Clonal instability of V region hypermutation in the Ramos Burkitt's lymphoma cell line

Affinity maturation of the humoral immune response is caused by single base changes that are introduced into the V regions of the Ig genes during a br ief period of B cell differentiation. It has recently become possible to st udy V region mutation in some human Burkitt's lymphoma cell lines that muta te their V regions and express surface markers that suggest they arose from the malignant transformation of germinal center B cells. Ramos Burkitt's c ells constitutively mutate their V regions at a rate of similar to2 x 10(-5 ) mutations/bp/generation. However, the sequencing of unselected V regions suggested that our Ramos cell line was progressively losing its ability to undergo V region hypermutation. To accurately quantify this process, subclo nes with different nonsense mutations in the mu heavy chain V region were i dentified. Reversion analysis and sequencing of unselected V regions were u sed to examine the clonal stability of V region hypermutation. Even after o nly 1 month in culture, stable and unstable subclones could be identified. The identification of mutating and non-mutating subclones of Ramos provided a unique opportunity to identify factors involved in the mutational proces s. Differential gene expression between mutating and non-mutating Ramos clo nes was examined by RT-PCR and cDNA microarray analyses. We found that the expression of activation-induced cytidine deaminase (AID), a putative cytid ine deaminase, correlated with mutation rates in Ramos subclones. These res ults suggest that the hypermutation phenotype is inherently unstable in Ram os and that long culture periods favor outgrowth of non-mutating cells that express lower levels of AID.