Human keratinocytes PTCH1 cell line
A cellular model is described for targeting dysregulation or inappropriate activation of the Sonic Hedgehog/Patched (SHH/PTCH) pathway. Also described, is a screening method using this cellular model to screen for pharmacological compounds that can treat or prevent skin cancer and, in particular, Basal Cell Carinoma (BCC) lesions.
1. An immortalized cell line of human keratinocytes with a natural exogenous sequence bearing at least one GLI binding site comprising a 5′-GACCAACCCA-3′ sequence (SEQ ID NO: 1) in a reporter cassette; and wherein the cell is produced in the absence of serum and without feeder cells.
2. The immortalized cell line of claim 1 , wherein the expression of the reporter cassette is the same as an initial level without recombination or loss of chromosomal material.
3. The immortalized cell line of claim 1 , wherein the reporter cassette is a PTCH1-luciferase construct.
4. The immortalized cell line of claim 3 , wherein the cell line expresses a PTCH protein.
5. The immortalized cell line of claim 1 , wherein the cell line is immortalized by retroviral transduction of pLE6/E7SN.
6. A process for obtaining immortalized human keratinocytes as described in claim 1 , the process comprising the following steps of:
isolating human primary keratinocytes from a healthy individual,
inserting a regulation region of PTCH1 upstream of a reporter gene in an appropriate plasmid, and
immortalizing the cell line by retroviral transduction with pLE6/E7SN.
7. A drug screening method, the method comprising screening a capacity of a drug to inhibit or down regulate expression or biological activity of PTCH with the immortalized cell line of claim 1 .
8. A drug screening method, the method comprising the following steps of:
a. contacting one sample of the immortalized cell line of claim 1 with at least one test compound or a mixture of compounds,
b. measuring expression or activity of a reporter gene and/or activity of a PTCH1 promoter, and
c. selecting at least one compound for which a modulation of the expression or of the activity of the reporter gene and/or a modulation of the expression of the PTCH1 gene thereof is measured in step b) when compared to the expression or activity in the absence of any test compounds or mixture of compounds.
9. The drug screening method of claim 8 wherein the reporter gene is a luciferase or a GFP reporter gene.
10. The drug screening method of claim 7 , wherein the method identifies an antitumor drug.
11. An In vitro method for screening candidate compounds for preventive and/or curative treatment of cutaneous cancer, the method comprising the following steps of:
a. contacting one sample of the immortalized cell line of claim 3 with at least one test compound or with a mixture of compounds,
b. measuring the expression or the activity of luciferase and/or the activity of at least one PTCH1 promoter thereof, and
c. selecting at least one compound for which a modulation of the expression or of the activity of the reporter cassette and/or a modulation of the expression of the PTCH1 gene, is measured in step b) when compared to the expression or activity in the absence of any test compounds or mixture of compounds.