mTOR is effective against multiple myeloma

[henry123]

Please give me a hand in interpreting in close to layman's tems this journal.....Thanks

 

Regulation of D-cyclin translation inhibition in myeloma cells treated with mammalian target of rapamycin inhibitors: rationale for combined treatment with extracellular signal–regulated kinase inhibitors and rapamycin

 

 

Abstract

We have shown that heightened AKT activity sensitized multiple myeloma cells to the antitumor effects of the mammalian target of rapamycin inhibitor CCI-779. To test the mechanism of the AKT regulatory role, we stably transfected U266 multiple myeloma cell lines with an activated AKT allele or empty vector. The AKT-transfected cells were more sensitive to cytostasis induced in vitro by rapamycin or in vivo by its analogue, CCI-779, whereas cells with quiescent AKT were resistant. The ability of mammalian target of rapamycin inhibitors to down-regulate D-cyclin expression was significantly greater in AKT-transfected multiple myeloma cells due, in part, to the ability of AKT to curtail cap-independent translation and internal ribosome entry site (IRES) activity of D-cyclin transcripts. Similar AKT-dependent regulation of rapamycin responsiveness was shown in a second myeloma model: the PTEN-null OPM-2 cell line transfected with wild-type PTEN. Because extracellular signal–regulated kinase (ERK)/p38 activity facilitates IRES-mediated translation of some transcripts, we investigated ERK/p38 as regulators of AKT-dependent effects on rapamycin sensitivity. AKT-transfected U266 cells showed significantly decreased ERK and p38 activity. However, only an ERK inhibitor prevented D-cyclin IRES activity in resistant “low-AKT” myeloma cells. Furthermore, the ERK inhibitor successfully sensitized myeloma cells to rapamycin in terms of down-regulated D-cyclin protein expression and G1 arrest. However, ectopic overexpression of an activated MEK gene did not increase cap-independent translation of D-cyclin in “high-AKT” myeloma cells, indicating that mitogen-activated protein kinase/ERK kinase/ERK activity was required, but not sufficient, for activation of the IRES. These data support a scenario where heightened AKT activity down-regulates D-cyclin IRES function in multiple myeloma cells and ERK facilitates activity. [Mol Cancer Ther 2009;8(1):83–93

 

 

[ecoli]

We have shown that heightened AKT activity sensitized multiple myeloma cells to the antitumor effects of the mammalian target of rapamycin inhibitor CCI-779.

 

Enhancing the activity of AKT - a protein that plays a key role in cellular processes - in specific kinds of cells (a cancer/myleloma cell line) in a culture/test tube setting makes these cells more sensitive to rapamycin inhibitor - an antitumor chemical with well studied protein targets.

 

To test the mechanism of the AKT regulatory role, we stably transfected U266 multiple myeloma cell lines with an activated AKT allele or empty vector.

What role does this protein AKT play in transducing the effects of this antitumor drug? To study this, the authors introduced a gene that encodes active AKT into cells, so that the cell over-expresses this gene. As a control, they also introduced DNA without this AKT to make sure the effects they see in the study are due to the AKT protein alone.

 

The AKT-transfected cells were more sensitive to cytostasis induced in vitro by rapamycin or in vivo by its analogue, CCI-779, whereas cells with quiescent AKT were resistant.

How do the authors measure the effect of AKT protein? By measuring cytostasis - the inhibition of cell growth. Since tumors divide rapidly, one way of stopping tumors is to suppress cell growth. Introducing extra and active AKT into the cell makes a cell less likely to divide uncontrollably when rapamycin or similar drugs are introduced. However, without active AKT, cells are not as likely to inhibit cell growth, even with rapamycin present. This indicates that AKT has an important role for rapamycin's antitumor effects.

 

The ability of mammalian target of rapamycin inhibitors to down-regulate D-cyclin expression was significantly greater in AKT-transfected multiple myeloma cells due, in part, to the ability of AKT to curtail cap-independent translation and internal ribosome entry site (IRES) activity of D-cyclin transcripts

D-cyclin is a protein that controls cell cycle progression. When cells move faster through the cell cycle, it means they're dividing more rapidly (and look more like a tumor cell). Similar to the above result, when active AKT is introduced into a cell, rapamycin inhibitors can better down-regular (turn off) the D-cyclin gene, thereby slowing down cell cycle progression. How does it do this? AKT turns off protein translation (making protein from a messenger RNA) of D-cyclin gene transcripts.

 

Similar AKT-dependent regulation of rapamycin responsiveness was shown in a second myeloma model: the PTEN-null OPM-2 cell line transfected with wild-type PTEN.

There are many kinds of mutations than can result in cancer. PTEN is a known regulator of the AKT pathway and the results are similar as above using a different type of cell.

 

Because extracellular signal–regulated kinase (ERK)/p38 activity facilitates IRES-mediated translation of some transcripts, we investigated ERK/p38 as regulators of AKT-dependent effects on rapamycin sensitivity.

ERK/p38 are proteins involved in signaling cascades... they take signals received on the outside of the cell and transduce them down to the level of gene control. Basically, they respond to external signals (through lots of intermediate steps) by controlling ribosome binding to messenger RNA of specific gene transcripts. The authors hypothesize that ERK/p38 could be involved in mediating expression of downstream genes in manner dependent on active AKT being expressed in the cell.

 

AKT-transfected U266 cells showed significantly decreased ERK and p38 activity. However, only an ERK inhibitor prevented D-cyclin IRES activity in resistant “low-AKT” myeloma cells. Furthermore, the ERK inhibitor successfully sensitized myeloma cells to rapamycin in terms of down-regulated D-cyclin protein expression and G1 arrest.

 

When active AKT is introduced into a cell, ERK/p38 activity is decreased. Direct inhibition of ERK/p38 without adding AKT, therefore, might have a similar effect of introducing AKT... however this only held true for a ERK inhibitor. ERK inhibitor plus adding rapamycin resulting in decreased D-cyclin and stoppage of cell cycle progression. This suggests that ERK is a necessary component of the action of rapamycin.

 

However, ectopic overexpression of an activated MEK gene did not increase cap-independent translation of D-cyclin in “high-AKT” myeloma cells, indicating that mitogen-activated protein kinase/ERK kinase/ERK activity was required, but not sufficient, for activation of the IRES.

MEK is a protein that activates ERK protein, so you'd expect inceasing MEK expression would increase ERK activity. However, increasing MEK activity -> ERK activity did not increase translation of D-cyclin (which would stop cell cycle progression) when AKT was introduced into the cell.

We saw earlier that D-cyclin translation in 'high AKT' cells required the signalling transduction pathway involving ERK, but clearly there are other factors at play.

 

These data support a scenario where heightened AKT activity down-regulates D-cyclin IRES function in multiple myeloma cells and ERK facilitates activity. [Mol Cancer Ther 2009;8(1):83–93

 

In conclusion, hight AKT downregulates D-cyclin translation in cancer cell model, in a manner dependent on ERK plus some unknown molecular players.

[henry123]

which enzyme adds the poly A tail the most efficiently?

1- eif4

2- PABP II

3- PABP I

4- poly A polymerase