BACKGROUND
since the discovery that balb/c mice predictably develop plasma cell tumors  following the induction of intraperitoneal  inflammatory granulomas by implanting plastic objects or injecting mineral oil or silicones, this model has been a valuable experimental system for studying plasma cell neoplasia  <cit> .
the ip inflammatory tissue produces interleukin  <dig> , insulin-like growth factor  <dig> , and other cytokines that support the growth of tepc-type pcts that appear slowly, starting no earlier than  <dig> days after ip pristane injection.
infection with retroviruses containing oncogenes, e.g., abelson virus , j3v <dig> virus , rim virus , ablmyc virus , accelerates pct formation, producing abpc, j3pcs, rimpcs and ablmycpcs, respectively, in as little as  <dig> days .
it has never been tested whether continued activity of these accelerating oncogenes was required subsequent to completion of transformation.
imyceμ  balb/c mice and il-6-transgenic balb/c mice also develop intraperitoneal pcts imyceμ pcs  <cit>  and kipcs  <cit> , respectively, at a slightly accelerated rate following pristane injection.
il-6-transgenic balb/c mice spontaneously develop pcts in lymph nodes  but with latent periods no shorter than tepcs.
as with human multiple myeloma , most of the tumors in the different groups of pcts consist of neoplasms of plasma cells that are morphologically indistinguishable, although the cells in the accelerated tumors occasionally appear larger and more blastic.
other forms of b-cell lymphomas  also appear in the lymph nodes of imyc mice, bcleμ  <cit>  and bclcα  <cit> .
chromosomal translocations that deregulate expression of the proto-oncogene c-myc constitute the earliest known tumorigenic step in pct formation.
more than 95% of pcts, have chromosomal translocations, either the typical  t or variants  t or t  <cit> .
t joins the immunoglobulin heavy chain locus and its strong enhancers to different regions: within the c-myc locus  or upstream of it .
t joins the immunoglobulin kappa light chain locus with its enhancers to a site in the pvt <dig> locus, between  <dig> and  <dig> kb 3' of the c-myc locus  <cit> .
myc activation appears to be the hallmark of all typical balb/c pcts, presumably via ig enhancer insertion near c-myc.
it has been assumed that the variant translocations also contribute to the transformation of plasma cells by upregulating c-myc by some mechanism akin to but different from the 12; <dig> translocations.
this notion has never been experimentally validated.
to investigate the mechanisms underlying myc-induced b-cell neoplasia  in balb/c mice, gene expression profiles for four groups of bcls and for pcts from six different induction protocols  were generated.
the analysis of the microarray gene expression data focused on four questions: 1) which genes differentiate bcls from pcts; 2) which genes and pathways are involved in pct acceleration by the v-abl oncogene; 3) are there genes that differ between pcts induced by typical and variant c-myc-activating chromosomal translocations; 4) how much similarity in gene expression profiles exists between mouse pcts and human mms?
to answer the final question, a meta-analysis was performed by combining our mouse tumor expression data with data published for human multiple myelomas to determine similarities and differences between these plasma cell tumors in different species.
RESULTS
unsupervised clustering yielded two main clusters: b cell lymphoma cluster and plasma cell tumor cluster
to assess the relative similarities or dissimilarities in global gene expression patterns from six different subtypes of mouse pct  and four different groups of mouse bcl , we first applied unsupervised hierarchical cluster analysis using correlation-based average linkage clustering.
characteristic expression signatures were searched for among the  <dig> genes remaining after filtering out genes showing more than 50% absence  calls among the  <dig> rna samples.
this analysis revealed two principal groups, composed of bcls and pcts, as shown in figures 1b and 1c.
with few exceptions, most of the pcts, regardless of transgenic differences or viral agents accelerating appearance of pcts, clustered together .
this cluster included both forms of pcts that arose in il-6-transgenic balb/c mice: kipcs, which arose intraperitoneally after ip pristane, and il6pcs, which spontaneously arose in hyperplastic lymph nodes, independent of ip pristane.
these results indicated that all pcts share a common genetic expression signature.
the il-6-pre-malignant transgenic lymph node samples  did not cluster with the il6pcs.
instead, these clustered with the bcls .
abls are pre-b cell lymphomas induced by abelson virus infection.
note that while the v-abl-containing retroviruses also produced two subtypes of pcts , the ablss clustered with bcleμ and bclcα, suggesting that cell type influenced gene expression more than v-abl, in this situation.
the nature of c-myc-activating chromosomal translocations, i.e., typical t vs. variant t  <cit> , did not affect their inclusion in the pct group, nor did it lead to subclusters within the abpc or tepc subgroups in this particular analysis .
instead, tumor sample subclustering was influenced more by the agents used to accelerate pct development, generating subclusters within the pct cluster, each of which contained virtually only one pct subclass, ablmycpc, abpc, il6pc, kipc or tepc.
note that abpcs and ablmycpcs clustered with each other before clustering with other pcts, indicating a greater degree of similarity between these two groups of pcts, both of which were accelerated by v-abl.
the two forms of pcts that arose in il-6-transgenic balb/c mice also clustered most closely with one another.
constitutive expression of il- <dig> in il-6-transgenic mice triggers polyclonal plasmacytosis in lymphoid organs such as lymph nodes and spleen  <cit> , and neoplastic transformation of these plasma cells occurs spontaneously over time  <cit> .
thus, rna samples from  <dig> enlarged lymph nodes in young il-6-transgenic balb/c mice were provisionally designated il6ln , while samples from  <dig> larger lymph nodes from older mice were designated il6pc .
as expected, hierarchical clustering grouped all  <dig> il6pcs into the "plasma cell tumor" cluster.
however, two of the "il6ln" samples, expected to be pre-malignant, owing to the younger ages of these transgenic mice, clustered with the il6pc samples in the pct group and are marked with asterisks in figure 1b.
since the remaining four samples of il6ln were tightly clustered with the bulk of the b-cell lymphomas in the bcl group, we took this finding as evidence that the two pct-like il6ln samples probably contained a significant percentage of transformed plasma cells.
class comparison of the two main clusters revealed major, consistent gene expression differences between pcts and bcls.
to gain biological insight into these differences, a two-sample t-test was applied to select the genes whose expression was significantly different between these two groups .
functional enrichment analysis  based on gene ontology  terms showed that these expression differences were heavily enriched for b cell-specific genes that were generally downregulated in plasma cells  <cit> .
figure  <dig> presents heat maps of a subset of those genes from categories of gene functions assigned by cancer genome anatomy project , illustrating that that pcts and bcls mobilize different signal transduction pathways.
it is interesting to note that the majority of the gene differences consist of downregulation of genes in pcts.
it is likely that this reflects the wholesale focusing of the protein synthesis machinery of these antibody-secreting "factories" on the production of their characteristic protein.
the genes with higher expression in pcts include cyclin d <dig> , syndecan <dig> , irf <dig>  and xbp <dig>  irf <dig> and syndecan <dig>  were previously reported to have increased expression in plasma cells  <cit> .
cyclin d <dig> was previously reported to be induced by c-myc activation  <cit> .
several pro-collagens  also showed higher expression in pcts than in bcls.
as expected, transcription factors previously known to have higher expression in plasma cells, xbp <dig>  cebpb, and irf <dig>  <cit>  also showed higher expression in pct groups.
b-cell surface markers  were highly expressed in bcl samples, as expected.
expression of caspases  was higher in the bcl group, as was expression of the chemokines , cytokines  and cytokine receptors .
an additional relevant gene that was more highly expressed in the bcl group was syk, a tyrosine kinase that is essential for b-cell antigen receptor triggering of cellular activation  <cit> .
genes involved in jak-stat signaling  and nfκb signaling  also showed higher expression in the bcl groups.
none of these functions are thought to be active after b cells differentiate into plasma cells, which have lost nearly all their surface receptors and downregulated many other b-cell-specific genes.
although whole lymph nodes were used as samples in il-6-transgenic pcts and ln, the lower expression of b-cell surface markers seen in the expression profiles of the il6pcs suggests that the main populations of cells in il-6-transgenic mouse lymph nodes are plasma cells, which typically lose expression of most surface markers.
our results confirm the histological finding that percentage of b- and t lymphocytes decreased as the lymph nodes fill with plasma cells and pcts  <cit> .
genes reflecting v-abl-accelerated pct induction
a direct comparison between two groups of pcts: the rapid onset pcts  and the slow onset pcts,  was performed to get information on genes that might have contributed to the acceleration.
a two-sample t-test between these two groups revealed  <dig> genes with significant  expression differences.
among these  <dig> genes,  <dig> genes showed greater than 2-fold higher expression in the abpc plus ablmyc group, while  <dig> genes showed greater than 2-fold higher expression in the tepc, kipc and il6pc group .
several genes involved in cellular growth and proliferation  showed higher expression in the abpc plus ablmyc group, as did many interferon-induced genes, presumably induced by the presence of retroviruses in this group.
several genes involved in cell death  were higher in tepcs, kipcs and il6pcs.
table  <dig> lists a subset of these genes arranged by their categories of gene functions assigned by cgap.
class comparison between slow-forming pcts  and rapid-forming pcts  by two-sample t-test generated  <dig> genes showing significant differences .
among these  <dig> genes, the genes showing more than  <dig>  fold increase and p-value <  <dig>  were selected in this table.
the genes in each category of gene function were assigned based on the annotation of cgap.
effects of v-abl
the gene expression levels of socs <dig> and socs <dig>  showed very high levels in all abelson virus-infected samples  compared to other pcts .
socs  <dig> &  <dig> are proteins that normally interrupt jak/stat signaling  <cit> , but their growth-inhibiting effects are abrogated by the v-abl protein  <cit> .
these high levels of socs expression are likely to reflect the continuous activation of jak-stat expected from v-abl  <cit> .
this hyperactive jak/stat pathway may be partly responsible for the rapid appearance of abpcs and ablmycpcs compared to tepcs, as shown in figure 1a.
sensitivity of v-abl accelerated pcts to treatment with sti-571
the main difference between abpcs and tepcs is the acceleration of plasma cell tumor formation by the v-abl gene.
the high levels of socs <dig> & <dig> expression in abpcs suggested that v-abl was active in the fully mature pcts.
to confirm this finding and to determine whether fully transformed pcts still retained a requirement for the v-abl kinase that accelerated its appearance, cell survival analysis was performed after treatment of tissue culture lines of abpc  <dig>  abpc  <dig>  tepc  <dig>  tepc  <dig> and pre b v-abl lymphoma with  <dig>  to  <dig>  μm sti- <dig>  an abl kinase inhibitor  <cit> .
figure 3a shows a clear difference in the susceptibility of those cell lines to sti- <dig>  abpcs  and pre b cell lymphoma  generated by abelson virus infection showed higher susceptibility to sti- <dig> with complete inhibition of cell growth at  <dig>  μm of sti- <dig> solution, while tepc  <dig> and tepc  <dig> showed no inhibition of cell growth even at  <dig> μm sti- <dig>  those data suggest that v-abl is still required for cell survival and proliferation even in fully transformed abpcs as was previously shown for v-abl-induced pre b lymphomas  <cit> .
sti- <dig> inhibits stat activation of v-abl accelerated pct and pre b lymphoma
the preceding analysis suggests that the v-abl-induced pcts utilize the jak/stat signaling pathways.
as shown in figure 3b, the pct cell lines studied showed highly activated  stat <dig> and stat <dig>  however treatment with  <dig> mm sti- <dig> led to decreased phosphorylation of stat <dig> and stat <dig>  but only in the cell lines induced with abelson virus, abpc  <dig> cell line and pre b v-abl lymphoma.
this dose of sti- <dig> had no effect on phosphorylation of stat <dig> or stat <dig> in tepc  <dig> and tepc  <dig>  presumably the latter pcts also utilized the jak/stat signaling pathways, but they were not stimulated by abl.
what is more,  <dig> μm sti- <dig> completely inhibited the stat <dig> phosphorylation in abpc  <dig> and pre b v-abl lymphoma and the stat <dig> tyrosine phosphorylation in pre b v-abl, and it virtually completely inhibited the tyrosine and serine phosphorylation of stat <dig> in abpc  <dig>  signals activating stat <dig> can also come from other different pathways .
in contrast, stat <dig> activation of the pre b v-abl line comes almost exclusively from v-abl, as was shown in v-abl-transformed pre b-cells  <cit> .
sti-571's selective inhibition of stat activation in abpc  <dig> and pre b v-abl lymphoma suggests that the v-abl signaling pathways are still critical for viability, even in fully transformed tissue-cultured tumors.
quantitative rt-pcr analysis
for validation of certain key findings in the microarray data, real time rt-pcr was performed for  <dig> selected genes that may play significant roles in pcts .
thirteen different tumor rna samples  were analyzed, using rna from the sample that showed the lowest expression as calibrator for each gene.
means for each of the three tumor types are shown in figure  <dig>  and individual results are presented in additional file  <dig>  these results paralleled the mrna expression levels acquired via microarray analysis.
c-myc expression was generally highest in the four tepc samples, while it showed lowest expression in the v-abl-expressing abls .
socs <dig> and socs <dig> expression was higher in abpc and abls than in tepc samples.
the genes previously known to be plasma cell-specific transcription factors  and surface marker  showed higher expression in pcts than in pre-b cell lymphomas .
jak <dig> expression was higher in the pre-b lymphomas than in the pcts.
differences of gene expressions based on c-myc gene chromosomal translocations
cluster analysis suggested that the site of chromosomal translocation on chromosome  <dig> and the nature of its translocation partner chromosome did not affect the gene expression profile as much as additional oncogenes play in pct induction.
to test this concept more stringently, a two-sample t-test was used to search for differences in gene expression between typical t and variant t chromosomal translocations within a single subgroup of pcts, abpcs.
only  <dig> genes, most of which had low levels of expression, showed significant difference in expression, suggesting that the site of translocation had little effect on the mechanism of transformation of the pcts .
another t-test was performed to compare the effect of two different forms of t, class i or ii, in tepcs .
this showed  <dig> genes with significant differences in expression, also relatively low in expression, consistent with the previous comparison and our interpretation that the different forms of chromosomal translocation did not affect significant differences in mechanism of transformations or the type of cell being transformed.
this analysis cannot rule out some differences in early neoplastic events, since we were only examining expression values from fully transformed tumors.
meta-analysis of the mouse expression data co-mingled with expression pattern data for human plasma cell neoplasms
having defined distinctive gene expression patterns among the pct subtypes as well as between pcts and bcls, we next sought to examine how well our mouse models recapitulate human phenotypes as measured by relative similarities of gene expression patterns.
mouse plasmacytomas form in the intraperitoneal cavity, while human multiple myelomas are found in bone marrow, very different locations but not without potentially meaningful similarities: richness in adipocytes, plentiful reactive myeloid cells, abundant blood supply, among others.
we used gene expression data from human b lymphocytes and human multiple myeloma isolates, and cross-compared them with the gene expression data from our models.
using genes that existed as orthologues in both human and mouse affymetrix chips, unsupervised cluster analysis showed that bcls  clustered separately from the pcts and mms .
since the b-cell components in this series were small in number, we focused our analysis on the plasma cell components, human and murine.
the human multiple myeloma samples had been previously assigned to mm1-mm <dig> subgroups, based on cluster analysis of gene expression patterns of bone-marrow plasma cells from patients with multiple myeloma  <cit> .
when pcts and mms were re-analyzed without the b-cell components, there was co-stratification of mouse and human tumors in such a way that the more aggressive mm <dig> and mm <dig> co-clustered with the accelerated pct- <dig> and - <dig> , while mm <dig> co-clustered with il- <dig> transgenic pcts  .
the slow-arising tepcs  co-clustered with the less aggressive mm1-mm <dig> patients.
this intriguing inter-species co-clustering of plasma cell neoplasms will be investigated in greater depth in another study that will incorporate more recent myeloma data  <cit>  that present a new molecular classification of myelomas into seven subgroups.
discussion
microarray expression profiling has been quite successful in differentiating subclasses within tumors with a similar histological/morphological diagnosis  <cit> .
tumors with common genetic lesions tend to cluster together, reflecting the fact that deregulation of one or more genes leads to downstream gene activation/inactivation cascades that are unique to that lesion.
it has also made it possible to predict the prognosis of cancer patients and the likelihood of their responding to chemotherapy, based on gene expression patterns  <cit> .
major progress in experimental cancer research will require animal model systems and in vitro cell culture systems.
experimentally induced pcts and related bcls have long been, and continue to be, useful for the study of plasma cell neoplasms.
in this study, we generated gene expression profiles of mouse b-lymphocytic neoplasms, including four groups of pcts that arose with different latent periods in chronic inflammatory tissue induced by ip pristane.
we also studied two subgroups of pcts that arose spontaneously in hyperplastic lymph nodes of il-6-transgenic mice and b-cell lymphomas from imyc gene-insertion mice.
statistical methods were employed to compare these expression profiles to search for specific gene expression signatures that might aid in understanding the molecular mechanisms at work in each subtype of b cell malignancy and the differences in tumor latent periods resulting from variation in the pct induction schemes.
classical unsupervised clustering analysis and statistical analysis of microarray data revealed a tight clustering of pcts regardless of subtype.
the expression patterns in this group showed major differences from those of the b-cell lymphoma group.
as seen in earlier analyses  <cit> , the stage of b cell differentiation strongly influenced the major gene expression differences.
it is important to note that virtually all of the pcts clustered together, including both intraperitoneal oil-induced pcts as well as those that arose spontaneously in il-6-transgenic lymph nodes, independent of ip pristane.
these results indicated that all pcts share a common genetic expression signature.
it can also be seen that many genes present in the b-cell signature have been downregulated in the pct signature, a well-known feature of the specialized plasma cell, which seems to reflect this cell's single-minded dedication to maximize synthesis of antibody ig.
many of the genes that emerged as part of the pct signature after unsupervised clustering analysis had been recognized in previous studies of gene expression patterns as characteristic of this stage of b cell differentiation, before and after neoplastic transformation  <cit> .
irf <dig> and syndecan <dig> are characteristically expressed in plasma cells, and cyclin d <dig> has been reported to have high expression in pct, presumably secondary to deregulated c-myc.
subclusters within the pct cluster were seen, chiefly reflecting the slow vs. rapid appearance of the pcts and the nature of the accelerating oncogenes.
unlike human myelomas, mouse plasma cell tumors have a common initiating event, a myc-activating chromosome translocation.
we found that unsupervised clustering of these tumors did not reveal major differences in gene expression that could be associated with position of breakpoint and translocation on chromosome  <dig>  that is, the nature of c-myc-activating chromosomal translocations  did not affect their inclusion in the pct group, nor did it lead to subclusters within the broad pct grouping or the abpc or tepc subgroups, in this particular analysis.
this supports our hypothesis that even t translocations that occur within the pvt- <dig> region initiate pcts via induction of constitutive c-myc expression, and that fine structural differences in the site of t within or near the c-myc locus itself do not materially affect the way myc influences pct induction.
of course, we know that additional genetic events are required for complete transformation of plasma cells, for myc-activating chromosome translocations have been found in normal cells and organs that did not develop plasma cell tumors  <cit> .
future studies will focus on identifying these additional events that occur during tumor progression.
our cluster analysis did, however, reflect differences in the cooperating genes used to accelerate tumorigenesis.
the most striking gene expression changes associated with tumors accelerated by abelson virus infection are the high levels of socs <dig> expression.
the socs proteins are inhibitors of signaling pathways and are generally expressed at low levels in unstimulated cells.
they become rapidly induced by cytokines, thereby inhibiting jak-stat signaling, forming a classic negative feedback loop  <cit> .
it was previously shown that the v-abl oncogene activates jak-stat signaling during transformation of pre-b cells in mice  <cit> .
although socs- <dig> is highly expressed in v-abl-transformed cells, it is unable to inhibit v-abl-mediated jak- stat signaling.
this is thought to be the result of one of the non-tyrosine kinase effects of v-abl, such as phosphorylation of socs- <dig> on non-tyrosine residues, leading to disruption of the interaction between socs- <dig> and the elongin b/c complex and inhibition of socs1-mediated proteasome targeting of activated jaks  <cit> .
sti-571/imatinib , at low concentrations, is a specific inhibitor of abl kinase and shows effectiveness in the treatment of chronic myelogenous leukemia  <cit> .
abpc <dig>  abpc <dig> and pre b v-abl are much more sensitive to killing by sti- <dig> than tepc <dig> or tepc <dig> , indicating that the abl kinase activity is still required for viability of pcts induced with the assistance of abelson virus.
since nearly all abpcs also show c-myc-activating chromosome translocations, some cooperation between the signaling pathways of v-abl and c-myc must be responsible for the rapid transformation of abpcs.
unexpectedly, however, this cooperation appears to be required even after full transformation is achieved and even after adaptation to growth in culture.
it will be interesting to see if resistance to sti- <dig> develops in these cultures, as is often seen in human cml  <cit> .
stat <dig> is more highly expressed in the bcl group than in pcts.
however, the level of stat <dig> expression does not differ significantly between bcls and pcts, both groups showing relatively high expression levels.
jak <dig> also showed higher expression in the bcl group compared to pcts, but jak <dig> is relatively highly expressed, even in pcts.
the accelerating mechanisms engaged after v-abl infection seems to utilize these pathways , despite the concomitant induction of the counteracting socs family of genes.
these pathways are currently being studied in greater depth at the translational and post-translational levels within the pct system, following up the leads afforded by our gene expression studies and the initial phosphorylation studies shown here, with the goals of understanding the mechanisms at work.
it has been illuminating to analyze our mouse expression data in conjunction with already published affymetrix data from human multiple myeloma.
cluster analysis showed that human mm <dig> clustered most closely with pct <dig> and pct <dig>  il6pc and kipc, the two groups of pcts from il-6-transgenic mice, while the more aggressive myeloma groups, mm3-mm <dig>  clustered more tightly with pct <dig> and pct <dig>  ablmycpc and abpc, those with appearance accelerated by v-abl activity.
this similarity includes differences in expression of genes associated with proliferation.
this was unexpected but significant, because plasma cell neoplasms are not generally associated with rapid proliferation.
instead, increased survival or escape from apoptosis is thought to be the chief mechanism responsible for the expansion of lymphocytes or plasma cells in lymph nodes or bone marrow, respectively.
this similarity brings to mind the possibility that imatinib, the activated abl inhibitor, might be effective in treating aggressive myeloma patients.
this co-clustering suggests that different pathways can be utilized to achieve a similar outcome, namely transformation of plasma cells.
thus, the mouse pct model, despite its biological differences from mm, offers an experimental model for studying the details of the etiology of plasma cell neoplasms with different degrees of aggressiveness, much as seen in human myelomas.
this aspect of our study will be broadened to include new data on additional myeloma patients  <cit>  in which expression data are used to define seven subgroups that differ in their molecular characteristics.
this study will be the subject of a separate manuscript.
CONCLUSIONS
lymphoid transformation and plasma cell tumor formation are complicated, multi-stage processes, so it is necessary to study these processes prospectively using research tools covering genome-wide changes in expression.
the present study shows that gene expression profiling can differentiate b-cell lymphomas from plasma cell tumors and also distinguish slow from accelerated plasma cell tumors.
these results and data obtained from the sensitivity of v-abl-accelerated plasma cell tumors and their phosphorylated stat proteins to the effects of sti- <dig> indicate that these otherwise similar tumors utilize different signaling pathways but share a common initiating genetic lesion, a c-myc-activating chromosome translocation.
this study of gene expression profiles of mouse b-cell lymphomas and several subclasses of plasma cell tumors provides data that offer clues for the understanding of b-cell neoplasia and plasma cell tumor formation and the interpretation of the prospective plasma cell tumor induction studies that are now under way.

 since the discovery that balb/c mice predictably develop plasma cell tumors following the induction of intraperitoneal inflammatory granulomas by implanting plastic objects or injecting mineral oil or silicones, this model has been a valuable experimental system for studying plasma cell neoplasia. the ip inflammatory tissue produces interleukin, insulin-like growth factor, and other cytokines that support the growth of tepc-type pcts that appear slowly, starting no earlier than days after ip pristane injection. infection with retroviruses containing oncogenes, e.g., abelson virus, j3v virus, rim virus, ablmyc virus, accelerates pct formation, producing abpc, j3pcs, rimpcs and ablmycpcs, respectively, in as little as days. it has never been tested whether continued activity of these accelerating oncogenes was required subsequent to completion of transformation. imyceμ balb/c mice and il-6-transgenic balb/c mice also develop intraperitoneal pcts imyceμ pcs and kipcs, respectively, at a slightly accelerated rate following pristane injection. il-6-transgenic balb/c mice spontaneously develop pcts in lymph nodes but with latent periods no shorter than tepcs. as with human multiple myeloma, most of the tumors in the different groups of pcts consist of neoplasms of plasma cells that are morphologically indistinguishable, although the cells in the accelerated tumors occasionally appear larger and more blastic. other forms of b-cell lymphomas also appear in the lymph nodes of imyc mice, bcleμ and bclcα. chromosomal translocations that deregulate expression of the proto-oncogene c-myc constitute the earliest known tumorigenic step in pct formation. more than 95% of pcts, have chromosomal translocations, either the typical t or variants t or t. t joins the immunoglobulin heavy chain locus and its strong enhancers to different regions: within the c-myc locus or upstream of it. t joins the immunoglobulin kappa light chain locus with its enhancers to a site in the pvt locus, between and kb 3' of the c-myc locus. myc activation appears to be the hallmark of all typical balb/c pcts, presumably via ig enhancer insertion near c-myc. it has been assumed that the variant translocations also contribute to the transformation of plasma cells by upregulating c-myc by some mechanism akin to but different from the 12; translocations. this notion has never been experimentally validated. to investigate the mechanisms underlying myc-induced b-cell neoplasia in balb/c mice, gene expression profiles for four groups of bcls and for pcts from six different induction protocols were generated. the analysis of the microarray gene expression data focused on four questions: 1) which genes differentiate bcls from pcts; 2) which genes and pathways are involved in pct acceleration by the v-abl oncogene; 3) are there genes that differ between pcts induced by typical and variant c-myc-activating chromosomal translocations; 4) how much similarity in gene expression profiles exists between mouse pcts and human mms? to answer the final question, a meta-analysis was performed by combining our mouse tumor expression data with data published for human multiple myelomas to determine similarities and differences between these plasma cell tumors in different species.  unsupervised clustering yielded two main clusters: b cell lymphoma cluster and plasma cell tumor cluster to assess the relative similarities or dissimilarities in global gene expression patterns from six different subtypes of mouse pct and four different groups of mouse bcl, we first applied unsupervised hierarchical cluster analysis using correlation-based average linkage clustering. characteristic expression signatures were searched for among the genes remaining after filtering out genes showing more than 50% absence calls among the rna samples. this analysis revealed two principal groups, composed of bcls and pcts, as shown in figures 1b and 1c. with few exceptions, most of the pcts, regardless of transgenic differences or viral agents accelerating appearance of pcts, clustered together. this cluster included both forms of pcts that arose in il-6-transgenic balb/c mice: kipcs, which arose intraperitoneally after ip pristane, and il6pcs, which spontaneously arose in hyperplastic lymph nodes, independent of ip pristane. these results indicated that all pcts share a common genetic expression signature. the il-6-pre-malignant transgenic lymph node samples did not cluster with the il6pcs. instead, these clustered with the bcls. abls are pre-b cell lymphomas induced by abelson virus infection. note that while the v-abl-containing retroviruses also produced two subtypes of pcts, the ablss clustered with bcleμ and bclcα, suggesting that cell type influenced gene expression more than v-abl, in this situation. the nature of c-myc-activating chromosomal translocations, i.e., typical t vs. variant t, did not affect their inclusion in the pct group, nor did it lead to subclusters within the abpc or tepc subgroups in this particular analysis. instead, tumor sample subclustering was influenced more by the agents used to accelerate pct development, generating subclusters within the pct cluster, each of which contained virtually only one pct subclass, ablmycpc, abpc, il6pc, kipc or tepc. note that abpcs and ablmycpcs clustered with each other before clustering with other pcts, indicating a greater degree of similarity between these two groups of pcts, both of which were accelerated by v-abl. the two forms of pcts that arose in il-6-transgenic balb/c mice also clustered most closely with one another. constitutive expression of il- in il-6-transgenic mice triggers polyclonal plasmacytosis in lymphoid organs such as lymph nodes and spleen, and neoplastic transformation of these plasma cells occurs spontaneously over time. thus, rna samples from enlarged lymph nodes in young il-6-transgenic balb/c mice were provisionally designated il6ln, while samples from larger lymph nodes from older mice were designated il6pc. as expected, hierarchical clustering grouped all il6pcs into the "plasma cell tumor" cluster. however, two of the "il6ln" samples, expected to be pre-malignant, owing to the younger ages of these transgenic mice, clustered with the il6pc samples in the pct group and are marked with asterisks in figure 1b. since the remaining four samples of il6ln were tightly clustered with the bulk of the b-cell lymphomas in the bcl group, we took this finding as evidence that the two pct-like il6ln samples probably contained a significant percentage of transformed plasma cells. class comparison of the two main clusters revealed major, consistent gene expression differences between pcts and bcls. to gain biological insight into these differences, a two-sample t-test was applied to select the genes whose expression was significantly different between these two groups. functional enrichment analysis based on gene ontology terms showed that these expression differences were heavily enriched for b cell-specific genes that were generally downregulated in plasma cells. figure presents heat maps of a subset of those genes from categories of gene functions assigned by cancer genome anatomy project, illustrating that that pcts and bcls mobilize different signal transduction pathways. it is interesting to note that the majority of the gene differences consist of downregulation of genes in pcts. it is likely that this reflects the wholesale focusing of the protein synthesis machinery of these antibody-secreting "factories" on the production of their characteristic protein. the genes with higher expression in pcts include cyclin d, syndecan, irf and xbp irf and syndecan were previously reported to have increased expression in plasma cells. cyclin d was previously reported to be induced by c-myc activation. several pro-collagens also showed higher expression in pcts than in bcls. as expected, transcription factors previously known to have higher expression in plasma cells, xbp cebpb, and irf also showed higher expression in pct groups. b-cell surface markers were highly expressed in bcl samples, as expected. expression of caspases was higher in the bcl group, as was expression of the chemokines, cytokines and cytokine receptors. an additional relevant gene that was more highly expressed in the bcl group was syk, a tyrosine kinase that is essential for b-cell antigen receptor triggering of cellular activation. genes involved in jak-stat signaling and nfκb signaling also showed higher expression in the bcl groups. none of these functions are thought to be active after b cells differentiate into plasma cells, which have lost nearly all their surface receptors and downregulated many other b-cell-specific genes. although whole lymph nodes were used as samples in il-6-transgenic pcts and ln, the lower expression of b-cell surface markers seen in the expression profiles of the il6pcs suggests that the main populations of cells in il-6-transgenic mouse lymph nodes are plasma cells, which typically lose expression of most surface markers. our results confirm the histological finding that percentage of b- and t lymphocytes decreased as the lymph nodes fill with plasma cells and pcts. genes reflecting v-abl-accelerated pct induction a direct comparison between two groups of pcts: the rapid onset pcts and the slow onset pcts, was performed to get information on genes that might have contributed to the acceleration. a two-sample t-test between these two groups revealed genes with significant expression differences. among these genes, genes showed greater than 2-fold higher expression in the abpc plus ablmyc group, while genes showed greater than 2-fold higher expression in the tepc, kipc and il6pc group. several genes involved in cellular growth and proliferation showed higher expression in the abpc plus ablmyc group, as did many interferon-induced genes, presumably induced by the presence of retroviruses in this group. several genes involved in cell death were higher in tepcs, kipcs and il6pcs. table lists a subset of these genes arranged by their categories of gene functions assigned by cgap. class comparison between slow-forming pcts and rapid-forming pcts by two-sample t-test generated genes showing significant differences. among these genes, the genes showing more than fold increase and p-value < were selected in this table. the genes in each category of gene function were assigned based on the annotation of cgap. effects of v-abl the gene expression levels of socs and socs showed very high levels in all abelson virus-infected samples compared to other pcts. socs & are proteins that normally interrupt jak/stat signaling, but their growth-inhibiting effects are abrogated by the v-abl protein. these high levels of socs expression are likely to reflect the continuous activation of jak-stat expected from v-abl. this hyperactive jak/stat pathway may be partly responsible for the rapid appearance of abpcs and ablmycpcs compared to tepcs, as shown in figure 1a. sensitivity of v-abl accelerated pcts to treatment with sti-571 the main difference between abpcs and tepcs is the acceleration of plasma cell tumor formation by the v-abl gene. the high levels of socs & expression in abpcs suggested that v-abl was active in the fully mature pcts. to confirm this finding and to determine whether fully transformed pcts still retained a requirement for the v-abl kinase that accelerated its appearance, cell survival analysis was performed after treatment of tissue culture lines of abpc abpc tepc tepc and pre b v-abl lymphoma with to μm sti- an abl kinase inhibitor. figure 3a shows a clear difference in the susceptibility of those cell lines to sti- abpcs and pre b cell lymphoma generated by abelson virus infection showed higher susceptibility to sti- with complete inhibition of cell growth at μm of sti- solution, while tepc and tepc showed no inhibition of cell growth even at μm sti- those data suggest that v-abl is still required for cell survival and proliferation even in fully transformed abpcs as was previously shown for v-abl-induced pre b lymphomas. sti- inhibits stat activation of v-abl accelerated pct and pre b lymphoma the preceding analysis suggests that the v-abl-induced pcts utilize the jak/stat signaling pathways. as shown in figure 3b, the pct cell lines studied showed highly activated stat and stat however treatment with mm sti- led to decreased phosphorylation of stat and stat but only in the cell lines induced with abelson virus, abpc cell line and pre b v-abl lymphoma. this dose of sti- had no effect on phosphorylation of stat or stat in tepc and tepc presumably the latter pcts also utilized the jak/stat signaling pathways, but they were not stimulated by abl. what is more, μm sti- completely inhibited the stat phosphorylation in abpc and pre b v-abl lymphoma and the stat tyrosine phosphorylation in pre b v-abl, and it virtually completely inhibited the tyrosine and serine phosphorylation of stat in abpc signals activating stat can also come from other different pathways. in contrast, stat activation of the pre b v-abl line comes almost exclusively from v-abl, as was shown in v-abl-transformed pre b-cells. sti-571's selective inhibition of stat activation in abpc and pre b v-abl lymphoma suggests that the v-abl signaling pathways are still critical for viability, even in fully transformed tissue-cultured tumors. quantitative rt-pcr analysis for validation of certain key findings in the microarray data, real time rt-pcr was performed for selected genes that may play significant roles in pcts. thirteen different tumor rna samples were analyzed, using rna from the sample that showed the lowest expression as calibrator for each gene. means for each of the three tumor types are shown in figure and individual results are presented in additional file these results paralleled the mrna expression levels acquired via microarray analysis. c-myc expression was generally highest in the four tepc samples, while it showed lowest expression in the v-abl-expressing abls. socs and socs expression was higher in abpc and abls than in tepc samples. the genes previously known to be plasma cell-specific transcription factors and surface marker showed higher expression in pcts than in pre-b cell lymphomas. jak expression was higher in the pre-b lymphomas than in the pcts. differences of gene expressions based on c-myc gene chromosomal translocations cluster analysis suggested that the site of chromosomal translocation on chromosome and the nature of its translocation partner chromosome did not affect the gene expression profile as much as additional oncogenes play in pct induction. to test this concept more stringently, a two-sample t-test was used to search for differences in gene expression between typical t and variant t chromosomal translocations within a single subgroup of pcts, abpcs. only genes, most of which had low levels of expression, showed significant difference in expression, suggesting that the site of translocation had little effect on the mechanism of transformation of the pcts. another t-test was performed to compare the effect of two different forms of t, class i or ii, in tepcs. this showed genes with significant differences in expression, also relatively low in expression, consistent with the previous comparison and our interpretation that the different forms of chromosomal translocation did not affect significant differences in mechanism of transformations or the type of cell being transformed. this analysis cannot rule out some differences in early neoplastic events, since we were only examining expression values from fully transformed tumors. meta-analysis of the mouse expression data co-mingled with expression pattern data for human plasma cell neoplasms having defined distinctive gene expression patterns among the pct subtypes as well as between pcts and bcls, we next sought to examine how well our mouse models recapitulate human phenotypes as measured by relative similarities of gene expression patterns. mouse plasmacytomas form in the intraperitoneal cavity, while human multiple myelomas are found in bone marrow, very different locations but not without potentially meaningful similarities: richness in adipocytes, plentiful reactive myeloid cells, abundant blood supply, among others. we used gene expression data from human b lymphocytes and human multiple myeloma isolates, and cross-compared them with the gene expression data from our models. using genes that existed as orthologues in both human and mouse affymetrix chips, unsupervised cluster analysis showed that bcls clustered separately from the pcts and mms. since the b-cell components in this series were small in number, we focused our analysis on the plasma cell components, human and murine. the human multiple myeloma samples had been previously assigned to mm1-mm subgroups, based on cluster analysis of gene expression patterns of bone-marrow plasma cells from patients with multiple myeloma. when pcts and mms were re-analyzed without the b-cell components, there was co-stratification of mouse and human tumors in such a way that the more aggressive mm and mm co-clustered with the accelerated pct- and -, while mm co-clustered with il- transgenic pcts. the slow-arising tepcs co-clustered with the less aggressive mm1-mm patients. this intriguing inter-species co-clustering of plasma cell neoplasms will be investigated in greater depth in another study that will incorporate more recent myeloma data that present a new molecular classification of myelomas into seven subgroups. discussion microarray expression profiling has been quite successful in differentiating subclasses within tumors with a similar histological/morphological diagnosis. tumors with common genetic lesions tend to cluster together, reflecting the fact that deregulation of one or more genes leads to downstream gene activation/inactivation cascades that are unique to that lesion. it has also made it possible to predict the prognosis of cancer patients and the likelihood of their responding to chemotherapy, based on gene expression patterns. major progress in experimental cancer research will require animal model systems and in vitro cell culture systems. experimentally induced pcts and related bcls have long been, and continue to be, useful for the study of plasma cell neoplasms. in this study, we generated gene expression profiles of mouse b-lymphocytic neoplasms, including four groups of pcts that arose with different latent periods in chronic inflammatory tissue induced by ip pristane. we also studied two subgroups of pcts that arose spontaneously in hyperplastic lymph nodes of il-6-transgenic mice and b-cell lymphomas from imyc gene-insertion mice. statistical methods were employed to compare these expression profiles to search for specific gene expression signatures that might aid in understanding the molecular mechanisms at work in each subtype of b cell malignancy and the differences in tumor latent periods resulting from variation in the pct induction schemes. classical unsupervised clustering analysis and statistical analysis of microarray data revealed a tight clustering of pcts regardless of subtype. the expression patterns in this group showed major differences from those of the b-cell lymphoma group. as seen in earlier analyses, the stage of b cell differentiation strongly influenced the major gene expression differences. it is important to note that virtually all of the pcts clustered together, including both intraperitoneal oil-induced pcts as well as those that arose spontaneously in il-6-transgenic lymph nodes, independent of ip pristane. these results indicated that all pcts share a common genetic expression signature. it can also be seen that many genes present in the b-cell signature have been downregulated in the pct signature, a well-known feature of the specialized plasma cell, which seems to reflect this cell's single-minded dedication to maximize synthesis of antibody ig. many of the genes that emerged as part of the pct signature after unsupervised clustering analysis had been recognized in previous studies of gene expression patterns as characteristic of this stage of b cell differentiation, before and after neoplastic transformation. irf and syndecan are characteristically expressed in plasma cells, and cyclin d has been reported to have high expression in pct, presumably secondary to deregulated c-myc. subclusters within the pct cluster were seen, chiefly reflecting the slow vs. rapid appearance of the pcts and the nature of the accelerating oncogenes. unlike human myelomas, mouse plasma cell tumors have a common initiating event, a myc-activating chromosome translocation. we found that unsupervised clustering of these tumors did not reveal major differences in gene expression that could be associated with position of breakpoint and translocation on chromosome that is, the nature of c-myc-activating chromosomal translocations did not affect their inclusion in the pct group, nor did it lead to subclusters within the broad pct grouping or the abpc or tepc subgroups, in this particular analysis. this supports our hypothesis that even t translocations that occur within the pvt- region initiate pcts via induction of constitutive c-myc expression, and that fine structural differences in the site of t within or near the c-myc locus itself do not materially affect the way myc influences pct induction. of course, we know that additional genetic events are required for complete transformation of plasma cells, for myc-activating chromosome translocations have been found in normal cells and organs that did not develop plasma cell tumors. future studies will focus on identifying these additional events that occur during tumor progression. our cluster analysis did, however, reflect differences in the cooperating genes used to accelerate tumorigenesis. the most striking gene expression changes associated with tumors accelerated by abelson virus infection are the high levels of socs expression. the socs proteins are inhibitors of signaling pathways and are generally expressed at low levels in unstimulated cells. they become rapidly induced by cytokines, thereby inhibiting jak-stat signaling, forming a classic negative feedback loop. it was previously shown that the v-abl oncogene activates jak-stat signaling during transformation of pre-b cells in mice. although socs- is highly expressed in v-abl-transformed cells, it is unable to inhibit v-abl-mediated jak- stat signaling. this is thought to be the result of one of the non-tyrosine kinase effects of v-abl, such as phosphorylation of socs- on non-tyrosine residues, leading to disruption of the interaction between socs- and the elongin b/c complex and inhibition of socs1-mediated proteasome targeting of activated jaks. sti-571/imatinib, at low concentrations, is a specific inhibitor of abl kinase and shows effectiveness in the treatment of chronic myelogenous leukemia. abpc abpc and pre b v-abl are much more sensitive to killing by sti- than tepc or tepc, indicating that the abl kinase activity is still required for viability of pcts induced with the assistance of abelson virus. since nearly all abpcs also show c-myc-activating chromosome translocations, some cooperation between the signaling pathways of v-abl and c-myc must be responsible for the rapid transformation of abpcs. unexpectedly, however, this cooperation appears to be required even after full transformation is achieved and even after adaptation to growth in culture. it will be interesting to see if resistance to sti- develops in these cultures, as is often seen in human cml. stat is more highly expressed in the bcl group than in pcts. however, the level of stat expression does not differ significantly between bcls and pcts, both groups showing relatively high expression levels. jak also showed higher expression in the bcl group compared to pcts, but jak is relatively highly expressed, even in pcts. the accelerating mechanisms engaged after v-abl infection seems to utilize these pathways, despite the concomitant induction of the counteracting socs family of genes. these pathways are currently being studied in greater depth at the translational and post-translational levels within the pct system, following up the leads afforded by our gene expression studies and the initial phosphorylation studies shown here, with the goals of understanding the mechanisms at work. it has been illuminating to analyze our mouse expression data in conjunction with already published affymetrix data from human multiple myeloma. cluster analysis showed that human mm clustered most closely with pct and pct il6pc and kipc, the two groups of pcts from il-6-transgenic mice, while the more aggressive myeloma groups, mm3-mm clustered more tightly with pct and pct ablmycpc and abpc, those with appearance accelerated by v-abl activity. this similarity includes differences in expression of genes associated with proliferation. this was unexpected but significant, because plasma cell neoplasms are not generally associated with rapid proliferation. instead, increased survival or escape from apoptosis is thought to be the chief mechanism responsible for the expansion of lymphocytes or plasma cells in lymph nodes or bone marrow, respectively. this similarity brings to mind the possibility that imatinib, the activated abl inhibitor, might be effective in treating aggressive myeloma patients. this co-clustering suggests that different pathways can be utilized to achieve a similar outcome, namely transformation of plasma cells. thus, the mouse pct model, despite its biological differences from mm, offers an experimental model for studying the details of the etiology of plasma cell neoplasms with different degrees of aggressiveness, much as seen in human myelomas. this aspect of our study will be broadened to include new data on additional myeloma patients in which expression data are used to define seven subgroups that differ in their molecular characteristics. this study will be the subject of a separate manuscript.  lymphoid transformation and plasma cell tumor formation are complicated, multi-stage processes, so it is necessary to study these processes prospectively using research tools covering genome-wide changes in expression. the present study shows that gene expression profiling can differentiate b-cell lymphomas from plasma cell tumors and also distinguish slow from accelerated plasma cell tumors. these results and data obtained from the sensitivity of v-abl-accelerated plasma cell tumors and their phosphorylated stat proteins to the effects of sti- indicate that these otherwise similar tumors utilize different signaling pathways but share a common initiating genetic lesion, a c-myc-activating chromosome translocation. this study of gene expression profiles of mouse b-cell lymphomas and several subclasses of plasma cell tumors provides data that offer clues for the understanding of b-cell neoplasia and plasma cell tumor formation and the interpretation of the prospective plasma cell tumor induction studies that are now under way. 
to elucidate the genes involved in the neoplastic transformation of b cells, global gene expression profiles were generated using affymetrix u74av microarrays, containing genes, for four different groups of mouse b-cell lymphomas and six subtypes of pristane-induced mouse plasma cell tumors, three of which developed much earlier than the others. when integrated with previously published affymetrix array data from human multiple myelomas, the il-6-transgenic subset of mouse plasma cell tumors clustered more closely with mm subsets of human myelomas, slow-appearing plasma cell tumors clustered together with mm while plasma cell tumors accelerated by v-abl clustered with the more aggressive mm3-mm myeloma subsets. these data and those obtained from the sensitivity of v-abl-accelerated plasma cell tumors and their phosphorylated stat proteins indicate that these similar tumors utilize different signaling pathways but share a common initiating genetic lesion, a c-myc-activating chromosome translocation. gene expression profiling differentiates not only b-cell lymphomas from plasma cell tumors but also distinguishes slow from accelerated plasma cell tumors. within a single subgroup, e.g., abpcs, plasma cell tumors that contained typical t chromosomal translocations did not display gene expression patterns distinct from those with variant t translocations, in which the breakpoint was in the pvt- locus, kb 3' of c-myc, suggesting that c-myc activation was the initiating factor in both. slow-appearing plasma cell tumors expressed socs and socs but v-abl-accelerated plasma cell tumors expressed 4– times as much. both v-abl-accelerated and non-v-abl-associated tumors exhibited phosphorylated stat and but only v-abl-accelerated plasma cell tumors lost viability and stat and phosphorylation when cultured in the presence of the v-abl kinase inhibitor, sti- these data suggest that the jak/stat pathway was critical in the transformation acceleration by v-abl and that v-abl activity remained essential throughout the life of the tumors, not just in their acceleration. a different pathway appears to predominate in the more slowly arising plasma cell tumors. this report represents the first step in using global gene expression to investigate molecular signatures related to the role of cooperating oncogenes in a model of myc-induced carcinogenesis. unsupervised hierarchical cluster analysis exhibited two main sub-clusters of samples: a b-cell lymphoma cluster and a plasma cell tumor cluster with subclusters reflecting mechanism of induction.
 to elucidate the genes involved in the neoplastic transformation of b cells, global gene expression profiles were generated using affymetrix u74av microarrays, containing genes, for four different groups of mouse b-cell lymphomas and six subtypes of pristane-induced mouse plasma cell tumors, three of which developed much earlier than the others.  unsupervised hierarchical cluster analysis exhibited two main sub-clusters of samples: a b-cell lymphoma cluster and a plasma cell tumor cluster with subclusters reflecting mechanism of induction. this report represents the first step in using global gene expression to investigate molecular signatures related to the role of cooperating oncogenes in a model of myc-induced carcinogenesis. within a single subgroup, e.g., abpcs, plasma cell tumors that contained typical t chromosomal translocations did not display gene expression patterns distinct from those with variant t translocations, in which the breakpoint was in the pvt- locus, kb 3' of c-myc, suggesting that c-myc activation was the initiating factor in both. when integrated with previously published affymetrix array data from human multiple myelomas, the il-6-transgenic subset of mouse plasma cell tumors clustered more closely with mm subsets of human myelomas, slow-appearing plasma cell tumors clustered together with mm while plasma cell tumors accelerated by v-abl clustered with the more aggressive mm3-mm myeloma subsets. slow-appearing plasma cell tumors expressed socs and socs but v-abl-accelerated plasma cell tumors expressed 4– times as much. both v-abl-accelerated and non-v-abl-associated tumors exhibited phosphorylated stat and but only v-abl-accelerated plasma cell tumors lost viability and stat and phosphorylation when cultured in the presence of the v-abl kinase inhibitor, sti- these data suggest that the jak/stat pathway was critical in the transformation acceleration by v-abl and that v-abl activity remained essential throughout the life of the tumors, not just in their acceleration. a different pathway appears to predominate in the more slowly arising plasma cell tumors.  gene expression profiling differentiates not only b-cell lymphomas from plasma cell tumors but also distinguishes slow from accelerated plasma cell tumors. these data and those obtained from the sensitivity of v-abl-accelerated plasma cell tumors and their phosphorylated stat proteins indicate that these similar tumors utilize different signaling pathways but share a common initiating genetic lesion, a c-myc-activating chromosome translocation. 