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Anita Roberts, PhD, a much-loved colleague and member of the NCI Intramural Community for 30 years, died peacefully at home Friday, May 26, 2006. With the rest of the world, we have always admired her work. But watching her battle with gastric cancer, we came to admire equally her grace and strength. —Robert Wiltrout, PhD, Director, CCR
Dr. Roberts came to the NCI in 1976 and served as chief of the LCRC from 1995 to 2004. In 2003, Science Watch listed her among the 50 most-cited scientists during 1982 to 2002. Her 344 publications and 36,397 citations made her the third most-cited female researcher in the world and one of five NCI scientists listed among the top 50. A graduate of Oberlin College, Dr. Roberts earned her PhD at the University of Wisconsin, was an NIH Postdoctoral Fellow at Harvard Medical School, and taught chemistry at Indiana University before coming to the NCI. Her work focused primarily on the cytokine, transforming growth factor-β (TGF-β). She collaborated for nearly 20 years with Michael Sporn, MD, formerly chief of NCI’s Laboratory of Chemoprevention. Together, they discovered and characterized TGF-β and established its role in autoimmune disease, fibrogenesis, carcinogenesis, and wound healing. They found that TGF-β was among the proteins that control growth in epithelial and lymphoid cells, which are involved in most cancers. This cytokine normally sends a signal to the inside of the cell that tells it to stop growing. But the signal is mediated by Smads and, when altered, can cause cells to run wild—actually promoting carcinogenesis. Some of the newest therapeutics, such as the breast cancer drug trastuzumab and the tumor inhibitor bevacizumab, are based on the work of Drs. Roberts and Sporn. Their collaboration was recognized when they received the 2005 Susan G. Komen Foundation Brinker Award for Distinguished Science. Dr. Roberts’ more recent work focused on piecing together the Smad puzzle. Dr. Roberts’ many other awards and honors include: 2005
2004
2001
Additionally, she served on numerous editorial boards and was past president of the Wound Healing Society. In March 2004, Dr. Roberts was diagnosed with aggressive stage IV gastric cancer. Suddenly, she was not only a cancer researcher; she was also a cancer patient. Faced with the emotional pain of a poor prognosis and the physical difficulties of chemotherapy, she lived her motto: “Take one day at a time and get the most out of it!” She made her disease more understandable to her grandchildren by creating a blog, which ultimately became a way for her to organize her thoughts and communicate her progress to family, friends, colleagues, and supporters everywhere. She found solace in and wrote about doing the things she loved: visiting her family; spending time with her husband, Bob, at their beach house in Bethany, Delaware; and gardening. “Happiness is having more dirt under your fingernails,” one blog entry reads. Despite the physical discomforts of the disease and the aggressive therapy, Dr. Roberts also continued her work. As Dr. Merlino put it, “For the last two years, Anita has had to live with the knowledge that her disease was likely fatal. Yet, her productivity did not diminish. She was in the lab nearly every day. She didn’t just go to big meetings and take part in them, she organized them. And she continued to take care of everybody in the lab, her science family.” Dr. Roberts leaves behind her husband, Robert E. Roberts, two sons, five grandchildren, and a sister. She also leaves a strong legacy at the NCI and to the science of medicine: Her work has already inspired much ongoing research and new treatment options. Her colleagues have all been touched by her unique gifts, and the clarity of her vision remains. As she told Cancer Research (Spring 2006), “Research takes a long, long time.… As basic scientists, we’re all driven by our excitement in finding answers. We hope it ends up as something that becomes therapy. But that doesn’t happen unless you have a basic understanding of the process. And that’s what my work is all about.”
Nanobiology Is Taking Off at the CCR: The First Nanobiology Think Tank Held
In his opening remarks, Travis Earles of the NCI Alliance for Nanotechnology in Cancer provided a general introduction to nanotechnology as an important tool that promises to revolutionize cancer diagnosis and treatment and described several important areas of research, including nanotechnology-based imaging techniques and the development of multifunctional nanoparticles. He also emphasized the role of the alliance in helping to reduce the burden of cancer by accelerating the application of the best innovative nanotechnologies currently available. Dr. Blumenthal described the CCRNP as a program with a firm research foundation in mathematics, physics, chemistry, and biology and one with theoretical, experimental, and practical application to clinical settings and nanotechnologies. The CCRNP’s mission is to understand the structure and function of biomolecules and their assemblies and, based on the knowledge gained, to design nanodevices, including biologically based nanoparticles, for in vivo imaging, diagnosis, and targeted therapy for cancer, AIDS, and other viral diseases. This Nanobiology Think Tank is one of the several outreach programs of the CCRNP to promote collaborations, disseminate knowledge, and enhance the development of novel creative ideas for research and biomedical applications. After the opening remarks and introduction by Dr. Blumenthal, 20 speakers presented various aspects of their nanobiology-related research and development. To view the list of invited speakers, click here. Notably, in vivo experiments were described that could lead to nanodevices for clinical use. Linda Molnar, PhD (SAIC-Frederick, Inc.) provided a stimulating overview of nanotechnology and how NCI contributes to the realization of its full potential. She presented examples of successful nanotechnology-based approaches for in vivo imaging. Steven Libutti, MD (NCI) described a novel nanoparticle based on the conjugation of gold nanoparticles with tumor necrosis factor (TNF) that dramatically increases the TNF concentration in tumors compared with native TNF. He presented data for MC-38 tumor growth in C57BL/6 mice demonstrating 100% survival of mice treated with the nanoparticles compared with 60% survival of mice treated with native TNF. The nanoparticle formulation was found safe in rabbits and will be tested in a phase I clinical trial with 36 patients. Gregory Lanza, MD, PhD (Washington University) described several nanoparticle-based approaches and a large amount of data for in vivo imaging and treatment of tumors in mice and humans, as well as fibrin-targeted streptokinase nanoparticles that rapidly lyse thrombi. He demonstrated the superiority of targeted nanoparticles compared with those that are non-targeted. Anu Puri, PhD (NCI) presented very interesting data for nanoliposome-based, temperature-triggered release of fluorescent probes as imaged in mice and data demonstrating increased stability of liposomes in rats after appropriate modifications of the phosphatidylcholine sn-2 fatty acid carbonyl ester conferring resistance to phospholipase A2. King Li, MD, MBA (Clinical Center, NIH) emphasized the role of effective biodistribution of polymerized lipid and other nanoparticles for imaging and therapy. He presented amazing data for the use of pulsed high-intensity focused ultrasound to enhance delivery of nanoparticles in murine cancer models. Mark Kester, PhD (Penn State College of Medicine) demonstrated how nanoparticles help to solve problems with the systematic delivery of the chemotherapeutic agent ceramide, which is limited by its cell impermeability, metabolism, and precipitation. He presented data that showed encapsulation of ceramide (as well as siRNA) in nanoliposomes significantly enhances its therapeutic activity in mouse models of cancer. Another exciting area of research he described involved new nanoparticles called molecular dots, which are based on calcium phosphate matrix material, and their use for encapsulation of ceramide to treat tumors in mice. Mansoor Amiji, PhD (Northeastern University) described the use of nanoemulsions for image-guided murine cancer therapy and the delivery of paclitaxel across the blood-brain barrier in mice, as well as multifunctional nanocarriers against drug resistance in cancer. Oxana Pickeral, PhD, MBA (NCI) provided an overview of the Joint Division of Cancer Treatment and Diagnosis (DCTD)–CCR Early Therapeutics Development Program. She emphasized the new U.S. Food and Drug Administration (FDA) Exploratory IND (Investigational New Drug) Guidance issued in 2005 and the concept of phase 0 trials, which are intended to accelerate clinical development of new therapeutic and imaging agents. This is a unique co-development opportunity open to NCI researchers, academics, and business-sector collaborators, who can benefit from NCI’s deep expertise in developing anticancer agents and its greater tolerance of developmental risk compared with the commercial sector. Scott McNeil, PhD (SAIC-Frederick, Inc.) described the objectives of the Nanotechnology Characterization Laboratory, which are to identify and characterize critical parameters related to the biocompatibility and structure-activity relationships of nanomaterials, establish and standardize an assay cascade for nanomaterial characterization, examine the biological characteristics of multicomponent and combinatorial platforms, and engage in and facilitate academic and industrial-based education and knowledge sharing. He emphasized the preclinical characterization of nanoparticles and described various equipment and assays available in the laboratory, as well as data obtained with those assays. Stimulating discussions dominated the first Nanobiology Think Tank, reflecting an intellectual atmosphere that is fruitful with novel ideas and excitement for science.
An RNA Regulator for Avoiding Sugar-phosphate Stress
A major class of bacterial non-coding RNAs conducts posttranscriptional regulatory functions by basepairing with target mRNAs and altering their translation or stability. Thus far, all of the members of this class in E. coli, such as the RNA described here, SgrS, bind to and require an RNA chaperone protein called Hfq to perform their regulatory functions. SgrS is encoded as a 227-nucleotide (nt) transcript in a region between two protein coding genes. The first clue to its physiological role came with the observation that cells overexpressing SgrS could not grow using glucose as the only energy source, but were capable of using many other sugars. A previous study from the laboratory of Hiroji Aiba (Nagoya University, Japan) (Morita T. et al. J Biol Chem 278: 15608–14, 2003) had documented an unexplained posttranscriptional regulatory phenomenon involving the ptsG mRNA, which encodes the major glucose transporter in E. coli. The PtsG protein transports glucose through the cytoplasmic membrane of the bacterial cell, phosphorylating it in the process (Figure 1, part A). Aiba and colleagues found that in cells that accumulated excess glucose-phosphate or the glucose-phosphate analog α-methyl glucoside (αMG)-phosphate, the ptsG mRNA became very unstable. We hypothesized that if SgrS were causing the ptsG transcript to become unstable, cells overexpressing SgrS might not be able to transport enough glucose to use it as a carbon source for growth. Non-metabolizable sugar-phosphate molecules are known to be growth inhibitory or sometimes lethal by mechanisms that are not completely understood. Normal E. coli cells that are exposed to αMG transport and phosphorylate this molecule via PtsG, but cannot metabolize the phosphorylated sugar further. As a result, their growth is transiently inhibited. On the other hand, growth in sgrS-negative mutant cells is more severely inhibited upon exposure to αMG, suggesting that (1) a greater amount of toxic phosphorylated sugar is produced and (2) SgrS is important to reduce the level of phosphorylated sugar, which helps reduce sugar-phosphate stress. The levels of SgrS RNA and ptsG mRNA were examined and were found to be reciprocal. In the absence of αMG, we could not detect SgrS, but the ptsG mRNA was present. Within 5 minutes after cells were exposed to αMG, large amounts of SgrS RNA appeared, and the ptsG message had disappeared. However, in sgrS mutant cells, the ptsG mRNA did not disappear. Since other Hfq-binding non-coding RNAs in E. coli act by basepairing with their targets, we examined the SgrS and ptsG RNAs for regions of complementarity. SgrS has the potential to basepair with the ptsG mRNA in the 5´ untranslated region in an area that overlaps with the ribosome binding site. This suggests that SgrS:ptsG mRNA basepairing may inhibit translation of the ptsG mRNA and lead to its rapid degradation by inhibiting ribosome binding, a mechanism that has been described for other non-coding RNAs in E. coli. Figure 1. A model for the cellular response to glucose-phosphate stress where SgrR and SgrS function to modulate the transporter for glucose. A) The PtsG transporter brings glucose into the cell, phosphorylating it in the process. B) Accumulated glucose-phosphate is proposed to interact with the regulatory protein SgrR to stimulate transcription of the regulatory RNA SgrS, which binds to the RNA chaperone Hfq. C) SgrS pairs with the mRNA encoding PtsG, leading to degradation of the small RNA and the ptsG mRNA. This downregulates further transport of glucose into the cell, limiting the accumulation of toxic sugar-phosphates. The pattern of αMG-inducible SgrS synthesis indicated that expression of SgrS was controlled by one or more factors that could respond to increased levels of sugar-phosphates. In bacteria, genes related in function are often found in close proximity to one another; frequently, a gene encoding a regulatory protein is positioned near the genes it regulates. The sgrR gene, divergently transcribed from sgrS, is present only in bacterial genomes that contain SgrS. Furthermore, the SgrR protein was predicted to contain an N-terminal DNA binding domain similar to those of other transcription factors. SgrR is in fact required for SgrS synthesis. In addition to its DNA-binding domain, SgrR also contains a domain similar to sugar-binding domains of other proteins. We propose that SgrR itself is the sensor of intracellular sugar-phosphates and the activator of SgrS synthesis.
We Keep Learning from Retroviruses
Research over approximately the past 20 years has revealed that many retroviruses depend on elaborate mechanisms for nucleocytoplasmic export of their unspliced, full-length RNA. These transcripts encode the Gag-pol polyprotein and, in addition, serve as genomic RNA to be packaged into progeny virions in the cytoplasm. Studies of the molecular biology of HIV-1 have been instrumental for major discoveries in the field of mRNA metabolism and macromolecule transport. In HIV-1 and other lentiviruses, this process depends on the Rev protein, which is essential for the production of structural proteins and infectious virions (Figure 1). Rev promotes export and expression of gag/pol and env mRNAs by binding to the cis-acting RNA recognition signal, the Rev-responsive element (RRE). A similar mechanism is essential for human T-cell leukemia virus (HTLV) and human endogenous retrovirus (HERV-K) expression. Rev and its functional homologs, as well as many cellular proteins, share a leucine-rich nuclear export signal, which is recognized by the cellular export receptor CRM1 thereby linking the mRNP (mRNA-protein) cargo to the nuclear pore complex. The discovery of the Rev export pathway paved the way to understanding the trafficking of cellular proteins such as MDM2, MAPKK, PKI, and others. Figure 1. Distinct export pathways from the nucleus. CRM1 and NXF1 represent two key export pathways from the nucleus. Studies of retroviruses and retroelements have been critical for their discovery. CRM1 is essential for the Rev-mediated HIV mRNA export and expression. NXF1 is the molecular link between the constitutive transport element (CTE)– and RNA transport element (RTE)–containing mRNAs and the nuclear pore complex. The key factors mediating mRNA export of retrovirus and retroelement mRNA export also promote transport of cellular mRNAs and proteins. Rev, HIV-1 protein essential for the production of structural proteins and infectious virions; RRE, Rev-responsive element; SRV, simian type D retrovirus. Expression of simian type D retrovirus (SRV/MPMV) depends on the cellular trans-acting factor TAP/NXF1, which binds to the cis-acting constitutive transport element (CTE) (Figure 1). The extended stem-loop structure of CTE is conserved among all type D species. A CTE-related element was found in a subgroup of rodent intracisternal A particle retroelements (IAP), and more than 100 CTE-related elements are present in the mouse genome. The cellular NXF1 is not only the export receptor of CTE-containing RNA, but most importantly, it is the key export factor for cellular mRNAs, a function that is conserved among eukaryotes. We discovered another potent RNA transport element (RTE, Figure 1), linked to a “fossilized” mouse IAP (Nappi F et al. J Virol 75: 4558–69, 2001). RTE is functionally similar, but structurally unrelated, to CTE and also functions in many cell types of different species, indicating that its export factor(s) are widely expressed and evolutionarily conserved. RTE does not bind the export factor NXF1 and mediates mRNA export via interactions with other still unknown cellular factor(s). Similar to CTE, RTE depends on a conserved cellular transport mechanism, which makes this mRNA export element a valuable tool for further understanding the mRNA nucleocytoplasmic transport. Here, using computer prediction supported by experimental RNA structure analysis, we found that RTE folds into a novel, extended RNA secondary structure (Figure 2) (Smulevitch S et al. J Virol 79: 2356–65, 2005). Detailed mutational analysis revealed that the minimal RTE contains four internal stem-loops that are indispensable for function in mammalian cells. Therefore, the RTE depends on a complex secondary structure, which is important for the interaction with cellular export factor(s). Sequence similarity analyses revealed that in addition to more than 100 identical RTEs, there are more than 3,000 RTE-related elements in the mouse genome, which share at least 70% sequence identity and which can be found on all the chromosomes. The predicted key structural features of RTE are preserved among these related elements, consistent with their functional importance. Based on their sequence and structure, these elements form four subgroups (Figure 2, structures A through D). Figure 2. Family of RTE-related elements in the mouse genome. The 226-nucleotide (nt)–spanning RTE was used to identify related elements in the mouse genome. Based on structure and sequence, these elements form four groups. The top panel shows the alignment of the RTE and a representative member of each group of RTE-related elements found in GenBank entries al663101 (Group B), al671215 (Group C), and ac003061 (Group D). The shaded areas indicate the loops defined for RTE. Phylogenetic tree and comparison of the identified RTE structure and the predicted secondary structures for the RTE-related elements are shown (bottom panel). SL I, II, III, and IV indicate the identified stem-loop (SL) structures with the RTE. Our research on posttranscriptional elements has further given new insights into the biology of retroelements and their potential effect to alter cellular gene expression. We found that IAP retroelements are more complex than previously thought and that they fall into at least two subfamilies depending on the presence of either the CTE- or the RTE-related RNA export elements. Using an active IAP, we recently found that removal of its RTE leads to abolishment of retrotransposition. This experiment showed for the first time that posttranscriptional control is essential not only for retroviruses but also for long terminal repeat (LTR) retroelements. Our findings suggest that active RNA export elements are inserted into genes via retrotransposition, and can thereby affect the posttranscriptional regulation of cellular gene expression. The presence of the many RTEs in the genome provides us with important new information about posttranscriptional regulation, genome organization, genome evolution, and the potential of IAPs to affect cellular gene expression, which may lead to carcinogenesis.
The Genome in Three Dimensions: From Basics to DiagnosticsMisteli T. Spatial positioning: A new dimension in genome function. Cell 119: 153–6, 2004.
All non-chloroplastic, non-mitochondrial eukaryotic genomes exist and function in the cell nucleus. Advanced imaging methods have revealed that the interior of the human nucleus is divided into distinct neighborhoods where various functions such as transcription and RNA processing occur in spatially separate subcompartments. Given the existence of such functionally specialized regions, it seems obvious to consider that the placement of chromosomes and genes within the nucleus might contribute to their proper function and regulation. Yet, the study of how genomes are spatially organized and what this organization means for function is only in its infancy. What we have learned is that genomes are indeed non-randomly organized. Some chromosomes have a tendency to localize toward the center of the nucleus, whereas others preferentially associate with the edges of the nucleus (Figure 1). This in turn places some chromosomes closer to others to form pairs and clusters, leading to the creation of defined genome neighborhoods. Since genes are located on chromosomes, it is not surprising to find that the position of genes is similarly non-random. The patterns of genome organization differ among cell types and tissues and might be related to the differential sets of genes, which are expressed in cells at various times during development and differentiation. Figure 1. High-throughput genome imaging. Genomes are non-randomly positioned within the cell nucleus. Automated high-throughput imaging systems and mining of positioning information allows exploitation of chromosome- and gene-positioning patterns for basic discovery and diagnostic applications. Efforts are currently under way in many laboratories to describe genome organization patterns and to ask how positioning is linked to gene function. The general approach in these studies is to correlate the expression pattern of a gene with its position relative to nuclear landmarks, such as the periphery, heterochromatin regions, or other genes. For example, we have shown that during T-cell differentiation, the CD4 locus moves from a peripheral position to a more internal nuclear position in correlation with its activity. This type of analysis can now be performed routinely for single genes; the next step is to map the positions of sets of genes (for example, those that have been identified as co-regulated) by use of microarray analysis. There is also reason to believe that spatial genome organization plays a role in cancer. Recent experimental data from several laboratories demonstrate that in many tumors, including leukemias and liver tumors, the most frequent translocations occur between chromosomes that are generally in close proximity. This means that the non-random, spatial arrangement of the genome might predispose cells to particular translocation events. The non-random position of genomic regions is of great interest as a fundamental mechanism in gene regulation, but analysis of spatial genome organization also has direct applications as a novel strategy in diagnostics. A disease-causing gene may be repositioned as its activity becomes aberrant. Similarly, gene markers in pre-malignant or pre-metastatic cells might already be misplaced prior to the cells becoming neoplastic. Spatial positioning might be a better early indicator than gene activity, because changes in positioning patterns often occur prior to changes in a gene’s activity. A particular advantage of such an interphase genome-positioning mapping method is its applicability to solid tumor samples, whose genomes currently cannot be easily analyzed. Since such solid tumors constitute the majority of all human tumors, positioning diagnostics would fill a significant gap in our diagnostic repertoire. The full exploration of the spatial organization of the genome and the development of three-dimensional diagnostic methods requires technology development. The strategy is to implement high-throughput microscopy systems that can acquire large amounts of positioning data (Figure 1). These images will be automatically processed and analyzed using dedicated three-dimensional positioning software tools. The resulting distributions will then be analyzed by advanced pattern-recognition tools to correlate expression with position and to define patterns that are characteristic of a particular physiological state. Efforts to create such systems are currently under way at the NCI. These systems will eventually be used to generate extensive three-dimensional maps of genomes, to follow the changes in genome organization patterns during differentiation, development, and disease progression. Most importantly, they have a high potential to translate what we learn about fundamental genome organization to disease-relevant therapeutic applications.
From T-Cell Antigen Receptor Engagement to Cytoskeleton Reorganization
In the present study, we focused on the dynamic interactions that occur between signaling molecules crucial for translating TCR engagement into localized actin polymerization. This goal was accomplished by using advanced molecular imaging techniques to observe T-cell spreading at the level of single activated T cells. During these events, we characterized the dynamic localization of the proteins regulating actin polymerization and demonstrated the molecular interactions involved in this process (Figure 1). We defined protein-protein interactions by performing quantitative characterization at the Angstrom level using fluorescence resonance energy transfer (FRET) techniques. Figure 1. Molecular interactions leading to T-cell receptor (TCR)–induced actin polymerization. T-cell activation is initiated by antigen presenting cells (APCs) containing stimulatory major histocompatibility complex (MHC)–peptide complexes. Phosphorylation of the TCR (black circles) is mediated by Src family protein tyrosine kinases. ZAP-70 is recruited to the phosphorylated TCR subunits through its SH2 domain. It is phosphorylated and activated by the Src family protein tyrosine kinases. The linker for the activation of T cells (LAT) is tyrosine phosphorylated by ZAP-70. LAT contains nine tyrosine residues that, when phosphorylated, act as docking sites for adapter proteins such as Grb2 and Gads. SLP-76 is recruited by the LAT-nucleated complex through its interaction with the SH3 domains of Gads. SLP-76 associates with the SH2 domain of Nck. Nck binds WASp, which in turn binds the Arp2/3 complex that mediates actin polymerization. Our studies demonstrated that actin polymerization began at the site of TCR engagement and then migrated to the cellular periphery. Actin polymerization was driven by the Wiskott-Aldrich Syndrome protein (WASp) and was dependent on its dynamic localization. We demonstrated the critical role of TCR-induced tyrosine phosphorylation of the adaptor proteins LAT and SLP-76 in recruiting other adaptors, Nck and WASp, that are required for actin polymerization. The use of mutant T-cell lines lacking LAT or SLP-76 revealed a complexity in the mechanism of actin polymerization. In LAT-deficient cells, Nck and WASp did not cluster at the TCR. Nck also failed to cluster at the TCR in SLP-76–deficient cells. Reconstitution of these cells with the appropriate cDNA (LAT or SLP-76) restored normal recruitment of the tagged molecules at the TCR. However, reconstitution of LAT-deficient cells with a mutant form of LAT lacking four critical tyrosine residues, the sites of important phosphorylation, failed to reconstitute Nck recruitment. In the deficient cells, analysis of actin status revealed significant, albeit incomplete, inhibition of polymerization. The role of Nck in T-cell antigen receptor action has recently received much attention. Gil et al. (Cell 109: 901–12, 2002) reported experiments indicating that Nck directly binds TCR/CD3ε chains upon TCR engagement due to induced exposure of proline-rich sequences within the CD3ε. Recruitment of Nck to CD3ε was found to precede phosphorylation of it and other TCR subunits. Also, this binding was found to be independent of SLP-76. The cellular reagents used in our current study enabled us to test these observations. Two of our results contradicted the study. First, the Src kinase inhibitor PP2 was used to block proximal tyrosine phosphorylation. Despite TCR-induced clustering, no activated ZAP-70, SLP-76, or Nck was detected in clusters at the cell membrane. We attribute this failure of recruitment to the lack of TCR phosphorylation induced by Lck or Fyn, the consequent lack of ZAP-70 recruitment and activation, followed by the failure of any ZAP-70 SH2–mediated interactions. Since our means of activation is via CD3ε-mediated clustering of TCRs, the failure of Nck recruitment argues against recruitment being dependent on CD3ε conformational changes exposing Nck binding sites. Also, the expression of YFP-Nck in the SLP-76–deficient cells was used to test whether SLP-76 is necessary for Nck recruitment. In the absence of SLP-76, Nck was not recruited to the TCR, whose activation was confirmed by the presence of pZAP-70 at the TCR. Restoration of Nck clustering in these cells was demonstrated following reexpression of SLP-76. Thus, Nck recruitment in our studies requires tyrosine phosphorylation and SLP-76. To confirm that the same protein recruitment and interactions occur in normal, non-transformed cells, we adapted the assay to human peripheral blood lymphocytes. Though the kinetics were slower, the pattern of protein localization in these cells was similar to that observed with Jurkat cells. Nck and WASp were initially recruited to the TCR, where they co-localized with activated ZAP-70 and then migrated to the periphery where they accumulated at an actin-rich circumferential ring. In conclusion, advances in confocal microscopy and imaging techniques, such as FRET and time-resolved imaging of fluorescent chimeras, enabled our study of actin polymerization and reorganization at the TCR. This process plays a major role in immunological synapse formation and amplification of the immune response.
Scientific Advisory CommitteeIf you have scientific news of interest to the CCR research community, please contact one of the scientific advisors (below) responsible for your areas of research.
CCR Frontiers in Science—StaffCenter for Cancer Research Robert H. Wiltrout, PhD, Director Deputy Directors Douglas R. Lowy, MD Editorial Staff Sue Fox, BA/BSW, Senior Editor * Palladian Partners, Inc. FOR INTERNAL USE ONLY |
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nita
Roberts was a friend and colleague, an exemplary and always supportive mentor,
and an impeccable scientist with an uncanny ability to balance her professional
and personal lives. As aptly stated by Glenn Merlino, PhD, her successor as
chief of the Laboratory of Cell Regulation and Carcinogenesis (LCRC), “Dr.
Roberts established a sort of scientific universe in her lab that met not only
intellectual needs, but was almost a family. All of her people, from postdoctoral
fellows to PIs, knew they could come to her for anything. It was a very nurturing
and incredibly productive environment.”
he
realignment of the former Laboratory of Experimental and Computational Biology
to the first intramural Center for Cancer Research Nanobiology Program (CCRNP)
created much excitement and provided new opportunities for the development of
innovative approaches to the diagnosis and treatment of cancer. The excitement
was felt in the enthusiastic response to the first Nanobiology Think Tank, organized
by the Director of the CCRNP, Robert Blumenthal, PhD. The event, held on June
2 in Frederick, was attended by more than 100 participants from within and outside
the NIH.
etroviruses
gave us oncogenes, and their study helped in the elucidation of molecular mechanisms
of carcinogenesis. However, this is not all. Retroviruses and retroelements
keep us busy in the discovery and the refining of our understanding of basic
mechanisms mediating gene expression. Posttranscriptional regulation is a critical
step in mRNA metabolism that controls the levels of gene expression of both
viral and cellular genes. The detailed analysis of the fundamental cellular
processes that guide the complex assembly of mRNA and proteins and their transport
from the nucleus to the cytoplasm is essential for understanding the regulation
of gene expression. 
hen
we hear about genomes, we usually think of genome sequences. However, if genome
sequencing projects have taught us one thing, it is that the sequence of a genome
is not everything. The answers to some of the most fundamental questions, such
as why humans have only four times as many genes as yeast and how the right
genes are turned on at the right time in the right place, will likely not come
from sequence information. Clearly, regulated genome function involves more
than sequence. One regulatory factor in genome function that is gaining increasing
recognition is the spatial organization of genomes within the interphase cell
nucleus.
nce
the T-cell receptor (TCR) binds foreign antigens on antigen presenting cells
(APC), multiple proteins redistribute to form the immunological synapse. Organization
of the synapse and the creation of a tight seal between the T cell and APC depend
on actin polymerization. Studies using biochemical and imaging techniques have
shown the formation of signaling assemblies at the TCR; however, studies of
the molecular interactions linking the TCR to the cytoskeleton proteins in live
cells have been rare.
