5-Aminouracil and other inhibitors of DNA replication induce biphasic interphase-mitotic cells in apical root meristems of Allium cepa

Abstract

Induction of biphasic interphase-mitotic cells and PCC is connected with an increased level of metabolism in root meristem cells of Allium cepa. Previous experiments using primary roots of Allium cepa exposed to low concentrations of hydroxyurea have shown that long-term DNA replication stress (DRS) disrupts essential links of the S-M checkpoint mechanism, leading meristem cells either to premature chromosome condensation (PCC) or to a specific form of chromatin condensation, establishing biphasic organization of cell nuclei with both interphase and mitotic domains (IM cells). The present study supplements and extends these observations by describing general conditions under which both abnormal types of M-phase cells may occur. The analysis of root apical meristem (RAM) cell proliferation after prolonged mild DRS indicates that a broad spectrum of inhibitors is capable of generating PCC and IM organization of cell nuclei. These included: 5-aminouracil (5-AU, a thymine antagonist), characterized by the highest efficiency in creating cells with the IM phenotype, aphidicolin (APH), an inhibitor of DNA polymerase α, 5-fluorodeoxyuridine (FUdR), an inhibitor of thymidylate synthetase, methotrexate (MTX), a folic acid analog that inhibits purine and pyrimidine synthesis, and cytosine arabinoside (Ara-C), which inhibits DNA replication by forming cleavage complexes with topoisomerase I. As evidenced using fluorescence-based click chemistry assays, continuous treatment of onion RAM cells with 5-AU is associated with an accelerated dynamics of the DNA replication machinery and significantly enhanced levels of transcription and translation. Furthermore, DRS conditions bring about an intensified production of hydrogen peroxide (H₂O₂), depletion of reduced glutathione (GSH), and some increase in DNA fragmentation, associated with only a slight increase in apoptosis-like programmed cell death events.

Keywords: 5-AU; Allium cepa; IM cells; Metabolic activity; ROS; Replication stress.

Fig. 1

Nuclear Feulgen staining of aberrant mitotic cells in A. cepa RAMs. Scale bars = 20 μm. A A variety of biphasic IM cells formed after 72 h treatment with 5-AU (a–c); note the IM cell nucleus combining biphasic and amitotic phenotype (c), APH (d, e), Ara-C (f, g), FUdR (h, i), and MTX (j, k). B Selected cell nuclei with evident PCC-like morphology during prophase (a–c), metaphase (d–f), anaphase (g–i) and telophase (j–l) following treatment with 5-AU (a, d, g, j), Ara-C (b, e, h, k), and MTX (c, f, i, l). Post-telophase cell nuclei with micronuclei formed after treatment with APH (m) and FUdR (n). Numerous micronuclei in 5-AU-treated RAM cells (o)

Fig. 2

Root meristem shortening during continuous incubation of A. cepa seedlings with 5-AU. Feulgen-stained root tips aligned according to the transition regions between RAMs and elongation zones (EZ), demarcated by dotted line (a); corresponding RAM lengths [μm] in control plants (c) and after 24, 48, and 72 h treatment periods (b). Scale bar = 500 μm. When compared with the control, statistically significant changes in mean RAM length (± SD) are marked by asterisks: * indicates p < 0.05 and *** indicates 0.001

Fig. 3

Analysis of DNA replication in A. cepa RAMs by EdU incorporation. A Schematic representation of copper(l)-catalyzed Alexa Fluor^® 555 azide-alkyne "click" reaction used to detect replicating DNA. B RAM cell populations labeled with EdU (a–c) and the same cells counterstained with DAPI (a–c) in the control seedlings (a, a), after 1 h (b, b) and 48 h (c, c) incubations with 5-AU. Scale bar = 20 μm. (C) EdU labeling of RAM cells at successive stages of interphase: G1 (a), early S (b), middle S (c), late S (d), G2 (e), with corresponding images of the same cells counterstained with DAPI (a–e). Scale bar = 10 μm. D Single molecule DNA fibers isolated from actively replicating cell nuclei after their 48 h treatment with 5-AU. Scale bar = 10 μm ≈ 20 kbp. E Mean percentage (± SD) of EdU-labeled cells (labeling index; dotted diagram) and mean (± SD) intensity of EdU labeling (Fluorescence intensity, lined diagram), calculated as a percentage of the maximum possible brightness (pixel value = 255) evaluated for selected groups of mid-S-phase cell nuclei (determined microfluorimetrically following DAPI staining). The data represent values obtained for the control and 5-AU-treated onion RAM cell nuclei at successive periods of incubation. When compared with the control, statistically significant changes in mean values (± SD) are marked by asterisks: * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001

Fig. 4

Analysis of nascent transcription level in A. cepa RAM cells using EU incorporation followed by Click-iT^® RNA Alexa Fluor^® 488 imaging. A Detection of RNA synthesis in the control root tip cells (a) and after 72 h 5-AU treatment (b); corresponding images of the same cells counterstained with PI (a and b); note faint fluorescence signal emitted in prophase (a, a; arrows) and metaphase cells (b, b; arrows). Scale bar = 20 μm. B Changes in nascent transcription level during successive stages of mitosis in the control onion cells at prophase (a), metaphase (b), anaphase (c), and telophase (d), with corresponding images of the same cells counterstained with PI (a–d, respectively). Scale bar = 10 μm. C Increased EU incorporation into nascent transcripts observed in elongated prophase (a) and the biphasic IM cells after 72 h treatment with 5-AU (b); corresponding images of the same cells counterstained with PI (a, b, respectively). Scale bar = 20 μm. D Microfluorimetric evaluation of mean nascent transcription levels measured for the nucleus and nucleolus in the control and 5-AU-treated onion RAM cells. When compared with the control, statistically significant changes in mean fluorescence levels (± SD) expressed in arbitrary units [a.u.] are marked by asterisks: * indicates p < 0.05 and ** indicates 0.005

Fig. 5

Nascent protein synthesis detected using Click-iT® HPG Alexa Fluor® 488 imaging in A. cepa RAM cells. (A) Immunofluorescence of proteins: control (a), 72 h treatment with 5-AU (b), negative control series performed using (50 µM) cycloheximide (c); corresponding images of the same cells counterstained with DAPI (a’–c’). Scale bars = 20 μm. (B) Microfluorimetric evaluation of protein synthesis levels (Fluorescence intensity [a.u.]) expressed in arbitrary units, calculated as mean pixel value (± SD) measured at the cytoplasm area. When compared with the control, the fluorescence intensity in 5-AU-treated is increased, with the statistical significance at p < 0.001

Fig. 6

Generation of ROS, changes in GSH levels, DNA fragmentation, and induction of AL-PCD in A. cepa RAM cells following prolonged (72 h) treatment with 5-AU. (A) DAB-stained cells in the control root meristem (a), in root cells after 72 h treatment with the mixture containing 5-AU and 1 mM ascorbic acid (b), and in root cells incubated for 72 h with 5-AU (c). Scale bar = 20 μm. Cytometric evaluation of the DAB staining intensities in the control (C) and 5-AU-treated RAM cells ([a.u.]—arbitrary units); statistical significance at p < 0.001. (B) Detection of reduced glutathione (GSH) using ThiolTracker™ Violet fluorescence assay: longitudinal thick section of onion root meristems from the control (a–c) and 5-AU-treated seedlings (d–f); individual RAM cells at large magnification: control (b), 5-AU treatment (e), and the same cells viewed under phase contrast (c, f, respectively); scale bars = 500 μm for a and d, and 20 μm for (b, c, e, and f). Microfluorimetric evaluation ([a.u.—arbitrary units) of the mean total GSH fluorescence (± SD; g) in the control (C) and 5-AU-treated RAM cells, with the statistical significance at p < 0.001. C Agarose gel electrophoresis assay for DNA extracted and purified from onion root meristem cells: lane 1—mass marker, lane 2—control, and lane 3—5-AU treatment for 72 h. Note some shift of DNA towards lower buoyant density due to DNA fragmentation after incubations with 5-AU. D Detection of AL-PCD by TUNEL assay in DNase I-treated RAM cells (positive control; a), in the untreated control root cells (b), and in root meristem cells exposed to 72 h treatment with 5-AU (c); the same cells are visualized after DAPI staining (a–c), scale bar = 20 μm; mean frequency [%] of TUNEL-positive RAM cells in the control (C) and 5-AU-treated onion meristems; statistical significance at p < 0.001 (d)