**5. Ectopic expression of DDB2 induces apoptosis in DDB2-deficient cells**

An extensive review of XP-E and DDB has been presented by Itoh who focused on XP-E and DDB2 as well as the classification of photosensitive diseases [Itoh, 2006]. Surprisingly, XP-E cell strains proved to be abnormally resistant to UV irradiation and possessed reduced caspase-3 activity. Since the apoptotic defect in XP-E strains could be rescued by exogenous p53 expression, DDB2 was also proposed to regulate p53-mediated apoptotic pathway after UV irradiation in human primary cell strains [Itoh et al, 2000; 2003]. Cells from DDB2 knockout mice also showed abnormal resistance and impaired p53 response to UV irradiation similar to human XP-E cell strains [Itoh et al., 2004]. Furthermore, a recent study has demonstrated that mouse embryonic fibroblasts and human HeLa that express DDB2 shRNA are resistant to apoptosis induced by a variety of DNA-damaging agents despite the activation of p53 and other pro-apoptotic genes [Stoyanova et al., 2009]. Also, these DDB2 deficient cells are resistant to E2F1-induced apoptosis, probably due to the observation that these cells undergo p21Waf1/Cip1-associated cell cycle arrest following DNA damage. Notably, DDB2 targets p21Waf1/Cip1 for proteolysis and this process involves Mdm2 in a manner that is distinct from the p53-regulatory activity of Mdm2 [Stoyanova et al., 2009]. These results suggest a new regulatory loop involving DDB2, Mdm2, and p21Waf1/Cip1 that is critical in determining the cellular fate between apoptosis and cell cycle arrest (for DNA repair) in response to DNA damage. The existence of this regulatory loop may be strengthened by showing that forced expression of DDB2 renders XP-E or DDB2-deficient cells sensitive to apoptotic stimuli.

#### **6. Cancer-prone DDB2-deficient mice**

DDB2-knockout mice have been shown to be prone to cancer formation [Itoh et al., 2004]. Importantly, mice with single DDB2 allele knockout showed enhanced skin cancer following UV-B exposure, suggesting that DDB2 heterozygotes may be predisposed to skin cancer [Itoh et al., 2004]. In addition, XP mouse models were reported to be prone to the formation of papillomas induced by 7,12-dimethylbenz[a]anthracene (DMBA) [de Bohr et al., 1999; Nakane et al., 1995; de Vries et al., 1995], a carcinogen that produces bulky DNA adducts usually repaired by the NER system. On the other hand, p53-knockout mice are prone to spontaneous tumors [Donehower et al., 1992; Jacks et al., 1994], but not to tumors induced by DMBA or 12-O-tetradecanoyl-phorbol-13-acetate (TPA) [Kemp et al.,1993]. Taken together, these observations suggest that DDB2 may be involved in cancer formation through p53-mediated pathways. However, it is unclear whether re-introducing DDB2 in DDB2-knockout mice may prevent cancer formation.

#### **7. Concluding remarks and future perspectives**

The various results cited above suggest that the genetic integrity or gene expression status of the cells may be critical in determining the regulatory effects of DDB2 in response to apoptotic stimuli. The level of DDB2, p53, E2F1, and other proteins such as anti-apoptotic cFLIP and cell-cycle arrest p21, for instance, should be considered. The pro-apoptotic

protect cells against UV-induced apoptosis, at least in HeLa cells, we could not exclude the possibility that other genes are also involved in mediating the anti-apoptotic effect of DDB2.

An extensive review of XP-E and DDB has been presented by Itoh who focused on XP-E and DDB2 as well as the classification of photosensitive diseases [Itoh, 2006]. Surprisingly, XP-E cell strains proved to be abnormally resistant to UV irradiation and possessed reduced caspase-3 activity. Since the apoptotic defect in XP-E strains could be rescued by exogenous p53 expression, DDB2 was also proposed to regulate p53-mediated apoptotic pathway after UV irradiation in human primary cell strains [Itoh et al, 2000; 2003]. Cells from DDB2 knockout mice also showed abnormal resistance and impaired p53 response to UV irradiation similar to human XP-E cell strains [Itoh et al., 2004]. Furthermore, a recent study has demonstrated that mouse embryonic fibroblasts and human HeLa that express DDB2 shRNA are resistant to apoptosis induced by a variety of DNA-damaging agents despite the activation of p53 and other pro-apoptotic genes [Stoyanova et al., 2009]. Also, these DDB2 deficient cells are resistant to E2F1-induced apoptosis, probably due to the observation that these cells undergo p21Waf1/Cip1-associated cell cycle arrest following DNA damage. Notably, DDB2 targets p21Waf1/Cip1 for proteolysis and this process involves Mdm2 in a manner that is distinct from the p53-regulatory activity of Mdm2 [Stoyanova et al., 2009]. These results suggest a new regulatory loop involving DDB2, Mdm2, and p21Waf1/Cip1 that is critical in determining the cellular fate between apoptosis and cell cycle arrest (for DNA repair) in response to DNA damage. The existence of this regulatory loop may be strengthened by showing that forced expression of DDB2 renders XP-E or DDB2-deficient

DDB2-knockout mice have been shown to be prone to cancer formation [Itoh et al., 2004]. Importantly, mice with single DDB2 allele knockout showed enhanced skin cancer following UV-B exposure, suggesting that DDB2 heterozygotes may be predisposed to skin cancer [Itoh et al., 2004]. In addition, XP mouse models were reported to be prone to the formation of papillomas induced by 7,12-dimethylbenz[a]anthracene (DMBA) [de Bohr et al., 1999; Nakane et al., 1995; de Vries et al., 1995], a carcinogen that produces bulky DNA adducts usually repaired by the NER system. On the other hand, p53-knockout mice are prone to spontaneous tumors [Donehower et al., 1992; Jacks et al., 1994], but not to tumors induced by DMBA or 12-O-tetradecanoyl-phorbol-13-acetate (TPA) [Kemp et al.,1993]. Taken together, these observations suggest that DDB2 may be involved in cancer formation through p53-mediated pathways. However, it is unclear whether re-introducing DDB2 in

The various results cited above suggest that the genetic integrity or gene expression status of the cells may be critical in determining the regulatory effects of DDB2 in response to apoptotic stimuli. The level of DDB2, p53, E2F1, and other proteins such as anti-apoptotic cFLIP and cell-cycle arrest p21, for instance, should be considered. The pro-apoptotic

**5. Ectopic expression of DDB2 induces apoptosis in DDB2-deficient cells** 

cells sensitive to apoptotic stimuli.

**6. Cancer-prone DDB2-deficient mice** 

DDB2-knockout mice may prevent cancer formation.

**7. Concluding remarks and future perspectives** 

activity of p53 could vary between primary and cultured cell lines. For example, p53 activity in HeLa cells is hijacked by the human papillomavirus (HPV) E6 protein, a process that weakens apoptotic signaling in these cells. High levels of DDB2 may up-regulate and potentiate p53 activity by up-regulating apoptotic proteins in p53-normal cells. As such, HeLa cells, which harbor nearly null-p53 activity and additional anti-apoptotic cFLIP activity elicited by DDB2, may become resistant to apoptosis in response to cytotoxic DNA damage. These cellular responses are not surprising if the cultured cell lines were transformed by viruses or chemical means. Unfortunately, the cell lines used for the studies mentioned above are often treated this way. Furthermore, the expression of DDB2 isoforms, including the inhibitory D1 isoform, is often overlooked and the differential expression of such isoforms may dictate the cellular responses observed. Accordingly, alternative splicing of DDB2 transcripts and alteration of these genetic factors by other means in cell lines must be considered while evaluating the role of DDB2 in regulating apoptosis. In fact, there is no evidence so far that the apoptotic resistance of DDB2-defective XP-E, DDB2-knockout mouse cells, or DDB2-deficient human cells could be rescued by re-introducing DDB2 expression. In this sense, DDB2 is required to suppress apoptosis, but it does not suffice to be apoptotic. Furthermore, DDB2 as a proteasome component can target various proteins, such as p21 which is involved in cell cycle arrest, subsequently dysregulating cell cycle arrest during stress repair and leading to apoptosis. The cisplatin-selected HeLa cells used in our study do not display G1 arrest following mild, repairable DNA damage [Lin-Chao & Chao, 1994], which may explain the negligible, pro-apoptotic influence of DDB2 found by others [Stoyanova et al., 2009]. Therefore, an updated model is proposed in Figure 3, in

Fig. 3. Updated model for the regulation of DNA damage-induced apoptosis by DDB2. In this model, DNA damage applied to cells was mild and reached repairable level, leading to inhibition of apoptosis and cell cycle arrest for stress repair. The regulatory effect of DDB2 can be pro-apoptotic in cells experiencing mild DNA damage through p21 degradation which is targeted by DDB2. On the other hand, DDB2 can also be anti-apoptotic in cells harboring non-DNA damage apoptotic stimuli (e.g., death receptor) with up-regulation of anti-apoptotic cFLIP. Accordingly, the final outcome may be influenced by intrinsic mutations or extrinsic viral hijacking that can impair checkpoint for G1 arrest via p53 and p21.

The Role of DDB2 in Regulating Cell Survival

protein. *J. Biol. Chem.*, 274, 20027-20033

checkpoint. *Nat. Cell Biol.,* 5, 1008-1015

*Proc. Natl. Acad. Sci. U.S.A.,* 96, 424-428

them. *J. Invest. Dermatol.,* 114,1022-1029

carcinogen. *Proc. Natl. Acad. Sci. USA,* 101, 2052-2057

278, 46906-46910

5014-5022

1009

and Apoptosis Following DNA Damage - A Mini-Review 53

Fitch, M.E., Nakajima, S., Yasui, A., Ford, J.M. (2003). In vivo recruitment of XPC to UV-

Friedberg, E.C., Walker, G.C., Siede, W., Wood, R.D., Schultz, R.A., Ellenberger, T. (2006). *DNA repair and mutagenesis*. Second Edition, ASM Press, Washington, DC Fujiwara, Y., Masutani, C., Mizukoshi, T., Kondo, J., Hanaoka, F., Iwai, S. (1999).

Gonzalez, V.M., Fuertes, M.A., Alonso, C., Perez, J.M. (2001). Is cisplatin-induced cell death

Groisman, R., Polanowska, J., Kuraoka, I., Sawada, J., Saijo, M., Drapkin, R., Kisselev, A.F.,

Guerrero-Santoro, J., Kapetanaki, M.G., Hsieh, C.L., Gorbachinsky, I., Levine, A.S., Rapic-

Hayes, S., Shiyanov, P., Chen, X., Raychaudhuri, P. (1998). DDB, a putative DNA repair

Higa, L.A., Mihaylov, I.S., Banks, D.P., Zheng, J., Zhang, H. (2003). Radiation-mediated

Hu, J., McCall, C.M., Ohta, T., Xiong, Y. (2004). Targeted ubiquitination of CDT1 by the

Hwang, B.J., Ford, J.M., Hanawalt, P.C., Chu, G. (1999). Expression of the p48 xeroderma

Inoki, T., Yamagami, S., Inoki, Y., Tsuru, T., Hamamoto, T., Kagawa, Y., Mori, T., Endo, H.

Itoh, T., O'Shea, C., Linn, S. (2003). Impaired regulation of tumor suppressor p53 caused by

Itoh, T. (2006). Xeroderma pigmentosum group E and DDB2, a smaller subunit of damage-

Jacks, T., Remington, L., Williams, B.O., Schmitt, E.M., Halachmi, S., Bronson, R.T.,

damaged DNA repair. *Biochem. Biophys. Res. Commun.,* 314, 1036-1043 Itoh, T., Linn, S., Ono, T., Yamaizumi, M. (2000). Reinvestigation of the classification of five

interactions between p48 DDB2 and p53. *Mol. Cell. Biol.,* 23, 7540-7553 Itoh, T., Cado, D., Kamide, Y., Linn, S. (2004). DDB2 disruption leads to skin tumors and

always produced by apoptosis? *Mol. Pharmacol*., 59, 657–663

COP9 signalosome in response to DNA damage. *Cell*, 113, 357-367

induced cyclobutane pyrimidine dimers by the DDB2 gene product. *J. Biol. Chem.,*

Characterization of DNA recognition by the human UV-damaged DNA-binding

Tanaka, K., Nakatani, Y. (2003). CSA complexes is differentially regulated by the

Otrin, V. (2008). The cullin 4B-based UV-damaged DNA-binding protein ligase binds to UV-damaged chromatin and ubiquitinates histone H2A. *Cancer Res.,* 68,

protein, can function as a transcriptional partner of E2F1. Mol. Cell Biol.,18, 240–249

proteolysis of CDT1 by CUL4-ROC1 and CSN complexes constitutes a new

DDB1-CUL4A-ROC1 ligase in response to DNA damage. *Nat. Cell Biol.,* 6, 1003–

pigmentosum gene is p53-dependent and is involved in global genomic repair.

(2004). Human DDB2 splicing variants are dominant negative inhibitors of UV-

cell strains of xeroderma pigmentosum group E with reclassification of three of

mutations in the xeroderma pigmentosum DDB2 gene: mutual regulatory

resistance to apoptosis after exposure to ultraviolet light but not a chemical

specific DNA binding protein: Proposed classification of xeroderma pigmentosum, Cockayne syndrome, and ultraviolet-sensitive syndrome. .*J Dermatol. Sc.i,* 41, 87-96

Weinberg, R.A. (1994).Tumor spectrum analysis in p53-mutant mice. *Curr Biol*, 4,1-7

which the regulatory effect of DDB2 can be either pro-apoptotic in cells that respond to mild DNA damage or anti-apoptotic in cells that respond to non-DNA damage apoptotic stimuli and that show up-regulation of the anti-apototic cFLIP. Notably, we found that human DDB2 may play a protective role against UV irradiation in the fruit fly *Drosophila* which does not express DDB2 as seen in the DDB2-defective cultured cell models. Therefore, the seemingly contrasting results mentioned above may be explained by our models, and primary cell cultures which are more representative of in vivo situations may represent a better choice for future studies of the biological functions of DDB2.
