Selective assays for thymidine kinase 1 and 2 and deoxycytidine kinase and their activities in extracts from human cells and tissues

Arnér, Elias S.J.; Spasokoukotskaja, Tatjana; Eriksson, Staffan

doi:10.1016/0006-291x(92)91114-6

Cited by 132 publications

(70 citation statements)

References 16 publications

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“…K m (mean 25.4 M) and V max (mean 794.2 nmol/min × mg) studies in PHA-stimulated T cells from a healthy donor showed that the dCK proteins obtained in the in vitro transcription-translation reaction exhibited similar enzyme kinetics as described by others (K m values ranging from 0.99-163 M and V max between 1-760 nmol/min × mg). 16,26,27 That the V max values we observed are relatively high as compared to the literature, may be explained by the fact that there are no inhibitory factors in the wheat germ extract mixture, which may be present in cellular extract. 16,17,23,27 Enrichment of the leukemic blasts is necessary in order to analyze dCK in leukemic blasts specifically, since bone marrow or peripheral blood samples of patients with AML contain a heterogeneous cell population consisting of leukemic blasts and non-leukemic cells.…”

Section: Discussioncontrasting

confidence: 43%

“…A disadvantage of the analysis of dCK activity in cellular extracts is the requirement of high amounts of cells. Moreover, it is known that dCK expression is strongly variable among different cell sources and moments of sampling, 25,26 resulting in changes in dCK activity measurements that are difficult to compare between various experiments. The cloning of dCK into the pET vector avoids these variables, but is very time-consuming since many cloned dCK-cDNA fragments will necessarily be tested to give a good representation of the whole sample when a heterogeneous cell population is present such as in leukemias.…”

Section: Discussionmentioning

confidence: 99%

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A novel RT-PCR-based protein activity truncation assay for direct assessment of deoxycytidine kinase in small numbers of purified leukemic cells

et al. 2000

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In vitro studies have demonstrated that deoxycytidine kinase (dCK) plays a crucial role in the mechanism of resistance to cytarabine (AraC). The resistant phenotype in vitro is always a result of mutational inactivation of dCK, leading to defects in the metabolic pathways of AraC. Although inactivation of dCK has shown to be one of the major mechanism of resistance to AraC in vitro, limited in vivo data are available. To improve research concerning the involvement of dCK inactivation in patients with acute myeloid leukemia (AML), we have set up a protocol that allows direct assessment of dCK expression and activity in primary human cells. In this protein activity truncation assay (PAT assay), the complete coding region of dCK is amplified by RT-PCR and a T7 RNA polymerase promoter sequence is introduced upstream of the coding region in a nested PCR reaction. After in vitro transcription-translation dCK proteins are analyzed for their molecular weight and phosphorylating capacities. We show that this relatively quick method can be used in purified, primary human leukemic blasts. In addition, inactivation of dCK by point mutations, deletions or genomic rearrangements can easily be detected in AraC-resistant cell lines. This novel assay may contribute to further elucidate the mechanism of AraC resistance in vivo.

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Section: Discussioncontrasting

confidence: 43%

Section: Discussionmentioning

confidence: 99%

A novel RT-PCR-based protein activity truncation assay for direct assessment of deoxycytidine kinase in small numbers of purified leukemic cells

et al. 2000

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“…TK expression under these experimental conditions was previously confirmed by performing an in vitro TK enzyme assay in cell lysates prepared 48 hours after infection with AdTK. 26 …”

Section: Recombinant Adenovirus and Fgf2 -Fab 0 Conjugatesmentioning

confidence: 99%

Targeting of suicide gene delivery in pancreatic cancer cells via FGF receptors

et al. 2002

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Pancreatic ductal adenocarcinomas ( PDACs ) overexpress various cell -surface tyrosine kinase receptors, including the type I highaffinity fibroblast growth factor receptor ( FGFR -1 ). The purpose of this study was to determine whether FGFR -targeted gene therapy is feasible in this disorder. Accordingly, the effects of a conjugate consisting of fibroblast growth factor ( FGF ) -2 linked to a Fab 0 fragment against the adenovirus knob region were evaluated in human pancreatic cancer cell lines treated with an adenoviral vector containing the herpes simplex virus thymidine kinase ( AdTK ) gene. An adenoviral vector containing the firefly luciferase reporter gene ( AdLuc ) served to assess infection efficiency, and was initially tested in L6 rat myoblasts. In parental L6 cells that express exceedingly low levels of high -affinity FGFRs, transduction with AdLuc was enhanced 7 -to 10 -fold with the FGF2 -Fab 0 conjugate, whereas in L6 cells transfected to express FGFR -1, it was enhanced 39 -to 52 -fold. The pancreatic cancer cell lines expressed variable levels of the four high -affinity FGF receptors, and exhibited 2 -to 34 -fold increases in gene transduction in the presence of the FGF2 -Fab 0 conjugate. In the absence of FGF2 -Fab 0 there was no correlation between surface binding of FGF2 and AdLuc transduction efficiency, whereas in the presence of FGF2 -Fab 0 , enhanced AdLuc transduction efficiency correlated with greater surface binding of FGF2. In the absence of AdTK, all the cell lines were insensitive to ganciclovir, whereas after AdTK transduction, only ASPC -1 and PANC -1 cells were resistant to ganciclovir even in the presence of FGF2 -Fab 0 . Ganciclovir -mediated inhibition was dependent on the conjugate in CAPAN -1 and COLO -357 cells, but was independent of the conjugate in T3M4 and MIA -PaCa -2 cells. Real -time quantitative PCR of laser -captured cancer cells revealed high levels of various FGFR mRNA species in six of seven PDAC tumor samples. These findings indicate that transduction efficiency with FGF2 -Fab 0 in pancreatic cancer cells is independent of native adenoviral transduction efficiency and is greatest in cells that exhibit concomitant expression of various highaffinity FGFRs. In view of the overexpression of high -affinity FGFRs in the cancer cells in PDAC, our findings also suggest that the combined use of AdTK, ganciclovir, and FGF2 -Fab 0 may ultimately be a promising therapeutic approach in a subgroup of patients with PDAC.

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“…Interestingly, while the U-937 cells produced the most TTP, the H9c2 cells produced the most phosphorylated AZT. Since TK1 phosphorylates AZT relative to thymidine much better than TK2 [10], these data suggest that the thymidine phosphorylation activity of U-937 depends more on TK2 than TK1 relative to the other cell lines. The amount of AZT in the media did not change significantly over time, and no phosphorylated AZT was detected in the media (data not shown).…”

Section: Time Courses Of Thymidine and Azt Phosphorylationmentioning

confidence: 83%

“…This discrepancy is likely to be related to the relative amounts of TK1 versus TK2 expressed. Both enzymes function well with thymidine as substrate, but TK1 is much better at phosphorylating AZT than TK2 [10]. Thus, it is possible that there is relatively more TK2 activity in the U-937 cell line while there is relatively more TK1 activity in the H9c2 cell line.…”

Section: Discussionmentioning

confidence: 99%

Effect of AZT on thymidine phosphorylation in cultured H9c2, U-937, and Raji cell lines

Lynx¹,

Kang²,

McKee³

2008

Biochemical Pharmacology

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Abstract3′-azido-3′-deoxythymidine (AZT) has been shown to be a potent inhibitor of thymidine kinase 2 in work from this laboratory. Inhibition results in decreased salvage of thymidine to TTP, which may lead to depletion of the TTP pool and result in the mitochondrial dysfunction and mt-DNA depletion observed with AZT toxicity. The effect of AZT on thymidine phosphorylation in growing cells expressing thymidine kinase 1 has not been shown. Three cell lines were used in these experiments: H9c2, derived from rat cardiomyoblasts; U-937, derived from human monocytes; and Raji, derived from human lymphoblasts. AZT inhibited growth in a concentration dependent manner in U-937 cells, but not the other cell lines. The phosphorylation of [3H]-thymidine or [3H]-AZT was determined during log growth. All cell lines salvaged and phosphorylated thymidine to TTP, with TTP the major product. The U-937 cells had a much more active salvage pathway than the other cells. All cell lines phosphorylated AZT to the triphosphate, but the major product was AZTMP. The AZT inhibition of growth of the U-937 cells did not correlate with levels of phosphorylated AZT. In contrast, pro-drug AZT was shown to inhibit thymidine phosphorylation in all lines with 50% inhibition concentrations (IC50) ranging from 4.4-21.9 μM. Since the U-937 cells expressed higher activity of the salvage pathway than the other cell lines, the U-937 cells may rely more heavily on the salvage pathway for TTP synthesis, accounting for AZT inhibition of growth.

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Selective assays for thymidine kinase 1 and 2 and deoxycytidine kinase and their activities in extracts from human cells and tissues

Cited by 132 publications

References 16 publications

A novel RT-PCR-based protein activity truncation assay for direct assessment of deoxycytidine kinase in small numbers of purified leukemic cells

A novel RT-PCR-based protein activity truncation assay for direct assessment of deoxycytidine kinase in small numbers of purified leukemic cells

Targeting of suicide gene delivery in pancreatic cancer cells via FGF receptors

Effect of AZT on thymidine phosphorylation in cultured H9c2, U-937, and Raji cell lines

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