Hiroyuki Kitao

FANCI phosphorylation functions as a molecular switch to turn on the Fanconi anemia pathway

In response to DNA damage or replication fork stress, the Fanconi anemia pathway is activated, leading to monoubiquitination of FANCD2 and FANCI and their colocalization in foci. Here we show that, in the chicken DT40 cell system, multiple alanine-substitution mutations in six conserved and clustered Ser/Thr-Gln motifs of FANCI largely abrogate monoubiquitination and focus formation of both FANCI and FANCD2, resulting in loss of DNA repair function. Conversely, FANCI carrying phosphomimic mutations on the same six residues induces constitutive monoubiquitination and focus formation of FANCI and FANCD2, and protects against cell killing and chromosome breakage by DNA interstrand cross-linking agents. We propose that the multiple phosphorylation of FANCI serves as a molecular switch in activation of the Fanconi anemia pathway. Mutational analysis of putative phosphorylation sites in human FANCI indicates that this switch is evolutionarily conserved.

Emergence of epidermal growth factor receptor T790M mutation during chronic exposure to gefitinib in a non-small cell lung cancer cell line

The epidermal growth factor receptor (EGFR)-specific tyrosine kinase inhibitor gefitinib may provide dramatic clinical responses in some patients with pulmonary adenocarcinoma carrying activating mutations of the EGFR. However, prolonged administration of gefitinib may eventually induce acquired resistance in such patients. To gain insight into the mechanisms of this phenomenon, we placed PC-9, a cell line derived from pulmonary adenocarcinoma that has a 15-bp deletion in EGFR exon 19, under the continuous selective pressure of low levels of gefitinib without any mutagen, and established a subline that was able to grow in the presence of 2 micromol/L of gefitinib (designated RPC-9). In this cell line, about half of the reverse transcription-PCR products from mutated EGFR also carried an additional mutation (T790M). In keeping with the proposed role of T790M in abrogating gefitinib binding with EGFR, gefitinib-treated RPC-9 hardly displayed any decrease in the constitutive phosphorylation of EGFR, Akt, or Erk1/2 unlike in PC-9 cells. Interestingly, transfection of the EGFR carrying only a 15-bp deletion reversed the resistance to gefitinib in RPC-9 cells. Thus, the balance of expression levels between gefitinib-sensitive or gefitinib-resistant EGFR may govern the response to gefitinib in lung cancer.

Fanconi anemia protein FANCD2 promotes immunoglobulin gene conversion and DNA repair through a mechanism related to homologous recombination.

ABSTRACT Recent studies show overlap between Fanconi anemia (FA) proteins and those involved in DNA repair mediated by homologous recombination (HR). However, the mechanism by which FA proteins affect HR is unclear. FA proteins (FancA/C/E/F/G/L) form a multiprotein complex, which is responsible for DNA damage-induced FancD2 monoubiquitination, a key event for cellular resistance to DNA damage. Here, we show that FANCD2-disrupted DT40 chicken B-cell line is defective in HR-mediated DNA double-strand break (DSB) repair, as well as gene conversion at the immunoglobulin light-chain locus, an event also mediated by HR. Gene conversions occurring in mutant cells were associated with decreased nontemplated mutations. In contrast to these defects, we also found increased spontaneous sister chromatid exchange (SCE) and intact Rad51 foci formation after DNA damage. Thus, we propose that FancD2 promotes a subpathway of HR that normally mediates gene conversion by a mechanism that avoids crossing over and hence SCEs.

Fanconi anemia: a disorder defective in the DNA damage response

Fanconi anemia (FA) is a cancer predisposition disorder characterized by progressive bone marrow failure, congenital developmental defects, chromosomal abnormalities, and cellular hypersensitivity to DNA interstrand crosslink (ICL) agents. So far mutations in 14 FANC genes were identified in FA or FA-like patients. These gene products constitute a common ubiquitin–phosphorylation network called the “FA pathway” and cooperate with other proteins involved in DNA repair and cell cycle control to repair ICL lesions and to maintain genome stability. In this review, we summarize recent exciting discoveries that have expanded our view of the molecular mechanisms operating in DNA repair and DNA damage signaling.

Activation of downstream epidermal growth factor receptor (EGFR) signaling provides gefitinib-resistance in cells carrying EGFR mutation

Patients with pulmonary adenocarcinoma carrying the epidermal growth factor receptor (EGFR) mutation tend to display dramatic clinical response to treatment with the EGFR tyrosine kinase inhibitor gefitinib. Unfortunately, in many cases the cancer cells eventually acquire resistance, and this limits the duration of efficacy. To gain insight into these acquired resistance mechanisms, we first prepared HEK293T cell line stably transfected with either wild‐type (WT) or mutant (L858R) EGFR, and then expressed oncogenic K‐Ras12V mutant in the latter transfectant. Although 293T cells expressing wild‐type EGFR did not show any growth inhibition by gefitinib treatment similarly to the non‐transfected cells, the cells expressing the EGFR‐L858R were exquisitely sensitive. Consistently, phospho‐Akt levels were decreased in response to gefitinib in cells expressing EGFR‐L858R but not in cells with EGFR‐WT. In contrast, 293T cells expressing both EGFR‐L858R and oncogenic K‐Ras were able to proliferate even in the presence of high concentration of gefitinib probably by inducing Erk1/2 activation. We also expressed K‐Ras12V in the gefitinib‐sensitive pulmonary adenocarcinoma cell line PC‐9, which harbors an in‐frame deletion in the EGFR gene. The activated K‐Ras inhibited the effects of gefitinib treatment on cell growth, cell death induction and levels of phospho‐Akt, as well as phospho‐Erk. These data indicate that activated Ras could substitute most of the upstream EGFR signal, and are consistent with the hypothesis that mutational activation of targets immediately downstream from the EGFR could induce the secondary resistance to gefitinib in patients with lung cancer carrying EGFR mutation. (Cancer Sci 2007; 98: 357–363)

Histone chaperone activity of Fanconi anemia proteins, FANCD2 and FANCI, is required for DNA crosslink repair

Fanconi anaemia (FA) is a rare hereditary disorder characterized by genomic instability and cancer susceptibility. A key FA protein, FANCD2, is targeted to chromatin with its partner, FANCI, and plays a critical role in DNA crosslink repair. However, the molecular function of chromatin‐bound FANCD2‐FANCI is still poorly understood. In the present study, we found that FANCD2 possesses nucleosome‐assembly activity in vitro. The mobility of histone H3 was reduced in FANCD2‐knockdown cells following treatment with an interstrand DNA crosslinker, mitomycin C. Furthermore, cells harbouring FANCD2 mutations that were defective in nucleosome assembly displayed impaired survival upon cisplatin treatment. Although FANCI by itself lacked nucleosome‐assembly activity, it significantly stimulated FANCD2‐mediated nucleosome assembly. These observations suggest that FANCD2‐FANCI may regulate chromatin dynamics during DNA repair.

Regulation of Ionizing Radiation-induced Rad52 Nuclear Foci Formation by c-Abl-mediated Phosphorylation

The RAD52 epistasis group of proteins, including Rad51, Rad52, and Rad54, plays an important role in the homologous recombination repair of double strand breaks. A well characterized feature associated with the ability of these proteins to repair double strand breaks is inducible nuclear foci formation at the sites of damage. How the process is functionally regulated in response to DNA damage, however, remains elusive. We show here that c-Abl tyrosine kinase associates with and phosphorylates Rad52 on tyrosine 104. Importantly, the very same site of Rad52 is phosphorylated on exposure of cells to ionizing radiation (IR). The functional significance of c-Abl-dependent phosphorylation of Rad52 is underscored by our findings that cells that express the phosphorylation-resistant Rad52 mutant, in which tyrosine 104 is replaced by phenylalanine, exhibit compromised nuclear foci formation in response to IR. Furthermore, IR-induced Rad52 nuclear foci formation is markedly suppressed by the expression of dominant-negative c-Abl. Together our data support a mode of post-translational regulation of Rad52 mediated by the c-Abl tyrosine kinase.

Fanconi anemia group J mutation abolishes its DNA repair function by uncoupling DNA translocation from helicase activity or disruption of protein-DNA complexes

Fanconi anemia (FA) is a genetic disease characterized by congenital abnormalities, bone marrow failure, and susceptibility to leukemia and other cancers. FANCJ, one of 13 genes linked to FA, encodes a DNA helicase proposed to operate in homologous recombination repair and replicational stress response. The pathogenic FANCJ-A349P amino acid substitution resides immediately adjacent to a highly conserved cysteine of the iron-sulfur domain. Given the genetic linkage of the FANCJ-A349P allele to FA, we investigated the effect of this particular mutation on the biochemical and cellular functions of the FANCJ protein. Purified recombinant FANCJ-A349P protein had reduced iron and was defective in coupling adenosine triphosphate (ATP) hydrolysis and translocase activity to unwinding forked duplex or G-quadruplex DNA substrates or disrupting protein-DNA complexes. The FANCJ-A349P allele failed to rescue cisplatin or telomestatin sensitivity of a FA-J null cell line as detected by cell survival or γ-H2AX foci formation. Furthermore, expression of FANCJ-A349P in a wild-type background exerted a dominant-negative effect, indicating that the mutant protein interferes with normal DNA metabolism. The ability of FANCJ to use the energy from ATP hydrolysis to produce the force required to unwind DNA or destabilize protein bound to DNA is required for its role in DNA repair.

Functional interplay between BRCA2/FancD1 and FancC in DNA repair

A rare hereditary disorder, Fanconi anemia (FA), is caused by mutations in an array of genes, which interact in a common FA pathway/network. These genes encode components of the FA “core” complex, a key factor FancD2, the familial breast cancer suppressor BRCA2/FancD1, and Brip1/FancJ helicase. Although BRCA2 is known to play a pivotal role in homologous recombination repair by regulating Rad51 recombinase, the precise functional relationship between BRCA2 and the other FA genes is unclear. Here we show that BRCA2-dependent chromatin loading of Rad51 after mitomycin C treatment was not compromised by disruption of FANCC or FANCD2. Rad51 and FancD2 form colocalizing subnuclear foci independently of each other. Furthermore, we created a conditional BRCA2 truncating mutation lacking the C-terminal conserved domain (CTD) (brca2ΔCTD), and disrupted the FANCC gene in this background. The fancc/brca2ΔCTD double mutant revealed an epistatic relationship between FANCC and BRCA2 CTD in terms of x-ray sensitivity. In contrast, levels of cisplatin sensitivity and mitomycin C-induced chromosomal aberrations were increased in fancc/brca2ΔCTD cells relative to either single mutant. Taken together, these results indicate that FA proteins work together with BRCA2/Rad51-mediated homologous recombination in double strand break repair, whereas the FA pathway plays a role that is independent of the CTD of BRCA2 in interstrand cross-link repair. These results provide insights into the functional interplay between the classical FA pathway and BRCA2.

High expression of BUBR1 is one of the factors for inducing DNA aneuploidy and progression in gastric cancer

(Cancer Sci 2010; 101: 639–645)