Insights into the transition between G2/M arrest and apoptosis triggering in HeLa cells DOI: 10.13140/RG.2.2.18707.66083, ISSN 2753-8176 (online)

Ana Pedro 1

1 Gwyntwr1386 Pharmacy, Regus Chester Business Park, Heronsway, Chester, CH49QR, UK info@gwyntwr1386.com

MTC (2-Methoxy-5-(2’, 3’, 4’-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one) is a bicyclic colchine analogue synthesized by Fitzgerald (1) and induces microtubule disruption quickly and reversibly (2, 3) leading to a G2/M cell cycle arrest what culminates in apoptosis in human leukemic cells (Gajate et al, 2000) and in HeLa cells (4). B-cell lymphoma-extra large (Bcl-xL), is a member of the Bcl-2 family of proteins. It is a transmembrane molecule localized in the mitochonria and acts as an anti-apoptotic protein by preventing the release of mitochondrial contents such as cytocrome c, which leads to caspase activation and ultimately, apoptosis triggering (5).

Here we analyse the events which occur during the transition between G2/M arrest and apoptosis triggering in HeLa cells and HeLa BCL-XL cells by using MTC to induce a reversible G2/M arrest.

As previously shown (4), PARP was broken down at 24 and 36h MTC 10-6 treatment corresponding to irreversible apoptosis triggering. Apoptosis was detected at 24h treatment and cells were completely apoptotic by 48h treatment (4). If HeLa cells are treated with MTC 10-6 during 12h and then the culture media is washed out and the cells are left to recover in media without MTC, HeLa cells can fully recover from MTC treatment (Fig.1A) . These corresponds to fully cytoskeleton recovery (Fig. 1B). However, at 18h treatment, cell recovery is not so complete, and it appears a subG1 cell population (Fig.1A). At 36h treatment, this recovery is very small, being that most of the cells are apoptotic (Fig. 1A) and the cytoskeleton does not show any recovery at all (Fig. 1B).

Finally, HeLa BCL-XL cells treated with MTC 10-6 during 48h are still arrest in G2/M (Fig.2).

Methods

This work was performed under the scope of a Masters thesis at University of Salamanca within the PhD program in Microbiology and Genetics (2002/2002).

Staining

DAPI (4’,6- diamino-2-phenylindole), Sigma

Flutax-2 (7-O-[N-(2,7-difluoro-4’-fluresceincarbonyl-L-alanyl] (with difluorofluorescein)- a kind gift of Drs AU Acuna and F Amat (CSIC, Madrid, Spain)

Culture media

Dulbecco’s MEM 25MM Hepes (DMEN), GibcoBRL, Life Technologies supplemented with 10% (v/v) heat-inactivated fetal calf (FCS), 2mM L-glutamine, 100 units/ml penicillin and 24 ug/ml gentamicin.

Cells

HeLa cells

HeLa BCLxl cells (F. Mollinedo Lab)

Drugs

MTC (2-methoxy-5-92’,3’4’-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one). This compound was kindly given by Dr. JM Andreu and Dr. Thomas L. Fitzgerald (Florida A&M University, USA).

Instruments

- Citometry – Becton Dickinson – FACS calibu

- Confocal microscopy – Zeiss LSM 510

Programmes

- INPLOT-4 – protein concentration calculation

- Winmdi – flow citometry

- LSM 5 Image Browser – confocal

References

1.Fitzgerald TJ (1976). Molecular features of colchicine associated with antimitotic activity and inhibition of tubulin polymerization. Biochem Pharmacol1976 Jun 15;25(12):1383-7

2. Mollinedo F et al (1989). Cytoplasmic microtubules in human neutrophil degranulation: reversible inhibition by the colchicine analogue 2-methoxy-5-(2',3',4'-trimethoxyphenyl)-2,4,6-cycloheptatrien-1- one. Mol Pharmacol 36(4), 547-55

3. Gajate et al (2000). Induction of apoptosis in leukemic cells by the reversible microtubule-disrupting agent 2-methoxy-5-(2',3',4'-trimethoxyphenyl)-2,4,6-cycloheptatrien-1 -one: protection by Bcl-2 and Bcl-X(L) and cell cycle arrest. Cancer Research 60, 2651-2659

4. Pedro A (2023).The effects of MTC in the cell cycle, cytoskeleton and apoptosis pathways in HeLa cells, ISSN 2753-8176 (online), DOI:10.13140/RG.2.2.21767.37283

5. Korsmeyer SJ (March 1995). "Regulators of cell death". Trends in Genetics. 11 (3): 101–105. doi:10.1016/S0168-9525(00)89010-1. PMID 7732571.