Exchange of intraoperative balloon occlusion of the internal iliac artery for the common iliac artery during cesarean hysterectomy in a patient with placenta percreta.

PATIENT: Female, 36 FINAL DIAGNOSIS: Pregnancy - placenta increta Symptoms: - MEDICATION: - Clinical Procedure: Cesarean hysterectomy Specialty: Obstetrics and Gynecology. OBJECTIVE: Unusual clinical course. BACKGROUND: The generally accepted treatment for placenta percreta is cesarean hysterectomy without attempts to detach the placenta. Preoperative internal iliac artery balloon occlusion (IIABO) has been widely performed to minimize blood loss during cesarean hysterectomy for an abnormal attachment of the placenta. Our case is the first reported case of common iliac artery balloon occlusion (CIABO) being more effective than IIABO for reducing blood loss during a cesarean hysterectomy in the same patient. CASE REPORT: We performed cesarean hysterectomy with IIABO in a 36-year-old Japanese female who had placenta percreta. However, there was still a large amount of blood loss. We immediately changed the balloon from the internal iliac artery to the common iliac artery, which visibly reduced the amount of blood loss. We finally achieved cesarean hysterectomy. CONCLUSIONS: CIABO was found to be more effective than IIABO for reducing blood loss during cesarean hysterectomy. Failure of IIABO can be explained by the presence of extensive anastomoses in the pelvic vasculature.

Mutagenicity and tumorigenicity of the four enantiopure bay-region 3,4-diol-1,2-epoxide isomers of dibenz[a,h]anthracene.

Each enantiomer of the diastereomeric pair of bay-region dibenz[a,h]anthracene 3,4-diol-1,2-epoxides in which the benzylic 4-hydroxyl group and epoxide oxygen are either cis (isomer 1) or trans (isomer 2) were evaluated for mutagenic activity. In strains TA 98 and TA 100 of Salmonella typhimurium, the diol epoxide with (1S,2R,3S,4R) absolute configuration [(-)-diol epoxide-1] had the highest mutagenic activity. In Chinese hamster V-79 cells, the diol epoxide with (1R,2S,3S,4R) absolute configuration [(+)-diol epoxide-2] had the highest mutagenic activity. The (1R,2S,3R,4S) diol epoxide [(+)-diol epoxide-1] also had appreciable activity, whereas the other two bay-region diol epoxide enantiomers had very low activity. In tumor studies, the (1R,2S,3S,4R) enantiomer was the only diol epoxide isomer tested that had strong activity as a tumor initiator on mouse skin and in causing lung and liver tumors when injected into newborn mice. This stereoisomer was about one-third as active as the parent hydrocarbon, dibenz[a,h]anthracene as a tumor initiator on mouse skin; it was several-fold more active than dibenz[a,h]anthracene as a lung and liver carcinogen when injected into newborn mice. (-)-(3R,4R)-3ß,4α-dihydroxy-3,4-dihydro-dibenz[a,h]anthracene [(-)-3,4-dihydrodiol] was slightly more active than dibenz[a,h]anthracene as a tumor initiator on mouse skin, whereas (+)-(3S,4S)-3α,4ß-dihydroxy-3,4-dihydro-dibenz[a,h]anthracene [(+)-3,4-dihydrodiol] had only very weak activity. The present investigation and previous studies with the corresponding four possible enantiopure bay-region diol epoxide enantiomers/diastereomers of benzo[a]pyrene, benz[a]anthracene, chrysene, benzo[c]phenanthrene, dibenz[c,h]acridine, dibenz[a,h]acridine and dibenz[a,h]anthracene indicate that the bay-region diol epoxide enantiomer with [R,S,S,R] absolute stereochemistry has high tumorigenic activity on mouse skin and in newborn mice.

Expression of p53 and p21(WAF-1), apoptosis, and proliferation of smooth muscle cells in normal myometrium during the menstrual cycle: implication of DNA damage and repair for leiomyoma development.

Uterine leiomyoma is the most common tumor in the female genital tract, although its pathogenesis remains unclear. Molecular analyses have demonstrated that each leiomyoma nodule is monoclonal and harbors various DNA abnormalities, suggesting that DNA damage in normal smooth muscle cells plays an important role in the pathogenesis of leiomyoma. The aim of this study is to evaluate precisely when and where DNA damage occurs in the myometrium. The localization of damaged, apoptotic, and proliferating cells was evaluated by immunohistochemical staining of p53, p21(WAF-1), TUNEL, and the cell proliferation marker, Ki-67, in normal myometrium during the menstrual cycle. p53-positive cells and p21(WAF-1)-positive cells were observed during the follicular phase, mostly in the submucosal layer of the myometrium. TUNEL-positive cells were sporadically identified in this layer during either the menstrual or follicular phase. In contrast, the number of Ki-67-positive cells was higher in the luteal phase. These results suggest that DNA damage, repair, and apoptosis occur cyclically in normal myometrium during the follicular phase. In addition, smooth muscle cells proliferate in the luteal phase, which may be a vulnerable period for DNA damage. Thus, these cyclic events during the menstrual cycle may contribute to a high incidence of leiomyoma development.

Extrapulmonary lymphangioleiomyomatosis in pelvic and paraaortic lymph nodes associated with uterine cancer: a report of 3 cases.

We report 3 cases of extrapulmonary lymphangioleiomyomatosis incidentally found in pelvic and paraaortic lymph nodes in association with uterine cancers. Three women, 47-year-old, 59-year-old, and 71-year-old, respectively, had uterine cancers and underwent hysterectomy, bilateral salpingo-oophorectomy, and pelvic and paraaortic lymph node excision. None of the 3 patients had tuberous sclerosis complex or lymphangioleiomyomatosis in other organs. None had any history of extrinsic hormonal administration. The postoperative pathologic diagnoses were uterine cervical squamous cell carcinoma for the first patient and endometrioid adenocarcinomas for the second and the third patients. Besides these malignant lesions, all 3 patients showed spindle cell proliferation, 2 to 5 mm in size, in 1 to 8 foci of the pelvic and paraaortic lymph nodes. The spindle cells having small polygonal nuclei and inconspicuous nucleoli with palely eosinophilic cytoplasm, reminiscent of immature smooth muscle cells, proliferated in nested and whorling patterns. Neither cellular atypia nor mitotic figures were observed. Immunohistochemically, these spindle cells were positive for α-Smooth Muscle Actin, Desmin, HMB45, Microphthalmia Transcription Factor, Estrogen receptor, and Progesterone receptor. And the network of the vascular-like channels surrounded by these spindle cells was positive for D2-40. From the pathologic and immunohistochemical findings, the spindle cell proliferation in the lymph nodes is best interpreted as lymphangioleiomyomatosis.

Classification using hierarchical clustering of tumor-infiltrating immune cells identifies poor prognostic ovarian cancers with high levels of COX expression.

Local immune status is influenced by the tumor microenvironment. This study aims to characterize the local immune/microenvironment status by examining tumor-infiltrating immune cells, as well as cyclooxygenase (COX) expression in tumor cells, and to analyze the relationship with the prognosis of ovarian cancers. Using immunohistochemical staining of 70 ovarian cancer specimens, the numbers of CD8+, CD57+, and CD1a+ cells infiltrating intraepithelial or stromal spaces were counted (six parameters). Hierarchical clustering was used to analyze the six parameters at one time. Expression of COX-1 and COX-2 in tumor cells was also analyzed by immunohistochemistry. Expression of both COX-1 and COX-2 was negatively correlated with intraepithelial CD8+ cells (P<0.05 for both). Hierarchical clustering using the six parameters classified ovarian cancers into three clusters. The overall and progression-free survival of cluster 1 with low CD8+ cell and high CD1a+ cell density was poorer than cluster 2 with high CD8+ cell density (P<0.05). The cluster classification did not correlate with clinical features, such as histology, stage, age, and amount of residual tumor. In a multivariate analysis, cluster 1 was an independent poor prognostic factor (P<0.05). Expression of both COX-1 and COX-2 was higher in cluster 1 than in cluster 2 (P<0.05, respectively). In conclusion, hierarchical clustering of tumor-infiltrating immune cells allows poor prognostic COX-high subgroup of ovarian cancer to be detected. COX may influence the pattern of tumor-infiltrating immune cells and prognosis in ovarian cancer.

Clinical significance of the NKG2D ligands, MICA/B and ULBP2 in ovarian cancer: high expression of ULBP2 is an indicator of poor prognosis.

OBJECTIVE: To investigate the clinical significance of the expression of the NKG2D ligands MICA/B and ULBP2 in ovarian cancer. METHODS: Eighty-two ovarian cancer patients and six patients without ovarian cancer from Department of Obstetrics and Gynecology of Kyoto University Hospital were enrolled in this study between 1993 and 2003. Expression of MICA/B, ULBP2, and CD57 in ovarian cancer tissue and normal ovary tissue was evaluated by immunohistochemical staining, and the relationship of these results to relevant clinical patient data was analyzed. Expression of MICs, ULBP2, and HLA-class I molecules in 33 ovarian cancer cell lines and two normal ovarian epithelial cell lines, as well as levels of soluble MICs and ULBP2 in the culture supernatants, were measured. RESULTS: Expression of MICA/B and ULBP2 was detected in 97.6 and 82.9% of ovarian cancer cells, respectively, whereas neither was expressed on normal ovarian epithelium. The expression of MICA/B in ovarian cancer was highly correlated with that of ULBP2. Strong expression of ULBP2 in ovarian cancer cells was correlated with less intraepithelial infiltration of T cells and bad prognoses for patients, suggesting that ULBP2 expression is a prognostic indicator in ovarian cancer. The expression of NKG2D ligands did not correlate with the levels of the soluble forms of the ligands. CONCLUSIONS: High expression of ULBP2 is an indicator of poor prognosis in ovarian cancer and may relate to T cell dysfunction in the tumor microenvironment.

Revised assignment of absolute configuration of the cis- and trans-N6-deoxyadenosine adducts at C14 of (+/-)-11beta,12alpha-dihydroxy-13alpha,14alpha-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene by stereoselective synthesis.

We have reassigned relative and absolute configurations by unambiguous stereoselective syntheses of the cis- (13s and 13R) and trans-N6-deoxyadenosine (dAdo) adduct diastereomers (14S and 14R) derived from (+/-)-11beta,12alpha-dihydroxy-13alpha,14alpha-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DB[a,l]P DE-2), previously reported by Li et al. [(1999) Chem. Res. Toxicol. 12, 758-767]. Two stereoselective methods, asymmetric aminohydroxylation of the (+/-)-trans-11,12-dihydrodiol (3) with 3',5'-di-O-(tert -butyldimethylsilyl)-2'-deoxyadenosine (4) and the highly stereoselective cis addition of 4 to (+/-)-DB[a,l]P DE-2 in hexafluoropropan-2-ol (HFP), were employed. Both afforded a 1:1 mixture of the cis-N6-dAdo adduct diastereomers, which were separated as triacetates (5S and 5R) in comparable yields (approximately 80%). The corresponding trans adduct diastereomers (10S and 10R) were obtained by coupling the aminotriol derived from trans opening of (+/-)-DB[a,l]P DE-2 with 6-fluoro-(2'-deoxy-3,5-di-tert-butyldimethylsilyloxy-beta-D-erythro-pentafuranosyl)purine (9) and subsequent acetylation in approximately 70% yield. The cis-5S and -5R and trans-10S and -10R were separately treated with 7% HF-pyridine followed by ammonolysis in NH3-saturated MeOH to give the dAdo adducts with all hydroxyl groups free (13S, 13R, 14S, and 14R). Comparison of the 1H NMR and CD spectra of these presently synthesized dAdo adducts with spectra of the previously reported compounds revealed that the interpretation of the 1H NMR and CD spectra and assignment of the relative stereochemistry (cis/trans) and absolute configuration made by Li et al. were at variance with our results. The above highly stereoselective syntheses of (+/-)-DB[a,l]P DE-2 adducted dAdo derivatives enabled efficient preparation of each of the four possible stereoisomeric 5'-dimethoxytrityl-3'-phosphoramidites for use in oligonucleotide synthesis.

3'-Intercalation of a N2-dG 1R-trans-anti-benzo[c]phenanthrene DNA adduct in an iterated (CG)3 repeat.

The conformation of the 1 R,2 S,3 R,4 S-benzo[ c]phenanthrene- N (2)-dG adduct, arising from trans opening of the (+)-1 S,2 R,3 R,4 S- anti-benzo[ c]phenanthrene diol epoxide, was examined in 5'- d(ATCGC XCGGCATG)-3'.5'-d(CATGCCG CGCGAT)-3', where X = 1 R,2 S,3 R,4 S-B[ c]P- N (2)-dG. This duplex, derived from the hisD3052 frameshift tester strain of Salmonella typhimurium, contains a (CG) 3 iterated repeat, a hotspot for frameshift mutagenesis. NMR experiments showed a disconnection in sequential NOE connectivity between X (4) and C (5), and in the complementary strand, they showed another disconnection between G (18) and C (19). In the imino region of the (1)H NMR spectrum, a resonance was observed at the adducted base pair X (4) x C (19). The X (4) N1H and G (18) N1H resonances shifted upfield as compared to the other guanine imino proton resonances. NOEs were observed between X (4) N1H and C (19) N (4)H and between C (5) N (4)H and G (18) N1H, indicating that base pairs X (4) x C (19) and C (5) x G (18) maintained Watson-Crick hydrogen bonding. No NOE connectivity was observed between X (4) and G (18) in the imino region of the spectrum. Chemical shift perturbations of greater than 0.1 ppm were localized at nucleotides X (4) and C (5) in the modified strand and G (18) and C (19) in the complementary strand. A total of 13 NOEs between the protons of the 1 R-B[ c]Ph moiety and the DNA were observed between B[ c]Ph and major groove aromatic or amine protons at base pairs X (4) x C (19) and 3'-neighbor C (5) x G (18). Structural refinement was achieved using molecular dynamics calculations restrained by interproton distances and torsion angle restraints obtained from NMR data. The B[ c]Ph moiety intercalated on the 3'-face of the X (4) x C (19) base pair such that the terminal ring of 1 R-B[ c]Ph threaded the duplex and faced into the major groove. The torsion angle alpha' [X (4)]-N3-C2-N2-B[ c]Ph]-C1 was calculated to be -177 degrees, maintaining an orientation in which the X (4) exocyclic amine remained in plane with the purine. The torsion angle beta' [X (4)]-C2-N2-[B[ c]Ph]-C1-C2 was calculated to be 75 degrees. This value governed the 3'-orientation of the B[ c]Ph moiety with respect to X (4). The helical rise between base pairs X (4) x C (19) and C (5) x G (18) increased and resulted in unwinding of the right-handed helix. The aromatic rings of the B[ c]Ph moiety were below the Watson-Crick hydrogen-bonding face of the modified base pair X (4) x C (19). The B[c]Ph moiety was stacked above nucleotide G (18), in the complementary strand.

Immunostimulatory effect of Fms-like tyrosine kinase 3 ligand on peripheral monocyte-derived dendritic cells and natural killer cells: utilization for ovarian cancer treatment.

Systemic administration of Fms-like tyrosine kinase 3 ligand (FLT3L) has been considered to be a major route of delivery for tumor immunotherapy because expression of its receptor, FLT3, was detected predominantly in hematopoetic progenitor cells. However, several studies indicate that FLT3L locally overexpressed in tumor or dendritic cells (DCs) also shows an anti-tumor effect. In the current study, we found that FLT3 expression is not present in monocytes but is instead induced in DCs through the differentiation process resulting from stimulation by GM-CSF and IL-4. Addition of FLT3L further augmented FLT3 induction and also increased CD40 expression in DCs, leading to enhanced induction of lymphoblastoid cell line-targeted cytotoxic T-lymphocyte response and CD107a mobilization in CD8+ T cells. Furthermore, FLT3L also induced FLT3 expression in peripheral blood NK cells that showed an enhanced response detected by CD107a mobilization. In a murine ovarian cancer model, locally expressed FLT3L showed anti-tumor effects. Collectively, the current study indicates that FLT3L has an immunostimulatory effect on peripheral blood cells and FLT3L targeted to mature peripheral blood cells may serve as a useful tool for cancer immunotherapy.

Fluorinated alcohol mediated displacement of the C10 acetoxy group of benzo[a]pyrene-7,8,9,10-tetrahydrotetraol tetraacetates: a new route to diol epoxide-deoxyguanosine adducts.

We describe a novel trifluoroethanol (TFE) or hexafluoropropan-2-ol (HFP) mediated substitution reaction of the bay-region C10 acetoxy group in four stereoisomeric 7,8,9,10-tetraacetoxy-7,8,9,10-tetrahydrobenzo[a]pyrenes (tetraol tetraacetates, two pairs of cis and trans isomers at the 9,10 positions) by the exocyclic N2-amino group of O6-allyl-3',5'-di-O-(tert-butyldimethylsilyl)-2'-deoxyguanosine (3). The tetraacetates are derived from cis and trans hydrolysis of (+/-)-7beta,8alpha-dihydroxy-9beta,10beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]P DE-1) and of (+/-)-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]P DE-2) at C-10 followed by acetylation. Excellent yields and high regioselectivity were observed. Similar cis/trans product ratios were observed for each set of cis and trans tetraol tetraacetates derived from DE-1 ( approximately 75/25) and from DE-2 (approximately 67/33) in HFP. This strongly suggests that the substitution proceeds via an SN1 mechanism involving a carbocation intermediate that is common to the cis and trans tetraacetates. Since it is likely that the cis and trans products from 3 arise from different conformations of the carbocation, its lifetime must be sufficiently long to permit conformational equilibration before its capture by the purine nucleophile. The corresponding reaction of (+/-)-9alpha-bromo-7beta,8alpha,10beta-triacetoxy-7,8,9,10-tetrahydrobenzo[a]pyrene with 3 in HFP was highly regio- and stereoselective and gave exclusively trans 10beta-adducts. This newly developed substitution reaction provides an attractive alternative synthetic strategy for the preparation of polycyclic hydrocarbon adducted oligonucleotide building blocks.

A structural gap in Dpo4 supports mutagenic bypass of a major benzo[a]pyrene dG adduct in DNA through template misalignment.

Erroneous replication of lesions in DNA by DNA polymerases leads to elevated mutagenesis. To understand the molecular basis of DNA damage-induced mutagenesis, we have determined the x-ray structures of the Y-family polymerase, Dpo4, in complex with a DNA substrate containing a bulky DNA lesion and incoming nucleotides. The DNA lesion is derived from an environmentally widespread carcinogenic polycyclic aromatic hydrocarbon, benzo[a]pyrene (BP). The potent carcinogen BP is metabolized to diol epoxides that form covalent adducts with cellular DNA. In the present study, the major BP diol epoxide adduct in DNA, BP-N(2)-deoxyguanosine (BP-dG), was placed at a template-primer junction. Three ternary complexes reveal replication blockage, extension past a mismatched lesion, and a -1 frameshift mutation. In the productive structures, the bulky adduct is flipped/looped out of the DNA helix into a structural gap between the little finger and core domains. Sequestering of the hydrophobic BP adduct in this new substrate-binding site permits the DNA to exhibit normal geometry for primer extension. Extrusion of the lesion by template misalignment allows the base 5' to the adduct to serve as the template, resulting in a -1 frameshift. Subsequent strand realignment produces a mismatched base opposite the lesion. These structural observations, in combination with replication and mutagenesis data, suggest a model in which the additional substrate-binding site stabilizes the extrahelical nucleotide for lesion bypass and generation of base substitutions and -1 frameshift mutations.

Fluorinated alcohol mediated control over cis vs trans opening of benzo[a]pyrene-7,8-diol 9,10-epoxides at C-10 by the exocyclic amino groups of O6-allyl protected deoxyguanosine and of deoxyadenosine.

A detailed study was carried out on the stereoselective control of cis- vs trans-opening of (+/-)-7beta,8alpha-dihydroxy-9beta,10beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene {B[a]P DE-1 (1)} and (+/-)-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene {B[a]P DE-2 (2)} at C-10 by the exocyclic amino groups of protected purine nucleosides in the fluorinated alcohols trifluoroethanol (TFE), hexafluoropropan-2-ol (HFP), and perfluoro-tert-butanol (PFTB). Addition of the 2-amino group of O6-allyl-3',5'-di-O-(tert-butyldimethylsilyl)-2'-deoxyguanosine (3) and of the 6-amino group of 3',5'-di-O-(tert-butyldimethylsilyl)-2'-deoxyadenosine (4) occurs at C-10 of the epoxides. The observed cis:trans ratio for the reaction of DE-1 (1) in the presence of 5 equiv of 3 over the range of 10-250 equiv of fluorinated alcohol varied from 53:47 to 87:13 for TFE, 60:40 to 92:8 for HFP, and 52:48 to 73:27 for PFTB. The corresponding ratios for DE-2 (2) varied from 22:78 to 72:28 for HFP under the same set of conditions. In contrast, the corresponding ratios for DE-2 (2) remained unchanged ( approximately 40:60) for TFE and for PFTB over the range of 25-250 molar equiv. Unlike the addition of the dGuo reactant 3, the corresponding addition of the dAdo reactant (4) to the DEs (1 or 2) in over 25 molar equiv of TFE occurred highly stereoselectively to afford only cis adducts for both DEs. A highly efficient HPLC separation of dGuo adduct diastereomers derived from DE-2 (2) was developed using acetone as a modifier in CH2Cl2 or in n-hexane. Through the use of varying molar ratios of the different fluorinated alcohols described above and the newly developed HPLC separation method, the four possible phosphoramidites (cis/trans, R/S) of the B[a]P DE-2 N2-dGuo adducts can be prepared in an efficient fashion on gram scale for use in oligonucleotide synthesis.

Synthesis and absolute configuration of cis- and trans-opened cyclopenta[cd]pyrene 3,4-oxide N2-deoxyguanosine adducts: conversion to phosphoramidites for oligonucleotide synthesis.

Fluorinated alcohols such as trifluoroethanol (TFE) or hexafluoropropan-2-ol (HFP) catalyze addition of the N2-amino group of O6-allyl-3',5'-di-O-(tert-butyldimethylsilyl)-2'-deoxyguanosine (3) to cyclopenta[cd]pyrene 3,4-oxide (CPPO). The reaction occurs via a carbocation intermediate at C-3 such that cis- and trans-opened dGuo adducts are formed in a combined yield of approximately 37% together with the 4-ketone and a cis-opened solvent adduct. Fluorinated alcohol-mediated regioselective substitution at C-3 of the CPP cis- (11) and trans-3,4-dihydrodiol diacetates (15) with the N2-amino group of 3 proceeded smoothly to give the O6-allyl di-(tert-butyldimethylsilyl) cis- and trans-opened dGuo-adduct acetates (8a,b and 9a,b) in 75-85% yields. The cis-opened adducts predominated (56-70%) from both 11 and 15. Interestingly, trans-3-acetoxy-4-bromo-3,4-dihydro-CPP and 3 in TFE or HFP gave a mixture of 8a,b and 9a,b in 75-85% yield with cis:trans adduct ratios similar to those observed for 11 and 15. This observation is consistent with initial formation of a cyclic acetoxonium intermediate followed by formation of the same carbocation as that derived from 11 or 15. Absolute configurations of 8a,b and 9a,b were assigned by using enantiomerically pure (+)-trans-[3S.4S]-dihydrodiol as the starting material, which afforded a single cis-[3R,4S]-dGuo adduct and a single trans-[3S,4S]-dGuo adduct. The optically active trans-3,4-dihydrodiols were obtained by HPLC separation of their bis-(-)-menthoxyacetates. Their absolute configuration was determined by several empirical methods in addition to application of the exciton chirality CD method to their bis-(p-N,N-dimethylamino)benzoates. Removal of all blocking groups from the protected cis- and trans-opened dGuo adducts (8a,b and 9a,b) in three steps (overall yields of >70%) gave the free dGuo adducts, which are useful markers for DNA-binding studies. Adducts 8a,b and 9a,b were also converted to the appropriately protected phosphoramidites in three steps (overall yields of 72-81%).

Programmed cell death 1 ligand 1 and tumor-infiltrating CD8+ T lymphocytes are prognostic factors of human ovarian cancer.

The ligands for programmed cell death 1 (PD-1), an immunoinhibitory receptor belonging to CD28/cytotoxic T lymphocyte antigen 4 family, are PD-1 ligand 1 and 2 (PD-Ls). Recent reports suggest that the aberrant expression of PD-Ls on tumor cells impairs antitumor immunity, resulting in the immune evasion of the tumor cells. Although an inverse correlation between the expression level of PD-Ls and patients' prognosis has been reported for several malignant tumors, the follow-up period was limited because of the lack of the antibody (Ab) applicable to paraffin-embedded specimens. Here we generated a new Ab against PD-1 ligand 1 (PD-L1) and analyzed the expression level of PD-Ls in human ovarian cancer using paraffin-embedded specimens. Patients with higher expression of PD-L1 had a significantly poorer prognosis than patients with lower expression. Although patients with higher expression of PD-1 ligand 2 also had a poorer prognosis, the difference was not statistically significant. A significant inverse correlation was observed between PD-L1 expression and the intraepithelial CD8(+) T lymphocyte count, suggesting that PD-L1 on tumor cells directly suppresses antitumor CD8(+) T cells. Multivariate analysis showed the expression of PD-L1 on tumor cells and intraepithelial CD8(+) T lymphocyte count are independent prognostic factors. The PD-1/PD-L pathway can be a good target for restoring antitumor immunity in ovarian cancer.

3'-H-phosphonate synthesis of chiral benzo[a]pyrene diol epoxide adducts at N(2) of deoxyguanosine in oligonucleotides.

A synthetic route to oligonucleotides containing N(2)-deoxyguanosine adducts at C-10 of the enantiomeric 7,8-diol 9,10-epoxides of 7,8,9,10-tetrahydrobenzo[a]pyrene in which the epoxide oxygen and the 7-hydroxyl group are trans is described. The present adducts result from the trans addition of N(2) of deoxyguanosine to the epoxide at C-10. Our synthesis proceeds via preparation of the 3'-H-phosphonate of a suitably protected deoxyguanosine N(2)-adduct. The blocking groups consisted of O(6)-allyl on the deoxyguanosine, acetates on the 7-, 8-, and 9-hydroxyl groups of the hydrocarbon moiety, and dimethoxytrityl on the 5'-hydroxyl group of the sugar. These blocking groups are well suited to oligonucleotide synthesis on solid supports. The free 3'-hydroxyl group of this nucleoside adduct was readily converted to its 3'-H-phosphonate with diphenyl phosphite in pyridine in high yield for both the 10R and 10S isomers. For synthesis of oligonucleotides, the first several nucleotides were incorporated onto the solid support with an automated synthesizer using standard phosphoramidite chemistry. The adducted deoxyguanilic acid residue was introduced as the H-phosphonate in a manual step (80% yield), followed by completion of the sequence on the synthesizer. Although a 10-fold excess of the 3'-H-phosphonate was used in the manual coupling step, as much as 70% of the reactant could be recovered. The 3'-H-phosphonate of the protected 10S nucleoside adduct was converted to the unblocked nucleotide adduct, various salts of which failed to form crystals suitable for X-ray analysis. Although submilligram quantities of this compound have been formed as mixed diastereomers by direct reaction of deoxyguanylic acid with racemic diol epoxide, the present study represents the first actual synthesis of the major DNA adduct formed from benzo[a]pyrene in mammals as its 3'-phosphate.

Structure of DNA polymerase beta with a benzo[c]phenanthrene diol epoxide-adducted template exhibits mutagenic features.

We have determined the crystal structure of the human base excision repair enzyme DNA polymerase beta (Pol beta) in complex with a 1-nt gapped DNA substrate containing a template N2-guanine adduct of the tumorigenic (-)-benzo[c]phenanthrene 4R,3S-diol 2S,1R-epoxide in the gap. Nucleotide insertion opposite this adduct favors incorrect purine nucleotides over the correct dCMP and hence can be mutagenic. The structure reveals that the phenanthrene ring system is stacked with the base pair immediately 3' to the modified guanine, thereby occluding the normal binding site for the correct incoming nucleoside triphosphate. The modified guanine base is displaced downstream and prevents the polymerase from achieving the catalytically competent closed conformation. The incoming nucleotide binding pocket is distorted, and the adducted deoxyguanosine is in a syn conformation, exposing its Hoogsteen edge, which can hydrogen-bond with dATP or dGTP. In a reconstituted base excision repair system, repair of a deaminated cytosine (i.e., uracil) opposite the adducted guanine was dramatically decreased at the Pol beta insertion step, but not blocked. The efficiency of gap-filling dCMP insertion opposite the adduct was diminished by >6 orders of magnitude compared with an unadducted templating guanine. In contrast, significant misinsertion of purine nucleotides (but not dTMP) opposite the adducted guanine was observed. Pol beta also misinserts a purine nucleotide opposite the adduct with ungapped DNA and exhibits limited bypass DNA synthesis. These results indicate that Pol beta-dependent base excision repair of uracil opposite, or replication through, this bulky DNA adduct can be mutagenic.

Effect of DNA modifications on DNA processing by HIV-1 integrase and inhibitor binding: role of DNA backbone flexibility and an open catalytic site.

Integration of the viral cDNA into host chromosomes is required for viral replication. Human immunodeficiency virus integrase catalyzes two sequential reactions, 3'-processing (3'-P) and strand transfer (ST). The first integrase inhibitors are undergoing clinical trial, but interactions of inhibitors with integrase and DNA are not well understood in the absence of a co-crystal structure. To increase our understanding of integrase interactions with DNA, we examined integrase catalysis with oligonucleotides containing DNA backbone, base, and groove modifications placed at unique positions surrounding the 3'-processing site. 3'-Processing was blocked with substrates containing constrained sugars and alpha-anomeric residues, suggesting that integrase requires flexibility of the phosphodiester backbone at the 3'-P site. Of several benzo[a]pyrene 7,8-diol 9,10-epoxide (BaP DE) adducts tested, only the adduct in the minor groove at the 3'-P site inhibited 3'-P, suggesting the importance of the minor groove contacts for 3'-P. ST occurred in the presence of bulky BaP DE DNA adducts attached to the end of the viral DNA suggesting opening of the active site for ST. Position-specific effects of these BaP DE DNA adducts were found for inhibition of integrase by diketo acids. Together, these results demonstrate the importance of DNA structure and specific contacts with the viral DNA processing site for inhibition by integrase inhibitors.

Facile interstrand migration of the hydrocarbon moiety of a dibenzo[a,l]pyrene 11,12-diol 13,14-epoxide adduct at N2 of deoxyguanosine in a duplex oligonucleotide.

When a synthesized deoxyribonucleotide duplex, 5'-CCATCGCTACC-3'.5'-GGTAGCGATGG-3', containing a trans 14R dibenzo[a,l]pyrene (DB[a,l]P) adduct, corresponding to trans opening of the (+)-(11S,12R)-diol (13R,14S)-epoxide by N (2) of the central G residue, was allowed to stand for 2-6 days at ambient temperature in neutral aqueous solution, three new products were observed on denaturing HPLC. One of these corresponded to loss of the DB[a,l]P moiety from the original adducted strand to give an 11-mer with an unmodified central dG. The other two products resulted from a highly unexpected migration of the hydrocarbon moiety to either dG5 or dG7 of the complementary strand, 5'-GGTAG5CG7ATGG-3'. Enzymatic hydrolysis of the two 11-mer migration products followed by CD spectroscopy of the isolated adducted nucleosides indicated that, in both cases, the hydrocarbon moiety had undergone configurational inversion at C14 to give the cis 14S DB[a,l]P dG adduct. MS/MS and partial enzymatic hydrolysis showed that the major 11-mer had the hydrocarbon at dG7. Two 11-mer oligonucleotides were synthesized with a single cis 14S DB[a,l]P dG adduct either at G7 or at G5 and were found to be chromatographically identical to the major and minor migration products, respectively. Although HPLC evidence suggested that a small extent of hydrocarbon migration from the trans 14S DB[a,l]P dG diastereomer also occurred, the very small amount of presumed migration products from this isomer precluded their detailed characterization. This interstrand migration appears unique to DB[a,l]P adducts and has not been observed for their fjord-region benzo[c]phenanthrene or bay-region benzo[a]pyrene analogues.

Foxp3+ CD25+ CD4+ natural regulatory T cells in dominant self-tolerance and autoimmune disease.

Naturally arising CD25+ CD4+ regulatory T (Treg) cells, most of which are produced by the normal thymus as a functionally mature T-cell subpopulation, play key roles in the maintenance of immunologic self-tolerance and negative control of a variety of physiological and pathological immune responses. Natural Tregs specifically express Foxp3, a transcription factor that plays a critical role in their development and function. Complete depletion of Foxp3-expressing natural Tregs, whether they are CD25+ or CD25-, activates even weak or rare self-reactive T-cell clones, inducing severe and widespread autoimmune/inflammatory diseases. Natural Tregs are highly dependent on exogenously provided interleukin (IL)-2 for their survival in the periphery. In addition to Foxp3 and IL-2/IL-2 receptor, deficiency or functional alteration of other molecules, expressed by T cells or non-T cells, may affect the development/function of Tregs or self-reactive T cells, or both, and consequently tip the peripheral balance between the two populations toward autoimmunity. Elucidation of the molecular and cellular basis of this Treg-mediated active maintenance of self-tolerance will facilitate both our understanding of the pathogenetic mechanism of autoimmune disease and the development of novel methods of autoimmune disease prevention and treatment via enhancing and re-establishing Treg-mediated dominant control over self-reactive T cells.

Intercalating polycyclic aromatic hydrocarbon-DNA adducts poison DNA religation by Vaccinia topoisomerase and act as roadblocks to digestion by exonuclease III.

Polycyclic aromatic hydrocarbon (PAH)-DNA adducts pervert the execution or fidelity of enzymatic DNA transactions and cause mutations and cancer. Here, we examine the effects of intercalating PAH-DNA adducts on the religation reaction of vaccinia DNA topoisomerase, a prototypal type IB topoisomerase (TopIB), and the 3' end-resection reaction of Escherichia coli exonuclease III (ExoIII), a DNA repair enzyme. Vaccinia TopIB forms a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate at a target site 5'-C(+5)C(+4)C(+3)T(+2)T(+1)p / N(-1) in duplex DNA. The rate of the forward cleavage reaction is suppressed to varying degrees by benzo[a]pyrene (BP) or benzo[c]phenanthrene (BPh) adducts at purine bases within the 3'-G(+5)G(+4)G(+3)A(+2)A(+1)T(-1)A(-2) sequence of the nonscissile strand. We report that BP adducts at the +1 and -2 N6-deoxyadenosine (dA) positions flanking the scissile phosphodiester slow the rate of DNA religation to a greater degree than they do the cleavage rate. By increasing the cleavage equilibrium constant > or = 10-fold, the BPdA adducts, which are intercalated via the major groove, act as TopIB poisons. With respect to ExoIII, we find that (i) single BPdA adducts act as durable roadblocks to ExoIII digestion, which is halted at sites 1 and 2 nucleotides prior to the modified base; (ii) single BPhdA adducts, which also intercalate via the major groove, elicit a transient pause prior to the lesion, which is eventually resected; and (iii) BPh adducts at N2-deoxyguanosine, which intercalate via the minor groove, are durable impediments to ExoIII digestion. These results highlight the sensitivity of repair outcomes to the structure of the PAH ring system and whether intercalation occurs via the major or minor groove.