Evidence for Allelic Exclusion of p53 within Single Sorted Human B Cells

Abstract 1122

The survival of replicating B cells, with DNA damage arising from oxidative stress and/or activation-induced cytosine deaminase (AID), appears in part to be under p53 control. Importantly, a common C>G single nucleotide polymorphism (SNP) within codon 72 of p53 influences p53 function. Among other differences, p53-72R (CGC=Arginine) is notably more effective than p53-72P (CCC=Proline) at inducing apoptosis. The SNP has been linked to clinical outcome in multiple settings, including malignancy. Most individuals in the US population display heterozygosity.

In this study, we have examined whether B cells, whose genomic DNA is heterozygous for the codon 72 SNP, exhibit allelic exclusion at the level of expressed RNA. This was suggested by reported evidence that p53 expression is strongly regulated by gene methylation status; mRNA of peripheral blood cells from p53-72P/R heterozygous individuals is skewed toward the representation of only one SNP, depending upon ethnic status; and a p53 intron 2 SNP, representing a potential methylation site, is in strong linkage disequilibrium with the codon 72 SNP (PLoS ONE 6:e15320, 2011). Evidence for allelic exclusion of this functionally relevant p53 SNP would suggest that not all identically-stimulated B cells have equal likelihoods of survival within p53-72P/R heterozygous individuals.

To investigate this issue, we first confirmed that p53 was expressed in non-transformed human B cells replicating in response to surrogate C3d-bound antigen, IL4 and BAFF. These physiologically-relevant stimuli synergize to induce a burst of T cell-independent clonal expansion, followed by apoptosis of many of the divided progeny (J. Immunol. 175:6143, 2005). Expression of p53 was monitored by immunoblotting and flow cytometry. Consistent with a role in regulating clonal burst size, p53 protein and mRNA/protein of p53-regulated pro-apoptotic genes were significantly elevated in blasts, prior to apoptosis. This contrasted with undectable p53 protein in non-stimulated B cells.

To assess whether expressed p53 within a single lymphoblast derives from one allele, i.e. demonstrates allelic exclusion, we first identified tonsil donors heterozygous for the codon 72 polymorphism. This involved PCR-restriction fragment length polymorphism (RFLP) analysis of genomic DNA, as described by others (Leukemia Research 30:1113, 2006). Subsequently, purified resting B2 cells from cryopreserved tonsil cell suspensions, determined to be heterozygous for p53-72P/R, were labeled with CFSE and stimulated as above. At d5, single B lymphoblasts were sorted into 96 well PCR plates containing lysis buffer and cDNA prepared using random hexamer primers. A p53 sequence comprising exons 2a, 3, and 4 was subjected to two rounds of PCR amplification with the following primers, Forward: 5-cagccagactgccttccg-3 & Reverse: 5-gcaagtcacagacttggctg-3. Nucleotide sequencing of PCR-amplified p53 was performed commercially (Applied Biosystems Big Dye Terminator v3.1 cycle sequencing) and analyzed by chromasPro software. In two experiments, only 39 cells of a total of 236 assayed were positive for p53. By contrast, β-actin cDNA from two rounds of PCR amplification yielded 88% of the single cell-containing wells positive for actin. These yields indicate that p53 mRNA levels within a single cell are significantly more limiting than those for actin and are consistent with quantitative PCR of cDNA obtained from activated cell pools.

Interestingly, analysis of the cDNA p53 chromatogram sequence of the p53-positive single cells (n=39), whose genomic DNA was heterozygous for the p53 codon 72 SNP, showed only a single homogenous sequence: either P (n=21) or R (n=18), but never both. This was in marked contrast to the chromatogram of cDNA derived from a pool of activated B cells within each experiment. In the latter cases, two overlapping peaks indicating co-expression of p72-P and p72-R were noted. Taken together, our findings suggest that p53 mRNA expression from a single non-transformed human B lymphocyte arises from the transcriptional activation of a single allele, i.e. shows allelic exclusion. Although the mechanism for this phenomenon requires further investigation, these results imply that B cells within individuals heterozygous for the functionally important p53-p72 polymorphism might vary considerably in their resistance to apoptosis.