Camptothecin (CPT) class of compounds has been demonstrated to be effective against a broad spectrum of tumors. Their molecular target has been firmly established to be human DNA topoisomerase I (topo I). CPT inhibits topo I by blocking the rejoining step of the cleavage/religation reaction of topo‐I, resulting in accumulation of a covalent reaction intermediate, the cleavable complex. The primary mechanism of cell killing by CPT is S‐phase‐specific killing through potentially lethal collisions between advancing replication forks and topo‐I cleavable complexes. Collisions with the transcription machinery have also been shown to trigger the formation of long‐lived covalent topo‐I DNA complexes, which contribute to CPT cytotoxicity. Two novel repair responses to topo‐I‐mediated DNA damage involving covalent modifications of topo‐I have been discovered. The first involves activation of the ubiquitin/26S proteasome pathway, leading to degradation of topo‐I (CPT‐induced topo‐I downregulation). The second involves SUMO conjugation to topo‐I. The potential roles of these new mechanisms for repair of topo‐I‐mediated DNA damage in determining CPT sensitivity/resistance in tumor cells are discussed.

Ubiquitin͞26S proteasome-dependent degradation of topoisomerase I (TOP1) has been suggested to be a unique repair response to TOP1-mediated DNA damage. In the current study, we show that treatment of mammalian cells or yeast cells expressing human DNA TOP1 with camptothecin (CPT) induces covalent modification of the TOP1 by SUMO-1͞Smt3p, a ubiquitin-like protein. This conclusion is based on the following observations: (i) Mammalian DNA TOP1 conjugates induced by CPT were cross-reactive with SUMO-1͞Smt3p-specific antibodies both in yeast expressing human DNA TOP1 as well as mammalian cells. (ii) The formation of TOP1 conjugates was shown to be dependent on UBC9, the E2 enzyme for SUMO-1͞Smt3p. (iii) TOP1 physically interacts with UBC9. (iv) Ubc9 mutant yeast cells expressing human DNA TOP1 was hypersensitive to CPT, suggesting that UBC9͞SUMO-1 may be involved in the repair of TOP1-mediated DNA damage.UBC9 ͉ Smt3p ͉ ubiquitin T opoisomerase-mediated DNA damage represents an unique type of DNA damage whose importance has become increasingly appreciated. Many antibiotics, anticancer drugs, toxins, carcinogens, and physiological stresses are known to abort the catalytic cycles of topoisomerases resulting in the formation of topoisomerase-mediated DNA damage (1-7). Despite its importance, little is known about the molecular basis for the repair of this unique type of DNA damage.Recent studies have demonstrated that topoisomerase I-(TOP1) mediated DNA damage activates a ubiquitin͞proteasome pathway, resulting in degradation of TOP1 (down-regulation of TOP1) (8). It has been speculated that this pathway may represent a unique repair mechanism for TOP1-mediated DNA damage because down-regulation of human TOP1 (hTOP1) is expected to result in resistance͞tolerance to TOP1 poisons (8).Human SUMO-1 (small ubiquitin-like modifier) is an 11-kDa protein that shares 18% sequence homology with ubiquitin (9-11). Smt3p, which is 48% identical to human SUMO-1, has been identified in yeast (12,13). SUMO-1͞Smt3p is activated by a heterodimeric E1 enzyme in both the yeast and mammalian systems (13)(14)(15). In addition, UBC9, an E2 enzyme, has been shown to specifically conjugate SUMO-1͞Smt3p to the target proteins (11,(16)(17)(18). Recently, an enzyme Ulp1p, which specifically cleaves proteins from SUMO-1͞Smt3p conjugates and is distinct from isopeptidases for ubiquitin conjugates, also has been identified in yeast (19). These results suggest that the SUMO-1͞Smt3p pathway is very similar to the ubiquitin pathway but distinct E1 and E2 enzymes as well as proteases are involved in these pathways. The biological function of the SUMO-1͞Smt3p pathway appears diverse. UBC9 was originally identified to play an essential function in G 2 -M cell cycle progression in yeast (20). However, UBC9 is also known to be important for DNA repair (21). It has been shown that SUMO-1 can be conjugated to a number of proteins such as PML (22), RanGAP1 (23, 24), I B␣ (25), FAS͞apolipoprotein-1 (26), p53 (27, 28), RAD51, and RAD52 (29). A growing number of pr...

SUMMARYA set of predictive rules governing the likelihood of generating a particular peptide±major histocompatibility complex (MHC) class II complex from an intact antigen has not been fully elucidated. We investigated the in¯uence of positional and structural constraints in the region of the epitope by designing a set of recombinant antigens that each contained the well-characterized T-cell epitope moth cytochrome c (MCC) (88±103), which is speci®cally recognized by the monoclonal antibody (mAb) D4 when complexed with H-2E k . Our model antigens contained MCC(88±103) either peripherally, at or towards the C-terminus, or internally. Their abilities to bind directly to soluble H-2E k , and the extent of D4 epitope formation from them by antigen processing-competent and -incompetent cell lines, were determined. Here we report that three of these four antigens yielded MCC(88±103)/H-2E k complexes independently of the conventional MHC class II antigenprocessing and presentation pathway, and in each case the epitope was carried peripherally; two bound directly as intact proteins, probably as a result of spatial separation of the epitope from the major globular domain, and one was processed to peptide by a cell-surface protease. One protein, which carried the epitope inserted into an internal loop, acted as a conventional processingdependent MCC(88±103) delivery vehicle. Thus, this epitope has different presentation requirements depending on its context. These antigens constitute a panel whose framework could be modi®ed to further de®ne predictive rules for antigen processing for presentation through the different MHC class II complex-generating pathways.

In our earlier studies we observed that cells treated with the anticancer drug camptothecin (CPT), topoisomerase I (topo I), is rapidly multi-ubiquitinated and destroyed by 26S proteasome. 1 Many tumor cells are found to be defective in this process. Recently, we have also reported that topoisomerase I is conjugated to another ubiquitin-like protein, SUMO-1 (small ubiquitin modifiers), in response to CPT treatment. 2 Here we report upregulation of yet another ubiquitin-like protein in many tumor cells. This protein is identified as the interferon-inducible protein called ISG15 (interferon-stimulatory gene product 15) or UCRP (ubiquitin cross-reactive protein) using antiserum specific to UCRP in Western blot analysis. Interestingly, UCRP was greatly elevated in the cell line transformed with SV40 T-antigen (2RA), but not in its untransformed parental cell line, WI38, suggesting that accumulation of UCRP might be related to oncogenic transformation. We also observed a correlation between CPT hypersensitivity and UCRP upregulation in a panel of colorectal and breast cancer cell lines. By contrast, SUMOylation of topo I is not significantly different in these cancer cell lines.UCRP is a 15-kD protein and is composed of two domains, each of which bears striking homology to ubiquitin. 3 These two domains are linked to each other with insertion of an extra proline residue at the junction. 3 It was found to be induced upon interferon treatment. 3 Induction of this protein is correlated with the appearance of resistance to viral infections. 3 Interestingly, UCRP is constitutively elevated in humans with the inherited disease ataxia telangiectasia as a result of constitutive activation of NFκB. 4 SUMO-1 is also a 15-kD protein and has an 18% sequence similarity to ubiquitin. 5 Both SUMO-1 and UCRP are conjugated to target substrates in a way similar but not identical to that of ubiquitin. 3,5 It is well demonstrated that ubiquitin-conjugated substrates are targeted for destruction by 26S proteasome, 5 but the function of SUMO-1 and UCRP conjugation to the target protein has not yet been understood. It has been proposed that SUMO-1 conjugation is required for cellular trafficking or for antagonizing ubiquitination in order to prevent its target destruction. 6 Whether SUMO-1 conjugation to topo I upon CPT treatment is to facilitate or to prevent its

Camptothecin (CPT) induces topoisomerase I (topo I)-mediated DNA damage by specifically blocking the religation step in the topo 1-catalyzed breakage/religation cycles on DNA. 1 Unlike other DNA-damaging agents, CPT causes a highly specific type of DNA damage; the protein-linked single-strand DNA breaks. Little is known about the repair mechanism of topo I-mediated DNA damage.Compact chromatin is a structural barrier to DNA processes such as DNA replication, RNA transcription, and DNA repair. It is assumed that in order to repair DNA damage, chromatin must undergo some changes for the access of repair proteins to the DNA-damaged site. 2 To monitor the state of chromatin upon CPT treatment, we measured the linking number of SV40 episomal DNA in COS cells. Topo I-mediated DNA damage induced by CPT resulted in a rapid and extensive increase in the linking number of SV40 episomal DNA in COS cells. 3 The linking number increase was shown to be due to topo I-mediated DNA damage induced by CPT, because the inactive S-isomer of CPT, which does not induce topo I-mediated DNA damage, was unable to affect the linking number. The CPT-induced change in linking number was reversible, as removal of CPT from the culture media resulted in a temperature-and time-dependent reversal of the linking number change. 3 CPT-induced linking number increase is probably independent of DNA replication, RNA transcription, and poly(ADP)ribosylation, because aphidicolin (an inhibitor of DNA polymerase α and δ), dichloro-D-ribofuranosylbenzimidazole (DRB) (an RNA polymerase II inhibitor), and 3-aminobenzamide (3-AB) (an inhibitor of poly(ADP)ribose polymerase) do not affect CPT-induced relaxation.To further study the mechanism of CPT-induced relaxation, we took advantage of yeast genetic system. CPT can similarly induce a large linking number increase in yeast expressing human topoisomerase I (htopoI). Within minutes of CPT treatment, an increase of over 10 linking number was observed on yeast 2µ plasmid DNA. Bleomycin but not etoposide, MMS and cisplatin can similarly induce linking number increase on yeast 2µ plasmid in yeast expressing human DNA topo I. Various yeast mutants have been screened to identify the genes affecting CPT-induced plasmid ree