Dose Dependent Effects on Cell Cycle Checkpoints and DNA Repair by Bendamustine

Abstract

Bendamustine (BDM) is an active chemotherapeutic agent approved in the U. S. for treating chronic lymphocytic leukemia and non-Hodgkin lymphoma. Its chemical structure suggests it may have alkylator and anti-metabolite activities; however the precise mechanism of action is not well understood. Here we report the concentration-dependent effects of BDM on cell cycle, DNA damage, checkpoint response and cell death in HeLa cells. Low concentrations of BDM transiently arrested cells in G2, while a 4-fold higher concentration arrested cells in S phase. DNA damage at 50, but not 200 µM, was efficiently repaired after 48 h treatment, suggesting a difference in DNA repair efficiency at the two concentrations. Indeed, perturbing base-excision repair sensitized cells to lower concentrations of BDM. Timelapse studies of the checkpoint response to BDM showed that inhibiting Chk1 caused both the S- and G2-arrested cells to prematurely enter mitosis. However, whereas the cells arrested in G2 (low dose BDM) entered mitosis, segregated their chromosomes and divided normally, the S-phase arrested cells (high dose BDM) exhibited a highly aberrant mitosis, whereby EM images showed highly fragmented chromosomes. The vast majority of these cells died without ever exiting mitosis. Inhibiting the Chk1-dependent DNA damage checkpoint accelerated the time of killing by BDM. Our studies suggest that BDM may affect different biological processes depending on drug concentration. Sensitizing cells to killing by BDM can be achieved by inhibiting base-excision repair or disrupting the DNA damage checkpoint pathway.

Centromere fragmentation is a common mitotic defect of S and G2checkpoint override

“…However, when cells were treated with 200 μM BDM, a concentration that induces S phase arrest, and thus unreplicated DNA/centromeres, forced entry into mitosis generated mitotic figures consistent with the generation of MUGs. 25 The simplest explanation for the overt difference in chromosome integrity after cells are treated with centromere, with its assembled kinetochore complex, from the bulk of the chromatin. A study using CHO cells demonstrated that microtubules were required for MUGs formation, suggesting that microtubules physically ripped the centromere/kinetochore complex away from the chromatin.…”

“…25 The concentration of UCN-01 was determined previously to induce checkpoint override 13,25 and not to have adverse effects on cell viability. 25 Cells were placed into a heated chamber and using a Nikon TE2000S microscope (Nikon) controlled by Metamorph software (Molecular Devices); brightfield and fluorescent images were taken every 5 min for up to 48 h. Images were compiled into movies, which were used to track fates of individual cells. A minimum of 100 cells were counted for each movie, and movies were conducted at least three times.…”

RXDX-107, A Dodecanol Alkyl Ester of Bendamustine, Demonstrates Greater Stability and Broad Antitumor Activity in Multiple Pre-Clinical Models of Solid Tumor

“…In our study, increased phosphorylation of H2AX and higher interstrand crosslinks suggest superior alkylating properties of RXDX-107. The interstrand crosslinks and phosphorylation of H2AX are known DNA damage markers, induced by multiple chemo agents, including bendamustine [4,[16][17][18]. However, the degree of DNA damage is rate limiting, the moderate DNA damage can be repaired in many cancer cell lines, [19][20][21].…”

“…However, the degree of DNA damage is rate limiting, the moderate DNA damage can be repaired in many cancer cell lines, [19][20][21]. Extensive DNA damage can induce apoptosis in cancer cells and lead to efficient therapy [4,5]. Thus we sought to evaluate induction of apoptosis in RXDX-107 treated cells compare to bendamustine treated cells.…”

“…Bendamustine contains three structural elements: 1) a chloroethylamine alkylating group, which is shared with other member of nitrogen mustard family, including cyclophosphamide, chlorambucil and melphalan; 2) a butyric acid side chain, which is shared with chlorambucil and melphalan; 3) a benzimidazole central ring system is unique to bendamustine [1][2][3]. The complete mechanism of action of bendamustine in humans has not been fully characterized, but unique mechanisms of action include DNA damage stress response, inhibition of mitotic checkpoint, activation of mitotic catastrophe, and induction of extensive and durable DNA damage [2][3][4][5].…”