Some cells (especially those with non- functional p53) are not able to arrest in G1 in response to treatment with for example DNA-damaging agents. Those cells often rely on the G2 checkpoint for DNA damage repair as excessive DNA damage may lead to mitotic catastrophe and apoptosis.
For instance, Que and Chen (DOI: 10.3892/mmr.2011.426) found that shRNA-mediated downregulation of c-met increases the percentage of cells in the G0/G1 phase in U266 cells. In addition to G1 cell cycle arrest, they also demonstrated that the apoptotic rate was higher upon c-met downregulation. Furthermore, they showed that downregulation of c-met increases sensitivity to Doxorubicin.
Are cells, in which c-met has been downregulated, more sensitive to doxorubicin due to increased G1 cell cycle arrest or due to increased apoptosis or both? Does G1 cell cycle arrest probably facilitate Doxorubicin-mediated apoptosis? If yes, why are G1-arrested cells more sensitive for apoptosis?
An other study by Gogolin et al. (DOI: 10.4161/cc.24091) demonstrated that “CDK4 inhibition by siRNAs, selective small compounds or p19(INK4D) overexpression partly restored G(1)-S arrest, delayed S-phase progression and reduced cell viability upon doxorubicin treatment”.
Why might G1 cell cycle arrest increase sensitivity to doxorubicin in these cell lines? What makes G1-arrested cells more sensitive? Gogolin et al. write that “high CDK4 in MYCN-amplified cells has an unexpected function during S phase, promoting not only S- phase progression but also further cell death resistance”. Does this mean that G1 cell cycle arrest does probably not directly increase doxorubicin sensitivity but that downregulation of factors (such as CDK4 in this case) important for G1 cell cycle progression leads to increased sensitivity? This would then mean that these factors (such as CDK4) increase resistance independently on their G1 cell cycle progression function.
Currently I am looking for reviews/studies explaining why G1 cell cycle arrest increases sensitivity to DNA damaging agents (especially in cells with non-functional p53 in which DNA damaging agents would induce G2 cell cycle arrest). But until no, I was not successful.
Does anyone have an idea or know reviews/ studies dealing with this topic?
In
https://www.researchgate.net/publication/7242037_Sleeping_Beauty_transposase_modulates_cell-cycle_progression_through_interaction_with_Miz-1
it is said that in the G1 phase chances for integration
of a transposon is biggest but also DNA repair
is very active due to a pathway named NHEJ.
Regards,
Joachim (not an expert)
Article Sleeping Beauty transposase modulates cell-cycle progression...
G2 cells have done S phase and therefore each cromosome has two sister cromatides. One cromatide can be used to repair the damage of the other. However, G1 cells have only one copy.
The answers above are both basically correct. In mammalian cells, the majority of repair is thought to be non-homologous end-joining (NHEJ), but homologous recombination (HR) is a backup mechanism. However, there is no second copy to use for HR in G1. Cells also tend to be more prone to apoptosis in G1, so if the damage is left un-repaired in G1, the chances that the cell will die are higher.
I'd say to stay away from the cell cycle regulation literature, it's confusing and indirect with regard to DNA repair. Look more directly at the literature on the repair machinery itself, and signaling from ATM, ATR, and DNA-PK. There are good reviews on this topic. I probably put some in my Mendeley account, now I don't remember. Ping me on there if you can't find what you need in my shared folders.
This is a quite complex question, because there are different types of DNA damages and they can be repaired by different mechanisms. In regards of DNA breaks, the answers above are quite right. Recombination happens exclusively in S and G2 because they require high CDK activity. There are many different recombination proteins that are CDK substrates. NHEJ is active during G1, S and G2. The same is true to another minor repair pathway called microhomology-mediated end-joining (MMEJ) or alternative end-joining. That is, in G1 you only have two repair pathways, in S and G2 you have three.
But more important, HR in S and G2 will use the sister chromatid as repair template and the repair will be mainly error-free. However, NHEJ in some cases and MMEJ always are error prone mechanisms. That means that the cells in G1 not only will repair less, but also in many cases the repair will lead to the appearance of mutations, even gross chromosomal rearrangements such as translocations and deletions. This mutations could lead to cell death themselves due to a loss of fitness, increasing the overall sensitivity to DNA damage.
Also, not only DNA repair but DNA damage checkpoint activation is different in G1, S and G2. There is also growing evidence that CDKs modulate the checkpoint. This also contribute to differential survival.
There are many recent reviews on the relationship of cell cycle and DNA break repair, as is a quite hot topic in the field.
The only reasonable explanation would be that Doxorubicin generates a type of DNA damage that would need to be fixed during S-phase. However, (to my knowledge) the only damage that can exclusively be fixed using the sister chromatid is run off synthesis from a nicked template. Doxorubicin indeed induces nicks by trapping Topo II in the cleavage complex state where the DNA strand is nicked. Depending on how close these nicks are together they can lead to either double strand breaks or just stay as nicks in the DNA until replication leads to run off synthesis. In example a similar inhibition of Topo I by camptothecin will usually lead to run off synthesis during replication, which can exclusively be repaired via HR (BIR) during S-Phase. That being said, this type of damage will only be a problem during replication, meaning in S-phase, since in G1 nicks can be religated and run of synthesis wouldn't occur.
So run off synthesis doesn't seem to be the issue here since the cells won't get into S-phase if arrested in G1. If DSB are generated instead by nicks in close proximity, NHEJ and Alt-EJ could still take care of it, which they do predominantly anyways in human cells during G1. Besides that, even without a sister chromatid the cells would still be very likely able to use the homologous chromosome to repair a DSB via HR instead (human cells are diploid after all). So it would be unreasonable to think a G1 arrest would have a negative impact on cell survival due to induction of DNA breaks, especially since one of the main purposes of a G1 arrest is to deal with DNA damages before entering S-Phase. Arresting cells and inducing a DNA damage response resulting in a higher sensitivity against DNA damages seems in general a little weird to me (which doesn't mean it wouldn't be possible though).
Taking this into account I would rather think the effect has something to do with transcription/translation inhibition and maybe apoptosis as an immediate result:
Doxirubicin intercalates into DNA and prevents proteins from binding to DNA at higher concentrations, including Topoisomerase II (See: http://www.sciencedirect.com/science/article/pii/S1074552110001614). This would be a significant problem in G1 where cells perform mostly biosynthesis. And being severely impaired in transcription/translation, cells might induce apoptosis (I have to admit I don't know enough about apoptosis induction, so this is just speculation here).
The first question therefore would be what concentration they used. If the concentration is high enough to prevent protein binding rather than inducing nicks, it seems very likely that the problem is rather linked to a problem with biosynthesis than to DNA lesions.
Hope this was helpful.
Cheers Chris.
p53-functional cells arrest in G1 in response to DNA damage. So, there they either repair or (if repair is unsuccessfull) undergo apoptosis. In this case, chances for survival are determined only by the repair capacity of this checkpoint (mostly by NHEJ).. If the cells skip G1- arrest and move to G2 (usually when p53 is not functional) they have another option to undergo repair by HR there but also the chance to post-pone repair and enter through mitotic slippage into polyploidy, where they have several matrices for repair. Each delay of repair increases chance for survival and on the contrary - repair versus apoptosis in the very first checkpoint decreases this chance (determined as increased sensitivity to apoptosis). It is rather important how sensitivity is measured - delayed apoptosis may peak from day 3-6 only, while in the first two days it may be absent. G1 phase is also a site of interplay between Cdk4 and its inhibitors, f.ex. senescence inducer p16inka4a, which targets the catalytic unit of Cdk4 cyclin D. So, up-regulation of Cdk4 will allow to overcome senescence (irreversible growth arrest) , while its down-regulation or activation of p16 and other kinase inhibitors( p19) will deepen senescence in G1. Senescence itself is associated with the secondary DNA damage followed b ypostponed cell death. For this inbterplay of the regulators I recommend to look at the review articles by Manuel Serrano and collegues, f.ex. Cancer: a lower bar for senescence. Nature 2010.
The homologous recombination based DNA repair pathway is not functioning in G1 phase cells and hence, those cells are sensitive to DNA damaging agents. I hope that The homologous recombination based DNA repair pathway is ultimate possibility to save the cell from DNA damage because while the genetic info is lost in both strands of a DNA molecule, The homologous recombination based DNA repair pathway is the only machinery to retrieve the lost genetic info from the homologous chromosome. Since there is no homologous chromosomes in the G1 cells, the homologous recombination based DNA repair pathway is not functioning there.
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