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1 Laboratory of Immunopathology, National Institute of Allergy and Infectious Diseases, Rockville, Maryland and 2 Mouse Cancer Genetics Program, National Cancer Institute, Frederick, Maryland
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although many lymphoma disease genes have been identified by proviral tagging in spontaneous mouse lymphomas (3, 4, 5, 6) , there has been no concerted effort to relate specific RIS to different diagnostic categories of mouse B-cell lineage lymphomas or to distinguish mutations involved in initiation from those contributing to progression. In this study, we sought to determine whether identification of RIS in mouse splenic marginal zone lymphoma (MZL) could lead to an understanding of the molecular pathogenesis of this disease.
Splenic MZL occurs at a high frequency in NFS/N mice congenic for high-expressing ecotropic murine leukemia virus (MuLV) genes from AKR and C58 mice (NFS.V+; 10
, 11
) but also occurs in other mice including AKXD RI strains (12)
and p53 knockout mice (13)
. MZL is a clonal disease that develops from B cells of the marginal zone (MZ), a histologically, phenotypically, and functionally distinct population (14)
, which lies just outside the splenic white pulp. The MZ in most strains of mice, including NFS.V+, is only about 3-cells thick, and MZ B cells represent 
5% of the splenic B-cell population or 
2% of all spleen cells.
A remarkable feature of MZL is the progression from low- to high-grade disease (10
, 15)
. The earliest manifestation of MZL is an expansion of the MZ by 
5-fold. Cytologically, the cells of this extended B-cell subset do not differ from the normal population, and they remain confined to the MZ. Southern blot hybridization analyses of immunoglobulin JH gene organization in spleens of mice at this stage of disease, designated early MZL (MZL-E), indicate the presence of clonal expansions and sometimes more than one. Densities of clonal bands are consistent with cellular contributions of 10% to the total number of spleen cells, and the disease is judged to be early lymphoma at this stage.
The transition between MZL-E and tumor cells presumed to be fully transformed is marked by both histological and molecular changes. The first stage of this change, designated MZL, is characterized by cells with increased cytoplasm (size) and nuclei with more open chromatin and obvious nucleoli that are still confined to the MZ (10 , 15) . Molecular studies of clonality indicate that the MZL tumor population now compromises on the order of 15–30% of spleen cells.
MZL with signs of further progression, designated MZL+, are characterized by a proliferative expansion that results in invasion of the red pulp and, sometimes, early compression of the white pulp. JH status by Southern blots is indicative of a fully clonal population comprising 40% of all spleen cells. Mitoses and apoptotic figures seen rarely in MZL are more common at this stage of disease (10)
.
The evidence for high-grade transition of MZL+ to MZL++ is, again, both morphological/histological and molecular. Cells with increased size, larger nuclei with open chromatin, and prominent nucleoli now occupy not only the MZ but extensively infiltrate the red pulp and compress the white pulp, leaving only a ragged periarteriolar lymphoid sheath around the central arteriole. The cytological features are basically indistinguishable from those of centroblastic lymphomas of follicular B-cell origin (10
, 15)
. It is only the residual affinity for their origins in the MZ and the remnants of white pulp that allow them to be distinguished from diffuse large B-cell lymphomas of centroblastic type that originate within the follicles. By Southern analyses, the transformed cells compromise 50% of spleen cells.
Two fundamental sets of observations permit MZL-E in NFS.V+ mice to be united in a progression to MZL++. The first is the occurrence in cohorts of mice of lymphomas that provide "bridges," through histological, cytologic, and molecular features, for progression of one stage into the next. This could be considered circumstantial because each putative stage of disease could be envisioned to have distinct cellular and molecular origins. A second approach involves splenic biopsy followed by examination of the same mouse at autopsy. These studies showed that a low-grade tumor, defined at biopsy, can progress to high-grade disease in the same mouse at autopsy with both being demonstrably from the same molecular clone (10) . It is noteworthy that whereas all grades of progression were observed in NFS.V+ mice, only-low grade MZL was seen in AKXD RI strains.
Recent studies have demonstrated the power of retroviral tagging in myeloid and lymphoid tumors in an era in which the mouse genome sequence is now known in exquisite detail (4 , 5 , 16, 17, 18, 19) . The sequence offers a powerful means for rapid identification of novel oncogenes that flank common integration sites (CIS) of MuLV. To develop an understanding of genes involved in splenic MZL, we applied the inverse PCR method to clone RIS coupled with a comparison to the University of California at Santa Cruz genome sequences to identify RIS in 43-splenic MZL arising in NFS.V+ mice with low numbers of endogenous ecotropic MuLV. Moreover, to determine whether the virus integration alters the expression of surrounding genes thereby contributing to the initiation or progression of splenic MZL, we examined the expression patterns for all genes identified in this study using DNA microarrays and real-time quantitative PCR (qPCR) analysis.
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MATERIALS AND METHODS |
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Inverse PCR Cloning.
Inverse PCR was performed as described previously (4)
with only slight modification. Briefly, 5 µg of tumor DNA was digested to completion with SacII and BamHI (New England BioLabs, Beverly, MA). Digested DNA was then self-circularized by dilution and ligation using T4 DNA ligase (3200 units; New England BioLabs, Beverly, MA) in a total volume of 600 µl at 16°C for 16 h. Circular DNA was precipitated with ethanol and dissolved in 40 µl of 10 mM Tris (pH 8)-1 mM EDTA buffer. Two µl were used in the primary PCR in a 50-µl PCR reaction volume containing 20 nmol each of deoxynucleoside triphosphate, 10 pmol each forward and reverse primer, 1x buffer 2, and 2.5 units enzyme mix in the Expand Long Template PCR System (Roche, Indianapolis, IN). We used a Perkin-Elmer thermocycler programmed at 94°C for 2 min, followed by 30 cycles of 94°C for 25 s, 60°C for 60 s, 68°C for 15 min, and a final extension step at 68°C for 30 min. Using electrophoresis on a 1% agarose gel, we quantified the primary PCR product and used 0.1–1.0 µl primary PCR product as the template in the secondary PCR reaction. The secondary PCR used the same conditions as the primary, except that secondary amplication primers were used. We separated the secondary PCR product on a 1% agarose gel, purified it using the QIAquick Gel Extraction kit (Qiagen, Valencia, CA), and directly cloned it using the Topo TA Cloning kit (Invitrogen, Carlsbad, CA). The primer sequences for inverse PCR have been described previously (4)
.
DNA Sequencing.
Clones were sequenced using the Thermo Sequenase Cycle Sequencing kit (USB, Cleveland, OH) and LI-COR Global Edition IR2 System Sequencer (LI-COR, Lincoln, NE) with the M13 forward and reverse primers (LI-COR).
Sequence Comparison.
We compared the RIS sequences against the publically available mouse genome database October 2003 draft assembly3
and identified annotated candidate genes located near each RIS. We compared the RIS with previously identified RIS in the web-accessible Mouse Retroviral Tagged Cancer Gene Database.4
To define common integration sites, we have adopted the statistical approach used in the publication by Suzuki et al. (18)
. In this algorithm, two or more integrations falling within a 30-kb window are considered as a common site. In the data presented, we identify the gene most proximal to that site, even if it is much >30-kb away from the common site.
Oligonucleotide Microarrays.
Microarray chips, printed by Microarray Research Facility, National Institute of Allergy and Infectious Diseases,5
comprised approximately 16,000 mouse gene targets represented by 70mer oligonucleotides purchased from Compugen (Jamesburg, NJ). These oligonucleotides were arrayed on poly-L lysine-coated slides, stored in a dry dark area, and used within 60 days. Total RNA was isolated from tumor using TRIzol Reagent (Invitrogen). Samples were processed using a modified version of the National Human Genome Research Institute RNA extraction protocol.6
A reference sample was created by extracting total RNA from ten well characterized hematopoetic cell lines and making a pool comprising equal amounts of RNA from each line. Tumor cell lines included T cell, macrophage, pre-B cell, mature B cell, and plasmacytoma. RNA was labeled using a modified version of a National Human Genome Research Institute-labeling protocol by direct incorporation of dUTP nucleotides labeled with fluorescent dyes (Cy3 and Cy5) during cDNA generation using reverse transcriptase. Tumor RNA was labeled with Cy3, and reference RNA was labeled with Cy5 to form a sample pair. Members of a sample pair were combined and applied to the prehybridized microarray chip. Chips were prehybridized in a sealed chamber immersed in a 42°C waterbath using established procedures.5
Chips were then washed twice for 2 min with agitation, first in ultra-pure water, then in isopropyl alcohol. After the last isopropyl alcohol wash, samples were quickly placed in a room-temperature centrifuge and spun dry (3000 rmp, 5 min). The chips were hybridized overnight in a sealed humidified slide chamber immersed in a light-protected 42°C waterbath, then washed, dried, and stored in the dark until scanned.
Chips were scanned using an Axon 4000B scanner and GenePix 4.0 software. Photomultiplier tube voltage settings were adjusted to allow 1% saturation of signal in both channels. Image data were extracted, and extracted data were analyzed using National Institute of Allergy and Infectious Diseases mAdb data storage and analysis programs.5
Real-Time qPCR.
Total RNA was isolated from tumor cells using TRIzol Reagent (Invitrogen). Reverse transcription was performed using 1 µg of RNA, random hexamer primer (Invitrogen), and Superscript II (Invitrogen). The primers for real-time PCR were designed using the Primer Express software (Applied Biosystems, Foster City, CA) and synthesized at MWG-Biotec (High Point, NC; Supplemental Table 1). Each real-time PCR reaction was performed in a mix of 10-µl reaction mixture containing 50 ng of cDNA, 2x SYBR Green PCR Master Mix (Applied Biosystems), and 3 µM of each primer. The reaction mixture was denatured for 10 min at 94°C and incubated for 40 cycles (denaturing for 15 s at 95°C and annealing and extending for 1 min at 60°C) using ABI PRISM 7900HT (Applied Biosystems). All samples were tested in triplicate, and average values were used for quantification. Analysis was performed using SDS v2.1 software (Applied Biosystems) according to the manufacturer’s instruction. Gapdh was used as an endogenous reference. The comparative CT method (
CT) was used for quantification of gene expression.
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RESULTS |
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Several viral integrations were found to target the same locus in independent tumors, thus defining a CIS and inferring the presence of a cancer-associated gene (1
, 19)
. We compared the 212 integrations with each other and with previously identified RIS in the Mouse Retroviral Tagged Cancer Gene Database4
yielding a total of 62 CIS (Table 1)
.
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. The 24 CIS that were not MZL specific were defined by one or more RIS identified in our study with an additional site defined in published work. The 7 new CIS that were MZL specific are unique to this study. Sixteen CIS were identified as targets of more than two viral integrations and of these, seven were specific to splenic MZL (Table 1)
. Sox4 (Evi16) was the most frequently targeted CIS (eight tumors). Most CIS genes encode proteins that fall into classes commonly associated with cancer, including transcription factors, cell cycle regulatory proteins, or proteins involved in signal transduction (Supplemental Table 3). The largest class in this study was transcription factor (Sox4, Bach2, Gfi1, Prdm1, Zfp36l1, Nfkb1, Bcl11a, Junb, and Runx3).
Six CIS genes (Brca2, Bach2, Bcl11a, Ccnd1, Lasp1, and Runx3) are known to be altered by translocation or point mutation in human leukemia or lymphoma (Table 2)
, indicating again the power of retroviral tagging for identifying candidate disease genes. Furthermore, two CIS genes (Klf5 and Rnf34) are deleted or improperly expressed in other human cancers (Table 2)
. In addition, the five following oncogenes were identified among the CIS: Grb2, Junb, Rel, Ccnd2, and Gfi1.
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. Most notably, Cdk6, Etv6, and Pou2af1 are dysregulated by translocation in human lymphoma and leukemia (Table 2)
. In addition, three putative tumor suppressor genes (Blnk, Cdkn1a, and Limd1) and one proto-oncogene (Rab3a) were identified in this panel. Because these genes are not located at CIS, however, their potential role in splenic MZL should be interpreted with caution (21)
.
Differential Patterns of Gene Expression during MZL Progression Determined by DNA Microarray and qPCR Analysis.
Previous studies of RIS have examined the expression patterns of only one or a few linked genes. This study takes advantage of an extensive archive of MZL at different stages of progression that can be used for analyzing global gene expression patterns using microarrays and independent, quantitative analyses of expression using qPCR. With microarrays, we studied two of MZL-E, eight of MZL, eight of MZL+, and seven of MZL++ (Fig. 1A
; Supplemental Fig. 2). As controls, comparisons were made with data from total nude spleen cells that contain on the order of 80% B cells. The qPCR studies examined five cases each of MZL, MZL+, and MZL++ with negatively selected normal splenic B cells serving as controls (Figs. 1B
and 2)
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; Supplemental Table 4). As noted above, Sox4 (Evi16) was the most common target with 8 insertions. Sox4 is a transcriptional activator that is restricted to immature B and T lymphocytes in adult mice and is critical for B-cell differentiation beyond the pro-B stage (22)
. All eight insertions interrupted 3' noncoding sequences from 0.9 to 9 kb downstream and might be suspected to enhance message stability (Supplemental Table 4). Of note, six of the eight insertions were recovered from MZL+. The Sox4 oligonucleotide on the microarrays failed to generate good data. qPCR was used to compare Sox4 transcript levels in the various stages of progression with levels in purified normal splenic B cells (Fig. 1B)
. Sox4 transcripts were greatly increased over normal only for MZL+. This suggests that heightened expression of Sox4 may contribute to the changes that distinguish MZL from MZL+.
Gfi1 encodes a nuclear zinc finger transcriptional repressor that collaborates with Myc for transformation of pre-B cells (23)
and with Myc and Pim1 for transformation of intrathymic T cells (24
, 25)
. In T-cell lymphomagenesis, heightened expression appears to reduce the requirement for extrinsic growth factors (24)
while enhancing proliferation and inhibiting activation-induced cell death (26)
. Mechanisms of Gfi1activity that might contribute to transformation of B lineage cells have not been defined in mouse and human lymphomas. In the present study, we identified two RIS in different tumors located within 1 kb of the transcriptional start site. Both integrations were recovered from MZL++ (Supplemental Table 4). Although RNA from these particular cases of MZL++ was not available for testing, eight others tested on the arrays exhibited substantially higher levels of expression than the controls for these studies prepared from normal nude spleen cells (Fig. 1A)
. In qPCR analyses of the five lymphomas representing each stage of progression, transcripts were significantly higher than in normal B cells for each category and increased in level with disease progression (Fig. 1B)
. Increasing expression of Gfi1 with progression most likely reflects increases in the proportions of tumor cells in spleen that occur with advancing disease rather than increased expression per cell. If true this would suggest a role for deregulated Gfi1 expression in MZL initiation rather than progression. To determine whether this relationship of Gfi1 to MZL was unique to this lymphoma type or was common to other distinct lymphoma classes, we used qPCR to compare Gfi1 levels in the three stages of MZL progression to levels in other B lineage lymphomas including centroblastic and immunoblastic diffuse large B-cell lymphomas, Burkitt-like lymphomas and Burkitt lymphoma (Fig. 3)
. The results of these studies showed that high-level expression of Gfi1 was unique to MZL.
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90 kb 5' whereas the second was 11 kb 3' (Supplemental Table 4). By both array and qPCR analyses, Snf1lk transcripts were expressed at high levels in MZL regardless of the stage of progression (Fig. 1)
.
Brca2 encodes a nuclear protein implicated in DNA repair, and Brca2 mutations resulting in inactivation are found in breast and ovarian cancers. It is suggested that Brca2 inactivation may be a key event leading to genomic instability and tumorigenesis (28)
. Both RIS lay 3' of the gene within 1 kb of each other. One was recovered from a MZL and the second from a MZL+ (Supplemental Table 4). Analyses of transcripts by both array and qPCR showed that Brca2 levels were in the range of normal for MZL but were significantly reduced for both MZL+ and MZL++ (Fig. 1)
. Down-regulation of Brca2 could potentially increase the frequency of unrepaired DNA breaks that would otherwise activate p53-dependent and other apoptotic pathways. Progression to MZL+ and MZL++ is clearly associated with increased mitotic and apoptotic activity as compared with MZL (10)
, and down-regulation of Brca2 may provide a salvage signal for cells destined to die without this change.
Analyses of other genes located at multi-hit CIS in MZL by array analyses (Fig. 1A)
and qPCR (data not shown) showed, when taken together, that levels of expression for Alas1, Stk10, Rarg, Ehd2, and Scl12a8 were not significantly altered with disease progression.
We also studied expression of genes at CIS and single-hit RIS and found substantial changes in gene expression patterns for 9 of 35 that were examined by both qPCR and arrays. Two general patterns were seen. First, expression of Blnk, Prdm1, and Il6ra was significantly increased in MZL over the levels found for normal splenic B cells and tended to decrease with disease progression (Fig. 2
; Supplemental Fig. 2). None of these genes are directly implicated in human cancers although all contribute in important ways to normal B-cell signaling and differentiation. By qPCR, transcripts for Pou2af1, Nfkb1, Prkce, Pik3r1, Cd37, and Stat6 were all substantially increased in MZL over normal B cells, and these expression levels were sustained to varying extents in MZL+ and MZL++ (Fig. 2)
. Pou2af1, through translocation (29)
, and Pik3r1, by mutation of regulatory sequences (30)
, have been directly implicated in cancer development whereas aberrant activation of the protein encoded by Nfkb1 is connected with multiple aspects of oncogenesis (31)
. To determine whether, like Gfi1, altered expression of Stat6, Nfkb1, Blnk, Snf1lk, Brca2, and Pou2af1 could be specially associated with this lymphoma type, we performed qPCR for these genes using lymphomas of different origin in relation to expression in normal B cells (Supplemental Fig. 3). The results showed that expression of these genes was variable among the various lymphoma types with no unique association with MZL being evident.
Relations of RIS Found among All Hematopoietic Tumors, B Lymphomas, and MZL from AKXD RI and NFS.V+ Mice.
All but one previous study of RIS in mouse lymphomas classified the tumors as T-cell or B-cell origin without attempting to identify subtypes of B-cell lymphomas. In a singular study of lymphomas occurring in AKXD RI strains, 96 RIS were cloned from a series of MZL (18)
. Importantly, the histological diagnoses in the latter study were made by the same pathologist as for the present study, and the targets were cloned using techniques identical to those used in this study. This minimizes variabilities that could arise from inter-observer bias in making histological diagnoses or from different technical approaches to identifying RIS. We reasoned that the same disease in the two genetic backgrounds (AKXD and NFS), with Akv-type ecotropic viruses as the common mutator, would share a substantial proportion of the sites identified in either setting. Remarkably, only 6 of 300 RIS were shared by the two sets of MZL (Fig. 4
; Supplemental Table 5).
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DISCUSSION |
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RIS at Gfi1 fall into a 50-kb region that comprises a series of CIS including Evi5, Eis1, and Pal1. Insertions at any of these sites result in enhanced transcription of Gfi1 (23)
. The insertions identified in this study fell into the Gfi1 CIS. Although studies involving selection for factor independence of lymphomas in vitro identified a role for Gfi1 in T-cell lymphoma progression (36)
, the gene is also involved in the initiation of tumorigenesis (23)
. Gfi1 is a transcriptional repressor that is thought to contribute to transformation by restricting the transcription of genes normally inhibitory for cell survival, such as Bax and Bak (37)
and proliferation, including p27Kip1 (38)
. Our study suggests a role for deregulated Gfi1 expression in lymphoma initiation rather than progression.
Gfi1 collaborates with Myc in accelerating the development of pre-B lymphomas in Eµ-Myc transgenic mice infected with Moloney MuLV (39) . In these tumors, integrations at Gfi1 are mutually exclusive with insertions at the Bmi1 locus. This indicates that Gfi1 and Bmi1 belong to the same complementation group (19 , 21) and, by extension, may act on similar downstream targets for lymphoid transformation. Insertions at Bmi1 result in enhanced expression of an unmutated protein (37 , 38) . Bmi1, like Gfi1, is a transcriptional repressor but belongs to the polycomb family of genes. Eµ-Bmi1-transgenic mice overexpressing the wild-type protein exhibit perturbed lymphoid development and succumb almost uniformly to B and T cell neoplasms (40 , 41) demonstrating activity as a directly transforming oncogene. The tumor suppressor genes p16and p19ARFare critical downstream targets of Bmi1-mediated repression (42) thereby providing separate, cooperative functions with Myc for transformation. Although the relevance for Bmi1-mediated transformation is not clear, recent studies have demonstrated markedly different profiles for B-cell expression of Bmi1in association with distinct stages of the human germinal cell reaction (43) ; features of MZ B-cell expression were not reported.
Sox4 was the most frequent CIS identified in earlier screens of mouse hematopoietic tumors, with B-cell lymphomas being targeted more frequently than T-cell or myeloid neoplasms.7
It was also the most common target found in our study. The great majority of insertions lie within 3-kb 3' to the gene. Sox4 belongs to a large family of transcription factors related by homology in their DNA-binding domains to the HMG-box region of the testis-determining gene, SRY. Expression in adult mice is limited to immature T and B lymphocytes, and B-cell development in mice lacking Sox4 is blocked at the pro-B-cell stage (22)
. Interestingly, Sox4 is a downstream target of the interleukin-5R
chain with syntenin functioning as an intermediate (44)
. The genes regulated by this specific pathway are not known.
The finding that heightened expression of Sox4 was restricted to MZL+, the tumor grade in which it was most frequently targeted, is intriguing. This could suggest that activation of Sox4 is required for progression of MZL to MZL+, but that expression need not be sustained for progression to MZL++. Possibly other genetic changes may substitute for Sox4 in maintaining these signaling requirements. A similar suggestion has been made for MYC-independent tumors that develop after MYC inactivation in a conditional transgenic model for MYC regulation (45) . Alternatively, not all MZL+ may develop by progression from MZL nor MZL++ by from progression from MZL+. Human diffuse large B-cell lymphomas can appear de novo or by progression from pre-existing follicular lymphoma. The possibility that Sox4insertions might distinguish de novo MZL+ from cases that progress from MZL will require additional study.
It is quite striking that the RIS identified in MZL of NFS.V+ mice showed so little overlap with RIS in MZL of AKXD RI strains, only 6 of 300 or 2% of sites were held in common. Remarkably, little concordance also was seen between data from two sets of mice with Moloney-induced thymic T-cell lymphomas (5 of 92 sites) and another two other sets of mice with spontaneous Burkitt-like lymphomas (8/295 sites). The differences cannot be ascribed to discrepancies in histological classification as all diagnoses of MZL and Burkitt-like lymphomas were made by the same pathologist, and thymic T lymphomas are uniformly lymphoblastic. The differences between the two sets of MZL and Burkitt-like lymphomas are also not attributable to technical differences because the same technique was used throughout both studies. RIS in the two sets of mice with T lymphomas were identified by different PCR techniques (32) . It seems unlikely that strain-specific polymorphisms could bias the integrations in a systematic way or select among a remarkably large range of pathways that can lead to the same pathological phenotype.
These considerations can be extended to question the degree to which the integrations in any study deviate from a pattern that could be considered random. Algorithms have been developed to assign parameters of significance to the definition of a CIS, but they are known to be compromised by the inability to account for preferential insertions determined by sequence, chromatin conformation, or gene expression. A recent publication provides unusual insights into this problem. In a study of over 900 different MuLV RIS in human HeLa cells, it was shown that there was preferential integration in proximity to the start sites of transcriptionally active genes (46) ; 34.2% of integrations were within genes, 11.2% were within 5-kb upstream of genes, and 3.4% were within 5-kb downstream of genes. The frequencies of integrations within and 5' to genes were significantly different from random whereas that for downstream hits was indistinguishable from random. The fact that these sites were recovered 48 h postinfection should exclude any bias based on cell growth or viability. In addition, they were likely to reflect only primary integrations because the data were mostly generated using recombinant retroviruses.
The data from the present study showed that 34.2% of the RIS in NFS.V+ MZL were within genes, 20.6% were within 5-kb upstream of genes, and 6.6% were within 5-kb downstream of genes. The frequency of RIS within genes does not distinguish this study from that of human cells, but the frequencies of both 5' and 3' integrations were significantly different from those described in HeLa (P = 0.004 for 5' and P = 0.016 for 3' integrations; Fisher exact test). Consequently, the 3' integrations also differed significant from the calculations for random placements in HeLa cells. Although extrapolation of data from a human carcinoma cell line to spontaneous MZL of mice is likely to have its limitations, we feel the differential features of RIS in NFS.V+ MZL reflect a bias toward survival and proliferative advantages conferred by these mutagenic events.
These findings place several demands on future studies designed to identify genes involved in specific lymphoid diseases such as MZL. First, it will be critical to devise means for distinguishing RIS that reflect "noise" from those truly involved in pathogenesis. This need is quite distinct from that required of sensitized screens designed to define complementation groups. Second, it will be necessary to develop mechanisms for expressing candidate disease genes or aborting their expression in restricted lymphoid subsets that differ but little from other cells of the same lineage in stage of differentiation or anatomical location. As an example, there is currently no promoter/enhancer cassette that could be used to reliably drive expression of a gene in MZ B cells. Our studies of MZL have established a firm foundation for approaching these issues.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Note: Supplemental data for this article can be found at Cancer Research Online (http://cancerres@aacrjournals.org).
Requests for reprints: Herbert C. Morse III, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, NIH, 5640 Fishers Lane, Rockville, MD 20852. Phone: (301) 496-6379; Fax: (301) 402-0077; E-mail: hmorse{at}niaid.nih.gov
4 http://genome2.ncifcrf.gov/RTCGD. 
6 http://research.nhgri.gov/microarray.
7 http://rtcgd.ncifcrf.gov/mm4/index.html.
Received 12/11/03. Revised 4/ 5/04. Accepted 4/22/04.
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S. J. Erkeland, R. G.W. Verhaak, P. J.M. Valk, R. Delwel, B. Lowenberg, and I. P. Touw Significance of Murine Retroviral Mutagenesis for Identification of Disease Genes in Human Acute Myeloid Leukemia Cancer Res., January 15, 2006; 66(2): 622 - 626. [Abstract] [Full Text] [PDF]
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 Z. Duan, A. Zarebski, D. Montoya-Durango, H. L. Grimes, and M. Horwitz Gfi1 Coordinates Epigenetic Repression of p21Cip/WAF1 by Recruitment of Histone Lysine Methyltransferase G9a and Histone Deacetylase 1 Mol. Cell. Biol., December 1, 2005; 25(23): 10338 - 10351. [Abstract] [Full Text] [PDF]
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W. Lowther, K. Wiley, G. H. Smith, and R. Callahan A New Common Integration Site, Int7, for the Mouse Mammary Tumor Virus in Mouse Mammary Tumors Identifies a Gene Whose Product Has Furin-Like and Thrombospondin-Like Sequences J. Virol., August 1, 2005; 79(15): 10093 - 10096. [Abstract] [Full Text] [PDF]
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 L. Moro, A. A. Arbini, E. Marra, and M. Greco Down-regulation of BRCA2 Expression by Collagen Type I Promotes Prostate Cancer Cell Proliferation J. Biol. Chem., June 10, 2005; 280(23): 22482 - 22491. [Abstract] [Full Text] [PDF]
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J. Q. Zhang, C. Okumura, T. McCarty, M. S. Shin, P. Mukhopadhyay, M. Hori, T. A. Torrey, Z. Naghashfar, J. X. Zhou, C. H. Lee, et al. Evidence for Selective Transformation of Autoreactive Immature Plasma Cells in Mice Deficient in Fasl J. Exp. Med., December 6, 2004; 200(11): 1467 - 1478. [Abstract] [Full Text] [PDF]
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