Polyploidy does not always trigger cell death. A growing number of normal tissues (including human epidermis, liver, etc ) have been shown to use polyploidization as a way to make big cells (and maybe as an anti-cancer barrier).

Flow cytometry with propidium iodide or 7-AAD should show cells with >4N DNA content in the case of polyploidy, although you may underestimate the number of polyploid cells because those that are in G1 will have the same amount of DNA as normoploid cells in G2/M.

Apoptotic cells should harbour a sub-G1 peak because of DNA fragmentation (although it's not at all the most sensitive assay for apoptosis).

If you are growing adherent cells, I would suggest that you also collect the floating cells, in case they have a different profile.

for the polyploidy the easiest thing to do woudl be to transfect the cells with something like H2a-GFP or the Invitrogen/Life Technologies Organeele Lights for DNA (which is also a histone tag). As ploidy increases the intenisty/area of positive signal will increase. Thats if you are doing live cells. If its all on fixed or soon to be dead cells using Hoechst 33342 woudl be the best. It is live cell premiable and stains DNA in a stoichiometric way. So more signal must equal more DNA

Polyploidy does not always trigger cell death. A growing number of normal tissues (including human epidermis, liver, etc ) have been shown to use polyploidization as a way to make big cells (and maybe as an anti-cancer barrier).

Flow cytometry with propidium iodide or 7-AAD should show cells with >4N DNA content in the case of polyploidy, although you may underestimate the number of polyploid cells because those that are in G1 will have the same amount of DNA as normoploid cells in G2/M.

Apoptotic cells should harbour a sub-G1 peak because of DNA fragmentation (although it's not at all the most sensitive assay for apoptosis).

If you are growing adherent cells, I would suggest that you also collect the floating cells, in case they have a different profile.

Thanks guys for your answers. Alright, if I know how to get some insights and hints about apoptosis pathways and which one did I activate using some markers in Western Blot. However, how can I know if I activate a polyploidy pathway instead of apoptosis?

Fawzi,

What make you say such things? I may not very up-dated on this but as far as I recall there is no such well defined pathway for polyploidy. If you take liver you will find a heterogeneous population of hepatocytes with different polyploidy numbers (2n, 4n, 8n, 16, 32n). What make dividing hepatocytes take different decisions is still unknown. Polyploidy is achieved through endo-replication whereby the cells cycle between S phase and G2/M. It is not well know how cells do not commit to mitosis after S phase. Normally, at least in hepatocytes, most polyploid cells are blocked in G2/M. The bigger is the N number the bigger the chance they will not be allowed to reenter the cell cycle, likely to prevent a mitotic disaster. Whether or not a mitotic disaster can signal to the cell through a checkpoint that the damage is irreparable that a different question.

Thanks Abdelhalim.

What makes me said that because indeed it is the fact that some cells undergo polyploidy and then die whereas some others trigger apoptosis from the beginning specially in cancer, WHY? Chemotherapeutic agents presumably will destroy cancer cells but via which pathway polyploidy or apoptosis? I know as clinician I don't really care which pathway will be chosen because the end of the story (in a cancer scenario) we're killing the cancerous cells but from a researcher point of view, why some initiate direct apoptosis whilst others go through polyploidy then apoptosis? :S

Hi Fawzi,

You cannot oppose polyploidy to apoptosis like if the cells were to commit to enter polyploidy or to die. It is possible that some disasters during the cell cycle activate the apoptosis pathway because of checkpoints. But a lot of cancer cells may have their checkpoints inactivated or missing. Hence, the importance of some biomarkers. Caner cells only accumulate all kind of errors and end up in a complete mess. Most cancer cells do not conserve their initial polyploidy numbers due to chromosomes loss, unequal segregation, etc…There is a lot of recombination going on, chromosome fragmentation, etc…Despite all that, they survive.

Some scientists may even question the word polyploidy applied to a cancer cell. It may change with the passage number. Most of the time we don’t know the polyploidy number of a cancer cell. If your chemotherapeutic drug targets a process that is down stream a checkpoint, you have to take into account that cells are asynchronously growing. Some cells will have to cycle before been stopped at that checkpoint or at that window of the cell cycle where the drug has a target. Please take into account that this response is only generic.

I would also check markers for cellular senescence since senescent cells can become polyploid, but these cells tend to be resistant to cell death.

It is not simple question. For example it is known that some of caspases such as caspase-2 and caspase-8 may be functional as tumor suupressor, but its central funcition is pro-apoptotic. So, cancer cells lose the expression of these caspases. Also is known that pro-apoptotic protein Bim interacts with microtubules (dynein) and when is stress on these microtubules like polyploidy or taxol treatment, Bim released and goes to mitochondria to induce pro-apoptotic response. So, i think for the begining needs to check the expression of caspases like caspase-3, 8, 9. Next to check interaction between Bim and Bcl-2 by IP that demonstrates apoptotic switch on in mitochondria.

The fate of polyploidy is not apoptosis. Generally, the polyploid cells do not undergo apoptosis. Polyploidy may be reversible as well. You can detect polyploidy by many ways. One way you can do this is by counting the number of chromosomes in the cells. The second way is identify the DNA status of the cells by using flow cytometer.upon Propidium Iodide staining. The normal cells will have at the most 4N status and the polyploid cells would have 6N, 8N, 16N and so on. Hope this helps

First, I would also suggest that you take Mitotic Catastrophe into account? Mitotic catastrophe is a type of cell death characterized as an aberrant mitosis results in the generation of aneuploid or mostly polyploid cell progeny.

Second, actually genetic background and protein content of the cells can determine the fate or type of cell death. Particularly, cell cycle status of the cells plays a key role for the drug effects, eg., in response to a same anti-cancer drug, the cells in G1 may undergo apoptosis whereas G2/M cells may induce polyploidy and subsequently mitotic catastrophe/ apoptosis. If you are doing an experiment it can be check through synchronizing the cells.

I am agree with the opinion of Dr. Neel Fofaria.

i think mitotic death, mitotic catastrophe and apoptosis are different mode of cell death. Cells which goes for mitotic catastrophe mediated cell death will not go for apoptosis. Flowcytometry may not be helpful, which will say only the stage or mitotic arrest, however cell death may occur by aneuploidy....

. "Cell death by mitotic catastrophe: a molecular definition". PMID 15077146

A mitotic catastrophe can be signaled to a checkpoint which can activate apoptotic signaling pathway. There are only two ways for a cell to die. Either by necrosis or by apoptosis. If the signals have been generated from inside the cell then it is a programmed cell death or apoptosis. If the signals have come from outside the cell then it is necrosis. The differences are not only relative to the signal but also to the way and how the remain of the cell are dealt with by the organism.

There are pathways for apoptosis (if detected then we will know cells may underwent this particular pathway) but are there pathways for polyploidy? We can detect polyploid cells simply by using Flow cytometry but what pathway was selected (by the cells) to undergo this particular polyploidy stream?

Dear Fawzi,

newer findings show that tumor formation is due to allowance of replication, this is the shortcut in cell cycle. Thereby polyploidy might be induced. Allowance of replication is combined with onset of the repair systems.

Replication can be monitored by 3HThymidin incorporation into nucleic acids. Apoptosis is visible in fast nucleic acid degradation, i.e. 3HT release from nucleic acids. Measuring uptake and release of 3HT in nucleic acids might reveal answers to your question.

I agree with all the statements above. I would only like to add a couple of suggestions:

1. Look at mitotic figures in your cell population. If there are mitotic defects, you should be able to see them.

2. Perform time-lapse, if you can, and look at your dying cells. Apoptotic cells have very defined features (i.e. nuclear blebbing, picnotic nuclei), easy to recognize, while cells that die by mitotic crisis usually struggle to divide before starting dying.

Polyploidy in healthy cells should cause usually apoptosis, but in others also could be a way to cancerogenesis or disease (syndroms) related to diversities in number of chromosomes. You can check simultaneously the apoptosis level by cytometric tests (e.g. Vybrant Apoptosis Assays) and chromosome aberration by FISH

Thank you all for you great contribution.

Thanks Mal, but see these two questions I thought about:

1- What pathways if activated cells will go for apoptosis (apoptosis pathways)? We as researchers pretty much know the answer to this question using different apoptosis and apoptotic pathways markers.

2- What pathway(s) if activated cells will go polyploidy? Are there pathways for polyploidy? Is polyploidy a stage of the apoptosis pathway (based on some answers above it is not), IF NOT, then that means there are pathways if activated cells will undergo polyploidy. :s

Polyploidy occurs when mistakes in chromatid segregation have place. It leads to chromosome aberration changing the number of chromosomes in cell as well as in fact could cause chromosome breakage that usually have an impact on gene expression. There are several factors from outside or inside of cell that could induce apoptosis. To date it is difficult to say why many cancerogen or factor related to cell division or metabolism will cause polyploidy.

Megakaryocytes for example are typical polyploid cells generating platelets. I was interested in such field few years ago. I read a paper where the authors mentioned the need of active caspase 3 not to induce apoptosis but to generate the platelets at the edges of cell dendrites. I also used to treat CML cell lines with staurosporine (PKC inhibitor) to induce polypoidy, staurosporine killed the half of the cells while the remaining differentiate to polyploid healthy cells.

When working on breast cancer cells and their response to pacletaxel I did notice a few cell lines respond to treatment by entering polyploidy, going from 2N to 4N after treatment, characterised by by a G2 arrest which one would expect. However the survivors then went on to 8N and then a "X"N. Usually these cells would become very very senscent-like, taking up a huge surface area, but with multiple nuclei. I kept the cultures, just to see what would happen, but they sat there for a few weeks not dividing. Interesting as this was, we all have projects to finish, and this would distract from my main work so I did not follow it up!

I sense that your question is more theoretical rather than technical at the moment, so here a few musings. The real problems that cells face in terms of division is that the gene dosage of a 2N nuclei can only support the functioning of a cytoplasm of a particular size, so for a cell to grow really large it must somehow increase that dosage. Typically this either occurs by the fusion of cells such as osteoclast and muscle development, or as Makram Essafi points out, during endoreplication cycles in megakaryocyte. In these cases the cells then have the gene dosage needed to maintain function in these highly active cell types.

In answering your question what you may want to try looking at is how come the cells do not undergo cytokinesis, and maybe the best models would be Makram's megakaryocytes, and cancer cells treated with taxols. The former might allow you to study the apoptosis more, but in the cancer cells you might be able to do a screen which allows you to detect a few cell lines which undergo polyploidy, but which do not die becuase they lack the final effectors of cell death, but maybe they do initiate signalling which should trigger apoptosis (which you could test for using standard assays), but due to the lack of final effectors, it does not actually occur- you can then "rescue" the pathway later to kill the cells.

This is an interesting question, but one that will be hard to answer, so good luck!

Xiao Quiang who is now Director of International Medical Studies at Guanzi Medical University China, did some excellent time lapse studies, following 8Gy of photon irradiation, in His MD Thesis (The Cytological Effects of Human Cancer Cells of Different Histogenetic Origin) whilst with me at Liverpool University. In these the tendency to apoptosis, necrosis, polyploidy or senescence appeared to be cell line dependent to some degree although some lines showed more than one type of cell death. For example HEP2, MGHU1 and HRT18 cells showed mainly classical apoptosis while RT112 showed polyploidy. The polyploidy, however, interestingly was both the result of failed cytoplasmic division or cell fusion. I407 and HT29 also formed polyploidy cells by cell fusion. RT112 also showed the morphological appearances of necrosis in some cases and OAW42/86 showed senescence.

This may at least give you some specific cell lines in which to investigate youevery interesting question.

Good luck

I would recommend you an assay of TUNEL-Cell cycle. This will allow you to see polyploidy and apoptosis. Furthermore, it allows you to answer whether the apoptotic cells come from G1, G2/M or from the polyploid population. If so, you can show that polyploidy occurs before apoptosis.

polyploidy is -as others have pointed out - quite normal under various conditions - e.g. rodent liver when aging or regenerating or after action of chemical carcinogens. In these experimental models, polyploid cells are not necessarily doomed to die. We may recognize polyploid cells just by nucleus size. I attach a pic of - in this case avian - hepatocytes. Obviously, some nuclei are (as the result of the carcinogen treatment ) much larger and ceratinly polyploid.

Dear Friends,

polyploidy is induced by a mutation in a proliferative gene, that allows replication without control. Cells with these mutations continuously replicate and repair their DNA. These cells cannot be brought to apoptosis.

Research Article J Cell Sci Ther 2012, 3: 133 pdf

doi: 10.4172/2157-7013.1000133

Inhibition of Apoptosis in ALL-1 Leukemic Cell Lines: Allowance of Replication, Constant Repair Replication, Defect DNA Damage Control

Well friends, in histological sections it is rather not cumbersome to identify polyploid cells from apoptotic cells. In the former, chromatin is increased and open , coarse and even several mitotic figures are visible, whereas in the latter, the cells are rather shruken, isolated from the rest, nuclei condensed and fragmented within cytoplasmic blebs or seen as apoptotic bodies. Similar criteria apply in cytologic preparations.

What can a veterinary oncologist add? In plants, polyploidy (here most often teraploidy) may form a (undeliberate) step to grow bigger plants. As said by others, normal liver cells may be polyploid (tetraploid). Megakaryocytes are often multinucleated which is not the same as polyploid (by definition, it is the nucleus to which ploidy is referred to, not the cell).

In many cancers, as already described by Shackney et al (Cancer Res 1989), Giaretti ea (Int J Cancer 1993) and Dutrillaux et al (Breast Cancer Res Treat 1991) tetraploidy appears an important step in carcinogenesis, followed by chromosome loss and yet further changes that lead to frequent hypertriploidy. Other cancers contain much lower quantity of chromosomal gains/losses. Tissue of origin, but also species, influences frequence and extent of aneuploidy in cancers. Tetraploidy in itself - I belief - does not form a stimulus for apoptosis. Mechanisms that contribute to aneuploidy and their consequences (death or cancer) have been nicely described in EMBO J 2012,13

Dear Gerard, in fact polploidy in amimal cells may result in aneuploidy and predisposition to cancer.It can also be measured in cancer cells by flow cytometry or NMR.

Dear Naresh,

Familiar to the issue: published on canine and feline mammary tumors, canine thyroid cancer, canine lymphoma, soft tissue sarcoma (latest: Cancers 2012: http://www.mdpi.com/2072-6694/4/4/1300): apart from similarities with such cancers in the human, the interspecies dissimilarities as well as the variation among cancers of one type are fascinating.

Dear friends,

you might want to know what a proliferative mutation does:

- It allows replication and thereby shortcuts the cell cycle.

- Thereby polyploidy can be induced, just by allowing replication twice before the mitosis can run.

- In Drosophila polyteny is induced by proliferative mutations.

- It can induce genome instability by inducing replication at one site of the genome, followed by repair (copy number control), misleading the whole system by repeated induction of replication at the same site.

- In Drosophila asymmetric giant chromosomes occur due to proliferative mutations.

More questions than answers?

Have a nice time!

Isolde