GliPR1 knockdown by RNA interference exerts anti-glioma effects in vitro and in vivo.

INTRODUCTION: In human glioblastomas, glioma pathogenesis-related protein1 (GliPR1) is overexpressed and appears to be an oncoprotein. We investigated whether GliPR1 knockdown in glioma cells by RNA interference exerts anti-glioma effects. METHODS: Experiments used human glioblastoma cell lines transduced with GliPR1 shRNA (sh#301, sh#258). Transduction produced stringent doxycycline-dependent GliPR1 knockdown in clones (via lentiviral "all-in-one" TetOn-shRNA vector) or stable GliPR1 knockdown in polyclonal cells (via constitutive retroviral-shRNA vector). In vitro assessments included cellular proliferation and clonogenic survival. In vivo assessments in tumor-bearing nude mice included tumor growth and survival. RESULTS: Using doxycycline-dependent GliPR1 knockdown, shGliPR1-transduced U87-MG clones demonstrated reductions in cellular proliferation in the presence versus absence of doxycycline. Using stable GliPR1 knockdown, polyclonal shGliPR1-transduced U87-MG, A172, and U343-MG cells consistently showed decreased clonogenic survival and induced apoptosis (higher proportion of early apoptotic cells) compared to control shLuc-transduced cells. In tumor-bearing nude mice, using doxycycline-dependent GliPR1 knockdown, subcutaneous and cranial transplantation of the U87-MG clone 980-5 (transduced with GliPR1 sh#301) resulted in reduced subcutaneous tumor volume and cerebral tumor area in doxycycline-treated mice versus those left untreated. Using stable GliPR1 knockdown, nude mice cranially transplanted with polyclonal U87-MG cells transduced with GliPR1 sh#258 had significantly prolonged survival compared to mice cranially transplanted with control shLuc-transduced cells (41 versus 26 days; P < 0.001). CONCLUSION: GliPR1 knockdown in glioma cells decreased cellular proliferation, decreased clonogenic survival, and induced apoptosis in vitro, and reduced glioblastoma tumor growth and prolonged survival in vivo. These findings support that GliPR1 may have potential value as a therapeutic target.

Comparison of central and local serial CT assessments of metastatic renal cell carcinoma patients in a clinical phase IIB study.

Background Clinical oncological studies attempt to improve precision of data by central radiological assessments. However, it is unclear, to which extent local and central assessments diverge. Purpose To quantify inter-reader variability and the deviation of local from central radiological assessments of computed tomography (CT) scans. Material and Methods This was a sub-study of a randomized clinical phase IIb trial in metastatic renal cell carcinoma (RCC), comparing first-line sorafenib with interferon-alpha-2a (IFN-α-2a). It analyzed agreements of local with central RECIST CT assessments by Cohen's kappa (κ), symmetry tests, deviations in waterfall plots, Bland-Altman plots, and parametric survival analyses. Results The concordance between local and central radiologic review was quantified by κ = 0.53. While local assessment yielded progressive disease (PD) in 18.6%, central assessment classified 22.5% of patient time points as PD exhibiting only a partial overlap with the 18.6% The tumor shrinkage rates in waterfall plots were 68.1% in local and 55.8% in central review (57.8% and 59% by Reader 1 and Reader 2). Bland-Altman plots identified a systematic shift of tumor change rates by -7.5% in local compared to central assessments, that may reflect a systematic tendency of more favorable results in local assessments. The discordance between local and central review was reflected by a time to progression (TTP) hazard ratio (HR) of 1.73 ( P = 0.0003). Conclusion These data suggest that central radiologic review may reduce technical measurement variability in clinical trials, which should be scrutinized in future studies compared to a volumetric reference.

HIV-1 infection suppresses expression of host cell cycle-associated gene PDS5A.

OBJECTIVE: To unravel the interplay between HIV-1 and its host cell, the effect of HIV-1 infection on cellular gene expression was investigated. METHODS: HIV-1(SF33)-infected and uninfected H9 T cells were screened by differential display and RNase protection assay. The finding (PDS5A) was confirmed in HIV-1(Lai)-infected P4-CCR5 HeLa cells, which were also examined after PDS5A siRNA knockdown in regard to HIV-1 replication by quantitative RT-PCR, p24 ELISA and LTR-driven ß-galactosidase expression. The PDS5A knockdown effect on cellular gene expressions was studied by microarray analysis. PDS5A tissue expression was determined by Northern blotting. RESULTS: Regulator of cohesion maintenance, homolog A (PDS5A) was found to be down-regulated by HIV-1. When PDS5A was suppressed by siRNA, HIV-1 replication was unaffected. PDS5A was found to be highly expressed in skeletal muscle tissue, and to lesser degrees in pancreas, heart, placenta, lung, kidney, liver and brain. Microarray analysis of PDS5A knockdown revealed 91 differential gene products over-representing cell cycle, transport and protein stability regulation, including 4 genes (PP2A, RANTES, PCAF, TCF7L2) previously reported to interact with HIV-1. CONCLUSION: The data show a downregulation of proliferation-associated host gene PDS5A and suggest a role of PDS5A in HIV-1-induced cellular pathogenesis but not viral replication.

Inhibition of HIV-1 replication by small interfering RNAs directed against glioma pathogenesis related protein (GliPR) expression.

BACKGROUND: Previously, we showed that glioma pathogenesis related protein (GliPR) is induced in CEM T cells upon HIV-1 infection in vitro. To examine whether GliPR plays a role as HIV dependency factor (HDF), we tested the effect of GliPR suppression by siRNA on HIV-1 replication. RESULTS: Induction of GliPR expression by HIV-1 was confirmed in P4-CCR5 cells. When GliPR was suppressed by siRNA, HIV-1 replication was significantly reduced as measured by HIV-1 transcript levels, HIV-1 p24 protein levels, and HIV-1 LTR-driven reporter gene expression, suggesting that GliPR is a cellular co-factor of HIV-1. Microarray analysis of uninfected HeLa cells following knockdown of GliPR revealed, among a multitude of gene expression alterations, a down-regulation of syndecan-1, syndecan-2, protein kinase C alpha (PRKCA), the catalytic subunit beta of cAMP-dependent protein kinase (PRKACB), nuclear receptor co-activator 3 (NCOA3), and cell surface protein CD59 (protectin), all genes having relevance for HIV-1 pathology. CONCLUSIONS: The up-regulation of GliPR by HIV-1 and the early significant inhibition of HIV-1 replication mediated by knockdown of GliPR reveal GliPR as an important HIV-1 dependency factor (HDF), which may be exploited for HIV-1 inhibition.

Proliferative arrest and cell cycle regulation in CD8(+)CD28(-) versus CD8(+)CD28(+) T cells.

CD8(+)CD28(-) T cells have been characterized by oligoclonal expansions, impaired proliferative responses, but preserved cytotoxicity and reduced telomeres. To examine this subset further and define the underlying mechanisms of proliferation arrest, we investigated several features of this cell type compared with CD8(+)CD28(+) controls. We analyzed expression of various activation markers, thymidine incorporation upon activation, T-cell receptor (TCR) zeta-chain phosphorylation, cell cycle characteristics, and cell cycle related gene expression. Flow cytometry revealed higher expression of CD11b, CD29, CD57, and CD94, and lower expression of CD25 in CD8(+)CD28(-) compared with CD8(+)CD28(+) T cells. Sorted CD8(+)CD16(-)CD28(-) cells exhibited decreased phosphorylation of the TCR zeta-chain in three of four probands. Proliferation of these T cells was impaired, even when activated with mitogens that bypass TCR signaling. Cell cycle profiles demonstrated a lower percentage of cycling cells and significantly higher levels of cyclin dependent kinase inhibitor p16(INK4a) in the CD28(-) subset compared with the CD28(+) control. These observations suggest that expanded CD8(+)CD28(-) T cells in normal elderly individuals have reduced proliferation concomitant with increased p16(INK4a) expression. Defects in TCR signaling were associated with altered TCR zeta-chain phosphorylation.

Characterization of ZC3H15 as a potential TRAF-2-interacting protein implicated in the NFκB pathway and overexpressed in AML.

The gene product of the zinc finger CCCH-type containing 15 (ZC3H15) gene, an immediate early erythropoietin response gene (synonymous: LEREPO4), was further characterized. ZC3H15 was expressed ubiquitously in all human tissues tested by northern blotting and showed mainly a diffuse cytoplasmic distribution by immune fluorescence microscopy and western blotting of subcellular protein fractions. The expression of ZC3H15 was downregulated effectively in HeLa cells to ≤13% of the control by transfection of specific small interfering RNA (siRNA). Subsequent Affymetrix microarray analysis revealed 202 differentially expressed genes including 114 induced (≥3-fold) genes and 88 suppressed (≤0.3-fold) genes. The gene ontology (GO) categories containing an over-representation of differentially expressed genes comprised cell growth, transcription, cell adhesion, regulation of NF-κB, regulation of MAPK, cell cycle arrest and immune response. ZC3H15 interacted with the signaling adapter protein tumor necrosis factor receptor associated factor 2 (TRAF-2) as shown by co-immunoprecipitation. ZC3H15 expression was found to be significantly increased in acute myeloid leukemia (AML) samples compared to MDS, CML, ALL and normal bone marrow samples using the Leukemia Gene Atlas (LGA) database. Based on these data, it is hypothesized that ZC3H15 may interact with TRAF-2 functionally within the NF-κB pathway, and may be explored as a potential target in AML.

Endoplasmic reticulum protein GliPR1 regulates G protein signaling and the cell cycle and is overexpressed in AML.

Glioma pathogenesis­related protein 1 (GliPR1) is a pleiotropic protein involved in cell proliferation, tumor growth and apoptosis. The aim of the present study was to further characterize GliPR1 in regard to its subcellular localization and its overall effect on cellular gene expression. Knockdown of GliPR1 and Affymetrix microarray mRNA expression analysis revealed 262 GliPR1­dependent differentially expressed genes, of which 40 were induced and 222 were suppressed. Differentially expressed genes were overrepresented in five Gene Ontology categories: G protein signaling pathways, regulation of cyclin­dependent protein kinase activity, ER to Golgi vesicle-mediated transport, axon guidance and dephosphorylation. GliPR1-EGFP fusion protein co­localized with the endoplasmic reticulum (ER) or with cytoplasmic vesicles as demonstrated by confocal microscopy. GliPR1 expression was found to be significantly increased in acute myeloid leukemia (AML) bone marrow samples, while markedly reduced in acute lymphoblastic leukemia, unchanged in myelodysplastic syndrome and slightly decreased in chronic lymphocytic leukemia as well as in chronic myelocytic leukemia (CML) when compared to normal samples. GliPR1 was localized and involved in the ER secretory protein pathway. GliPR1 affects G protein signaling and cell cycle regulation. Based on the observed overexpression in AML samples, GliPR1 should be further explored as a potential target for AML.

Knockdown of ERM family member moesin in host cells increases HIV type 1 replication.

Moesin is a member of the ERM (ezrin, radixin, moesin) family of cytoskeleton/membrane structure organizing and signal transduction proteins. Previously, we found an increased expression of moesin during HIV-1 infection. Moesin was also reported to be incorporated into HIV-1 virions. To analyze whether moesin is a host factor affecting the replication cycle of human immunodeficiency virus type 1 (HIV-1), we used small interfering RNAs (siRNAs) to evaluate the effect of moesin knockdown on HIV-1 replication in P4-CCR5 cells. Moesin's knockdown did not affect the cell viability or cell phenotype. Interestingly, we observed a marked increase in viral replication, as demonstrated by enhanced HIV-1 RNA, p24 antigen, and ß-galactosidase reporter expression. Moesin-dependent enhancement of HIV-1 replication was confirmed in lymphocytic host cells (Jurkat). These results suggest an overall rather restrictive role of moesin for HIV-1 replication in host cells in vitro.

Inhibition of X4-tropic HIV type 1 replication by knockdown of the cellular protein LEREPO4.

Human immunodeficiency virus 1 (HIV-1) and host cell factors show important mutual interactions. We found that HIV-1 infection induced expression of a likely ortholog of mouse immediate early response erythropoietin 4 (LEREPO4) in vitro. When LEREPO4 expression was suppressed by siRNA in P4-CCR5 cells, HIV-1 replication showed significantly reduced HIV-1 transcript and p24 protein levels as measured by quantitative PCR and ELISA, respectively. The LEREPO4 knockdown also had an inhibitory effect on HIV-1-LTR-driven reporter plasmid expression of ß-galactosidase. Furthermore, the inhibitory effect of LEREPO4 silencing on HIV-1 replication was confirmed in Jurkat T cells. The up-regulation of LEREPO4 by HIV-1 and the inhibition of HIV-1 replication mediated by knockdown of LEREPO4 may point to an important functional role of LEREPO4 as a novel HIV-1 dependency factor.

Randomized phase II trial of first-line treatment with sorafenib versus interferon Alfa-2a in patients with metastatic renal cell carcinoma.

PURPOSE: An open-label, phase II study to evaluate progression-free survival (PFS), overall best response, adverse events (AEs), and patient-reported outcomes with sorafenib versus interferon alfa-2a (IFN-alpha-2a) in patients with untreated, advanced renal cancer. PATIENTS AND METHODS: A total of 189 patients were randomly assigned to oral sorafenib 400 mg twice daily or to subcutaneous IFN-alpha-2a 9 million U three times weekly (period 1). Sorafenib patients who progressed were dose-escalated to 600 mg twice daily; IFN-alpha-2a patients who progressed were switched to sorafenib 400 mg twice daily (period 2). RESULTS: In period 1 PFS was similar for sorafenib-treated (n = 97; 5.7 months) and IFN-alpha-2a-treated patients (n = 92; 5.6 months); more sorafenib-treated patients had tumor shrinkage (68.2% v 39.0%). Common drug-related AEs (Grades > or = 3) for sorafenib were hand-foot skin reaction (11.3%), diarrhea (6.2%), and rash/desquamation (6.2%); for IFN-alpha-2a, these were fatigue (10.0%), nausea (3.3%), flu-like syndrome (2.2%), and anorexia (2.2%). Sorafenib-treated patients reported fewer symptoms, better quality of life (QOL), and greater treatment satisfaction. In period 2, 41.9% of patients who received sorafenib 600 mg twice daily (n = 43) experienced tumor reduction (median PFS, 3.6 months). After the switch to sorafenib 400 mg twice daily, tumors were reduced in 76.2% of 50 patients (median PFS, 5.3 months). AEs were mostly grade 1 to 2; no increase in AEs of grades > or = 3 occurred after sorafenib dose escalation. CONCLUSION: In this study, sorafenib resulted in similar PFS as IFN-alpha-2a in patients with untreated RCC. However, sorafenib-treated patients experienced greater rates of tumor size reduction, better QOL, and improved tolerability. Both dose escalation of sorafenib after progression and a switch to sorafenib after progression on IFN-alpha-2a resulted in clinical benefit.

Early molecular response to posttransplantation imatinib determines outcome in MRD+ Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL).

In adult Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL), minimal residual disease (MRD) after stem cell transplantation (SCT) is associated with a relapse probability exceeding 90%. Starting imatinib in the setting of MRD may decrease this high relapse rate. In this prospective multicenter study, 27 Ph+ ALL patients received imatinib upon detection of MRD after SCT. Bcr-abl transcripts became undetectable in 14 (52%) of 27 patients, after a median of 1.5 months (0.9-3.7 months) ((early)CR(mol)). All patients who achieved an (early)CR(mol) remained in remission for the duration of imatinib treatment; 3 patients relapsed after imatinib was discontinued. Failure to achieve polymerase chain reaction (PCR) negativity shortly after starting imatinib predicted relapse, which occurred in 12 (92%) of 13 patients after a median of 3 months. Disease-free survival (DFS) in (early)CR(mol) patients is 91% +/- 9% and 54% +/- 21% after 12 and 24 months, respectively, compared with 8% +/- 7% after 12 months in patients remaining MRD+ (P < .001). In conclusion, approximately half of patients with Ph+ ALL receiving imatinib for MRD positivity after SCT experience prolonged DFS, which can be anticipated by the rapid achievement of a molecular complete remission (CR). Continued detection of bcr-abl transcripts after 2 to 3 months on imatinib identifies patients who will ultimately experience relapse and in whom additional or alternative antileukemic treatment should be initiated.

Early minimal residual disease (MRD) analysis during treatment of Philadelphia chromosome/Bcr-Abl-positive acute lymphoblastic leukemia with the Abl-tyrosine kinase inhibitor imatinib (STI571).

The Abl kinase inhibitor imatinib mesylate (STI571) has significant and rapid antileukemic activity in Philadelphia chromosome/Bcr-Abl-positive acute lymphoblastic leukemia (Ph(+) ALL) but such activity is usually of short duration except for a small proportion of patients. To determine the prognostic significance of early Bcr-Abl levels and changes in peripheral blood (PB) and bone marrow (BM), serial samples of 56 patients with relapsed or refractory Ph(+) ALL treated in phase 2 trials of imatinib were analyzed by quantitative polymerase chain reaction (PCR). Imatinib induced a complete hematologic response (CHR) or complete marrow response (marrow-CR) in 40 patients (good responders) and a partial (n = 2) or no (n = 14) remission in the remaining patients (poor responders). Compared with baseline, the median Bcr-Abl/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) ratios decreased significantly in PB by 2.65, 2.64, and 3.11 log steps after 2 weeks, 4 weeks, and at the time of best response, respectively. In BM, the decline of median Bcr-Abl/GAPDH was 0.75, 1.37, and 2.78 logs, respectively. Thus, Bcr-Abl levels decreased more rapidly in PB than in BM (median time to best level 31 vs 39 days). Low Bcr-Abl/GAPDH ratios below 10(-4) in PB and below 10(-2) in BM after 2 weeks were significantly associated with good responses after 4 weeks. Moreover, Bcr-Abl levels (< 10(-2)) in BM of good responders after 4 weeks discriminated between 2 groups of patients with significantly different median time to progression (139 vs 22 days). The data show that Bcr-Abl levels in PB and BM after 2 weeks of imatinib treatment and in BM after 4 weeks have predictive relevance and may guide the application of additional therapies.

Long-term benzimidazole treatment of alveolar echinococcosis with hematogenic subcutaneous and bone dissemination.

We report a case of advanced alveolar echinococcosis (AE) that presented like a malignant tumor. It was diagnosed histologically from a subcutaneous nodule with skin inflammation on the right leg. Additionally, the patient showed bone metastases in the lower thoracic spine and the left third toe. This is the first case with proven hematogenic spread of AE to a subcutaneous site. The patient was treated with albendazole and remained stable for 6 years. When progression of AE occurred the therapy was changed to mebendazole, resulting in a stable condition for further 4 years.

Early prediction of response in patients with relapsed or refractory Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) treated with imatinib.

Imatinib has pronounced but brief antileukemic activity in advanced Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(+)ALL). We assessed the prognostic impact of pretreatment disease features and the early bone marrow (BM) response in 68 consecutive patients with Ph(+)ALL receiving imatinib salvage therapy. A complete hematologic or marrow response was achieved by 92% of patients with BM blasts below 5% on day 14, whereas 62.5% of patients with more than 5% BM blasts on day 14 were nonresponders. Similarly, time to progression (TTP) was superior in patients with a good day 14 response (5.2 versus 0.9 months; P <.0001). Prior complete remission of less than 6 months, white blood cell count of more than 10 x 10(9)/L, circulating peripheral blood blasts at diagnosis, additional Philadelphia chromosomes, or at least 2 Bcr-Abl fusion signals were associated with significantly inferior remission rate and response duration. In patients without poor prognostic features, single-agent imatinib may be appropriate before transplant salvage therapy. Conversely, patients with clinically or cytogenetically defined poor-risk features are candidates for trials of upfront imatinib in combination with other agents.