Identification of cell lines CL-14, CL-40 and CAL-51 as suitable models for SARS-CoV-2 infection studies.

The SARS-CoV-2 pandemic is a major global threat that sparked global research efforts. Pre-clinical and biochemical SARS-CoV-2 studies firstly rely on cell culture experiments where the importance of choosing an appropriate cell culture model is often underestimated. We here present a bottom-up approach to identify suitable permissive cancer cell lines for drug screening and virus research. Human cancer cell lines were screened for the SARS-CoV-2 cellular entry factors ACE2 and TMPRSS2 based on RNA-seq data of the Cancer Cell Line Encyclopedia (CCLE). However, experimentally testing permissiveness towards SARS-CoV-2 infection, we found limited correlation between receptor expression and permissiveness. This underlines that permissiveness of cells towards viral infection is determined not only by the presence of entry receptors but is defined by the availability of cellular resources, intrinsic immunity, and apoptosis. Aside from established cell culture infection models CACO-2 and CALU-3, three highly permissive human cell lines, colon cancer cell lines CL-14 and CL-40 and the breast cancer cell line CAL-51 and several low permissive cell lines were identified. Cell lines were characterised in more detail offering a broader choice of non-overexpression in vitro infection models to the scientific community. For some cell lines a truncated ACE2 mRNA and missense variants in TMPRSS2 might hint at disturbed host susceptibility towards viral entry.

The effect of 17ß-estradiol on sex-dimorphic cytochrome P450 expression patterns induced by hyperoxia in the liver of male CBA/H mice.

The aim of this study was to determine whether treatment of male CBA/H mice with 17ß-estradiol (E2) had protective effect on survival and hepatic oxidative damage of lipids and proteins against hyperoxia. Furthermore, we wanted to explore the effect of E2 treatment on the expression of sex-specific cytochrome P450 isoforms, and their possible involvement in E2-induced resistance to hyperoxia. Lipid peroxidation and protein carbonylation were analysed spectrophotometrically and were used as a measure of lipid and protein oxidative damage. Real-time PCR and western blot analysis were used to measure both gene and protein expression levels of Cyp2E1, Cyp7B1 and Cyp2A4, respectively. We found that treatment of male CBA/H mice with E2 increased survival upon hyperoxia exposure, and provided protection against hepatic lipid and protein oxidative damage. Hyperoxia had feminizing effect on the expression of sex-specific CYPs, which resembled the lifespan-promoting conditions. E2 administration had the opposite effect on the expression pattern of these CYPs in hyperoxic versus normoxic conditions. Results of this research proposed possible male strategy in adaptive response to oxidative stress, which may finally result in their longer lifespan.

Prominent role of exopeptidase DPP III in estrogen-mediated protection against hyperoxia in vivo.

A number of age-related diseases have a low incidence in females, which is attributed to a protective effect of sex hormones. For instance, the female sex hormone estrogen (E2) has a well established cytoprotective effect against oxidative stress, which strongly contributes to ageing. However, the mechanism by which E2 exerts its protective activity remains elusive. In this study we address the question whether the E2-induced protective effect against hyperoxia is mediated by the Nrf-2/Keap-1 signaling pathway. In particular, we investigate the E2-induced expression and cellular distribution of DPP III monozinc exopeptidase, a member of the Nrf-2/Keap-1 pathway, upon hyperoxia treatment. We find that DPP III accumulates in the nucleus in response to hyperoxia. Further, we show that combined induction of hyperoxia and E2 administration have an additive effect on the nuclear accumulation of DPP III. The level of nuclear accumulation of DPP III is comparable to nuclear accumulation of Nrf-2 in healthy female mice exposed to hyperoxia. In ovariectomized females exposed to hyperoxia, supplementation of E2 induced upregulation of DPP III, Ho-1, Sirt-1 and downregulation of Ppar-γ. While other cytoprotective mechanisms cannot be excluded, these findings demonstrate a prominent role of DPP III, along with Sirt-1, in the E2-mediated protection against hyperoxia.

Response to hyperoxia is associated with similar ho-1 gene expression level in lungs of aging CBA mice of both sexes.

Beside classical antioxidative enzymes, the response to hyperoxia might be mediated via regulation of other systems, such as heme oxygenase (HO). Ho-1 gene expression is found to be upregulated by hyperoxia in all groups of mice, while HO-1 protein isoform was increased only in 4 months old male mice. In steady-state conditions ho-1 and ho-2 gene expression remained unchanged irrespective of sex or age, which was not the case with protein level of both isoforms. This study suggests that in lungs of CBA mice the response to oxidative stress may be mediated through the interaction of other systems such as heme oxygenase, primarily via upregulation of ho-1 gene expression in both sexes. Contrary to our previous study in liver of hyperoxia treated mice, current results might imply that at conventional oxygen conditions lungs of female mice with the emphasis on aging females, are better prepared for oxidative stress conditions through the increase of HO-activity.

Female headstart in resistance to hyperoxia-induced oxidative stress in mice.

Increased oxygen concentration (hyperoxia) induces oxidative damage of tissues and organs. Oxygen toxicity in hyperoxia is controlled by factors such as sex, age, tissue, strain and hormones. In most species females show lower incidence of some age-related pathologies linked with oxidative stress, which has been attributed to a beneficial effect of ovarian hormones. In this study we found that hyperoxia induced hepatic oxidative damage exclusively in male CBA/H mice, followed by their decreased survival. Histopathological examination revealed that the observed differences in survival were not the consequence of acute lung injury induced by hyperoxia. Next, we observed that an increased Sirt1 protein level in hyperoxia-exposed female CBA/H mice correlated with their lower PPAR-γ and higher eNOS and Sod2 protein levels. In males, higher PPAR-γ and lower Sod2 protein levels were associated with unchanged Sirt1 expression. Although these results are of a correlative nature only, they clearly show that females show better survival, increased resistance to hyperoxia and have generally more efficient defense systems, which suggests that their headstart in resistance to hyperoxia could be a consequence of the beneficial effect of ovarian hormones.