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Molecular crosstalk between apoptosis and autophagy induced by a 2-methoxyestradiol analogue (C19) in HeLa cellsTheron, A.E. (Anne Elisabeth) 30 July 2012 (has links)
Cervical cancer is reported by the World Health Organisation to be the second most
common type of cancer to affect women in poorer socioeconomic countries. Treatment of
this pathology remains sub-optimal at advanced stages and continues to be of importance
on the research agenda.
Previous studies have reported cytotoxic and antiproliferative effects of 2-methoxyestradiol
(2-ME) in vitro on a HeLa cervical cancer cell line. These results were promising but use of
2-ME itself is limited due to pharmacodynamic constraints. In an attempt to overcome
these, a sulphamoylated analogue of 2-ME, namely 2-ethyl-3-O-sulphamoyl-estra-
1,3,5(10)16-tetraene or compound 19 (C19), was synthesised.
In this in vitro study, the induction of a block in mitosis with subsequent culmination of
apoptosis and autophagy as types of cells death was investigated after HeLa cells were
exposed for 24 hours to a 0.5 μM C19 solution. This was achieved by morphological
assessment (fluorescent, Polarization-optical transmitted light differential interference
contrast microscopy (PlasDIC) and transmission electron microscopy (TEM)) and flow
cytometry (cell cycle progression, cyclin B1 analysis, phosphatidylserine (PS) flip and
aggresome formation). Spectrophotometric quantification of the apoptotic initiator and
executioner caspases 8 and 3 respectively was done to determine their involvement in the
crosstalk between apoptosis and autophagy.
Results included the following: (i) PlasDIC microscopy illustrated the appearance of an
increased number of cells blocked in metaphase, stress signaling, premature cell shrinkage,
hypercondensed chromatin and the presence of apoptotic bodies after C19 exposure. The
presence of ghost cells, cell debris and decreased cell density of the treated cells correlated
with the autophagy control. (ii) Fluorescence microscopy employing triple staining
highlighted an increased lysosomal activity and staining of C19-exposed cells when
compared to the control, as well as evidence of apoptotic and metaphase-blocked cells. This is indicative of both the autophagic and apoptotic cell death process. (iii) TEM allowed
for examination of the ultrastructure of the intracellular processes, and revealed that
apoptotic cells have hallmarks of both autophagy and apoptosis, confirming the results of
light microscopy. (iv) Cell cycle analysis demonstrated more cells present in the sub-G1 and
G2/M populations, indicating the induction of apoptosis (confirmed with PS fip flow
cytometric quantification) and a metaphase block (corroborated by an increased cyclin B1
fluorescence). (v) The increase in autophagosome formation seen on fluorescence- and
transmission electron microscopy was confirmed by flow cytometry demonstrating an
upregulation of aggresome formation in C19-exposed cells. This investigation
demonstrated induction of both types of cells death by this novel compound. (vi) The
upregulation of caspases 8 and 3 was demonstrated in the C19-treated cells, indicating
apoptosis induction via the extrinsic pathway. (vii) Confocal microscopy demonstrated
complete microtubule disintegration in the C19-exposed HeLa cells.
Both apoptotic and autophagic cell death mechanisms were induced in C19-treated HeLa
cells after spindle abrogation kept the cells in metaphase block. Insight gained into the
molecular effect of C19 on HeLa cells may be used as a springboard for in vivo studies,
furthering the development of this promising anticancer agent toward clinical application. / Dissertation (MSc)--University of Pretoria, 2012. / Physiology / MSc / Unrestricted
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Molecular remodelling of the spindle architecture during metaphase arrest in oocytesCosta, Mariana Fernandes Alves January 2018 (has links)
Oocytes of most species assemble and maintain a functional bipolar spindle in the absence of centrosomes. Strikingly, after bipolar spindle formation, oocytes arrest in metaphase for several hours before fertilisation. How the dynamic spindle maintains its bipolarity during this long arrest is poorly understood. I hypothesise that the bipolar spindle is stably maintained by changes in the distribution of microtubule-associated proteins (MAPs) on the spindle during the long oocyte arrest. To test this, I generated transgenic flies expressing GFP-tagged microtubule-associated proteins (MAPs), and found that 13 out of 24 proteins change localisation between early and late oocytes. I refer to these changes in MAP localisation after establishment of bipolarity as 'spindle maturation'. In order to identify the molecular mechanisms triggering MAP relocalisation, I manipulated the kinase activity of the cell cycle regulator Cdk1 by over-expressing non-degradable cyclin A or B, the major activators of Cdk1. Their expression prevented re-localisation of distinct sets of MAPs, and disrupted spindle bipolarity and accurate chromosome segregation in oocytes. Kinesin-6 Pavarotti/MKlp1 localised strongly to the spindle equator in late oocytes, whilst nearly always absent from this region in early oocytes. The localisation of Pavarotti to the spindle equator in late oocytes was reduced when cyclin B is over-expressed in oocytes, suggesting a role for Cdk1/cyclin B complex in regulating Pavarotti localisation. Indeed, a Pavarotti/Mklp1 mutant non-phosphorylatable by Cdk1 prematurely localised to the meiotic spindle and disrupted spindle bipolarity. Moreover, removal of Pavarotti from the metaphase-I spindle by RNAi induced spindle defects in oocytes. Therefore, it is likely that the microtubule cross-linking activity of Pavarotti enhances the stability of the metaphase-I spindle during the long arrest. Consistent with this, I found that the microtubule density in the spindle equator is higher in late oocytes. Altogether, I propose that remodelling the molecular architecture of the spindle during the long oocyte arrest is important to stabilise the bipolar spindle without centrosomes.
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