Table 1.

Analysis of p53 mutation frequency in progeny of TI B-cell clones stimulated by BCR:CD21-L, IL-4, and BAFF with or without supplementary PGE₂

Experiment	Donor B cells cultureda	Total cells screened		Total cells p53 seq+		Total nucleotides screenedb		Total mutations detected		Mutation frequency/base pairc		Mutation frequency/celld
		No pulse	+PGE2	No pulse	+PGE2	No pulse	+PGE2	No pulse	+PGE2	No pulse	+PGE2	No pulse	+PGE2
1	T602	28	28	4	9	1320	2970	0	0	0	0	0	0
2	T606	90	90	1	5	330	1650	0	0	0	0	0	0
3	T612	90	90	3	10	990	3300	2	4	20 × 10−4	12 × 10−4	0.022	0.044
4	T613	90	90	0	13	0	4290	0	0	0	0	0	0
5	T587	12	12	4	8	840	1680	0	0	0	0	0	0
6	T597	12	12	3	6	630	1260	3	3	48 × 10−4	24 × 10−4	0.25	0.25
Total		322	322	15	51	4110	15,150	5	7	12 × 10−4	5 × 10−4e	0.015	0.022e

^aBased on our prior findings that PGE₂ can have both prosurvival and proapoptotic functions on B lymphoblasts, in part dependent on PGE₂ dose (34), cultures were monitored for viability during sorting of PGE₂-pulsed/nonpulsed single cells for p53-specific RT-PCR. Percentage viability in the d 5 cultures (excepting T612 on d 6) at time of sorting was T602 = 67/74% with/without PGE₂, respectively; and similarly, T606 = 72/73%; T612 = 36/40%; T613 = 58/60%; T587 = 64/63%; T597 = 65/68% [mean of 60±5 vs. 63±5% viability in PGE₂⁻ and PGE₂⁺ cultures, respectively; P=0.06 (nonsignificant)]. There is a high likelihood that PGE₂ prosurvival effects were minimized due to the relatively high cell density of the 24-well cultures from which the sorted cells originated (10⁶ cells/2 ml). Notably, other analyses involving cells cultured at varying densities (0.01–0.4×10⁶ cells/0.2 ml in 96-well plates) show that supplementary PGE₂ boosts lymphoblast viability only in cultures of low to moderate cell density (unpublished results). This may reflect sufficient levels of autocrine PGE₂ at higher cell density. It is also possible that at the higher culture density, prosurvival effects of PGE₂ on one subset of dividing blasts are masked by opposing proapoptotic effects on another subset. Consistent with the latter, viable cells with PGE₂-up-regulated levels of phospho-H2AX are prominent only when cultures are pulsed with the pan-caspase inhibitor Z-VAD (see Fig. 8). In an effort to reduce cell death, cultures T612, T613, T587, and T597 received Z-VAD (40 μm) on d 3.5–4 following activation. Nevertheless, as compared to previously noted significant prosurvival effects of Z-VAD in less dense cultures (within 96-well plates; ref. 24), significant apoptosis remained. The less-than-expected Z-VAD function might reflect limited Z-VAD infiltration into the substantially larger clusters of activated cells present in the 24-well cultures seeded with 10⁶ cells, a need for counteracting caspase function at an earlier interval in these cultures, or alternatively, activation of non-caspase-mediated death.

^bIn experiments 1–4, single-cell amplicons from cDNA were generated using primers for exon 2 and exon 4 (400-bp amplicon), whereas in experiments 5 and 6, amplicons were generated using primers for exon 4 alone (280-bp amplicon; 199 bp were common to both). In sequencing these amplicons, the most 5′ nucleotides were not considered due to sequence noise near the binding region for the sequencing primer.

^cDetermining the background rate of p53 mutation in unstimulated cells is not possible due to insufficient recovery of p53 mRNA for this single-cell assay. Nevertheless, a background rate based on the frequency of introduced mutations from 2× PCR amplification of the p53 exon segment from cDNA and 1× amplification for sequencing was assessed by calculating the total nucleotides sequenced in the 4 of 6 experiments that exhibited no p53 mutations. This indicates that background error rate is <0.8 × 10⁻⁴/bp.

^dIn an effort to avoid the bias of calculating mutation frequency on the basis of only cells with high detectable levels of p53 mRNA, values for mutation frequency per number of single cells screened were determined. While this assessment is compromised by the fact that most cells screened did not display sufficient p53 mRNA for evaluation, the calculated frequencies might be more reliable given that it is highly likely that those cells with low p53 mRNA transcription are unlikely candidates for mutation.

^eThere was no significant association between the frequency distribution of mutations (per base pair) and culture (PGE₂⁺ vs. PGE₂⁻); P < 0.15 by Fisher's exact test. In addition, there was no significant association between the frequency of mutations (per cell) and culture (PGE₂⁺ vs. PGE₂⁻); P < 0.56 by χ² analysis.