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Life Sciences Division E-Newsletter

October, 2009

In this issue:

DOE Scientific Focus Area News
Scientific & Divisional News
Awards
Recent Publications

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DOE scientific focus area notes

Low Dose Radiation Research

New Clues to Why Stem Cells Stop Dividing
Berkeley Lab scientists have pieced together a mechanism that causes a type of human adult stem cell to permanently stop dividing after being exposed to ionizing radiation. Their research can be used to help refine cancer treatments that utilize ionizing radiation, and may help inform future work to protect the health of astronauts on missions to deep space. It also sheds light on cellular senescence, a process in which cells permanently stop dividing that is linked to cancer and aging. Daojing Wang of Berkeley Lab’s Life Sciences Division is the principal investigator of the study that is published in the October 15, 2009 issue of the journal Cancer Research. More> http://newscenter.lbl.gov/feature-stories/2009/10/28/stem-cells-stop-dividing/

Wang D, Jang DJ. Protein kinase CK2 regulates cytoskeletal reorganization during ionizing radiation-induced senescence of human mesenchymal stem cells. Cancer Research 2009 Oct 15;69(20):8200-7. PMID: 19826041
Today at Berkeley lab, CG, 10/30/09

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GTL-Genomics

SAXS Nature Methods Paper Second in Top Ten Chart
A recent paper from IDAT and MAGGIE, "Robust, high-throughput solution structural analyses by small angle X-ray scattering (SAXS)," covering research funded by the Department of Energy, was listed on the Nature website in October as number two of the top ten chart of research articles that have been downloaded most often, in PDF format, from the Nature Methods website in the weeks prior. The paper:
Hura GL, Menon AL, Hammel M, Rambo RP, Poole Ii FL, Tsutakawa SE, Jenney Jr FE, Classen S, Frankel KA, Hopkins RC, Yang SJ, Scott JW, Dillard BD, Adams MW, Tainer JA. Robust, high-throughput solution structural analyses by small angle X-ray scattering (SAXS). Nature Methods, 2009 Jul 20. [Epub ahead of print] PMID: 19620974

Abstract: We present an efficient pipeline enabling high-throughput analysis of protein structure in solution with small angle X-ray scattering (SAXS). Our SAXS pipeline combines automated sample handling of microliter volumes, temperature and anaerobic control, rapid data collection and data analysis, and couples structural analysis with automated archiving. We subjected 50 representative proteins, mostly from Pyrococcus furiosus, to this pipeline and found that 30 were multimeric structures in solution. SAXS analysis allowed us to distinguish aggregated and unfolded proteins, define global structural parameters and oligomeric states for most samples, identify shapes and similar structures for 25 unknown structures, and determine envelopes for 41 proteins. We believe that high-throughput SAXS is an enabling technology that may change the way that structural genomics research is done.
John Tainer, 10/09

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Radiochemistry & Instrumentation

Nuclear Science Symposium and Medical Imaging Conference
Scientists and engineers in the Department of Radiotracer Development and Imaging Technology were co-authors of five oral presentations and seventeen poster presentations at the annual IEEE Nuclear Science Symposium and Medical Imaging Conference (NSS/MIC), held October 25–31, 2009 in Orlando, Florida. Department members also presented a workshop at the conference entitled “Improving optical Monte Carlo simulations with measured optical reflectance” led by Martin Janecek and William Moses. While Department Head Stephen Derenzo was the Short Course Program Chair for the Symposium, Jennifer Huber chaired the Short Course Program for the Conference. The courses, given at the start of the programs, covered a wide range of nuclear and medical imaging technology.

The Nuclear Science Symposium’s attendees work in the fields of nuclear science, radiation instrumentation, software and their applications, and they meet and discuss the latest developments in technology and instrumentation and their implementation in experiments for space, accelerators, other radiation environments and homeland security. The Medical Imaging Conference, simultaneously held with the Nuclear Science Symposium, is a leading international scientific meeting on the physics, engineering and mathematical aspects of nuclear medicine–based imaging.
Bob Smith, 10/09

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Collaboration with Earth Sciences Division
In the field of environmental remediation, there is a strong interest in understanding how contaminants, especially heavy metals and radioactive ions, are transported through soils and groundwater aquifers. Members of the Department of Radiotracer Development and Imaging Technology and the Earth Sciences Division met on October 1 and October 7, 2009 to discuss methods for developing radiotracer techniques to image contaminant flow in soils. Radiotracer imaging allows very sensitive measurements of migrating contaminants without physically removing and measuring numerous soil samples.
Bill Moses, 10/09

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Scientific & divisional news

Cover Article on Mass spectrometry

An article on Mass spectrometry, authored by Richard Baran, Wolfgang Reindl, and Trent Northen of the Life Sciences Division was selected for the cover of the October issue of Current Opinion in Microbiology : Baran R, Reindl W, Northen TR. Mass spectrometry based metabolomics and enzymatic assays for functional genomics. Current Opinion in Microbiology, 2009 Aug 18. [Epub ahead of print] PMID: 19695948

From the authors: The exponential growth in the number of sequenced microorganisms versus the relative slow influx of direct biochemical characterization of microbes is limiting the utility of sequence information. High-throughput experimental approaches to functionally characterize microbial metabolism are urgently needed to leverage genome sequences for example: to understand host–microbe interactions, microbial communities, to utilize microbes for bioenergy, bioremediation, etc. Mass spectrometry based small molecule metabolite analysis is rapidly becoming a method of choice to meet these needs and enables multiple paths to discovering and validating the functional assignments. Approaches range from the targeted in vitro screening of small sets of metabolic transformations to define enzymatic activities to global metabolic profiling (metabolomics) to define metabolic pathways and gain insights into microbial responses to environmental and genetic perturbations. The combination of metabolite profiling with genome-scale models of metabolism and other -omic approaches provides opportunities to expand our understanding of microbial metabolic networks, stress responses, and to identify genes associated with specific enzymatic and regulatory activities.
Trent Northen, 10/09

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Missing Piece of DNA Repair Machine Deciphered

Scientists from Berkeley Lab and the Scripps Research Institute have uncovered the role played by the least-understood part of a first-responder molecule that rushes in to bind and repair breaks in DNA strands, a process that helps people avoid cancer. With this final piece of the puzzle in place, scientists can better understand how the repair mechanism fends off cancer in healthy people, and conversely, how it helps cancer cells resist chemotherapy. This could enable researchers to develop more effective therapies with fewer side effects. Their work is published in the October 2, 2009 issue of the journal Cell.

“This not only reveals how life works at a fundamental level, but also promises to guide the development of cancer treatments,” says John Tainer of Berkeley Lab’s Life Sciences Division and the Scripps Research Institute in La Jolla, CA. Tainer co-led the research with Paul Russell of the Scripps Research Institute. More > http://newscenter.lbl.gov/press-releases/2009/10/01/dna-repair-uncovered/
Today at Berkeley Lab, 10/2/09

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Silence of the Genes

Jennifer Doudna (right) of the Physical Biosciences Division and Eva Nogales of the Life Sciences Division teamed up to produce the first image that shows the molecular architecture of a protein complex that helps determine the fate of human cells. Known as a human RISC-loading complex, this structure consists of snippets of ribonucleic acid (RNA) that control whether genetic messages, such as "turn cancerous," are silenced or expressed. More > http://newscenter.lbl.gov/press-releases/2009/10/12/silence-of-the-genes/
Today at Berkeley Lab, 10/13/09

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Jeffress Chairs Junior Investigators Meeting

The annual National Cancer Institute's Integrative Cancer Biology Program's (ICBP) Meeting of the Junior Investigators 2009 was chaired by Mara Jeffress, a Postdoctoral Fellow in Paul Yaswen's lab and part of the local ICBP lead by Joe Gray. The meeting, held in Berkeley, CA on October 11-13, 2009, was attended by NCI staff, invited speakers, and postdoctoral fellows and students from the nine ICBP centers: Berkeley Lab, Duke, Stanford, MIT, Vanderbilt, Harvard, Broad Institute, Case Western, and Ohio State.

The program included opening remarks by Jeffress and Betty Tarnowski, ICBP Education and Outreach Committee Chair, an overview of the ICBP by Program Manager Dan Gallahan, and diverse Science talks from each of the nine ICBP centers. The program also included a Career Development Panel, invited talks by Berkeley Lab scientists, Mina Bissell and Adam Arkin, and a keynote address by Steve Wiley of the Pacific Northwest National Lab on "Using a systems approach for reconstructing signaling networks in normal and transformed cells."

The Integrative Cancer Biology Program focuses on the analysis of cancer as a complex biological system. A cornerstone of the program is the development and implementation of computational models of processes relevant to cancer prevention, diagnostics and therapeutics. The integration of experimental biology with mathematical modeling will result in new insights in the biology and new approaches to the management of cancer. The program brings clinical and basic cancer researchers together with researchers from mathematics, physics, information technology, imaging sciences, and computer science to work on key questions in cancer biology. More> http://icbp.nci.nih.gov/
CG, 10/09

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Berkeley Blog Features Numerous Lab Experts

UC Berkeley's best and brightest are often asked to share their insights at the White House, on Wall Street and with the media worldwide. Now, they are furthering that conversation in a new format — The Berkeley Blog (http://blogs.berkeley.edu/). The blog features experts fielding a wide spectrum of questions about the hottest current events. The blog appears to be the first such enterprise based at a major university in the United States. Lab researchers include Interim Lab Director Paul Alivisatos, Jamie Cate and David Shaffer (Physical Biosciences), Michael Eisen (Genomics), Inez Fung (Earth Sciences), William Jagust and Randy Schekman (Life Sciences), Rich Muller (Physics), William Nazaroff (EETD), and David Patterson (Computing Sciences). More> http://www.berkeley.edu/news/media/releases/2009/10/26_berkeley_blog_launch.shtml
Today at Berkeley Lab, 10/28/09

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Awards

Lab Part of New $16 Million Center to Battle Cancer

The National Cancer Institute has awarded UC Berkeley $15.7 million over five years to allow physical scientists and engineers to open a new front in the war on cancer. UC Berkeley's Physical Sciences-Oncology Center, a collaboration with Berkeley Lab, UC San Francisco, San Francisco's Helen Diller Comprehensive Cancer Center, is one of 12 centers announced October 26 "to bring new perspectives to the mechanisms of cancer." Lab researchers include Jan Liphardt (principal investigator), Joe Gray, Jay Groves, James Sethian, and Mina Bissell. More> http://www.berkeley.edu/news/media/releases/2009/10/26_natl_cancer_institute.shtml
Today at Berkeley Lab, 10/28/09

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Obama Supports TCGA Projects in Life Sciences Division

In a visit to the National Institutes of Health (NIH) campus, President Barack Obama announced $5 billion in grant awards under the American Recovery and Reinvestment Act of 2009 (ARRA) to fund cutting-edge medical research in every state across America. More than $1 billion of the grant funding is dedicated to research applying the technology produced by the Human Genome Project between 1990 and 2003. This new funding will allow researchers to make quantum leaps forward in studying the genomic changes linked to cancer, heart, lung, and blood disease and autism– potentially leading to new treatments and cures. The investment includes $175 million for The Cancer Genome Atlas (TCGA) to collect more than 20,000 tissue samples from approximately 25 cancer types, and to determine in detail all of the genetic changes in these tumor samples.

One of seven data analysis centers being set up as part of this effort is the "TCGA Data Analysis Center at Berkeley," which was awarded $650,000 annually for five years. The center, proposed and lead by Life Sciences Division Computational Scientist Paul Spellman, focuses on integrating data from the TCGA project. The data analysis center will work closely with the other six data analysis centers and the data production centers to make critical conclusions regarding the 25 cancer types that are being analyzed by TCGA. The Berkeley center is co-directed by University of California, Berkeley Professor, Terry Speed. Bahram Parvin of the Life Sciences Division is also a key contributor. Together, they will (1) systematically characterize images of human cancers to relate them to the molecular underpinnings of the disease with the goal of increasing the fidelity of pathological classification, and (2) examine genetic links between the architecture of tumors and the patient in which they arise.

The TCGA Data Analysis Center at Berkeley is a continuation of the Berkeley Lab Cancer Genome Characterization Center (BCGC), lead by Joe Gray and Paul Spellman. The BCGC has been part of a 3 year pilot program funded by the NIH National Cancer Institute (NCI) and the National Human Genome Research Institute (NHGRI) launched in December 2005 to test the feasibility of a team science approach to cancer genome analysis. It was awarded an ARRA supplement of $278,332 this year to finish work on characterization of gene expression in the three cancer (glioblastoma, lung and ovarian) selected for analysis in the TCGA pilot program. The pilot project has been deemed a success and the overall project will move from pilot to full program status beginning this year. The TCGA Data Analysis Center at Berkeley will continue analytic work initiated in the BCGC as part of the ongoing TCGA project. More > http://cancergenome.nih.gov/media/news_overview.asp
Joe Gray, 10/09

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Life Scientists Receive NASA Award

NASA's Space Radiation Program (SRP) has selected individual investigator awards for Ground-Based Studies in Space Radiobiology and has announced that Paul Yaswen and Co-investigators Amy Kronenberg and Bahram Parvin have had their project selected for funding. The project is entitled "Epigenetic effects of radiation on epithelial cell self-renewal." The goal of the Space Radiation Program (SRP) is to enable humans to explore space without exceeding an acceptable level of risk from exposure to space radiation.

The intent of the awarded project (3 yrs.; $385K/yr) is to develop a risk model of major processes in radiation carcinogenesis using epithelial cell culture models. Aberrant self-renewal is a fundamental property of cancer cells that can be conferred by non-mutational (epigenetic) means. It is proposed that early events in radiation-induced carcinogenesis must either cause the expansion of pre-existing cells with extensive self-renewal potential or the acquisition of extensive self-renewal potential by cells that have repressed it. A current weakness in cancer risk assessment models is the lack of consideration of epigenetic factors that influence malignant transformation.

Recent data from the Yaswen lab indicate that radiation can promote changes in the epigenetic properties of cultured human cell populations. The proposed experimental studies of this project will employ new assays and advanced quantitative and modeling methods to improve the mechanistic understanding of radiation quality effects on the gain of aberrant self-renewal capacity and loss of responsiveness to differentiation signals - two related cancer development processes.
CG, 10/09

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New Subcontract for PET Research

Woon-Seng Choong received funding for a subcontract, “New, Solid-State Photosensor for PET,” to Phase 2 of an NIH SBIR grant to Radiation Monitoring Devices (RMD), Inc. in Watertown, Massachusetts. RMD received the grant to explore the fabrication of a novel photodetector, the solid-state photomultiplier (SSPM), with the ultimate goal of using it for nuclear medical imaging applications such as PET and SPECT. Choong is designing and fabricating a prototype SSPM readout application-specific integrated circuit (ASIC), and he will test and characterize the performance properties of PET detector modules made from the SSPMs that RMD will fabricate.
Bob Smith, 10/09

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Wang Awarded "Discovery" Track LDRD

Daojing Wang was awarded a Berkeley Lab “Discovery” track Laboratory Directed Research & Development (LDRD) award for his project entitled “Multinozzle Arrays for Single Cell Metabolomics”. It was the first time “Discovery” LDRDs were offered at Berkeley Lab, starting in the FY 2010 cycle. In his project, Wang proposed to develop breakthrough multinozzle array-based electrospray mass spectrometry for single cell metabolomics. The new technologies to be developed will help elucidation of single cell metabolome and may contribute to future bioenergy, cancer research, regenerative medicine, and DOE Low Dose Program.

All proposals for the “Discovery” track review were self-contained scientific projects that do not augment existing funding. Funding recommendations were made by external scientific reviewers based on the following criteria: Boldness and innovation, with strong potential for impact on the field; Quality of science; and potential of post-project funding, and relevance to DOE missions and national needs.
CG, 10/09

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Bissell Receives MERIT Award

A grant application of Mina Bissell was selected by the National Cancer Institute (NCI) for conversion to a MERIT (Method to Extend Research in Time) award. This National Institutes of Health (NIH) program is for selected investigators who have demonstrated superior competence and outstanding productivity during their previous research endeavors and who have received exceptional scores in the last two cycles.

Bissell’s MERIT Award, the converted NCI R01 grant application entitled “Definition of microenvironment in breast cancer,” proposes to continue to examine the hypothesis that architectural integrity is crucial for maintenance of normal breast function s well as for suppression of neoplasia. One of the earliest manifestations of malignant progression is loss of tissue organization. Bissell and colleagues have postulated that elucidation of a “signaling integration plan” that establishes and maintains polarity and structure within the acini and ducts of breast tissue will uncover prognostic and therapeutically-relevant markers of intermediary steps in malignancy. Collectively, the proposed experiments address the importance of MEP/LEP interactions in maintenance of polar acinar structures in breast tissue, and could also provide a proof of principle for other tissues. Since loss of appropriate alance and/or integration of these signals leads to malignancy, our results will both advance fundamental knowledge and yield novel markers for early diagnosis and therapeutic strategies to limit and/or reverse tumor progression.

The MERIT award is expected to be an investigator’s principal scientific endeavor; hence, investigators generally should have one MERIT award from the NIH. Candidates should be the principal investigator (PI) on an un-amended competing continuation (Type 2) R01 research grant application that has a history of continuous NCI support for at least seven years, and has been approved by the National Cancer Advisory Board (NCAB) for five years of additional support with a priority score within the 5th percentile. The grant should represent the PI's principal area of research, and be in an area of special importance or promise. More > http://grants.nih.gov/grants/guide/notice-files/not96-033.html
CG, 10/09

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Rothschild-Yvette Mayent-Institut Curie Award for Bissell

Mina Bissell was awarded the Rothschild-Yvette Mayent-Institut Curie Award, a Fellowship to support Sabbatical studies at the Curie Institute, Paris, and she consequently joined Dr. Olivier Delatttre's laboratory in Paris, France, from April 1 - June 30, 2009.

Thanks to the E. de Rothschild and Y. Mayent foundations, since 1992, the Institut Curie has been able to award grants enabling scientists of international renown to stay at one of the Paris or Orsay Research Center laboratories. The Institut Curie's Board of Directors each year selects about ten senior fellows to participate in the research work within a research unit, give a series of lectures on their work and visit the Orsay laboratories to meet the site's researchers.

The Institut Curie continues, since 1909, according to the will of Marie Curie, a mission of treatment and research against cancer. The continuity of research to the care constitutes the originality of the Curie model to support the excellence, the innovation and the quality of patient management. A foundation accredited as a public service body since 1921, the Institut Curie profits from the generosity of the public by donation, legacy and sponsorship.
CG, 10/09

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Hu Receives Research Scholars Award

Zhi Hu, a Postdoctoral Fellow in Joe Gray’s laboratory, has been selected by the San Antonio Breast Cancer symposium to receive an American Association of Cancer Research Translational Research Scholars-in-Training Award, funded by Susan G. Komen for the Cure. The scholarship, in the amount of $1,200, was given in recognition of her “meritorious proffered” abstract on translational breast cancer research.

The selection committee noted that Hu’s "research was considered to be of significant quality” and “the applicant pool was highly competitive". She has been invited to attend the 32nd Annual CTRC-AACR San Antonio Breast Cancer Symposium and to present her poster abstract entitled "A Systems Analysis of Mitotic Apparatus Inhibitors Defines a Response Network for Breast Cancer," at the Symposium on December 11, 2009. Hu also received an invitation to attend a special reception for award recipients.

From the abstract: We (Zhi Hu and Jian-hua Mao) have defined a 54 gene mitotic apparatus network that is transcriptionally upregulated in a subset of tumors including breast tumors, and we have shown that transcriptional upregulation is associated with reduced survival duration. We also present evidence that high activity may be driven by genetic/genomic determinants on chromosomes 1, 4, 6, 14, 16, 19 and 22. We have demonstrated that the small molecule inhibitors GSK461364, GSK923295 and GSK1070916 that target the network genes PLK1, CENPE and AURKB are preferentially effective in cell lines in which transcriptional activity of the mitotic network is upregulated and we have defined a mitotic network index (MNAI) that can be used to identify patients most likely to respond to drugs that attack within the mitotic network. In breast cancer, basal subtype cancers tend to have high MNAIs.
CG, 10/09

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Student Intern Chosen to Participate in DOE Competition

Sarah Lauder, a summer 2009 student intern mentored by Life Sciences Division scientist Yetta Eagleman, was chosen to participate in the 2009 Science and Energy Research Challenge (SERCh) Poster Competition. Lauder is an undergraduate student at Contra Costa College in San Pablo, California. In her poster, “Scintillation of cerium doped Ba9La5Br33 and NaBaLaBr6,” she describes her investigation of whether either of these scintillation compounds could be useful substitutes for LaBr3:Ce3+, which is expensive to produce. Further work is needed to optimize these materials and to determine if low-cost crystals can be produced. The other authors on her poster are Yetta Eagleman, Edith Bourret-Courchesne and Stephen Derenzo.

The Competition, hosted by the Department of Energy, showcases the research projects of DOE-funded undergraduate students and interns at the national laboratories. One-hundred student researchers were selected to convene November 8–9, 2009 at Oak Ridge National Laboratory to present their research posters and to compete for scholarship prizes. During the competition weekend, students and a designated university faculty advisor, along with laboratory personnel, attended seminars and toured user facilities at the National Laboratory.
Bob Smith, 10/09

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Recent publications (selected)

Bazarov AV, Hines WC, Mukhopadhyay R, Beliveau A, Melodyev S, Zaslavsky Y, Yaswen P. Telomerase activation by c-Myc in human mammary epithelial cells requires additional genomic changes. Cell Cycle, 2009 Oct 15;8(20):3373-8. PMID: 19806010

A central question in breast cancer biology is how cancer cells acquire telomerase activity required for unlimited proliferation. According to one model, proliferation of telomerase(-) pre-malignant cells leads to telomere dysfunction and increased genomic instability. Such instability leads in rare cases to reactivation of telomerase and immortalization. The mechanism of telomerase reactivation remains unknown. We have studied immortalization of cultured human mammary epithelial cells by c-Myc, a positive transcriptional regulator of the hTERT gene encoding the catalytic subunit of telomerase. Retrovirally introduced c-Myc cDNA resulted in immortalization of human mammary epithelial cells in which the cyclin dependent kinase inhibitor, p16(INK4A), was inactivated by an shRNA-encoding retrovirus. However, while c-Myc introduction immediately resulted in increased activity of transiently transfected hTERT promoter reporter constructs, endogenous hTERT mRNA levels did not change until about 60 population doublings after c-Myc introduction. Increased endogenous hTERT transcripts and stabilization of telomeric DNA in cells expressing exogenous c-Myc coincided with telomere dysfunction-associated senescence in control cultures. Genome copy number analyses of immortalized cells indicated amplifications of some or all of chromosome 5, where hTERT genes are located. hTERT gene copy number, however, was not increased in one case. The results are consistent with the hypothesis that changes in chromosome 5, while not necessarily increasing hTERT gene copy number, resulted in removal of repressive chromatin structures around hTERT loci, allowing induction of hTERT transcription. These in vitro results model one possible sequence of events leading to immortalization of breast epithelial cells during cancer progression.

Simian M, Bissell MJ, Barcellos-Hoff MH, Shyamala G. Estrogen and progesterone receptors have distinct roles in the establishment of the hyperplastic phenotype in PR-A transgenic mice. Breast Cancer Research, 2009 Sep 29;11(5):R72. [Epub ahead of print] PMID: 19788752

ABSTRACT: Expression of the A and B forms of progesterone receptor (PR) in an appropriate ratio is critical for mammary development. Mammary glands of PR-A transgenic mice, carrying an additional A form of PR as a transgene, exhibit morphological features associated with the development of mammary tumors. Our objective was to determine the roles of estrogen (E) and progesterone (P) in the genesis of mammary hyperplasias/preneoplasias in PR-A transgenics. METHODS: We subjected PR-A mice to hormonal treatments and analyzed mammary glands for the presence of hyperplasias and used BrdU incorporation to measure proliferation. Quantitative image analysis was carried out to compare levels of latency-associated peptide and transforming growth factor beta 1 (TGFbeta1) between PR-A and PR-B transgenics. Basement membrane disruption was examined by immunofluorescence and proteolytic activity by zymography. RESULTS: The hyperplastic phenotype of PR-A transgenics is inhibited by ovariectomy, and is reversed by treatment with E + P. Studies using the antiestrogen ICI 182,780 or antiprogestins RU486 or ZK 98,299 show that the increase in proliferation requires signaling through E/estrogen receptor alpha but is not sufficient to give rise to hyperplasias, whereas signaling through P/PR has little impact on proliferation but is essential for the manifestation of hyperplasias. Increased proliferation is correlated with decreased TGFbeta1 activation in the PR-A transgenics. Analysis of basement membrane integrity showed loss of laminin-5, collagen III and collagen IV in mammary glands of PR-A mice, which is restored by ovariectomy. Examination of matrix metalloproteases (MMPs) showed that total levels of MMP-2 correlate with the steady-state levels of PR, and that areas of laminin-5 loss coincide with those of activation of MMP-2 in PR-A transgenics. Activation of MMP-2 is dependent on treatment with E and P in ovariectomized wild-type mice, but is achieved only by treatment with P in PR-A mice. CONCLUSIONS: These data establish a link between hormonal response, proliferation, modulation of MMP activity and maintenance of basement membrane integrity that depend on a balance in the expression levels of PR-A and PR-B isoforms. Notably, concomitant increased proliferation, due to inhibition of TGFbeta1 activation, and loss of basement membrane integrity, via increased MMP-2 activity, appear to be prerequisites for the PR-A hyperplastic phenotype.

Garbe JC, Bhattacharya S, Merchant B, Bassett E, Swisshelm K, Feiler HS, Wyrobek AJ, Stampfer MR. Molecular distinctions between stasis and telomere attrition senescence barriers shown by long-term culture of normal human mammary epithelial cells. Cancer Research, 2009 Oct 1;69(19):7557-68.. PMID: 19773443

Normal human epithelial cells in culture have generally shown a limited proliferative potential of approximately 10 to 40 population doublings before encountering a stress-associated senescence barrier (stasis) associated with elevated levels of cyclin-dependent kinase inhibitors p16 and/or p21. We now show that simple changes in medium composition can expand the proliferative potential of human mammary epithelial cells (HMEC) initiated as primary cultures to 50 to 60 population doublings followed by p16-positive, senescence-associated beta-galactosidase-positive stasis. We compared the properties of growing and senescent pre-stasis HMEC with growing and senescent post-selection HMEC, that is, cells grown in a serum-free medium that overcame stasis via silencing of p16 expression and that display senescence associated with telomere dysfunction. Cultured pre-stasis populations contained cells expressing markers associated with luminal and myoepithelial HMEC lineages in vivo in contrast to the basal-like phenotype of the post-selection HMEC. Gene transcript and protein expression, DNA damage-associated markers, mean telomere restriction fragment length, and genomic stability differed significantly between HMEC populations at the stasis versus telomere dysfunction senescence barriers. Senescent isogenic fibroblasts showed greater similarity to HMEC at stasis than at telomere dysfunction, although their gene transcript profile was distinct from HMEC at both senescence barriers. These studies support our model of the senescence barriers encountered by cultured HMEC in which the first barrier, stasis, is retinoblastoma-mediated and independent of telomere length, whereas a second barrier (agonescence or crisis) results from telomere attrition leading to telomere dysfunction. Additionally, the ability to maintain long-term growth of genomically stable multilineage pre-stasis HMEC populations can greatly enhance experimentation with normal HMEC.

Zeng GL, Gullberg GT. Exact iterative reconstruction for the interior problem. Physics in Medicine and Biology, 2009 Oct 7;54(19):5805-14. PMID: 19741279

There is a trend in single photon emission computed tomography (SPECT) that small and dedicated imaging systems are becoming popular. For example, many companies are developing small dedicated cardiac SPECT systems with different designs. These dedicated systems have a smaller field of view (FOV) than a full-size clinical system. Thus data truncation has become the norm rather than the exception in these systems. Therefore, it is important to develop region of interest (ROI) reconstruction algorithms using truncated data. This paper is a stepping stone toward this direction. This paper shows that the common generic iterative image reconstruction algorithms are able to exactly reconstruct the ROI under the conditions that the convex ROI is fully sampled and the image value in a sub-region within the ROI is known. If the ROI includes a sub-region that is outside the patient body, then the conditions can be easily satisfied.

Huber J, Peng Qiyu, and Moses W. Multi-modality phantom development. IEEE Transactions on Nuclear Science, 56(5):2722-2727 (Oct 2009).

Multi-modality imaging has an increasing role in the diagnosis and treatment of a large number of diseases, particularly if both functional and anatomical information and images are accurately registered together. Hence, there is a resulting need for multi-modality phantoms (objects used to mimic human tissue) in order to validate mutual image registration and to calibrate the imaging systems. We present our PET-ultrasound phantom development, including PET and ultrasound images of a simple prostate phantom. We use agar and gelatin mixed with a radioactive solution in the phantom. We also present our development of custom multi-modality phantoms that are compatible with PET, transrectal ultrasound (TRUS), MRI and CT imaging. We describe both our selection of tissue-mimicking materials and phantom construction procedures. Our custom PET-TRUS-CT-MRI prostate phantoms use agar-gelatin radioactive mixtures with additional contrast agents and preservatives. We show multi-modality images of these custom prostate phantoms and discuss phantom construction alternatives. Although we are currently focused on prostate imaging, this phantom development is applicable to many multi-modality imaging applications.

Dairkee SH, Sayeed A, Luciani G, Champion S, Meng Z, Jakkula LR, Feiler HS, Gray JW, Moore DH. Immutable functional attributes of histologic grade revealed by context-independent gene expression in primary breast cancer cells. Cancer Research, 2009 Oct 1;69(19):7826-34. PMID: 19789341

Inherent cancer phenotypes that are independent of fluctuating cross-talk with the surrounding tissue matrix are highly desirable candidates for targeting tumor cells. Our novel study design uses epithelial cell lines derived from low versus high histologic grade primary breast cancer to effectively diminish the breadth of transient variability generated within the tumor microenvironment of the host, revealing a "paracrine-independent expression of grade-associated" (PEGA) gene signature. PEGA members extended beyond "proliferation-driven" signatures commonly associated with aggressive, high-grade breast cancer. The calcium-binding protein S100P was prominent among PEGA genes overexpressed in high-grade tumors. A three-member fingerprint of S100P-correlated genes, consisting of GPRC5A, FXYD3, and PYCARD, conferred poor outcome in multiple breast cancer data sets, irrespective of estrogen receptor status but dependent on tumor size (P < 0.01). S100P silencing markedly diminished coregulated gene transcripts and reversed aggressive tumor behavior. Exposure to pathway-implicated agents, including the calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, phenothiazine, and chlorpromazine, resulted in rapid apoptotic cell death in high-grade tumor cells resistant to the chemotherapeutic drug cisplatin. This is the first comprehensive study describing molecular phenotypes intimately associated with histologic grade whose expression remains relatively fixed despite an unavoidably changing environment to which tumor cells are invariably exposed.

Williamson A, Wickliffe KE, Mellone BG, Song L, Karpen GH, Rape M. Identification of a physiological E2 module for the human anaphase-promoting complex. Proceedings of the National Academy of Sciences U S A, 2009 Oct 27;106(43):18213-8. PMID: 19822757

Ubiquitination by the anaphase-promoting complex (APC/C) is essential for proliferation in all eukaryotes. The human APC/C promotes the degradation of mitotic regulators by assembling K11-linked ubiquitin chains, the formation of which is initiated by its E2 UbcH10. Here, we identify the conserved Ube2S as a K11-specific chain elongating E2 for human and Drosophila APC/C. Ube2S depends on the cell cycle-dependent association with the APC/C activators Cdc20 and Cdh1 for its activity. While depletion of Ube2S already inhibits APC/C in cells, the loss of the complete UbcH10/Ube2S-module leads to dramatic stabilization of APC/C substrates, severe spindle defects, and a strong mitotic delay. Ube2S and UbcH10 are tightly co-regulated in the cell cycle by APC/C-dependent degradation. We conclude that UbcH10 and Ube2S constitute a physiological E2-module for APC/C, the activity of which is required for spindle assembly and cell division.

Rodriguez EM, Dunham EE, Martin GS. Atypical protein kinase C activity is required for extracellular matrix degradation and invasion by Src-transformed cells. Journal of Cellular Physiology, 2009 Oct;221(1):171-82. PMID: 19492416

Atypical protein kinase C (aPKC) isoforms have been shown to mediate Src-dependent signaling in response to growth factor stimulation. To determine if aPKC activity contributes to the transformed phenotype of cells expressing oncogenic Src, we have examined the activity and function of aPKCs in 3T3 cells expressing viral Src (v-Src). aPKC activity and tyrosine phosphorylation were found to be elevated in some but not all clones of mouse fibroblasts expressing v-Src. aPKC activity was inhibited either by addition of a membrane-permeable pseudosubstrate, by expression of a dominant-negative aPKC, or by RNAi-mediated knockdown of specific aPKC isoforms. aPKC activity contributes to morphological transformation and stress fiber disruption, and is required for migration of Src-transformed cells and for their ability to polarize at the edge of a monolayer. The lambda isoform of aPKC is specifically required for invasion through extracellular matrix in Boyden chamber assays and for degradation of the extracellular matrix in in situ zymography assays. Tyrosine phosphorylation of aPKClambda is required for its ability to promote cell invasion. The defect in invasion upon aPKC inhibition appears to result from a defect in the assembly and/or function of podosomes, invasive adhesions on the ventral surface of the cell that are sites of protease secretion. aPKC was also found to localize to podosomes of v-Src transformed cells, suggesting a direct role for aPKC in podosome assembly and/or function. We conclude that basal or elevated aPKC activity is required for the ability of Src-transformed cells to degrade and invade the extracellular matrix. Copyright 2009 Wiley-Liss, Inc.

Orjalo AV, Bhaumik D, Gengler BK, Scott GK, Campisi J.Cell surface-bound IL-1alpha is an upstream regulator of the senescence-associated IL-6/IL-8 cytokine network. Proceedings of the National Academy of Sciences U S A, 2009 Oct 6;106(40):17031-6. PMID: 19805069

Inflammation underlies most age-related diseases, including cancer, but the etiology is poorly understood. One proposed factor is the presence of senescent cells, which increase with age. The senescence response arrests the proliferation of potentially oncogenic cells, and most senescent cells secrete high levels of proinflammatory cytokines and other proteins. The complex senescence-associated secretory phenotype is likely regulated at multiple levels, most of which are unknown. We show that cell surface-bound IL-1alpha is essential for signaling the senescence-associated secretion of IL-6 and IL-8, 2 proinflammatory cytokines that also reinforce the senescence growth arrest. Senescent human fibroblasts expressed high levels of IL-1alpha mRNA, intracellular protein, and cell surface-associated protein, but secreted very little protein. An IL-1 receptor (IL1R) antagonist, neutralizing IL-1alpha antibodies, and IL-1alpha depletion by RNA interference all markedly reduced senescence-associated IL-6/IL-8 secretion. Depletion of the key IL-1R signaling component IRAK1 also suppressed this secretion, and IL-1alpha neutralizing antibodies prevented IRAK1 degradation, indicating engagement of the IL-1R signaling pathway. Furthermore, IL-1alpha depletion reduced the DNA binding activity of NF-kappaB and C/EBPbeta, which stimulate IL-6/IL-8 transcription. IL-1alpha was a general regulator of senescence-associated IL-6/IL-8 secretion because IL-1alpha blockade reduced IL-6/IL-8 secretion whether cells senesced owing to DNA damage, replicative exhaustion, oncogenic RAS, or chromatin relaxation. Furthermore, conditioned medium from IL-1alpha-depleted senescent cells markedly reduced the IL-6/IL-8-dependent invasiveness of metastatic cancer cells, indicating that IL-1alpha regulates the biological effects of these cytokines. Thus, cell surface IL-1alpha is an essential cell-autonomous regulator of the senescence-associated IL-6/IL-8 cytokine network.

Wang D, Jang DJ. Protein kinase CK2 regulates cytoskeletal reorganization during ionizing radiation-induced senescence of human mesenchymal stem cells. Cancer Research, 2009 Oct 15;69(20):8200-7. PMID: 19826041

See also Low Dose Highlight.
Human mesenchymal stem cells (hMSC) are critical for tissue regeneration. How hMSC respond to genotoxic stresses and potentially contribute to aging and cancer remain underexplored. We showed that ionizing radiation induced cellular senescence of hMSC over a period of 10 days, showing a critical transition between days 3 and 6. This was confirmed by senescence-associated beta-galactosidase staining, protein expression profiles of key cell cycle regulators (retinoblastoma protein, p53, p21(waf1/Cip1), and p16(INK4A)), and senescence-associated secretory phenotypes (interleukin-8, interleukin-12, GRO, and MDC). We observed dramatic cytoskeletal reorganization of hMSC through reduction of myosin-10, redistribution of myosin-9, and secretion of profilin-1. Using a SILAC-based phosphoproteomics method, we detected significant reduction of myosin-9 phosphorylation at Ser(1943), coinciding with its redistribution. Importantly, through treatment with cell-permeable inhibitors (4,5,6,7-tetrabromo-1H-benzotriazole and 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole) and gene knockdown using RNA interference, we identified CK2, a kinase responsible for myosin-9 phosphorylation at Ser(1943), as a key factor contributing to the radiation-induced senescence of hMSC. We showed that individual knockdown of CK2 catalytic subunits CK2alpha and CK2alpha' induced hMSC senescence. However, only knockdown of CK2alpha resulted in morphologic phenotypes resembling those of radiation-induced senescence. These results suggest that CK2alpha and CK2alpha' play differential roles in hMSC senescence progression, and their relative expression might represent a novel regulatory mechanism for CK2 activity.