FASEB J.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 QUICK SEARCH:   [advanced]


     


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.
Published as doi: 10.1096/fj.00-0611fje.
This Article
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
15/7/1224
00-0611fjev1    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by POOT, M.
Right arrow Articles by RABINOVITCH, P. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by POOT, M.
Right arrow Articles by RABINOVITCH, P. S.
(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.



View larger version (11K):
[in this window]
[in a new window]
 
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).



View larger version (29K):
[in this window]
[in a new window]
 
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.



View larger version (25K):
[in this window]
[in a new window]
 
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




This article has been cited by other articles:


Home page
Mol. Biol. CellHome page
W.-H. Cheng, D. Muftic, M. Muftuoglu, L. Dawut, C. Morris, T. Helleday, Y. Shiloh, and V. A. Bohr
WRN Is Required for ATM Activation and the S-Phase Checkpoint in Response to Interstrand Cross-Link-Induced DNA Double-Strand Breaks
Mol. Biol. Cell, September 1, 2008; 19(9): 3923 - 3933.
[Abstract] [Full Text] [PDF]


Home page
J. Biol. Chem.Home page
K. Li, A. Casta, R. Wang, E. Lozada, W. Fan, S. Kane, Q. Ge, W. Gu, D. Orren, and J. Luo
Regulation of WRN Protein Cellular Localization and Enzymatic Activities by SIRT1-mediated Deacetylation
J. Biol. Chem., March 21, 2008; 283(12): 7590 - 7598.
[Abstract] [Full Text] [PDF]


Home page
Mol. Cell. Biol.Home page
B. Li, S. P. Jog, S. Reddy, and L. Comai
WRN Controls Formation of Extrachromosomal Telomeric Circles and Is Required for TRF2{Delta}B-Mediated Telomere Shortening
Mol. Cell. Biol., March 15, 2008; 28(6): 1892 - 1904.
[Abstract] [Full Text] [PDF]


Home page
JCBHome page
L. M. Pirzio, P. Pichierri, M. Bignami, and A. Franchitto
Werner syndrome helicase activity is essential in maintaining fragile site stability
J. Cell Biol., January 28, 2008; 180(2): 305 - 314.
[Abstract] [Full Text] [PDF]


Home page
Nucleic Acids ResHome page
J. Grillari, H. Katinger, and R. Voglauer
Contributions of DNA interstrand cross-links to aging of cells and organisms
Nucleic Acids Res., December 14, 2007; (2007) gkm1065v1.
[Abstract] [Full Text] [PDF]


Home page
Nucleic Acids ResHome page
N. Saydam, R. Kanagaraj, T. Dietschy, P. L. Garcia, J. Pena-Diaz, I. Shevelev, I. Stagljar, and P. Janscak
Physical and functional interactions between Werner syndrome helicase and mismatch-repair initiation factors
Nucleic Acids Res., September 27, 2007; 35(17): 5706 - 5716.
[Abstract] [Full Text] [PDF]


Home page
Nucleic Acids ResHome page
A. Machwe, L. Xiao, R. G. Lloyd, E. Bolt, and D. K. Orren
Replication fork regression in vitro by the Werner syndrome protein (WRN): Holliday junction formation, the effect of leading arm structure and a potential role for WRN exonuclease activity
Nucleic Acids Res., September 27, 2007; 35(17): 5729 - 5747.
[Abstract] [Full Text] [PDF]


Home page
Sci Aging Knowl EnvironHome page
F. M. Hisama, V. A. Bohr, and J. Oshima
WRN's Tenth Anniversary
Sci. Aging Knowl. Environ., June 28, 2006; 2006(10): pe18 - pe18.
[Abstract] [Full Text]


Home page
Nucleic Acids ResHome page
J. A. Harrigan, D. M. Wilson III, R. Prasad, P. L. Opresko, G. Beck, A. May, S. H. Wilson, and V. A. Bohr
The Werner syndrome protein operates in base excision repair and cooperates with DNA polymerase {beta}
Nucleic Acids Res., January 30, 2006; 34(2): 745 - 754.
[Abstract] [Full Text] [PDF]


Home page
Nucleic Acids ResHome page
W.-H. Cheng, R. Kusumoto, P. L. Opresko, X. Sui, S. Huang, M. L. Nicolette, T. T. Paull, J. Campisi, M. Seidman, and V. A. Bohr
Collaboration of Werner syndrome protein and BRCA1 in cellular responses to DNA interstrand cross-links.
Nucleic Acids Res., January 1, 2006; 34(9): 2751 - 2760.
[Abstract] [Full Text] [PDF]


Home page
J. Biol. Chem.Home page
N. Zhang, R. Kaur, X. Lu, X. Shen, L. Li, and R. J. Legerski
The Pso4 mRNA Splicing and DNA Repair Complex Interacts with WRN for Processing of DNA Interstrand Cross-links
J. Biol. Chem., December 9, 2005; 280(49): 40559 - 40567.
[Abstract] [Full Text] [PDF]


Home page
Nucleic Acids ResHome page
S. Sharma, J. A. Sommers, R. K. Gary, E. Friedrich-Heineken, U. Hubscher, and R. M. Brosh Jr
The interaction site of Flap Endonuclease-1 with WRN helicase suggests a coordination of WRN and PCNA
Nucleic Acids Res., December 2, 2005; 33(21): 6769 - 6781.
[Abstract] [Full Text] [PDF]


Home page
Nucleic Acids ResHome page
S. Richards, S.-T. Liu, A. Majumdar, J.-L. Liu, R. S. Nairn, M. Bernier, V. Maher, and M. M. Seidman
Triplex targeted genomic crosslinks enter separable deletion and base substitution pathways
Nucleic Acids Res., September 25, 2005; 33(17): 5382 - 5393.
[Abstract] [Full Text] [PDF]


Home page
Hum Mol GenetHome page
S. Sharma, J. A. Sommers, and R. M. Brosh Jr
In vivo function of the conserved non-catalytic domain of Werner syndrome helicase in DNA replication
Hum. Mol. Genet., October 1, 2004; 13(19): 2247 - 2261.
[Abstract] [Full Text] [PDF]


Home page
J. Biol. Chem.Home page
S. Choudhary, J. A. Sommers, and R. M. Brosh Jr.
Biochemical and Kinetic Characterization of the DNA Helicase and Exonuclease Activities of Werner Syndrome Protein
J. Biol. Chem., August 13, 2004; 279(33): 34603 - 34613.
[Abstract] [Full Text] [PDF]


Home page
DevelopmentHome page
S.-J. Lee, J.-S. Yook, S. M. Han, and H.-S. Koo
A Werner syndrome protein homolog affects C. elegans development, growth rate, life span and sensitivity to DNA damage by acting at a DNA damage checkpoint
Development, June 1, 2004; 131(11): 2565 - 2575.
[Abstract] [Full Text] [PDF]


Home page
J. Biol. Chem.Home page
W.-H. Cheng, C. von Kobbe, P. L. Opresko, L. M. Arthur, K. Komatsu, M. M. Seidman, J. P. Carney, and V. A. Bohr
Linkage between Werner Syndrome Protein and the Mre11 Complex via Nbs1
J. Biol. Chem., May 14, 2004; 279(20): 21169 - 21176.
[Abstract] [Full Text] [PDF]


Home page
Sci Aging Knowl EnvironHome page
R. J. Monnat Jr. and Y. Saintigny
Werner Syndrome Protein--Unwinding Function to Explain Disease
Sci. Aging Knowl. Environ., March 31, 2004; 2004(13): re3 - re3.
[Abstract] [Full Text] [PDF]


Home page
Mol. Cell. Biol.Home page
W.-H. Cheng, C. von Kobbe, P. L. Opresko, K. M. Fields, J. Ren, D. Kufe, and V. A. Bohr
Werner Syndrome Protein Phosphorylation by Abl Tyrosine Kinase Regulates Its Activity and Distribution
Mol. Cell. Biol., September 15, 2003; 23(18): 6385 - 6395.
[Abstract] [Full Text] [PDF]


Home page
Sci Aging Knowl EnvironHome page
W.-H. Cheng and V. A. Bohr
Diverse Dealings of the Werner Helicase/Nuclease
Sci. Aging Knowl. Environ., August 6, 2003; 2003(31): pe22 - 22.
[Abstract] [Full Text]


Home page
J. Biol. Chem.Home page
S. Sharma, J. A. Sommers, H. C. Driscoll, L. Uzdilla, T. M. Wilson, and R. M. Brosh Jr.
The Exonucleolytic and Endonucleolytic Cleavage Activities of Human Exonuclease 1 Are Stimulated by an Interaction with the Carboxyl-terminal Region of the Werner Syndrome Protein
J. Biol. Chem., June 20, 2003; 278(26): 23487 - 23496.
[Abstract] [Full Text] [PDF]


Home page
CarcinogenesisHome page
P. L. Opresko, W.-H. Cheng, C. von Kobbe, J. A. Harrigan, and V. A. Bohr
Werner syndrome and the function of the Werner protein; what they can teach us about the molecular aging process.
Carcinogenesis, May 1, 2003; 24(5): 791 - 802.
[Abstract] [Full Text] [PDF]


Home page
Mol. Cell. Biol.Home page
Y. Saintigny, K. Makienko, C. Swanson, M. J. Emond, and R. J. Monnat Jr.
Homologous Recombination Resolution Defect in Werner Syndrome
Mol. Cell. Biol., October 15, 2002; 22(20): 6971 - 6978.
[Abstract] [Full Text] [PDF]


Home page
Hum Mol GenetHome page
A. Franchitto and P. Pichierri
Protecting genomic integrity during DNA replication: correlation between Werner's and Bloom's syndrome gene products and the MRE11 complex
Hum. Mol. Genet., October 1, 2002; 11(20): 2447 - 2453.
[Abstract] [Full Text] [PDF]


Home page
Nucleic Acids ResHome page
P. Karmakar, C. M. Snowden, D. A. Ramsden, and V. A. Bohr
Ku heterodimer binds to both ends of the Werner protein and functional interaction occurs at the Werner N-terminus
Nucleic Acids Res., August 15, 2002; 30(16): 3583 - 3591.
[Abstract] [Full Text] [PDF]


Home page
J. Biol. Chem.Home page
R. M. Brosh Jr., J. Waheed, and J. A. Sommers
Biochemical Characterization of the DNA Substrate Specificity of Werner Syndrome Helicase
J. Biol. Chem., June 21, 2002; 277(26): 23236 - 23245.
[Abstract] [Full Text] [PDF]


Home page
J. Biol. Chem.Home page
P. Karmakar, J. Piotrowski, R. M. Brosh Jr., J. A. Sommers, S. P. L. Miller, W.-H. Cheng, C. M. Snowden, D. A. Ramsden, and V. A. Bohr
Werner Protein Is a Target of DNA-dependent Protein Kinase in Vivo and in Vitro, and Its Catalytic Activities Are Regulated by Phosphorylation
J. Biol. Chem., May 17, 2002; 277(21): 18291 - 18302.
[Abstract] [Full Text] [PDF]


Home page
Sci Aging Knowl EnvironHome page
M. Fry
The Werner Syndrome Helicase-Nuclease--One Protein, Many Mysteries
Sci. Aging Knowl. Environ., April 3, 2002; 2002(13): re2 - 2.
[Abstract] [Full Text] [PDF]


Home page
Cancer Res.Home page
J. Oshima, S. Huang, C. Pae, J. Campisi, and R. H. Schiestl
Lack of WRN Results in Extensive Deletion at Nonhomologous Joining Ends
Cancer Res., January 1, 2002; 62(2): 547 - 551.
[Abstract] [Full Text] [PDF]


This Article
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
15/7/1224
00-0611fjev1    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by POOT, M.
Right arrow Articles by RABINOVITCH, P. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by POOT, M.
Right arrow Articles by RABINOVITCH, P. S.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS