Supervisors info:
Κουτσιλιέρης Μιχαήλ, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Χαβάκη Σοφία, Επίκουρη Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Πατέρας Ιωάννης, Επίκουρος Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Summary:
The current dissertation was conducted in the frame of the study of our laboratory regarding the oncogenic effect of the prolonged induction of p21CIP1/WAF1 gene (abbreviated p21) on Li-Fraumeni cell line, clone MDAH041 (fibroblasts which arise from skin biopsy of a Li-Fraumeni syndrome patient), for further ultrastructural study. In this specific clone MDAH041 of the Li-Fraumeni cell line the one allele of TP53 is absent, while the other one carries a mutation which alters the open reading frame of this gene resulting in the production of a truncated p53 protein with a totally different conformation. The utilized cellular induction system was Li-Fraumeni-TetON p21 where transcription is inversely activated in the presence of the antibiotic doxycycline (Dox). The cyclin-dependent kinase inhibitor p21WAF1/CIP1 manifests anti-proliferative activities through p53-dependent and p53-independent pathways. However, p21 can also promote tumorigenesis. In pRb/p53 mutant tumors a detected number of large cancer cells unexpectedly presented mutual expression of p21 and the mitotic marker Ki67. Based on a possible hypothesis that was made, prolonged expression of p21 may lead to a selective procedure which allows these cells to retrieve their proliferative capacity. This biological paradox was initially transferred for study on the cellular system Saos2-TetON p21 and then on the system Li-Fraumeni-TetON p21. Upon the induction of p21, cells inserted in a senescence phase which was peaked in the 10th day in Saos2 and in the 6th day in MDAH041 (Li-Fraumeni syndrome). Prolonged induction of p21 on both these cell lines drove to the emergence of a sub-population of proliferating p21-positive cells (“escaped” cells) with a more aggressive phenotype than before (Galanos, Vougas et al. 2016). The aim of this study was the ultra-structural study of Li-Fraumeni cells on three different time-points of p21 inducing expression (0, 6 and 12 days), since p21 forces cells to senescence during the 6th day and to escape from senescence during the12th day according to Galanos et al. 2016. For the materialization of this aim, cell culture and electron microscopy techniques were applied.
Initially, in the selected Li-Fraumeni clone employed for the experiments of optical and electronic microscopy, the expression of p21 was confirmed through the immunofluorescence technique. Specifically, MDAH041 cells without Dox treatment were used as control cell line (Li-Fraumeni OFF) and were compared to cells treated with Dox for 6 and 12 days (Li-Fraumeni ON 6days and ON 12days respectively). Indeed, increased p21 protein expression was detected both in Li-Fraumeni ON 6days (“senescent” cells) and 12days (“escaped” cells) in contrast with Li-Fraumeni OFF where there was no visible signal (Section 3.1).
Under phase contrast inversion microscope, a change in morphology of Li-Fraumeni ON 6days was observed, having larger size and proportion of cytoplasm/nucleus. They also seem to contain pronounced nuclei and nucleoli along with a granulated cytoplasm compared to Li-Fraumeni OFF. The latter are presented more spindle and elongated, some of them with a more flattened shape. Furthermore, they seem to have less pronounced nuclei, nucleoli and be smaller in size (Section 3.2).
In the majority of Li-Fraumeni ON 6days, a senescent phenotype was identified due to the detection of lipofuscin bodies in their cytoplasm by means of GL13 histo-immunohistochemical stain (SenTraGor), whereas no similar detection was observed in the OFF cells (Section 3.3). It is worth noting that the induction of aging is due solely to overexpression of p21 and is independent of the presence of p53, which is mutated and could not be detected by both Western blot and immunofluorescence in the studied cells (Galanos, Vougas et al. 2016). After Dox administration for 12 days, the majority of escaped cells are smaller in size compared to senescent cells and spindle, with a sub-rounded cell body, distinct nuclei and numerous nucleoli approaching the morphology of the OFF cells (Section 3.2). Senescent cells are much less at day 12 overexpression of p21 compared to day 6 where the aging phenotype is predominant. In addition, after 12 days of inducible expression of p21, a specific distribution pattern of cells was observed under phase contrast, where giant-senescent cells were surrounded by escaped cells (having the morphological characteristics described above). The latter seem to communicate with the neighboring senescent cells via cytoplasmic projections (Section 3.2).
According to the ultrastructural study of Li-Fraumeni cells in Transmission Electron Microscope (TEM) the OFF cells are mainly spindle, they host a lot of vesicles in their cytoplasm while it seems that some of them are engulfed via thin cytoplasmic extensions. Moreover, these cells form spherical protrusions of the cytoplasm which encompass microvesicles. Some of them seem to be released to the intercellular space, probably addressed to neighboring cells, in the context of communication. The observed autophagosomes are few, while a quite interesting finding is the predominant presence of mitochondria with abnormal morphology. In particular, mitochondria appear elongated with irregular arrangement of their cristae (Section 3.4).
On the other hand, large and multi-lobular nuclei, as well as the extended-dilated endoplasmic reticulum predominate in Li-Fraumeni ON 6days cells. In addition, much more mitochondria appear more swollen and rounded and are deprived of cristae in comparison to those in OFF cells. Autophagy seems to be increased due to the elevated number of autophagosomes observed in various formation stages. Increased autophagy is further supported by the augmented number of lysosomes in the cytoplasm. Senescence-associated secretory phenotype (SASP) is another profound feature of these cells which communicate each other via either thin cytoplasmic projections or intercellular bridges in the formation of which microtubules may be implicated (Section 3.4).
“Escaped” cells are more rounded than ON 6days cells and are characterized by multi-lobular nuclei with numerous nucleoli. Fewer autophagosomes are observed compared to senescent cells indicating reduced autophagic activity. Their mitochondria are spherical in their majority, swollen –some of them giant- with disorganized or completely absent cristae. Interestingly, there was observed their accumulation at the periphery of the cells and their encapsulation in cytoplasmic vesicles, which probably appear to be removed from the rest of the cellular body (mitoptotic bodies). This phenomenon is named “mitoptosis” (programmed mitochondrial death) whereby cells dispose of mitochondria with deregulated morphology and function (Tinari, Garofalo et al. 2007, Mijaljica, Prescott et al. 2010, Jangamreddy and Los 2012). Furthermore, the enlarged/giant size of the observed mitochondria may indicate that fusion is occurred possibly between mitochondria in these cells in order to limit the autophagy-mitophagy, restore the respiratory capacity of mitochondria and preserve the homogeneity of their genetic material (Pernas and Scorrano 2016, Bordi, Nazio et al. 2017). Therefore, the above indications support survival following escape from senescence and the acquisition of cancerous properties by the particular emerging cell subpopulation (Galanos, Vougas et al. 2016).
Keywords:
Cancer, Li-Fraumeni syndrome, p21, Senescence, Cell Communication, Autophagy, Transmission Electron Microscope
