nanoparticles to increase their local concentrations. The bipedal DNA walking on the surface of NPs accelerates reaction kinetics using the proximity effect. Taking advantage of the signal amplification of P-CHA as well as the rapid reaction rate of the DNA walker in 80min, the proposed sensor detects TK1 mRNA with a low detection limit of 14pM and may then be applied toclinical diagnosis.

Strand displacement-triggered FRET nanoprobe tracking TK1 mRNA in living cells for ratiometric fluorimetry of nucleic acid biomarker.

Aprecisely designed dual-color biosensor has realized a visual assessment of thymidine kinase 1 (TK1) mRNA in both living cells and cell lysates. The oligonucleotide probe is constructed by hybridizing the antisense strand of the target and two recognition sequences, in which FAM serves as the donor and TAMRA as the acceptor. Once interacting with the target, two recognition strands are replaced, and then the antisense complementary sequence forms a more stable double-stranded structure. Due to the increasing spatial distance between two dyes, the FRET is attenuated, leading to a rapid recovery of FAM fluorescence and a reduction of TAMRA fluorescence. A discernible color response from orange to green could be observed by the naked eye, with a limit of detection (LOD) of 0.38 nM and 5.22 nM for spectrometer- and smartphone-based assays, respectively. The proposed ratiometric method transcends previous reports in its capacities in visualizing TK1 expression toward reliable nucleic acid biomarker analysis, which might establish a general strategy for ratiometric biosensing via strand displacement.

Construction of an autofluorescence interference-free phosphorescence biosensor for the specific detection of TK1 mRNA

The anti-interference ability of biosensors is critical for detection in biological samples. Fluorescence-based sensors are subject to interference from self-luminescent substances in biological matrices. Therefore, phosphorescent sensors stand out among biosensors due to their lack of self-luminescence background. In this study, a phosphorescent sensor was constructed, which can accurately detect thymidine kinase 1 (TK1) mRNA in biological samples and avoid autofluorescence interference. When there is no target, polydopamine (PDA) is used as the phosphorescence resonance energy transfer (PRET) acceptor to quench the phosphorescence of the persistently luminescent (PL) nanomaterial. When there is a target, the DNA modified by the PL nanomaterial is replaced by the hairpin H and removed away from the PDA, resulting in a rebound in phosphorescence. The phosphorescent sensor exhibits a good linear relationship in the TK1 mRNA concentration range of 0–200 nM, and the detection limit was 1.74 nM. The sensor fabricated in this study can effectively avoid interference from spontaneous fluorescence in complex biological samples, and sensitively and precisely detect TK1 mRNA in serum samples, providing a powerful tool to more accurately detect biomarkers in biological samples.

Self-assembly of DNA-gold nanoaggregate for visual detection of thymidine kinase 1 (TK1) mRNA via lateral flow assay.

Nucleic acid lateral flow assay (NALFA) with gold nanoparticles (AuNPs) as colorimetric probes have been extensively adopted for point-of-care testing (POCT). However, the sensitivity of NALFA still needs to be improved. Herein, DNA-gold nanoaggregate (DNA-AuNA) was assembled as a signal amplification probe of NALFA for sensitive detection of tumor marker TK1 mRNA. Four functional oligonucleotides with complementary pairs were assembled to form DNA-AuNA that coupled more AuNPs to improve sensitivity. Thus, the limit of detection (LOD) was 0.36pM, which is lower than that of conventional AuNPs-based NALFA. Moreover, the bioassay showed good reproducibility, stability, and specificity for detecting TK1 mRNA. The detection of TK1 mRNA in human serum was also satisfactory. Therefore, DNA-AuNA-based NALFA provides a sensitive method for portable detection of TK1 mRNA.

M5C-dependent cross-regulation between nuclear reader ALYREF and writer NSUN2 promotes urothelial bladder cancer malignancy through facilitating RABL6/TK1 mRNAs splicing and stabilization

The significance of 5-methylcytosine (m5C) methylation in human malignancies has become an increasing focus of investigation. Here, we show that m5C regulators including writers, readers and erasers, are predominantly upregulated in urothelial carcinoma of the bladder (UCB) derived from Sun Yat-sen University Cancer Center and The Cancer Genome Atlas cohort. In addition, NOP2/Sun RNA methyltransferase family member 2 (NSUN2) as a methyltransferase and Aly/REF export factor (ALYREF) as a nuclear m5C reader, are frequently coexpressed in UCB. By applying patient-derived organoids model and orthotopic xenograft mice model, we demonstrate that ALYREF enhances proliferation and invasion of UCB cells in an m5C-dependent manner. Integration of tanscriptome-wide RNA bisulphite sequencing (BisSeq), RNA-sequencing (RNA-seq) and RNA Immunoprecipitation (RIP)-seq analysis revealed that ALYREF specifically binds to hypermethylated m5C site in RAB, member RAS oncogene family like 6 (RABL6) and thymidine kinase 1 (TK1) mRNA via its K171 domain. ALYREF controls UCB malignancies through promoting hypermethylated RABL6 and TK1 mRNA for splicing and stabilization. Moreover, ALYREF recognizes hypermethylated m5C site of NSUN2, resulting in NSUN2 upregulation in UCB. Clinically, the patients with high coexpression of ALYREF/RABL6/TK1 axis had the poorest overall survival. Our study unveils an m5C dependent cross-regulation between nuclear reader ALYREF and m5C writer NSUN2 in activation of hypermethylated m5C oncogenic RNA through promoting splicing and maintaining stabilization, consequently leading to tumor progression, which provides profound insights into therapeutic strategy for UCB.

Boron Carbon Nitride Nanosheets-Ru Nanocomposite Self-Enhancement Electrochemiluminescence Emitter with a Three-Dimensional DNA Network Structure as a Signal Amplifier for Ultrasensitive Detection of TK1 mRNA.

In this study, a neoteric self-enhanced nanocomposite boron carbon nitride nanosheets (BCN NSs)-Ru obtained by chemical crosslinking between boron carbon nitride nanosheets (BCN NSs) and tris (4,4'-dicarboxylicacid-2,2'-bipyridyl) ruthenium(II) dichloride (Ru(dcbpy)3Cl2) was used as an emitter to build an electrochemiluminescence (ECL) biosensor for ultrasensitive detection of the cancer marker human thymidine kinase 1 (TK1) mRNA. Importantly, the self-enhanced BCN NSs-Ru could exhibit strong ECL emission because boron radicals and amine groups derived from BCN NSs could significantly enhance the ECL response of Ru(dcbpy)3Cl2, which avoided the defects of the long electron transfer path and large energy loss between the emitter and coreactant in the traditional coreaction ECL system. Impressively, in the presence of target TK1 mRNA, three-dimensional DNA network structure-labeled numerous ferrocene probes could be assembled to quickly quench the ECL signal of BCN NSs-Ru, resulting in improved biosensor sensitivity. The obtained "on-off" biosensor showed excellent stability and high sensitivity with a detection limit of 32.3 aM. In general, the developed strategy provided a new biosensing way for ultrasensitive detection of biomolecules in early disease diagnosis.

Thymidine Kinase 1 Mediates the Synergistic Antitumor Activity of Ubenimex and Celecoxib via Regulation of Cell Cycle in Colorectal Cancer.

Colorectal cancer (CRC) is a common clinical malignant tumor of the digestive system that seriously affects the health and life of patients. Because it is difficult to cure CRC, the strategy of drug combination is often used in clinical therapy. This study mainly revealed that ubenimex and/or celecoxib exerted anti-colon cancer effects in vitro and in vivo, and the efficacy was significantly enhanced when the two drugs were combined. The combination of the two drugs induced significantly stronger cell-cycle arrest than did the single drug, and also enhanced the antitumor efficacy of 5-fluorouracil and its derivatives. At the same time, the expression of thymidine kinase 1 (TK1) protein was decreased through regulating the level of TK1 mRNA treated with celecoxib and/or ubenimex, but the combination drugs exhibited much more reduction of TK1 mRNA and protein as compared with the single agent alone. TK1 may be the molecular target of the combination of two drugs to exert the anti-colorectal cancer effect. In summary, this research demonstrates that celecoxib combined with ubenimex inhibits the development of colorectal cancer in vitro and in vivo, making them a viable combination regimen. SIGNIFICANCE STATEMENT: In this study, our data reveal the great potential of celecoxib combined with ubenimex in the treatment of colorectal cancer, providing new ideas for clinical antitumor drug regimens and theoretical reference for drug development.

Abstract 1322: The potential effects of thymidine kinase 1 on breast cancer invasion

Abstract Breast cancer is the most common cancer in women and is largely treatable within the early stages of the disease. However, patient mortality drastically declines as the tumor begins to invade other tissues and metastasize, making aggressive phenotypes especially problematic to treat. Such treatment typically requires an aggressive and decisive multidisciplinary approach. The recent expansion of immunotherapy as a viable treatment option has greatly improved treatment outcomes, especially with aggressive breast cancer phenotypes. Thymidine kinase 1 (TK1) is a DNA salvage pathway enzyme that is highly expressed during S phase and involved in cell cycle repair. Past studies indicated that TK1 is secreted into the serum of cancer patients and that its upregulation is an early event in tumorigenesis, thus suggesting that TK1 upregulation may have potential importance in tumor progression. This study’s objective was to further elucidate TK1’s role in tumor progression and metastasis of breast cancer specifically. We hypothesized that TK1 overexpression in breast cancer may affect invasion. To investigate this, TK1 mRNA transcript levels were analyzed between normal and BRCA patient tissue samples. The wild-type breast cancer cell line HCC 1806 was used, as well as a TK1 knockdown (L133) which was produced using CRISPR-Cas9. Successful TK1 knockdown was verified through western blot and qPCR. Bioinformatics was also performed to analyze the relationship between our breast cancer cell lines and various cell adhesion factors. The cell lines HCC 1806 and L133 were then tested with an invasive scratch assay. Additional western blots were run to further investigate the potential relationship TK1 may have with other cell cycle checkpoint proteins. Results showed that TK1 mRNA transcript levels are higher in BRCA tissues compared to normal controls. Western blot and qPCR indicated that TK1 was successfully knocked down in L133 cells. Bioinformatic differential gene analysis revealed several correlations with cell adhesion factors, with contrasting correlations when compared between the HCC 1806 and L133 cell lines. The invasive scratch assay results indicated that the TK1 knockdown L133 cells were less invasive than HCC 1806 cells. In conclusion, results verified that TK1 is highly expressed in breast cancer and that it may help tumor invasion by influencing cell adhesion factors. Further investigation to understand TK1’s potential interactions with important cell adhesion factors, such as FHL1 or GAS6, may help elucidate other therapeutic targets for preventing disease progression. Citation Format: Eliza E. Bitter, Rachel M. Morris, Toni Mortimer, Kai Barlow, Abigail Schekall, Michelle H. Townsend, Jonathan Skidmore, Brett E. Pickett, Kim L. O'Neill. The potential effects of thymidine kinase 1 on breast cancer invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1322.

Overexpression of TK1 and CDK9 in plasma-derived exosomes is associated with clinical resistance to CDK4/6 inhibitors in metastatic breast cancer patients.

Cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) improve progression-free survival (PFS) in patients with hormone receptor-positive (HR+) advanced breast cancer. However, a better knowledge of predictive biomarkers of response and resistance to CDK4/6i is needed. Therefore, the present article addresses the role of the mRNA expression of thymidine kinase 1 (TK1), CDK4, 6 and 9 in plasma-derived exosomes and their relevance in the pharmacologic activity of CDK4/6i. Blood samples of 40 HR+/HER2- advanced breast cancer patients were collected before (T0) the administration of palbociclib plus hormonal therapy and after 3 months (T1). RNA was isolated from exosomes and analysed for the expression of TK1, CDK 4, 6 and 9 by digital droplet PCR (ddPCR). A higher value of TK1 copies/ml at baseline (T0) was significantly associated with the number of previous lines of chemotherapy (p = 0.009). In patients with PD, a significant increase was observed in the number of copies/ml of TK1 (p = 0.01) and CDK9 (p = 0.03) comparing T1 vs. T0 values. No significant correlations between response to treatment and clinical parameters were found at univariate analysis. High baseline CDK4 expression was significantly correlated with longer PFS in patients treated with fulvestrant + palbociclib (low versus high: 6.45months vs. not reached, p = 0.01). The present study demonstrates that, in plasma-derived exosomes, highbaseline CDK4 mRNA levels are associated with response to palbociclib plus hormonal therapy, while the increase in TK1 and CDK9 mRNA copies/ml is associated with clinical resistance.

Y-shaped DNA-Mediated hybrid nanoflowers as efficient gene carriers for fluorescence imaging of tumor-related mRNA in living cells

Organic-inorganic hybrid nanomaterial has gained much attention due to its excellent performances in bioanalysis and biomedicine. However, the preparation of DNA-inorganic hybrid nanomaterial with suitable size for cell uptake remains a huge challenge. Herein, a moderate biomineralization strategy for synthesis of Y-DNA@Cu3(PO4)2 (Y-DNA@CuP) hybrid nanoflowers is reported. Y-DNA with a loop structure is used as both the biomineralization template and the recognition unit for thymidine kinase 1 (TK1) mRNA. The Y-DNA probe can linearly response to TK1 mRNA target sequence in a range from 2 nM to 150 nM with the limit of detection as low as 0.56 nM. Interestingly, the presence of Y-DNA significantly decreases the size of Cu3(PO4)2 (CuP) particles, which allows them suitable for intracellular applications as gene nanocarriers. Once inside the cells, the hybrid nanoflowers dissolve and release the Y-DNA probes. Then, the intracellular TK1 mRNA hybridizes with the loop region of Y-DNA, which dissociates the Cy3-labeled loop strand and turns on the red fluorescence. Through the real-time imaging of the intracellular TK1 mRNA, the assessment of tumor cells before and after the treatment of drugs including β-estradiol and tamoxifen is achieved.

Low Background Cascade Signal Amplification Electrochemical Sensing Platform for Tumor-Related mRNA Quantification by Target-Activated Hybridization Chain Reaction and Electroactive Cargo Release.

Herein a low background cascade signal amplification electrochemical sensing platform has been proposed for the ultrasensitive detection of mRNA (mRNA) by coupling the target-activated hybridization chain reaction and electroactive cargo release from mesoporous silica nanocontainers (MSNs). In this sensing platform, the 5'-phosphate-terminated DNA (5'-PO4 cDNA) complement to target mRNA is hybridized with the trigger DNA and anchor DNA on the surface of the MSNs, aiming at forming a double-stranded DNA gate molecule and sealing the methylene blue (MB) in the inner pores of the MSNs. In the presence of target mRNA, the 5'-PO4 cDNA is displaced from the MSNs and competitively hybridizes with mRNA, which led to the liberation of the trigger DNA and the opening of the MSNs pore. The liberated trigger DNA can be then immobilized onto the electrode surface through hybridization with the capture DNA, triggering HCR on the electrode surface. At the same time, the MB released from the MSNs will selectively intercalate into the HCR long dsDNA polymers, giving rise to significant electrochemical response. In addition, due to the λ-exonuclease (λ-Exo) cleavage reaction-assisted target recycling, more amounts of trigger DNA will be liberated and trigger HCR, and numerous MB are uncapped and intercalate into the HCR products. As proof of concept, thymidine kinase 1 (TK1) mRNA was used as a model target. Featured with amplification efficiency, label-free capability, and low background signal, the strategy could quantitatively detect TK1 mRNA down to 2.0 aM with a linear calibration range from 0.1 fM to 1 pM. We have also demonstrated the practical application of our proposed sensing platform for detecting TK1 mRNA in real samples, opening up new avenues for highly sensitive quantification of biomarkers in bioanalysis and clinical diagnosis.

Spiny Nanorod and Upconversion Nanoparticle Satellite Assemblies for Ultrasensitive Detection of Messenger RNA in Living Cells

Quantitation and in situ monitoring of target mRNA (mRNA) in living cells remains a significant challenge for the chemical and biomedical communities. To quantitatively detect mRNA expression levels in living cells, we have developed DNA-driven gold nanorod coated platinum-upconversion nanoparticle satellite assemblies (termed Au NR@Pt-UCNP satellites) for intracellular thymidine kinase 1 (TK1) mRNA analysis. The nanostructures were capable of recognizing target mRNA in a sequence-specific manner as luminescence of UCNPs was effectively quenched by Au NR@Pt within the assemblies. Following recognition, UCNPs detached from Au NR@Pt, resulting in luminescence restoration to achieve effective in situ imaging and quantifiable detection of target mRNA. The upconversional luminescence intensity of confocal images showed a good linear relationship with intracellular TK1 mRNA ranging from 1.17 to 65.21 fmol/10 μg RNA and a limit of detection (LOD) of 0.67 fmol/10 μg RNA. We believe that our present assay can be broadly applied for detection of endogenous biomolecules at the cellular and tissue levels and restoration of tissue homeostasis in vivo.

Identification of Fhit as a post-transcriptional effector of Thymidine Kinase 1 expression

FHIT is a genome caretaker gene that is silenced in >50% of cancers. Loss of Fhit protein expression promotes accumulation of DNA damage, affects apoptosis and epithelial-mesenchymal transition, though molecular mechanisms underlying these alterations have not been fully elucidated. Initiation of genome instability directly follows Fhit loss and the associated reduced Thymidine Kinase 1 (TK1) protein expression. The effects on TK1 of Fhit knockdown and Fhit induction in the current study confirmed the role of Fhit in regulating TK1 expression. Changes in Fhit expression did not impact TK1 protein turnover or transcription from the TK1 promoter, nor steady-state levels of TK1 mRNA or turnover. Polysome profile analysis showed that up-regulated Fhit expression resulted in decreased TK1 RNA in non-translating messenger ribonucleoproteins and increased ribosome density on TK1 mRNA. Fhit does not bind RNA but its expression increased luciferase expression from a transgene bearing the TK1 5′-UTR. Fhit has been reported to act as a scavenger decapping enzyme, and a similar result with a mutant (H96) that binds but does not cleave nucleoside 5′,5′-triphosphates suggests the impact on TK1 translation is due to its ability to modulate the intracellular level of cap-like molecules. Consistent with this, cells expressing Fhit mutants with reduced activity toward cap-like dinucleotides exhibit DNA damage resulting from TK1 deficiency, whereas cells expressing wild-type Fhit or the H96N mutant do not. The results have implications for the mechanism by which Fhit regulates TK1 mRNA, and more broadly, for its modulation of multiple functions as tumor suppressor/genome caretaker.

Theranostic Upconversion Nanobeacons for Tumor mRNA Ratiometric Fluorescence Detection and Imaging-Monitored Drug Delivery.

Remote optical detection and imaging of specific tumor-related biomarkers and simultaneous activation of therapy according to the expression level of the biomarkers in tumor site with theranostic probes should be an effective modality for treatment of cancers. Herein, an upconversion nanobeacon (UCNPs-MB/Dox) is proposed as a new theranostic nanoprobe to ratiometrically detect and visualize the thymidine kinase 1 (TK1) mRNA that can simultaneously trigger the Dox release to activate the chemotherapy accordingly. UCNPs-MB/Dox is constructed with the conjugation of a TK1 mRNA-specific molecular beacon (MB) bearing a quencher (BHQ-1) and an alkene handle modified upconversion nanoparticle (UCNP) through click reaction and subsequently loading with a chemotherapy drug (Dox). With this nanobeacon, quantitative ratiometric upconversion detection of the target with high sensitivity and selectivity as well as the target triggered Dox release in vitro is demonstrated. The sensitive and selective ratiometric detection and imaging of TK1 mRNA under the irradiation of near infrared light (980 nm) and the mRNA-dependent release of Dox for chemotherapy in the tumor MCF-7 cells and A549 cells are also shown. This work provides a smart and robust platform for gene-related tumor theranostics.

Effects of CO₂ pneumoperitoneum on the expression of thymidine kinase 1 and Ki67 in colorectal carcinoma cells.

To investigate thymidine kinase 1(TK-1) and Ki67 expression levels of human colorectal carcinoma cells line SW480 after exposure to a simulated laparoscopic carbon dioxide (CO2) pneumoperitoneum environment at different pressures and lengths of exposure time. The effects of the simulated laparoscopic CO2 pneumoperitoneum environment at different CO2 pressures (6, 9, 12, and 15 mmHg) and exposure times (2 and 4 h) on TK-1 and Ki67 of SW480 cells were assessed by flow cytometry and reverse transcription (RT-PCR). Cells cultured in a standard environment were used as the control group (at 37 °C, 5% CO2). In this study, TK-1 and Ki67 in SW480 cells tended to decrease with the increase of CO2 pressure and exposure time. Significantly lower expression levels were observed at 0 and 24 h of culture after exposure to both at 12 and 15 mmHg, as compared with the control group at 6 and 9 mmHg (p < 0.05). The expression of TK-1 and Ki67 levels increased up to a plateau of the control group after 48 and 72 h (p > 0.05). With the CO2 pneumoperitoneum exposure time prolonging, the expression of TK-1 and Ki67 levels in 12 or 15 mmHg was lower than in 2 h (p < 0.05). In the same exposure time, the transcription level of TK-1 and Ki67 decreased significantly in 12 and 15 mmHg CO2 pneumoperitoneum groups (p < 0.05) and returned to the basal level of control group after being cultivated for 48 h (p > 0.05). In the same pressure, the difference of TK-1 mRNA between the groups of 2 and 4 h was also significant. The expression levels of TK-1 and Ki67 were suppressed temporarily after the continuous CO2 insufflation in higher pressure (at 12 and 15 mmHg). The higher the pressure of CO2 insufflation, the more the inhibiting effects of TK-1 and Ki67 will be. The longer the time of CO2 insufflation, the more significantly their expression decreased.

Thymidine kinase and mtDNA depletion in human cardiomyopathy: epigenetic and translational evidence for energy starvation

This study addresses how depletion of human cardiac left ventricle (LV) mitochondrial DNA (mtDNA) and epigenetic nuclear DNA methylation promote cardiac dysfunction in human dilated cardiomyopathy (DCM) through regulation of pyrimidine nucleotide kinases. Samples of DCM LV and right ventricle (n = 18) were obtained fresh at heart transplant surgery. Parallel samples from nonfailing (NF) controls (n = 12) were from donor hearts found unsuitable for clinical use. We analyzed abundance of mtDNA and nuclear DNA (nDNA) using qPCR. LV mtDNA was depleted in DCM (50%, P < 0.05 each) compared with NF. No detectable change in RV mtDNA abundance occurred. DNA methylation and gene expression were determined using microarray analysis (GEO accession number: GSE43435). Fifty-seven gene promoters exhibited DNA hypermethylation or hypomethylation in DCM LVs. Among those, cytosolic thymidine kinase 1 (TK1) was hypermethylated. Expression arrays revealed decreased abundance of the TK1 mRNA transcript with no change in transcripts for other relevant thymidine metabolism enzymes. Quantitative immunoblots confirmed decreased TK1 polypeptide steady state abundance. TK1 activity remained unchanged in DCM samples while mitochondrial thymidine kinase (TK2) activity was significantly reduced. Compensatory TK activity was found in cardiac myocytes in the DCM LV. Diminished TK2 activity is mechanistically important to reduced mtDNA abundance and identified in DCM LV samples here. Epigenetic and genetic changes result in changes in mtDNA and in nucleotide substrates for mtDNA replication and underpin energy starvation in DCM.

Evaluation of 3′-deoxy-3′-[18F]-fluorothymidine (18F-FLT) kinetics correlated with thymidine kinase-1 expression and cell proliferation in newly diagnosed gliomas

The thymidine analog 3'-deoxy-3'-[(18)F]fluorothymidine ((18)F-FLT) has been developed as a positron emission tomography (PET) tracer to assess the proliferation activity of tumors in vivo. The present study investigated the relationship between the kinetic parameters of (18)F-FLT in vivo and thymidine kinase-1 (TK-1) expression and cell proliferation rate in vitro, and blood-brain barrier (BBB) breakdown in human brain gliomas. A total of 21 patients with newly diagnosed gliomas were examined by (18)F-FLT PET kinetic analysis. Maximum standardized uptake value (SUVmax) and tumor-to-normal (T/N) ratio of (18)F-FLT in the tumor and (18)F-FLT kinetic parameters in the corresponding contralateral region were determined. The expression levels of TK-1 protein and mRNA were determined by immunohistochemistry (IHC) and real-time polymerase chain reaction (PCR), respectively, using surgical specimens. The cell proliferation rate of the tumor was determined in terms of the Ki-67 labeling index. BBB breakdown was evaluated on MR images with contrast enhancement. (18)F-FLT SUVmax and T/N ratio were significantly correlated with the influx rate constant (K (1); P = 0.001 and P < 0.001, respectively), but not with the phosphorylation rate constant (k (3)). IHC and real-time PCR studies demonstrated a significant correlation between K (1) and TK-1 mRNA expression (P = 0.001), but not between k (3) and TK-1 protein and mRNA expression. Linear regression analysis revealed a significant correlation between K (1) and the Ki-67 index (P = 0.003), but not between k (3) and the Ki-67 index. TK-1 mRNA expression was significantly correlated with the Ki-67 index (P = 0.009). (18)F-FLT SUVmax and T/N ratio were significantly correlated with BBB breakdown evaluated by contrast enhancement in MR images (P = 0.003 and P = 0.011, respectively). These results indicate that (18)F-FLT uptake in the tumor is significantly related to transport through the disrupted BBB, but not through phosphorylation activity. Although the tissue TK-1 expression reflects tumor proliferation activity, the phosphorylation rate constant k (3) determined by (18)F-FLT PET kinetic analysis does not accurately reflect TK-1 expression in the tissue and should not be used as a surrogate biomarker of cell proliferation activity in human brain gliomas.

Correlation of 18F-FLT uptake with equilibrative nucleoside transporter-1 and thymidine kinase-1 expressions in gastrointestinal cancer

We examined equilibrative nucleoside transporter-1 (ENT1) and thymidine kinase-1 (TK1) messenger ribonucleic acid (mRNA) expressions in cancer tissue samples to elucidate the mechanism of 3'-deoxy-3'-F-fluorothymidine (FLT) uptake by positron emission tomography (PET) scan in gastrointestinal cancer. A total of 21 patients with newly diagnosed gastrointestinal cancer were examined with FLT PET. Tumor lesions were identified as areas of focally increased uptake, exceeding that of surrounding normal tissue. For semiquantitative analysis, the maximal standardized uptake value (SUV) was calculated. The expressions of ENT1 and TK1 in cancer tissue samples were compared with that of FLT SUV. Of all gastrointestinal cancer lesions only one gastric cancer showed focally increased uptake of FLT PET. The mean (±standard deviation) FLT SUV in gastrointestinal cancer was 5.48±1.87. There was no significant correlation between FLT SUV and ENT1 (P=0.90) mRNA expression. There was a significant correlation between FLT SUV and TK1 mRNA expression (P<0.05). Results of this preliminary study indicate that TK1 activity is an important determinant of FLT uptake in gastrointestinal cancer. In this study, it was found that ENT1 activity and FLT uptake were not related.

Acute cytotoxicity of arabinofuranosyl nucleoside analogs is not dependent on mitochondrial DNA

The nucleoside analogs 9-β- D-arabinofuranosylguanine (araG) and 1-β- d-arabinofuranosylthymine (araT) are substrates of mitochondrial nucleoside kinases and have previously been shown to be predominantly incorporated into mtDNA of cells, but the pharmacological importance of their accumulation in mtDNA is not known. Here, we examined the role of mtDNA in the response to araG, araT and other anti-cancer and anti-viral agents in a MOLT-4 wild-type (wt) T-lymphoblastoid cell line and its petite mutant MOLT-4 ρ 0 cells (lacking mtDNA). The mRNA levels and activities of deoxyguanosine kinase (dGK), deoxycytidine kinase (dCK), thymidine kinase 1 (TK1) and thymidine kinase 2 (TK2) were determined in the two cell lines. Compared to that in the MOLT-4 wt cells the mRNA level of the constitutively expressed TK2 was higher ( p < 0.01) in the ρ 0 cells, whereas the TK1 mRNA level was lower ( p < 0.05). The enzyme activity of the S-phase restricted TK1 was also lower ( p < 0.05) in the MOLT-4 ρ 0 cells, whereas the activities of dGK, dCK and TK2 were similar in MOLT-4 wt and ρ 0 cell lines. The sensitivities to different cytotoxic nucleoside analogs were determined and compared between the two cell lines. Interestingly, we found that the acute cytotoxicity of araG, araT and other anti-viral and anti-cancer agents is independent of the presence of mtDNA in MOLT-4 T-lymphoblastoid cells.

The modulation of radiation-induced cell death by genistein in K562 cells: Activation of thymidine kinase 1

Ionizing radiation is one of the most effective tools in cancer therapy. In a previous study, we reported that protein tyrosine kinase (PTK) inhibitors modulate the radiation responses in the human chronic myelogenous leukemia (CML) cell line K562. The receptor tyrosine kinase inhibitor, genistein, delayed radiation-induced cell death, while non-recepter tyrosine kinase inhibitor, herbimycin A (HMA) enhances radiation-induced apoptosis. In this study, we focused on the modulation of radiation-induced cell death by genistein and performed PCR-select suppression subtractive hybridization (SSH) to understand its molecular mechanism. We identified human thymidine kinase 1 (TK1), which is cell cycle regulatory gene and confirmed expression of TK1 mRNA by Northern blot analysis. Expression of TK1 mRNA and TK 1 enzymatic activity were parallel in their increase and decrease. TK1 is involved in G1-S phase transition of cell cycle progression. In cell cycle analysis, we showed that radiation induced G2 arrest in K562 cells but it was not able to sustain. However, the addition of genistein to irradiated cells sustained a prolonged G2 arrest up to 120 h. In addition, the expression of cell cycle-related proteins, cyclin A and cyclin B1, provided the evidences of G1/S progression and G2-arrest, and their relationship with TK1 in cells treated with radiation and genistein. These results suggest that the activation of TK1 may be critical to modulate the radiation-induced cell death and cell cycle progression in irradiated K562 cells.