Autocrine regulation of human urothelial cell proliferation and migration during regenerative responses in vitro.

Regeneration of the urothelium is rapid and effective in order to maintain a barrier to urine following tissue injury. Whereas normal human urothelial (NHU) cells are mitotically quiescent and G0 arrested in situ, they rapidly enter the cell cycle upon seeding in primary culture and show reversible growth arrest at confluency. We have used this as a model to investigate the role of EGF receptor signaling in urothelial regeneration and wound-healing. Transcripts for HER-1, HER-2, and HER-3 were expressed by quiescent human urothelium in situ. Expression of HER-1 was upregulated in proliferating cultures, whereas HER-2 and HER-3 were more associated with a growth-arrested phenotype. NHU cells could be propagated in the absence of exogenous EGF, but autocrine signaling through HER-1 via the MAPK and PI3-kinase pathways was essential for proliferation and migration during urothelial wound repair. HB-EGF was expressed by urothelium in situ and HB-EGF, epiregulin, TGF-alpha, and amphiregulin were expressed by proliferating NHU cells. Urothelial wound repair in vitro was attenuated by neutralizing antibodies against HER-1 ligands, particularly amphiregulin. By contrast, the same ligands applied exogenously promoted migration, but inhibited proliferation, implying that HER-1 ligands provoke differential effects in NHU cells depending upon whether they are presented as soluble or juxtacrine ligands. We conclude that proliferation and migration during wound healing in NHU cells are mediated through an EGFR autocrine signalling loop and our results implicate amphiregulin as a key mediator.

Effects of loss of p53 and p16 function on life span and survival of human urothelial cells.

Human urothelial cell carcinoma evolves via the accumulation of numerous genetic alterations, with loss of p53 and p16 function representing important stages in the development of superficial lesions and their progression to malignant disease. To investigate the effects of disabling either or both proteins in otherwise normal human urothelial cells, we performed retroviral transductions with a dominant negative p53 miniprotein and/or mutant cyclin-dependent kinase 4 (CDK4R24C) in 3 independent cell lines. Although cells with disabled p53 function showed a higher proliferation rate, inactivation of neither p53 nor p16 function resulted in any extension of life span and the double-transductants failed to flourish, demonstrating that further genetic alterations are required to attain an immortalised phenotype. However, CDK4R24C transductants showed a marked increase in apoptotic susceptibility to membrane-presented CD40 ligand, being intermediate between normal cells (nonsusceptible) and transformed cells (highly susceptible). By contrast, loss of p53 function alone only slightly increased the apoptotic susceptibility of urothelial cells. These results demonstrate that loss of p16 function, while insufficient to immortalise urothelial cells, nevertheless renders the cells more vulnerable to apoptosis induced by CD40 ligation.

Synthesis of jaspaquinol and effect on viability of normal and malignant bladder epithelial cell lines.

The synthesis of jaspaquinol 1, a monocyclic diterpene-benzenoid, is reported. Two synthetic routes to this natural product have been developed. The first, utilises a difunctional terpene derivative containing different leaving groups, facilitating the selective introduction of the cyclohexenyl and benzenoid fragments. The alternative route employs a regiospecific Stille cross-coupling reaction to introduce the cyclohexenyl fragment, which occurs without allylic transposition. Preliminary data shows the cell viability of 1 against normal and malignant human bladder epithelial cell lines.