Abstract
CBS human colon carcinoma cells are poorly tumorigenic in athymic nude mice, whereas FET colon carcinoma cells are non-tumorigenic. Both cell lines have well differentiated properties in tissue culture. Transforming growth factor alpha (TGF-alpha) was ectopically expressed by stable transfection of a TGF-alpha cDNA under repressible tetracycline control. The TGF-alpha-transfected cells showed enhanced clonal initiation and shortened lag phase growth in tissue culture without an alteration in doubling time in exponential phase relative to untransfected cells. Furthermore, the TGF-alpha transfectants showed increased independence from exogenous growth factors in clonal growth assays and induction of DNA synthesis after release from quiescence. Growth factor independence was associated with sustained epidermal growth factor receptor activation in quiescent TGF-alpha-transfected cells and the requirement of exogenous insulin for stimulation of quiescent cells to re-enter the cell cycle. Higher cloning, reduced lag time in tissue, and the acquisition of growth factor independence for DNA synthesis without a change in doubling time of TGF-alpha-transfected cells indicate that autocrine TGF-alpha functions by facilitating re-entry into the cell cycle from sub-optimal growth states rather than promoting or controlling the proliferation of actively cycling cells. The modulation of growth regulation by autocrine TGF-alpha was associated with increased malignant properties as TGF-alpha transfectants showed increased tumorigenicity in athymic nude mice. The administration of tetracycline reversed the effects of TGF-alpha expression in these cells both in vivo and in vitro, indicating that the alterations of the biological properties were due to the expression of TGF-alpha. Since these cells are continuously grown in a completely chemically defined medium without serum supplementation, it was possible to assign the mechanism underlying the generation of growth factor independence to the replacement of a requirement for exogenous insulin in parental cells by autocrine TGF-alpha.
Highlights
§ Both authors contributed to this work. ** To whom correspondence should be addressed: Dept. of Surgery and Biochemistry, the University of Texas, Health Science Center at San Antonio, 7703 Floyd Curl Dr, San Antonio, TX 78284-7840
It is clear that TGF-␣ promotes tumorigenicity and malignant progression in a variety of in vitro and in vivo assays, it is not apparent what specific growth advantages it imparts to cells in the various contexts of proliferative function and what specific growth functions it replaces in generating the independence from exogenous growth factors associated with malignant progression
These growth factor-dependent cell lines were stably transfected with a human TGF-␣ cDNA under repressible control by tetracycline in order to generate a strong autocrine TGF-␣ loop so that specific growth requirements assumed by autocrine TGF-␣ could be determined and evaluated in the context of malignant progression as well
Summary
Cell Culture—The human colon carcinoma cell lines were originally isolated from primary tumors as described previously [18] and continuously maintained in a chemically defined serum-free medium consisting of McCoy’s 5A medium (Sigma) supplemented with pyruvate, vitamins, amino acids, antibiotics, insulin (20 g/ml, Sigma), transferrin (4 g/ml, Sigma), and EGF (10 ng/ml, R & D Systems, Minneapolis, MN) [20]. TGF-␣ transfectants and control cells were routinely maintained in serum-free medium containing 650 g/ml active geneticin (Life Technologies, Inc.). Conditioned Medium and TGF-␣ Enzyme-linked Immunosorbent Assay—Cells were plated at 120,000 cells per well in 6-well plates in serum-free medium lacking EGF in the presence or absence of tetracycline at a final concentration of 0.1 g/ml. Growth Assays—Cells were plated at a clonal density of 300 cells/well into 24-well tissue culture plates in serum-free medium in the presence or absence of EGF. Immunoprecipitates were subsequently incubated with 50% protein A-agarose suspension (Life Technologies, Inc.) for 30 min at 4 °C and were washed twice with cold lysis buffer 3 times followed by centrifugation. 3,000 cells suspended in serum-free growth medium minus EGF containing 0.4% agarose (Sigma) were plated per well into 6-well tissue culture plates containing 0.8% agarose underlayers. The cell cycle phase distribution was performed using a FACScan flow cytometer (BectonDickinson, San Jose, CA), and cell cycle parameters were obtained using a ModFit LT program (Verity Software House Inc.)
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