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1.  Impact of age and vector construct on striatal and nigral transgene expression 
Therapeutic protein delivery using viral vectors has shown promise in preclinical models of Parkinson’s disease (PD) but clinical trial success remains elusive. This may partially be due to a failure to include advanced age as a covariate despite aging being the primary risk factor for PD. We investigated transgene expression following intracerebral injections of recombinant adeno-associated virus pseudotypes 2/2 (rAAV2/2), 2/5 (rAAV2/5), 2/9 (rAAV2/9), and lentivirus (LV) expressing green fluorescent protein (GFP) in aged versus young adult rats. Both rAAV2/2 and rAAV2/5 yielded lower GFP expression following injection to either the aged substantia nigra or striatum. rAAV2/9-mediated GFP expression was deficient in the aged striatonigral system but displayed identical transgene expression between ages in the nigrostriatal system. Young and aged rats displayed equivalent GFP levels following LV injection to the striatonigral system but LV-delivered GFP was deficient in delivering GFP to the aged nigrostriatal system. Notably, age-related transgene expression deficiencies revealed by protein quantitation were poorly predicted by GFP-immunoreactive cell counts. Further, in situ hybridization for the viral CβA promoter revealed surprisingly limited tropism for astrocytes compared to neurons. Our results demonstrate that aging is a critical covariate to consider when designing gene therapy approaches for PD.
doi:10.1038/mtm.2016.82
PMCID: PMC5142515  PMID: 27933309
2.  Intrastriatal injection of pre-formed mouse α-synuclein fibrils into rats triggers α-synuclein pathology and bilateral nigrostriatal degeneration 
Neurobiology of disease  2015;82:185-199.
Previous studies demonstrate that intrastriatal injections of fibrillar alpha-synuclein (α-syn) into mice induce Parkinson’s disease (PD)-like Lewy body (LB) pathology formed by aggregated α-syn in anatomically interconnected regions and significant nigrostriatal degeneration. The aim of the current study was to evaluate whether exogenous mouse α-syn pre-formed fibrils (PFF) injected into the striatum of rats would result in accumulation of LB-like intracellular inclusions and nigrostriatal degeneration. Sprague Dawley rats received unilateral intrastriatal injections of either non-fibrillized recombinant α-syn or PFF mouse α-syn in 1- or 2- sites and were euthanized at 30, 60 or 180 days post-injection (pi). Both non-fibrillized recombinant α-syn and PFF α-syn injections resulted in phosphorylated α-syn intraneuronal accumulations (i.e., diffuse Lewy neurite (LN)- and LB-like inclusions) with significantly greater accumulations following PFF injection. LB-like inclusions were observed in several areas that innervate the striatum, most prominently the frontal and insular cortices, the amygdala, and the substantia nigra pars compacta (SNpc). α-Syn accumulations co-localized with ubiquitin, p62, and were thioflavin-S-positive and proteinase-k resistant, suggesting PFF-induced pathology exhibits properties similar to human LBs. Although α-syn inclusions within the SNpc remained ipsilateral to striatal injection, we observed bilateral reductions in nigral dopamine neurons at the 180-day time point in both the 1- and 2-site PFF injection paradigms. PFF injected rats exhibited bilateral reductions in striatal dopaminergic innervation at 60 and 180 days and bilateral decreases in homovanillic acid; however, dopamine reduction was observed only in the striatum ipsilateral to PFF injection. Although the level of dopamine asymmetry in PFF injected rats at 180 days was insufficient to elicit motor deficits in amphetamine-induced rotations or forelimb use in the cylinder task, significant disruption of ultrasonic vocalizations was observed. Taken together, our findings demonstrate that α-syn PFF are sufficient to seed the pathological conversion and propagation of endogenous α-syn to induce a progressive, neurodegenerative model of α-synucleinopathy in rats.
doi:10.1016/j.nbd.2015.06.003
PMCID: PMC4640952  PMID: 26093169
alpha-synuclein; Parkinson’s disease; rat model; pre-formed fibrils; neurodegeneration; propagation; rats
3.  Animal models of chemical carcinogenesis: driving breakthroughs in cancer research for 100 years 
Cold Spring Harbor protocols  2015;2015(10):865-874.
Chemical carcinogenesis studies in animals have directly contributed to a reduction of cancer burden in the human population through their ability to identify carcinogens from the workplace, diet, and environment. Reduced exposure to these carcinogens through lifestyle changes, government regulation, or change in industry practices has reduced cancer incidence in exposed populations. In addition to providing the first experimental evidence for the link between chemical and radiation exposure and cancer, animal models of environmentally induced cancer have and will continue to provide important insight into the causes, mechanisms, and conceptual frameworks of cancer. More recently, combining chemical carcinogens with genetically engineered mouse models (GEMMs) has emerged as an invaluable approach to study the complex interaction between genotype and environment that contributes to cancer development. In the future, animal models of environmentally induced cancer are likely to provide insight into areas such as the epigenetic basis of cancer, genetic modifiers of cancer susceptibility, the systems biology of cancer, inflammation and cancer, and cancer prevention.
doi:10.1101/pdb.top069906
PMCID: PMC4949043  PMID: 26430259
4.  Identification of a novel E-box binding PI polyamide inhibiting MYC-driven cell-proliferation 
Cancer science  2015;106(4):421-429.
The MYC transcription factor plays a crucial role in the regulation of cell cycle progression, apoptosis, angiogenesis, and cellular transformation. Due to its oncogenic activities and over expression in a majority of human cancers, it is an interesting target for novel drug therapies. MYC binding to the E-box (5’-CACGTGT-3’) sequence at gene promoters contributes to more than 4,000 MYC-dependent transcripts. Owing to its importance in MYC regulation, we designed a novel sequence-specific DNA-binding Pyrrole-Imidazole (PI) polyamide, Myc-5, that recognizes E-box consensus sequence. Bioinformatics analysis revealed that Myc-5 binding sequence appeared in 5′ MYC binding E-box sequences at the eIF4G1, CCND1 and CDK4 gene promoters. Furthermore Chromatin immunoprecipitation (ChIP) coupled with detection by qPCR demonstrated that Myc-5 has the ability to inhibit MYC-binding at the target gene promoters and thus cause down-regulation at m-RNA level and protein expression of its target gene in human Burkitt's lymphoma model cell line, P493.6, carrying an inducible MYC repression system and K562 (human chronic myelogenous leukaemia) cell lines. Single intravenous injection of Myc-5 at 7.5 mg/kg dose caused a significant tumor-growth inhibition in a MYC-dependent tumor xenograft model without evidence of toxicity. We report here a compelling rationale for the identification of a PI polyamide that inhibits a part of an E-box-mediated MYC downstream gene expression and is a model for showing that phenotype-associated MYC downstream gene targets consequently inhibits MYC dependent tumor growth.
doi:10.1111/cas.12610
PMCID: PMC4406810  PMID: 25611295
cell cycle; E-Box; MYC; pyrrole-imidazole polyamide; transcription therapy
5.  Anthracyclines induce double-strand DNA breaks at active gene promoters 
Mutation research  2015;773:9-15.
Doxorubicin is a widely used chemotherapeutic drug that intercalates between DNA base-pairs and poisons Topoisomerase II, although the mechanistic basis for cell killing remains speculative. Doxorubicin and related anthracycline compounds have been shown to increase nucleosome turnover and/or eviction around promoters, which suggests that the resulting enhanced exposure of DNA might underlie cell killing. Previously, we showed that low doses of anthracyclines increase nucleosome turnover around active gene promoters, which suggests that loss of nucleosomes might contribute to cancer cell killing. Here we apply a genome-wide method to precisely map DNA double-strand breaks (DSBs) in cancer cells. We find that spontaneous DSBs occur preferentially around promoters of active genes, and that both anthracyclines and etoposide, a Topoisomerase II poison, increase DSBs around promoters, although CpG islands are conspicuously protected from DSBs. We propose that torsion-based enhancement of nucleosome turnover by anthracyclines exposes promoter DNA, ultimately causing DSBs around promoters.
doi:10.1016/j.mrfmmm.2015.01.007
PMCID: PMC4332850  PMID: 25705119
DNA double-strand breaks; Doxorubicin; Etoposide; Nucleosome turnover; Squamous cell carcinoma
6.  ARF Inhibits the Growth and Malignant Progression of Non-Small Cell Lung Carcinoma 
Oncogene  2013;33(20):2665-2673.
Non-small cell lung carcinoma (NSCLC) is among the deadliest of human cancers. The CDKN2A locus, which houses the INK4a and ARF tumor suppressor genes, is frequently altered in NSCLC. However, the specific role of ARF in pulmonary tumorigenesis remains unclear. KRAS and other oncogenes induce the expression of ARF, thus stabilizing p53 activity and arresting cell proliferation. To address the role of ARF in Kras-driven NSCLC, we compared the susceptibility of NIH/Ola strain wild-type and Arf knockout mice to urethane-induced lung carcinogenesis. Lung tumor size, malignancy, and associated morbidity were significantly increased in Arf−/− compared to Arf+/+ animals at 25 weeks post-induction. Pulmonary tumors from Arf knockout mice exhibited increased cell proliferation and DNA damage compared to wild-type. A subgroup of tumors in Arf−/− animals presented as dedifferentiated and metastatic, with many characteristics of pulmonary sarcomatoid carcinoma, a neoplasm previously undocumented in mouse models. Our finding of a role for ARF in NSCLC is consistent with the observation that benign adenomas from Arf+/+ mice robustly expressed ARF, while ARF expression was markedly reduced in malignant adenocarcinomas. ARF expression also frequently co-localized with expression of p21CIP1, a transcriptional target of p53, arguing that ARF induces the p53 checkpoint to arrest cell proliferation in vivo. Together, these findings demonstrate that induction of ARF is an early response in lung tumorigenesis that mounts a strong barrier against tumor growth and malignant progression.
doi:10.1038/onc.2013.208
PMCID: PMC3839253  PMID: 23752194
p19Arf; p14ARF; ethyl carbamate; metastasis
7.  Recombinant adeno-associated virus 2/5-mediated gene transfer is reduced in the aged rat midbrain 
Neurobiology of aging  2014;36(2):1110-1120.
Clinical trials are examining the efficacy of viral vector-mediated gene delivery for treating Parkinson’s disease (PD). While viral vector strategies have been successful in preclinical studies, to date clinical trials have disappointed. This may be due to the fact that preclinical studies fail to account for aging. Aging is the single greatest risk factor for developing PD and age alters cellular processes utilized by viral vectors. We hypothesized that the aged brain would be relatively resistant to transduction when compared to the young adult. We examined recombinant adeno-associated virus 2/5 mediated green fluorescent protein (rAAV2/5 GFP) expression in the young adult and aged rat nigrostriatal system. GFP overexpression was produced in both age groups. However, following rAAV2/5 GFP injection to the substantia nigra (SN) aged rats displayed 40-60% less GFP protein in the striatum, regardless of rat strain or duration of expression. Further, aged rats exhibited 40% fewer cells expressing GFP and 4-fold less GFP mRNA. rAAV2/5-mediated gene transfer is compromised in the aged rat midbrain, with deficiencies in early steps of transduction leading to significantly less mRNA and protein expression.
doi:10.1016/j.neurobiolaging.2014.07.047
PMCID: PMC4315740  PMID: 25457558
8.  Functional kinomics identifies candidate therapeutic targets in head and neck cancer 
Purpose
To identify novel therapeutic drug targets for p53 mutant head and neck squamous cell carcinoma (HNSCC).
Experimental Design
RNAi kinome viability screens were performed on HNSCC cells including autologous pairs from primary tumor and recurrent/metastatic lesions, and in parallel on murine squamous cell carcinoma (MSCC) cells derived from tumors of inbred mice bearing germline mutations in Trp53, and p53 regulatory genes: Atm, Prkdc, and p19Arf. Cross-species analysis of cell lines stratified by p53 mutational status and metastatic phenotype was utilized to select 38 kinase targets. Both primary and secondary RNAi validation assays were performed on additional HNSCC cell lines to credential these kinase targets utilizing multiple phenotypic endpoints. Kinase targets were also examined via chemical inhibition utilizing a panel of kinase inhibitors. A preclinical study was conducted on the WEE1 kinase inhibitor, MK-1775.
Results
Our functional kinomics approach identified novel survival kinases in HNSCC involved in G2/M cell cycle checkpoint, SFK, PI3K and FAK pathways. RNAi mediated knockdown and chemical inhibition of the WEE1 kinase with a specific inhibitor, MK-1775, had a significant effect on both viability and apoptosis. Sensitivity to the MK-1775 kinase inhibitor is in part determined by p53 mutational status, and due to unscheduled mitotic entry. MK-1775 displays single-agent activity and potentiates the efficacy of cisplatin in a p53 mutant HNSCC xenograft model.
Conclusions
WEE1 kinase is a potential therapeutic drug target for HNSCC. This study supports the application of a functional kinomics strategy to identify novel therapeutic targets for cancer.
doi:10.1158/1078-0432.CCR-13-2858
PMCID: PMC4135446  PMID: 25125259
HNSCC; functional genomics; WEE1; p53; MK-1775 (a.k.a, AZD-1775)
9.  High-Frequency Stimulation of the Rat Entopeduncular Nucleus Does Not Provide Functional or Morphological Neuroprotection from 6-Hydroxydopamine 
PLoS ONE  2015;10(7):e0133957.
Deep brain stimulation (DBS) is the most common neurosurgical treatment for Parkinson’s disease (PD). Whereas the globus pallidus interna (GPi) has been less commonly targeted than the subthalamic nucleus (STN), a recent clinical trial suggests that GPi DBS may provide better outcomes for patients with psychiatric comorbidities. Several laboratories have demonstrated that DBS of the STN provides neuroprotection of substantia nigra pars compacta (SNpc) dopamine neurons in preclinical neurotoxin models of PD and increases brain-derived neurotrophic factor (BDNF). However, whether DBS of the entopeduncular nucleus (EP), the homologous structure to the GPi in the rat, has similar neuroprotective potential in preclinical models has not been investigated. We investigated the impact of EP DBS on forelimb use asymmetry and SNpc degeneration induced by 6-hydroxydopamine (6-OHDA) and on BDNF levels. EP DBS in male rats received unilateral, intrastriatal 6-OHDA and ACTIVE or INACTIVE stimulation continuously for two weeks. Outcome measures included quantification of contralateral forelimb use, stereological assessment of SNpc neurons and BDNF levels. EP DBS 1) did not ameliorate forelimb impairments induced by 6-OHDA, 2) did not provide neuroprotection for SNpc neurons and 3) did not significantly increase BDNF levels in any of the structures examined. These results are in sharp contrast to the functional improvement, neuroprotection and BDNF-enhancing effects of STN DBS under identical experimental parameters in the rat. The lack of functional response to EP DBS suggests that stimulation of the rat EP may not represent an accurate model of clinical GPi stimulation.
doi:10.1371/journal.pone.0133957
PMCID: PMC4519335  PMID: 26222442
10.  The mutational landscapes of genetic and chemical models of Kras-driven lung cancer 
Nature  2014;517(7535):489-492.
SUMMARY
Next-generation sequencing of human tumours has refined our understanding of the mutational processes operative in cancer initiation and progression, yet major questions remain regarding factors that induce driver mutations, and the processes that shape their selection during tumourigenesis. We performed whole-exome sequencing (WES) on adenomas from three mouse models of non-small cell lung cancer (NSCLC), induced by exposure to carcinogens (Methylnitrosourea (MNU) and Urethane), or by genetic activation of Kras (KrasLA2). Although the MNU-induced tumours carried exactly the same initiating mutation in Kras as seen in the KrasLA2 model (G12D), MNU tumours had an average of 192 non-synonymous, somatic single nucleotide variants (SNVs), compared to only 6 in tumours from the KrasLA2 model. In contrast, the KrasLA2 tumours exhibited a significantly higher level of aneuploidy and copy number alterations (CNAs) compared to the carcinogen-induced tumours, suggesting that carcinogen and genetically-engineered models adopt different routes to tumour development. The wild type (WT) allele of Kras has been shown to act as a tumour suppressor in mouse models of NSCLC. We demonstrate that urethane-induced tumours from WT mice carry mostly (94%) Q61R Kras mutations, while those from Kras heterozygous animals carry mostly (92%) Q61L mutations, indicating a major role of germline Kras status in mutation selection during initiation. The exome-wide mutation spectra in carcinogen-induced tumours overwhelmingly display signatures of the initiating carcinogen, while adenocarcinomas acquire additional C>T mutations at CpG sites. These data provide a basis for understanding the conclusions from human tumour genome sequencing that identified two broad categories based on relative frequency of SNVs and CNAs1, and underline the importance of carcinogen models for understanding the complex mutation spectra seen in human cancers.
doi:10.1038/nature13898
PMCID: PMC4304785  PMID: 25363767
11.  The mutational landscapes of genetic and chemical models of Kras-driven lung cancer 
Nature  2014;517(7535):489-492.
SUMMARY
Next-generation sequencing of human tumours has refined our understanding of the mutational processes operative in cancer initiation and progression, yet major questions remain regarding factors that induce driver mutations, and the processes that shape their selection during tumourigenesis. We performed whole-exome sequencing (WES) on adenomas from three mouse models of non-small cell lung cancer (NSCLC), induced by exposure to carcinogens (Methyl-nitrosourea (MNU) and Urethane), or by genetic activation of Kras (KrasLA2). Although the MNU-induced tumours carried exactly the same initiating mutation in Kras as seen in the KrasLA2 model (G12D), MNU tumours had an average of 192 non-synonymous, somatic single nucleotide variants (SNVs), compared to only 6 in tumours from the KrasLA2 model. In contrast, the KrasLA2 tumours exhibited a significantly higher level of aneuploidy and copy number alterations (CNAs) compared to the carcinogen-induced tumours, suggesting that carcinogen and genetically-engineered models adopt different routes to tumour development. The wild type (WT) allele of Kras has been shown to act as a tumour suppressor in mouse models of NSCLC. We demonstrate that urethane-induced tumours from WT mice carry mostly (94%) Q61R Kras mutations, while those from Kras heterozygous animals carry mostly (92%) Q61L mutations, indicating a major role of germline Kras status in mutation selection during initiation. The exome-wide mutation spectra in carcinogen-induced tumours overwhelmingly display signatures of the initiating carcinogen, while adenocarcinomas acquire additional C>T mutations at CpG sites. These data provide a basis for understanding the conclusions from human tumour genome sequencing that identified two broad categories based on relative frequency of SNVs and CNAs1, and underline the importance of carcinogen models for understanding the complex mutation spectra seen in human cancers.
doi:10.1038/nature13898
PMCID: PMC4304785  PMID: 25363767
12.  Arf Suppresses Hepatic Vascular Neoplasia in a Carcinogen-Exposed Murine Model 
The Journal of pathology  2012;227(3):10.1002/path.4024.
Hepatic haemangiosarcoma is a deadly malignancy whose aetiology remains poorly understood. Inactivation of the CDKN2A locus, which houses the ARF and p16INK4a tumour suppressor genes, is a common event in haemangiosarcoma patients, but the precise role of ARF in vascular tumourigenesis is unknown. To determine the extent to which ARF suppresses vascular neoplasia, we examined the incidence of hepatic vascular lesions in Arf-deficient mice exposed to the carcinogen urethane (i.p. 1 mg/g). Loss of Arf resulted in elevated morbidity and increased the incidence of both haemangiomas and incipient haemangiosarcomas. Suppression of vascular lesion development by ARF was heavily dependent on both Arf gene-dosage and the genetic strain of the mouse. Trp53-deficient mice also developed hepatic vascular lesions after exposure to urethane, suggesting that ARF signals through a p53-dependent pathway to inhibit the development of hepatic haemangiosarcoma. Our findings provide strong evidence that inactivation of Arf is a causative event in vascular neoplasia and suggest that the ARF pathway may be a novel molecular target for therapeutic intervention in haemangiosarcoma patients.
doi:10.1002/path.4024
PMCID: PMC3871210  PMID: 22430984
p19Arf; p14ARF; ethyl carbamate; liver; endothelium; haemorrhage; tumour progression
13.  Doxorubicin enhances nucleosome turnover around promoters 
Current biology : CB  2013;23(9):782-787.
Summary
Doxorubicin is an anthracycline DNA intercalator that is among the most commonly used anti-cancer drugs [1]. Doxorubicin causes DNA double-strand breaks in rapidly dividing cells, although whether it also affects general chromatin properties is unknown. Here, we use a metabolic labeling strategy to directly measure nucleosome turnover [2] to examine the effect of doxorubicin on chromatin dynamics in squamous cell carcinoma cell lines derived from genetically defined mice. We find that doxorubicin enhances nucleosome turnover around gene promoters, and turnover correlates with gene expression level. Consistent with a direct action of doxorubicin, enhancement of nucleosome turnover around promoters gradually increases with time of exposure to the drug. Interestingly, enhancement occurs both in wild-type cells and in cells lacking either the p53 tumor suppressor gene or the master regulator of the DNA damage response, Atm, suggesting that doxorubicin action on nucleosome dynamics is independent of the DNA damage checkpoint. In addition, another anthracycline drug, aclarubicin, shows similar effects on enhancing nucleosome turnover around promoters. Our results suggest that anthracycline intercalation promotes nucleosome turnover around promoters by its effect on DNA topology, with possible implications for mechanisms of cell killing during cancer chemotherapy.
doi:10.1016/j.cub.2013.03.043
PMCID: PMC3678987  PMID: 23602475
CATCH-IT; Squamous cell carcinoma; p53; Atm
14.  CTCF haploinsufficiency destabilizes DNA methylation and predisposes to cancer 
Cell reports  2014;7(4):1020-1029.
SUMMARY
Epigenetic alterations, particularly in DNA methylation, are ubiquitous in cancer, yet the molecular origins and the consequences of these alterations are poorly understood. The DNA binding protein CTCF regulates a diverse array of epigenetic processes and is frequently altered by hemizygous deletion or mutation in human cancer. To date, a causal role for CTCF in cancer has not been established. Here we show that Ctcf hemizygous knockout mice are markedly susceptible to spontaneous, radiation, and chemically induced cancer in a broad range of tissues. Ctcf+/− tumors are characterized by increased aggressiveness including invasion, metastatic dissemination, and mixed epithelial/mesenchymal differentiation. Molecular analysis of Ctcf+/− tumors indicates that Ctcf is haploinsufficient for tumor suppression. Tissues with hemizygous loss of CTCF exhibit increased variability in CpG methylation genome-wide. These findings establish CTCF as a prominent tumor suppressor gene and point to CTCF mediated epigenetic stability as a major barrier to neoplastic progression.
doi:10.1016/j.celrep.2014.04.004
PMCID: PMC4040130  PMID: 24794443
15.  A Mouse Model Repository for Cancer Biomarker Discovery 
Journal of proteome research  2008;7(8):3613-3618.
Early detection of cancer using biomarkers obtained from blood or other easily accessible tissues would have a significant impact on reducing cancer mortality. However, identifying new blood-based biomarkers has been hindered by the dynamic complexity of the human plasma proteome, confounded by genetic and environmental variability, and the scarcity of high quality controlled samples. In this report we discuss a new paradigm for biomarker discovery through the use of mouse models. Inbred mouse models of cancer recapitulate many critical features of human cancer, while eliminating sources of environmental and genetic variability. The ability to collect samples from highly matched cases and controls under identical conditions further reduces variability which is critical for successful biomarker discovery. We describe the establishment of a repository containing tumor, plasma, urine, and other tissues from ten different mouse models of human cancer, including two breast, two lung, two prostate, two gastro-intestinal, one ovarian, and one skin tumor model. We present the overall design of this resource and its potential use by the research community for biomarker discovery.
doi:10.1021/pr800210b
PMCID: PMC3727967  PMID: 18624399
16.  Identification of a novel E-box binding pyrrole-imidazole polyamide inhibiting MYC-driven cell proliferation 
Cancer Science  2015;106(4):421-429.
The MYC transcription factor plays a crucial role in the regulation of cell cycle progression, apoptosis, angiogenesis, and cellular transformation. Due to its oncogenic activities and overexpression in a majority of human cancers, it is an interesting target for novel drug therapies. MYC binding to the E-box (5′-CACGTGT-3′) sequence at gene promoters contributes to more than 4000 MYC-dependent transcripts. Owing to its importance in MYC regulation, we designed a novel sequence-specific DNA-binding pyrrole–imidazole (PI) polyamide, Myc-5, that recognizes the E-box consensus sequence. Bioinformatics analysis revealed that the Myc-5 binding sequence appeared in 5′- MYC binding E-box sequences at the eIF4G1, CCND1, and CDK4 gene promoters. Furthermore, ChIP coupled with detection by quantitative PCR indicated that Myc-5 has the ability to inhibit MYC binding at the target gene promoters and thus cause downregulation at the mRNA level and protein expression of its target genes in human Burkitt's lymphoma model cell line, P493.6, carrying an inducible MYC repression system and the K562 (human chronic myelogenous leukemia) cell line. Single i.v. injection of Myc-5 at 7.5 mg/kg dose caused significant tumor growth inhibition in a MYC-dependent tumor xenograft model without evidence of toxicity. We report here a compelling rationale for the identification of a PI polyamide that inhibits a part of E-box-mediated MYC downstream gene expression and is a model for showing that phenotype-associated MYC downstream gene targets consequently inhibit MYC-dependent tumor growth.
doi:10.1111/cas.12610
PMCID: PMC4406810  PMID: 25611295
Cell cycle; E-Box; MYC; pyrrole-imidazole polyamide; transcription therapy
17.  Doxorubicin, DNA torsion, and chromatin dynamics 
Biochimica et biophysica acta  2013;1845(1):84-89.
Doxorubicin is one of the most important anti-cancer chemotherapeutic drugs, being widely used for the treatment of solid tumors and acute leukemias. The action of doxorubicin and other anthracycline drugs has been intensively investigated during the last several decades, but the mechanisms that have been proposed for cell killing remain disparate and controversial. In this review, we examine the proposed models for doxorubicin action from the perspective of the chromatin landscape, which is altered in many types of cancer due to recurrent mutations in chromatin modifiers. We highlight recent evidence for effects of anthracyclines on DNA torsion and chromatin dynamics that may underlie basic mechanisms of doxorubicin-mediated cell death and suggest new therapeutic strategies for cancer treatment.
doi:10.1016/j.bbcan.2013.12.002
PMCID: PMC3927826  PMID: 24361676
doxorubicin; anthracycline; cancer; DNA torsion; chromatin dynamics; chemotherapy
18.  Proteome and Transcriptome Profiles of a Her2/Neu-driven Mouse Model of Breast Cancer 
Proteomics. Clinical applications  2011;5(3-4):179-188.
Purpose
We generated extensive transcriptional and proteomic profiles from a Her2-driven mouse model of breast cancer that closely recapitulates human breast cancer. This report makes these data publicly available in raw and processed forms, as a resource to the community. Importantly, we previously made biospecimens from this same mouse model freely available through a sample repository, so researchers can obtain samples to test biological hypotheses without the need of breeding animals and collecting biospecimens.
Experimental design
Twelve datasets are available, encompassing 841 LC-MS/MS experiments (plasma and tissues) and 255 microarray analyses of multiple tissues (thymus, spleen, liver, blood cells, and breast). Cases and controls were rigorously paired to avoid bias.
Results
In total, 18,880 unique peptides were identified (PeptideProphet peptide error rate ≤1%), with 3884 and 1659 non-redundant protein groups identified in plasma and tissue datasets, respectively. Sixty-one of these protein groups overlapped between cancer plasma and cancer tissue.
Conclusions and clinical relevance
These data are of use for advancing our understanding of cancer biology, for software and quality control tool development, investigations of analytical variation in MS/MS data, and selection of proteotypic peptides for MRM-MS. The availability of these datasets will contribute positively to clinical proteomics.
doi:10.1002/prca.201000037
PMCID: PMC3069718  PMID: 21448875
Breast cancer; Her2; mouse; proteome; transcriptome
19.  Systematic Screen Identifies miRNAs that Target RAD51 and RAD51D to Enhance Chemosensitivity 
Molecular cancer research : MCR  2013;11(12):10.1158/1541-7786.MCR-13-0292.
Homologous recombination (HR) mediates error-free repair of DNA double-strand breaks (DSBs). RAD51 is an essential protein for catalyzing HR and its recruitment to DSBs is mediated by many factors including RAD51, its paralogs, and breast/ovarian cancer susceptibility gene products BRCA1/2. Deregulation of these factors leads to impaired DNA repair, genomic instability, and cellular sensitivity to chemotherapeutics like cisplatin and poly (ADP-ribose) polymerase (PARP) inhibitors. microRNAs (miRs) are short, non-coding RNAs that post-transcriptionally regulate gene expression; however, the contribution of miRs in the regulation of HR is not well understood. To address this, a library of human miR mimics was systematically screened to pinpoint several miRs that significantly reduce RAD51 foci formation in response to ionizing radiation (IR) in human osteosarcoma cells. Subsequent study focused on two of the strongest candidates, miR-103 and miR-107, as they are frequently deregulated in cancer. Consistent with the inhibition of RAD51 foci formation, miR-103 and miR-107 reduced homology-directed repair and sensitized cells to various DNA damaging agents, including cisplatin and a PARP inhibitor. Mechanistic analyses revealed that both miR-103 and miR-107 directly target and regulate RAD51 and RAD51D, which is critical for miR-103/107-mediated chemosensitization. Furthermore, endogenous regulation of RAD51D by miR-103/107 was observed in several tumor subtypes. Combined, these data show that miR-103 and miR-107 overexpression promotes genomic instability and may be used therapeutically to chemosensitize tumors.
Implications
These findings demonstrate a role for miR-103 and -107 in regulating DNA damage repair, thereby identifying new players in the progression of cancer and response to chemotherapy.
doi:10.1158/1541-7786.MCR-13-0292
PMCID: PMC3869885  PMID: 24088786
microRNA; cisplatin; PARP inhibitor; RAD51; RAD51D; homologous recombination; Fanconi anemia
20.  Tumors induce coordinate growth of artery, vein, and lymphatic vessel triads 
BMC Cancer  2014;14:354.
Background
Tumors drive blood vessel growth to obtain oxygen and nutrients to support tumor expansion, and they also can induce lymphatic vessel growth to facilitate fluid drainage and metastasis. These processes have generally been studied separately, so that it is not known how peritumoral blood and lymphatic vessels grow relative to each other.
Methods
The murine B16-F10 melanoma and chemically-induced squamous cell carcinoma models were employed to analyze large red-colored vessels growing between flank tumors and draining lymph nodes. Immunostaining and microscopy in combination with dye injection studies were used to characterize these vessels.
Results
Each peritumoral red-colored vessel was found to consist of a triad of collecting lymphatic vessel, vein, and artery, that were all enlarged. Peritumoral veins and arteries were both functional, as detected by intravenous dye injection. The enlarged lymphatic vessels were functional in most mice by subcutaneous dye injection assay, however tumor growth sometimes blocked lymph drainage to regional lymph nodes. Large red-colored vessels also grew between benign papillomas or invasive squamous cell carcinomas and regional lymph nodes in chemical carcinogen-treated mice. Immunostaining of the red-colored vessels again identified the clustered growth of enlarged collecting lymphatics, veins, and arteries in the vicinity of these spontaneously arising tumors.
Conclusions
Implanted and spontaneously arising tumors induce coordinate growth of blood and lymphatic vessel triads. Many of these vessel triads are enlarged over several cm distance between the tumor and regional lymph nodes. Lymphatic drainage was sometimes blocked in mice before lymph node metastasis was detected, suggesting that an unknown mechanism alters lymph drainage patterns before tumors reach draining lymph nodes.
doi:10.1186/1471-2407-14-354
PMCID: PMC4045915  PMID: 24886322
Melanoma; Carcinoma; Lymphangiogenesis; Lymphatic vessel; Angiogenesis; Blood vessel
21.  Palmitic acid mediates hypothalamic insulin resistance by altering PKC-θ subcellular localization in rodents 
The Journal of Clinical Investigation  2009;119(9):2577-2589.
Insulin signaling can be modulated by several isoforms of PKC in peripheral tissues. Here, we assessed whether one specific isoform, PKC-θ, was expressed in critical CNS regions that regulate energy balance and whether it mediated the deleterious effects of diets high in fat, specifically palmitic acid, on hypothalamic insulin activity in rats and mice. Using a combination of in situ hybridization and immunohistochemistry, we found that PKC-θ was expressed in discrete neuronal populations of the arcuate nucleus, specifically the neuropeptide Y/agouti-related protein neurons and the dorsal medial nucleus in the hypothalamus. CNS exposure to palmitic acid via direct infusion or by oral gavage increased the localization of PKC-θ to cell membranes in the hypothalamus, which was associated with impaired hypothalamic insulin and leptin signaling. This finding was specific for palmitic acid, as the monounsaturated fatty acid, oleic acid, neither increased membrane localization of PKC-θ nor induced insulin resistance. Finally, arcuate-specific knockdown of PKC-θ attenuated diet-induced obesity and improved insulin signaling. These results suggest that many of the deleterious effects of high-fat diets, specifically those enriched with palmitic acid, are CNS mediated via PKC-θ activation, resulting in reduced insulin activity.
doi:10.1172/JCI36714
PMCID: PMC2735917  PMID: 19726875
22.  VOLUNTARY CONSUMPTION OF ETHYL OLEATE REDUCES FOOD INTAKE AND BODY WEIGHT IN RATS 
Physiology & behavior  2007;93(4-5):912-918.
Previous studies have shown that administration of the fatty acids, linoleic and oleic acid, either by intragastric or intraintestinal infusion, suppresses food intake and body weight in rats. While still not fully understood, gut-mediated satiety mechanisms likely are potential effectors of this robust response to gastrointestinal fatty acid infusions. The objective of this study was to assess the effects of voluntary access to an oleic acid derivative, ethyl oleate (EO), on subsequent food intake and body weight in rats. Animals were randomized either to a 12.5% EO diet or a soybean oil diet as a “breakfast,” followed either by two one-hour or one five-hour access periods to standard rodent diet, and food intake and body weights were collected. Across 14 days access, rats consuming EO on both feeding schedules gained less weight and consumed less total kilocalories than rats consuming the SO diet. Further, plasma levels of glucose and insulin were comparable in both EO and SO diet groups. In summary, EO was found to increase weight loss in rats maintained on a 75% food-restriction regimen, and attenuate weight-gain upon resumption of an ad-libitum feeding regimen. These data indicate that voluntary access to EO promoted short-term satiety, compared to SO diet, and that these effects contributed to a important and novel attenuated weight gain in EO-fed animals.
doi:10.1016/j.physbeh.2007.12.008
PMCID: PMC2372161  PMID: 18234242
Linoleic Acid; Obesity; Oleic Acid; Ethyl Oleate; Satiety; Triglyceride
23.  TRAIL-R deficiency in mice enhances lymph node metastasis without affecting primary tumor development 
TRAIL is a promising anticancer agent due to its ability to selectively induce apoptosis in established tumor cell lines but not nontransformed cells. Herein, we demonstrate a role for the apoptosis-inducing TRAIL receptor (TRAIL-R) as a metastasis suppressor. Although mouse models employing tumor transplantation have shown that TRAIL can reduce tumor growth, autochthonous tumor models have generated conflicting results with respect to the physiological role of the TRAIL system during tumorigenesis. We used a multistage model of squamous cell carcinoma to examine the role of TRAIL-R throughout all steps of tumor development. DMBA/TPA-treated TRAIL-R–deficient mice showed neither an increase in number or growth rate of benign papillomas nor an increase in the rate of progression to squamous cell carcinoma. However, metastasis to lymph nodes was significantly enhanced, indicating a role for TRAIL-R specifically in the suppression of metastasis. We also found that adherent TRAIL-R–expressing skin carcinoma cells were TRAIL resistant in vitro but were sensitized to TRAIL upon detachment by inactivation of the ERK signaling pathway. As detachment from the primary tumor is an obligatory step in metastasis, this provides a possible mechanism by which TRAIL-R could inhibit metastasis. Hence, treatment of cancer patients with agonists of the apoptosis-inducing receptors for TRAIL may prove useful in reducing the incidence of metastasis.
doi:10.1172/JCI33061
PMCID: PMC2129237  PMID: 18079967
24.  Morphological and Behavioral Impact of AAV2/5-Mediated Overexpression of Human Wildtype Alpha-Synuclein in the Rat Nigrostriatal System 
PLoS ONE  2013;8(11):e81426.
The discovery of the involvement of alpha-synuclein (α-syn) in Parkinson’s disease (PD) pathogenesis has resulted in the development and use of viral vector-mediated α-syn overexpression rodent models. The goal of these series of experiments was to characterize the neurodegeneration and functional deficits resulting from injection of recombinant adeno-associated virus (rAAV) serotype 2/5-expressing human wildtype α-syn in the rat substantia nigra (SN). Rats were unilaterally injected into two sites in the SN with either rAAV2/5-expressing green fluorescent protein (GFP, 1.2 x 1013) or varying titers (2.2 x 1012, 1.0 x 1013, 5.9 x 1013, or 1.0 x 1014) of rAAV2/5-α-syn. Cohorts of rats were euthanized 4, 8, or 12 weeks following vector injection. The severity of tyrosine hydroxylase immunoreactive (THir) neuron death in the SN pars compacta (SNpc) was dependent on vector titer. An identical magnitude of nigrostriatal degeneration (60-70% SNpc THir neuron degeneration and 40-50% loss of striatal TH expression) was observed four weeks following 1.0 x 1014 titer rAAV2/5-α-syn injection and 8 weeks following 1.0 x 1013 titer rAAV2/5-α-syn injection. THir neuron degeneration was relatively uniform throughout the rostral-caudal axis of the SNpc. Despite equivalent nigrostriatal degeneration between the 1.0 x 1013 and 1.0 x 1014 rAAV2/5-α-syn groups, functional impairment in the cylinder test and the adjusting steps task was only observed in rats with the longer 8 week duration of α-syn expression. Motor impairment in the cylinder task was highly correlated to striatal TH loss. Further, 8 weeks following 5.9 x 1013 rAAV2/5-α-syn injection deficits in ultrasonic vocalizations were observed. In conclusion, our rAAV2/5-α-syn overexpression model demonstrates robust nigrostriatal α-syn overexpression, induces significant nigrostriatal degeneration that is both vector and duration dependent and under specific parameters can result in motor impairment that directly relates to the level of striatal TH denervation.
doi:10.1371/journal.pone.0081426
PMCID: PMC3842242  PMID: 24312298
25.  Fundamental differences in promoter CpG island DNA hypermethylation between human cancer and genetically engineered mouse models of cancer 
Epigenetics  2013;8(12):1254-1260.
Genetic and epigenetic alterations are essential for the initiation and progression of human cancer. We previously reported that primary human medulloblastomas showed extensive cancer-specific CpG island DNA hypermethylation in critical developmental pathways. To determine whether genetically engineered mouse models (GEMMs) of medulloblastoma have comparable epigenetic changes, we assessed genome-wide DNA methylation in three mouse models of medulloblastoma. In contrast to human samples, very few loci with cancer-specific DNA hypermethylation were detected, and in almost all cases the degree of methylation was relatively modest compared with the dense hypermethylation in the human cancers. To determine if this finding was common to other GEMMs, we examined a Burkitt lymphoma and breast cancer model and did not detect promoter CpG island DNA hypermethylation, suggesting that human cancers and at least some GEMMs are fundamentally different with respect to this epigenetic modification. These findings provide an opportunity to both better understand the mechanism of aberrant DNA methylation in human cancer and construct better GEMMs to serve as preclinical platforms for therapy development.
doi:10.4161/epi.26486
PMCID: PMC3933486  PMID: 24107773
cancer; DNA methylation; epigenomics; genetically engineered mouse models; medulloblastoma

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