| Abstract |
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.
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