The rabbit lens epithelial cell line N/N1003A requires 12-lipoxygenase activity for DNA synthesis in response to EGF.
OBJECTIVE
Cultured rat lenses and primary human lens epithelial cells (HLECs) express12-lipoxygenase (12-LOX) and require a 12-LOX metabolite of arachidonic acid for growth in response to EGF and insulin. This study seeks to identify an established cell line with these characteristics.


METHODS
Immunoblotting was used to screen eight lens epithelial cell lines for 12-LOX expression: the human line, HLE-B3; mouse lines alphaTN4, 17EM15, 21EM15, and MLE6, and rabbit lines N/N1003A, LEP2 and B3. DNA synthesis was measured as incorporation of 3H-thymidine into DNA. Expression of c-fos mRNA was detected by RT-PCR. The involvement of 12-lipoxygenase metabolites was determined using the lipoxygenase inhibitors baicalein, cinnamyl 3,4-dihydroxy-alpha-cyanocinnamate (CDC), or nordihydroguiairetic acid (NDGA).


RESULTS
12-LOX was detected only in the rabbit lines N/N1003A, LEP2 and B3. N/N1003A cells were chosen for further study. 12-LOX inhibitors blocked DNA synthesis in response to EGF with or without insulin. Inhibition of EGF-stimulated DNA synthesis was reversed by 0.3 microM to 3 microM 12(S)hydroxyeicosatetraenoic acid (HETE), but not by equivalent concentrations of 5(S)HETE, 8(S)HETE, 15(S)HETE, or 12(R)HETE. Baicalein prevented EGF induction of c-fos mRNA. The transformed HLEC line, HLE-B3, showed little stimulation of DNA synthesis in response to EGF and was unaffected by the presence of 12-LOX inhibitors.


CONCLUSIONS
N/N1003A cells, like primary cultured human lens epithelial cells or neonatal rat lenses, require 12-LOX activity for EGF dependent growth. This line will be useful for studies of the mechanism of action of 12(S)HETE.
Haque
M S
MS
Laboratory of Molecular and Developmental Biology, National Eye Institute, Bethesda, MD 20892, USA. haque@helix.nih.gov
Arora
J K
JK
Dikdan
G
G
Lysz
T W
TW
Zelenka
P S
PS
D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid / Q000096:biosynthesis / Q000494:pharmacology; D000818:Animals; D001092:Arachidonate 12-Lipoxygenase / Q000096:biosynthesis*; D002109:Caffeic Acids / Q000494:pharmacology; D002460:Cell Line; D004247:DNA / Q000096:biosynthesis; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D004847:Epithelial Cells / Q000166:cytology* / Q000201:enzymology; D044950:Flavanones; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000378:metabolism / Q000494:pharmacology; D015151:Immunoblotting; D007328:Insulin / Q000494:pharmacology; D007908:Lens, Crystalline / Q000166:cytology / Q000201:enzymology*; D016859:Lipoxygenase Inhibitors; D009637:Masoprocol / Q000494:pharmacology; D016760:Proto-Oncogene Proteins c-fos / Q000096:biosynthesis; D011817:Rabbits; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction
D002109:Caffeic Acids; D004791:Enzyme Inhibitors; D044950:Flavanones; D005419:Flavonoids; D006893:Hydroxyeicosatetraenoic Acids; D007328:Insulin; D016859:Lipoxygenase Inhibitors; D016760:Proto-Oncogene Proteins c-fos; C099255:cinnamyl-3,4-dihydroxycyanocinnamate; C006680:baicalein; D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D004815:Epidermal Growth Factor; D009637:Masoprocol; D004247:DNA; D001092:Arachidonate 12-Lipoxygenase