Hello everyone, I would like to know if anyone knows if a knockdown of p53 in primary luminal epithelial cells of mouse mammary glands does support the growth of these cells in cleared fat pad injections? Usually, primary luminal epithelial cells do not grow well in cleared fat pad injections since they do no longer contain stem cell reneval activity. Nevertheless, I am wondering if you can get growth or even probably breast cancer development when you knock-down mouse p53? This knockdown can either be already genetically (p53+/- mice) or by infection of the fresh isolated cells (Adeno-shP53 or Lentiviral-shP53). I would really appreciate any ideas or even better if someone have done that experiment. Thanks, Nicole
PS: I am very familiar with the fact that knockdown of p53 enhance or induce tumorigenesis, respectively. Furthermore, I also know that p53-/- mice do develop breast tumorigenesis. Nevertheless, I would like to know if in this specific context it will do it as well: Normal luminal epithelial cells from a female pregnant mouse (NO tumors at this point) after parturition of the pups with knockdown of p53 injected into cleared fat pad. Usually, you do not get an outgrowth of luminal epithelial cells, but do you get one when you knockdown p53?
The knockdown of p53 shoud increase the sensibility to tumorigenesis
Yes, Nicole. Go ahead with the experiment and the practice will confirm your theory.
Thanks guys. I was more asking if anyone has seen any publication showing it or even if someone of you published it and can tell me about it. Searching by Pubmed is quite complicated for this respect.
Hi Manish, you mean more than 80% chance of them to go into tumorigenesis or to just show me an outgrowth (which ususally does not occur with luminal cells)? In p53 +/- mice or after knockdown ex vivo?
Dear Nicole,
Are this sort of paper usefull for your search ?
Regards
Didier J
(1)
Dear Nicole, Are this sort of paper usefull for your search ? Regards Didier J (2)
The knockdown of p53 shoud increase the sensibility to tumorigenesis
As i know, p53 -/- female mice spontaneously develops mammary tumors (apart from others) during their lifespan and p53+/- mice is an acceptable model for mammary tumorigenesis studies. Usually, in p53 mutated/dysfunctional tumors its associated with both transformation as well as in tumor growth. However, since the tumor development is a multistep process the transient Knockdown of p53 might not lead to tumor development. You can select a combination of p53+/- model with some chemical/viral carcinogen, which can increase the mammary tumorigenesis leading to higher rate of spontaneous tumor development.
Thanks to all of you!
@Didier, thank for the two publications. I will take a look at them but after a short read through the abstract, it did not seem to be specifically for fat pad injection outgrowth of ONLY luminal epithelial cells.
@Shubhankar: I do want to combine my gene of interest knockdown with p53 knockdown to see if my gene of interest in changing the behaviour, but I am not sure if p53 knockdown in ONLY LUMINAL epithelial cells of the mammary gland and injection of these cells into cleared fat pad is changing their normal behaviour.
@Alejandro: My experiment would be the following - I have a knockdown mouse for my gene of interest which shows me more luminal progenitor cells at the end of pregnancy and therefore a defect in differentiation to milk producing mature luminal cells. It is already known that specific breast cancer types derive from luminal progenitor cells (which are increased in my mouse model). Now, my question would be: Can I drive these cells into tumorigenesis by isolating the luminal cells in control and knockdown mouse, infect them ex vivo with shRNA against p53 and then transplant them into cleared fat pad? Therefore, I am trying to figure out first, if my control luminal cells with p53 knockdown will go into tumorigenesis, show me outgrowth at all (because usually luminal cells do no longer have self renewal capacity) or no change at all in comparison to control luminal cells without p53 knockdown. The easier way would for sure be crossing my mice to p53+/- mice and then monitor the tumor development in comparison to controls, but it will be too long term (I do not have enough time for this). Did this make it clear to you?
You will definitly increase the probability to develop cancer when you inactivate p53, but if your mouse devellop cancer you will identify for sure other mutations than p53 disruption in the tumor
Hey Nicole,
p53 knock down will definitely result into tumourogenesis and basically impacts expression of pro apoptotic genes like BAX, PC3 or PC9. p53 silencing leads to higher proliferative phenotype due to downregulation of cell cycle check point proteins.
Refer Fig. 4.Effects of p53 knockdown on single cells during the reprogramming, Dynamic single-cell imaging of direct reprogramming reveals an early specifying event, Nature Biotechnology, Volume:28, Pages: 521–526
A single nucleotide polymorphism in the HDM-2 gene regulates the p53 apoptotic response and influences the age of onset of cancers in humans: the SNP 309 HDM-2 polymorphism
GL Bond1 and AJ Levine1, 2
(1) Cancer Institute of New Jersey, New Brunswick, New Jersey, USA
(2) Institute for Advanced Study, School of Natural Sciences, Princeton, New Jersey, USA
Published online: 17 June 2005
Without Abstract
The HDM-2 gene in humans has two promoters for transcription. 5' to the first exon is a maintenance promoter providing low levels of HDM-2 in the cell. In the first intron are the P53 DNA binding sites and the p53 inducible promoter that yields threefold to 10-fold more HDM-2 mRNA after a p53 activation and response. When this intronic promoter is employed, transcriptional initiation starts at the second exon and this mRNA is translated more efficiently than mRNA that starts at the first exon. The coding region of the HDM-2 protein starts in the third exon. At residue 309 in this first intron is a single nucleotide polymorphism, with 12% of people being a G/G homozygote, 40% being a G/T heterozygote and 48% of people being T/T wild-type homozygotes (the G/G genotype is lower in black Americans and the sample size is now over 300 people). We have found that the G/G genotype creates a better SP-1 transcription factor binding site, raises the level of m-RNA in unstressed cells and produces threefold to sixfold more HDM-2 protein in cells (cancer cells in culture) with the G/G genotype. This mRNA starts at the second exon, and is probably translated better in unstressed cells. After DNA damage or other stresses, P53 activity in cells with the G/G genotype is lower and the percentage of cells undergoing apoptosis is lower when compared with cells in culture with T/T genotypes. We have reproduced these observations with lymphocytes taken from human volunteers and placed in culture, with EBV-immortalized B cells in culture, with primary fibroblasts in cell culture and with cancer cell lines in culture. In 92 individuals that have donated lymphocytes we see individuals forming a distribution of apoptotic responses between 20% and 60% after gamma radiation, with individuals being quite reproducible in repeated experiments. The lower half of the distribution is heavily weighted with the G/G genotype, while the upper half of the distribution has mainly the T/T genotype. The higher HDM-2 levels in cells thus result in a lower apoptotic index in cells from these volunteers. It has become clear in recent studies that SNP 309 has a clinical impact. We have genotyped two cancer cohorts, one at MD Anderson and one in Germany, containing patients with sarcomas and breast cancers. The results have been statistically significant (P = 0.01-0.02) and clear in both cohorts, and the average age of onset of these cancers is 10–15 years earlier in people with the G/G genotype than in people with the same cancer with the T/T genotype. The interpretation is then that the probability of eliminating pre-cancerous clones of cells via a p53 mechanism is lower in people with a G/G genotype (high HDM-2 levels) and the probability of developing a cancer at an earlier time in life is higher. In addition, in patients that have a germline mutation in the p53 gene (this yields one-half of the p53 protein level in a cell) those individuals that have a G/G genotype or a G/T genotype develop multiple cancers (three, four or five cancers) over their lifetimes, while no T/T homozygotes develop that many independent cancers.
Dear Nicole, I have not direct experience with a such type of experiments as you propose. It seems a challenge idea that simple deletion of p53 leads to cell transformation, or at least to acquiring cell growth capacity. I believe that p53 deletion could be not enough, since the occurrence of redundancy with other tumor suppresors (e.g., p73 and p63). On the other hand, other citostatic factors (e.g., p21, Foxo, p16, etc) are not only depend on p53 regulation. The lack of stem cells self-renewal could be overcome by re-programming techniques. It seems that some other questions related with the switch from quiescence to proliferation stage (e.g., reversion /activation of some epigenetic regulated genes) should be considered as well. Best regards. Daniel
A single nucleotide polymorphism in the HDM-2 gene regulates the p53 apoptotic response and influences the age of onset of cancers in humans: the SNP 309 HDM-2 polymorphism
GL Bond1 and AJ Levine1, 2
(1) Cancer Institute of New Jersey, New Brunswick, New Jersey, USA
(2) Institute for Advanced Study, School of Natural Sciences, Princeton, New Jersey, USA
Published online: 17 June 2005
Without Abstract
The HDM-2 gene in humans has two promoters for transcription. 5' to the first exon is a maintenance promoter providing low levels of HDM-2 in the cell. In the first intron are the P53 DNA binding sites and the p53 inducible promoter that yields threefold to 10-fold more HDM-2 mRNA after a p53 activation and response. When this intronic promoter is employed, transcriptional initiation starts at the second exon and this mRNA is translated more efficiently than mRNA that starts at the first exon. The coding region of the HDM-2 protein starts in the third exon. At residue 309 in this first intron is a single nucleotide polymorphism, with 12% of people being a G/G homozygote, 40% being a G/T heterozygote and 48% of people being T/T wild-type homozygotes (the G/G genotype is lower in black Americans and the sample size is now over 300 people). We have found that the G/G genotype creates a better SP-1 transcription factor binding site, raises the level of m-RNA in unstressed cells and produces threefold to sixfold more HDM-2 protein in cells (cancer cells in culture) with the G/G genotype. This mRNA starts at the second exon, and is probably translated better in unstressed cells. After DNA damage or other stresses, P53 activity in cells with the G/G genotype is lower and the percentage of cells undergoing apoptosis is lower when compared with cells in culture with T/T genotypes. We have reproduced these observations with lymphocytes taken from human volunteers and placed in culture, with EBV-immortalized B cells in culture, with primary fibroblasts in cell culture and with cancer cell lines in culture. In 92 individuals that have donated lymphocytes we see individuals forming a distribution of apoptotic responses between 20% and 60% after gamma radiation, with individuals being quite reproducible in repeated experiments. The lower half of the distribution is heavily weighted with the G/G genotype, while the upper half of the distribution has mainly the T/T genotype. The higher HDM-2 levels in cells thus result in a lower apoptotic index in cells from these volunteers. It has become clear in recent studies that SNP 309 has a clinical impact. We have genotyped two cancer cohorts, one at MD Anderson and one in Germany, containing patients with sarcomas and breast cancers. The results have been statistically significant (P = 0.01-0.02) and clear in both cohorts, and the average age of onset of these cancers is 10–15 years earlier in people with the G/G genotype than in people with the same cancer with the T/T genotype. The interpretation is then that the probability of eliminating pre-cancerous clones of cells via a p53 mechanism is lower in people with a G/G genotype (high HDM-2 levels) and the probability of developing a cancer at an earlier time in life is higher. In addition, in patients that have a germline mutation in the p53 gene (this yields one-half of the p53 protein level in a cell) those individuals that have a G/G genotype or a G/T genotype develop multiple cancers (three, four or five cancers) over their lifetimes, while no T/T homozygotes develop that many independent cancers.
A mule-simple public search of p53 knockdown yields 550,000 results, most relevant scientific articles. Pubmed may be using the keyword search terms gene silencing or regulation or reduced expression, rather than knockdown, a relatively new term. It would be wasteful not to check first that this line of experimentation hasn’t already been done— getting work already done by others published is difficult. I’d include knockout literature to be sure. For example, one hit described “conditional knockout”—you’d have to read it to know whether they described a knockdown.
Also be sure you haven’t been too restricted in how you query luminal epithelium—keep it wide with only mammary and epithel$ (truncated if database allows) to sweep in all variants of how the authors describe the tissue. Better to eliminate the wrong ones later than to eliminate the rights ones from the start.
For mule-simple search of knockdown luminal epithelium "mammary gland" p53, there were 32,200 hits. A significant drill-down topic suggestion was “Altering p53.” An example of a probable miss from your prior search includes, “….derived from mammary transplants of p53 null epithelium, to test whether Id1 ...” (R. Nair, 2010). I’d certainly follow up all the papers citing Blackburn and Jerry (Breast Cancer Res 2002, 4:101-111). Also, “p53null mammary epithelium was transplanted into cleared mammary fat pad…” Also by Jerry, in Oncogene 8: 1052-1058 (2000), A mammary-specific model demonstrates the role of the p53 tumor suppressor gene in tumor development.
Do a more thorough search of publications before launching on this; looks rather obvious (not to mention old!), you just have to find the right papers. You may find Google Scholar a more useful starting point than Pubmed in using natural language to point you to relevant literature; using too many qualifiers can keep you from learning the lore of the field. You owe it at least to the mice.
Dear Nicole,
I am Niccola Funel from Pisa University (Surgical Pathology). I have been involving in Pancreatic Ductal Adenocarcinoma PDAC. So far the findings of PDAC tumor is close to those observed in the brest. Indeed We found that p53 mutazions increased the presence of pre-neoplastic leasions in PDAC tissues (when associated with loss other TSG -Tumor Suppressor Gene such as KLF4 (please see Funel et al. (pancreatology, 2011) . So far, I personally think taht p53+/- model, can be useful for your study.
Have a Nice day!
Niccola
Well, what I have seen in literature is a pattern regarding p53 knockdown in epithelial tissues, especially concerning late-generation telomerase-null (normally TERC-/-) mice: the tissue impairment is rescued [1-3], resulting in proliferation of genomically unstable cells, thus favoring cancer. Importantly, both apoptosis and senescence seems to be important for efficiently inhibiting carcinomas according to studies using a skin cancer mice model [4,5]. However, p53-dependent senescence deficient late-generation telomerase-null mice (with regular apoptosis) presented extended lifespan and rescued proliferative deffects, and were not reported to have increased cancer incidence (which does not mean the risk is not increased) [6]. These and other studies lead me to hypothesize the best alternative would be knocking down p53-dependent senescence only (Cdkn1a-/-, for example), which might result in a slight increase in cancer risk but would greatly increase the proliferative potential of your primary cell culture. An alternative (wich I particularly prefer) is to target other pathways, such as promoting TERT expression in vitro by either cell transfection or telomerase activators in the media. This way you would not decrease the tumor suppression capacity of your cells and would extended their lifespan, as shown by several studies in the past [7,8]. But things can get a bit more complicated if we want... Mice with increased tumor suppression capacity live longer than wild-type mice, and this is not totally due to cancer resistance. The super-M mice experiment showed that this genetic background, when added to constitutive telomerase activity along life, greatly increases fitness and lifespan [reviwed in 9].
Depending on what you are testing and other aspects of your experiment, you could pick the most suitable strategy (p53-dependet senescence knockdown, promote TERT expression or engineer "super" cells) and try it. Unfortunatelly, most of the times we do not really know which of the options is the best until we test them all...
Best of luck! Remeber to tell us the results once!! I guess we all got curious :-)
1. Flores I, Blasco MA. A p53-dependent response limits epidermal stem cell functionality and organismal size in mice with short telomeres. PLoS One 2009;4:e4934.
2. Begus-Nahrmann Y, Lechel A, Obenauf AC et al. p53 deletion impairs clearance of chromosomal-instable stem cells in aging telomere-dysfunctional mice. Nat Genet 2009;41:1138-43.
3. Artandi SE, Chang S, Lee SL et al. Telomere dysfunction promotes non-reciprocal translocations and epithelial cancers in mice. Nature 2000;406:641-5.
4. Gonzalez-Suarez E, Samper E, Flores JM, Blasco MA. Telomerase-deficient mice with short telomeres are resistant to skin tumorigenesis. Nat Genet 2000;26:114-7.
5. Cosme-Blanco W, Shen MF, Lazar AJ et al. Telomere dysfunction suppresses spontaneous tumorigenesis in vivo by initiating p53-dependent cellular senescence. EMBO Rep 2007;8:497-503.
6. Choudhury AR, Ju Z, Djojosubroto MW et al. Cdkn1a deletion improves stem cell function and lifespan of mice with dysfunctional telomeres without accelerating cancer formation. Nat Genet 2007;39:99-105.
7. Weinrich SL, Pruzan R, Ma L et al. Reconstitution of human telomerase with the template RNA component hTR and the catalytic protein subunit hTRT. Nat Genet 1997;17:498-502.
8. Bodnar AG, Ouellette M, Frolkis M et al. Extension of life-span by introduction of telomerase into normal human cells. Science 1998;279:349-52.
9. Donate LE, Blasco MA. Telomeres in cancer and ageing. Philos Trans R Soc Lond B Biol Sci 2011;366:76-84.
In our experience in canine mammary tumour tissues, by advaced polymer based IHC method, mutated expression of p53 was recorded only in 25 to 30% cases, which by Kaplan Meirer analysis had worst prognosis. But what would happen to remaining 70 to 75% cases is really a question that is unanswered. It seems that although p53 is an important anti-oncogene with multifarious functions, yet for neoplastic transformation in at least in vivo model, several more genes and epigenetic pathways may have to be up or down regulated i. e. again corroborating the multi-hit hypothesis of oncogenesis.
James E. Trosko, Ph.D., Michigan State University.
Our published results on normal human breast epithelial stem cells indicate that , by rendering the p53 inactive by transfection with the SV40 large T gene, we could isolate stem cells that were kept in their immortalized state, they grew in soft agar, but did not grow into tumors in nude mice. After X ray irradiation, we isolated weakly tumorigenic clones.that grew into large colonies in soft agar. If we transfected these clones with the neu oncogene, they became highly tumorigenic. See these papers: Kao, C.Y., K. Nomata, C.S. Oakley, C.W. Welsch and C.C. Chang, 1995. Two types of normal human breast epithelial cells derived from reduction mammoplasty: Phenotypic characterization and response to SV40 transfection. Carcinogenesis 16:531-538;253. Kang, K.-S. M. Ikue, A. Cruz, Y.J. Jeon, J.E. Trosko and C.C. Chang: “Expression of estrogen receptors in a normal human breast epithelial cell type with luminal and stem cell characteristics and its neoplastic transformed cell lines”. Carcinogenesis 18:251-257, 1997;268. Kang, K.S., W. Sun, K. Nomata, I. Morita, A. Cruz, C.J. Liu, J.E. Trosko and C.C. Chang: “Involvement of tyrosine phosphorylation of p185c-erbB2/neu in tumorigenicity induced by x rays and the neu oncogene in human breast epithelial cells”. Molecular Carcinogenesis 21:225-233, 1998;280. Sun, W., K.S. Kang, I. Morita, J.E. Trosko and C.C. Chang: “High susceptibility of a human breast epithelial cell type with stem cell characteristics to telomerase activation and immortalization”. Cancer Res. 59: 6118-6123, 1999;298. Chang, C.C., W. Sun, A. Cruz, M. Saitoh, M.-H. Tai and J.E. Trosko: “A human breast epithelial cell type with stem cell characteristics as target cells for carcinogenesis”. Radiation Res. 155: 201-207, 2001;349. Park, J.S., Noh, D. Y.,Kim, S. H., Kong, G.,Chang, C.C.,Lee, Y.S., Trosko J.E., and Kang, K.S. Gene expression analysis in SV40-immortalized human breast luminal epithelial cells with stem cell characteristics using a cDNA microarray, International J. of Oncology, 24:1545-1558,2004;356. Tai, M.H., Chang, C.C., kiupel, M., Webster, J.D., Olson, L.K. and Trosko, J.E., “Oct-4 expression in adult human stem cells: evidence in support of the stem cell theory of carcinogenesis. Carcinogenesis 26: 495-502, 2005; Several more will be appearing soon.
Therefore should we conclude Dr. Trosko that in vitro and in in vivo tumorigenesis are partially and not fully dependent variables. It is indeed a great poser for futuristic research!
Enhancing tumorigenicity and driving solely by simple knockout by a single gene into tumorogenesis in vitro should be looked into as again partially dependent variables.
TP53 mutation is seen in more than 50% of all human malignancies. Also this mutation is common finding in epithelial tumors and it is proved for example in clorectal adenocarcinomas p53 mutation is a commponent of gentic alteration that lead to adenocarcinoma or today the role of P53 mutation is confirmed in tumor progresion and agressiveness in malignant melanoma, In pancreatic ductal adenocarcinomas in about 50% of case p53 mutation is present . However,non malignant pancreatic epithelial proliferation this not occur at all. Therefore , we can predict that P53 mutation could not only cause tumorigenesis in breast epithelial cells but also could have effect on tumor invasiveness as well.One of the Hot topics in breast cancer is triple negative breast cancer and new targeted therapies in the major signalling pathways. I hope it could help. good luck in you research
Hi, Nicole,
The first thing you need to know is what is the biological role of P53 gene in cells. P53 normally is not expressed however, once cell is “sick”, then P53 will be activated. The activated P53 thus activates apoptosis pathway and lead cell death. If you want to culture “normal” cells but this type cells can not divide rapidly, so if you knock out P53, this type cells will develop into cancer cell soon or later, and the pathway of this cell may change also.
p53 knock-out is what we find in Li-Fraumeni....but this does not lead to fast tumor formation. it takes years. HPV infection also leads to p53 dysfunction but again, cancer does not always follow. there must be something that knocks out effective immune surveillance.....one the develops over so many years on top of a p53 knock-down or knock-out that finally leads to clinical cancer formation.
correction on my previous post:........."one THAT develops over so many years on top of a p53 knock-down or...."
p53-deficiency can befound in more than 50% of all cancer caises. But there are two types of cancer regarding p53 - when the p53 protein is mutated and when it does not get activated...
knock out of p53 in a situation where the NK cells work normally will lead to the killing of that p53-KO-cell....... should this cell not go into apoptosis.
http://works.bepress.com/cgi/viewcontent.cgi?article=1031&context=djoseph_jerry&sei-redir=1&referer=http%3A%2F%2Fwww.google.com.eg%2Furl%3Fsa%3Dt%26rct%3Dj%26q%3DDoes%2Ba%2Bknockdown%2Bof%2Bp53%2Bin%2Bluminal%2Bepithelial%2Bcells%2Bsupport%2Btheir%2Bgrowth%2Bor%2Bmight%2Beven%2Bdrive%2Bthem%2Binto%2Btumorigenesis%253F%26source%3Dweb%26cd%3D17%26ved%3D0CGQQFjAGOAo%26url%3Dhttp%253A%252F%252Fworks.bepress.com%252Fcgi%252Fviewcontent.cgi%253Farticle%253D1031%2526context%253Ddjoseph_jerry%26ei%3DW1CAT7LDE8PmtQbW4cjKBA%26usg%3DAFQjCNE-zppZvIi0pY-AIqj21RKbHnptpw#search=%22Does%20knockdown%20p53%20luminal%20epithelial%20cells%20support%20their%20growth%20or%20might%20even%20drive%20them%20into%20tumorigenesis%3F%22
http://www.carcinogenesis.com/article.asp?issn=1477-3163;year=2011;volume=10;issue=1;spage=35;epage=35;aulast=Mohibi
http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1000121
lacking in all these publications, especially those on breast cancers, is the eventual cellular profile of this disease: the proliferation of Tregs. and the NK-Treg cross-talk finally defines the clinical outcome and prognosis of breast cancer. so combining the initial p53 loss of function with the final TIL picture must make us wonder how these phenomena evolved....in continuity.
TP53 is a tumor suppressor genelocated at the short arm of chromosome 17.The protein product acts as a transcription factor for cell differentiation,proliferation and death.The role of this gene in salivary gland tumorgenesis remains unknown.Studiesof different tumors have yielded variable results.The incidence of P53 expression in other benign,malignant and hybrid tumors is low and does not correlate with recurrence.At present there is in sufficient information on the correlation between p53 and outcome.These unsetting results reflect the lack of technical and interpretative uniformity in assesing this marker.In some malignancy of salivary glands TP53 point mutation have been reported,for example: salivary duct carcinoma,Adenoid cystic carcinoma
maybe, then, this lack of correlation between p53 (dys)function and clinical tumor expression (specifically cancer formation) is due to the lack of knowledge on why the NK cancer surveillance mechanism fails in some instances.
Hi Nicole. you have very invaluable tools in your hands now. what ever you want to try to find out... proceed. Carcinogenesis is a multistep process and has to go through various stages which you might have known already.. initiation, promotion and progression. and mutation of p53 is one of the processes whereby the cells acquired theis 'immortality' status but it does not mean that tumour will be formed. unless you have another specific promoter to promote specific tumour, the your p53 knocked down mice will for sure develop the tumour.. not sure about proliferating in the foot pad of the mouse though. but, as far as the materials you have... you can almost anything that you want to do and any findings will be a significant findings. Good job!!!!
Leonardo Jr. Mangahas
Yes, by only if the macrophages detect specific surface markers
"..only if the macrophages detect specific surface markers." in clinical cancer, the malignant cells produce no MHC I; while all the others, dendritic cells, macrophages, NK cells, etc produce corresponding MHCs as they respond to environmental stimuli. this MHC I mismatch is what the KIRs of the NK cells watch. so when this immune surveillance works efficiently, then no malignant cell will prosper in its p53-deficient ways.
p53 is the guardian of the genome.......for only as long as it is intact. HPV can easily disrupt its integrity. once p53 is dysfunctional, the NK cells take over in halting the proliferation of these transformed cells.... or p53-independent pathways to apoptosis may take place.
in the analysis of these processes that link tumorigenesis with cancer formation, the only thing lacking now, in my opinion, is the problem of why the NK cells are paralyzed when confronted with transformed cells....and why it takes so many years for this process to manifest clinically.
Tumorgenesis is a multistep process and affected by many factors ,the most importants are imbalance between oncogenes and oncosupressor genes , p53 consider the guardian gene of the cell which forced it twords apoptosis during damage or stress . Its mutation is associated with 90% of cancer cases , so there is 10% not associated with p53 mutation, so down regulation of p53 not enugh for inducing tumorgenesis .
You can improve that in vitro by culturing normal cells with normal wild type p53, and other with sielencing p53
Hi there Nicole.
To my opinion which i think agrees with most of the above mentioned answers you have to reach your answer in multiple ways. Due to the multifunctional character of p53 even if you get a phenotype by just deleting the whole gene or just one allele you need more evidence to be sure if it is a direct or p53 mediated effect you get. In most cases with proteins such as p53 which play pivotal roles in cellular stress responses you need to check the levels (in various assays) of other known up- or downstream effectors that can "paint" the phenotype you get. It's a try and error process but with the right literature and guiding you will get to the bottom line.
Good luck!
Hi Nicole, I have not done the experiment but I would like very much to know the result if you perform it. This problem is closely related to a point which I raised in my theoretical paper "cancer origin in committed versus stem cells:hypothetical antineoplastic mechanism7s associated with stem cells. Crit. Rev. Oncol. Hematol. (2011) 80, 209-224
Thanks to all of you guys for your comments. I really appreciate it. Also thank you very much Antonio, I will post it here in case I am going to do the experiment. Thanks again!
Hi Nicole, In Li-Fraumeni syndrome (LFS), the most common tumour that occurs is Breast cancer. (In LFS, the normal allele of p53 is either lost of mutated). Somehow, the p53 knockout mice does not develop breast cancer. (Lang et al, 2004), (Olive et al., 2004). Do let me know if i'm wrong. Thanks!
it takes more than a p53 knockout to develop cancer whether you are a mouse or a man. show me how you knock out the immune surveillance mechanism and i'll tell you that surely, sooner rather than later, you will get cancer.....on top of the earlier p53 knockout, of course. knocking out the NK cells needs no genetic defect. it needs an external force to do it. now go figure what that is.
Hi, P53 knockdown should decrease apopotosis, so may increase tumorigenesis
Hi, P53 knockdown should decrease apopotosis, so may increase tumorigenesis
not only during G1/S cell cycle arrest p53 can be activated, also G2/M cell cycle arrested cells have increased p53 level (also coming out from my own results - vinblastine treated murine lymphoma cell do have a high p53 level)
Hi, Nicole! I think Pelicci's 2009 Cell paper (V138 Issue 6) somehow answers your question. In such a case if your freshly-isolated primary luminal epithelial cells contain SC-like cells (about ~1 in 46k cells as determined by their serial dilution transplantation assays) and happened to be efficiently knocked down for p53, you could have a remarkable boost in self-renewal of SC-like cells in culture and they do replenish cleared fat pad once transplanted.
For efficient spontaneous tumorigenesis in your xenografted mice, however, I think you may need 1 or 2 driver mutation(s) to give the cells growth advantage and ability to escaping immune surveillance. As you mentioned, p53-/+ or p53 null mice do develop spontaneous mammary tumors. I am not in the p53 field and wonder how potent this is. Just happened to find this early paper that suggests mammary tumorigenesis due to p53 deficiency exists but quite rare (as compared to lymphoma). See http://www.nature.com/nature/journal/v356/n6366/pdf/356215a0.pdf.
Thank you guy, specifically to Jimin for your helpful comment.
P53 is a gurdian protein in cell cycle against genome damage .If any genome damage introduced the cell ,then P53 imped progress of cell cycle in G1 stage of the cell cycle.Then in P53 knockdown this system of cell controller is removed and the cell cycle progress and tumorigenesis increased.
Roghayeh: not necessarily: that p53-knocked-down-cell will be knocked out by NK cells once it starts to downregulate MHC I expression.
Leonardo :You mean p53-knocked-down-cell that knocked out by NK cells caused decresed expression of MHC I and finally decreased tumorigenesis.
yes, in real life situation, this is what is termed immune surveillance. the HPV model exactly fits into this.
and no. the process goes this way: p53 ko by HPV, then decrease in MHC I expression by the p53-ko'd cell, NK attack leading to p53-ko-cell lysis. it must be hard to create this complete scenario in any lab.
Dear Friends, I don't really know how much immune surveillance propagated mainly by NK cells, could work against the overt cancer. It is thought to work only against initially transformed cells or during occult and latent phases of tumorigenesis.In vivo, it has been shown that CD8 cells , macrophages and ADCC take over during later stages of cancer progression. The role of p53 is just one of many, including help in killing by NK cells, but again how effective needs to be proven in vivo.
overt cancer means immune surveillance (NK, CD8, macrophage functions are lost) is replaced by immnosuppression. this is where the tumor microenvironment takes over. am not sure about ADCC function since i have yet to see an antibody being produced in cancer. my impression is that cancer is an innate immunity problem.
I think that the real problem today that remains unanswered in overt cancer is why immune surveillance mechanisms fail. What paralyzes the cancer cell killers? Why the overwhelming immunosuppression that initially is localized in the area of the primary tumor.....but has the capacity to be transported to another area in the later stages of cancer progression. What in the world is driving this phenomenon? I believe that there is an external force behind all this...rather than our own body trying to destroy our own defenses. In other words, there is no genetic defect that our body suddenly creates so that the whole body gets to self-destruct.
Dear Colleagues,
In response to Leonardo, and to my mind, probably permanently some cells, in all our body processes, are able to trans form in cancer cells. And our immune system (except particular situation ie VHI) is able to recognize them and destroy. NK are less specific cells which can do it, and in the case of specialized immune cells, MHC is implicated for a recognition of the "non-ourself".
But the cancerisation process implicates differents steps with addition of abnormalities in time. The process can be stop by immune sytem at a specific level.
For me the first response allowing cancer cell to avoid natural immune defenses to destroy them is quantitative: under a certain threshold of abnormal cells, immune system is able to eliminatee cancer cells, and if abnormalities accumulated (in term of number of cells, mutations in cells increasing resistance to apoptosis ...) , it surpassess the capacity of immune system to cure oursefl.
Added to that, if environmental conditions render immun system less efficient (whatever the mechanism is), it increases the possibility of cancer occuring.
Regards
Didier J
Once again, let us try to get out of this riddle, called as cancer. First of all let us be clear that both innate and adaptive immune responses are required for killing cancer cells and they usually compliment each other. Enough literature has piled up on this issue. Secondly, It is not that bodily defences totally fail, rather as I said earlier, they are quite successful during initial transformation and latent phases of cancerogenesis. However, during later phases they fail only by a narrow margin as is evident from the fact that out all the tumor cells that enter the lymphatic or blood circulation, only 0.001% are able to survive and form metastasis. Therefore, the crux of the story is that it is the struggle of existence between the tumor and the host, and in that tug of war, the narrow margin of growth advantage and better adaptability coupled with continuous evolution of new clones of wide heterogenity play a key role. The survivor is thus the fittest.
Right Naresh.
But if we are finalists, cancer cells spreading out the body compromise their own survival as the killed the body where they grow. Compared to parasitism, it's not a good adaptation because the two "fighters" die.
We could say that "egoism" of cancer cells (anthropomorphism; because they choose not to work in a coordinate manner to the functionning of a pluricellular organism) leads also to their death.
Regards
Didier
The external force in the mechanism that renders the human immune system unresponsive when it comes to the killing of "nonself' cancer cells does not die in the process. It will exist as a 'live entity" and revert back to its homeostatic state where it does not activate its own immune system: for its enemy, the human immune system has been effectively disabled. That is the final riddle.
p53+MOMP impairment must be the key. This should help Article Molecular genetics and cellular events of K-Ras-driven tumorigenesis
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