I want to make cancer cells and normal cells devoid of mitochondria, keeping their nuclear DNA intact. Then I would like to use those cells for introduction of new mitochondria by cybrid technique.
If you maintain the cells in glucose, they should be ok. Their proliferation rates will probably be reduced though, so d mito-null cells will be rapidly out-competed by any cells in the culture still containing mitochondria. Do make sure you change the medium regularly as well, because these cells will generate plenty of lactate. How are you planning to deplete the mitochondria? Are you going to use EtBr, if so this has been done for many many years now, so methods ought to be available on the web.
I agree with JL and AM above. These are surely what we used to call "rho zero" cells? (q.v.). In passing I would like to point out that all cells (rho-zero and cybrid and 'normal') grown on 25 mM glucose will be in a sense "diabetic". For 25 mM glucose would be regarded as an horrendous concentration in clinical practice. Just a thought.
Rho-zero though, if I am not mistaken, retain mitochondria, it is the mtDNA that is depleted (e.g. by EtBr as suggested above). You have an ATP issue to tackle without mitochondria I am afraid!
King and Attardi at CalTech in the 80s created the first rho-zero cells, A pubmed search will pull up their paper(s). Might have been in JMB or PNAS. Another place to ask is on the Mitochondria Interest Group (MIG) list serv. see their website for information on how to join the listserv (http://sigs.nih.gov/mito).
Steve
Disclaimer: I coordinate MIG at NIH.
correct, the only thing lost in rho zero cells are the mtDNAs, which code for only 13 proteins, 22 tRNAs, and 2 ribosomal RNAs, so protein synthesis is shut down in the mitochondria (synthesis of the 13 proteins) and since all 13 are involved in the electron transport enzyme complexes and ATPase, no OXPHOS happens, the cells only run glycolysis. And I think uridine is needed because of one fo the uridine pathways going thru the mito, which are nonfunctional in this case. The mitochondria though contain multitude of proteins from the nucleus, I am not sure if anyone has examined what nuclear-encoded proteins are missing from rho-zero cell mitochondria. And of course you have the differing protein composition of the mitochondria depending on their cell type. You might want to examin the literature concering your idea for moving mitochondria around, it has been done in cancer cells and in stem cells too I believe. Current projects funded by the NIH can be searched in the congressionally mandated NIH RePorter whcih lists extramural and intramural projects, you will only see publicaly available information like the abstract and the PI and the NIH Program Directors, and other non-confidential information, but it is a start in knowing what is being done in your area currently funded by NIH, and with PubMed and Google searches you can glean alot about rho-zero cells.
Steve
Disclaimer, I work at NIH
I agree with others. Rho null cells still have mitochondria. Only respiratory oxidative phosphorylation is lost. The mitochondria still carry out and are needed for a variety of other cellular/metabolic functions in addition to OXPHOS and ATP production. They also still have and need at least some level of mitochondrial membrane potential to import proteins from the nucleus. This is no longer made by OXPHOS but it is believed by ATP/ADP exchange through the adenine nucleotide transporter.....ATP has more negative charges than ADP so an exchange driven for example by relative concentration gradients (ATP being made in the cytosol) can drive generation of a membrane potential. Others may be able to comment more knowledgeably on how the membrane potential is created in rho null mitochondria. Cybrids can be made because the mitochondria that are introduced that way have mitochondria that do contain DNA and they can then reestablish OXPHOS for the cell.
We did work with this type of cells call rho zero cells. They are fully glycolytic.. But after that they grow well in presence of glucose 25 mM and have a normal mitochondrial membrane potential since the electron transport even if reduced is enough to built up a potential. We described also that they undergo a normal apoptosis linked to an early drop of their mitochondrial membrane potential.
Have fun with them they merit your attention....
Hello all. A very interesting discussion. I wish I had joined it earlier. I have three questions here. Firstly given the multiple exogenous growth requirements of human tumor cells that lack mtDNA (I am a yeast person and not used to such complexicity) does this mean that they are likely underrepresented in established tumor cell lines if these metabolites haven't been added to the growth medium? Secondly do these Rho zero cells from different tumors have a mutator or hyper-recombination phenotype due to increased ROS production? And lastly as these tumor cells in aerobic glycolysis will likely result in localized type B lactic acidosis do they all show increased invasiveness and metastatic potential?
While I agree that 25mM glucose in media will allow for growth of these cells in culture what I worry about are selection artifacts and that these growth conditions while convenient experimentally are not informative. They might be if all cancer patients were diabetic but they are not.
The recent papers from Lisanti group are very interesting and address issues relating to glycolysis / OXPHOS pathways operating in cancer cells. They present intersting data showing that human breast cancers have two distinct metabolic compartments:
1) a glycolytic tumor stroma,
2) surrounded by oxidative epithelial cancer cells (that are rich in mitochondria).
They propose a kind of metabolic symbiosis (between two compartments) between epithelial cancer cells and their surrounding stroma. Also, cancer cells act as parasites by stealing high energy mito fuel from adjacent cells. In summary, cancer cells utilize both OXPHOS and glycolysis pathways as opposed to only using glucose via glycolysis pathway. As though for a long time also called as Warburg effect.
Ref:
Mitochondria "fuel" breast cancer metabolism: fifteen markers of mitochondrial biogenesis label epithelial cancer cells, but are excluded from adjacent stromal cells.
Sotgia F, Whitaker-Menezes D, Martinez-Outschoorn UE, Salem AF, Tsirigos A, Lamb R, Sneddon S, Hulit J, Howell A, Lisanti MP.
Cell Cycle. 2012 Dec 1;11(23):4390-401. doi: 10.4161/cc.22777. Epub 2012 Nov 21.
Mitochondrial biogenesis in epithelial cancer cells promotes breast cancer tumor growth and confers autophagy resistance.
Salem AF, Whitaker-Menezes D, Howell A, Sotgia F, Lisanti MP.
Cell Cycle. 2012 Nov 15;11(22):4174-80. doi: 10.4161/cc.22376. Epub 2012 Oct 15.
This is an excellent answer to this question Suresh as it speaks to two important issues of tumor biology. Firstly what matters in models of early carcinogenesis is how tumors behave in vivo and maybe even in silico in 3D tissue culture monolayers in petri dishes or in batch cultures not so much. And secondly we now know that many very early tumors have complex histopathology and may well consist of cell lineages derived from different tissue types. In the case of early breast cancer cells with damaged mitochondria (The Warburg epithelial compartment as suggested in this paper) may well have a complex pleiotropic phenotype and require the collaboration of adjacent stromal cells to act much like nurse cells feeding the former glucose pyruvate and uridine to survive. Cooperation at the tissue level can now begin to drive further disease progression.
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