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My major research interest is the prevention and control of esophageal cancer - a cancer that kills 10,000 Americans annually and is the fourth most common cause of cancer death among African American men. Less than 10% of patients with esophageal cancer survive for five years after diagnosis, largely because most of these tumors do not produce symptoms until it is too late for surgery and nonsurgical treatments are usually not curative. In this setting, we will probably need to develop successful primary-prevention and/or early-detection strategies to significantly reduce esophageal cancer mortality. At NCI, I have participated in two large nutritional-intervention clinical trials in Linxian, China, a region with extraordinarily high rates of esophageal and gastric cardia cancer. During these trials, my collaborators and I performed several studies that were relevant to an early-detection approach to squamous esophageal cancer. We carried out two prospective follow-up studies that documented the predictive value of esophageal cytology, another follow-up study that showed that high-grade squamous dysplasia is the only important near-term histologic precursor of squamous esophageal cancer, and two endoscopic studies that demonstrated that this histologic precursor lesion is usually associated with visible mucosal abnormalities that can be biopsed. Our discovery that squamous dysplasia can usually be identified through an endoscope has important implications for research and clinical practice. In research, endoscopic biopsies should be an accurate gold standard for validating less-invasive diagnostic techniques such as esophageal cytology, and endoscopic protocols should be able to evaluate future intervention studies that use squamous dysplasia as an intermediate endpoint. In the clinic, endoscopic biopsies should be able to confirm and localize screening-detected abnormalities, primary endoscopic screening may be feasible in certain high-risk groups, and focal endoscopic therapy may be possible for controlling precursor and early invasive disease. Building upon these results, I have recently begun a series of studies to evaluate and possibly improve some techniques that may be useful in a practical early-detection program for squamous esophageal cancer. One study is aimed at evaluating the sensitivity and specificity of the currently available esophageal cytologic samplers and at developing improved models of these samplers. Another study focuses on whether mucosal staining can improve endoscopic localization of squamous dysplasia and cancer, thereby optimizing the visualization of such lesions for focal therapy. A third study is designed to evaluate how accurately endoscopic ultrasonography can stage early squamous cancers so that focal therapy will not be attempted on tumors that are already too advanced. A fourth study is aimed at assessing the safety, acceptability, and preliminary efficacy of several methods of focal endoscopic therapy, including endoscopic mucosal resection and thermal coagulation. I am also currently involved in etiologic studies of the roles of human papillomavirus and certain fungal toxins in the development of squamous esophageal cancer, and other studies of the role of Helicobacter pylori in the development of gastric cardia cancer. In addition, I am participating in a group of new genetic studies of esophageal and gastric cancers in high-risk Chinese populations. We are hopeful that our etiologic and genetic studies will contribute to the development of additional promising strategies for the prevention and control of esophageal cancer.
My lab's recent research has centered on glial cells of the mammalian brain. Glial cells do not directly participate in synaptic transmission, and their precise role in the developing and adult brain is yet to be defined. Using oligodendroglial progenitor (O-2A) cells purified from the embryonic rat cerebral cortex as a model system, we are trying to understand the regulation and physiological role of neurotransmitter receptors in glia during development. In the embryonic mammalian brain, oligodendroglial cells divide, migrate, and differentiate later than neurons. This observation has given rise to the hypothesis that neurotransmitters released by neurons may play an important role in the development of the oligodendroglial lineage-a hypothesis that we are testing by focusing on the main excitatory neurotransmitter of the mammalian brain, glutamate, and its receptors. Our previous work, which demonstrated that O-2A cells express glutamate-receptor (GluR) genes and genes that encode functional glutamate-gated channels, led to two important findings. First, we characterized two subtypes of GluRs with distinct molecular composition and function in cells of the oligodendrocyte lineage. Second, we identified a set of genes that are induced by GluR-activation in a calcium-dependent fashion in O-2A progenitors. In more recent experiments, we demonstrated that activation of GluRs in O-2A cells reversibly inhibits their proliferation and prevents lineage progression through the indirect blockage of delayed-rectifier potassium channels. Our future work will focus on the molecular analysis of intracellular events crucial to glial-cell development that are triggered by GluR activation. My lab is also using glial cells to study how GluR genes are regulated in the mammalian brain. Our specific goal is to determine whether the DNA regulatory elements and transcription factors that regulate GluR gene transcription in glia and in neurons are the same. We have cloned the entire rat gene encoding the GluR subunit KA2. This gene, named GRIK5, is abundantly expressed in both glia and neurons. GRIK5 spans approximately 70 kilobases of genomic DNA and comprises 20 exons. We identified multiple transcription-start sites in its 5' flanking region, and also found that GRIK5 displays features of a housekeeping gene. Our analysis in rat neural cells and in nonneural rat and human cells, as well as in transgenic mice, demonstrated that a region of GRIK5's 5'-flanking sequence restricts tissue-specific expression of this GluR gene in vitro and in vivo. Now, we are working on characterizing the mechanisms of transcriptional regulation of GRIK5 during development and identifying the DNA-binding sites involved. Finally, I am also collaborating with Mark Mayer and Chris McBain of NICHD's Laboratory of Cellular and Molecular Neurophysiology on projects to determine whether GluRs can be regulated at the transcriptional level by growth factors that are known to modulate glial development and to define the precise role of other membrane ion channels in glial development.
The major focus of my research program over the past five years has been to establish the genetic architecture of one human chromosome by constructing a complete physical map of its DNA and then determining the DNA sequence. My lab's efforts have centered on chromosome 7, which spans an estimated 170 million base pairs (bp) and accounts for roughly 5% of the human genome. Our mapping approach uses yeast artificial chromosomes (YACs) as the cloned DNA fragments and sequence-tagged sites (STSs) as the landmarks for establishing the overlapping relationships among the YACs. STSs are short stretches of DNA that can be specifically detected using the polymerase chain reaction (PCR). We have developed and implemented strategies for generating STSs specific to chromosome 7 and for identifying YACs containing each of these STSs. This has involved performing an average of 1,000 to 2,000 PCR assays per day for nearly three years. We reached a major milestone recently when we completed construction of one of the most detailed maps of a human chromosome to date-a physical map of chromosome 7 that provides YAC coverage across the chromosome as well as a mapped STS every 80,000 bp. This achievement reflects the development of more than 2,000 STSs unique to chromosome 7, the mapping of each of these STSs to individual YACs, the rigorous integration of our physical map with the genetic and cytogenetic maps, and the mapping of hundreds of gene sequences. These results also provide support for an experimental paradigm, termed YAC-based STS-content mapping, that we proposed in 1991 for building a physical map of the human genome. As a result of our mapping efforts, chromosome 7 is among the first targets for large-scale DNA sequencing within the Human Genome Project. In collaboration with the genome centers at Washington University in St. Louis and the University of Washington in Seattle, we have begun genomic sequencing of chromosome 7. While the notion of sequencing an entire human chromosome may seem daunting, remember that only five years ago the idea of making a complete physical map of a human chromosome was equally intimidating. On the basis of preliminary data and previous experience, we expect that our collective efforts will yield a first-pass sequence of chromosome 7 within three to four years. The availability of an evolving genetic blueprint for 5% of the human genome is already providing spectacular opportunities to explore human biology. Our geographic map of chromosome 7 is now yielding serendipitous research opportunities that cut across biology. We are actively engaged in several projects to study the molecular basis of cancer susceptibility, cardiovascular disease, immune response, and neural development. In many of these projects, we are in pursuit of genes that cause human disease. In every case, our detailed maps, DNA-based reagents, and growing body of sequence data are enhancing our ability to study complex biological processes. These limited examples-which reflect only the tip of the future genetic iceberg-illustrate how the fruits of the genome project are creating a new era for biomedical research.
Trained as an applied mathematician specializing in the analysis and development of methods for numerical solution of ordinary and partial differential equations, I came to NIDDK to work in the Mathematical Research Branch-a leading force in theoretical biology and computational neuroscience since the 1950s. I was particularly attracted by the group's reputation for fostering collaboration between theoreticians and experimental biologists. I was assigned to model the electrical activity of pancreatic beta-cells in the islet of Langerhans using elaborated Hodgkin-Huxley equations that describe neural action potentials. Beta-cells exhibit rhythmic electrical activity, similar to that observed in many neurons, that plays an important role in insulin secretion. An as-yet-unidentified defect in beta-cell response to blood plasma glucose is thought to be central to the development of Type II diabetes. Together with fellow theoreticians John Rinzel and Joel Keizer, I tested the hypothesis of two NIDDK experimentalists, Illani Atwater and Eduardo Rojas, that the bursting electrical rhythm of beta-cells is an emergent property of the gap-junction-coupled network of cells in the islet of Langerhans. Atwater and Rojas developed their "channel-sharing" hypothesis after they found that isolated beta-cells rarely displayed the bursting rhythm. We demonstrated that electrical coupling could not only synchronize the activity of inherently oscillatory units, but also play a role in generating oscillations. More recently, Richard Bertram and I have worked with Atwater, Rojas, and others on parasympathetic regulation of beta-cell electrical activity. We proposed that the inositol-1,4,5-trisphosphate- and aceytylcholine-mediated release of calcium from the endoplasmic reticulum leads to depolarization via calcium-release activated current (CRAC) channels. Unexpectedly, our mathematical model revealed that the important first phase of insulin secretion following a glucose challenge might also be governed by CRAC, a prediction supported by follow-up experiments. Bertram and I have also been collaborating with Elis Stanley of NINDS on mechanisms of synaptic release. We have developed a mathematical model of Stanley's hypothesis that facilitation by high-frequency stimulation is due to accumulation of calcium bound to release sites. We hope this work will help resolve long-standing controversies about synaptic facilitation and also shed light on endocrine secretion. My long-term goals are to continue studying the mechanisms and dynamics of insulin secretion, delving deeper into its regulation by metabolic and hormonal signals. The current flood of detailed biochemical information on vesicle exocytosis should also open up exciting opportunities for the mathematical modeling of this final step in secretion in both neural and endocrine cells.
My research is directed toward understanding the interaction between genes and environmental exposures in human carcinogenesis. There are two main elements to this work: investigating the role of environmental exposure in critical-target gene mutation and investigating the role of genetic susceptibility and environmental exposure in cancer risk. The research on critical-target genes addresses the hypothesis that different environmental exposures cause different patterns of mutation in genes that are important in carcinogenesis. My initial focus has been on mutational activation of oncogenes and deactivation of tumor-suppressor genes. Such patterns can be used to identify novel critical-target genes and to suggest mutational mechanisms by which an environmental agent causes cancer. If specific carcinogens produce characteristic patterns of gene mutation in tumors, detection of such patterns would be a powerful tool in studies of environmental risk and in prevention and early diagnosis. Most of my work has been on lung and bladder cancer-two tumors that have strong environmental determinants. In a recent study done with Teddy Devereux at NIEHS and Geno Saccomanno at St. Mary's Hospital in Grand Junction, Colo., we showed that roughly one-third of large- and squamous-cell lung tumors from uranium miners had an identical mutation in the tumor-suppressor gene p53. This is one of only four known examples of an exposure-specific pattern of critical-target gene mutation in human tumors. It is a provocative result because alpha-particle radiation, although known to cause single base-pair mutations, might not be expected to produce such a highly specific DNA lesion. My research on genetic susceptibility tests the hypothesis that commonly inherited allelic variants of selected candidate genes, in conjunction with environmental exposures, affect a person's risk of developing cancer. Working with genetically susceptible subgroups may allow us to identify the environmental exposures that cause disease and the true risks associated with exposure. It could also lead to programs for protecting susceptible populations and for targeted screening of high-risk groups. We are studying inherited polymorphisms in selected genes that have potential links to bladder cancer risk: genes involved in carcinogen metabolism, proto-oncogenes, tumor-suppressor genes, and genes involved in DNA synthesis and repair. Doug Bell at NIEHS and I have looked at a polymorphism in the gene GSTM1, which is involved in detoxification of certain carcinogens. Interestingly, roughly half of the U.S. population has no working copy of this gene (homozygous null). We have found evidence of a gene-environment interaction on risk: people with the homozygous null GSTM1 genotype have twice the risk of developing bladder cancer as people with at least one working copy of the gene-but only if they also are exposed to a carcinogen, such as cigarette smoke. Although the increased risk is fairly small, particularly compared with the risk posed by genes responsible for familial clusters of cancer, such a gene polymorphism can still be important to public health because both the polymorphism and the exposure are common. We calculate that 25% of bladder cancer may be attributable to the heritable defect in GSTM1. My two research areas also overlap: critical-target genes are often polymorphic and their inherited allelic variants may affect susceptibility; conversely, the inherited variant alleles of susceptibility genes may ultimately affect the pattern of mutation in critical-target genes found within a tumor. By combining epidemiology and molecular biology, my long-term goal is to develop a more integrated view of how exposure, genetic susceptibility, and critical-target gene damage interact in lung and bladder cancers. |
