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Biomedical subjects

Bert W O'Malley

Publications and source records attributed to Bert W O'Malley.

At least 19 recordsLinked to original sources
PubMed2007

Radiosensitization of head/neck squamous cell carcinoma by adenovirus-mediated expression of the Nbs1 protein.

PURPOSE: Local failure and toxicity to adjacent critical structures is a significant problem in radiation therapy of cancers of the head and neck. We are developing a gene therapy based method of sensitizing head/neck squamous cell carcinoma (HNSCC) to radiation treatment. As patients with the rare hereditary disorder, Nijmegen breakage syndrome, show radiation sensitivity we hypothesized that tumor-specific disruption of the function of the Nbs1 protein would lead to enhanced cellular sensitivity to ionizing radiation. EXPERIMENTAL PROCEDURES: We constructed two recombinant adenoviruses by cloning the full-length Nbs1 cDNA as well as the C-terminal 300 amino acids of Nbs1 into an adenovirus backbone under the control of a CMV promoter. The resulting adenoviruses were used to infect HNSCC cell line JHU011. These cells were evaluated for expression of the viral based constructs and assayed for clonogenic survival following radiation exposure. RESULTS: Exposure of cells expressing Nbs1-300 to ionizing radiation resulted in a small reduction in survival relative to cells infected with control virus. Surprisingly, expression of full-length Nbs1 protein resulted in markedly enhanced sensitivity to ionizing radiation. Furthermore, the use of a fractionated radiation scheme following virus infection demonstrates that expression of full-length Nbs1 protein results in significant reduction in cell survival. CONCLUSIONS: These results provide a proof of principle that disruption of Nbs1 function may provide a means of enhancing the radiosensitivity of head and neck tumors. Additionally, this work highlights the Mre11 complex as an attractive target for development of radiation sensitizers.

Adenoviridae↗
PubMed2006

Specific amino acid residues in the basic helix-loop-helix domain of SRC-3 are essential for its nuclear localization and proteasome-dependent turnover.

SRC-3/AIB1/ACTR/pCIP/RAC3/TRAM-1 is a primary transcriptional coactivator for the estrogen receptor. Here we report that deletion of the SRC-3 basic helix-loop-helix (bHLH) domain blocks its proteasome-dependent turnover. We further identified two residues (K17 and R18) in the SRC-3 bHLH domain that are essential for its stability. Moreover, we found that the bHLH domain contains a bipartite nuclear localization signal (NLS). SRC-3 NLS mutants block its translocation into the nucleus, and this correlates with its insensitivity to proteasome-dependent turnover. SRC-3 shows a time-dependent decay in the presence of cycloheximide which is not apparent for the cytoplasmic mutant. Fusion of a simian virus 40 T antigen NLS to the cytoplasmic localized SRC-3 mutant drives it back into the nucleus and restores its proteasomal sensitivity. In addition, the cytoplasmic mutants are inactive for transcriptional coactivation and cancer cell growth. Taken together, our data indicate that proteasome-dependent turnover of SRC-3 occurs in the nucleus and that two amino acid residues in the bHLH domain provide a signal for its nuclear localization and proteasome-dependent degradation as well as for regulation of SRC-3 transcriptional coactivator capacity.

Active Transport, Cell Nucleus↗
PubMed2006

Oncogenic steroid receptor coactivator-3 is a key regulator of the white adipogenic program.

The white adipocyte is at the center of dysfunctional regulatory pathways in various pathophysiological processes, including obesity, diabetes, inflammation, and cancer. Here, we show that the oncogenic steroid receptor coactivator-3 (SRC-3) is a critical regulator of white adipocyte development. Indeed, in SRC-3(-/-) mouse embryonic fibroblasts, adipocyte differentiation was severely impaired, and reexpression of SRC-3 was able to restore it. The early stages of adipocyte differentiation are accompanied by an increase in nuclear levels of SRC-3, which accumulates to high levels specifically in the nucleus of differentiated fat cells. Moreover, SRC-3(-/-) animals showed reduced body weight and adipose tissue mass with a significant decrease of the expression of peroxisome proliferator-activated receptor gamma2 (PPARgamma2), a master gene required for adipogenesis. At the molecular level, SRC-3 acts synergistically with the transcription factor CAAT/enhancer-binding protein to control the gene expression of PPARgamma2. Collectively, these data suggest a crucial role for SRC-3 as an integrator of the complex transcriptional network controlling adipogenesis.

3T3 Cells↗
PubMed2006

Haploinsufficiency of the corepressor of estrogen receptor activity (REA) enhances estrogen receptor function in the mammary gland.

Estrogen receptor (ER)-mediated gene expression plays an essential role in mammary gland morphogenesis, function, and carcinogenesis. The repressor of ER activity (REA) is an ER-interactive protein that counterbalances estrogen-induced ER transcriptional activity. Our previous study showed that genetic deletion of both REA alleles resulted in embryonic lethality. This study demonstrates that REA and ERalpha are coexpressed in mammary epithelial cells. REA heterozygous (REA(+/-)) mutant mice exhibit faster mammary ductal elongation in virgin animals, increased lobuloalveolar development during pregnancy, and delayed mammary gland involution after weaning. These morphological phenotypes of REA(+/-) mice are associated with significantly increased cell proliferation and ER transcriptional activities, as indicated by the estrogen response element (ERE)-luciferase reporter in the WT/ERE-Luc and REA(+/-)/ERE-Luc bigenic mice and by the higher expression levels of estrogen-responsive genes such as progesterone receptor and cyclin D1 in the mammary gland. Our analysis also revealed that REA is an important repressor of ER transcriptional activity in the mammary gland under natural, as well as ovariectomized and estrogen-replaced, hormonal conditions. Our results indicate that REA is a physiological modulator of ER function in the mammary gland and that its correct gene dosage is required for maintenance of normal ER activity and normal mammary gland development. Consequently, a reduction or loss of REA function may cause overactivation of ER and increase breast cancer risk in humans.

Animals↗
PubMed2006

Steroid receptor coactivator 2 is essential for progesterone-dependent uterine function and mammary morphogenesis: insights from the mouse--implications for the human.

While the indispensability of the progesterone receptor (PR) in female reproduction and mammary morphogenesis is acknowledged, the coregulators preferentially recruited by PR to mediate its in vivo effects have yet to be fully delineated. To further parse the roles of steroid receptor coactivator (SRC)/p160 family members in P-dependent physiological processes, genetic approaches were employed to generate a mouse model (PR(Cre/+)SRC-2(flox/flox)) in which SRC-2 function was ablated specifically in cell-types that express the PR. Fertility evaluation revealed that while ovulation occurred normally in the PR(Cre/+)SRC-2(flox/flox) mouse, uterine function was markedly affected. Absence of SRC-2 in PR positive uterine cells contributed to an early block in embryo implantation, a phenotype not shared by knockouts for SRC-1 or -3. Although the PR(Cre/+)SRC-2(flox/flox) uterus could mount a partial decidual response, removal of SRC-1 in the PR(Cre/+)SRC-2(flox/flox) uterus resulted in a complete block in decidualization, confirming that uterine SRC-2 and -1 are both required for P-initiated transcriptional programs which lead to full decidualization. In the case of the mammary gland, whole-mount and histological analyses revealed the absence of significant branching morphogenesis in the hormone-treated PR(Cre/+)SRC-2(flox/flox) mammary gland, reinforcing an important role for mammary SRC-2 in cellular proliferative events that require PR. Based on the above and the observation that SRC-2 is expressed in many of the uterine and mammary cell-lineages in the human as observed in the mouse, we suggest that further investigations are warranted to gain additional insights into SRC-2's involvement in normal (and possibly abnormal) uterine and mammary cellular responses to progestins.

Animals↗
PubMed2006

Signaling within a coactivator complex: methylation of SRC-3/AIB1 is a molecular switch for complex disassembly.

Recent studies indicate that steroid receptor-mediated transcriptional initiation is a cyclical process involving multiple rounds of coactivator assembly and disassembly. Steroid receptor coactivator 3 (SRC-3) coactivator phosphorylation has been shown to regulate coactivator complex assembly, but the mechanisms by which coactivator disassembly is triggered are not well understood. In this study, we provide in vitro and in vivo evidence that members of the SRC coactivator family serve as substrates for the enzymatic coactivator coactivator-associated arginine methyltransferase 1 (CARM1). Methylation of SRC-3 was localized to an arginine in its CARM1 binding region and correlated with decreased estrogen receptor alpha-mediated transcription, as seen with both cell-based and in vitro transcription assays. Consistent with this finding, we demonstrated that methylation promotes dissociation of the SRC-3/CARM1 coactivator complex. Methylation of SRC-3 is regulated by estrogen signaling in MCF7 cells and serves as a molecular switch for disassembly of the SRC-3 transcriptional coactivator complex. We propose that CARM1 is a dual-function coactivator, as it not only activates transcription by modifying core histone tails but also terminates hormone signaling by disassembly of the coactivator complex.

Amino Acid Sequence↗
PubMed2006

Stimulation of steroid receptor coactivator-3 (SRC-3) gene overexpression by a positive regulatory loop of E2F1 and SRC-3.

Steroid receptor coactivator 3 (SRC-3, amplified in breast cancer 1, or ACTR) is a transcriptional coactivator for nuclear receptors and certain other transcription factors such as E2F1. SRC-3 is overexpressed in breast cancers, and its overexpression is sufficient to cause mammary carcinomas in vivo. However, the mechanisms controlling endogenous SRC-3 overexpression are unknown. In this study, we identified the first exon and analyzed the 5' regulatory sequence of the SRC-3 gene. We found three evolutionarily conserved regions (ECRs) in the 5' SRC-3 regulatory sequence, and ECR2 makes a major contribution to the SRC-3 promoter activity. The ECR2 region (bp -250/+350) contains several specificity protein 1 (Sp1) binding sites and two E2F1 binding sites. We show that E2F1 can significantly activate the ECR2 promoter activity in a dose-dependent manner. Furthermore, overexpression of E2F1 significantly increases the promoter activity of the endogenous SRC-3 gene and boosts SRC-3 expression in vivo. Conversely, knockdown of E2F1 reduces SRC-3 expression. We demonstrate that the mechanism of E2F1 activity on SRC-3 promoter is independent of the E2F binding sites but relies on the Sp1 element located at bp +150/+160. Sp1, E2F1, and SRC-3 are specifically recruited to this Sp1 site and the interaction between E2F1 and Sp1 is essential to modulate SRC-3 expression. Moreover, SRC-3 coactivates E2F1 activity and thereby additively stimulates a further increase in SRC-3 expression in vivo. These results suggest that in cells with hyperactive E2F1, such as the case encountered in breast cancer cells, there is a positive feedback regulatory loop consisting of E2F1 and SRC-3 to maintain high levels of SRC-3 and E2F1 activity, which may partially interpret the oncogenic role of SRC-3 overexpression.

Amino Acid Motifs↗
PubMed2006

A scoring system for the follow up study of nuclear receptor coactivator complexes.

We have systematically isolated a variety of coactivator complexes from HeLa S3 cells using proteomic approaches. In the present report, we have evaluated twelve coactivator complexes involved in nuclear receptor-dependent gene transcription that have been purified by using an immunoprecipitation method. The twelve purified coactivator complexes are SRC-1, SRC-2, SRC-3, CBP, p300, CAPER, E6-AP, ASC-1, CoREST, CRSP3, CRSP2, and CDK7 containing complexes. We have identified 153 protein components associated with these coactivator complexes using mass spectrometry. In order to systematically characterize the functional roles for these components in nuclear receptor-dependent gene transcription and their investigative potential, we have developed a scoring system. This scoring system is comprised of biological and experimental parameters. The biological evaluation considers aspects such as intrinsic enzymatic activity of a protein component, cellular signaling processes in which protein components may be involved, associations with human disease, specific protein motifs, and the known biological roles of other interacting partners of the identified protein. In the experimental evaluation, we include parameters, such as the availability of research materials for the functional study of the identified protein component; such as full-length cDNA clones, antibodies, and commercially available knock-out embryonic stem (ES) cells. Each scoring parameter has been assigned an arbitrary number of points according to perceived relative importance. On the basis of this scoring system, we prioritized each of the protein components in terms of the likelihood of their importance for coactivator complex networking in nuclear receptor-dependent gene transcription.

Journal Article↗
PubMed2006

WW domain binding protein-2, an E6-associated protein interacting protein, acts as a coactivator of estrogen and progesterone receptors.

WW domain binding protein-2 (WBP-2) was cloned as an E6-associated protein interacting protein, and its role in steroid hormone receptors functions was investigated. We show that WBP-2 specifically enhanced the transactivation functions of progesterone receptor (PR) and estrogen receptor (ER), whereas it did not have any significant effect on the androgen receptor, glucocorticoid receptor, or the activation functions of p53 and VP-16. Depletion of endogenous WBP-2 with small interfering RNAs indicated that WBP-2 was required for the proper functioning of PR and ER. We also demonstrated that WBP-2 contains an intrinsic activation domain. Moreover, chromatin immunoprecipitation assays demonstrate the hormone-dependent recruitment of WBP-2 onto an estrogen-responsive promoter. Mutational analysis suggests that one of three polyproline (PY) motifs of WBP-2 is essential for its coactivation and intrinsic activation functions. We show that WBP-2 and E6-associated protein each enhance PR function, and their effect on PR action are additive when coexpressed, suggesting a common signaling pathway. In this study, we also demonstrate that the WBP-2 binding protein, Yes kinase-associated protein (YAP) enhances PR transactivation, but YAP's coactivation function is absolutely dependent on WBP-2. Taken together, our data establish the role of WBP-2 and YAP as coactivators for ER and PR transactivation pathways.

Adaptor Proteins, Signal Transducing↗
PubMed2006

SLIRP, a small SRA binding protein, is a nuclear receptor corepressor.

Steroid receptor RNA activator (SRA), the only known RNA coactivator, augments transactivation by nuclear receptors (NRs). We identified SLIRP (SRA stem-loop interacting RNA binding protein) binding to a functional substructure of SRA, STR7. SLIRP is expressed in normal and tumor tissues, contains an RNA recognition motif (RRM), represses NR transactivation in a SRA- and RRM-dependent manner, augments the effect of Tamoxifen, and modulates association of SRC-1 with SRA. SHARP, a RRM-containing corepressor, also binds STR7, augmenting repression with SLIRP. SLIRP colocalizes with SKIP (Chr14q24.3), another NR coregulator, and reduces SKIP-potentiated NR signaling. SLIRP is recruited to endogenous promoters (pS2 and metallothionein), the latter in a SRA-dependent manner, while NCoR promoter recruitment is dependent on SLIRP. The majority of the endogenous SLIRP resides in the mitochondria. Our data demonstrate that SLIRP modulates NR transactivation, suggest it may regulate mitochondrial function, and provide mechanistic insight into interactions between SRA, SLIRP, SRC-1, and NCoR.

Amino Acid Sequence↗
PubMed2006

The expanding cosmos of nuclear receptor coactivators.

About 200 coactivators play a central role in promoting gene expression mediated by nuclear receptors. This diverse group of proteins are key integrators of signals from steroid hormones and have been implicated in cancer and other diseases.

Active Transport, Cell Nucleus↗
PubMed2006

Absence of the steroid receptor coactivator-3 induces B-cell lymphoma.

Steroid receptor coactivator 3 (SRC-3/ACTR/AIB-1/pCIP/RAC3/TRAM-1) is a member of the p160 family of nuclear receptor coactivators that plays an important role in mammary gland growth, development, and tumorigenesis. We show that deletion of SRC-3 gene decreases platelet and increases lymphocytes numbers, leading to the development of malignant B-cell lymphomas upon aging. The expansion of the lymphoid lineage in SRC-3(-/-) mice is cell autonomous, correlates with an induction of proliferative and antiapoptotic genes secondary to constitutive NF-kappaB activation, and can be reversed by restoration of SRC-3 expression. NF-kappaB activation is explained by the degradation of IkappaB, consequent to increases in free IkappaB kinase, which is no longer inhibited by SRC-3. These results demonstrate that SRC-3 regulates lymphopoiesis and in combination with previous studies indicate that SRC-3 has vastly diverging effects on cell proliferation depending on the cellular context, ranging from proliferative and tumorigenic (breast) to antiproliferative (lymphoid cells) effects.

Animals↗
PubMed2006

Tumor metastasis in an orthotopic murine model of head and neck cancer: possible role of TGF-beta 1 secreted by the tumor cells.

In an orthotopic murine model of head and neck cancer, combined subcutaneous and intratumoral vaccination with recombinant vaccinia virus expressing interleukin-2 (rvv-IL-2) induced significant tumor regression early on therapy. However, its efficacy was restricted by recurrent tumor growth and loco-regional metastases. In this study, we explored the mechanism of tumor metastasis. We compared the levels of expression of a number of molecules involved in tumor metastasis, which included transforming growth factor-beta1 (TGF-beta1), E-cadherin, matrix metalloproteinases (MMPs): MT1-MMP, MMP-2, MMP-9, their tissue inhibitors (TIMPs): TIMP-1/TIMP-2, and pro-angiogenic factors CD31, VEGF-R2, and iNOS between primary and metastatic tumors by real-time RT-PCR and immunohistochemistry. We detected spontaneous lymph node and tongue metastasis. Metastasis was delayed in rvv-IL-2 treated mice. Cultured tumor cells expressed negligible amount of TGF-beta1. Untreated or metastatic tumors, on the other hand, expressed high levels of TGF-beta1 and secreted TGF-beta1 in the sera of tumor-bearing mice. Levels of TGF-beta1 in the sera suddenly jumped at the time when tumor metastasis started. In the metastatic tumors, levels of MT1-MMP, MMP-2, and MMP-9 were significantly elevated (P < 0.001), while levels of TIMP-1/TIMP-2 and E-cadherin were decreased (P < 0.001) compared to control or primary tumors. Levels of CD31, VEGF-R2, and iNOS were also significantly elevated in the metastatic lesions (P < 0.001). The concurrence of high levels of TGF-beta1 in the sera, expression of proteins involved in metastasis and initiation of metastasis suggested possible role of TGF-beta1 in on setting the metastatic cascade in this model.

Animals↗
PubMed2006

Transoral robotic surgery (TORS): glottic microsurgery in a canine model.

OBJECTIVES/HYPOTHESIS: We hypothesize that bimanual, three-dimensional robotic surgery will prove valuable for glottic microsurgical procedures. STUDY DESIGN: To test this hypothesis, we developed and optimized a canine model for glottic microsurgery using a commercially available surgical robot. Methods Using a da Vinci Surgical Robot (Intuitive Surgical, Inc., Sunnyvale, CA), glottic microsurgery was performed with a hydrodissection technique in a canine model. The experiments were performed on two orotracheal intubated mongrel dogs under general anesthesia in the supine position on a standard operating room table. A videoscope and two, 360-degree rotating, 5- and 8-mm, wrested-end effector instruments were introduced transorally with three robotic arms. The surgeon performed the actual procedures while positioned at a robotic system console that was located across the operating room suite. The procedure was performed in duplicate and was documented with still and video photography. RESULTS: Glottic microsurgery was successfully performed using the da Vinci Surgical Robot, with both 5- and 8-mm instrumentation. The smaller, 5-mm instruments afforded greater visualization of the operative site and increased maneuverability, which resulted in reduced operative time. The robotic system provided excellent visualization as well as controlled and delicate microdissection at the vocal cord level. CONCLUSIONS: Robotic laryngeal microsurgery is technically feasible in the canine model and warrants consideration for evaluation in controlled human trials.

Animals↗
PubMed2006

The SRC-3/AIB1 coactivator is degraded in a ubiquitin- and ATP-independent manner by the REGgamma proteasome.

Steroid receptor coactivator-3 (SRC-3/AIB1) is an oncogene frequently amplified and overexpressed in breast cancers. Here we report that SRC-3 interacts with REGgamma, a proteasome activator known to stimulate the trypsin-like activity of the 20S proteasome. RNAi knockdown and gain-of-function experiments suggest that REGgamma promotes SRC-3 protein degradation. Cellular levels of REGgamma expression affect estrogen-receptor target-gene expression and cell growth as a result of its ability to promote degradation of the SRC-3 protein. In vitro proteasome proteolysis assays using purified REGgamma, SRC-3, and the 20S proteasome reinforce these conclusions and demonstrate that REGgamma promotes the degradation of SRC-3 in a ubiquitin- and ATP-independent manner. This work demonstrates the first example of a physiologically relevant endogenous cellular target for the REGgamma-proteasome complex. It also highlights the fact that an alternative mode of proteasome-mediated protein degradation, independent of the 19S proteasome regulatory cap, targets the SRC-3 protein for degradation.

Acetyltransferases↗
PubMed2006

The genomic analysis of the impact of steroid receptor coactivators ablation on hepatic metabolism.

Members of the steroid receptor coactivator (SRC) family, which include SRC-1 (NcoA-1/p160), SRC-2(TIF2/GRIP1/NcoA-2) and SRC-3(pCIP/RAC3/ACTR/pCIP/ AIB1/TRAM1), are critical mediators of steroid receptor action. Gene ablation studies previously identified SRC-1 and SRC-2 as being involved in the control of energy homeostasis. A more precise identification of the molecular pathways regulated by these coactivators is crucial for understanding the role of steroid receptor coactivators in the control of energy homeostasis and obesity. A genomic approach using microarray analysis was employed to identify the subsets of genes that are altered in the livers of SRC-1-/-, SRC-2-/-, and SRC-3-/- mice. Microarray analysis demonstrates that gene expression changes are specific and nonoverlapping for each SRC member in the liver. The overall pattern of altered gene expressions in the SRC-1-/- mice was up-regulation, whereas SRC-2-/- mice showed an overall down-regulation. Several key regulatory enzymes of energy metabolism were significantly altered in the liver of SRC-2-/- mice, which are consistent with the prior observation that SRC-2-/- mice have increased energy expenditure. This study demonstrates that the molecular targets of SRC-2 regulation in the murine liver stimulate fatty acid degradation and glycolytic pathway, whereas fatty acid, cholesterol, and steroid biosynthetic pathways are down-regulated.

Animals↗
PubMed2006

Microvascular reconstruction of cranial base defects: An evaluation of complication and survival rates to justify the use of this repair.

OBJECTIVES: To describe our experience, including selection criteria and complication rates, using microvascular free tissue transfer to repair large skull base defects and to determine if complication rate and posttreatment survival justify the use of this technique. DESIGN: Retrospective review of clinical cohort. SETTING: Tertiary care medical center. PATIENTS: A consecutive sample of patients undergoing ablative surgery with repair of cranial base defects with free tissue transfer from 1995 to 2004. All the patients' defects involved intracranial exposure. MAIN OUTCOME MEASURES: Rate of local and systemic complications, postoperative survival, and recurrence rate. RESULTS: The study population comprised 40 patients. Fifteen (38%) of the patients' defects were in the anterior cranial base, and 26 (65%) were in the middle cranial base. We used 5 types of free tissue flap, with a success rate of 95%. Our rate of perioperative mortality, meningitis, stroke, cerebrospinal fluid leak, epidural abscess, and osteomyelitis was 0%. We had 7 local complications and 4 systemic complications requiring increased length of hospital stay. Including microvascular problems, 12 patients had complications, for an overall complication rate of 30%. Follow-up ranged from 1 to 96 months, with a mean of 24 months. The tumor recurrence rate was 30%, and disease-specific survival was 81% at a mean 24-month follow-up. CONCLUSIONS: We did not experience any perioperative mortality or intracranial morbidity. Our low complication rate in combination with our tumor recurrence rate and rate of patient survival justify the use of free tissue transfer as an option in the closure of appropriate cranial base defects.

Adolescent↗