Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Primate cell – per se
Reexamination Certificate
1997-12-23
2001-09-04
Houtteman, Scott W. (Department: 1656)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Primate cell, per se
C435S320100, C424S130100
Reexamination Certificate
active
06284537
ABSTRACT:
FIELD OF THE INVENTION
The subject of the present invention is new immortalized human corneal epithelial cell lines as well as their use in processes for the identification of agents which are mutagenic, toxic or beneficial to the metabolism of the corneal cells.
BACKGROUND ART
For many years, efforts have been made to develop human cell lines adapted to the study of human diseases such as infections, inflammations or cancers, for example. Among the cells often involved in the onset of diseases, there are the epithelial cells which are sensitive to the surroundings of the human body.
The epithelial cells differ from other cells of the human body in the expression of compounds or structures which are found only in the epithelial cells, such as, for example, cytokeratins (Moll et al., Cell, 31, 11-24, 1982), connections between the cells (Gumbiner et al., Cell, 69, 385-387, 1992), and vimentin (Richard et al., Arch. Dematol. Res., 282, 512-515, 1990).
Other compounds are also generally found in human epithelial cells, but not only in the epithelial cells, such as the cytochrome P4505 (Mercurio et al., Biochem. Biophys. Research Communications, 210, No.2, 350-355, 1995; McKinnon et al., Gut, 36, 259-267, 1995), the enzymes involved in the defence against cellular oxidation (Cu/Zn-superoxide dismutase, glutathione peroxidase, glutathione reductase and catalase: Albers et al., Toxicology and Applied Pharmacology, 109, 507-513, 1991) and/or in the detoxification of electrophiles (&agr;-, &mgr;- or &pgr;-glutathione-S-transferases: Singh et al. Exp. Eye Res., 40, 431-437, 1985; aldehyde reductase: Ellis et al., Biochemical J., 312, 535-541, 1995). The epithelial cells of the human cornea also differ from other human epithelial cells in the expression of compounds which are found only in the epithelial cells of the cornea, such as for example the cytokeratin of 64 kD and the glutathione-S-transferase hGST 5.8 which are found in the ocular tissues (hGST 5.8: Singhal et al., Invest. Ophthalmol. Vis. Sci., 36, 142-150, 1995; 64 kD: Kiritoshi et al., Invest. Ophthalmol. Vis. Sci., 32, 3073-3077, 1991).
Analysis of the expression of certain cytokines and growth factors in the epithelial cells of human cornea, without inflanimatory stimulation, has already been determined by Cubitt et al. (1993, Invest. Ophthanol. & Vis. Sci. 34:3199-3206; and 1995, Invest. Ophthamol. & Vis. Sci. 36:330-336), Wilson et al. (1992, Invest. Ophthamol. & Vis. Sci. 33:1756-1762), Kennedy et al. (1995, J. Clinical Invest. 95:82-88), and DeQuan et al. (1995, J. Cellular Physiol. 163:61-79). Moreover, Rosenbaum et al. have suggested that the cytokine profile of the corneal cells could be a means for effectively detecting the presence of certain diseases of the cornea (Invest. Ophthalmol. Vis. Sci., 36, 2151-2155, 1995). The profile of expression of certain cytokines and growth factors in the epithelial cells of the cornea therefore makes it possible to characterize and differentiate the normal corneal epithelial cells from the other cells, and in particular from the abnormal comeal epithelial cells.
Moreover, the methods of screening for molecules which are noninflammatory for the eyes involve laboratory animals. To overcome the fact that there are substantial morphological and biochemical differences between the human eyes and those of these animals, Hainsworth et al. propose to carry out these screening tests on primary epithelial cell cultures of cornea (J. Tissue Culture Meth., 13, 45-48, 1991). Unfortunately, primary cell cultures of cornea stop multiplying after one or two passages, each passage consisting of subculturing the cells in fresh medium after growing until they become confluent. Furthermore, these primary cells do not survive freezing in liquid nitrogen.
To overcome these disadvantages, human corneal epithelial cell lines have been developed by Kahn et al. (Invest. Opthalmol. Vis Sci., 34, 3429-3441, 1993) and Araki-Sasaki et al. (Invest. Opthalmol. Vis Sci., 36, 614-621, 1995). Unfortunately, these lines are not yet fully satisfactory.
Indeed, the lines developed by Kahn et al. are not completely immortalized because they retain certain original differentiation characteristics up to about 25 successive culture passages (see Kahn et al.). These lines also release SV40 viruses into the culture medium (see the Araki-Sasaki et al. analysis). Finally, it is not known if they effectively express other differentiation markers, such as glutathione-S-transferase hGST 5.8, and an appropriate cytokine and growth factor profile, as well as markers which are specific or necessary for an inflammatory reaction.
The immortalized lines developed by Araki-Sasaki et al. are, on the other hand, incapable of becoming stratified in comeal reconstruction trials in vivo (see Araki-Sasaki et al.), and it is not known if they express certain differentiation markers, such as glutathione-S-transferase hGST 5.8, and an appropriate cytokine and growth factor profile, as well as markers which are specific or necessary for an inflammatory reaction.
The aim of the invention is to provide new human comeal epithelial cell lines which are genetically and physiologically similar to the normal epithelial cells of the human cornea, to the extent that they can be used effectively in trials for screening potentially inflammatory molecules.
SUMMARY OF THE INVENTION
To this end, the invention relates to any immortalized human comeal epithelial cell lines capable of becoming stratified, and capable of expressing metabolic differentiation markers specific for non-immortalized human epithelial cells, metabolic markers specific for non-immortalized human corneal epithelial cells, and markers specific for an inflammatory reaction.
Another aspect of the invention relates to a new process for identifying the mutagenic, toxic or beneficial effect of an agent on the metabolism of the cells of the cornea, in which (1) a culture comprising a cell line according to the invention is reacted with an agent suspected of being a mutagenic, toxic or beneficial agent for the metabolism of the cells of the human cornea, and (2) the effects of the said agent on the said cell line are determined.
The invention also relates to a diagnostic kit comprising the immortalized human corneal epithelial cells according to the invention and reagents for determining a metabolic response of the said cells to mutagenic, toxic or beneficial agents for the said cells.
Finally, the subject of the invention is also any uses of the cell lines according to the invention, in processes for the identification of mutagenic, toxic or beneficial agents for the metabolism of the corneal cells, as well as any uses of these lines as active pharmaceutical agent.
REFERENCES:
patent: 5585265 (1996-12-01), Kahn et al.
patent: 5672498 (1997-09-01), Walker et al.
patent: 0 802 257 (1997-10-01), None
patent: WO 94/05472 (1994-03-01), None
patent: WO 96/07750 (1996-03-01), None
GeneBank databank(Accession No. M64753).
Albers et al., 1991, “In vivo intestinal metabolism of 7-ethoxycoumarin in the rat: Production and distribution of phase I and II metabolites in the isolated, perfused intestinal loop”,Toxicol.&Applied Pharmacol.109:507-513.
Araki et al., 1993, “Immortalization of rabbit corneal epithelial cells by a recombinant SV40-adenovirus vector”,Invest Ophthalmol. Visual Sci.34:2665-2671.
Araki-Sasaki et al., 1995, “An SV40-immortalized human corneal epithelial cell line and its characterization”,Invest Ophth.&Visual Sci.36:614-621.
Bazan et al., 1991, “Platelet-activating factor (PAF) accumulation correlates with injury in the cornea”,Exp. Eye Res.52:481-491.
Bazan et al., 1993, “Platelet-activating factor induces collagenase expression in corneal epithelial cells”,Proc. Natl. Acad. Sci. USA90:8678-8682.
Chen et al., 1987, “High-efficiency transformation of mammalian cells by plasmid DNA”,Mol.&Cell. Biol.7:2745-2752.
Conners et al., 1995, “A closed eye contact lens model of corneal inflammation”,Inves. Ophth.&Visu Sci.36:828-840.
Cubitt et al., 1993, “IL-8 gene expression in cultures of h
Offord Cavin Elizabeth
Pfeifer Andrea M. A.
Sharif Najam A.
Tromvoukis Yvonne
Houtteman Scott W.
Nestec S.A.
Pennie & Edmonds LLP
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