The xeroderma pigmentosum group E gene product DDB2, a protein involved in nucleotide excision repair (NER), associates with the E3 ubiquitin ligase complex Cul4A-DDB1. But the precise role of these interactions in the NER activity of DDB2 is unclear. Several models, including DDB2-mediated ubiquitination of histones in UV-irradiated cells, have been proposed. But those models lack clear genetic evidence. Here we show that DDB2 participates in NER by regulating the cellular levels of p21 Waf1/Cip1 . We show that DDB2 enhances nuclear accumulation of DDB1, which binds to a modified form of p53 containing phosphorylation at Ser18 (p53 S18P ) and targets it for degradation in low-dose-UV-irradiated cells. Waf1/Cip1 to the NER activity of DDB2.The UV rays in sunlight are considered to be the major cause of skin cancers. UV causes DNA damage by generating cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts, which are repaired mainly by the nucleotide excision repair (NER) pathway (reviewed in reference 41). NER involves excision of the strand containing CPDs or 6-4 photoproducts, followed by repair synthesis to fill the gap. Several genes involved in NER are mutated in xeroderma pigmentosum, a rare repair deficiency disease in which the patients are sun sensitive and develop skin cancer at a high frequency (see references 11 and 16 for reviews). Eight complementation groups, XPA through XPG and XPV, have been characterized in xeroderma pigmentosum. While XPV encodes an error-prone DNA polymerase, the other XP genes encode proteins that participate in the excision of the damaged DNA strand. For example, XPC participates in the recognition of damaged DNA. Subsequently, XPB and XPD participate in unwinding the strands at the damaged site; XPA is critical for positioning the endonucleases XPF and XPG with respect to the damaged site, leading to excision of the damaged strand. The gap generated following excision is filled by DNA polymerase delta that involves PCNA to carry out the repair synthesis (41). Thus, while all other XP genes have been functionally characterized, the mechanism by which the XPE gene participates in NER has remained controversial.The XPE gene encodes DDB2, a subunit of the damaged-DNA-binding protein DDB, which possesses a high affinity for CPDs and 6-4 photoproducts (reviewed in reference 48). Cells from XP-E patients exhibit a deficiency in NER. The NER deficiency in XP-E cells, along with several other in vivo studies, suggested a role for DDB2 in NER (25,36,48). Studies with DDB2Ϫ/Ϫ mice provided further evidence of a role for DDB2 in inhibiting UV-induced skin carcinogenesis (3,22,54), a characteristic of the XP gene products.Because of the high affinity of DDB, a complex of DDB1 and DDB2, for damaged DNA, several studies have implicated DDB2 and DDB in the early damaged-DNA recognition step of NER. However, a direct role for DDB2 or DDB in NER is a point of controversy. Initial studies reported a stimulatory activity of DDB in NER assays in vitro (51). However, two recent studies carri...
In this study, we show that administration of low-dose melphalan (l-PAM, l-phenylalanine mustard) to mice bearing a large MOPC-315 plasmacytoma led to a rapid up-regulation of B7-1 (CD80), but not B7-2 (CD86), expression on the surface of MOPC-315 tumor cells. This l-PAM-induced preferential up-regulation of B7-1 surface expression was due, at least in part, to a direct effect of l-PAM on the tumor cells, as in vitro exposure of MOPC-315 tumor cells to l-PAM led to the preferential up-regulation of B7-1 surface expression. Moreover, in vitro exposure of MOPC-315 tumor cells to two other anticancer modalities, γ-irradiation and mitomycin C, resulted in the preferential up-regulation of B7-1 surface expression. This effect was not restricted to MOPC-315 tumor cells, as preferential up-regulation of B7-1 surface expression was observed also following in vitro exposure of the P815 mastocytoma (that is negative for both B7-1 and B7-2 surface expression) to any of the three anticancer modalities. The up-regulation of B7-1 surface expression following in vitro exposure of tumor cells to l-PAM, γ-irradiation, or mitomycin C required de novo protein and RNA synthesis, and was associated with the accumulation of mRNA for B7-1 within 4–8 h, indicating that the regulation of B7-1 expression is at the RNA transcriptional level. These results have important implications for an additional immune-potentiating mechanism of these anticancer modalities in clinical setting.
Following inoculation with 1 X 10(6) MOPC-315 tumor cells, a single injection of a very low dose of melphalan (L-PAM, L-phenylalanine mustard), 0.75 mg/kg, cured most of the mice bearing a day 11 large primary tumor (20 mm) and metastases, but failed to cure mice bearing a day 4 nonpalpable tumor. Treatment of mice bearing a nonpalpable tumor with the very low dose of drug compromised the ability of the mice to respond effectively to the same low dose of drug when the tumor became large (day 12). However, a nonpalpable tumor could be eradicated by treatment of tumor bearers with a low dose of L-PAM, if it was present concomitantly with a large tumor on the contralateral side. A high dose of L-PAM, 15 mg/kg, cured mice bearing either a nonpalpable or a large tumor. The eradication of the tumor induced by the high dose of L-PAM appeared to be due solely to the tumoricidal effect of the drug. On the other hand, the eradication of the tumor by the low dose of L-PAM also required the participation of antitumor immunity of the host, since subsequent injection of antithymocyte serum abrogated the curative effect of the drug in most mice. Mice cured by a high dose of L-PAM were not resistant to subsequent lethal tumor challenge. In contrast, mice cured by the low dose of L-PAM were able to reject a tumor challenge of 300 times the minimal lethal tumor dose. The results obtained with L-PAM therapy are similar to the results that we had previously reported with cyclophosphamide therapy. Thus, the timing of therapy with a low dose of drug for mice bearing a MOPC-315 tumor is critical for successful therapy. Moreover, the selection of a low dose rather than a high dose of drug to eradicate a large tumor offers the advantage that it results in long-lasting potent antitumor immunity as a consequence of the participation of host antitumor immunity in the eradication of the tumor.
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