UHRF2 regulates cell cycle, epigenetics and gene expression to control the timing of retinal progenitor and ganglion cell differentiation.

Ubiquitin-like, containing PHD and RING finger domains 2 (UHRF2) regulates cell cycle and binds 5-hydroxymethylcytosine (5hmC) to promote completion of DNA demethylation. Uhrf2-/- mice are without gross phenotypic defects; however, the cell cycle and epigenetic regulatory functions of Uhrf2 during retinal tissue development are unclear. Retinal progenitor cells (RPCs) produce all retinal neurons and Müller glia in a predictable sequence controlled by the complex interplay between extrinsic signaling, cell cycle, epigenetic changes and cell-specific transcription factor activation. In this study, we find that UHRF2 accumulates in RPCs, and its conditional deletion from mouse RPCs reduced 5hmC, altered gene expressions and disrupted retinal cell proliferation and differentiation. Retinal ganglion cells were overproduced in Uhrf2-deficient retinae at the expense of VSX2+ RPCs. Most other cell types were transiently delayed in differentiation. Expression of each member of the Tet3/Uhrf2/Tdg active demethylation pathway was reduced in Uhrf2-deficient retinae, consistent with locally reduced 5hmC in their gene bodies. This study highlights a novel role of UHRF2 in controlling the transition from RPCs to differentiated cell by regulating cell cycle, epigenetic and gene expression decisions.

Retinoblastoma tumor cell proliferation is negatively associated with an immune gene expression signature and increased immune cells.

This study focuses on gene expression differences between early retinal states that ultimately lead to normal development, late onset retinoblastoma, or rapid bilateral retinoblastoma tumors. The late-onset and early-onset retinoblastoma tumor cells are remarkably similar to normally proliferating retinal progenitor cells, but they fail to properly express differentiation markers associated with normal development. Further, early-onset retinoblastoma tumor cells express a robust immune gene expression signature followed by accumulation of dendritic, monocyte, macrophage, and T-lymphocyte cells in the retinoblastoma tumors. This characteristic was not shared by either normal retinae or late-onset retinoblastomas. Comparison of our data with other human and mouse retinoblastoma tumor gene expression significantly confirmed, that the immune signature is present in tumors from each species. Strikingly, we observed that the immune signature in both mouse and human tumors was most highly evident in those with the lowest proliferative capacity. We directly assessed this relationship in human retinoblastoma tumors by co-analyzing proliferation and immune cell recruitment by immunohistochemistry, uncovering a significant inverse relationship between increased immune-cell infiltration in tumors and reduced tumor cell proliferation. Directly inhibiting proliferation with a PI3K/mTOR inhibitor significantly increased the number of CD45+ immune cells in the retina. This work establishes an in vivo model for the rapid recruitment of immune cells to tumorigenic neural tissue.

Co-deleting Pten with Rb in retinal progenitor cells in mice results in fully penetrant bilateral retinoblastomas.

BACKGROUND: Rb1 is the most frequently mutated gene in the pediatric cancer retinoblastoma, and its loss causes E2F transcription factors to induce proliferation related genes. However, high E2F levels following pRB loss also induce apoptosis-promoting genes as a safeguard mechanism to suppress emergent tumors. Although p53 accumulation and apoptosis induction is believed to be a primary mechanism to eliminate cells with excess E2F activity, p53 deletion doesn't suppress RB/E2F induced apoptosis in vivo in the retina. This prompted us to test the PTEN/PI3K/AKT signaling pathway on RB/E2F apoptosis suppression in vivo, to ascertain if the PI3K pathway may provide a potential avenue for retinoblastoma therapy. METHODS: We developed a mouse model in which Rb1 and Pten were conditionally deleted from retinal progenitor cells using Chx10-Cre, whereas Rbl1 (p107) was constitutively deleted. Pathway components were also tested individually by in vivo electroporation into newborn retinas for an effect on apoptosis and tumor initiation. Mouse retinal tissues were analyzed by immunohistochemistry (IHC) for proliferation, apoptosis, and pathway activation. ShRNAs were used in vitro to assess effects on apoptosis and gene expression. RESULTS: Co-deleting Pten with Rb1 and Rbl1 in mouse retinal progenitor cells (RPCs) causes fully penetrant bilateral retinoblastomas by 30 days and strongly suppresses Rb/E2F-induced apoptosis. In vivo electroporation of constitutively active (ca)-Pik3ca, ca-Akt, or dominant-negative (dn)-Foxo1 into apoptosis prone newborn murine retina with deleted Rb/p107 eliminate Rb/E2F induced apoptosis and induce retinoblastoma emergence. Retinal deletion of Pten activates p-AKT and p-FOXO1 signaling in incipient retinoblastoma. An unbiased shRNA screen focusing on Akt phosphorylation targets identified FOXOs as critical mediators of Rb/E2F induced apoptosis and expression of Bim and p73 pro-apoptotic genes. CONCLUSIONS: These data indicate that we defined a key molecular trigger involving E2F/FOXO functioning to control retinal progenitor cell homeostasis and retinoblastoma tumor initiation. We anticipate that our findings could provide contextual understanding of the proliferation of other progenitor cells, considering the high frequency of co-altered signaling from RB/E2F and PTEN/PI3K/AKT pathways in a wide variety of normal and malignant settings.

The nuclear protein UHRF2 is a direct target of the transcription factor E2F1 in the induction of apoptosis.

The E2F1 transcription factor is active in many types of solid tumors and can function as either an oncogene or tumor suppressor in vivo. E2F1 activity is connected with a variety of cell fates including proliferation, apoptosis, senescence, differentiation, and autophagy, and these effects are mediated through differential target gene expression. E2F1-induced cell death is an innate anti-cancer mechanism to kill cells with a spontaneous oncogenic mutation that might otherwise form a cancer. Relatively little is known about the molecular circuitry that tips E2F1 balance toward proliferation during normal growth versus apoptosis during oncogenic stress, and which pathways mediate this decision. To further explore these mechanisms, we utilized an unbiased shRNA screen to identify candidate genes that mediate E2F1-induced cell death. We identified the ubiquitin-like with PHD and ring finger domains 2 (UHRF2) gene as an important mediator of E2F1-induced cell death. UHRF2 encodes a nuclear protein involved in cell-cycle regulation. Several of these domains have been shown to be essential for the regulation of cell proliferation, and UHRF2 has been implicated as an oncogene in some settings. Other reports have suggested that UHRF2 causes growth arrest, functions as a tumor suppressor, and is deleted in a variety of tumors. We show that UHRF2 is a transcriptional target of E2F, that it directly interacts with E2F1, and is required for E2F1 induction of apoptosis and transcription of a number of important apoptotic regulators.

UHRF2 accumulates in early G1-phase after serum stimulation or mitotic exit to extend G1 and total cell cycle length.

Ubiquitin like with PHD and ring finger domains 2 (UHRF2) regulates the cell cycle and epigenetics as a multi-domain protein sharing homology with UHRF1. UHRF1 functions with DNMT1 to coordinate daughter strand methylation during DNA replication, but UHRF2 can't perform this function, and its roles during cell cycle progression are not well defined. UHRF2 role as an oncogene vs. tumor suppressor differs in distinct cell types. UHRF2 interacts with E2F1 to control Cyclin E1 (CCNE1) transcription. UHRF2 also functions in a reciprocal loop with Cyclin E/CDK2 during G1, first as a direct target of CDK2 phosphorylation, but also as an E3-ligase with direct activity toward both Cyclin E and Cyclin D. In this study, we demonstrate that UHRF2 is expressed in early G1 following either serum stimulation out of quiescence or in cells transiting directly out of M-phase, where UHRF2 protein is lost. Further, UHRF2 depletion in G2/M is reversed with a CDK1 specific inhibitor. UHRF2 controls expression levels of cyclins and CDK inhibitors and controls its own transcription in a negative-feedback loop. Deletion of UHRF2 using CRISPR/Cas9 caused a delay in passage through each cell cycle phase. UHRF2 loss culminated in elevated levels of cyclins but also the CDK inhibitor p27KIP1, which regulates G1 passage, to reduce retinoblastoma phosphorylation and increase the amount of time required to reach G1/S passage. Our data indicate that UHRF2 is a central regulator of cell-cycle pacing through its complex regulation of cell cycle gene expression and protein stability.

Inhibition of ovarian cancer by RGD-P125A-endostatin-Fc fusion proteins.

Previous studies have shown that a single point mutation in endostatin at position 125 (P125A) can improve the biological activity of endostatin. Addition of an integrin-targeting moiety, R-G-D, resulted in better localization to tumor vasculature and improved the antiangiogenic activity of endostatin. Because endostatin has relatively shorter serum half-life, frequent dosing was required for inhibiting tumor growth. In our study, we have genetically fused RGD-P125A-endostatin to Fc of IgG4 isotype and evaluated its antiangiogenic and antitumor effects in athymic mice. Two genetic constructs were made, RGD-P125A-endostatin-Fc (RE-Fc) and P125A-endostatin-RGD-Fc (ER-Fc). Both constructs were cloned and expressed in mammalian cells. Purified fusion proteins inhibited endothelial cell migration and proliferation better than yeast-derived P125A-endostatin. Both RE-Fc and ER-Fc inhibited ovarian cancer growth and were found to be as effective as Bevacizumab treatment. Fusion protein showed marked increased half-life. Combination treatment with Bevacizumab and ER-Fc showed additive inhibition of ovarian cancer growth. These studies demonstrate that genetic fusion with human IgG4-Fc increases the half-life of P125A-endostatin and can be used along with Bevacizumab to improve antiangiogenic and antitumor activities.

Fhit and CHK1 have opposing effects on homologous recombination repair.

Fragile histidine triad (FHIT) gene deletion or promoter methylation and reduced Fhit protein expression occur in approximately 70% of human epithelial tumors and, in some cancers, are clearly associated with tumor progression. Specific Fhit signal pathways have not been identified. We previously reported that compared with Fhit+/+ cells, Fhit-/- cells with an overactivated ATR/CHK1 pathway show increased mutation frequency and resistance to DNA damage-induced killing, indicating that Fhit and the CHK1 pathway have opposing roles in cells responding to DNA damage. In this study, we show that cells, with or without Fhit expression, have similar DNA double-strand break induction levels and similar rejoining rates following ionizing radiation, indicating that the effect of Fhit on cell radiosensitivity is independent of nonhomologous end-joining. By combining I-SceI-induced-DNA double-strand break system and small interfering RNA approach, we also show that knocking down Fhit increases the efficiency of homologous recombination repair of cells, but knocking down Chk1 decreases the efficiency of homologous recombination repair, associated with the sensitivity to ionizing radiation-induced killing. Taken together, the results show that the role of Fhit in affecting the sensitivity of cells to ionizing radiation-induced killing is through the CHK1 pathway linked to homologous recombination repair. These results also illustrate the importance of balanced checkpoint activation in genomic stability and suggest a connection between the radioresistance and mutagenesis, carcinogenesis, as well as tumor progression in Fhit-deficient cells or tissue.

Retinoblastoma cells activate the AKT pathway and are vulnerable to the PI3K/mTOR inhibitor NVP-BEZ235.

Retinoblastoma is a pediatric cancer of the retina most often caused by inactivation of the retinoblastoma (RB1) tumor suppressor gene. We previously showed that Rb1 loss cooperates with either co-activating the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, or co-deleting Pten, to initiate retinoblastoma tumors in mice. The objectives of this study were to determine if the AKT pathway is activated in human retinoblastomas and the extent that anti-PI3K therapy induces apoptosis in retinoblastoma cells, alone or in combination with the DNA damaging drugs carboplatin and topotecan. Serial sections from human retinoblastoma tissue microarrays containing 27 tumors were stained with antibodies specific to p-AKT, Ki-67, forkhead box O1 (p-FOXO1), and ribosomal protein S6 (p-S6) using immunohistochemistry and each tumor sample scored for intensity. Human retinoblastoma tumors displayed significant correlation between p-AKT intensity with highly proliferative tumors (p = 0.008) that were also highly positive for p-FOXO1 (p = 0.002). Treatment with BEZ235, a dual PI3K/mTOR inhibitor, reduced phosphorylation levels of the AKT targets p-FOXO and p-S6 and effectively induced apoptosis the Y79 and Weri-1 human retinoblastoma cell lines and in vivo in our retinoblastoma mouse model. Long-term treatment with BEZ235 in vivo using our retinoblastoma-bearing mice induced apoptosis but did not significantly extend the lifespan of the mice. We then co-administered BEZ235 with topotecan and carboplatin chemotherapeutics in vivo, which more effectively induced apoptosis of retinoblastoma, but not normal retinal cells than either treatment alone. Our study has increased the variety of potentially effective targeted treatments that can be considered for human retinoblastoma.

Loss of UHRF2 expression is associated with human neoplasia, promoter hypermethylation, decreased 5-hydroxymethylcytosine, and high proliferative activity.

Ubiquitin-like with PHD and ring finger domains 2 (UHRF2) binds to 5-hydroxymethylcytosine (5hmC), a DNA base involved in tissue development, but it is unknown how their distribution compares with each other in normal and malignant human tissues. We used IHC on human tumor specimens (160 from 19 tumor types) or normal tissue to determine the expression and distribution of UHRF2, Ki-67, and 5hmC. We also examined UHRF2 expression in cord blood progenitors and compared its expression to methylation status in 6 leukemia cell lines and 15 primary human leukemias. UHRF2 is highly expressed, paralleling that of 5hmC, in most non-neoplastic, differentiated tissue with low Ki-67 defined proliferative activity. UHRF2 is expressed in common lymphoid progenitors and mature lymphocytes but not common myeloid progenitors or monocytes. In contrast, UHRF2 immunostaining in human cancer tissues revealed widespread reduction or abnormal cytoplasmic localization which correlated with a higher Ki-67 and reduced 5hmC. UHRF2 expression is reduced in some leukemia cell lines, this correlates with promoter hypermethylation, and similar UHRF2 methylation profiles are seen in primary human leukemia samples. Thus, UHRF2 and 5hmC are widely present in differentiated human tissues, and UHRF2 protein is poorly expressed or mislocalized in diverse human cancers.

DNA damage, c-myc suppression and apoptosis induced by the novel topoisomerase II inhibitor, salvicine, in human breast cancer MCF-7 cells.

Salvicine, a diterpenoid quinone compound, possesses potent in vitro and in vivo antitumor activity. Salvicine is a novel non-intercalative topoisomerase II poison. In this study salvicine induced evident DNA damage, which was further characterized as double-strand breaks mainly in MCF-7 human breast cancer cells. The degree of damage was highly correlated with growth inhibition of MCF-7. Using a PCR-stop assay we demonstrated that this damage was selective. Preferential damage occurred in the p2 promoter region, but not the 3'-end of the protooncogene c-myc. The expression of oncogenes, such as c-myc and c-jun, was additionally investigated. Salvicine induced a dose-dependent decrease in c-myc gene transcription, concomitant with an increase in c-jun expression. Furthermore, reverse-transcription PCR and Western blotting data revealed that salvicine failed to stimulate the mRNA and protein levels of p53 and its downstream targets p21 and bax. The phosphorylation degree of serine 15 of p53, which is thought to be an active form of p53 in response to cellular DNA damage, remained in a steady state. In view of these results, we propose that the downregulation of c-myc resulting from selective damage plays a role in apoptosis signaling. Moreover, salvicine-induced apoptosis in MCF-7 subsequent to DNA damage seems to be mediated through a p53-independent pathway.

Rb1 and Pten Co-Deletion in Osteoblast Precursor Cells Causes Rapid Lipoma Formation in Mice.

The Rb and Pten tumor suppressor genes are important regulators of bone development and both are frequently mutated in the bone cancer osteosarcoma (OS). To determine if Rb1 and Pten synergize as tumor suppressor genes for osteosarcoma, we co-deleted them in osteoprogenitor cells. Surprisingly, we observed rapid development of adipogenic but not osteosarcoma tumors in the ΔRb1/Pten mice. ΔPten solo deleted mice also developed lipoma tumors but at a much reduced frequency and later onset than those co-deleted for Rb1. Pten deletion also led to a marked increase in adipocytes in the bone marrow. To better understand the function of Pten in bone development in vivo, we conditionally deleted Pten in OSX(+) osteoprogenitor cells using OSX-Cre mice. µCT analysis revealed a significant thickening of the calvaria and an increase in trabeculae volume and number in the femur, consistent with increased bone formation in these mice. To determine if Pten and Rb1 deletion actively promotes adipogenic differentiation, we isolated calvarial cells from Pten(fl/fl) and Pten(fl/fl); Rb1(fl/fl) mice, infected them with CRE or GFP expressing adenovirus, treated with differentiation media. We observed slightly increased adipogenic, and osteogenic differentiation in the ΔPten cells. Both phenotypes were greatly increased upon Rb1/Pten co-deletion. This was accompanied by an increase in expression of genes required for adipogenesis. These data indicate that Pten deletion in osteoblast precursors is sufficient to promote frequent adipogenic, but only rare osteogenic tumors. Rb1 hetero- or homo-zygous co-deletion greatly increases the incidence and the rapidity of onset of adipogenic tumors, again, with only rare osteosarcoma tumors.

Sensitivity to TOP2 targeting chemotherapeutics is regulated by Oct1 and FILIP1L.

Topoisomerase II (TOP2) targeting drugs like doxorubicin and etoposide are frontline chemotherapeutics for a wide variety of solid and hematological malignancies, including breast and ovarian adenocarcinomas, lung cancers, soft tissue sarcomas, leukemias and lymphomas. These agents cause a block in DNA replication leading to a pronounced DNA damage response and initiation of apoptotic programs. Resistance to these agents is common, however, and elucidation of the mechanisms causing resistance to therapy could shed light on strategies to reduce the frequency of ineffective treatments. To explore these mechanisms, we utilized an unbiased shRNA screen to identify genes that regulate cell death in response to doxorubicin treatment. We identified the Filamin A interacting protein 1-like (FILIP1L) gene as a crucial mediator of apoptosis triggered by doxorubicin. FILIP1L shares significant similarity with bacterial SbcC, an ATPase involved in DNA repair. FILIP1L was originally described as DOC1, or "down-regulated in ovarian cancer" and has since been shown to be downregulated in a wide variety of human tumors. FILIP1L levels increase markedly through transcriptional mechanisms following treatment with doxorubicin and other TOP2 poisons, including etoposide and mitoxantrone, but not by the TOP2 catalytic inhibitors merbarone or dexrazoxane (ICRF187), or by UV irradiation. This induction requires the action of the OCT1 transcription factor, which relocalizes to the FILIP1L promoter and facilitates its expression following doxorubicin treatment. Our findings suggest that the FILIP1L expression status in tumors may influence the response to anti-TOP2 chemotherapeutics.

FOXO1: a potential target for human diseases.

The forkhead box O (FoxO) transcription factors are known to be involved in many physiological and pathological processes including apoptosis, cell cycle arrest, stress resistance, glucose metabolism, cellular differentiation and development, and tumor suppression. The environmental cues, such as growth factors, nutrients, oxidative stress and irradiation, can either positively or negatively modulate FoxO proteins' activities, thereby ensuring distinctive transcription programs in the cell. The potent activities of FoxOs are tightly controlled by multiple mechanisms, which include posttranslational modification such as phosphorylation, acetylation, methylation and ubiquitination, subcellular localization, and direct protein-protein interaction. Mounting evidence suggests that the human FOXO1 protein, a founding member of the FoxO family is likely involved in carcinogenesis, diabetes and other human diseases. Here we give an overview of most recent findings regarding the regulation and function of FoxO1, its potential role in human diseases and useful animal models for functional studies on FoxO1. Prospective ways in which the discoveries from the basic research of FoxO1 can be utilized for drug targeting and development of novel therapeutics for human diseases are also discussed.

Inhibition of cyclin-dependent kinase phosphorylation of FOXO1 and prostate cancer cell growth by a peptide derived from FOXO1.

Increasing evidence suggests that FOXO1 possesses a tumor suppressor function. Inactivation of FOXO1 has been documented in many types of human cancer, and restoring the activity of FOXO1 holds promise for cancer treatment. In this study, we identified a FOXO1-derived peptide termed FO1-6nls that inhibits cyclin-dependent kinases 1 and 2 (CDK1/2)-mediated phosphorylation of FOXO1 at the serine 249 residue in vitro and in vivo. Overexpression of FO1-6nls in prostate cancer (PCa) cells not only blocked CDK1-induced cytoplasmic localization of FOXO1 but also augmented FOXO1's transcriptional activity. This effect of FO1-6nls requires its binding to CDK1 and CDK2. Moreover, the ectopic expression of FO1-6nls inhibited the growth of PTEN-positive DU145 PCa cells. Importantly, the growth-inhibitory function of FO1-6nls is dependent on FOXO1. Finally, the ectopic expression of FO1-6nls overcame CDK1-mediated inhibition of FOXO1-induced apoptosis of PCa cells. These results indicate that the FOXO1-derived peptide FO1-6nls can restore FOXO1's tumor suppressor function by specifically opposing CDK1/2-mediated phosphorylation and inhibition of FOXO1 and hence may have a therapeutic potential for the treatment of PCa.

Jab1/CSN5 mediates E2F dependent expression of mitotic and apoptotic but not DNA replication targets.

The E2F transcription factors are critical regulators of cell cycle and cell fate control. Several classes of E2F target genes have been categorized based on their roles in DNA replication, mitosis, apoptosis, DNA repair, etc. How E2Fs coordinate the appropriate and timely expression of these functionally disparate gene products is poorly understood at a molecular level. We previously showed that the E2F1 binding partner Jab1/CSN5 promotes E2F1-dependent induction of apoptosis but not proliferation. To better understand how Jab1 regulates E2F1 dependent transcription, we performed gene expression analysis to identify E2F target genes most and least affected by shRNA depletion of Jab1. We find that a significant number of apoptotic and mitotic E2F target genes are poorly expressed in cells lacking Jab1/CSN5, whereas DNA replication genes are generally still highly expressed. Chromatin immunoprecipitation analysis indicates that both Jab1 and E2F1 co-occupy apoptotic and mitotic, but not DNA replication target genes. We explored a potential connection between PI3K activity and Jab1/E2F1 target gene induction, and found that E2F1/Jab1 co-induction of apoptotic target genes can be inhibited by activated PI3K. Furthermore, PI3K activity interferes with formation of the E2F1/Jab1 complex by co-immunoprecipitation. Jab1/CSN5 is upregulated in a variety of human tumors, but it's unclear how its pro-proliferatory and apoptotic functions are regulated in this context. We explored the link between increased Jab1 levels and PI3K function in tumors and detected a highly significant correlation between elevated Jab1/CSN5 levels and PI3K activity in breast, ovarian, lung and prostate cancers.

A stronger DNA damage-induced G2 checkpoint due to over-activated CHK1 in the absence of PARP-1.

Poly(ADP-ribose) polymerase 1 (PARP-1) is involved in multi-pathways to respond to DNA damage. Lack of or inhibition of PARP-1 activity leads to slow progress of cell cycle and sensitization of cells to different stresses. Recently, it was reported that besides the Ku dependent main nonhomologous end joining (NHEJ) pathway, there is a PARP-1 dependent complementary NHEJ pathway to repair DNA double strand break (DSB). Here we show that compared with PARP-1+/+ cells, PARP-1-/- cells display a much stronger G2 checkpoint response following ionizing radiation (IR). Treatment with Chk1 siRNA abolishes the stronger G2 checkpoint response and sensitizes PARP-1-/- cells to IR. These data indicate that the stronger G2 checkpoint response in PARP-1-/- cells is CHK1 dependent, which protects cells from IR induced killing. We also show that 4-Amino-1,8-naphthalimide (4-AN, inhibitor of PARP) but not methoxyamine (inhibitor of base excision repair (BER)), affects IR induced G2 arrest and cell sensitivity in PARP-1+/+ cells, resulting in the phenotypes similar to those of PARP-1-/- cells. These results indicate that DSB repair from the complementary NHEJ pathway of PARP-1, but not single strand break (SSB) repair from the BER function of PARP-1, may play an essential role in the over-activated CHK1 regulated G2 checkpoint response and radiosensitivity in PARP-1-/- cells.

Reactive oxygen species elicit apoptosis by concurrently disrupting topoisomerase II and DNA-dependent protein kinase.

Reactive oxygen species (ROS) are produced by all aerobic cells and have been implicated in the regulation of diverse cellular functions, including intracellular signaling, transcription activation, proliferation, and apoptosis. Salvicine, a novel diterpenoid quinone compound, demonstrates a broad spectrum of antitumor activities. Although salvicine is known to trap the DNA-topoisomerase II (Topo II) complex and induce DNA double-strand breaks (DSBs), its precise antitumor mechanisms remain to be clarified. In this study, we investigated whether salvicine altered the levels of ROS in breast cancer MCF-7 cells and whether these ROS contributed to the observed antitumoral activity. Our data revealed that salvicine stimulated intracellular ROS production and subsequently elicited notable DSBs. The addition of N-acetyl cysteine (NAC), an antioxidant, effectively attenuated the salvicine-induced ROS enhancement and subsequent DNA DSBs. Heat treatment reversed the accumulation of DNA DSBs, and the addition of NAC attenuated the Topo II-DNA cleavable complexes formation and the growth inhibition of salvicine-treated JN394top2-4 yeast cells, collectively indicating that Topo II is a target of the salvicine-induced ROS. On the other hand, when examining the impact of salvicine on DNA repair pathways, we unexpectedly observed that salvicine selectively down-regulated the catalytic subunit of DNA-dependent protein kinase (DNA-PK(CS)) protein levels and repressed DNA-PK kinase activity; both of these effects were attenuated by NAC pretreatment of MCF-7 cells. Finally and most importantly, NAC attenuated salvicine-induced apoptosis and cytotoxicity in MCF-7 cells. These results indicate that apart from its direct actions, salvicine generates ROS that modulate DNA damage and repair, contributing to the comprehensive biological consequences of salvicine treatment, such as DNA DSBs, apoptosis, and cytotoxicity in tumor cells. The finding of salvicine-induced ROS provides new evidence for the molecular mechanisms of this compound. Moreover, the effects of salvicine-induced ROS on Topo II and DNA-PK give new insights into the diverse biological activities of ROS.