TRIM27 expression is associated with poor prognosis in sinonasal mucosal melanoma.

BACKGROUND: Tripartite motif-containing 27 (TRIM27) has been implicated in the progression of various cancers. However, the role of TRIM27 in sinonasal mucosal melanoma (SNMM) remains poorly understood. MATERIALS & METHODS: We retrospectively examined 28 patients with SNMM treated with between 2003 and 2021. We undertook immunohistochemical analysis of TRIM27, Ki-67, and p-Akt1 expression in SNMM tissues. We also investigated the relationship between TRIM27 expression and clinical characteristics, prognosis, Ki-67 as a tumor growth potential marker, and p-Akt1 as one of the prognostic factors in mucosal melanoma. RESULTS: TRIM27 expression was significantly higher in T4 disease than in T3 disease and was higher in stage IV than in stage III. Patients with high-TRIM27 SNMM had a significantly poorer prognosis in terms of overall survival (OS) and disease-free survival.There was also a significantly higher rate of distant metastasis. Univariate analysis for OS revealed that TRIM27 and T classification were significant poor prognostic factors. In addition, the Ki-67 positive score and the p-Akt1 total staining score were significantly higher in the high-TRIM27 group than in the low-TRIM27 group. CONCLUSIONS: High TRIM27 expression in SNMM was associated with advanced T classification, poor prognosis and distant metastasis. We suggest that TRIM27 has potential as a novel biomarker for prognosis in SNMM.

Autoantibodies to killer cell immunoglobulin-like receptor 3DL1 in patients with systemic lupus erythematosus.

A genetic variant of the killer immunoglobulin-like receptor 3DL1 (KIR3DL1) has been found in patients with systemic lupus erythematosus (SLE). Herein, we investigated the presence of autoantibodies to KIR3DL1 in a cohort of patients with SLE. We tested sera from 28 patients with SLE, 11 patients with rheumatoid arthritis (RA) and 17 healthy control subjects for anti-KIR3DL1 activity by an enzyme-linked immunosorbent assay (ELISA) using recombinant KIR3DL1-enhanced green fluorescent protein (EGFP) and EGFP proteins. Anti-KIR3DL1 antibodies were detected in 22 (79%) of the 28 patients with SLE, whereas they were present in only three (27%) of the 11 patients with RA examined. Notably, 10 (91%) of the 11 samples from patients with SLE prior to therapy had anti-KIR3DL1 antibodies. None of the samples from healthy donors were positive for the antibodies. Here, we report the presence of anti-KIR3DL1 antibodies in the sera of patients with SLE for the first time. Anti-KIR3DL1 autoantibodies may be involved in the pathogenesis of autoimmune diseases.

Integrating quantitative morphological and intratumoural textural characteristics in FDG-PET for the prediction of prognosis in pharynx squamous cell carcinoma patients.

AIM: To assess potential prognostic factors in pharynx squamous cell carcinoma (SCC) patients by quantitative morphological and intratumoural characteristics obtained by 2-[18F]-fluoro-2-deoxy-d-glucose positron-emission tomography/computed tomography (FDG-PET/CT). MATERIALS AND METHODS: The cases of 54 patients with pharynx SCC who underwent chemoradiation therapy were analysed retrospectively. Using their FDG-PET data, the quantitative morphological and intratumoural characteristics of 14 parameters were calculated. The progression-free survival (PFS) and overall survival (OS) information was obtained from patient medical records. Univariate and multivariate analyses were performed to assess the 14 quantitative parameters as well as the T-stage, N-stage, and tumour location data for their relation to PFS and OS. When an independent predictor was suggested in the multivariate analysis, the parameter was further assessed using the Kaplan-Meier method. RESULTS: In the assessment of PFS, the univariate and multivariate analyses indicated the following as independent predictors: the texture parameter of homogeneity and the morphological parameter of sphericity. In the Kaplan-Meier analysis, the PFS rate was significantly improved in the patients who had both a higher value of homogeneity (p=0.01) and a higher value of sphericity (p=0.002). With the combined use of homogeneity and sphericity, the patients with different PFS rates could be divided more clearly. CONCLUSION: The quantitative parameters of homogeneity and sphericity obtained by FDG-PET can be useful for the prediction of the PFS of pharynx SCC patients, especially when used in combination.

Superselective intra-arterial cisplatin infusion and concomitant radiotherapy for maxillary sinus cancer.

BACKGROUND: The purpose of this study was to evaluate the efficacy of superselective cisplatin infusion with concomitant radiotherapy (RADPLAT) for previously untreated patients with the squamous cell carcinoma of maxillary sinus (SCC-MS). METHODS: Between 1999 and 2010, 54 patients were given superselective intra-arterial infusions of cisplatin (100-120 mg m(-2) per week) with simultaneous intra-venous infusions of thiosulfate to neutralise cisplatin toxicity and conventional radiotherapy (65-70 Gy). RESULTS: One patient (1.9%) was diagnosed with T2, 14 (25.9%) with T3, 27 (50%) with T4a, and 12 (22.2%) with T4b disease. Lymph-node involvement was present in 12 patients (22.2%). During the median follow-up period of 6.4 years, the 5-year local progression-free and overall survival rates were 65.8 and 67.9% for all patients, respectively. No patient died as a result of treatment toxicity or experienced a cerebrovascular accident. Osteonecrosis (n=5), brain necrosis (n=1), and ocular/visual problems (n=14) were observed as late adverse reactions. CONCLUSION: We have shown excellent overall survival and local progression-free rate in SCC-MS patients treated by RADPLAT with acceptable rates of acute and late toxicity. A multi-institutional trial is needed to prove that this strategy is a feasible and effective approach for the treatment of SCC-MS.

The production of vesicular stomatitis virus by antigen- or mitogen-stimulated lymphocytes and continuous lymphoblastoid lines.

A variety of lymphoid cell populations were examined in terms of their ability to replicate vesicular stomatitis virus (VSV), a lytic, RNA-containing virus maturing at the cell surface. The number of cells capable of producing VSV was estimated in terms of infectious centers by the virus plaque assay (VPA), and morphologically by electron microscopy (EM). The lymphoid cells examined in this study included: (a) lymph node cells from delayed hypersensitive guinea pigs stimulated by specific antigen, (b) mouse spleen cells activated by selective bone marrow-derived (B) cell and thymus derived (T) cell mitogens, and (c) cells of human and murine continuous lymphoblastoid or lymphoma lines. In unstimulated cultures of guinea pig lymph node cells there is a background of approximately 1 in 1,000 cells which produces VSV; in purified protein derivative (PPD)-stimulated cultures the number of cells producing virus was 1.6% in the VPA and 1.9% by EM. These cells were large lymphocytes with some morphological features of transformed lymphocytes but were not typical blast cells. A few macrophages were associated with virus in both stimulated and control cultures. These observations indicate that (a) cells responsive to antigens, as detected by a marker virus, were lymphocytes; (b) cells other than lymphocytes (macrophages) were capable of replicating VSV even without antigenic stimulation; and (c) the correlation of results obtained by VPA and morphologic examination was usually quite good. Of the total number of mouse spleen cells stimulated with concanavalin (Con A), a T cell mitogen, 4.5 (EM)-5.7% (VPA) were associated with VSV. These were characteristic transformed lymphocytes, similar to phytohemagglutinin (PHA)-stimulated human lymphocytes. In contrast Escherichia coli lipopolysaccharide (LPS)-treated mouse spleen cultures contained lower numbers of virus plaque-forming cells. The majority of such cells associated with virus displayed extensive rough endoplasmic reticulum. Two cultured murine lymphomas containing lymphocytes with the theta surface marker (L5178Y and EL-4) showed a 15-100-fold higher incidence of virus-producing cells than leukemias (L1210 and C57Bl/6) which did not carry this marker. Similarly, the L2C guinea pig leukemia, a known B cell leukemia, yielded a low percent of virus plaque-forming cells (<2%). However, MOPC-104, a plasma cell tumor presumed to be of B cell origin, was found to be an efficient virus producer. There was a wide variation in the efficiency of VSV replication among human lymphoblastoid lines. One line, Wil-2, produced 80% infectious centers after 24 h of exposure to VSV, and all cells were associated with virus at the EM level. The relationship between the virus-producing cells and different lymphocyte subpopulations as well as the efficiency of the two assays for studying virus-producing lymphocytes is discussed.

Regulation of the growth and functions of cloned murine large granular lymphocyte lines by resident macrophages.

Using cloned lines with the morphology of large granular lymphocytes (LGL) from BALB/c mice, we studied the exact requirements for proliferation and their functional characteristics, as well as their regulation. Although these cloned LGL lines were interleukin 2 (IL-2) dependent for growth, experiments using human recombinant IL-2 (rIL-2), known to be active on murine cells, indicated that IL-2 was a necessary but not sufficient factor. Coexistance of normal macrophages in addition to rIL-2 was found to support continuous proliferation of cloned LGL in vitro. This role of macrophages could be replaced by partially purified IL-1 derived from macrophage-conditioned medium. An IL-2 binding assay using 125I-rIL-2 suggested that the role of normal macrophages was to selectively induce and/or maintain high affinity IL-2 receptors (IL-2R) (Kd, 0.2-0.5 nM) without affecting low affinity ones (Kd, 10-30 nM). Functional studies indicated that most of the LGL clones killed various combinations of representative groups of natural killer (NK)-susceptible target cells, including leukemic cells (YAC-1, RL male 1), virus-infected cells (HeLa-measles, HeLa-herpes simplex virus), and normal bone marrow cells (BMC), whereas none of them affected any of NK-resistant target cells, including uninfected HeLa cells. Some of these clones also suppressed in vitro hematopoiesis. Such characteristic cytotoxic spectra, as well as serological phenotypes (Thy-1+, Lyt-1-2-, asialo GM1-positive, T200+, TdT-, Fc receptor-positive) indicated that these LGL clones exactly represent endogenous NK cells, rather than a variety of anomalous killer cells generated in various culture conditions. Although there was significant heterogeneity of cytotoxic spectrum among LGL clones, no clonotypic distribution of specificities was observed. Normal macrophages were found to modulate the functional expression of LGL clones. They augmented the cytotoxic potential of the clones against leukemic and virus-infected targets, but suppressed intrinsic reactivity against normal BMC. Similarly, LGL clones maintained with macrophages showed much less suppressive effect on in vitro hematopoiesis. The present observations on the interaction of cloned LGL and normal macrophages provide a basic explanation for the mechanisms by which the immediate responsiveness to IL-2 of the NK effector system, without exogenous stimulation, and the functional selectivity toward abnormal rather than normal cells, are actively maintained in vivo.

Differentiation in vitro of T3+ large granular lymphocytes with characteristic cytotoxic activity from an isolated hematopoietic progenitor colony.

Blast colonies were developed by rIL-3 from the spleen cells of mice pretreated with 5-fluorouracil (5-FU) in the methylcellulose cultures. When such IL-3-induced blast colonies were individually lifted up and recultured in the presence of rIL-3 and recombinant erythropoietin (rEpo), a variety of hematopoietic colonies developed from every single colony, including neutrophils, macrophages, eosinophils, megakaryocytes, mast cells, and erythroblasts. The results indicated that IL-3-induced blast colonies consisted of multipotential hematopoietic progenitor cells. By culturing individual IL-3-induced blast colonies in the presence of rIL-2 and irradiated peritoneal macrophages, on the other hand, the proliferation of homogeneous lymphoid cells was observed in 5 of 24 wells, each of which received a single blast colony. Morphologically, they were typical large granular lymphocytes (LGL), and thus it was indicated that LGL could be differentiated directly from the hematopoietic progenitor cells utterly in vitro by rIL-2 and accessory macrophages. From one of these culture wells, a continuous LGL line, IL3B1, was successfully obtained. The proliferation of IL3B1 was dependent on IL-2 in the presence of accessory macrophages, but they no longer responded to IL-3, nor to another T cell growth factor, IL-4. Flow cytometric analysis indicated that the phenotype of IL3B1 was Thy-1+,T3+,L3T4-,Lyt-2-,T200+ Asialo GM1+, whereas that of original IL-3-induced blast cells was Thy-1+,T3-,L3T4-,Lyt-2-,B220-. The results suggested that the expression of T3 molecules was induced in the process of LGL differentiation from the hematopoietic progenitor cells in vitro. Conforming to this, it was revealed that both gamma and beta chain genes of the TCR were rearranged in IL3B1. Northern blot analysis indicated that IL3B1 had abundant mRNA for gamma chain, while mRNA for beta chain was rather faint. Functionally, IL3B1 exhibited typical NK-patterned cytotoxic activity against a panel of tumor cell targets. In addition, they showed significant cytotoxic activity against normal bone marrow cells, as well as various factor-dependent myelogenous progenitor cell lines, all of which were resistant to endogenous NK activity of the fresh spleen cells. These results indicated that at least a set of T3+ LGL with rearranged TCR genes could be directly differentiated from isolated hematopoietic progenitor cells in vitro. Results also suggested that such a prethymically differentiated subset of T-lineage lymphocytes, namely T3+ double-negative LGL, had particular cytotoxic activity in addition to conventional NK activity, which might well contribute to feedback regulation of hematopoiesis.

Expression and rearrangement of the alpha, beta, and gamma chain genes of the T cell receptor in cloned murine large granular lymphocyte lines. No correlation with the cytotoxic spectrum.

Using cloned murine large granular lymphocyte (LGL) lines, the expression and the rearrangement of the alpha, beta, and gamma chain genes of the T cell receptor (TCR) were analyzed. Morphological, phenotypical, as well as functional studies indicated that the LGL lines were identical to normal, endogenous NK cells. Northern blot hybridization analysis indicated that the full-length transcripts of all the alpha, beta, and gamma chain genes were expressed in most of the LGL lines, including two lines derived from athymic nude mice. In one line, SPB, however, no transcript of the gamma chain gene was detected, whereas the alpha and beta chain genes were clearly expressed. In every LGL line studied, all of the alpha, beta, and gamma chain genes were rearranged. Conforming to the results of Northern blot hybridization study, the gamma chain gene of the SPB line was aberrantly rearranged, whereas those of all the other lines were productively rearranged. The results clearly revealed that NK cells represented a population of lymphocytes genetically committed to the T cell lineage. It was also suggested that the expression and rearrangement of the TCR genes of NK cells might occur in a thymus-independent fashion. An SPB line without expression of the gamma chain gene could exhibit NK activity indistinguishable from other NK lines. Furthermore, the rearrangement patterns of the beta chain gene did not correlate with the specificity of the cytotoxic activity. These results strongly suggested that the cytotoxic activity in NK cells was not directly mediated by TCR on them. We particularly noted that the beta chain gene of most independently established LGL lines showed identical patterns of rearrangement, indicating that they used the same V beta and J beta gene segments. The significance of the restricted pattern of rearrangement of the beta chain gene in LGL lines, as well as the possible functional roles of TCR on NK cells, was discussed.

Generation of continuous large granular lymphocyte lines by interleukin 2 from the spleen cells of mice infected with Moloney leukemia virus. Involvement of interleukin 3.

Continuous cell lines could be reproducibly established by culturing spleen cells from adult mice injected with MLV-producer cells or directly infected with Mo-MLV with rIL-2, whereas the culture of normal splenic cells with rIL-2 induced only transient and limited proliferation resulting in no such lines. All of the lines showed morphological characteristics as LGL with Thy-1+,Lyt-1-,L3T4-,Lyt-2-,AsGM1+,FcR gamma+ phenotype without exception, and most of them exhibited typical NK-patterned cytotoxicity. Analysis of reverse transcriptase activity of the culture supernatants as well as Southern hybridization of the DNA from the lines using an Mo-MLV-specific cDNA probe indicated no evidence of retroviral replication or proviral integration, suggesting that the generation of cell lines reflected a reactive process and viral infection was not directly responsible. It was subsequently revealed that Thy-1+,Lyt-1+,Lyt-2- spleen cells from mice infected with Mo-MLV in vivo spontaneously produced surprising amounts of IL-3 in vitro, leading to the possibility that IL-3 was responsible for the generation of lines. The possibility was directly supported by the observation that continuous lines with identical characteristics could be generated completely in vitro by sequential stimulation with rIL-3 and rIL-2 from normal spleen cells without any involvement of Mo-MLV. The C beta gene of TCR was shown to be rearranged in all the lines examined, indicating the LGL lines were all genetically committed to T cell lineage. Unlike the situation in normal splenic populations expanded by rIL-2, where the expression of IL-2-R was progressively lost, constitutive expression of high-affinity-IL-2-R was observed in all the lines and thus, this was considered to explain the unlimited proliferation of them in response to rIL-2 alone. These results suggested the probable role of IL-3 in the regulation of growth and differentiation of a set of LGL committed to T cell lineage. The possible implications of the phenomenon in the regulation of hematopoiesis as well as in the control of Mo-MLV-induced leukemogenesis were discussed.

Characterization of the 4C8 antigen involved in transendothelial migration of CD26(hi) T cells after tight adhesion to human umbilical vein endothelial cell monolayers.

In extravasation of T cells, little is known about the mechanisms of transendothelial migration subsequent to the T cells' tight adhesion to endothelium. To investigate these mechanisms, we developed a monoclonal antibody (mAb), termed anti-4C8, that blocks transmigration but not adhesion in a culture system in which high CD26-expressing (CD26(hi)) T cells preferentially migrate through human umbilical vein endothelial cell (HUVEC) monolayers cultured on collagen gels. Anti-4C8 reacted with all CD3(+) T cells and monocytes but not neutrophils or HUVECs. The structure defined by this antibody was an 80-kD molecule. The mAb at 1 mug/ml inhibited 80-90% of migration of CD3(+) T cells through unstimulated and interferon gamma-stimulated HUVEC monolayers without interfering with adhesion and cell motility. When added to the cultures after the adhesion, anti-4C8 completely blocked subsequent transmigration of adherent T cells. Phase-contrast and electron microscopy revealed that T cells are arrested at the intercellular junctions of HUVECs in the presence of anti-4C8. Anti-4C8 exhibited agonistic effects on resting T cells without other stimuli under culture conditions in which anti-4C8 can stimulate T cells. First, in the checkerboard assay using collagen gels, the antibody promoted chemokinetic migration of the cells in a dose-dependent manner from 0.1 to 10 mug/ml. The predominant population of T cells that migrated into collagen gels with impregnated anti-4C8 were CD26(hi). Second, solid-phase-immobilized anti-4C8 induced adhesion of T cells to the substrate, often with polarizations in cell shape and large pseudopods rich in filamentous (F-) actin. Third, soluble anti-4C8 augmented F-actin content preferentially in CD26(hi) T cells when added to T cells at a high dose of 10 mug/ml. Finally, both anti-4C8-induced chemokinetic migration and transendothelial migration were inhibited by pretreatment of T cells with pertussis toxin. These findings suggest that stimulation via the 4C8 antigen increases cell motility of CD26(hi) cells with profound cytoskeletal changes through signaling pathways including G proteins. The 4C8 antigen may be involved in preferential transmigration of CD26(hi) cells adherent to HUVECs.

Mechanisms of lymphocyte adhesion to human vascular endothelial cells in culture. T lymphocyte adhesion to endothelial cells through endothelial HLA-DR antigens induced by gamma interferon.

The effects of interferons (IFNs) on lymphocyte adhesion to cultured human vascular endothelial cells (EC) were investigated using an in vitro assay. Endothelial cells obtained from umbilical vein were first cultured at a low density with a conditioned medium (CM) from 12-O-tetra decanoylphorbol 13-acetate-concanavalin A (TPA-Con A) stimulated human peripheral blood lymphocytes (PBL), or with recombinant (r) gamma interferon (IFN-gamma) or r alpha interferon (IFN-alpha), and then were incubated with freshly isolated PBL. Natural IFN-gamma in the TPA-Con A CM and rIFN-gamma (12.5-500 U/ml) induced major histocompatibility complex-class II antigens (HLA-DR, HLA-DP, and HLA-DQ) and significant lymphocyte adhesion to the EC, whereas rIFN-alpha did not. The lymphocyte adhesion to the EC and the expression of DR antigens on the EC were well correlated in terms of both kinetics and the dose-response pattern of rIFN-gamma. When EC expressing I region associated (Ia) antigen were preincubated with monoclonal anti-DR antibody before the addition of lymphocytes, the lymphocyte adhesion was significantly inhibited in both allogeneic and syngeneic combinations, whereas anti-HLA-DP, anti-HLA-DQ, and anti-HLA-ABC antibodies did not inhibit the binding at all. Cell fractionation experiments indicated that the majority of lymphocytes adhering to Ia-expressed EC were Leu-3+ T cells, whose binding was again almost completely inhibited by anti-DR antibody. Moreover, anti-Leu-3a, but not anti-Leu-2a, antibody effectively inhibited the T cell adhesion to the EC. These results strongly suggest that the interaction of the Leu-3(T4) receptor of T cells with IFN-gamma-induced DR antigens on EC plays a central role in the selective adhesion of Leu-3+ T cell to EC.

Studies of the function of natural killer-interferon system in patients with Sjögren syndrome.

The natural killer (NK)-interferon (IFN) system is shown to be significantly involved in the resistance of host to viral infections and to tumours in numbers of animal models (1-4). The patients with Sjögren syndrome (SS) as well as those with collagen diseases were systematically investigated for the functions of NK-IFN system, including endogenous and augmented NK activity, IFN production, and responsiveness of NK cells to IFN stimulation, using virus persistently infected cells (heLa-measles cells) as target and stimulator cells. Although endogenous NK activity was not reduced, augmented NK activity by HeLa-measles cells in vitro was significantly depressed in patients with SS compared with that in age-matched normal controls. The patients with SS had also impaired capacity to produce IFN, which is shown to be a major factor regulating NK activity (5,6) in response to HeLa-measles cells in vitro. In three patients with SS who showed severely depressed NK activity, the effect of exogenous IFN was examined, and virtually no augmentation of NK activity was observed in all cases. Under the same condition, the normal controls demonstrated a dramatic increase in NK activity. The reduced IFN production was observed in all examined patients with SS, whereas impaired augmentation of NK activity by the stimulation with HeLa-measles cells as well as IFN seemed to be more striking in patients with the systemic manifestations of the disease, such as hypergammaglobulinemia and lymphoid hyperplasia. The possible involvement of dysfunction of NK-IFN system in the systemic manifestations of SS is discussed.

Molecular phylogeny of Trypanosoma cruzi from Central America (Guatemala) and a comparison with South American strains.

Molecular phylogenetic analysis was carried out for 21 strains of Trypanosoma cruzi, nine of which were obtained from Guatemala and 12 from South America. Phylogenetic trees were constructed using the nucleotide sequences of two nuclear gene regions, dihydrofolate reductase-thymidylate synthase (DHFR-TS) and trypanothione reductase (TR), and contiguous portions of two mitochondrial genes, cytochrome oxidase subunit II (COII) and reduced nicotinamide adenine dinucleotide dehydrogenase subunit 1 (ND1). Possible genetic exchange between the rather divergent lineages of T. cruzi II from South America was suggested in the trees of the two nuclear genes. T. cruzi I strains obtained from Guatemala and Colombia were identical in all the genes examined, but other T. cruzi I isolates from South America were rather polymorphic in the DHFR-TS and mitochondrial genes. No genetic exchange was identified between T. cruzi I populations from Central and South America in the present study.

[Current problems in the diagnosis of malignant pleural mesothelioma].

The diagnosis of malignant pleural mesothelioma (MPM) is challenging although MPM is highly aggressive tumor. The current diagnostic gold standard is principally based on light microscopic examination of hematoxylin-eosin and immunohistochemical stains of large tissue sections. However, pathological diagnosis of MPM and classification of histological findings into 1 of the 3 subtypes (epithelial, sarcomatoid, biphasic) are difficult. We studied correlation between initial and final histological diagnosis retrospectively from the records of 21 cases with MPM from 1989 to 2005. The diagnosis of MPM was confirmed by histopathological examination of pleural tissue samples obtained by closed biopsy under computed tomography (CT) or ultrasonography-guided (5 cases), by biopsy under thoracoscopy with local anesthesia (9), by open biopsy via thoracotomy (2), and by video-assisted thoracoscopic surgery (VATS) [5] . Pleural biopsy under those diagnostic methods led to initial diagnosis of MPM in 15 of 21 cases (71.4%) . In 6 cases (28.6%) , initial diagnosis of MPM were not confirmed because of missing malignant tissue (1 case) and relatively small and sarcomatous element (5). In 2 cases examined by closed biopsy and in 3 examined by thoracoscopy under local anesthesia, initial diagnosis of MPM were not confirmed. To get the accurate diagnosis of MPM, obtaining large tissue samples in the initial examination by less invasive thoracoscopy is recommended.

Augmentation of mouse natural killer cell activity by a streptococcal preparation, OK-432.

Streptococcal immunopotentiator OK-432 (NSC-B116209) augmented the natural killer (NK) cell activity of peritoneal exudate cells (PEC) in inbred C57BL/6 mice given ip injections of 0.1 mg OK-432 per mouse. The cytotoxic activity of PEC increased as early as 1 day after inoculation, reached its peak on day 3, and gradually declined thereafter, YAC-1, K562, and MOLT-4 target cells were more sensitive to PEC than were EL 4 and P815 target cells. The elimination of adherent cells by a nylon wool column enriched the proportion of cytotoxic cells among PEC. Nylon wool column-passed PEC were resistant to treatment with anti-Thy 1.2 antibody plus complement and sensitive to anti-asialo GM1 serum plus complement. Because 1:40-diluted rabbit antiserum against glycosphingolipid asialo GM1 is capable of eliminating mouse NK cell activity and is not cytotoxic to killer T-cells, the above results strongly suggest that OK-432 augments the NK cell activity in mice.

Decreased activation of carcinogens in the liver of carcinogen-resistant rats.

We have developed a new strain of rats (resistant rat) which exhibit resistance against the carcinogenic action of several carcinogenic compounds. In the present study, we compared the ability of resistant rat liver to activate 3'-methyl-4-dimethylaminoazobenzene and 2-acetylaminofluorene to highly reactive metabolites, which can covalently bind to DNA, with that of carcinogen-sensitive parent strain rats (sensitive rat), using in vitro DNA binding assay. The covalent binding of these carcinogens to calf thymus DNA, catalyzed by the hepatic 9000 x g supernatant fraction from resistant rats pretreated with 3-methylcholanthrene, was about one-half of that catalyzed by the 9000 x g supernatant from sensitive rats which had received the same treatment. These results suggest that the ability of resistant rat liver to activate the carcinogens decreases compared to sensitive rat liver. Further experiments revealed that this decreased activation of the carcinogens in resistant rat livers is due to a low inducibility of 3-methylcholanthrene-inducible forms of cytochrome P-450 mRNA. The hepatic cytosolic Ah receptor concentrations in resistant rats were shown to be significantly lower than those of sensitive rats. Scatchard plot analysis demonstrated, however, that there is no significant difference between the affinity of Ah receptors for 2,3,7,8-tetrachlorodibenzo-p-dioxin in these two strains. These data implicate that the hepatic Ah receptor level may be an important factor in determining carcinogen sensitivity in rats.

Ultraconserved region-containing Transformer 2ß4 controls senescence of colon cancer cells.

Ultraconserved regions (UCRs) are >200 bp genomic segments with perfect human-to-rodent sequence identity. Transcribed UCRs constitute a new category of noncoding RNAs whose functions remain poorly understood. The human transformer 2ß (TRA2B) gene contains a 419-bp UCR spanning the 276-bp exon 2 and its neighboring introns. TRA2B exon 2 has premature stop codons, whereas an exon 2-containing splice variant (TRA2ß4) was expressed preferentially in the nuclei of human colon cancer cells. TRA2ß4 knockdown p53-independently stimulated CDKN1A transcription and increased p21, resulting in the appearance of senescent cells. Biotin pull-down and RNA immunoprecipitation assays revealed that TRA2ß4 interacted with Sp1 through a Sp1-binding sequence (485-GGGG-488) in a stem-loop structure of exon 2. Mutation of this sequence (485-AAGG-488) disrupted the stem-loop structure, blocked the interaction with Sp1 and increased CDKN1A transcription. Overexpression of TRA2ß4 significantly decreased CDKN1A mRNA levels and accelerated cell growth, but the introduction of the mutation in the Sp1-binding sequence completely canceled these effects. Taken together, TRA2ß4 may sequester Sp1 from occupying promoters of target genes including CDKN1A, promoting cell growth by interrupting the senescence-related gene expression program. This novel function of TRA2ß4 may uncover an oncogenic function of transcribed UCRs.

Soybean phospholipid dependent reductions in triacylglycerol concentration and synthesis in the liver of fasted-refed rats.

The effect of dietary soybean phospholipid on the activities of hepatic triacylglycerol-synthesizing enzymes was compared with soybean oil in fasted-refed rats. Soybean oil at the dietary level corresponding to 20% but not at 5% fatty acid level (21.2 and 5.3% on weight bases, respectively) significantly decreased liver microsomal diacylglycerol acyltransferase activities measured with the endogenous diacylglycerol substrate. Dietary soybean phospholipid even at the dietary level corresponding to 2% fatty acids (3.4% on weight base) significantly decreased the acyltransferase activities measured with endogenous substrate. The dietary phospholipid further decreased the parameter as the dietary level increased, and at the 5% fatty acid level, it was lower than that obtained with soybean oil at 20% fatty acid level. Soybean oil and phospholipid decreased the diacylglycerol acyltransferase activities measured with the saturating concentration of exogenous dioleoylglycerol substrate only when the activities were expressed in terms of total activity (mumol/min per liver) but to much lesser extents. Dietary phospholipid compared to the oil profoundly decreased not only hepatic triacylglycerol but also microsomal diacylglycerol levels. It was indicated that the availability of microsomal diacylglycerol as the substrate for diacylglycerol transferase is the critical determinant in regulating hepatic triacylglycerol synthesis and concentration in this experimental situation. Alterations in the activities of microsomal glycerol 3-phosphate acyltransferase and of the enzymes in fatty acid synthesis could account for the phospholipid-dependent decrease in the microsomal concentration of this intermediate in triacylglycerol synthesis.