The overall goal of the following experiment is to observe the cytotoxic effect of the philanthr derivative PJ 34 during mitosis in multicenter somal human cancer cells. This is achieved by labeling centrosomes with GFP gamma tubulin and chromosomes with H two B read by transfection. The goal of transfection is to enable tracing migration and clustering of centrosomes and arrangement of chromosomes during mitosis in live multicenter somal cancer cells.
As a second step, some of the transfected cells are exposed to PJ 34, a molecule that acts as an extra centrosome decluttering agent. Next transfected live cancer cells are scanned by confocal microscopy in order to capture events during mitosis associated with centrosome and chromosome migration. Rearrangement or clustering sequential images that are obtained show the decluttering and cytotoxic effects of treatment with PJ 34 in cancer cells during mitosis based on confocal imaging of live cells.
The implications of this technique extend towards a targeted cancer therapy exclusively eradicating autosomal cancer cells during mitosis. Demonstrating of this technique will be our collaborator, Dr.Asher Castillo from the Cancer Research Center in Sheba Medical Center, and Dr.Leonid middleman from the imaging unit of Sacko School of Medicine in the Telaviv University To prepare M-D-A-M-B 231 breast cancer cells for live confocal imaging seed two times 10 to the fifth cells in 35 millimeter diameter. Poly de lysine coated glass bottom culture dishes into liters of complete medium incubate in 5%carbon dioxide at 37 degrees Celsius.
The cell culture is ready for transfection when it reaches a confluence of 60 to 70%The cells will be transfected with two plasmids, one encoding gamma tubulin, GFP for fluorescent detection of centrosomes and the other encoding histone red for fluorescent detection of chromosomes, transection is accomplished using the liposomal transfection reagent jet PI following the manufacturer's protocol. Wash the cells once with PBS and then replace the cell medium with two milliliters of warm DMEM with no supplements. Gently add the transfection mixture to the cells in DMEM and then return the cells to the incubator for eight hours after eight hours.
Replace the DMEM with two milliliters of complete medium and incubate the cells in the incubator for 24 hours. 24 hours. After transfection, replace the medium with two milliliters of complete medium containing 20 micromolar PGA 34.
Incubate the cells for an additional 18 hours prior to live confocal imaging. The following are the technical parameters of the live confocal imaging scanner settings. Scan mode XYZT pinhole, airy 1.00 zoom 3.5 resolution eight bits, laser DPSS 561 nanometers argon visible laser 488 nanometers helium neon visible laser 633 nanometers.
Objective H-C-X-P-L-A-P-O-C-S 63 times 1.40 Oil UV numerical aperture 1.4. Scan speed, 700 hertz and refraction index 1.52. Subject the cells to live confocal imaging for at least 16 hours in an imaging chamber, keeping the cells at 5%carbon dioxide and 37 degrees Celsius.
Transfection efficacy will be examined by fluorescence microscopy. Begin this procedure by seeding two times 10 to the fifth MDA MB 231 cells in a six well plate containing one cover slip per well in two milliliters of complete medium. Transfect the cells in the same way as before using the liposomal transfection reagent jet PI following the manufacturer's protocol.
36 hours post transfection. Remove the medium from each well after washing transfected cells mounted on a cover slip with PBS. Fix the cells by incubation in a cold solution of methanol and acetone at a ratio of one to one for seven minutes at negative 20 degrees Celsius.
After seven minutes, aspirate the fixation solution and let the cover slip with the mounted cells dry in a chemical hood for 30 minutes. Next, apply prolonged gold anti fade reagent with DPI and let the cover slip dry in the dark for six hours. Examine the slide under a fluorescent microscope and calculate the percentage of the transfected cells with red and green signals.
From the total population of cells, the desired transfection percentage is about 20 to 40%When 100 to 200 cells are counted. To begin this procedure, see two times 10 to the fifth M-D-A-M-B 231 breast cancer cells on glass cover slips in a six well plate in two milliliters of complete medium. Add PJ 34 to the medium and incubate the cells for the required period.
Upon completion of the incubation period, wash the cover slips once with PBS and fix the cells by incubation in a solution of ice cold methanol and acetone at a one-to-one ratio for seven minutes at negative 20 degrees Celsius. Next, aspirate the fixation solution and let the cover slips dry in a chemical hood for 30 minutes. Wash the cover slips once with PBST to perme the cell membranes and then block cells with 10%normal donkey serum in PBST for one hour ad room temperature.
Create the first antibody mixture by adding antifa tubulin and anti gamma tubulin primary antibodies to blocking solution and vortexing. Then apply 100 microliters of antibody mixture for each cover slip on a six well plate cover. Turn the cover slip upside down and gently place it on the antibody drop incubate for two hours.
Add room temperature. This will stain spindles and centrosomes. Return the cover slips to the wells and wash the cells three times with PBST.
Then add the secondary antibodies, LOR 488 and LOR 568 1 drop per cover slip on a six well plate cover. Then place the cover slips face down on the antibody drops, incubate for one hour at room temperature in the dark at the completion of the incubation with secondary antibodies. Return the cover slips to the wells and wash the cells three times with PBST.
Lastly, mount the cover slips using prolonged gold anti fade reagent with dappy incubate overnight at room temperature in the dark to dry before imaging the fixed cells by confocal microscopy. PJ 34 is a stable water-soluble phant rodine that was previously observed to exclusively eradicate cancer cells with multi centrosomes without impairing normal cells undergoing mitosis with two centrosomes and bifocal spindles. In this study, the cytotoxic activity of PJ 34 was examined using live human breast cancer M-D-A-M-B 231 cells, which have a high occurrence of extra centrosomes.
The cells were transfected with gamma tubulin GFP to label the gamma tubulin foci and histone H two B red to label chromosomes. Scanning of the M-D-A-M-B 231 cells for at least 16 hours by confocal imaging revealed bifocal clustering of centrosomes representing extra centrosome bifocal clustering in the majority of cells in mitosis as illustrated in the following video clips. This first video shows a randomly selected live cell during mitosis with the centrosomes labeled green by gamma tubulin.
GFP in the second video chromosome rearrangement during mitosis is visualized in a randomly selected M-D-A-M-B 231 cell transfected with histone H two B red. This third video is of an MDA MB 231 cell undergoing mitosis where bifocal clustered extra risomes foci are labeled green and chromosomes are labeled red. In contrast, unclustered centrosomes and aberrant arrangement of chromosomes were detected in live transfected M-D-A-M-B 231 cells incubated with 20 micromolar PJ 34 and mitosis in these cells ended in cell death as depicted in this movie.
This real-time documentation of cell death during mitosis strongly supports a previously defined positive correlation between the number of human malignant cells with multipolar spindles in mitosis and the percentage of cell death in cells incubated with PJ 34. Since PJ 34 acts as a potent PARP one inhibitor, the possibility of PARP one inhibition causing cell death associated with mitotic failure was investigated. The cytotoxic activity of increasing concentrations of PJ 34 was tested in normal and PARP one deficient mouse embryonic fibroblasts and the results are shown here.
The graph on the left shows the percentage of multifocal spindles calculated in normal mouse embryonic fibroblasts represented by the black line and PARP one deficient fibroblasts represented by the gray line incubated for 48 hours with PJ 34 at the indicated concentrations. These percentages were calculated out of 20 total spindles to detected in three different experiments. The graph on the right shows survival of normal and PARP one deficient mouse embryonic fibroblasts incubated for 72 hours with 20 micromolar.
PJ 34 cell survival was assay by the cells A TP production. The mean values of four measurements for each cell line in three different experiments are presented. These results show that PJ 34 doses dependently caused distorted spindles and cell death in PARP one deficient mouse embryonic fibroblasts, but not in normal fibroblasts.
The fact that PJ 34 eradicated PARP one deficient meth despite their PARP one deficiency and the correlation between the formation of multifocal spindles and cell eradication in PARP one deficient mouse embryonic fibroblasts incubated with PJ 34 at concentrations higher than those required for PARP one inhibition is not consistent with a causal linkage between centrosomes decluttering in PARP one deficient mouse embryonic fibroblasts and PARP one inhibition. These confocal images show normal and PARP one deficient mouse embryonic fibroblasts in mitosis, fixed permeated and immuno labeled for alpha and gamma tubulin that labeled their spindles green and centrosomes red respectively. Chromosomes were labeled blue with DPI reagent.
The cells were either untreated or incubated with PJ 34 for 48 hours at the indicated concentrations. Unclustered gamma tubulin ne foci distorted spindles and cell death were observed in PARP one deficient mouse embryonic fibroblasts treated with PJ 34 but not in normal mouse embryonic fibroblasts treated with PJ 34. Some of the examined cell cultures were treated with two potent non arine PARP one inhibitors A BT 8 88 and a G 0 1 4 6 9 which inhibit the enzymatic activity of PARP one.
Neither of the tested PARP one inhibitors impaired normal mouse embryonic fibroblasts at concentrations inhibiting PARP one activity. These PARP one inhibitors also did not affect centrosome clustering in PARP one deficient mouse embryonic fibroblasts. These results indicate that the cytotoxic activity of PJ 34 on cells with multi centrosomes was not shared by other potent PARP one inhibitors Once mastered.
This technique can be used in other living cell types for a variety of proposals.
