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Supplementary Figure 1 from Functional Interplay of p53 and Mus81 in DNA Damage Responses and Cancer

Supplementary Figure 4 from Functional Interplay of p53 and Mus81 in DNA Damage Responses and Cancer

Supplementary Tables 1-2 from Functional Interplay of p53 and Mus81 in DNA Damage Responses and Cancer

Supplementary Figure Legends 1-4 from Functional Interplay of p53 and Mus81 in DNA Damage Responses and Cancer

Supplementary Figure 3 from Functional Interplay of p53 and Mus81 in DNA Damage Responses and Cancer

Supplementary Figure 2 from Functional Interplay of p53 and Mus81 in DNA Damage Responses and Cancer

Mus81 plays an integral role in the maintenance of genome stability and DNA repair in mammalian cells. Deficiency of Mus81 in human and mouse cells results in hypersensitivity to interstrand cross-linking (ICL) agents and elevated levels of genomic instability. Furthermore, Mus81-mutant mice are susceptible to spontaneous lymphomas. The role of cellular checkpoints in mediating the phenotypes observed in Mus81-deficient cells and mice is currently unknown. In this study, we have observed increased activation of p53 in Mus81−/− cells in response to ICL-induced DNA damage. In addition, p53 inactivation completely rescued the ICL hypersensitivity of Mus81−/− cells, signifying p53 is essential for the elimination of ICL-damaged cells in the absence of Mus81. Confirming that p53 acts as a critical checkpoint for the Mus81 repair pathway, a synergistic increase of spontaneous and ICL-induced genomic instability was observed in Mus81−/−p53−/− cells. To clarify the genetic interactions of Mus81 and p53 in tumor suppression, we monitored Mus81−/−p53−/− and control mice for the development of spontaneous tumors. Significantly, we show that loss of even a single allele of Mus81 drastically modifies the tumor spectrum of p53-mutant mice and increases their predisposition to developing sarcomas. Our results reveal a key role for p53 in mediating the response to spontaneous and ICL-induced DNA damage that occurs in the absence of Mus81. Furthermore, our data show that loss of Mus81, in addition to p53, is a key step in sarcoma development. [Cancer Res 2007;67(18):8527–35]

DNA damage triggers Atm- and/or Atr-dependent signaling pathways to control cell cycle progression, apoptosis, and DNA repair. However, how Atm and Atr are activated is not fully understood. One of the downstream targets of Atm is non-receptor tyrosine kinase c-Abl, which is phosphorylated and activated by Atm. The current view is that c-Abl relays pro-apoptotic signals from Atm to p73 and p53. Here we show that c-Abl deficiency resulted in a broad spectrum of defects in cell response to genotoxic stress, including activation of Chk1 and Chk2, activation of p53, nuclear foci formation, apoptosis, and DNA repair, suggesting that c-Abl might also act upstream of the DNA damage-activated signaling cascades in addition to its role in p73 and p53 regulation. Indeed, we found that c-Abl is required for proper activation of both Atm and Atr. c-Abl is bound to the chromatin and shows enhanced interaction with Atm and Atr in response to DNA damage. c-Abl can phosphorylate Atr on Y291 and Y310 and this phosphorylation appears to have a positive role in Atr activation under genotoxic stress. These findings suggest that Atm-mediated c-Abl activation in cell response to double-stranded DNA breaks might facilitate the activation of both Atm and Atr to regulate their downstream cellular events.

Ataxia Telangiectasia Mutated Protein (ATM) is one of the first DNA damage sensors and is involved in telomere repair. Telomeres help maintain the stability of our chromosomes by protecting their ends from degradation. AT patients lacking the gene ATM are susceptible to acquire chromosomal anomalies and show heightened susceptibility to cancer. Here we show that cells from AT patients display considerable telomere attrition. Further, induced DNA damage and genomic instability were found to be more in DNA repair deficient ATM-/- cells (treated and untreated) than in normal cells. Results demonstrate that the cells ATM- deficient (heterozygous and homozygous) cells are sensitive to arsenite- and radiation-induced oxidative stress. Elevated numbers of chromosome alterations was seen in arsenic-treated and irradiated ATM-/- cells. The results might help in understanding the extent of susceptibility of AT patients to oxidative stress., Acta medica Nagasakiensia, 53(supl.), pp.45-48 ; 2009

Cancer stem cells have been shown to initiate and sustain tumor growth. In many instances, clinical material is limited, compounded by a lack of methods to preserve such cells at convenient time points. Although brain tumor-initiating cells grown in a spheroid manner have been shown to maintain their integrity through serial transplantation in immune-compromised animals, practically, it is not always possible to have access to animals of suitable ages to continuously maintain these cells. We therefore explored vitrification as a cryopreservation technique for brain tumor-initiating cells. Tumor neurospheres were derived from five patients with glioblastoma multiforme (GBM). Cryopreservation in 90% serum and 10% dimethyl sulfoxide yielded greatest viability and could be explored in future studies. Vitrification yielded cells that maintained self-renewal and multipotentiality properties. Karyotypic analyses confirmed the presence of GBM hallmarks. Upon implantation into NOD/SCID mice, our vitrified cells reformed glioma masses that could be serially transplanted. Transcriptome analysis showed that the vitrified and nonvitrified samples in either the stem-like or differentiated states clustered together, providing evidence that vitrification does not change the genotype of frozen cells. Upon induction of differentiation, the transcriptomes of vitrified cells associated with the original primary tumors, indicating that tumor stem-like cells are a genetically distinct population from the differentiated mass, underscoring the importance of working with the relevant tumor-initiating population. Our results demonstrate that vitrification of brain tumor-initiating cells preserves the biological phenotype and genetic profiles of the cells. This should facilitate the establishment of a repository of tumor-initiating cells for subsequent experimental designs.

A study was undertaken to correlate telomere dysfunction and genomic instability with the histopathological grades and the estrogen and progesterone receptor status in breast cancer. Sixty-one archived breast tissues (38 cancer tissues and 23 paired normal tissues) were used in the study. The breast tumor tissues showed significantly shorter telomeres (7.7 kb) compared with the paired adjacent tissues (9.0 kb) by Southern blot analysis. Moreover, telomere shortening was more significant in Grade III tumors than in the Grade II tumors (P 5 0.05). Quantitative fluorescence in situ hybridization on paraffin tissue sections revealed a similar trend in telomere shortening. Telomere attrition was associated with telomere dysfunction as revealed by the presence of significantly higher anaphase bridges in tumor cells which was tumor grade dependent. Furthermore, estrogen receptive negative tumors displayed higher anaphase and internuclear bridges. Selected samples from each grade showed greater genomic imbalances in the higher grades than the lower grade tumors as detected by array-comparative genomic hybridization. Telomerase activity was found to be higher in the higher grades (Grade II and III) compared with the lower grade (Grade I). The average mRNA expression of TRF1 and POT1 was lower in the tumor tissues than in the normal tissues. Tankyrase 1 mRNA expression showed a grade-dependent increase in tumor tissues and its expression was also high in estrogen and progesterone negative tumors. The data support the notion that telomere dysfunction might be of value as a marker of aggressiveness of the tumors in breast cancer patients. V C 2008 Wiley-Liss, Inc.

Mus81 plays an integral role in the maintenance of genome stability and DNA repair in mammalian cells. Deficiency of Mus81 in human and mouse cells results in hypersensitivity to interstrand cross-linking (ICL) agents and elevated levels of genomic instability. Furthermore, Mus81-mutant mice are susceptible to spontaneous lymphomas. The role of cellular checkpoints in mediating the phenotypes observed in Mus81-deficient cells and mice is currently unknown. In this study, we have observed increased activation of p53 in Mus81−/− cells in response to ICL-induced DNA damage. In addition, p53 inactivation completely rescued the ICL hypersensitivity of Mus81−/− cells, signifying p53 is essential for the elimination of ICL-damaged cells in the absence of Mus81. Confirming that p53 acts as a critical checkpoint for the Mus81 repair pathway, a synergistic increase of spontaneous and ICL-induced genomic instability was observed in Mus81−/−p53−/− cells. To clarify the genetic interactions of Mus81 and p53 in tumor suppression, we monitored Mus81−/−p53−/− and control mice for the development of spontaneous tumors. Significantly, we show that loss of even a single allele of Mus81 drastically modifies the tumor spectrum of p53-mutant mice and increases their predisposition to developing sarcomas. Our results reveal a key role for p53 in mediating the response to spontaneous and ICL-induced DNA damage that occurs in the absence of Mus81. Furthermore, our data show that loss of Mus81, in addition to p53, is a key step in sarcoma development. [Cancer Res 2007;67(18):8527–35]

Telomeres and telomerase appear to participate in the repair of broken DNA ends produced by oxidative damage. Arsenite is an environmental contaminant and a potent human carcinogen, which induces oxidative stress on cells via the generation of reactive oxygen species affecting cell viability and chromosome stability. It promotes telomere attrition and reduces cell survival by apoptosis. In this study, we used mouse embryonic fibroblasts (MEFs) from mice lacking telomerase RNA component (mTERC(-/-) mice) with long (early passage or EP) and short (late passage or LP) telomeres to investigate the extent of oxidative damage by comparing the differences in DNA damage, chromosome instability, and cell survival at 24 and 48 h of exposure to sodium arsenite (As3+; NaAsO2). There was significantly high level of DNA damage in mTERC(-/-) cells with short telomeres as determined by alkaline comet assay. Consistent with elevated DNA damage, increased micronuclei (MN) induction reflecting gross genomic instability was also observed. Fluorescence in situ hybridization (FISH) analysis revealed that increasing doses of arsenite augmented the chromosome aberrations, which contributes to genomic instability leading to possibly apoptotic cell death and cell cycle arrest. Microarray analysis has revealed that As3+ treatment altered the expression of 456 genes of which 20% of them have known functions in cell cycle and DNA damage signaling and response, cell growth, and/or maintenance. Results from our studies imply that short dysfunctional telomeres impair the repair of oxidative damage caused by arsenite. The results will have implications in risk estimation as well as cancer chemotherapy.

Previously, we demonstrated that undifferentiated human embryonic stem cells (hESC) displayed higher resistance to oxidative and genotoxic stress compared to somatic cells, but did not further probe the underlying mechanisms. Using H₂O₂-induced genotoxicity as a model, this study investigated whether higher resistance of hESC to oxidative and genotoxic stress could be due to lower innate basal intracellular levels of reactive oxygen species (ROS), as compared to their differentiated fibroblastic progenies (H1F) and two other somatic cell types - human embryonic palatal mesenchymal (HEPM) cells and peripheral blood lymphocytes (PBL). Comet assay demonstrated that undifferentiated hESC consistently sustained lower levels of DNA damage upon acute exposure to H₂O₂ for 30 min, compared to somatic cells. DCFDA and HE staining with flow cytometry showed that undifferentiated hESC had lower innate basal intracellular levels of reactive oxygen species compared to somatic cells, which could lead to their higher resistance to genotoxic stress upon acute exposure to H₂O₂.

Some immortal cells use the alternative lengthening of telomeres (ALT) pathway to maintain their telomeres instead of telomerase. Previous studies revealed that homologous recombination (HR) contributes to the ALT pathway. To further elucidate molecular mechanisms, we inactivated Rad54 involved in HR, in mouse ALT embryonic stem (ES) cells. Although Rad54-deficient ALT ES cells showed radiosensitivity in line with expectation, cell growth and telomeres were maintained for more than 200 cell divisions. Furthermore, although MMC-stimulated sister chromatid exchange (SCE) was suppressed in the Rad54-deficient ALT ES cells, ALT-associated telomere SCE was not affected. This is the first genetic evidence that mouse Rad54 is dispensable for the ALT pathway.

Arsenite (As3+) has long been known to induce cancer and other degenerative diseases. Arsenite exerts its toxicity in part by generating reactive oxygen species. Identification of genetic factors that contribute to arsenic mutagenicity and carcinogenicity is critical for the treatment and prevention of arsenic exposure in human population. As poly(ADP-ribose) polymerase (PARP) is critical for genomic DNA stability, role of PARP-1 was evaluated in arsenic-induced cytotoxic and genotoxic effects. Our study revealed that telomere attrition, probably owing to arsenite-induced oxidative stress, was much more pronounced in PARP-1−/− mouse embryonic fibroblasts (MEF; 40%) compared with PARP-1+/+ MEFs (10-20%). Correlation observed between telomere reduction and apoptotic death in PARP-1 null cells strongly indicates that the telomere attrition might be a trigger for enhanced apoptotic death after arsenite treatment. Elevated DNA damage detected by alkaline comet assay points to an impaired repair ability of arsenite-induced DNA lesions in PARP-1−/− MEFs. Consistent with elevated DNA damage, increased micronuclei induction reflecting gross genomic instability was also observed in arsenite-treated PARP-1−/− MEFs. Microarray analysis has revealed that arsenite treatment altered the expression of about 311 genes majority of which have known functions in cellular responses to stress/external stimulus and cell growth and/or maintenance. Our results suggest an important role for PARP-1 gene product in the maintenance of chromosome-genome stability in response to arsenite-induced DNA damage.

Neurospheres are widely used to propagate and investigate neural stem cells (NSCs) and neural progenitors (NPs). However, the exact cell types present within neurospheres are still unknown. To identify cell types, we used single-cell mRNA profiling of 48 genes in 187 neurosphere cells. Using a clustering algorithm, we identified 3 discrete cell populations within neurospheres. One cell population [cluster unsorted (US) 1] expresses high Bmi1 and Hes5 and low Myc and Klf12. Cluster US2 shows intermediate expression of most of the genes analyzed. Cluster US3 expresses low Bmi1 and Hes5 and high Myc and Klf12. The mRNA profiles of these 3 cell populations correlate with a developmental timeline of early, intermediate, and late NPs, as seen in vivo from the mouse brain. We enriched the cell population for neurosphere-forming cells (NFCs) using morphological criteria of forward scatter (FSC) and side scatter (SSC). FSC/SSC(high) cells generated 2.29-fold more neurospheres than FSC/SSC(low) cells at clonal density. FSC/SSC(high) cells were enriched for NSCs and Lewis-X(+ve) cells, possessed higher phosphacan levels, and were of a larger cell size. Clustering of both FSC/SSC(high) and FSC/SSC(low) cells identified an NFC cluster. Significantly, the mRNA profile of the NFC cluster drew close resemblance to that of early NPs. Taken together, data suggest that the neurosphere culture system can be used to model central nervous system development, and that early NPs are the cell population that gives rise to neurospheres. In future work, it may be possible to further dissect the NFCs and reveal the molecular signature for NSCs.

Understanding how autocrine/paracrine factors regulate neural stem cell (NSC) survival and growth is fundamental to the utilization of these cells for therapeutic applications and as cellular models for the brain. In vitro, NSCs can be propagated along with neural progenitors (NPs) as neurospheres (nsphs). The nsph conditioned medium (nsph-CM) contains cell-secreted factors that can regulate NSC behavior. However, the identity and exact function of these factors within the nsph-CM has remained elusive. We analyzed the nsph-CM by mass spectrometry and identified DSD-1-proteoglycan, a chondroitin sulfate proteoglycan (CSPG), apolipoprotein E (ApoE) and cystatin C as components of the nsph-CM. Using clonal assays we show that CSPG and ApoE are responsible for the ability of the nsph-CM to stimulate nsph formation whereas cystatin C is not involved. Clonal nsphs generated in the presence of CSPG show more than four-fold increase in NSCs. Thus CSPG specifically enhances the survival of NSCs. CSPG also stimulates the survival of embryonic stem cell (ESC)-derived NSCs, and thus may be involved in the developmental transition of ESCs to NSCs. In addition to its role in NSC survival, CSPG maintains the three dimensional structure of nsphs. Lastly, CSPG's effects on NSC survival may be mediated by enhanced signaling via EGFR, JAK/STAT3 and PI3K/Akt pathways.

The central nervous system (CNS) is a large network of interconnecting and intercommunicating cells that form functional circuits. Disease and injury of the CNS are prominent features of the healthcare landscape. There is an urgent unmet need to generate therapeutic solutions for CNS disease/injury. To increase our understanding of the CNS we need to generate cellular models that are experimentally tractable. Neural stem cells (NSCs), cells that generate the CNS during embryonic development, have been identified and propagated in vitro. To develop NSCs as a cellular model for the CNS we need to understand more about their genetics and cell biology. In particular, we need to define the mechanisms of self-renewal, proliferation and differentiation--i.e. NSC behavior. The analysis of pluripotency of embryonic stem cells through mapping regulatory networks of transcription factors has proven to be a powerful approach to understanding embryonic development. Here, we discuss the role of transcription factors in NSC behavior.

The use of human embryonic stem (hES) cells in genotoxicity screening can potentially overcome the deficiencies associated with using immortalized cell lines, primary explanted somatic cells, and live animal models. Hence this study sought to compare the responses of hES cells and primary explanted somatic cells (IMR-90 cells, human fetal lung fibroblasts) to genotoxic stress, to evaluate whether hES cells can accurately reflect the normal physiology of human somatic cells. The effects of mitomycin C (MMC) on the chromosomal stability of hESC and IMR-90 was assayed and compared by fluorescence in situ hybridization (FISH) with telomere-specific peptide nucleic acid and multicolor (m) FISH techniques. The results showed that, the percentage of aberrant cells increased from 6% in the untreated control to 57.5% at the higher dose of 0.06 microg/ml MMC (9.6-fold increase) group in the case of IMR-90 cells, whereas hES cells displayed a corresponding increase from 6% to 28% (4.6-fold increase). Telomere FISH ascertained that the main types of damage induced by MMC are chromosomal breaks and the loss of telomeric signals. No fusions were observed in all samples analyzed. This was further confirmed by mFISH, which showed that fusions and translocations were not the type of aberration induced by MMC, with no such aberrations being observed in all samples analyzed. Hence, hES cells of the H1 line are apparently more resistant to MMC-induced DNA damage, as compared to the IMR-90 cells. These results highlight possible intrinsic differences in response to damaging agents between hES cells and normal somatic cells.

We compared cryopreservation of mammalian neural stem cells (NSCs) cultured as neurospheres by slow-cooling (1 C/min) in 10% (v/v) DMSO and cryopreservation by immersion into liquid nitrogen in ethylene glycol (EG)-sucrose solutions that support vitrification (40% (v/v) EG, 0.6 M sucrose) or that do not (37% v/v) EG, 0.6 M sucrose and 30% (v/v) EG, 0.6 M sucrose); the concentration of penetrating cryoprotectant in the last two solutions was lowered with the intention to reduce their toxicity towards NSCs. To protect against contamination a straw-in-straw technique was employed. Vitrification offered the best combination of preservation of structural integrity of neurospheres, cell viability (96%), multipotency and karyotype. Rapid cooling in 37% (v/v) EG, 0.6 M sucrose afforded good viability but did not preserve structural integrity. Rapid cooling in 30% (v/v) EG, 0.6 M sucrose additionally reduced cell viability to 77%. Slow-cooling reduced cell viability to 65% and damaged the neurospheres. This study suggests that, in contrast to freezing, vitrification has immense potential for the cryopreservation of stem cells cultured as neurospheres or in other structured cultures.

Understanding the molecular etiology and heterogeneity of disease has a direct effect on cancer therapeutics. To identify novel molecular changes associated with breast cancer progression, we conducted phosphoproteomics of the MCF10AT model comprising isogenic, ErbB2- and ErbB3-positive, xenograft-derived cell lines that mimic different stages of breast cancer. Using in vitro animal model and clinical breast samples, our study revealed a marked reduction of epidermal growth factor receptor (EGFR) expression with breast cancer progression. Such diminution of EGFR expression was associated with increased resistance to Gefitinib/Iressa in vitro. Fluorescence in situ hybridization showed that loss of EGFR gene copy number was one of the key mechanisms behind the low/null expression of EGFR in clinical breast tumors. Statistical analysis on the immunohistochemistry data of EGFR expression from 93 matched normal and breast tumor samples showed that (a) diminished EGFR expression could be detected as early as in the preneoplastic lesion (ductal carcinoma in situ) and this culminated in invasive carcinomas; (b) EGFR expression levels could distinguish between normal tissue versus carcinoma in situ and invasive carcinoma with high statistical significance (P < 0.001, n = 81). However, no significant correlation of EGFR expression with disease-free survival and overall survival was observed. This is the first time EGFR expression has been tracked meaningfully and developmentally from the normal condition through disease progression using in vitro, xenograft, and matched normal and tumor samples. Thus, our study provides a new insight into the role of EGFR in breast cancer development. Although no value of EGFR expression in prognosis was found, our findings are likely to have implications in the design of clinical trials targeting the EGFR family of proteins in breast cancer. [Mol Cancer Ther 2007;6(11):2828–42]

The role of BRCA1 in breast and ovarian tumor suppression has been primarily ascribed to the maintenance of genome integrity. BRCA1 interacts with components of the non-homologous end-joining pathway previously shown to play a role in telomere maintenance in yeast. Here, we provide evidence that links Brca1 with telomere integrity. Brca1(-/-) T-cells display telomere dysfunction in both loss of telomere repeats as well as defective telomere capping. Loss of Brca1 synergizes with p53 deficiency in the onset and frequency of tumorigenesis. Karyotyping of tBrca1(-/-)p53(-/-) thymic lymphomas revealed the presence of telomere dysfunction accompanied by clonal chromosomal translocations. The telomere dysfunction phenotype in Brca1-deficient cells suggests that loss of telomere integrity might contribute to chromosome end dysfunction and permit the formation of potentially oncogenic translocations.

Mus81-Eme1 endonuclease has been implicated in the rescue of stalled replication forks and the resolution of meiotic recombination intermediates in yeast. We used gene targeting to study the physiological requirements of Mus81 in mammals. Mus81 –/– mice are viable and fertile, which indicates that mammalian Mus81 is not essential for recombination processes associated with meiosis. Mus81-deficient mice and cells were hypersensitive to the DNA cross-linking agent mitomycin C but not to γ-irradiation. Remarkably, both homozygous Mus81 –/– and heterozygous Mus81 +/– mice exhibited a similar susceptibility to spontaneous chromosomal damage and a profound and equivalent predisposition to lymphomas and other cancers. These studies demonstrate a critical role for the proper biallelic expression of the mammalian Mus81 in the maintenance of genomic integrity and tumor suppression.