Hi Imran,

any evidence that your cells are expressing the insulin receptor (they seem to do: http://www.ncbi.nlm.nih.gov/pubmed/20463885)? If so, then just go ahead. As you will have to establish a dose/response/time curve anyway, why not simply test it with 10-7M insulin after starving the cells with serum free medium? Then check by western blotting for Akt Ser 473 phosphorylation (as ABs you may use eg the rabbit monoclonals from Cell Signaling, the worked perfectly for us)

Hi Imran:

I am not aware of much physiological/pathological contribution of insulin to leukemia. I would not bother with insulin.

Virtually all growth factors and many cancer mutations contribute to Akt activation. Thus, there is a good possibility that Akt is constitutively active (i.e. phosphorylated) in your cells.

Here is my experimental suggestion:

Grow your cells for 1-4 hrs with either fresh serum/growth factor medium and serum-free medium (you do not want to kill cells). Then treat your cells with 1uM-5uM of Akt allosteric inhibitor (MK2206 or inhibitor VIII) for 15min.

Probe the blot with either pS473 and pT308 Akt antibody from cell signaling technlogy. In my lab, we use a dual-color digital western system. So, I use a rabbit pT308 antibody and a mouse pS473 antibody at the same time.

Inhibitor will determine the extend of basal Akt phosphorylation in your cells. If only inhibitor, but not serum starvation, decreases Akt phosphorylation, then Akt is constitutively active in your cells.

good luck with your studies.

tung

Wolfgang Schechinger and Tung Chan Thanks for your suggestions. I'm interested in a gene, which is positively regulated by MAPK pathway while Akt significantly decreases its RNA level. I have determined the mechanism for MAPK-mediated regulation, and now clarifying the mechanism for Akt-mediated inhibition. I believe both the pathways follow distinct regulatory mechanisms. I have already used Akt inhibitor (LY294002) in complete media as well as in serum deprived media and that worked perfectly well. Now I want to activate Akt to further confirm this finding. Using growth factors apparently doesn't seem to be a good option because GF can activate MAPK pathway also and I want to avoid MAPK activation, so in such a case insulin seems to be a good option. Jus as a final note, I am using HL-60, NB4 and THP-1 cells, which are of leukemia origin.

The K562 myeloid leukaemia cell line also express the Insulin receptor

Hi Imran Khan

Wolfgang Schechinger is right, ·Insulin is one of growth factore cytokines and all of human cells express insuli receptor at differrent level ( hepatocyte much and RBC low), Therefore it possible that your cell has responce to insuline.

Margaret Gee and Saeid Abroun....Thanks for explaination. I am going to start with HL-60 cells....and I will share the information once I get the data....Thank you all again...

I used to work with the Myelomonocytic Cell line Mono Mac 6, they require insulin in the medium. In general, I'd say there is a good chance for your cells to respond. I say, give it a shot. make sure to include a positive control, which, according to Margaret above could be K562. thats a pretty standard cell line and you should be able to obtain it easily from a lab in your neighborhood.

Thomas Sternsdorf ·Thank you...........My first trial with HL-60 cells did not work... but I guess I need to do time and dose dependent study...and additionally, as you mentioned, including a positive control (that I missed in my first attempt) will be a good idea....