When PEL cells are treated with 5-aza-2-dc, how does it affect PDLIM2 expression
ID: 210814 • Letter: W
Question
When PEL cells are treated with 5-aza-2-dc, how does it affect PDLIM2 expression? Pick a panel in figure 4 that helps to support your reasoning. In your explanation describe with bars or lines in the panel you picked help to support your reasoning.
PDLIM2 Repression Involves Its Promoter Methylation and Can Be Reversed by 5-aza-dC and Vitamin D to Inhibit Growth of KSHV-associated Tumor Cells—Given the role of PDLIM2 in KSHV pathogenesis and its potential application in the therapy of KSHV-associated tumors, it is of interest and importance to determine the molecular mechanism by which KSHV represses PDLIM2 expression. Our previous studies showed that PDLIM2 is epigenetically repressed in several cancer types (37– 39). Thus, we examined the methylation status of the pdlim2 promoter in PEL cells. We found that the pdlim2 promoter was indeed hyper-methylated in PEL cells, which could be reversed by the demethylation drug 5-aza-dC (Fig. 4A). Moreover, the treatment of 5-aza-dC resulted in significant recovery of PDLIM2 expression in the PEL cells (Fig. 4B). Interestingly, 5-aza-dC had a dose-dependent effect on PDLIM2 recovery at a concentration lower than 0.5 M. These data are consistent with the fact that 5-aza-dC induces expression of methylated genes at low doses and mainly exerts its cytotoxicity through inducing DNA damage at high doses. To determine how KSHV induces PDLIM2 promoter hyper-methylation for its repression, we examined the expression of all three DNA methyltransferases in PEL cells. We found that DNMT3a, but not DNMT1 or DNMT3b, was dramatically induced in BCBL-1 and BC-1 cells when compared with the virus-free BJAB cells (Fig. 4C). These data suggest that KSHV represses PDLIM2 expression through inducing DNMT3a expression and pdlim2 promoter methylation. A recent study showed that vitamin D can induce PDLIM2 re-expression in human breast cancer cells through both DNA methylation-dependent and DNA methylation-independent mechanisms (50). As expected, 1,25(OH)2D3, the biologically active form of vitamin D3, induced PDLIM2 re-expression in PEL cells (Fig. 4D). Interestingly, 5-aza-dC and 1,25(OH)2D3 could synergize in inducing PDLIM2 re-expression in PEL cells (Fig. 4E). Of note, re-induction of PDLIM2 by 5-aza-dC and 1,25(OH)2D3 was associated with decreased transcriptional activity of NF-B and STAT3, as well as growth inhibition of PEL cells (Fig. 4, F and G). To determine whether PDLIM2 re-induction is involved in the antitumor activity of 5-aza-dC and 1,25(OH)2D3, we generated PEL cells stably expressing PDLIM2 shRNA, in which PDLIM2 re-induction by 5-aza-dC and 1,25(OH)2D3 could be sufficiently blocked. Remarkably, the growth inhibition of PEL cells by 5-aza-dC and 1,25(OH)2D3 was significantly inhibited by PDLIM2 knockdown (Fig. 4H). These data not only provide the mechanistic insights into the KSHV-mediated repression of PDLIM2, but also suggest an immediate strategy to target PDLIM2 for the prevention and treatment of KSHV-associated tumors.
FIGURE 4. PDLIM2 repression by KSHV involves its promoter methylation and can be reversed by 5-aza-2-dC and vitamin D to inhibit growth of KSHV-associated tumor cells. A, bisulfite genomic DNA sequencing showing epigenetic repression of PDLIM2 in BCBL-1 cells. Each circle represents a CpG site, and the ratio of the filled area in each circle represents the percentile of methylation in the CpG site. The position of each CpG nucleotide relative to the PDLIM2 transcription initiation site (1) is indicated at the side. 5-aza-dC treatment: 0.2 M for 4 days. DMSO, dimethyl sulfoxide. B, qPCR analyses showing dose- and time-dependent re-induction of PDLIM2 by 5-aza-dC in BCBL-1 cells. 5-aza-dC treatment: left, 4 days; right, 0.2 M. C, qPCR analyses showing increased RNA level of DNMT3a, but not DNMT1 or DNMT3b in PEL cells. D, qPCR analyses showing dose- and time-dependent re-induction of PDLIM2 by vitamin D in BCBL-1 cells. 1,25(OH)2D3 treatment: left, 4 days; right, 0.1 M. E, qPCR analyses showing cooperation of 5-aza-dC and vitamin D in PDLIM2 re-induction in BCBL-1 cells. F, gene reporter assays showing inhibition of transcriptional activity of NF-B and STAT3 by 5-aza-dC and vitamin D in BCBL-1 cells. G, cell counting assays showing synergistic growth inhibition of BCBL-1 cells by 5-aza-dC and vitamin D. H, cell counting assays showing that growth inhibition of BCBL-1 cells by 5-aza-dC and/or vitamin D (3-day treatment) can be blocked by PDLIM2 knockdown. For E–H, 5-aza-dC, 0.05 M; 1,25(OH)2D3, 0.1 M. Data are mean S.D. *, p 0.05; **, p 0.01.
5-aza kx 160 BC-1 OBJAB ** 1,25(0H)2D3 BCBL- 1-1011 . 12 E -1009 G3 988 G02 1-959 928 o-923 1-915 -o FO DMSO ODMSO H vector 5-aza-dC -o-5-aza-dC 01,25(OH)2D3 .shPDLIM2 E 1,25(OH)2D3 120r Combination a, 11,25(OH)2D3 Combination 0T -Combination 10 2 10 1,25(0H)2D3 25("Con Day 3 NF-KB STAT3 5-aza-dC Day0 1 2 3 4 5 Combination 3 3 1 1 4 -d 208642 543210 5 7195898354 2100852218 0000999998Explanation / Answer
From the given data Figure B and Figure D gives the complete analysis that 5-azadC shows effect and good results on PDLIM2 and also showd that they could get re expressed in PEL cells.
Interestingly, 5-aza-dC had a dose-dependent effect on PDLIM2 recovery at a concentration lower than 0.5 M. These data are consistent with the fact that 5-aza-dC induces expression of methylated genes at low doses and mainly exerts its cytotoxicity through inducing DNA damage at high doses. To determine how KSHV induces PDLIM2 promoter hyper-methylation for its repression, we examined the expression of all three DNA methyltransferases in PEL cells. We found that DNMT3a, but not DNMT1 or DNMT3b, was dramatically induced in BCBL-1 and BC-1 cells when compared with the virus-free BJAB cells . These data suggest that KSHV represses PDLIM2 expression through inducing DNMT3a expression and pdlim2 promoter methylation. A recent study showed that vitamin D can induce PDLIM2 re-expression in human breast cancer cells through both DNA methylation-dependent and DNA methylation-independent mechanisms . As expected, 1,25(OH)2D3, the biologically active form of vitamin D3, induced PDLIM2 re-expression in PEL cells.
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