Biomedical subjects

S M Hanash

Publications and source records attributed to S M Hanash.

At least 73 records · Page 4Linked to original sources

PubMed1993

Elevated proliferating cell nuclear antigen levels in immature thymocytes. Dissociation from cell cycle progression.

Entry into and progression through the cell cycle is associated with a tightly regulated program of gene expression in mature T cells. One such gene product, proliferating cell nuclear Ag (PCNA), is the auxiliary protein of DNA polymerase delta, and is induced during late G1 and early S phase after stimulation of resting (G0) cells. Blockade of PCNA production has been found to inhibit cell division suggesting that PCNA plays an important role in cell proliferation. The extent to which PCNA and other proliferation-related gene products are similarly regulated in thymocytes has been largely undetermined. Here, we report that immature double positive (CD4+CD8+) thymocytes express high levels of PCNA protein and mRNA relative to mature single positive (CD4+CD8- or CD4-CD8+) thymocytes or peripheral blood T cells. Elevation of PCNA expression among double positive thymocytes is not the result of increased numbers of cycling cells in this subpopulation, being specifically observed in double positive thymocytes with normal diploid DNA content and resting (G0) levels of RNA. Unlike mature thymocytes and T cells, mitogenic stimulation did not induce an increase in PCNA expression in double positive thymocytes. These data indicate that immature thymocytes express high levels of PCNA in the absence of cell cycle progression, thus providing evidence for differential regulation of proliferation related pathways during lymphoid development. Altered expression patterns of these genes in immature vs mature thymocytes may contribute to the process of thymic selection.

Animals↗

PubMed1993

Mapping of the gene for interferon-inducible dsRNA-dependent protein kinase to chromosome region 2p21-22: a site of rearrangements in myeloproliferative disorders.

Recent evidence suggests that the human interferon-inducible double-stranded RNA-dependent protein kinase may function as a tumor suppressor. Here we describe the mapping of the gene for this kinase to chromosome region 2p21-22 by fluorescence in situ hybridization. A combined analysis of cytogenetic data from a series of 341 patients with hematologic disorders that exhibited cytogenetic abnormalities and from published reports indicates that abnormalities involving 2p21-22 occur nonrandomly and are observed among patients with acute myelogenous leukemia, raising the possibility of a role for this protein kinase in leukemogenesis.

Bone Marrow↗

PubMed1993

PCNA levels in neuroblastoma are increased in tumors with an amplified N-myc gene and in metastatic stage tumors.

N-myc oncogene amplification in neuroblastoma has been found to be significantly associated with advanced stage disease and tumor progression. However, there is a lack of data on tumors, regarding the relationship between N-myc gene amplification and proliferation activity. Proliferating cell nuclear antigen (PCNA) is a proliferation-induced 36 kD nuclear protein that is the auxiliary component of DNA polymerase delta. PCNA levels in tissues have been found to correlate with proliferative activity. We have examined PCNA levels in neuroblastomas in relation to N-myc gene amplification and tumor stage. Statistically, significantly higher levels of PCNA were observed in tumors with an amplified N-myc gene relative to tumors with a single gene copy. The highest levels of PCNA were observed in advanced stage tumors with an amplified N-myc gene. Treatment of neuroblastoma cells in culture with retinoic acid, which induces differentiation, resulted in a substantial decrease in PCNA. Our results suggest that PCNA levels may reflect differences in proliferative activity between neuroblastomas, related to stage of the disease and to N-myc gene copy number.

Amino Acid Sequence↗

PubMed1992

Similarity of spontaneous germinal and in vitro somatic cell mutation rates in humans: implications for carcinogenesis and for the role of exogenous factors in "spontaneous" germinal mutagenesis.

The rate of spontaneous mutation resulting in electrophoretic variants per cell generation in a human lymphoblastoid cell line, on the basis of experiments described in this paper, is found to be 7.2 x 10(-8) per locus. A review of similar data on electrophoretic variants resulting from spontaneous mutation in the human germ line leads to an estimate of 3.3 x 10(-8) per locus per cell generation. It is argued that the similarity of these two estimates, despite an average cell generation time of 18.5 hr for the cultured somatic cells but about 26 days in the germ line, suggests that spontaneous mutation involving nucleotide substitutions is much more dependent on cell generation than on time. This finding permits the inference that environmental (exogenous) variables make a relatively small contribution to the rate of this type of human germinal spontaneous mutation. While in vitro somatic-cell mutation rates, such as derived in this study, provide a basis for modeling the contribution of nucleotide substitutions in multihit/clonal theories of carcinogenesis, it is also argued that the complex of events involved in carcinogenesis, including chromosomal rearrangements and mitotic recombination, could have very different individual probabilities. Estimates for the rates of these other types of mutation are needed to provide a better understanding of the manner in which multiple mutations accumulate in malignant cells.

Alleles↗

PubMed1992

Cell cycle progression is associated with distinct patterns of phosphorylation of Op18.

Op18 is a highly conserved major cytosolic phosphoprotein which has been implicated in signal transduction in a wide variety of cell types. Freshly isolated peripheral blood lymphocytes (PBL) constitutively express low levels of mostly unphosphorylated Op18. Following mitogenic stimulation of PBL, Op18 synthesis is induced at a time when cells are entering S-phase. In this study we have characterized Op18 phosphorylation during progression of freshly isolated PBL through the cell cycle. Transition from G0 to G1 following activation with OKT3 was associated with an increase in a phosphorylated form designated Op18c. Progression of cells through G1 into S resulted in an increase in phosphorylated Op18 forms, designated Op18a and Op18b, which paralleled new Op18 synthesis. Transition of cells into G2 + M resulted in the appearance of the more acidic phosphorylated forms Op18d and Op18e. Calphostin C, a specific inhibitor of protein kinase C, dramatically decreased all forms of phosphorylated Op18 in OKT3 treated Jurkat cells. Our results suggest that Op18 phosphorylation is mediated in part by PKC activation as well as by other kinases yielding different phosphorylated forms at specific stages of the cell cycle.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1992

Phenotypic and functional similarities between 5-azacytidine-treated T cells and a T cell subset in patients with active systemic lupus erythematosus.

OBJECTIVE: Antigen-specific CD4+ T cells treated with DNA methylation inhibitors become autoreactive, suggesting a novel mechanism for autoimmunity. To test whether this mechanism might be involved in systemic lupus erythematosus (SLE), phenotypic markers for the autoreactive cells were sought. METHODS: Cloned normal T cells were treated with the DNA methylation inhibitor 5-azacytidine (5-azaC) and studied for altered gene expression. T cells from patients with active SLE were then studied for a similar change in gene expression, and cells expressing the marker were tested for autoreactivity. RESULTS: 5-azaC-treated normal T cells had increased CD11a (leukocyte function-associated antigen 1 alpha) expression relative to other membrane molecules. A T cell subset with similar CD11a expression was found in patients with active SLE. This subset contained cells that spontaneously lysed autologous macrophages, with a specificity similar to that of 5-azaC-treated cells.

Antigens, CD↗

PubMed1992

Proliferation-related expression of p19/nm23 nucleoside diphosphate kinase.

High level expression of the nm23-H1 gene, which encodes for a nucleoside diphosphate kinase, has been found to correlate with diminished metastasis in some tumors but not in others. We have previously identified the protein product of the nm23-H1 gene in two-dimensional electrophoretic gels and have designated it p19/nm23. In neuroblastoma, higher levels of p19/nm23, which are associated with amplification of the N-myc oncogene, large tumor mass, and metastasis, were observed in advanced stage tumors compared with limited stage disease. Because of the variable expression of nm23-H1 in different tumors, we have investigated the relationship between amounts of the protein and cell proliferation. The levels of p19/nm23 were compared between resting and mitotically stimulated normal human PBLs and in leukemia cells. The amount of p19/nm23 increased in normal lymphocytes in response to mitotic stimulation and paralleled the increase in DNA synthesis. In leukemia cells obtained from patients with different subtypes of acute leukemia, p19/nm23 levels were also increased relative to resting normal lymphocytes. Treatment of mitotically stimulated lymphocytes with cyclosporin, which inhibits proliferation, blocked the increase in p19/nm23; treatment of the leukemia cell line HL-60 with dimethylsulfoxide, which induces terminal differentiation, resulted in diminished levels of p19/nm23. Our data therefore provide evidence that nm23-H1 expression is related to cell proliferative activity.

Flow Cytometry↗

PubMed1991

Involvement of OP18 in cell proliferation.

Op18 is a highly conserved major cytosolic phosphoprotein that is expressed at high levels in acute leukemia and in neuroblastoma. In this study we present evidence pointing to a role for Op18 in cellular proliferation. Blocking of Op18 mRNA translation using antisense oligonucleotides delayed entrance of mitotically stimulated normal peripheral blood lymphocytes into the S phase. Moreover treatment of HL-60 promyelocytic leukemia cells with DMSO or PMA which induced terminal differentiation resulted in a decrease in the level of Op18 RNA and protein. Inhibition of lymphoid proliferation with cyclosporin also resulted in reduced Op18 levels.

Base Sequence↗

PubMed1991

Characterization of the gene for a proliferation-related phosphoprotein (oncoprotein 18) expressed in high amounts in acute leukemia.

The oncoprotein 18 (Op18) gene encodes a proliferation-related cytosolic phosphoprotein, which is induced in normal lymphocytes following mitogenic stimulation. Studies of the Op18 gene are of particular interest because of the proposed role of Op18 protein in signal transduction and because of its occurrence in markedly increased amounts in acute leukemia cells. We have recently reported the cloning and sequencing of two cDNA clones for Op18 (1 and 1.5 kilobases). Both clones code for the same 149-amino acid polypeptide; however, they differ in their 3'-region as a result of alternative polyadenylation. We report here the sequencing of the Op18 gene and describe its expression in leukemia. The Op18 gene, which is 6.3 kilobases in length, is comprised of five exons and four introns and exhibits features that are common to other genes involved in cellular growth and proliferation. The increase in Op18 polypeptide in leukemia is associated with increased RNA transcription without gene amplification or rearrangement. Treatment of K562 leukemia cell line with hemin that induces terminal differentiation resulted in decreased expression of Op18. Our findings suggest that the high amount of Op18 protein in acute leukemia results from increased expression of a structurally unaltered gene.

Acute Disease↗

PubMed1991

Highly resolving two-dimensional gels for protein sequencing.

Two-dimensional (2D) PAGE, using carrier ampholytes for the first-dimension separation, has provided a tool for the simultaneous analysis of cellular proteins. To extend the utility of 2D PAGE to the preparative level, we have investigated the use of immobilized pH gradients (IPG) for the first-dimension separation. The results we have obtained indicate that as much as 1 mg of cellular protein can be loaded onto a single IPG gel without loss of resolution. Mutant polypeptides previously detected in carrier ampholyte-based 2D gels were equally detectable in IPG-based 2D gels. With IPG gels several hundred cellular polypeptides can be isolated, from as few as 10 gels, in sufficient amount for sequencing with current sequencing technology. We therefore conclude that IPG greatly enhances the prospects for the large-scale sequencing of cellular proteins for the development of 2D gel-related protein data bases and for the identification of new polypeptide gene products, with the attendant implications for a genome sequencing effort.

Amino Acid Sequence↗

PubMed1991

Diminished phosphorylation of a heat shock protein (HSP 27) in infant acute lymphoblastic leukemia.

We have previously reported lack of expression of a polypeptide designated L3 in infant acute lymphoblastic leukemia (ALL). Expression of L3 occurred predominantly in older children with pre-B ALL. We have recently reported the expression during B cell ontogeny of two other polypeptides, designated L2 and L4 with a similar Mr as L3, which were identified as phosphorylated and non-phosphorylated forms respectively of the low Mr heat shock protein. hsp27. In this study we have characterized L3 and identified it as another phosphorylated form of hsp27. The two phosphorylated forms appear to be differentially expressed in acute leukemia. L3 levels in infants who expressed hsp27 isoforms L2 and L4 were significantly diminished compared to levels in older children with an equivalent amount of hsp27. We conclude that leukemic cells in infant ALL exhibit a unique pattern of phosphorylation of hsp27 expressed at a pre-B cell stage of differentiation.

Cell Line↗

PubMed1991

Temperature-dependent spot positional variability in two-dimensional polypeptide patterns.

The effect of temperature, at which isoelectric focusing with immobilized pH gradients is performed, on spot positions and pattern quality in two-dimensional (2-D) electrophoresis was examined. Increased temperatures revealed improved 2-D patterns with respect to sample entry, resolution, and background staining. Focusing at 20 degrees C was superior to focusing at 10 and 15 degrees C. Even at 30 degrees C, a pattern of well-resolved polypeptide spots with a minimum amount of horizontal streaking at the basic end was observed. A computer-based analysis showed that a substantial proportion of polypeptides assumed altered positions in the 2-D pattern in relation to temperature. Mobility shifts of polypeptides were more variable on the neutral part than on the acidic or basic end. The mobility shifts were not restricted to one direction for all the spots whose migration was altered. However, for any given spot, the direction was the same with subsequent increments of temperature. The results clearly demonstrate that a defined temperature for first-dimensional isoelectric focusing is a requirement for the reproducibility of 2-D electrophoresis. After elimination of the cathodic drift, a major source of variability in 2-D patterns, associated with carrier ampholytes, temperature control becomes a critical parameter.

Electrophoresis, Gel, Two-Dimensional↗

PubMed1991

A two-dimensional electrophoresis-related laboratory information processing system: spot matching.

An approach for the computer-assisted analysis of two-dimensional gels has been developed as a part of our laboratory information processing system (LIPS). This approach relies in part on an algorithm for the pairwise matching of protein spots. The matching process initially matches spots based on a cross-correlational measure of how well neighboring spots align. While this first pass correctly determines most spot correspondences and noncorrespondences, it can make errors. Higher accuracy is obtained by monitoring the consistency of spot match decisions in a second pass, which demands that neighboring spot pairs that align spatially must also have been found to match in the first pass. Pairwise comparisons of gels are combined into n-way comparisons by matching spot lists of gels to "master" gel spot lists, which in turn are matched to higher level masters, resulting in a hierarchy of matched spots. After each pairwise match the results are reviewed and corrected with the assistance of a graphical match-editor. Results are given for 19 single-cell-derived lymphoid clones in which the presence of a mutation had previously been established, each processed in duplicate. Only one of 46 spot changes failed to be detected, which demonstrates that the strategy is sensitive and efficient for detecting qualitative spot differences.

Algorithms↗

PubMed1991

Implementation of a two-dimensional electrophoresis-related laboratory information processing system: database aspects.

A laboratory information processing system (LIPS) has been developed to provide support for various laboratory activities related to two-dimensional electrophoresis. In this paper we present the relational schema for each level of activity and the application development of LIPS, in terms of the relational database management system being used. We also discuss our experience with the current system.

Algorithms↗

PubMed1990

Proliferating cell nuclear antigen expression in childhood acute leukemia.

Proliferating cell nuclear antigen (PCNA) is a 36-Kd nuclear protein, identified as the auxiliary protein of DNA polymerase delta, that is upregulated in activated proliferating cells from a variety of tissues and species, including human lymphocytes. We have examined by two-dimensional polyacrylamide gel electrophoresis the expression of PCNA in various subtypes of childhood acute leukemia and have found differences in its expression according to subtype. These differences were not related to the initial peripheral white blood count, age, or sex, and appeared to reflect differences in proliferative activity between subtypes of acute leukemia.

Acute Disease↗

PubMed1990

A gene that encodes for a leukemia-associated phosphoprotein (p18) maps to chromosome bands 1p35-36.1.

The cytosolic protein p18 which is expressed in increased amounts in acute leukemia cells is variably phosphorylated as a function of growth and differentiation. Proteins with identical amino acid sequence were independently found to be highly expressed in normal brain tissue and neuroendocrine tumor cells. Here we described the mapping of the recently cloned p18 gene to chromosome 1, band p35-36.1 by Southern blot analysis of human-rodent somatic cell hybrid DNA and by chromosome in situ hybridization using a p18 genomic probe. This region of the distal short arm of chromosome 1 is a frequent site of deletions or loss of heterozygosity in tumors derived from neural crest cells, particularly neuroblastomas and melanomas. The high levels of expression of p18 in brain and neuroendocrine tumor cells, its possible role in growth regulation, and its chromosomal location in a region frequently deleted in neuroectodermal tumors suggest that this gene may be involved in common genetic events occurring in these tumors.

Animals↗

PubMed1989

Molecular cloning of a novel human leukemia-associated gene. Evidence of conservation in animal species.

We have recently described an 18-kilodalton polypeptide (p18) that is present in much greater abundance in acute leukemic blast cells (myeloid and lymphoid) than in resting or proliferating nonleukemic lymphoid cells or chronic lymphoid and myeloid leukemic cells. In this report we describe the cloning of two different sized full-length cDNAs that code for p18. The two cDNAs differ in their 3'-noncoding regions as a result of alternative polyadenylation. Analysis of the complete nucleotide sequence and the corresponding amino acid sequence did not reveal significant homology to any previously described sequences. We show evidence that this gene is highly conserved in several animal species and low stringency hybridization studies suggest that the p18 gene may be a member of a family of partially homologous genes in the human genome.

Amino Acid Sequence↗

PubMed1989

Advances in two-dimensional electrophoresis.

New developments in the first dimension step of two-dimensional electrophoresis have expanded the utility of the technique in cell and molecular biology.

Cell Physiological Phenomena↗