Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells

Summary

The cytotoxic activity of the phenanthridine PJ-34 in cancer cells undergoing mitosis was documented in real time by live confocal imaging. PJ-34 eradicated human breast cancer MDA-MB-231 cells harboring extra-centrosomes in mitosis. Unlike normal bi-focal mitosis, the extra-centrosomes were not clustered in the two spindle poles in the presence of PJ-34.

Abstract

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human cancer cells in mitosis. The phenanthridine PJ-34 was the most potent molecule. Declustering of extra-centrosomes causes mitotic failure and cell death in multi-centrosomal cells. Most solid human cancers have high occurrence of extra-centrosomes. The activity of PJ-34 was documented in real-time by confocal imaging of live human breast cancer MDA-MB-231 cells transfected with vectors encoding for fluorescent γ-tubulin, which is highly abundant in the centrosomes and for fluorescent histone H2b present in the chromosomes. Aberrant chromosomes arrangements and de-clustered γ-tubulin foci representing declustered centrosomes were detected in the transfected MDA-MB-231 cells after treatment with PJ-34. Un-clustered extra-centrosomes in the two spindle poles preceded their cell death. These results linked for the first time the recently detected exclusive cytotoxic activity of PJ-34 in human cancer cells with extra-centrosomes de-clustering in mitosis, and mitotic failure leading to cell death. According to previous findings observed by confocal imaging of fixed cells, PJ-34 exclusively eradicated cancer cells with multi-centrosomes without impairing normal cells undergoing mitosis with two centrosomes and bi-focal spindles. This cytotoxic activity of PJ-34 was not shared by other potent PARP1 inhibitors, and was observed in PARP1 deficient MEF harboring extracentrosomes, suggesting its independency of PARP1 inhibition. Live confocal imaging offered a useful tool for identifying new molecules eradicating cells during mitosis.

Introduction

Phenanthrene derived PARP1 inhibitors, including PJ-34, were designed to protect quiescent cells from apoptotic cell death induced by the energy consuming PARP1 mediated DNA-repair under stress conditions (stroke or myocardial infarction)¹. However, recently we discovered that PJ-34, at twice higher concentration than that inducing PARP1 inhibition, can exclusively cause cell death in human cancer cells^2,3. The more rapid the proliferation of the cell was, the more efficient the eradication of the cells was. The cytotoxic activity of PJ-34 was attributed to extra-centrosomes de-clustering in mitosis². Many human cancer cells harbor mul....

Protocol

1. Cell Culture Preparation

MDA-MB-231 cells were purchased from ATCC (American Type Culture Collection) and stored in liquid nitrogen.

1. Seed 10⁶ MDA-MB-231 cells in 92 mm diameter Petri dish in 10 ml complete medium containing Dulbeco Modified Eagle Medium (DMEM), 10% horse serum, 1% L-glutamine, and 1% Penstrep-Amphotericine B. Allow cells to proliferate to about 80-100% confluence.
2. Remove culture medium from dish and discard.
3. Wash the cell layer briefly with 0.25% (w/v) Trypsin-EDTA solution to remove all traces of serum.
4. Add 2.0 ml of Trypsin-EDTA solution to dish and observe ....

Results

PJ-34 is a stable water soluble phenanthridine¹ (Figure 1). Our previous results revealed cell death and de-clustered extra-centrosomes in several types of fixed multi-centrosomal cancer cells that were treated with PJ-34. In contrast, normal proliferating cells were not impaired^2,3. Centrosomes were identified by double labeling with antibodies directed against centrine1 and γ-tubulin in the fixed extra-centrosomal cells².

Here, the cytotoxic .......

Discussion

Live confocal imaging provided a real-time documentation of the cytotoxic effect of PJ-34 in live multi-centrosomal cells during mitosis (Figure 3 and Supplementary information). This was the first live documentation attributing the cytotoxicity of PJ-34 in human cancer cells to extra centrosomes de-clustering and cell death, suggesting induction of Mitotic Catastrophe cell death by PJ-34^5-9. In contrast, bi-focal clustering of super-numerary centrosomes was observed in live untreated MDA-MB-2.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
REAGENTS
DMEM	Invitrogen (GIBCO)	41965
FBS (Fetal bovine Serum)	Invitrogen (GIBCO)	12657
Pen-Strep-Ampho solution	Biological Industries, Israel	03-033-1B
L-glutamine	Invitrogen (GIBCO)	25030-024
0.25% Tripsin-EDTA	Invitrogen (GIBCO)	25200
92 mm Petri dishes	Nunc,Thermo scientific	150350
35 mm poly-D-lysine coated glass bottom culture dishes	MatTek Corporation, USA	P35GC-0-14-C
Luminescent ATP detection assay kit	Abcam	ab113849
NDS (Normal Donkey Serum)	Jackson ImmunoResearch	017-000-121
Anti α-tubulin antibody	Sigma	T9026	1:250 dilution (IF)
Anti γ-tubulin antibody	Sigma	T5192	1:200 dilution (IF)
Alexa Fluor 488 Goat Anti-Mouse IgG	Invitrogen	A-11017	1:1,000 dilution (IF)
Alexa Fluor 568 Donkey Anti-Rabbit IgG	Invitrogen	A-10042	1:1,000 dilution (IF)
ProLong Gold antifade reagent with DAPI (mounting)	Invitrogen	P36935
JetPEI (liposomal transfection reagent )	Polyplus	101-10
EQUIPMENT
Confocal microscope	Leica (Mannheim, Germany)	TCS SP5II