Expression of carbohydrate binding protein 35 in human fibroblasts: variations in the levels of mRNA, protein, and isoelectric species as a function of replicative competence.
We have compared the expression and localization of carbohydrate binding protein 35 (CBP35) in human SL66 fibroblasts of different replicative capacities. When serum-starved, quiescent young (passage 11, corresponding to approximately 18 cumulative population doublings) SL66 cells were treated with serum, there was a marked stimulation in the expression of CBP35. This was revealed both by an increase in the percentage of cells positively stained with anti-CBP35 under immunofluorescence and by an increase in the amount of the protein in immunoblots, as well as by an increase in the level of accumulated mRNA in Northern blots. The rise in the expression of CBP35 in proliferating cells was manifested most clearly in the nuclear fraction, with elevation in the levels of the nonphosphorylated (pI8.7) protein, as well as the phosphorylated (pI8.2) derivative. In contrast, older (passage 27-35, 55-68 cumulative population doublings) cultures of SL66 fibroblasts appear to have lost the normal proliferation-dependent regulation of CBP35 expression. The level of CBP35 was high in quiescent high-passage cells and decreased somewhat after serum stimulation. Furthermore, the unphosphorylated (pI 8.7) form of the lectin could not be detected in either the nucleus or the cytoplasm of high-passage SL66 cells. Finally, the level of the mRNA for CBP35 was high in quiescent cultures of high-passage cells, but undetectable 17 h after serum stimulation. These results establish that the expression of CBP35 becomes altered as human fibroblasts acquire reduced replicative capacity.
Hamann
K K
KK
Department of Biochemistry, Michigan State University, East Lansing 48824.
Cowles
E A
EA
Wang
J L
JL
Anderson
R L
RL
D000943:Antigens, Differentiation / Q000235:genetics*; D001798:Blood Proteins / Q000494:pharmacology; D015152:Blotting, Northern; D002352:Carrier Proteins / Q000235:genetics*; D002478:Cells, Cultured; D004261:DNA Replication; D015180:Electrophoresis, Gel, Two-Dimensional; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D037502:Galectin 3; D015870:Gene Expression / Q000187:drug effects; D006388:Hemagglutinins / Q000235:genetics*; D006801:Humans; D007525:Isoelectric Focusing; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*
D000943:Antigens, Differentiation; D001798:Blood Proteins; D002352:Carrier Proteins; D037502:Galectin 3; D006388:Hemagglutinins; D012333:RNA, Messenger