Immortalized human corneal epithelial cells for ocular toxicity and inflammation studies.

PURPOSE: To develop a metabolically competent, human immortalized corneal epithelial cell line for use in toxicity and inflammation studies. METHODS: Primary corneal epithelial cells (P-CEPI) were immortalized by a recombinant simian virus (SV)40 T antigen retroviral vector defective for viral replication. The cells were grown in serum-free medium with the addition of bovine pituitary extract, cloned at passage 15 and one of the best-growing clones, CEPI-17-CL4, was extensively characterized for differentiation and metabolic characteristics of the human corneal epithelium. Methods used were immunostaining, reverse transcription-polymerase chain reaction (RT-PCR), northern blot analysis, and enzyme assays. RESULTS: The CEPI-17-CL4 cells showed a typical cobblestone morphology, grew to more than 200 passages and expressed the SV40 T antigen in the nucleus of every cell. Immunofluorescence staining for CEPI-17-CL4 cells was strongly positive for keratins (K)8, K18, and K19 and vimentin; weakly positive for K3, K13, and K17; and negative for K4, K7, and K14. Expression of cytokines (interleukin [IL]-1alpha, IL-1beta, IL-6, IL-8, tumor necrosis factor-alpha, and IL-ra), growth factors (transforming growth factor [TGF]-alpha, epidermal growth factors [EGF], EGF receptor [EGFR], TGF-beta1, TGF-beta2, and platelet-derived growth factor-beta) and cytochrome P450 enzymes (1A1, 2C, 2E1, and 3A5) was similar in CEPI-17-CL4 cells and human corneal epithelial samples obtained in biopsy. The CEPI-17-CL4 cells were metabolically competent for enzymes glutathione S-transferase, quinone reductase, aflatoxin aldehyde reductase, glutathione peroxidase, glutathione reductase, superoxide dismutase, and catalase. CONCLUSIONS: The CEPI-17-CL4 cells are truly immortal and express an extensive array of cytokines, growth factors, and metabolic enzymes that resemble the original tissue. These characteristics, which remain stable up to high passage, will allow reproducible, mechanistic studies on toxicity, inflammation, and wound healing.

The proximal promoter region of the human heme oxygenase gene contains elements involved in stimulation of transcriptional activity by a variety of agents including oxidants.

The rate of transcription of the heme oxygenase gene is enhanced by a variety of agents including oxidants such as hydrogen peroxide and UVA (320-380 nm) radiation and the sulfhydryl reagent, sodium arsenite. To further analyze the inducible response, we have isolated genomic clones of the human heme oxygenase gene. A 1.44 kb fragment corresponding to a region extending from 1416 bp upstream of the mRNA cap site to 24 bp into the 5' untranslated region of the mRNA has been further subcloned and sequenced and used as the basis for the construction of recombinant CAT transient expression vectors. By deleting large portions of this fragment, we have established that elements within 121 bp of sequence immediately upstream of the mRNA cap site respond to various agents (sodium arsenite, hydrogen peroxide, hemin, cadmium chloride and 12-O-tetradecanoyl-phorbol-13-acetate) to give a 3- to 5-fold enhancement in transient expression of the reporter gene. Under the assay conditions employed, induction can only be detected when a SV40 enhancer element is present upstream of the promoter sequence. However, control experiments show that the SV40 sequences serve to amplify the response and are not directly involved in the induction itself. Only a small induction occurs when the entire 1.44 kb fragment is present. The results are consistent with the possibility that additional inducible enhancer elements lie outside of the sequence under study and that a silencer or negative regulatory element occurs upstream of the mRNA cap site within the 1.44 kb fragment.

Oxidant stress leads to transcriptional activation of the human heme oxygenase gene in cultured skin fibroblasts.

Treatment of cultured human skin fibroblasts with near-UV radiation, hydrogen peroxide, and sodium arsenite induces accumulation of heme oxygenase mRNA and protein. In this study, these treatments led to a dramatic increase in the rate of RNA transcription from the heme oxygenase gene but had no effect on mRNA stability. Transcriptional activation, therefore, appears to be the major mechanism of stimulation of expression of this gene by either oxidative stress or sulfydryl reagents.

Conservation of the paired domain in metazoans and its structure in three isolated human genes.

Sequences homologous to the paired domain of Drosophila melanogaster have been conserved in species as distantly related as nematodes, sea urchins, or man. In particular, paired domains of three human genes, HuP1, HuP2 and HuP48, have been isolated and sequenced. Together with four Drosophila paired domains, they fall into two separate paired domain classes named according to their Drosophila members, paired--gooseberry and P29 class. The P29 class includes the mouse Pax 1 and the human HuP48 gene which are nearly identical in their sequenced portions and hence might be true homologues. In addition to the paired domain, the two human genes HuP1 and HuP2 share the highly conserved octapeptide HSIAGILG with the two gooseberry genes of Drosophila. Possible functions of the paired domain are discussed in the light of a predicted helix-turn-helix structure in its carboxy-terminal portion.