bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 3870: Distinct and cooperative functions of the RB and p53 tumor suppressors in genotoxic response and tumorigenesis
Authors: McClendon Kathleen , Dean Jeffry L. , Ertel Adam , Reed Christopher A. , Yang Xiaoping , Knudsen Erik S. 
Published: February 2011

Abstract
The compound inactivation of RB and p53 is a frequent occurrence in human cancers. At a molecular level, the RB and p53 pathways intersect at multiple points that have critical implications for cell cycle control and tumorigenesis. To examine the cooperation of these proteins in the response to genotoxic damage, we used a combination of genetic deletion and dominant negative strategies in vitro, and liver-specific genetic deletion in vivo. In vitro, RB proficiency was sufficient to maintain cell cycle checkpoint in response to genotoxic stress, independent of p53 status. In contrast, RB-deficient cells displayed deregulated cell growth, resulting in aggressive recovery from DNA damage. Strikingly, while disruption of the RB and p53 pathways alone does not result in transformation, the RB-deficient, recurred cell populations were tumorigenic. These in vitro findings indicate that RB is a critical suppressor of tumorigenesis, beyond that of p53, and a significant factor in the evolution of cells during genotoxic challenge. To further examine the cooperation of RB and p53 in vivo, a model of liver tumorigenesis was employed. Interestingly, these in vivo studies confirmed that while compound inactivation of the RB and p53 pathways in liver tissue promoted deregulated cell cycle control, this was not sufficient for transformation. However, loss of RB and p53 cooperated in promoting aberrant cell proliferation and mitosis in the presence of genotoxic challenge by DEN (diethyl-nitrosamine), ultimately resulting in enhanced tumorigenesis. Strikingly, loss of RB alone promoted S-phase entry as a result of genotoxic challenge, but there was an uncoupling of this event from productive mitosis. In contrast, the combined loss of RB/p53 resulted in further deregulation of DNA replication that was accompanied by a productive mitotic entry. These data indicate that, while both RB and p53 can contribute to appropriate replicative control, p53 plays a dominant role in mediating G2/M regulation. Furthermore, RB/p53-deficient livers displayed rapid progression to highly aggressive HCC within the span of six months, which was not observed in either RB-deficient or p53-deficient livers. Combined, these studies indicate that RB and p53 play distinct and cooperating roles in modulating response to genotoxic stress and tumorigenesis. Discovering the mechanism behind this cooperation could uncover a critical facet of tumor etiology and provide a means by which to treat this complex disease.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3870.
Abstract 3876: Cell cycle checkpoint in vivo in developing mouse embryo liver cells
Authors: Chan Kwok Kin , Wang XiaoQi 
Published: February 2011

Abstract
Embryonic stem (ES) cells have a distinct cell cycle distribution and cyclins/CDKs expression. ES cells are characterized by a short G1 phase and a high proportion of cell in S and G2 phase, maintain consistent level of cyclins expression seems not to be phase dependent. However, most cell cycle studies are on mouse or human ES cell line, whether that it is really reflective in vivo is not well defined. By extracting fresh liver cells from mouse embryo, we have investigated cell cycle distribution, correspondent cyclins and CDK expression in vivo in mouse embryo from embryonic days 10.5(E10.5, liver start to develop at E9.5) to post-natal 21 days(P21). We found that most of the cells (62%) were in S and G2 phase, 38% of cells in G1 at E11.5. Upon embryo development, the proportion of cells in G1 increased, although there were still large proportion of cells in S and G2 phase (55%) at P0. The short G1 phase in early embryonic day suggests that cell phase distribution may be related with embryo cells’ pluripotency in vivo. Distinct high levels of cyclin D1, E1, A, B1 and CDK1(cdc2) expressed at E10.5, which seemed to be pluripotency related but not cell phase dependent. The cyclins and CDK level started to drop from E11.5 to post-natal (P0) suggesting that cyclins/CDKs expression reduced and became regulated with differentiation. In ES cell line, where G1 population increased with differentiation, G1 DNA-damage checkpoint was still absent. However, in response to ionizing irradiation, P0 mouse liver cells showed a short G2 population increase followed by G1 cell phase arrest indicating that at P0 or even earlier stage, G1 DNA-damage checkpoint had been established in vivo. Thus, cell cycle and cell cycle checkpoint in vivo in mouse embryo liver cells displayed both similar and differential pattern compared to that in mES cell line in vitro. Underlying mechanism such as DNA-damage checkpoint patterns and its regulation will be further discussed.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3876.
Abstract 3879: YAP (Yes-associated protein), a proposed tumor suppressor in breast cancer, is regulated by cyclin D1
Authors: Lehn Sophie , Tobin Nicholas P. , Landberg Göran 
Published: February 2011

Abstract
In breast cancer, cyclin D1 over-expression and amplification has been linked to a worse prognosis. Conversely, lower levels of cyclin D1 have also been correlated to an unfavorable outcome. We have in our previous studies observed that lower levels of cyclin D1 correlate to a more migratory phenotype of breast cancer cells. In order to specifically delineate the possible effectors downstream of cyclin D1, we performed a whole-genome microarray experiment where cyclin D1 was downregulated in MDA-MB-231 cells. To avoid selecting for pure cell cycle-dependent changes, the cell cycle-related binding partners of cyclin D1; cyclin-dependent kinases 4 and 6 (Cdk4/6), were also downregulated and included in the microarray experiment.
One of the top downregulated genes upon cyclin D1 silencing was YAP, a recently proposed tumor suppressor gene implicated in regulation of breast cancer cell migration. We set out to clarify the link between cyclin D1 and YAP, both in an in vitro setting and by examining the expression of the two proteins in a clinical material of 179 primary invasive breast cancers.
To validate the microarray results, cyclin D1 and Cdk4/6 protein levels were transiently silenced up to 96 h in MDA-MB-231 and MCF-7 breast cancer cells using siRNA, and YAP protein levels were observed by western blot. The clinical material was stained using YAP and cyclin D1 antibodies, and intensity of the signal was scored. Also, chromogenic in situ hybridization was performed allowing for assessment of amplification of the CCND1 gene.
Transient downregulation of cyclin D1 in MDA-MB-231 and MCF-7 cells resulted in decreased YAP protein levels, as predicted by the microarray results. Downregulation of Cdk4/6 did not result in decreased YAP levels. In the clinical material, cyclin D1 intensity was inversely correlated to YAP intensity (Spearman's rho −0.220, P=0.031). Amplification of CCND1 was significantly correlated to none or low intensity of YAP expression.
We conclude that on protein level, the silencing of cyclin D1 decreases levels of YAP independently of cyclin D1's cell cycle-dependent association with Cdk4/6. On the chromosomal level, CCND1 gene amplification is correlated to loss of YAP expression which might explain the inverse correlation seen between cyclin D1 and YAP in the clinical material. Studies to further characterize the link between cyclin D1, YAP and migration are ongoing.
Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3879.
Abstract 3890: Induction of cell cycle arrest and apoptosis in mantle cell lymphoma by AZD5438, a novel cyclin-dependent kinase inhibitor
Authors: Lo Wann-Cheng , Growcott Jim , LeBrun David P. , Greer Peter A. 
Published: February 2011

Abstract
Mantle cell lymphoma (MCL) is recognized as a distinct subtype of B-cell lymphoma and it is characterized by cyclin D1 over-expression, which is caused by t(11;14)(q13;q32) translocation and unregulated cell cycle progression driven by increased cyclin-dependent kinase (CDK) activity. Accordingly, small inhibitors for CDKs represent a potential therapy for MCL. We are investigating a selective small molecule CDK inhibitor, AZD5438, which has nanomolar ranges of IC50 for cyclin E/CDK2, cyclin A/CDK2, cyclin B1/CDK1 and cyclin T/CDK9 and comparing its anti-tumor effects with a pan-CDK inhibitor, Flavopiridol. We hypothesize that inhibition of CDK1/2 by AZD5438 may lead to G1/S and G2 arrest, and therefore inhibit tumor cell proliferation and induce apoptosis in MCL. In Jeko-1 MCL cell lines, AZD5438 has shown both time- and dose-dependent inhibition of cell proliferation, inhibition of the phosphorylation of CDK substrates, and cell cycle arrest in G1 phase. The EC50 for AZD 5438 is 1μM as determined by inhibition of cell viability. We further developed an in vivo MCL xenograft assay: first, to guide the optimization of dosage and dosing schedule for clinical use; and second, to explore the selectivity and potency of this drug using phospho-histone H3 (Ser10) flow cytometry and phosopho-Rb (Ser249/Thr252) immunoblotting as our drug efficacy monitoring system. Recently we have established GFP transduced Jeko-1 cells and inoculated them into 8 to 12 week-old, immune-compromised NOD SCID mice. Investigations of AZD5438 drug dosage in this xenograft model are ongoing. Ultimately, this project is aimed at improving the treatment for MCL patients and reducing the toxic responses to the standard chemotherapeutic regimes.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3890.
Abstract 3891: Reduced tumor formation by forced expression of cyclin-dependent kinase 6 in mouse epidermis
Authors: Wang Xian , Sistrunk Christopher , Rodriguez-Puebla Marcelo 
Published: February 2011

Abstract
The Cyclin-Dependent kinases (CDKs) are a family of essential serine-threonine kinases that are cell cycle regulated by association with cyclins at specific time-points during the cell cycle. Specifically, D-type cyclins form complexes and activate CDK4 and CDK6. We have previously determined that overexpression of CDK4 in mouse epidermis increases malignant progression in chemically induced skin tumors. In order to study the role of CDK6 in squamous epithelial tissues, we generated transgenic mice overexpressing CDK6, driven by the regulatory sequence of the keratin 5 promoter (K5CDK6). Keratinocytes from K5CDK6 mice have similar levels of proliferation compared to wild type mice, however, contrary to the role of CDK4 in mouse skin tumorigenesis, forced expression of CDK6 results in strong reduction of the multiplicity (papillomas per mouse) and incidence of skin tumors. Tumors derived from K5CDK6 or the wild type sibling show no histological differences nor changes in the rate of malignant progression, suggesting that CDK6 expression affects early stages of skin carcinogenesis. Our previous results have also shown that CDK6 preferentially binds to cyclin D3, which overexpression also decreases tumor development in mouse epidermis. Interestingly, lack of Cyclin D1 produces similar phenotype upon chemical carcinogenesis treatment. Thus, we hypothesize that overexpression of Cyclin D3 or CDK6 in a Cyclin D1-null background should synergize with the reduced tumor development previously observed in cyclin D1-null mice. As expected, a lower number of tumors were found in K5-cyclin D3/cyclin D1−/− compound mice compared to cyclin D1−/− siblings. Thus, we hypothesized that the reduced tumor development observed in these mice is a result of the CDK6/Cyclin D3 complex activity. Consequently, K5-CDK6/cyclin D3−/− compound mice are being generated to investigate whether CDK6-associated tumor inhibition depends on cyclin D3 expression.
Only in few cases, CDK6 has been associated with tumorigenesis as compared to CDK4 and CDK2, in particular, no association of CDK6 with epidermal tumors has been reported so far. We propose that CDK6/cyclin D3 complexes play an important role in maintaining normal epidermal homeostasis. Therefore we speculate that upregulation of one or both of its components will block tumor development which could be use as a therapeutic approach.
Supported by NIH grant CA116328.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3891.
Abstract 3920: EphA2 transcription is regulated by activation of Ras / MEK2 / cytoplasmic ERK pathway in pancreatic cancer
Authors: Levin Pavel A. , Arumugam Thiruvengadam , McConkey David J. , Reddy Shrikanth , Logsdon Craig D. 
Published: February 2011

Abstract
Background: We have previously shown that the EphA2 receptor is overexpressed in pancreatic cancer. Silencing of EphA2 receptor in combination with gemcitabine decreases tumor growth and metastases in a mouse model of gemcitabine resistant pancreatic cancer. However, regulation of EphA2 expression in pancreatic cancer is unknown. The objective of this study was to determine a possible relationship between Ras/MAPK pathway and EphA2. Methods: Pancreatic cancer cell lines, MPanc96 and MiaPaca-2, were used in this study. Ras was silenced with 2 different siRNA constructs; EphA2 protein and RNA expression levels were measured. ERK involvement in EphA2 regulation was analyzed by treating cells with 2 inhibitors for MEK (U0126 and PD 98059) and siRNA specific for either MEK1 or MEK2. Effect of MEK inhibition on EphA2 mRNA stability was examined by blocking mRNA synthesis with actinomycin D and measuring mRNA degradation by Q-PCR. In order to investigate the role of subcellular localization of ERK in regulation of EphA2 expression, we overexpressed cytoplasmic anchor protein PEA-15, which binds to ERK and sequesters it to the cytoplasm. Results: Silencing of mutant K-Ras decreased EphA2 mRNA and protein levels. EphA2 mRNA and protein levels decreased in a time- and dose-dependent manner after treatment with MEK chemical inhibitors. Inhibition of MEK activity did not affect EphA2 mRNA stability. Silencing of MEK2 but not MEK1 decreased EphA2 mRNA levels. Overexpression of PEA-15 depleted nuclear ERK, indicated by the absence of phosphorylated Fra-1. Depletion of the nuclear ERK did not affect EphA2 transcription, suggesting that EphA2 is regulated through the cytoplasmic ERK. Conclusions: These data suggest that EphA2 levels are regulated by the RAS / MEK2 / cytoplasmic ERK pathway via regulation of EphA2 mRNA transcription.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3920.
Abstract 3925: RNF138, a novel ubiquitin E3 ligase is overexpressed in colon cancer cell lines and interacts with RAD51D
Authors: Yard Brian D. , Bedenbaugh Michael K. , Pittman Douglas L. 
Published: February 2011

Abstract
The RAD51 protein family provides core components of the Homologous Recombination (HR) complex, which is critical for repairing DNA double-strand breaks and maintaining genome stability. The purpose of this study was to identify proteins that regulate HR processes through post-translational modification of the RAD51 paralogs. A yeast two-hybrid screen was performed using the RAD51 paralogs RAD51C, RAD51D, and XRCC2 as bait against a normalized universal mouse cDNA library. Fifty-eight primary candidates were isolated, and five are predicted to function in the ubiquitination pathway. Within this group, Ring Finger Protein 138 (RNF138) is of particular interest since RNF138 is a novel E3-ubiquitin ligase having both an N-terminal RING finger domain and a putative C-terminal ubiquitin interaction motif. Presence of both domains indicates RNF138 induces the transfer of ubiquitin to target substrates and also recognizes ubiquitin moieties on other proteins. Expression of RNF138 is increased by 12-fold in the HCC38 breast cancer cell line and 4-fold in SW-48, RKO, and Wi-DR colon cancer cell lines compared to non-tumorigenic controls. Yeast two-hybrid results indicate that RNF138 interaction is unique to RAD51D, as RNF138 failed to directly interact with RAD51C and XRCC2. Further analysis using N- and C-terminal truncated RAD51D constructs revealed that the RAD51D core domain rather than the N-terminal linker region that governs the interaction between RAD51D and XRCC2 mediates the association with RNF138. Moreover, K113A and K113R mutations of the highly conserved RAD51D Walker A ATP binding motif abolished RAD51D-RNF138 association, suggesting that ATP binding and hydrolysis may regulate the interaction. As further demonstration of this association, Myc-RNF138 co-immunoprecipitated with HA-RAD51D following transient transfections. Future studies will determine whether RNF138 is directly required for HR repair and if the mechanism specifically involves RAD51D ubiquitination. Understanding the regulation of HR proteins by ubiquitination processes will provide important insights into cancer progression and guide the identification of new therapeutic strategies.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3925.
Abstract 3928: KU-0060019, a novel small molecule inhibitor of ATM
Authors: Shea Kerry F. , Albertella Mark , Bárdos Julia , Batey Michael A. , Cranston Aaron , Curtin Nicola , Griffin Roger , Lau Alan , Menear Keith , Newell David , O'Connor Mark , Richardson Caroline , Jones Louise , Smith Graeme , Thömmes Pia , Zhao Yan , Martin Niall 
Published: February 2011

Abstract
Ataxia telangiectasia mutated (ATM) is an important serine/threonine kinase involved in the DNA damage response and cell cycle checkpoint control. The 350kDa protein is a member of the PI3-kinase like kinase (PIKK) family and is mutated in the neurodegenerative disorder, Ataxia telangiectasia.
ATM exists in the inactive form as a dimer but following DNA damage is phosphorylated at a serine residue at position 1981 and dissociates to become an active monomer. It detects DNA double strand breaks (DSBs) through the Mre11/Rad50/NBS1 (MRN) complex and signals this information downstream to the cell cycle machinery, for example by the phosphorylation of p53 (Ser15) and Chk2 (Thr68).
ATM is a key signalling molecule for DNA double strand breaks that can regulate the homologous recombination repair pathway. Inhibition of ATM activity sensitises cells to the cytotoxic effects of DNA damaging agents such as some chemotherapies or ionising radiation that either directly or indirectly induce double stand breaks.
Here we present the biological profile of KU-0060019, a potent and specific novel small molecule inhibitor of ATM kinase activity. The compound has an IC50 value against the enzyme of 5 nM with above 1 μM IC50 value against DNA-PK and inhibits cellular phosphorylation of p53 at serine 15 with an EC50 value of 150 nM. It sensitises cells to chemotherapeutics, in particular topoisomerase II inhibitors, as well as radiotherapeutics in vitro in a variety of cancer cell lines. We also demonstrate potentiation of chemotherapeutics in in vivo mouse xenograft tumour models. Using the novel ATM inhibitor KU-0060019 these studies demonstrate the potential of targeting ATM as a sensitising mechanism by a small molecule in cancer therapy.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3928.
Abstract 3940: Determining the mitochondrial DNA repair capacity in peripheral blood mononuclear cells using QPCR
Authors: Fonseca-Williams Sharon , Ayala-Torres Sylvette , El-Zein Randa , Torres-Ramos Carlos A. 
Published: February 2011

Abstract
Mutations in mitochondrial DNA (mtDNA) have been described in many cancer types. The functional significance of these mutations is not totally understood, but it has been suggested that mtDNA mutations may lead to increased ROS production, aerobic glycolysis and growth promotion in cancer cells. Increased mutagenesis in mtDNA may be due to decreased mtDNA repair capacity. However, there are a limited number of methods to specifically determine mtDNA repair capacity. The goal of this work is to apply a gene-specific PCR-based assay that can detect the presence of a variety of lesions in the mtDNA as a tool to determine the mtDNA repair capacity of peripheral blood mononuclear cells (PBMCs). The rationale of the QPCR assay is that lesions that stall the thermostable DNA polymerase on the DNA template during the PCR reaction will lead to a decrease in amplification of the fragment of interest. Because the DNA polymerase amplifies only undamaged templates, amplification is inversely proportional to the presence of damage. We performed dose-response and time-course experiments using the oxidizing agent H2O2 to define the optimal conditions of the QPCR assay to be used to determine mtDNA repair capacity in PBMCs from healthy human subjects. Our dose-response experiments show a dose-dependent reduction in the amplification of the mtDNA amplicon. In these experiments, exposure of PBMCs to 0.1 mM H2O2 for 1-hour resulted in a 50% decrease in the amplification of the mtDNA fragment. Time course experiments showed that 24 hours after treatment there was still a 50% reduction in the amplification of the mtDNA amplicon, suggesting that mtDNA lesions were still present. However, we observed that 48 hours after treatment, the amplification of the mtDNA amplicon reached levels similar to those from untreated control cells. These results indicate that human PBMCs take 48 hours to repair the mtDNA damage induced by 1-hour exposure to 0.1 mM H2O2. These results define the conditions to compare mtDNA repair capacity in PBMCs from cancer patients and healthy subjects. Sponsored by 5U54CA096297-07.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3940.