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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.

Most conventional semi-blind channel estimation schemes for MU-MIMO systems are based on eigenval decomposition (EVD) or singular value decomposition (SVD). However, EVD- or SVD-based channel estimation would impose a high computational complexity when the base station is equipped with large number antennas. Those methods are not well suited for real-time processing, especially for Channel State Information (CSI) tracking in time-varying environments. In order to reduce the computational complexity, a PASTd-based (Projection Approximation Subspace Tracking with deflation) CSI tracking algorithm is proposed. The PASTd-based algorithm converges fast, has low computational complexity, and can operate with very small training overhead. Simulation results show that the proposed algorithm can effectively track the CSI with mild Doppler spreads. Only one pilot symbol per user at the beginning of transmission session is needed to resolve the multiplicative factor ambiguity.

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.

Previous clinical trials of dendritic cell (DC)-based immunotherapy in patients with glioblastoma multiforme (GBM) have reported induction of systemic immune responses and prolonged survival. From 2003 to 2005, we performed a clinical trial in which patients with malignant glioma underwent surgery for maximal cytoreduction followed by a 6-month 10-injection course of autologous DC-tumor vaccine therapy, each injection containing 1-6×10(7) DC. Of the 17 treated patients (16 with World Health Organization grade IV and one with grade III glioma), eight (47.1%) had an initial transient elevation in aspartate aminotransferase (AST)/alanine aminotransferase (ALT). Vaccination caused some tumor shrinkage and increased concentration of tumor-infiltrating CD8(+) lymphocytes. Median survival and 5-year survival were 525 days and 18.8%, respectively, for 16 patients with grade IV glioma (381 days and 12.5% for eight newly diagnosed; 966 days and 25% for eight relapsed patients) compared to 380 days and 0% for 63 historical control patients. We concluded that autologous DC-tumor immunotherapy benefits patients with malignant glioma but may cause transient but reversible elevation of serum AST/ALT levels.

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.

The main purpose of this paper is to investigate the retailer’s inventory policy under two-level delay permitted to reflect the supply chain management situation. In this paper, we assume that the retailer maintains a powerful position. So, it is assumed that the retailer can obtain the full trade credit offered by the supplier yet the retailer just offers the partial trade credit to his customers. Under these conditions, the retailer can obtain the most benefits. Then, an algebraic approach is provided to investigate the retailer’s inventory system as a cost minimization problem to determine the retailer’s optimal inventory policy under the supply chain management. One ease-to-use theorem is developed to efficiently determine the optimal inventory policy for the retailer. Finally, numerical examples are given to illustrate the theorem.

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

Various human colon cancer cell lines tested in vitro differed significantly in susceptibility to growth inhibition of recombinant human interferon-beta (rHuIFN-beta). Two p53-mutant lines, COH and CC-M2, derived from high-grade colon adenocarcinoma, showed signs of apoptosis after treatment with 250 IU/ml of HuIFN- beta in the culture medium. The similarly p53-mutated HT-29 line from a grade I adenocarcinoma showed no apoptosis, however, and only cell cycle G1/G0 or S phase retardation with 1000 IU/ml HuIFN-beta. After HuIFN-beta exposure, COH and CC-M2 cells showed increased levels of Fas and FasL proteins, alteration of mitochondrial membrane potential, and activation of caspase-9, caspase-8, and caspase-3 in a time-dependent manner. Treatment of COH and CC-M2 cells with anti-FasL antibodies or rFas/Fc fusion protein, however, could not prevent the apoptosis induced by HuIFN-beta. In contrast, cell-permeable specific inhibitors of the three caspases could inhibit the DNA fragmentation and cell death but not the mitochondrial membrane potential changes. Treatment with mitochondria-stabilizing reagents could significantly abrogate the apoptosis and caspase activation induced by HuIFN-beta. These results suggest that in COH and CC-M2 colon cancer cell lines, HuIFN-beta induces apoptosis mainly through mitochondrial membrane alteration and subsequent activation of the caspase cascade pathway, but not by the Fas/FasL interaction or the p53-dependent apoptotic mechanism.

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.

In this study, the effectiveness of a 188Re labeled sulfur colloid with two particle size ranges was used to evaluate the effectiveness of this agent on melanoma tumors in mice in terms of animal lifespan.Two separate group of animals were used for investigating biodistribution and survival time. A total of 188 B16F10-melanoma-bearing BDF(1) mice were injected intraperitoneally with 3.7 MBq (0.1mCi)/2mL of radiolabeled sulfur colloid ten days after intraperitoneal inoculation of 5x10(5) B16F10 melanoma cells/2ml. For group 1, 30 mice were sacrificed at 1, 4, 24, 48 and 72 hours for biodistribution studies. In group 2, 158 mice were divided into 9 groups (n=16 approximately 18/groups)each receiving respectively tumor alone, tumor with normal saline, cold colloid or hot colloid with 16, 23, 31, 46, 62, or 124 MBq activity. Each of these colloid groups was further divided into two groups, one receiving smaller particle sizes (3 microm:80.4 +/-7.2%, colloid 1) and the other receiving larger particle sizes (3 microm:12.3+/-1.0%, colloid 2). The animals were checked daily until death and their survival recorded.Colloid 2 showed higher accumulation in almost all tissues, the highest accumulation organ was tumor ( approximately 40%), then spleen ( approximately 20%), stomach ( approximately 15%), diaphragm ( approximately 3%), and liver ( approximately 2%). There was a significant increase in survival time with increasing amount of the larger-particle-size colloid. Administered levels of 16-31 MBq/mouse were most efficacious and with higher amounts the survival times decreased significantly below that of the controls. There was a significant difference in the dose-response curves for the two preparations. Protection factors (1/Relative-risk) of nearly 5 were achieved using the larger colloid size, and nearly 30 using the smaller colloid size. An amount of 16-31 MBq of the colloid 2 was the optimal activity in these studies. On the one hand, the survival data agreed well with the biodistribution data, where higher accumulation was found in tumor with colloid 2.Rhenium-188 offers on-site availability, medium half-life, higher beta-particle energy of 2.12 MeV for therapy and emission of 155keV gamma photon suitable for imaging. The present study demonstrated that 188Re-sulfur colloid is an effective agent in controlling tumor cells in the abdominal cavity in animals.

Non-steroidal anti-inflammatory drugs (NSAIDs) have been reported to reduce the risk and mortality of colorectal cancer (CRC). Although the exact mechanisms remain unclear, the inhibition of cyclooxygenase (COX) by NSAIDs appears to abort, if not prevent, CRC carcinogenesis or metastatic tumor progression. The aim of our study was to investigate the association between COX-2 expression and CRC tumor cell invasiveness. The differences in immunoblot-detectable COX-2 protein contents in primary CRCs, metastatic hepatic lesions and corresponding normal mucosa from the same individual were evaluated in 17 patients. Three different colon cancer cell lines, SW620, Lovo, HT-29 and a metastatic variant of HT-29, HT-29/Inv3, were employed to evaluate COX-2 expression and prostaglandin E2 (PGE2) production in relation to their invasive abilities in vitro. The effects of a COX-2–selective inhibitor, etodolac, on cell proliferation and invasive activity were also determined. The results showed that 15 of 17 (88%) metastatic CRC cells from the liver and 14 of 17 (82%) primary CRC tissue exhibited much higher levels of COX-2 than corresponding adjacent normal mucosa from the same patient. Among those patients with relatively high COX-2 expression in the primary tumors, almost all exhibited even higher levels of COX-2 in their hepatic metastases. Among the 4 colon cancer cell lines, HT-29/Inv3 manifested the highest COX-2 expression, PGE2 production and in vitro invasive activity. The selective COX-2 inhibitor, etodolac, could especially exert cytotoxicity and markedly suppress the invasive property and PGE2 production, although not the COX-2 protein level, in HT-29/Inv3 cells. Our results imply that COX-2 expression may be associated with the invasive and metastatic properties of CRC tumor cells. © 2001 Wiley-Liss, Inc.

The present study was designed to ascertain whether or not the pleural effusion and serum cytokine levels (granulocyte-macrophage colony-stimulating factor [GM-CSF], interleukin-10 [IL-10], and interferon-gamma [IFN gamma]) in lung cancer patients differ from tuberculous (TB) pleural effusion, in which a strong cellular immune reaction is found; and, whether cytokine levels are a prognostic factor in lung cancer patients with malignant effusion. A total of 202 lung cancer patients with malignant pleural effusion and 26 patients with TB pleural effusion were studied consecutively between 1995 and 1998. Serum and effusion cytokine levels were analyzed with ELISA assays. The results showed that pleural effusion GM-CSF and IL-10 levels were significantly higher than serum levels in both cancer and TB patients. Pleural effusion IFN gamma levels were significantly higher than serum levels in TB patients. IFN gamma levels in both pleural effusion and serum were significantly higher in TB patients than in those with cancer. No significant difference was found, between TB and cancer patients, in the serum or pleural effusion levels of either IL-10 or GM-CSF. The ratio of pleural effusion IFN gamma to serum IFN gamma, effusion IFN gamma to effusion IL-10, and effusion IL-10 to serum IL-10, were all significantly higher in TB than in cancer patients, suggesting a higher cellular activity and T-helper 1 (Th1) reaction in TB pleural effusion than in malignant effusions, which were predominantly Th2 type. Survival analysis showed no significant difference in lung cancer patients with different levels of these cytokines. It was concluded that lung cancer patients with malignant pleural effusion had poorer immune profiles than those with TB pleurisy, both locally and systemically; and the cytokine profiles were not prognostic factors for lung cancer patients with malignant pleural effusion.

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).

There is strong evidence that tyrosine kinases are involved in the regulation of cellular growth and tumor progression. Over-expressions of tyrosine kinases have been documented in a number of neoplasms. To study the roles of tyrosine kinases in colon cancer, we developed a tyrosine-kinase-expression profile for each of the four different stages of colon carcinogenesis, using normal colon mucosa, adenomatous polyps, primary carcinoma and hepatic metastases collected from the same patient. We identified 30 tyrosine kinases expressed in these tissues: they include 10 non-receptor tyrosine kinases (yes, fyn, lyn, brk, abl, arg, jak1, jak3, tyk2 and itk), 17 receptor tyrosine kinases (erbB2, PDGF-Ralpha, PDGF-Rbeta, kit, c-fms, met, ron, FGF-R1, FGF-R2, FGF-R3, FGF-R4, cek5, tie-1, tkt, axl, sky and Ins-R), 2 dual kinases (mek and sek) and one possible novel kinase. Among these kinases, arg kinase appears to be expressed at a higher level in primary carcinoma and metastatic tumor than in adjacent normal mucosa or adenomatous polyp. This result was confirmed by extensive analysis of 50 additional matched sets of normal colon and colon-tumor specimens, using arg-specific primers and RT-PCR reactions. This study identifies a possible role for arg tyrosine kinase in colon carcinogenesis, especially in the transition from adenoma to carcinoma.

E6/E7 genes of human papilloma virus type 16 were used to immortalize a primary culture of marginal cells (MC) from gerbils. One of the cloned lines was selected which demonstrated preservation of the main characteristics of the MC, both morphologically and physiologically. Electron microscopic examination showed well-developed junctional complexes and apical microvilli which suggested its epithelial origin. Polymerase chain reaction (PCR) demonstrated the incorporation of E6/E7 genes with the genome. Reverse transcription PCR revealed the existence of mRNA of the IsK channel, a unique marker of MC among the inner ear cells, in this clone. Flow cytometric analysis of this cell line's DNA content was diploid. Numerous large domes formed after confluence of the cell monolayer. Electrophysiologic studies displayed evidence of apical K+ and Na+ channels which were blocked by Ba2+ (2 mM) and amiloride (10(-5) M), respectively. Existence of basolateral Na,K-ATPase and Na+/Cl-/K+ cotransporter was shown by blockage by ouabain (10(-3) M) and bumetanide (50 microM), individually. Injection of the cell line to nude mice failed to induce growth of tumors. This cell line was serum-, density- and anchorage-dependent when cultured in plastic dishes. In conclusion, this cell line shows characteristics of well-differentiated MC maintaining the major ionic transport processes, and provides us a good model to study the possible mechanisms and regulating factors of endolymph production.

Mechanism of cell killing by transfer of Herpes simplex virus type-1 thymidine kinase ( HSVtk ) and subsequent ganciclovir (GCV) treatment was examined in B16F10 murine melanoma model. While parental B16F10 melanoma cells were resistant to GCV at 100 μM or higher, HSVtk -transduced B16F10 melanoma cell clones became susceptible to GCV with IC 50 of 0.1 to 0.3 μM. By means of various parameters including characteristic morphological changes, in situ DNA end-labeling, DNA ladder pattern, flow cytometric detection of sub-G1 DNA content, and annexin V binding of inverted cell surface phosphatidylserine, apoptosis was shown to be associated with the cell killing of ganciclovir on HSVtk -transduced melanoma B16F10 cells. Kinetic analysis showed that the signs of apoptosis were observed not until 60 h of continued GCV treatment and preceded first by a rise in p53 protein level in 12 h and then by S-phase/G2-phase cell cycle arrest associated with corresponding increases in the level of cyclin B1 protein but no apparent change in protein level of Bax or Cdc2. These results suggest that apoptosis occurred as a result of ganciclovir-induced cell cycle arrests rather than direct chemical effect on HSVtk -transduced B16F10 melanoma cells.

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

Glial fibrillary acidic protein (GFAP) is an astrocytic lineage-specific intermediate filament protein, and its expression or non-expression is inversely correlated with the tumourigenecity of astrocytoma cells. To estimate the GFAP levels of astrocytes in intracranial tumour tissues, we established primary cultures from six astrocytic tumour specimens and used a double-staining flow cytometric method to detect the different levels of GFAP among these primary cultures. Although these primary cultures exhibited the same Matrigel invasiveness, their GFAP expression is inversely related to the rate of cell growth and the histologic grade of the original tumour. Phenylacetate, 12-O-tetradecanoylphorbol-13-acetate (TPA) and sodium butyrate, which are potent inducers of differentiation in various cancer cells, have been examined for their effects on these primary cultures. Cytostasis was more or less caused by these compounds in all six primary cultures, but induction of GFAP was observed only in the primary culture derived from a less malignant astrocytoma specimen having the highest intrinsic GFAP level. Interestingly, this primary culture, but not others, also exhibited increased HRG-α expression after phenylacetate or sodium butyrate treatment. Loss of the inducibility of differentiation-related gene expression could be one of the events involved in the malignant progression of astrocytomas. In addition, the chemotherapeutic agent BiCNU has a killing effect on all six primary culture cells, with LD50 less than 60nM. The underlying mechanism was through the induction of apoptosis in these primary culture cells regardless of their varying malignancies of original tumours. However, unlike colon cancer and leukaemia cells, sodium butyrate could not induce apoptosis within 4 days in these astrocytic tumour cells, indicating that the cell context of different cell types indeed determined the ability of sodium butyrate to induce apoptosis.

The present study investigates the nature of the immunosuppressed state of the lymphocytes obtained from the malignant pleural effusion (effusion associated lymphocytes, EAL) of lung cancer patients. The immunocompetence of EAL from 13 patients was assessed by determining their T-helper cell phenotype, proliferative response to alpha CD3-activation, and their cytolytic activity against three tumor targets: the autologous tumor, Daudi, and K562. Flow cytometry analysis showed that the lymphocytes in EAL were predominantly T cells with < 1% natural killer cells. The T-helper cell phenotype was found to be predominantly of Th2 type, but could be readily converted to Th1 type by culturing the EAL in vitro, and this conversion was augmented by interleukin-2 (IL-2) or IL-2 plus alpha CD3. To test the cytolytic activity of EAL, it was found that after 6-day culturing, the EAL remained in an immunosuppressed state so that they failed to kill any of the three tumor targets. Stimulation with IL-2 partially restored the immunocompetence of EAL. Further engagement of TCR-CD3 by alpha CD3 fully restored the cytolytic activity of the EAL to kill the autologous tumor target but not Daudi or K562 tumor cells, and thus seemed to be tumor specific. The specificity was further confirmed by testing the activated EAL and normal donor peripheral blood lymphocytes against a variety of tumor targets and control targets. Furthermore, the killing by EAL against the autologous tumor target seemed to be major histocompatibility complex-restricted and was inhibited by anti-human leukocyte antigen class I antibody. The EAL from lung cancer patients also showed much reduced responsiveness to the alpha CD3 stimulation to induce proliferation, and addition of IL-2 restored the responsiveness. These results suggest that, through close contact with tumor cells, anergy of cytotoxic T lymphocytes (CTLs) was induced in vivo at a localized site. IL-2 stimulation and TCR-CD3 engagement could reverse the anergic state and restored the full competence of CTLs in EAL to mediate the specific anti-tumor killing against the autologous tumor. Proper manipulation of EAL may prove useful as a source of anti-tumor effectors for cancer adoptive immunotherapy.

Microsatellite instability (MSI) is intrinsic to most colorectal carcinomas (CRC) from patients with hereditary non-polyposis colorectal cancer (HNPCC), reflecting germline mutations in the mismatch-repair (MMR) genes. Its occurrence and chronological sequence of development in sporadic CRC appears less well defined. To explore the time sequence in acquisition of MSI, and the role it plays during tumor progression in sporadic CRC, we compared the incidence of MSI in tissue samples from 40 Dukes'-B and 30 Dukes'-D CRC patients with liver metastases, at 4 different microsatellite loci, representing sites on the APC, DCC and p53 genes respectively as well as the D2S123 site. Among the 30 patients with hepatic metastases, MSI was found in 9 (30%) of the primary, and 13 (43.3%) of the metastatic tumors. In comparison, among the 40 Dukes'-B CRC, MSI was found in only 8 cases (20%). CRC with MSI were more frequently located in the right colon, less frequently on the left side, and seldom in the rectum. Tumor ploidy analysis shows that 46.2% of Dukes'-D primary tumors with MSI are diploid (χ2 = 4.46, p = 0.035). With a mean follow-up time of 4.2 years for the Dukes'-B CRC, there were no recurrences in the 8 patients with MSI, whilst 6 (18.8%) relapses occurred amongst the 32 patients without MSI, average time to recurrence being 15 months. In Dukes'-D CRC, mean survival time for patients with MSI was 37 months (95% CI, 24 to 51 months), for those without MSI 26 months (95% CI, 18 to 35 months), although this was not statistically significant. Our data suggest that tumor progression may involve increased genetic instability. Int. J. Cancer 74:470–474, 1997. © 1997 Wiley-Liss, Inc.

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.

GL331, a new homologue of etoposide (VP-16), was developed to cope with the multiple drug resistance occurring in certain malignant tumours. We previously indicated that GL331, like VP-16 and other major cancer chemotherapeutic agents, induced apoptosis in a variety of human cancer cell lines including nasopharyngeal carcinoma (NPC) NPC-TW01 and NPC-TW04 cells. In this study, we further explored the effect of GL331 on the cell cycle progression of NPC cells. Flow cytometric analysis of DNA content was first used to demonstrate the ability of GL331 to induce cell growth arrest at S-G2 phase in most NPC cells. Besides acting as a topoisomerase II inhibitor, GL331 inhibited cellular cyclin B1-associated CDC 2 kinase activity 6 h after treatment, accounting partly at least for its induction of the cell cycle arrest. As with cyclin A, D1, E, CDK 2 and PCNA, the levels of cyclin B1 and CDC 2 proteins were not changed after GL331 treatment; however, the ability to form complex between cyclin B1 and CDC 2 was obviously affected in GL331-treated NPC cells, which associates with the inhibition of cyclin B1/CDC 2 kinase activity elicited by GL331. These data could provide more principal bases for future therapeutic application of this potential anti-cancer agent.

Interleukin-3 (Il-3) is a glycoprotein produced by a CD4+CD8- subpopulation of T-lymphocytes. Il-3 has been associated with the proliferation of bone marrow stem cells and their differentiation to granulocytes, macrophages, basophil/mast cells, megakaryocytes, erythroid cells, and neutrophils. The pBOR-Il-3 transgenic mice were developed by pronuclear microinjection to study how chemical insults modulate transcription of the Il-3 gene driven by a long-terminal repeat (LTR) of an endogenous retrovirus and to determine the biological consequences of interleukin-3 expression. We injected 5-azacytidine, a demethylating agent, to increase the LTR-driven expression of Il-3. Upon 5-azacytidine treatment, both the pBOR-Il-3 and the FVB/N nontransgenic controls developed thymic lymphomas. The pBOR-Il-3 mice developed thymic lymphomas at a higher frequency than the FVB/N mice. The thymic lymphoma cells were of a T-cell origin, as determined by T-cell receptor gene rearrangement analysis, and, in most cases, were of monoclonal origin. According to flow cytometric analysis of CD3, CD4, and CD8 cell surface markers, the thymic lymphoma cells did not lose their ability to differentiate, but the differentiation process was aberrant. Flow cytometric analyses also revealed that in pBOR-Il-3 mice the thymic lymphomas are mostly of a CD8+CD4- origin, whereas in the FVB/N group, the predominant type of thymic lymphoma is of a CD4+CD8- origin.

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.

Transduction of murine B16 melanoma cells with a GM-CSF gene, the B16-MG tumor line, showed reduced tumorigenicity. In vitro studies demonstrated no remarkable difference between the parent and transduced tumor lines in their ability to induce secondary response to generate the anti-tumor killer cells (immunogenicity), or in their susceptibility to the killing by anti-tumor killer cells (immunosensitivity). Both CD4(+) and CD8(+) cells were required for the generation of the effecters. Nevertheless the effecters were determined to be Thy1.2(+), CD8(-), and NK1.1(-). At least two antigenic specificities could be defined in the cytolytic reactions. One was a broadly cross-reactive antigen shared by a variety of tumor cells, and the other apparently a tumor-specific antigen which was only present in B16 tumors. Cold target inhibition experiment confirmed these specificities. In the in vivo tumor transplantation study, the B16-MG cell line was not only more immunogenic but also was more immunosensitive than the parent line. More than 50% of the mice which were immunized with B16-MG remained tumor free after challenge with the parent tumor B16, indicating that GM-CSF gene transfer makes an effective tumor vaccine. The in vivo protective effect was specific for B16 tumor, thus only the tumor-specific antigen could function as transplantation antigen. Both CD4(+) and CD8(+) cells were required for providing the in vivo protection. Both the B16 and B16-MG tumor bearing hosts could generate anti-tumor killer cells, hence the development of progressive growth of B16 tumor was not due to the lack of anti-tumor immune response. It appears that the overall effect of in vivo tumor immunity is determined by a complex network of interactions among different compartments of host immune cells and different compartments of host immune cells and different immune-regulatory molecules derived from the host and from the tumor.

This study sought to evaluate effectiveness of autologous dendritic cell vaccine (immunotherapy) for glioblastoma multiforme (GBM).Patients 14 to 70 years of age with newly diagnosed GBM and Karnofsky Performance Scale (KPS) score70 who were receiving initial treatment were enrolled and were randomized into 2 groups during the 5-year study period. Eighteen patients underwent conventional treatment (surgery, radiotherapy, and chemotherapy) and received adjuvant autologous dendritic cell vaccine, and 16 patients (control group) underwent conventional treatment only. Administration of the vaccine was begun within 1 to 2 months postoperatively, with 10 inoculations given over 6 months. Outcome measures were overall survival (OS); progression-free survival (PFS); 1-, 2-, and 3-year survival rates, and quality of life (QoL).Follow-up time ranged from 14 to 56 months (median, 33 months). The 1-, 2-, and 3-year survival rates were 88.9%, 44.4%, and 16.7% for the vaccine group, respectively, and 75.0%, 18.8%, and 0%, respectively, for the control group, (P = 0.299, 0.0035, 0.0014, respectively). The median OS for the vaccine group was 31.9 months and for the control group was 15.0 months (P0.002). The median progression-free survival (PFS) for the vaccine group was 8.5 months, and 8.0 months for the control group (P = 0.075). The surviving fraction was significantly higher in the vaccine group based on Kaplan-Meier analysis.Adjuvant immunotherapy with whole-cell lysate dendritic cell vaccine may improve short-term survival. It seems to be safe, and its long-term effectiveness is worthy of further investigation.

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.

As one of the most intensively studied probes for imaging of the cellular proliferation, [ 18 F]FLT was investigated whether the targeting specificity of thymidine kinase 1 (TK1) dependency could be enhanced through a synergistic effect mediated by herpes simplex type 1 virus (HSV1) tk gene in terms of the TK1 or TK2 expression. 5-[ 123 I]Iodo arabinosyl uridine ([ 123 I]IaraU) was prepared in a radiochemical yield of 8% and specific activity of 21 GBq/μmol, respectively. Inhibition of the cellular uptake of these two tracers was compared by using the arabinosyl uridine analogs such as 5-iodo, 5-fluoro and 5-( E )-iodovinyl arabinosyl uridine along with 2′-fluoro-5-iodo arabinosyl uridine (FIAU). Due to potential instability of the iodo group, accumulation index of 1.6 for [ 123 I]IaraU by HSV1-TK vs. control cells could virtually be achieved at 1.5 h, but dropped to 0.2 compared to 2.0 for [ 18 F]FLT at 5 h. The results from competitive inhibition by these nucleosides against the accumulation of [ 18 F]FLT implied that FLT exerted a mixed TK1- and TK2-dependent inhibition with HSV1- tk gene transfection because of the shifting of thymidine kinase status. Taken together, the combination of [ 18 F]FLT and HSV1-TK provides a synergistic imaging potency.

Diabetes is often associated with atherothrombosis. It is unknown whether high glucose can modulate the expression of tissue factor (TF) and thrombomodulin (TM) in human aortic endothelial cells (HAECs). HAECs were treated with a lower-degree high glucose condition (LG, 11.2 mM) for 8 days and a higher-degree high glucose condition (HG, 30 mM) for 4–6 h. Methoxyphenyl tetrazolium inner salt assay, real-time polymerase chain reaction, western blot, and TF activity assay were performed. In HAECs, both LG and HG conditions were nontoxic. LG caused a 74 ± 20% decrease (P = 0.273) and HG caused a 57 ± 5% decrease in TF mRNA expression (P = 0.001). LG caused a 53 ± 13% decrease (P = 0.036) and HG caused a 75 ± 10% decrease in TF protein expression (P = 0.096). TF activity was not significantly changed by LG (127 ± 13%, P = 0.40) or HG treatments (120 ± 42%, P = 0.70). In contrast, LG caused a 153 ± 16% increase (P = 0.03) and HG caused a 211 ± 20% increase in TM mRNA expression (P = 0.005). LG caused a 131 ± 31% increase (P = 0.35) and HG caused a 140 ± 9% increase in TM protein expression (P = 0.006). Different high glucose conditions do not provide the sufficient stress required to induce TF expression in HAECs. In contrast, high glucose conditions can induce TM expression in HAECs.

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.

Spinal muscular atrophy (SMA) is caused by homozygous deletions/mutations of SMN1 gene. All SMA patients carry a nearly identical SMN2 gene. A nucleotide change in SMN2 results in exon 7 exclusion in the majority of SMN2 mRNA, thus producing low level of SMN protein. Extracellular pH change has been shown to modulate alternative splicing of several pre-mRNAs. In this study, we showed that extracellular pH change can also modulate SMN2 exon 7 splicing in SMA cells. Low extracellular pH enhances SMN2 exon 7 skipping, whereas high extracellular pH promotes its inclusion. Low extracellular pH also reduces SMN protein expression but increases hnRNP A1 expression. In addition, we tested whether intracellular pH-modulating genes could be the modifier of SMA in a SMA discordant family and found that the mRNA levels of ATP6V1B2 gene are significantly higher in two affected siblings than the unaffected one. In conclusion, our results suggest that extracellular pH change modulates SMN2 exon 7 splicing through regulation of hnRNP A1 expression in SMA cells.

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.

The present study attempts to define the role of interleukin-15 (IL-15), as compared with IL-2, in generating cytotoxic T lymphocytes (CTL) from the malignant effusions of cancer patients. Effusion-associated lymphocytes (EAL) from malignant effusion were incubated with IL-15 or IL-2 with or without alphaCD3. Proliferation and cytotoxicity assays were performed. IL-15 was found to have at least an equivalent, if not higher, activity to IL-2 in terms of lymphocyte proliferation and generation of CTL from EAL. The proliferative response of EAL, cocultured with IL-15, with or without alphaCD3, was partly inhibited by pretreatment with an anti-IL2 receptor beta chain monoclonal antibody (mAb). The proliferative response of EAL, cocultured with alphaCD3, IL-2, or both, was partly inhibited by pretreatment with an anti-IL-2 receptor alpha chain mAb. Overnight [5lCr] release assays against K562, Daudi, and the patients' autologous tumor cells were done to evaluate EAL's cytolytic activity. MHC class I Ab blocked the stimulated cytolytic activity of EAL against autologous tumors. An mAb depletion assay showed that the phenotype of the restored EAL was CD16-CD4-CD8+; thus, the restored activity of EAL was CTL activity. The results suggest that both IL-15 and IL-2 can restore CTL activity from EAL in the presence of T cell receptor (TCR)-CD3 engagement, but the effect of IL-15 was superior.

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.

Study objective Our previous report demonstrated that there was impairment of local cellular immunity with elevated interleukin-10 (IL-10) and undetectable IL-12 in neoplastic pleural effusion. These findings suggest that the local immune reactions favor the T-helper type 2 (Th2) pathway instead of Th1 pathway. The present study was designed to examine whether local cellular immunity could be manipulated by IL-2 and/or IL-12 treatment, and to determine their effect on the helper T-cell pathways and the cytolytic activity of the effusion-associated lymphocytes (EALs). Design Using malignant pleural effusions obtained from four patients suffering from adenocarcinoma of lung, we separated the tumor cells from the EALs with Ficol-Hypaque centrifugation, followed by Percoll density centrifugation. To test whether the cytolytic function of lymphocytes could be enhanced by culturing with IL-2 and/or IL-12, lymphocytes were incubated with recombinant IL-2 with/without IL-12 for 6 days. Following this, the tumoricidal activity was assessed in an overnight 51 chromium-release assay. Autologous tumor cells for measuring specific antitumor activity, Daudi cells susceptible to lymphokine-activated killer cells, and NK-susceptible K562 cells were used as target cells. Measurements and results After treatment in vitro with IL-2, IL-12, or IL-2 plus IL-12, the Th pathway shifted from Th2 to Thl type (increased γ-interferon production). To further study the effect of cytokine treatment on the cytolytic activity of EALs, it was found that after 6-day culturing, the EALs failed to kill any of the three tumor targets, whereas the 6-day cultured peripheral blood lymphocytes (PBLs) gave low level of cytotoxicity against all three tumor targets. Stimulation with IL-2 alone partially restored the immunocompetence of EALs to kill the tumor targets. Stimulation with IL-12 alone showed no significant effect on their cytolytic activity. However, IL-12 synergized with IL-2 to increase the cytolytic activity of EALs and PBLs against autologous tumor targets. This synergistic effect was not found for Daudi cells and K562 cells. Conclusions These results suggest that EALs activated with IL-12 in the presence of a low concentration of IL-2, which converted the EALs from Th2 pathway to Thl pathway, could be an alternative source of antitumor effectors for adoptive immunotherapy of cancer.

Paclitaxel is a potential anti-cancer agent for several malignancies including ovary, breast, and head and neck cancers. This study investigated the kinetics of paclitaxel-induced cell cycle perturbation in two human nasopharyngeal carcinoma (NPC) cell lines, NPC-TW01 and NPC-TW04. NPC cells treated with higher concentrations (0.1 or 1 micro M) of paclitaxel showed obvious G2/M arrest and then converted to a cell population with reduced DNA content, which was detected as a sub-G2 peak in the flow cytometric histographs. If a low concentration (5 nM) of paclitaxel was used instead, transient G2/M arrest was observed in NPC cells, which subsequently converted to a sub-G1 form during the treatment period. Internucleosomal fragmentation and chromatin condensation were detectable in these sub-G1 and sub-G2 cells, suggesting that persistent or transient G2/M arrest is a prerequisite step for apoptosis elicited by varying doses of paclitaxel. The levels of cyclins A, B1, D1, E, CDK 1 (CDC 2), CDK 2 and proliferating cell nuclear antigen (PCNA) were unchanged in NPC cells following treatment with any concentration of paclitaxel; however, apoptosis-related cyclin B1-associated CDC 2 kinase was highly activated by paclitaxel even at concentrations as low as 5 nM, which is consistent with the finding that low-dose paclitaxel is also able to induce apoptosis in NPC cells. Activation of cyclin B1-associated CDC 2 kinase seems to be an important G2/M event required for paclitaxel-induced apoptosis, and this activation of cyclin B1/CDC 2 kinase could be attributed to the increased activity of CDK 7 kinase.

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/

GL331 is a semisynthetic topoisomerase II inhibitor derived from a plant toxin podophyllotoxin. In 72-h exposure assays, LD50 values of GL331 range from 0.5 to 2 microM, which are three- to ten-fold lower than those of its homologous compound etoposide (VP-16), depending on different cancer cell lines including nasopharyngeal, hepatocellular, gastric, cervical and colon cancer types. Apoptotic DNA ladders could be detected when cancer cells were treated with GL331 for 24 h even if the Bcl-2 and Bax protein levels were not altered during the period. Besides acting as topoisomerase II inhibitors, both GL331 and VP-16 decrease the cellular protein tyrosine kinase (PTK) activities in cancer cells. The activities of protein tyrosine phosphatase (PTP) are significantly increased after GL331 treatment but are not affected by VP-16. GL331-induced internucleosomal cleavage can be efficiently prevented by two inhibitors of PTP, sodium orthovanadate and zinc chloride, but not by okadaic acid, which inhibits serine/threonine phosphatase activity. These results indicate that GL331 may induce apoptotic cell death, and that activation of protein tyrosine phosphatases may be involved in this process.

Paclitaxel is a potential cancer chemotherapeutic agent for ovary, breast, and head and neck cancers; its effects on nasopharyngeal carcinoma (NPC) have not been reported previously. This study investigated the cytotoxic mechanism of paclitaxel in two NPC cell lines, NPC-TW01 and NPC-TW04. NPC cells treated with pacli-taxel showed convoluted nuclei, condensed chromatin and decreased cellular and nuclear volume, and also exhibited genomic DNA degradation into multiple oligonucleosomal fragments, suggesting that pacli-taxel induced apoptosis in these cells. The effects of paclitaxel on apoptosis-related proteins including Bcl-2, Bax and CDC 2 were also detected. Although the levels of Bcl-2 and Bax were not changed in NPC cells following treatment with 5 nM-1 μM of paclitaxel, phosphorylation of Bcl-2 was significantly observed in the cells treated with 1 μM of paclitaxel for 12 hours. In addition, cyclin B1-associated CDC 2 kinase was highly activated in the NPC cells exposed to paclitaxel even at low (5 nM) concentration, and this result is associated with the finding that low concentration of paclitaxel is able to induce apoptosis in NPC cells.

Study objective Human immunity has been found to have two major components, cellular and humoral immunity. T-helper type 1 (Th1) pathway favors cellular immunity and Th2 pathway favors humoral immunity. Early determination toward Th1 and Th2 cells in the immune response is dependent on the balance between interleukin-12 (IL-12), which favors Th1 responses, and IL-4, which favors Th2 responses. IL-2 and interferon-γ (IFN-γ) are produced in the Th1 pathway, and IL-4 and IL-10 are produced in the Th2 pathway. Lack of cellular immunity, IL-2, and IFN-γ had been reported in malignant pleural effusions. However, to our knowledge, there are no previous reports on other cytokine components involving Th1 or Th2 pathway. The present study was designed to answer these questions. Design Cytokine levels in peripheral blood and pleural fluid of 21 patients with malignant pleural effusion, including IL-4, IL-10, and IL-12, were analyzed with enzyme-linked immunosorbent assays. Lymphocyte subpopulations of peripheral blood and pleural effusion were also studied by using flow cytometry. Measurements and results The results showed a significant increase in IL-10 level as compared with blood samples. IL-4 and IL-12 were below minimal detectable concentrations both in the blood and the effusion. The ratio of pleural helper T cells was significantly higher than in the blood (p=0.0002). The ratio of pleural natural killer (NK) cells was significantly lower than in the blood (p=0.0001). The ratio of pleural suppressor T cells was lower than blood with borderline significance (p=0.0522). No significant change in B-lymphocyte ratio between blood and pleural effusion was found (p=0.2471). There was no correlation between difference in IL-10 level and lymphocyte subpopulation of pleural effusion and blood samples. Conclusions Helper T-cell subpopulations were increased while NK and suppressor T-cell subpopulations were decreased in malignant pleural effusions. The decrease in NK cell subpopulations with elevated IL-10 and minimal IL-12 concentration in neoplastic pleural effusion would suggest the usage of IL-12 or antibody of IL-10 to improve local cellular immunity. Further study is needed.

Treatment of mice with hepatic carcinogens, including CCl4, has been shown to rapidly enhance the transcription of endogenous murine leukemia virus—related proviral sequences in the liver. To understand the mechanism for this transcriptional stimulation, we used nuclear protein preparations from mouse livers to perform DNase I protection analyses and identified nuclear protein binding on approximately 20 individual sequences within the regulatory regions of the long terminal repeat (LTR) of a polytropic-class endogenous provirus clone. From 3 to 144 h after treatment with CCl4, the livers of FVB/N mice were analyzed for specific nuclear protein binding to the LTR DNA. Three to nine hours after CCI4 treatment, decreased protection was seen at potential regulatory cis-elements throughout the LTR, including specific sites within the putative negative regulatory element (located 5′ of the consensus enhancer sequences) and the 3′ terminal portion of the polytropic class-specific enhancer-like inserted sequence element and around the CCAA(C/T) box in the promoter region. In addition, by 3–6 h after treatment, a transient increase in protection activity for the transcription initiation site occurred. The loss of cis-element protection expanded to other binding sites and became most marked by 48 h after treatment. As the regenerating liver recovered, the nuclear protein binding activities for these LTR sequences also recovered, but protection at the TATAA and transcription initiation sites remained deprotected at 144 h after treatment. Nuclear protein protection of other sites, particularly in the conserved LTR enhancer sequences, was minimally affected by CCl4 treatment. Three nuclear protein binding sites that showed rapid CCI4-induced kinetic changes were homologous to the consensus sequence for the binding of the transcription factor families MEF-2, HNF-1, and C/EBP. The complex kinetic changes in factors that may contribute to the rapid and transient induction of endogenous retroviral gene expression in the liver after CCI4 exposure are discussed.

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.