Genomic alterations reveal potential for higher grade transformation in follicular lymphoma and confirm parallel evolution of tumor cell clones.

Our aim was to examine the genetics of clonal evolution in follicular lymphoma (FL) and to identify genetic alterations associated with disease progression. A total of 100 biopsies from 44 patients diagnosed with t(14;18)-positive FL were examined by array comparative genomic hybridization. In 20 patients the patterns of somatic hypermutations (SHMs) in the variable region of heavy chain gene were additionally analyzed. Gain of chromosome X in male samples was a marker for poor outcome (P < .01). Gains involving chromosome 2, 3q, and 5 were exclusively present in FL biopsies from cases with higher grade transformation and were among the copy number alterations (CNAs) associated with inferior survival. Although we noted a trend for increasing genomic complexity in initial versus late FL samples, the overall frequencies of CNAs in initial and late FL biopsies showed a surprisingly stable pattern through the course of the disease. In 27 of cases the initial samples harbored CNAs that were absent in relapse samples, indicating that tumor cell clones at relapse were not direct descendants of initially dominating clones. The pattern of SHMs confirmed parallel development of tumor cell clones in 14 cases. Our findings support the hypothesis of common progenitor cells in FL.

Bone marrow stroma cells regulate TIEG1 expression in acute lymphoblastic leukemia cells: role of TGFbeta/BMP-6 and TIEG1 in chemotherapy escape.

The bone marrow microenvironment regulates early B lymphopoiesis and protects leukemia cells against chemotherapy treatment, thus the microenvironment may serve as a sanctuary site for these cells. Yet, few factors that contribute to this process are known. We have explored the role of transforming growth factor beta (TGFbeta) and bone morphogenetic protein-6 (BMP-6) and one target gene, TGFbeta inducible early gene 1 (TIEG1), in the communication between stroma cells and acute lymphoblastic leukemia (ALL) cell lines and their escape from chemotherapy. Here, we have demonstrated TIEG1 expression in both normal B progenitor cells and ALL cells, which increased rapidly upon TGFbeta and BMP-6 treatment. Stimulation with TGFbeta or BMP-6, as well as overexpression of TIEG1 inhibited proliferation. Furthermore, interaction with stroma cells induced TIEG1 expression in ALL cells, inhibited their proliferation and protected the cells against chemotherapeutic treatment. Similarly, treatment with TGFbeta or BMP-6, as well as overexpression of TIEG1, protected ALL cells against chemotherapy-induced cell death. These data suggest that TGFbeta and BMP-6 in the bone marrow microenvironment allow leukemia cells to escape therapy. Further, the data indicate that TIEG1 might be involved in mediating this effect from the microenvironment onto the leukemia cells.

Inhibitory effects and target genes of bone morphogenetic protein 6 in Jurkat TAg cells.

Bone morphogenetic proteins (BMP) are multifunctional cytokines that belong to the TGF-beta superfamily. BMP have been shown to regulate haematopoietic stem cells, B lymphopoiesis and early thymocyte differentiation. In the present study we explored the role of BMP-6 in Jurkat TAg cells. BMP-6 rapidly induced phosphorylation of Smad1/5/8, p38 and ERK1/2, followed by a potent up-regulation of ID1, ID2 and ID3. ID1 and ID3 were also induced at the protein level. Genome-wide expression profiling of cells treated with BMP-6 compared to medium confirmed that ID1-ID3 were target genes of BMP-6 together with Noggin and Smad6. Furthermore, several genes involved in transcriptional regulation were also identified, including NFKBIA, HEY1, DLX2, KLF10 and early growth response 1. Stimulation with BMP-6 exerted an antiproliferative effect that was counteracted by inhibitor of DNA binding (Id)1 siRNA, indicating that Id1 is an important downstream mediator in Jurkat TAg cells. A subset of CD4(+) T cells were found to express the BMP receptors Alk-2 and Alk-3 (type I), in addition to BMPRII (type II). BMP-6 also induced phosphorylation of Smad1/5/8, followed by transcriptional increase in ID1-ID3 mRNA expression. However, we did not observe significant changes in Id protein expression in CD4(+) T cells. Altogether, the data indicate a role for BMP-6 in human T lineage cells.

Characterization of early stages of human B cell development by gene expression profiling.

We have characterized several stages of normal human B cell development in adult bone marrow by gene expression profiling of hemopoietic stem cells, early B (E-B), pro-B, pre-B, and immature B cells, using RNA amplification and Lymphochip cDNA microarrays (n = 6). Hierarchical clustering of 758 differentially expressed genes clearly separated the five populations. We used gene sets to investigate the functional assignment of the differentially expressed genes. Genes involved in VDJ recombination as well as B lineage-associated transcription factors (TCF3 (E2A), EBF, BCL11A, and PAX5) were turned on in E-B cells, before acquisition of CD19. Several transcription factors with unknown roles in B lymphoid cells demonstrated interesting expression patterns, including ZCCHC7 and ZHX2. Compared with hemopoietic stem cells and pro-B cells, E-B cells had increased expression of 18 genes, and these included IGJ, IL1RAP, BCL2, and CD62L. In addition, E-B cells expressed T/NK lineage and myeloid-associated genes including CD2, NOTCH1, CD99, PECAM1, TNFSF13B, and MPO. Expression of key genes was confirmed at the protein level by FACS analysis. Several of these Ags were heterogeneously expressed, providing a basis for further subdivision of E-B cells. Altogether, these results provide new information regarding expression of genes in early stages of human B cell development.

Wnt3A activates canonical Wnt signalling in acute lymphoblastic leukaemia (ALL) cells and inhibits the proliferation of B-ALL cell lines.

Acute lymphoblastic leukaemia (ALL) is the most common malignancy in children. Recently, there has been a growing interest in Wnt signalling in several aspects of cellular development, including cancer formation. Little is known about Wnt signalling in B-ALL. We investigated whether activation of canonical Wnt signalling could occur in B-ALL cells and thereby play a potential role in cellular growth and/or survival. This study found that Wnt3A induced beta-catenin accumulation in both primary B-ALL cells and B-ALL leukaemia cell lines. Further, Wnt3A was shown to induce nuclear translocation of beta-catenin and TCF/Lef-1 dependent transcriptions in the B-ALL cell line Nalm-6. Examination of the mRNA expression pattern of WNT ligands, FZD receptors and WNT antagonists in Nalm-6 cells identified a set of ligands and receptors available for signalling, as well as antagonists potentially available for modulating the response. Functional analyses showed that Wnt3A inhibited the proliferation of several, but not all, B-ALL cell lines studied. Finally, microarray analysis was used to identify several Wnt3A target genes involved in a diverse range of cellular activities, which are potential mediators of the Wnt3A-restrained proliferation.

BMP-6 inhibits human bone marrow B lymphopoiesis--upregulation of Id1 and Id3.

OBJECTIVE: In mammals, factors produced by bone marrow (BM) stromal cells are instrumental in orchestrating the developmental process of B lymphocytes. Bone morphogenetic proteins (BMPs) are multifunctional cytokines previously found to regulate hematopoietic stem cells. In the present study, we have explored the role of BMP-6 in human B progenitor cells. MATERIALS AND METHODS: In vitro B lymphopoiesis of CD10(+) B progenitor cells from human BM was evaluated in the presence or absence of BMP-6 in short- or long-term coculture on MS-5 stromal cells, by tracking CFSE-labeled CD10(+) B progenitor cells or by quantification of CD19(+) cells. DNA synthesis in the pre-B cell line Nalm-6 was measured by (3)H-thymidine incorporation. BMP-6-induced phosphorylation of Smad1/5/8 was determined by Western blot analysis, whereas elevation of Id1-Id4 mRNA levels and basal BMP-6 mRNA levels were measured by real-time and conventional RT-PCR, respectively. RESULTS: By in vitro coculture of CD10(+) B progenitor cells or monoculture of Nalm-6 cells, we found that BMP-6 inhibited B lymphopoiesis by impeding cell proliferation. Furthermore, in CD10(+) B progenitors as well as in Nalm-6 cells, BMP-6 rapidly induced phosphorylation of Smad1/5/8, followed by an upregulation of Id1 and Id3 mRNA levels. Finally, we demonstrated that human bone marrow stromal cells express BMP-6 mRNA whereas B progenitor cells did not. CONCLUSIONS: We suggest that BMP-6, produced by the BM, may participate to fine-tune the balance between proliferation, apoptosis, and differentiation in human B progenitor cells during BM B lymphopoiesis.

BMP-6 inhibits growth of mature human B cells; induction of Smad phosphorylation and upregulation of Id1.

BACKGROUND: Bone morphogenetic proteins (BMPs) belong to the TGF-beta superfamily and are secreted proteins with pleiotropic roles in many different cell types. A potential role of BMP-6 in the immune system has been implied by various studies of malignant and rheumatoid diseases. In the present study, we explored the role of BMP-6 in normal human peripheral blood B cells. RESULTS: The B cells were found to express BMP type I and type II receptors and BMP-6 rapidly induced phosphorylation of Smad1/5/8. Furthermore, Smad-phosphorylation was followed by upregulation of Id1 mRNA and Id1 protein, whereas Id2 and Id3 expression was not affected. Furthermore, we found that BMP-6 had an antiproliferative effect both in naive (CD19+CD27-) and memory B cells (CD19+CD27+) stimulated with anti-IgM alone or the combined action of anti-IgM and CD40L. Additionally, BMP-6 induced cell death in activated memory B cells. Importantly, the antiproliferative effect of BMP-6 in B-cells was completely neutralized by the natural antagonist, noggin. Furthermore, B cells were demonstrated to upregulate BMP-6 mRNA upon stimulation with anti-IgM. CONCLUSION: In mature human B cells, BMP-6 inhibited cell growth, and rapidly induced phosphorylation of Smad1/5/8 followed by an upregulation of Id1.

Cloning, pharmacological characterisation and tissue distribution of a novel 5-HT4 receptor splice variant, 5-HT4(i).

5-HT4 receptor pre-mRNA is alternatively spliced in human (h) tissue to produce several splice variants, called 5-HT4(a) to 5-HT4(h) and 5-HT4(n). Polymerase chain reaction (PCR) with primers designed to amplify both 5-HT4(a) and 5-HT4(b) amplified three additional bands in different tissues, two representing different mRNA species both encoding 5-HT4(g) and one representing mRNA for a novel splice variant named 5-HT4(i), cloned from testis and pancreas respectively. Primary and nested PCR detected both 5-HT4(g) and 5-HT4(i) in multiple tissues. Whereas 5-HT4(i), was found in all cardiovascular tissues analysed, 5-HT4(g) was mainly present in atria. However, quantitative RT-PCR indicated 5-HT4(g) expression also in cardiac ventricle. The pharmacological profiles and ability to activate adenylyl cyclase (AC) were compared between four recombinant h5-HT4 splice variants (a, b, g and i) expressed transiently and stably in HEK293 cells. Displacement of [(3)H]GR113808 with ten ligands revealed identical pharmacological profiles (affinity rank order: GR125487, SB207710, GR113808>SB203186>serotonin, cisapride, tropisetron>renzapride, 5-MeOT>5-CT). In transiently transfected HEK293 cells cisapride was a partial agonist compared to serotonin at 5-HT4(b), 5-HT4(g) and 5-HT4(i) receptors. In membranes from HEK293 cells stably expressing 5-HT4(g) (3,000 fmol/mg protein) or 5-HT4(i) (500 fmol/mg protein), serotonin and 5-MeOT were full agonists while cisapride was full agonist at 5-HT4(g) and partial agonist at 5-HT4(i), probably due to different receptor expression levels. At both 5-HT4(g) and 5-HT4(i), the behaviour of 5-HT4 receptor antagonists was dependent on receptor level. At high receptor levels, tropisetron and SB207710 and to a variable extent SB203186 and GR113808 displayed some partial agonist activity, whereas GR125487 and SB207266 reduced the AC activity below basal, indicating both receptors to be constitutively active. We conclude that the novel 5-HT4(i) receptor splice variant is pharmacologically indistinguishable from other 5-HT4 splice variants and that the 5-HT4(i) C-terminal tail does not influence coupling to AC.