Endocrine Abstracts

During early embryogenesis, the olfactory axons and gonadotrophin-releasing hormone(GnRH)-secreting neurons undergo dynamic processes of differentiation, migration and proliferation, regulated by a complex network of molecular and cellular mechanisms responding to various hormonal and developmental signals. Mutations in anosmin-1 and FGFR1 disrupt this pathway, causing Kallmann's syndrome(KS), a human hereditary disorder with GnRH deficiency and inability to smell. A primary neuroblast culture FNCB4, originated from human foetal olfactory epithelium, provided a useful model to study normal olfactory-GnRH neurogenesis and the molecular pathogenesis of KS. We have previously shown that anosmin-1 induces neurite outgrowth and cytoskeletal rearrangements in FNCB4 cells through FGFR1 signalling pathway. However, the limited life span of the primary culture has hindered more versatile application of this unique cell system. Although a few cell lines have been generated from mouse neuronal tumours and hypothalamic neurons, none of them truly represents differentiated human GnRH neurons. The major barrier for immortalisation of human cells is telomere-dependent senescence. By using ecotrophic retroviral vector expressing the catalytic subunit of human telomerase (hTERT), we have established an immortal FNCB4 derivative. The cells were first infected with amphotrophic retrovirus delivering ecotrophic receptor and neomycin resistance marker. The G418 selected cells were then infected with pBabe-puro ecotrophic retrovirus containing hTERT. For both infections, BOSC packaging cell line was used to provide the essential retroviral proteins in trans. FNCB4-hTERT cells have been continuously growing far beyond their normal life span with a much shorter doubling time, while the empty vector infected cells entered senescence. Expression of specific neuronal markers, receptors and neuropeptides, cellular morphology and responsiveness to GnRH and calcium transient inducer metastin will be examined to see if FNCB4-hTERT retains the GnRH olfactory neuronal characteristics. It will also be analysed for any tumorigenic changes such as chromosomal abnormalities and mutated tumour suppressors/oncogenes.