REGULATION OF TRANSFORMING-GROWTH-FACTOR-ALPHA EXPRESSION IN A GROWTHFACTOR-INDEPENDENT CELL-LINE

Aberrant transcriptional regulation of transforming growth factor alph a (TGF alpha) appears to be an important contributor to the malignant phenotype and the growth factor independence with which malignancy is frequently associated. However, little is known about the molecular me chanisms responsible for dysregulation of TGF alpha expression in the malignant phenotype, In this paper, we report on TGF alpha promoter re gulation in the highly malignant growth factor-independent cell line H CT116. The HCT116 cell line expresses TGF alpha and the epidermal grow th factor receptor (EGFR) but is not growth inhibited by antibodies to EGFR or TGF alpha. However, constitutive expression of TGF alpha anti sense RNA in the HCT116 cell line resulted in the isolation of clones with markedly reduced TGF alpha mRNA and which were dependent on exoge nous growth factors for proliferation, We hypothesized that if TGF alp ha autocrine activation is the major stimulator of TGF alpha expressio n in this cell line, TGF alpha promoter activity should be reduced in the antisense TGF alpha clones in the absence of exogenous growth fact or, This was the case, Moreover, transcriptional activation of the TGF alpha promoter was restored in an antisense-TGF alpha-mRNA-expressing clone which had reverted to a growth factor-independent phenotype, Us ing this model system, we were able to identify a 25-bp element within the TGF alpha promoter which conferred TGF alpha autoregulation to th e TGF alpha promoter in the HCT116 cell line. In the TGF alpha-antisen se-RNA-expressing clones, this element was activated by exogenous EGF, This 25-bp sequence contained no consensus sequences of known transcr iption factors so that the TGF alpha or EGF regulatory element within this 25-bp sequence represents a unique element, Further characterizat ion of this 25-bp DNA sequence by deletion analysis revealed that regu lation of TGF alpha promoter activity by this sequence is complex, as both repressors and activators bind in this region, but the overall ex pression of the activators is pivotal in determining the level of resp onse to EGF or TGF alpha stimulation, The specific nuclear proteins bi nding to this region are also regulated in an autocrine-TGF alpha-depe ndent fashion and by exogenous EGF in EGF-deprived TGF alpha antisense clone 33, This regulation is identical to that seen in the growth fac tor-dependent cell line FET, which requires exogenous EGF for optimal growth. Moreover, the time response of the stimulation of trans-acting factor binding by EGF suggests that the effect is directly due to gro wth factor and not mediated by changes in growth state, We conclude th at this element appears to represent the major positive regulator of T GF alpha expression in the growth factor-independent HCT116 cell line and may represent the major site of transcriptional dysregulation of T GF alpha promoter activity in the growth factor-independent phenotype.