Cancer Research Laboratory 2003-2004

In 2003-2004, the Cancer Research Laboratory provided services to 88 Principal Investigators and 20
Divisions and Departments within the University. Six research companies and hospitals also used the
facilities of the CRL.

CRL Significance: The facilities operated by the Cancer Research Laboratory have been an
incalculable and indispensable part of the UC Berkeley research community. It is safe to say that
without these facilities it would be much harder, if not impossible, to recruit the best and brightest
researchers in biomedicine and cancer biology to Berkeley. The CRL has also produced a number of
antibodies that have been licensed to several commercial laboratories and distributed to the research
community. The CRL’s commitment to service and the ability to evolve and remain at the forefront of research technology make it the vital ORU it is today.

DIRECTOR'S PROGRESS REPORT

The Cancer Research Laboratory spent the past year providing expert technical support and state-of-the-art equipment from its several recharge units.

1. The Flow Cytometry Facility underwent a major upgrade with the acquisition of a Dakocytomation
MoFlo high-speed sorter in 2003. This is a new, state of the art instrument that has many features that were unavailable on our old sorter. These include triple laser-based ultra high-speed analysis and sorting with a triple laser beam excitation optical path and eight photo multiplier tubes. This new instrument was funded in large part by a shared instrumentation grant from the N.I.H. (P.I. Jim Allison, $500,000). In order to accommodate the new instrument the Flow Cytometry Facility moved into remodeled space on the 4th floor of LSA that became available upon the closure of the CRL Microchemical Facility in 2002. This instrument has been a real success, not only greatly increasing the capacity of the facility for sorting in general, but also for the first time allowing the high speed sorting required for isolation of rare cell populations, while also providing for multi-parameter analysis not previously available on campus. During the calendar year of 2003 the old Coulter ELITE sorter was used 699 hours and the new Dakocytomation

MoFlo highspeed sorter was used 338
hours. The combined sorter use time for 2003 totaled
1,037 hours. The three Coulter XL analyzers were
used by 52 Principal investigators from 14 departments
on the UCB campus and LBNL. In addition, the flow
cytometry facility was used by six biotechnology
companies.

2. The Cancer/Immunology DNA Microarray Consortium
(http://biology.berkeley.edu/crl/cancerimmunologymicroarray/index.html) has the following missions: to
generate and maintain supplies of mouse cancer/immune system custom microarray slides, to perform DNA microarray experiments for graduate students and postdoctoral fellows in molecular biology labs and to gain access to other arrays through collaboration with other microarray facilities. This effort started three years ago and culiminated last year with publication of two papers in international peer reviewed journals (see Publications at the end of this report). Commercial microarrays such as those made by Affymetrix are either not affordable by most research laboratories (especially for projects that require a large number of arrays) or containing too many ESTs that are not useful for our particular research purpose. A collection of clones that focus on cancer and immunology would go a long way toward global assessement of gene expression in immune- and cancer-related samples. Selected clones that represent genes involved in the signal transduction pathways, apoptosis, transcription factors, immune system and cancer were purchased from RZPD – a German research institute. In addition, we exchanged our clones with the UCSF consortium to obtain a final of 2700 clones. These clones were sequenced individually and printed onto glass slides using the microarray robots at the College of Natural Resources (UC Berkeley).

Commercial microarrays such as those made by Affymetrix are either not affordable by most research
laboratories (especially for projects that require a large number of arrays) or containing too many ESTs that are not useful for our particular research purpose. A collection of clones that focus on cancer and immunology would go a long way toward global assessement of gene expression in immune- and cancer-related samples. Selected clones that represent genes involved in the signal transduction pathways, apoptosis, transcription factors, immune system and cancer were purchased from RZPD – a German research institute. In addition, we exchanged our clones with the UCSF consortium to obtain a final of 2700 clones. These clones were sequenced individually and printed onto glass slides using the microarray robots at the College of Natural Resources (UC Berkeley).

3. The Gene Targeting Facility completed 32 projects supplying 1 chimeric and 20 transgenic mouse
strains with 11 ES cell electroporations. Researchers assisted were from MCB Immunology, MCB Cell &
Developmental Biology, MCB Genetics and the Department of Nutritional Science.

4. The Molecular Imaging Center has been open over two years now in 251 LSA and usage has
continued to expand. Users are now obtaining high quality confocal, 2-photon, and spectral images on the
upright Zeiss 510 META Confocal/NLO (non-linear optics) system. The META system was installed in late 2003, which provides the spectral imaging capabilities. Users are doing 3-dimensional, time-lapse, FRAP, and FRET studies. The inverted Zeiss 510 NLO system is now equipped to do either 1-photon confocal lifetime imaging or 2-photon fluorescent lifetime imaging, as well as single-channel confocal imaging and singlechannel multi-photon imaging. Due to the large increase in user base (over 80 users from over 40 different labs, including 7 different campus departments, LBNL, and 1 industry), we added an off-line computer workstation in October 2003 for analyzing data post acquisition. This workstation allows users to view their data in the native Zeiss format, as well as backup data, and analyze data. Recently, a license for Bitplane’s Imaris Imaging Suite software was added to allow users to view and quantitate their 3D and 4D datasets. The Imaris software includes packages for colocalization, filament tracing, and measurement in x, y, z, and time. We are currently also evaluating deconvolution software, but are unsure if the need is great enough for the cost of the packages. The Center is in the process of evaluating another major imaging system, which would provide relief for the current overworked systems. The systems we have been evaluating include real-time spinning disk confocal systems, fast laser-scanning confocal systems and fast-scanning multi-photon systems. All of these systems are capable of acquiring 3-D data at video rates or faster, which will provide a unique compliment to the current slow-scanning systems. These systems are still under evaluation and we hope to make a decision on which one to purchase at the beginning of next year.

In January 2004, the Molecular Imaging Center hosted the first annual Fluorescent Microscopy Advanced
Techniques Workshop. The workshop was a 2-day event, comprised of lectures and demonstrations. It was well attended by nearly 50 participants. We had corporate sponsorship from 3 industry partners. We had 15 speakers, including internationally renowned scientists from UC Berkeley and abroad. In January 2005, we will host the 2nd Annual Fluorescent Microscopy Workshop. This will also be a 2-day event and feature speakers who are on the leading edge of imaging techniques. We have 20 speakers lined up and commitments from 4 industry partners for sponsorship. This year we expect to have 80 participants.

5. The CRL administrative office continued to provide administrative assistance for both the CRL and the
FGL. Even though the CRL and FGL took 10% permanent budget cuts for 2003-2004 most services and
programs were successfully continued. The CRL continued to provide a half-time photographer. A digital
camera was purchased and he began producing web page designs for researchers as part of his service.
Director Jim Allison resigned the directorship of the CRL June 30, 2004 necessitating budget help from the Vice Chancellor for Research for 2004-2005. Professor Astar Winoto was appointed Director for the next five years. The affiliated faculty of the CRL continued its contribution to life at Berkeley and maintained its impact in the world. Former Director James P. Allison was appointed Chairman of the Immunology Program at Memorial Sloan Kettering Cancer Center, New York City. Professor Astar Winoto was appointed Director of the CRL and immediately expanded the CRL by adding a Mass Spectrometry Facility. The professors affiliated with the Cancer Research Laboratory had many roles in the Berkeley, National and International communities. Steven Martin continued as the Head of the Division of Cell and Developmental Biology, Astar Winoto became the Immunology Division Head in 2003-2004. and continued to serve in that capacity after being named director of CRL. Michael Botchan was the Head of the Division of Biochemistry and Molecular Biology. Gary Firestone was the co-chair of the Graduate Group in Endocrinology and served on the Campus Committee of Classroom Policy Management. He continued at Principal Investigator of the Cancer Research Lab’s NIH Training Grant directing the production of a competing renewal application to NIH. Satyabrata Nandi continued to serve as the Chair of the International Association for Breast Cancer Research but announced his retirement. David Raulet was elected Fellow of the American Association for the Advancement of Science in 2003.

IDS282 “Tumor Biology Seminar” is offered each fall and is attended by students at all levels. The two cancerrelated training grants (CRL and MCB) continued to sponsor the class and, in addition to presentations by affiliated grad students, postdocs and professors, a concerted effort to present translational/clinical topics was continued. Dr. Meri Firpo, UCSF, spoke on, “Therapeutic Applications of Human Embryonic Stem Cells.” Recently appointed Assistant Professor Laurent Coscoy presented, “Immune evasion by Kaposi’s Sarcoma associated Herpes virus.” Peter Vogt, internationally known researcher on oncovirology presented the CRL Distinguished Lecture titled, “Transcription and Translation in Oncogenesis.” The CRL Training Grant funded 4 graduate students and 7 postdoctoral fellows. . Major awards were received by the CRL faculty. Jeremy Thorner received the 2004 Award for distinguished Research Mentoring of Undergraduates in the Biological Sciences, College of Letters and Science at Berkeley. In July 2003 Thorner was appointed Editor of Molecular and Cellular Biology. Gary Firestone received the Rhoda H. Goldman Award for Distinguished Faculty Advising of Undergraduates in 2004.

There are 12 Principal Investigators affiliated with the Cancer Research Laboratory. Their research projects address cancer in its many forms and aspects. Jim Allison’s lab (MCB-IMM) is engaged in the understanding of T cells in immunology and in particular tumor immunotherapy. Ideas from Allison’s lab are now in phase I and II clinical trials for the treatment of prostate and ovarian cancer and malignant melanoma. David Raulet’s (MCB-IMM) newest research demonstrated that the NKG1D-ligand interaction plays an important role in lysis of many susceptible tumor targets cells, and can therefore be considered a major NK receptor for tumor cells. Steve Martin’s Lab (MCB-CDB has observed that pharmacological inhibition of endogenous Src causes the phenotypic reversion of human mammary epithelial cells. Satyabrata Nandi’s (MCB-CDB) laboratory has continued to focus on the induction of refractoriness to breast cancer by short-term hormonal treatment using an experimental rat model. The lab has discovered that short-term chemoprevention treatment is as effective as full term pregnancy, ovariectomy or long-term tamoxifen treatment and there is no permanent loss of ovarian function. This treatment can be used as a paradigm for developing strategies for human breast cancer prevention. Gary Firestone’s (MCB-CDB) research is focused on the characterization of cell signaling pathways that inhibit the growth and regulate differentiated properties of reproductive epithelial tumor cells, especially from human breast and prostate cancers. Intracellular components or extracellular signals of the studied regulated pathways are being developed as unique therapeutic agents that may potentially be used to control reproductive cancers. Astar Winoto’s (MCB-IMM) lab continues to focus on apoptosis or programmed cell death, widely believed to play a key role in regulation of the immune system and other biological processes. They also studied the role of survivin, the universal tumor antigen, in proliferation and apoptosis. Jeremy Thorner’s (MCB-BMB) research focus continues to be on the mechanisms of transmembrane and intracellular signal transduction especially how extracellular stimuli control gene expression, cell growth, cell morphology, and cell division at the biochemical level. Bill Sha’s (MCB-IMM) lab is developing novel retroviral strategies for functional indentification of genes that regulate lymphocyte function. Ellen Robey’s (MCB-IMM) lab is studying thymic development and trying to learn how T cells adopt their appropriate developmental fates. Bruce Ames’ (MCB-BMB) primary research interest involves various aspects of tuning-up metabolism to optimize health. His research shows that an optimum intake of micronutrients and metabolites, which varies with age and genetics, should tune up metabolism and markedly increase health at little cost, particularly for the poor and elderly. Mike Botchan’s (MCB-BMB) lab is doing research to understand how papilloma viruses replicate their DNA and regulate gene expression in order to develop drugs that effectively block viral persistence in oncogenically transformed cells. The specific viruses under study continue to be the high-risk human virus HPV-18 and the Bovine virus BPV-1. Kathy Collins’ (MCB-BMB) lab has shown that high-risk human virus HPV-18 and the Bovine virus BPV-1. Kathy Collins’ (MCB-BMB) lab has shown that human disease can result from insufficient telomerase activity. Cancer cells escape proliferative limitation by activating telomerase. The lab is intensely studying the biochemical mechanisms that underlie specialized features of the telomerase enzyme.

In summary, the Cancer Research Laboratory continues to serve as the focal point for cancer research on the Berkeley campus and to support the research needs of the biological scientific community through the latest equipment, quality technical and administrative staff and support of students working on cancer research projects.

CRL PUBLICATIONS 2003-2004

Principal Investigators and their labs:

Lee, S. R., Wong, J. M. and Collins, K. Human telomerase reverse transcriptase motifs
required for elongation of a telomeric substrate. Journal of Biological Chemistry 278: 52531-
52536 (2003).

Strahl, T., B. Grafelmann, J. Dannenberg, J. Thorner and O. Pongs (2003) Conservation of
regulatory function in calcium-binding proteins: human frequenin (neuronal calcium sensor-1)
associates productively with yeast phosphatidylinositol 4-kinase isoform, Pik1. J. Biol.
Chem.278(49):49589-49599.

Firestone, G.L. (2003) Sgk Protein (Serum- and glucocorticoid-inducible protein kinase, In:
Encyclopedia of Hormones (Eds. Henry, H.L. and Norman, A.W.), Elsevier Science, USA., pp.
362-371.

Zhang, J., Hsu, J.C., Kinseth, M.A., Bjeldandes, L.F., and Firestone, G.L. (2003) Indole-3-
carbinol (I3C) induces a G1 cell cycle arrest and inhibits prostate specific antigen production in
human LNCaP prostate cancer cells. Cancer 98: 2511-2520.

Chesnokov, I.N., Chesnokov, O.N. and Botchan, M. 2003. A cytokinetic function of Drosophila
ORC6 protein resides in a domain distinct from its replication activity. Proc. Natl. Acad. Sci.
USA. 100: 9150-9155.

Raulet, D. H. Roles of the NKG2D immunoreceptor and its ligands. Nature Rev. Immunol.
3:781-790 (2003).

Raulet, D. H. Natural Killer Cells. In the textbook Fundamental Immunology, Fifth Edition. W.
E. Paul, Ed. Lippincott Williams & Wilkens. Baltimore (2003).

Martin, G.S. (2003). Activation of oncogenic protein kinases. In: Handbook of Cellular
Signaling (R.Bradshaw and E. Dennis, eds.), Academic Press, pp. 441-449.

Webb, B.L., Díaz, B., Martin, G.S. and Lai, F. (2003). A reporter system for reverse
transfection cell arrays. J. Biomolec. Screening 8: 620-623.

Martin, G.S. (2003). Cell signaling and cancer. Cancer Cell 4: 167-174.
Rajkumar, L., R.C. Guzman, J. Yang, G. Thordarson, F. Talamantes and S. Nandi. Prevention
of mammary carcinogenesis by short-term estrogen and progestin treatments. Breast Cancer
Research 6:R31-R37, 2003.

DeYoung, R.A., Baker, J.C., Cado, D. and Winoto, A. (2003) The orphan steroid receptor
Nur77 family member Nor-1 is essential for early mouse embryogenesis. J. Biol. Chem. 278,
47104-47109.

Rajpal, A., Cho, Y.A., Yelent, B., Koza-Taylor, P.H., Li, D., Chen, E., Whang, M., Kang, C.,
Turi, T.G. and Winoto, A. (2003) Transcriptional activation of several known and novel
apoptotic pathways by Nur77 orphan steroid receptor. EMBO J. 22, 6526-6536.

Witkin, K. L. and Collins, K. Holoenzyme proteins required for the physiological assembly
and activity of telomerase. Genes & Development 18:1107-1118 (2004).

Truckses, D.M., J.E. Bloomekatz and J. Thorner (2004) Yeast Ste50 delivers Ste11 MAPKKK
to the plasma membrane via guanine nucleotide-independent interaction with Cdc42.
Submitted for publication.

Versele, M., B. Gullbrand, M.J. Shulewitz, V.J. Cid, S. Bahmanyar, R.E. Chen, P. Barth, T.
Alber and J. Thorner (2004) Protein-protein interactions governing septin heteropentamer
assembly and septin filament organization in Saccharomyces cerevisiae. Mol. Biol. Cell, in
press.

Roelants, F.M., P.D. Torrance and J. Thorner (2004) Differential roles of PDK1- and PDK2-
phosphorylation sites in the yeast AGC kinases, Ypk1, Pkc1 and Sch9. Microbiology, in press.

Thorner, J. (2004) "Signal Transduction", In Landmark Papers in Yeast Biology (Linder, P., D.
Shore, and M. Hall, eds.) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, in
press.

Konopka JB, Thorner JW (2004) Yeast pheromone receptors, In Encyclopedia of Biological
Chemistry (eds., Lennarz WJ, Lane MD), Elsevier Science, Inc., San Diego, CA, in press.

Sakchaisri, K., S. Asano, L.-R. Yu, M.J. Shulewitz, C.J.Park, J.-E. Park, Y.-W. Cho, T.D.
Veenstra, J. Thorner and K.S. Lee (2004) Coupling morphogenesis to mitotic entry. Proc. Natl.
Acad. Sci. USA 101: 4124-4129.

Versele, M. and J. Thorner (2004) Septin collar formation in budding yeast requires GTP
binding and direct phosphorylation by the PAK, Cla4. J. Cell Biol. 164: 701-715.

Rockwell N.C. and J. Thorner (2004) The kindest cuts of all: crystal structures of Kex2 and
furin reveal secrets of precursor processing. Trends Biochem. Sci. 29: 80-87.

Rudge, S.A, V.A. Sciorra, M. Iwamoto, C. Zhou, T. Strahl, A.J. Morris, J. Thorner and J.
Engebrecht (2004) Roles of phosphoinositides and of Spo14p (phospholipase D)-generated
phosphatidic acid during yeast sporulation. Mol. Biol. Cell 15: 207-218.

Guan, Y., Rubenstein, N.M., Failor, K.L., Woo, P.L., and Firestone, G. L. (2004)
Glucocorticoids control β-catenin protein expression and localization through distinct pathways
that can be uncoupled by disruption of signaling events required for tight junction formation in
rat mammary epithelial tumor cells. Molecular Endocrinol., 18: 214-227.

Paruthiyil, S., Parmar, H. Kerekatte, V., Cunha, G.R., Firestone, G.L., and Leitman, D.C. (2004)
Estrogen receptor-beta inhibits human breast cancer cell proliferation and tumor formation by
causing a G2 cell cycle arrest. Cancer Research, 64: 423-428.

Leong H, Riby JE, Firestone GL, Bjeldanes LF. (2004) Potent ligand-independent estrogen
receptor activation by 3,3'-diindolylmethane is mediated by cross-talk between the PKA and
MAPK signaling pathways. Molecular Endocrinol. 18, 291-302.

Chatterji, U., Riby, J.E., Taniguchi, T., Bjeldanes, E. L., Bjeldanes, L.F., and Firestone, G.L.
(2004) Indole-3-carbinol stimulates transcription of the interferon-gamma receptor 1 gene and
augments interferon responsiveness in human breast cancer cells. Carcinogenesis, 25, 1119-
1128.

Bousso, P and Robey, E (2004) Visualizing Thymocyte Motility Using 2-Photon Microscopy.
Immunity, in press

Witt, C and Robey, E (2004) The Ins and Outs of CCR7 in the Thymus J. of Exp. Med, in
press.

Remus, D., Beall, E.L. and Botchan, M.R. 2004. DNA topology, not DNA sequence, is a critical
determinant for Drosophila ORC-DNA binding. EMBO J. 23: 897-907.

Botchan, M. and Levine, M. 2004. A genome analysis of endoreplication in the Drosophila ovary.
Dev. Cell 6: 4-5.

Botchan, M. 2004. Hitchhiking without covalent integration. Cell 117: 1-20.

Beall, E.L., Bell, M., Georlette, D. and Botchan, M.R. 2004. Dm-myb mutant lethality in
Drosophila is dependent upon mip130: positive and negative regulation of DNA replication.
Genes & Dev. 18: 1667-1680.

Abbate, E.A., Berger, J. and Botchan, M.R. 2004. The X-ray structure of the
papillomavirus helicase in complex with its molecular matchmaker E2. Genes & Dev.
18: 1981-1996.

Jamieson, A. M., Isnard, P., Dorfman, J. R., Coles, M. C. and Raulet, D. H. Turnover and
proliferation of natural killer cells in steady state and lymphopenic conditions. J. Immunol.
172:864-70 (2004).

Tanamachi, D., Moniot, D. C., Cado, D, Liu, S. D., Hsia, J. K. and Raulet, D. H. Genomic
Ly49A transgenes: Basis of variegated Ly49A gene expression and identification of a critical
regulatory element. J. Immunol. 172:1074-82 (2004).

Xiong, N., Baker, J. E., Kang, C. and Raulet, D. H. The genomic arrangement of T cell receptor
variable genes is a determinant of the developmental rearrangement pattern. Proc. Natl. Acad.
Sci. USA. 101:260-5 (2004).

Carlyle, J. R., Jamieson, A. M., Gasser, S., Clingan, C. S., Arase, H. and Raulet, D. H. Missing
self recognition of Ocil/Clr-b by Inhibitory NKR-P1 Natural Killer Cell Receptors. Proc. Natl.
Acad. Sci. USA 101:3527-32 (2004).

Xiong, N., Kang, C. and Raulet, D. H. Positive selection of dendritic epidermal gd T cell
precursors in the fetal thymus determines expression of skin-homing receptors. Immunity
21:121-31 (2004).

Berdeaux, R., Diaz, B., Kim, L.C., Tu, A., Martin, G.S. (2004). Active Rho is localized to
podosomes induced by oncogenic Src and is required for their assembly and function. J. Cell
Biol. 166: 317-323.

Martin, G.S. (2004). The road to Src. Oncogene (in press).

Prathapam, T., Tegen, S., Oskarsson, T., Trumpp, A. and Martin, G.S. The Src-Myc pathway
regulates Cyclin E- and Cyclin A-dependent kinases. Submitted.

Ge. Y., L. Rajkumar, R.C. Guzman, S. Nandi, W.F. Patton and B.J. Agnew. Multiplexed
fluorescence detection of phosphorylation, glycosylation, and total protein in the proteomic
analysis of breast cancer refractoriness. Proteonomics 4:0000-0000, 2004.

Thordarson, G., N. Slusher, H. Leong, D. Ochoa, L. Rajkumar, R.Guzman, S. Nandi and F.
Talamantes. Insulin-like growth factor (IGF)-I obliterates the pregnancy-associated protection
against mammary carcinogenesis in rats: evidence that IGF-I enhances cancer progression
through estrogen receptor-a activation via the mitogen-activated protein kinase pathway. Breast
Cancer Research 6:R423-436, 2004.

Xing, Z., Conway, E.M., Kang, C. and Winoto, A. (2004) Essential role of survivin, an inhibitor
of apoptosis (IAP) in T cell development, maturation and homeostasis. J. Exp. Med. 199, 69-80.

Huang, Y.H., Li, D., Winoto, A., and Robey, E. A. (2004) Distinct transcriptional programs in
thymocytes responding to T cell receptor, Notch and positive selection signals. Proc. Natl. Acad.
Sci. USA, 101, 4936-4941.

Loke, P. and J.P. Allison. Differential regulation of PD-L1 and PD-L2 on macrophages by
classical vs alternative activation and Th1 vs Th2 cells. Proc. Natl. Acad. Sci., USA 100:5336-
5341, 2003.

Hodi, F.S., M.C. Mihn, R.J. Soiffer, F.G. Haluska, M. Butler, M. Seiden, T. Davis, R. Henry-
Spires, S. MacRae, A. Willman, R. Padera, M.T. Jaklitsch, S. Shankar, T.C. Chen, A. Korman,
J.P. Allison and G. Dranoff. Biologic activity of CTLA-4 antibody blockade in previously
vaccinated metastatic melanoma and ovarian carcinoma patients. Proc. Natl. Acad. Sci., USA
100:4712-4717, 2003.

Taylor, P.A., C.I. Lees, S. Fournier, J.P. Allison, A.H. Sharpe and B.R. Blazar. Brief definitive
report: B7 expression on T cells downregulates immune responses through CTLA-4 ligation via
R-T interactions. J Immunol 172:34-39, 2004

Gregor, P.D., J.D. Wolchok, C.R. Ferrone, H. Buchinshky, J.A. Guevara-Patiño, M.A. Perales,
F. Mortazavi, D. Bacich, W. Heston, J.B. LaTouche, M. Sadelain, J.P. Allison, H.I. Scher and
A.N. Houghton. CTLA-4 blockade in combination with xenogeneic DNA vaccines enhances
T-cell responses, tumor immunity and autoimmunity to self antigens in animal and cellular
models systems. J Vaccine 22:1700-1708, 2004

Stohl, W., D. Xu, K.S. Kim, C.S. David and J.P. Allison. MHC class II-independent and
–dependent T cell expansion and B cell hyperactivity in vivo in mice deficient in CD152
(CTLA-4). Int. Immunol. 16:895-904, 2004.

Rajkumar, L., Hirschberg D.L., Hartnett M.D., Loh K.C., Guzman, R.C., Thordson G and
Nandi S. Microarray analysis of Estrogen Induced Protection against mammary cancer. In
Hormonal Carcinogenesis, vol 4 (J.J. Li et al., eds), Springer-Verlag (in press 2004).

Guzman, R.C., Rajkumar, L., Thordarson, G. and Nandi, S. Pregnancy levels of estrogen
prevents mammary cancers. In Hormonal Carcinogenesis, vol 4 (J.J. Li et al., eds.),
Springer-Verlag (In Press; 2004).

Nandi, S., Guzman R.C., Rajkumar, L. and Thordarson, G. Estrogen can prevent breast
cancer by mimicking the protective effect of pregnancy. In Hormonal Carcinogenesis, vol
4 (J.J. Li et al., eds.), Springer-Verlag (In Press; 2004).

Thordarson, G., Semaan, S., Low, C., Ochoa, D., Leong, H., Rajkumar, L., Guzman,
R.C., Nandi, S. and Talamantes, F. Mammary tumorigenesis in growth hormone deficient
spontaneous dwarf rats; effects of hormonal treatments.
Breast Cancer Research and Treatment (In Press; 2004)

Gene Targeting Facility: CRL Specialists Dragana Cado and Chulho Kang

Hsu,LY; Lauring,J; Liang,HE; Greenbaum,S; Cado,D; Zhung,Y; Schlissel, MS. DH. A
conserved transcriptional enhancer regulates RAG gene expression in developing B cells
Immunity 19(1):105-17 (2003).

DeYoung,RA; Baker,JC; Cado, D; Winoto, A. DH. The orphan steroid receptor Nur77 family
member Nor-1 is essential for early mouse embryogenesis. J Biol Chem 278(47):47104-9
(2003).

Hua, Z.C., Sohn, J.A., Kang, C.,Cado, D. and Winoto, A. (2003) A Function of Fas-Associated
Death Domain Proteion in Cell Cycle Progression Localized to a Single Amino Acid at Its CTerminal
Region. Immunity, 18, 513-521.

Lee, K., Villena, J.A., Moon, Y.S., Kim, K.H., Lee, S., Kang, C. and Sul, H.S. (2003) Inhibition
of adipogenesis and development of glucose intolerance by soluble preadipocyte factor-1 (Pref-
1). J. Clin. Invest., 111, 453-461.

Schwab, S.R., Li, K.C., Kang, C. and Shastri, N. (2003) Constitutive display of cryptic
translation products by MHC class I molecules. Science, 301, 1367-1371.

Hsu, L.Y., Liang, H.E., Johnson, K., Kang, C. and Schlissel, M.S. (2004) Pax5 Activates
Immunoglobulin Heavy Chain V to DJ Rearrangement in Transgenic Thymocytes. J. Exp. Med.,
199, 825-830.

Kim, K.H., Zhao, L., Moon, Y., Kang, C. and Sul, H.S. (2004) Dominant inhibitory adipocytespecific
secretory factor (ADSF)/resistin enhances adipogenesis and improves insulin sensitivity.
Proc. Natl. Acad. Sci. USA, 101, 6780-6785.

Xing, Z., Conway, E.M., Kang, C. and Winoto, A. (2004) Essential Role of Survivin, an
Inhibitor of Apoptosis Protein, in T Cell Development, Maturation, and Homeostasis. J. Exp.
Med., 199, 69-80.

Xiong, N., Baker, J.E., Kang, C. and Raulet, D.H. (2004) The genomic arrangement of T cell
receptor variable genes is a determinant of the developmental rearrangement pattern. Proc. Natl.
Acad. Sci. USA, 101, 260-265.

Tanamachi, DM; Moniot, DC; Cado, D; Liu, SD; Hsia, JK; Raulet, DH. Genomic Ly49A
transgenes: basis of variegated Ly49A gene expression and identification of a critical regulatory
element. J. Immunol 172(2):1074-82 (2004).

Itoh,T; Cado, D; Kamide, R; Linn, S. DDB2 gene disruption leads to skin tumors and resistance
to apoptosis after exposure to ultraviolet light but not a chemical carcinogen. Proc Natl Acad
Sci USA 101(7): 2052-7 (2004).

Liang, HE; Hsu, LY: Cado, D; Schlissel, MS. DH. variegated transcriptional activation of the
immunoglobulin kappa locus in pre-b cells contributes to the allelic exclusion of light-chain
expression. Cell 118(1):19-29 (2004).

CRL Immunology DNA Microarray Consortium: SRAII Dongling Li

Rajpal, A., Cho, Y.A., Yelent, B., Koza-Taylor, P.H., Li, D., Chen, E., Whang, M., Kang, C.,
Turi, T.G. and Winoto, A. (2003) Transcriptional activation of known and novel apoptotic
pathways by Nur77 orphan steroid receptor. EMBO J., 22, 6526-6536.

Huang, Yina H., Dongling Li, Astar Winoto, and Ellen A. Robey Distinct transcriptional
programs in thymocytes responding to T cell receptor, Notch, and positive selection signals.
Proc. Natl. Acad. Sci. USA, May 2004; Vol. 101, No. 14, 4936-4941 (2004).

CANCER RESEARCH LABORATORY	Director 2003-2004 James P. Allison Incoming Director 2004 Astar Winoto Address 447 Life Sciences Addition University of California, Berkeley Berkeley, CA 94720-2751 Telephone (510)642-4714 Fax (510)642-5741 Email crl@uclink.berkeley.edu Web site http:biology/Berkeley.edu/crl/ Established 1951



Mission and Overview The Cancer Research Laboratory began in 1951 when the California state legislature provided funds to establish a colony of mice of reliable genetic background to be made available to cancer researchers throughout the state and elsewhere. Since that time as research support needs changed the CRL acquired other technical support staff and equipment so that the CRL now operates a Flow Cytometry Facility, Gene Targeting Facility and the Molecular Imaging Facility. The mission of the CRL has always been to further communication among members of the U.C. Berkeley cancer research community, to provide training in cancer research for graduate students and postdoctoral fellows, and to enhance the research environment at U.C. Berkeley by providing investigators with access to methods and technologies which are either too expensive or too specialized to be acquired by the laboratories of individual faculty. Information about the Cancer Research Laboratory including services available, recharge information, personnel and links to affiliated faculty research programs is available on the web page: http://biology.berkeley.edu/crl/

		Gene Targeting Facility Blastocyst injection
		Gene Targeting Facility Blastocyst injection

Communication: The CRL sponsors a seminar course, IDS 282 “Tumor Biology Seminar Series,” in which students, postdoctoral trainees, and faculty present and discuss their current research findings. In an effort to give student researchers an understanding of the goal of their research, at least one seminar highlights the clinical side of cancer. The Cancer Research Laboratory also sponsors the annual "CRL Distinguished Lectureship". These lectures feature prestigious researchers from other universities and the format allows interaction between the speaker and the trainees. Training: The second goal of the CRL is to further training of undergraduate students, graduate students and postdoctoral trainees. Training for graduate students and postdoctoral fellows is supported by stipends and research funds provided by a training grant entitled "Regulatory Biology of Cancer and Growth Control" from the National Cancer Institute now in its 30th year. This grant was competitively renewed in 2000 and provides support in the amount of $428,709 (direct annual costs) for stipends and partial research support to 4 predoctoral and 7 postdoctoral trainees. The Flow Cytometry Facility is involved in the undergraduate laboratory course (MCB 150L) required for MCB Immunology majors and taken as an elective by students from several departments. Students are trained in the use and applications of the flow cytometer analyzers. Graduate students and postdoctoral fellows using flow cytometry or cell sorting in their research receive individualized training in sample preparation, instrument operation and data analysis. The Molecular Imaging Center also offers training in new imaging techniques to graduate students, postdocs and senior researchers. Research Facilities: The CRL currently operates three research facilities: the Flow Cytometry Facility, the Molecular Imaging Center and the Gene Targeting Facility. The CRL also administers the Immunology Genomics Consortium that supports a technician devoted to gene chip manufacture for the immunology researchers. The CRL has remained a fluid unit acquiring new equipment and personnel to meet the latest research needs of the biological research community. The Molecular Imaging Center features 2 confocal microscopes, 2 calcium imaging microscopes, 1 deconvolution microscope. Two two-photon microscope systems, funded by NSF, were installed in the Spring of 2002. Technical personnel provide user training, acquire new microscopy systems and offer consultations on experimental design. In July 2002, the CRL was awarded $500,000 for the purchase of a high-speed cell sorter (MoFlo) for the Flow Cytometry Facility. In cooperation with the Department of Molecular & Cell Biology, a new facility was constructed to house the MoFlo, three Coulter XL analyzers and the Coulter Elite cell sorter. The Gene Targeting Facility has made many transgenic and chimeric mice for the use of researchers in MCB. CRL also sponsors a photographer, one of the few remaining on campus. Real-time imaging of CD8 T cells interacting with DC in intact lymph nodes
In 2003-2004, the Cancer Research Laboratory provided services to 88 Principal Investigators and 20 Divisions and Departments within the University. Six research companies and hospitals also used the facilities of the CRL. CRL Significance: The facilities operated by the Cancer Research Laboratory have been an incalculable and indispensable part of the UC Berkeley research community. It is safe to say that without these facilities it would be much harder, if not impossible, to recruit the best and brightest researchers in biomedicine and cancer biology to Berkeley. The CRL has also produced a number of antibodies that have been licensed to several commercial laboratories and distributed to the research community. The CRL’s commitment to service and the ability to evolve and remain at the forefront of research technology make it the vital ORU it is today. DIRECTOR'S PROGRESS REPORT The Cancer Research Laboratory spent the past year providing expert technical support and state-of-the-art equipment from its several recharge units. 1. The Flow Cytometry Facility underwent a major upgrade with the acquisition of a Dakocytomation MoFlo high-speed sorter in 2003. This is a new, state of the art instrument that has many features that were unavailable on our old sorter. These include triple laser-based ultra high-speed analysis and sorting with a triple laser beam excitation optical path and eight photo multiplier tubes. This new instrument was funded in large part by a shared instrumentation grant from the N.I.H. (P.I. Jim Allison, $500,000). In order to accommodate the new instrument the Flow Cytometry Facility moved into remodeled space on the 4th floor of LSA that became available upon the closure of the CRL Microchemical Facility in 2002. This instrument has been a real success, not only greatly increasing the capacity of the facility for sorting in general, but also for the first time allowing the high speed sorting required for isolation of rare cell populations, while also providing for multi-parameter analysis not previously available on campus. During the calendar year of 2003 the old Coulter ELITE sorter was used 699 hours and the new Dakocytomation MoFlo highspeed sorter was used 338 hours. The combined sorter use time for 2003 totaled 1,037 hours. The three Coulter XL analyzers were used by 52 Principal investigators from 14 departments on the UCB campus and LBNL. In addition, the flow cytometry facility was used by six biotechnology companies. GENE CHIP FROM CRL/IMM DNA MICROARRAY CONSORTIUM 2. The Cancer/Immunology DNA Microarray Consortium (http://biology.berkeley.edu/crl/cancerimmunologymicroarray/index.html) has the following missions: to generate and maintain supplies of mouse cancer/immune system custom microarray slides, to perform DNA microarray experiments for graduate students and postdoctoral fellows in molecular biology labs and to gain access to other arrays through collaboration with other microarray facilities. This effort started three years ago and culiminated last year with publication of two papers in international peer reviewed journals (see Publications at the end of this report). Commercial microarrays such as those made by Affymetrix are either not affordable by most research laboratories (especially for projects that require a large number of arrays) or containing too many ESTs that are not useful for our particular research purpose. A collection of clones that focus on cancer and immunology would go a long way toward global assessement of gene expression in immune- and cancer-related samples. Selected clones that represent genes involved in the signal transduction pathways, apoptosis, transcription factors, immune system and cancer were purchased from RZPD – a German research institute. In addition, we exchanged our clones with the UCSF consortium to obtain a final of 2700 clones. These clones were sequenced individually and printed onto glass slides using the microarray robots at the College of Natural Resources (UC Berkeley). Commercial microarrays such as those made by Affymetrix are either not affordable by most research laboratories (especially for projects that require a large number of arrays) or containing too many ESTs that are not useful for our particular research purpose. A collection of clones that focus on cancer and immunology would go a long way toward global assessement of gene expression in immune- and cancer-related samples. Selected clones that represent genes involved in the signal transduction pathways, apoptosis, transcription factors, immune system and cancer were purchased from RZPD – a German research institute. In addition, we exchanged our clones with the UCSF consortium to obtain a final of 2700 clones. These clones were sequenced individually and printed onto glass slides using the microarray robots at the College of Natural Resources (UC Berkeley). 3. The Gene Targeting Facility completed 32 projects supplying 1 chimeric and 20 transgenic mouse strains with 11 ES cell electroporations. Researchers assisted were from MCB Immunology, MCB Cell & Developmental Biology, MCB Genetics and the Department of Nutritional Science. 4. The Molecular Imaging Center has been open over two years now in 251 LSA and usage has continued to expand. Users are now obtaining high quality confocal, 2-photon, and spectral images on the upright Zeiss 510 META Confocal/NLO (non-linear optics) system. The META system was installed in late 2003, which provides the spectral imaging capabilities. Users are doing 3-dimensional, time-lapse, FRAP, and FRET studies. The inverted Zeiss 510 NLO system is now equipped to do either 1-photon confocal lifetime imaging or 2-photon fluorescent lifetime imaging, as well as single-channel confocal imaging and singlechannel multi-photon imaging. Due to the large increase in user base (over 80 users from over 40 different labs, including 7 different campus departments, LBNL, and 1 industry), we added an off-line computer workstation in October 2003 for analyzing data post acquisition. This workstation allows users to view their data in the native Zeiss format, as well as backup data, and analyze data. Recently, a license for Bitplane’s Imaris Imaging Suite software was added to allow users to view and quantitate their 3D and 4D datasets. The Imaris software includes packages for colocalization, filament tracing, and measurement in x, y, z, and time. We are currently also evaluating deconvolution software, but are unsure if the need is great enough for the cost of the packages. The Center is in the process of evaluating another major imaging system, which would provide relief for the current overworked systems. The systems we have been evaluating include real-time spinning disk confocal systems, fast laser-scanning confocal systems and fast-scanning multi-photon systems. All of these systems are capable of acquiring 3-D data at video rates or faster, which will provide a unique compliment to the current slow-scanning systems. These systems are still under evaluation and we hope to make a decision on which one to purchase at the beginning of next year. In January 2004, the Molecular Imaging Center hosted the first annual Fluorescent Microscopy Advanced Techniques Workshop. The workshop was a 2-day event, comprised of lectures and demonstrations. It was well attended by nearly 50 participants. We had corporate sponsorship from 3 industry partners. We had 15 speakers, including internationally renowned scientists from UC Berkeley and abroad. In January 2005, we will host the 2nd Annual Fluorescent Microscopy Workshop. This will also be a 2-day event and feature speakers who are on the leading edge of imaging techniques. We have 20 speakers lined up and commitments from 4 industry partners for sponsorship. This year we expect to have 80 participants. 5. The CRL administrative office continued to provide administrative assistance for both the CRL and the FGL. Even though the CRL and FGL took 10% permanent budget cuts for 2003-2004 most services and programs were successfully continued. The CRL continued to provide a half-time photographer. A digital camera was purchased and he began producing web page designs for researchers as part of his service. Director Jim Allison resigned the directorship of the CRL June 30, 2004 necessitating budget help from the Vice Chancellor for Research for 2004-2005. Professor Astar Winoto was appointed Director for the next five years. The affiliated faculty of the CRL continued its contribution to life at Berkeley and maintained its impact in the world. Former Director James P. Allison was appointed Chairman of the Immunology Program at Memorial Sloan Kettering Cancer Center, New York City. Professor Astar Winoto was appointed Director of the CRL and immediately expanded the CRL by adding a Mass Spectrometry Facility. The professors affiliated with the Cancer Research Laboratory had many roles in the Berkeley, National and International communities. Steven Martin continued as the Head of the Division of Cell and Developmental Biology, Astar Winoto became the Immunology Division Head in 2003-2004. and continued to serve in that capacity after being named director of CRL. Michael Botchan was the Head of the Division of Biochemistry and Molecular Biology. Gary Firestone was the co-chair of the Graduate Group in Endocrinology and served on the Campus Committee of Classroom Policy Management. He continued at Principal Investigator of the Cancer Research Lab’s NIH Training Grant directing the production of a competing renewal application to NIH. Satyabrata Nandi continued to serve as the Chair of the International Association for Breast Cancer Research but announced his retirement. David Raulet was elected Fellow of the American Association for the Advancement of Science in 2003. IDS282 “Tumor Biology Seminar” is offered each fall and is attended by students at all levels. The two cancerrelated training grants (CRL and MCB) continued to sponsor the class and, in addition to presentations by affiliated grad students, postdocs and professors, a concerted effort to present translational/clinical topics was continued. Dr. Meri Firpo, UCSF, spoke on, “Therapeutic Applications of Human Embryonic Stem Cells.” Recently appointed Assistant Professor Laurent Coscoy presented, “Immune evasion by Kaposi’s Sarcoma associated Herpes virus.” Peter Vogt, internationally known researcher on oncovirology presented the CRL Distinguished Lecture titled, “Transcription and Translation in Oncogenesis.” The CRL Training Grant funded 4 graduate students and 7 postdoctoral fellows. . Major awards were received by the CRL faculty. Jeremy Thorner received the 2004 Award for distinguished Research Mentoring of Undergraduates in the Biological Sciences, College of Letters and Science at Berkeley. In July 2003 Thorner was appointed Editor of Molecular and Cellular Biology. Gary Firestone received the Rhoda H. Goldman Award for Distinguished Faculty Advising of Undergraduates in 2004. There are 12 Principal Investigators affiliated with the Cancer Research Laboratory. Their research projects address cancer in its many forms and aspects. Jim Allison’s lab (MCB-IMM) is engaged in the understanding of T cells in immunology and in particular tumor immunotherapy. Ideas from Allison’s lab are now in phase I and II clinical trials for the treatment of prostate and ovarian cancer and malignant melanoma. David Raulet’s (MCB-IMM) newest research demonstrated that the NKG1D-ligand interaction plays an important role in lysis of many susceptible tumor targets cells, and can therefore be considered a major NK receptor for tumor cells. Steve Martin’s Lab (MCB-CDB has observed that pharmacological inhibition of endogenous Src causes the phenotypic reversion of human mammary epithelial cells. Satyabrata Nandi’s (MCB-CDB) laboratory has continued to focus on the induction of refractoriness to breast cancer by short-term hormonal treatment using an experimental rat model. The lab has discovered that short-term chemoprevention treatment is as effective as full term pregnancy, ovariectomy or long-term tamoxifen treatment and there is no permanent loss of ovarian function. This treatment can be used as a paradigm for developing strategies for human breast cancer prevention. Gary Firestone’s (MCB-CDB) research is focused on the characterization of cell signaling pathways that inhibit the growth and regulate differentiated properties of reproductive epithelial tumor cells, especially from human breast and prostate cancers. Intracellular components or extracellular signals of the studied regulated pathways are being developed as unique therapeutic agents that may potentially be used to control reproductive cancers. Astar Winoto’s (MCB-IMM) lab continues to focus on apoptosis or programmed cell death, widely believed to play a key role in regulation of the immune system and other biological processes. They also studied the role of survivin, the universal tumor antigen, in proliferation and apoptosis. Jeremy Thorner’s (MCB-BMB) research focus continues to be on the mechanisms of transmembrane and intracellular signal transduction especially how extracellular stimuli control gene expression, cell growth, cell morphology, and cell division at the biochemical level. Bill Sha’s (MCB-IMM) lab is developing novel retroviral strategies for functional indentification of genes that regulate lymphocyte function. Ellen Robey’s (MCB-IMM) lab is studying thymic development and trying to learn how T cells adopt their appropriate developmental fates. Bruce Ames’ (MCB-BMB) primary research interest involves various aspects of tuning-up metabolism to optimize health. His research shows that an optimum intake of micronutrients and metabolites, which varies with age and genetics, should tune up metabolism and markedly increase health at little cost, particularly for the poor and elderly. Mike Botchan’s (MCB-BMB) lab is doing research to understand how papilloma viruses replicate their DNA and regulate gene expression in order to develop drugs that effectively block viral persistence in oncogenically transformed cells. The specific viruses under study continue to be the high-risk human virus HPV-18 and the Bovine virus BPV-1. Kathy Collins’ (MCB-BMB) lab has shown that high-risk human virus HPV-18 and the Bovine virus BPV-1. Kathy Collins’ (MCB-BMB) lab has shown that human disease can result from insufficient telomerase activity. Cancer cells escape proliferative limitation by activating telomerase. The lab is intensely studying the biochemical mechanisms that underlie specialized features of the telomerase enzyme. In summary, the Cancer Research Laboratory continues to serve as the focal point for cancer research on the Berkeley campus and to support the research needs of the biological scientific community through the latest equipment, quality technical and administrative staff and support of students working on cancer research projects. CRL PUBLICATIONS 2003-2004 Principal Investigators and their labs: Lee, S. R., Wong, J. M. and Collins, K. Human telomerase reverse transcriptase motifs required for elongation of a telomeric substrate. Journal of Biological Chemistry 278: 52531- 52536 (2003). Strahl, T., B. Grafelmann, J. Dannenberg, J. Thorner and O. Pongs (2003) Conservation of regulatory function in calcium-binding proteins: human frequenin (neuronal calcium sensor-1) associates productively with yeast phosphatidylinositol 4-kinase isoform, Pik1. J. Biol. Chem.278(49):49589-49599. Firestone, G.L. (2003) Sgk Protein (Serum- and glucocorticoid-inducible protein kinase, In: Encyclopedia of Hormones (Eds. Henry, H.L. and Norman, A.W.), Elsevier Science, USA., pp. 362-371. Zhang, J., Hsu, J.C., Kinseth, M.A., Bjeldandes, L.F., and Firestone, G.L. (2003) Indole-3- carbinol (I3C) induces a G1 cell cycle arrest and inhibits prostate specific antigen production in human LNCaP prostate cancer cells. Cancer 98: 2511-2520. Chesnokov, I.N., Chesnokov, O.N. and Botchan, M. 2003. A cytokinetic function of Drosophila ORC6 protein resides in a domain distinct from its replication activity. Proc. Natl. Acad. Sci. USA. 100: 9150-9155. Raulet, D. H. Roles of the NKG2D immunoreceptor and its ligands. Nature Rev. Immunol. 3:781-790 (2003). Raulet, D. H. Natural Killer Cells. In the textbook Fundamental Immunology, Fifth Edition. W. E. Paul, Ed. Lippincott Williams & Wilkens. Baltimore (2003). Martin, G.S. (2003). Activation of oncogenic protein kinases. In: Handbook of Cellular Signaling (R.Bradshaw and E. Dennis, eds.), Academic Press, pp. 441-449. Webb, B.L., Díaz, B., Martin, G.S. and Lai, F. (2003). A reporter system for reverse transfection cell arrays. J. Biomolec. Screening 8: 620-623. Martin, G.S. (2003). Cell signaling and cancer. Cancer Cell 4: 167-174. Rajkumar, L., R.C. Guzman, J. Yang, G. Thordarson, F. Talamantes and S. Nandi. Prevention of mammary carcinogenesis by short-term estrogen and progestin treatments. Breast Cancer Research 6:R31-R37, 2003. DeYoung, R.A., Baker, J.C., Cado, D. and Winoto, A. (2003) The orphan steroid receptor Nur77 family member Nor-1 is essential for early mouse embryogenesis. J. Biol. Chem. 278, 47104-47109. Rajpal, A., Cho, Y.A., Yelent, B., Koza-Taylor, P.H., Li, D., Chen, E., Whang, M., Kang, C., Turi, T.G. and Winoto, A. (2003) Transcriptional activation of several known and novel apoptotic pathways by Nur77 orphan steroid receptor. EMBO J. 22, 6526-6536. Witkin, K. L. and Collins, K. Holoenzyme proteins required for the physiological assembly and activity of telomerase. Genes & Development 18:1107-1118 (2004). Truckses, D.M., J.E. Bloomekatz and J. Thorner (2004) Yeast Ste50 delivers Ste11 MAPKKK to the plasma membrane via guanine nucleotide-independent interaction with Cdc42. Submitted for publication. Versele, M., B. Gullbrand, M.J. Shulewitz, V.J. Cid, S. Bahmanyar, R.E. Chen, P. Barth, T. Alber and J. Thorner (2004) Protein-protein interactions governing septin heteropentamer assembly and septin filament organization in Saccharomyces cerevisiae. Mol. Biol. Cell, in press. Roelants, F.M., P.D. Torrance and J. Thorner (2004) Differential roles of PDK1- and PDK2- phosphorylation sites in the yeast AGC kinases, Ypk1, Pkc1 and Sch9. Microbiology, in press. Thorner, J. (2004) "Signal Transduction", In Landmark Papers in Yeast Biology (Linder, P., D. Shore, and M. Hall, eds.) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, in press. Konopka JB, Thorner JW (2004) Yeast pheromone receptors, In Encyclopedia of Biological Chemistry (eds., Lennarz WJ, Lane MD), Elsevier Science, Inc., San Diego, CA, in press. Sakchaisri, K., S. Asano, L.-R. Yu, M.J. Shulewitz, C.J.Park, J.-E. Park, Y.-W. Cho, T.D. Veenstra, J. Thorner and K.S. Lee (2004) Coupling morphogenesis to mitotic entry. Proc. Natl. Acad. Sci. USA 101: 4124-4129. Versele, M. and J. Thorner (2004) Septin collar formation in budding yeast requires GTP binding and direct phosphorylation by the PAK, Cla4. J. Cell Biol. 164: 701-715. Rockwell N.C. and J. Thorner (2004) The kindest cuts of all: crystal structures of Kex2 and furin reveal secrets of precursor processing. Trends Biochem. Sci. 29: 80-87. Rudge, S.A, V.A. Sciorra, M. Iwamoto, C. Zhou, T. Strahl, A.J. Morris, J. Thorner and J. Engebrecht (2004) Roles of phosphoinositides and of Spo14p (phospholipase D)-generated phosphatidic acid during yeast sporulation. Mol. Biol. Cell 15: 207-218. Guan, Y., Rubenstein, N.M., Failor, K.L., Woo, P.L., and Firestone, G. L. (2004) Glucocorticoids control β-catenin protein expression and localization through distinct pathways that can be uncoupled by disruption of signaling events required for tight junction formation in rat mammary epithelial tumor cells. Molecular Endocrinol., 18: 214-227. Paruthiyil, S., Parmar, H. Kerekatte, V., Cunha, G.R., Firestone, G.L., and Leitman, D.C. (2004) Estrogen receptor-beta inhibits human breast cancer cell proliferation and tumor formation by causing a G2 cell cycle arrest. Cancer Research, 64: 423-428. Leong H, Riby JE, Firestone GL, Bjeldanes LF. (2004) Potent ligand-independent estrogen receptor activation by 3,3'-diindolylmethane is mediated by cross-talk between the PKA and MAPK signaling pathways. Molecular Endocrinol. 18, 291-302. Chatterji, U., Riby, J.E., Taniguchi, T., Bjeldanes, E. L., Bjeldanes, L.F., and Firestone, G.L. (2004) Indole-3-carbinol stimulates transcription of the interferon-gamma receptor 1 gene and augments interferon responsiveness in human breast cancer cells. Carcinogenesis, 25, 1119- 1128. Bousso, P and Robey, E (2004) Visualizing Thymocyte Motility Using 2-Photon Microscopy. Immunity, in press Witt, C and Robey, E (2004) The Ins and Outs of CCR7 in the Thymus J. of Exp. Med, in press. Remus, D., Beall, E.L. and Botchan, M.R. 2004. DNA topology, not DNA sequence, is a critical determinant for Drosophila ORC-DNA binding. EMBO J. 23: 897-907. Botchan, M. and Levine, M. 2004. A genome analysis of endoreplication in the Drosophila ovary. Dev. Cell 6: 4-5. Botchan, M. 2004. Hitchhiking without covalent integration. Cell 117: 1-20. Beall, E.L., Bell, M., Georlette, D. and Botchan, M.R. 2004. Dm-myb mutant lethality in Drosophila is dependent upon mip130: positive and negative regulation of DNA replication. Genes & Dev. 18: 1667-1680. Abbate, E.A., Berger, J. and Botchan, M.R. 2004. The X-ray structure of the papillomavirus helicase in complex with its molecular matchmaker E2. Genes & Dev. 18: 1981-1996. Jamieson, A. M., Isnard, P., Dorfman, J. R., Coles, M. C. and Raulet, D. H. Turnover and proliferation of natural killer cells in steady state and lymphopenic conditions. J. Immunol. 172:864-70 (2004). Tanamachi, D., Moniot, D. C., Cado, D, Liu, S. D., Hsia, J. K. and Raulet, D. H. Genomic Ly49A transgenes: Basis of variegated Ly49A gene expression and identification of a critical regulatory element. J. Immunol. 172:1074-82 (2004). Xiong, N., Baker, J. E., Kang, C. and Raulet, D. H. The genomic arrangement of T cell receptor variable genes is a determinant of the developmental rearrangement pattern. Proc. Natl. Acad. Sci. USA. 101:260-5 (2004). Carlyle, J. R., Jamieson, A. M., Gasser, S., Clingan, C. S., Arase, H. and Raulet, D. H. Missing self recognition of Ocil/Clr-b by Inhibitory NKR-P1 Natural Killer Cell Receptors. Proc. Natl. Acad. Sci. USA 101:3527-32 (2004). Xiong, N., Kang, C. and Raulet, D. H. Positive selection of dendritic epidermal gd T cell precursors in the fetal thymus determines expression of skin-homing receptors. Immunity 21:121-31 (2004). Berdeaux, R., Diaz, B., Kim, L.C., Tu, A., Martin, G.S. (2004). Active Rho is localized to podosomes induced by oncogenic Src and is required for their assembly and function. J. Cell Biol. 166: 317-323. Martin, G.S. (2004). The road to Src. Oncogene (in press). Prathapam, T., Tegen, S., Oskarsson, T., Trumpp, A. and Martin, G.S. The Src-Myc pathway regulates Cyclin E- and Cyclin A-dependent kinases. Submitted. Ge. Y., L. Rajkumar, R.C. Guzman, S. Nandi, W.F. Patton and B.J. Agnew. Multiplexed fluorescence detection of phosphorylation, glycosylation, and total protein in the proteomic analysis of breast cancer refractoriness. Proteonomics 4:0000-0000, 2004. Thordarson, G., N. Slusher, H. Leong, D. Ochoa, L. Rajkumar, R.Guzman, S. Nandi and F. Talamantes. Insulin-like growth factor (IGF)-I obliterates the pregnancy-associated protection against mammary carcinogenesis in rats: evidence that IGF-I enhances cancer progression through estrogen receptor-a activation via the mitogen-activated protein kinase pathway. Breast Cancer Research 6:R423-436, 2004. Xing, Z., Conway, E.M., Kang, C. and Winoto, A. (2004) Essential role of survivin, an inhibitor of apoptosis (IAP) in T cell development, maturation and homeostasis. J. Exp. Med. 199, 69-80. Huang, Y.H., Li, D., Winoto, A., and Robey, E. A. (2004) Distinct transcriptional programs in thymocytes responding to T cell receptor, Notch and positive selection signals. Proc. Natl. Acad. Sci. USA, 101, 4936-4941. Loke, P. and J.P. Allison. Differential regulation of PD-L1 and PD-L2 on macrophages by classical vs alternative activation and Th1 vs Th2 cells. Proc. Natl. Acad. Sci., USA 100:5336- 5341, 2003. Hodi, F.S., M.C. Mihn, R.J. Soiffer, F.G. Haluska, M. Butler, M. Seiden, T. Davis, R. Henry- Spires, S. MacRae, A. Willman, R. Padera, M.T. Jaklitsch, S. Shankar, T.C. Chen, A. Korman, J.P. Allison and G. Dranoff. Biologic activity of CTLA-4 antibody blockade in previously vaccinated metastatic melanoma and ovarian carcinoma patients. Proc. Natl. Acad. Sci., USA 100:4712-4717, 2003. Taylor, P.A., C.I. Lees, S. Fournier, J.P. Allison, A.H. Sharpe and B.R. Blazar. Brief definitive report: B7 expression on T cells downregulates immune responses through CTLA-4 ligation via R-T interactions. J Immunol 172:34-39, 2004 Gregor, P.D., J.D. Wolchok, C.R. Ferrone, H. Buchinshky, J.A. Guevara-Patiño, M.A. Perales, F. Mortazavi, D. Bacich, W. Heston, J.B. LaTouche, M. Sadelain, J.P. Allison, H.I. Scher and A.N. Houghton. CTLA-4 blockade in combination with xenogeneic DNA vaccines enhances T-cell responses, tumor immunity and autoimmunity to self antigens in animal and cellular models systems. J Vaccine 22:1700-1708, 2004 Stohl, W., D. Xu, K.S. Kim, C.S. David and J.P. Allison. MHC class II-independent and –dependent T cell expansion and B cell hyperactivity in vivo in mice deficient in CD152 (CTLA-4). Int. Immunol. 16:895-904, 2004. Rajkumar, L., Hirschberg D.L., Hartnett M.D., Loh K.C., Guzman, R.C., Thordson G and Nandi S. Microarray analysis of Estrogen Induced Protection against mammary cancer. In Hormonal Carcinogenesis, vol 4 (J.J. Li et al., eds), Springer-Verlag (in press 2004). Guzman, R.C., Rajkumar, L., Thordarson, G. and Nandi, S. Pregnancy levels of estrogen prevents mammary cancers. In Hormonal Carcinogenesis, vol 4 (J.J. Li et al., eds.), Springer-Verlag (In Press; 2004). Nandi, S., Guzman R.C., Rajkumar, L. and Thordarson, G. Estrogen can prevent breast cancer by mimicking the protective effect of pregnancy. In Hormonal Carcinogenesis, vol 4 (J.J. Li et al., eds.), Springer-Verlag (In Press; 2004). Thordarson, G., Semaan, S., Low, C., Ochoa, D., Leong, H., Rajkumar, L., Guzman, R.C., Nandi, S. and Talamantes, F. Mammary tumorigenesis in growth hormone deficient spontaneous dwarf rats; effects of hormonal treatments. Breast Cancer Research and Treatment (In Press; 2004) Gene Targeting Facility: CRL Specialists Dragana Cado and Chulho Kang Hsu,LY; Lauring,J; Liang,HE; Greenbaum,S; Cado,D; Zhung,Y; Schlissel, MS. DH. A conserved transcriptional enhancer regulates RAG gene expression in developing B cells Immunity 19(1):105-17 (2003). DeYoung,RA; Baker,JC; Cado, D; Winoto, A. DH. The orphan steroid receptor Nur77 family member Nor-1 is essential for early mouse embryogenesis. J Biol Chem 278(47):47104-9 (2003). Hua, Z.C., Sohn, J.A., Kang, C.,Cado, D. and Winoto, A. (2003) A Function of Fas-Associated Death Domain Proteion in Cell Cycle Progression Localized to a Single Amino Acid at Its CTerminal Region. Immunity, 18, 513-521. Lee, K., Villena, J.A., Moon, Y.S., Kim, K.H., Lee, S., Kang, C. and Sul, H.S. (2003) Inhibition of adipogenesis and development of glucose intolerance by soluble preadipocyte factor-1 (Pref- 1). J. Clin. Invest., 111, 453-461. Schwab, S.R., Li, K.C., Kang, C. and Shastri, N. (2003) Constitutive display of cryptic translation products by MHC class I molecules. Science, 301, 1367-1371. Hsu, L.Y., Liang, H.E., Johnson, K., Kang, C. and Schlissel, M.S. (2004) Pax5 Activates Immunoglobulin Heavy Chain V to DJ Rearrangement in Transgenic Thymocytes. J. Exp. Med., 199, 825-830. Kim, K.H., Zhao, L., Moon, Y., Kang, C. and Sul, H.S. (2004) Dominant inhibitory adipocytespecific secretory factor (ADSF)/resistin enhances adipogenesis and improves insulin sensitivity. Proc. Natl. Acad. Sci. USA, 101, 6780-6785. Xing, Z., Conway, E.M., Kang, C. and Winoto, A. (2004) Essential Role of Survivin, an Inhibitor of Apoptosis Protein, in T Cell Development, Maturation, and Homeostasis. J. Exp. Med., 199, 69-80. Xiong, N., Baker, J.E., Kang, C. and Raulet, D.H. (2004) The genomic arrangement of T cell receptor variable genes is a determinant of the developmental rearrangement pattern. Proc. Natl. Acad. Sci. USA, 101, 260-265. Tanamachi, DM; Moniot, DC; Cado, D; Liu, SD; Hsia, JK; Raulet, DH. Genomic Ly49A transgenes: basis of variegated Ly49A gene expression and identification of a critical regulatory element. J. Immunol 172(2):1074-82 (2004). Itoh,T; Cado, D; Kamide, R; Linn, S. DDB2 gene disruption leads to skin tumors and resistance to apoptosis after exposure to ultraviolet light but not a chemical carcinogen. Proc Natl Acad Sci USA 101(7): 2052-7 (2004). Liang, HE; Hsu, LY: Cado, D; Schlissel, MS. DH. variegated transcriptional activation of the immunoglobulin kappa locus in pre-b cells contributes to the allelic exclusion of light-chain expression. Cell 118(1):19-29 (2004). CRL Immunology DNA Microarray Consortium: SRAII Dongling Li Rajpal, A., Cho, Y.A., Yelent, B., Koza-Taylor, P.H., Li, D., Chen, E., Whang, M., Kang, C., Turi, T.G. and Winoto, A. (2003) Transcriptional activation of known and novel apoptotic pathways by Nur77 orphan steroid receptor. EMBO J., 22, 6526-6536. Huang, Yina H., Dongling Li, Astar Winoto, and Ellen A. Robey Distinct transcriptional programs in thymocytes responding to T cell receptor, Notch, and positive selection signals. Proc. Natl. Acad. Sci. USA, May 2004; Vol. 101, No. 14, 4936-4941 (2004).