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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 5, 2001 as doi:10.1096/fj.00-0611fje. |
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Department of Pathology, University of Washington, Seattle, Washington 98195, USA
2Correspondence: Department of Pathology, University of Washington, 1959 NE Pacific Ave., Health Sciences Building, Room K-081, Seattle, WA 98195-7705, USA. E-mail: mpoot{at}u.washington.edu
SPECIFIC AIMS
To define the type of chromatin lesion(s) that require the Werner syndrome (WRN) helicase/exonuclease activity to prevent cytotoxicity and S phase prolongation/arrest, we exposed lymphoblastoid cell lines (LCLs) from WRN patients with LCLs from WRN wild-type family members to drugs that damage DNA or interfere with DNA metabolism via biochemically defined mechanisms. After drug exposure, WRN -/- LCLs were compared with WRN +/+ LCLs with respect to induction of apoptosis, S phase arrest, and decrease in proliferative survival.
PRINCIPAL FINDINGS
1. Differential induction of apoptosis in
WRN -/- vs. WRN
+/+ LCLs in response to drugs that cause DNA interstrand
cross-links
WRN -/- LCLs respond to DNA
topoisomerase I trapping by exposure to camptothecin with S phase
specific apoptosis. To define the type of chromatin lesion(s) that
leads to camptothecin sensitivity of WRN-deficient cells, we
exposed LCLs to drugs with a spectrum of biochemically defined
mechanisms. WRN -/- LCLs exposed to melphalan,
chlorambucil, mitomycin C, and cis-platinum(II)diamine
dichloride (CDDP), but not to trans-platinum(II)diammine
dichloride (TDDP), etoposide, berenil, daunomycin, adriamycin,
mitoxantrone, and echinomycin, showed increased apoptosis during
the S phase of the cell cycle (Fig. 1
). Drugs known to inhibit WRN helicase activity by
intercalation between DNA base pairs (daunomycin, adriamycin,
mitoxantrone, and echinomycin) or by blocking enzyme access to the
minor groove of the DNA (berenil) do not elicit increased S phase
apoptosis. Etoposide, which interferes with DNA topoisomerase II
activity and results in a DNA double-strand break, does not induce
increased S phase apoptosis in WRN -/- cells.
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Mitomycin C, CDDP, melphalan, and chlorambucil preponderantly
cause formation of DNA interstrand cross-links, whereas TDDP mainly
generates DNA intrastrand cross-links. Mitomycin C induces both
DNA intrastrand and, to a greater extent, interstrand DNA
cross-links, but it does not produce DNA–protein cross-links.
Figure 2A
B
C
shows a dose-dependent increase in apoptosis after exposure to
mitomycin C in WRN -/- LCLs from three families with WRN
patients, but no induction of apoptosis in the respective
WRN +/+ family members. Although the
WRN -/- cells from the SYR family (Fig. 2A
) showed the strongest response, their counterparts from
the TUR and the SY family also showed significant differences in
apoptosis between the WRN -/- and the WRN +/+
LCLs (P<0.0001 for the SYR and TUR families;
P=0.0014 for the SY family).
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CDDP induces more DNA interstrand cross-links than
DNA–proteincross-links. WRN -/- LCLs from each of three
families undergo apoptosis after exposure to CDDP whereas the LCLs from
the WRN +/+ family members do not (Fig. 2D
E
F
). Thus, WRN-deficient cells show elevated sensitivity
toward CDPP, melphalan, chlorambucil, and mitomycin C, but not to TDDP
(Fig. 1)
. Of all drugs tested, mitomycin C, the drug that does not lead
to protein–DNA cross-links, showed the strongest differential effect
on WRN-deficient cells. The magnitude of this differential sensitivity
of WRN cells to mitomycin C is substantially greater than the effects
previously shown for 4NQO or camptothecin (Fig. 1)
. Taken together,
these results support the conclusion that deficiency for WRN
helicase/exonuclease activity leads to sensitivity toward DNA
interstrand cross-links resulting from DNA cross-linking drugs or
from covalent binding to DNA of proteins such as DNA topoisomerase I
that ‘wrap around’ the DNA.
2. Greater inhibition of proliferative survival after
exposure to DNA interstrand cross-linking drugs in WRN
-/- vs. WRN +/+ LCLs
To confirm the differential sensitivities of
WRN -/- LCLs to the apoptogenic DNA
cross-linking drugs described above, we exposed WRN -/-
and WRN +/+ LCLs to these drugs and measured
proliferative survival. WRN -/- LCLs showed
significantly stronger decrements in proliferative survival after
exposure to melphalan, CDDP and mitomycin C, whereas chlorambucil
elicited a near to significant response (P=0.111; two-sided
Student’s t test).
3. Similar increase in S phase arrest in
WRN -/- vs. WRN
+/+ LCLs exposed to DNA interstrand cross-linking drugs
It is conceivable that induction of apoptosis after exposure to
DNA cross-linking drugs is a direct consequence of arrest of cells in
the S phase of the cell cycle. A higher level of apoptosis in
WRN-deficient vs. wild-type cells could thus be a
consequence of a greater arrest of cells in the S phase. To test this
hypothesis, we determined whether exposure to DNA interstrand
cross-linking drugs led to systematic differences in the proportion of
cells arrested in S phase in WRN -/- and WRN
+/+ LCLs. We found similar increases in % S phase cells
after CDDP and mitomycin C treatment of WRN -/-
and WRN +/+ LCLs.
CONCLUSIONS
The increased sensitivity of WRN -/- vs. WRN +/+ LCLs toward DNA interstrand cross-linking drugs during S phase indicates that WRN helicase/exonuclease activity is required when the DNA replication complex encounters an interstrand DNA cross-link. Our finding that inhibitors of the WRN helicase activity did not elicit elevated S phase apoptosis or arrest suggests that the WRN helicase activity is not involved in the phenotype of S phase prolongation/arrest/apoptosis. In contrast, WRN exonuclease activity may, in cooperation with protein complexes such as Ku70/80 and RPA, be involved in the removal and/or bypass of DNA interstrand cross-links during DNA replication. Fanconi anemia (FANC) cells have also been shown to be sensitive to DNA cross-linking drugs. These similar patterns of sensitivity are consistent with the hypothesis that the WRN and the FANC proteins cooperate in a pathway that processes DNA interstrand cross-links.
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FOOTNOTES
1 To read the full text of this article, go to
http://www.fasebj.org/cgi/doi/10.1096/fj.00-0611fje ; to cite this
article, use FASEB J. (March 5, 2001)
10.1096/fj.00-0611fje 
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