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DNA-microarrays as tools for the identification of tumor specific gene expression profiles: applications in tumor biology, diagnosis and therapy.

DNA-microarrays allow the analysis of almost the complete gene expression program of tumor samples and normal control samples in a single experiment. This allows the processing of a large number of samples in a reasonable short time. Tumor specific gene expression profiles can be used for molecular tumor classification and as a new diagnostic tool. In addition, the identification of tumor specific genes can help to understand the biology of tumor cells and identified genes can be used for the development of new therapeutic strategies. However, the huge amount of data generated by DNA-microarrays creates new challenges for data analysis. In addition, accuracy and reproducibility of the available techniques require complementary methods for verification of DNA-microarray data.

Child↗

Cyclin A1 and gametogenesis in fertile and infertile patients: a potential new molecular diagnostic marker.

BACKGROUND: The study aim was to evaluate cyclin A1 mRNA expression levels as a potential molecular diagnostic parameter in the work-up of testicular tissue from fertile versus infertile patients. METHODS: Cyclin A1 expression was quantified in 55 cryopreserved testicular tissue specimens by fluorescence real-time RT-PCR. A conventional histological work-up was performed concomitantly in all tissue specimens with additional semi-thin sectioning in all cases of non-obstructive azoospermia (n = 12), maturation arrest (n = 17) and Sertoli cell-only syndrome (SCOS; n = 9). RESULTS: The mean (+/- SD) normalized cyclin A1 expression (N(CyclinA1)) was 3.82 +/- 2.23 relative gene expression (RGE) in tissue specimens with normal spermatogenesis, and 0.625 +/- 0.221 RGE in those with maturation arrest at the level of early spermatids. Only minimal N(CyclinA1) was detected in tissue specimens with spermatogonia only or maturation arrest at the level of primary spermatocytes (0.005 +/- 0.008). Cyclin A1 expression was absent in the majority of SCOS specimens (0.002 +/- 0.002). CONCLUSIONS: These investigations suggested that cyclin A1 expression is altered in cases of spermatogenic disorders. Moreover, the level of cyclin A1 mRNA expression correlates with gametogenic disorders and seems well suited for a molecular-diagnostic classification supplementing the histopathological evaluation of spermatogenic disorders.

Biomarkers↗

KDBI: Kinetic Data of Bio-molecular Interactions database.

Understanding of cellular processes and underlying molecular events requires knowledge about different aspects of molecular interactions, networks of molecules and pathways in addition to the sequence, structure and function of individual molecules involved. Databases of interacting molecules, pathways and related chemical reaction equations have been developed. The kinetic data for these interactions, which is important for mechanistic investigation, quantitative study and simulation of cellular processes and events, is not provided in the existing databases. We introduce a new database of Kinetic Data of Bio-molecular Interactions (KDBI) aimed at providing experimentally determined kinetic data of protein-protein, protein-RNA, protein-DNA, protein-ligand, RNA-ligand, DNA-ligand binding or reaction events described in the literature. KDBI contains information about binding or reaction event, participating molecules (name, synonyms, molecular formula, classification, SWISS-PROT AC or CAS number), binding or reaction equation, kinetic data and related references. The kinetic data is in terms of one or a combination of the following quantities as given in the literature of a particular event: association/dissociation or on/off rate constant, first/second/third/. order rate constant, equilibrium rate constant, catalytic rate constant, equilibrium association/dissociation constant, inhibition constant and binding affinity constant. Each entry can be retrieved through protein or nucleic acid or ligand name, SWISS-PROT AC number, ligand CAS number and full-text search of a binding or reaction event. KDBI currently contains 8273 entries of biomolecular binding or reaction events involving 1380 proteins, 143 nucleic acids and 1395 small molecules. Hyperlinks are provided for accessing references in Medline and available 3D structures in PDB and NDB. This database can be accessed at http://xin.cz3.nus.edu.sg/group/kdbi/kdbi.asp.

DNA↗

Phosphoproteomics delineates hepatocellular carcinoma subtypes and pinpoints therapeutic targets.

BACKGROUND AND AIMS: Only a minority of patients could benefit from systemic therapy owing to the high heterogeneity of HCC. Therefore, a deeper understanding of the pathogenesis of HCC is essential for precision therapy. Genomic and proteomic studies of HCC have enhanced our understanding of HCC. However, the phosphoproteomic characterization of HCC remains poorly understood. APPROACH AND RESULTS: We conducted an in-depth analysis of a clinical cohort of HCC using high-coverage phosphoproteomic. Effective therapeutic targets were validated using liver cancer cell lines and HCC patient-derived xenograft mouse models that correspond to the phosphoproteomic subtypes of HCC. Phosphoproteomic analysis classified HCC into 3 subtypes, A, B, and C, with increasing malignancy and correlation with clinical features, including patient prognosis, tumor staging, serum alpha-fetoprotein levels, tumor thrombus, and tumor size. Phosphoproteomic subtyping deeply reflected the biological characteristics and clinical features of patients with HCC​​​​​​. The profiles of HCC-dysregulated kinase activities inferred from the different phosphoproteomic subtypes consistently identify increased kinase activity related to cell proliferation. Subtype-C HCC patients showed the most significant dysregulation, indicating a potential therapeutic target. The corresponding drug, bosutinib, demonstrated efficacy in inhibiting the growth of subtype C tumors in liver cancer cell lines and HCC patient-derived xenograft mouse models representative of the phosphoproteomic HCC subtypes. CONCLUSIONS: Our study provides a comprehensive exploration of the phosphoproteomic landscape of HCC, establishing new subtypes that match clinical features and identifying potential therapeutic targets for the most malignant C subtype.

Carcinoma, Hepatocellular↗

Nosological considerations of the neurofibromatoses.

We are on the threshold of evaluating the NF1 and NF2 loci with respect to variant forms of the neurofibromatoses. Genetic mapping of NF1, gene cloning and characterization of its encoded product, neurofibromin, provides a framework for the evaluation of the variant forms of NF. This may also apply to NF2 variant forms in the near future. The mapping approach in evaluating variant forms of NF should begin with the rigorous clinical assessment of familial cases whereby the establishment of genetic linkage in families with overlap syndromes might determine if either NF1, NF2, or a separate locus is involved in the phenotype. Conditions mapping to the NF1 locus could then be screened for mutations in hopes of identifying the etiologies of the variant forms of NF. Mutation identification should provide a molecular-based classification scheme for the variant forms of NF, now tentatively divided into alternative and related forms. It is expected that the nosology of the neurofibromatoses will most certainly change as more is learned of the NF1 and NF2 loci.

Diagnosis, Differential↗

Determination of 23S rRNA sequences from members of the genus Streptococcus and characterization of genetically distinct organisms previously identified as members of the Streptococcus anginosus group.

The 23S rRNA gene sequences of eight type strains of the genus Streptococcus were determined. Three species-specific probes for the three members of the Streptococcus anginosus group and one group-specific probe for these three species were designed. The PCR-amplified 23S rRNA gene sequences of 28 clinical strains were examined by hybridization with the four oligonucleotide probes. They were suspected to be members of the S. anginosus group by biochemical tests; however, only 21 strains (75%) hybridized to one of the three species probes. Seven biochemically atypical strains did not hybridize to these species-specific probes. Of the seven strains, three hybridized to the group-specific probe. The 23S ribosomal RNA sequences of these three strains differed by three bases within the 20-base probe area of S. anginosus. The phylogenetic tree of the 16S rRNA of these three strains located them within the branch of S. anginosus. The remaining four strains, which did not hybridize to group- or species-specific probes, had a raffinose-positive and VP-negative phenotype. The probe hybridization results showed that these four strains did not belong to the S. anginosus group and were closely related to S. parasanguis according to partial sequences of their 16S rRNA genes.

Base Sequence↗

A temperate phage of Streptomyces azureus.

A new phage, SAt1, was isolated from soil on Streptomyces azureus ATCC 14921. This phage was able to lysogenize S. azureus. The percentage of lysogenic responses was ca. 10%. Electron microscopic observation showed that this phage belonged to group B of Bradley's morphological classification. The molecular mass of SAt1 DNA was ca. 24 megadaltons. The guanine-plus-cytosine content and the density of SAt1 DNA were ca. 71% and 1.724 g/cm3, respectively. A cleavage map of SAt1 DNA was constructed with restriction endonucleases BanIII, BglII, HindIII, and XbaI. Furthermore, some other characteristics of this phage were investigated.

Bacteriophages↗

Genomic classification of fish nodaviruses by molecular phylogenetic analysis of the coat protein gene.

A molecular phylogenetic analysis of 25 isolates of fish nodaviruses, the causative agents of viral nervous necrosis of marine fish, was performed based on the nucleotide sequences (427 bases) of the coat protein gene. These fish nodaviruses were classified into four clusters: tiger puffer nervous necrosis virus, striped jack nervous necrosis virus, berfin flounder nervous necrosis virus, and red-spotted grouper nervous necrosis virus.

Amino Acid Sequence↗

The Montreal classification of inflammatory bowel disease: controversies, consensus, and implications.

In recent years, investigators have readdressed the complex issues involved in the classification of inflammatory bowel diseases. In 2003, a Working Party of investigators with an interest in the issues involved in disease subclassification was formed with the aim of summarising recent developments in disease classification and establishing an integrated clinical, molecular, and serological classification of inflammatory bowel disease. The results of the Working Party were reported at the 2005 Montreal World Congress of Gastroenterology. Here we highlight the key issues that have emerged from discussions of the Montreal Working Party and the relevance to clinical practice and research activities.

Biomarkers↗

Chronic granulomatous disease.

Chronic granulomatous disease (CGD) encompasses a group of rare inherited disorders characterized by defects in a phagocyte-specific NADPH-oxidase complex that forms the superoxide radical during the respiratory burst. In this chapter, the protein components and cellular biochemistry of the oxidase are reviewed in light of recent genetic and biochemical studies of CGD. The classification and molecular genetic analysis of CGD is discussed. Finally, the use of recombinant human interferon-gamma as a new therapeutic agent for management of the disorder is reviewed.

Free Radicals↗

Functional gene expression analysis uncovers phenotypic switch in aggressive uveal melanomas.

Microarray gene expression profiling is a powerful tool for generating molecular cancer classifications. However, elucidating biological insights from these large data sets has been challenging. Previously, we identified a gene expression-based classification of primary uveal melanomas that accurately predicts metastatic death. Class 1 tumors have a low risk and class 2 tumors a high risk for metastatic death. Here, we used genes that discriminate these tumor classes to identify biological correlates of the aggressive class 2 signature. A search for Gene Ontology categories enriched in our class-discriminating gene list revealed a global down-regulation of neural crest and melanocyte-specific genes and an up-regulation of epithelial genes in class 2 tumors. Correspondingly, class 2 tumors exhibited epithelial features, such as polygonal cell morphology, up-regulation of the epithelial adhesion molecule E-cadherin, colocalization of E-cadherin and beta-catenin to the plasma membrane, and formation of cell-cell adhesions and acinar structures. One of our top class-discriminating genes was the helix-loop-helix inhibitor ID2, which was strongly down-regulated in class 2 tumors. The class 2 phenotype could be recapitulated by eliminating Id2 in cultured class 1 human uveal melanoma cells and in a mouse ocular melanoma model. Id2 seemed to suppress the epithelial-like class 2 phenotype by inhibiting an activator of the E-cadherin promoter. Consequently, Id2 loss triggered up-regulation of E-cadherin, which in turn promoted anchorage-independent cell growth, a likely antecedent to metastasis. These findings reveal new roles for Id2 and E-cadherin in uveal melanoma progression, and they identify potential targets for therapeutic intervention.

Animals↗

Dementia with lewy bodies: molecular pathogenesis and implications for classification.

Dementia with Lewy bodies results from the accumulation from Lewy-type pathology (Lewy bodies, Lewy neurites), secondary cellular injury, and apoptotic neurodegeneration. The severity of dementia correlates with the abundance of Lewy bodies in the cortex. Dementia with Lewy bodies co-occurs with 2 specific syndromes, one beginning with dementia complicated by visual hallucinations and parkinsonism; the other beginning with Parkinson's disease and progressing to a parkinsonian-dementia syndrome. Clinical syndromes associated with these 2 pathways to dementia share many clinical features including the type of cognitive impairment, fluctuating attentional disturbances, prominent visual hallucinations and psychosis, depression, and rapid eye movement sleep behavior disorder. Lewy pathology results from protein misfolding and the accumulation of alpha-synuclein in the cell cytoplasm. Dementia with Lewy bodies is one of many neurodegenerative disorders linked to protein misfolding. Identification of clinical symptoms indicative of the presence of a specific protein disturbance will assist in choosing therapies when protein-specific disease-modifying treatments are available. Classification systems based on symptom complexes related to the presence of protein misfolding will assist therapeutic decisions.

Diagnosis, Differential↗

Clinical and biologic implications of recurrent genomic aberrations in myeloma.

Nonrandom recurrent chromosomal abnormalities are ubiquitous in multiple myeloma (MM) and include, among others, translocations of the immunoglobulin heavy chain locus (IgH). IgH translocations in MM result in the up-regulation of oncogenes, and include more commonly t(11;14)(q13;q32), t(4;14)(p16;q32), and t(14;16)(q32;q23). Based on the recurrent nature of these translocations and their finding since the early stages of the plasma cell (PC) disorders, we hypothesized that they would confer biologic and clinical variability. In addition, deletions of 13q14 and 17p13 have also been associated with a shortened survival. We used cytoplasmic Ig-enhanced interphase fluorescent in situ hybridization to detect deletions (13q14 and 17p13.1), and translocations involving IgH in 351 patients treated with conventional chemotherapy entered into the Eastern Cooperative Oncology Group clinical trial E9486/9487. Translocations were frequently unbalanced with loss of one of the derivative chromosomes. The presence of t(4; 14)(p16;q32) (n = 42; 26 vs 45 months, P <.001), t(14;16)(q32;q23) (n = 15; 16 vs 41 months, P =.003), - 17p13 (n = 37; 23 vs 44 months, P =.005), and - 13q14 (n = 176; 35 vs 51 months, P =.028) were associated with shorter survival. A stratification of patients into 3 distinct categories allowed for prognostication: poor prognosis group (t(4;14)(p16;q32), t(14; 16)(q32;q23), and - 17p13), intermediate prognosis (- 13q14), and good prognosis group (all others), with median survivals of 24.7, 42.3, and 50.5 months, respectively (P <.001). This molecular cytogenetic classification identifies patients into poor, intermediate, and good risk categories. More importantly it provides further compelling evidence that MM is composed of subgroups of patients categorized according to their underlying genomic aberrations.

Adult↗

A CpG island hypermethylation profile of primary colorectal carcinomas and colon cancer cell lines.

BACKGROUND: Tumor cell lines are commonly used as experimental tools in cancer research, but their relevance for the in vivo situation is debated. In a series of 11 microsatellite stable (MSS) and 9 microsatellite unstable (MSI) colon cancer cell lines and primary colon carcinomas (25 MSS and 28 MSI) with known ploidy stem line and APC, KRAS, and TP53 mutation status, we analyzed the promoter methylation of the following genes: hMLH1, MGMT, p16INK4a (CDKN2A alpha-transcript), p14ARF (CDKN2A beta-transcript), APC, and E-cadherin (CDH1). We compared the DNA methylation profiles of the cell lines with those of the primary tumors. Finally, we examined if the epigenetic changes were associated with known genetic markers and/or clinicopathological variables. RESULTS: The cell lines and primary tumors generally showed similar overall distribution and frequencies of gene methylation. Among the cell lines, 15%, 50%, 75%, 65%, 20% and 15% showed promoter methylation for hMLH1, MGMT, p16INK4a, p14ARF, APC, and E-cadherin, respectively, whereas 21%, 40%, 32%, 38%, 32%, and 40% of the primary tumors were methylated for the same genes. hMLH1 and p14ARF were significantly more often methylated in MSI than in MSS primary tumors, whereas the remaining four genes showed similar methylation frequencies in the two groups. Methylation of p14ARF, which indirectly inactivates TP53, was seen more frequently in tumors with normal TP53 than in mutated samples, but the difference was not statistically significant. Methylation of p14ARF and p16INK4a was often present in the same primary tumors, but association to diploidy, MSI, right-sided location and female gender was only significant for p14ARF. E-cadherin was methylated in 14/34 tumors with altered APC further stimulating WNT signaling. CONCLUSIONS: The present study shows that colon cancer cell lines are in general relevant in vitro models, comparable with the in vivo situation, as the cell lines display many of the same molecular alterations as do the primary carcinomas. The combined pattern of epigenetic and genetic aberrations in the primary carcinomas reveals associations between them as well as to clinicopathological variables, and may aid in the future molecular assisted classification of clinically distinct stages.

Adaptor Proteins, Signal Transducing↗

Differences in biological features of gastric dysplasia, indefinite dysplasia, reactive hyperplasia and discriminant analysis of these lesions.

AIM: To investigate the differences in biological features of gastric dysplasia (Dys), indefinite dysplasia (IDys) and reactive hyperplasia (RH) by studying the biomarker alterations in cell proliferation, cell differentiation, cell cycle control and the expression of house-keeping genes, and further to search for markers which could be used in guiding the pathological diagnosis of three lesions. METHODS: Expressions of MUC5AC, MUC6, adenomatous polyposis coli (APC), p53, Ki-67, proliferation cell nuclear antigen (PCNA) and EGFR were studied by immunohistochemistry with a standard Envision technique in formalin-fixed and paraffin-embedded specimens from 43 RH, 35 IDys, 35 Dys and 36 intestinal type gastric carcinomas (IGC). In addition, Bayes discriminant analysis was used to investigate the value of markers studied in differential diagnosis of RH, IDys, Dys and IGC. RESULTS: The MUC5AC and MUC6 antigen expressions in RH, IDys, Dys and IGC decreased gradually (MUC5AC: 86.04%, 77.14%, 28.57%, 6.67%; MUC6: 65.15%, 54.29%, 20.00%, 25.00%, respectively). The expressions of the two markers had no significant difference between RH and IDys, but were all significantly higher than those of the other two lesions (MUC5AC: chi2=27.607, 38.027 and 17.33, 26.092; MUC6: chi2=16.54, 12.665 and 9.282, 6.737, P<0.01). There was no significant difference between RH and IDys, Dys and IGC in MUC6 expression. The APC gene expression in the four lesions had a similar decreasing tendency (RH 69.76%, IDys 68.57%, Dys 39.39%, IGC 22.86%), and it was significantly higher in the first two lesions than in the last two (chi2=7.011, 16.995 and 14.737, 19.817, P<0.05). The p53 expression in RH, IDys, Dys and IGC was 6.98%, 20%, 57.14% and 50%, respectively. There was no significant difference between RH and IDys or Dys and IGC, but the p53 expression in RH and IDys was significantly lower than that in Dys and IGC (chi2=7.011, 16.995 and 14.737, 19.817, P<0.01). The Ki-67 label index was significantly different among four lesions (RH: 0.298+/-8.92%, IDys: 0.358+/-9.25%, Dys: 0.498+/-9.03%, IGC: 0.620+/-10.8%, P<0.001). Positive immunostaining of PCNA was though observed in all specimens, significant differences were detected among four lesions (F=95.318, P<0.01). In addition, we used Bayes discriminant analysis to investigate molecular pathological classification of the lesions, and obtained the best result with the combination of MUC5AC, Ki-67 and PCNA. The overall rate of correct classification was 67.4% (RH), 68.6% (IDys), 70.6% (Dys) and 84.8% (IGC), respectively. CONCLUSION: Dys has neoplastic biological characteristics, while RH and IDys display hyperplastic characteristics. MUC5AC and proliferation-related biomarkers (Ki-67, PCNA) are more specific in distinguishing Dys from RH and IDys.

Biomarkers↗

Study on the classification of chronic gastritis at molecular biological level.

AIM: To explore the pathophysiologibasis for the fact that patients with digestive tract symptoms do not necessarily have gastric mucosal pathology and those without clinical symptoms do not necessarily have no gastric mucosal pathology. METHODS: The ultrastructure, trace elements, cAMP, DNA, SOD and LPO in the gastric mucosa and its epithelial cells of 188 patients without organic lesions of heart, lung, liver, gallbladder, pancreas, kidney or intestine and basically histopathological normal persons (F) were detected synchronously by SEM, TEM, EDAX, Image analysis system RIA and (3)H-TdR Lymphocyte Transfer Test. RESULTS: The content of Zn, Cu, cAMP and (3)H-TdR LCT in gastric mucosa and the content of Zn, Cu, DNA and LPO in gastric mucosa epithelial nuclei of each group were shown as belows: Normal control (4.1+/-1.0, 5.2+/-0.8, 15.9+/-1.5, 1079.7+/-227.4, 7.6+/-0.4, 58.4+/-0.3, 12.6+/-2.7, 2.6+/-0.6); CSG without symptoms group (3.7+/-1.2, 5.1+/-1.8, 15.6+/-0.9, 924.5+/-234.9, 7.8+/-0.3, 58.6+/-0.4, 13.0+/-3.1, 2.9+/-0.4); CAG without symptoms group (3.3+/-1.0, 4.8+/-0.9, 14.9+/-0.7, 887.7+/-243.6, 7.8+/-0.3, 58.7+/-0.3, 14.3+/-2.8, 3.1+/-0.4); F type with symptoms group (3.5+/-1.4, 4.5+/-1.0, 15.7+/-1.4, 932.1+/-2449.3, 7.9+/-0.4, 58.7+/-0.5, 13.5+/-4.6, 2.9+/-0.7); CSG with symptoms group (2.8+/-1.9, 4.0+/-1.5, 14.2+/-1.8, 867.3+/-240.5, 8.1+/-0.5, 58.9+/-0.5, 15.2+/-3.2, 4.2+/-0.7); CAG with symptoms group (2.0+/-1.8, 3.4+/-1.5, 13.4+/-1.8, 800.9+/-221.8, 8.6+/-0.4, 59.3+/-0.5, 16.5+/-3.1, 4.5+/-0.6). The contents of Zn, Cu in mitochonondria and SOD in gastric mucosa of each group were shown as belows: Normal control group (9.2+/-0.5, 58.3+/-0.3, 170.5+/-6.1), CSG without symptoms group (8.9+/-0.5, 58.2+/-0.3, 167.2+/-5.3), CAG without symptoms group (8.8+/-0.4, 57.5+/-0.2, 166.1+/-4.2); F type with symptoms group (8.9+/-0.5, 58.0+/-0.3, 167.9+/-5.7), CSG with symptoms group (8.6+/-0.5, 57.8+/-0.3, 163.3+/-5.6); CAG with symptoms group (8.3+/-0.4, 57.5+/-0.3, 161.2+/-4.3). There were significant differences in these cases, P<0.05-0.001. There were synchronous changes of gastric mucosa epithelial cellular ultrastructure. The "background lesions" (focal atrophic gastritis, focal intestinal metaplasia, micro-ulcer) in nonfocal gastric mucosa of all groups had significant differences (P<0.05-0.001). CONCLUSION: Disease with symptoms, disease without symptoms, nondisease with symptoms occur on the basis of the quantitative changes of gastric mucosa epithelial cellular ultrastructure and related bioactive substances.

Cell Nucleolus↗

Primary cutaneous lymphomas other than mycosis fungoides.

Primary cutaneous lymphomas present in and are confined to the skin with no evidence of extracutaneous disease. The skin is the second most common extranodal site involved by primary lymphoma; 50% are mycosis fungoides (MF)-type cutaneous T-cell lymphoma, with the remainder being peripheral T-cell lymphoma (25%) and B-cell lymphoma (25%). The diagnosis of non-MF primary cutaneous lymphomas differs from that of nodal lymphomas: (1) presentation in the skin more often predicts outcome than histology, (2) immunophenotyping and immunogenotyping studies show differences in chromosomal translocations, cell-surface antigen expression (T-cell receptor [TCR] and immunoglobulin [Ig] heavy and light chains), and oncogene expression, (3) involvement of structural compartments of the skin (epidermis, periadnexal or adventitial dermis, interstitial dermis, and subcutis) aids differential diagnosis in place of nodal architecture, and (4) cytokine and extracellular matrix environments may influence behavior of cutaneous lymphomas. Diagnosis often requires coordinated evaluation of clinical history, immunohistochemistry on paraffin and frozen sections of skin biopsies, and molecular analysis. Classification of primary cutaneous lymphomas by a combined histologic type and clinical behavior is useful.

Humans↗