Lifetime extension of humpback whale skin fibroblasts and their response to lipopolysaccharide (LPS) and a mixture of polychlorinated biphenyls (Aroclor)

Abstract

Marine mammals, such as whales, have a high proportion of body fat and so are susceptible to the accumulation, and associated detrimental health effects, of lipophilic environmental contaminants. Recently, we developed a wild-type cell line from humpback whale fibroblasts (HuWa). Extensive molecular assessments with mammalian wild-type cells are typically constrained by a finite life span, with cells eventually becoming senescent. Thus, the present work explored the possibility of preventing senescence in the HuWa cell line by transfection with plasmids encoding the simian virus large T antigen (SV40T) or telomerase reverse transcriptase (TERT). No stable expression was achieved upon SV40 transfection. Transfection with TERT, on the other hand, activated the expression of telomerase in HuWa cells. At the time of manuscript preparation, the transfected HuWa cells (HuWa_TERT) have been stable for at least 59 passages post-transfection. HuWa_TERT proliferate rapidly and maintain initial cell characteristics, such as morphology and chromosomal stability. The response of HuWa_TERT cells to an immune stimulant (lipopolysaccharide (LPS)) and an immunotoxicant (Aroclor1254) was assessed by measurement of intracellular levels of the pro-inflammatory cytokines interleukin (IL)-6, IL-1β and tumour necrosis factor (TNF)-α. HuWa_TERT cells constitutively express IL-6, IL-1β and TNFα. Exposure to neither LPS nor Aroclor1254 had an effect on the levels of these cytokines. Overall, this work supports the diverse applicability of HuWa cell lines in that they display reliable long-term preservation, susceptibility to exogenous gene transfer and enable the study of humpback whale-specific cellular response mechanisms.

Keywords: Cell line transfection; Humpback whale; Immunotoxicity; Inflammatory cytokines; Megaptera novaeangliae; Relative telomerase activity.

Fig. 1

Characterisation of the new strain: HuWa_TERT. TERT-transfected cells were characterised for five different features. For morphological appearance (1), HuWa_TERT cells were cultivated over time and the morphological appearance at different levels of confluency were assessed after 1 day (a), 3 days (b) and 7 days (c). Growth (2) was estimated as cell numbers at 1, 3, 5 and 7 days after plating and the population doubling time calculated during the log phase (d). For the karyotype (3), chromosomes of HuWa_TERT cells were visualised by G-banding; chromosome pairs of autosomes (1–21), sex chromosomes (X, Y) and unidentified chromosomes are displayed (e). The expression of telomerase (4) was measured in HuWa_TERT cells at different passages post-transfection (F). Data represent the mean of three technical replicates, and the error bars represent the SD for 10,000 cell equivalents. Sub-cellular structures (5) of cells were visualised by  SEM (G); the inset shows a cross section of one intracellular lipid body. Arrows indicate lipid bodies as  seen by confocal microscopy (H, green and arrows). Other sub-cellular structures visualised are the endoplasmic reticulum (magenta) and cell nucleus (blue)

Fig. 2

Expression of inflammatory cytokines in HuWa_TERT either stimulated with LPS or exposed to Aroclor. For cell viability assessment, HuWa_TERT cells were treated with different LPS (A.1) and Aroclor (B.1) concentrations. After 24 h of exposure, metabolic activity using AlamarBlue (LPS and Aroclor) and membrane integrity using CFDA-AM (Aroclor only) were measured. The data in A.1 represents the mean of three technical and, B.1, three biological replicates; the error bars represent SD and are shown as percentage of non-treated cells. The cellular expression of selected inflammatory cytokines (IL-1β, IL-6, TNFα and HSP70) was measured upon treatment with non-cytotoxic concentrations (indicated by red arrow) of LPS (10 μg/ml) (A.2) or Aroclor (1700 μg/l) (B.2) for 24 h. The data is shown as percentage of control. Boxplots indicate median, 5 and 95 percentiles and min/max of three independent biological replicates. No statistical difference was found between single markers and unexposed control using one-way ANOVA and Bonferroni’s post hoc test (p < 0.05)