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 (HuWaTERT) have been stable for at least 59 passages post-transfection. HuWaTERT proliferate rapidly and maintain initial cell characteristics, such as morphology and chromosomal stability. The response of HuWaTERT 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. HuWaTERT 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.
Highlights
Long-lived cetaceans, such as humpback whales (Megaptera novaeangliae), are continuously exposed to lipophilic and bioaccumulative environmental contaminants
HuWawild-type cells were transfected with plasmids containing simian virus 40 large T antigen (SV40T) or telomerase reverse transcriptase (TERT), and successful clones were characterised regarding their growth, chromosomal stability and sub-cellular structure
Because fetal bovine serum (FBS) contains hormones and growth factors which can influence the expression of cytokines in cells, we reduced the influence of these undefined factors by performing experiments in DMEM/F12 supplemented with 1% FBS, rather than the usual 10% FBS
Summary
Long-lived cetaceans, such as humpback whales (Megaptera novaeangliae), are continuously exposed to lipophilic and bioaccumulative environmental contaminants. There is little information regarding the toxicological sensitivity of the species (Bengtson Nash et al 2014; Waugh et al 2011), and no information related to the influence of contaminants on the immune health of humpback whales; studies are limited to postmortem examination (Apprill et al 2014; Holyoake et al 2012) precluding controlled experimentation. Various transfection strategies have been developed to introduce foreign DNA into mammalian wild-type cells in order to control the cell cycle and cellular senescence. Expression of SV40T antigen inactivates tumour suppressor genes, such as p53 and the Rb family, and enables continuous mitosis and long-term survival (Ali and DeCaprio 2001; Ahuja et al 2005; Wright and Shay 1992)
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