AACR: PCR Assays, Enzyme Activity Measurements in Purine Analogs in CLL

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Abstract Number: 388

Real-time quantitative PCR assay together with activity measurements

for enzymes involved in purine analogues metabolism in previously

untreated symptomatic B-cell chronic lymphocytic leukemia

Kourosh Lotfi, Nilsson, Karin Karlsson, Gunnar Juliusson, Curt

, Staffan sson, Freidoun Albertioni. Medicine and Care,

Clinical Pharmacology, Linkoping, Sweden, Dept of Hematology,

Linkoping, Sweden, Agricultur, Uppsala, Sweden.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in

Europe and North America. The purine analogues fludarabine and

cladribine have clearly been shown to provide effective therapy in

CLL.

In this study 62 patients included in the international Phase III

Trial for untreated B-cell CLL were randomized in order to evaluate

the efficacy of chlorambucil, fludarabine, and cladribine, as primary

treatment of previously untreated patients with symptomatic B-cell

CLL. Both fludarabine and cladribine are prodrugs and must be

phosphorylated intracellularly to monophosphates by the

nuclear/cytosol enzyme deoxycytidine kinase (dCK) and possibly by the

mitochondrial enzyme deoxyguanosine kinase (dGK). DCK plays a pivotal

roll in the activation of fludarabine and cladribine and it has

previously been reported that resistance to these drugs is mainly due

to deficiency of the dCK.

Before treatment, peripheral blood cells were isolated by

centrifugation on Lymphoprep® and vitally frozen. In this study the

activity of dCK and dGK enzymes were analyzed in patient cells

together with the dCK and dGK mRNA levels using the real-time

quantitative PCR method. The activity of dCK was determined with

cladribine, cytarabine, deoxycytidine and fludarabine and dGK

activity with deoxyguanosine, cladribine and 9-beta-D-

arabinofuranosylguanine (with an excess of deoxycytidine added) as

substrates.

A good correlation (r2 & #8805;0.7) between activities measured with the

substrates was observed both for dCK and dGK. The dGK activity was

significantly lower (mean 69 pmol/mg protein/min) than the dCK

activity (mean 367 pmol/mg protein/min) using cladribine and

deoxyguanosine as substrates, respectively. The enzyme activities in

samples from CLL patients varied from 41 to 741 pmol/mg protein/min

(18 folds) for dCK using cladribine and from 8 to 216 pmol/mg

protein/min (28 folds) for dGK using deoxyguanosine as substrate.

The mRNA expression for dCK and dGK (expressed as the ratio of the

respective gene and the reference gene) in patients showed a large

inter individual variability from 0,006 to 0,167 and non-detectable

to 0,785 respectively.

There was no correlation between enzyme activity and mRNA levels in

the studied CLL patients.

Studies of the relationship between enzyme activities and clinical

response to chemotherapy are underway and will be reported.

Presenter: Kourosh Lotfi

Affiliation: Medicine and Care, Clinical Pharmacology, Linkoping,

Sweden; E-mail: koulo@...

Copyright © 2004 American Association for Cancer Research. All rights

reserved. Citation information: Proceedings of the AACR, Volume 45,

March 2004.