Molecular Basis for G2 Arrest Induced by 2′-C-Cyano-2′-Deoxy-1-β-d-Arabino-Pentofuranosylcytosine and Consequences of Checkpoint Abrogation

Abstract

Abstract2V -C -cyano-2V -deoxy-1-B-D-arabino -pentofuranosylcytosine (CNDAC) is a nucleoside analogue with a novel mechanism of action that is currently being evaluated in clinical trials. Incorporation of CNDAC triphosphate into DNA and extension during replication leads to single-strand breaks directly caused by B-elimination. These breaks, or the lesions that arise from further processing, cause cells to arrest in G 2 . The purpose of this investigation was to define the molecular basis for G 2 checkpoint activation and to delineate the sequelae of its abrogation. Cell lines derived from diverse human tissues underwent G 2 arrest after CNDAC treatment, suggesting a common mechanism of response to the damage created. CNDAC-induced G 2 arrest was instituted by activation of the Chk1-Cdc25C-Cdk1/cyclin B checkpoint pathway. Neither Chk2, p38, nor p53 was required for checkpoint activation. Inhibition of Chk1 kinase with 7-hydroxystaurosporine (UCN-01) abrogated the checkpoint pathway as indicated by dephosphorylation of checkpoint proteins and progression of cells through mitosis and into G 1 . Cell death was first evident in hematologic cell lines after G 1 entry. As indicated by histone H2AX phosphorylation, DNA damage initiated by CNDAC incorporation was transformed into double-strand breaks when ML-1 cells arrested in G 2 . Some breaks were manifested as chromosomal aberrations when the G 2 checkpoint of CNDAC-arrested cells was abrogated by UCN-01 but also in a minor population of cells that escaped to mitosis during treatment with CNDAC alone. These findings provide a mechanistic rationale for the design of new strategies, combining CNDAC with inhibitors of cell cycle checkpoint regulation in the therapy of hematologic malignancies. (Cancer Res 2005; 65(15): 6874-81)

Chk1-dependent slowing of S-phase progression protects DT40 B-lymphoma cells against killing by the nucleoside analogue 5-fluorouracil

“…Since these effects of 5FU should trigger the DNA damage and replication checkpoints, respectively, both of which are controlled primarily by the Chk1 protein kinase in vertebrate cells (Bartek et al, 2003), we investigated how genetic inactivation of Chk1 by gene targeting affects the cell cycle response and survival of DT40 B-lymphoma cells exposed to 5FU . Our findings differ in several respects from those of other recent studies which have used pharmacological or siRNA approaches to assess the effect of Chk1 inhibition on tumour cell responses to 5FU (Xiao et al, 2005) and other antimetabolite drugs (Shi et al, 2001;Liu et al, 2005;Morgan et al, 2005). The exact consequences of Chk1 inhibition appear to vary according to the nature of the genotoxic agent examined, however, mitotic division with damage or premature entry to mitosis with incompletely replicated DNA combined in each case with an increased frequency of apoptosis has been documented in a variety of cell types (Shi et al, 2001;Liu et al, 2005;Morgan et al, 2005;Xiao et al, 2005).…”

Antiproliferative effects of sapacitabine (CYC682), a novel 2′-deoxycytidine-derivative, in human cancer cells

“…Our data are consistent with the hypothesis that cells treated by sapacitabine or CNDAC may directly progress into G 2 /M phase of cell cycle. Other authors indicated that after incubation with cytostatic concentrations of CNDAC, cell cycle progression was delayed during S phase, but that cells arrested predominantly in the G 2 phase (Azuma et al, 2001;Liu et al, 2005). This unique cell cycle arrest pattern was related to the strandbreaking action of CNDAC caused by the b-elimination-mediated mechanism.…”

“…DNA strand breaks that arise from further processing initiate signals that activate the G 2 checkpoint pathway, ultimately resulting in cellular apoptosis. After incubation with cytostatic concentrations of CNDAC, cell cycle arrest in G 2 occurs following a delayed S phase (Liu et al, 2005). This differs from other deoxycytidine analogues such as ara-C or gemcitabine for which the predominant biological alterations consist of cell cycle arrest in S phase (Azuma et al, 2001).…”

“…Sapacitabine is primarily metabolised by plasma, gut and liver amidases into the active metabolite CNDAC, which is subsequently transported inside cells and then phosphorylated by deoxycytidine kinase (dCK) into CNDAC triphosphate before being incorporated into cellular DNA ( Figure 1B). Extension during DNA replication leads to single-strand breaks directly caused by b-elimination (Liu et al, 2005). DNA strand breaks that arise from further processing initiate signals that activate the G 2 checkpoint pathway, ultimately resulting in cellular apoptosis.…”

Ataxia-telangiectasia and Rad3-related and DNA-dependent protein kinase cooperate in G2 checkpoint activation by the DNA strand-breaking nucleoside analogue 2′-C-cyano-2′-deoxy-1-β-d-arabino-pentofuranosylcytosine

“…The DNA nick results from the rearrangement of the CNDAC molecule to form 2 ¶-C -cyano-2 ¶,3 ¶-didehydro-2 ¶,3 ¶-dideoxycytidine (CNddC), which is a de facto DNA chain terminator (16). Unlike the nucleoside analogues 1-h-D-arabinofuranosylcytosine, gemcitabine, and fludarabine that cause S-phase arrest, cells respond to this damage by activating the G 2 checkpoint (17). Although CNDAC-induced cell cycle arrest is instituted through activation of the Chk1-Cdc25C-cyclin-dependent kinase 1 (Cdk1)/cyclin B checkpoint pathway, participants in the signaling events upstream of Chk1 kinase remain obscure.…”