Detection of Cellular Senescence in Human Primary Melanocytes and Malignant Melanoma Cells In Vitro

Abstract

Detection and quantification of senescent cells remain difficult due to variable phenotypes and the absence of highly specific and reliable biomarkers. It is therefore widely accepted to use a combination of multiple markers and cellular characteristics to define senescent cells in vitro. The exact choice of these markers is a subject of ongoing discussion and usually depends on objective reasons such as cell type and treatment conditions, as well as subjective considerations including feasibility and personal experience. This study aims to provide a comprehensive comparison of biomarkers and cellular characteristics used to detect senescence in melanocytic systems. Each marker was assessed in primary human melanocytes that overexpress mutant BRAFV600E, as it is commonly found in melanocytic nevi, and melanoma cells after treatment with the chemotherapeutic agent etoposide. The combined use of these two experimental settings is thought to allow profound conclusions on the choice of senescence biomarkers when working with melanocytic systems. Further, this study supports the development of standardized senescence detection and quantification by providing a comparative analysis that might also be helpful for other cell types and experimental conditions.

Knockdown of Lamin B1 and the Corresponding Lamin B Receptor Leads to Changes in Heterochromatin State and Senescence Induction in Malignant Melanoma

“…The increased amount of heterochromatin foci in LMNB1 knockdown cells could indicate the need for LMNB1 in melanoma cells to prevent cellular ageing or senescence. To confirm this, we investigated the functional influence of LMNB1 knockdown in MEL-JUSO and SK-MEL-28 cells using established senescence assays [ 37 , 38 ] and revealed an increase in senescent cells as determined by SA β-Gal staining ( Figure 2 A). Furthermore, immunofluorescence staining of the promyelocytic leukaemia protein (PML) expression after LMNB1 knockdown indicated a significant induction of the PML ( Figure 2 B).…”

“…Interestingly, we revealed that LMNB1 is downregulated in BRAF V600E - compared to mock-transduced NHEMs ( Figure 3 A). For a proof of concept, we induced senescence in melanoma cells with the cytostatic drug etoposide, which was described previously [ 38 ]. LMNB1 was downregulated in MEL-JUSO and SK-MEL-28 cells treated with etoposide for 48 h ( Figure 3 A).…”

“…Similar to LMNB1, the low senescence induction may also be related to compensation by EMD ( Figure S2A,B ). Since we could show that LBR has similar functional effects regarding senescence in melanoma-like LMNB1, we additionally investigated the expression of the LBR on the mRNA level in the senescence models described above [ 23 , 38 , 39 ]. We observed LBR downregulation in the NHEM/BRAF V600E senescence model as well as in etoposide-treated MEL-JUSO cells ( Figure 6 D).…”

LPA receptor 1 (LPAR1) is a novel interaction partner of Filamin A that promotes Filamin A phosphorylation, MRTF-A transcriptional activity and oncogene-induced senescence

“…1E , S1E ). We next investigated well-known markers of oncogene-induced senescence [ 28 ] and observed phosphorylation of ERK, hypophosphorylation of Retinoblastoma (Rb) protein and accumulation of histone H3 methylated on lysine 9 upon LPAR1 depletion in HuH7 cells (Fig. 1F ).…”

Current Understanding of the Role of Senescent Melanocytes in Skin Ageing

“…The mechanisms of melanocyte senescence have been extensively explored in the context of melanoma, particularly BRAF V600E OIS within naevi (moles) [ 22 , 25 , 80 , 81 ]. Although the focus of this review is on the contribution of senescent melanocytes to skin ageing, mechanistic insights can be gained from melanoma research.…”