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 multicentrosomes^4,5. Incubation of human breast cancer cells MDA-MB-231, which harbor supernumerary centrosomes, with 20 μM PJ-34 efficiently eradicated these cells within 72-96 hr without impairing quiescent cells or some benign proliferating cells harboring two centrosomes in mitosis^2,3. Benign cells included human mammary epithelial cells MCF-10, Human endothelial cells (huvec) and primary mesenchymal cells prepared from human thymus. These cells were resistant to the cytotoxic activity of PJ-34. PJ-34 did not interfere with their cell cycle during 96 hr incubation or affect their centrosomes and bi-focal spindle formation^2,3.

Bipolar centrosome assembly is crucial for bipolar spindle formation in mitosis^4,5. Therefore, cells with more than two centrosomes have developed a scarcely understood molecular mechanism, clustering their extra centrosomes at two poles ^4-9. Failure of bipolar assembly of their centrosomes may cause multipolar distorted spindles and aberrant chromosomes segregation that arrests the cell-cycle in G2/M arrest, and leads to cell death attributed to mitotic failure^4,5. The molecular mechanisms underlying extra-centrosomes de-clustering are intensively investigated¹⁰. Understanding this death mechanism will enable exclusive eradication of cancer cells while sparing healthy tissues ^5,10.

Thus, compounds that activate mitotic catastrophe cell death offer a new mode of a selective cancer therapy, which may be efficient in a wide range of human solid cancers.Our results suggest that confocal imaging can be used to identify molecules affecting extra-centrosomes clustering in mitosis^2,3, rendering these compounds cancer targeting drug candidates.

We have documented the cytotoxic activity of the phenanthridine PJ-34 by scanning fixed and live human cancer cells (with high occurrence of extra-centrosomes in mitosis) versus normal cells. A step-by step description of the imaging procedures used to identify the cytotoxic activity of PJ-34 in human cancer cells is included below.

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 cells by inverted microscope until cell layer is dispersed (usually within 5 to 15 min).
5. Add 18 ml complete growth medium and gently aspirate the cells by pipette. Transfer to a tube.
6. Centrifuge the cell suspension at 1,200 rpm.
7. Re-suspend the cell pellet in 24 ml of culture medium.
8. Add 2 ml of cell suspension to 35 mm glass bottom dishes (about 25% confluence) and place in incubator (5% CO₂ , 37 °C).
9. Solutions:
   1. Complete Medium for cells proliferation: DMEM with 10%FBS, 1% Antibiotics (100 units/ml penicillin G, 100 μg/ml streptomycin, Pen-Strep-Ampho solution) and 2 mM L-glutamine.
   2. Trypsin-EDTA solution, containing 0.25%Trypsin-EDTA.
10. Dishes:
    92 mm diameter Petri dishes.
    35 mm diameter poly-D-lysine coated glass bottom culture dishes.

2. Preparation of Cells for Live Confocal Imaging

1. Seed 2 x 10⁵ MDA-MB-231 cells in glass bottom culture dishes in 2 ml complete medium as mentioned in section 1. When cell culture reaches confluence of 60-70% (about 3-4 x 10⁵ cells per dish), proceed with transfection.
2. Transfect the cells with two plasmids encoding the fusion proteins γ-Tubulin-GFP (for fluorescent detection of centrosomes) and Histone-RED (H2b-RED, for fluorescent detection of chromosomes) using the liposomal transfection reagent Jet-PI, following the manufacture protocol. Briefly, mix 2 μg from each plasmid in a tube with 100 μl NaCl (150 mM). Mix the transfection reagent (100 μl) with 100 μl NaCl (150 mM) in a second tube, and incubate 5 min at room temperature (RT). Then combine the two solutions, mix (using mild vortex) and spin-down. Incubate for 30 min at RT.
3. During the incubation of the transfection mixture, wash the cells once with PBS and replace the cell medium with 2 ml of warm DMEM with no supplements (37 °C).
4. Gently add the transfection mixture to the cells in DMEM and then return the cells to the incubator (37 °C, 5% CO₂) for 8 hr.
5. After 8 hr of incubation, replace the DMEM with 2 ml complete medium and incubate the cells in the incubator for 24 hr.
6. 24 hr after transfection, replace the medium of the cells with 2 ml complete medium containing 20 μM PJ-34.
7. Incubate the cells for additional 18 hr (37 °C, 5% CO₂).
8. Subject the cells to live confocal imaging for at least 16 hr in imaging chamber keeping the cells at 5% CO₂ and 37 °C.
9. In parallel, examine transfection efficacy 36 hr post transfection using fluorescent microscopy as follow:
   1. Seed 2 x 10⁵ MDA-MB-231 cells in 6-well plate containing 1 coverslip per well in 2 ml complete medium.
   2. Transfect the cells as mentioned in sections 2.2-2.5.
   3. 36 hr post transfection fix the transfected cells mounted on a coverslip by incubation in cold methanol:acetone (1:1) solution, 7 min, -20 °C.
   4. Aspirate the fixation solution and let the coverslip with the mounted cells to dry in a chemical hood.
   5. Apply ProLong Gold antifade reagent with DAPI and let the coverslip to dry in the dark for 6 hr.
   6. Examine the slide under fluorescent microscope and calculate the percentage of the transfected cells (red and green signals) from the total population of cells (DNA staining by DAPI). The desired transfection percentage is about 20-40% when 100-200 cells are counted.

3. Technical Parameters of the Live Confocal Imaging Scanner Settings

1. ScanMode XYZT; Pinhole [airy] 1.00; Zoom 3.5; Resolution 8 bits; Laser DPSS 561 nm; Argon, visible laser 488 nm; Laser He/Ne visible 633 nm; Objective HCX PL APO CS 63X 1.40 OIL UV; Numerical aperture 1.4; Scan speed 700 Hz; Refraction index 1.52.
2. Image 3-D presentation were prepared by IMARIS imaging software 7.0.

4. Confocal Imaging of Mitosis in Fixed Cells

1. Seed 2 x 10⁵ MDA-MB-231 breast cancer cells (ATCC), normal mouse embryonic fibroblasts (MEF), or PARP1 deficient MEF (PARP^-/-, prepared by Dr Francoise Dantzer) on glass coverslips in 6-well plate in 2 ml complete medium. Coverslips were washed with 96% ethanol, following by wash with sterile DD water, dried for 2 hr, and placed in each well of 6-well dish.
2. Add PJ-34 (10-30 μM) to the medium and incubate the cells for the required period (usually up to 96 hr).
3. Wash the coverslips once with PBS (phosphate buffered saline), and fix the cells using incubation in ice-cold methanol:acetone (1:1) solution, 7 min, -20 °C.
4. Aspirate the fixation solution and let the coverslips to dry in chemical hood (at this stage, the coverslips can be kept in -20 °C for several weeks).
5. Wash the coverslips once with PBST (PBS supplemented with 0.1% Tween-20) to permeabilize the cell membranes and block the cells with 10% NDS (Normal Donkey Serum) in PBST ('blocking solution') for 1 hr at RT.
6. Incubate the permeabilized fixed cells with primary antibodies for 2 hr at RT (for spindles and centrosomes staining). The antibodies are diluted in the blocking solution as follow: anti-α tubulin (1:250 dilution) and anti-γ tubulin (1:200 dilution). Primary antibodies are applied as follows: apply 100 μl (in a drop) of a mixture of the antibodies in blocking solution for each coverslip on 6-well plate cover (the cover is upside down). Gently put the coverslip on the antibodies drop, seeded cells facing the drop. Incubate the coverslips facing the antibodies for 2 hr at room temperature.
7. Place the coverslips back in the wells and wash the cells 3 times with PBST. Then use the same procedure described in 4.6 for labeling cells on the coverslips with the fluorescent secondary antibodies. Incubate the cells on the coverslips with the secondary antibodies for 1 hr, RT, in the dark. The antibodies are diluted in the blocking solution as follow: Alexa Fluor 488 (1:1,000 dilution; green) and Alexa Fluor 568 (1:1,000 dilution; red).
8. Mount the coverslips using ProLong Gold antifade reagent with DAPI (for chromosomes staining) and incubate overnight at RT in dark to dry.
9. Examine the cover slips by confocal microscopy.

5. Cell Viability Measured by ATP Production

ATP production is measured by a luminescent ATP detection assay kit.

1. Seed the cells in 96-well plate, approximately 20,000 cells in 800 μl medium in each well. Three blank wells should be used for determination of the background luminescence of the medium.
2. Prepare ATP standard dilution series from approximately 10 μM to 100 μM and keep on ice.
3. Add 50 μl of detergent to each well and shake the plate for 5 min in orbital shaker, 700 rpm.
4. Reconstitute each vial of the' lyophilized substrate' with 5 ml of 'substrate buffer' in the kit.
5. Add 50 μl of the reconstituted substrate solution to the wells, and shake the plate for 5 min on orbital shaker, 700 rpm.
6. Keep the plate in dark for 10 min.
7. Measure luminiscence of each well by ELISA microplate reader.

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