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. 2006 Aug 21;95(4):496-505.
doi: 10.1038/sj.bjc.6603283. Epub 2006 Aug 1.

Cellular senescence in naevi and immortalisation in melanoma: a role for p16?

V C Gray-Schopfer  1 , S C Cheong, H Chong, J Chow, T Moss, Z A Abdel-Malek, R Marais, D Wynford-Thomas, D C Bennett
Affiliations

Cellular senescence in naevi and immortalisation in melanoma: a role for p16?

V C Gray-Schopfer et al. Br J Cancer. .

Abstract

Cellular senescence, the irreversible proliferative arrest seen in somatic cells after a limited number of divisions, is considered a crucial barrier to cancer, but direct evidence for this in vivo was lacking until recently. The best-known form of human cell senescence is attributed to telomere shortening and a DNA-damage response through p53 and p21. There is also a more rapid form of senescence, dependent on the p16-retinoblastoma pathway. p16 (CDKN2A) is a known melanoma susceptibility gene. Here, we use retrovirally mediated gene transfer to confirm that the normal form of senescence in cultured human melanocytes involves p16, since disruption of the p16/retinoblastoma pathway is required as well as telomerase activation for immortalisation. Expression (immunostaining) patterns of senescence mediators and markers in melanocytic lesions provide strong evidence that cell senescence occurs in benign melanocytic naevi (moles) in vivo and does not involve p53 or p21 upregulation, although p16 is widely expressed. In comparison, dysplastic naevi and early (radial growth-phase, RGP) melanomas show less p16 and some p53 and p21 immunostaining. All RGP melanomas expressed p21, suggesting areas of p53-mediated senescence, while most areas of advanced (vertical growth-phase) melanomas lacked both p16 and p21, implying escape from both forms of senescence (immortalisation). Moreover, nuclear p16 but not p21 expression can be induced in human melanocytes by oncogenic BRAF, as found in around 80% of naevi. We conclude that cell senescence can form a barrier to melanoma development. This also provides a potential explanation of why p16 is a melanoma suppressor gene.

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Figures

Figure 1
Figure 1
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.
Figure 2
Figure 2
Typical expression of senescence-associated proteins in benign compound naevi and normal epidermis. Immunostained proteins appear red and nuclei blue (haematoxylin counterstain, except where noted). Acidic β-galactosidase gives a blue colour, with eosin (pink) counterstain. (A) Top and second rows, detection of melanocytic marker MART1 to show melanocytes and for p16 in parallel sections of the same biopsy. Top panels, low magnification of compound naevus (N), showing intense p16 immunostain (without haematoxylin). E, epidermis. Scale bar, 200 μm. Second row, higher magnification of another naevus to show detail. Contrast slightly enhanced in right image to clarify chromatin detail. Scale bar, 20 μm. Levels of p16 expression vary, with some cells not visibly positive. Immunostain where present is both nuclear and cytoplasmic. Arrows: large multinucleate cells; arrowheads: large nucleoli. Third row, parallel sections of same naevus as top panels, immunostained for p53 and p21 as indicated. No p53 or p21 was detected. Brown material (arrowheads) is melanin pigment. Bottom row, high magnification of haematoxylin-stained areas of a benign compound naevus (left), showing heterochromatic foci in nuclei (arrows), and of a VGP melanoma (right) showing absence of such foci. Note that lesions are heterogeneous and not all areas showed such clear differences. Contrast digitally enhanced as sections were lightly stained. Scale bar, 20 μm. (B) Top, normal adjacent epidermis from same sections as (A) top. Scale bar, 50 μm. MART1 (left) shows distribution of normal melanocytes (arrows), which have no detectable p16 (right). Middle and bottom, MART1 and acidic β-galactosidase in parallel sections of a benign congenital naevus. All cells of naevus (N) show both markers. Higher magnification (bottom) shows some acidic β-galactosidase reactivity in whole basal epidermis (arrowheads), but faint compared to naevus cells. Note: frozen sections were needed for this stain, and were available only for congenital naevi. Arrows, melanocytes. Scale bars, 150 μm (middle), 50 μm (bottom).
Figure 3
Figure 3
Immunostaining of senescence effectors in dysplastic naevi. (A) lesions from normal patients. Top and middle, MART1 shows naevus cells throughout basal epidermis in this lesion, yet there is little or no p16, little p53 and no p21 detected in parallel sections. Scale bar, 100 μm. Bottom left, detail of p53 immunostaining in same lesion showing a few positive nuclei (arrowheads). Scale bar, 50 μm. Bottom right, detail of p16 reaction in a different dysplastic naevus. Scale bar, 50 μm. Most naevus cells here are strongly positive for p16, yet the reaction appears generally cytoplasmic (arrowheads: unstained nuclei). Arrow: other parts of this naevus had little or no p16. (B) lesion from a patient lacking active p16 because of two germline mutations. MART1 and p16 immunostaining (top) show similar patterns: nearly all naevus cells were prominently positive for p16. Many naevus cells are also positive for p53 and p21 (middle). Scale bar, 100 μm. Bottom left: p16 reaction is cytoplasmic only (arrowheads: unstained nuclei). Scale bar, 30 μm. Bottom right: p53-positive naevus cells in dermis, in same lesion, surrounded by pigmented macrophages (arrows) and lymphocytes (arrowheads). Scale bar, 50 μm.
Figure 4
Figure 4
Typical immunostaining of senescence mediators in melanomas and naevi. (A) RGP melanomas. Top and middle: The same area of one melanoma is positive for MART1, p16, p53 and p21, showing co-localisation of p16, p53 and p21 in the same area, together with some pigment. Melanophages are also seen. The unreactive cells in this area appear to be melanoma cells; many areas of this tumour were unpigmented and negative for all of these markers. (MART1 can be lost in advanced melanomas). Scale bar, 100 μm. Bottom left, p16-positive cells are often large and sometimes have more than one nucleus (arrowheads). Scale bar, 60 μm. Bottom right: nuclear p53 reaction in another RGP melanoma. Again, some positive cells are multinucleate (arrowheads). Scale bar, 30 μm. (B) Typical VGP melanoma. Left: rare area of p16 expression. The positive cells (arrows) are large and well-pigmented (melanin, brown). It is unclear whether all pigmented cells have p16, but some have prominent nucleoli (arrowheads). Scale bar, 30 μm. Right: p53 reaction is also rare. Here, the positive cells are again large and colocalised with pigment, and the p53 is cytoplasmic (arrows, cells with clearly negative nuclei). Scale bar, 30 μm. (C) CHK2 expression in various lesion types. Top left: benign compound naevus. There is nonspecific reaction and occasional stronger reaction (arrowheads). Top right: dysplastic naevus (same area as in Figure 3A, top). Little reaction is visible. Scale bar, 100 μm. Bottom left: RGP melanoma (same area as in A, top). Prominent CHK2 immunostain (arrowheads) colocalises with p16, p53 and p21. Bottom right, VGP melanoma (same area as in B), showing prominent specific reaction (arrowheads). Some nonspecific reaction is also seen. Scale bar, 100 μm.
Figure 5
Figure 5
Effects of transfected BRAF sequences on cultured normal human melanocytes. Assays were performed 10 days (A) or 5 days (B, C) after transfection of cells. (A) Expression of growth-related proteins as shown by immunoblotting. (NHM), normal human melanocytes, transfected with the vector only (CONT, control), or with WTBRAF (WT) or V600EBRAF (VE) sequences. WM266-4 human melanoma cells and HeLa cells were used as positive controls for p21 and p16 expression, respectively. mBRAF: detection of Myc tag present on both WT and VE transfected BRAF sequences. pERK: phospho-ERK as a marker of active MAPK signalling. ERK: total ERK protein. (B) Counts of nuclei positive for proliferative marker Ki67 or for p16, by immunostaining of cells transfected with vector only (0), WTBRAF or V600EBRAF. Means and SEM are shown. p16 positivity is shown in both the total culture including any nontransfected cells, and (*) as % of double-stained cells positive for exogenous BRAF (Myc tag), that also showed nuclear p16. After only 5 days, Ki67 positivity was significantly reduced in the total culture with V600EBRAF (P<0.05). Nuclear p16 expression was greater in V600EBRAF-transfected (P<0.001) but not WTBRAF-transfected cells relative to control (whole culture), and in V600EBRAF-expressing cells (over 45% positive for p16) relative to WTBRAF-expressing cells (P<0.001). Significance testing was by Student's t-test. (C) Typical examples of immunostained cells, showing nuclear p16 staining in melanocytes expressing V600EBRAF but not WTBRAF. Upper label on each panel indicates transfected sequence, lower label indicates stain. Both BRAF proteins were immunostained with anti-Myc tag. This gave no staining in cultures transfected with the vector only (not shown). DAPI staining showed nuclei.
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