Biomedical subjects

Mary Helen Barcellos-Hoff

Publications and source records attributed to Mary Helen Barcellos-Hoff.

At least 19 recordsLinked to original sources

PubMed2006

Inhibition of transforming growth factor-beta1 signaling attenuates ataxia telangiectasia mutated activity in response to genotoxic stress.

Ionizing radiation causes DNA damage that elicits a cellular program of damage control coordinated by the kinase activity of ataxia telangiectasia mutated protein (ATM). Transforming growth factor beta (TGFbeta)-1, which is activated by radiation, is a potent and pleiotropic mediator of physiologic and pathologic processes. Here we show that TGFbeta inhibition impedes the canonical cellular DNA damage stress response. Irradiated Tgfbeta1 null murine epithelial cells or human epithelial cells treated with a small-molecule inhibitor of TGFbeta type I receptor kinase exhibit decreased phosphorylation of Chk2, Rad17, and p53; reduced gammaH2AX radiation-induced foci; and increased radiosensitivity compared with TGFbeta competent cells. We determined that loss of TGFbeta signaling in epithelial cells truncated ATM autophosphorylation and significantly reduced its kinase activity, without affecting protein abundance. Addition of TGFbeta restored functional ATM and downstream DNA damage responses. These data reveal a heretofore undetected critical link between the microenvironment and ATM, which directs epithelial cell stress responses, cell fate, and tissue integrity. Thus, Tgfbeta1, in addition to its role in homoeostatic growth control, plays a complex role in regulating responses to genotoxic stress, the failure of which would contribute to the development of cancer; conversely, inhibiting TGFbeta may be used to advantage in cancer therapy.

Activin Receptors, Type I↗

PubMed2006

Quantitative in vivo microscopy: the return from the 'omics'.

The confluence of recent advances in microscopy instrumentation and image analysis, coupled with the widespread use of GFP-like proteins as reporters of gene expression, has opened the door to high-throughput in vivo studies that can provide the morphological and temporal context to the biochemical pathways regulating cell function. We are now able to quantify the concentration and three-dimensional distribution of multiple spectrally resolved GFP-tagged proteins. Using automatic segmentation and tracking we can then measure the dynamics of the processes in which these elements are involved. In this way, parallel studies are feasible where multiple cell colonies treated with drugs or gene expression repressors can be monitored and analyzed to study the dynamics of relevant biological processes.

Animals↗

PubMed2006

A systems biology approach to multicellular and multi-generational radiation responses.

Recent studies have highlighted crosstalk between irradiated cells and non-irradiated bystander cells and have uncovered high-frequency phenotypes of genomic instability in the progeny of irradiated cells that cannot be solely explained by radiation-induced mutation. It is difficult to explain these multicellular and multi-generational phenomena using the current paradigm of radiation biology. Radiation-induced bystander effect is a type of multicellular response to radiation that illustrates that the unit of function in multicellular organisms is neither the genome nor the cell. Cell function in complex three-dimensional tissues is coordinated by soluble signaling peptides and by small molecules within the context of insoluble scaffolding provided by the extracellular matrix. Adaptive response and radiation-induced genomic instability could thus result from persistent signaling perturbations following radiation exposures. A model of radiation response based on the systems biology principles of network interconnectivity and spatial organization should reconcile the apparent contradiction of these cellular phenotypes within the higher order structure of tissues and organisms.

Animals↗

PubMed2006

Intensity-based signal separation algorithm for accurate quantification of clustered centrosomes in tissue sections.

Centrosomes are small organelles that organize the mitotic spindle during cell division and are also involved in cell shape and polarity. Within epithelial tumors, such as breast cancer, and some hematological tumors, centrosome abnormalities (CAs) are common, occur early in disease etiology, and correlate with chromosomal instability and disease stage. In situ quantification of CA by optical microscopy is hampered by overlap and clustering of these organelles, which appear as focal structures. CA has been frequently associated with Tp53 status in premalignant lesions and tumors. Here the authors described an approach to accurately quantify centrosome frequencies in tissue sections and tumors, independently of background or noise levels. Applying simple optical rules in nondeconvolved conventional 3D images of stained tissue sections, the authors showed that they could evaluate more accurately and rapidly centrosome frequencies than with traditional investigator-based visual analysis or threshold-based techniques. The resulting population-based frequency of centrosomes per nucleus could then be used to approximate the proportion of cells with CA in that same population. This was done by taking into account baseline centrosome amplification and proliferation rates measured in the tissue. Using this technique, the authors showed that 20-30% of cells have amplified centrosomes in Tp53 null mammary tumors.

Algorithms↗

PubMed2006

Isoform-specific activation of latent transforming growth factor beta (LTGF-beta) by reactive oxygen species.

The three mammalian transforming growth factor beta (TGF-beta) isoforms are each secreted in a latent complex in which TGF-beta homodimers are non-covalently associated with homodimers of their respective pro-peptide called the latency-associated peptide (LAP). Release of TGF-beta from its LAP, called activation, is required for binding of TGF-beta to cellular receptors, making extracellular activation a critical regulatory point for TGF-beta bioavailability. Our previous work demonstrated that latent TGF-beta1 (LTGF-beta1) is efficiently activated by ionizing radiation in vivo and by reactive oxygen species (ROS) generated by Fenton chemistry in vitro. In the current study, we determined the specific ROS and protein target that render LTGF-beta1 redox sensitive. First, we compared LTGF-beta1, LTGF-beta2 and LTGF-beta3 to determine the generality of this mechanism of activation and found that redox-mediated activation is restricted to the LTGF-beta1 isoform. Next, we used scavengers to determine that ROS activation was a function of OH(.) availability, confirming oxidation as the primary mechanism. To identify which partner of the LTGF-beta1 complex was functionally modified, each was exposed to ROS and tested for the ability to form a latent complex. Exposure of TGF-beta1 did not alter its ability to associate with LAP, but exposing LAP-beta1 to ROS prohibited this phenomenon, while treatment of ROS-exposed LAP-beta1 with a mild reducing agent restored its ability to neutralize TGF-beta1 activity. Taken together, these results suggest that ROS-induced oxidation in LAP-beta1 triggers a conformational change that releases TGF-beta1. Using site-specific mutation, we identified a methionine residue at amino acid position 253 unique to LAP-beta1 as critical to ROS-mediated activation. We propose that LTGF-beta1 contains a redox switch centered at methionine 253, which allows LTGF-beta1 to act uniquely as an extracellular sensor of oxidative stress in tissues.

Dose-Response Relationship, Drug↗

PubMed2006

Imaging features that discriminate between foci induced by high- and low-LET radiation in human fibroblasts.

In this study, we investigated the formation of radiation-induced foci in normal human fibroblasts exposed to X rays or 130 keV/mum nitrogen ions using antibodies to phosphorylated protein kinase ataxia telangiectasia mutated (ATMp) and histone H2AX (gamma-H2AX). High-content automatic image analysis was used to quantify the immunofluorescence of radiation-induced foci. The size of radiation-induced foci increased for both proteins over a 2-h period after nitrogen-ion irradiation, while the size of radiation-induced foci did not change after exposure to low-LET radiation. The number of radiation-induced ATMp foci showed a more rapid rise and greater frequency after X-ray exposure and was resolved more rapidly such that the frequency of radiation-induced foci decreased by 90% compared to 60% after exposure to high-LET radiation 2 h after 30 cGy. In contrast, the kinetics of radiation-induced gamma-H2AX focus formation was similar for high- and low-LET radiation in that it reached a plateau early and remained constant for up to 2 h. High-resolution 3D images of radiation-induced gamma-H2AX foci and dosimetry computation suggest that multiple double-strand breaks from nitrogen ions are encompassed within large nuclear domains of 4.4 Mbp. Our work shows that the size and frequency of radiation-induced foci vary as a function of radiation quality, dose, time and protein target. Thus, even though double-strand breaks and radiation-induced foci are correlated, the dynamic nature of both contradicts their accepted equivalence for low doses of different radiation qualities.

Ataxia Telangiectasia Mutated Proteins↗

PubMed2005

Proliferation of estrogen receptor-alpha-positive mammary epithelial cells is restrained by transforming growth factor-beta1 in adult mice.

Transforming growth factor (TGF)-beta1 is a potent inhibitor of mammary epithelial proliferation. In human breast, estrogen receptor (ER)-alpha cells rarely co-localize with markers of proliferation, but their increased frequency correlates with breast cancer risk. To determine whether TGF-beta1 is necessary for the quiescence of ER-alpha-positive populations, we examined mouse mammary epithelial glands at estrus. Approximately 35% of epithelial cells showed TGF-beta1 activation, which co-localized with nuclear receptor-phosphorylated Smad 2/3, indicating that TGF-beta signaling is autocrine. Nuclear Smad co-localized with nuclear ER-alpha. To test whether TGF-beta inhibits proliferation, we examined genetically engineered mice with different levels of TGF-beta1. ER-alpha co-localization with markers of proliferation (ie, Ki-67 or bromodeoxyuridine) at estrus was significantly increased in the mammary glands of Tgf beta1 C57/bl/129SV heterozygote mice. This relationship was maintained after pregnancy but was absent at puberty. Conversely, mammary epithelial expression of constitutively active TGF-beta1 via the MMTV promoter suppressed proliferation of ER-alpha-positive cells. Thus, TGF-beta1 activation functionally restrains ER-alpha-positive cells from proliferating in adult mammary gland. Accordingly, we propose that TGF-beta1 dysregulation may promote proliferation of ER-alpha-positive cells associated with breast cancer risk in humans.

Animals↗

PubMed2005

Radiation and the microenvironment - tumorigenesis and therapy.

Radiation rapidly and persistently alters the soluble and insoluble components of the tissue microenvironment. This affects the cell phenotype, tissue composition and the physical interactions and signalling between cells. These alterations in the microenvironment can contribute to carcinogenesis and alter the tissue response to anticancer therapy. Examples of these responses and their implications are discussed with a view to therapeutic intervention.

Animals↗

PubMed2005

A tool for the quantitative spatial analysis of complex cellular systems.

Spatial events largely determine the biology of cells, tissues, and organs. In this paper, we present a tool for the quantitative spatial analysis of heterogeneous cell populations, and we show experimental validation of this tool using both artificial and real (mammary gland tissue) data, in two and three dimensions. We present the refined relative neighborhood graph as a means to establish neighborhood between cells in an image while modeling the topology of the tissue. Then, we introduce the M function as a method to quantitatively evaluate the existence of spatial patterns within one cell population or the relationship between the spatial distributions of multiple cell populations. Finally, we show a number of examples that demonstrate the feasibility of our approach.

Algorithms↗

PubMed2004

Integrative radiation carcinogenesis: interactions between cell and tissue responses to DNA damage.

Tissue function requires coordinated multicellular behavior as a consequence of diverse signals integrated through the tissue microenvironment; importantly, these cell-cell and cell-microenvironment interactions also actively suppress cancer. Ionizing radiation (IR) elicits a well-defined cellular response to DNA damage that mediates the fate of the individual cell, concomitantly with a less well-characterized overarching tissue stress response that coordinates the response of multiple cell types via microenvironment signaling. We have now shown that these programs to reestablish homeostasis intersect via mutual regulation by transforming growth factor beta1 (TGF beta 1), which acts as an extracellular sensor and signal of stress. In this review, the concept that this type of functional integration of cell and tissue stress response programs is essential to cancer suppression will be discussed. Our experiments using IR, and several recent studies that experimentally manipulate stromal TGF beta, show that disruption of microenvironment signaling actively promotes malignant progression. Understanding the dynamic interactions between tissue and cell stress responses will be necessary for an accurate assessment of cancer risk and may also provide targets for prevention.

Animals↗

PubMed2004

Conditional overexpression of active transforming growth factor beta1 in vivo accelerates metastases of transgenic mammary tumors.

To address the role of transforming growth factor (TGF) beta in the progression of established tumors while avoiding the confounding inhibitory effects of TGF-beta on early transformation, we generated doxycycline (DOX)-inducible triple transgenic mice in which active TGF-beta1 expression could be conditionally regulated in mouse mammary tumor cells transformed by the polyomavirus middle T antigen. DOX-mediated induction of TGF-beta1 for as little as 2 weeks increased lung metastases >10-fold without a detectable effect on primary tumor cell proliferation or tumor size. DOX-induced active TGF-beta1 protein and nuclear Smad2 were restricted to cancer cells, suggesting a causal association between autocrine TGF-beta and increased metastases. Antisense-mediated inhibition of TGF-beta1 in polyomavirus middle T antigen-expressing tumor cells also reduced basal cell motility, survival, anchorage-independent growth, tumorigenicity, and metastases. Therefore, induction and/or activation of TGF-beta in hosts with established TGF-beta-responsive cancers can rapidly accelerate metastatic progression.

Animals↗

PubMed2004

New highlights on stroma-epithelial interactions in breast cancer.

Although the stroma in which carcinomas arise has been previously regarded as a bystander to the clonal expansion and acquisition of malignant characteristics of tumor cells, it is now generally acknowledged that stromal changes are required for the establishment of cancer. In the present article, we discuss three recent publications that highlight the complex role the stroma has during the development of cancer and the potential for targeting the stroma by therapeutic approaches.

Animals↗

PubMed2004

Quantitative image analysis in mammary gland biology.

In this paper we present a summary of recent quantitative approaches used for the analysis of macro and microscopic images in mammary gland biology. The advantages and disadvantages of whole mount analysis, reconstruction of serial tissue sections and nucleus/cell segmentation of either conventional and confocal images are discussed, as are applications of quantitative image analysis, such as quantification of protein levels or vasculature measurements in normal tissue and cancer. Integration of quantitative imaging into the further study of the mammary gland holds the promise of better understanding its tissue complexity that evolves during development, differentiation and disease.

Animals↗

PubMed2004

Models for evaluating agents intended for the prophylaxis, mitigation and treatment of radiation injuries. Report of an NCI Workshop, December 3-4, 2003.

To develop approaches to prophylaxis/protection, mitigation and treatment of radiation injuries, appropriate models are needed that integrate the complex events that occur in the radiation-exposed organism. While the spectrum of agents in clinical use or preclinical development is limited, new research findings promise improvements in survival after whole-body irradiation and reductions in the risk of adverse effects of radiotherapy. Approaches include agents that act on the initial radiochemical events, agents that prevent or reduce progression of radiation damage, and agents that facilitate recovery from radiation injuries. While the mechanisms of action for most of the agents with known efficacy are yet to be fully determined, many seem to be operating at the tissue, organ or whole animal level as well as the cellular level. Thus research on prophylaxis/protection, mitigation and treatment of radiation injuries will require studies in whole animal models. Discovery, development and delivery of effective radiation modulators will also require collaboration among researchers in diverse fields such as radiation biology, inflammation, physiology, toxicology, immunology, tissue injury, drug development and radiation oncology. Additional investment in training more scientists in radiation biology and in the research portfolio addressing radiological and nuclear terrorism would benefit the general population in case of a radiological terrorism event or a large-scale accidental event as well as benefit patients treated with radiation.

Animals↗

PubMed2003

Ionizing radiation induces heritable disruption of epithelial cell interactions.

Ionizing radiation (IR) is a known human breast carcinogen. Although the mutagenic capacity of IR is widely acknowledged as the basis for its action as a carcinogen, we and others have shown that IR can also induce growth factors and extracellular matrix remodeling. As a consequence, we have proposed that an additional factor contributing to IR carcinogenesis is the potential disruption of critical constraints that are imposed by normal cell interactions. To test this hypothesis, we asked whether IR affected the ability of nonmalignant human mammary epithelial cells (HMEC) to undergo tissue-specific morphogenesis in culture by using confocal microscopy and imaging bioinformatics. We found that irradiated single HMEC gave rise to colonies exhibiting decreased localization of E-cadherin, beta-catenin, and connexin-43, proteins necessary for the establishment of polarity and communication. Severely compromised acinar organization was manifested by the majority of irradiated HMEC progeny as quantified by image analysis. Disrupted cell-cell communication, aberrant cell-extracellular matrix interactions, and loss of tissue-specific architecture observed in the daughters of irradiated HMEC are characteristic of neoplastic progression. These data point to a heritable, nonmutational mechanism whereby IR compromises cell polarity and multicellular organization.

Cadherins↗

PubMed2003

The not-so innocent bystander: the microenvironment as a therapeutic target in cancer.

The microenvironment in which cancer arises is often regarded as a bystander to the clonal expansion and acquisition of malignant characteristics of the tumour. However, a major function of the microenvironment is to suppress cancer, and its disruption is required for the establishment of cancer. In addition, tumour cells can further distort the microenvironment to promote growth, recruit non-malignant cells that provide physiological resources, and facilitate invasion. In this review, the authors discuss the contribution of the microenvironment, i.e., the stroma and its resident vasculature, inflammatory cells, growth factors and the extracellular matrix (ECM), in the development of cancer, and focus on two components as potential therapeutic targets in breast cancer. First, the ECM, which imparts crucial signalling via integrins and other receptors, is a first-line barrier to invasion, modulates aggressive behaviour and may be manipulated to provide novel impediments to tumour growth. Second, the authors discuss the involvement of TGF-beta1 as an example of one of many growth factors that can regulate ECM composition and degradation and that play complex roles in cancer. Compared to the variable routes taken by cells to become cancers, the response of tissues to cancer is relatively consistent. Therefore, controlling and eliminating cancer may be more readily achieved indirectly via the tissue microenvironment.

Breast Neoplasms↗

PubMed2003

Education and training for radiation scientists: radiation research program and American Society of Therapeutic Radiology and Oncology Workshop, Bethesda, Maryland, May 12-14, 2003.

Current and potential shortfalls in the number of radiation scientists stand in sharp contrast to the emerging scientific opportunities and the need for new knowledge to address issues of cancer survivorship and radiological and nuclear terrorism. In response to these challenges, workshops organized by the Radiation Research Program (RRP), National Cancer Institute (NCI) (Radiat. Res. 157, 204-223, 2002; Radiat. Res. 159, 812-834, 2003), and National Institute of Allergy and Infectious Diseases (NIAID) (Nature, 421, 787, 2003) have engaged experts from a range of federal agencies, academia and industry. This workshop, Education and Training for Radiation Scientists, addressed the need to establish a sustainable pool of expertise and talent for a wide range of activities and careers related to radiation biology, oncology and epidemiology. Although fundamental radiation chemistry and physics are also critical to radiation sciences, this workshop did not address workforce needs in these areas. The recommendations include: (1) Establish a National Council of Radiation Sciences to develop a strategy for increasing the number of radiation scientists. The strategy includes NIH training grants, interagency cooperation, interinstitutional collaboration among universities, and active involvement of all stakeholders. (2) Create new and expanded training programs with sustained funding. These may take the form of regional Centers of Excellence for Radiation Sciences. (3) Continue and broaden educational efforts of the American Society for Therapeutic Radiology and Oncology (ASTRO), the American Association for Cancer Research (AACR), the Radiological Society of North America (RSNA), and the Radiation Research Society (RRS). (4) Foster education and training in the radiation sciences for the range of career opportunities including radiation oncology, radiation biology, radiation epidemiology, radiation safety, health/government policy, and industrial research. (5) Educate other scientists and the general public on the quantitative, basic, molecular, translational and applied aspects of radiation sciences.

Curriculum↗

PubMed2002

System for combined three-dimensional morphological and molecular analysis of thick tissue specimens.

We present a new system for simultaneous morphological and molecular analysis of thick tissue samples. The system is composed of a computer-assisted microscope and a JAVA-based image display, analysis, and visualization program that allows acquisition, annotation, meaningful storage, three-dimensional reconstruction, and analysis of structures of interest in thick sectioned tissue specimens. We describe the system in detail and illustrate its use by imaging, reconstructing, and analyzing two complete tissue blocks that were differently processed and stained. One block was obtained from a ductal carcinoma in situ (DCIS) lumpectomy specimen and stained alternatively with Hematoxilyn and Eosin (H&E), and with a counterstain and fluorescence in situ hybridization (FISH) to the ERB-B2 gene. The second block contained a fully sectioned mammary gland of a mouse, stained for histology with H&E. We show how the system greatly reduces the amount of interaction required for the acquisition and analysis and is, therefore, suitable for studies that require morphologically driven, wide-scale (e.g., whole gland) analysis of complex tissue samples or cultures.

Animals↗