Susan L. Naylor - US grants

Of the 40,000 new cases yearly of small cell lung cancer (SCLC), a severe form of cancer, only 10% of the affected individuals will respond to chemotherapy. In this proposal DNA markers will be used to study the molecular pathology of a specific and consistent chromosome aberration in SCLC. SCLC has been demonstrated cytogenetically to possess a deletion in the short arm of chromosome 3 (bands p14 to p23). Molecular probes have been used to extend the cytogenetic findings, and have shown a high frequency of allele loss on the short arm of chromosome 3. The long-range goal is to identify the gene(s) affected by the 3p deletion in SCLC and to determine if there is a relationship between the deletion in SCLC and the fragile site at chromosome 3p14 and also the chromosome 3p deletion seen in other cancers. Additional informative polymorphic DNA markers specific for the 3p deletion are required for initiating a fine structural analysis of this chromosomal region, and for identifying genes that may play key roles in the pathogenesis of SCLC. Using existing polymorphic DNA markers specific for the deleted region, junction fragment, Lambda, and cosmid libraries will be screened to identify additional polymorphic DNA probes. New probes will be characterized, chromosomally mapped, and used to screen for polymorphisms i) to analyze paired normal and tumor SCLC samples, ii) to identify the limits of the SCLC deletion, iii) to analyze the fragile site and the boundaries of the SCLC deletion by pulsed field electrophoresis, and iv) to analyze the 3p deletion in other cancers. Factors that may predispose individuals to SCLC that will be examined are the unusual frequency of a DNA polymorphism D3S3 in SCLC and the susceptibility to breakage of the fragile site on chromosome 3p. The data obtained should provide molecular insight into the chromosomal changes in SCLC and other cancers and information about their relationship to the fragile site at chromosome 3p14.

Chromosome 3 is a large metacentric chromosome encompassing 7% of the human genome, about 210 megabases. The long range goal of this project is to construct a physical map of chromosome 3 and to isolate overlapping clones for the entire chromosome. First, a battery of useful markers will be isolated consisting of highly polymorphic VNTR (variable number of tandem repeat) probes and potentially transcribed regions of chromosome 3. These markers will be mapped to broad regions of chromosome 3 using a panel of somatic cell hybrids. A panel of radiation hybrids will be examined to produce a refined map of the chromosome. Radiation hybrids clones will bridge the gap between conventional somatic cell mapping and the level of resolution obtained by recombinant DNA clones and pulsed field gel electrophoresis. The radiation hybrid clones will also be the source of nested cosmid and YAC (yeast artificial chromosome) clones for a given region of the chromosome. A rudimentary linkage map of the most polymorphic markers will be used to compare the distances on the physical and linkage maps of chromosome 3.

Chromosome 3 is 7% of the human genome and encompasses 230 megabases of DNA. Despite its size, chromosome 3 has been slow to accumulate markers. Recently, a large number of new markers have been generated for chromosome 3, as evidenced by the large number of DNA segments assigned to chromosome 3: there has been an increase from 100 markers to 1600 over the past 3 years. Consequently, now we will be able to begin to construct maps for chromosome 3. The purpose of this workshop is to consolidate data from many labs to construct physical, linkage and radiation maps for chromosome 3. These maps will establish the common bases for the maps, identify overlap, identify areas that need resolution, and set up a resource for common reagents. The workshop will be two days in march, 1992 at the Eisai Convention Room, Bunkyo-ku, Tokyo, Japan. The structure of the meeting will be a series of short presentations by each of the laboratories involved followed by an intense working session to resolve inconsistencies in the map and to produce consensus maps. Relevant material generated at this meeting will be presented in a published report.

Chromosome 3 has 7% of the human genome and is approximately 210 megabases. The construction of integrated physical and genetic maps for this chromosome, based on a common set of DNA markers, will be a powerful tool for studying the genetics of man. A consortium of 5 laboratories all with excellent resources for chromosome 3 has been formed to develop a 10 cM map of highly polymorphic markers for chromosome 3. To date there have been only a limited number of highly polymorphic markers developed for this chromosome. Four of the laboratories will isolate highly polymorphic markers from cosmids, YACs, and flow sorted libraries as well as minilibraries for specific regions of the chromosome. Polymorphisms will be predominantly based on (CA)n repeats which will be regionally localized on a physical map. The polymorphisms will be detected by a PCR based assay from sequence information flanking the (CA)n repeats. In addition, well established loci that are only slightly or moderately polymorphic will be analyzed for single stranded conformational polymorphisms and gradient gel electrophoresis to increase the level of polymorphism detected. These reagents will be typed in CEPH and Venezuelan families and the data will be analyzed to produce a genetic map. By two years we will generate a 10 cM map of markers that have a heterozygosity of 0.7 or greater. In the third year we will expand the map and fill in gaps towards the 2-5 cM map that is the 5 year goal of the Genome Project. The development of a genetic linkage map of human chromosome 3 will significantly facilitate the identification of disease genes in germline and malignant disorders. A set of markers that have been placed on both physical and genetic maps and are based on sequence will greatly accelerate the progress of mapping chromosome 3.

Chromosome 2 is 7% of the human genome and encompasses 270 megabases of DNA. Despite its size, chromosome 2 has been slow to accumulate markers. Recently, a large number of new markers have been generated for chromosome 2, as evidenced by the number of DNA segments assigned to chromosome 2: at HGM9 there were only 50 single copy DNA segments and at HGM10.5 there were 81 firmly assigned markers. Consequently, now we will be able to begin to construct maps for chromosome 2. The purpose of this workshop is to consolidate data from many labs to construct physical, linkage and radiation maps for chromosome 2. These maps will establish the common bases for the maps, identify overlap, and identify areas that need resolution. The workshop will be two days in October, 1991 (just following the International Congress of Human Genetics) in Washington, D.C. The structure of the meeting will be a series of short presentations by each of the laboratories involved followed by an intense working session to resolve inconsistencies in the map and to produce consensus maps.

information systems; genetic mapping; biomedical facility; artificial chromosomes; sequence tagged sites; genetic markers; nucleic acid probes;

The long term goal of this project to produce a battery of polymorphic markers for chromosome 3 which will serve as a framework for mapping and sequencing the chromosome. Currently, the chromosome 3 maps is sparse and only a handful of gene have been mapped to this large chromosome. Approximately 250 markers will be isolated for each chromosome and placed on radiation hybrid and linkage maps. The net result of this project will be a 2 cM map and physical markers approximately every megabase. The markers to be developed will be highly useful as they are highly polymorphic and PCR based. The first specific aim of the project is to produce highly polymorphic markers base on simple repeats. Repeats of 2, 3, and 4 base pairs will be isolated from chromosome specific libraries, and their sequence used to develop a PCR based assay. A second aim is to develop polymorphisms within known gene loci mapped to chromosome 3. The method of single strand conformational polymorphism (SSCP) and sequencing will identify polymorphism which will be detected in individuals by an allele specific oligonucleotide. Further markers will be developed in specific aim three for those regions of the chromosomes which lack markers. ALU PCR will be used on radiation reduced hybrids to generate region specific clones. These clones will be sequenced and analyzed for polymorphism. All the above mentioned markers will be placed on the physical map by radiation hybrids and the genetic map by analyzing CEPH and other family DNA. Because this project will generate PCR based polymorphisms, the markers will be immediately useful to the scientific community. Investigators seeking linkage markers will have a large reservoir of precisely mapped markers to draw upon. Our approach assimilates all the known data on chromosome 3 by incorporating known sequences into detailed physical and genetic maps. These markers are a first step towards a directed aligning of YAC and cosmid clones and will facilitate closure on this chromosome in the future.

The goal of this program project is to produce detailed physical and genetic maps of polymorphic markers for human chromosomes 2 and 3. The current maps of chromosomes 2 and 3 only have a limited number of markers and there are several large gaps on both the physical and genetic maps. The physical map generated by the program project will be based on radiation reduction hybrids and the genetic map a result of analyzing CEPH family DNA. The genetic markers generated will all be PCR based and consist of highly polymorphic markers such as found with simple di-, tri-, and tetranucleotide repeats and in the poly A tract of L1 sequences as well as transcribed sequences specific for these chromosomes. In addition the program will devise a method for globally mapping each chromosome with Line elements and correlating this map to the genetic and radiation hybrid map. To accomplish these goals, the program consists of 4 projects and a core for linkage studies as well as an administrative core. These highly interdependent projects will result in the placement of 600 highly useful markers on chromosomes 2 and 3 over the grant period. The markers are termed highly useful since they will be highly polymorphic, be based on PCR and available to all investigators, and be mapped on both physical and genetic maps. The physical map generated will be complete at the 2 cM level and in many regions 1 cM distances will be achieved. The physical map of each chromosome will have approximately 1 marker per megabase. This framework panel of markers and cell reagents will be used to direct the isolation of ordered clones for chromosomes 2 and 3 and will be especially useful for closure on these chromosomes. The polymorphic markers will be immediately applicable for identifying markers adjacent to disease loci.

The short arm of human chromosome 3 in the region 3p21.3 is deleted in many cancers including lung cancer which is the cause of 40,000 deaths in the U.S. each year. A fragment of chromosome 3 from this region suppresses tumor formation of A9 mouse fibrosarcoma cells in a nude mouse assay. Furthermore, this same fragment of chromosome 3 changes the response of A9 cells to chemotherapeutic drugs from apoptosis to growth arrest. This study is designed to identify the gene inducing tumor suppression in A9 and to determine whether it is the same gene imparting the differential response to chemotherapeutic drugs. The cDNA which suppresses tumor growth of mouse A9 fibrosarcoma cells will be identified using an inducible expression system. The candidate cDNA will be tested to determine if it is the same gene which gives a differential response to chemotherapeutic drugs. The cDNA which induces tumor suppression and differential drug response in mouse A9 cells will be tested for these effects in human tumor cells including small call lung cancer. The inactivation of this gene in human tumors will be explored by both mutation analysis and by methylation assays. The mechanism of action of the tumor suppressor gene and the basis of the switch from apoptosis to growth arrest will be determined relating this gene to other genes in apoptotic pathways. This study will define a gene which already has been implicated in a number of cancers by deletion and define its functioning in normal cells and during tumorigenesis. Status of this gene in a tumor may provide clues about that tumor's response to chemotherapeutic drugs.

DESCRIPTION (provided by applicant): The Latino/Hispanic population is the largest minority in the US and the highest concentration of Latinos is along the US-Mexico Border. To address the specific needs of this population in cancer research and education, a partnership between The University of Texas-Pan American and the Cancer Center at The University of Texas Health Science Center at San Antonio is proposed. The goals of this application are to develop research and training partnerships to increase the cancer research base on the US-Mexico border. The projects proposed will not only develop translational research in basic science but also address cancer related health disparities in the Hispanic border population. The specific goals of the application are: 1. to develop and foster cancer research at UTPA;2. to foster research dedicated towards reducing the cancer burden of the Hispanic population with emphasis on the US-Mexico border, and 3. to increase the number of cancer researchers coming from South Texas. To accomplish these goals, two pilot projects and a training program are proposed. The first pilot project is a basic science project which will take advantage of the expertise at UTPA to synthesize polycyclic aromatic compounds and expertise at UTHSCSA to test these compounds as anticancer agents. The second pilot project is directed toward understanding the attitudes and decision making processes in genetic testing for increased breast cancer risk. The third project is a summer undergraduate program for UTPA students to work in laboratories and to learn about cancer biology with the goal of increasing the number of students from communities in South Texas interested in cancer research. This multidimensional approach will heighten cancer research and education in the Hispanic/Latino population along the Texas border.

DESCRIPTION (provided by applicant): The Latino/Hispanic population is the largest minority in the US and the highest concentration of Latinos is along the US-Mexico Border. To address the specific needs of this population in cancer research and education, a partnership between The University of Texas-Pan American and the Cancer Center at The University of Texas Health Science Center at San Antonio is proposed. The goals of this proposal are to develop research and training partnerships to increase the cancer research base on the US-Mexico border. The projects proposed will not only develop translational research in basic science but also address cancer related health disparities in the Hispanic border population. The specific goals of the application are: 1. To develop and foster cancer research at UTPA;2. To foster research dedicated towards reducing the cancer burden of the Hispanic population with emphasis on the US-Mexico border, and 3. To increase the number of cancer researchers coming from South Texas. To accomplish these goals, two pilot projects and a training program are proposed. The first pilot project is a basic science project which will take advantage of the expertise at UTPA to synthesize polycyclic aromatic compounds and expertise at UTHSCSA to test these compounds as anticancer agents. The second pilot project is directed toward understanding the attitudes and decision making processes in genetic testing for increased breast cancer risk. The third project is a summer undergraduate program for UTPA students to work in laboratories and to learn about cancer biology with the goal of increasing the number of students from communities in South Texas interested in cancer research. This multidimensional approach will heighten cancer research and education in the Hispanic/Latino population along the Texas border.

The Genomics Shared Resource provides state-of-the-art services to the members of the Cancer Center by providing access to two complementary platforms, the lllumina platform, used for primarily for highthroughput genotyping of single nucleotide polymorphisms, and the Agilent Technologies platform, used primarily for array comparative genomic hybridization and expression microarray analysis. In addition to these genomic services, the shared resource offers Cancer Center members assistance in automated nucleic acid isolation, real-time quantitative PCR, and sample banking. The banking of samples range from isolating DNA, processing blood specimens, establishing lymphoblastoid cell lines and long term storage of samples. The shared resource has been reorganized with a single overall coordinator and two co-directors, one per platform. All three have extensive experience in genomics and provide Cancer Center members with consulting services to assist in experimental design. This shared resource combines two previous funded cancer center shared resources, the Cytogenetic and Genetics Shared Resource and the Microarray Shared Resource.

DESCRIPTION (provided by applicant): The long term objective of this research is to identify the molecular mechanism that accounts for extended lifespan. Two mouse models of extended lifespan will be studied: Caloric restriction and the Ames dwarf mouse. It has been known for decades that caloric restriction in rodents leads to significantly increased lifespan. Likewise, dwarf mice such as the Ames mouse also have a prolonged lifespan. MicroRNAs are likely potential candidates for molecules that regulate lifespan as one of these small RNAs (~22 nt) can control the expression of multiple genes. To determine if miRNAs have a role in aging three specific aims are proposed. Specific aim 1 is to establish the miRNA profile of normal mice at young adulthood, middle age and old age using microarray technology to determine if specific miRNAs correlate with age. miRNA microarray data will be validated by real-time reverse transcriptase PCR. Specific aim 2 will determine the miRNA profile of the long lived caloric restricted mouse to examine if there are specific miRNA correlated with longer lifespan. Specific aim 3 will examine the miRNA profile of the Ames dwarf mouse which is not considered to be a mimetic of caloric restriction. Comparison of the extended lifespan models will indicate if there are specific miRNAs in common that contribute to increased lifespan. The identification of such a regulatory molecule will help unlock the mechanism of controlling lifespan in mammals PUBLIC HEALTH RELEVANCE: Aging is a normal process of life but it is still unknown what mechanism instigates intrinsic aging. If we understand this process, we may be able to delay age- related changes especially those related to age-dependent disease. Understanding models of prolonged lifespan will yield clues into the process and may give insight into how we can age in a more healthy manner.

DESCRIPTION (provided by applicant): The proposed Cancer Biology Training Program is a resubmission of an application for an institutional training grant. We request funds to support 2 predoctoral and 2 postdoctoral trainees. Despite all the advances made in cancer research, it has become the number one killer of people under 85 in the U.S. The goal of the Cancer Biology Training Program is to educate the next generation of cancer researchers to meet the growing demands for scientists trained in the multiple facets of cancer biology. The training program has 35 faculty mentors (22 men and 13 women) from 12 departments who are working together in the graduate program as well as in the Cancer Therapy and Research Center (CTRC), the NCI Designated Cancer Center of the University of Texas Health Science Center. The mentors have a strong record of funded research as well as one in training pre- and postdoctoral fellows. This training program is uniquely poised to train minority students because of our rich cultural heritage in South Texas. Predoctoral trainees will be recruited from students in the Integrated Multidisciplinary Graduate Program who have selected the cancer biology track. Trainees will be selected for their academic excellence and their dedication to cancer research. A key component of the program is the broad scope of training from molecular biology to clinical problems. In addition, the students will participate in cancer journal club as well as attend the cancer seminar series. Our program emphasizes training in scientific communication with opportunities to improve both oral and written skills. Postdoctoral candidates will have a Ph.D. or M.D. degree or equivalent. The postdoctoral trainees will have the same opportunities to obtain a diverse background in cancer research as well as training in communication. The broad training program and the large number of minority trainees attracted to our program make this a unique opportunity to enrich the diversity in the pool of future cancer researchers. PUBLIC HEALTH RELEVANCE: A steady stream of new researchers is necessary to continue the fight against cancer, the primary cause of death in the U.S. The training of a diverse workforce is a key component to the next generation of scientist. Giving trainees a broad exposure to the many areas of cancer research will help them become part of the multidisciplinary approach to disease.