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Most of the artifacts I see from induced blocks stem from blocking the cells
for too long at excessive drug concentrations. This can induce apoptosis
or cells may fail to begin cycling after the block is removed.
When establishing the starting conditions for a synchrony, many people
expect to see a >90% G1 (aphidicolin) or G2/M (nocadazole) population.
In most cases this is not necessary or desirable. It's much more important
to establish conditions where the cells respond quickly (i.e., resume cycling)
after drug removal.
One investigator here uses a low dose of aphidicolin for 24 hours. The
first time point after removal of the block (t=0hr) appears as a normal
cell cycle distribution (G1=67%, S=26%, G2/M=7%). At t=2hrs more than 80%
of the cells are in S-phase and most of these are in early S. At t=4hrs
the cells are still >80% S-phase and most are in late S. Yet, you would never
suspect the cells were synchronized from the t=0hr data.
Zbigniew Darzynkiewicz wrote:
>For example after double thymidine block we have observed "unscheduled"
>expression of cyclins B1 and A in cells at the G1/S boundary, over five-fold
>increase in expression of cyclin E, and 40% increased total protein content
>[Gong et al., Cell Growth & Differentiation, 6: (November issue) 1485-93,
>1995].
>Kinetic and metabolic properties of so synchronized cells are much different
>compared to the cells from asynchronous, exponentially growing cultures.
I see nothing unusual about a five-fold increase in cyclin E expression or
a 40% increase in total protein for a population of cells synchronized at
the G1/S border. Both phenomena can be due to the increase in the percentage
of late G1 cells. Protien content increases throughout G1 as cells prepare
for S and since the cells can not proceed with DNA synthesis cyclin E is not
degraded as it normally would be.
Forced synchronization can produce artifact but they can also yield valuable,
relevant information. Drug induced blocks are convenient and can be done
without
specialized equipment (elutriators) and are not as labor intensive as mitotic
shake-offs. Data from induced blocks can be corroborated with alternative
methods.
I also contend that valid cell cycle kinetic information can be obtained from
blocks when properly done.
Well I'm just average, common too,
just like him and the same as you.
I'm everybody's brother and son
and I'm no different than anyone.
Ain't no sense in talking to me,
it's the same as talking to you.
- RZ aka BD
-- ============================================================================== Thomas Delohery | Internet: t-delohery@ski.mskcc.org Manager, Flow Cytometry Core Facility | Phone: (212) 639-8729 Memorial Sloan-Kettering Cancer Center | Fax: (212) 794-4019 1275 York Ave. Box 98 | New York, NY 10021 | ==============================================================================
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