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1.
Am J Clin Oncol ; 35(3): 255-60, 2012 Jun.
Article in English | MEDLINE | ID: mdl-21358293

ABSTRACT

BACKGROUND: Despite the efficacy of gemcitabine-platinum regimen, the outcome of patients with recurrent and/or metastatic nasopharyngeal carcinoma (RM NPC) is poor. A phase II trial was conducted to determine the efficacy of erlotinib, given as maintenance therapy after gemcitabine-platinum chemotherapy in patients with RM NPC. PATIENTS AND METHODS: Patients were treated with gemcitabine 1000 mg/m on days 1 and 8 as well as cisplatin 70 mg/m on day 1 (or carboplatin area under curve 5 on day 1, if contraindication to cisplatin) 3 weeks. After 6 chemotherapy cycles (or before in case of progression), patients were switched to erlotinib 150 mg/d 4 weeks. Primary end point was time to progression in patients without progressive disease after 6 chemotherapy cycles and treated with maintenance erlotinib. Epstein-Barr virus DNA plasma levels, measured using quantitative real-time polymerase chain reaction, were correlated with outcome. RESULTS: Of 20 enrolled patients, 19 patients received 96 chemotherapy cycles. Fifteen patients were switched to erlotinib and received 36 cycles (range: 1 to 6 cycles). Safety profiles observed with the chemotherapy combination and erlotinib were those expected. Of 12 patients evaluable for response to erlotinib, all progressed except 3 patients (25%) who had stable disease for 3, 4, and 7 months, respectively. Median time to progression was 6.9 months for 13 patients without progressive disease after 6 chemotherapy cycles and treated with erlotinib. No correlation was identified between Epstein-Barr virus DNA plasma levels and clinical outcome. CONCLUSIONS: Maintenance or second-line therapy with erlotinib after chemotherapy was not effective in RM NPC. Historical comparison with patients treated with the same chemotherapy alone until progression suggests that it may be detrimental to stop chemotherapy after 6 cycles if disease did not progress.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Nasopharyngeal Neoplasms/drug therapy , Neoplasm Recurrence, Local/drug therapy , Salvage Therapy , Adult , Aged , Carboplatin/administration & dosage , Cisplatin/administration & dosage , Deoxycytidine/administration & dosage , Deoxycytidine/analogs & derivatives , Disease Progression , Erlotinib Hydrochloride , Female , Follow-Up Studies , Humans , Male , Middle Aged , Nasopharyngeal Neoplasms/mortality , Nasopharyngeal Neoplasms/pathology , Neoplasm Metastasis , Neoplasm Recurrence, Local/mortality , Neoplasm Recurrence, Local/pathology , Neoplasm Staging , Prognosis , Quinazolines/administration & dosage , Survival Rate , Gemcitabine
2.
Cancer ; 113(11): 3169-80, 2008 Dec 01.
Article in English | MEDLINE | ID: mdl-18991282

ABSTRACT

BACKGROUND: The authors have previously shown that overexpression of claudin 1 (CLDN1) is associated with advanced disease stage in oral squamous cell carcinomas (OSCCs). Their goal was to examine CLDN1 expression in a large series of primary OSCCs and to further investigate whether CLDN1 overexpression plays a role in invasion in OSCC. METHODS: CLDN1 gene expression levels were determined by quantitative real-time reverse transcription polymerase chain reaction (QRT-PCR) in 100 primary OSCCs. CLDN1 protein expression was examined by immunohistochemistry in 70 of 100 OSCCs. E-Cadherin protein levels were also assessed in 58 OSCCs. The authors performed a transwell Matrigel invasion assay for assessment of the invasive potential of CLDN1 overexpressing oral carcinoma cells. Western blotting and QRT-PCR were used to assess CLDN1 expression in transfected cells and controls. RESULTS: CLDN1 mRNA was increased (median = 18.5) in 79 of 100 OSCCs, compared with normal oral mucosa (expression = 1.0). CLDN1 overexpression was associated with angiolymphatic (P = .037) and perineural invasion (P = .051). CLDN1 was highly expressed in 48 of 70 (68%) OSCCs. E-Cadherin was lost or underexpressed in 49 of 58 (84%) OSCCs. The invasion assay showed that cells overexpressing CLDN1 have increased invasive potential, whereas small interfering RNA-mediated depletion of CLDN1 decreased the invasive potential of cells. CONCLUSIONS: CLDN1 overexpression is associated with angiolymphatic and perineural invasion, consistent with aggressive tumor behavior. Overexpression of CLDN1 protein is associated with increased invasiveness of oral carcinoma cells.


Subject(s)
Carcinoma, Squamous Cell/genetics , Membrane Proteins/genetics , Mouth Neoplasms/genetics , Adult , Aged , Aged, 80 and over , Carcinoma, Squamous Cell/metabolism , Claudin-1 , DNA-Binding Proteins , Female , Gene Expression , Humans , Immunohistochemistry , Male , Membrane Proteins/metabolism , Middle Aged , Neoplasm Invasiveness/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction
3.
PLoS Genet ; 4(4): e1000051, 2008 Apr 11.
Article in English | MEDLINE | ID: mdl-18404216

ABSTRACT

The Locus Control Region (LCR) requires intronic elements within beta-globin transgenes to direct high level expression at all ectopic integration sites. However, these essential intronic elements cannot be transmitted through retrovirus vectors and their deletion may compromise the therapeutic potential for gene therapy. Here, we systematically regenerate functional beta-globin intron 2 elements that rescue LCR activity directed by 5'HS3. Evaluation in transgenic mice demonstrates that an Oct-1 binding site and an enhancer in the intron cooperate to increase expression levels from LCR globin transgenes. Replacement of the intronic AT-rich region with the Igmu 3'MAR rescues LCR activity in single copy transgenic mice. Importantly, a combination of the Oct-1 site, Igmu 3'MAR and intronic enhancer in the BGT158 cassette directs more consistent levels of expression in transgenic mice. By introducing intron-modified transgenes into the same genomic integration site in erythroid cells, we show that BGT158 has the greatest transcriptional induction. 3D DNA FISH establishes that induction stimulates this small 5'HS3 containing transgene and the endogenous locus to spatially reorganize towards more central locations in erythroid nuclei. Electron Spectroscopic Imaging (ESI) of chromatin fibers demonstrates that ultrastructural heterochromatin is primarily perinuclear and does not reorganize. Finally, we transmit intron-modified globin transgenes through insulated self-inactivating (SIN) lentivirus vectors into erythroid cells. We show efficient transfer and robust mRNA and protein expression by the BGT158 vector, and virus titer improvements mediated by the modified intron 2 in the presence of an LCR cassette composed of 5'HS2-4. Our results have important implications for the mechanism of LCR activity at ectopic integration sites. The modified transgenes are the first to transfer intronic elements that potentiate LCR activity and are designed to facilitate correction of hemoglobinopathies using single copy vectors.


Subject(s)
Genetic Therapy/methods , Globins/genetics , Anemia, Sickle Cell/genetics , Anemia, Sickle Cell/therapy , Animals , Base Sequence , Binding Sites/genetics , Cell Line , DNA Primers/genetics , Enhancer Elements, Genetic , Female , Gene Expression , Genetic Complementation Test , Genetic Vectors , Introns , Locus Control Region , Male , Mice , Mice, Transgenic , Octamer Transcription Factor-1/metabolism , Pregnancy , Recombinant Proteins/genetics , Reverse Transcriptase Polymerase Chain Reaction
4.
Mol Ther ; 10(1): 27-36, 2004 Jul.
Article in English | MEDLINE | ID: mdl-15233939

ABSTRACT

Retrovirus silencing in stem cells produces silent or variegated provirus. Additional memory and extinction mechanisms act during differentiation. Here we show that retrovirus is silent or variegated in mouse embryonic stem (ES) cells that are de novo methyltransferase (dnmt3a and dnmt3b) null. Memory is maintained during differentiation, and extinction occurs on variegated retrovirus, indicating that DNA methylation is dispensable for all forms of retrovirus silencing. Silent and variegated provirus are marked by hypoacetylated histone H3 and bound H1. In wild-type ES cells, silent and variegated proviruses are methylated and bound by hypoacetylated H3, MeCP2, and less H1. Silencing, variegation, and extinction are partially reactivated by 5-AzaC in this context. Lentivirus vectors are also silent or variegated, marked by silent chromatin, and exhibit memory and extinction. We conclude that the universal epigenetic mark of retrovirus silencing is silent chromatin established via the dynamic interplay of multiple epigenetic modifications that include but do not require DNA methylation. A molecular mechanism of competitive H1 and MeCP2 binding may account for this epigenetic interplay, and a model for variegation is discussed.


Subject(s)
Epigenesis, Genetic , Gene Expression Regulation, Viral , Gene Silencing , Retroviridae/genetics , Animals , Azacitidine/pharmacology , Chromatin/genetics , Chromatin/metabolism , Chromatin Immunoprecipitation , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , DNA/chemistry , DNA/metabolism , DNA Methylation/drug effects , DNA Restriction Enzymes/chemistry , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Genetic Therapy/methods , Green Fluorescent Proteins/analysis , Green Fluorescent Proteins/genetics , Histones/genetics , Histones/metabolism , Methyl-CpG-Binding Protein 2 , Mice , Phosphoglycerate Kinase/analysis , Phosphoglycerate Kinase/genetics , Phosphoglycerate Kinase/metabolism , Proviruses/genetics , Proviruses/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , Retroviridae/metabolism , Stem Cells/metabolism , Stem Cells/virology
5.
Nucleic Acids Res ; 31(18): 5317-23, 2003 Sep 15.
Article in English | MEDLINE | ID: mdl-12954767

ABSTRACT

Silencing of retrovirus vectors poses a significant obstacle to genetic manipulation of stem cells and their use in gene therapy. We describe a mammalian silencer blocking assay using insulator elements positioned between retrovirus silencer elements and an LCRbeta-globin reporter transgene. In transgenic mice, we show that retrovirus silencers are blocked by the cHS4 insulator. Silencer blocking is independent of the CTCF binding site and is most effective when flanking the internal reporter transgene. These data distinguish silencer blocking activity by cHS4 from its enhancer blocking activity. Retrovirus vectors can be created at high titer with one but not two internal dimer cHS4 cores. cHS4 in the LTRs has no effect on expression in transduced F9 cells, suggesting that position effect blocking is not sufficient to escape silencing. The Drosophila insulators gypsy and Scs fail to block silencing in transgenic mice, but gypsy stimulates vector expression 2-fold when located in the LTRs of an infectious retrovirus. The silencer blocking assay complements existing insulator assays in mammalian cells, provides new insight into mechanisms of insulation and is a valuable tool to identify additional silencer blocking insulators that cooperate with cHS4 to improve stem cell retrovirus vector design.


Subject(s)
DNA-Binding Proteins/metabolism , Enhancer Elements, Genetic/genetics , Gene Expression Regulation/genetics , Repressor Proteins/metabolism , Animals , Binding Sites/genetics , CCCTC-Binding Factor , Chickens , DNA/chemistry , DNA/genetics , DNA/metabolism , Dimerization , Genetic Vectors/genetics , Globins/genetics , Humans , Mice , Mice, Transgenic , Retroelements/genetics , Retroviridae/genetics
6.
Nucleic Acids Res ; 31(12): 3257-66, 2003 Jun 15.
Article in English | MEDLINE | ID: mdl-12799453

ABSTRACT

The human beta-globin locus is home to five genes that are regulated in a tissue-specific and developmental stage-specific manner. While the exact mode of expression remains somewhat enigmatic, a significant effort has been focused at the locus control region (LCR). The LCR is marked by five DNase I-hypersensitive sites (HS) approximately 15 kb upstream of the epsilon-globin gene. Nuclear matrix-associated regions (MARs) organize chromatin into functional domains and at least one of the HS appears bound to the nuclear matrix. We have employed an in vivo based PCR MAR assay to investigate the role of MAR-mediated regulation of the beta-globin locus. This was facilitated with a novel reaction efficiency based quantitative real-time PCR analysis software tool, Target Analysis Quantification. Using a log-linear regression strategy, discordances were eliminated. This allowed us to reliably estimate the relative amount of initial template associated with the nuclear matrix at 15 unique regions spanning the beta-globin locus in both non-expressing and expressing cell lines. A dynamic association dependent on expression status was revealed both at the LCR/5'HS region and within the second intron of the beta-globin gene. These results provide the first evidence that nuclear matrix association dynamically mediates the looping of the beta-globin locus to achieve transcriptional control.


Subject(s)
Globins/genetics , Locus Control Region , Nuclear Matrix/metabolism , Polymerase Chain Reaction/methods , Regulatory Sequences, Nucleic Acid , Software , Cell Line , Humans , K562 Cells , Models, Genetic , Time Factors
7.
Blood ; 101(4): 1603-10, 2003 Feb 15.
Article in English | MEDLINE | ID: mdl-12393478

ABSTRACT

Human beta-globin transgenes regulated by the locus control region (LCR) express at all integration sites in transgenic mice. For such LCR activity at ectopic sites, the 5'HS3 element requires the presence of the AT-rich region (ATR) in beta-globin intron-2. Here, we examine the dependence of 5'HS3 LCR activity on transcription factor binding sites in the ATR. In vitro DNaseI footprint analysis and electrophoretic mobility shift assays of the ATR identified an inverted double Gata-1 site composed of 2 noncanonical sequences (GATT and GATG) and an Oct-1 consensus site. Mutant Oct-1, Gata-1, or double mutant sites were created in the ATR of the BGT50 construct composed of a 5'HS3 beta/gamma-globin hybrid transgene. Transgenes with double mutant sites expressed at all sites of integration, but mean expression levels in transgenic mice were reduced from 64% per copy (BGT50) to 37% (P <.05). Mutation of the inverted double Gata-1 site had no effect at 61% per copy expression levels. In contrast, mutation of the Oct-1 site alone reduced per-copy expression levels to 31% (P <.05). We conclude that the ability of 5'HS3 to activate expression from all transgene integration sites is dependent on sequences in the ATR that are not bound at high affinity by transcription factors. In addition, the Oct-1 site in the ATR is required for high-level 5'HS3 beta/gamma-globin transgene expression and should be retained in LCRbeta-globin expression cassettes designed for gene therapy.


Subject(s)
AT Rich Sequence/genetics , DNA-Binding Proteins/metabolism , Globins/genetics , Locus Control Region/physiology , Transcription Factors/metabolism , Animals , Binding Sites , DNA/chemistry , DNA/metabolism , DNA Footprinting , Electrophoretic Mobility Shift Assay , Erythroid-Specific DNA-Binding Factors , GATA1 Transcription Factor , Gene Expression Regulation , Host Cell Factor C1 , Introns , Locus Control Region/genetics , Mice , Mice, Transgenic , Mutagenesis, Site-Directed , Octamer Transcription Factor-1 , Polymerase Chain Reaction
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