Encapsulation of selenium in chitosan nanoparticles improves selenium availability and protects cells from selenium-induced DNA damage response

Selenium, an essential mineral, plays important roles in optimizing human health. Chitosan (CS) is an effective, naturally oriented material for synthesizing nanoparticles with preferable properties such as biocompatibility, biodegradation and resistance to certain enzymes. We have recently shown that cellular exposure to selenium compounds activates ataxia-telangiectasia mutated (ATM)-dependent DNA damage responses, a tumorigenesis barrier. To test whether nanoencapsulation of selenium modulates the cellular response to selenium compounds, the HCT 116 cancerous and the MRC-5 normal cells were treated with Na₂SeO₃ and methylseleninic acid (MSeA) encapsulated in CS/polyphosphate nanoparticles. Analyses of cellular selenium levels demonstrate that (1) the nanoencapsulation enhances selenium levels in cells after exposure to Na₂SeO₃ and MSeA (1-10 μM); (2) cells retained more selenium when treated with Na₂SeO₃ than with MSeA; (3) selenium levels are greater in HCT 116 than in MRC-5 cells after Na₂SeO₃, but not MSeA, exposure. Survival analysis shows that CS encapsulation desensitizes HCT 116 and MRC-5 cells to Na₂SeO₃ or MSeA exposure. Immunofluorescent analysis demonstrates that CS encapsulation attenuates the selenium-induced ATM phosphorylation on Ser-1981, and the extent is greater in HCT 116 than in MRC-5 cells. Our results reveal features of selenium nanoencapsulation in CS, including increased selenium retention in cells and decreased cellular sensitivity and DNA damage response to selenium exposure.     

Growth inhibition due to complementation of transforming growth factor-beta receptor type II-defect by human chromosome 3 transfer in human colorectal carcinoma cells

The transforming growth-beta receptor type II (TGF-beta RII) gene is one of the target genes of the DNA mismatch repair (MMR) defect. The human colorectal carcinoma cell line HCT116 has mutations in the hMLH1 gene and in the microsatellite region of the TGF-beta RII gene, both located on the short arm of chromosome 3. Introduction of the wild-type hMLH1 gene on transferred human chromosome 3 restores many characteristics of MMR-deficiency in HCT116. In this study, we determined whether transfer of chromosome 3 into HCT116 also complements the TGF-beta RII gene defect. We compared in vitro growth characteristics between HCT116 and HCT116 with a transferred chromosome 3 (HCT116 + ch3). The growth was suppressed in HCT116 + ch3 compared with parental HCT116. This suppression was abolished by frequent replacement with fresh medium, suggesting that the autocrine TGF-beta-TGF-beta RII system may be responsible for growth suppression. To explore this possibility, we determined several characteristics essential for the autocrine system. We found that HCT116 + ch3 expresses wild-type as well as mutated TGF-beta RII mRNA. In addition, phosphorylation of TGF-beta RI and growth inhibition were observed in HCT116 + ch3 but not in HCT116 by exposure to exogenous TGF-beta. The amount of TGF-beta1 in HCT116 + ch3 cultures was remarkably less than that in the HCT116, suggesting that TGF-beta produced by HCT116 + ch3 cells may be consumed by the cells. The conditioned medium from HCT116 cultures inhibits HCT116 + ch3 growth. This inhibition was neutralized by the anti-TGF-beta antibody. Taken together, these results strongly suggest that the TGF-beta RII gene defect in HCT116 is complemented by a wild-type gene on the transferred chromosome 3 and that HCT116 + ch3 gained the ability to respond to TGF-beta. Simultaneous complementation of defects of a responsible gene and a major target gene by the chromosome transfer is useful to prove the inactivated phenotypes acquired during colorectal tumorigenesis.     

ATM-mediated stabilization of hMutL DNA mismatch repair proteins augments p53 activation during DNA damage

Human DNA mismatch repair (MMR) proteins correct DNA errors and regulate cellular response to DNA damage by signaling apoptosis. Mutations of MMR genes result in genomic instability and cancer development. Nonetheless, how MMR proteins are regulated has not yet been determined. While hMLH1, hPMS2, and hMLH3 are known to participate in MMR, the function of another member of MutL-related proteins, hPMS1, remains unclear. Here we show that DNA damage induces the accumulation of hPMS1, hPMS2, and hMLH1 through ataxia-telangiectasia-mutated (ATM)-mediated protein stabilization. The subcellular localization of PMS proteins is also regulated during DNA damage, which induces nuclear localization of hPMS1 and hPMS2 in an hMLH1-dependent manner. The induced levels of hMLH1 and hPMS1 are important for the augmentation of p53 phosphorylation by ATM in response to DNA damage. These observations identify hMutL proteins as regulators of p53 response and demonstrate for the first time a function of hMLH1-hPMS1 complex in controlling the DNA damage response.     

ATM-Deficient Colorectal Cancer Cells Are Sensitive to the PARP Inhibitor Olaparib

The ataxia telangiectasia mutated (ATM) protein kinase plays a central role in the cellular response to DNA damage. Loss or inactivation of both copies of the ATM gene (ATM) leads to ataxia telangiectasia, a devastating childhood condition characterized by neurodegeneration, immune deficiencies, and cancer predisposition. ATM is also absent in approximately 40% of mantle cell lymphomas (MCLs), and we previously showed that MCL cell lines with loss of ATM are sensitive to poly-ADP ribose polymerase (PARP) inhibitors. Next-generation sequencing of patient tumors has revealed that ATM is altered in many human cancers including colorectal, lung, prostate, and breast. Here, we show that the colorectal cancer cell line SK-CO-1 lacks detectable ATM protein expression and is sensitive to the PARP inhibitor olaparib. Similarly, HCT116 colorectal cancer cells with shRNA depletion of ATM are sensitive to olaparib, and depletion of p53 enhances this sensitivity. Moreover, HCT116 cells are sensitive to olaparib in combination with the ATM inhibitor KU55933, and sensitivity is enhanced by deletion of p53. Together our studies suggest that PARP inhibitors may have potential for treating colorectal cancer with ATM dysfunction and/or colorectal cancer with mutation of p53 when combined with an ATM kinase inhibitor.     

The p53-p21WAF1 checkpoint pathway plays a protective role in preventing DNA rereplication induced by abrogation of FOXF1 function

We previously identified FOXF1 as a potential tumor suppressor gene with an essential role in preventing DNA rereplication to maintain genomic stability, which is frequently inactivated in breast cancer through the epigenetic mechanism. Here we further addressed the role of the p53-p21^WAF1 checkpoint pathway in DNA rereplication induced by silencing of FOXF1. Knockdown of FOXF1 by small interference RNA (siRNA) rendered colorectal p53-null and p21^WAF1-null HCT116 cancer cells more susceptible to rereplication and apoptosis than the wild-type parental cells. In parental HCT116 cells with a functional p53 checkpoint, the p53-p21^WAF1 checkpoint pathway was activated upon FOXF1 knockdown, which was concurrent with suppression of the CDK2-Rb cascade and induction of G₁ arrest. In contrast, these events were not observed in FOXF1-depleted HCT116-p53−/− and HCT116-p21−/− cells, indicating that the p53-dependent checkpoint function is vital for inhibiting CDK2 to induce G₁ arrest and protect cells from rereplication. The pharmacologic inhibitor (caffeine) of ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3 related (ATR) protein kinases abolished activation of the p53-p21^WAF1 pathway upon FOXF1 knockdown, suggesting that suppression of FOXF1 function triggered the ATM/ATR-mediated DNA damage response. Cosilencing of p53 by siRNA synergistically enhanced the effect of FOXF1 depletion on the stimulation of DNA rereplication and apoptosis in wild-type HCT116. Finally, we show that FOXF1 expression is predominantly silenced in breast and colorectal cancer cell lines with inactive p53. Our study demonstrated that the p53-p21^WAF1 checkpoint pathway is an intrinsically protective mechanism to prevent DNA rereplication induced by silencing of FOXF1.     

BRCA1 is required for hMLH1 stabilization following doxorubicin-induced DNA damage

Human DNA mismatch repair (MMR) is involved in the removal of DNA base mismatches that arise either during DNA replication or are caused by DNA damage. In this study, we show that the activation of the MMR component hMLH1 in response to doxorubicin (DOX) treatment requires the presence of BRCA1 and that this phenomenon is mediated by an ATM/ATR dependent phosphorylation of the hMLH1 Ser-406 residue. BRCA1 is an oncosuppressor protein with a central role in the DNA damage response and it is a critical component of the ATM/ATR mediated checkpoint signaling. Starting from a previous finding in which we demonstrated that hMLH1 is able to bind to BRCA1, in this study we asked whether BRCA1 might be the bridge for ATM/ATR dependent phosphorylation of the hMLH1 molecular partner. We found that: (i) the negative modulation of BRCA1 expression is able to produce a remarkable reversal of hMLH1 stabilization, (ii) BRCA1 is required for post-translational modification produced by DOX treatment on hMLH1 which is, in turn, attributed to the ATM/ATR activity, (iii) the serine 406 phosphorylatable residue is critical for hMLH1 activation by ATM/ATR via BRCA1. Taken together, our data lend support to the hypothesis suggesting an important role of this oncosuppressor as a scaffold or bridging protein in DNA-damage response signaling via downstream phosphorylation of the ATM/ATR substrate hMLH1.     

Structure/Function Analysis of PARP-1 in Oxidative and Nitrosative Stress-Induced Monomeric ADPR Formation

Poly adenosine diphosphate-ribose polymerase-1 (PARP-1) is a multifunctional enzyme that is involved in two major cellular responses to oxidative and nitrosative (O/N) stress: detection and response to DNA damage via formation of protein-bound poly adenosine diphosphate-ribose (PAR), and formation of the soluble 2^nd messenger monomeric adenosine diphosphate-ribose (mADPR). Previous studies have delineated specific roles for several of PARP-1′s structural domains in the context of its involvement in a DNA damage response. However, little is known about the relationship between the mechanisms through which PARP-1 participates in DNA damage detection/response and those involved in the generation of monomeric ADPR. To better understand the relationship between these events, we undertook a structure/function analysis of PARP-1 via reconstitution of PARP-1 deficient DT40 cells with PARP-1 variants deficient in catalysis, DNA binding, auto-PARylation, and PARP-1′s BRCT protein interaction domain. Analysis of responses of the respective reconstituted cells to a model O/N stressor indicated that PARP-1 catalytic activity, DNA binding, and auto-PARylation are required for PARP-dependent mADPR formation, but that BRCT-mediated interactions are dispensable. As the BRCT domain is required for PARP-dependent recruitment of XRCC1 to sites of DNA damage, these results suggest that DNA repair and monomeric ADPR 2^nd messenger generation are parallel mechanisms through which PARP-1 modulates cellular responses to O/N stress.     

Mismatch repair (MMR) efficiency and <Emphasis Type="Italic">MSH2</Emphasis> gene mutation in human colorectal carcinoma cell line COLO320HSR

Colorectal cancer (CC) is one of two diseases, in which the link between cancer proneness and DNA repair deficiency appears to be proved. A strict relationship between mismatch repair (MMR) gene mutations, microsatellite instability (MSI) has been found in familiar colorectal cancer (Lynch syndrome). Tumorigenesis at familiar cancer is initiated by biallelic mutations in the major MMR genes, namely MSH2 or MLH1. One of these mutations is an inherited germline alteration and the other is a somatic one. The initiating mutation in sporadic colorectal tumors was not still identified although biochemical and genetic signs of MMR deficiency are observed in tumor cells. Two currently used colorectal tumor cell lines HCT116 and COLO320HSR were derived from hereditary and sporadic tumors accordingly. HCT116 cell line exhibits MMR-deficiency due to biallelic deletion in MLH1. As a consequence this shows MSI phenotype and a near-diploid karyotype. COLO320HSR cell line is characterized by MSS phenotype with mostly imbalanced aberrations. This indicates MMR proficiency in these cells. However, both MMR-deficient HCT116 and COLO320HSR cells reveal near-diploid karyotype. Earlier we have shown that the number of secondary DNA double strand breaks, induced by methylnitrosourea (MNU), represent functional activity of cellular MMR. In the present study, using this approach we evaluated sensitivity to MNU and MMR activity in two colorectal tumor cell lines (HCT116, COLO320HSR) and compared them to that in the HeLa cell line, which have MMR-proficient phenotype. We showed that cell line COLO320HSR exhibits low MMR activity, close to the level of MMR-activity in HCT116 cell line. We found a mutation in MSH2-G520A gene in COLO320HSR. This neutral mutation apparently is not related to polymorphism as we failed to identify the same mutation in any of MSH2 gene sequences of lymphocytes from 30 patients with sporadic colorectal cancer.     

Novel DNA Variants and Mutation Frequencies of hMLH1 and hMSH2 Genes in Colorectal Cancer in the Northeast China Population

Research on hMLH1 and hMSH2 mutations tend to focus on Lynch syndrome (LS) and LS-like colorectal cancer (CRC). No studies to date have assessed the role of hMLH1 and hMSH2 genes in mass sporadic CRC (without preselection by MSI or early age of onset). We aimed to identify novel hMLH1 and hMSH2 DNA variants, to determine the mutation frequencies and sites in both sporadic and LS CRC and their relationships with clinicopathological characteristics of CRC in Northeast of China. 452 sporadic and 21 LS CRC patients were screened for germline and somatic mutations in hMLH1 and hMSH2 genes with PCR–SSCP sequencing. We identified 11 hMLH1 and seven hMSH2 DNA variants in our study cohort. Six of them were novel: four in hMLH1 gene (IVS8-16 A>T, c.644 GAT>GTT, c.1529 CAG>CGG and c.1831 ATT>TTT) and two in hMSH2 gene (−39 C>T, insertion AACAACA at c.1127 and deletion AAG at c.1129). In sporadic CRC, germline and somatic mutation frequencies of hMLH1/hMSH2 gene were 15.59% and 17.54%, respectively (p = 0.52). Germline mutations present in hMLH1 and hMSH2 genes were 5.28% and 10.78%, respectively (p<0.01). Somatic mutations in hMLH1 and hMSH2 genes were 6.73% and 11.70%, respectively (p = 0.02). In LS CRC, both germline and somatic mutation frequencies of hMLH1/hMSH2 gene were 28.57%. The most prevalent germline mutation site in hMSH2 gene was c.1168 CTT>TTT (3.90%), a polymorphism. Somatic mutation frequency of hMLH1/hMSH2 gene was significantly different in proximal, distal colon and rectal cancer (p = 0.03). Our findings elucidate the mutation spectrum and frequency of hMLH1 and hMSH2 genes in sporadic and LS CRC, and their relationships with clinicopathological characteristics of CRC.     

Measurement of DNA mismatch repair activity in live cells

Loss of DNA mismatch repair (MMR) function leads to the development and progression of certain cancers. Currently, assays for DNA MMR activity involve the use of cell extracts and are technically challenging and costly. Here, we report a rapid, less labor-intensive method that can quantitatively measure MMR activity in live cells. A G–G or T–G mismatch was introduced into the ATG start codon of the enhanced green fluorescent protein (EGFP) gene. Repair of the G–G or T–G mismatch to G–C or T–A, respectively, in the heteroduplex plasmid generates a functional EGFP gene expression. The heteroduplex plasmid and a similarly constructed homoduplex plasmid were transfected in parallel into the same cell line and the number of green cells counted by flow cytometry. Relative EGFP expression was calculated as the total fluorescence intensity of cells transfected with the heteroduplex construct divided by that of cells transfected with the homoduplex construct. We have tested several cell lines from both MMR-deficient and MMR-proficient groups using this method, including a colon carcinoma cell line HCT116 with defective hMLH1 gene and a derivative complemented by transient transfection with hMLH1 cDNA. Results show that MMR-proficient cells have significantly higher EGFP expression than MMR-deficient cells, and that transient expression of hMLH1 alone can elevate MMR activity in HCT116 cells. This method is potentially useful in comparing and monitoring MMR activity in live cells under various growth conditions.     

Defective Mre11-dependent activation of Chk2 by ataxia telangiectasia mutated in colorectal carcinoma cells in response to replication-dependent DNA double strand breaks

The Mre11.Rad50.Nbs1 (MRN) complex binds DNA double strand breaks to repair DNA and activate checkpoints. We report MRN deficiency in three of seven colon carcinoma cell lines of the NCI Anticancer Drug Screen. To study the involvement of MRN in replication-mediated DNA double strand breaks, we examined checkpoint responses to camptothecin, which induces replication-mediated DNA double strand breaks after replication forks collide with topoisomerase I cleavage complexes. MRN-deficient cells were deficient for Chk2 activation, whereas Chk1 activation was independent of MRN. Chk2 activation was ataxia telangiectasia mutated (ATM)-dependent and associated with phosphorylation of Mre11 and Nbs1. Mre11 complementation in MRN-deficient HCT116 cells restored Chk2 activation as well as Rad50 and Nbs1 levels. Conversely, Mre11 down-regulation by small interference RNA (siRNA) in HT29 cells inhibited Chk2 activation and down-regulated Nbs1 and Rad50. Proteasome inhibition also restored Rad50 and Nbs1 levels in HCT116 cells suggesting that Mre11 stabilizes Rad50 and Nbs1. Chk2 activation was also defective in three of four MRN-proficient colorectal cell lines because of low Chk2 levels. Thus, six of seven colon carcinoma cell lines from the NCI Anticancer Drug Screen are functionally Chk2-deficient in response to replication-mediated DNA double strand breaks. We propose that Mre11 stabilizes Nbs1 and Rad50 and that MRN activates Chk2 downstream from ATM in response to replication-mediated DNA double strand breaks. Chk2 deficiency in HCT116 is associated with defective S-phase checkpoint, prolonged G2 arrest, and hypersensitivity to camptothecin. The high frequency of MRN and Chk2 deficiencies may contribute to genomic instability and therapeutic response to camptothecins in colorectal cancers.     

Nuclear reorganization of DNA mismatch repair proteins in response to DNA damage

The DNA mismatch repair (MMR) system is highly conserved and vital for preserving genomic integrity. Current mechanistic models for MMR are mainly derived from in vitro assays including reconstitution of strand-specific MMR and DNA binding assays using short oligonucleotides. However, fundamental questions regarding the mechanism and regulation in the context of cellular DNA replication remain. Using synchronized populations of HeLa cells we demonstrated that hMSH2, hMLH1 and PCNA localize to the chromatin during S-phase, and accumulate to a greater extent in cells treated with a DNA alkylating agent. In addition, using small interfering RNA to deplete hMSH2, we demonstrated that hMLH1 localization to the chromatin is hMSH2-dependent. hMSH2/hMLH1/PCNA proteins, when associated with the chromatin, form a complex that is greatly enhanced by DNA damage. The DNA damage caused by high doses of alkylating agents leads to a G₂ arrest after only one round of replication. In these G₂-arrested cells, an hMSH2/hMLH1 complex persists on chromatin, however, PCNA is no longer in the complex. Cells treated with a lower dose of alkylating agent require two rounds of replication before cells arrest in G₂. In the first S-phase, the MMR proteins form a complex with PCNA, however, during the second S-phase PCNA is missing from that complex. The distinction between these complexes may suggest separate functions for the MMR proteins in damage repair and signaling. Additionally, using confocal immunofluorescence, we observed a population of hMSH6 that localized to the nucleolus. This population is significantly reduced after DNA damage suggesting that the protein is shuttled out of the nucleolus in response to damage. In contrast, hMLH1 is excluded from the nucleolus at all times. Thus, the nucleolus may act to segregate a population of hMSH2–hMSH6 from hMLH1–hPMS2 such that, in the absence of DNA damage, an inappropriate response is not invoked.     

Honokiol radiosensitizes colorectal cancer cells: enhanced activity in cells with mismatch repair defects

DNA mismatch repair is required for correcting any mismatches that are created during replication and recombination, and a defective mismatch repair system contributes to DNA damage-induced growth arrest. The colorectal cancer cell line HCT116 is known to have a mutation in the hMLH1 mismatch repair gene resulting in microsatellite instability and defective mismatch repair. Honokiol is a biphenolic compound that has been used in traditional Chinese medicine for treating various ailments including cancer. This study was designed to test the hypothesis that honokiol enhances the radiosensitivity of cancer cells with mismatch repair defect (HCT116) compared with those that are mismatch repair proficient (HCT116-CH3). We first determined that the combination of honokiol and γ-irradiation treatment resulted in dose-dependent inhibition of proliferation and colony formation in both cell lines. However, the effects were more pronounced in HCT116 cells. Similarly, the combination induced higher levels of apoptosis (caspase 3 activation, Bax to Bcl2 ratio) in the HCT116 cells compared with HCT116-CH3 cells. Cell cycle analyses revealed higher levels of dead cells in HCT116 cells. The combination treatment reduced expression of cyclin A1 and D1 and increased phosphorylated p53 in both cell lines, although there were significantly lower amounts of phosphorylated p53 in the HCT116-CH3 cells, suggesting that high levels of hMLH1 reduce radiosensitivity. These data demonstrate that honokiol is highly effective in radiosensitizing colorectal cancer cells, especially those with a mismatch repair defect.     

<Emphasis Type="SmallCaps">l</Emphasis>-carnosine induces apoptosis/cell cycle arrest via suppression of NF-κB/STAT1 pathway in HCT116 colorectal cancer cells

l-carnosine, a dipeptide of the amino acids β-alanine and histidine, is found in various tissues, such as the central nervous system and skeletal muscles. Recently, l-carnosine has been reported to possess anti-tumor activity; however, the molecular mechanism underlying its activity in colorectal cancer is still unknown. Therefore, we investigated the effect of l-carnosine using a human colorectal cancer cell line, HCT116. Treatment with l-carnosine (0, 100, or 200 mM) for 24 h gradually reduced cellular proliferation according to immunochemistry and 7-aminoactinomycin D (7-AAD) analyses and induced G0/G1 phase arrest. In the RT-PCR analysis, l-carnosine decreased the mRNA levels of cell cycle-related genes in HCT116 cells. In the Western blot analysis, levels of the cyclin D1, BAX/Bcl-2, cleaved caspase-3, p21, and p53 proteins were significantly increased in cells treated with l-carnosine. We next determined whether STAT1/NF-κB pathway is involved in regulation of cell cycle arrest- and cell death-associated gene in HCT116. The l-carnosine treatment significantly inhibited the phosphorylation of STAT1 on Tyr701 and NF-κB p65 on Ser276 and Ser536, and then, we exogenously blocked the NF-κB phosphorylation using Bay 11-7082. Based on our findings, l-carnosine induces cell cycle arrest and apoptosis in human colorectal cancer cells by suppressing of NF-κB/STAT1 signaling.     

siRNA‐induced CD44 knockdown suppresses the proliferation and invasion of colorectal cancer stem cells through inhibiting epithelial–mesenchymal transition

CD44 has shown prognostic values and promising therapeutic potential in multiple human cancers; however, the effects of CD44 silencing on biological behaviors of cancer stem cells (CSCs) have not been fully understood in colorectal cancer. To examine the contribution of siRNA‐induced knockdown of CD44 to the biological features of colorectal CSCs, colorectal CSCs HCT116‐CSCs were generated, and CD44 was knocked down in HCT116‐CSCs using siRNA. The proliferation, migration and invasion of HCT116‐CSCs were measured, and apoptosis and cell‐cycle analyses were performed. The sensitivity of HCT116‐CSCs to oxaliplatin was tested, and xenograft tumor growth assay was performed to examine the role of CD44 in HCT116‐CSCs tumorigenesis in vivo. In addition, the expression of epithelial–mesenchymal transition (EMT) markers E‐cadherin, N‐cadherin and vimentin was quantified. siRNA‐induced knockdown of CD44 was found to inhibit the proliferation, migration and invasion, induce apoptosis, promote cell‐cycle arrest at the G1/G0 phase and increase the sensitivity of HCT116‐CSCs to oxaliplatin in HCT116‐CSCs, and knockdown of CD44 suppressed in vivo tumorigenesis and intrapulmonary metastasis of HCT116‐CSCs. Moreover, silencing CD44 resulted in EMT inhibition. Our findings demonstrate that siRNA‐induced CD44 knockdown suppresses the proliferation, invasion and in vivo tumorigenesis and metastasis of colorectal CSCs by inhibiting EMT.     

c-Myc Quadruplex-forming Sequence Pu-27 Induces Extensive Damage in Both Telomeric and Nontelomeric Regions of DNA

Quadruplex-forming DNA sequences are present throughout the eukaryotic genome, including in telomeric DNA. We have shown that the c-Myc promoter quadruplex-forming sequence Pu-27 selectively kills transformed cells (Sedoris, K. C., Thomas, S. D., Clarkson, C. R., Muench, D., Islam, A., Singh, R., and Miller, D. M. (2012) Genomic c-Myc quadruplex DNA selectively kills leukemia. Mol. Cancer Ther. 11, 66–76). In this study, we show that Pu-27 induces profound DNA damage, resulting in striking chromosomal abnormalities in the form of chromatid or chromosomal breaks, radial formation, and telomeric DNA loss, which induces γ-H2AX in U937 cells. Pu-27 down-regulates telomeric shelterin proteins, DNA damage response mediators (RAD17 and RAD50), double-stranded break repair molecule 53BP1, G₂ checkpoint regulators (CHK1 and CHK2), and anti-apoptosis gene survivin. Interestingly, there are no changes of DNA repair molecules H₂AX, BRCA1, and the telomere maintenance gene, hTERT. ΔB-U937, where U937 cells stably transfected with deleted basic domain of TRF2 is partially sensitive to Pu-27 but exhibits no changes in expression of shelterin proteins. However, there is an up-regulation of CHK1, CHK2, H₂AX, BRCA1, and survivin. Telomere dysfunction-induced foci assay revealed co-association of TRF1with γ-H2AX in ATM deficient cells, which are differentially sensitive to Pu-27 than ATM proficient cells. Alt (alternating lengthening of telomere) cells are relatively resistant to Pu-27, but there are no significant changes of telomerase activity in both Alt and non-Alt cells. Lastly, we show that this Pu-27-mediated sensitivity is p53-independent. The data therefore support two conclusions. First, Pu-27 induces DNA damage within both telomeric and nontelomeric regions of the genome. Second, Pu-27-mediated telomeric damage is due, at least in part, to compromise of the telomeric shelterin protein complex.