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S M Hanash

Publications and source records attributed to S M Hanash.

At least 37 records · Page 2Linked to original sources
PubMed2000

Biomedical applications of two-dimensional electrophoresis using immobilized pH gradients: current status.

There is currently much interest, as we enter the postgenome era, in studying gene expression at the protein level. Two-dimensional electrophoresis (2-DE) using immobilized pH gradients (IPG), coupled with mass spectrometry (MS), is currently the most widely utilized approach for the analysis of whole tissue proteins. The methodology for IPG-based 2-DE, since the introduction of the technique in the 1980s, is reviewed. In its present form the IPG methodology is mostly useful as a research tool. In general, high reproducibility and high resolution have been achieved. However, the lack of substantial automation and the limited sensitivity of the current overall methodology continue to represent drawbacks for biomedical applications. Further developments to increase throughput and to reduce sample requirement would substantially benefit the application of IPG-based 2-DE to biomedicine and would enhance the prospects for introducing the methodology into the clinical laboratory.

Animals↗
PubMed2000

Impaired translational response and increased protein kinase PKR expression in T cells from lupus patients.

Activation of peripheral blood T cells results in a rapid and substantial rise in translation rates and proliferation, but proliferation in response to mitogen stimulation is impaired in systemic lupus erythematosus (SLE). We have investigated translation rates and initiation factor activities in T cells from SLE patients in response to activating signals. Activation by PMA plus ionomycin strongly increased protein synthesis in control T cells but not in T cells from SLE patients. The rate of protein synthesis is known to be strongly dependent on the activity of two eukaryotic translation initiation factors, eIF4E and eIF2alpha. We show that following stimulation, eIF4E expression and phosphorylation increased equivalently in control and SLE T cells. Expression of eIF4E interacting proteins - eIF4G, an inducer, and 4E-BP1 and 4E-BP2, two specific repressors of eIF4E function - and the phosphorylation level of 4E-BP1, were all identical in control and SLE T cells. In contrast, the protein kinase PKR, which is responsible for the phosphorylation and consequent inhibition of eIF2alpha activity, was specifically overexpressed in activated SLE T cells, correlating with an increase in eIF2alpha phosphorylation. Therefore, high expression of PKR and subsequent eIF2alpha phosphorylation is likely responsible, at least in part, for impaired translational and proliferative responses to mitogens in T cells from SLE patients.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Operomics: molecular analysis of tissues from DNA to RNA to protein.

The identification of coding sequences in a number of species, including human in the near future, has ushered in the post-genome era. In this era, technologies are becoming available that allow the profiling of tissues and cell populations at the genomic, transcriptomic and proteomic levels. The molecular analysis of tissues at all three levels has been referred to as operomics. This review covers some basic technologies for operomics and their application to some lymphoid disorders. It is proposed that no one type of analysis is fully informative and that information that can be derived from the different compartments encompassed in operomics is complementary. Prospects for introducing such profiling technologies into the clinical laboratory will depend on their robustness, their user friendliness and the clinical relevance of the added information they provide, which cannot be captured through other technologies in use in the clinical laboratory.

Genomics↗
PubMed2000

Identification of beta-tubulin isoforms as tumor antigens in neuroblastoma.

There is currently substantial interest in the identification of human tumor antigens for diagnosis and immunotherapy of cancer. We have implemented a proteomic approach for the identification of tumor proteins that elicit a humoral response in cancer patients, which we have applied to neuroblastoma. Proteins from neuroblastoma tumors and cell lines were separated by two-dimensional PAGE and transferred to poly(vinylidene difluoride) membranes. Sera from 23 newly diagnosed patients with neuroblastoma, from 12 newly diagnosed children with other solid tumors, and from 13 normal individuals were screened for IgG and IgM autoantibodies against neuroblastoma proteins by means of Western blot analysis. Sera from 11 patients with neuroblastoma and from 1 patient with a primitive neuroectodermal tumor, but none of the other controls exhibited IgG-based reactivity against a protein constellation with an estimated Mr 50,000. NH2-terminal sequence and mass spectrometric analysis identified the major constituents of this constellation as beta-tubulin isoforms I and III. The IgG antibodies were additionally characterized to be of the subclass IgG1. Neuroblastoma patient sera that contained anti-beta-tubulin IgG antibodies also contained IgM antibodies specific against the full-length beta-tubulin molecule and against COOH-terminal beta-tubulin cleavage products. Neuroblastoma patient sera that reacted with beta-tubulin I and III isoforms in neuroblastoma tissues did not react with beta-tubulin I and III isoforms found in normal brain tissue. Our findings indicate the occurrence of beta-tubulin peptides in neuroblastoma, which are immunogenic. The occurrence of immunogenic peptides in neuroblastoma may have utility in diagnosis and in immunotherapy of this aggressive childhood tumor.

Adolescent↗
PubMed1999

Co-amplification of a novel gene, NAG, with the N-myc gene in neuroblastoma.

Substantial evidence implicates amplification of the N-myc gene with aggressive tumor growth and poor outcome in neuroblastoma. However some evidence suggests that this gene alone is not the sole determinant of outcome in N-myc amplified tumors. We have searched for genes that co-amplify with N-myc in neuroblastoma by means of two-dimensional analysis of genomic restriction digests. Using this approach, we have identified and cloned a novel genomic fragment which is co-amplified with N-myc in neuroblastomas. This fragment was mapped in close vicinity to N-myc on chromosome arm 2p24. It was amplified in 5/8 N-myc amplified neuroblastoma cell lines and in 9/13 N-myc amplified tumors. Using a PCR-based approach we isolated a 4.5 kb c-DNA sequence that is partly contained in the genomic fragment. The open reading frame of the cDNA encodes a predicted protein of 1353 amino acids (aa). The homology of the predicted protein, which we designated NAG (neuroblastoma amplified gene), to a C. elegans protein of as yet unknown function, and its ubiquitous expression suggest that NAG may serve an essential function. By Northern blot analysis we showed that amplification of the cloned gene correlates with over-expression in neuroblastoma cell lines. Amplification and consequent over-expression of NAG may, therefore, contribute to the phenotype of a subset of neuroblastomas.

Chromosome Mapping↗
PubMed1999

Identification of proteins from two-dimensional gel electrophoresis of human erythroleukemia cells using capillary high performance liquid chromatography/electrospray-ion trap-reflectron time-of-flight mass spectrometry with two-dimensional topographic map analysis of in-gel tryptic digest products.

Protein spots from two-dimensional (2-D) gel electrophoresis of a human erythroleukemia cell line have been identified by analysis of the in-gel tryptic digests using capillary high performance liquid chromatography (HPLC) separation with on-line detection using electrospray ionization mass spectrometry (ESI-MS). This is performed using an electrospray/ion trap storage/reflectron time-of-flight mass spectrometer system (ESI-IT-reTOFMS). A 2-D topographic mapping display developed to process the on-line data acquired with this TOF system has been used to obtain mass identification of each peptide, even though the capillary HPLC only provides limited separation capability of the tryptic peptide mixtures studied herein. Using this method, a substantial fraction of the protein sequence can be covered and identified using the tryptic map. It is demonstrated that by entering the cell species, the approximate MW and pI range as determined by 2-D gel electrophoresis, and the tryptic peptide map into the database a unique match for identification of the protein generally results. It is also demonstrated that a much improved coverage of the protein sequence is obtained by this method relative to matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS).

Amino Acid Sequence↗
PubMed1999

Capillary electrophoresis/tandem mass spectrometry for analysis of proteins from two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Capillary electrophoresis/time-of-flight mass spectrometry(CE/TOFMS) has been used for analysis of in-gel digests of protein spots excised from two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2-D SDS-PAGE). An off-line purification and preconcentration procedure with a Zip Tip is used before CE/TOFMS analysis which allows for detection of protein spots with <1 picomole of material from 2-D gels. The off-line procedure provides sufficient purification for analysis while maintaining the quality of the CE separation. Using this procedure, several proteins from Coomassie Blue and zinc negatively stained gels are identified by the peptide maps generated and database searching. CE/TOF tandem mass spectrometry is used for the confirmation of database searching results and structural analysis of peptides that do not match the expected peptide maps obtained from the database in order to identify structural modifications. Several modifications were pinpointed and identified by this method.

Adenocarcinoma↗
PubMed1999

Horizontal two-dimensional electrophoresis of complex DNA samples using precast gel systems.

We have developed a simplified procedure for the separation of enzyme-digested genomic DNA, or of complex mixtures of cloned DNA, into two dimensions. The procedure relies on the use of precast gels for horizontal electrophoretic separations. Precast agarose-type gels are used for the first-dimensional separation of fragments based on size. Precast polyacrylamide gels are used for the second-dimensional separation of fragments. The separated fragments are subjected to enzymatic digestion in situ prior to their transfer to the second-dimensional gel. Applications of this procedure include the analysis of DNA libraries, analysis of yeast artificial chromosomes (YACs) as well as other similar preparations, and the screening of genomic DNA for the occurrence of multi-copy DNA fragments as in the case of genomic amplifications in cancer.

Acrylic Resins↗
PubMed1999

Differential expression of Hsp27 in normal oesophagus, Barrett's metaplasia and oesophageal adenocarcinomas.

The protein expression patterns of normal, metaplastic and malignant oesophageal tissues were analysed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) to identify changes associated with Barrett's metaplasia and transformation to oesophageal adenocarcinoma. Heat-shock protein 27 (Hsp27), a small heat-shock protein which is protective against cytotoxic stresses, was abundant in normal oesophagus. However, Hsp27 expression was markedly lower in Barrett's metaplasia and oesophageal adenocarcinomas. This was confirmed by immunohistochemical analysis. Hsp27 protein was most highly expressed in the upper layers of squamous epithelium and exhibited a pattern of expression that corresponded with the degree of squamous maturation. Northern and Southern analysis demonstrated Hsp27 to be regulated at the level of mRNA transcription or abundance. Normal oesophageal tissues were examined for gender differences in Hsp27 expression. Women expressed fourfold higher levels of Hsp27 mRNA, however, this difference was not appreciable in protein expression. Hsp27 protein was inducible by heat shock in Barrett's adenocarcinoma cell lines and an immortalized oesophageal epithelial cell line (HET-1A), but not by oestradiol. These results demonstrate abundant constitutive expression of the stress-response protein Hsp27 in the normal oesophagus, and suggest that low-level expression in Barrett's metaplasia may be one factor which may influence susceptibility to oesophageal adenocarcinoma development.

Adenocarcinoma↗
PubMed1998

Differential regulation of translation and eIF4E phosphorylation during human thymocyte maturation.

Activation of peripheral blood T cells by cross-linking of CD3 results in a rapid and substantial rise in translation rates and proliferation, which coincides with an increase in the cap-binding protein, eIF4E activity. In contrast, immature CD4+ CD8+ double-positive (DP) thymocytes undergo apoptosis in response to anti-CD3 mAb. We have investigated translation initiation in the response of immature thymocytes to activating signals. Activation by anti-CD3 + anti-CD4 of immature CD4+ CD8+ DP thymocytes results in a rapid decrease in protein synthesis. In contrast, similar treatment of CD4+ or CD8+ single-positive (SP) thymocytes results in an increase in protein synthesis. The rate of protein synthesis is linked to the phosphorylation status of eIF4E. Following anti-CD3 + anti-CD4 stimulation, eIF4E phosphorylation strongly decreases in immature DP thymocytes, whereas it increases in mature SP thymocytes. The expression of 4E-BP2, a specific repressor of eIF4E function, is high in DP cells but decreases during maturation, raising the possibility of a role for 4E-BP2 in repressing eIF4E phosphorylation. These data provide evidence for differential regulation of the translational machinery during T cell development.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Mining the human proteome: experience with the human lymphoid protein database.

We have undertaken an effort in the past five years aimed at developing a database of lymphoid proteins detectable by two-dimensional (2-D) polyacrylamide gel electrophoresis. The database contains 2-D patterns and derived information pertaining to: (i) polypeptide constituents of unstimulated and stimulated mature T cells and immature thymocytes; (ii) cultured T cells and cell lines that have been manipulated by transfection with a variety of constructs or by treatment with specific agents; (iii) single cell-derived T and B cell clones; (iv) cells obtained from patients with lymphoproliferative disorders and leukemia; and (v) a variety of other relevant cell populations. The database has experienced a substantial expansion in 2-D patterns it contains, numbering currently 9167 individual 2-D patterns. This number represents a fraction of the 30,682 2-D patterns maintained in our databases. The capacity to design and undertake experiments, produce high-quality 2-D patterns, and to undertake simple or rudimentary analyses of 2-D patterns to meet the basic needs of the experiments for which the 2-D gels were produced has exceeded the capacity to fully and uniformly integrate information generated from any gel image or experiment, across all images and experiments. While only a fraction of the information in the 2-D patterns contained in the lymphoid database has been mined, novel findings derived from querying the database point to the merits of this protein based approach. Additional resources have recently been put into place to mine more effectively data pertaining to protein expression in lymphoid cells.

Cell Cycle↗
PubMed1997

Effect of mitogenic stimulation and DNA methylation on human T cell DNA methyltransferase expression and activity.

DNA methylation, a mechanism modifying gene expression, is mediated in part by the enzyme DNA methyltransferase. Reduced levels of T cell DNA methyltransferase have been observed in lupus-like diseases, and increased levels have been reported in malignancies. Little is known concerning the regulation of human DNA methyltransferase. In this report we demonstrate that mitogenic T cell stimulation causes an increase in DNA methyltransferase mRNA and enzyme activity. We also show that pharmacologic inhibition of T cell DNA methylation causes an increase in the rate of DNA methyltransferase mRNA transcription and a corresponding increase in mRNA levels and enzyme activity. This suggests that DNA methyltransferase is itself regulated in part by DNA methylation status, possibly representing a feedback mechanism. DNA methylation inhibition also resulted in an increase in Ha-ras and c-jun mRNA levels, overexpression of which increases DNA methyltransferase in murine systems. These results thus identify two mechanisms regulating levels of human T cell DNA methyltransferase and raise the possibility that abnormalities in either could contribute to disorders associated with altered DNA methylation.

Adult↗
PubMed1997

p53 mutation as a prognostic marker in advanced laryngeal carcinoma. Department of Veterans Affairs Laryngeal Cancer Cooperative Study Group.

OBJECTIVE: To determine the relationship of p53 mutations in advanced laryngeal carcinomas to p53 immunohistochemistry, organ preservation, and patient survival. DESIGN: Paraffin-embedded tumor specimens were obtained from patients enrolled in the Department of Veterans Affairs Laryngeal Cancer Cooperative Study, a multi-institutional randomized clinical trial comparing induction chemotherapy (cisplatin and fluorouracil) plus radiation therapy surgery plus postoperative radiation therapy. Tumor specimens were analyzed for p53 mutations in exons 5 through 8 by using single-strand conformational polymorphism (SSCP) analysis followed by DNA sequencing of all variants. Five-year follow-up data were available for all patients studied. SUBJECTS: Forty-four patients enrolled in the Department of Veterans Affairs Laryngeal Cancer Cooperative Study from whom paraffin-embedded tumor specimens were readily available. RESULTS: p53 immunostaining did not correlate with p53 SSCP and DNA sequencing results. More than half (62% [16/26]) of the tumors that overexpressed p53 immunohistochemically did not have a detectable p53 gene mutation. Similarly, 39% (7/18) of tumors that did not overexpress p53 did have a p53 gene mutation. p53 mutations were present in 39% of tumors tested. Mutations within exon 5 made up 41% of p53 gene mutations in laryngeal carcinomas. Transitions were the most common type of mutation in this study (92% of mutations). CONCLUSIONS: The presence of a p53 mutation as detected by SSCP is associated with decreased patient survival. Further study is required to confirm this relationship and to determine whether specific p53 mutations predict organ preservation.

Carcinoma, Squamous Cell↗
PubMed1997

Quantitative analysis of Op18 phosphorylation in childhood acute leukemia.

Oncoprotein 18 (Op18) is a major cystolic phosphoprotein constituent of leukemia cells. There is cumulative evidence that suggests a role for Op18 in integrating signals from diverse pathways involved in cell growth. Op18 phosphorylation is induced with proliferation in a variety of cell types, and is essential for cell cycle progression. In this study we analyzed the level of unphosphorylated Op18 and of its major phosphorylated forms, Op18a and Op18b, in a series of 177 childhood acute leukemias by means of quantitative two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Op18 phosphorylation was significantly correlated with the white blood count at the time of diganosis, and with a high percentage of cells in the S phase. Our findings suggest that strategies to inhibit Op18 expression or phosphorylation may be effective in inhibiting leukemia cell proliferation.

Acute Disease↗
PubMed1997

Monocyte-dependent regulation of T lymphocyte activation through CD98.

CD98 is a 125 kDa heterodimer, which is strongly expressed on the surface of activated and proliferating cells. Its expression is strikingly regulated during T cell differentiation and activation, but the role of CD98 during T lymphocyte responses is not yet understood. We report here that proliferation of resting peripheral blood mononuclear cells (PBMC) induced by lectin, superantigen (SAg) or conventional antigens was blocked by anti-CD98 heavy chain (CD98hc) mAb. In contrast, anti-CD98hc did not block responses of T cell clones or lines. Anti-CD98hc inhibited IL-2 receptor expression and progression of T cells from G1 to S phase, but did not reduce expression of the IL-2 gene. Anti-CD98hc mAb did not regulate the initial activation events involving the TCR and co-receptor structures, but instead inhibited T lymphocyte responses even when added 18 h or more after the activation stimulus. Further experiments demonstrated that anti-CD98 was not directly affecting T cells in this system, but was instead acting on accessory cells. This was supported using a novel xenogeneic system that takes advantage of the lack of xenoreactivity of purified human T cells against mouse splenocytes. Despite absence of a direct xenoresponse to murine spleen cells, human T cells were activated by SAg presented by murine splenic antigen-presenting cells (APC). Murine anti-human CD98hc did not block T cell proliferation in this system. Furthermore, responses using monocyte-depleted PBMC as APC were not blocked by anti-CD98hc. Taken together, the present data suggests that triggering of human monocyte CD98 can suppress T cell proliferation by a process that halts progression through the cell cycle of recently activated T lymphocytes. This may represent a novel pathway for monocyte regulation of T cell activation.

Antibodies, Blocking↗
PubMed1996

Tissue distribution, genomic structure, and chromosome mapping of mouse and human eukaryotic initiation factor 4E-binding proteins 1 and 2.

Two related eukaryotic initiation factor-4E binding proteins (4E-BP1 and 4E-BP2) were recently characterized for their capacity to bind specifically to eIF4E and inhibit its function. Here, we determined the cDNA sequence, tissue distribution, genomic structure, and chromosome localization of murine and human 4E-BP1 and 4E-BP2. Mouse 4E-BP1 and 4E-BP2 consist of 117 and 120 amino acids and exhibit 91. 5 and 95.0% identity, respectively, to their human homologues. 4E-BP1 mRNA is expressed in most tissues, but is most abundant in adipose tissue, pancreas, and skeletal muscle, while 4E-BP2 mRNA is ubiquitously expressed. The structures of the mouse 4E-BP1 and 4E-BP2 were determined. The 4E-BP1 gene consists of three exons and spans approximately 16 kb. In addition, two 4E-BP1 pseudogenes exist in the mouse genome. The 4E-BP2 gene spans approximately 20 kb and exhibits an identical genomic organization to that of 4E-BP1, with the protein coding portion of the gene divided into three exons. There are no pseudogenes for 4E-BP2. The chromosomal locations of 4E-BP1 and 4E-BP2 were determined in both mice and humans by fluorescence in situ hybridization analysis. Mouse 4E-BP1 and 4E-BP2 map to chromosomes 8 (A4-B1) and 10 (B4-B5), respectively, and human 4E-BP1 and 4E-BP2 localize to chromosomes 8p12 and 10q21-q22, respectively.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Expression of the protein kinase PKR in modulated by IRF-1 and is reduced in 5q- associated leukemias.

The transcription factor IRF-1 (interferon regulatory factor 1) is an activator of type I interferon and interferon-inducible genes. IRF-1 manifests tumor suppressor activity. Its overexpression results in inhibition of cell growth, and deletions of the IRF-1 gene were demonstrated in a number of human leukemias and myelodysplasias. Although the mechanism by which IRF-1 affects cell growth is unknown, it is believed that IRF-1 activates a set of genes that negatively regulate cell growth. The double-stranded RNA-dependent protein kinase (PKR), which is an interferon-inducible gene, contains a promoter element for the binding of IRF-1 and exhibits antiproliferative properties. Consequently, we investigated the role of IRF-1 in PKR expression. Here, we show that in IRF-1-deficient embryonic fibroblasts, PKR expression is reduced relative to wild-type cells. This result predicts diminished expression of PKR as a potential consequence of deletion of the IRF-1 gene in human leukemias. We show that cells of the human leukemic U937 cell line contain a deletion of one IRF-1 gene and express low levels of PKR. We demonstrate that upregulation of IRF-1 expression in U937 cells by transfection is sufficient to induce PKR expression. We also found a marked reduction in the expression of PKR in blood samples from two patients with myelodysplasias, carrying a deletion of chromosome 5q, a locus to which IRF-1 was mapped. These results show that IRF-1 activates PKR expression and suggest that loss of one allele of the IRF-1 gene is sufficient to affect PKR expression. Therefore, PKR is a strong candidate for a mediator of the tumor suppressor activity of IRF-1.

Chromosomes, Human, Pair 5↗