T H E   N I H   C A T A L Y S T     J A N U A R Y  –  F E B R U A R Y  2002



Martin Brechbiel

Martin Brechbiel received his Ph.D. from the American University in Washington, D.C., in 1988 after having joined the Radioimmune and Inorganic Chemistry Section, NCI, in 1983, where he is now a senior investigator and section chief.

My interests are in metal chelating agents used in medicinal chemistry. My primary focus is on the design, synthesis, and evaluation of bifunctional chelating agents for sequestering metallic radionuclides in vivo. My research at NCI has applied these interests to create useful reagents and methodologies that can be translated directly into clinical trials for performing tumor-targeted imaging (g-scintigraphy, SPECT, PET) in conjunction with targeting particle-emitting therapeutic radionuclides (b- and a-emitters).

We have been studying the preclinical and clinical potential of the chelating agents created in our lab with the array of available radionuclides. We primarily use monoclonal antibody radioimmunoconjugates to target the agents. Parallel to this research is a complementary area of interest stemming from the fact that many of the chelators created for radionuclides are of equal utility for complexing Gd(III). This permits the simultaneous creation of novel MRI contrast agents.

In my early work at NIH, my colleagues and I created numerous radioimmunoconjugates using bifunctional acyclic and macrocyclic chelating agents. These were evaluated in preclinical animal model studies. From these studies, we identified a DTPA derivative that proved suitable for clinical use. We used this ligand—1B4M-DTPA, also known as MX-DTPA—as the chelating agent in two clinical trials at NCI in collaboration with the Metabolism Branch and the Laboratory of Molecular Biology. This agent, commercially known as Tiuxetan, is now a component in the commercial anti-CD20 agent, Zevalin, for the treatment of non-Hodgkin’s lymphoma. These advances and successes provided the platform for my subsequent and ongoing studies.

Building on what we learned from MX-DTPA, I developed methodology for the creation of the CHX DTPA family of chelating agents that have since found use at NIH and many other institutions. Of particular importance is the use of this agent in the first clinical trial with an a-emitting radionuclide, 213Bi, in the treatment of acute myelogenous leukemia.

Subsequent preclinical and in vitro studies of the family of CHX DTPA chelating agents revealed a highly significant finding. The effects of stereochemistry on the in vivo stability of the metal complexes formed with radioimmunoconjugates had previously been dismissed as unimportant. We found that stereochemistry has a profound influence on complex stability that can only be detected via in vivo studies. We also recognized that this result would have to be addressed in all future studies and could be exploited to create novel agents for future applications. Examination of stereochemical components of chelating agents has since become a key aspect of my lab’s work.

Carrying our chemistry forward into preclinical evaluations, we have recently initiated studies of the suitability of various particle emitters in the treatment of disseminated intraperitoneal disease. We have chosen two different model systems, pancreatic cancer and a colorectal model previously used as a model for ovarian cancer. We plan to investigate the effects of targeting multiple isotopes using at least two targeting proteins. We will also look at fractionation of dose and the inclusion of either DNA repair inhibitors and/or radiosensitizers.

Preliminary results have already revealed that the use of a-emitting radionuclides has significant therapeutic effects for the treatment of disseminated disease, permitting selective cell-by-cell targeted therapy. We now have ongoing experiments using multiple doses of 212Pb and 212Bi, and future studies based on the results are being planned.

In addition to targeted radiation therapies, these same chelating agents hold promise as MRI contrast agents based on polymeric dendrimeric cores. Dendrimers not only permit control of molecular size and shape but also allow large molar amounts of Gd(III) to be sequestered, thereby creating contrast agents of high relaxivity and superior contrast.

We have recently demonstrated the utility of these agents by imaging vasculature in mice. Having investigated variables of dendrimer size, character, and PEG conjugation, we have recently initiated studies to evaluate the effects of radiation on tumor vasculature. These studies can provide real-time MRI images to assess the effects of either external or systemic radiation.

We hope these parallel areas of research will be used in the clinic in the future to eradicate residual cancer cells while allowing physicians to monitor the progress of this therapy via targeted macromolecular MRI contrast agents.

Jeff Duyn

Jeff Duyn received his Ph.D. from Delft University of Technology in the Netherlands in 1988 and did postdoctoral work at the University of Trento, Italy, and the University of California at San Francisco before joining the Clinical Center in 1992. He is now a senior investigator in the Laboratory of Functional and Molecular Imaging, NINDS.

The main emphasis of my career has been the development of magnetic resonance (MR) methodology for the study of the human brain in vivo. My interest in this field originated in the early 1980s, when MR imaging (MRI) was just starting to show promise in the detection of pathology in humans. Since then, I have witnessed and been involved in the tremendous growth in capability and application of MR in vivo. I am intrigued and fascinated with the versatility of MRI and the great variety of contrasts that can be generated to elucidate biological processes.

During my initial years at NIH, I was involved in the development of MR spectroscopic imaging methods for the detection of metabolic abnormalities in brain infarction and brain tumors. By mapping the spatial distribution of metabolite levels and their evolution over the course of the disease, we found that metabolites such as N-acetyl aspartate, choline, and lactate could serve as markers for the severity and state of disease.

Subsequently, our research focus shifted towards MRI rapid imaging techniques that detect the water signal. Specifically, we improved on techniques that could follow a bolus of contrast agent as it passed through the brain vasculature.

This work established that bolus arrival time is a sensitive marker of brain areas at risk of oxygen deprivation in patients with carotid artery disease and acute stroke. In addition, my group has designed rapid imaging techniques to detect perfusion levels without administration of contrast agents, allowing sensitive detection of the perfusion changes associated with brain activation.

More recently, we have worked on the improvement of MRI sensitivity and resolution through multichannel signal detection. Using dedicated receiver antennae that independently receive NMR signals, we found we could substantially improve sensitivity throughout the brain. This allows MRI with a spatial resolution approaching the scale of cortical columns and layers.

We expect to obtain even further improvements in MRI of human brain with the high field (7.0 tesla) MRI scanner that will be installed at NIH in 2002—only the third of its kind. It is expected to open up new avenues in the already very exciting area of brain research.

David Lovinger

David Lovinger received his Ph.D. from Northwestern University, Evanston, Ill., in 1987 and did postdoctoral work at NIAAA before joining the Department of Molecular Physiology and Biophysics at Vanderbilt University School of Medicine in Nashville, Tenn. He advanced to the rank of professor in that department before returning to NIAAA in February 2002 as chief of the Laboratory of Integrative Neuroscience.

My interests are in the area of modulation and plasticity of synaptic transmission in the brain, as well as the molecular basis of acute intoxication.

One area of emphasis has been on synaptic transmission within the basal ganglia, a brain region with crucial roles in movement patterning and habit formation. My laboratory has focused especially on short- and long-term regulation of synaptic transmission at synapses connecting the cerebral cortex to the striatum (the so-called cortico-striatal synapses). These synapses constitute the entryway for information flow into the basal ganglia circuitry and are an important point for regulation of the function of the entire circuit.

We have examined how neurotransmitters modulate transmission at these synapses through G-protein–coupled receptors. We have also characterized long-lasting changes in the efficacy of cortico-striatal synapses, such as long-term depression (LTD) and long-term potentiation that occur during development and can be mimicked by persistent activation of cortico-striatal inputs.

Dopamine is a key neurotransmitter in the striatum, and our studies have helped to demonstrate important roles for this neurotransmitter in striatal synaptic plasticity. We have gathered evidence indicating that striatal LTD involves a long-lasting decrease in release of the neurotransmitter glutamate from the axon terminals of cortical neurons.

The modulatory agents known as endocannabinoids coordinate communication between postsynaptic and presynaptic elements in the induction of the long-lasting decrease in transmitter release. The receptors activated by endocannabinoids are the targets of the psychoactive compounds present in marijuana and hashish. Thus, our studies are becoming intertwined with efforts to understand the mechanism of action of drugs of abuse in this brain region.

In future studies, we will continue to examine the mechanisms underlying the long-lasting decrease in synaptic function, as well as characterizing the sequence of molecular events involved in initiation of such plasticity. Ultimately, our studies may aid in the development of treatments for disorders of the basal ganglia such as Huntington’s and Parkinson’s diseases, and we are using animal models of these disorders to determine whether cortico-striatal transmission might be disrupted in these pathological states.

Another emphasis of research in my laboratory has been the acute actions of alcohol on ligand-gated ion channels. Work that my colleagues and I began when I was a postdoctoral fellow at NIAAA demonstrated acute actions of alcohol on different ligand-gated ion channel subtypes. These receptor channels mediate fast synaptic transmission throughout the brain, and thus their function is central to proper communication in the brain. Alcohol effects on these channels are believed to contribute to many aspects of acute intoxication.

My laboratory is interested in the role of particular subunit proteins in conferring alcohol sensitivity on the receptors. We are examining these roles in a variety of ways—from heterologous expression systems to gene-targeted mice. We hope to develop the capability in our lab to examine how these receptors and their subunits are affected by ethanol at the molecular and cellular levels. We also aim to determine the role of the receptors and subunits in acute intoxication in the behaving organism. This research may lead to new treatments of alcohol abuse and alcoholism.

Beverly Mock

Beverly Mock received her Ph.D. from the University of Maryland, College Park, in 1983 and was a National Research Council Research Associate in the Department of Immunology at Walter Reed Army Institute of Research, Washington, D.C., before joining the Laboratory of Genetics of NCI in 1986 as a Hall-Shields Fellow. As an active member of the Mammalian Genome Society, she has been responsible for collating maps of mouse chromsome 4. She is now a senior investigator in the Laboratory of Genetics, Center for Cancer Research (CCR), NCI, and serves as CCR associate director of scientific policy.

My research interests are concentrated on the genetics of susceptibility and resistance to cancer. I am working on mapping, cloning, and functional characterization of a set of genes involved in controlling whether certain strains of mice will, when exposed to an exogenous inducer, develop plasmacytomas—hematologic tumors of the B cell lineage. We have found that susceptibility or resistance (S or R) to this tumor is controlled by multiple genetic loci and have initiated molecular identification of these. In contrast to diseases controlled by strong gain-of-function or loss-of-function alleles, the S/R lesions in mice that develop plasmacytomas appear to be examples of "efficiency alleles" that display relatively modest divergence from the activity of the wild-type allele.

The mouse plasmacytoma tumor system represents an excellent experimental model in which tumor S/R is inherited as a complex genetic trait. Most tumor susceptibility models in humans and in experimental animals have focused on the inherited abnormality of a single gene, such as germline mutations of p53 or mutations of the Apc gene in familial polyposis of the colon and in the homologous min gene of the mouse. These particular single-locus lesions predispose to tumor formation because they harbor strong loss-of-function alleles. Because it is estimated that such strong germline alleles may account for only about 10 percent of human cancer, another paradigm is required to explain the other 90 percent of human cancers. Either individuals in whom these cancers arise must lack a germ-line genetic component, or tumor development in these individuals represents a complex, genetically inherited trait. Most cancers are believed to arise after exposure to environmental factors, but it is likely that genetic factors play a role in determining which exposed individuals develop tumors.

The long-term goal of my research is to elucidate the molecular and biological basis for how S/R genes determine neoplastic development. The animals we use do not have deliberately introduced genes, in contrast to transgenic and knockout or knock-in mice. Instead, we take widely used mouse strains, such as BALB/c and DBA/2, which are ostensibly normal in most respects, and analyze their genetic susceptibility and resistance to pristane-induced plasmacytomas.

The most complete part of our work has been genetic identification of multiple modifier loci that contribute to the S or R phenotype. We have used classical genetic approaches involving backcrosses of (BALB/c X DBA/2)F1 hybrids to BALB/c, genome scanning, existing congenic strains, and new congenic strains developed in our lab. The loci were then identified by correlating genotype with plasmacytoma incidence.

These studies revealed that mice harbor five or more different genes affecting susceptibility or resistance and that BALB/c is susceptible at most of these loci. Three of these loci, designated Pctr1, Pctr2, and Pctr3, are located on chromosome 4. We have also found that the introduction of specific onco-genes, via retroviruses, can convert DBA/2 mice from being resistant to being susceptible to pristane-induced plasmacytomas. The combination of Ras and Myc is particularly active.

We have pursued the molecular identification of the modifier loci via positional cloning as well as candidate gene approaches to test for the presence of polymorphic alleles between BALB/c and DBA/2. Using the candidate gene approach, we have shown that the Ink4a locus (also called Cdkn2a) is a candidate for this modifier locus. This gene is located within the interval to which we mapped Pctr1. The Ink4a locus is complex, as it encodes two unrelated regulatory proteins, p16INK4a and p19ARF. Comparison of the coding sequences of Ink4a for the BALB/c alleles vs. the alleles found in most other mouse strains showed that the p16INK4a in BALB/c contained two missense mutations, whereas p19ARF in BALB/c contained a single missense mutation. Furthermore, compared with the 16INK4a protein encoded by the common allele, the BALB/c p16INK4a protein is less efficient in binding CDK4 and inhibiting its kinase activity.

To genetically determine whether INK4a was a modifier locus, we bred an Ink4a knockout (with a genetic lesion that disrupts both p16INK4a and p19ARF) onto a C57BL/6 plasmacytoma-resistant background and tested these mice for their susceptibility to pristane-induced plasmacytomas. We found that although the p16INK4a/p19ARF heterozygotes were still resistant, the mice that were homozygous null developed these tumors even faster than BALB/c mice. In addition, the Ink4a locus remained tightly linked to the resistant interval on chromosome 4 when the Pctr1 interval was shortened by further congenic breeding of the resistant DBA locus on a BALB/c background.

The biological activity of the coding sequences from the BALB/c and DBA alleles for p16INK4a and p19ARF were also compared experimentally. Similar results were obtained with two different bioassays—growth inhibition of BALB/c plasmacytoma cell lines and inhibition of ras-induced focal transformation of NIH 3T3 cells. The BALB/c p16INK4a allele was less efficient than its DBA counterpart, while the efficiency of both p19ARF alleles was similar. These results establish Ink4a as the Pctr1 modifier locus and strongly suggest that it is primarily the gene encoding p16INK4a that is responsible for the BALB/c locus being a susceptibility allele. Although formal proof of this hypothesis will require analysis of mice with isolated defects in only p16INK4a or p19ARF, analysis of p19ARF in BALB/c plasmacytomas suggests this gene may not have a major role in tumor formation, because it continues to be expressed in most of these tumors.

By contrast, p16INK4a is not expressed in the majority of the plasmacytomas, which is consistent with the in vitro studies noted above, indicating that the BALB/c allele possesses some biological activity. To this end, we have also observed sequence variation in the promoter region of p16 and have identified a transcription factor that may influence the expression of the protein in susceptible vs. resistant strains of mice. Taken together, the results establish that the Pctr1 modifier locus in BALB/c represents an efficiency locus, rather than a strong gain- or loss-of-function locus as described for many familial cancer syndromes.

In addition, we are currently evaluating the candidacy of a kinase involved in detecting DNA damage for the Pctr2 locus. Once again, a single base pair change affects the efficiency of the protein in BALB/c compared with DBA/2. These observations have led us to propose that the other modifier loci will also turn out to be efficiency alleles of pathways that are critical for pristane-induced plasmacytomas. By extension, we speculate that many human cancers will prove to be complex genetic traits determined by analogous efficiency alleles.

Jerrel Yakel

Jerrel Yakel received his Ph.D. from the University of California–Los Angeles in 1988 and did postdoctoral work at the École Normale Supérieure (Paris, France) and Vollum Institute (Portland, Oregon) before joining the Laboratory of Cellular and Molecular Pharmacology of NIEHS in 1993. He is now a senior investigator in the Laboratory of Signal Transduction, NIEHS.

My interests are in the area of neuronal communication at the synapse, where the neurotransmitter released by the presynaptic terminal diffuses across the synaptic cleft and binds to and activates various ligand-gated ion channels on the postsynaptic membrane. My research at NIEHS has focused on the nicotinic acetylcholine receptor (nAChR) and the serotonin 5-HT3 receptor channel, both of which are known to mediate rapid (on the order of milliseconds) synaptic transmission in the brain. Changes in the function of these channels have profound effects on neuronal excitability and synaptic plasticity of the cell and learning and memory in the organism. Dysfunction in these channels has been linked to various neurological diseases, such as Alzheimer’s, Parkinson’s, epilepsy, schizophrenia, and depression.

Nicotine is one of the most prevalent and potent neurotoxins to which we are exposed. Exposure to nicotine in utero or in early childhood has been implicated in a variety of developmental abnormalities, including brain damage and cognitive impairment. Interestingly, in adults, particularly patients with Alzheimer’s disease who have been shown to express significantly fewer nAChRs in the brain, nicotine may have positive physiological effects, such as enhancing cognition and alleviating some symptoms of Alzheimer’s disease. Nicotine exerts all its actions in the brain by acting on the nAChR.

To better understand the basic mechanisms and regulation of neuronal excitability by nAChRs and 5-HT3 receptors, my lab is focusing on the function and regulation of these channels in the hippocampus, a region of the brain known to be important for learning and memory. In 1997, my colleagues and I first identified the selective expression of functional nAChRs on a subset of neurons within the hippocampus–the hippocampal interneurons. Hippocampal interneurons are inhibitory because they are known to release the inhibitory neurotransmitter GABA. A single interneuron can innervate and regulate the activity of hundreds of excitatory cells in the hippocampus. Although the nAChR and 5-HT3 receptor channels are known to be involved in a variety of physiological processes, the precise nature of these actions is not currently known and is the major focus of investigation in my lab.

Using a variety of physiological and molecular techniques, we have been investigating which of many known nAChR and 5-HT3 receptor subunits are forming functional channels in the hippocampus. We are also investigating whether the subunits from these two neurotransmitter receptor channels co-assemble into a single, novel type of ligand-gated channel. In 1999, my colleagues and I were the first to discover that nAChR and 5-HT3 receptor subunits can co-assemble in heterologous expression systems, and our recent data suggest that such an interaction may be occurring in the hippocampus. Such information is extremely important in understanding the role of these channels in the brain.

Alzheimer’s disease, a neurodegenerative disorder that is the leading cause of dementia, affects an estimated 4 million persons in the United States and 15 million worldwide at a staggering cost, both in quality of life and in medical care. Further, with the aging of the population, the prevalence and impact of neurodegenerative diseases such as Alzheimer’s are expected to increase dramatically. Alzheimer’s disease is characterized by the extensive accumulation in the brain of the b-amyloid peptide (Ab1-42), the formation of senile plaques, and a progressive loss of cognitive function. Whether Ab1-42 leads to the loss of cognitive function, and what the mechanism involved in such action might be, is unknown.

My colleagues and I recently discovered that Ab1-42 directly inhibits nAChRs in the hippocampus, an effect that might help to explain the cognitive deficits associated with Alzheimer’s disease and lead to the development of therapeutic agents to treat patients with this condition.




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