Cancer Prevention and Population Sciences (Genetics)
Director, DNA Microarray and Genomics Facility
Roswell Park Cancer Institute
Elm and Carlton Streets
Buffalo, NY 14263
Telephone : (716) 845-8966
Fax : (716) 845-1698
Email: Norma.Nowak@RoswellPark.org

Current Program

The working draft of sequence for the human genome has been completed. Yet we are still facing the biggest challenge in determining the function of the vast majority of genes that will be uncovered. Described below are projects for global comparative genomic and differential gene expression analyses.

Laboratory Personnel

Michael Bianchi BS
Jeffrey Conroy BS
W. Michael Henry MS
Devin McQuaid BS
Paul Quinn MS

Description of Research

A Resource of Arrayed BAC Clones for FISH Mapping the Human Genome Cancer is a heterogeneous disorder, encompassing more than 100 diseases, thought to progress as a result of cumulative genetic alterations at numerous loci controlling growth and proliferation. Our knowledge of the mechanisms for the initiation and progression of cancer has increased dramatically during the past decade. Yet cancer remains a formidable health challenge with one of every four deaths in the United States attributable to cancer.

To assist in the effort to discover the genetic alterations that result in cancer, we are generating a genome-wide resource of mapped BAC clones from the RPCI-11 human BAC library, for their application as tools for FISH (Fluorescence In Situ Hybridization) analysis of chromosomal rearrangements in human cancer. Each clone in the resource will have been assigned a sequence tag and mapped relative to cytogenetic bands. The short-term goal is to provide cytogeneticists with an evenly spaced set of clones with which to analyze tumor rearrangements. This set can be used as probes for FISH analyses of tumor chromosomes and as immobilized DNA targets for high-resolution CGH (Comparative Genomic Hybridization) analysis of tumor DNA versus normal DNA. The information from these analyses will define the genetic signature for each tumor type and stage analyzed. The sequence tags of these clones will afford investigators direct access to detailed genomic information, including the (predicted) sequences of candidate genes.

In addition to the impact this resource will have on clinical cytogenetics, these studies are also providing valuable information on the structure, organization, and evolution of the human genome. This project provides a valuable assessment of the "golden path" or predicted tiling path of the human sequence draft. Each clone can be positioned via its STS, EST, BAC-end, or entire sequence on the draft sequence. FISH analyses provide an independent validation of the order of contigs in the sequence assembly predicted by other mapping methods. Furthermore, many clones in our set will be positive for more than one marker, allowing high-resolution validation of sequence assembly. In addition, our efforts will facilitate analyses of duplicated regions in the human genome. Duplications are important in the genesis of multi-gene families and as potential sites of recombination events that can result in chromosome abnormalities, and they can also pose challenges to sequence assembly. FISH readily detects these regions, in sharp contrast to other molecular techniques. Ultimately, use of this integrated resource should also lead to a better understanding of the organization of the cell nucleus, the process resulting in the compaction of the DNA into mitotic chromosomes, and the molecular basis of chromosomal banding patterns.

High Throughput Analysis of Gene Expression Patterns in the Nervous System

The development of databases chronicling the spatial, and temporal patterns of gene expression will greatly aid in defining gene function. Model organisms such as the mouse afford invaluable research tools for elucidating gene function. This proposal will provide the mouse genomic clone resources to generate, through homologous recombination/excision, modified BAC clones carrying the marker genes lacZ and EGFP. These tagged BACs will be utilized to map gene expression in the central nervous system through the creation of transgenic mouse strains. We will carry out high throughput screening of the RPCI BAC libraries, and identify 2000 BAC clones representing 1000 CNS genes/per year to generate the modified BACs. These 2000 BACs/year will be validated through PCR confirmation as well as HindIII fingerprint analysis. The timely contribution of genomic clone resources plays a vital role in the success of this intensive screen for novel CNS gene expression profiles and phenotypes as proposed by Dr. Nathaniel Heintz. This will very likely culminate in a major landmark study for mammalian functional genomics.

Sequence Connected, Mapped BACs for Array Based Analysis of the Mouse Genome

Advances in technology, fostered through the genomics revolution, in conjunction with a greater understanding of the genetic mechanisms of tumorigenesis, have set the stage for capitalizing on the mouse as a powerful model to dissect the complexity of cancer. The human/mouse comparative map is highly sophisticated due to the comprehensive genetic analyses that have been conducted for these two mammalian species. Greater than 1,800 orthologous gene pairs have been mapped in both species with indications for several hundred conserved genomic segments (Nadeau et al., 1995). The mouse is already a rich source of potential models for human disease as hundreds of existing mutant loci have already been well characterized. More recently, the ability to create transgenic mice using large-insert DNA clones such as bacterial artificial chromosomes (BACs) and P1 artificial chromosomes (PACs) and targeted gene knockouts allows the specific manipulation of any genomic segment (Bedell et al., 1997a, Yang et al 1999). Hundreds of homologous mouse/human genes have been identified in which mutations cause a disease state in both species, often producing similar phenotypes (Bedell et al., 1997b).

A gene-based map of the mouse genome has been constructed, following the development of a suitable RH mapping panel. This RH panel was constructed using a hamster recipient cell line and has an average retention frequency of 27%, similar to that of the human whole genome RH panels (Walter, et al., 1994). The dosage used was 3000 rad with providing an approximate resolution of 1000-1500 bins. Since a common RH panel was used among the three groups for RH mapping, comprised of the Whitehead Institute and two European efforts, allowing the resulting data to be integrated. Thus, the combined effort is expected to provide a mouse gene-based RH map consisting of 40,000 to 50,000 transcripts.

Techniques for high resolution genome-wide analysis such as BAC array CGH (Comparative Genomic Hybridization) will detect chromosomal imbalances in human and mouse tumors. Since non-random, recurrent abnormalities are best identified through massive screening of specific tumor types these resources and their application in array based technology will greatly advance our genotyping and phenotyping ability towards comprehensive profiling of tumors. This will inevitably allow us to identify disease subtypes within traditional pathological classifications that likely will be be diagnostically and prognostically useful. While tumor rearrangements in mice tend to be less complex than those observed in humans, they do mirror the affected conserved chromosomal regions observed in humans. Application of large insert BAC clone resources for generation of transgenics and as tools for studying at high resolution mouse models of human cancer will provide insight into the mechanisms directing and controlling development, differentiation and proliferation within the human and mouse genomes (Oeltjen, et al. 1997, Yang et al 1999). Should the sequencing effort move towards a shotgun based approach, these clones will be anchored to the sequence by virtue of their STS content making them an invaluable to the scientific community for downstream functional genomics projects.

The establishment of the DNA Microarray and Genomics Facility has afforded the RPCI scientific community with the ability to globally perform comparative analysis at both the DNA and RNA levels as well access to the RPCI BAC resources for downstream functional studies. During the past 9 months we have:

Generated spotted human gene expression arrays representing 4, 6, and 10 thousand human genes. Similarly for the mouse, we have generated spotted gene expression arrays containing 1 and 4 thousand mouse genes.

Designed a human cancer gene array with ~2,500 genes associated with cancer, and are designing a similar array for the mouse. Successfully applied the human and mouse expression arrays for 20 NIH/NCI funded investigators.

Generated a genome wide human genomic BAC array for Comparative Genomic Hybridization or Genome Mismatch Scanning at an average of ~500 kb resolution. This array has been successfully applied to detect submicroscopic numerical alterations in a leukemia cell line not previously detected by classic karyotype analysis.

Generated a similar array for the mouse as part of the NCI Mouse Models of Cancer Program.

Performed high-throughput screening for Dr. Nathaniel Heintz (Howard Hughes Investigator, Rockefeller University) of the RPCI-23 library to provide BAC resources that are essential to the creation of a public database globally profiling central nervous system gene expression during development.

Key Publications 

• Chung I, Karpf, AR, Muindi JR, Conroy JM, Nowak NJ, Johnson CS, Trump DL.  Epigenetic silencing of CYP24 in tumor-derived endothelial cells contribute to selective growth inhibition by calcitriol.  J Biol Chem 2007 Jan 22, [Epub ahead of print]

• Hicks DG, Yoder BJ, Short S, Tarr S, Prescott N, Crowe JPO, Dawson AE, Budd GT, Sizemore S, Cicek M, Choueiri T, Tubbs RR, Gaile D, Nowak N, Accacvitti-Loper MA, Frost AR, Welch DR, Casey G.  Loss of BRMS1 protein expression predicts reduced disease-free survival in hormone receptor neative and HER2 positive ubsets of breast cancer.  Clin Cancer Res 12:6702-6708, 2006.

• Shankar G, Rossi MR, McQuaid D, Conroy JM, Gaile DG, Cowell JK, Nowak NJ, Liang P.  A CGH viewer:  A general visualization tool for aCGH data.  Cancer Informatics 2:36-43, 2006.

• Nowak NJ, Snijders A, Conroy J, Albertson D.  The BAC Resources.  Curr Protocols in Human Genet 4.13 (1-31, 2005).

• Rossi MR, LaDuca J, Matsui S-I, Nowak NJ, Hawthorn L, Cowell JK.   Novel amplicons on the short arm of chromosome 7 identified using high resolution array CGH contain over expressed genes in addition to EGFR in glioblastoma multiiforme. Genes Chromosomes Cancer 44:392-404, 2005.
• Miliaras D, Grimbizis G, Conroy J, Psarra N, Miliaras S, Nowak N, Bontis J. Novel karyotypic changes detected by comparative genomic hybridization in a case of congenital cervical immature teratoma. Birth Defects Res A Clin Mol Teratol 73(8):572-576, 2005.
• Matsui SI, LaDuca J, Rossi MR, Nowak NJ, Cowell JK.  Molecular characterization of a consistent 4.5 megabase deletion in 4q28 in prostate cancer cells. Cancer Genet Cytogenet 159: 18-26, 2005.
• Snijders AM, Nowak NJ, Huey B, Fridlyand J, Law S, Conroy J, Tokyuasu T, Demir K, Chiu R, Mao J-H,  Jain AN, Jones SJM, Balmain A, Pinkel D, Albertson GH.  Mapping segmental and sequence variations among laboratory mice using BAC array CGH.  Genome Res 15:302-311, 2005.
• Kimura MT, Mori T, Conroy J, Nowak, NJ, Satomi S, Katsuyuki T, Nagase H.  Two functional coding single nucleotide polymorphisms in STK15 (Aurora-A) coordinately increase esophageal cancer risk.  Cancer Res. 65:3548-3554, 2005.
• Minderman H, Conroy J, Loughlin KL, McQuaid D, Quinn P, Li S, Pendyala L,  Nowak N, Baer MR.  In vitro and in vivo irinotecan-induced changes in expression profiles of cell cycle and apoptosis-associated genes in acute myeloid leukemia cells. Mol Cancer Ther 4:885-900, 2005.
• Rossi MR, Gaile D, LaDuca J, Matsui SI, Conroy J, McQuaid D, Chervinsky D, Eddy R, Chen H-S, Barnett G, Nowak NJ, Cowell JK.  Identification of consistent novel megabase deletions in low-grade oligodendrogliomas using array-based comparative genomic hybridization.  Genes Chromosomes Cancer 44:85-96, 2005.
• Nowak NJ, Gaile D, Conroy JM, McQuaid D, Cowell J, Carter R, Goggins MG, Hruban RH, Maitra A. Genome wide aberrations in pancreatic adenocarcinoma. Cancer Genet Cytogenet 161:36-50, 2005.
• Varma G, Varma R, Huang H, Pryshchepava A, Groth J, Fleming D, Nowak NJ, McQuaid D, Conroy J, Mahoney M, Moysich K, Falkner KL, Geradts J.  Array comparative genomic hybridisation (aCGH) analysis of premenopausal breast cancers from a nuclear fallout area and matched cases from Western New York. Br J Cancer 93(6):699-708, 2005.
• Rossi MR, La Duca J, Matsui S, Nowak NJ, Hawthorn L, Cowell JK.  Novel amplicons on the short arm of chromosome 7 identified using high resolution array CGH contain over expressed genes in addition to EGFR in glioblastoma multiforme. Genes Chromosomes Cancer 44(4):392-404, 2005.
• Deeb G, Baer MR, Gaile DP, Sait SN, Barcos M, Wetzler M, Conroy JM, Nowak NJ, Cowell JK, Cheney RT.  Genomic profiling of myeloid sarcoma by array comparative genomic hybridization. Genes Chromosomes Cancer 44(4):373-383, 2005.
• Drazinic CM, Ercan-Sencicek AG, Gault LM, Hisama FM, Qumsiyeh MB, Nowak NJ, Cubells JF, State MW.  Rapid array-based genomic characterization of a subtle structural abnormality: a patient with psychosis and der(18)t(5;18)(p14.1;p11.23.  Am J Med Genet  134(3):282-289, 2005.
• Cowell JK, Matsui SI, Wang YD, LaDuca J, Conroy J, McQuaid D, Nowak NJ. Application of bacterial artificial chromosome array-based comparative genomic hybridization and spectral karotyping to the analysis of glioblastoma multiforme. Cancer Genet Cytogenet 151:1:36-51, 2004.
• Collins Y, Tan DF, Pejovic T, Mor G, Qian F, Rutherford T, Varma R, McQuaid D, Driscoll D, Jiang M, Deeb G, Lele S, Nowak N, Odunsi, K. Identification of differentially expressed genes in clinically distinct groups of serous ovarian carcinomas using cDNA microarray. Int J Molecular Med 14:1:43-53, 2004.
• Cowell J, Wang Y, Head K, Conroy J, McQuaid D, Nowak N. Identification and characterization of chromosome abnormalities using arrays of bacterial artificial chromosomes. Br J Cancer 90:860-865, 2004.
• Cowell J, Barnett G, Nowak N. Characterization of the 1p/19q chromosomal loss in oligodendrogliomas using CGHa. J Neuropathol and Experimental Neurology 63:2:151-158, 2004.
• Ionov Y, Nowak N, Markowitz S, Perucho M, Cowell J. Manipulation of nonsense mediated decay identifies gene mutations in colon cancer cell lines with microsatellite instability. Oncogene 23:639-645, 2004.
• Cowell JK, Nowak NJ. High resolution analysis of genetic events in cancer cells using BAC arrays and CGHa. Adv Cancer Res 90:91-125, 2003.
• Hackett C, Hodgson J, Law M, Fridlyand J, Osoegawa K, deJong P, Nowak N, Albertson D, Jain A, Jenkins R, Gray J, Weiss W. Genome-wide array CGH analysis of murine neuroblatoma reveals distinct genomic aberrations which parallel those in human tumors. Cancer Res 63:5266-5273, 2003.
• Bruce CK, Howard P, Nowak NJ, Hoban PR. Molecular analysis of region t(5;6)(q21;q21) in Wilms tumor. Cancer Genet Cytogenet 141:106-113, 2003.
• Gong S, Zheng C, Doughty ML, Losos K, Didkovsky N, Schambra UB, Nowak NJ, Joyner A, Leblanc G, Hatten ME, Heintz N. A gene expression atlas of the central nervous system based on bacterial artificial chromosomes. Nature 425:917-925, 2003.
• Sossey-Alaoui K, Head K, Nowak N, Cowell J. Genomic organization and expression profile of the human and mouse WAVE gene family. Mamm Genome 14(5):314-322, 2003.
• Sait S, Qadir M, Conrol J, Matsui S, Nowak N, Baer M. Double minute chromosomes in acute myeloid leukemia and myelodysplastic syndrome: Identification of new amplification regions by fluorescence in situ hybridization and spectral karotyping. Genes, Chromosomes and Cancer 34:42-47, 2002.
• Matsui S, Sait S, Jones C, Nowak N, Gross K. Rapid location of transgenes in mouse chromosomes with a combined spectral karotyping/FISH technique. Mamm Genome 13:680-685, 2002.
• Snijders A, Nowak N, Segraves R, Blackwood S, Brown N, Controy J, Hamilton G, Hindle A, Huey B, Kimura K, Law S, Myambo K, Palmer J, Yistra B, Yue J, Gray J, Jain Am Pinkel , Albertson D. Assembly of microarrays for genome-wide measurement of DNA copy number. Nat Genet 29:263-264, 2001.
• Zhang J, Qin S, Sait S, aley L, Henry W, Higgins M, Nowak N, Shows T, Gerhard D. The pericentromeric region of human chromosome 11: evidence for a chromosome-specific duplicatoin. Cytogenet Cell Genet 94:137-141, 2001.
• Hodgson G, Hager JH, Volik S, Hariono S, Wernick M, Moore D, Nowak N, Albertson DG, Pinkel D, Collins C, Hanahan D, Gray JW. Genome scanning with arrary CGH delineates regional alterations in murine islet carcinomas. Nature Genetics 29:459-464, 2001.
• Cheung V*, Nowak N.* *Co-first authors, et al. Integration of cytogenetic landmarks into the draft sequence of the human genome. Nature 409:953-958, 2001.
• The International Human Genome Mapping Consortium. A physical map of the human genome suitable for systematic sequencing. Nature 409:934-939, 2001.
• Zhao XF, Nowak NJ, Shows TB, Aplan PD. MAGOH interacts with a novel RNA-binding protein. Genomics 63:145-148, 2000.
• van Schie R, Conroy J, Marras SAE, Nowak NJ, de Jong PJ. Semiautomated clone verification by real-time PCR using molecular beacons. BioTechniques 29:1296-1308, 2000.