Specifically in breast cancer human (MCF-7 , MDA MB 231) and mouse mammary epithelial cells.
I would treat the cells with either different concentrations of drug or do a time-dependent treatment with one concentration (or both, but then it get´s more and more complicated) and afterwards isolate mRNA and read-out the expression of c-myc target genes (must be several, Tert for example and others from cell cycle/apoptosis gene groups) using qRT-PCR.
ALternatively, transfect the cells with a kind of reporter plasmid containing several "E boxes" upstream of lucifearse reporter gene, treat cells with drug and read-out the luciferase activity which shoudl reflect the transcriptional actiity of c-myc.
Hi Parash
I am afraid that you need to be a bit more specific in your question. c-myc functionality in relation to different target genes is going to be different in different cell types, but if you want a "general" assay you could try transfecting a consensus c-myc reporter construct, Qiagen do a kit commercially (http://www.sabiosciences.com/reporter_assay_product/HTML/CCS-012L.htmland), and I believe that there is a new(ish) ELISA kit that measures myc complex binding to a consensus nucleotide. (ELISA http://www.activemotif.com/catalog/188/transam-c-myc).
You could also try band shift (EMSA) assays as well, which will let you know that active complexes are present in you cell extract.
However if you are interested in the role of c-myc in breast cancer, or indeed targeting c-myc with you drug in breast cancer, I would choose a few reporter constructs for myc target genes that are important in whatever aspect of breast cancer you are studying.
Rather than just c-myc, why not look at cell cycling in generalised way. So many ways of doing this one to fit your hypothesis.
BrdU growth medium can be used to compare rate of DNA synthesis to untreated controls.
You could use a IHC targeting c-Myc, Ki67, and whole myriad of gene products which c-Myc acts as transcription factor for.
Best bet is to find some key signalling pathway positions which you can diferentiate between pro/activated forms.
You may get interesting results just using c-Myc and DAPI to look at its position in the cell, i.e pro/uncleaved in cytosol and active/cleaved in nucleus, depending on whether you can get an antibody polyclonal for boh forms (or could use 2 mono's) .
- Badges
- Science topic