Reports
The expression of p16INK4a, the product of a tumor suppressor gene for melanoma, is upregulated in human melanocytes by UVB irradiation,☆☆

https://doi.org/10.1067/mjd.2000.103988Get rights and content

Abstract

Background: The genetic locus CDKN2A has been linked to familial melanoma, and mutations or deletions in its coding sequence are seen in some cases of sporadic and familial melanomas. The protein encoded by CDKN2A, p16INK4a, functions as a negative regulator of cell cycle progression and as a tumor suppressor, but the regulatory mechanisms involved in controlling its expression remain poorly defined. Objective: This study tested the hypothesis that UVB irradiation, which transiently inhibits the growth of human melanocytes, is one of the regulators of p16INK4a expression. Methods: Cultured human melanocytes were irradiated with UVB over a sublethal dosage range, and p16INK4a protein and mRNA levels were quantified at varying times thereafter by quantitative immunostaining and by Western and Northern blotting. Results: Levels of p16INK4a protein in melanocytes increased significantly after sublethal UVB irradiation as compared with nonirradiated cells. Northern analysis indicated that p16INK4a messenger RNA coordinately increased in a dose-dependent manner more than 2-fold in irradiated cells at the tested doses. Conclusion: UVB irradiation transcriptionally activates the expression of p16INK4a in cultured human melanocytes. Therefore the growth arrest that occurs with irradiation of melanocytes could be mediated, in part, by upregulation of p16INK4a. This transient arrest may allow repair of UVB-induced DNA damage before cell division. Conversely, hereditary or acquired defects in CDK4A that give rise to functional insufficiency of p16INK4a could permit the premature propagation of melanocytes harboring potentially carcinogenic DNA damage. (J Am Acad Dermatol 2000;42:741-5.)

Section snippets

Melanocyte culture and UVB irradiation

Primary melanocyte cultures were established from human neonatal foreskins (from whites) with selective melanocyte growth medium (MGM; Clonetics, San Diego, Calif), as reported.9 At near confluence of the asynchronous monolayers, the cells were washed with warm HEPES-buffered saline, leaving but a thin film over the cells. Single UVB (290-320 nm) exposure was administered with a UltraLum UVB-28 lamp, calibrated with a UltraLum CDR-2 All Wave UV Intensity Meter (UltraLum, Inc, Paramount, Calif).

RESULTS

After irradiation at several UVB doses, or no irradiation, melanocyte cultures were harvested at increasing incubation times thereafter for analysis of p16INK4a protein expression by immunostaining. Cellular labeling intensities of the immunoperoxidase-stained cells fixed in situ on slide chambers were quantified by digital image analysis, as previously described.9 It was found that expression of p16INK4a protein increased over time after irradiation and was maximal at approximately 5 to 12

DISCUSSION

With these studies we have evaluated a rule for UVB irradiation in regulating the expression of p16INK4a in cultured human neonatal melanocytes. Trends in p16INK4a protein expression after dosing with UVB were analyzed by quantitative immunolabeling and by Western blotting, and complementary Northern blotting was used to assess coordinate changes in the levels of p16INK4a mRNA. The data demonstrated significant upregulation of p16INK4a protein and a greater than 2-fold increase in melanocyte p16

Acknowledgements

Note added in proof: A recent report showed that UV irradiation of skin organ cultures upregulates p16INK4a expression in epidermal melanocytes and keratinocytes. These results are consistent with those reported herein (Pavey S, Conroy S, Russell T, Gabrielli B. Ultraviolet radiation induces p16CDKN2A expression in human skin. Cancer Res 1999;59:4185-9).

The technical assistance of Linda Foy, Terrance Peterson, and Dr Deniz Seçkin is gratefully acknowledged.

References (18)

There are more references available in the full text version of this article.

Cited by (33)

  • Role of CDKN2A/p16 expression in the prognostication of oral squamous cell carcinoma

    2017, Oral Oncology
    Citation Excerpt :

    Telomere attrition in the malignant tumor results in induction of CDKN2A/p16 and stabilization of telomeres by telomerase activation thus suppresses the expression of CDKN2A/p16 [19–23]. CDKN2A/p16 is also reported to respond to other telomere independent stress signal such as constitutive oncogenic signalling and DNA damage signalling [24–26]. Telomerase activity is reported to be upregulated in solid tumors including OSCC [4,27].

  • Suppression of autophagy dysregulates the antioxidant response and causes premature senescence of melanocytes

    2015, Journal of Investigative Dermatology
    Citation Excerpt :

    The changes in the morphology of autophagy-deficient MC were accompanied by significantly higher expression of p16Ink4a (CDKN2A) and p21 (CDKN1A) mRNAs (Figure 4e, P<0.05), and a significantly higher proportion of mutant cells exhibited nuclear p16Ink4a protein as determined by immunofluorescence staining (Figure 4f and g). Nuclear translocation of p16Ink4a increased upon UVB irradiation (20 mJ cm-2) (Piepkorn, 2000) in autophagy-competent cells, whereas it could not be elevated above the high basal level in autophagy-deficient cells (Figure 4f and g). Taken together these data suggest that the absence of autophagy leads to premature senescence of MC.

  • Molecular pathology of malignant melanoma: Changing the clinical practice paradigm toward a personalized approach

    2014, Human Pathology
    Citation Excerpt :

    INK4 encodes the protein p16, which is also located in the 9p21 region. This gene is upregulated after ultraviolet B exposure in human melanocytes [105]. Progressive loss of p16 is seen in transformation of nevi toward melanoma.

  • Lentigines, nevi, and melanomas

    2009, Weedon's Skin Pathology: Third Edition
  • Photochemoprevention of ultraviolet B signaling and photocarcinogenesis

    2005, Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
View all citing articles on Scopus

Supported in part by US Public Health Service grant AR 21557 from the National Institutes of Health, Department of Health and Human Services.

☆☆

Reprint requests: Michael Piepkorn, MD, PhD, Division of Dermatology, Box 356524, Seattle, WA 98195-6524.

View full text