Natalia F. Krynetskaia

Thioguanine substitution alters DNA cleavage mediated by topoisomerase II

Thiopurines and topoisomerase II‐targeted drugs (e.g., etoposide) are widely used anticancer drugs. However, topoisomerase II‐targeted drugs can cause acute myeloid leukemia, with the risk of this secondary leukemia linked to a genetic defect in thiopurine catabolism. Chronic thiopurines result in thioguanine substitution in DNA. The effect of these substitutions on DNA topoisomerase II activity is not known. Our goal was to determine whether deoxythioguanosine substitution alters DNA cleavage stabilized by human topoisomerase II. We studied four variations of a 40 mer oligonucleotide with a topoisomerase II cleavage site, each with a single deoxythioguanosine in a different position relative to the cleavage site (‐1 or +2 in the top and +2 or +4 in the bottom strand). Deoxythioguanosine substitution caused position‐dependent quantitative effects on cleavage. With the ‐ 1 or + 2 top and + 2 or+ 4 bottom substitutions, mean topoisomerase II‐induced cleavage was 0.6‐, 2.0‐, 1.1‐, and 3.3‐fold that with the wild‐type substrate (P=0.011, < 0.008, 0.51, and < 0.001, respectively). In the presence of 100 µ? etoposide, cleavage was enhanced for wild‐type and all thioguanosine‐modified substrates relative to no etoposide, with the + 4 bottom substitution showing greater etoposide‐induced cleavage than the wild‐type substrate (P=0.015). We conclude that thioguanine incorporation alters the DNA cleavage induced by topoisomerase II in the presence and absence of etoposide, providing new insights to the mechanism of thiopurine effect and on the leukemogenesis of thiopurines, with or without topoisomerase inhibitors.—Krynetskaia, N. F., Cai, X., Nitiss, J. L., Krynetski, E. Y., Relling, M. V. Thioguanine substitution alters DNA cleavage mediated by topoisomerase II. FASEB J. 14, 2339‐2344 (2000)

Rapid genotyping of common deficient thiopurine S-methyltransferase alleles using the DNA-microchip technique

Thiopurine drugs are metabolized, in part, by S-methylation catalyzed by thiopurine S-methyltransferase (TPMT). Patients with very low or undetectable TPMT activity are at high risk of severe, potentially fatal hematopoietic toxicity when they are treated with standard doses of thiopurines. As human TPMT activity is controlled by a common genetic polymorphism, it is an excellent candidate for the clinical application of pharmacogenetics. Here, we report a new molecular approach developed to detect point mutations in the TPMT gene that cause the loss of TPMT activity. A fluorescently labeled amplified DNA is hybridized with oligonucleotide DNA probes immobilized in gel pads on a biochip. The specially designed TPMT biochip can recognize six point mutations in the TPMT gene and seven corresponding alleles associated with TPMT deficiency: TPMT*2; TPMT*3A, TPMT*3B, TPMT*3C, TPMT*3D, TPMT*7, and TPMT*8. The effectiveness of the protocol was tested by genotyping 58 samples of known genotype. The results showed 100% concordance between the biochip-based approach and the established PCR protocol. The genotyping procedure is fast, reliable and can be used for rapid screening of inactivating mutations in the TPMT gene. The study also provides the first data on the frequency of common TPMT variant alleles in the Russian population, based on a biochip analysis of 700 samples. TPMT gene mutations were identified in 44 subjects; genotype *1/*3A was most frequent.

Deoxythioguanosine triphosphate impairs HIV replication: a new mechanism for an old drug

Inhibition of HIV‐1 reverse transcriptase (RT) and HIV protease are effective mechanisms for anti‐retroviral agents, and the combined use of mechanistically different medications has markedly improved the treatment of HIV infected patients. The active metabolite of mercaptopurine and thioguanine (TG), deoxythioguanosine triphosphate, was shown to be incorporated into DNA by the polymerase function of HIV‐1 RT and then to abrogate RNA cleavage by HIV‐1 RNaseH. Treatment of human lymphocyte cultures with thioguanine produced substantial inhibition of HIV replication (IC50 = 0.035 μM, IC95=15.4 μM), with minimal toxicity to host lymphocytes (< 10% at 15.4 μM TG, P < 0.000005). Furthermore, low concentrations of TG and zidovudine were synergistic in inhibiting HIV replication in human lymphocytes (synergy volume = 19 μM2 %), without additive cytotoxicity to host lympho‐cytes. Thus, thiopurines are novel anti‐retroviral agents that alter the DNA‐RNA substrates for HIV RNaseH, thereby abrogating early stages of HIV replication.

Synthesis of modified thiopurine nucleosides for structural characterization of human thiopurine S-methyltransferase.

Synthesis of a number of photoactive thiopurine-containing nucleosides was described. S-methylation of the synthesized compounds in the course of the reaction catalyzed by recombinant human thiopurine S-methyltransferase was studied by UV-spectroscopy.