Properties of cellular and serum forms of thymidine kinase 1 (TK1) in dogs with acute lymphocytic leukemia (ALL) and canine mammary tumors (CMTs): implications for TK1 as a proliferation biomarker

BACKGROUND: Thymidine kinase 1 (TK1) is a deoxyribonucleic acid (DNA) precursor enzyme and a proliferation biomarker used for prognosis and treatment monitoring of breast cancer in humans. The aim was to determine if serum thymidine kinase 1 (sTK1) activity and sTK1 protein levels in dogs with mammary tumors could be useful in veterinary medicine. RESULTS: Serum samples from 20 healthy dogs and 27 dogs with mammary tumors were analyzed for sTK1 activity, using an [(3)H]-deoxythymidine (dThd) phosphorylation assay, and for sTK1 protein levels by immune affinity/Western blot assay. The molecular forms of sTK1 in acute lymphocytic leukemia (ALL), canine mammary tumor (CMT), and healthy sera were determined by size exclusion chromatography. Mean sTK1 activities in CMT were 1.0 ± 0.36 pmol/min/mL, differing significantly from healthy dogs (mean ± SD = 0.73 ± 0.26 pmol/min/mL). Serum TK1 protein (26 kDa polypeptide) levels were also significantly higher in CMTs compared to healthy dogs (mean ± SD = 28.5 ± 11.4, and 8.5 ± 4 ng/mL, respectively). Cellular TK1 isolated from ALL tumor cells was predominantly a dimer, while the serum TK1 activity eluted as a high molecular weight (MW) oligomer. In analyses of CMT tissue extracts, TK1 activity eluted in two peaks, a minor peak with a high MW oligomer and a major tetramer peak. Western blot analysis of chromatographic fractions showed that cellular TK1 protein in both ALL and CMT dogs, and to some extent serum TK1 from ALL dogs, correlated with activity profiles, but a large fraction of inactive TK1 protein was detected in CMT. CONCLUSIONS: Serum TK1 protein and activity levels were significantly higher in CMT than in healthy dogs. Size exclusion chromatography demonstrated major differences in the molecular forms of sTK1 in ALL, healthy, and CMT dogs, with a large fraction of inactive TK1 protein in CMT. Our results showed that the sTK1 protein assay can differentiate benign tumors (early stage tumors) from healthy more efficiently than sTK1 activity assay. This preliminary data supports that sTK1 protein assay is clinically useful. Further studies are needed to evaluate the diagnostic or prognostic role of serum TK1 protein in CMTs

Deoxythymidine kinase in the tumour cells and serum of patients with non-Hodgkin lymphomas

The levels of deoxythymidine kinase in tumour cells (C-TK) and in serum (S-TK) were investigated and the tumour volume calculated in 89 patients with non-Hodgkin lymphoma (NHL), in order to ascertain the importance of C-TK and tumour burden as regards the S-TK levels. Among all patients, a correlation was seen between S-TK and tumour volume but not between S-TK and C-TK. However, within different tumour volume categories (small, medium-sized and large), there was a correlation between S-TK and C-TK. Multiple regression analysis supported this notion. C-TK correlated with the proliferation-associated parameters, S-phase fraction and mitotic index. As already known, S-TK was found to have a strong prognostic value. C-TK and tumour burden were also of prognostic value. In multivariate analyses, C-TK and tumour volume did not provide prognostic information in addition to S-TK, whereas, in the absence of S-TK, C-TK and tumour volume did provide additional information. It is concluded that the serum level of TK depends on both the tumour burden and the tumour cell proliferation rate. Based upon estimations of S-TK in patients assessed shortly after chemotherapy, we also suggest that S-TK reflects the number of proliferating cells that have died during the period immediately before sampling

A Non-Radioactive Microtitre Plate Reverse Transcriptase (RT) Assay, Based on Immobilized Template, for Screening of RT Activity Inhibitors and Evaluation of their Mode of Action

A new sensitive colorimetric reverse transcriptase (RT) activity assay utilizing a 96-well microtitre plate format, with solid phase-conjugated polyadenylic acid (prA), was investigated for simple analyses of the RT inhibiting capacity and mode of action of various substances. Three different technical procedures using the assay were evaluated: (i) direct lC50determinations with various substances, using four different combinations of primer and dNTP amounts; (ii) analyses of the capacity of the substances to interfere with the binding of RT to template or template-primer (BIC50); (iii) analyses of the capacity of the substances to destroy the template-primer in presence or absence of RT (TDC50). The assay was found to be useful for all three purposes using small amounts of recombinant RT. In the IC50analyses, the test substances gave values similar to those reported for soluble RT assays, and the values varied in accordance with their known mode of action in relation to the combination of primer and dNTP amount used. Only one of the substances, prG, in addition to DNA and RNA gave true RT binding inhibition. The template destruction assay showed that chain terminating substances gave destruction at low inhibitor concentrations. Furthermore, this destruction was RT-dependent, in contrast to the destruction obtained with substances that can base-pair with the template or primer. For optimum information on mode of action of a given substance all three assay procedures should be used. The use of the assay in relation to the screening and analyses of new RT inhibitory substances and characterization of RT in primary isolates or plasma is discussed.</jats:p

Differences in reverse transcriptase activity versus p24 antigen detection in cell culture, when comparing a homogeneous group of HIV type 1 subtype B viruses with a heterogeneous group of divergent strains

Failure to detect infection with HIV-1 non-B subtypes in some antibody screening assays has been shown. To date, however, no studies have been published evaluating the capacity of standard tests to quantify replication of divergent HIV-1 in cell culture. Reverse transcriptase (RT) activity and p24 antigen assays are the two methods most commonly used for this purpose. A homogeneous panel of HIV-1 subtype B viruses from northern Italy and a heterogeneous panel of diverse genetic subtypes (A to F and O) from different regions of the world were cultured under identical conditions. A new nonradioactive RT assay was used as a basis for comparison to evaluate the capacity of two p24 assays to quantify viral growth in both panels. Comparison of the p24 amount/RT activity (p24/RT) ratios showed that ratios in the subtype B panel tended to be markedly higher than in the diverse subtype panel. Greatest variation was seen with one of the subtype O isolates, where up to a 400 times lower ratio was obtained compared with the average ratio for the subtype B panel. In addition, one Thai subtype B virus also gave a markedly reduced ratio. Furthermore, comparison between the two p24 assays showed different abilities to detect p24 from different HIV-1 isolates. We discuss limitations for the use of anti-HIV-1 p24 antibodies produced by immunization with subtype B p24 in p24 assays

Serum TK levels in CLL identify Binet stage A patients within biologically defined prognostic subgroups most likely to undergo disease progression

Objective: Serum thymidine kinase (TK) levels have been shown to be correlated with survival in many malignancies, including chronic lymphocytic leukaemia (CLL). This study was designed to investigate associations between TK levels and other prognostic markers, in newly and previously diagnosed Binet stage A patients. Furthermore, the use of serum TK measurement to identify subcategories of disease within those defined by IgVH mutational status, gene usage and chromosomal aberrations was investigated. Methods: Ninety-one CLL patients were enrolled. Serum TK levels were measured using a radioenzyme assay. IgVH mutational status and VH gene usage were determined using BIOMED-2 primers and protocol. Recurring chromosomal abnormalities were detected by interphase fluorescent in situ hybridisation (FISH). Flow cytometry and reverse transcriptase polymerase chain reaction (RT-PCR) determined CD38 and Zap-70 expression, respectively. Results: Significantly higher serum TK levels were found in IgVH unmutated, compared with IgVH mutated, patients (P 8.5 U/L best identified patients with progressive disease. Elevated TK levels could identify patients categorised, at diagnosis, into good prognosis subgroups by the various biological markers (mutated IgVH, good prognosis chromosomal aberrations, Zap-70− and CD38−) who subsequently showed disease progression. Additionally, patients with VH3-21 gene usage showed high TK levels, irrespective of mutational status, and serum TK measurement retained predictive power as disease progressed in all subcategories studied