No abstract

Elevated expression of glial fibrillary acidic protein (GFAP) is associated with astrocyte activation during responses to injury in the CNS. Because transforming growth factor-␤1 (TGF-␤1) and interleukin-1␤ (IL-1␤) are released during neural responses to injury and because these cytokines also modulate GFAP mRNA levels, it is of interest to define their role in GFAP transcription. The increases of GFAP mRNA in response to TGF-␤1 and decreases in response to IL-1␤ were shown to be transcriptionally mediated in rat astrocytes transfected with a luciferase-reporter construct containing 1.9 kb of 5Ј-upstream rat genomic DNA. Constructs containing sequential deletions of the rat GFAP 5Ј-upstream promoter identified a short region proximal to the transcription start (Ϫ106 to Ϫ53 bp) that provides full responses to TGF-␤1 and IL-1␤. This region contains an unusual sequence motif with overlapping nuclear factor-1 (NF-1)-and nuclear factor-B (NF-B)-like binding sites and homology to known TGF-␤ response elements. Mutagenesis (3-bp exchanges) in Ϫ70 to Ϫ68 bp blocked the induction of GFAP by TGF-␤1 and the repression by IL-1␤. Gel shift experiments showed that the DNA segment Ϫ85 to Ϫ63 bp was bound by a factor(s) in nuclear extracts from astrocytes. The concentrations of these DNA binding factors were increased by treatment of astrocytes with TGF-␤1 and decreased by IL-1␤. Binding of these nuclear factors was blocked by mutation of Ϫ70 to Ϫ68 bp. Despite homology to NF-1 or NF-B binding sites in the GFAP promoter at segment Ϫ79 to Ϫ67 bp, anti-NF-B or anti-NF-1 antibodies did not further retard the gel shift of the nuclear factors/DNA complex. Moreover, astrocytic nuclear proteins do not compete for the specific binding to NF-1 consensus sequence. Thus, nuclear factors from astrocytes that bind to the Ϫ85-to Ϫ63-bp promoter segment might be only distantly related to NF-1 or NF-B. These findings are pertinent to the use of GFAP promoter constructs in transgenic animals, because cisacting elements in the GFAP promoter are sensitive to cytokines that may be elaborated in response to expression of transgene products. Key Words: Glial fibrillary acidic protein promoter-Transforming growth factor-␤1-Interleukin-1␤-Nuclear factor-1 binding site.

BSO (75 gram/m(2) ) with melphalan (125 mg/m(2) ) is tolerable with stem cell support and active in recurrent/refractory neuroblastoma. Further dose escalation is feasible and may increase responses.