The effects of PGA2 on cell cycle progression in oesophageal carcinoma cells
The molecular mechanisms causing anti-mitotic and cytotoxic effects of prostaglandin .A2 [PGA2 (CzoHroOa)] treatment of human oesophageal carcinoma (WHCO3) cells were investigated. WHCO3 cells, incubated with cytotoxic doses of PGAz (20 and 50pg/ml) for 3, 24 and 48 hrs showed characteristic morphological features of apoptosis such as chromatin condensation, nuclear fragmentation, as well the formation of apoptotic bodies. Chromatin condensation was more prevalent in cells exposed to 201tglml PGAz for 24 hrs. Apoptosis induction was found at both concentrations and cells exposed to 50pg/ml showed apoptosis at 3 hrs already. An increase in abnormal metaphases was observed in cells exposed to 2\pg/ml PGAz for 24 hrs. This outspoken increase in metaphases could be due to the activation of the spindle assembly checkpoint. Activation of this checkpoint blocks the onset of anaphase and arrests cells in metaphase. The flow cytometry studies also showed an increase of cells in metaphase. Metaphase arrest occurs when any of the following situations have effect: unattachment of a single kinetochore to the microtubules, unbalanced tension on chromosomes or if there is abnormal dynamic behaviour of the microtubules attached to the kinetochore (Bunz et al., 1998; Gorbsky, 1997; Gorbsky, 2001; Murray, 1994). WHCO3 cells exposed to 20pglml PGAz for 24 hrs showed an accumulation of cells with condensed chromosomes and according to Cahill et aL.,1998, this accumulation is characteristic of a mitotic block. After 48 hrs, cells exposed to 2}pglml PGAz showed a decrease in the number of apoptotic cells and it appeared that the cells were able to overcome the arrest. Indirect immunofluorescent studies of otubulin showed that PGAz had varied effects on the cytoskeleton. Fewer but intact microtubules were observed when cells were exposed to 201tglml PGA2 for 24 hrs, whereas the results obtained from cells exposed to z}p/ml PGA2 for 48 hrs showed some normal spindles, a few abnormal arranged spindles and others disrupted microtubules. Cotter et al., 1992 showed that disrupted microtubules prevent the formation of apoptosis. The results of the 2lpglml PGAz studies for 24 and 48 hrs can possibly be explained by this phenomenon. The cells exposed to 20pglml PGAz for 24 hrs showed increased chromatin condensation and apoptosis formation, whilst the cells exposed to the same concentration for 48 hrs showed a decrease in apoptosis formation. This result can possibly be ascribed to either the ability of the cell to overcome the spindle assembly checkpoint or an increase in the disruption of the microtubule arrangement, thus preventing the induction of apoptosis. In cells exposed to 50pg/ml PGAz total rearrangement of o-tubulin was very much evident and this rearrangement could be a cause of apoptosis induction as indicated by the large amount of apoptotic cells (28.93%o and 5l.63oh during 24 and 48 hrs respectively) with flow cytometry. Silver staining of PGAz treated WHCO3 cells showed segregation of granular and fibrillar components. This segregation is usually associated with non-functional nucleoli and indicative of an inhibition in protein synthesis (Hadjiolov, 1985). DNA fragmentation was also observed with the two higher concentrations of PGAz. On agarose gel electrophoresis, DNA laddering was evident in the cells exposed to 50pg/ml for 3 hrs and 20pglml for 24 hrs. It was furthermore found that caspase 3 could possibly play a role in the induction of apoptosis, internucleosomal DNA degradation, chromatin condensation, membrane blebbing and disassembly into membrane bound vesicles at the higher concentrations.