Aberrant expression of the c-myc immediate-early gene (IEG) has been shown to play a causal role in the development of several cancers. Another IEG is the CC-chemokine gene MCP-1, whose protein product has been suggested to be a potentially effective agent in """"""""tumor vaccination"""""""" efforts. Strategies designed to alter transcription of IEGs might, therefore, be effective in the future treatment of cancer via at least two different mechanisms. A major inducer of MCP-1 transcription is platelet-derived growth factor (PDGF). We have shown that PDGF-mediated induction of MCP-1 requires a 240bp fragment present in the distal 5' flanking sequences of the MCP-1 gene.
One aim of this proposal focuses on the major enhancer element present in the 240bp fragment (to be called element IV herein). Element IV binds both a PDGF-activated form of NF-kappaB, and a previously unidentified 90 kDa phosphoprotein (p9O). Binding of both NF-kappaB and p90 to element IV are required for optimum PDGF induction of MCP-1, suggesting that p90 is a transcriptional coactivator with NF-kappaB in PDGF-mediated induction of MCP-1. We have screened a cDNA expression library, using a probe corresponding to the unique p90 binding sequence, and have obtained a fragment of a candidate p90 gene. Planned studies include in vitro translation of full-length p90, and truncated forms of p90, in order to study the DNA binding properties/domains of p90. The effects of overexpression of a transfected antisense p90 construct on MCP-1 induction, and c-myc induction, will be used as one means to study the functions of p90 directly.
The second aim of this proposal is based on our finding that PDGF induction of MCP-1 requires the 7 nucleotide motif, TTTTGTA, present in the proximal 3' untranslated sequences of numerous IEGs. More recently we have demonstrated PDGF-regulated interactions between this 7-mer and two distinct classes of """"""""inhibitory"""""""" DNA control elements. The net effects of the 7-mer/inhibitory element interactions are to remove a block to PDGF-stimulated induction of MCP-1. We will use DNase I footprinting to delineate the sequences of one class of inhibitory element. DNA-protein mobility shift assays will detect and characterize the nuclear regulatory protein(s) binding to the inhibitory element. These studies are a necessary prelude to obtaining the gene(s) coding for the inhibitory element-binding protein(s). A dual DNA control element system like this has not been described previously for this clinically important group of genes.
| Sridhar, P; Liu, Y; Chin, L D et al. (1999) Platelet-derived growth factor-stimulated expression of the MCP-1 immediate-early gene involves an inhibitory multiprotein complex. Mol Cell Biol 19:4219-30 |
