- Top of page
- Materials and Methods
When a replicative DNA polymerase encounters a lesion on the template strand and stalls, it is replaced with another polymerase(s) with low processivity that bypasses the lesion to continue DNA synthesis. This phenomenon is known as translesion replication or replicative bypass. Failing this, the cell is increasingly likely to undergo apoptosis. In this study, we found that proteasome inhibitors prevent translesion replication in human cancer cells but not in normal cells. Three proteasome inhibitors, MG-132, lactacystin, and MG-262, inhibited UV-induced translesion replication in a wide range of cancer cell lines, including HeLa, HGC-27, MCF-7, HepG2, WiDr, a malignant melanoma, an acute lymphoblastic leukemia, and a multiple myeloma cell line; irrespective of cell origin, histological type, or p53 status. In contrast, these inhibitors had little or no influence on normal fibroblasts (NB1RGB and TIG-1) or a normal liver mesenchymal (LI90) cell line. Among the DNA-damaging antineoplastic agents, cisplatin caused a UV-type translesion reaction; the proteasome inhibitors delayed cisplatin-induced translesion replication in cancer cell lines but had only a weak effect on normal cell lines. Therefore, translesion replication would be an effective target of proteasome inhibitors for cancer chemotherapy by which cancer cells can be efficiently sensitized to DNA-damaging antineoplastic agents, such as cisplatin. (Cancer Sci 2008; 99: 863–871)
Patients with the autosomal recessive disorder XP-V are predisposed to skin cancer, and XP-V cells demonstrate hyper-mutability after UV irradiation (for reviews, see(1,2)). The defective gene in XP-V encodes one of the Y-family DNA polymerases (polη), which catalyzes translesion synthesis past the cyclobutane dimer of UV-lesions instead of the replicative polymerase(s) that have stalled just before the lesion. Polη then incorporates nucleotides relatively accurately in a manner similar to AA opposite the cis-syn TT-dimer (error-free bypass)(3) as proved by in vitro lesion-bypass assays. By pulse-labeling cells with [14C]thymidine and using a modified protocol of ASDG,(4) we precisely detected translesion replication in vivo, which indicated that it is delayed in XP-V cells but not completely abolished.(5) Taking the above results together, error-prone (= mutagenic) and inefficient polymerase(s) are plausibly involved in the UV-mutagenesis in XP-V cells devoid of polη (for a review, see(6)). To learn more about the in vivo function of the polymerase(s), specific DNA polymerase inhibitors were added. Butylphenyldeoxyguanosine (BuPGdR) inhibited the elongation of pulse-labeled replication products in the UV-irradiated XP-V cells(5) suggesting that the ‘mismatch extender’(7) polζ, is involved in this polη-independent bypass. Evidence that polζ causes mutagenesis is being accumulated (for a review, see(8)).
Caffeine at concentrations of the order of mM significantly prevents elongation in UV-exposed XP-V cells but not in normal cells,(9) indicating that polη-independent and error-prone bypass is caffeine-sensitive and is regulated differently from error-free bypass. Caffeine has pleiotropic effects on DNA metabolism or cell-cycle checkpoint.(10) Although this inhibitory mechanism remains unexplained, the target of the inhibition must be a Ser/Thr kinase, which functions during the DNA damage response. Even at 10 mM, caffeine did not delay the bypass in normal cells (Fig. 1c), indicating that error-free UV-bypass predominates exclusively in normal cells.
Figure 1. Alkaline sucrose density gradient centrifugation (ASDG) profiles of replication products in UV-irradiated (a,c) NB1RGB (normal) and (b,d) HeLa cells. (a,b) Time-course of elongation, (c,d) effect of caffeine. Cells synchronized in the mid-S phase were UV (10 J/m2)-irradiated, incubated in normal medium for 30 min, pulse-labeled with 10 µCi/mL of [14C]thymidine for 1 h, washed twice with phosphate-buffered saline (PBS), and incubated for 1–5 h at 37°C in a normal medium containing (c,d) 1.25–10 mM caffeine, or (a,b) no inhibitor. (c) Some of these profiles overlap. Sedimentation is from right to left. The arrow indicates the position of the T4 phage DNA (166 kb, i.e. approximately 5.5 × 107 Da/single strand). Labeled E. coli DNA (approximately 4 Mb) sedimented near the bottom (fractions 3–6).(4) The average fragment length (in Mb) of each profile is shown in square brackets. cpm, counts per minute.
Download figure to PowerPoint
Error-free bypass is reportedly initiated by RAD6/RAD18, a protein complex that conjugates monoubiquitins to PCNA (a DNA polymerase sliding clamp) arrested at the lesion-site and recruits polη.(11,12) Polη is also recruited to the site via direct physical interaction with RAD18.(12) Monoubiquitinated PCNA was observed in UV-irradiated XP-V cells(11) suggesting that unaccomplished error-free bypass is later switched to polη-independent (error-prone) bypass via unknown mechanisms. Several models have been proposed for the ‘polymerase switching’ (for reviews, see(13,14)).
In this study, we detected UV-induced translesion replication in cancer cell lines. Similar to that observed in XP-V cells, the translesion in cancer cells was considerably slower than that in normal cells, and it was sensitive to caffeine, indicating that UV-bypass in cancer cells is predominantly of the polη-independent type.
Additionally, we found that proteasome inhibitors prevent the UV-bypass in cancer cells. Three different types of proteasome inhibitors (for a review, see(15)) were used. MG-132 is of the peptide-aldehyde type, and it blocks the proteolytic activity of the 26S proteasome but also inhibits lysosomal cysteine proteases and calpains. Lactacystin is a natural product derived from actinomycetes, and it differs in structure from peptide aldehydes; it is substantially more specific to the proteasome. The very potent MG-262 (Cbz-LLL-B(OH)2) has an inhibitory boronate group attached to the peptide moiety; it does not inhibit any cellular proteases and is therefore highly specific.
Usually, translesion replication is detected using the ASDG technique. After UV irradiation, the pulse-labeled replication products in XP-V cells are smaller than those in non-irradiated cells, but on prolonged incubation, they eventually attain a high molecular weight similar to that in non-irradiated cells. This conversion is interpreted that DNA synthesis is temporarily retarded by a pyrimidine dimer and then continues beyond the dimer, leaving a gap that is subsequently sealed.(9) The initial size of the newly synthesized DNA is approximately equal to the average distance between dimers in the template strands.(16) This means that the gaps in the newly synthesized DNA are opposite the dimers (for a review, see(17)). Therefore, the mechanism by which the gaps are sealed (by translesion or other postreplication repair mechanisms) can be observed by assessing the molecular weight of the labeled DNA. Using the ASDG technique, we actually detected postreplication repair other than translesion in camptothecin-treated RAD18-/- chicken DT40 cells.(18)
Postreplication repair was designated to be the mechanisms by which the stalling of the replication fork is avoided, including translesion replication and template switching. The latter is also known as ‘strand displacement and branch migration’;(19) however, its real nature is yet unclear. By analogy with postreplication repair mechanisms in E. coli, homologous recombination (HR)-mediated mechanisms have long been considered.
In order to determine whether the gaps in the newly synthesized strands are sealed by a recombinational exchange or translesion synthesis, A.R. Lehmann used a bromodeoxyuridine (BrUdR)-313 nm light photolysis method(16) in UV-irradiated L5178Y cells. The results indicated that the gaps in the newly synthesized strands opposite the pyrimidine dimers are sealed by translesion. (Reportedly, post-UV conversion of replication products in the mouse lymphoma L5178Y cell is slow in kinetics and is caffeine-sensitive,(17) similar to that in human cancer cells observed in this study.)
We also tried to detect these recombinational exchanges after UV-irradiation using BrUdR-density labeling followed by isopycnic sedimentation analysis. Human cells are labeled together with 14C-cytidine and BrUdR after UV irradiation; their DNAs are isolated, sheared, and then analyzed by neutral CsCl equilibrium density gradient. If the strand exchange occurs between sister chromatids in the vicinity of UV-lesion, two newly synthesized strands ought to be paired there. The resulting peak will be observed in the full heavy position (HH, i.e. both strands are density-labeled). But such an HH peak could be seen neither in XP-V cells nor in HeLa cells (unpublished observations). Therefore, the conversion of post-UV replication products that we observed is exclusively the result of translesion.
Additionally, many groups have reported a mutation spectrum after UV irradiation not only in normal cells, but also in XP-V cells or cancer cells including HeLa. They agreed that the hot spot of mutation is the dipyrimidine sequence. This implies that translesion is mainly executed in UV-irradiated human cells to resume the stalled replication, because HR-mediated postreplication repair is considered to be error-free (this has been confirmed in yeast at least).
- Top of page
- Materials and Methods
We found that translesion replication after UV irradiation was very slow in cancer cells, compared to that in normal cells. In addition, caffeine delayed UV-induced translesion replication in cancer cells as well as in XP-V cells.(5) We also revealed that proteasome inhibitors prevent UV-bypass not only in XP-V cells (manuscript in preparation) but also in cancer cells. In contrast, these agents have no effect on error-free bypass in normal cells. These results imply that the polη-independent (error-prone) pathway of translesion reaction is executed in cancer cells.
Our results clearly suggest the involvement of the 26S proteasome in translesion replication. By conducting an epistatic analysis, Podlaska et al. found a link between 20S proteasomal activity and postreplication repair in the yeast Saccharomyces cerevisiae.(27,28) However, the target protein that is probably signaled by polyubiquitins and degraded by the proteasome remains to be identified.
In yeast, genotypes in mutants of postreplication repair are classified into the RAD6 epistasis group (for a review, see(29)). In this group, two sets of ubiquitination complex exist: RAD6/RAD18 and UBC13/MMS2/RAD5. RAD6/RAD18 catalyzes the monoubiquitination of PCNA at lysine 164(30) following DNA damage, such as that resulting from UV and MMS. The alternate ubiquitination complex, UBC13/MMS2/RAD5(31,32) is inferred to further extend the single ubiquitin moiety with a polyubiquitin chain(30,33) in a non-canonical lysine 63-linked fashion.(34) The polyubiquitinated PCNA is postulated to function in error-free ‘template switching’ (for reviews, see(35,36)), not in proteolysis.(34) Accordingly, no candidate targets of proteasomal disruption have thus far been presented in yeast.
Polyubiquitinated PCNA is rarely detected in human cells.(11) RAD18 protein is autopolyubiquitinated, but this is for the regulation of the protein itself by proteolysis; besides, a mutant that lacks autoubiquitination has normal sensitivity to UV.(37) Less information is available about other candidate targets, for example, the small or large subunit of polδ, RPA, and topoisomerases. The mechanism by which proteasome inhibitors inhibit error-prone translesion replication remains to be elucidated.
In this paper, we presented evidence that translesion replication is the most effective target of proteasome inhibitors for cancer chemotherapy, with cancer cells being efficiently attacked with minimal damage to normal tissues. Translesion replication was particularly prevented in an undifferentiated gastric carcinoma cell line (HGC-27), a malignant melanoma cell line (Mewo), and a multiple myeloma cell line (RPMI8226); patients with these cancers are difficult to convalesce. Although UV is rarely used in medicine, cisplatin, one of the most frequently used antineoplastic agents, has been revealed to induce a UV-type translesion reaction, which proteasome inhibitors prevent in cancer cells.
Bortezomib (Velcade; Millennium Pharmaceuticals, MA, US) is the first proteasome inhibitor in antineoplastic use (for a review, see(38)). This agent has recently been approved for use in the United States for the treatment of multiple myeloma, and it has demonstrated promising results. Furthermore, phase II studies of the agent are underway in patients with other hematologic malignancies and solid tumors, in combination with other agents. Bortezomib has a modified peptidyl boronic acid structure, similar to MG-262. Our findings should contribute to the application of bortezomib and similar drugs to clinical chemotherapy, thereby relieving unendurable pain for a large number of patients.
Proteasome inhibitors may be considerably cytotoxic and non-specific to translesion replication because the 26S proteasome is involved in the recycling of various unfolded or damaged proteins. Nonetheless, our results are worth reporting. The finding that proteasomal disruption is necessary for translesion replication in cancer cells, which differs from that in normal cells, provides a novel clue regarding the development of chemotherapy with fewer side-effects.