p16/Cyclin-Dependent Kinase Inhibitor 2A Deficiency in Human Melanocyte Senescence, Apoptosis, and Immortalization: Possible Implications for Melanoma Progression

Sviderskaya, Elena V.; Gray-Schopfer, Vanessa C.; Hill, Simon P.; Smit, Nico P.M.; Evans-Whipp, Tracy J.; Bond, Jane A.; Hill, L. J.; Bataille, Véronique; Peters, Gordon; Kipling, David; Wynford-Thomas, David; Bennett, Dorothy C.

doi:10.1093/jnci/95.10.723

Cited by 108 publications

(164 citation statements)

References 60 publications

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“…Primary human melanocyte strains Nohm1 (Bennett et al, 1985), 830c (Scott et al, 2002) and HM303CN (Minwalla et al, 2001) were grown as described (Bennett et al, 1985), except in the following melanocyte medium (Sviderskaya et al, 2003): RPMI 1640 medium, 10% foetal calf serum, 200 nM 12-O-tetradecanoyl phorbol 13-acetate (Sigma Chemical Co., Poole, UK), 200 pM cholera toxin (Sigma), 10 ng ml À1 human stem cell factor (R&D Systems, Abingdon, UK) and 10 nM endothelin 1 (Sigma), gassed with 10% CO 2 . Supplements shortly after retroviral infection also included 1 mM insulin, 40 pM fibroblast growth factor 2 (R&D Systems, Abingdon, UK) and 1 mg ml À1 a-tocopherol.…”

Section: Melanocyte Culture and Gene Transfermentioning

confidence: 99%

“…WM266.4 melanoma cells (ATCC), all producer cells and HeLa cells were grown in DMEM with 10% calf serum. Retroviral gene transfer into melanocytes was largely as described (Sviderskaya et al, 2003). All vectors, with or without inserts, were from Dr CJ Jones (Pathology, University of Wales College of Medicine, Cardiff, UK); the HPV16-E7 vector was originally from D Galloway (Fred Hutchinson Cancer Research Center, Seattle, WA, USA).…”

Section: Melanocyte Culture and Gene Transfermentioning

confidence: 99%

“…Moreover, a study with melanoma cell lines found that 100% of lines with normal p16 had a different alteration in the RB pathway (Bartkova et al, 1996). It is not clear how many other cancer types show such high total frequencies of somatic p16 alteration; but even if the specific relation between p16 and melanoma is restricted to germline mutations, it is interesting that there is evidence for the p16-dependent form of senescence in human melanocytes, even in a rich culture medium and with feeder cells (Bennett and Medrano, 2002;Sviderskaya et al, 2003). Two human melanocyte strains lacking functional p16 both showed a greatly extended lifespan, p53-dependent senescence, and immortalisation by hTERT alone (Sviderskaya et al, 2003).…”

mentioning

confidence: 99%

“…Radial growth phase (RGP) melanomas are thin, growing only in or near the epidermis, while vertical growth phase (VGP) melanomas invade more deeply and are competent for metastasis and immortal in cell culture (Clark et al, 1984;Herlyn et al, 1985;Clark, 1991). We proposed a genetic model, in which benign and dysplastic naevi represent p16 and p53-dependent senescence, while melanomas arise by immortalisation of melanocytes (Bennett, 2003;Sviderskaya et al, 2003;Bennett, 2006). Published biological and molecular data (Talve et al, 1997;Keller-Melchior et al, 1998;Glaessl et al, 1999;Rudolph et al, 2000;Hussein and Wood, 2002;Bennett, 2003;Sviderskaya et al, 2003) were consistent with this model, but not conclusive, although a convincing report of cell senescence in melanocytic naevi has appeared recently (Michaloglou et al, 2005).…”

mentioning

confidence: 99%

“…We proposed a genetic model, in which benign and dysplastic naevi represent p16 and p53-dependent senescence, while melanomas arise by immortalisation of melanocytes (Bennett, 2003;Sviderskaya et al, 2003;Bennett, 2006). Published biological and molecular data (Talve et al, 1997;Keller-Melchior et al, 1998;Glaessl et al, 1999;Rudolph et al, 2000;Hussein and Wood, 2002;Bennett, 2003;Sviderskaya et al, 2003) were consistent with this model, but not conclusive, although a convincing report of cell senescence in melanocytic naevi has appeared recently (Michaloglou et al, 2005). We have now identified the genetic requirements for immortalisation of human melanocytes, and systematically studied molecular markers and mediators of cell senescence in clinical pigmented lesions of increasing malignancy.…”

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confidence: 99%

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

et al. 2006

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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 growthphase) 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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Section: Melanocyte Culture and Gene Transfermentioning

confidence: 99%

Section: Melanocyte Culture and Gene Transfermentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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

et al. 2006

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Isolation, Culture, and Transfection of Melanocytes

et al. 2014

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Located in the basal epidermis and hair follicles, melanocytes of the integument are responsible for its coloration through production of melanin pigments. Melanin is produced in lysosomal‐like organelles called melanosomes. In humans, this skin pigmentation acts as an ultraviolet radiation filter. Abnormalities in the division of melanocytes are quite common, with potentially oncogenic growth usually followed by cell senescence producing benign naevi (moles), or occasionally melanoma. Therefore, melanocytes are a useful model for studying melanoma, as well as pigmentation and organelle transport and the diseases affecting these mechanisms. This chapter focuses on the isolation, culture, and transfection of human and murine melanocytes. The first basic protocol describes the primary culture of melanocytes from human skin and the maintenance of growing cultures. The second basic protocol details the subculture and preparation of mouse keratinocyte feeder cells. The primary culture of melanocytes from mouse skin is described in the third basic protocol, and, lastly, the fourth basic protocol outlines a technique for transfecting melanocytes and melanoma cells. Curr. Protoc. Cell Biol. 63:1.8.1‐1.8.20. © 2014 by John Wiley & Sons, Inc.

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