Thymidine block and PI staining

DARZYNKIEWICZ ZBIGNIEW Z_DARZYNKIEWICZ at nymc.edu
Wed Jun 30 16:06:06 EST 2004


Cell synchronization in the cycle by inhibition of DNA replication (e.g.
by thymidine block or DNA polymerase inhibitors such as aphidicolin)
leads to severe growth imbalance (cell growth continues in the absence
of cycle progression - DNA replication). There is a gross change in
composition of chromosomal proteins; for example the G2 cyclins A and B
become strongly expressed in these cells, which actually are
synchronized not in G1 but at the entrance to S (Gong et al.,Cell Growth
& Differentiation,  6: 1485-1493,1995). Because accessibility of DNA to
the intercalating dyes varies depending on chromatin structure
(Cytometry, 5:355-363, 1984) one may expect the change in stainability
with PI of the cells characterized by the growth imbalance. The second
reason for the altered DNA stainability could be that in these arrested
cells DNA replication is already initiated but is not progressing.
Initiation of DNA replication involves local denaturation (melting) of
DNA sections at the replication points, the sites of DNA polymerase
attachment. The denatured (ss) DNA is unstainable with PI or other
intercalation dyes. Thus, if there is a large number of the initiation
points in the synchronized cells, there may be a detectable deficit in
extent of dsDNA (stainable with PI), compared to the non-synchronized
cells.	 

Zbigniew Darzynkiewicz, M.D., Ph.D.
Director, Brander Cancer Research Institute at NYMC


-----Original Message-----
From: Frederic.Grosjean at isrec.unil.ch
[mailto:Frederic.Grosjean at isrec.unil.ch] 
Sent: Wednesday, June 30, 2004 4:49 AM
To: cyto-inbox
Subject: Thymidine block and PI staining

Dear Flowers,

Someone in our facility is synchronizing cells using double thymidine
block (BJAB and
HeLa cells), but while performing cell cycle analysis post
synchronization release a
clear shift towards lower PI fluorescence intensity is visible compared
to
non-synchronized control. According to that same person, the cells were
all stained at
the same time with equivalent cell concentration exposed to similar PI
quantity (for 15
minutes at 37C). Cells were fixed with 70% EtOH. Could it be that
thymidine treatment
affect stainability ?  Could PI react with thymidine alone and thus
lower the amount that
will stain DNA ?

Any help is welcome.

Best regards,

Fred

Dr Frederic Grosjean
Ludwig Institute for Cancer Research
Lausanne Branch
Chemin des Boveresses 155
1066 Epalinges
Switzerland
Tel. +41-21-692 58 66
Fax. +41-21-653 44 74
www.licr.org






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