Jay,
I assume the theory is that the FITC-VAD-FMK binds to activated caspases in
much the same was as the Z-VAD-FMK inhibitor binds. The peptide portion
presumably recognizes the active site of caspases and I think the FMK is
involved in forming a covlent link responsible for the irreversibe binding.
It should be pointed out that Z-VAD-FMK is a general inhibitor and I assume
FITC version also is not specific for caspase-3. Some of the early work
that was done using biotinylated VAD-FMK compounds such as these for
affinity purification bound more than just caspases to the column.
The commercial source for FITC-VAD-FMK is Promega Corporation as G7461. You
can see more information on their web site
at...http://catalog.promega.com/catalog/catinfo.asp?idx=1414
I do not have direct experience myself; but, I understand it can be used for
flow cytometry. Perhaps you could get additional information from their
technical services people.
Regarding your note of caution, I fully agree that more than one method
should be used to confirm the presence of apoptosis. Caspase-3 (or any other
method) should not be relied on solely. Morphology or DNA fragmentation
(TUNEL) are generally good methods to confirm apoptosis; but, it should be
noted that it may be possible to detect early markers before you see TUNEL
staining so there may not be a direct one-to-one correlation among different
methods. I am interested in following-up and looking at the publications
you listed, especially in how you defined the nonapoptotic proliferating
cells and how you measured caspase-3 activity. Was caspase-3 active in all
of the cells or was this measurement as an activity present in an extract
from the population? In some cases, I think the proliferation/apoptosis
issue may be clouded because there may be an association between the ability
to undergo apoptosis and the phase of the cell cycle.
Just to clarify an issue, when you refer to PARP as "activated", I assume
you really meant the appearance of caspase-cleaved PARP. It was my
understanding that PARP is mostly "inactivated" by caspase cleavage and
although the p89 fragment may still retain biological activity, without the
DNA binding domain attached, its "normal" activity is dimished.
Ty
-----Original Message-----
From: Jayaraman, Sundararajan <SJayaram@med.miami.edu>
To: cyto-inbox
Date: Friday, March 03, 2000 8:32 PM
Subject: RE: FITC-VAD-FMK & Apoptosis Markers / Re: apoptosis in neuronal
cells
>
> Ty Lee mentioned FITC-VAD-FMK usage for detection of apoptosis. I am
>wondering if it is known whether this binds to activated caspases. What is
>the commercial source for this? Finally, can it be used in a flow cytometry
>assay?
> A note of caution about solely relying on activated caspase-3 and/or
>PARP as indicators of apoptosis. Recent studies have shown that caspase-3
>and PARP are activated in nonapoptotic proliferating human peripheral blood
>T cells in vitro. We have confirmed that caspase-3 is constitutively
>activated in human T cells that have been activated with allogeneic
>antigens. However, Fas-mediated apoptosis does not accompany enhancement in
>caspase-3 activity. All these observations were made in human T cells. This
>may or not be the case in neuronal or other type of cells. The following
>references may be helpful:
> 1. Miosse et al. 1997. J. Biol. Chem. 272: 13459.
> 2. Wilheim et al. 1998. Eur. J. Immunol. 28: 891.
> 3. Alam et al. 1999. J. Exp. Med. 190: 1879.
>
>Jay
>Sundararajan Jayaraman, Ph.D.
>Research Assistant Professor
>Dept. of Pathology
>University of Miami Medical School
>DRI Building Room 5018 (R-134)
>1450 NW 10th Avenue
>Miami, FL 33136
>Phone: (305) 243-6100
>Fax: (305) 243-4553
>
>> -----Original Message-----
>> From: tylee [SMTP:tylee@itis.com]
>> Sent: Thursday, March 02, 2000 11:01 PM
>> To: Cytometry Mailing List
>> Subject: FITC-VAD-FMK & Apoptosis Markers / Re: apoptosis in neuronal
>> cells
>>
>> Snezna,
>>
>> Another approach you might try is to use FITC-VAD-FMK which is a
>> fluorescently labeled cell permeable peptide inhibitor of caspases. FITC
>> is the fluorescent tag; VAD is valine-alanine-aspartic acid, and FMK is
>> fluoromethly ketone (the irreversible protease inhibitor "business end"
of
>> the molecule. The FITC-VAD-FMK can enter live cells undergoing apoptosis
>> and irreversibly bind to active caspase enzymes.
>>
>> I am not sure what GD 12-14 cells are or how they will be removed from
>> fetal brain samples; however, for another approach, I might recommend
>> trying immunohistochemistry as an alternative to flow. There are a few
>> marker antibodies that are becomming available to stain active caspases
or
>> the protein fragments that result from their protease activity. I have
>> done immunocytochemistry on human cells using an antibody from Promega
>> that is specific for the cleaved form of PARP (i.e. a marker of
>> apoptosis); but I'm not sure if it will work on rat neuronal cells. They
>> also have an antibody against the active form of caspase-3 that should be
>> much more species cross reactive because of the conserved amino acid
>> sequence of that protein. Caspase-3 activity has been shown to be an
>> earlier marker than Annexin-V staining because caspase activity is
>> required for the phosphatidyl serine translocation to the outer membrane
>> that Annexin V binds to.
>>
>> My two cents worth.
>>
>> Ty Lee
>>
>> -----Original Message-----
>> From: Vanhoek, Monique {BERK~Berkeley} < MONIQUE.VANHOEK@roche.com
>> <mailto:MONIQUE.VANHOEK@roche.com>>
>> To: Cytometry Mailing List < cytometry@flowcyt.cyto.purdue.edu
>> <mailto:cytometry@flowcyt.cyto.purdue.edu>>
>> Date: Thursday, March 02, 2000 5:47 PM
>> Subject: RE: apoptosis in neuronal cells
>>
>>
>> Yes, we (Roche Molecular Biochemicals) offer many kits for studying
>> apoptosis. The Annexin-V kits are designed to be done by FACS. I
>> reccommend the Annexin-V staining kit and the Alexa dye. See
>> <http://biochem.roche.com/apoptosis/prod07.htm> for more details. You
can
>> order by phone (1-800-262-1640) or at www.IbuyRMB.com
>> <http://www.IbuyRMB.com>
>>
>> -----Original Message-----
>> From: Snezna Rogelj [ <mailto:snezna@nmt.edu>]
>> Sent: Tuesday, February 29, 2000 4:04 PM
>> To: Cytometry Mailing List
>> Subject: apoptosis in neuronal cells
>>
>>
>> Wise (wo)men of Flowland -
>>
>> A colleague of mine would like to know if flow cytometry can
>> be used to determine whether a subset of fetal brain cells (rat, GD
12-14)
>> is undergoing apoptosis. Yes? No? Any and all information is bound to be
>> useful.
>>
>> With many thanks,
>> Snezna
>>
>>
>
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