Establishment of stable immortal differentiated cell lines for the development of new cell lines for in vitro tests

• Research unit: EU RFP
• Project number: 95.0857

Key words (English)

Immortalization; differentiation; oncogenes; in vitro models

Alternative project number (English)

EU project number: BIO4CT960052

Research programs (English)

EU-programme: 4. Frame Research Programme - 4.1 Biotechnology

Short description (English)

See abstract

Partners and International Organizations (English)

C. Guillouzo (F), E. Gerard (UK), A. Khan (F), M. Adolphe (F), M. Gomez-Lechon (E)

Abstract (English)

Development of new constructs for the immortalization of human cells

Functional telomeres are essential for chromosomal integrity in eukaryotes, and consequently, telomere maintenance is required for long-term proliferation. Interestingly, ectopic expression of the telomerase reverse transcriptase protein (TERT) in human fibroblasts before scenescence elicits telomere lengthening and indefinite cell proliferation. Consequently, to these new findings we have cloned the human TERT (hTERT) in the pLHXSD and pRetro-Tet-off (Clontech AG) retroviral vectors. Recombinant retroviral particles were generated by a 'ping-pong' infection protocole involvig co-cultivation of packaging cell lines with different host ranges. By testing the 2 retrovirus in the 3T3 mouse fibroblast cell line it appeared that the pLHXSD-hTERT but not the pRetro-off-hTERT system was effective to transduce the express of the hTERT gene, confirming previous results showing that the pRetro-off retroviral vector does not work properly. The pLHXSD-hTERT virus has been recently used on two newly SV40 T-antigen immortalized human cell lines, the Pc-3 cells (osteoblasts) and HMC cells (muscle), to test whether the ectopic expression of the hTERT can avoid these cell lines to go in scenescence (around passage 16) as previously described for a keratinocyte cell line. In the case of positive results,, the hTERT will be used to immortalize human mesenchymal cells.

Development of in vitro and corneal cellular models for inflammation/carcinogenesis

Cyclooxygenase-2 (COX-2), a key enzyme for the formation of prostaglandins form arachidonic acid, is an early and central event in colon carcinogenesis and consequently provides an important target for chemoprevention. The previously non tumorigenic SV40 T-antigen immortalized human colonic cell lines (HCEC ce1ls) was evaluated for ist capacity to express COX-2 after stimulation by phorbol esters and pro-inflammatory cytokines. Either 50 ng/ml PMA or 10 ng/ml of TNF-alpha were able to strongly stimulate both COX-2 mRNA and protein expression in the HCEC cells but not in the CaCO-2 cells suggesting different COX-2 regulation pathways between non tumorgenic and tumorgenic cell lines. Therefore, the HCEC cell line constitutes an interesting tool for studying and discovering anti-inflammatory as well as anti-carcinogenic compounds.

References in databases (English)

Swiss Database: Euro-DB of the
State Secretariat for Education and Research
Hallwylstrasse 4
CH-3003 Berne, Switzerland
Tel. [phone]
Swiss Project-Number: 95.0857