Background: Ultraviolet radiation (UV) damaged-DNA binding (DDB) activity comprises two major components: damaged-DNA binding protein 1 (DDB1) and 2 (DDB2). Although the function of DDB is unclear, mutation on DDB2 is associated with cellular sensitivity to a variety of genotoxic agents including UV. It has been suggested that DDB2 may play a role in UV-induced DNA repair. However, evidence that DDB2 involves in DNA repair and UV sensitivity is lacking.
Methods: To examine the role of DDB2, we established DDB2-overexpressing hamster V79 cell lines, V79ddb2, by stable transfection with full-length open reading frame of human ddb2 cDNA. Cells were irradiated with UV and determined its DNA repair activity by testing the remaining photoproducts on the chromatin and measuring the plasmid reactivation, respectively. UV induced cytotoxicity was determined by the colorimetric assay (MTT assay), and apoptotic cells exhibiting morphological features of chromatin condensation and nuclear fragmentation were counted after 4-diamidino-2-phenylindole (DAPI) staining.
Results: DDB activity was increased in DDB2-overexpressing cell lines. Analysis on DNA repair indicated that UV photoproducts were removed in a time-dependent manner and there was greater than 50% of damage removed within 12 h in DDB2-overexpressing cells. In contrast, nearly all the damage remained unrepaired in V79 cells. However, using bacterial CAT gene as a reporter, both V79 and V79ddb2 cells demonstrated no difference in the reactivation of plasmid DNA carrying UV damage. These results suggest that DDB2 may involve in repair of bulky genomic DNA damage. Although a maximum of only 30% of apoptosis was induced, UV irradiation caused a dose-dependent apoptosis and cytotoxicity in these cell lines. V79ddb2 cells displayed resistance to UV-induced apoptosis and cytotoxicity.
Conclusion: Our findings indicate that overexpression of DDB2 in V79 cell potentiates DNA repair and protects cells from UV-induced cytotoxicity. These results also suggest that DDB2 may be involved in the development of UV resistance.
(Chang Gung Med J 2002;25:723-33)

Key words: apoptosis, DDB2, DNA repair, resistance, ultraviolet radiation.

The recognition and incision of damaged DNA are crucial in nucleotide excision repair and cell sensitivity to genotoxic agents including ultraviolet radiation (UV). Nucleotide excision repair is a versatile strategy for defending against genotoxic insults to chromosome DNA in organisms ranging from microplasma to mammals, and represents a major mechanism of DNA repair in mammalian cells. Cells from the heritable human disorder xeroderma pigmentosum (XP) complementation groups A through G have reduced nucleotide excision repair.(1) Defects in DNA repair of XP cells often occur in the recognition and incision of damaged DNA. Indeed, there is as yet only indirect evidence which suggests that damaged-DNA-binding (DDB) activity may be involved in DNA repair in mammalian cells.(2-5) UV-DDB activity has been purified to apparent homogeneity and characterized from human placenta and HeLa cells,(6-8) and is identical to the activity originally identified from human placenta.(9) In vitro reconstitution studies indicate that UV-DDB proteins, although not essential, can stimulate nucleotide excision repair.(10) UV-DDB activity appears to be enhanced in UV-resistant human cells(2-4,11) and to be lost or reduced in several XP-E cell lines.(5,12-14) Microinjection of purified UV-DDB into XP-E cells restores the repair defect.(15,16)
UV-DDB was originally identified as a complex with 2 subunits of ~127 kDa (i.e., DDB1) and ~48 kDa (i.e., DDB2).(7,17,18) DDB1 cDNA was isolated from both monkeys(19) and humans.(20,21) Although DDB1 was proposed to be involved in nucleotide excision repair,(22) several studies have shown that DDB1 is not essential for this step.(23) Human DDB2 cDNA was isolated,(20) and together with DDB1 is required for the recognition of UV damage.(24) Since a subset of XP-E cells, which lack DDB activity, retains mutations in DDB2,(18) DDB2 may contribute to nucleotide excision repair and cell sensitivity. UV irradiation has been shown to induce apoptosis, genetically programmed cell death, in a variety of cell types. Although UV-induced cytotoxicity and resistance have been extensively reported, the mechanism remains unclear. In the present study, we found that overexpression of DDB2 in cells can potentiate DNA repair and apoptotic resistance in response to UV.

METHODS

Reagents and cell lines
Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and penicillin/streptomycin were obtained from GIBCO (Gaithersburg, MD). [32P]dCTP (3000 Ci/mmol) was obtained from Amersham (Arlington Heights, IL). Cisplatin, vincristine, DAPI, G418, and other were purchased from Sigma (St. Louis, MO). Chinese hamster V79 cells were obtained originally from American Type Culture Collection.
To generate DDB2-overexpressing cell variants, V79 cells were transfected with pcDNA3ddb2, a pcDNA3 plasmid (Invitrogen, Carlsbad, CA) that contains a full-length ORF of human DDB2 cDNA and a neomycin-resistant gene. Potential cell candidates were selected after being cultured in media containing G418 (for selecting resistance to geneticin) for 2 weeks. The G418 concentration (400 mg/ml), which killed all cells without plasmid transfection, was empirically determined in the cell line.

DDB2 cDNA and antibody
An 1820-nucleotide segment of human DDB2 cDNA was isolated from a placenta cDNA library (Quick-Clone cDNA, Clontech) by polymerase chain reaction (PCR)(25) according to the human DDB2 cDNA sequence (GenBank database accession no. U18300).(20) PCR primer pairs were designed according to the reported DDB2 sequence to cover the full-length open reading frame. For convenience of plasmid construction, the recognition sequences of Sma I and Hind III were added to each primer at the 5' and 3' ends, respectively. The PCR products were cloned into a plasmid pGEM-T Easy vector (Promega), designated as pGTddb2. Plasmid DNA was sequenced by the dideoxynucleotide method(26) using a T7 or SP6 primer complementary to a vector region immediately outside the cDNA insert. To test for its continuity in the predicted ORF, proteins were made using the TNT reticulocyte lysate system (Promega) from pGTddb2 as described.(27)
To produce DDB2 antibodies, pGTddb2 was digested with the restriction enzymes Sma I and Hind III, and inserted in frame into pET15bDH, a modified form of pET15b (Invitrogen), to produce pETddb2. Proteins were made in bacteria and purified with a nickel column according the specifications of the supplier (Qiagen). Polyclonal DDB2 antibodies were generated in New Zealand rabbits according to the described methods.(28)

Cell extracts and immunoblot analysis
For whole-cell extracts, cells were washed with phosphate-buffered saline (PBS) twice and lysed in 1 ml of modified radio immunoprecipitation buffer (RIPA; 50 mM Tris HCl, pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM PMSF, 1 mg/ml each of aprotinin, leupeptin, and pepstatin, 1 mM Na3VO4, and 1 mM NaF) on ice for 30 min. The insoluble material was removed by centrifugation at 14,000 rpm for 10 min at 4 oC. The protein concentration was measured with the Bradford assay using the BioRad dye reagent,(29) and separated by SDS-polyacrylamide gel electrophoresis.(30) Equivalent amounts of protein from each sample were separated by SDS-polyacrylamide electrophoresis (PAGE), transferred onto PVDF membranes and incubated with antibodies. Antigen-antibody complexes were visualized by the standard enhanced chemiluminescence reaction according to the specifications of the supplier (Pierce, Rockford, IL).

Nuclear extracts and gel mobility shift assay
Crude nuclear extracts were prepared according to Dignam et al.(31) Protein-DNA binding was performed in 15 ml of buffer containing 12% glycerol, 12 mM Hepes (pH 7.9), 100 mM KCl, 5 mM MgCl2, 4 mM Tris HCl, 1 mM EDTA, 1 mM dithiothreitol, and 300 mg/ml BSA at 25 oC for 30 min.(32) DNA probes were prepared as described previously.(33) Hind III- and EcoR I-generated f103 fragments were [32P]dCTP-labeled (3×104 cpm/ng DNA) using Klenow DNA polymerase and purified using column chromatography by standard methods.(27) DNA at a concentration of 100 mg/ml was exposed to a fluence rate of 25 J/m2/s from a VL-100C UV irradiation unit (Vilbert Lourmat, France). UV exposures were measured with a VLX-254 radiometer (Vilbert Lourmat, France). The reaction mixtures were then subjected to 5% polyacrylamide gel electrophoresis under low ionic strength (6.7 mM Tris HCl, pH 7.9, 3.3 mM sodium acetate, 1 mM EDTA) at 30 oC and a constant current of 15 mA. The resolved gel was dried and exposed to Kodak XAR-5 X-ray film at -70 oC with an intensifying screen.

DNA repair assays
To measure DNA repair, cells were irradiated with 10 J/m2 of UV and incubated to allow repair of the photoproducts as described.(34) The relative number of UV-induced DNA photoproducts was determined by a radioimmunoassay as previously described.(35) Antiserum was raised in rabbits by injection with UV-irradiated, denatured calf thymus DNA coupled to methylated bovine serum albumin.(36) Two grams of heat-denatured sample was allowed to compete against 32P-labeled poly(dA): poly(dT) irradiated with 40 kJ/m2 UV for antibody-binding sites. Using a standard curve, inhibition in the repaired samples was converted to dose equivalents, and the removal of antibody-binding sites was determined.
To measure plasmid reactivation,(33) cells were seeded at 3×106 cells per 100-mm plate 1 day before electroporation. One milliliter of cell suspensions, in Hepes buffer, was added to a sterile cuvette containing 20 mg pRSVcat and 10 mg pSVb (Clontech) plasmids. This was gently mixed and subjected to electroporation(27) as described in the manufacturer's instructions (GenePulser, BioRad). The following day, fresh medium was provided to the cells, and they were incubated for another 48 h. Cells were then harvested for CAT assay as previously described.(37) The b-gal activity of the same preparation for CAT assay was also analyzed as an internal control.(27) After autoradiography, the density on the X-ray film corresponding to the modified chloramphenicol was quantitated with a scanning densitometer (Personal Densitometer SI, Molecular Dynamics). Being normalized to b-gal activity, the CAT activity was calculated as the percent of chloramphenicol converted into acetylated derivatives.

Analysis of cytotoxicity and apoptosis
Cytotoxicity was determined by MTT assay.(38) Cells were plated in 96-well dishes in a total volume of 100 ml. Cells were exposed to UV for 12 hours after plating, and the culture was incubated for 72 hours at 37 oC. The percentage of cells surviving UV was determined by the ability of the cells to convert the tetrazolium MTT salt into a formazan product solubilized in acid-propanol.
For analysis of apoptosis, cells growing in 6-well plates were exposed to UV for 24 hours at 37 oC. Cells were fixed with methanol and incubated with a DAPI (4-diamidino-2-phenylindole) solution for 30 min in the dark. Floating cells from each well were also fixed and then placed back into the respective wells and analyzed using a microscope at 420 nm. The apoptotic cells exhibiting morphological features of chromatin condensation and nuclear fragmentation(39) were counted in 6-8 randomly selected fields. Exactly 500 nuclei were examined from each sample, and the results were calculated as the number of apoptotic nuclei over the total number of nuclei counted from 3 independent experiments.

RESULTS

Overexpression of DDB2 potentiates UV-
damaged DNA binding
To examine the function of DDB2, DDB2-overexpressing cell lines were established from a Chinese hamster V79 cell line, which displays either none or only trace amounts of the protein. Two typical candidate clones, V79ddb2#1 and #2, were isolated for detection of the DDB2 protein (Fig. 1). Using an antibody for DDB2, both DDB2-overexpressing cell lines displayed several times the specific DDB2 proteins (around 47 kDa) compared to parental V79 cells. In contrast, no protein was detected for the preimmune serum on the same protein membrane blot. The same membrane blot, after being stained with Amido black, revealed equal amounts of proteins for each cell extract. The DDB activities of the selected DDB2-overexpressing cell lines were compared (Fig. 2A). On an electrophoretic mobility shift assay (EMSA), V79 nuclear extracts displayed no DDB activity, whereas both V79ddb2 cell lines displayed dose-dependent DDB activity (indicated by "bound"). However, the damage-binding activity was much higher in V79ddb2#2 than in V79ddb2#1. It should be noted that both lines overexpressed similar amounts of DDB2 (see Fig. 1). The data suggest that DDB activity is not solely determined by the concentration of DDB2 in these cells. To ensure that the DDB activity was specific to UV damage, competitive EMSA was carried out (Fig. 2B). An increasing molar excess of a specific competitor (f103/UV) inhibited the DDB activity. In contrast, there was no measurable inhibition of DDB activity by a 100-fold increase in untreated f103 or single-stranded f103 (ssf103). However, a majority of the DDB activity was inhibited by a 100-fold increase in UV-irradiated ssf103 (ssf103/UV). Hence, the DDB2-overexpressing cell lines displayed enhanced DDB activity preferential for UV damage.

Overexpression of DDB2 potentiates DNA repair
To examine the role of DDB2 in DNA repair, the DDB2-overexpressing cell lines were irradiated with UV and assayed for DNA repair. UV induces mainly cyclobutane pyrimidine dimers and (6-4) photoproducts on cellular DNA.(1) Using an antibody against UV-induced photoproducts, we measured the majority of DNA remaining in V79 cells (Fig. 3). In contrast, 50% or more of the photoproducts were removed within 12 hours in a time-dependent manner in the DDB2-overexpressing cell lines. Repair became saturated at 12 hours since no more repairs occurred with a longer incubation. There was essentially no detectable repair at 4 oC in these cells, suggesting that an enzymatic reaction may be required. These results indicate that overexpression of DDB2 potentiates the repair of UV damage in cells. Since V79 cells lack photoreactivating enzyme activity, which removes the major dimer photoproducts,(1) enhanced DNA repair detected in DDB2-overexpressing cells is likely preferential for cyclobutane pyrimidine dimers.

Overexpression of DDB2 did not potentiate plasmid reactivation
Plasmid reactivation using CAT enzymatic activity as a reporter can be used in certain conditions to quantify DNA repair.(37) Using this assay, the reporter plasmids carrying UV damage were transfected into V79 cells, and the relative CAT activity was measured (Fig. 4). There was dose-dependent inhibition of CAT activity in each cell line. By assuming that plasmid reactivation or CAT activity is expressed by intact DNA, DNA damage on plasmid DNA was apparently repaired in the cells. However, there was no difference between parental and DDB2-overexpressing cells. A typical CAT activity pattern is shown in the top panel of Fig. 4. These results indicate that overexpression of DDB2 did not potentiate plasmid reactivation in V79 cells.

Overexpression of DDB2 protects cells from UV-induced apoptosis
To examine the role of DDB2 in UV sensitivity, we further investigated the effects of DDB2 overexpression in UV-treated hamster V79 cells, which express no of only trace amounts of DDB2. Cytotoxicity measured by the MTT assay indicated dose dependence. The cytotoxicity was partially reduced in V79ddb2 cells (Fig. 5A). Apoptotic cells were induced, to a maximum of 30%, in a dose-dependent manner in both V79 and V79ddb2 cells. V79ddb2 cells displayed reduced UV-induced apoptosis compared to V79 cells (Fig. 5B). Overexpression of DDB2 inhibited UV-induced apoptosis by more than 50%. These results indicate that overexpressing DDB2 might protect cells from UV-induced apoptosis and cytotoxicity.

DISCUSSION

In the present study, we found that DDB2 is expressed only in trace amounts, if any, in hamster V79 cells, and that overexpression of DDB2 potentiates cell resistance to UV. Independently selected DDB2-overexpressing cell lines displayed increased, but different, levels of UV-DDB activity. In addition, V79 cells overexpressing DDB2 repaired UV damage more efficiently than did parental cells. Other investigators have also shown that damaged-DNA-binding activity is potentiated by transient transfection of DDB2 in V79 and human 293T cells, but not by mutated DDB2 from several XP-E cell lines.(24) The involvement of DDB2 in the response to UV is also supported by the findings that a subset of XP-E cells, although containing DDB1, did not exhibit damaged-DNA-binding activity due to a mutation in DDB2.(18) Further, overexpression of DDB1 by transient transfection in human 293T cells failed to induce damaged-DNA-binding activity.(24) In addition, DDB activity is associated with and probably responsible for enhanced DNA repair in a HeLa cell model.(3) It is likely that increased DDB activity in DDB2-overexpressing V79 cells may be attributed to enhanced DNA repair. However, different DDB2 activities occur in DDB2-overexpressing V79 cell lines, whereas the levels of DNA repair capacity were the same in these DDB2-overexpressing cell lines. The efficacy of DNA repair in this cell system, therefore, cannot be fully explained by enhanced DDB activity. In fact, 3 DDB proteins have now been implicated in nucleotide DNA repair: the XPA protein,(40) the XPC/HR23B heterodimer,(41) and UV-DDB. Although there may be cell-type dependence, UV-DDB is probably not the only factor affecting DNA repair in V79 cells. Since insertion of exogenous DNA into cellular chromosomes occurs randomly during establishment of DDB2-overexpressing cell lines, other genes involved in DDB activity may also be affected. Recently, DNA repair detected with damage-specific antibodies has been demonstrated, and the results revealed that DDB2 enhances global genomic repair of cyclobutane pyrimidine dimers, but not (6-4) photoproducts.(42) In addition, microinjection of purified DDBs into XP-E cells, but not in an in vitro system, restored repair synthesis. These results suggest that the DDB protein in its DDB1 or DDB1-DDB2 form may play a specific role in the repair of chromosomal DNA in the nuclear environment, which is not easily revealed in vitro.(15,16) Since V79 cells contain minimal photoreactivating enzyme activity,(43) which removes the major cyclobutane dimer photoproducts, enhanced DNA repair detected in DDB2-overexpressing cells is likely preferential for cyclobutane dimer photoproducts. Taken together, DDB2 is involved in cyclobutane dimer repair that may regulate cell responses to UV. Interestingly, overexpression of DDB2 in cells did not improve repair of naked DNA damage. DDB2 contains a WD motif with some identity to the WD motifs in a subfamily of WD repeat proteins involved in the reorganization of chromatin.(24) The results strongly suggest that the regulation of DNA repair, and maybe cytotoxicity, is affected by other chromosome-binding proteins. This may explain why DDB2 alone cannot potentiate DNA excision repair in vitro and naked DNA repair in cells.
In this study, we also found that overexpression of DDB2 protected cells from UV-induced cytotoxicity and apoptosis. This is expected because DNA repair is enhanced in DDB2-overexpressing cells. It has been well demonstrated that cells with improved DNA repair are often associated with resistance to DNA damage.(22,44,45) Our results suggest that apoptotic resistance may represent acquired UV resistance, and that apoptotic resistance may be due to overexpression of DDB2. Overexpression of DDB2 thereby establishes an opportunity for cells to develop resistance to UV. Indeed, resistant HeLa cells, which exhibit enhanced DNA repair,(37,46) became sensitive to UV-induced apoptosis by inhibiting DDB2 expression.(47) It is likely that the role of DDB2 in modulating UV-induced sensitivity in hamster cells may also occur in other mammalian cells. However, inhibition of DDB2 did not affect cisplatin- and mitomycin c-induced cytotoxicity or apoptosis (data not shown), suggesting that modulation of apoptosis by DDB2 may depend on the stimulus involved.
Activation of caspases and their regulators is required for apoptosis, and this plays a predominant role in cell sensitivity or resistance to a variety of DNA-damaging agents.(45,48) Using commercial antibodies, we had difficulty detecting apoptotic molecules in V79 cells. However, 80 J/m2 of UV induced a maximum of 30% apoptosis in V79 cells. In contrast, 40 J/m2 of UV induced more than 80% apoptosis in human cells.(39) Although the induction of apoptosis in V79 cells was less effective, the apoptotic phenotype (chromosome fragmentation and chromatin condensation) was the same as that in human cells. Incubation of V79 cells for longer than 24 hours to allow repair showed no greater elimination of DNA damage. In addition, components of apoptosis are conserved between Caenorhabditis elegans, Drosophila melanogaster, and humans.(19) There may be a different epitope on hamster apoptotic proteins. A recent report by other investigators revealed that overexpression of DDB2 by stable transfection in hamster V79 cells also potentiated damaged-DNA-binding activity.(42) However, they detected no protection against UV toxicity (by colony-forming assay) by DDB2 overexpression.(42) In contrast to that report, we found that overexpression of DDB2 protected V79 cells from apoptosis and thus enhanced cell survival. The contradiction is probably due to different cell clones and/or assays employed. Therefore, the association of DDB2 with DDB1 and DDB2's effect on DNA repair and apoptosis deserve further investigation.

Acknowledgments

This work was supported by an intramural fund from Chang Gung University (CMRP743 and CMRP1025) and a grant from the National Science Council of Taiwan, R.O.C. (NSC87-2314-B182-009 and NSC89-2316-B182-007). C.C.K.C. is a holder of the Yuan-Tche Lee Distinguished Chair from the Foundation for the Advancement of Outstanding Scholarship.

REFERENCES

1. Friedberg EC, Walker GC, Siede W. DNA Repair and Mutagenesis. ASM Press, Washington, D.C. 1995.
2. Chao CCK Damage-recognition proteins as a potential indicator of DNA-damage- mediated sensitivity or resistance of human cells to ultraviolet radiation. Biochem J 1992;282:203-7.
3. Chao CCK Huang SL, Huang HM, Lin-Chao S. Cross-resistance to UV radiation of a cisplatin-resistant human cell line: overexpression of cellular factors that recognize UV-modified DNA. Mol Cell Biol 1991a;11:2075-80.
4. Chu G, Chang E. Cisplatin-resistant cells express increased levels of a factor that recognizes damaged DNA. Proc Natl Acad Sci USA 1990;87:3324-8.
5. Hirschfeld S, Levine AS, Ozato K, Protic M. A constitutive damage-specific DNA-binding protein is synthesized at higher levels in UV-irradiated primate cells. Mol Cell Biol 1990;10:2041-8.
6. Hwang BJ, Chu G. Purification and characterization of a human protein that binds to damaged DNA. Biochemistry 1993;32:1657-66.
7. Keeney S, Chang GJ, Linn S. Characterization of a human DNA damage binding protein implicated in xeroderma pigmentosum E. J Biol Chem 1993;268:21293- 300.
8. van Assendelft GB, Rigney EM, Hickson ID. Purification of a HeLa cell nuclear protein that binds selectively to DNA irradiated with ultra-violet light. Nucleic Acids Res 1993;21:3399-404.
9. Feldberg RS, Grossman L. A DNA binding protein from human placenta specific for ultraviolet damaged DNA. Biochemistry 1976;15:2402-8.
10. Aboussekhra A, Biggerstaff M, Shivji MK, Vilpo JA, Moncollin V, Podust VN, Protic M, Hubscher U, Egly JM, Wood RD. Mammalian DNA nucleotide excision repair reconstituted with purified protein components. Cell 1995;80:859-68.
11. Chao CCK. Huang SL, Lee LY, Lin-Chao S. Identification of inducible damage- recognition proteins that are overexpressed in HeLa cells resistant to cis-diaminedichloroplatinum (II). Biochem J 1991b;277:875-8.
12. Chu G, Chang E. Xeroderma pigmentosum group E cells lack a nuclear factor that binds to damaged DNA. Science 1988;242:564-7.
13. Kataoka H, Fujiwara Y. UV damage-specific DNA-binding protein in xeroderma pigmentosum complementation group E. Biochem Biophys Res Comm 1991;175:1139-43.
14. Keeney S, Wein H, Linn S. Biochemical heterogeneity in xeroderma pigmentosum complementation group E. Mutat Res 1992;273:49-56.
15. Keeney S, Eker AP, Brody T, Vermeulen W, Bootsma D, Hoeijmakers JH, Linn S. Correction of the DNA repair defect in xeroderma pigmentosum group E by injection of a DNA damage-binding protein. Proc Natl Acad Sci USA 1994;91:4053-6.
16. Rapic Otrin V, Kuraoka I, Nardo T, McLenigan M , Eker AP, Stefanini M, Levine AS, Wood RD. Relationship of the xeroderma pigmentosum group E DNA repair defect to the chromatin and DNA binding proteins UV-DDB and replication protein A. Mol Cell Biol 1998;18:3182-90.
17. Abramic M, Levine AS, Protic M. Purification of an ultraviolet-inducible, damage-specific DNA-binding protein from primate cells. J Biol Chem 1991;266:22493-500.
18. Nichols AF, Ong P, Linn S. Mutations specific to the xeroderma pigmentosum group E Ddb-phenotype. J Biol Chem 1996;271:24317-20.
19. Takao M, Abramic M, Moos M Jr, Otrin VR, Wootton JC, McLenigan M, Levine AS, Protic M. A 127 kDa component of a UV-damaged DNA-binding complex, which is defective in some xeroderma pigmentosum group E patients, is homologous to a slime mold protein. Nucleic Acids Res 1993;21:4111-8.
20. Dualan R, Brody T, Keeney S, Nichols AF, Admon A, Linn S. Chromosomal localization and cDNA cloning of the genes (DDB1 and DDB2) for the p127 and p48 subunits of a human damage-specific DNA binding protein. Genomics 1995;29:62-9.
21. Hwang BJ, Liao JC, Chu G. Isolation of a cDNA encoding a UV-damaged DNA binding factor defective in xeroderma pigmentosum group E cells. Mutat Res 1996;362:105-17.
22. Chu G. Cellular responses to cisplatin. The roles of DNA-binding proteins and DNA repair. J Biol Chem 1994;269:787-90.
23. Sancar A. DNA excision repair. Ann Rev Biochem 1996;65:43-81
24. Hwang BJ, Toering S, Francke U, Chu G. p48 Activates a UV-damaged-DNA binding factor and is defective in xeroderma pigmentosum group E cells that lack binding activity. Mol Cell Biol 1998;18:4391-9.
25. Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA. Primer- directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 1998; 239:487-91.
26. Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 1977;74:5463-7.
27. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, 1989.
28. Harlow E, Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, 1988.
29. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248-54.
30. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680-5.
31. Dignam JD, Lebovitz RM, Roeder RG. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 1983;11:1475-89.
32. Hennighausen L, Lubon H. Interaction of protein with DNA in vitro. Methods Enzymol 1987;152:721-35.
33. Chao CCK. Lee YL, Lin-Chao S. Phenotypic reversion of cisplatin resistance in human cells accompanies reduced host cell reactivation of damaged plasmid. Biochem Biophys Res Comm 1990;170:851-9.
34. Chao CCK. Rosenstein BS. Induction of sister-chromatid exchanges in ICR 2A frog cells exposed to 254 nm and solar UV wavelengths. Photochem Photobiol 1985;41:625-7.
35. Mitchell DL, Clarkson JM, Chao CC, Rosenstein BS. Repair of cyclobutane dimers and (6-4) photoproducts in ICR 2A frog cells. Photochem Photobiol 1986;43:595-7.
36. Mitchell DL, Clarkson JM. The development of a radioimmunoassay for the detection of photoproducts in mammalian cell DNA. Biochem. Biophys Acta 1981;655:54-60.
37. Chao CCK. Lee YL, Cheng PW, Lin-Chao S. Enhanced host cell reactivation of damaged plasmid DNA in HeLa cells resistant to cis-diamminedichloroplatinum(II). Cancer Res 1991c;51:601-5.
38. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983;89:55-63.
39. Kamarajan P, Chao CCK. UV-induced apoptosis in resistant HeLa cells. Bioscience Reports 2000;20:99-108.
40. Jones CJ, Wood RD. Preferential binding of the xeroderma pigmentosum group A complementing protein to damaged DNA. Biochemistry 1993;32:12096-104.
41. Sugasawa K, Ng JM, Masutani C, Iwai S, van der Spek PJ, Eker AP, Hanaoka F, Bootsma D, Hoeijmakers JH. Xeroderma pigmentosum group C protein complex is the initiator of global genome nucleotide excision repair. Mol Cell 1998;2:223-32.
42. Tang JY, Hwang BJ, Ford JM, Hanawalt PC, Chu G. Xeroderma pigmentosum p48 gene enhances global genomic repair and suppresses UV-induced mutagenesis. Mol Cell 2000;5:737-44.
43. Sutherland BM, Runge P, Sutherland JC. DNA photoreactivating enzyme from placental mammals. Origin and characteristics. Biochemistry 1974;13:4710-5.
44. Chao CCK. Molecular basis of cis-diamminedichloroplatinum(II) resistance: a review. J Formos Med Assoc 1996;95:893-900.
45. Zunino F, Perego P, Pilotti S, Pratesi G, Supino R, Arcamone F. Role of apoptotic response in cellular resistance to cytotoxic agents. Pharmacol Ther 1997;76:177-85.
46. Chao CCK. Huang SL. Inhibition of UV-damaged DNA recognition activity in HeLa cells by calcium ionophore A23187: potential involvement of cytosolic proteins. Biochem Biophys Res Comm 1993;193:764-70.
47. Sun NK, Kamarajan P, Huang H, Chao CCK. Restoration of UV sensitivity in UV-resistant HeLa cells by antisense-mediated depletion of damaged DNA-binding protein 2 (DDB2). FEBS Lett 2002;512:168-72.
48. Houghton JA. Apoptosis and drug response. Curr Opin Oncol 1999;11:475-81.

From the Tumor Biology Laboratory, Department of Biochemistry, Chang Gung University, Taoyuan.
Received: Jul. 1, 2002; Accepted: Aug. 13, 2002
Address for reprints: Chuck C.-K. Chao, PhD, Tumor Biology Laboratory, Department of Biochemistry, Chang Gung University. 259,Wen-Hwa 1st Road, Kweishan 333, Taoyuan, Taiwan, R.O.C. Tel.: 886-3-3283016 ext. 5157; Fax: 886-3-3283031; E-mail: cckchao@mail.cgu.edu.tw