Preferential inhibition of DNA polymerasesα,δ, andε from Novikoff hepatoma cells by inhibitors of cell proliferation

Abstract

DNA polymerases α, δ and ε from normal regenerating rat liver and Novikoff hepatoma cells were purified about 300-fold, characterized, and checked for sensitivity towards drugs known to inhibit cell proliferation. Characterization included (a) identification of associated proteins, (b) measurement of physicochemical constants (including sedimentation coefficients, diffusion coefficients, calculation of relative molecular masses), (c) quantification of catalytic activities using specific DNA primer templates (K _m values) and specific inhibitors (K _i values), and (d) discrimination between DNA polymerases from normal cells and those from malignant cells using inhibitors of cell proliferation. (a) DNA primase associated with DNA polymerase α, and 3′–5′ exonuclease accompanying DNA polymerases δ and ε had similar activities. (b) Comparison of physicochemical and catalytic properties of DNA polymerases from both sources revealed similarities but also some important differences. Sedimentation and diffusion coefficients of DNA polymerases α and ε from malignant cells differed significantly. (c) The DNA-binding domain of DNA polymerases α and ε from hepatoma cells was altered sinceK _m values, determined with several specific DNA primer-templates, were higher. Furthermore, dNTP-binding sites of DNA polymerases from malignant cells; when probed with specific inhibitors (aphidicolin, butylphenyl-dGTP, carbonyldiphosphonate, and dideoxy-TTP) showed significantly lowerK _i values, indicating lower affinity to deoxyribonucleoside 5′-triphosphates. (d) Sixteen drugs representative of various modes of interaction with DNA and protein were chosen. Dose/response experiments were performed and the concentration at which the polymerizing activity was reduced to 50% was calculated (K ₅₀ values). Preferential inhibition of DNA polymerases α,δ, and ε from Novikoff hepatoma cells was found for: the intercalating drugs doxorubicin, daunorubicin, amsacrine, mitoxantrone, quinacrine and ethidium bromide, the minor-groove binders distamycin and netropsin, the ATPase-blocking agents novobiocin and coumamycin, and the topoisomerase I inhibitors camptothecin and topotecan. When the sensitivity of polymerases δ and ε was measured using poly(dA·dT) as a primer-template, the preferential inhibition of the enzymes from malignant cells was even more pronounced. Drugs known to trap the DNA-topoisomerase-II complex, etoposide, nalidixic acid, teniposide, and merbarone did not affect DNA polymerases irrespective of the source. Since the majority of the inhibitors used, particularly intercalators and minor-groove binders, act by modification of the primer-template, inhibition of DNA synthesis must have occurred through weakening of non-covalent bonds between DNA and catalytic polypeptides. Consequently, preferential inhibition of DNA polymerases from malignant cells seems to be indicative of abnormally diminished binding of the enzymes to their primer-templates. This effect may be caused by conformational alterations in polymerases from malignant cells which affect the DNA binding domains. Similarly, changes in physicochemical and kinetic constants are indicative of alterations of dNTP-binding domains.

Abbreviations

butylphenyl-dGTP

N ²-(p-n-butylphenyl)-2′-deoxyguanosine 5′-triphosphate

dideoxy-TTP

2′,3′-dideoxyribosylthymine 5′-triphosphate

HEPES

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

etoposide

4′-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-β-D-glucopyranoside)

amsacrine

4′-(9-acridinylamino)methanesulfon-m-ansidide

mitoxantrone

1,4-dihydroxy-5,8-bis{2-[(2-hydroxyethyl)amino]ethyl} amino)-9,10-anthracenedione

merbarone

5-(N-phenylcarboxamido)-2-thiobarbituric acid

PCNA

proliferating-cell nuclear antigen

teniposide

4′-demethylepipodophyllotoxin-9-(4,6-O-thionylidine-β-d-glucopyranoside)

topotecan

(S)-10-[(dimethylamino) methyl]-4-ethyl-4,9-dihydroxy-1H-pyrano[3′, 4′∶6, 7] indolizino[1, 2-b]quinoline-3,14(4H, 12H)-dione hydrochloride