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S Hanash

Publications and source records attributed to S Hanash.

36 records · Page 2Linked to original sources
PubMed1998

A novel amplicon at 8p22-23 results in overexpression of cathepsin B in esophageal adenocarcinoma.

Cathepsin B (CTSB) is overexpressed in tumors of the lung, prostate, colon, breast, and stomach. However, evidence of primary genomic alterations in the CTSB gene during tumor initiation or progression has been lacking. We have found a novel amplicon at 8p22-23 that results in CTSB overexpression in esophageal adenocarcinoma. Amplified genomic NotI-HinfI fragments were identified by two-dimensional DNA electrophoresis. Two amplified fragments (D4 and D5) were cloned and yielded unique sequences. Using bacterial artificial chromosome clones containing either D4 or D5, fluorescent in situ hybridization defined a single region of amplification involving chromosome bands 8p22-23. We investigated the candidate cancer-related gene CTSB, and potential coamplified genes from this region including farnesyl-diphosphate farnesyltransferase (FDFT1), arylamine N-acetyltransferase (NAT-1), lipoprotein lipase (LPL), and an uncharacterized expressed sequence tag (D8S503). Southern blot analysis of 66 esophageal adenocarcinomas demonstrated only CTSB and FDFT1 were consistently amplified in eight (12.1%) of the tumors. Neither NAT-1 nor LPL were amplified. Northern blot analysis showed overexpression of CTSB and FDFT1 mRNA in all six of the amplified esophageal adenocarcinomas analyzed. CTSB mRNA overexpression also was present in two of six nonamplified tumors analyzed. However, FDFT1 mRNA overexpression without amplification was not observed. Western blot analysis confirmed CTSB protein overexpression in tumor specimens with CTSB mRNA overexpression compared with either normal controls or tumors without mRNA overexpression. Abundant extracellular expression of CTSB protein was found in 29 of 40 (72. 5%) of esophageal adenocarcinoma specimens by using immunohistochemical analysis. The finding of an amplicon at 8p22-23 resulting in CTSB gene amplification and overexpression supports an important role for CTSB in esophageal adenocarcinoma and possibly in other tumors.

Adenocarcinoma↗
PubMed1998

Amplification of DNA sequences from chromosome 19q13.1 in human pancreatic cell lines.

Conventional cytogenetics and comparative genomic hybridization (CGH) were utilized to identify recurrent chromosomal imbalances in 12 pancreatic adenocarcinoma cell lines. Multiple deletions and gains were observed in all cell lines. Losses affecting chromosomes or chromosome arms 9p, 13, 18q, 8p, 4, and 10p and gains involving chromosome arms or bands 19q13.1, 20q, 5p, 7p, 11q, 3q25-qter, 8q24, and 10q were commonly observed. Interestingly, 19 distinct sites of high-level amplification were found by CGH. Recurrent sites involved 19q13.1 (6 cases), 5p (3 cases), and 12p and 16p (2 cases). Amplification of KRAS2 was demonstrated in 2 cell lines and that of ERBB2 in another. To define the occurrence of chromosome 19 amplification further, two-dimensional analysis of NotI genomic restriction digests and fluorescence in situ hybridization using probes from band 19q13.1 were utilized. High-level amplification of overlapping sets of chromosome 19 NotI fragments was exhibited in 3 cell lines of which 2 showed amplification of both OZF and AKT2 genes and 1 that of AKT2 alone. In these 3 cell lines, amplification of chromosome 19 sequences was associated with the presence of a homogeneously staining region. Our results provide evidence of heterogeneity in the extent of chromosome 19 amplification and suggest the existence of yet unknown amplified genes that may play a role in pancreatic carcinogenesis.

Chromosome Aberrations↗
PubMed1998

GAC1, a new member of the leucine-rich repeat superfamily on chromosome band 1q32.1, is amplified and overexpressed in malignant gliomas.

We have used two-dimensional electrophoresis of enzyme-digested genomic DNA to identify a novel gene GAC1, which maps at 1q32.1 and which is overexpressed in malignant gliomas in which it is amplified. GAC1 encodes a protein which belongs to the leucine-rich repeat superfamily. Amplification and overexpression of GAC1 was demonstrated in two of eight tumors where amplifications were previously evidenced by comparative genomic hybridization (one glioblastoma multiforme and one anaplastic astrocytoma), and in one of eight unselected glioblastomas multiforme. GAC1 exhibits sequence homology with other proteins which function as cell-adhesion molecules or as signal transduction receptor and is a likely candidate for the target gene in the 1q32.1 amplicon in malignant gliomas.

Amino Acid Sequence↗
PubMed1996

Two-dimensional separation and cloning of chromosome 1 NotI-EcoRV-derived genomic fragments.

The two-dimensional (2-D) separation of genomic digests has provided the means to analyze over 2000 unique restriction fragments simultaneously in a single gel, for genetic variation as well as for genomic alterations in cancer. By utilizing different combinations of restriction enzymes or different electrophoretic conditions, the number of analyzable fragments in multiple 2-D patterns can be augmented. We have previously shown the feasibility of distinguishing between spot intensities representing fragments from one allele and from two alleles and have implemented approaches for the cloning of fragments of interest in 2-D gels. In this study, the 2-D separation and cloning of chromosome 1 NotI-EcoRV-derived genomic fragments was performed. Three hundred forty-six NotI fragments in whole genomic preparations were assigned to chromosome 1. To verify the reliability of the assignment, two of the NotI fragments attributed to chromosome 1 were cloned and sequenced. The fragments that contained CpG islands were mapped by FISH to 1p35-p36.1 and to 1p13.3-p21, respectively. Our study indicates the feasibility of analyzing 2-D separations of whole genomic digests for the detection of alterations in specific chromosomes. The large number of restriction fragments attributed to chromosome 1 provides the means to screen 2-D patterns for chromosome 1 deletions and amplifications with a high marker density.

Cell Line↗
PubMed1996

A methylated human 9-kb repetitive sequence on acrocentric chromosomes is homologous to a subtelomeric repeat in chimpanzees.

We have implemented an approach for the detection of DNA alterations in cancer by means of computerized analysis of end-labeled genomic fragments, separated in two dimensions. Analysis of two-dimensional patterns of neuroblastoma tumors, prepared by first digesting DNA with the methylation-sensitive restriction enzyme Not I, yielded a multicopy fragment which was detected in some tumor patterns but not in normal controls. Cloning and sequencing of the fragment, isolated from two-dimensional gels, yielded a sequence with a strong homology to a subtelomeric sequence in chimpanzees and which was previously reported to be undetectable in humans. Fluorescence in situ hybridization indicated the occurrence of this sequence in normal tissue, for the most part in the satellite regions of acrocentric chromosomes. A product containing this sequence was obtained by telomere-anchored PCR using as a primer an oligonucleotide sequence from the cloned fragment. Our data suggest demethylation of cytosines at the cloned Not I site and in neighboring DNA in some tumors, compared with normal tissue, and suggest a greater similarity between human and chimpanzee subtelomeric sequences than was previously reported.

Animals↗
PubMed1996

Demethylation of repetitive DNA sequences in neuroblastoma.

Altered genomic methylcytosine content has been described for a number of tumor types, including neuroblastoma. However, it remains to be determined for different tumor types whether specific loci or chromosomal regions are affected by a methylation change or whether the change is random. We have implemented a computer-based approach for the analysis of two-dimensional separations of human genomic restriction fragments. Through the use of methylation-sensitive restriction enzymes, methylation differences in genomic DNA between tumor and normal tissues can be detected. We report the cloning and sequencing of two fragments detectable in two-dimensional separations of genomic DNA of neuroblastomas. These fragments were found to be a part of repetitive units that exhibited demethylation in neuroblastoma relative to other tumor types. Our finding of a distinct pattern of methylation of repetitive units in neuroblastoma suggests that altered methylation at certain loci may contribute to the biology of this tumor.

Base Sequence↗
PubMed1995

Proliferating cell nuclear antigen expression in sheep infected with bovine leukemia virus.

Proliferating cell nuclear antigen (PCNA) or cyclin (C), a major nuclear protein, has been shown to be associated with human leukemia and malignancies. PCNA protein was quantitated in this study, in lymphocytes from bovine leukemia virus (BLV) infected and non-infected sheep, using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and silver staining. The PCNA mean levels in lymphocytes of BLV-infected sheep (27 months post-infection) was significantly (P < 0.001) higher than in the lymphocytes of the non-infected sheep. The mean of PCNA levels in lymphocytes of sheep, 21 days after BLV infection, showed a two-fold increase compared with the non-infected sheep. Phytohemagglutinin (PHA) (3 days) treatment of lymphocytes from the non-infected and from the BLV-infected sheep resulted in a significant (P < 0.01) increase in the mean of PCNA levels only in the non-infected sheep. The mean lymphocyte counts of the BLV-infected sheep were not significantly different from the mean counts of the non-infected sheep at the time of lymphocyte protein analysis. Thus, these findings showed, similar to human leukemia and malignancies, that high levels of PCNA were found in lymphocytes from BLV-infected sheep compared with those from the non-infected sheep, and this was independent of high cell count. Our results also suggest that PCNA protein may play a role in the process of lymphoid transformation as a result of BLV infection of sheep.

Amino Acid Sequence↗
PubMed1992

Differential expression of Op18 phosphoprotein during human thymocyte maturation.

Op18 (also termed prosolin/stathmin) is a highly conserved 18-kD cytosolic phosphoprotein expressed in low levels in mature resting G0 lymphocytes, but induced in late G1 and S phases after entry into the cell cycle. In addition to its induction in normal proliferating lymphocytes, Op18 has been found to occur at high levels in acute leukemias and in neuroendocrine tissue. The presence and rapid phosphorylation of Op18 after stimulation of proliferating cells correlates with subsequent functional responses of the cells, and, therefore, Op18 has been suggested to play a key role in signal transduction. The pattern of expression of Op18 during lymphoid development is of interest in view of its high levels of expression in acute leukemias, representing cells arrested at an immature stage, thus raising the possibility that Op18 may be regulated differently in mature and immature lymphoid cells. We report here that immature human thymocytes bearing the cortical double positive phenotype (CD4+CD8+) constitutively express high levels of Op18 protein. In contrast, in mature single positive thymocytes (CD3+CD4+ or CD3+CD8+), Op18 protein is expressed at a lower level, comparable to that seen in peripheral blood T cells. Cell cycle analysis demonstrated that most of the cells in the double positive thymocyte population expressing high levels of Op18 were noncycling and arrested in G0. Furthermore, there was no correlation between Op18 levels and the proportion of cycling cells in double positive thymocyte populations isolated from different thymuses. Interestingly, although Op18 protein levels did not increase any further after mitogenic stimulation of double positive thymocytes, an increase in Op18 phosphorylation was observed, thus coupling of Op18 phosphorylation to cell activation remained intact. Our results show that during lymphoid maturation Op18 expression is uncoupled from cell proliferation. These data also suggest that the ordered expression of proliferation-associated genes seen in mature T cells may be disrupted during T cell maturation.

Animals↗
PubMed1991

Expression of a novel nuclear protein in activated and in tat-I expressing T cells.

The intracellular events that occur in T lymphoid cells after activation or after infection with HIV-1 are not well defined. In the case of HIV-1 infection, it is unknown whether the tat-I gene, an essential gene for viral replication, affects host cell nuclear factors. Using two-dimensional PAGE, we have identified a novel nuclear protein, designated nuclear protein-28,000 (NP-28), which is induced in Jurkat T cells by stimulation with PMA and/or PHA or ionomycin. This nuclear protein has an apparent molecular mass of 28,000 Da and an isoelectric point of 4.6. Interestingly, Jurkat cells transfected with tat-I express higher levels of NP-28 constitutively, without added stimulation. Incubation of Jurkat cells expressing tat-I with PMA and/or PHA or ionomycin causes superinduction of NP-28. We have therefore identified a novel lymphoid nuclear protein induced by T cell activation that occurs in tat-I expressing cells in the absence of activating agents.

Genes, tat↗
PubMed1990

Evidence for impaired T cell DNA methylation in systemic lupus erythematosus and rheumatoid arthritis.

Procainamide and hydralazine inhibit T cell DNA methylation and induce autoreactivity in cloned CD4+ T cells. These drugs also induce an autoimmune syndrome, suggesting a possible relationship between DNA hypomethylation, T cell autoreactivity, and certain autoimmune diseases. To test this relationship, DNA methylation was studied in T cells from patients with rheumatoid arthritis and patients with systemic lupus erythematosus, and was found to be impaired. These results support a relationship between DNA hypomethylation and some forms of autoimmune disease.

Adult↗
PubMed1990

N-myc gene amplification in neuroblastoma is associated with altered phosphorylation of a proliferation related polypeptide (Op18).

We have recently identified and cloned the gene for a cytosolic polypeptide, designated oncoprotein 18 (Op18), which is expressed in acute lymphocytic leukemia and some solid tumors including neuroblastoma. Op18 is phosphorylated upon treatment of lymphoid cells with phorbol myristate acetate. We have proposed that unphosphorylated Op18 plays a role in cellular proliferation, and that its phosphorylated forms, namely Op18a and Op18b, are associated with diminished cell proliferation. In this study, we report that in neuroblastoma tumors, the phosphorylation of Op18 was substantially diminished with increasing N-myc gene copy number. Treatment of the neuroblastoma cell line SMS-KCNR, which contains 75 copies of the N-myc gene, with retinoic acid for ten days resulted in an increase in Op18 phosphorylation. Our findings provide evidence for distinct patterns of Op18 phosphorylation in neuroblastoma tumors with and without N-myc gene amplification.

Cell Division↗
PubMed1988

Hydralazine and procainamide inhibit T cell DNA methylation and induce autoreactivity.

Inhibitors of DNA methylation, such as 5-azacytidine, induce gene expression. We have previously reported that cloned T cells treated with 5-azacytidine lose the requirement for Ag and can be activated by autologous HLA-D molecules alone, thus becoming auto-reactive. This phenomenon could potentially mediate an autoimmune disease in vivo. Inasmuch as several drugs are known to cause autoimmune disease, we asked whether they exert the same effects on T cells as 5-azacytidine. We report that hydralazine and procainamide, two drugs associated with a lupus-like autoimmune disease, also inhibit DNA methylation and induce self-reactivity in cloned T cell lines. These results suggest that drug-induced autoimmune disease may be due to activation of as yet unidentified genes through mechanisms involving DNA methylation.

Autoimmune Diseases↗
PubMed1988

N-acetylprocainamide is a less potent inducer of T cell autoreactivity than procainamide.

We have reported that an inhibitor of DNA methylation, 5-azacytidine, makes cloned, antigen-specific CD4+ T cells autoreactive, and that procainamide and hydralazine mimic this effect. Those results suggested that procainamide and hydralazine may induce autoimmunity by inhibiting DNA methylation and causing T cell autoreactivity. We report now that N-acetylprocainamide, a procainamide derivative that does not induce lupus, is also a DNA methylation inhibitor, but it is 100 times less potent than procainamide in inducing T cell autoreactivity.

Acecainide↗
PubMed1983

Separation of transferrin types in human plasma by anion-exchange high-performance liquid chromatography.

An anion-exchange high-performance liquid chromatographic procedure was developed for the separation of transferrin in human plasma. The procedure allowed the four molecular forms of transferrin, which differ with respect to bound iron, to be separated from each other and from other plasma proteins. Transferrin variants, including B and D types, could also be identified using anion-exchange high-performance liquid chromatography. The approach followed for optimizing the separation of transferrin included identification of the peaks in the chromatogram by two-dimensional polyacrylamide gel electrophoresis. This approach could be extended to other proteins in plasma or biological fluids in order to optimize their separation.

Blood Proteins↗
PubMedDate not supplied

Role of the ras-MAPK signaling pathway in the DNA methyltransferase response to DNA hypomethylation.

Our group reported that inhibiting DNA methylation in human T cells increases DNA methyltransferase expression and activity, and suggested that this may represent a response to DNA hypomethylation. The increase correlates with increases in Ha-ras and c-jun, suggesting that increased signaling through the ras-MAPK pathway, due to overexpression of some elements, may be responsible. However, whether human DNA MTase is regulated by the ras-MAPK pathway, and whether overexpression of elements in this pathway will increase DNA MTase, is unknown. We report that treating cells with a DNA methylation inhibitor increases transcription regulated by a putative DNA MTase promoter, and that this increase requires AP-1 sites. Additional studies demonstrate that overexpression of an unmutated Ha-ras causes an increase in DNA MTase, and that human T cell DNA MTase can be decreased by inhibiting signaling through the ras-MAPK pathway. Together, these studies suggest that human T cell DNA MTase is regulated through the ras-MAPK pathway, and that overexpression of Ha-ras is sufficient to increase DNA MTase expression. These results thus provide a mechanism for the increase in DNA MTase observed after inducing DNA hypomethylation, a response which may have relevance to some disease states.

Animals↗
PubMedDate not supplied

Frequency of polymorphisms for alleles encoding for liver proteins of domesticated mice.

Three different inbred strains of mice have been crossed with a lethal albino line (cch/c3H) and the liver polypeptides of the parents and offspring examined by two-dimensional polyacrylamide gel electrophoresis for evidences of protein polymorphisms, different alleles of which have gone to fixation in different strains. In the battery of polypeptides considered most favorable for scoring, 3.3 +/- 1.6 percent of the battery exhibited paired variants and 1.6 +/- 1.2 percent, unpaired. An adjustment for the fact the same allele of a biallelic polymorphism may go to fixation in two inbred lines of common ancestry leads to the suggestion that in the stock from which these inbred lines were ultimately derived, there were some 11.0 percent paired and 5.3 percent unpaired polymorphisms in the average mouse. This is about half the frequency of polymorphisms observed in wild European Mus musculus musculus and Mus musculus domesticus with one-dimensional electrophoresis of blood plasma and erythrocyte proteins. Three explanations were considered for the lower estimated frequency for liver protein polymorphisms: the difference is real, the apparent difference is due to the lower resolving power of two-dimensional gels, or the mouse strains from which the present inbred lines were drawn had already, lost through inbreeding, a considerable amount of their genetic variation before the inbreeding leading to the present strains commenced.

Alleles↗