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1.
Nat Commun ; 13(1): 1028, 2022 03 01.
Article in English | MEDLINE | ID: mdl-35232966

ABSTRACT

Prime editing is a versatile genome-editing technique that shows great promise for the generation and repair of patient mutations. However, some genomic sites are difficult to edit and optimal design of prime-editing tools remains elusive. Here we present a fluorescent prime editing and enrichment reporter (fluoPEER), which can be tailored to any genomic target site. This system rapidly and faithfully ranks the efficiency of prime edit guide RNAs (pegRNAs) combined with any prime editor variant. We apply fluoPEER to instruct correction of pathogenic variants in patient cells and find that plasmid editing enriches for genomic editing up to 3-fold compared to conventional enrichment strategies. DNA repair and cell cycle-related genes are enriched in the transcriptome of edited cells. Stalling cells in the G1/S boundary increases prime editing efficiency up to 30%. Together, our results show that fluoPEER can be employed for rapid and efficient correction of patient cells, selection of gene-edited cells, and elucidation of cellular mechanisms needed for successful prime editing.


Subject(s)
CRISPR-Cas Systems , Gene Editing , CRISPR-Cas Systems/genetics , Gene Editing/methods , Genome , Humans , Mutation , RNA, Guide, Kinetoplastida/genetics
2.
Oncogene ; 35(26): 3465-75, 2016 06 30.
Article in English | MEDLINE | ID: mdl-26549027

ABSTRACT

Epithelial-mesenchymal transition (EMT) is a key event that is involved in the invasion and dissemination of cancer cells. Although typically considered as having tumour-suppressive properties, transforming growth factor (TGF)-ß signalling is altered during cancer and has been associated with the invasion of cancer cells and metastasis. In this study, we report a previously unknown role for the cytoplasmic promyelocytic leukaemia (cPML) tumour suppressor in TGF-ß signalling-induced regulation of prostate cancer-associated EMT and invasion. We demonstrate that cPML promotes a mesenchymal phenotype and increases the invasiveness of prostate cancer cells. This event is associated with activation of TGF-ß canonical signalling pathway through the induction of Sma and Mad related family 2 and 3 (SMAD2 and SMAD3) phosphorylation. Furthermore, the cytoplasmic localization of promyelocytic leukaemia (PML) is mediated by its nuclear export in a chromosomal maintenance 1 (CRM1)-dependent manner. This was clinically tested in prostate cancer tissue and shown that cytoplasmic PML and CRM1 co-expression correlates with reduced disease-specific survival. In summary, we provide evidence of dysfunctional TGF-ß signalling occurring at an early stage in prostate cancer. We show that this disease pathway is mediated by cPML and CRM1 and results in a more aggressive cancer cell phenotype. We propose that the targeting of this pathway could be therapeutically exploited for clinical benefit.


Subject(s)
Epithelial-Mesenchymal Transition , Promyelocytic Leukemia Protein/metabolism , Prostatic Neoplasms/metabolism , Transforming Growth Factor beta/metabolism , Active Transport, Cell Nucleus , Cell Line, Tumor , Cell Movement , Cell Nucleus/metabolism , Cytoplasm/metabolism , Humans , Immunoblotting , Kaplan-Meier Estimate , Karyopherins/genetics , Karyopherins/metabolism , Male , Neoplasm Invasiveness , Phosphorylation , Promyelocytic Leukemia Protein/genetics , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , RNA Interference , Receptor, Transforming Growth Factor-beta Type I , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Transforming Growth Factor beta/genetics , Receptors, Transforming Growth Factor beta/metabolism , Signal Transduction , Smad2 Protein/metabolism , Smad3 Protein/metabolism , Exportin 1 Protein
3.
Br J Cancer ; 110(10): 2450-61, 2014 May 13.
Article in English | MEDLINE | ID: mdl-24755885

ABSTRACT

BACKGROUND: HAGE protein is a known immunogenic cancer-specific antigen. METHODS: The biological, prognostic and predictive values of HAGE expression was studied using immunohistochemistry in three cohorts of patients with BC (n=2147): early primary (EP-BC; n=1676); primary oestrogen receptor-negative (PER-BC; n=275) treated with adjuvant anthracycline-combination therapies (Adjuvant-ACT); and primary locally advanced disease (PLA-BC) who received neo-adjuvant anthracycline-combination therapies (Neo-adjuvant-ACT; n=196). The relationship between HAGE expression and the tumour-infiltrating lymphocytes (TILs) in matched prechemotherapy and postchemotherapy samples were investigated. RESULTS: Eight percent of patients with EP-BC exhibited high HAGE expression (HAGE+) and was associated with aggressive clinico-pathological features (Ps<0.01). Furthermore, HAGE+expression was associated with poor prognosis in both univariate and multivariate analysis (Ps<0.001). Patients with HAGE+did not benefit from hormonal therapy in high-risk ER-positive disease. HAGE+and TILs were found to be independent predictors for pathological complete response to neoadjuvant-ACT; P<0.001. A statistically significant loss of HAGE expression following neoadjuvant-ACT was found (P=0.000001), and progression-free survival was worse in those patients who had HAGE+residual disease (P=0.0003). CONCLUSIONS: This is the first report to show HAGE to be a potential prognostic marker and a predictor of response to ACT in patients with BC.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Biomarkers, Tumor/analysis , Breast Neoplasms/chemistry , Carcinoma/chemistry , DEAD-box RNA Helicases/analysis , Drug Resistance, Neoplasm , Neoplasm Proteins/analysis , Antineoplastic Agents, Hormonal/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Breast Neoplasms/drug therapy , Breast Neoplasms/mortality , Breast Neoplasms/therapy , Carcinoma/drug therapy , Carcinoma/mortality , Carcinoma/therapy , Combined Modality Therapy , Cyclophosphamide/administration & dosage , Female , Fluorouracil/administration & dosage , Humans , Kaplan-Meier Estimate , Lymphocytes, Tumor-Infiltrating , Mastectomy , Menopause , Methotrexate/administration & dosage , Mitotic Index , Neoplasm Invasiveness , Neoplasms, Hormone-Dependent/chemistry , Neoplasms, Hormone-Dependent/drug therapy , Neoplasms, Hormone-Dependent/mortality , Neoplasms, Hormone-Dependent/therapy , Prognosis , Proportional Hazards Models , Receptor, ErbB-2/analysis , Receptors, Estrogen/analysis , Receptors, Progesterone/analysis , Tamoxifen/administration & dosage , Treatment Outcome
4.
Cell Death Dis ; 5: e1061, 2014 Feb 13.
Article in English | MEDLINE | ID: mdl-24525737

ABSTRACT

The tumour suppressor PML (promyelocytic leukaemia protein) regulates several cellular pathways involving cell growth, apoptosis, differentiation and senescence. PML also has an important role in the regulation of stem cell proliferation and differentiation. Here, we show the involvement of the helicase HAGE in the transcriptional repression of PML expression in ABCB5+ malignant melanoma-initiating cells (ABCB5+ MMICs), a population of cancer stem cells which are responsible for melanoma growth, progression and resistance to drug-based therapy. HAGE prevents PML gene expression by inhibiting the activation of the JAK-STAT (janus kinase-signal transducers and activators of transcription) pathway in a mechanism which implicates the suppressor of cytokine signalling 1 (SOCS1). Knockdown of HAGE led to a significant decrease in SOCS1 protein expression, activation of the JAK-STAT signalling cascade and a consequent increase of PML expression. To confirm that the reduction in SOCS1 expression was dependent on the HAGE helicase activity, we showed that SOCS1, effectively silenced by small interfering RNA, could be rescued by re-introduction of HAGE into cells lacking HAGE. Furthermore, we provide a mechanism by which HAGE promotes SOCS1 mRNA unwinding and protein expression in vitro. Finally, using a stem cell proliferation assay and tumour xenotransplantation assay in non-obese diabetic/severe combined immunodeficiency mice, we show that HAGE promotes MMICs-dependent tumour initiation and tumour growth by preventing the anti-proliferative effects of interferon-α (IFNα). Our results suggest that the helicase HAGE has a key role in the resistance of ABCB5+ MMICs to IFNα treatment and that cancer therapies targeting HAGE may have broad implications for the treatment of malignant melanoma.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Antineoplastic Agents/pharmacology , DEAD-box RNA Helicases/metabolism , Interferon-alpha/pharmacology , Melanoma/drug therapy , Neoplasm Proteins/metabolism , Neoplastic Stem Cells/drug effects , Nuclear Proteins/metabolism , Skin Neoplasms/drug therapy , Suppressor of Cytokine Signaling Proteins/metabolism , Transcription Factors/metabolism , Tumor Suppressor Proteins/metabolism , ATP Binding Cassette Transporter, Subfamily B , Animals , Cell Line, Tumor , Cell Proliferation , DEAD-box RNA Helicases/genetics , Drug Resistance, Neoplasm , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Humans , Janus Kinase 1/metabolism , Melanoma/enzymology , Melanoma/genetics , Melanoma/pathology , Mice , Mice, Inbred NOD , Mice, SCID , Neoplasm Proteins/genetics , Neoplastic Stem Cells/enzymology , Neoplastic Stem Cells/pathology , Nuclear Proteins/genetics , Phosphorylation , Promyelocytic Leukemia Protein , RNA Interference , RNA, Messenger/metabolism , STAT Transcription Factors/metabolism , Signal Transduction/drug effects , Skin Neoplasms/enzymology , Skin Neoplasms/genetics , Skin Neoplasms/pathology , Spheroids, Cellular , Suppressor of Cytokine Signaling 1 Protein , Suppressor of Cytokine Signaling Proteins/genetics , TYK2 Kinase/metabolism , Time Factors , Transcription Factors/genetics , Transfection , Tumor Burden , Tumor Suppressor Proteins/genetics , Ubiquitination
5.
Parasite Immunol ; 35(12): 441-56, 2013 Dec.
Article in English | MEDLINE | ID: mdl-23869911

ABSTRACT

The effect of alterations in virulence and transformation by long-term in vitro culture of Leishmania mexicana promastigotes on infectivity and immune responses was investigated. Fresh parasite cultures harvested from Balb/c mice were passaged 20 times in vitro. Infectivity was decreased and was completely avirulent after 20 passages. The qPCR results showed a down-regulation of GP63, LPG2, CPC, CPB2, CPB2.8, CHT1, LACK and LDCEN3 genes after passage seven concomitant with a reduced and absence of infectivity by passages seven and 20, respectively. Parasites at passages one and 20 are referred to as virulent and avirulent, respectively. The growth of avirulent and virulent parasite was affected by conditioned media derived from macrophages or monocytes infected with parasites for 2 h. Giemsa staining showed the failure of avirulent but not virulent parasites to transform to the amastigote stage in infected host cells with both virulent and avirulent modulating the expression of CCL-22, Tgad51, Cox2, IL-1, IL-10, TGF-ß, TNF-α, Rab7, Rab9 and A2 genes; virulent but not avirulent L. mexicana significantly up-regulated Th2-associated cytokines, but down-regulated Rab7 and Rab9 gene expression. In conclusion, a model for L. mexicana is reported, which is of potential value in studying host-parasite interaction.


Subject(s)
Host-Parasite Interactions , Leishmania mexicana/immunology , Leishmania mexicana/pathogenicity , Macrophages/immunology , Macrophages/parasitology , Animals , Culture Media, Conditioned , Cytokines/genetics , Gene Expression Regulation , Genes, Protozoan , Humans , Leishmania mexicana/genetics , Leishmania mexicana/growth & development , Leishmaniasis, Cutaneous/parasitology , Mice , Mice, Inbred BALB C , Monocytes/parasitology , Phagocytosis , Serial Passage , Transcriptome , U937 Cells , Virulence/genetics
6.
Breast Cancer Res Treat ; 120(1): 83-93, 2010 Feb.
Article in English | MEDLINE | ID: mdl-19347577

ABSTRACT

Gene expression microarrays allow for the high throughput analysis of huge numbers of gene transcripts and this technology has been widely applied to the molecular and biological classification of cancer patients and in predicting clinical outcome. A potential handicap of such data intensive molecular technologies is the translation to clinical application in routine practice. In using an artificial neural network bioinformatic approach, we have reduced a 70 gene signature to just 9 genes capable of accurately predicting distant metastases in the original dataset. Upon validation in a follow-up cohort, this signature was an independent predictor of metastases free and overall survival in the presence of the 70 gene signature and other factors. Interestingly, the ANN signature and CA9 expression also split the groups defined by the 70 gene signature into prognostically distinct groups. Subsequently, the presence of protein for the principal prognosticator gene was categorically assessed in breast cancer tissue of an experimental and independent validation patient cohort, using immunohistochemistry. Importantly our principal prognosticator, CA9, showed that it is capable of selecting an aggressive subgroup of patients who are known to have poor prognosis.


Subject(s)
Biomarkers, Tumor/genetics , Breast Neoplasms/genetics , Gene Expression Profiling , Neoplasm Metastasis/genetics , Neural Networks, Computer , Adult , Aged , Antigens, Neoplasm/biosynthesis , Area Under Curve , Breast Neoplasms/pathology , Carbonic Anhydrase IX , Carbonic Anhydrases/biosynthesis , Computational Biology/methods , Female , Humans , Immunohistochemistry , Middle Aged , Prognosis , ROC Curve , Sensitivity and Specificity , Tissue Array Analysis
7.
J BUON ; 14 Suppl 1: S97-102, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19785076

ABSTRACT

Since van der Bruggen and colleagues first identified specific human tumour-associated antigens of the MAGE family, numerous potential immunotherapeutic targets have been discovered, often belonging to the so-called cancer/ testis gene family. Several members of this group have been described as immunogenic and have been utilised in clinical trials. In a search for interesting targets within this family, our laboratory has focussed its works for a number of years on two novel cancer/testis antigens called T21 and HAGE. In this article, we will focus our discussion on their levels of expression in a wide variety of both normal and cancer tissues, their possible role in tumour cell development and proliferation, and their immunogenic potential.


Subject(s)
Antigens, Neoplasm/therapeutic use , Testicular Neoplasms/genetics , Testicular Neoplasms/immunology , Antigens, Neoplasm/genetics , Antigens, Neoplasm/immunology , Chromosome Mapping , Female , Humans , Immunotherapy/methods , Liver Neoplasms/drug therapy , Liver Neoplasms/genetics , Liver Neoplasms/immunology , Male , Testicular Neoplasms/drug therapy , Testis/immunology , Uterine Cervical Neoplasms/drug therapy , Uterine Cervical Neoplasms/genetics , Uterine Cervical Neoplasms/immunology
8.
Parasite Immunol ; 31(7): 373-83, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19527453

ABSTRACT

Immunity to Leishmania is believed to be strongly dependent upon the activation of Th1 immune responses, although the exact role of cytotoxic T lymphocytes (CTLs) has not yet been determined. The aims of this study were to establish a suitable cytotoxicity assay to measure CTL activity and to compare immunity induced by Leishmania mexicana gp63 cDNA via i.m. injection and gene gun immunization in the BALB/c mouse model. The CTL activity was evaluated by short-term (51)Cr-release cytotoxicity assays against CT26 tumour cells transfected with L. mexicana gp63 cDNA and dendritic cells (DCs) loaded with soluble Leishmania antigen (SLA) as targets. The results clearly demonstrated that higher protection to L. mexicana infection was induced by gene gun DNA-immunization vs. i.m. injection. Cytotoxic T lymphocyte activity of splenocytes was observed in mice immunized either with L. mexicana gp63 cDNA or SLA and long-lived CTL activity was observed in immunized and/or re-challenged mice but not naïve mice infected with the parasite.


Subject(s)
Cytotoxicity, Immunologic , Leishmania mexicana/immunology , Leishmaniasis Vaccines/immunology , Metalloendopeptidases/immunology , T-Lymphocytes, Cytotoxic/immunology , Vaccines, DNA/immunology , Animals , Antibodies, Protozoan/blood , Biolistics , Cytotoxicity Tests, Immunologic , DNA, Complementary/genetics , Enzyme-Linked Immunosorbent Assay/methods , Immunoglobulin G/blood , Injections, Intramuscular , Leishmania mexicana/genetics , Leishmaniasis Vaccines/administration & dosage , Leishmaniasis Vaccines/genetics , Leishmaniasis, Cutaneous/pathology , Leishmaniasis, Cutaneous/prevention & control , Metalloendopeptidases/genetics , Mice , Mice, Inbred BALB C , Severity of Illness Index , Spleen/immunology , Vaccines, DNA/administration & dosage , Vaccines, DNA/genetics
9.
Prostate ; 68(2): 210-22, 2008 Feb 01.
Article in English | MEDLINE | ID: mdl-18092350

ABSTRACT

INTRODUCTION: Epigenetic silencing mechanisms are increasingly thought to play a major role in the development of human cancers, including prostate cancer. Promoter CpG island hypermethylation and histone hypoacetylation, catalyzed by DNA methyltransferase (DNMT) and histone deacetylase (HDAC), respectively, are associated with transcriptional repression in a number of cancers. Evidence is accumulating the two mechanisms are dynamically linked, yet few studies have examined a potential interaction in prostate cancer. METHODS: LNCaP, DU-145, and PC-3 prostate cancer cells were co-treated with a DNMT inhibitor, 5'-aza-2'-deoxycytidine (5-AZAC), and an HDAC inhibitor, trichostatin A (TSA). Following treatment cells were processed for cell proliferation/apoptosis assays, or harvested for real-time RT-PCR. Assessed target genes were estrogen receptor beta (ERbeta), estrogen receptor alpha (ERalpha), androgen receptor (AR), progesterone receptor (PGR), and prostate specific antigen (PSA). RESULTS: In all cell-lines, co-treatment was associated with reduced cell proliferation compared with control groups (P<0.05). A reciprocal rise in caspase activation was identified, indicating apoptosis was the major mechanism of cell death. Most marked effects were seen in the androgen-dependent, AR-positive LNCaP cell-line. In all cell-lines, an additive re-expression of ERbeta was identified in the co-treatment group, a finding not seen for either AR or PSA. CONCLUSION: At concentrations associated with gene re-expression, the DNA demethylating agent 5-AZAC and the HDAC inhibitor TSA co-operate to induce apoptosis in prostate cancer cell-lines. Increased apoptosis in the co-treatment group was associated with marked re-expression of ERbeta, raising the possibility of further targeting of prostate cancer cells with ERbeta-selective agents.


Subject(s)
Apoptosis/physiology , DNA Modification Methylases/antagonists & inhibitors , Estrogen Receptor beta/metabolism , Histone Deacetylase Inhibitors , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Apoptosis/drug effects , Azacitidine/analogs & derivatives , Azacitidine/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , DNA Methylation/drug effects , Decitabine , Enzyme Inhibitors/pharmacology , Estrogen Receptor alpha/metabolism , Histones/drug effects , Humans , Hydroxamic Acids/pharmacology , Male , Prostate-Specific Antigen/metabolism , Receptors, Androgen/metabolism , Receptors, Progesterone/metabolism
10.
Protein Pept Lett ; 14(5): 455-60, 2007.
Article in English | MEDLINE | ID: mdl-17584170

ABSTRACT

The immunogenicity of "novel" MART-1 and Tyrosinase class-II peptides was assessed in transgenic mice. Tyrosinase(141-161) peptide was found to be immunogenic and endogenously processed in the HLA-DRbeta1*0101 and HLA-DRbeta1*0401 transgenic mice with peptide specific production of IFNgamma or IL-5 respectively. The MART-1(29-43) peptide was only found immunogenic in HLA-DRbeta1*0101 mice.


Subject(s)
HLA Antigens/immunology , Isoantigens/isolation & purification , Monophenol Monooxygenase/metabolism , T-Lymphocytes/immunology , Animals , Cells, Cultured , Granulocytes , HLA-DR1 Antigen/immunology , HLA-DR4 Antigen/immunology , Melanoma/immunology , Mice , Mice, Transgenic , Monophenol Monooxygenase/immunology , Peptide Fragments/immunology
11.
Analyst ; 131(8): 892-4, 2006 Aug.
Article in English | MEDLINE | ID: mdl-17028721

ABSTRACT

The transduction signals from the immobilisation of a class I heavy chain, HLA-A2, on a layer guided acoustic plate mode device, followed by binding of beta(2)-microglobulin and subsequent selective binding of a target peptide are reported.


Subject(s)
Biosensing Techniques/methods , HLA-A2 Antigen/metabolism , Streptavidin/metabolism , Biosensing Techniques/instrumentation , Humans , Microscopy, Acoustic , Protein Folding , Protein Interaction Mapping , beta 2-Microglobulin/metabolism
12.
Leuk Lymphoma ; 43(6): 1313-21, 2002 Jun.
Article in English | MEDLINE | ID: mdl-12153001

ABSTRACT

AT helper 1 (Th1) immune response is considered more effective than T helper 2 (Th2) for anti-tumor immunity, but either response could potentially stimulate tumor cell growth in lymphomas. Moreover, both IL-4 and IL-2/IL-12 are used in experimental treatment models for non-Hodgkin's lymphoma (NHL) despite their differing ability to elicit Th2 or Th1 responses, respectively. Here, we investigate which T helper cytokines (Th1 or Th2) predominate in B cell NHL tissue and determine whether cytokine expression correlates with tumor cell growth, cell death, and survival in a series of 44 NHL patients. Overall, we observed both Th1 and Th2 cytokine expression at the mRNA level, detecting high levels of IFN-gamma, IL6 and IL-10 expression in the majority of tumors. Transcripts for the IL-12 subunits p35 (38 of 38) and p 40 (23 of 38) were frequently detected in NHL tissue, and high p40 levels were common in patients with a good prognosis. Furthermore, high IL-4 levels correlated with greater survival duration (P < 0.0024) but nor overall survival. Cytokine expression of IL-2, IFNgamma and IL-4 was significantly reduced in the high grade tumor group. Interestingly, there was a strong correlation between high IL-4 levels and reduced levels of apoptosis (P < 0.006) or proliferation (P < 0.0001), which has also been reported in leukemic models. This has important implications for the success of IL-4 as a treatment for low and high grade tumors.


Subject(s)
Cytokines/biosynthesis , Lymphoma, B-Cell/pathology , Th1 Cells/metabolism , Th2 Cells/metabolism , Adolescent , Adult , Aged , Aged, 80 and over , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Apoptosis , Cell Division , Combined Modality Therapy , Cyclophosphamide/administration & dosage , Cytokines/genetics , Cytokines/metabolism , Doxorubicin/administration & dosage , Female , Gene Expression Regulation, Neoplastic , Humans , Interleukin-12/biosynthesis , Interleukin-12/genetics , Interleukin-12 Subunit p40 , Lymphoma, B-Cell/genetics , Lymphoma, B-Cell/metabolism , Lymphoma, B-Cell/mortality , Lymphoma, B-Cell/therapy , Lymphoma, Non-Hodgkin/metabolism , Lymphoma, Non-Hodgkin/pathology , Male , Middle Aged , Prednisone/administration & dosage , Protein Subunits/biosynthesis , Protein Subunits/genetics , Reverse Transcriptase Polymerase Chain Reaction , Survival Analysis , Vincristine/administration & dosage
13.
Br J Cancer ; 86(11): 1824-30, 2002 Jun 05.
Article in English | MEDLINE | ID: mdl-12087473

ABSTRACT

Serological identification of tumour antigens by recombinant expression cloning has proved to be an effective strategy for the identification of cancer-associated genes having a relevance to cancer aetiology and progression, and for defining possible targets for immunotherapeutic intervention. In the present study we applied this technique to identify immunogenic proteins for gastric cancer that resulted in isolation of 14 distinct serum-reactive antigens. In order to evaluate their role in tumourigenesis and assess the immunogenicity of the identified antigens, we characterised each cDNA clone by DNA sequence analysis, mRNA tissue distribution, comparison of mRNA levels in cancerous and adjacent non-cancerous tissues and the frequency of antibody responses in allogeneic patient and control sera. Previously unknown splice variants of TACC1 and an uncharacterised gene Ga50 were identified. The expression of a newly identified TACC1 isoform is restricted to brain and gastric cancer tissues. Comparison of mRNA levels by semi-quantitative RT-PCR revealed a relative overexpression of three genes in cancer tissues, including growth factor granulin and Tbdn-1--an orthologue of the mouse acetyltransferase gene which is associated with blood vessel development. An unusual DNA polymorphism--a three-nucleotide deletion was found in NUCB2 cDNA but its mRNA level was consistently decreased in gastric tumours compared with that in the adjacent non-cancerous tissues. This study has revealed several new gastric cancer candidate genes; additional studies are required to gain a deeper insight into their role in the tumorigenesis and their potential as therapeutic targets.


Subject(s)
Antigens, Neoplasm/genetics , Chromosome Mapping , Stomach Neoplasms/genetics , Antigens, Neoplasm/blood , Gene Library , Humans , Molecular Sequence Data , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNA , Stomach Neoplasms/surgery
14.
J Immunol Methods ; 262(1-2): 5-19, 2002 Apr 01.
Article in English | MEDLINE | ID: mdl-11983215

ABSTRACT

Electrospray ionisation (ESI) mass spectrometry (MS) has been used extensively for the detection of peptides presented by major histocompatibility complex (MHC) molecules. This review focuses on the optimisation of electrospray mass spectrometry and the use of tandem mass spectrometry to sequence MHC class I peptides. We review the isolation of MHC class I peptides from the surface of cells with particular reference to tumour cells. In addition, we also discuss the advantages and disadvantages of the methods available to concentrate and fractionate the peptides prior to analysis by electrospray mass spectrometry.


Subject(s)
Histocompatibility Antigens Class I/analysis , Neoplasms/chemistry , Neoplasms/immunology , Peptides/analysis , Animals , Histocompatibility Antigens Class I/chemistry , Histocompatibility Antigens Class I/immunology , Humans , Peptides/chemistry , Peptides/immunology , Spectrometry, Mass, Electrospray Ionization
15.
Bioinformatics ; 18(3): 395-404, 2002 Mar.
Article in English | MEDLINE | ID: mdl-11934738

ABSTRACT

MOTIVATION: MALDI mass spectrometry is able to elicit macromolecular expression data from cellular material and when used in conjunction with Ciphergen protein chip technology (also referred to as SELDI-Surface Enhanced Laser Desorption/Ionization), it permits a semi-high throughput approach to be taken with respect to sample processing and data acquisition. Due to the large array of data that is generated from a single analysis (8-10000 variables using a mass range of 2-15 kDa-this paper) it is essential to implement the use of algorithms that can detect expression patterns from such large volumes of data correlating to a given biological/pathological phenotype from multiple samples. If successful, the methodology could be extrapolated to larger data sets to enable the identification of validated biomarkers correlating strongly to disease progression. This would not only serve to enable tumours to be classified according to their molecular expression profile but could also focus attention upon a relatively small number of molecules that might warrant further biochemical/molecular characterization to assess their suitability as potential therapeutic targets. RESULTS: Using a multi-layer perceptron Artificial Neural Network (ANN) (Neuroshell 2) with a back propagation algorithm we have developed a prototype approach that uses a model system (comprising five low and seven high-grade human astrocytomas) to identify mass spectral peaks whose relative intensity values correlate strongly to tumour grade. Analyzing data derived from MALDI mass spectrometry in conjunction with Ciphergen protein chip technology we have used relative importance values, determined from the weights of trained ANNs (Balls et al., Water, Air Soil Pollut., 85, 1467-1472, 1996), to identify masses that accurately predict tumour grade. Implementing a three-stage procedure, we have screened a population of approximately 100000-120000 variables and identified two ions (m/z values of 13454 and 13457) whose relative intensity pattern was significantly reduced in high-grade astrocytoma. The data from this initial study suggests that application of ANN-based approaches can identify molecular ion patterns which strongly associate with disease grade and that its application to larger cohorts of patient material could potentially facilitate the rapid identification of validated biomarkers having significant clinical (i.e. diagnostic/prognostic) potential for the field of cancer biology. AVAILIBILITY: Neuroshell 2 is commercially available from ward systems.


Subject(s)
Astrocytoma/classification , Biomarkers, Tumor/analysis , Central Nervous System Neoplasms/classification , Neural Networks, Computer , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Feasibility Studies , Humans , Models, Neurological , Sensitivity and Specificity
16.
Blood ; 98(10): 2887-93, 2001 Nov 15.
Article in English | MEDLINE | ID: mdl-11698267

ABSTRACT

The BCR-ABL oncogene is central in the pathogenesis of chronic myeloid leukemia (CML). Here, tandem nanospray mass spectrometry was used to demonstrate cell surface HLA-associated expression of the BCR-ABL peptide KQSSKALQR on class I-negative CML cells transfected with HLA-A*0301, and on primary CML cells from HLA-A3-positive patients. These patients mounted a cytotoxic T-lymphocyte response to KQSSKALQR that also killed autologous CML cells, and tetramer staining demonstrated the presence of circulating KQSSKALQR-specific T cells. The findings are the first demonstration that CML cells express HLA-associated leukemia-specific immunogenic peptides and provide a sound basis for immunization studies against BCR-ABL.


Subject(s)
Antigen Presentation , Antigens, Neoplasm/immunology , Antigens, Surface/immunology , Fusion Proteins, bcr-abl/immunology , HLA-A3 Antigen/immunology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/immunology , Neoplasm Proteins/immunology , Neoplastic Stem Cells/immunology , Peptide Fragments/immunology , Adult , Amino Acid Sequence , Antigens, Neoplasm/chemistry , Antigens, Surface/chemistry , Female , Fusion Proteins, bcr-abl/chemistry , HLA-A3 Antigen/genetics , Humans , K562 Cells/immunology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Male , Middle Aged , Neoplasm Proteins/chemistry , Peptide Fragments/chemistry , Recombinant Fusion Proteins/immunology , Spectrometry, Mass, Electrospray Ionization , T-Lymphocytes, Cytotoxic/immunology , Transfection
17.
Med Hypotheses ; 57(1): 56-8, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11421625

ABSTRACT

H2O2, a highly reactive agent, can react under certain conditions with a variety of cellular components. These reactions include the lipid peroxidation of membrane and hydroxylation of proteins and DNA. The reactions can take place in the presence of oxygen and are fairly rapid, the H2O2 being converted to water and oxygen. Experiments were carried out in vitro to assess the ability of this agent to destroy cancer cells without generating dangerous by-products. The direct administration of aqueous H2O2 into solid tumours has the potential to cause tumour cell death. The efficacy of the use of H2O2 for treating 'solid' cancers will necessitate its delivery to the tumour site, for example by direct special multiple injection of H2O2 into a detectable tumour mass. We anticipate that, if suggested mode of delivery can be obtained, H2O2 can act as an anti-cancer drug with two distinct advantages over conventional chemotherapeutic agents: to produce minimal short- and long-term side-effects and is relatively cheap and cost effective.


Subject(s)
Antineoplastic Agents/pharmacology , Hydrogen Peroxide/pharmacology , Neoplasms/drug therapy , Animals , Antineoplastic Agents/therapeutic use , Humans , Hydrogen Peroxide/therapeutic use , Mice , Neoplasms/pathology , Tumor Cells, Cultured
18.
Cancer Immunol Immunother ; 49(8): 417-25, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11043848

ABSTRACT

The tumour-suppressor gene p53 is pivotal in the regulation of apoptosis, and point mutations within p53 are the commonest genetic alterations in human cancers. Cytotoxic T lymphocytes (CTL) recognise peptide-MHC complexes on the surface of tumour cells and bring about lysis. Therefore, p53-derived peptides are potential candidates for immunisation strategies designed to induce antitumour CTL in patients. Conformational changes in the p53 protein, generated as a result of point mutations, frequently expose the 240 epitope, RHSVV (amino acids 212-217), which may be processed differently from the wild-type protein resulting in an altered MHC-associated peptide repertoire recognised by tumour-specific CTL. In this study 42 peptides (37 overlapping nonameric peptides, from amino acids 193-237 and peptides 186-194, 187-197, 188-197, 263-272, 264-272, possessing binding motifs for HLA-A2) derived from the wild-type p53 protein sequence were assayed for their ability to stabilise HLA-A2 molecules in MHC class I stabilisation assays. Of the peptides tested, 24 stabilised HLA-A2 molecules with high affinity (fluorescence ratio >1.5) at 26 degrees C, and five (187-197, 193-200, 217-224, 263-272 and 264-272) also stabilised the complexes at 37 degrees C. Peptides 188-197, 196-203 and 217-225 have not previously been identified as binders of HLA-A2 molecules and, of these, peptide 217-225 stabilised HLA-A2 molecules with the highest fluorescence ratio. Peptide 217-225 was chosen to generate HLA-A2-restricted CTL in vitro; peptide 264-272 was used as a positive control. The two primary CTL thus generated (CTL-217 using peptide 217 225; and CTL-264 using peptide 264-272) were capable of specifically killing peptide-pulsed T2 or JY cells. In order to determine whether these peptides were endogenously processed and to test the hypothesis that mutants expressing different protein conformations would generate an alternative peptide repertoire at the cell surface, a panel of target cells was generated. HLA-A2+ SaOs-2 cells were transfected with p53 cDNA containing point mutations at either position 175 (R-->H) or 273 (R-->H) (SaOs-2/175 and SaOs-2/273). Two HLA-A2-negative cell lines, A431 and SKBr3, naturally expressing p53 mutations at positions 273 and 175 respectively, were transfected with a cDNA encoding HLA-A2. The results showed that primary CTL generated in response to both peptides were capable of killing SaOs-2/175 and SKBr3-A2 cells, which possess the same mutation, but not SaOs-2/273, A431-A2 or SKBr3 cells transfected with control vector. This suggests that these peptides are presented on the surface of SaOs-2/175 and SKBr3-A2 cells in a conformation-dependent manner and represent potentially useful target peptides for immunotherapy.


Subject(s)
Genes, p53 , Mutation , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/metabolism , Tumor Suppressor Protein p53/chemistry , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cytokines/pharmacology , DNA, Complementary/metabolism , Dendritic Cells/metabolism , HLA-A2 Antigen/genetics , Humans , Immunoglobulin G/immunology , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/metabolism , Peptides/chemical synthesis , Peptides/metabolism , Point Mutation , Protein Conformation , Recombinant Proteins/chemistry , Transfection , Tumor Cells, Cultured , Tumor Suppressor Protein p53/metabolism
19.
Br J Cancer ; 83(8): 1061-8, 2000 Oct.
Article in English | MEDLINE | ID: mdl-10993655

ABSTRACT

RENCA-IL-2 (Murine Renal Cell Carcinoma transfected with murine IL-2 gene) cells were rejected by immunocompetent (but not T-cell deficient) Balb/c mice, which developed 'immunity' to subsequent parental RENCA tumour cell challenge. Splenocytes adoptively transferred this immunity. CD4(+)and CD8(+)T-lymphocytes prepared from the spleens of 'tumour immune' mice were evaluated for their ability to traffic into the tumour environment using an in vivo model that enables visualization of events within the microvasculature. RENCA cells were implanted into the mouse cremaster muscle and the trafficking of syngeneic lymphocyte subpopulations, derived from naive and 'immune' animals, into both the RENCA tumour and the surrounding normal cremaster muscle microcirculation was measured by in vivo microscopy. Fluorescently labelled CD4(+)and CD8(+)T lymphocytes taken from the spleens of naive mice or mice previously immunized with RENCA-IL-2 were injected systemically into tumour-bearer mice. Naive effector cells migrated to, and flowed through both the tumour and the normal microcirculation, with negligible adhesion. However we observed the selective recruitment, localization and arrest of immune CD4(+)and CD8(+)T lymphocytes (P< 0.05) into the tumour microcirculation, and in some instances the subsequent extravasation of cells into the tumour interstitium. Lymphocyte rolling by 'immune' CD4(+)and CD8(+)T-cells in the tumour microcirculation was greatly reduced, suggesting impaired adhesion molecule expression on the tumour endothelium. This study clearly demonstrates, by direct in vivo microscopy assessment, the localization of effector cells, CD4(+)and CD8(+)lymphocytes into tumours.


Subject(s)
Adenocarcinoma/immunology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Carcinoma, Renal Cell/immunology , Kidney Neoplasms/blood supply , Kidney Neoplasms/immunology , Microcirculation/immunology , Adenocarcinoma/blood supply , Adenocarcinoma/pathology , Animals , Carcinoma, Renal Cell/blood supply , Carcinoma, Renal Cell/pathology , Cell Division , Flow Cytometry , Interleukin-2/genetics , Interleukin-2/physiology , Kidney Cortex , Kidney Neoplasms/pathology , Lymphocyte Transfusion , Mice , Mice, Inbred BALB C , Spleen/immunology , Transfection
20.
Analyst ; 125(4): 599-603, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10892016

ABSTRACT

The formation of copper/peptide complex ions by nano-electrospray and microbore HPLC-electrospray mass spectrometry has been investigated for major histocompatibility complex (MHC) class I and class II restricted peptides. Post-column addition of copper(II) acetate following microbore HPLC-MS separation was carried out using a mixing T-piece or via the sheath flow inlet of the electrospray source. Optimal analytical conditions for copper complex ion formation were determined by variation of copper concentration, pH, nebulization gas supply and spray voltage. Tandem mass spectrometry of copper/peptide complex ions provides peptide sequence information and insight into the peptide chelation sites. Copper associated y fragment ions dominate the product ion spectrum for non-histidine containing peptides, but both b and y copper complex ions were observed for the histidine containing MHC class I associated peptide gp70.


Subject(s)
Chelating Agents , Copper , Histocompatibility Antigens/analysis , Animals , Humans , Mass Spectrometry/methods , Mice
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