BL Cells Research Articles

Internalisation of uncross-linked rituximab is not essential for the induction of caspase-independent killing in Burkitt lymphoma cell lines

Characterising the mechanisms underpinning caspase-independent programmed cell death (CI-PCD) induction by uncross-linked rituximab in B-cells may positively impact upon the treatment of disease states in which the classical apoptotic pathway is disabled. The necessity of rituximab internalisation for CI-PCD induction was investigated by flow cytometry and confocal microscopy in human BL cell lines with (e.g. Mutu I) and without (Mutu III) susceptibility to rituximab-induced killing. Flow cytometry demonstrated small, significant and similar amounts of rituximab internalisation by Mutu I cells after 1, 2, 4 and 24 h (p < 0.03, n = 5) and Mutu III cells after 0.5, 2, 4 and 24 h (p < 0.05, n = 4). Confocal microscopy confirmed this. Cytochalasin B and latrunculin A significantly inhibited rituximab-induced CI-PCD (p ≤ 0.04, n = 6 and p = 0.01, n = 6, respectively) and internalisation (p = 0.02, n = 5 and p = 0.0002, n = 6, respectively) in Mutu I cells, but confocal microscopy showed no correlation between internalised rituximab and phosphatidylserine exposure. We conclude that rituximab internalisation is not essential for CI-PCD induction in BL cell lines.

Gene-expression analysis identifies novel RBL2/p130 target genes in endemic Burkitt lymphoma cell lines and primary tumors

AbstractBurkitt lymphoma (BL) is a B-cell tumor whose characteristic gene aberration is the translocation t(8;14), which determines c-myc overexpression. Several genetic and epigenetic alterations other than c-myc overexpression have also been described in BL. It has been demonstrated that the RBL2/p130 gene, a member of the retinoblastoma family (pRbs), is mutated in BL cell lines and primary tumors. The aim of this study was to investigate the biologic effect of RBL2/p130 in BL cells and its possible role in lymphomagenesis. Therefore, we reintroduced a functional RBL2/p130 in BL cell lines where this gene was mutated. Our results demonstrated that RBL2/p130-transfected cells regain growth control. This suggests that RBL2/p130 may control the expression of several genes, which may be important for cell growth and viability. Gene-expression analysis revealed a modulation of several genes, including CGRRF1, RGS1, BTG1, TIA1, and PCDHA2, upon RBL2/p130 reintroduction. We then monitored their expression in primary tumors of endemic BL as well, demonstrating that their expression resembled those of the BL cell lines. In conclusion, these data suggest that, as RBL2/p130 modulates the expression of target genes, which are important for cell growth and viability, its inactivation may be relevant for the occurrence of BL.

Epstein-Barr Virus-Encoded Latent Membrane Protein 1 (LMP1) Induces the Expression of the Cellular MicroRNA miR-146a

MicroRNAs (miRNAs) are involved in sequence-specific cleavage, translational repression or deadenylation of specific target mRNAs resulting in post-transcriptional gene silencing. Epstein-Barr Virus (EBV) infection induces cellular non-coding (nc)RNAs e.g. the “vault” RNAs or miRNAs such as miR-21, miR-155 or miR-146a. MiR-146a is up-regulated in various tumours and plays a role in innate immunity. We show that the EBV-encoded latent membrane protein 1 (LMP1) induces the expression of miR-146a via NF-κB. LMP1 activates the miR-146a promoter but not a promoter with a mutation of the NF-κB-response elements. Conversely, a LMP1-mutant deficient in NF-κB-activation failed to activate the promoter. The “CAO”-LMP1 variant which has an increased potential to induce NF-κB also showed a higher ability to activate the miR-146a promoter as compared to standard B95.8-LMP1. Northern blotting revealed high levels of miR-146a and miR-155 in the Burkitt’s lymphoma cell line Jijoye which expresses LMP1 while the LMP1-deficient P3HR1 mutant derived from Jijoye expresses less miR-146a or miR-155. Likewise, EBV-latency type I Burkitt’s lymphoma cells with low LMP1 levels also contain low levels of either miR-146a or miR-155 while their levels are increased in LMP1-expressing EBV-latency type III BL cells. Expression of LMP1 in P3HR1 cells up-regulates miR-146a levels. Neither miR-146a nor miR-155 are detectable in BCBL-1 cells transformed by the Kaposi-Sarcoma Herpes virus (KSHV/HHV8). It is possible that the induction of miR-146a plays a role in the induction or maintenance of EBV latency by modulating innate immune responses to the virus infected host cell.

Latent Membrane Protein 1 of Epstein Barr Viruses Activates the PI3K Pathway to Induce Production of IL-10 (96.5)

Abstract Epstein Barr virus is a B lymphotrophic gammaherpes virus that is associated with multiple human malignancies. The EBV-encoded protein, latent membrane protein 1 (LMP1), is required for transformation of human B cells by EBV in vitro. LMP1 usurps multiple cellular signal transduction pathways to promote cell survival and to induce expression of cellular IL-10, an autocrine growth factor for EBV+ B cell lymphomas. However, the mechanisms by which LMP1 induces IL-10 are not understood. We previously showed that rapamycin, an mTOR inhibitor, could suppress IL-10 production by EBV-infected B cell lines. To test whether LMP1 activates rapamycin-sensitive signaling pathways to induce IL-10, we stably transfected EBV− BL41 cells to express a chimeric NGFR.LMP1 protein that mimics LMP1 signaling upon crosslinking. Treatment with either the p38 inhibitor SB203580 or with inhibitors that affect PI3K/AKT/mTOR signaling diminished (25–75% decrease) IL-10 production in EBV+ B cell lines. While induction of LMP1 signaling through NGFR.LMP1 resulted in phosphorylation of MAPK and PI3K pathway intermediates and production of IL-10, inhibition of either the p38 or PI3K/AKT/mTOR signaling axis markedly suppressed LMP1-mediated IL-10 production. These data indicate that p38 and PI3K/AKT/mTOR signaling contributes to LMP1-induced IL-10 production. Moreover, targeting these pathways may be an effective strategy to prevent IL-10 production and inhibit the growth of EBV+ B cell lymphomas.

Human Follicular Lymphoma Cells Contain Oligomannose Glycans in the Antigen-binding Site of the B-cell Receptor

Expression of surface immunoglobulin appears critical for the growth and survival of B-cell lymphomas. In follicular lymphoma, we found previously that the Ig variable (V) regions in the B-cell receptor express a strikingly high incidence of N-glycosylation sequons, NX(S/T). These potential glycosylation sites are introduced by somatic mutation and are lymphoma-specific, pointing to their involvement in tumor pathogenesis. Analysis of the V region sugars from lymphoma-derived IgG/IgM reveals that they are mostly oligomannose and, remarkably, are located in the antigen-binding site, possibly precluding conventional antigen binding. The Fc region contains complex glycans, confirming that the normal glycan processing pathway is intact. Binding studies indicate that the oligomannose glycans occupying the V regions are accessible to mannose-binding lectin. These findings suggest a potential contribution to lymphoma pathogenesis involving antigen-independent interaction of surface immunoglobulin of the B-cell receptor with mannose-binding molecules of innate immunity in the germinal center.

Known components of the immunoglobulin A:T mutational machinery are intact in Burkitt lymphoma cell lines with G:C bias

The basis for mutations at A:T base pairs in immunoglobulin hypermutation and defining how AID interacts with the DNA of the immunoglobulin locus are major aspects of the immunoglobulin mutator mechanism where questions remain unanswered. Here, we examined the pattern of mutations generated in mice deficient in various DNA repair proteins implicated in A:T mutation and found a previously unappreciated bias at G:C base pairs in spectra from mice simultaneously deficient in DNA mismatch repair and uracil DNA glycosylase. This suggests a strand-biased DNA transaction for AID delivery which is then masked by the mechanism that introduces A:T mutations. Additionally, we asked if any of the known components of the A:T mutation machinery underscore the basis for the paucity of A:T mutations in the Burkitt lymphoma cell lines, Ramos and BL2. Ramos and BL2 cells were proficient in MSH2/MSH6-mediated mismatch repair, and express high levels of wild-type, full-length DNA polymerase η. In addition, Ramos cells have high levels of uracil DNA glycosylase protein and are proficient in base excision repair. These results suggest that Burkitt lymphoma cell lines may be deficient in an unidentified factor that recruits the machinery necessary for A:T mutation or that AID-mediated cytosine deamination in these cells may be processed by conventional base excision repair truncating somatic hypermutation at the G:C phase. Either scenario suggests that cytosine deamination by AID is not enough to trigger A:T mutation, and that additional unidentified factors are required for full spectrum hypermutation in vivo.

332 EFFECT OF DIFFERENT PIG OOCYTE ACTIVATION PROTOCOLS ON EMBRYO DEVELOPMENT IN SOF AND NCSU-23

Several factors affect nuclear transfer success. These include efficient parthenogenetic activation and embryo culture medium that should efficiently support pre-implantation development of good quality blastocysts. We investigated pig oocyte activation and embryo development in SOFaa in response to ionomycin (Io = 5 µM Io for 4 min; Io° = 15 µM Io for 20 min) and electric impulse (EL; one 30-µs pulse of DC 1.5 kV cm−1 in the presence of 50 µM Ca) in combination with 2 mM 6-DMAP or 10 µg mL−1 cycloheximide (CHX) +5 µg mL−1 cytochalasin B (CB) for 4 h. In addition, we studied the effect of elevated (1 mM) (Cheong et al. 2002 Mol. Reprod. Dev. 61, 488) in comparison with 50 µM Ca during EL activation on embryo development in SOFaa and NCSUaa-23. Porcine oocytes were recovered from slaughtered donors and matured in vitro for 44 h in DMEM-F12 supplemented with 10% FCS, 0.05 IU LH and FSH (Menogon®, Ferring, Milan, Italy), 0.3 mM cystine, 0.5 mM cysteamine, 50 ng mL−1 long-EGF, 100 ng mL−1 long-IGF1, 5 ng mL−1 bFGF (Sigma-Aldrich, Milan, Italy) in 5% CO2 at 38.5°C. The rates of cleavage, blastocyst formation (BL) and BL cell number on Day 7 (BL-D7) were recorded. All experiments were done with 3 replicates. The data were compared by chi-square test. There was no difference in the ability of Io (all groups) and EL + CB activated oocytes to cleave, whereas the additional treatment of EL-activated oocytes with DMAP and CHX + CB significantly increased cleavage. Io activation resulted in poor blastocyst development in comparison with all EL-activated groups (see Table 1). When calcium levels were elevated during EL activation, significantly more embryos developed in SOFaa (35.6%, n = 191 vs. 26%, n = 192; P &amp;lt; 0.05), but no differences were observed with culture in NCSUaa-23 (about 56%). The BL rate was significantly higher in NCSUaa-23 vs. SOFaa (55.9%, n = 68 vs. 34.8%, n = 69, respectively); however, the BL total cell number was significantly higher in SOFaa (58 ± 18, n = 40 vs. 86 ± 35, n = 56, respectively; P &amp;lt; 0.05). In conclusion, we have found that SOFaa and NCSUaa-23 differ in ability to support pig parthenogenetic embryo development. EL activation combined with elevated Ca significantly increased the embryo developmental capacity in SOFaa but not in NCSUaa-23. NCSUaa-23 was more efficient for embryo culture, whereas SOF produced BLs of higher quality. Table 1.Effect of activation protocol on the development of pig parthenogenetic embryos in SOFaa This work was supported by grants ISS-CS11 and Fondazione Cariplo.

Down-regulation of proteolytic complexes following EBV activation in BL cells

In Burkitt’s lymphoma cells, Epstein Barr virus (EBV) latency products interact with the ubiquitin–proteasome system to promote episomal maintenance and immunological evasion while the tripeptidylpeptidase II (TPPII) functions as an alternative protease. In the present study, we have examined the activities and levels of the proteasome and TPPII complex in Raji and in Akata cells after induction of EBV lytic cycle. The results show that the chymotrypsin-like and caspase-like activities of the proteasome were substantially reduced in Raji and Akata cells. Similarly, TPPII activity was diminished in both cell lines but was recovered in Akata cells at longer time after induction. Protein levels of the α/β subunits of the 20S proteasome and TPPII concentration decreased to different extents after EBV activation, whereas the ubiquitin binding S6’ subunit of the 19S regulatory complex increased three to fourfold along with the levels of ubiquitin-conjugates. Collectively, these observations demonstrate impairment of two major cellular proteolytic systems at the onset of EBV lytic infection.

Down-regulation of DNA polymerase β accompanies somatic hypermutation in human BL2 cell lines

Somatic hypermutation (SHM) is a fundamental process in immunoglobulin gene maturation that results in increased affinity of antibodies toward antigens. In one hypothesis explaining SHM in human B cells, the process is initiated by enzymatic deamination of cytosine to uracil in the immunoglobulin gene V-region and this in turn triggers mutation-prone forms of uracil-DNA base excision repair (BER). Yet, an uncertainty with this model is that BER of uracil-DNA in mammalian cells is generally error-free, wherein DNA polymerase β (pol β) conducts gap-filling synthesis by insertion of bases according to Watson–Crick rules. To evaluate this inconsistency, we examined pol β expression in various SHM proficient human BL2 cell line subclones. We report that expression of pol β in SHM proficient cell lines was strongly down-regulated. In contrast, in other BL2 subclones, we found that SHM was deficient and that pol β expression was much higher than in the SHM proficient subclones. We also found that overexpression of recombinant human pol β in a SHM proficient subclone abrogated its capacity for SHM. These results suggest that down-regulation of the normal BER gap-filling DNA polymerase, pol β, accompanies induced SHM in BL2 cells. This is consistent with the hypothesis that normal error-free BER must be silenced to make way for an error-prone BER process that may be required during somatic hypermutation.

Human Mesenchymal Stem Cells Isolated from Bone Marrow and Lymphoid Organs Support Tumor B-Cell Growth: Implications in Follicular Lymphoma Pathogenesis.

There is accumulating evidence that cellular microenvironment plays a key role in follicular lymphoma (FL) pathogenesis, both within tumor lymph nodes (LN) and in infiltrated bone marrow (BM) where ectopic LN-like reticular cells are integrated within malignant B-cell nodular aggregates. In normal secondary lymphoid organs, specific stromal cell subsets provide a highly specialized microenvironment that supports immune response. In particular, fibroblastic reticular cells (FRC) mediate immune cell migration, adhesion, and reciprocal interactions. The role of FRC and their postulated progenitors, i.e. bone marrow mesenchymal stem cells (MSC), in FL remains unexplored. In this study, we have investigated the relationships between FRC and MSC and their capacity to sustain malignant B-cell growth. Our findings strongly suggest that secondary lymphoid organs contain bona-fide MSC able to give rise at single-cell level to adipocytes, chondrocytes, and osteoblasts. These LN-derived MSC could also differentiate, in response to a combination of tumor necrosis factor-α (TNF) and lymphotoxin-α1β2 (LT), into fully functional FRC, able to construct a dense extracellular reticular meshwork positive for transglutaminase and fibronectin staining, to produce inflammatory (CXCL9, CXCL10, CCL5, CCL2) and LN-specific (CCL19) chemokines, and to favour lymphoma B-cell growth. Bone marrow-derived MSC (BM-MSC) acquire in vitro a complete FRC phenotype in the same culture conditions. As an exemple, BM-MSC had a strong, although not complete, protective effect on serum deprivation-induced apoptosis of BL2 cell line (mean percentage of CD20posCaspase-3pos cells: 24.8 +/− 17.5% in coculture with BM-MSC versus 80.7 +/- 10.4% in medium alone; P < .05; n =5) and pretreatment with TNF/LT fully restored BL2 viability (mean percentage of CD20posCaspase-3pos cells: 7.4 +/− 4.7%; P < .05; n = 5). Moreover, stimulation of stromal cells by TNF/LT before coculture enhanced the number of viable CD19pos primary FL B cells by 2.4-fold for BM-MSC and 2.3 fold for LN-MSC compared with the culture without stromal cells (P < .05; n = 6). Interestingly, cell contact with lymphoma B-cell lines or purified FL B cells trigger the differentiation of BM-MSC into FRC that, in turn, support malignant B-cell migration, adhesion and survival. Altogether, these new insights into the interactions between lymphoma cells and their microenvironment could offer original therapeutic strategies.

OEP37 Is a New Member of the Chloroplast Outer Membrane Ion Channels

The chloroplast outer envelope protein OEP37 is a member of the growing beta-barrel protein family of the outer chloroplast membrane. The reconstituted recombinant protein OEP37 from pea forms a rectifying high conductance channel with a main conductance (lambda) of Lambda= 500 picosiemens (symmetrical 250 mm KCl). The OEP37 channel is cation-selective (P(K+)/P(K-) = 14:1) with a voltage-dependent open probability maximal at V(mem) = 0 mV. The channel pore reveals an hourglass-shaped form with different diameters for the vestibule and restriction zone. The diameters of the vestibule at the high conductance side were estimated by d = 3.0 nm and the restriction zone by d = 1.5 nm. The OEP37 channel displayed a nanomolar affinity for the precursor of the chloroplast inner membrane protein Tic32, which is imported into the chloroplast through a yet unknown pathway. Pre-proteins imported through the usual Toc pathway and synthetic control peptides, however, did not show a comparable block of the OEP37 channel. In addition to the electrophysiological characterization, we studied the gene expression of OEP37 in the model plant Arabidopsis thaliana. Here, transcripts of AtOEP37 are ubiquitously expressed throughout plant development and accumulate in early germinating seedlings as well as in late embryogenesis. The plastid intrinsic protein could be detected in isolated chloroplasts of cotyledons and rosette leaves. However, the knock-out mutant oep37-1 shows that the proper function of this single copy gene is not essential for development of the mature plant. Moreover, import of Tic32 into chloroplasts of oep37-1 was not impaired when compared with wild type. Thus, OEP37 may constitute a novel peptide-sensitive ion channel in the outer envelope of plastids with function during embryogenesis and germination.

IL-10 can induce the expression of EBV-encoded latent membrane protein-1 (LMP-1) in the absence of EBNA-2 in B lymphocytes and in Burkitt lymphoma- and NK lymphoma-derived cell lines

AbstractEBV-positive nasopharyngeal carcinoma and Hodgkin, T, and natural killer (NK) lymphomas express EBNA-1 and the latent membrane proteins (LMP1-2; type II latency). In contrast to type III EBV-transformed lymphoblastoid cell lines, in these cells the LMPs are expressed in the absence of EBNA-2. We have previously reported that exposure to CD40 ligand and IL-4 could induce LMP-1 in an in vitro EBV-infected Hodgkin lymphoma-derived cell line, which expressed only EBNA-1. We show now that both human and EBV-encoded IL-10 can induce LMP-1 in the absence of EBNA-2 in the Daudi, P3HR1, and other BL cell lines. Interestingly, induction of LMP-1 was not accompanied by the downregulation of BCL-6. IL-10 could also induce LMP-1 in the conditional lymphoblastoid cell line ER/EB2-5 where EBNA-2 was downregulated in the absence of estrogen. Moreover, IL-10 could induce the expression of LMP-1 in tonsillar B cells infected with the nontransforming, EBNA-2-deficient EBV strain P3HR1 and enhance LMP-1 expression in 2 EBV-positive NK lymphoma lines. The demonstration that IL-10 can induce the expression of LMP-1 in an EBNA-2-independent manner shows that the major transforming EBV gene LMP-1 can be induced by extracellular signals in lymphoid cells, and IL-10 might contribute to the establishment of type II EBV latency.

Normal hypermutation in antibody genes from congenic mice defective for DNA polymerase ι

Several low fidelity DNA polymerases participate in generating mutations in immunoglobulin genes. Polymerase η is clearly involved in the process by causing substitutions of A:T base pairs, whereas polymerase ι has a controversial role. Although the frequency of mutations was decreased in the BL2 cell line deficient for polymerase ι, hypermutation was normal in the 129 strain of mice, which has a natural nonsense mutation in the Poli gene. It is possible that the mice compensated for the defect over time, or that polymerase η substituted in the absence of polymerase ι. To examine polymerase ι in a genetically defined background, we backcrossed the 129 nonsense mutation to the C57BL/6 strain for six generations. Class switch recombination and hypermutation were studied in these mice and in congenic mice doubly deficient for both polymerases ι and η. The absence of both polymerases did not affect production of IgG1, indicating that these enzymes are not involved in switch recombination. Poli −/− F6 mice had the same types of nucleotide substitutions in variable genes as their C57BL/6 counterparts, and mice doubly deficient for polymerases ι and η had the same mutational spectrum as Polh −/− mice. Thus, polymerase ι did not contribute to the mutational spectra, even in the absence of polymerase η.

Antitumor Genotoxic Agents, Such as Fludarabine*, Doxorubincin or Cis-Platine, Induce Activation of STAT1 in a p53 Protein Dependent Manner.

Introduction:Chemotherapeutic drug such as Fludarabine*; doxorubicin or cis-platine induce cell cycle arrest and apoptosis via activation of p53. Convergent studies suggest that p53 and STAT1 cooperate in the induction of apoptosis, and that STAT1 favors p53 activation. However, to our knowledge, the role of p53 in the activation of STAT1 is not documented. We present our results suggesting that (i) genotoxic agents are STAT1 inducers, (ii) STAT1 activation depends on the presence of p53 protein, and (iii) this phenomenon is modulated by the tyrosine kinase inhibitor STI571.Materials and Methods:To analyse the role of p53 in STAT1 activation, we have used different cellular models with different p53 status: PRI (p53wt), BL2 (p53wt), BL41 (p53 mutated on Arg248, resulting in the loss of p53 DNA binding activity (p53mut)), Jurkat, HL60 and MEF (the 3 latter being p53 null). The following cDNAs were used for functional studies: p53wt, p53mut, MDM2 and MTBP (MDM2 transforming protein). These cDNAs were cloned either in a pcDNA3 vector or a pRT-1 inducible vector (in the latter, the gene of interest is expressed from a bidirectional doxycycline regulatable promoter allowing simultaneous expression of truncated NGF receptor, used as a surrogate marker of inducibility).Results:Treatment of the different cell lines with the 3 genotoxic drugs Fludarabine*, doxorubicin or cis-platine induced STAT1 activation in p53wt BL2 or PRI cells and in p53mut BL41 cells, but not in Jurkat cells neither in HL60 or MEF cells. Induction of STAT1 was also obtained in presence of the RNA synthesis inhibitor Actinomycin D or in presence of secretion inhibitor Brefeldine A. Over-expression of p53wt or p53mut markedly increased STAT1 activation in PRI cells. This effect was reversed by over-expression of MTBP. Complementation of both HL60 and MEF cells with both p53wt and p53mut cDNA induced constitutive STAT1 activation, an effect that was increased by treatment with doxorubine in transfected HL60 cells. This effect was reversed by over-expression of MDM2 in HL60 cells. Finally, we found that treatment of cells with the inhibitor STI 571 of c-Abl tyrosine kinase, a kinase known to be associated with ATM during p53 activation, decreased STAT1 activation by genotoxic drugs.Conclusion:Our results show that genotoxic agents are inducers of STAT1, that p53 protein but not p53 transcriptional activity is responsible for this STAT1 activation, and suggest a possible involvement the cABL tyrosine kinase.

Involvement of PKC-δ in the Regulation of Notch2 in B-CLL.

We have recently shown that NOTCH2 signaling is involved in the overexpression of CD23 in B-cell chronic lymphocytic leukemia (B-CLL) cells (Hubmann et al., BLOOD 2002). There is an increasing evidence that NOTCH2 plays a determining role in the development/homeostasis of self-reactive CD5+ B-cells, suggesting a potential function of NOTCH2 in B-cell leukemogenesis. Here we study the regulation of NOTCH2 signaling in B-CLL cells and its possible function in B-lymphocytes using NOTCH2 transduced BL41 cells as a model system. Cultured B-CLL samples (n=30) lose their nuclear NOTCH2 (N2IC) activity within one day as demonstrated by electrophoretic mobility shift assays (EMSA). However, DNA-bound NOTCH2 complexes could be maintained in culture by exposure to the phorbolester TPA and is accompained by increased cell viability. The effect of TPA is prevented by the PKC-δ inhibitor Rottlerin indicating that PKC-δ is involved in the regulation of NOTCH2 signaling in B-CLL cells. The activity of N2IC in the leukemic cells appeared to be resistant to the γ-secretase inhibitors DAPT and compound E, two substances known to block ligand mediated release of the NOTCH2 intracellular domain (N2IC). Since B-CLL cells are locked in an anergic state, we next asked whether NOTCH2 modulates B-cell receptor (BCR) signaling and found that retrovirally transduced N2IC rescues the B-cell line BL41 from surface immunoglobulin M (sIgM) mediated apoptosis, a mechanism proposed to prevent the uncontrolled expansion of self-reactive CD5+ B-cells. In summary, our data suggest that B-CLL cells express an activated form of NOTCH2 which might be involved in the protection of the malignant clone from peripheral negative selection.

Identification of Surrogate Prognostic Markers and Genes Implicated in Immunoglobulin Gene (IgVH) Somatic Hypermutation in Small B-Cell Lymphomas.

Immunoglobulin (Ig) gene somatic hypermutation (SHM) is a prognostic factor in small B-cell lymphomas, as demonstrated by studies reporting that hypermutated chronic lymphocytic leukemia (CLL) and splenic marginal zone lymphoma (SMZL) cases show a better prognosis, while hypermutated mantle cell lymphoma (MCL) cases display specific clinicopathological features (leukemic course, increased survival).However both the mechanisms and markers of SHM are poorly characterized, with the partial exception of the role of the genes ZAP70 and AID. With the purpose of identifying SHM surrogate markers in small B-cell lymphomas, we analyzed IgVH mutational status and expression profiles of 93 small B-cell lymphoma patient samples including SMZL (24 cases), MCL (33 cases) and CLL (36 cases). Patients were classified into two groups: high SHM (>5% mutations) and low SHM (<5% mutations). T-test analysis with 100,000 permutations was performed and 39 genes were identified whose expression is significantly different (p<0.005, FDR<0.05) between cases with high and low SHM burdens.To dissect the molecular mechanisms of Ig somatic hypermutation and validate the observed findings, SHM was induced in a model system and compared to results in patient samples. The BL2 cell line was used, and SHM was induced by treating the cells with IL4 and CD40. After 24 hours, active SHM was confirmed by western blot using an anti-AID monoclonal antibody on treated and untreated cells. These same cells were analyzed using microarrays, to identify the genes that were induced or downregulated during the SHM activation. A total of 29 genes (18 upregulated and 11 downregulated) were identified which are significantly differentially expressed in the cell line model during induction of SHM (expression change >0.4 Log2 scale) and are also differentially expressed between cases with high and low SHM (p<0.001, FDR<0.1). In both analyses, the upregulated genes are implicated in transcription, DNA repair and replication and chromosome maintenance, correlating well with previous hypotheses indicating that active transcription is necessary for SHM. Based on these observations, a group of 10 key genes, implicated in DNA repair, replication and transcription, were selected and protein expression was analyzed in a set of tissue microrrays containing 150 paraffin embedded small B-cell lymphoma cases, for which clinical data is available and SHM status is known. These proteins may provide a surrogate marker for prognosis and/or analysis of IgVH SHM in patient samples using paraffin-embedded tissue samples.

A novel biological assay to detect the active form of TGF-β in urine to monitor renal allograft rejection

Transforming growth factor-beta (TGF-beta) plays an important role in renal fibrosis. Measurement of the concentration of the active form of TGF-beta particularly in urine may help our understanding of the mechanism of chronic allograft nephropathy and could be used as a diagnostic tool. However, TGF-beta release and activation are complex and, consequently, there is currently no accurate way to measure TGF-beta activity. TGF-beta-sensitive BL41 cells were stably transfected with a reporter plasmid harboring a synthetic TGF-beta-inducible DNA sequence upstream from the luciferase gene. Cells were incubated with urine samples from normal donors or transplanted recipients with or without patent nephropathy, and the active form of TGF-beta was determined as luciferase activity. We have established a cell line which expresses luciferase activity in response to active TGF-beta in a dose-dependent manner. Moreover, the use of a histone deacetylase inhibitor greatly increased sensitivity to TGF-beta and also stabilized luciferase inductibility. This test is highly specific to active TGF-beta. Detectable levels of TGF-beta were found in urine from patients with renal dysfunction due to acute or chronic renal allograft rejection (P < 0.001), but not in that from patients with stable, correctly functional kidneys. We describe a highly sensitive and specific assay for active TGF-beta. We also show that, in cases of renal allograft, TGF-beta expression is highly and significantly correlated with acute or chronic rejections.

Effect of focal adhesion kinase (FAK) downregulation with FAK antisense oligonucleotides and 5-fluorouracil on the viability of melanoma cell lines

Inhibition of focal adhesion kinase (FAK), a non-receptor tyrosine kinase linked to tumour cell survival, causes cell rounding, loss of adhesion and apoptosis in human cancer cell lines. In this study, we tested antisense oligonucleotide inhibitors of FAK, in combination with 5-fluorouracil (5-FU), to increase its sensitivity in human melanoma cell lines. Antisense oligonucleotides directed to the 5' mRNA sequence of FAK and missense control oligonucleotides were used. In BL melanoma cells, treatment with FAK antisense oligonucleotide was associated with a 2.5-fold increase in cell death compared with treatment with control oligonucleotide (33+/-2% vs. 13+/-3%, P<0.0001). 5-FU alone had no effect on BL cells (4.4% cell death, P=0.15). The addition of 5-FU after antisense oligonucleotide resulted in a significant synergistic increase in cell death equal to 69+/-2% compared with treatments with antisense oligonucleotide alone, 5-FU alone and control oligonucleotide (P<0.0001). Similar results were found in the C8161 melanoma cell line. In both cell lines, reduction in cell viability was accompanied by an increased loss of adhesion and increased apoptosis that was proportional to the decrease in viability. Treatment with antisense oligonucleotide plus 5-FU resulted in significantly decreased p125FAK expression in both C8161 and BL melanoma cell lines, demonstrated by Western blot analyses. These data show that the downregulation of FAK by antisense oligonucleotide combined with 5-FU chemotherapy results in a greater loss of adhesion and greater apoptosis in melanoma cells than treatment with either agent alone, suggesting that the combination may be a potential therapeutic agent for human melanoma in vivo.

Azidothymidine inhibits NF-κB and induces Epstein-Barr virus gene expression in Burkitt lymphoma

The antiviral compound azidothymidine (AZT), alone or in combination with other agents, induces apoptosis in early-passage, Epstein-Barr virus-positive Burkitt lymphoma (EBV+ BL) lines and has clinical activity in EBV+ BL. We report here a mechanism of AZT's antitumor activity. The nuclei of these cells contain activated nuclear factor-kappaB (NF-kappaB) subunits p50, c-Rel, RelB, and p52, but not p65. Treatment of primary EBV+ BL lines with AZT inhibited NF-kappaB within 1 to 2 hours. This was followed by up-regulation of EBV gene expression including viral thymidine kinase (vTK) and apoptosis. Subclones of EBV+ BL cells that demonstrated activated p65 were resistant to AZT. In EBV+ BLs, AZT but not ganciclovir (GCV) was highly phosphorylated to its monophosphate form (AZT-MP). Phosphorylation, as well as apoptosis, was markedly enhanced in the presence of hydroxyurea. AZT inhibits NF-kappaB and up-regulates EBV gene expression in primary EBV+ BLs. AZT with hydroxyurea may represent an inexpensive, targeted regimen for endemic BL.

Telomere size and telomerase activity in Epstein-Barr virus (EBV)-positive and EBV-negative Burkitt’s lymphoma cell lines

The telomere repeat lengths of BL cell lines were quantified by measuring terminal restriction fragment (TRF). Epstein-Barr virus (EBV)-positive Namalwa, Raji, and EB-3 cell lines have long telomeres, i.e. TRFs 10-19 kbp, whereas the Daudi cell line, producing a transformation-defective EBV mutant, has TRFs approximately 2.2 kbp. EBV-negative BJAB and DG75 cell lines have short TRFs 3.9-5.4 kbp, shorter than the approximately 12 kbp TRFs in PBLs. Telomerase activities of these BL cell lines are similar. TRFs of non-BL lymphoma cell lines are 2.3-5.5 kbp. Fluorescent in situ hybridization (FISH) studies of these cell lines showed remarkable heterogeneity of telomere size in chromosomes in the same BL cell. These results suggest that EBV-positive and EBV-negative BL cell lines have experienced various telomere dynamics.