Rh123 uptake in staurosporine treated cells

Witherspoon, Sam sw11527 at glaxowellcome.com
Sun Sep 10 21:21:48 EST 2000

Greetings Anna, Mark,
	I found a paper indicating that
"The MRD1 (P-glycoprotein) and MRP (P-190) transporters do not play a major
    role in the intrinsic multiple drug resistance of Jurkat T lymphocytes"
Source :    Leukemia Research. 21(8):743-52, 1997 Aug.

	That said, the paper indicated that these cells produced message for
both MDR-1 and MRP, but that functional assays indicated that perhaps other
mechanisms were at work.

	It is likely that staurosporine is inhibiting one or more of these
pumps to some extent, giving you a differential in Rh123 loading.  It is
known for example that staurosporine is an inhibitor of p-glyocoprotein:

Budworth J. Davies R. Malkhandi J. Gant TW. Ferry DR. Gescher A. Comparison
of staurosporine and four analogues: their effects on growth, rhodamine 123
retention and binding to P-glycoprotein in multidrug-resistant MCF-7/Adr
cells. British Journal of Cancer. 73(9):1063-8, 1996 May.

Measurement of mitochondrial membrane potential in intact cells is not
always straightforward.
I refer you to: Practical Flow Cytometry. 3rd Edition. Howard M Shapiro.
Alan R Liss, Inc. ISBN 0-471-30376-3 Pages 329-342.

	I wish I could offer you a workable solution.	Perhaps others can
add to the discussion.

Sam Witherspoon
sw11527 at glaxowellcome.com

Dept. of Receptor Biochemistry	Tel. 919-483-3078
Glaxo Wellcome R&D		Page 919-857-7768
5 Moore Dr.			Fax  919-483-0585
RTP, NC  27709

> -----Original Message-----
> From: Mark Cozens [SMTP:M.A.Cozens at utas.edu.au]
> Sent: Friday, September 08, 2000 12:40 AM
> To:	Cytometry Mailing List
> Subject:	Rh123 uptake in staurosporine treated cells
> I'm studying honours in the Dept. Pathology and am looking at
> depolarisation of the mitochondrial membrane as an early event in
> apoptosis of Jurkat T cells. I'm staining cells with the
> mitochondrial specific dye Rh123 and am using  5microM staurosporine
> treated cells as a positve control to show mitochondrial
> depolarisation. However, staurosporine treatment appears to be
> causing an increased uptake of Rh123. I'm wondering if this is due to
> mitochondrial changes or another property of staurosporine treatment
> that is increasing uptake of the dye.
> If anyone has any experience with this area or any ideas, feedback
> would be muchly appreciated.
> Thanks,
> Anna Hearps
> (ahearps at postoffice.utas.edu.au)
> --
> Mark A Cozens
> Division of Pathology
> University of Tasmania
> ph	03 62264828
> fax	03 62264833

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