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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)

Enhanced expression of dihydrofolate reductase by bovine kidney epithelial cells results in altered cell morphology, IGF-I responsiveness, and IGF binding protein-3 expression.

The kidney epithelial cell line (MDBK) secretes primarily insulin-like growth factor binding protein (IGFBP)-2 under basal conditions, but exposure to forskolin decreases the synthesis of and induces IGFBP-3. Since IGFBP-3 has been shown to both potentiate and inhibit insulin-like growth factor (IGF) bioactivity, MDBK cells were transfected with an expression vector containing bovine IGFBP-3 cDNA and the dihydrofolate reductase (DHFR) gene as a selectable marker, with the goal of obtaining an epithelial cell line which constitutively secreted IGFBP-3. Stable clones which secreted greater than 100 ng/ml of IGFBP-3 were obtained and designated MDBKpMONBP-3. Northern blotting indicated that endogenous IGFBP-3 mRNA, which was undetectable in wild-type (WT) MDBK cells, was expressed in MDBKpMONBP-3 cells while the IGFBP-3 transgene did not appear to be expressed. DHFR mRNA transcripts were also expressed by MDBKp-MONBP-3 cells, whereas these transcripts were not detected in WT MDBK cells, suggesting that gene amplification of DHFR may have allowed cells to survive in methotrexate (MTX) without taking up the expression vector. In addition to the altered pattern of IGFBP-3 secretion, a marked alteration in cell morphology was observed. MDBKpMONBP-3 cells grew in distinct islands and exhibited dome formation (a characteristic of differentiated epithelial cells) whereas the WT cells did not. The alterations in morphology and IGFBP-3 expression were irreversible, since MDBKpMONBP-3 cells failed to revert to the WT phenotype upon removal of MTX and dialyzed serum. Since vectorial secretion of proteins is often associated with epithelial cell differentiation, cells were plated on tissue culture inserts which allowed conditioned media (CM) to be collected from both the apical and basal surfaces of confluent monolayers. Release of IGFBP-2 was approximately equal from apical and basal surfaces in WT MDBK cells. In contrast, release of both IGFBP-2 and IGFBP-3 was greater (3.1-fold and 3.5-fold, respectively) from basal as compared to apical surfaces of the MDBKpMONBP-3 cells. To determine if cells which were secreting IGFBP-3 had altered growth responses to IGF-I, cells were grown in serum-free media in the presence of IGF-I (0 to 100 ng/ml). Treatment of MDBKpMONBP-3 cells with 100 ng/ml of IGF-I increased cell number 138 +/- 37% above serum-free controls compared to 73 +/- 10% in WT MDBK cells.(ABSTRACT TRUNCATED AT 400 WORDS)[1]


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