Preferential targeting of somatic hypermutation to hotspot motifs and hypermutable sites and generation of mutational clusters in the IgVH alleles of a rheumatoid factor producing lymphoblastoid cell line.

Epstein-Barr virus transforms human peripheral B cells into lymphoblastoid cell lines (LCL) that secrete specific antibodies. Our previous studies showed that a monoclonal LCL that secretes a rheumatoid factor expressed activation-induced cytidine deaminase (AID) and displayed an ongoing process of somatic hypermutation (SHM) at a frequency of 1.7×10⁻³ mut/bp in its productively rearranged IgVH gene. The present work shows that SHM similarly affects the nonproductive IgVH allele of the same culture. Sequencing of multiple cDNA clones derived from cellular subclones of the parental culture, showed that both alleles exhibited an ongoing mutational process with mutation rates of 2-3×10⁻5 mut/bp×generation with a high preference for C/G transition mutations and lack of a significant strand bias. About 50% of the mutations were targeted to the underlined C/G bases in the WRCH/DGYW and RCY/RGY hotspot motifs, indicating that they were due to the initial phase of AID activity. Mutations were targeted to the VH alleles and not to the Cµ or to the GAPDH genes. Genealogical trees showed a stepwise accumulation of only 1-3 mutations per branch of the tree. Unexpectedly, 27% of all the mutations in the two alleles occurred repeatedly and independently within certain sites (not necessarily the canonical hotspot motifs) in cellular clones belonging to different branches of the lineage tree. Furthermore, some of the mutations seem to arise as recurrent mutational clusters, independently generated in different cellular clones. Statistical analysis showed that it is very unlikely that these clusters were due to random targeting of equally accessible hotspots, indicating the presence of 'hypermutable sites' that generate recurring mutational clusters in the IgVH alleles. Intrinsic hypermutable sites may enhance affinity maturation and generation of effective mutated antibody repertoires against invading pathogens.

Ongoing somatic hypermutation of the rearranged VH but not of the V-lambda gene in EBV-transformed rheumatoid factor-producing lymphoblastoid cell line.

Epstein-Barr virus (EBV) transforms human peripheral B cells into lymphoblastoid cell lines (LCLs) that secrete specific antibodies. In contrast to peripheral blood B cells, LCLs express the activation-induced cytidine deaminase (AID) gene, a key enzyme in the generation of somatic hypermutation (SHM) in immunoglobulin variable genes. We have previously studied an LCL that secretes a rheumatoid factor (RF: an IgM(lambda) anti-IgG antibody) and identified the accumulation of SHM at a frequency of 1.5 x 10(-3)mut/bp in the rearranged variable region heavy chain gene (VH) of its RF sub-culture (i.e., RF-2004). The aim of the present work was to find out whether SHM was initiated as an early event following EBV transformation. Our results show that already the earliest RF-culture (RF-1983) mutates its VH at a somewhat higher frequency of 1.9 x 10(-3). Overall, we detected 17 point mutations in the RF-2004 culture and in 26 cellular clones derived from the RF-1983 and RF-2004 cultures. Most of the mutations were due to C to T or G to A transitions, with preferential targeting to WRCH/DGYW hotspot motifs, indicating that they were due to the initial phase of AID-directed mutations. A genealogical tree demonstrates that mutations were accumulated in a stepwise manner with 1-2 mutations per cell division. However, no mutations were found in the rearranged V-lambda (Vlambda) gene in the same RF-cultures and their subclones (i.e., <1.2 x 10(-4)mut/bp). To our knowledge this is the first reported clonal cell line that generates SHM in the VH, but not in the Vlambda. It may be due to abrogation of a cis-regulatory element(s) in the Vlambda or to a lack of a specific trans-acting factor which differentially direct the SHM machinery to this gene. Out of the 17 point mutations detected in both cell lines there were, 1 stop codon, 3 mutations which obliterated the binding of the RF antibody to its IgG antigen and 1 or 2 mutations which enhanced antigen-binding affinity. These results show that the evolutionary developed germline encoded antibody combining site is highly sensitive to amino acid replacements. Our combined findings that the RF cells accumulate in a stepwise manner up to 1-2 point mutations/sequence per cell division and the generation of high percentage of functionally deleterious mutations, are in accord with the 'multiphase-recycling model' of SHM, which states that B cells in the germinal center are subjected to multiple rounds of somatic mutations interchanged with periods of antigenic selection.

Cytotoxicity of NF-kappaB inhibitors Bay 11-7085 and caffeic acid phenethyl ester to Ramos and other human B-lymphoma cell lines.

OBJECTIVE: The viability of normal and malignant B-cells was shown to depend on the constitutive activation of the nuclear factor (NF)- kappaB pathway. Thus, attempts to find efficient inhibitors of NF-kappaB play a central role in the search for novel anti-B lymphoma therapies. We studied the effects of two NF-kappaB inhibitors, Bay 11-7085 (BAY) and caffeic acid phenethyl ester (CAPE), on the viability of B-lymphoma cell lines. METHODS: We investigated the mechanism(s) of the cytotoxic effect of the NF-kappaB inhibitors, BAY, and CAPE on human-lymphoma and nonhematological cell lines. RESULTS: BAY and CAPE were shown to kill Ramos-Burkitt's lymphoma cells with IC(50) values of 0.7 microM and 4 microM, respectively. The rapid killing by BAY (h) vs the slower killing by CAPE (1-3 days), and their differential effects on the stages of the cell cycle, indicated that these drugs induce killing by different mechanisms. BAY and CAPE induced a loss of the cytoplasmic compartment and generated pyknotic nuclei, which lacked nuclear or nucleosomal fragmentation, features characteristic of necrosis rather than apoptosis. BAY also induced a rapid loss of the mitochondrial potential and rapid inhibition of p65 NF-kappaB binding to its kappaB motif without reducing the level of nuclear p65. CONCLUSION: Our results indicate that BAY causes a necrotic rather than apoptotic cell death, either through its effect on the NF-kappaB pathway and/or by affecting additional molecular targets. The high sensitivity of B-lymphoma cell lines to the cytotoxicity of BAY, justify further research to explore its potential therapeutic effect on human B lymphomas.

Somatic mutations and activation-induced cytidine deaminase (AID) expression in established rheumatoid factor-producing lymphoblastoid cell line.

Epstein-Barr virus (EBV) transforms human peripheral B cells into lymphoblastoid cell lines (LCLs), allowing the production of specific antibody-secreting cell lines. We and others have previously found that in contrast to peripheral blood B cells, EBV-transformed lymphoblastoid cell lines express the activation-induced cytidine deaminase (AID) gene. The opposite is true for the germinal center-specific BCL6 gene: it is expressed in adult peripheral blood B cells and is no longer expressed in LCLs. The present work extends our findings and shows that whereas AID expression is rapidly induced following EBV infection, BCL6 expression is gradually down-regulated and is fully extinguished in already established LCLs. The question of whether AID activation induces the process of somatic hypermutation (SHM) was investigated in adult-derived LCLs. It was found that the VH gene from the rheumatoid factor-producing RF LCL (derived from a rheumatoid arthritis patient), accumulated somatic point mutations in culture. Overall, nine unique mutations have accumulated in the rearranged VH gene since the generation of the RF cell line. Four additional intraclonal mutations were found among 10 cellular clones of the RF cells. One out of the four was in CDR1 and could be correlated with loss of antigen-binding activity in three out of the 10 clones. Altogether, these 13 mutations were preferentially targeted to the DGYW motifs and showed preference for CG nucleotides, indicating that they were AID-mediated. By contrast, mutations were not detected among 3700-4000 nucleotides each of the Vlambda, Cmu and GAPDH genes derived from the same RF cell cultures and the cellular clones. Our results thus show that AID may generate point mutations in the rearranged Ig VH during in vitro cell culture of adult-LCLs and that these mutations may be responsible, at least in part, for the known instability and occasional loss of antigen-binding activity of antibody-secreting LCLs.

Tumor necrosis factor-alpha and CD40L modulate cell surface morphology and induce aggregation in Ramos Burkitt's lymphoma cells.

Interaction of CD40L and its cognate receptor is an essential component of B-lymphocyte signaling, affecting various aspects of B-cell differentiation pathways and immunoglobulin gene expression. However, much less is known about the biological consequences of B-cell signaling through tumor necrosis factor (TNF)-alpha and its cognate receptors TNF-R1 and 2. We used Ramos Burkitt's lymphoma cell line as a model system to study the direct effects of these cytokines on B cells. Treatment of Ramos cells with either TNF-alpha or CD40L, but not with interleukin (IL)- 4, interferon (IFN)-gamma and transforming growth factor (TGF)-beta, resulted in enhanced cell aggregation and enhancement of adherence to glass cover-slips. Scanning electron microscopy showed that Ramos cells have a polarized cell surface morphology and exhibit at least 3 cell surface morphological domains: microvilli, filopodia and ruffled membranes. The cells adhered to the glass matrix through multiple filopodia/podopodia-like cell processes and demonstrated distinct ruffled-like membrane projections on their opposite pole. Induction by TNF-alpha or CD40L, but not with IL-4, IFN-gamma and TGF-beta, resulted in increased number and complexity of both types of membrane projections. TNF-alpha and CD40L upregulated the expression of the adhesion molecule intercellular adhesion molecule-1 and the Fas receptor on Ramos cells, without affecting the expression levels of membrane immunoglobulin M or its secretion rate. Reverse transcriptase-polymerase chain reaction, and flow cytometry demonstrated that Ramos cells expressed TNF-R1 but very little if any TNF-R2, indicating that TNF-alpha exerted its effects on Ramos cells through the former receptor.

Differential effects of tumor necrosis factor-alpha and CD40L on NF-kappa B inhibitory proteins I kappa B alpha, beta and epsilon and on the induction of the Jun amino-terminal kinase pathway in Ramos Burkitt lymphoma cells.

Interaction between the CD40 ligand and its cognate receptor is known to affect various aspects of B-cell biology. Less is known about the biological consequences of B-cell signaling through tumor necrosis factor alpha (TNF-alpha) and its two receptors. We have used Ramos germinal center (GC)-derived Burkitt's lymphoma (BL) cells as a model system to compare some of the early signaling events of TNF-alpha and CD40L on the NF-kappaB and c-Jun amino-terminal kinase (JNK) pathways. We have previously found that both TNF-alpha and CD40L induced enhanced cell aggregation, adherence and modified cell surface morphology of Ramos cells. In the present report, it was found that treatment with either TNF-alpha or CD40L resulted in a rapid degradation (within 15 min) of IkappaBalpha, followed by a recovery period lasting up to a few hours. The level of IkappaBbeta, another inhibitory molecule of the NF-kappaB pathway, also decreased following treatment with CD40L or TNF-alpha. However, whereas CD40L induced a rapid drop without significant recovery within 2 h, TNF-alpha caused a slow and gradual decline of IkappaBbeta. In addition, treatment with CD40L resulted in a gradual and modest decline of up to 60% of the level of IkappaBepsilon within 2 h, whereas a much smaller decline was seen with TNF-alpha (approx. 20%) Our results thus show that in Ramos cells, TNF-alpha and CD40L have common, as well as differential, signaling effects on the IkappaBalpha, IkappaBbeta and IkappaBepsilon, which form inhibitory complex(es) with the NF-kappaB cytosolic proteins. We also found that CD40L, but not TNF-alpha activates the JNK pathway through transient phosphorylation of its threonine183/tyrosine185 residues. As expected, c-Jun, which is known to be a substrate of JNK, was also phosphorylated at serine residue 73 by treatment with CD40L, but not by TNF-alpha.