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Full-length version of this article is also available, published online March 5, 2001 as doi:10.1096/fj.00-0611fje.
Published as doi: 10.1096/fj.00-0611fje.
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(The FASEB Journal. 2001;15:1224-1226.)
© 2001 FASEB

Werner syndrome cells are sensitive to DNA cross-linking drugs 1

MARTIN POOT2, JIN SONG YOM, SUSAN H. WHANG, JACKSON T. KATO, KATHERINE A. GOLLAHON and PETER S. RABINOVITCH

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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Figure 1. Differences in apoptosis in WRN-deficient vs. WRN wild-type. Cultures of SYR family WRN-deficient vs. WRN wild-type LCLs were exposed to three increasing drug concentrations (including the LD50), analyzed by Hoechst 33342/SYTO 11/propidium iodide flow cytometry, and the ratio of cells in apoptosis in exposed vs. unexposed cell cultures was determined. The difference between WRN-deficient and WRN wild-type of these ratios is plotted. A positive bar indicates more apoptosis in WRN-deficient relative to WRN wild-type cells; a negative bar reflects less apoptotic cells in WRN-deficient than in WRN wild-type cells. Three- or four-letter codes indicate the drugs tested: ADR = adriamycin; DAU = daunomycin; ETO = etoposide; ECH = echinomycin; BER = berenil; MIT = mitoxantrone; TDDP = trans-platin; CHL = chlorambucil; MEL = melphalan; CDDP = cisplatin; MMC = mitomycin C; CAM = camptothecin.

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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Figure 2. Apoptosis after 24 h of exposure to mitomycin C (A–C) and CDDP (D–F) as a function of WRN genotype. Closed squares (SYR, A, D), circles (TUR, B, E), and triangles (SY, C, F) represent WRN-deficient cells; open symbols represent cells from corresponding family members with wild-type WRN alleles. Each data point represents the mean and standard deviation of triplicate samples.

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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Scheme 1. No caption available.

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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