Note for LIG4 mediates Wnt signalling-induced radioresistance
Note: LIG4 mediates Wnt signaling-induced radioresistance
(doi: 10.1038/ncomms10994)
This work is very nice piece and easy to follow step by step to test their hypothesis.
Focus tumor:
CRC
Cell line used (endogenous environment):
SW620
HCT116
HT15
COLO205
HCC2998
KM12
Cellular event marker:
g-H2Ax --> DNA double-strand break
Reporter for wnt-acivity:
7TGP
Reporter for functional stem cell (using the genetic engineered approach):
telomerase reverse transcriptase (TERT) --> expressed only in self-renewing cells
Inhibitor:
iCRT14 -- break the interaction between b-catenin-TCF--> no transcription occur (less LIG4)
SCR7 -- break the interaction between LIG4-DNA-->no DNA repair by LIG4
b-catenin targeted genes (transcript product):
CD44
CD133
Axin2
Wnt is related to radioresistant but the underly mechanism is unk --> therefore, this study want to investigate. How it links to DNA repair? --> Wnt regulates the stemness and this type of cell have very good DNA repair system. However, previous studies showed that mutation in LIG4 causing the cell to lose the stemness, therefore, Wnt might be involved in since its job is to maintain the stemness, but, how in the term of molecular level?
1.Using colorectal cancer cell line to investigate by putting the reporter gene (b-catenin-GFP; 7TGP) into the CRC cell line
2.Sort out with the one having the high fluorescence as well as wnt target gene (CD44 and CD133)
3.Check the "stemness" by using sphere formation with the highest fluorescence population; giving more and large colonies
4.Test each different population with various wnt signaling properties by radioresistant (test hypothesis) --> using clonogenic survival assay.
a --> sorting cell which showed wnt high and wnt low (my observation is even it was from the same cell line but the population have heterogeneity); b--> clonogenic formation assay after the cells have been irradiated.
c-->effect of IR dose toward the cell line which contains different magnitude of wnt signaling.
5.using the inhibitor to validate that b-catenin is involved in the resistant (not yet know which gene is related to the resistant)
e.-->effect of IR toward different types of CRC cell lines, using inhibitor inform us about wnt is related "resistant" -- no information on gene yet
6.explore more on the gene that related to wnt and contributing to radioresistant-->come up with the idea b-catenin regulate "DNA repair"--> test this by using microarray to detect the gene expression with and w/o inhibitor
a--not sure which cell line (HCT116?) is subjected to the microarray, pool cell lines? finding LIG4 is in the overlap region. b-->testing to confirm a. that which gene is regulated by b-catenin by using inhibitor, iCRT14, to validate the gene (HCT116)
7.since the function of b-catenin working through the binding of TF, and one of them is TCF --> next the team investigated whether TCF control the expression of LIG4 by finding the promoter region where TCF could bind
8.investigate more on highly expression of LIG4 linked to the radioresistant --> damage the HCT116 with 4Gy IR --> looking at the foci formation of g-H2Ax in DMSO/iCRT14 treated cell. iCRT14 --> less LIG4 --> foci formations were retain at 24 hrs (picture below).
9.using SCR7 to confirm the involvement of LIG4 expression and radioresistant.
wnt3A --> more activated b-catenin--> more LIG4 -->the damages are resolved quicker, SCR7 inhibits the function of LIG4-->less LIG4-->less DNA repair-->prolong H2Ax
Ref: doi: 10.1242/dmm.024232 (remind the molecular cell anatomy of intestine).
10.investigate the dynamic of LIG4 expression in the normal intestine when exposed to the radiation, observe how each cell type responses toward the damage.
Each cell types have a different level of LIG4 expression --> this reflects the activity of DNA repair.
tracking the self-renewing cell by using telomerase reverse transcriptase that is tagged by tdTomato, it said that LIG4 was higher in +4 ISC than CBCISC --> this experiment has done in the normal intestine of the mouse to see the expression of LIG4 in each cell type in the intestine.
irradiated mouse--cut out intestine and IF staining; arrow head-->CBCISC-->dead after radiation
Ref: 10.1016/j.stem.2012.09.009 (remind the molecular cell anatomy of intestine)
11.making use available database to investigate on DNA repair genes in CRC//then the team did IHC on CRC tissue using LIG and b-catenin Abs
found out LIG4 is upregulated
confirmation from IHC
12.confirm and point out the potential of therapeutic treatment of CRC that resists to the radiation bc of aberration of b-catenin causing the LIG4 upregulation
SCR7 causing less LIG4-->DNA damage not fixed-->cell death, but SCR7 does not affect the clonal growth, therefore, cell death is the pure effect from IR (different ways to show the result)
13.come up with the model of how ISC and CRC develop the resistant upon the radiation treatment and how to deal with it (see below). normal situation--> both CBCISC and +4 ISC have high level of LIG4 expression, however, when exposed to IR -- CBCISC death (to prevent abnormal genetic transfer) but +4 ISC resists and survives (to renew the tissue that damaged from IR).
Remarks:
LIG4 depending on the cell type whether it could be benefit or toxic (upon exposing to genotoxic stress)
(doi: 10.1038/ncomms10994)
This work is very nice piece and easy to follow step by step to test their hypothesis.
Focus tumor:
CRC
Cell line used (endogenous environment):
SW620
HCT116
HT15
COLO205
HCC2998
KM12
Cellular event marker:
g-H2Ax --> DNA double-strand break
Reporter for wnt-acivity:
7TGP
Reporter for functional stem cell (using the genetic engineered approach):
telomerase reverse transcriptase (TERT) --> expressed only in self-renewing cells
Inhibitor:
iCRT14 -- break the interaction between b-catenin-TCF--> no transcription occur (less LIG4)
SCR7 -- break the interaction between LIG4-DNA-->no DNA repair by LIG4
b-catenin targeted genes (transcript product):
CD44
CD133
Axin2
Wnt is related to radioresistant but the underly mechanism is unk --> therefore, this study want to investigate. How it links to DNA repair? --> Wnt regulates the stemness and this type of cell have very good DNA repair system. However, previous studies showed that mutation in LIG4 causing the cell to lose the stemness, therefore, Wnt might be involved in since its job is to maintain the stemness, but, how in the term of molecular level?
1.Using colorectal cancer cell line to investigate by putting the reporter gene (b-catenin-GFP; 7TGP) into the CRC cell line
2.Sort out with the one having the high fluorescence as well as wnt target gene (CD44 and CD133)
3.Check the "stemness" by using sphere formation with the highest fluorescence population; giving more and large colonies
4.Test each different population with various wnt signaling properties by radioresistant (test hypothesis) --> using clonogenic survival assay.
a --> sorting cell which showed wnt high and wnt low (my observation is even it was from the same cell line but the population have heterogeneity); b--> clonogenic formation assay after the cells have been irradiated.
c-->effect of IR dose toward the cell line which contains different magnitude of wnt signaling.
5.using the inhibitor to validate that b-catenin is involved in the resistant (not yet know which gene is related to the resistant)
e.-->effect of IR toward different types of CRC cell lines, using inhibitor inform us about wnt is related "resistant" -- no information on gene yet
6.explore more on the gene that related to wnt and contributing to radioresistant-->come up with the idea b-catenin regulate "DNA repair"--> test this by using microarray to detect the gene expression with and w/o inhibitor
a--not sure which cell line (HCT116?) is subjected to the microarray, pool cell lines? finding LIG4 is in the overlap region. b-->testing to confirm a. that which gene is regulated by b-catenin by using inhibitor, iCRT14, to validate the gene (HCT116)
7.since the function of b-catenin working through the binding of TF, and one of them is TCF --> next the team investigated whether TCF control the expression of LIG4 by finding the promoter region where TCF could bind
8.investigate more on highly expression of LIG4 linked to the radioresistant --> damage the HCT116 with 4Gy IR --> looking at the foci formation of g-H2Ax in DMSO/iCRT14 treated cell. iCRT14 --> less LIG4 --> foci formations were retain at 24 hrs (picture below).
9.using SCR7 to confirm the involvement of LIG4 expression and radioresistant.
wnt3A --> more activated b-catenin--> more LIG4 -->the damages are resolved quicker, SCR7 inhibits the function of LIG4-->less LIG4-->less DNA repair-->prolong H2Ax
Ref: doi: 10.1242/dmm.024232 (remind the molecular cell anatomy of intestine).
10.investigate the dynamic of LIG4 expression in the normal intestine when exposed to the radiation, observe how each cell type responses toward the damage.
Each cell types have a different level of LIG4 expression --> this reflects the activity of DNA repair.
tracking the self-renewing cell by using telomerase reverse transcriptase that is tagged by tdTomato, it said that LIG4 was higher in +4 ISC than CBCISC --> this experiment has done in the normal intestine of the mouse to see the expression of LIG4 in each cell type in the intestine.
irradiated mouse--cut out intestine and IF staining; arrow head-->CBCISC-->dead after radiation
Ref: 10.1016/j.stem.2012.09.009 (remind the molecular cell anatomy of intestine)
11.making use available database to investigate on DNA repair genes in CRC//then the team did IHC on CRC tissue using LIG and b-catenin Abs
found out LIG4 is upregulated
confirmation from IHC
12.confirm and point out the potential of therapeutic treatment of CRC that resists to the radiation bc of aberration of b-catenin causing the LIG4 upregulation
SCR7 causing less LIG4-->DNA damage not fixed-->cell death, but SCR7 does not affect the clonal growth, therefore, cell death is the pure effect from IR (different ways to show the result)
13.come up with the model of how ISC and CRC develop the resistant upon the radiation treatment and how to deal with it (see below). normal situation--> both CBCISC and +4 ISC have high level of LIG4 expression, however, when exposed to IR -- CBCISC death (to prevent abnormal genetic transfer) but +4 ISC resists and survives (to renew the tissue that damaged from IR).
Remarks:
LIG4 depending on the cell type whether it could be benefit or toxic (upon exposing to genotoxic stress)
- Benefit when cells are required for tissue homeostasis
- Toxic when cells are not required for tissue homeostasis, toxic fixing by LIG4?
There might be the factors underneath which requires to be explored more!
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