FAAP20: a novel ubiquitin-binding FA nuclear core-complex protein required for functional integrity of the FA-BRCA DNA repair pathway.

Fanconi anemia (FA) nuclear core complex is a multiprotein complex required for the functional integrity of the FA-BRCA pathway regulating DNA repair. This pathway is inactivated in FA, a devastating genetic disease, which leads to hematologic defects and cancer in patients. Here we report the isolation and characterization of a novel 20-kDa FANCA-associated protein (FAAP20). We show that FAAP20 is an integral component of the FA nuclear core complex. We identify a region on FANCA that physically interacts with FAAP20, and show that FANCA regulates stability of this protein. FAAP20 contains a conserved ubiquitin-binding zinc-finger domain (UBZ), and binds K-63-linked ubiquitin chains in vitro. The FAAP20-UBZ domain is not required for interaction with FANCA, but is required for DNA-damage-induced chromatin loading of FANCA and the functional integrity of the FA pathway. These findings reveal critical roles for FAAP20 in the FA-BRCA pathway of DNA damage repair and genome maintenance.

MHF1-MHF2, a histone-fold-containing protein complex, participates in the Fanconi anemia pathway via FANCM.

FANCM is a Fanconi anemia nuclear core complex protein required for the functional integrity of the FANC-BRCA pathway of DNA damage response and repair. Here we report the isolation and characterization of two histone-fold-containing FANCM-associated proteins, MHF1 and MHF2. We show that suppression of MHF1 expression results in (1) destabilization of FANCM and MHF2, (2) impairment of DNA damage-induced monoubiquitination and foci formation of FANCD2, (3) defective chromatin localization of FA nuclear core complex proteins, (4) elevated MMC-induced chromosome aberrations, and (5) sensitivity to MMC and camptothecin. We also provide biochemical evidence that MHF1 and MHF2 assemble into a heterodimer that binds DNA and enhances the DNA branch migration activity of FANCM. These findings reveal critical roles of the MHF1-MHF2 dimer in DNA damage repair and genome maintenance through FANCM.

Impaired FANCD2 monoubiquitination and hypersensitivity to camptothecin uniquely characterize Fanconi anemia complementation group M.

FANCM is a component of the Fanconi anemia (FA) core complex and one FA patient (EUFA867) with biallelic mutations in FANCM has been described. Strikingly, we found that EUFA867 also carries biallelic mutations in FANCA. After correcting the FANCA defect in EUFA867 lymphoblasts, a "clean" FA-M cell line was generated. These cells were hypersensitive to mitomycin C, but unlike cells defective in other core complex members, FANCM(-/-) cells were proficient in monoubiquitinating FANCD2 and were sensitive to the topoisomerase inhibitor camptothecin, a feature shared only with the FA subtype D1 and N. In addition, FANCM(-/-) cells were sensitive to UV light. FANCM and a C-terminal deletion mutant rescued the cross-linker sensitivity of FANCM(-/-) cells, whereas a FANCM ATPase mutant did not. Because both mutants restored the formation of FANCD2 foci, we conclude that FANCM functions in an FA core complex-dependent and -independent manner.

BLAP18/RMI2, a novel OB-fold-containing protein, is an essential component of the Bloom helicase-double Holliday junction dissolvasome.

Bloom Syndrome is an autosomal recessive cancer-prone disorder caused by mutations in the BLM gene. BLM encodes a DNA helicase of the RECQ family, and associates with Topo IIIalpha and BLAP75/RMI1 (BLAP for BLM-associated polypeptide/RecQ-mediated genome instability) to form the BTB (BLM-Topo IIIalpha-BLAP75/RMI1) complex. This complex can resolve the double Holliday junction (dHJ), a DNA intermediate generated during homologous recombination, to yield noncrossover recombinants exclusively. This attribute of the BTB complex likely serves to prevent chromosomal aberrations and rearrangements. Here we report the isolation and characterization of a novel member of the BTB complex termed BLAP18/RMI2. BLAP18/RMI2 contains a putative OB-fold domain, and several lines of evidence suggest that it is essential for BTB complex function. First, the majority of BLAP18/RMI2 exists in complex with Topo IIIalpha and BLAP75/RMI1. Second, depletion of BLAP18/RMI2 results in the destabilization of the BTB complex. Third, BLAP18/RMI2-depleted cells show spontaneous chromosomal breaks and are sensitive to methyl methanesulfonate treatment. Fourth, BLAP18/RMI2 is required to target BLM to chromatin and for the assembly of BLM foci upon hydroxyurea treatment. Finally, BLAP18/RMI2 stimulates the dHJ resolution capability of the BTB complex. Together, these results establish BLAP18/RMI2 as an essential member of the BTB dHJ dissolvasome that is required for the maintenance of a stable genome.

[The combination of recombinant rAd-p53 and adriamycin for management of primary drug resistance in chemotherapy of lung squamous cell cancer].

OBJECTIVE: To investigate the combination of wild-type p53 (wtp53) gene substitution and adriamycin (ADM) on the lung cancer in vivo. METHODS: The effect of combination of recombinant Adeno-wtp53 (rAd-p53) and ADM on reversing primary drug resistance to ADM was studied for the non-small cell lung cancer (NSCLC) in a nude mice model developed by subcutaneously transplanting with the 16HBE lung cancer cell line. Twenty four nude mice with loaded tumors were randomly divided into 4 groups (no treatment, rAd-p53 treatment alone, ADM treatment alone, combination of rAd-p53 plus ADM chemotherapy). The effect of rAd-p53 substitution and ADM on the drug sensitivity was evaluated. RESULTS: In vivo studies on nude mice model transplanted with NSCLC showed that, rAd-p53 treatment alone suppressed tumor growth by 60.11% of the tumor weight and 85.4% by the volume (n = 6), while the combination of rAd-p53 with ADM suppressed tumor growth more significantly (82.32%, 99.5%, respectively, n = 6). The treatment with Adriamycin alone was less effective (35.4%, 73.9%, respectively, n = 6). CONCLUSION: Combination of rAd-p53 and ADM significantly increased the sensitivity of NSCLC tumor graft to ADM, suggesting that the combination of replication-deficient wild p53 adenovirus with DNA-damaging drugs may increase the efficacy of chemotherapy in NSCLC and overcome primary drug resistance.

[Prediction of the possible tertiary structure alterations of p53 protein following point mutation in p53 gene condon 282 in lung cancer cells].

This study was carried out to predict the possible tertiary structure alterations of p53 protein after point mutation of p53 gene condon 282 in lung cancer cells based on the latest 3D structure analysis platform series of Phyre software. It was found that the p53 gene condon 282 mutation (Arg/Leu) may destabilize the H2 helix and DNA binding in the major groove by compromising the contacts of p53 protein with the beta-hairpin of DNA binding surface.

[Roles of mutant p53 gene in malignant phenotypes and resistance to drugs in anti-7,8-dihydrodiol-9,10-epoxide benzo(a)pyrene-induced lung cancer cells].

OBJECTIVE: To study the role of mutant p53 gene induced by anti-7,8-dihydrodiol-9,10-epoxide benzo(a)pyrene (BPDE) in the development of lung cancer and the effects of wild-type p53 substitution on malignant phenotype and resistance to drugs. METHODS: BPDE-induced human lung cancer cells (16HBE, a human bronchial epithelial cell line, treated by BPDE) with mutant p53 gene were transfected with pShuttle-cmv-wild p53, followed by soft-agar colony formation assay and cell growth assay. Genome DNAs were extracted from those cells after transfection for detection of apoptosis. Sensitivity of the cells to drugs was also detected simultaneously. RESULTS: It was shown that exogenous wild-type p53 transfection inhibited colony formation of the lung cancer cells with mutant p53 gene and induced cell growth arrest and cell apoptosis. In addition, the lung cancer cells transfected with wild-type p53 gene showed enhanced sensitivity to adriamycin (ADM) to which the cells before transfection was resistant. CONCLUSIONS: p53 mutation plays an important role in the development and progress of lung cancer, as well as in multi-drug resistance in the management of lung cancer chemotherapy. Wild-type p53 substitution therapy may enhance the sensitivity of lung cancer to chemical drugs.