We previously discovered that the natural product byrostatm (bryo), a potent acm ator of protein kmaseC (PKC), stimulates the protifcraiion of both adult human and mouse hematopoietic cells. To study the effects of bryo on developmental hcmatopoiesis. we examined the proliferat-ve potential and lineage commitment of munne ES cells in a twostep culture system. ES cells (6000/mI) were deprived of LIF factor and incubated with various concentrations of bryo( 10-10 I3M) in primary (1 ) methyîcellulose cultures for 6 days to form embryoid bodies <EB) EB were disrupted and IxlO5 cells/ml were plated in secondary (2C) methylccllulose cultures with or without bryo m the presence of purified munnc hematopoietic growth factors (SCF, IL- la, IL-3, GM-CSF) and EPÖ for another 7 days Results showed that when 10 M bryo was present in either the I:> or both 1!1 and 2 cultures, a significant increase m the BFU-c from 6± 2.2 to 16 3 3 1, CFU-GEMM from 5.5 ±I 9 to 24.3 ±4 6 andCFU-GM from 57 8 ±10.9 to 146.3 ±25 3 colonies was observed, indicating 3 stimulation of both early multipotential and lineage restricted colonies Interestingly, it was only necessary to add bryo in the 1 ' cultures to observe enhanced colony stimulation, indicating a potential requirement for stimulation of PKC during the early developmental stages injvitES- To determine which PKC isoforms may be activated, ES cells were screened for expression using isoform specific antibodies Results demonstrate that PKCcc. 0 and e were down regulated, while pi, C/i were not affected by bryo and βll and v isoforms were not expressed in ES cells. Collectively, these data indicate that ES cells are developmcntally sensitive to bryo during m vitro hematopoiesis and specific PKC isoforms may represent the targets) for the agonist. Thus, bryo represents a useful agent for studying the molecular/biochemical signaling during developmental hematopoiesis.
|Original language||English (US)|
|Number of pages||1|
|State||Published - Dec 1 1996|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology
- Cancer Research