Abstract 2101: Comparison of transcriptional response to phorbol ester, bryostatin 1, and bryostatin analogue in LNCaP and U937 cells provides insight into their differential mechanism.

Bryostatin 1 (bryo 1) is a promising cancer chemotherapeutic agent with a unique mechanism of action. Whereas bryo 1 binds to and activates protein kinase C (PKC) like the phorbol esters, it paradoxically antagonizes most phorbol ester responses. Previously, we compared the changes in transcription in response to the phorbol ester phorbol 12-myristate 13-acetate (PMA), to bryo 1, and to the synthetic bryo 1 derivative Merle 23 in the human cancer cell lines LNCaP and U937. Bryo1 failed to induce a typical phorbol ester biological response in either cell line, whereas the bryo 1 analog Merle 23 resembled PMA in the U937 cells and bryo 1 in the LNCaP cells. In both cell lines, bryo 1 differed from PMA in inducing a shorter duration of the transcriptional responses for a panel of PKC-regulated genes as quantitated by qPCR. Merle 23, in contrast, induced a pattern very similar to that of PMA. Here, we analyzed by microarray the genome wide transcriptional changes in LNCaP and U937 cells treated with maximally effective doses of PMA, bryo 1 and Merle 23 for 6 and 8 hours, respectively. Several thousand genes were significantly up- or down-regulated by PMA (3131 and 3206), bryo 1 (1569 and 2454) and Merle 23 (3533 and 3207) compared to control in LNCaP and U937 cells, respectively. As seen previously for selected genes, the magnitude of the changes was much greater for LNCaP cells than for U937 cells. The great majority of the genes followed a pattern in which bryo 1 induced changes similar to PMA but to a significantly smaller extent (3096 and 1610 genes significantly different in LNCaP and U937 cells), whereas Merle 23 was very similar to PMA (only 230 and 30 genes different between them). A detailed qPCR analysis (dose and time courses) of representative outlier genes revealed that the shorter duration of bryo 1 induced response was responsible for the differences between bryo 1 and PMA effect in all cases. The dose response experiments did not reveal a difference in bryo 1 potency linked to any specific pattern of response. Analysis of transcription factors revealed overrepresentation of the same families of transcription factors (13 in the LNCaP cells; 12 in the U937 cells) among the genes differentially regulated by PMA and bryo 1. These included AP2F, E2F, EGRF, KLF, NRF1, SP1F. However, the target genes of the transcription factor families were largely different between the two cell lines. The analysis of 45 signaling pathways using reporter assays identified significant changes in 7 pathways induced both by PMA and bryo 1 at 6 hours. While the analysis of the microarray data is ongoing, we conclude that early turn-off of the induced responses by bryo 1 is the central feature accounting for its difference from PMA in its transcriptional response. Understanding the basis for this transiency of response may reveal a more general strategy for inhibition of PKC pathways. Citation Format: Noemi Kedei, Aleksandra M. Michalowski, Mark E. Petersen, Gary E. Keck, Peter M. Blumberg. Comparison of transcriptional response to phorbol ester, bryostatin 1, and bryostatin analogue in LNCaP and U937 cells provides insight into their differential mechanism. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2101. doi:10.1158/1538-7445.AM2013-2101