Abstract 3234

Prostaglandin E2 (PGE2) plays a pivotal role in inflammation. It is produced by COX-2-positive cells of the innate immune system, pre-neoplastic and neoplastic tissue, and activated B cells. p53 is a tumor suppressor with broad effects that include regulation of the cell cycle, DNA repair, and induction of apoptosis. A common single nucleotide polymorphism (SNP) at codon 72, within the proline rich domain of p53, can alter p53 protein structure, phosphorylation state, and function. The p53-72R (arginine) form is significantly more effective than p53-72P (proline) at promoting apoptosis in several cell lineages, but its function in B lymphocytes is unknown. COX-2 and p53 are often found co-expressed. A mechanistic link between the two might help explain why a COX-2/PGE2 axis augments the viability of cycling non-transformed human B lymphocytes (J. Immunol. 176:6736, 2006).

This study tested the hypothesis that apoptosis-prone p53-72R-positive B lymphoblasts are preferentially rescued by PGE2. This analysis was aided by our discovery that p53 expression in activated B cells exhibits allelic exclusion. Thus, individual B cells whose DNA is heterozygous (HET) for p53-p72P/R express only p72P mRNA or p72R mRNA, but never both (Haque et al., submitted ASH abstract, 2011). In the present study, single cell RT-PCR of activated B cells bearing p72P/R HET DNA was used to examine whether expression of the p72P or p72R allelic forms is influenced by level of PGE2 exposure.

Purified HET quiescent tonsillar B2 cells were activated in vitro by a surrogate for C3dg-coated antigen (anti-IgM:anti-CD21:dextran), IL4 and BAFF. To permit later differentiation of undivided and dividing subsets, cells were pre-labeled with CFSE. To supplement the low levels of PGE2 produced in density-limiting cultures, one set of parallel cultures received exogenous PGE2 (50 nM) on d2 and d4. On d5, harvested cells were analysed by FACS and sorted as one cell per well into 96-well PCR plates containing lysis buffer. RNA was isolated and cDNA prepared, using random hexamer primers. Two rounds of amplification of a sequence comprising p53 exons 2a, 3, and 4 involved primers, Forward 5-cagccagactgccttccg-3 & Reverse 5-gcaagtcacagacttggctg-3, and yielded a 400bp product upon semi-quantitative gel electrophoresis. Sequencing of PCR-amplified p53 cDNA from single cells was performed commercially (Applied Biosystems Big Dye Terminator v3.1 cycle sequencing) with analysis by chromasPro software. Within each experiment, an equivalent number of single sorted cells was analyzed for each cell subset (undivided or divided ± exogenous PGE2).

The frequency of single sorted cells yielding p53 amplicons from cultures without added PGE2 was 6.1 ± 6.0 % and 12.8 ± 4.9 % for the undivided and divided subsets, respectively. In contrast, the corresponding frequency from PGE2-pulsed cultures was 8.1 ± 4.9 % and 23.8 ± 8.1 %, respectively. The higher yield of p53+ divided cells upon greater PGE2-exposure is in agreement with quantitative RT-PCR of p53 mRNA isolated from cell pools. PGE2-pulsed cultures had a 2.26 ± 0.15 fold increase p53 mRNA, as compared to non-pulsed cultures (p = 0.001).

Sequence chromatograms of PCR-amplified p53 cDNA revealed the identity of the expressed p53 SNP. Consistent with conclusions of p53 allelic exclusion in activated human B cells (Haque et al, submitted ASH abstract), individual B cells of p72P/R heterozygous donors manifest either p72P or p72R, but never both. While the reportedly more pro-apoptotic p72R allele was expressed in 0% (0 of 5) p53+ divided blasts from cultures without supplementary PGE2, the allele was manifest in 62% (13 of 21) of the p53+ divided blasts from PGE2-supplemented cultures. PGE2 did not promote p72R allele representation in the undivided subset. Within p53+ undivided cells, p72R was found a frequency of 50% (4 of 8) and 25% (1 of 4) in PGE2 unsupplemented and supplemented cultures respectively. Thus, the p72R allele is underrepresented in replicating B cell blasts exposed to autocrine PGE2 alone, but expressed comparably to the p72P allele when higher viability-promoting concentrations of PGE2 are present. Taken together, the observations suggest that the p72R allele promotes greater activation-induced B cell apoptosis; PGE2 has a role in dampening p53-driven apoptosis within activated B cells; and the recovery of B cell blasts expressing the p53 p72R allele will be heightened in inflammatory settings.

Disclosures:

No relevant conflicts of interest to declare.

Author notes

*

Asterisk with author names denotes non-ASH members.