Low-dose etoposide-treatment induces endoreplication and cell death accompanied by cytoskeletal alterations in A549 cells: Does the response involve senescence? The possible role of vimentin

Abstract

BackgroundSenescence in the population of cells is often described as a program of restricted proliferative capacity, which is manifested by broad morphological and biochemical changes including a metabolic shift towards an autophagic-like response and a genotoxic-stress related induction of polyploidy. Concomitantly, the cell cycle progression of a senescent cell is believed to be irreversibly arrested. Recent reports suggest that this phenomenon may have an influence on the therapeutic outcome of anticancer treatment. The aim of this study was to verify the possible involvement of this program in the response to the treatment of the A549 cell population with low doses of etoposide, as well as to describe accompanying cytoskeletal alterations.MethodsAfter treatment with etoposide, selected biochemical and morphological parameters were examined, including: the activity of senescence-associated ß-galactosidase, SAHF formation, cell cycle progression, the induction of p21Cip1/Waf1/Sdi1 and cyclin D1, DNA strand breaks, the disruption of cell membrane asymmetry/integrity and ultrastructural alterations. Vimentin and G-actin cytoskeleton was evaluated both cytometrically and microscopically.Results and conclusionsEtoposide induced a senescence-like phenotype in the population of A549 cells. Morphological alterations were nevertheless not directly coupled with other senescence markers including a stable cell cycle arrest, SAHF formation or p21Cip1/Waf1/Sdi1 induction. Instead, a polyploid, TUNEL-positive fraction of cells visibly grew in number. Also upregulation of cyclin D1 was observed. Here we present preliminary evidence, based on microscopic analyses, that suggest a possible role of vimentin in nuclear alterations accompanying polyploidization-depolyploidization events following genotoxic insults.

Unr defines a novel class of nucleoplasmic reticulum involved in mRNA translation

“…Our hypothesis, linking Unr-NRs to polyploidy, was correct since Unr-NRs were inducible in the hepatoblastic Hep3B cell line treated with etoposide, and in the trophoblastic BeWo cell line treated with Taxol (Litwiniec et al, 2013;Marth et al, 1995). Unr-NRs were preferentially formed in cells with enlarged nuclei, their frequency in the surviving cell population reaching up to 23% in etoposide-treated Hep3B cells and 70% in Taxol-treated BeWo cells.…”

“…To this end, we selected two human cancer cell lines, BeWo and Hep3B − derived from trophoblasts and hepatocytes, respectively − to test the capacity of cytotoxic treatments known to induce polyploidy to trigger UnrNRs formation. The drugs we used included etoposide − a DNAdamaging drug, and Taxol − a spindle toxin (Litwiniec et al, 2013;Marth et al, 1995). Whereas Unr-NRs were detectable in untreated cultures, their incidence markedly increased following drug treatment in both cell cultures (Figs 5E and 6A).…”

Expression of cyclin D1 after treatment with doxorubicin in the HL‐60 cell line

“…Therefore, it would be tempting to hypothesize that increased levels of cyclin D1 may somehow reflect or even promote these cells to enter the S phase and proliferate further, especially as we have shown in our cyclin D1/PI double staining experiment that the highest levels of cyclin D1 are indicative of G2/M population and particularly of the fraction of polyploid cells. In previous studies we also found that the levels/fluorescence of cyclin D1 increased in the G2/M and polyploidy after treatment with doxorubicin in the Jurkat and A549 cell lines ( _ Zury n et al, 2012; Litwiniec et al, 2013). The percentage of subG 1 Cyclin D1 after treatment with doxorubicin A.…”

“…Therefore, it would be tempting to hypothesize that increased levels of cyclin D1 may somehow reflect or even promote these cells to enter the S phase and proliferate further, especially as we have shown in our cyclin D1/PI double staining experiment that the highest levels of cyclin D1 are indicative of G2/M population and particularly of the fraction of polyploid cells. In previous studies we also found that the levels/fluorescence of cyclin D1 increased in the G2/M and polyploidy after treatment with doxorubicin in the Jurkat and A549 cell lines (Żuryń et al, ; Litwiniec et al, ). The percentage of subG 1 fraction as well as early and late apoptotic cells, as evidenced by annexin V/PI staining, is visibly increased starting from this concentration and the number of G0/G1‐cyclin D1‐positive cells decreased, whereas the respective values were also reduced in the S‐ and G2/M‐phases with 0.3 µM doxorubicin, which suggests that induction of cell death pathways most probably contribute to observed reductions in the number of polyploid cells.…”

Common Chemical Inductors of Replication Stress: Focus on Cell‐Based Studies

“…Indeed, several studies reported that higher doses of the compound (25–100 µM) activate apoptosis, mostly in a manner dependent on p53 [325,326,327,329]. Prolonged treatment at lower concentrations of ETP can also lead to induction of the p53 pathway, cell cycle arrest, senescence and apoptosis [145,325,330,335,337]. ETP induces the formation of irreversible DNA–TopoII cleavage complexes (TopoIIcc) and DNA damage regardless of concentration or incubation time [323,324,329,330,331,332,334,353].…”