Decatenation Checkpoint Research Articles

Rad9A Is Required for G2 Decatenation Checkpoint and to Prevent Endoreduplication in Response to Topoisomerase II Inhibition

The Rad9A checkpoint protein interacts with and is required for proper localization of topoisomerase II-binding protein 1 (TopBP1) in response to DNA damage. Topoisomerase II (Topo II), another binding partner of TopBP1, decatenates sister chromatids that become intertwined during replication. Inhibition of Topo II by ICRF-193 (meso-4,4'-(3,2-butanediyl)-bis-(2,6-piperazinedione)), a catalytic inhibitor that does not induce DNA double-strand breaks, causes a mitotic delay known as the G(2) decatenation checkpoint. Here, we demonstrate that this checkpoint, dependent on ATR and BRCA1, also requires Rad9A. Analysis of different Rad9A phosphorylation mutants suggests that these modifications are required to prevent endoreduplication and to maintain decatenation checkpoint arrest. Furthermore, Rad9A Ser(272) is phosphorylated in response to Topo II inhibition. ICRF-193 treatment also causes phosphorylation of an effector kinase downstream of Rad9A in the DNA damage checkpoint pathway, Chk2, at Thr(68). Both of these sites are major targets of phosphorylation by the ATM kinase, although it has previously been shown that ATM is not required for the decatenation checkpoint. Examination of ataxia telangectasia (A-T) cells demonstrates that ATR does not compensate for ATM loss, suggesting that phosphorylation of Rad9A and Chk2 by ATM plays an additional role in response to Topo II inhibition than checkpoint function alone. Finally, we have shown that murine embryonic stem cells deficient for Rad9A have higher levels of catenated mitotic spreads than wild-type counterparts. Together, these results emphasize the importance of Rad9A in preserving genomic integrity in the presence of catenated chromosomes and all types of DNA aberrations.

Metnase mediates chromosome decatenation in acute leukemia cells

After DNA replication, sister chromatids must be untangled, or decatenated, before mitosis so that chromatids do not tear during anaphase. Topoisomerase IIalpha (Topo IIalpha) is the major decatenating enzyme. Topo IIalpha inhibitors prevent decatenation, causing cells to arrest during mitosis. Here we report that acute myeloid leukemia cells fail to arrest at the mitotic decatenation checkpoint, and their progression through this checkpoint is regulated by the DNA repair component Metnase (also termed SETMAR). Metnase contains a SET histone methylase and transposase nuclease domain, and is a component of the nonhomologous end-joining DNA double-strand break repair pathway. Metnase interacts with Topo IIalpha and enhances its decatenation activity. Here we show that multiple types of acute leukemia cells have an attenuated mitotic arrest when decatenation is inhibited and that in an acute myeloid leukemia (AML) cell line this is mediated by Metnase. Of further importance, Metnase permits continued proliferation of these AML cells even in the presence of the clinical Topo IIalpha inhibitor VP-16. In vitro, purified Metnase prevents VP-16 inhibition of Topo IIalpha decatenation of tangled DNA. Thus, Metnase expression levels may predict AML resistance to Topo IIalpha inhibitors, and Metnase is a potential therapeutic target for small molecule interference.

Metnase Mediates Resistance to Topoisomerase II Inhibitors in Breast Cancer Cells

DNA replication produces tangled, or catenated, chromatids, that must be decatenated prior to mitosis or catastrophic genomic damage will occur. Topoisomerase IIα (Topo IIα) is the primary decatenating enzyme. Cells monitor catenation status and activate decatenation checkpoints when decatenation is incomplete, which occurs when Topo IIα is inhibited by chemotherapy agents such as the anthracyclines and epididophyllotoxins. We recently demonstrated that the DNA repair component Metnase (also called SETMAR) enhances Topo IIα-mediated decatenation, and hypothesized that Metnase could mediate resistance to Topo IIα inhibitors. Here we show that Metnase interacts with Topo IIα in breast cancer cells, and that reducing Metnase expression significantly increases metaphase decatenation checkpoint arrest. Repression of Metnase sensitizes breast cancer cells to Topo IIα inhibitors, and directly blocks the inhibitory effect of the anthracycline adriamycin on Topo IIα-mediated decatenation in vitro. Thus, Metnase may mediate resistance to Topo IIα inhibitors, and could be a biomarker for clinical sensitivity to anthracyclines. Metnase could also become an important target for combination chemotherapy with current Topo IIα inhibitors, specifically in anthracycline-resistant breast cancer.

Topoisomerase IIα controls the decatenation checkpoint

Topoisomerase II (Topo II) is required to separate intertwined sister chromatids before chromosome segregation can occur in mitosis. However, it remains to be resolved whether Topo II has any role in checkpoint control. Here we report that when phosphorylated, Ser 1524 of Topo IIalpha acts as a binding site for the BRCT domain of MDC1 (mediator of DNA damage checkpoint protein-1), thereby recruiting MDC1 to chromatin. Although Topo IIalpha-MDC1 interaction is not required for checkpoint activation induced by DNA damage, it is required for activation of the decatenation checkpoint. Mutation of Ser 1524 results in a defective decatenation checkpoint. These results reveal an important role of Topo II in checkpoint activation and in the maintenance of genomic stability.

Mitotic Bypass Via An Occult Cell Cycle Phase Following DNA Topoisomerase II Inhibition In p53 Functional Human Tumor Cells

Cell cycle checkpoints guard against the inappropriate commitment to critical cell events such as mitosis. The bisdioxopiperazine ICRF-193, a catalytic inhibitor of DNA topoisomerase II, causes a reversible stalling of the exit of cells from G2 at the decatenation checkpoint (DC) and can generate tetraploidy via the compromising of chromosome segregation and mitotic failure. We have addressed an alternative origin – endocycle entry - for the tetraploidisation step in ICRF-193 exposed cells. Here we show that DC-proficient p53-functional tumour cells can undergo a transition to tetraploidy and subsequent aneuploidy via an initial bypass of mitosis and the mitotic spindle checkpoint. DC-deficient SV40-tranformed cells move exclusively through mitosis to tetraploidy. In p53-functional tumour cells, escape through mitosis is enhanced by dominant negative p53 co-expression. The mitotic bypass transition phase (termed G2endo) disconnects cyclin B1 degradation from nuclear envelope breakdown and allows cells to evade the action of Taxol. G2endo constitutes a novel and alternative cell cycle phase - lasting some 8 h - with distinct molecular motifs at its boundaries for G2 exit and subsequent entry into a delayed G1 tetraploid state. The results challenge the paradigm that checkpoint breaching leads directly to abnormal ploidy states via mitosis alone. We further propose that the induction of bypass could: facilitate the covert development of tetraploidy in p53 functional cancers, lead to a misinterpretation of phase allocation during cell cycle arrest and contribute to tumour cell drug resistance.

The decatenation checkpoint

The decatenation checkpoint delays entry into mitosis until the chromosomes have been disentangled. Deficiency in or bypass of the decatenation checkpoint can cause chromosome breakage and nondisjunction during mitosis, which results in aneuploidy and chromosome rearrangements in the daughter cells. A deficiency in the decatenation checkpoint has been reported in lung and bladder cancer cell lines and may contribute to the accumulation of chromosome aberrations that commonly occur during tumour progression. A checkpoint deficiency has also been documented in cultured stem and progenitor cells, and cancer stem cells are likely to be derived from stem and progenitor cells that lack an effective decatenation checkpoint. An inefficient decatenation checkpoint is likely to be a source of the chromosome aberrations that are common features of most tumours, but an inefficient decatenation checkpoint in cancer stem cells could also provide a potential target for chemotherapy.

Decatenation checkpoint deficiency in stem and progenitor cells

The decatenation checkpoint normally delays entry into mitosis until chromosomes have been disentangled through the action of topoisomerase II. We have found that the decatenation checkpoint is highly inefficient in mouse embryonic stem cells, mouse neural progenitor cells, and human CD34+ hematopoietic progenitor cells. Checkpoint efficiency increased when embryonic stem cells were induced to differentiate, which suggests that the deficiency is a feature of the undifferentiated state. Embryonic stem cells completed cell division in the presence of entangled chromosomes, which resulted in severe aneuploidy in the daughter cells. The decatenation checkpoint deficiency is likely to increase the rates of chromosome aberrations in progenitor cells, stem cells, and cancer stem cells.

BRCA1 Regulates Gene Expression for Orderly Mitotic Progression

Germline mutations of the BRCA1 confer an increased risk for breast cancer, ovarian cancer, and several other tumor types, including prostate cancer. In sporadic (non-hereditary) breast and ovarian cancers, BRCA1 expression is frequently decreased or absent, implicating BRCA1 in the etiology of these cancers. To study the contribution of BRCA1 to sporadic cancers, we tested the effect of knocking down endogenous BRCA1 on the gene expression profiles of human prostate (DU-145) and breast (MCF-7) cancer cell lines. DNA microarray and independent confirmatory RNA analyses revealed that treatment with BRCA1 small interfering (si) RNA caused decreased expression of genes involved in cell cycle regulation and in DNA replication and repair. In particular, BRCA1-siRNA caused down-regulation of multiple genes implicated in the mitotic spindle checkpoint (eg., BUB1, BUB1B, HEC, STK6, and BIRC5), chromosome segregation (eg., ESPL1, NEK2, PTTG1, and multiple kinesins and kinesin-like proteins), centrosome function (eg., ASPM), cytokinesis (eg., PRC1, PLK, MPHOSPH1, and KNSL2), and the transition into and progression through mitosis (eg., B-type cyclins, CDC2, and CDC20). Consistent with these findings, cells treated with BRCA1-siRNA showed attenuation of the mitotic spindle checkpoint, demonstrated by a failure of cells to arrest in metaphase following treatment with nocodazole, an agent that activates the spindle checkpoint. On the other hand, BRCA1-siRNA did not attenuated the ionizing radiation-inducible G2 checkpoint or the G2 decatenation checkpoint. Finally, BRCA1 knockdown caused or promoted the accumulation of binucleated and multinucleated cells, suggesting a defect in the coordination of cytokinesis and karyokinesis; and BRCA1-siRNA caused centrosome amplification. These findings suggest that BRCA1 transcriptionally regulates gene expression for orderly mitotic progession.

Telomerase expression is sufficient for chromosomal integrity in cells lacking p53 dependent G1 checkpoint function

BackgroundSecondary cultures of human fibroblasts display a finite lifespan ending at senescence. Loss of p53 function by mutation or viral oncogene expression bypasses senescence, allowing cell division to continue for an additional 10 – 20 doublings. During this time chromosomal aberrations seen in mitotic cells increase while DNA damage and decatenation checkpoint functions in G2 cells decrease.MethodsTo explore this complex interplay between chromosomal instability and checkpoint dysfunction, human fibroblast lines were derived that expressed HPV16E6 oncoprotein or dominant-negative alleles of p53 (A143V and H179Q) with or without the catalytic subunit of telomerase.ResultsCells with normal p53 function displayed 86 – 93% G1 arrest after exposure to 1.5 Gy ionizing radiation (IR). Expression of HPV16E6 or p53-H179Q severely attenuated G1 checkpoint function (3 – 20% arrest) while p53-A143V expression induced intermediate attenuation (55 – 57% arrest) irrespective of telomerase expression. All cell lines, regardless of telomerase expression or p53 status, exhibited a normal DNA damage G2 checkpoint response following exposure to 1.5 Gy IR prior to the senescence checkpoint. As telomerase-negative cells bypassed senescence, the frequencies of chromosomal aberrations increased generally congruent with attenuation of G2 checkpoint function. Telomerase expression allowed cells with defective p53 function to grow >175 doublings without chromosomal aberrations or attenuation of G2 checkpoint function.ConclusionThus, chromosomal instability in cells with defective p53 function appears to depend upon telomere erosion not loss of the DNA damage induced G1 checkpoint.

89 hRad9 is required for the G2 decatenation checkpoint and to prevent endoreplication in response to topoisomerase II inhibition

89 hRad9 is required for the G2 decatenation checkpoint and to prevent endoreplication in response to topoisomerase II inhibition

Small molecule modulation of the human chromatid decatenation checkpoint.

After chromosome replication, the intertwined sister chromatids are disentangled by topoisomerases. The integrity of this process is monitored by the chromatid decatenation checkpoint. Here, we describe small molecule modulators of the human chromatid decatenation checkpoint identified using a cell-based, chemical genetic modifier screen. Similar to 1,2,7-trimethylyxanthine (caffeine), these small molecules suppress the G(2)-phase arrest caused by ICRF-193, a small molecule inhibitor of the enzymatic activity of topoisomerase II. Analysis of specific suppressors, here named suptopins for suppressor of Topoisomerase II inhibition, revealed distinct effects on cell cycle progression, microtubule stability, nucleocytoplasmic transport of cyclin B1, and no effect on the chromatin deacetylation checkpoint induced by trichostatin A. The suptopins provide new molecular tools for dissecting the role of topoisomerases in maintaining genomic stability and determining whether inhibiting the chromatid decatenation checkpoint sensitizes tumor cells to chemotherapeutics.

Degradation of ATM-Independent Decatenation Checkpoint Function in Human Cells is Secondary to Inactivation of p53 and Correlated with Chromosomal Destabilization

DNA topoisomerase II is required in the cell cycle to decatenate intertwined daughter chromatids prior to mitosis. To study the mechanisms that cells use to accomplish timely chromatid decatenation, the activity of a catenation-responsive checkpoint was monitored in human skin fibroblasts with inherited or acquired defects in the DNA damage G2 checkpoint. G2 delay was quantified shortly after a brief incubation with ICRF-193, which blocks the ability of topoisomerase II to decatenate chromatids, or treatment with ionizing radiation (IR), which damages DNA. Both treatments induced G2 delay in normal human fibroblasts. Ataxia telangiectasia fibroblasts with defective G2 checkpoint response to IR displayed normal G2 delay after treatment with ICRF-193, demonstrating that ATM kinase was not required for signaling when chromatid decatenation was blocked. The G2 delay induced by ICRF-193 was reversed by caffeine, indicating that active checkpoint signaling was involved. ICRF-193-induced G2 delay also was independent of p53 function, being evident in cells expressing HPV16E6 to inactivate p53. However, as fibroblasts expressing HPV16E6 aged in culture, they lost the ability to delay entry to mitosis, both after DNA damage and when decatenation was blocked. This age-related loss of G2 delay in response to ICRF-193 and IR in E6-expressing cells was blocked by induction of telomerase. Expression of telomerase also prevented chromosomal destabilization in aging E6-expressing cells. These observations lead to a new model of genetic instability, in which attenuation of G2 decatenatory checkpoint function permits cells to enter mitosis with insufficiently decatenated chromatids, leading to aneuploidy and polyploidy. Key Words:Checkpoints, DNA damage, Decatenation, Topoisomerase II, ICRF-193, Radiation