A patient in the clinical division presents with an aggressive ductal
breast tumor, the molecular cause of which is not readily discernable.
 After isolating some of the tumor cells, you discover by
morphological, flow cytometry/cell marker studies, and resistance to
several common chemotherapeutic drugs, that the tumor cells are
monoclonal in nature (meaning all the cells probably arose from a
common progenitor cell).  Flow cytometry analysis indicates also that
the tumor cells are rapidly dividing, i.e., in a virtually constant
state of growth

You culture the cells in increasingly lower concentrations of serum
and realize that there is
probably a mutation somewhere in their signal transduction pathways,
based on their ability to continue growing in 1/50 the serum normally
required for non-transformed cells.  An oncogene inclusive cDNA Array
Analysis, reveals that a unique sequence is present only in the tumor
cells when compared to normal breast tissue.  Using the cDNA Array
manufacturer’s cloned fragment as a probe, you screen a cDNA library
and isolate several possible clones.   You then isolate the cDNA
insert, send it to the sequencing facility, and they send back the
best candidate open reading frame amino acid translation shown below.
To analyze the sequence, GOTO * http://www.ncbi.nlm.nih.gov:80/BLAST/
 then scan down to Genomic blast pages and select Human genome option.
  Be sure to set the Database window to Protein and the Program window
to BlastP.   You should look closely for any mismatch or gaps in the
sequence, which may significantly affect the function of the protein.


MELAALCRWG LLLALLPPGA ASTQVCTGTD MKLRLPASPE THLDMLRHLY QGCQVVQGNL
ELTYLPTNAS LSFLQDIQEV QGYVLIAHNQ VRQVPLQRLR IVRGTQLFED NYALAVLDNG
DPLNNTTPVT GASPGGLREL QLRSLTEILK GGVLIQRNPQ LCYQDTILWK DIFHKNNQLA
LTLIDTNRSR ACHPCSPMCK GSRCWGESSE DCQSLTRTVC AGGCARCKGP LPTDCCHEQC
AAGCTGPKHS DCLACLHFNH SGICELHCPA LVTYNTDTFE SMPNPEGRYT FGASCVTACP
YNYLSTDVGS CTLVCPLHNQ EVTAEDGTQR CEKCSKPCAR VCYGLGMEHL REVRAVTSAN
IQEFAGCKKI FGSLAFLPES FDGDPASNTA PLQPEQLQVF ETLEEITGYL YISAWPDSLP
DLSVFQNLQV IRGRILHNGA YSLTLQGLGI SWLGLRSLRE LGSGLALIHH NTHLCFVHTV
PWDQLFRNPH QALLHTANRP EDECVGEGLA CHQLCARGHC WGPGPTQCVN CSQFLRGQEC
VEECRVLQGL PREYVNARHC LPCHPECQPQ NGSVTCFGPE ADQCVACAHY KDPPFCVARC
PSGVKPDLSY MPIWKFPDEE GACQPCPINC THSCVDLDDK GCPAEQRASP LTSIISAVVG
ILLEVVLGVV FGILIKRRQQ KIRKYTMRRL LQETELVEPL TPSGAMPNQA QMRILKETEL
RKVKVLGSGA FGTVYKGIWI PDGENVKIPV AIKVLRENTS PKANKEILDE AYVMAGVGSP
YVSRLLGICL TSTVQLVTQL MPYGCLLDHV RENRGRLGSQ DLLNWCMQIA KGMSYLEDVR
LVHRDLAARN VLVKSPNHVK ITDFGLARLL DIDETEYHAD GGKVPIKWMA LESILRRRFT
HQSDVWSYGV TVWELMTFGA KPYDGIPARE IPDLLEKGER LPQPPICTID VYMIMVKCWM
IDSECRPRFR ELVSEFSRMA RDPQRFVVIQ NEDLGPASPL DSTFYRSLLE DDDMGDLVDA
EEYLVPQQGF FCPDPAPGAG GMVHHRHRSS STRSGGGDLT LGLEPSEEEA PRSPLAPSEG
AGSDVFDGDL GMGAAKGLQS LPTHDPSPLQ PTAENPEYLG LDVPV

Next, knowing that the ras oncogene is activated in many tumor cells,
you use RT-PCR to amplify that gene in these same cells.  The
sequencing results reveal the following sequence for ras in the tumor
cells.

MTEYKLVVVGARGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILD
TAGVEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHQYREQIKRVKDSDDVPMV
LVGNKCDLAARTVESRQAQDLARSYGIPYIETSAKTRQGVEDAFYTLVREIRQHKL
RKLNPPDESGPGCMSCKCVLS.
Describe an experimental protocol that would enable you to determine
if you actually have a constitutively activated signaling pathway
which is affecting the activity of the AP-1 transcription factor

Should a mutation to the C-jun gene encoding Activation Protein 1
(AP-1) result in these uncontrolled proliferation of these cells then
specifically blocking AP-1 activation should in turn reduce or inhibit
proliferation.  This may be done by addition of an AP-1 inhibitor to
the culture media.  Several new synthetic retinoids have been shown to
selectively inhibit AP-1 transactivation thereby inhibit the growth of
breast cancer cells. Retinyl methyl ether (RME) and related compounds
prevent the development of mammary tumors induced in rats by different
carcinogens

http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=9270011&form=6&db=m&Dopt=b

Use a reported plasmid construct - say green fluoresnect protein under
control of an AP1 promoter.  Transfect it into cultured tumour cells,
and look for the level of expression.  Since you indicate availability
of flow cytometry - this would be the simplest assay technique.