Abstract
Autocrine transforming growth factor alpha (TGFalpha) is an important positive growth effector in malignant cells and plays a significant role in generating the growth factor-independent phenotype associated with malignant progression. However, the molecular mechanisms by which TGFalpha confers a growth advantage in progression is poorly understood. The highly tumorigenic cell line HCT116 up-regulates TGFalpha mRNA expression during growth arrest, whereas the poorly tumorigenic growth factor-dependent FET cell line down-regulates TGFalpha mRNA expression as it becomes quiescent. We have identified a 25-bp sequence at -201 to -225 within the TGFalpha promoter which mediates the differential regulation of TGFalpha expression during quiescence establishment in these two cell lines. This same sequence confers TGFalpha promoter responsiveness to exogenous growth factor or autocrine TGFalpha. The abberant upregulation of TGFalpha mRNA in quiescent HCT116 cells may allow them to return to the dividing state under more stringent conditions (nutrient replenishment alone) then quiescent FET cells (requires nutrients and growth factors). Antisense TGFalpha approaches showed that the dysregulated TGFalpha expression in quiescent HCT116 cells is a function of the strong TGFalpha autocrine loop (not inhibited by blocking antibodies) in these cells.
Highlights
Malignant progression is often associated with the loss of dependence on exogenous growth factors for growth control
To begin to understand the mechanism underlying this differential regulation, we studied whether the alterations in TGF␣ mRNA expression were transcriptionally regulated
The growth factor-dependent FET cell line down-regulates endogenous TGF␣ mRNA expression during growth factor and nutrient deprivation leading to growth arrest and quiescence [4, 5, 22]
Summary
Malignant progression is often associated with the loss of dependence on exogenous growth factors for growth control. HCT116 cells are poorly differentiated and highly tumorigenic While this cell line expresses both TGF␣ and EGFR, it is not growth inhibited by blocking antibodies to the EGFR and is growth factor independent as it does not require any exogenous growth factors for DNA synthesis and growth [21, 22]. TGF␣ Regulation during Acquisition of Quiescence notype appears to be via enhanced movement of non-dividing, quiescent cells back into the cell cycle due to inappropriate expression rather than TGF␣ mRNA overexpression in logarithmic phase growth In support of this hypothesis, overexpression of TGF␣ in poorly tumorigenic colon carcinoma cells by stable transfection of a constitutively active sense TGF␣ expression vector generates a more tumorigenic phenotype, which shows enhanced clonal initiation and a decreased lag phase rather than an altered exponential growth phase doubling time [8]. We show TGF␣ expression is not up-regulated during the transition from exponential growth to quiescence in the TGF␣ antisense transfected cells, further implicating TGF␣ autocrine function in the regulation of TGF␣ expression
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