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. 2006 Aug 1;95(4):496–505. doi: 10.1038/sj.bjc.6603283

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Copyright © 2006 Cancer Research UK

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Genetic requirements for immortalisation of normal human melanocytes. (A) Growth of a representative human melanocyte strain (830c) following transduction of the indicated experimental or control sequences with pBABE amphotropic retroviruses. hTERT was added two weeks before the other sequences. Time since the second infection is shown. Immortalisation was seen following infection with hTERT in combination with each of the other experimental sequences. (▪) HPV16-E7+hTERT. (•) CDK4+hTERT. (▴) Antisense p16+hTERT. (□) pBABEpuro+pBABEneo. (⧫) pBABEpuro+pBABEpuro. Two other human melanocyte strains, Nohm-1 (Bennett et al, 1985) and HM303CN gave similar results (not shown). The resulting immortal melanocyte lines were generically called Hermes 3 (from Nohm-1), Hermes 4 (from 830c) and Hermes 5 (from HM303CN) (see (Sviderskaya et al, 2003) for Hermes 1 and 2). Growth was monitored until cells either stopped growing or achieved at least twice the number of population doublings for that cell strain's normal lifespan (full curve not necessarily shown), when they were deemed immortal. (B) Growth of 830c melanocytes following infection with hTERT only. In 2/6 cases (one shown here), immortalisation was seen after a lag, with hTERT only. (▴) hTERT; (•) pBABEpuro. (C) Immunoblot analysis for p16, showing negligible p16 expression in the two normal melanocyte cultures that immortalised with hTERT only. First lane: HM303CN melanocytes with hTERT. Second lane: 830c melanocytes with hTERT. 3rd and 4th lanes: Nohm-1 melanocytes with hTERT and with HPV16-E7 or CDK4 as indicated. HeLa: HeLa cells as positive control for p16 expression.