RE: Proliferating lymphocytes in peripheral blood and CD25 expression

Let me add a note of caution regarding the use of pyronin Y as a marker
of proliferating cells or RNA. Both Howard and Edward are right that a
bivariate analysis of Hoechst vs pyronin Y fluorescence allows one to
discriminate between Go vs G1 cells. Edward is also correct stating that
pyronin Y is toxic to cells. I would argue, however, that when used
supravitally pyronin Y preferentially stains mitochondria and not RNA
(see e.g. "Cytostatic and cytotoxic properties of pyronin Y: Relation to
mitochondrial localization of the dye and its interaction with RNA",
Cancer Res., 46: 5760-5766, 1986). In fact, the cytotoxic and cytostatic
properties of pyronin Y remind very much rhodamine 123 and relate to its
interaction with mitochondria. The cytotoxicity is enhanced when cells
stained with pyronin Y are illuminated with white light - e.g. under
light microscopy one can easily see that mitochondria containing pyronin
Y swell and rupture within seconds after turning on light.
Noncycling (Go) cells, have much fewer mitochondria and their
stainability with Rhodamine 123, similar as with pyronin Y, is many-fold
lower that that of G1 -S- G2M lymphocytes ("Increased mitochondrial
uptake of Rhodamine 123 during lymphocyte stimulation" Proc. Natl Acad.
Sci. USA 78: 2383-2387, 1981)
 The photosensitizing effects of pyronin Y through mitochondria was
described in another publication ("Photosensitizing effect of tricyclic
heteroaromatic cationic dyes pyronin Y and toluidine blue O (Tolonium
chloride" Cancer Res., 48: 1295-1299, 1988)"). Perhaps to lower the
cytotoxicity of pyronin Y one has to carefully screen cells from the
ambient light. Rhodamine 123 may be less toxic than pyronin Y and both
discriminate Go from G1 cells.
Of course, pyronin Y and Hoechst can be used to stain differentially RNA
and DNA in fixed cells. Pyronin Y can also be used to discriminate
between ds and ssRNA ("Application of pyronin Y in cytochemistry of
nucleic acids". Cytometry, 8:138-145,1987).

Happy New Year to all FLOWERS !!!!!   

Zbigniew Darzynkiewicz, M.D., Ph.D.
Brander Cancer Research Institute at NYMC
19 Bradhurst Avenue, Suite 2400
Hawthorne, N.Y. 10532
darzynk@nymc.edu
http://www.darzynkiewicz.com/zbigniew/


-----Original Message-----
From: Edward F. Srour [mailto:esrour@iupui.edu] 
Sent: Tuesday, December 27, 2005 10:50 AM
To: cyto-inbox
Subject: Proliferating lymphocytes in peripheral blood and CD25
expression

This exchange took place between Petros Christopoulos and Howard Shapiro


Petros Christopoulos wrote:

>I would like to ask if anybody knows about the relationship of
>expression of classical
>proliferation markers (like Ki-67 or the transferrin receptor) and
>CD25 (which is also an activation marker) in peripheral blood
lymphocytes.
>Do they give similar results, do they correlate or are they totally
>independent of each
>other ?

Time to start another winning streak for Mailing List submissions...

See Figure 10-7, p.460, in the 4th Edition of Practical Flow
Cytometry (available without charge on the Invitrogen/Molecular
Probes web site, http://probes.invitrogen.com/) for plots relating
transferrin receptor (CD71) expression and RNA content to DNA
content. I have speculated in the book and on this Mailing List that
Ki-67 and CD71 should correlate almost absolutely in proliferating
lymphocytes and probably in most or all other cell types, but, when I
just looked on PubMed, I could not find any publication suggesting
that both had been measured simultaneously in the same cells at the
same time. This really needs to be done; there have been over 100
papers published referring to Ki-67 in the past year, representing a
lot of work by many people. Detecting Ki-67 requires permeabilization
and is only possible in fixed cells, whereas detecting CD71 requires
only surface staining, meaning that live cells can easily be analyzed
and sorted based on CD71 status, but not based on Ki-67 status. If
the two markers are as well correlated as I suspect, a switch from
Ki-67 to CD71 staining will save both time and money, and, more
importantly, allow researchers to do further functional studies on
live cells precisely characterized as to cell cycle position. Such
studies can, in at least some cases (see the work of Edward Srour et
al), be done using Hoechst 33342/Pyronin Y staining for DNA and RNA,
but pyronin Y can be toxic.

_________________________________________________________

So, my input on this topic is the following. Howard's assumption is
right on
the money. expression of CD71 and Ki67 correlate well with one another.
we
have isolated murine cells lacking the expression of CD71 versus cells
that
are CD71+. upon PCR analysis of these cells, we found that CD71- cells
do
not express Ki67 while CD71+ cells do. the separation between CD71+ and
CD71- cells is a little tricky depending on the cell type since many
cell
groups present a continuum of CD71 staining such that separation between
positive and negative is rather subjective. As others argued on this
forum
that an isotype control is in general useless, this may be a case where
the
use of an isotype may be helpful.  we have not published this yet.

Also another point of agreement with Howard. Pyronin Y is toxic
especially
to murine cells while it is well tolerated by human cells.
unfortunately,
some people are publishing the use of Pyronin Y with murine
hematopoietic
cells without recognizing that although the data presented clearly
indicate
something wrong was going on in these cells.

Happy Holidays

edward f. srour



Edward F. Srour, Ph.D.
Professor of Medicine, Pediatrics, Micro/Immunol.
Indiana University School of Medicine
Cancer Research Institute
1044 West Walnut Street. R4-202
Indianapolis, IN 46202-5121

Phone: 317-274-0343
	    317-274-3589
Fax:	 317-274-0396
email: esrour@iupui.edu






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