In situ localization of S-phase-specific histone (H3) mRNA in Bowen's disease

PCNA and Ki-67 immunohistochemistry has been used to assess cell proliferation in place of tritiated thymidine or BrdU labeling of S-phase cells. Recently, it has been possible to reliably demonstrate histone H3 mRNA by in situ hybridization in formalin-fixed and paraffin-embedded tissue sections. We have compared this new proliferation marker with Ki-67 and PCNA with regard to distribution of positive cells and labeling indices (LI%) for 22 cases of Bowen's disease. In normal skin, Ki-67-IHC positive cells and histone mRNA positive cells were observed in the basal and suprabasal layers of the epidermis. In Bowen's disease, positive cells with each marker were more frequent in upper neoplastic epidermis than in suprabasal layers, and the average LI%s were markedly elevated with all markers, the scores decreasing in the following order: PCNA-IHC, Ki-67-IHC and H3mRNA-ISH. However, the results of double staining demonstrated that S-phase cells do not necessarily show exactly the same distributions as with PCNA and Ki-67-IHC labeling. H3mRNA-ISH showed three different degrees of reaction with significantly different LI%s, whereas PCNA and Ki-67 LI% did not vary essentially in the same areas. These results strongly suggest that Bowen's disease, which is well known as a low-grade neoplastic state with malignant potential, also demonstrates clear intratumoral heterogeneity of S-phase cells using the H3mRNA-ISH method.     

Retrotransposon-adenovirus hybrid vectors: efficient delivery and stable integration of transgenes via a two-stage mechanism

It has long been recognized that the mechanisms mediating retrotransposition might be adapted for genomic integration and long-term expression of foreign genes. In particular, long interspersed nuclear elements (LINEs), an abundant class of retrotransposons that are the most active mobile genetic elements in the human genome, have been largely ignored as candidates for development as an integrating vector system because there has been no suitable method for efficiently introducing them into target cells. We have recently developed a LINE-based retrotransposon-adenovirus hybrid vector, in which a helper-dependent adenovirus (HDAd) is utilized as the platform for delivery of a human L1 element and its linked heterologous transgene cassette into the host cell nuclei. While a major drawback to the use of HDAd vectors has been their lack of specific mechanisms to achieve permanent integration into the host genome, the inserted retrotransposon sequences overcome this limitation. The L1-HDAd hybrid thus represents a single vector capable of mediating long-term gene expression by a two-stage mechanism: in the first (adenovirus) stage, the helper-dependent adenovirus serves as a carrier for efficient delivery and transient expression of its encoded L1/transgene cassette, and in the second (retrotransposon) stage, the L1 retro-element and its associated transgene then permanently integrate into the genome of the adenovirus-transduced cells. We propose that this novel retrotransposon-adenovirus hybrid vector system will be useful both as a vehicle for efficient delivery and long-term stable transduction of therapeutic genes, as well as a tool to elucidate aspects of retrotransposon biology that have previously been difficult to study.     

Expression profiles and functional implications of p53-like transcription factors in thymic epithelial cell subtypes

In this study, we investigated the localization and functional significance of p53 tumor suppressor-like molecules, p63 and p73, in human thymic epithelial cells (TECs). Immunohistochemical studies showed particular distribution profiles of p63 and p73 in thymic epithelium, in which cortical TECs preferentially expressed p63 in their nuclei whereas subcapsular and medullary TECs expressed both p63 and p73 in their nuclei. The wide distribution of p63 in TECs was further suggested by studies using TECs of primary culture. In vitro studies using two human TEC lines demonstrated that p63 was capable of up-regulating intercellular adhesion molecule-1 (ICAM-1) and enhancing the production of IL-6 and IL-8. Moreover, in vitro studies also indicated that p73, but not p63, had the capacity to induce granulocyte macrophage colony stimulating factor (GM-CSF) and granulocyte colony stimulating factor (G-CSF) in the TEC lines. These findings suggest that p63 would regulate the cell adhesive property through ICAM-1/LFA-1 interaction and the production of IL-6 and IL-8, probably in all TEC subtypes. p73 in subcapslar and medullary TECs was suggested to play a role in the regulation of the production of GM-CSF and G-CSF, which might stimulate other stromal cells such as dendritic cells, macrophages and endothelial cells around these regions.     

Evidence of the monoclonal composition of human endometrial epithelial glands and mosaic pattern of clonal distribution in luminal epithelium

The endometrium is a highly regenerative tissue that plays a crucial role in implantation. We examined the clonal constitution of glandular cells as well as the luminal epithelium of this unique tissue. Using collagenase-based digestion techniques with microscopic manipulation, we isolated individual human endometrial glands and examined their clonality using a polymerase chain reaction-based assay for nonrandom X chromosome inactivation with an X-linked androgen receptor gene. Most of the glands analyzed were composed of monoclonal populations of epithelial cells and one of the glands exhibited a loss of heterogeneity in the androgen receptor gene. In addition, adjacent glands within a 1-mm(2) area shared clonality, suggesting that clonality of the luminal epithelium is regionally defined. The clonality of endometrium was further confirmed in a study of female mice that harbor the green fluorescent protein gene on either the maternal or paternal X chromosome. Fluorescent microscopy of uterine sections revealed that individual endometrial glands consisted completely of either fluorescent or nonfluorescent cells and that the surface epithelium exhibited a clear boundary between these cell types. These findings suggest that single or multiple stem cells with uniform clonality exist on the bottom of each endometrial gland and genetic alterations occurring in such cells may play a critical role in endometrial carcinogenesis. The possible association between area-specific X inactivation of the endometrial surface and the endometrial receptivity of embryo implantation remains to be clarified.     

Detection of genetic alterations in advanced prostate cancer by comparative genomic hybridization

In this study, we examined nine cases of advanced Japanese prostate cancer by comparative genomic hybridization (CGH) to detect chromosomal imbalances across the entire genome and to identify several new regions likely to contain genes important to the development and progression of this disease. These cases had been previously examined for numerical chromosomal aberrations by fluorescence in situ hybridization (FISH). By CGH, the following regions were found to be over-represented (gains), with fluorescence ratio values higher than the threshold: 4p, 6p, 8q, 11q, 12q, 15q, 16p, 17q, 20, and 21 (>4 cases); underrepresentation (losses) involved: 1q, 4q, 5q, 6q, 13q, 14q, and 22 (>4 cases). The shortest regions of overlap (SRO) of gains were noted at 8q24.1 through q24.3, 12q23, and 17q23 through q24 (>5 cases). The SRO of losses were seen at 5q14 through q21, 6q16.1 through q21, 13q21.3 through q22, and 14q21 (>5 cases). Notably, the gain of chromosomes 8 and 12 by CGH was in agreement with the FISH data, suggesting that the gain of chromosomes 8 and 12 may play an important role in prostate carcinogenesis. The genes on the SRO regions were also discussed in relation to oncogenes and bone metastases.     

Antiinflammatory actions of ephedrines in acute inflammations1

Ephedrine (EP), pseudoephedrine (PEP), ephedroxane (EX) and pseudoephedroxane (PEX) inhibited carrageenin-induced hind-paw edema in sham-operated mice as well as adrenalectomized mice. Hind-paw edema induced by histamine, serotonin, bradykinin and prostaglandin E (1) was suppressed by these alkaloids, showing that they exert the antiinflammatory activity at the early exudative stage of inflammation. Although tolazoline and propranolol had no effects on the inhibitory activity of EX and PEX, treatment with tolazoline decreased the antiinflammatory activity of EP and PEP on carrageenin-induced hind-paw edema. Antiinflammatory activity of PEP was reduced by previous treatment with reserpine, indicating the antiinflammatory activity of EP and PEP to be partly concerned with the sympathetic nervous system. Although these alkaloids injected I.C.V. elicited no antiinflammatory actions on carrageenin-induced hind-paw edema and the inhibitory activity of morphine on carrageenin-induced hind-paw edema was potentiated by the concurrent administration of PEP and EX, demonstrating that the mechanism of antiinflammatory activity does not involve the central nervous system. Further, these alkaloids inhibited prostaglandin E (2) biosynthesis.     

High HNRNPA3 expression is associated with lymph node metastasis and poor prognosis in patients treated with radical cystectomy

ObjectiveWe sought to identify heterogeneous nuclear ribonucleoprotein A3 (HNRNPA3) expression in bladder cancer and its relationship to clinicopathological findings and prognosis.MethodsImmunohistochemical staining for HNRNPA3 was performed on 122 archived radical cystectomy specimens, with immunoreactivity being stratified on a 0 to 3 scale. The percentage of HNRNPA3 expressing tumor cells was calculated and multiplied by the staining score over an average of 5 areas to obtain a semiquantitative H-score (maximum value: 300). HNRNPA3 expression was categorized as high (≥80) or low (<80).ResultsThe patients’ median age was 70 years, and the median follow-up period was 39.4 months. High HNRNPA3 expression was significantly associated with lymph node metastasis (P= 0.014) and S100A8, S100A9 and uroplakin III expression (P= 0.028, 0.002, and 0.047, respectively). Log-rank tests indicated that high HNRNPA3 expression was significantly associated with disease progression and cancer-specific death (P= 0.013 and 0.006, respectively). In the Cox proportional hazards regression analysis, only lymph node metastasis was associated with disease progression and cancer-specific survival.ConclusionHNRNPA3 may be a new biomarker to predict biologically aggressive cancers and determine the appropriate treatment modality in patients after radical cystectomy.     

Analysis of acute-phase toxicities of intensity-modulated proton therapy using a model-based approach in pharyngeal cancer patients

Pharyngeal cancer patients treated with intensity-modulated proton therapy (IMPT) using a model-based approach were retrospectively reviewed, and acute toxicities were analyzed. From June 2016 to March 2019, 15 pharyngeal (7 naso-, 5 oro- and 3 hypo-pharyngeal) cancer patients received IMPT with robust optimization. Simulation plans for IMPT and intensity-modulated X-ray therapy (IMXT) were generated before treatment. We also reviewed 127 pharyngeal cancer patients with IMXT in the same treatment period. In the simulation planning comparison, all of the normal-tissue complication probability values for dysphagia, dysgeusia, tube-feeding dependence and xerostomia were lower for IMPT than for IMXT in the 15 patients. After completing IMPT, 13 patients completed the evaluation, and 12 of these patients had a complete response. The proportions of patients who experienced grade 2 or worse acute toxicities in the IMPT and IMXT cohorts were 21.4 and 56.5% for dysphagia (P < 0.05), 46.7 and 76.3% for dysgeusia (P < 0.05), 73.3 and 62.8% for xerostomia (P = 0.43), 73.3 and 90.6% for mucositis (P = 0.08) and 66.7 and 76.4% for dermatitis (P = 0.42), respectively. Multivariate analysis revealed that IMPT was independently associated with a lower rate of grade 2 or worse dysphagia and dysgeusia. After propensity score matching, 12 pairs of IMPT and IMXT patients were selected. Dysphagia was also statistically lower in IMPT than in IMXT (P < 0.05). IMPT using a model-based approach may have clinical benefits for acute dysphagia.     

Cytotoxicity of a novel indoloquinone eo9 in hypoxic non-small-cell lung-cancer cell-lines

3-Hydroxymethyl-5-aziridinyl-1-methyl-[1H-indole-4,7-dione]-prop-beta-en -alpha-ol (EO9) is a bioreductive anticancer agent active for non-small cell lung cancer (NSCLC) and structurally related to mitomycin C (MMC). DT-diaphorase (DTD) is regarded as a two electron reductase that plays an important role in the biotransformation of MMC to antitumor metabolites. To evaluate the role of DTD as a bioactivator of EO9 in NSCLC cell lines under oxic and hypoxic conditions, we examined the inhibitory effect of dicumarol which was regarded as a selective inhibitor of DTD on the sensitivity to EO9 in vitro. In this study, we used an MMC-resistant NSCLC cell line (PC-9/MC4) which was established from a PC-9 cell line as a parent cell line by continuous exposure to MMC in our laboratory. We reported previously that the subline PC-9/MC4 was 6.7-fold more resistant to MMC than PC-9 with decreased DTD activity. The IC50 value of PC-9 against EO9 was significantly increased by co-incubation with dicumarol under oxic conditions. EO9 was more cytotoxic against PC-9/MC4 than against PC-9 cells and the enhancement was impaired by tempol under hypoxic conditions. These findings suggest a suppressive role of DTD against one-electron reduction pathway in the bioactivation of EO9 under hypoxic conditions and EO9 may be more active against oxygen-deficient solid tumors especially in MMC-resistant NSCLC cells with low levels of DTD activity.