Am. J. Respir. Cell Mol. Biol., Vol 10, No. 4, Apr 1994, 437-447.
Cell cycle-dependent expression of cyclin D1 and a 45 kD protein in human A549 lung carcinoma cells
F Wu, KC Bui, S Buckley and D Warburton
Division of Neonatology and Pediatric Pulmonology, Childrens Hospital of Los Angeles, University of Southern California School of Medicine 90027.
Cyclin D1, which is suggested to have a role in G1 control during the cell
cycle, is genetically linked to BCL-1 and is widely overexpressed in
parathyroid, breast, and squamous cancer cells. We postulated that cyclin
D1 regulation may also be important in lung cancer. Therefore, we
characterized the cell cycle-dependent expression of cyclin D1 at both mRNA
and protein levels in synchronized human A549 lung carcinoma cells.
Monospecific anti-cyclin D1 C-terminal peptide antibodies recognized both
p36cyclinD1 and an as-yet uncharacterized 45 kD protein (p45). A549 cells
were synchronized with well-studied drugs. Cyclin D1 mRNA expression
remained relatively constant, with less than a twofold fluctuation during
the cell cycle and with a minor peak at M phase. However, the p36cyclinD1
protein fluctuated during the A549 cell cycle and was expressed at very low
levels in late G1 and at the G1/S boundary, but then increased in S phase
and peaked at M phase. In contrast, p45 protein was expressed at relatively
high levels in late G1 and reached maximal levels at the G1/S boundary, was
expressed at decreased levels in S phase, and then had disappeared by M
phase. Moreover, p45 was highly expressed only in transformed alveolar
epithelial cells, but not in normal rat alveolar epithelial cells or fetal
rat lung fibroblasts in primary cultures. In mink Mv1Lu cells, the
expression of p45 was totally blocked by transforming growth factor- beta 1
treatment or contact inhibition. p45 protein was phosphorylated on serine,
threonine, and tyrosine residues in A549 cells in culture. The
phosphorylation of the p45 protein was cell cycle-regulated and reached its
maximal levels at G2/M phase. The p45 protein had a different peptide map
from p36cyclinD1 after cleavage with N- chlorosuccinimide.
Immunoprecipitation studies showed that p45 was also anti-ubiquitin
immunoreactive during the cell cycle. We conclude that p36cyclinD1 and the
p45 protein are differentially regulated in a cell cycle-dependent manner
in A549 cells. Although p45 is antigenically related to p36cyclinD1, it is
probably not a closely cyclin-related protein. We speculate that p45 may be
associated with malignant transformation and may play a distinct role from
p36cyclinD1 in regulation of the cell cycle in A549 cells.
