[Protein-analysis] Re: How many passages takes to obtain mutants in a cell line?

Dr Engelbert Buxbaum via proteins%40net.bio.net (by engelbert_buxbaum At hotmail.com)
Mon Dec 18 15:02:41 EST 2006


Nancy E Caceres wrote:

> 
> 
> Hello:
> 
> We have a drug resistant cell (murine) line that routinely is grown in
> presence of the drug.
> 
> When these cell were grown in absence of the drug the phenotype changed to
> sensible after 90 passages (we compare levels of accumulation of drug in WT
> and resistant cell, the resistant accumulate less).
> 
> Can we conclude that there may be single mutation event involved in the
> generation of resistance and that after 90 passages in absence of selection
> pressure reverted?
> 
> Or is it just matter of regulation of expression of genes under that
> stress? And that in absence of drug everything goes back to normal levels?
> Is 90 passages too long to restore normal levels of expressions and
> actually it is a mutation?

Presumably you are talking about a cancer cell line, which is aneuploid.
In other words, single chromosomes, or even part of them, occur more
frequently than they should. Other non-required sequences may be chucked
out completely. So part of your drug resistance may be due to genetic
amplification of Mdr1, MRP, sPGP and similar genes. Do a chromosome
spread from your cells, it is something one has to see for oneself to
believe it.

In addition the regulation of transcription, translation and mRNA- and
protein degradation can be affected.

So in some multi-drug resistant cell lines for example Mdr1 may account
for more than 10% of all membrane proteins. That's  a considerable
expense for the cell, not only for the production of the protein but
also to supply the ATP it requires to run, which may accout for 1/3 of
the ATP-turnover of a cell. If selection pressure is released, that will
drop fairly quickly to normal levels. 


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