Optimization of Class I Histone Deacetylase PROTACs Reveals that HDAC1/2 Degradation is Critical to Induce Apoptosis and Cell Arrest in Cancer Cells.

Class I histone deacetylase (HDAC) enzymes 1, 2, and 3 organize chromatin as the catalytic subunits within seven distinct multiprotein corepressor complexes and are established drug targets. We report optimization studies of benzamide-based Von Hippel-Lindau (VHL) E3-ligase proteolysis targeting chimeras (PROTACs) and for the first time describe transcriptome perturbations resulting from these degraders. By modifying the linker and VHL ligand, we identified PROTACs 7, 9, and 22 with submicromolar DC50 values for HDAC1 and/or HDAC3 in HCT116 cells. A hook effect was observed for HDAC3 that could be negated by modifying the position of attachment of the VHL ligand to the linker. The more potent HDAC1/2 degraders correlated with greater total differentially expressed genes and enhanced apoptosis in HCT116 cells. We demonstrate that HDAC1/2 degradation by PROTACs correlates with enhanced global gene expression and apoptosis, important for the development of more efficacious HDAC therapeutics with reduced side effects.

Comet assay measures of DNA damage as biomarkers of irinotecan response in colorectal cancer in vitro and in vivo.

The use of irinotecan to treat metastatic colorectal cancer (CRC) is limited by unpredictable response and variable toxicity; however, no reliable clinical biomarkers are available. Here, we report a study to ascertain whether irinotecan-induced DNA damage measures are suitable/superior biomarkers of irinotecan effect. CRC-cell lines (HCT-116 and HT-29) were treated in vitro with irinotecan and peripheral blood lymphocytes (PBL) were isolated from patients before and after receiving irinotecan-based chemotherapy. Levels of in vitro-, in vivo-, and ex vivo-induced DNA damage were measured using the Comet assay; correlations between damage levels with in vitro cell survival and follow-up clinical data were investigated. Irinotecan-induced DNA damage was detectable in both CRC cell-lines in vitro, with higher levels of immediate and residual damage noted for the more sensitive HT-29 cells. DNA damage was not detected in vivo, but was measurable in PBLs upon mitogenic stimulation prior to ex vivo SN-38 treatment. Results showed that, following corrections for experimental error, those patients whose PBLs demonstrated higher levels of DNA damage following 10 h of SN-38 exposure ex vivo had significantly longer times to progression than those with lower damage levels (median 291 vs. 173 days, P = 0.014). To conclude, higher levels of irinotecan-induced initial and residual damage correlated with greater cell kill in vitro and a better clinical response. Consequently, DNA damage measures may represent superior biomarkers of irinotecan effect compared to the more often-studied genetic assays for differential drug metabolism.

Comet assay measures of DNA damage are predictive of bladder cancer cell treatment sensitivity in vitro and outcome in vivo.

Bladder cancer patients suffer significant treatment failure, including high rates of recurrence and poor outcomes for advanced disease. If mechanisms to improve tumour cell treatment sensitivity could be identified and/or if tumour response could be predicted, it should be possible to improve local-control and survival. Previously, we have shown that radiation-induced DNA damage, measured by alkaline Comet assay (ACA), correlates bladder cancer cell radiosensitivity in vitro. In this study we first show that modified-ACA measures of cisplatin and mitomycin-C-induced damage also correlate bladder cancer cell chemosensitivity in vitro, with essentially the same rank order for chemosensitivity as for radiosensitivity. Furthermore, ACA studies of radiation-induced damage in different cell-DNA substrates (nuclei, nucleoids and intact parent cells) suggest that it is a feature retained in the prepared nucleoids that is responsible for the relative damage sensitivity of bladder cancer cells, suggestive of differences in the organisation of DNA within resistant vs. sensitive cells. Second, we show that ACA analysis of biopsies from bladder tumours reveal that reduced DNA damage sensitivity associates with poorer treatment outcomes, notably that tumours with a reduced damage response show a significant association with local recurrence of non-invasive disease and that reduced damage response was a better predictor of recurrence than the presence of high-risk histology in this cohort. In conclusion, this study demonstrates that mechanisms governing treatment-induced DNA damage are both central to and predictive of bladder cancer cell treatment sensitivity and exemplifies a link between DNA damage resistance and both treatment response and tumour aggression.

Simplified method for the collection, storage, and comet assay analysis of DNA damage in whole blood.

Single-cell gel electrophoresis (comet assay) is one of the most common methods used to measure oxidatively damaged DNA in peripheral blood mononuclear cells (PBMC), as a biomarker of oxidative stress in vivo. However, storage, extraction, and assay workup of blood samples are associated with a risk of artifactual formation of damage. Previous reports using this approach to study DNA damage in PBMC have, for the most part, required the isolation of PBMC before immediate analysis or freezing in cryopreservative. This is very time-consuming and a significant drain on human resources. Here, we report the successful storage of whole blood in ~250 µl volumes, at -80°C, without cryopreservative, for up to 1 month without artifactual formation of DNA damage. Furthermore, this blood is amenable for direct use in both the alkaline and the enzyme-modified comet assay, without the need for prior isolation of PBMC. In contrast, storage of larger volumes (e.g., 5 ml) of whole blood leads to an increase in damage with longer term storage even at -80°C, unless a cryopreservative is present. Our "small volume" approach may be suitable for archived blood samples, facilitating analysis of biobanks when prior isolation of PBMC has not been performed.

DNA strand breaks and hypoxia response inhibition mediate the radiosensitisation effect of nitric oxide donors on prostate cancer under varying oxygen conditions.

Prostate cancer cells can exist in a hypoxic microenvironment, causing radioresistance. Nitric oxide (NO) is a radiosensitiser of mammalian cells. NO-NSAIDs are a potential means of delivering NO to prostate cancer cells. This study aimed to determine the effect and mechanism of action of NO-sulindac and radiation, on prostate cancer cells and stroma, under normoxia (21% oxygen) and chronic hypoxia (0.2% oxygen). Using clonogenic assays, at a surviving fraction of 10% the sensitisation enhancement ratios of radiation plus NO-sulindac over radiation alone on PC-3 cells were 1.22 and 1.42 under normoxia and hypoxia, respectively. 3D culture of PC-3 cells revealed significantly reduced sphere diameter in irradiated spheres treated with NO-sulindac. Neither NO-sulindac nor sulindac radiosensitised prostate stromal cells under normoxia or hypoxia. HIF-1α protein levels were reduced by NO-sulindac exposure and radiation at 21 and 0.2% oxygen. Alkaline Comet assay analysis suggested an increased rate of single strand DNA breaks and slower repair of these lesions in PC-3 cells treated with NO-sulindac prior to irradiation. There was a higher level of γ-H2AX production and hence double strand DNA breaks following irradiation of NO-sulindac treated PC-3 cells. At all radiation doses and oxygen levels tested, treatment of 2D and 3D cultures of PC-3 cells with NO-sulindac prior to irradiation radiosensitised PC-3, with minimal effect on stromal cells. Hypoxia response inhibition and increased DNA double strand breaks are potential mechanisms of action. Neoadjuvent and concurrent use of NO-NSAIDs have the potential to improve radiotherapy treatment of prostate cancer under normoxia and hypoxia.

Pediatric brain tumor cancer stem cells: cell cycle dynamics, DNA repair, and etoposide extrusion.

Reliable model systems are needed to elucidate the role cancer stem cells (CSCs) play in pediatric brain tumor drug resistance. The majority of studies to date have focused on clinically distinct adult tumors and restricted tumor types. Here, the CSC component of 7 newly established primary pediatric cell lines (2 ependymomas, 2 medulloblastomas, 2 gliomas, and a CNS primitive neuroectodermal tumor) was thoroughly characterized. Comparison of DNA copy number with the original corresponding tumor demonstrated that genomic changes present in the original tumor, typical of that particular tumor type, were retained in culture. In each case, the CSC component was approximately 3-4-fold enriched in neurosphere culture compared with monolayer culture, and a higher capacity for multilineage differentiation was observed for neurosphere-derived cells. DNA content profiles of neurosphere-derived cells expressing the CSC marker nestin demonstrated the presence of cells in all phases of the cell cycle, indicating that not all CSCs are quiescent. Furthermore, neurosphere-derived cells demonstrated an increased resistance to etoposide compared with monolayer-derived cells, having lower initial DNA damage, potentially due to a combination of increased drug extrusion by ATP-binding cassette multidrug transporters and enhanced rates of DNA repair. Finally, orthotopic xenograft models reflecting the tumor of origin were established from these cell lines. In summary, these cell lines and the approach taken provide a robust model system that can be used to develop our understanding of the biology of CSCs in pediatric brain tumors and other cancer types and to preclinically test therapeutic agents.

Protection of cells in physiological oxygen tensions against DNA damage-induced apoptosis.

Oxygen availability has important effects on cell physiology. Although hyperoxic and hypoxic stresses have been well characterized, little is known about cellular functions in the oxygen levels commonly found in vivo. Here, we show that p53-dependent apoptosis in response to different DNA-damaging agents was reduced when normal and cancer cells were cultured at physiological oxygen tensions instead of the usual atmospheric levels. Different from what has been described in hypoxia, this was neither determined by decreases in p53 induction or its transactivation activity, nor by differences in the intracellular accumulation of reactive oxygen species. At these physiological oxygen levels, we found a constitutive activation of the ERK1/2 MAPK in all the models studied. Inhibition of this signaling pathway reversed the protective effect in some but not all cell lines. We conclude that a stress-independent constitutive activation of prosurvival pathways, including but probably not limited to MAPK, can protect cells in physiological oxygen tensions against genotoxic stress. Our results underscore the need of considering the impact of oxygen levels present in the tissue microenvironment when studying cell sensitivity to treatments such as chemotherapy and radiotherapy.

DNA damage in earthworms from highly contaminated soils: assessing resistance to arsenic toxicity by use of the Comet assay.

Earthworms native to the former mine site of Devon Great Consols (DGC), UK reside in soils highly contaminated with arsenic (As). These earthworms are considered to have developed a resistance to As toxicity. The mechanisms underlying this resistance however, remain unclear. In the present study, non-resistant, commercially sourced Lumbricus terrestris were exposed to a typical DGC soil in laboratory mesocosms. The earthworms bio-accumulated As from the soil and incurred DNA-damage levels significantly above those observed in the control mesocosm (assessed using the Comet assay). A dose response was observed between DNA damage (% tail DNA) and As concentration in soil (control, 98, 183, 236, 324 and 436mgkg(-1)). As-resistant earthworms (Lumbricus rubellus, Dendrodrilus rubidus and L. terrestris) collected from contaminated soils at DGC (203 to 9025mgkg(-1) As) had also bio-accumulated high levels of As from their host soils, yet demonstrated low levels of DNA damage compared with earthworms from uncontaminated sites. The results demonstrate that the As-contaminated soils at DGC are genotoxic to non-native earthworms and much less so to earthworms native to DGC, thus providing further evidence of an acquired resistance to As toxicity in the native earthworms.

Introducing a true internal standard for the Comet assay to minimize intra- and inter-experiment variability in measures of DNA damage and repair.

The Comet assay (CA) is a sensitive/simple measure of genotoxicity. However, many features of CA contribute variability. To minimize these, we have introduced internal standard materials consisting of 'reference' cells which have their DNA substituted with BrdU. Using a fluorescent anti-BrdU antibody, plus an additional barrier filter, comets derived from these cells could be readily distinguished from the 'test'-cell comets, present in the same gel. In experiments to evaluate the reference cell comets as external and internal standards, the reference and test cells were present in separate gels on the same slide or mixed together in the same gel, respectively, before their co-exposure to X-irradiation. Using the reference cell comets as internal standards led to substantial reductions in the coefficient of variation (CoV) for intra- and inter-experimental measures of comet formation and DNA damage repair; only minor reductions in CoV were noted when the reference and test cell comets were in separate gels. These studies indicate that differences between individual gels appreciably contribute to CA variation. Further studies using the reference cells as internal standards allowed greater significance to be obtained between groups of replicate samples. Ultimately, we anticipate that development will deliver robust quality assurance materials for CA.

SIP1 protein protects cells from DNA damage-induced apoptosis and has independent prognostic value in bladder cancer.

The epithelial-mesenchymal transition (EMT) contributes to cancer metastasis. Two ZEB family members, ZEB1 and ZEB2(SIP1), inhibit transcription of the E-cadherin gene and induce EMT in vitro. However, their relevance to human cancer is insufficiently studied. Here, we performed a comparative study of SIP1 and ZEB1 proteins in cancer cell lines and in one form of human malignancy, carcinoma of the bladder. Whereas ZEB1 protein was expressed in all E-cadherin-negative carcinoma cell lines, being in part responsible for the high motility of bladder cancer cells, SIP1 was hardly ever detectable in carcinoma cells in culture. However, SIP1 represented an independent factor of poor prognosis (P = 0.005) in a series of bladder cancer specimens obtained from patients treated with radiotherapy. In contrast, ZEB1 was rarely expressed in tumor tissues; and E-cadherin status did not correlate with the patients' survival. SIP1 protected cells from UV- and cisplatin-induced apoptosis in vitro but had no effect on the level of DNA damage. The anti-apoptotic effect of SIP1 was independent of either cell cycle arrest or loss of cell-cell adhesion and was associated with reduced phosphorylation of ATM/ATR targets in UV-treated cells. The prognostic value of SIP1 and its role in DNA damage response establish a link between genetic instability and metastasis and suggest a potential importance for this protein as a therapeutic target. In addition, we conclude that the nature of an EMT pathway rather than the deregulation of E-cadherin per se is critical for the progression of the disease and patients' survival.

Phosphate alkylation in different DNA substrates: the role of local DNA sequence and electrophile character in determining the nonrandom nature of phosphotriester adduct formation.

DNA phosphate oxygens are sites for alkylation leading to DNA phosphotriester adduct (PTE) formation. Previously, we have reported that the manifestation of PTEs was nonrandom in mouse liver DNA treated in vivo [Guichard et al. (2000) Cancer Res. 60, 1276-1282], and while further studies revealed possible PTE repair, this was determined not to play a role in the observed nonrandom manifestation in vivo [Le Pla et al. (2004) Chem. Res. Toxicol. 17, 1491-1500]. In the present study, to determine whether the nonrandom manifestation of PTEs in vivo was specifically due to their nonrandom formation, we have compared the in vitro formation of diethylsulfate (DES)-induced PTEs in h2E1/OR human B-lymphoblastoid cells, their isolated nuclei, and their isolated DNA, using the 5' nearest neighbor analysis postlabeling procedure developed by Le Pla et al.. Furthermore, to determine the role of electrophile character in PTE manifestation, prepared oligonucleotides ([dT](20)[dG](20):[dC](20)[dA](20)) were treated with three alkylating agents of differing electrophilic character (DES, methylnitrosourea, and ethylnitrosourea), and PTE manifestation was assessed by postlabeling. The formation of PTEs was determined to be nonrandom in the whole cells, nuclei, and DNA, with PTEs being formed to a greater extent 3' to pyrimidine moieties than 3' to purine moieties. The studies with the oligonucleotides confirm these observations and demonstrate that the nonrandom formation of PTEs is primarily determined by DNA sequence, and not by DNA packaging/chromatin factors, and that the extent of the nonrandom formation of PTEs is also governed by electrophile reactivity, with the more reactive electrophiles yielding a more random formation of PTEs. From our observations, we propose a model for the nonrandom formation of PTEs, which is governed by (i) the phosphate oxygens having to compete with adjacent nucleophilic sites for the alkylating electrophile and (ii) the electrophile's inherent reactivity.

Further development of 32P-postlabeling for the detection of alkylphosphotriesters: evidence for the long-term nonrandom persistence of ethyl-phosphotriester adducts in vivo.

DNA phosphate oxygens are sites for alkylation leading to phosphotriester adducts (PTEs). PTEs are reported to be both abundant and persistent and so may serve as long-term markers of genotoxicity. Previously, we reported a 32P-postlabeling assay for the specific detection of PTEs plus identification of nucleosides located 5' to PTEs. Using this, we demonstrated the nonrandom nature of ethyl-PTEs (Et-PTEs) in vivo, these results being suggestive of either the nonrandom formation of Et-PTEs in vivo or sequence specific Et-PTE repair. Presently, we report the further development and validation of the 32P-postlabeling assay, to permit the more straightforward determination of nucleosides 5' to PTEs and, using this, have investigated the long-term persistence of PTEs in vivo. Analysis of liver DNA of mice treated in vivo with N-nitrosodiethylamine reveals an initial decline in the level of Et-PTEs (t1/2<24 h) as well as their nonrandom persistence for the duration of the time course, with approximately 37 and approximately 15% of the initial Et-PTEs remaining 4 and 56 days after treatment, respectively. From this, we conclude that Et-PTEs are suitable as long-term markers of genotoxic exposure and that putative PTE repair is not responsible for their nonrandom manifestation. However, the possibility of active repair contributing to the initial decline of Et-PTEs is considered.

Resistance-modifying agents. 11.(1) Pyrimido[5,4-d]pyrimidine modulators of antitumor drug activity. Synthesis and structure-activity relationships for nucleoside transport inhibition and binding to alpha1-acid glycoprotein.

The cardiovascular and antithrombotic agent dipyridamole (DP) has potential therapeutic utility as a modulator of the activity of antimetabolite antitumor agents by virtue of its inhibition of nucleoside transport. However, the activity of DP can be compromised by binding to the acute phase serum protein, alpha(1)-acid glycoprotein (AGP). Analogues of DP were synthesized and evaluated as inhibitors of (3)H-thymidine uptake into L1210 leukamia cells in the presence and absence of 5 mg/mL AGP. Compounds with potency similar to that of DP were identified where the piperidino substituents at the 4,8-positions were replaced by 4'-methoxybenzylamino, 3',4'-dimethoxybenzylamino, or piperonylamino groups. Replacement of the diethanolamino groups at the 2,6-positions of DP by alkylamino or alkoxy substituents was tolerated, although at least one oxygen-bearing function (hydroxyl or alkoxy) was required in the side chain for activity comparable to that of DP. Whereas AGP completely ablated the activity of DP, the majority of the newer compounds synthesized retained significant activity in the presence of excess AGP, although replacement of the piperidino groups at the 4,8-positions by N-methylbenzylamino substituents did, in some cases, restore susceptibility to AGP. Selected compounds have been demonstrated to prevent rescue from antifolate cytotoxicity, mediated by nucleoside salvage.

Improving and predicting radiosensitivity in muscle invasive bladder cancer.

PURPOSE: Muscle invasive bladder cancer is a common urological malignancy with a relatively poor prognosis and 5-year survival rates ranging from 20% to 90%. We review methods of improving the outcome of this condition, with particular emphasis on the principal bladder preserving treatment modality of radiation therapy. MATERIALS AND METHODS: We performed a literature search using MEDLINE and the ISI Web of Science using the keywords radiotherapy, radiosensitization and bladder neoplasia to ascertain the current status of radiation therapy and radiosensitizing agents in the treatment of muscle invasive bladder cancer. RESULTS: Several methods aimed at improving outcome following radiation therapy for muscle invasive bladder cancer are described. These methods range from modifications in the application of radiation therapy to use of conventional radiosensitizing agents, such as accelerated radiotherapy with carbon dioxide, oxygen and nicotinamide, and finally to use of more novel agents that interact with oncogenic products. The use of assays that predict tumor sensitivity on an individual basis represents an additional potential method to improve prognosis following radiation therapy. CONCLUSIONS: The ability to predict tumor radiosensitivity and the subsequent implementation of radiosensitizing techniques are likely to improve the results of treatment centered on radiation therapy, suggesting that bladder sparing approaches will remain a treatment option for muscle invasive bladder cancer.