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PGE 2 -promoted rise in p53 mRNA is not explained by PGE 2 -induced mRNA stabilization. In order to assess
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Within T-cell-dependent germinal centers, p53 gene transcription is repressed by Bcl-6 and is thus less vulnerable to mutation. Malignant lymphomas within inflamed extranodal sites exhibit a relatively high incidence of p53 mutations. The latter might originate from normal B-cell clones manifesting activation-induced cytosine deaminase (AID) and up...
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Context 1
... (24, 35), Fig. 1 A shows that both p53 and AID protein rise in a division-linked manner following the activation of resting follicular B cells from human tonsils with BCR:CD21-L, IL-4, and BAFF. The present simulta- neous assessment of both proteins in the same experiments shows that p53 reaches maximal expression at earlier divisions than AID (Fig. 1 B ) and suggests that AID-induced DNA damage, and ensuing p53 protein stabilization, do not solely explain the elevation in p53 protein. Transcription of p53 is reported to rise prior to proliferation (within 6 h) following human B-cell activation with anti-IgM and growth factors (43). We were interested in determining whether p53 transcription remains high during a TI B-cell proliferative response characterized by high AID expression (35). To do so, p53 mRNA levels in freshly isolated naive B cells were compared with that in proliferating cultures on d 4 following TI activation. Semiquantitative PCR ( Fig. 2 A ) and qPCR (Fig. 2 B ) show that p53 RNA levels are notably elevated at this activation interval. Because not all CD86 , CD27 B lymphoblasts uniformly divide in these cultures (36), we examined whether blasts representing 0 and 3– 4 divisions differ in p53 mRNA expression. Viable CFSE-labeled blasts were sorted according to division status, and cDNA was assessed by qPCR (Fig. 2 C ). Notably, blasts representing 3– 4 divisions display significantly greater p53 mRNA than undivided blasts ( P ϭ 0.006). Virtually all of these divided blasts express elevated AID protein (Fig. 1 A and ref. 35). One notable characteristic of cycling B lymphoblasts is an increase in COX-2 and linked proteins (34, 35, 44). The elevated p53 mRNA levels in cycling blasts and evidence of substantial crosstalk between the p53 and COX-2/PGE 2 axes in other cells (45–50) led us to examine whether PGE 2 signaling influences p53 expression. For these experiments, exogenous PGE 2 (50 nM), or vehicle alone, was pulsed into d 4 activated B cell cultures, and p53 RNA levels were subsequently assessed by qPCR. Following an early insignificant decline, PGE 2 -pulsed cultures showed significantly greater p53 mRNA than controls, with the peak increase at 8 h following PGE 2 ( P 0.0004; Fig. 3 A , B ). The PGE 2 mediated increase was noted, regardless of whether  -actin, GAPDH, RPS20, RPS13, or RPL27 was used as housekeeping reference gene (ref. 51 and Supplemental Fig. S1). Interestingly, a comparison of undivided and divided blasts from the same cultures for sensitivity to this PGE 2 effect (Fig. 3 C ) showed that divided blasts uniquely displayed PGE 2 -boosted p53 mRNA. The effects of PGE 2 on p53 protein appear to be considerably more complex, as might be expected, given the many mechanisms for post-translational regulation of p53 following cell stress and DNA damage (52). Dividing d 5 lymphoblasts display significantly greater levels of p53 protein when cultures are exposed to PGE 2 , ranging from 10 to 1000 nM, on d 2 and 4 after TI activation (Supplemental Fig. S2). Nevertheless, if TI-activated cultures receive a similar pulse with PGE 2 solely on d 4, p53 protein levels reproducibly decline by 24 h (data not shown). Additional work is needed to define the mechanisms involved. Given the rising expression of adenylate cyclase-activating PGE 2 receptors in cycling B cells (35), the drop in p53 protein following an acute pulse with p53 on d 4 may involve cAMP-facilitated Mdm2 ubiquitination of p53 protein (53). We were interested in assessing whether the PGE 2 driven increase in p53 mRNA involved EP2, the PGE 2 receptor that increases most significantly with succes- sive divisions (ref. 35 and Fig. 4 A ). This was accom- plished through testing the effects of a structural analog of PGE 2 that is highly selective for EP2, butaprost. Figure 4 B shows that d 4 activated cultures pulsed with butaprost from 2 to 30 h earlier express significantly greater levels of p53 mRNA than vehicle- pulsed cultures. The dose response in Fig. 4 C is consistent with butaprost’s significantly lesser affinity for EP2, as compared to PGE (54). A series of experiments was performed to discern whether the PGE 2 -mediated boost in p53 mRNA was explained by effects on transcription and/or mechanisms that stabilize message (55–57). As a first step, we asked whether RNA polymerase activity is needed for sustained p53 mRNA levels in proliferating d 4 cultures. Figure 5 shows that this is so: p53 mRNA levels dropped significantly following 8 h exposure to actinomycin D. In addition, treatment with actinomycin D just prior to the PGE 2 pulse abrogates the PGE 2 -induced increase in p53 mRNA. While the above results indicate that p53 transcription is required during the postpulse period, the PGE 2 mediated boost might reflect effects on mRNA stability, rather than transcription. To address this, mRNA decay rates were compared in PGE 2 -pulsed and parallel nonpulsed cultures, beginning at 8 h after the pulse ( Fig. 6 ). Any new transcription was blocked by treatment with actinomycin D. Pooled values from 4 replicate experiments show that the half-life of p53 message is ϳ 8 h. Notably, the decay rates (slope of plots over time) in PGE 2 ϩ and PGE 2 Ϫ cultures were not statistically different (Fig. 6). Thus, PGE 2 does not promote greater p53 mRNA stability. Unexpectedly, p53 mRNA levels in actinomycin D- treated PGE 2 ϩ cultures were consistently lower than those in parallel PGE 2 Ϫ cultures. This was most evidenced early after the block in RNA synthesis (Fig. 6). One possibility is that PGE 2 -induced signals concomi- tantly enhance synthesis of negative regulators of p53 transcript stability, e.g. , miR125a and miR125b (58), but further experiments are needed to resolve this. Single-cell p53-specific RT-PCR analyses were initiated for the purpose of discerning whether PGE 2 increases the proportion of cells with a transcriptionally active p53 gene or whether it uniformly elevates p53 mRNA transcription in all cells. Figure 7 A shows representative results from the PCR amplification of p53 and  -actin cDNA from 56 sorted individual B-cell progeny from TI cultures with or without PGE 2 . While ϳ 90% of the wells containing a single B cell scored positive for  -actin, the frequency of p53 ϩ wells was notably lower, indicating that individual progeny vary in their level of p53 transcripts. Of interest, the exogenous PGE 2 pulse notably augmented the proportion of p53 ϩ single cells (Fig. 7 B ). This PGE 2 -boosting effect was observed in each of 6 total experiments and was highly significant ( P Ͻ 0.001; Fig. 7 C ). Thus, PGE 2 clearly amplifies the proportion of B lymphoblasts producing relatively high levels of p53 mRNA. Since a threshold level of p53 mRNA may be necessary for p53 amplification (detec- tion), we cannot exclude the possibility that PGE 2 positively influences p53 mRNA transcription in all progeny. Multiple alternatively spliced transcripts of p53 are reported (59). Because identification of p53 mRNA ϩ cells in Fig. 7 involved primers for exons 2 and 4, we were concerned that the effects of PGE 2 might be restricted to certain splice forms. Nevertheless, results from an early experiment suggest otherwise. PGE 2 increased the frequency of p53 mRNA ϩ cells regardless of whether PCR amplification involved primers span- ning p53 exons 2– 4 (region A), the 3 = end of exon 4 through the 5 = end of exon 7 (region B), or exons 6 –11 (region C). Within this experiment, ratios for frequency of p53 ϩ cells with or without added PGE 2 using primers for regions A, B, and C were 1.8, 4.0, and 2.0, respectively. The prospect that elevated p53 transcription during a period of active replication and high AID expression might foster p53 mutations led us to sequence the purified p53 amplicons from each positive lymphoblast. We focused on upstream exons 2 through 4 because AID-induced mutations are more frequent near transcription start sites (60). This screen revealed p53 mutations, but only in the progeny of 2 of 6 experimen- tal cultures, each involving distinct B-cell donors ( Table 1 ). The total frequency of p53 mutations in cultures with or without exogenous PGE 2 (average of 6.2 ϫ 10 Ϫ 4 mutations/bp sequenced) was greater than an esti- mated background amplification error rate of Ͻ 0.8 ϫ 10 Ϫ 4 /bp in our assays (Table 1). Somewhat surprisingly, when divided progeny of PGE 2 -pulsed and control cultures were compared for p53 mutations, we found no evidence that p53 mutations were more frequent in cultures pulsed with PGE 2 (Table 1, columns 11–14). This was noted on the basis of mutations per total p53 mRNA ϩ cells sequenced (5/15 vs. 7/51 for nonpulsed vs. PGE -pulsed cultures, respectively) as well as mutations per total cells under study (Table 1, columns 13, 14). There is a plausible reason why PGE 2 -pulsed cultures did not reveal heightened p53 mutagenesis despite the concomitant elevation in AID protein and p53 transcription. Cells with PGE 2 -promoted DNA alterations (including p53 mutations) might have been excluded from analysis due to prior DNA damage-induced death. The data in Fig. 8 support this explanation. Notably, viable-gated blasts of PGE 2 -pulsed cultures expressed higher pH2AX (a measure of DNA double-strand breaks; ref. 24) than did control blasts, but only in the presence of pan-caspase inhibitor Z-VAD. This rein- forces past suggestions that PGE 2 promotes DNA damage in TI-activated B lymphoblasts (35) and shows that those with DNA double-strand breaks are prone to death. Thus, if p53-mutated cells were enriched in the subset with high levels of DNA damage, they would have been deleted, unless, of course, the mutation promoted survival. Next, we closely examined the type of p53 mutations in these TI cultures, both as a whole and with or without supplementary PGE 2 . Although numbers were limited, as seen in Table 2 , a majority of the total mutations are missense, resulting in ...
Context 2
... (within 6 h) following human B-cell activation with anti-IgM and growth factors (43). We were interested in determining whether p53 transcription remains high during a TI B-cell proliferative response characterized by high AID expression (35). To do so, p53 mRNA levels in freshly isolated naive B cells were compared with that in proliferating cultures on d 4 following TI activation. Semiquantitative PCR ( Fig. 2 A ) and qPCR (Fig. 2 B ) show that p53 RNA levels are notably elevated at this activation interval. Because not all CD86 , CD27 B lymphoblasts uniformly divide in these cultures (36), we examined whether blasts representing 0 and 3– 4 divisions differ in p53 mRNA expression. Viable CFSE-labeled blasts were sorted according to division status, and cDNA was assessed by qPCR (Fig. 2 C ). Notably, blasts representing 3– 4 divisions display significantly greater p53 mRNA than undivided blasts ( P ϭ 0.006). Virtually all of these divided blasts express elevated AID protein (Fig. 1 A and ref. 35). One notable characteristic of cycling B lymphoblasts is an increase in COX-2 and linked proteins (34, 35, 44). The elevated p53 mRNA levels in cycling blasts and evidence of substantial crosstalk between the p53 and COX-2/PGE 2 axes in other cells (45–50) led us to examine whether PGE 2 signaling influences p53 expression. For these experiments, exogenous PGE 2 (50 nM), or vehicle alone, was pulsed into d 4 activated B cell cultures, and p53 RNA levels were subsequently assessed by qPCR. Following an early insignificant decline, PGE 2 -pulsed cultures showed significantly greater p53 mRNA than controls, with the peak increase at 8 h following PGE 2 ( P 0.0004; Fig. 3 A , B ). The PGE 2 mediated increase was noted, regardless of whether  -actin, GAPDH, RPS20, RPS13, or RPL27 was used as housekeeping reference gene (ref. 51 and Supplemental Fig. S1). Interestingly, a comparison of undivided and divided blasts from the same cultures for sensitivity to this PGE 2 effect (Fig. 3 C ) showed that divided blasts uniquely displayed PGE 2 -boosted p53 mRNA. The effects of PGE 2 on p53 protein appear to be considerably more complex, as might be expected, given the many mechanisms for post-translational regulation of p53 following cell stress and DNA damage (52). Dividing d 5 lymphoblasts display significantly greater levels of p53 protein when cultures are exposed to PGE 2 , ranging from 10 to 1000 nM, on d 2 and 4 after TI activation (Supplemental Fig. S2). Nevertheless, if TI-activated cultures receive a similar pulse with PGE 2 solely on d 4, p53 protein levels reproducibly decline by 24 h (data not shown). Additional work is needed to define the mechanisms involved. Given the rising expression of adenylate cyclase-activating PGE 2 receptors in cycling B cells (35), the drop in p53 protein following an acute pulse with p53 on d 4 may involve cAMP-facilitated Mdm2 ubiquitination of p53 protein (53). We were interested in assessing whether the PGE 2 driven increase in p53 mRNA involved EP2, the PGE 2 receptor that increases most significantly with succes- sive divisions (ref. 35 and Fig. 4 A ). This was accom- plished through testing the effects of a structural analog of PGE 2 that is highly selective for EP2, butaprost. Figure 4 B shows that d 4 activated cultures pulsed with butaprost from 2 to 30 h earlier express significantly greater levels of p53 mRNA than vehicle- pulsed cultures. The dose response in Fig. 4 C is consistent with butaprost’s significantly lesser affinity for EP2, as compared to PGE (54). A series of experiments was performed to discern whether the PGE 2 -mediated boost in p53 mRNA was explained by effects on transcription and/or mechanisms that stabilize message (55–57). As a first step, we asked whether RNA polymerase activity is needed for sustained p53 mRNA levels in proliferating d 4 cultures. Figure 5 shows that this is so: p53 mRNA levels dropped significantly following 8 h exposure to actinomycin D. In addition, treatment with actinomycin D just prior to the PGE 2 pulse abrogates the PGE 2 -induced increase in p53 mRNA. While the above results indicate that p53 transcription is required during the postpulse period, the PGE 2 mediated boost might reflect effects on mRNA stability, rather than transcription. To address this, mRNA decay rates were compared in PGE 2 -pulsed and parallel nonpulsed cultures, beginning at 8 h after the pulse ( Fig. 6 ). Any new transcription was blocked by treatment with actinomycin D. Pooled values from 4 replicate experiments show that the half-life of p53 message is ϳ 8 h. Notably, the decay rates (slope of plots over time) in PGE 2 ϩ and PGE 2 Ϫ cultures were not statistically different (Fig. 6). Thus, PGE 2 does not promote greater p53 mRNA stability. Unexpectedly, p53 mRNA levels in actinomycin D- treated PGE 2 ϩ cultures were consistently lower than those in parallel PGE 2 Ϫ cultures. This was most evidenced early after the block in RNA synthesis (Fig. 6). One possibility is that PGE 2 -induced signals concomi- tantly enhance synthesis of negative regulators of p53 transcript stability, e.g. , miR125a and miR125b (58), but further experiments are needed to resolve this. Single-cell p53-specific RT-PCR analyses were initiated for the purpose of discerning whether PGE 2 increases the proportion of cells with a transcriptionally active p53 gene or whether it uniformly elevates p53 mRNA transcription in all cells. Figure 7 A shows representative results from the PCR amplification of p53 and  -actin cDNA from 56 sorted individual B-cell progeny from TI cultures with or without PGE 2 . While ϳ 90% of the wells containing a single B cell scored positive for  -actin, the frequency of p53 ϩ wells was notably lower, indicating that individual progeny vary in their level of p53 transcripts. Of interest, the exogenous PGE 2 pulse notably augmented the proportion of p53 ϩ single cells (Fig. 7 B ). This PGE 2 -boosting effect was observed in each of 6 total experiments and was highly significant ( P Ͻ 0.001; Fig. 7 C ). Thus, PGE 2 clearly amplifies the proportion of B lymphoblasts producing relatively high levels of p53 mRNA. Since a threshold level of p53 mRNA may be necessary for p53 amplification (detec- tion), we cannot exclude the possibility that PGE 2 positively influences p53 mRNA transcription in all progeny. Multiple alternatively spliced transcripts of p53 are reported (59). Because identification of p53 mRNA ϩ cells in Fig. 7 involved primers for exons 2 and 4, we were concerned that the effects of PGE 2 might be restricted to certain splice forms. Nevertheless, results from an early experiment suggest otherwise. PGE 2 increased the frequency of p53 mRNA ϩ cells regardless of whether PCR amplification involved primers span- ning p53 exons 2– 4 (region A), the 3 = end of exon 4 through the 5 = end of exon 7 (region B), or exons 6 –11 (region C). Within this experiment, ratios for frequency of p53 ϩ cells with or without added PGE 2 using primers for regions A, B, and C were 1.8, 4.0, and 2.0, respectively. The prospect that elevated p53 transcription during a period of active replication and high AID expression might foster p53 mutations led us to sequence the purified p53 amplicons from each positive lymphoblast. We focused on upstream exons 2 through 4 because AID-induced mutations are more frequent near transcription start sites (60). This screen revealed p53 mutations, but only in the progeny of 2 of 6 experimen- tal cultures, each involving distinct B-cell donors ( Table 1 ). The total frequency of p53 mutations in cultures with or without exogenous PGE 2 (average of 6.2 ϫ 10 Ϫ 4 mutations/bp sequenced) was greater than an esti- mated background amplification error rate of Ͻ 0.8 ϫ 10 Ϫ 4 /bp in our assays (Table 1). Somewhat surprisingly, when divided progeny of PGE 2 -pulsed and control cultures were compared for p53 mutations, we found no evidence that p53 mutations were more frequent in cultures pulsed with PGE 2 (Table 1, columns 11–14). This was noted on the basis of mutations per total p53 mRNA ϩ cells sequenced (5/15 vs. 7/51 for nonpulsed vs. PGE -pulsed cultures, respectively) as well as mutations per total cells under study (Table 1, columns 13, 14). There is a plausible reason why PGE 2 -pulsed cultures did not reveal heightened p53 mutagenesis despite the concomitant elevation in AID protein and p53 transcription. Cells with PGE 2 -promoted DNA alterations (including p53 mutations) might have been excluded from analysis due to prior DNA damage-induced death. The data in Fig. 8 support this explanation. Notably, viable-gated blasts of PGE 2 -pulsed cultures expressed higher pH2AX (a measure of DNA double-strand breaks; ref. 24) than did control blasts, but only in the presence of pan-caspase inhibitor Z-VAD. This rein- forces past suggestions that PGE 2 promotes DNA damage in TI-activated B lymphoblasts (35) and shows that those with DNA double-strand breaks are prone to death. Thus, if p53-mutated cells were enriched in the subset with high levels of DNA damage, they would have been deleted, unless, of course, the mutation promoted survival. Next, we closely examined the type of p53 mutations in these TI cultures, both as a whole and with or without supplementary PGE 2 . Although numbers were limited, as seen in Table 2 , a majority of the total mutations are missense, resulting in amino acid changes. These include changes in proline and cytosine that could alter protein structure. In addition, transition mutations (83% of total) significantly outnumber transversions under all conditions ( Fig. 9 and Table 3 ; P ϭ 0.04). Both the latter finding and several characteristics concerning mutation location and type are con- sistent with a role for AID in p53 mutagenesis within TI-activated clones. First, the majority of p53 mutations are localized at cytosine (C) or guanine (G) residues (50% for mutated cells in experiment 3 and 83% ...
Context 3
... that p53 reaches maximal expression at earlier divisions than AID (Fig. 1 B ) and suggests that AID-induced DNA damage, and ensuing p53 protein stabilization, do not solely explain the elevation in p53 protein. Transcription of p53 is reported to rise prior to proliferation (within 6 h) following human B-cell activation with anti-IgM and growth factors (43). We were interested in determining whether p53 transcription remains high during a TI B-cell proliferative response characterized by high AID expression (35). To do so, p53 mRNA levels in freshly isolated naive B cells were compared with that in proliferating cultures on d 4 following TI activation. Semiquantitative PCR ( Fig. 2 A ) and qPCR (Fig. 2 B ) show that p53 RNA levels are notably elevated at this activation interval. Because not all CD86 , CD27 B lymphoblasts uniformly divide in these cultures (36), we examined whether blasts representing 0 and 3– 4 divisions differ in p53 mRNA expression. Viable CFSE-labeled blasts were sorted according to division status, and cDNA was assessed by qPCR (Fig. 2 C ). Notably, blasts representing 3– 4 divisions display significantly greater p53 mRNA than undivided blasts ( P ϭ 0.006). Virtually all of these divided blasts express elevated AID protein (Fig. 1 A and ref. 35). One notable characteristic of cycling B lymphoblasts is an increase in COX-2 and linked proteins (34, 35, 44). The elevated p53 mRNA levels in cycling blasts and evidence of substantial crosstalk between the p53 and COX-2/PGE 2 axes in other cells (45–50) led us to examine whether PGE 2 signaling influences p53 expression. For these experiments, exogenous PGE 2 (50 nM), or vehicle alone, was pulsed into d 4 activated B cell cultures, and p53 RNA levels were subsequently assessed by qPCR. Following an early insignificant decline, PGE 2 -pulsed cultures showed significantly greater p53 mRNA than controls, with the peak increase at 8 h following PGE 2 ( P 0.0004; Fig. 3 A , B ). The PGE 2 mediated increase was noted, regardless of whether  -actin, GAPDH, RPS20, RPS13, or RPL27 was used as housekeeping reference gene (ref. 51 and Supplemental Fig. S1). Interestingly, a comparison of undivided and divided blasts from the same cultures for sensitivity to this PGE 2 effect (Fig. 3 C ) showed that divided blasts uniquely displayed PGE 2 -boosted p53 mRNA. The effects of PGE 2 on p53 protein appear to be considerably more complex, as might be expected, given the many mechanisms for post-translational regulation of p53 following cell stress and DNA damage (52). Dividing d 5 lymphoblasts display significantly greater levels of p53 protein when cultures are exposed to PGE 2 , ranging from 10 to 1000 nM, on d 2 and 4 after TI activation (Supplemental Fig. S2). Nevertheless, if TI-activated cultures receive a similar pulse with PGE 2 solely on d 4, p53 protein levels reproducibly decline by 24 h (data not shown). Additional work is needed to define the mechanisms involved. Given the rising expression of adenylate cyclase-activating PGE 2 receptors in cycling B cells (35), the drop in p53 protein following an acute pulse with p53 on d 4 may involve cAMP-facilitated Mdm2 ubiquitination of p53 protein (53). We were interested in assessing whether the PGE 2 driven increase in p53 mRNA involved EP2, the PGE 2 receptor that increases most significantly with succes- sive divisions (ref. 35 and Fig. 4 A ). This was accom- plished through testing the effects of a structural analog of PGE 2 that is highly selective for EP2, butaprost. Figure 4 B shows that d 4 activated cultures pulsed with butaprost from 2 to 30 h earlier express significantly greater levels of p53 mRNA than vehicle- pulsed cultures. The dose response in Fig. 4 C is consistent with butaprost’s significantly lesser affinity for EP2, as compared to PGE (54). A series of experiments was performed to discern whether the PGE 2 -mediated boost in p53 mRNA was explained by effects on transcription and/or mechanisms that stabilize message (55–57). As a first step, we asked whether RNA polymerase activity is needed for sustained p53 mRNA levels in proliferating d 4 cultures. Figure 5 shows that this is so: p53 mRNA levels dropped significantly following 8 h exposure to actinomycin D. In addition, treatment with actinomycin D just prior to the PGE 2 pulse abrogates the PGE 2 -induced increase in p53 mRNA. While the above results indicate that p53 transcription is required during the postpulse period, the PGE 2 mediated boost might reflect effects on mRNA stability, rather than transcription. To address this, mRNA decay rates were compared in PGE 2 -pulsed and parallel nonpulsed cultures, beginning at 8 h after the pulse ( Fig. 6 ). Any new transcription was blocked by treatment with actinomycin D. Pooled values from 4 replicate experiments show that the half-life of p53 message is ϳ 8 h. Notably, the decay rates (slope of plots over time) in PGE 2 ϩ and PGE 2 Ϫ cultures were not statistically different (Fig. 6). Thus, PGE 2 does not promote greater p53 mRNA stability. Unexpectedly, p53 mRNA levels in actinomycin D- treated PGE 2 ϩ cultures were consistently lower than those in parallel PGE 2 Ϫ cultures. This was most evidenced early after the block in RNA synthesis (Fig. 6). One possibility is that PGE 2 -induced signals concomi- tantly enhance synthesis of negative regulators of p53 transcript stability, e.g. , miR125a and miR125b (58), but further experiments are needed to resolve this. Single-cell p53-specific RT-PCR analyses were initiated for the purpose of discerning whether PGE 2 increases the proportion of cells with a transcriptionally active p53 gene or whether it uniformly elevates p53 mRNA transcription in all cells. Figure 7 A shows representative results from the PCR amplification of p53 and  -actin cDNA from 56 sorted individual B-cell progeny from TI cultures with or without PGE 2 . While ϳ 90% of the wells containing a single B cell scored positive for  -actin, the frequency of p53 ϩ wells was notably lower, indicating that individual progeny vary in their level of p53 transcripts. Of interest, the exogenous PGE 2 pulse notably augmented the proportion of p53 ϩ single cells (Fig. 7 B ). This PGE 2 -boosting effect was observed in each of 6 total experiments and was highly significant ( P Ͻ 0.001; Fig. 7 C ). Thus, PGE 2 clearly amplifies the proportion of B lymphoblasts producing relatively high levels of p53 mRNA. Since a threshold level of p53 mRNA may be necessary for p53 amplification (detec- tion), we cannot exclude the possibility that PGE 2 positively influences p53 mRNA transcription in all progeny. Multiple alternatively spliced transcripts of p53 are reported (59). Because identification of p53 mRNA ϩ cells in Fig. 7 involved primers for exons 2 and 4, we were concerned that the effects of PGE 2 might be restricted to certain splice forms. Nevertheless, results from an early experiment suggest otherwise. PGE 2 increased the frequency of p53 mRNA ϩ cells regardless of whether PCR amplification involved primers span- ning p53 exons 2– 4 (region A), the 3 = end of exon 4 through the 5 = end of exon 7 (region B), or exons 6 –11 (region C). Within this experiment, ratios for frequency of p53 ϩ cells with or without added PGE 2 using primers for regions A, B, and C were 1.8, 4.0, and 2.0, respectively. The prospect that elevated p53 transcription during a period of active replication and high AID expression might foster p53 mutations led us to sequence the purified p53 amplicons from each positive lymphoblast. We focused on upstream exons 2 through 4 because AID-induced mutations are more frequent near transcription start sites (60). This screen revealed p53 mutations, but only in the progeny of 2 of 6 experimen- tal cultures, each involving distinct B-cell donors ( Table 1 ). The total frequency of p53 mutations in cultures with or without exogenous PGE 2 (average of 6.2 ϫ 10 Ϫ 4 mutations/bp sequenced) was greater than an esti- mated background amplification error rate of Ͻ 0.8 ϫ 10 Ϫ 4 /bp in our assays (Table 1). Somewhat surprisingly, when divided progeny of PGE 2 -pulsed and control cultures were compared for p53 mutations, we found no evidence that p53 mutations were more frequent in cultures pulsed with PGE 2 (Table 1, columns 11–14). This was noted on the basis of mutations per total p53 mRNA ϩ cells sequenced (5/15 vs. 7/51 for nonpulsed vs. PGE -pulsed cultures, respectively) as well as mutations per total cells under study (Table 1, columns 13, 14). There is a plausible reason why PGE 2 -pulsed cultures did not reveal heightened p53 mutagenesis despite the concomitant elevation in AID protein and p53 transcription. Cells with PGE 2 -promoted DNA alterations (including p53 mutations) might have been excluded from analysis due to prior DNA damage-induced death. The data in Fig. 8 support this explanation. Notably, viable-gated blasts of PGE 2 -pulsed cultures expressed higher pH2AX (a measure of DNA double-strand breaks; ref. 24) than did control blasts, but only in the presence of pan-caspase inhibitor Z-VAD. This rein- forces past suggestions that PGE 2 promotes DNA damage in TI-activated B lymphoblasts (35) and shows that those with DNA double-strand breaks are prone to death. Thus, if p53-mutated cells were enriched in the subset with high levels of DNA damage, they would have been deleted, unless, of course, the mutation promoted survival. Next, we closely examined the type of p53 mutations in these TI cultures, both as a whole and with or without supplementary PGE 2 . Although numbers were limited, as seen in Table 2 , a majority of the total mutations are missense, resulting in amino acid changes. These include changes in proline and cytosine that could alter protein structure. In addition, transition mutations (83% of total) significantly outnumber transversions under all conditions ( Fig. 9 and Table 3 ; P ϭ 0.04). Both the latter finding and several ...
Citations
... It is important to note that, unlike cycling CD8 T cells, cycling B-CLL express elevated levels of activation-induced cytosine deaminase (AICDA) (118). Although the latter DNA-cleaving enzyme primarily targets Ig loci, it can also induce off-target mutations in B cells (119,120). ...
Clonal expansion of B cell chronic lymphocytic leukemia (B-CLL) occurs within lymphoid tissue pseudofollicles. IL-15, a stromal cell-associated cytokine found within spleens and lymph nodes of B-CLL patients, significantly boosts in vitro cycling of blood-derived B-CLL cells following CpG DNA priming. Both IL-15 and CpG DNA are elevated in microbe-draining lymphatic tissues, and unraveling the basis for IL-15-driven B-CLL growth could illuminate new therapeutic targets. Using CpG DNA-primed human B-CLL clones and approaches involving both immunofluorescent staining and pharmacologic inhibitors, we show that both PI3K/AKT and JAK/STAT5 pathways are activated and functionally important for IL-15→CD122/ɣc signaling in ODN-primed cells expressing activated pSTAT3. Furthermore, STAT5 activity must be sustained for continued cycling of CFSE-labeled B-CLL cells. Quantitative RT-PCR experiments with inhibitors of PI3K and STAT5 show that both contribute to IL-15-driven upregulation of mRNA for cyclin D2 and suppression of mRNA for DNA damage response mediators ATM, 53BP1, and MDC1. Furthermore, protein levels of these DNA damage response molecules are reduced by IL-15, as indicated by Western blotting and immunofluorescent staining. Bioinformatics analysis of ENCODE chromatin immunoprecipitation sequencing data from cell lines provides insight into possible mechanisms for STAT5-mediated repression. Finally, pharmacologic inhibitors of JAKs and STAT5 significantly curtailed B-CLL cycling when added either early or late in a growth response. We discuss how the IL-15-induced changes in gene expression lead to rapid cycling and possibly enhanced mutagenesis. STAT5 inhibitors might be an effective modality for blocking B-CLL growth in patients.
... In this study, single cells were sorted by flow cytometry into polymerase chain reaction (PCR) tubes, and then lysed by adding lysing buffer in the same PCR tube, with subsequent direct conversion of isolated RNA into cDNA. The p53 mRNA expression was confirmed by using the obtained cDNA template in a two steps PCR [11]. Since p53 is present at low copy number in the cell, two rounds of PCR amplification were necessary for detection in this so-called two step PCR method. ...
Exosomes are cell derived lipid nanoparticle with a size of 30-100 nm in diameter, found in almost all biological fluids. The composition of the exosomes is mainly lipid, proteins, RNA, DNA, and non-coding RNAs. Currently, most available methods and commercial kits for exosomal-RNA (Exo-RNA) isolation have limitations and shortcomings. Small starting volume of exosomes and the use of extraction/filtration columns results usually insufficient yield of exosomal RNA after isolation. The majority of RNA contained in purified exosomes range in size from 15-500 nucleotides. Some RNA isolation kits are well suited for small RNA transcripts isolation but larger mRNA transcripts are hard to detect. For all of the kits, the cost prize per sample analyzed is very high. Our current method provides a novel way for direct conversion of exosomes into cDNA synthesis (Exo-cDNA) and subsequent gene detection by polymerase chain reaction (PCR). This method has several advantages compared to established available kits. No extraction column is utilized in this procedure which means total recovery of exosomal RNA with maximal yield. In addition, this method is fast and uses a minimal amount of lab supplies, thereby reducing the overall working costs. Our findings suggest that direct conversion of exosomes into cDNA and subsequent gene amplification by two step PCR is a most efficient and reproducible technique. This novel method can be applied to and is useful to advance molecular research of exosomes by solving the problem of low molecular yields.
... For example, Prostaglandin E2 (PGE2) is a downstream metabolite of the AA pathway and could induce apoptosis in a dose-dependent manner [18,19]. Specifically, PGE2 can increase P53 transcriptional activity by binding with the subtype receptor EP2, thereby inducing apoptosis [20]. Activation of EP2/EP4 receptors by PGE2 can also promote apoptosis by increasing expressions of FasL, cas-pase8 and caspase9 [21]. ...
Background
Qi-shen-yi-qi (QSYQ), one of the most well-known traditional Chinese medicine (TCM) formulas, has been shown to have cardioprotective effects in rats with heart failure (HF) induced by acute myocardial infarction (AMI). However, the mechanisms of its therapeutic effects remain unclear. In this study, we aim to explore the mechanisms of QSYQ in preventing left ventricular remodelling in rats with HF. The anti-apoptosis an anti-inflammation effects of QSYQ were investigated.
Methods
Sprague–Dawley (SD) rats were randomly divided into 4 groups: sham group, model group, QSYQ treatment group and aspirin group. Heart failure model was induced by ligation of left anterior descending (LAD) coronary artery. 28 days after surgery, hemodynamics were detected. Echocardiography was adopted to evaluate heart function. TUNEL assay was applied to assess myocardial apoptosis rates. Protein expressions of cyclooxygenase1 and 2 (COX1and COX2), Fas ligand (FasL), P53 and MDM2 were measured by western-blot. RT-PCR was applied to detect expressions of our subtype receptors of PGE2 (EP1, 2, 3, and 4).
Results
Ultrasonography showed that EF and FS values decreased significantly and abnormal hemodynamic alterations were observed in model group compared to sham group. These indications illustrated that HF models were successfully induced. Levels of inflammatory cytokines (TNF-α and IL-6) in myocardial tissue were up-regulated in the model group as compared to those in sham group. Western-blot analysis showed that cyclooxygenase 2, which is highly inducible by inflammatory cytokines, increased significantly. Moreover, RT-PCR showed that expressions of EP2 and EP4, which are the receptors of PGE2, were also up-regulated. Increased expressions of apoptotic pathway factors, including P53 and FasL, might be induced by the binding of PGE2 with EP2/4. MDM2, the inhibitor of P53, decreased in model group. TUNEL results manifested that apoptosis rates of myocardial cells increased in the model group. After treatment with QSYQ, expressions of inflammatory factors, including TNF-α, IL-6 and COX2, were reduced. Expressions of EP2 and EP4 receptors also decreased, suggesting that PGE2-mediated apoptosis was inhibited by QSYQ. MDM2 was up-regulated and P53 and FasL in the apoptotic pathway were down-regulated. Apoptosis rates in myocardial tissue in the QSYQ group decreased compared with those in the model group.
Conclusions
QSYQ exerts cardiac protective efficacy mainly through inhibiting the inflammatory response and down-regulating apoptosis. The anti-inflammatory and anti-apoptosis efficacies of QSYQ are probably achieved by inhibition of COXs-induced P53/FasL pathway. These findings provide experimental evidence for the beneficial effects of QSYQ in the clinical application for treating patients with HF.
... During numerous cycles of proliferation, new mutations can be generated through oxidation, enzymatic activity of activation-induced cytosine deaminase, and/or chemotherapy, even in normal human B cells (151). Thus, it is expected that a B-CLL population's greater intrinsic potential for growth, facilitated by certain common chromosomal anomalies, will promote the continued generation of further pathogenic variants. ...
Clinical progression of B cell chronic lymphocytic leukemia (B-CLL) reflects the clone's Ag receptor (BCR) and involves stroma-dependent B-CLL growth within lymphoid tissue. Uniformly elevated expression of TLR-9, occasional MYD88 mutations, and BCR specificity for DNA or Ags physically linked to DNA together suggest that TLR-9 signaling is important in driving B-CLL growth in patients. Nevertheless, reports of apoptosis after B-CLL exposure to CpG oligodeoxynucleotide (ODN) raised questions about a central role for TLR-9. Because normal memory B cells proliferate vigorously to ODN+IL-15, a cytokine found in stromal cells of bone marrow, lymph nodes, and spleen, we examined whether this was true for B-CLL cells. Through a CFSE-based assay for quantitatively monitoring in vitro clonal proliferation/survival, we show that IL-15 precludes TLR-9-induced apoptosis and permits significant B-CLL clonal expansion regardless of the clone's BCR mutation status. A robust response to ODN+IL-15 was positively linked to presence of chromosomal anomalies (trisomy-12 or ataxia telangiectasia mutated anomaly + del13q14) and negatively linked to a very high proportion of CD38(+) cells within the blood-derived B-CLL population. Furthermore, a clone's intrinsic potential for in vitro growth correlated directly with doubling time in blood, in the case of B-CLL with Ig H chain V region-unmutated BCR and <30% CD38(+) cells in blood. Finally, in vitro high-proliferator status was statistically linked to diminished patient survival. These findings, together with immunohistochemical evidence of apoptotic cells and IL-15-producing cells proximal to B-CLL pseudofollicles in patient spleens, suggest that collaborative ODN and IL-15 signaling may promote in vivo B-CLL growth.
Copyright © 2015 by The American Association of Immunologists, Inc.
... 48,49 For instance, COX-2 overexpression is associated with worse survival among colon cancer patients. 50 This effect of COX-2 on clinical outcome may be modified by p53 status. 49 In addition, the cyclooxygenase product PGE2, via the EP2 PGE 2 receptor, displays heightened p53 transcription and increased risk of p53 mutagenesis. ...
... 49 In addition, the cyclooxygenase product PGE2, via the EP2 PGE 2 receptor, displays heightened p53 transcription and increased risk of p53 mutagenesis. 50 As a result, COX-2 inhibition has the potential to decrease p53 and tumour formation. ...
There are increasing links between cardiovascular disease and cancer, starting with optimal lifestyle, including diet, and extending to statin-induced reductions in the incidences of both cardiovascular disease and cancer. Furthermore, there are plausible mechanisms that are being explored.
CLL B cells express elevated pro-survival BCL2, and its selective inhibitor, venetoclax, significantly reduces leukemic cell load, leading to clinical remission. Nonetheless, relapses occur. This study evaluates the hypothesis that progressively diminished BCL2 protein in cycling CLL cells within patient lymph node niches contributes to relapse. Using CFSE-labeled, purified CLL populations known to respond with vigorous cycling in d6 cultures stimulated with TLR9-activating ODN (oligodeoxynucleotide) + IL15, we show that BCL2 protein progressively declines during consecutive cell divisions. In contrast, MCL1 and survivin are maintained/slightly elevated during cycling. Delayed pulsing of quiescent and activated CLL cultures with selective inhibitors of BCL2 or survivin revealed selective targeting of noncycling and cycling populations, respectively, raising implications for therapy. To address the hypothesis that BCL2-repressive miRs (miR15a/miR16-1), encoded in Chr13, are mechanistically involved, we compared BCL2 protein levels within ODN + IL15-stimulated CLL cells, with/without del(13q), yielding results suggesting these miRs contribute to BCL2 reduction. In support, within ODN-primed CLL cells, an IL15-driven STAT5/PI-3K pathway (required for vigorous cycling) triggers elevated p53 TF protein known to directly activate the miR15a/miR16-1 locus. Furthermore, IL15 signaling elicits the repression of BCL2 mRNA within 24 h. Additional comparisons of del(13q)+ and del(13q)−/− cohorts for elevated p53 TF expression during cycling suggest that a documented miR15a/miR16-1-mediated negative feedback loop for p53 synthesis is active during cycling. Findings that robust CLL cycling associates with progressively decreasing BCL2 protein that directly correlates with decreasing venetoclax susceptibility, combined with past findings that these cycling cells have the greatest potential for activation-induced cytosine deaminase (AICDA)-driven mutations, suggest that venetoclax treatment should be accompanied by modalities that selectively target the cycling compartment without eliciting further mutations. The employment of survivin inhibitors might be such an approach.
Simple Summary
Cancer is an incredibly smart disease, so much so that it can entirely reprogram our cells’ metabolism to ensure its survival and dissemination. Lymphomas are a particularly interesting group of cancers because they are very diverse and exhibit complex pathogenic mechanisms that can make them clinically challenging. Many of these mechanisms involve the dysregulation of a biologically relevant family of lipids known as sphingolipids. These molecules are involved in virtually every cellular process and therefore studying them in this context is crucial. However, sphingolipid biochemistry is intricate and the synergistic and antagonic effects of different sphingolipid species with one another has long puzzled scientists. This duality is also observed in cancer, but research specifically focusing on lymphomas is limited. Could novel biomarkers and therapies against lymphomas be hiding within this pathway?
Abstract
Lymphomas are a highly heterogeneous group of hematological neoplasms. Given their ethiopathogenic complexity, their classification and management can become difficult tasks; therefore, new approaches are continuously being sought. Metabolic reprogramming at the lipid level is a hot topic in cancer research, and sphingolipidomics has gained particular focus in this area due to the bioactive nature of molecules such as sphingoid bases, sphingosine-1-phosphate, ceramides, sphingomyelin, cerebrosides, globosides, and gangliosides. Sphingolipid metabolism has become especially exciting because they are involved in virtually every cellular process through an extremely intricate metabolic web; in fact, no two sphingolipids share the same fate. Unsurprisingly, a disruption at this level is a recurrent mechanism in lymphomagenesis, dissemination, and chemoresistance, which means potential biomarkers and therapeutical targets might be hiding within these pathways. Many comprehensive reviews describing their role in cancer exist, but because most research has been conducted in solid malignancies, evidence in lymphomagenesis is somewhat limited. In this review, we summarize key aspects of sphingolipid biochemistry and discuss their known impact in cancer biology, with a particular focus on lymphomas and possible therapeutical strategies against them.
DNMT1 (DNA-methyltransferase 1) is an enzyme which contributes in the process of normal embryonic development, and aberrant expression of DNMT1 leads to tumor/leukemia progression by significant change in DNA methylation and epigenetics. We found that DNMT1 mRNA transcript is elevated in Exo-PALL compared to Exo-HD. We also confirmed and showed heightened level of DNMT1 mRNA transcript in Exo-CM of leukemia cell lines. Co-culture of Exo-PALL with target cells showed transfer of exosomal DNMT1 mRNA transcript into the target cells, which may reprogram biological nature of normal healthy cells and leukemia cells.
Thousands of dollars are spent today with policies encouraging physical activity and healthy eating, but nutritional consultation per se has continuously failed to yield consistent and lasting results. The aim of this case report is to detail and evaluate nutritional coaching (employing health coaching techniques) in promoting lifestyle changes, enabling improvement of nutritional and body composition associated parameters. The patient in this study had previously engaged in a series of different diet regimens, all of which failed in achieving the proposed aim. After 12 nutritional coaching sessions (one per week) with the strategy presented herein, reductions in body fat mass and in total body weight were attained. Nutritional habits also improved, as the patient showed decreased total energy intake, decreased fat intake, and increased fiber ingestion. Daily physical activity and energy expenditure were enhanced. The coaching program was able to induce immediate health benefits using a strategy with the patient at the core of promoting his own lifestyle changes. In conclusion, the nutritional coaching strategy detailed was effective at helping our patient develop new eating patterns and improve related health parameters.
