Faculty Publications

Large homozygous deletions of 9p21 that inactivate CDKN2A, ARF, and MTAP are common in a wide variety of human cancers. The role for CDKN2A and ARF in tumorigenesis is well established, but whether MTAP loss directly affects tumorigenesis is unclear. MTAP encodes the enzyme methylthioadenosine phosphorylase, a key enzyme in the methionine salvage pathway. To determine if loss of MTAP plays a functional role in tumorigenesis, we have created an MTAP-knockout mouse. Mice homozygous for a MTAP null allele (Mtap(lacZ)) have an embryonic lethal phenotype dying around day 8 postconception. Mtap/Mtap(lacz) heterozygotes are born at Mendelian frequencies and appear indistinguishable from wild-type mice during the first year of life, but they tend to die prematurely with a median survival of 585 days. Autopsies on these animals reveal that they have greatly enlarged spleens, altered thymic histology, and lymphocytic infiltration of their livers, consistent with lymphoma. Immunohistochemical staining and fluorescence-activated cell sorting analysis indicate that these lymphomas are primarily T-cell in origin. Lymphoma-infiltrated tissues tend to have reduced levels of Mtap mRNA and MTAP protein in addition to unaltered levels of methyldeoxycytidine. These studies show that Mtap is a tumor suppressor gene independent of CDKN2A and ARF. [Cancer Res 2009;69(14):5961-9]

In the past 3 years, altered expression of the HEF1/CAS-L/NEDD9 scaffolding protein has emerged as contributing to cancer metastasis in multiple cancer types. However, whereas some studies have identified elevated NEDD9 expression as prometastatic, other work has suggested a negative role in tumor progression. We here show that the Nedd9-null genetic background significantly limits mammary tumor initiation in the MMTV-polyoma virus middle T genetic model. Action of NEDD9 is tumor cell intrinsic, with immune cell infiltration, stroma, and angiogenesis unaffected. The majority of the late-appearing mammary tumors of MMTV-polyoma virus middle T;Nedd9(-/-) mice are characterized by depressed activation of proteins including ART, Src, FAK, and extracellular signal-regulated kinase, emphasizing an important role of NEDD9 as a scaffolding protein for these prooncogenic proteins. Analysis of cells derived from primary Nedd9(+/+). and Nedd9(-/-) tumors showed persistently reduced FAK activation, attachment, and migration, consistent with a role for NEDD9 activation of FAK in promoting tumor aggressiveness. This study provides the first in vivo evidence of a role for NEDD9 in breast cancer progression and suggests that NEDD9 expression may provide a biomarker for tumor aggressiveness. [Cancer Res 2009;69(18):7198-206]

The subdivision of bone marrow (BM) with surface markers and reporter systems and the use of multiple culture and transplantation assays to assess differentiation potential have led to extraordinary progress in defining stages of B lymphopoiesis between the hematopoietic stem cell and B cell receptor (BCR)-expressing lymphocytes. Despite the lack of standard nomenclature and a series of technical issues that still need to be resolved, there seems to be a general consensus regarding the major route to becoming a B cell. Nevertheless, evidence that additional, minor pathways through which B lineage cells are generated exists, and a new appreciation that lymphoid progenitors are protean and able to alter their differentiation potential during embryogenesis and after birth in response to infections suggests that a full understanding of B cell development and how it is regulated has not yet been attained.

One of the key end-points for understanding the molecular basis of the breast in its normal and cancer status is the quantitation of gene expression in specific cell populations. Microdissection techniques allow extraction of morphologically distinct cells for molecular analysis. The objective of this study was to determine the optimal RNA isolation and amplification to perform genomic expression analysis using the microarray technique from normal breast paraffin-embedded tissue samples using laser capture microdissection (LCM). We isolated epithelial and interlobular stroma cells from normal breast tissue and the total RNA was amplified using a PCR methodology developed by us, and in parallel the same starting material was used for amplification using the linear methodology. After two rounds of RNA amplification, we checked the quality of each amplified RNA and carried out the hybridization with cDNA glass-microarrays employing 15,000 genes for each replicate. In conclus!

B-1 (CD5+) B cells constitute a phenotypic and functionally distinct population of B cells in mouse that show enriched expression of autoreactive B-cell antigen receptors and that produce several types of natural autoantibodies. Recently, there has been much progress in this field of research. Evidence has appeared for the existence of distinctive B-cell precursors that preferentially generate B-1 B cells, and the crucial requirement for strong B-cell antigen receptor signaling in the maturation of B-1 B cells has been established. Other work focuses on a phenotypically similar population that lacks CD5, termed 'B-1b', which shows similarities and differences from most CD5+ B cells in both development and function. The relationship of normal B-1 cells with B-cell lymphomas and leukemias continues to be a subject of interest and debate.

Although CD5+ B-1 B cells have been recognized as an infrequent B cell subset in mice for many years, attempts to identify their histol. location in normal mouse spleen have proven difficult due to both their paucity and low level expression of CD5. In this study the authors have studied VH11/DH/JH gene-targeted mice, VH11t, that develop elevated nos. of CD5+ VH11/Vk9 B cells with an anti-phosphatidylcholine (anti-PtC) autoreactive specificity, allowing B-1 B cell detection by anti-PtC Id-specific Abs in spleen section staining. Using this approach the authors found that anti-PtC B-1 cells first appear within the white pulp in neonates, expand in assocn. with follicular dendritic cells (FDC), and localize more centrally than other (non-B-1) IgDhigh follicular B cells in adults. Among neonatal B cells, CD5+ B-1 cells in both normal and VH11t mouse spleen and peritoneal cavity express the highest levels of CXCR5, which is important for FDC development. Injection of purified spleen or peritoneal B-1 cells into RAG knockout mice resulted in B-1 cell follicle formation in spleen, inducing FDC development and plasma cell generation. These results indicate that B-1 B cells are the first B cells to express fully mature levels of CXCR5, thereby promoting the development of FDC. [on SciFinder (R)]

Natural antibodies produced by CD5(+) B- I B cells include those with specificity for senescent erythrocytes (anti-BrMRBC, anti-PtC) and for thymocytes (anti -thymocyte autoantibody, ATA). Here we describe work from our laboratories Studying two prototypic examples, V(H)11V(kappa)9-encoded anti-BrMRBC and V(H)3609V(kappa)2 I c-encoded ATA. Using V(H)11 -mu transgenic mice, we discovered that certain natural autoantibodies utilize V-H genes that are selected against in bone marrow B cell development, but not fetal liver, effectively restricting their generation to fetal/neonatal life. Studies with ATA-mu transgenic mice demonstrated a critical requirement for self antigen ill the accumulation of B cells with this specificity and for the production of high levels of serum ATA. Finally, analysis of B cell development in ATA-mu kappa transgenic mice revealed two distinct responses by B cells to expression of this B cell receptor (BCR): most developing B cells in spleen of adult mice were blocked at an immature stage and only escaped apoptosis by editing their BCR to eliminate the ATA specificity; nevertheless, high levels of serum ATA were observed, indicating that some B cells differentiated to antibody-forming cells without altering their specificity. Thus, our studies reveal mechanisms for restricting the generation of B cells producing natural autoantibodies, demonstrate a key positive selection step in their development, and show that most developing B cells in adult mice bearing such specificities fail to reach a mature stage.

Natural antibodies produced by CD5+ B-1 B cells include those with specificity for senescent erythrocytes (anti-BrMRBC, anti-PtC) and for thymocytes (anti-thymocyte autoantibody, ATA). Here we describe work from our laboratories studying two prototypic examples, VH11Vkappa9-encoded anti-BrMRBC and VH3609Vkappa21c-encoded ATA. Using VH11-mu transgenic mice, we discovered that certain natural autoantibodies utilize VH genes that are selected against in bone marrow B cell development, but not fetal liver, effectively restricting their generation to fetal/neonatal life. Studies with ATA-mu transgenic mice demonstrated a critical requirement for self antigen in the accumulation of B cells with this specificity and for the production of high levels of serum ATA. Finally, analysis of B cell development in ATA-mukappa transgenic mice revealed two distinct responses by B cells to expression of this B cell receptor (BCR): most developing B cells in spleen of adult mice were blocked at an immature stage and only escaped apoptosis by editing their BCR to eliminate the ATA specificity; nevertheless, high levels of serum ATA were observed, indicating that some B cells differentiated to antibody-forming cells without altering their specificity. Thus, our studies reveal mechanisms for restricting the generation of B cells producing natural autoantibodies, demonstrate a key positive selection step in their development, and show that most developing B cells in adult mice bearing such specificities fail to reach a mature stage.

Natural autoantibodies constitute a large portion of serum immunoglobulin M (IgM) and bridge the adaptive and innate immune systems, serving as a rapid response to common pathogens. Many arise from a distinctive subset of B cells, termed B-1, that express CD5. Here, we describe our studies with a representative CD5(+) B-cell-derived natural autoantibody, the V(H)11V(kappa)9 B-cell receptor (BCR) that binds a determinant on senescent erythrocytes. This specificity represents 5-10% of the CD5(+) B-cell subset, with a large portion accounted for by two novel BCRs, V(H)11V(kappa)9 and V(H)12V(kappa)4. We have found that the development of B-lineage cells with a V(H)11 rearrangement is surprisingly restricted at several crucial bottlenecks: (i) one of the most common V(H)11 rearrangements generates a heavy-chain protein that only inefficiently assembles a pre-BCR, key for recombinase-activating gene downregulation/allelic exclusion and pre-B-clonal expansion; (ii) cells containin! g V(H)11-mu chains lacking N-addition a-re favored for progression to the B-cell stage, eliminating most bone marrow V(H)11 rearrangements; and (iii) only a subset of V.-light chains combine with V(H)11 heavy chain to foster progression to the mature B-cell stage. Together, these constrain V(H)11 generation to fetal development and may favor production of B cells with the prototype V(H)11V(kappa)9 BCR.