Toshiaki Kogame

Collaborative Action of Brca1 and CtIP in Elimination of Covalent Modifications from Double-Strand Breaks to Facilitate Subsequent Break Repair

Topoisomerase inhibitors such as camptothecin and etoposide are used as anti-cancer drugs and induce double-strand breaks (DSBs) in genomic DNA in cycling cells. These DSBs are often covalently bound with polypeptides at the 3′ and 5′ ends. Such modifications must be eliminated before DSB repair can take place, but it remains elusive which nucleases are involved in this process. Previous studies show that CtIP plays a critical role in the generation of 3′ single-strand overhang at “clean” DSBs, thus initiating homologous recombination (HR)–dependent DSB repair. To analyze the function of CtIP in detail, we conditionally disrupted the CtIP gene in the chicken DT40 cell line. We found that CtIP is essential for cellular proliferation as well as for the formation of 3′ single-strand overhang, similar to what is observed in DT40 cells deficient in the Mre11/Rad50/Nbs1 complex. We also generated DT40 cell line harboring CtIP with an alanine substitution at residue Ser332, which is required for interaction with BRCA1. Although the resulting CtIPS332A/−/− cells exhibited accumulation of RPA and Rad51 upon DNA damage, and were proficient in HR, they showed a marked hypersensitivity to camptothecin and etoposide in comparison with CtIP+/−/− cells. Finally, CtIPS332A/−/−BRCA1−/− and CtIP+/−/−BRCA1−/− showed similar sensitivities to these reagents. Taken together, our data indicate that, in addition to its function in HR, CtIP plays a role in cellular tolerance to topoisomerase inhibitors. We propose that the BRCA1-CtIP complex plays a role in the nuclease-mediated elimination of oligonucleotides covalently bound to polypeptides from DSBs, thereby facilitating subsequent DSB repair.

Crystal Structure of Human REV7 in Complex with a Human REV3 Fragment and Structural Implication of the Interaction between DNA Polymerase ζ and REV1

DNA polymerase ζ (Polζ) is an error-prone DNA polymerase involved in translesion DNA synthesis. Polζ consists of two subunits: the catalytic REV3, which belongs to B family DNA polymerase, and the noncatalytic REV7. REV7 also interacts with REV1 polymerase, which is an error-prone Y family DNA polymerase and is also involved in translesion DNA synthesis. Cells deficient in one of the three REV proteins and those deficient in all three proteins show similar phenotype, indicating the functional collaboration of the three REV proteins. REV7 interacts with both REV3 and REV1 polymerases, but the structure of REV7 or REV3, as well as the structural and functional basis of the REV1-REV7 and REV3-REV7 interactions, remains unknown. Here we show the first crystal structure of human REV7 in complex with a fragment of human REV3 polymerase (residues 1847–1898) and reveal the mechanism underlying REV7-REV3 interaction. The structure indicates that the interaction between REV7 and REV3 creates a structural interface for REV1 binding. Furthermore, we show that the REV7-mediated interactions are responsible for DNA damage tolerance. Our results highlight the function of REV7 as an adapter protein to recruit Polζ to a lesion site. REV7 is alternatively called MAD2B or MAD2L2 and also involved in various cellular functions such as signal transduction and cell cycle regulation. Our results will provide a general structural basis for understanding the REV7 interaction.

Differential and Common DNA Repair Pathways for Topoisomerase I- and II-Targeted Drugs in a Genetic DT40 Repair Cell Screen Panel

Clinical topoisomerase I (Top1) and II (Top2) inhibitors trap topoisomerases on DNA, thereby inducing protein-linked DNA breaks. Cancer cells resist the drugs by removing topoisomerase-DNA complexes, and repairing the drug-induced DNA double-strand breaks (DSB) by homologous recombination and nonhomologous end joining (NHEJ). Because numerous enzymes and cofactors are involved in the removal of the topoisomerase-DNA complexes and DSB repair, it has been challenging to comprehensively analyze the relative contribution of multiple genetic pathways in vertebrate cells. Comprehending the relative contribution of individual repair factors would give insights into the lesions induced by the inhibitors and genetic determinants of response. Ultimately, this information would be useful to target specific pathways to augment the therapeutic activity of topoisomerase inhibitors. To this end, we put together 48 isogenic DT40 mutant cells deficient in DNA repair and generated one cell line deficient in autophagy (ATG5). Sensitivity profiles were established for three clinically relevant Top1 inhibitors (camptothecin and the indenoisoquinolines LMP400 and LMP776) and three Top2 inhibitors (etoposide, doxorubicin, and ICRF-193). Highly significant correlations were found among Top1 inhibitors as well as Top2 inhibitors, whereas the profiles of Top1 inhibitors were different from those of Top2 inhibitors. Most distinct repair pathways between Top1 and Top2 inhibitors include NHEJ, TDP1, TDP2, PARP1, and Fanconi Anemia genes, whereas homologous recombination seems relevant especially for Top1 and, to a lesser extent, for Top2 inhibitors. We also found and discuss differential pathways among Top1 inhibitors and Top2 inhibitors. Mol Cancer Ther; 13(1); 214–20. ©2013 AACR.

A novel approach using DNA-repair-deficient chicken DT40 cell lines for screening and characterizing the genotoxicity of environmental contaminants

Background Many bacterial or mammalian cell-based test systems, such as the Ames test, chromosomal aberration assays, or gene mutation assays, are commonly used in developed countries to detect the genotoxicity of industrial chemicals. However, the specificity is generally limited and the sensitivity is not sufficiently high. In addition, most assays cannot provide information on mechanisms of genotoxicity of a given chemical. Objectives We aimed to establish a sensitive and fast screening method that is also capable of characterizing mechanisms of genotoxicity. Methods We developed a novel bioassay employing gene-disrupted clones of the chicken DT40 B-lymphocyte line, which are designed to be deficient in several specific DNA repair pathways. Genotoxic chemicals can delay cellular proliferation in DNA-repair–deficient clones more significantly than in wild-type cells by interfering with DNA replication, thereby inducing DNA damage. In addition, we verified the validity of this assay by analyzing the genotoxicity of γ-rays, ultraviolet (UV) light, and sodium metaarsenite (NaAsO2). We also characterized DNA lesions induced by NaAsO2. Results Genotoxicity of given stressors was successfully screened based on a comparison of proliferation kinetics between wild-type and DNA-repair–deficient mutants in 48 hr. We also found that NaAsO2 apparently induces at least two types of damage: chromosomal breaks and UV photoproduct-like DNA lesions. Conclusion This bioassay is a reliable and sensitive screening tool for environmental mutagens as well as for further characterizing the nature of detected genotoxicity.

GEMIN2 promotes accumulation of RAD51 at double-strand breaks in homologous recombination

RAD51 is a key factor in homologous recombination (HR) and plays an essential role in cellular proliferation by repairing DNA damage during replication. The assembly of RAD51 at DNA damage is strictly controlled by RAD51 mediators, including BRCA1 and BRCA2. We found that human RAD51 directly binds GEMIN2/SIP1, a protein involved in spliceosome biogenesis. Biochemical analyses indicated that GEMIN2 enhances the RAD51–DNA complex formation by inhibiting RAD51 dissociation from DNA, and thereby stimulates RAD51-mediated homologous pairing. GEMIN2 also enhanced the RAD51-mediated strand exchange, when RPA was pre-bound to ssDNA before the addition of RAD51. To analyze the function of GEMIN2, we depleted GEMIN2 in the chicken DT40 line and in human cells. The loss of GEMIN2 reduced HR efficiency and resulted in a significant decrease in the number of RAD51 subnuclear foci, as observed in cells deficient in BRCA1 and BRCA2. These observations and our biochemical analyses reveal that GEMIN2 regulates HR as a novel RAD51 mediator.

Palmoplantar keratosis in oculodentodigital dysplasia with a GJA1 point mutation out of the C-terminal region of connexin 43

Gap junction proteins are composed of 21 genes of the connexin (Cx) family. They play important roles in cell–cell contact by exchange of small molecules through hemichannels. Hence, mutations of Cx family genes affect various tissues of a body. The mutation of the GJA1 gene, which codes Cx43, causes oculodentodigital dysplasia (ODDD), commonly in an autosomal dominant manner with phenotypic variability. It has been believed that gene mutations causing truncation of the Cx43 C‐terminus is necessary and sufficient for palmoplantar keratosis (PPK) development in ODDD patients. Here, we report a case of an ODDD patient developing PPK with a GJA1 gene mutation (c.412G>A/p.Gly138Ser), which was previously reported in a case of ODDD without PPK and expected not to result in C‐terminal truncation of Cx43. This case suggests not only C‐terminal truncation, but also that a point mutation in the cytoplasmic region of Cx43 can cause PPK in ODDD patients.

Possible inducible skin-associated lymphoid tissues (iSALT)-like structures with CXCL13(+) fibroblast-like cells in secondary syphilis.

DEAR EDITOR, Nonlymphoid organs are not merely the sites of effector T-cell function. Ectopic accumulations of lymphoid cells arise in nonlymphoid organs under long-lived self-perpetuating chronic inflammation. These accumulations are called tertiary lymphoid organs (TLOs), and they function as antigen-presenting sites. TLOs are characterized by their cellular, organizational, chemokine and vascular similarity to lymph nodes. In well-developed TLOs, B and T cells are compartmentalized by CXCL13 from follicular dendritic cells (FDCs) and CCL19/CCL21 from fibroblastic reticular cells, respectively. The vascular system consisting of peripheral lymph node addressin (PNAd)-expressing high endothelial venules (HEVs) and lymphatic vessels is highly organized in TLOs as in lymph nodes. In mucosal areas, mucosa-associated lymphoid tissues function as TLOs. By analogy, the concept of skin-associated lymphoid tissue (SALT) was proposed in the 1980s, on the basis that cells in the skin can capture, process and present antigens. Recently, we identified inducible lymphoid structures composed of macrophages, dermal dendritic cells and effector T cells in mice, which we termed inducible SALT (iSALT). The formation of iSALT is essential to the efficient activation of effector cells in mice. However, it remains unknown whether the concept of iSALT can be applied to the human skin. This question can be addressed by investigating the characteristics of TLOs in chronic inflammatory skin lesions. We speculated that a skin lesion produced by infection with Treponema pallidum would suffice for this purpose for the following reasons. Firstly, plasmacyte infiltration in the cutaneous lesions of secondary syphilis suggests terminal differentiation of B cells locally in the skin. Secondly, CXCL13, a pivotal cytokine for TLO formation, is enriched in cerebrospinal fluid of neurosyphilitic patients. These lines of evidence imply that a lymphoid tissue organizing event could be engendered in syphilitic lesions. Here we studied the cutaneous lesions of secondary syphilis caused by persistent infection with T. pallidum. A 62-year-old man with membranous nephropathy was referred to us for concurrent disseminated noncoalescing asymptomatic macules of 3–5 mm in size on the torso and extremities. Serological tests indicated treponemal infection. The biopsied abdominal skin showed vacuolar lymphocytic interface dermatitis (Fig. 1a, b). Numerous plasma cells were scattered in the papillary dermis and the perivascular region (Fig. 1b). Spirochetes were visualized in the epidermis and lymphoid clusters by anti-T. pallidum polyclonal antibody (Fig. 1c). The perivascular infiltrate consisted of CD20 B cells and CD3 T cells (Fig. 1d, e). In the upper dermis, CXCL13 was detected in the fibroblast-like cells, which were negative for the FDC marker CNA.42 (Fig. 1f, g). CXCL13 cells were not detected in normal skin (Fig. 1j). Some but not all blood vascular endothelial cells (BECs) expressed PNAd (detected by MECA-79), indicating differentiation towards HEVs (Fig. 1h, i). The number of infiltrating lymphocytes was lower around the PNAd-negative BECs. Treatment with oral amoxicillin 1 5 g per day for 8 weeks successfully cleared the eruptions and proteinuria. Two additional cases of secondary syphilis showed essentially the same findings in immunohistochemistry (data not shown). Skin biopsies of secondary syphilis showed basal vacuolar changes with interface dermatitis accompanied by perivascular infiltration of CD4 T cells, CD8 T cells, CD20 B cells, spirochetes and plasma cells. These findings were consistent with those of secondary syphilis. We also found perivascular clusters of T cells that were reminiscent of iSALT, which we observed in a murine model. Moreover, some BECs expressed PNAd, indicating differentiation towards HEVs, a feature of TLOs. HEVs in lymphoid organs function as entry sites for naive lymphocytes and other immune cells in the circulation. Thus, iSALT-like structures in syphilitic lesions may be fuelled by migrating immune cells across these ectopic HEVs. We identified numerous CXCL13 fibroblast-like cells in the upper dermis. It is known that cytokines (such as CCL21, CXCL12 and CXCL13) contribute to TLO formation through recruitment of T cells and dendritic cells. Therefore, we speculate that CXCL13 helped the formation of iSALT-like structures in the upper dermis. The presence of dermal CXCL13 fibroblast-like cells and ectopic HEVs, but not compartmentalization of B and T cells, may indicate that iSALT is an early stage of TLO. The origin of CXCL13 fibroblast-like cells is unclear. In lymph nodes, FDCs express CXCL13 and recruit CXCR5 B cells to form B-cell follicles. However, the CXCL13 fibroblast-like cells in the syphilitic lesions did not react with CNA.42, a monoclonal antibody against FDC. Moreover, B cells were not colocalized with CXCL13 fibroblast-like cells.

Extraocular sebaceous carcinoma accompanied by invasive squamous cell carcinoma: The first case report and consideration of histogenesis

A 61‐year‐old man presented with a dome‐shaped nodule, 1.2 cm in size, with a central crater covered by keratinous material near the left lateral malleolus. Histological findings demonstrated a basophilic circular cone in the center, surrounded and sharply demarcated by a broad eosinophilic area. The central conical mass was composed mainly of atypical basaloid cells intermingled with scattered atypical sebaceous cells with scalloped nuclei and microvesicular cytoplasms, suggesting sebaceous carcinoma. The peripheral area consisted of atypical keratinizing squamoid cells without sebaceous cells, suggesting invasive squamous cell carcinoma. Atypical sebaceous cells were positive for adipophilin. Atypical basaloid cells were positive for 34βE12 and CAM5.2. Peripheral squamoid cells were positive for 34βB4 and 34βE12 throughout, and were positive for LHP1 in the superficial layer. We herein describe the first case of extraocular sebaceous carcinoma accompanied by invasive squamous cell carcinoma, which might have arisen from biphasic differentiation of cancer stem cells.

Improvement of erosive pustular dermatosis of the scalp following discontinuation of chemotherapy with afatinib

Erosive pustular dermatosis of the scalp (EPDS) is an idiopathic pustulosis that affects the scalp of the elderly. The onset of EPDS is often preceded by chemical or mechanical trauma such as sun damage, surgery, radiation, herpes zoster, and topical agents including tretinoin, imiquimod, latanoprost, ingenol mebutate, and minoxidil [1]. Here, we present a case of EPDS preceded by herpes zoster, which promptly resolved after the discontinuation of afatinib, an epidermal growth factor receptor (EGFR) inhibitor (EGFR-I) that the patient had been receiving for the treatment of lung cancer. A 73-year-old Japanese woman with non-small-cell lung carcinoma and multiple metastases was initially treated with whole-brain radiotherapy without notable adverse effects. Six months later, she received four courses of a combination therapy consisting of carboplatin, pemetrexed, and bevacizumab, followed by four additional courses of pemetrexed, with no effect. She was switched to oral afatinib at 20 mg/day on Day 0. On Day 47, she developed herpes zoster involving the right ophthalmic nerve dermatome, and was referred to our department (figure 1A). Acyclovir for seven days was effective and she was discharged on Day 59. On Day 119, she revisited us complaining of a folliculitis-like eruption without comedos and thick crusts on the area previously afflicted by zoster (figure 1B). The lesions were refractory to topical steroids. Neither bacterial nor fungal infection was detected in the lesions, except for methicillin-sensitive Staphylococcus aureus which we regarded as colonization since topical and oral antibiotics exerted no effect. A punch skin biopsy specimen on Day 263 showed a dense infiltration of neutrophils, lymphocytes, and plasma cells (figure 1E-G). The hair follicles were destroyed. Immunohistochemistry ruled out continuous herpes zoster infection (data not shown). Neurological examination did not reveal perception defects, excluding herpes zoster-induced trigeminal trophic syndrome. Considering the course of the disease, we diagnosed the patient with EPDS. On Day 363, afatinib was discontinued due to its inefficacy. Within a few months, the pustules and erythema of the scalp improved and crusts disappeared, leaving scarring alopecia (figure 1D). EPDS is diagnosed by excluding other pustular conditions. It is difficult to rule out EGFR-I-induced eruption, which resembles EPDS [2-4]. However, in our patient, the lesions were limited to the right ophthalmic nerve dermatome, where herpes zoster had developed two months prior. Furthermore, neither very thick crusts nor scarring alopecia are common in eruptions due to EGFR-I. Based on these clinical, histological, and microbial findings, we diagnosed the case as EPDS. A B

Maculopapular rash during a nadir period in a patient with acute myeloid leukaemia

Maculopapular rash (MPR) is caused by T cell-mediated hypersensitivity to infection or drugs [1]. The skin infiltrate consists largely of CD4+ and CD8+ T cells in the upper dermis. Little is known about the origin of these pathogenic T cells. Here, we report a case of MPR that occurred during a nadir period in a patient with acute myeloid leukaemia (AML) treated by chemotherapy. Despite the severe reduction of circulating leukocytes, T cells remained in the affected skin. Our case suggests the capability of developing effector memory T cells (TEM) to cause MPR even during a nadir period. A 53-year-old man had complaints of general fatigue, haematuria, and gingival bleeding. Complete blood count showed an increased number of white blood cells (WBCs; 358,000/ L [normal range: 3,200-9,600]), decreased haemoglobin (Hb; 6.6 g/dL [12.2-16.8]), and thrombocytopenia (43,000/ L [139,000-360,000]). The patient was hospitalised in our hospital. On Day 0, upon diagnosis of AML by a bone marrow examination, induction chemotherapy was commenced with daunorubicin (95 mg/day for two days) and Ara-C (100 mg/m2 for four days). Intravenous cefepime dihydrochloride (2 g/day), oral trimethoprim-sulfamethoxazole (150 mg/day), oral acyclovir (200 mg/day), and oral lansoprazole (20 mg/day) were concurrently administered. On Day 10, he experienced eruption on the bilateral thighs. On Day 11, he was referred to us for the exacerbating rash. On examination, maculopapular eruption had spread from the lower waist to the lower extremities. We suspected drug eruption and discontinued trimethoprim-sulfamethoxazole and acyclovir as possible culprits. A topical corticosteroid (0.05% betamethasone) was commenced. However, the erythematous lesion kept spreading and eventually covered 70% of the body surface on Day 17 (figures 1A, B). A blood test showed further elimination of WBCs (190/ L). Microscopic examination showed lymphocyte infiltration in the epidermis (figures 1C, D). There was slight spongiosis of the epidermis and perivascular infiltrates of lymphocytes in the upper dermis (figure 1C). This observation was consistent with MPR. Immunohistochemistry showed that the dermal infiltrates expressed either CD4 or CD8 (figures 1E, F). The infiltrating cells were dominated by CD45RO+ cells, but not CD45RA+ cells (data not shown). Most of the dermal T cells did not express CD69 or CD103 (data not shown). We stopped lansoprazole on Day 17, which eventually resulted in the recovery from MPR. MPR is a T cell-mediated skin disorder [1]. Naïve T cells in the peripheral blood play a major role in the induction of MPR [2]. However, a recent study showed that toxic epidermal necrolysis (TEN) can be caused by resident memory T cells (TRM) even during the nadir period [3]. In our case, the patient developed MPR during the nadir period. Although the white blood cell count decreased, the MPR became worse. This progression was intractable to topical steroid treatment, but the MPR was immediately cured after the cessation of lansoprazole. Thus, based on the clinical course, we suspect that the MPR in our case occurred in response to lansoprazole. A B