Expression of growth control and differentiation genes in human lens epithelial cells with extended life span.
OBJECTIVE
Peptide growth factors including hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), and epidermal growth factor (EGF) are mitogens for many cell types and may act as regulators of lens epithelial cell growth and differentiation. The present study was undertaken to investigate the expression of growth factor receptors and crystallin genes in the human lens epithelial cell line HLE B-3, created by infection with Adeno12-simian virus 40 (Ad12-SV40) hybrid virus.


METHODS
Reverse transcriptase-polymerase chain reaction (RT-PCR) with gene-specific primers was used to detect transcripts, and Southern and western blot analyses were used for identification of gene products. Functional analysis of PDGF receptor was performed by measuring the effect of PDGF-BB on Ca2+ release, cell growth, and western blot analysis, by using an antiphosphotyrosine antibody.


RESULTS
Human lens epithelial B-3 cells expressed the growth factor receptors HGF-R, EGF-R, and PDGF-Rbeta, but not PDGF-Ralpha, and also expressed the oncogenes H-ras and raf and the growth inhibitor transforming growth factor-beta1. Stimulation of PDGF-Rbeta with PDGF-BB in HLE B-3 cells increased phosphorylation of the receptor, was associated with an increase in intracellular Ca2+ levels, and produced a small increase in cell growth. In addition, HLE B-3 cells expressed transcripts for alphaA-, alphaB-, and betaB2-crystallins, and expressed the corresponding proteins. The transcripts for alphaA-crystallin decreased markedly at higher passages.


CONCLUSIONS
The above findings suggest that the increased growth potential of human lens epithelial cells by Ad12-SV40 infection maintained certain lens-specific properties and response to PDGF.
Fleming
T P
TP
Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Song
Z
Z
Andley
U P
UP
D000328:Adult; D000077214:Becaplermin; D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics; D002455:Cell Division / Q000235:genetics; D002472:Cell Transformation, Viral; D002675:Child, Preschool; D003459:Crystallins / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007223:Infant; D007908:Lens, Crystalline / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism; D020574:Proto-Oncogene Proteins c-sis; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D017978:Receptors, Growth Factor / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D003459:Crystallins; D017931:DNA Primers; D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D012333:RNA, Messenger; D017978:Receptors, Growth Factor; D000077214:Becaplermin; D019908:Proto-Oncogene Proteins c-raf; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D002118:Calcium