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Supplementary Figure 1 from Amiloride Modulates Alternative Splicing in Leukemic Cells and Resensitizes Bcr-AblT315I Mutant Cells to Imatinib

X-inactive-specific transcript (XIST), a long non-coding RNA, is essential for the initiation of X-chromosome inactivation. However, little is known about other roles of XIST in the physiological process in eukaryotic cells. In this study, the bioinformatics approaches revealed XIST could be processed into a small non-coding RNA XPi2. The XPi2 RNA was confirmed by a northern blot assay; its expression was gender-independent, suggesting the role of XPi2 was beyond X-chromosome inactivation. The pull-down assay combined with LC-MS-MS identified two XPi2-associated proteins, nucleolin and hnRNP A1, connected to the formation of G-quadruplex. Moreover, the microarray data showed the knockdown of XPi2 down-regulated the KRAS pathway. Consistently, we tested the expression of ten genes, including KRAS, which was correlated with a G-quadruplex formation and found the knockdown of XPi2 caused a dramatic decrease in the transcription level of KRAS among the ten genes. The results of CD/NMR assay also supported the interaction of XPi2 and the polypurine-polypyrimidine element of KRAS. Accordingly, XPi2 may stimulate the KRAS expression by attenuating G-quadruplex formation. Our present work sheds light on the novel role of small RNA XPi2 in modulating the G-quadruplex formation which may play some essential roles in the KRAS- associated carcinogenesis.

In our translational research, an immunotherapeutic, ADCTA-G, has been developed to emphasize autologous tumor antigens, DC antigen processing/presentation, enhanced tumor immunogenicity and CTL induction. Two clinical trials, Phase I/II [2003-2005Taiwan DOH/MA 0910072504] and phase II [2005–2016 NIH {"type":"clinical-trial","attrs":{"text":"NCT02772094","term_id":"NCT02772094"}}NCT02772094], “Dendritic Cell(DC)-Based Tumor Vaccine Adjuvant Immunotherapy of Human Glioblastoma Multiforme”, respectively enrolled 17 and 42 WHO Grade-IV glioblastomas. Every patient received peripheral blood apheresis for PBMNCs. Monocytes were used for derivation of 3-7x108 phagocytic dendritic cells (iDC). Autologous glioma cells grown out of surgical tumor specimen were irradiated and co-cultivated 1 to 2:1 with iDC to make a ADCTA-G lot. After surgical tumor de-bulking, 10 vaccinations were given, each with 2-5x107 mature DC from the ADCTA lot, in a [4x biweekly and 6x monthly] scheduled s.c. injection in both axillar regions. The follow-up period has been 15 years. Primary endpoint is the overall survival (OS); phenotypes of tumor infiltrating T cells and tumor IDH mutation were also analyzed for long-term survivors. The ADCTA-G inoculations were tolerated well, and curtailed the grade-3/4 lymphopenia adverse effect of the temozolomide CCRT. The median OS is 22.9 months for the total 59 grade IV patients in the two trials. The median OS is 21.8 months for the 44 newly diagnosed patients and 28.1 months for the 15 recurrent patients; the difference is insignificant statistically. In this study, vaccinations were initiated early in the recurrent GBM patients while the newly diagnosed GBM patients had to wait till after external radiation therapy, leading to a comparable OS benefit of the ADCTA vaccination. Also, we demonstrated in vitro the earlier vaccinations in the recurrent decrease CD133+ tumor stem-like cells. In addition, the CD8(+) cells were susceptible to ionizing radiation. A phase 3 open-labelled randomized study will be initiated to improve the survival of this aggressive malignancy.

Glioblastoma multiforme (GBM) is the most prevalent and high malignant primary brain tumors, and contains a small cohort of self-renewing, tumorigenic glioblastoma stem cells (GSCs) that contribute to tumor relapse and therapeutic resistance. GSCs are typically recognized by the expression of cell surface marker, CD133, encoded from prominin-1 (PROM1) gene. Recent studies have indicated CD133+ cancer stem-like cells are enriched after the treatment of ionizing radiation (IR). To investigate the effect of cytotoxic TILs to glioblastoma cells experienced irradiation, we used post-vaccination TILs treat with glioblastoma cells derived from patient’s tissue specimens without experienced and experienced irradiation respectively. The effect of cytotoxic TILs was not evident in glioblastoma cells experienced IR until 120 hours later. To further investigate whether immune cytokines have cytotoxicity in CD133+ glioblastoma cells, we observed that TNF-α and IFN-γ have the cytotoxic effects in CD133+ glioblastoma cells, implicating the reason why the cytotoxic of CD8+ TILs were delayed to 120 hours. These data provide the framework for understanding the radioresistance in glioblastoma cells that can be used to develop an efficient vaccine.

Pseudogenes, especially those that are transcribed, may not be mere genomic fossils, but their biological significance remains unclear. Postulating that in the human genome, as in animal models, pseudogenes may function as gene regulators through generation of endo-siRNAs (esiRNAs), antisense RNAs or RNA decoys, we performed bioinformatic and subsequent experimental tests to explore esiRNA-mediated mechanisms of pseudogene involvement in oncogenesis. A genome-wide survey revealed a partial retrotranscript pseudogene ψPPM1K containing inverted repeats capable of folding into hairpin structures that can be processed into two esiRNAs; these esiRNAs potentially target many cellular genes, including NEK8. In 41 paired surgical specimens, we found significantly reduced expression of two predicted ψPPM1K-specific esiRNAs, and the cognate gene PPM1K, in hepatocellular carcinoma compared with matched non-tumour tissues, whereas the expression of target gene NEK8 was increased in tumours. Additionally, NEK8 and PPM1K were downregulated in stably transfected ψPPM1K-overexpressing cells, but not in cells transfected with an esiRNA1-deletion mutant of ψPPM1K. Furthermore, expression of NEK8 in ψPPM1K-transfected cells demonstrated that NEK8 can counteract the growth inhibitory effects of ψPPM1K. These findings indicate that a transcribed pseudogene can exert tumour-suppressor activity independent of its parental gene by generation of esiRNAs that regulate human cell growth.

The antihypertensive drug amiloride is being considered as a tactic to improve cancer therapy including that for chronic myelogenous leukemia. In this study, we show that amiloride modulates the alternative splicing of various cancer genes, including Bcl-x, HIPK3, and BCR/ABL, and that this effect is not mainly related to pH alteration, which is a known effect of the drug. Splice modulation involved various splicing factors, with the phosphorylation state of serine-arginine–rich (SR) proteins also altered during the splicing process. Pretreatment with okadaic acid to inhibit protein phosphatase PP1 reversed partially the phosphorylation levels of SR proteins and also the amiloride-modulated yields of Bcl-xs and HIPK3 U(-) isoforms. Genome-wide detection of alternative splicing further revealed that many other apoptotic genes were regulated by amiloride, including APAF-1, CRK, and SURVIVIN. Various proteins of the Bcl-2 family and MAPK kinases were found to be involved in amiloride-induced apoptosis. Moreover, the effect of amiloride on mRNA levels of Bcl-x was demonstrated to translate to the protein levels. Cotreatment of K562 and BaF3/Bcr-AblT315I cells with amiloride and imatinib induced more loss of cell viability than either agent alone. Our findings suggest that amiloride may offer a potential treatment option for chronic myelogenous leukemia either alone or in combination with imatinib. Cancer Res; 71(2); 383–92. ©2011 AACR.

Human endogenous retroviruses (HERVs), abundantly inter-dispersed in the genome, carry long terminal repeats (LTRs) that may potentially retro-transpose to new genomic sites and deregulate the neighboring cellular genes. However, normally HERVs are either structurally defective or inactive due possibly to stringent negative control mechanisms. To study the possible negative regulation of HERV, we isolated the LTR of RTVL-Ia and constructed site-specific mutations for analysis of the promoter and enhancer functions by using chloramphenicol acetyl transferase (CAT) reporter assay. Our results showed that in most transfected human cells the LTR-mediated CAT expression was negligible unless a sequence segment at the AGTAAA polyadenylation site was deleted. In addition, we have found that the wild type p53 may inhibit whereas a p53 mutant (V143A) stimulate the transcriptional activity of HERV-I LTR. Our results imply that HERV-I LTR, while under negative control by its LTR cis-elements and by wild type p53, may become active upon p53 mutation.

The aim of this study was to assess the efficacy human mesenchymal stem cells (hMSC) for targeting microscopic tumors and suicide gene or cytokine gene therapy. Immunodeficient mice were transplanted s.c. with human colon cancer cells of HT-29 Inv2 or CCS line, and 3 to 4 days later, i.v. with “tracer” hMSCs expressing herpes simplex virus type 1 thymidine kinase (HSV1-TK) and enhanced green fluorescent protein (EGFP) reporter genes. Subsequently, these tumors were examined for specificity and magnitude of HSV1-TK+, EGFP+ stem cell engraftment and proliferation in tumor stroma by in vivo positron emission tomography (PET) with 18F-labeled 9-(4-fluoro-3-hydroxymethylbutyl)-guanine ([18F]-FHBG). In vivo PET images of tumors growing for 4 weeks showed the presence of HSV1-TK+ tumor stroma with an average of 0.36 ± 0.24% ID/g [18F]-FHBG accumulation. In vivo imaging results were validated by in situ correlative histochemical, immunofluorescent, and cytometric analyses, which revealed EGFP expression in vWF+ and CD31+ endothelial cells of capillaries and larger blood vessels, in germinal layer of dermis and hair follicles proximal to the s.c. tumor site. These differentiated HSV1-TK+, GFP+ endothelial cells had limited proliferative capacity and a short life span of

hMSCs derived from bone marrow are useful as a species-specific cell culture system for studying cell lineage differentiation and tissue remodeling. However, hMSCs usually have a short in vitro life span due to replicative senescence. We therefore used a high dose of retroviral vector LXSN-16E6E7 to transduce hMSCs of an aging donor and obtained an actively proliferating cell line, designated KP-hMSCs, which expressed HPV16 E6/E7 mRNA. Whereas parental hMSCs ceased to grow after 30 PDs, KP-hMSCs could be propagated beyond 100 PDs. With culture procedures to avoid selection pressure and crowded cell growth, KP-hMSCs showed no signs of neoplastic transformation as examined by soft-agar anchorage-independent growth and NOD-SCID mouse tumorigenicity assays. KP-hMSCs gave similar cytofluorimetric profiles of 31 CD markers to those of the parental primary hMSCs, except with some morphologic changes and expansion of an originally very minor CD34(dim)CD38(+)CD50+ cell population. Upon exposure to specific stimulating conditions in vitro, KP-hMSCs could respond and differentiate along the mesenchymal (bone, fat and cartilage) and nonmesenchymal (neuron) cell lineages. Our results indicated that hMSCs could be immortalized by transduction with HPV16 E6/E7, maintained without neoplastic transformation by careful culture procedures and thus useful for stem cell research and clinical application.

The first microRNA, let-7, and its family were discovered in Caenorhabditis elegans and are functionally conserved from worms to humans in the regulation of embryonic development and stemness. The let-7 family has been shown to have an essential role in stem cell differentiation and tumor-suppressive activity. Deregulating expression of let-7 is commonly reported in many human cancers. Emerging evidence has accumulated and suggests that reestablishment of let-7 in tumor cells is a valuable therapeutic strategy. However, findings reach beyond tumor therapeutics and may impinge on stemness and differentiation of stem cells. In this review, we discuss the role of let-7 in development and differentiation of normal adult stem/progenitor cells and offer a viewpoint of the association between deregulated let-7 expression and tumorigenesis. The regulation of let-7 expression, cancer-relevant let-7 targets, and the application of let-7 are highlighted.

Amplification of chromosome arm 3q is the most consistent aberration in cervical cancer, and is implicated in the progression of dysplastic uterine cervical cells into invasive cancer. The present study employed the 'positional candidate gene' strategy to determine the contribution of PIK3CA, which is located in 3q26.3, in cervical tumorigenesis. PIK3CA is known to be involved in the PI 3-kinase/AKT signaling pathway, which plays an important role in regulating cell growth and apoptosis. The results of comparative genomic hybridization show that the 3q26.3 amplification was the most consistent chromosomal aberration in primary tissues of cervical carcinoma, and a positive correlation between an increased copy number of PIK3CA (detected by competitive PCR) and 3q26.3 amplification was found in tumor tissues and in cervical cancer cell lines. In cervical cancer cell lines harboring amplified PIK3CA, the expression of gene product (p110alpha) of PIK3CA was increased, and was subsequently associated with high kinase activity. In addition, transformation phenotypes in these lines, including increased cell growth and decreased apoptosis, were found to be significantly affected by the treatment of specific PI 3-kinase inhibitor, suggesting that increased expression of PIK3CA in cervical cancer may result in promoting cell proliferation and reducing apoptosis. These evidences support that PIK3CA is an oncogene in cervical cancer and PIK3CA amplification may be linked to cervical tumorigenesis. Oncogene (2000).

The effect of granulocyte/macrophage colony-stimulating factor (GM-CSF) gene transfer on the tumorigenicity and immunogenicity of 2 different murine tumor lines was determined. Transduction of B16 melanoma cells with the GM-CSF gene rendered the cells more immunogenic. In contrast, transduction of NG4TL4 fibrosarcoma in FVB/N mice (NG) with the GM-CSF gene showed increased tumorigenicity in a high producer line (NG-MGh). The parent NG or NG-MG cells induced the same level of cytotoxic T-lymphocyte (CTL) response and the same magnitude of tumor transplantation immunity. However, the proliferation of the NG-MGh cells was increased 2- to 10-fold. There was no increase in apoptosis in the NG cells and there was no increase of NG-MGh cells in S-phase, hence the increase of the proliferative activity appeared to be indeed inherent to the cells. Mixing the splenocytes from the NG-MGh tumor bearers with the NG tumor cells did not increase tumorigenicity but totally inhibited the growth of the NG tumor, indicating that suppressor cells were not present. Mixing 10,000 rad X-irradiated NG-MGh cells with viable NG tumor cells resulted in 3- to 10-fold increased NG tumor growth rate. The in vitro proliferation of NG cells was increased by adding both GM-CSFs and macrophages and not by either one alone, suggesting that interaction between macrophages and GM-CSFs resulted in the production of tumor growth enhancing factor(s). Our findings suggest that transduction of NG tumor cells with the GM-CSF gene increases tumorigenicity, which is attributed both to an increased inherent proliferative ability of the tumor cells and to the in vivo production of a tumor growth enhancing factor(s) at the tumor site. Int. J. Cancer 75:459–466, 1998. Published 1998 Wiley-Liss, Inc.1

Cancer stem cells (CSCs) in glioblastoma multiforme (GBM) are radioresistant and chemoresistant, which eventually results in tumor recurrence. Targeting CSCs for treatment is the most crucial issue. There are five methods for targeting the CSCs of GBM. One is to develop a new chemotherapeutic agent specific to CSCs. A second is to use a radiosensitizer to enhance the radiotherapy effect on CSCs. A third is to use immune cells to attack the CSCs. In a fourth method, an agent is used to promote CSCs to differentiate into normal cells. Finally, ongoing gene therapy may be helpful. New therapeutic agents for targeting a signal pathway, such as epidermal growth factor (EGF) and vascular epidermal growth factor (VEGF) or protein kinase inhibitors, have been used for GBM but for CSCs the effects still require further evaluation. Nonsteroidal anti-inflammatory drugs (NSAIDs) such as cyclooxygenase-2 (Cox-2) inhibitors have proven to be effective for increasing radiation sensitivity of CSCs in culture. Autologous dendritic cells (DCs) are one of the promising immunotherapeutic agents in clinical trials and may provide another innovative method for eradication of CSCs. Bone-morphogenetic protein 4 (BMP4) is an agent used to induce CSCs to differentiate into normal glial cells. Research on gene therapy by viral vector is also being carried out in clinical trials. Targeting CSCs by eliminating the GBM tumor may provide an innovative way to reduce tumor recurrence by providing a synergistic effect with conventional treatment. The combination of conventional surgery, chemotherapy, and radiotherapy with stem cell-orientated therapy may provide a new promising treatment for reducing GBM recurrence and improving the survival rate.

Alternative splicing involves differential exon selection of a gene transcript to generate mRNA and protein isoforms with structural and functional diversity. Abnormal alternative splicing has been shown to be associated with malignant phenotypes of cancer cells, such as chemo-resistance and invasive activity. Screening small molecules and drugs for modulating RNA splicing in human hepatocellular carcinoma cell line Huh-7, we discovered that amiloride, distinct from four pH-affecting amiloride analogues, could “normalize” the splicing of BCL-X, HIPK3 and RON/MISTR1 transcripts. Our proteomic analyses of amiloride-treated cells detected hypo-phosphorylation of splicing factor SF2/ASF, and decreased levels of SRp20 and two un-identified SR proteins. We further observed decreased phosphorylation of AKT, ERK1/2 and PP1, and increased phosphorylation of p38 and JNK, suggesting that amiloride treatment down-regulates kinases and up-regulates phosphatases in the signal pathways known to affect splicing factor protein phosphorylation. These amiloride effects of “normalized” oncogenic RNA splicing and splicing factor hypo-phosphorylation were both abrogated by pre-treatment with a PP1 inhibitor. Global exon array of amiloride-treated Huh-7 cells detected splicing pattern changes involving 584 exons in 551 gene transcripts, many of which encode proteins playing key roles in ion transport, cellular matrix formation, cytoskeleton remodeling, and genome maintenance. Cellular functional analyses revealed subsequent invasion and migration defects, cell cycle disruption, cytokinesis impairment, and lethal DNA degradation in amiloride-treated Huh-7 cells. Other human solid tumor and leukemic cells, but not a few normal cells, showed similar amiloride-altered RNA splicing with devitalized consequence. This study thus provides mechanistic underpinnings for exploiting small molecule modulation of RNA splicing for cancer therapeutics.

Malignant gliomas, particularly glioblastoma multiforme (GBM) tumors, are very difficult to treat by conventional approaches. Although most of the tumor mass can be removed by surgical resection, radiotherapy, and chemotherapy, it eventually recurs. There is growing evidence that cancer stem cells (CSCs) play an important role in tumor recurrence. These stem cells are radioresistant and chemoresistant. The most commonly used tumor marker for CSCs is CD133. The amount of CSC component is closely correlated with tumor malignancy grading. Isolating, identifying, and treating CSCs as the target is crucial for treating malignant gliomas. CSC-associated vascular endothelial growth factor (VEGF) promotes tumor angiogenesis, tumor hemorrhage, and tumor infiltration. Micro-RNA (miRNA) plays a role in CSC gene expression, which may regulate oncogenesis or suppression to influence tumor development or progression. The antigenesis of CSCs and normal stem cells may be different. The CSCs may escape the T-cell immune response. Identifying a new specific antigen from CSCs for vaccine treatment is a key point for immunotherapy. On the other hand, augmented treatment with radiosensitizer or chemosensitizer may lead to reduction of CSCs and lead to CSCs being vulnerable to radiotherapy and chemotherapy. The control of signaling pathway and cell differentiation to CSC growth is another new hope for treatment of malignant gliomas. Although the many physiological behavioral differences between CSCs and normal stem cells are unclear, the more we know about these differences the better we will be able to treat CSCs effectively.

Immunotherapy is a new light of hope for the treatment of malignant gliomas. The brain is no longer believed to be an immunologically privileged organ. The major advantage of immunotherapy is the tumor-specific cytotoxic effect on the tumor cells with minimal side effects. Autologous dendritic cells (DCs)-based immunotherapy is a promising and feasible method. DCs are the most potent antigen-presenting cells (APCs). DCs prime T lymphocytes by epitopic major histocompatibility (MHC) class I and II for CD8+cytotoxic T lymphocytes (CTLs) and CD4+T helper cells, respectively. From the tissue specimen examination after DCs-based immunotherapy, CD8+CTLs have replaced T regulatory cells (Tregs) as the major dominant tissue infiltrating lymphocytes (TILs). CD8+CTLs play a key role in the tumor response, which may also be effective against cancer stem cells. DCs themselves also produce many cytokines including interferon-γ and interleukin (IL-2) to kill the tumor cells. From the preliminary better outcomes in the literature for malignant gliomas, DC-based immunotherapy may improve tumor response by increasing the survival rate and time. It is recommended that DC-based immunotherapy is applied as soon as possible with conjunctive radiotherapy and chemotherapy. Malignant gliomas have heterogeneity of tissue-associated antigens (TAAs). To find universal common antigens through different kinds of tumor culture may be the essential issue for tumor vaccine development in the future.

Objective Spinal muscular atrophy (SMA) is a common inherited neuromuscular disorder caused by homozygous loss of function of the survival motor neuron 1 (SMN1) gene. All SMA patients carry at least one copy of a nearly identical SMN2 gene. However, a critical nucleotide change in SMN2 results in alternative splicing and exclusion of exon 7 in the majority of SMN2 messenger RNA (mRNA), thus producing a low level of functional SMN protein. Increasing SMN protein production by promoting SMN2 exon 7 inclusion could be a therapeutic approach for SMA. It has been shown that cellular pH microenvironment can modulate pre-mRNA alternative splicing in vivo. In this study, we tested whether inhibitors of the Na+/H+ exchanger can modulate the exon 7 splicing of SMN2 mRNA Methods We treated SMA lymphoid cell lines with Na+/H+ exchanger inhibitors and then measured SMN2 exon 7 splicing by reverse transcriptase polymerase chain reaction and SMN protein production by Western blotting and immunofluorescence Results We found that treatment with an Na+/H+ exchanger inhibitor, 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), significantly enhances SMN2 exon 7 inclusion and SMN protein production in SMA cells. In addition, EIPA increases the number of nuclear gems in SMA cells. We further explored the underlying mechanism, and our results suggest that EIPA may promote SMN2 exon 7 inclusion through upregulation of the splicing factor SRp20 in the nucleus Interpretation Our finding that EIPA, an inhibitor of the Na+/H+ exchanger, can increase SMN protein expression in SMA cells provides a new direction for the development of drugs for SMA treatment. However, further translational studies are needed to determine whether this finding is applicable for SMA treatment or just a proof of cellular pH effect on SMN splicing. Ann Neurol 2007

SUMMARY Tartrate-resistant acid phosphatase (TRACP) is a cytochemical marker for hairy cell leukemia, macrophages, dendritic cells, and osteoclasts. Our purpose was to develop multicolor cytofluorometric methods to evaluate intracellular TRACP enzymic activity using a fluorogenic cytochemical reaction in combination with immunochemical stains for distinct surface membrane antigens. Monocyte-derived dendritic cells (DCs) were the model TRACP-expressing cells studied. Intracellular TRACP activity was disclosed using naphthol-ASBI phosphate as substrate with fast red-violet LB salt as coupler for the reaction product. Before the TRACP enzymic reaction, surface antigens, CD86 and CD11c of DCs, were bound with specific fluorescent antibodies to test compatibility of surface labeling and intracellular staining. TRACP activity varied in DCs from donor to donor but was reproducible on repeated examinations of each sample. Samples could be stained for simultaneous analysis of surface antigens and intracellular TRACP activity, provided certain technical details were observed. The TRACP reaction time should not exceed 9 min and the cell number should not exceed 2 � 10 5 /100 � l test. Fluorescent surface labels did not affect the intensity of the TRACP stain, but the intensity of some surface labels may be diminished by elution of low-affinity antibodies during the TRACP reaction. Readjustment of the threshold settings in triple-labeled cells is needed to compensate for this phenomenon. Intracellular TRACP activity can be quantitated in subpopulations of cells within mixed cell populations by flow cytofluorometry using simple cytochemical methods in combination with fluorescent antibodies to cell-surface and other differentiation antigens. The cytochemical method should be useful for basic investigations of differentiation, maturation, and function of macrophages, DCs, and osteoclasts, and for diagnosis and management of hairy cell leukemia. (J Histochem Cytochem 51:1131–1137, 2003)

Cyclooxygenase-2 (COX-2) expression has been shown to correlate with the invasiveness of colon cancer cells. To further investigate this positive correlation and its possible therapeutic implications, a selective COX-2 inhibitor, etodolac, was tested on three variants of HT-29 colon cancer cell lines, HT-29/Inv1, HT-29/Inv2 and HT-29/Inv3, with graded increases of in vitro Matrigel invasive potential and COX-2 expression levels. HT-29 variants with higher invasive potential were found to be more sensitive to etodolac by in vitro growth inhibition assays, the estimated LD(50) being 0.5 mM for highly invasive HT-29/Inv2 and HT-29/Inv3 cells, 0.6 mM for slightly less invasive HT-29/Inv1, and 1.8 mM for the parental HT-29. Treatment of the highly invasive HT-29/Inv2 and Inv3 variants with as little as 0.1 mM etodolac in the growth medium produced signs of apoptosis, as detected by DNA fragmentation and TUNEL (terminal deoxynucleotidyl transferase dUTP-biotin nick end labeling) assay. In vivo experiments in SCID mice showed that etolodac inhibited the growth of subcutaneous tumors induced by HT-29/Inv3 cells significantly more than those by the parental HT-29 cells. These results suggest that COX-2 inhibitors have a potential role in prevention of tumor invasion in colon cancer patients.

Transduction of cancer cells with herpes simplex virus thymidine kinase gene (HSVtk) followed by prodrug ganciclovir (GCV) treatment has been shown to induce apoptosis. In this study, four murine tumors including B16F10 melanoma, NG4TL4 sarcoma, H6 hepatoma and 1MEA 7R.1 hepatoma were found to vary in sensitivity to this gene therapy strategy in vitro but, at effective doses of GCV, the HSVtk-transduced cells of all four tumors showed similar kinetics of early rise in p53 protein levels, then cell cycle S-/G2-phase arrest and finally signs of apoptosis. Immunoblot analyses revealed that Fas (CD95/APO-1), Fas ligand (FasL) and two downstream mediators, RIP and caspase-3, (CPP32, YAMA, Apopain) were increased in GCV-treated HSVtk-transduced tumor cells the cell cycle arrest and before apoptosis. Increased expression of FasL could also be observed in vivo in HSVtk-transduced tumors induced to regress by GCV treatment. Enzyme measurements using specific substrate showed that the caspase-3 activation followed kinetically the FasL expression. More than half of the HSVtk/GCV-induced cell death could be abrogated by addition to the cell culture medium of a specific antisense oligonucleotide to block FasL synthesis, a recombinant Fas/Fc chimeric protein to compete with Fas receptor for FasL binding, or cell-permeable specific tetrapeptide inhibitors of caspase-3 or caspase-8.

RNA interference (RNAi) is a gene silencing process within living cells, which is controlled by the RNA-induced silencing complex with a sequence-specific manner. In flies and mice, the pseudogene transcripts can be processed into short interfering RNAs (siRNAs) that regulate protein-coding genes through the RNAi pathway. Following these findings, we construct an innovative and comprehensive database to elucidate siRNA-mediated mechanism in human transcribed pseudogenes (TPGs). To investigate TPG producing siRNAs that regulate protein-coding genes, we mapped the TPGs to small RNAs (sRNAs) that were supported by publicly deep sequencing data from various sRNA libraries and constructed the TPG-derived siRNA-target interactions. In addition, we also presented that TPGs can act as a target for miRNAs that actually regulate the parental gene. To enable the systematic compilation and updating of these results and additional information, we have developed a database, pseudoMap, capturing various types of information, including sequence data, TPG and cognate annotation, deep sequencing data, RNA-folding structure, gene expression profiles, miRNA annotation and target prediction. As our knowledge, pseudoMap is the first database to demonstrate two mechanisms of human TPGs: encoding siRNAs and decoying miRNAs that target the parental gene. pseudoMap is freely accessible at http://pseudomap.mbc.nctu.edu.tw/. Database URL: http://pseudomap.mbc.nctu.edu.tw/

DNA polymerase activities were studied in rabbit endometria at estrus and through the first seven days of pregnancy. Extracts of total endometrial homogenates by high salt gave two peaks of polymerase activity by sucrose gradient centrifugation; the 6-8S polymerase showed considerable increase in specific activity which reached a maximum at 5-7 days post-coitum, whereas the 3-4S polymerase showed only a slight increase after 2 days of pregnancy. A 5-6S RNA-dependent DNA polymerase activity, extracted by combined use of hypertonic salt solution and non-ionic detergent from particulate subcellular functions, appeared in the endometrium after coitus, reached a maximum at Day 3-4 and declined thereafter. This RNA-dependent DNA polymerase activity, isolated by phosphocellulose chromatography at distinct regions of salt gradient, showed preference for template-primers such as (rA)/sub n/.(dT)/sub 9/ and (rC)/sub n/.(dG)/sub 6/ but did not utilize 70S RNA of RNA tumor viruses. Attempts to demonstrate oncorna virus expression by other means have been unsuccessful with endometrium of 3 day pregnant rabbits. Interrelation between the changes of these DNA polymerase activities and the DNA replication in the endometrial cells is discussed.

A purification procedure is described for two major toxic proteins from seeds of Abrus precatorius by successive chromatography on diethylaminoethyl-Sephadex A-50, carboxymethyl-cellulose, and DEAE-cellulose. The two toxins obtained both appeared homogeneous as judged by electrophoresis and analytical ultracentrifugation. Abrin A, the more positively charged protein obtained from the DEAE-cellulose chromatography, can be crystallized as needles from ammonium sulfate solution by the free interface diffusion technique; under various similar conditions, the other fraction, abrin C, failed to yield crystals. X-ray precession photographs have indicated that abrin A crystallizes in an orthorhombic unit cell of symmetry P212121 and dimensions a = 74.9, b = 269.8, and c = 70.3 A. The amino acid composition of abrins A and C are different. Molecular weights determined by sedimentation equilibrium are 60,100 for A as compared to 63,800 for C. After A has been treated with 2-mercaptoethanol at 100° for 2 min, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate gives rise to one strong (corresponding to apparent subunit molecular weight of 32,000) and two much weaker bands. This is in sharp contrast with the pattern for C, two bands of approximately equal intensity, corresponding to apparent subunit molecular weights of 33,000 and 28,000. The two proteins also behave differently in affinity chromatography on a Sepharose 4B column. Abrin C at 1 µg per ml concentration level agglutinates sheep erythrocytes, whereas abrin A shows no such activity even at 40 µg per ml concentration level. Abrin C exhibits a more toxic effect on mice than abrin A. These differences in physicochemical and biological properties for the two abrins are presented in detail.

Fv-1 gene-specific resistance to ecotropic murine retroviruses can be transferred to permissive cells with RNA prepared from restrictive cells. The active RNA has a sedimentation coefficient of 20-22S at low ionic strength and multiple regions of activity (5-6S, 10-12S, 24-27S) at high ionic strength. The maximum level of resistance transfer cannot be enhanced with noninhibitory carrier RNA or facilitators of RNA uptake, and the maximum inhibition (40-60%) is independent of the MOI. The low efficiency of resistance transfer is, therefore, probably the result of a fraction of cells resistant to RNA uptake.

RFM/Un mice express an endogenous type C retrovirus throughout their life span in many tissues; primary or established embryo fibroblast cell cultures do not express a virus but can be induced by exposure to 5-iodo-2'-deoxyuridine. All of our sources yielded a single ecotropic virus (RFV) which appeared to be related more closely to the endogenous N-tropic virus (WN1802N) of BALB/c mice than to Gross leukemia virus on the basis of two-dimensional gel electropherograms of virion proteins. No xenotropic or recombinant viruses were isolated by cocultivation techniques. RFV is N-tropic, and RFM/Un cells possess the Fv-1n allele, as indicated by restriction of B-tropic virus and susceptibility to Gross strain N-tropic virus. However, RFM cells are highly resistant to RFV and other endogenous N-tropic viruses. This resistance is expressed by two-hit titration kinetics and by inhibition of viral linear duplex DNA formation. This is similar to the effects of the Fv-1 locus, but preliminary work has shown no apparent genetic linkage between the two restrictions. The relative strength of the restriction, the presence of a single class of ecotropic virus, and the absence of recombinant viruses suggest that in RFM mice virus is expressed only in cells in which it is induced and not by cell-to-cell transmission.

We molecularly cloned unintegrated viral DNA of the BALB/c endogenous N-tropic and B-tropic murine leukemia retroviruses and in vitro passaged N-tropic Gross (passage A) murine leukemia retroviruses. Recombinant genomes were constructed in vitro by exchanging homologous restriction enzyme fragments from N- or B-tropic parents and subsequent recloning. Infectious virus was recovered after transfection of these recombinant genomes into NIH-3T3 cells and cocultivation with the Fv-1 nonrestrictive SC-1 cells. XC plaque assays of recombinant virus progeny on Fv-ln and Fv-lb cells indicated that the Fv-l host range was determined by sequences located between the BamHI site in the p30 region of the gag gene (1.6 kilobase pairs from the left end of the map) and the HindIII site located in the pol gene (2.9 kilobase pairs from the left end of the map).

Specific antisera were prepared against the Bowman-Birk trypsin inhibitor and four other trypsin inhibitors of low molecular weight isolated from soybeans (Glycine max L. cv. Tracy). These antisera were used to detect the presence and amount of the inhibitors in: (a) seeds and protein extracts of soybean meal; (b) seedlings; and (c) the water surrounding the seeds and roots of seedlings. Lectin activities in seeds, seedlings, and water were also determined at the same time as the protease inhibitor activities. By competitive inhibition of immunoprecipitation, the combined five low molecular weight protease inhibitors were found to constitute the following percentages of proteins (w/w): 6.3% in defatted soybean meal; 8.1% of the protein extracted from the meal by a buffer of pH 8.6; 8.3, 14.7, 15.2, 16.1, 17.2, and 18.9% of the protein in a lyophilisate of water in which seeds were incubated for 4, 8, 12, 16, 20, and 24 hours, respectively; 8.2% in a lyophilisate of water in which roots of seedlings grew for 20 days; 1.5% in cotyledons; and less than 0.1% in epicotyls, hypocotyls, and roots of 12-day-old seedlings. Hemagglutination activities, expressed as the lowest amount of protein required to give a positive agglutination of 0.2 ml of 2% rabbit red blood cells, were as follows: purified soybean lectin, 0.08 μg; lyophilisate of water in which seeds were incubated for 4, 8, 12, 16, 20, and 24 hours, 10, 2.5, 5, 5, and 2.5 μg, respectively; lyophilisate of water in which roots grew for 20 days, 5 μg; 12-day-old cotyledons, roots, epicotyls, and hypocotyls, 12.5, 100, >1,000, and >500 μg, respectively. The results indicate that a large amount of protease inhibitors as well as lectins are released from seeds during the first 8 hours of imbibition. Neither lima bean trypsin inhibitor (mol wt, 10,000) nor Kunitz soybean trypsin inhibitor (mol wt, 21,500) showed competitive inhibition in tests with antisera against low molecular weight soybean protease inhibitors.

We molecularly cloned and analyzed an N-tropic ecotropic retrovirus induced with iododeoxyuridine from RFM/Un mouse cell cultures. Based on the restriction map, the RFM/Un virus appears to be indistinguishable from other induced N-tropic retroviruses. A nucleotide sequence analysis of the long terminal repeat of an infectious clone revealed structural features characteristic of murine type C retrovirus long terminal repeats. The U3 region of the RFM/Un virus long terminal repeat, however, contained no short sequence duplication or insertion found in other murine leukemia virus isolates.

Formation of viral closed circular supercoiled DNA duplexes and production of progeny virus were both inhibited in cultured mouse cells treated with cycloheximide in the first 4 h of type C retrovirus infection. With different doses of cycloheximide to cause different degrees of inhibition, the number of viral supercoiled DNA duplexes detected in the cells at 11 h showed an apparent correlation with the amount of progeny virus produced in the 12- to 22-h period of infection. A slight accumulation of the full-genome linear duplex and an open circular duplex of viral DNA intermediate was observed in the cycloheximide-treated cells. Cycloheximide given to the cells during the time of conversion of viral DNA from linear to supercoiled duplex forms (6 to 11 h after virus inoculation) did not inhibit the conversion. These kinetic data suggest that a cycloheximide-sensitive metabolic process, probably early viral protein synthesis, is required for retrovirus replication and supercoiled viral DNA formation in the cell.

Nucleotide sequences of the region that corresponds to the site of tRNA primer binding for a functional retrovirus were determined in five murine leukemia virus-related sequence clones from mouse chromosomal DNA, which contain a unique 170 to 200-base-pair additional internal segment in the long terminal repeats. The 3'-terminal 18-nucleotide sequence of a major glutamine tRNA isoacceptor was found to match well with the putative primer binding site: 18 of 18 in three clones, 17 of 18 in one clone, and 16 of 18 in one clone. This implies that most of these endogenous proviral sequences of the mouse genome, if replicated as retroviruses, will be different from ecotropic murine leukemia viruses and most mammalian type C retroviruses in using glutamine tRNA, rather than proline tRNA, as a primer.

A recombinant DNA clone, named AL10, that contains murine leukemia virus (MuLV) related sequences was isolated from BALB/c mouse chromosomal DNA and examined in detail. Restriction endonuclease mapping revealed that the 10.5 kbp EcoRI insert consists of a 3.6 kbp left flanking cellular DNA region and a 6.9 kbp MuLV-related region that has a typical proviral LTR-gag-pol-env structure up to the EcoRI site in the env gene region. Comparison of the AL10 map with ecotropic and xenotropic virus isolates revealed many common restriction sites in the LTR and pol gene regions, but much fewer in the leader and gag regions. A stretch of 1,700 nucleotides containing the cellprovirus junctional region was sequenced and revealed transcriptional consensus signals and other structural features characteristic of MuLV LTRs, as well as two distinctive features: (a) a sequence of approximately 170 bp with direct and inverted terminal repeats not seen in infectious MuLV LTRs was identified in the U3 region between the "enhancer" region and the "CAT" box. This novel segment or its homologous sequences appear to be present in most of the endogenous MuLV-related LTRs and in other chromosomal locations of the mouse (b) The tRNA primer binding site is not complementary to proline tRNA, the primer for all known MuLVs, but is a 17/18 match with rat glutamine tRNA. The integration site of AL10 provirus was in a unique DNA region but contained an "Alu"-like short interdispersed repeat in the 5' adjacent cellular region. The AL10 proviral integration found in BALB/c was also apparent in RFM, AKR and SENCAR mouse cells but not in cells of NFS/N, C3H, HRS/J, SC-1, and a California Lake Casitas wild mouse.

Lectin is released from soybean seeds during water uptake. Hemagglutination activity data show that the lectin is a preferential release product within the first 8 hours of hydration. A qualitative filter-paper assay for detection of lectin released by single seeds is used to show that the release phenomenon is independent of seed viability and insensitive to azide.

Tryptophan transfer RNA from chick cells chromatographs differently in the reversed-phase column chromatographic system 5 depending upon whether it is aminoacylated or not. This property was utilized to prepare pure tryptophan tRNA. Oligonucleotide fingerprints of tryptophan tRNA purified in this manner are identical with those reported for "spot 1" RNA isolated from Rous sarcoma virus.

Selected species of 4S RNA of chick embryo cells will hybridize in vitro with 35S RNA of avian myeloblastosis virus. A major tRNA component of the hybridizable 4S RNA is tryptophan tRNA. A hybrid prepared from purified tryptophan tRAN and 35S RNA of avian myeloblastosis virus in vitro is an efficient templateprimer for DNA synthesis catalyzed by reverse transcriptase (RNA-dependent DNA polymerase).

Soluble extracts of mouse cells with Fv-1 n or Fv-1 b gene alleles specifically and reciprocally inhibit infection of B- or N-tropic mouse leukemia viruses in permissive cell cultures. NB-tropic virus infection was not inhibited by either cell extract. Extracts from Fv-1 - cells did not inhibit infection by the three virus host-range types, but N- or B-tropic virus infection of Fv-1 - cells was inhibited by extracts of the nonpermissive cells, and Fv-1 nb cell extracts inhibited both viruses. The maximum degree of inhibition was 50-80% as determined by immunofluorescent or plaque assays, with extracts containing up to 500 μg/ml of nonpermissive cell protein. The inhibitor(s) is relatively unstable since activity is lost after 2 hr at 37° or 30 min at 56°. The inhibitor(s) was most effective if added 2 hr before or within 2 hr after infection, did not react with the virus directly, inhibit virus attachment, or inhibit the normal cell functions tested. These results indicate that nonpermissive mouse cells contain a product, possibly determined by the Fv-1 gene, which inhibits some early postpenetration event(s) in leukemia virus infection.

A standardized bioassay for transfer of Fv-1 gene-specific resistance to N-tropic and B-tropic murine retroviruses was developed using X plaque reduction in SC-1 (Fv-1-) cells inoculated with virus. Testing of subcellular fractions of restrictive cells showed that the resistance transfer activity was present in the cytoplasmic (microsomal and cytosol) fractions. The activity of the cytoplasmic extract was destroyed by treatment with ribonuclease, but not with deoxyribonuclease or proteases. RNA prepared by phenol-chloroform extraction of mouse tissues, including embryos and livers of weanling mice, transferred Fv-1 locus-specific resistance into DEAE-dextran-treated SC-1 cells. The activity of isolated RNA preparations against virus of the appropriate host-range type has been demonstrated to correspond to the Fv-1 genotypes of the cell sources. The specific transfer of resistance with cellular RNA was effective within a 5- to 6-h period from 2 h before to 4 to 5 after virus infection. Sucrose gradient centrifugation of the RNA showed that the activity sedimented as a broad peak, with an apparent maximum in the 22S region. Affinity chromatography of whole-cell RNA on polyuridylic acid-Sepharose tended to separate more activity into the polyadenylic acid RNA fraction than the non-polyadenylic acid RNA fraction. Except for the reciprocal inhibitory activity for the two host-range virus types, the RNAs of Fv-1n and Fv-1b specificities showed similar properties in all aspects studied.

Previously, in vitro recombinant DNA studies demonstrated that genetic determinants of N-tropism and B-tropism, or Fv-1-related host range properties of murine leukemia viruses, were located in a BamHI-HindIII DNA segment derived from the 5' portion of the cloned viral genome. We sequenced this segment and its immediate 5' region from cloned DNA of two BALB/c mouse C-type viruses (WN1802N and WN1802B) and found base differences at 12 positions out of the otherwise identical 1,390-base-pair sequences. Analysis of the most likely reading frame showed that 6 of the 12 base differences would result in four encoded amino acid changes, three of which occur at positions 109 (glutamine in WN1802N versus threonine in WN1802B), 110 (arginine in WN1802N versus glutamic acid in WN1802B), and 159 (glutamic acid in WN1802N versus glycine in WN1802B) of the p30 protein. The remaining one is located at position 36 (threonine in WN1802N versus isoleucine in WN1802B) of the viral polymerase protein. Significant conformational alteration of the p30 protein could be predicted from these amino acid changes.

We analyzed 15 recombinant DNA clones of the unintegrated closed circular DNA intermediate of the BALB/c endogenous ecotropic murine leukemia virus WN1802N. Thirteen of these clones had an insert which corresponded to the complete murine leukemia virus genome. Of these, six contained a single long terminal repeat (LTR) and seven contained two LTRs. The viral genomes in nine clones had an LTR of 520 base pairs (bp), one had an LTR of 570 bp, three had an LTR of 600 bp, and one had an LTR of 670 bp. Restriction endonuclease analysis demonstrated that the size variability resides in the U3 region. Seven of eight clones which yielded infectious virus by DNA transfection had the 520-bp LTR, and the other had a 600-bp LTR. More detailed examination of plasmid subclones of three isolates with different-sized LTRs revealed that the approximate position which varies in the U3 region corresponds to the 72-bp repeat region of Moloney sarcoma virus. Possible consequences of these variations are discussed.

Retrovirus infection of cultured murine teratocarcinoma cells depends upon the state of differentiation. We have used two cell lines derived from a teratocarcinoma of mouse, strain 129. One, an undifferentiated pluripotential cell line (PCC4), is restrictive to viral infection, while the other, a differentiated myoblast-derived cell line (PCD1), is fully permissive to virus replication. We have shown that no virus RNA expression can be found in PCC4 cells 48 h post-infection and that no nucleic acid sequences can be found in an integrated form in PCC4 cells. However, the kinetics of formation of free proviral intermediates show that the three forms (I, II and III) of free virus DNA are synthesized in both PCC4 and PCD1. Free proviral DNA disappears gradually after 24 h in PCC4 cells while all forms increase in PCD1. These results suggest that the viral multiplication restriction occurs somewhere between the proviral DNA synthesis and integration of DNA in the cellular genome.