Transformation of human trabecular meshwork cells with SV40 TAg alters promoter utilization.
OBJECTIVE
To compare promoter usage in primary differentiated and SV40 TAg transformed human trabecular meshwork cells (HTM and TM1 cells).


METHODS
Cultured HTM and TM1 cells were transfected with vectors expressing MYOC/TIGR from the CMV-IE, IE4/5 (HSV immediate early 4/5), ICP6 (early gene ICP6 of HSV), EF-1 alpha (human elongation factor 1 alpha-subunit), or the UB6 (human ubiquitin) promoters, respectively. Immunoblotting was used to measure MYOC/TIGR protein expression. MYOC/TIGR expression at the RNA level was detected by Northern blotting.


RESULTS
In primary HTM cells, CMV was the only promoter displaying substantial activity. In TM1 cells, several promoters were functional with the order in decreasing activity being EF-1 alpha > or = CMV > or = UB6 >> IE4/5.


CONCLUSIONS
The difference between the normal and transformed HTM cells suggests that the latter cell type has alterations that influence cellular promoter function. The type of cell used is likely to be a crucial factor in evaluating the functions of promoter elements for genes expressed in the trabecular meshwork and in screening promoters for use in gene delivery studies, especially for evaluations of the MYOC/TIGR gene in relation to glaucoma mechanisms.
Liu
Xuyang
X
Department of Ophthalmology & Visual Sciences, University of Wisconsin, Madison, WI 53706, USA.
Huang
Chun-Yuan
CY
Cai
Suping
S
Polansky
Jon R
JR
Kaufman
Paul L
PL
Brandt
Curtis R
CR
D000328:Adult; D000957:Antigens, Viral, Tumor / Q000502:physiology*; D002454:Cell Differentiation; D002460:Cell Line; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral / Q000502:physiology*; D003598:Cytoskeletal Proteins; D005136:Eye Proteins / Q000378:metabolism / Q000502:physiology; D017781:Genes, Immediate-Early / Q000502:physiology; D006023:Glycoproteins / Q000378:metabolism / Q000502:physiology; D006706:Homeostasis; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics; D020648:Peptide Elongation Factor 1 / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000502:physiology; D012313:RNA / Q000378:metabolism; D013539:Simian virus 40 / Q000276:immunology*; D018139:Simplexvirus / Q000235:genetics; D014129:Trabecular Meshwork / Q000166:cytology / Q000502:physiology* / Q000821:virology*; D025801:Ubiquitin / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics; D000099179:Myocilin
D000957:Antigens, Viral, Tumor; D003598:Cytoskeletal Proteins; D005136:Eye Proteins; D006023:Glycoproteins; D017874:Immediate-Early Proteins; D020648:Peptide Elongation Factor 1; D012313:RNA; D025801:Ubiquitin; D014764:Viral Proteins; D000099179:Myocilin; C088645:IE1 protein, cytomegalovirus; C054366:herpes simplex virus type 1-protein ICP6