Frequent altered expression of fragile histidine triad protein in human colorectal adenomas.

Fragile histidine triad (FHIT) gene is involved in deletions on the short arm of chromosome 3 in various human cancers. We found that 47% of colorectal adenomas, which is a higher frequency than that of K-ras, showed altered expression of the Fhit protein by Western blot analysis. The amount of Fhit protein was inversely correlated with the degree of dysplasia.

Involvement of CD95-independent caspase 8 activation in arsenic trioxide-induced apoptosis.

Arsenic trioxide (As2O3)-treatment is effective in acute promyelocytic leukemia (APL) patients with t(15;17). Clinically achievable concentrations of As2O3 induce apoptosis in NB4, an APL cell line, in vitro. Here, to study the mechanism of As2O3-induced apoptosis, we established an As2O3-resistant subline, NB4/As.

Arsenic-induced apoptosis in malignant cells in vitro.

Arsenic trioxide-induced apoptosis was identified by morphological change and nucleosomal DNA fragmentation in hematopoietic malignant cells and neuroblastoma cells. Arsenic trioxide directly induced apoptosis in the acute promyelocytic cell line NB4 cells at a low dose of 1 microM, whereas all-trans-retinoic acid caused the cells to differentiate and finally induced apoptosis. In addition to the involvement of caspase 3 in arsenic trioxide-induced apoptosis of NB4 cells, the activation of caspase 8 was also shown to be involved by Western blot analysis or by apoptosis inhibition assay using caspase 8 inhibitor Ac-IETD-CHO.

Molecular analysis of the rearranged genome and chimeric mRNAs caused by the t(6;11)(q27;q23) chromosome translocation involving MLL in an infant acute monocytic leukemia.

Chromosomal analysis of acute monocytic leukemia cells in a female infant revealed a t(6;11)(q27;q23) translocation. Southern blot analysis with a cDNA probe of the MLL gene at chromosome band 11q23 indicated that the breakpoint was in an 8.3-kb BamHI fragment that contained exons 5-11 of the MLL gene.

Arsenic trioxide induces apoptosis in neuroblastoma cell lines through the activation of caspase 3 in vitro.

Arsenic trioxide (As2O3) induces clinical remission in acute promyelocytic leukemia, even in all-trans retinoic acid-refractory cases, with minimal toxicity at low (1-2 microM) concentration. We exposed various neuroblastoma cell lines to As2O3 at a concentration of 2 microM: as a result, seven of 10 neuroblastoma cell lines underwent apoptosis characterized by morphological changes and nucleosomal DNA fragmentation. As2O3-induced apoptosis in neuroblastoma cells was shown to occur through the activation of caspase 3, as judged from Western blot analysis and apoptosis inhibition assay.

Apoptosis induced by an endogenous neurotoxin, N-methyl(R)salsolinol, is mediated by activation of caspase 3.

An endogenous neurotoxin, N-methyl(R)salsolinol, has been proved to be involved in the pathogenesis of Parkinson's disease. Increased level of N-methyl(R)salsolinol in the cerebrospinal fluid and high activity of its synthesizing (R)salsolinol N-methyltransferase in lymphocytes were confirmed in the majority of parkinsonian patients. Recently this neurotoxin was found to induce apoptosis in human dopaminergic neuroblastoma SH-SY5Y cells.

Overexpression of rck/p54, a DEAD box protein, in human colorectal tumours.

The RCK gene is a target of the t(11;14)(q23;q32) chromosomal translocation observed in human B-cell lymphoma, and the overexpression of its protein (rck/p54) by the translocation was shown to cause malignant transformation. The rck/p54 protein belongs to the DEAD box protein/RNA helicase family, which has a variety of functions such as translation initiation, pre-mRNA splicing and ribosome assembly. The expression of rck p54 in colorectal adenocarcinoma cells was examined by immunohistochemistry and Western blot analysis.

Expression of two dead box genes (DDX1 and DDX6) is independent of that of MYCN in human neuroblastoma cell lines.

To examine whether two DEAD box genes, DDX1 and DDX6, would have some roles in the progression of tumors, we investigated the correlation of the expression of these genes with that of MYCN in neuroblastomas either with or without MYCN amplification. The mRNA of MYCN was observed only in the cell lines with amplification of MYCN. The mRNAs of DDX1 and DDX6 were found in all the cell lines examined, but the correlation between the mRNA levels of DDX1 or DDX6 and MYCN was poor.

Induction of apoptosis in cultured colon cancer cells by transfection with human interferon beta gene.

The growth of SW480 colon cancer cells following the transfection with the human interferon beta (hIFNbeta) gene entrapped in cationic multilamellar liposomes was effectively inhibited, but not that of the cells transfected with the gene from which the secretion signal sequence of hIFNbeta had been deleted. The amount of hIFNbeta secreted in the medium from SW480 cells transfected with hIFNbeta gradually increased and became maximum 3 days after the transfection, but no hIFNbeta was detected in the medium of the cells transfected with the secretion signal-deleted hIFNbeta. These findings indicate that the growth inhibition of SW480 cells after the transfection with hIFNbeta was caused by hIFNbeta secreted from the transfected cells.

Arsenic induces apoptosis in B-cell leukaemic cell lines in vitro: activation of caspases and down-regulation of Bcl-2 protein.

We showed that arsenic inhibited the cell growth of four B-cell leukaemia cell lines of 11 various cell lines in vitro. In two of these four lines, KOCL44 and LyH7, apoptosis was identified by morphological and nucleosomal DNA fragmentation studies. Three of the four B-cell lines that were growth inhibited were acute infantile leukaemia with t(11;19)(q23;p13) translocations involving the MLL gene that encodes the transcriptional factor Drosophila trithorax.

Antisense oligodeoxyribonucleotide against the MLL-LTG19 chimeric transcript inhibits cell growth and induces apoptosis in cells of an infantile leukemia cell line carrying the t(11;19) chromosomal translocation.

To clarify the role of the multiple lineage leukemia gene-leukemia translocation gene of chromosome 19 (MLL-LTG19) protein in leukemogenesis, we synthesized antisense oligodeoxyribonucleotide (ODN) against the fused region of the MLL-LTG19 chimeric transcript and treated KOCL33 cells carrying the t(11;19) translocation with antisense ODN. The antisense ODN inhibited cell growth and induced apoptosis in KOCL33 cells but not in Daudi cells, which have no t(11;19). The levels of MLL-LTG19 mRNA and MLL-LTG19 protein in KOCL33 cells treated with antisense ODN were shown to decrease with time by reverse transcription-PCR and Western blot analysis.

Growth inhibition by overexpression of human DEAD box protein rck/p54 in cells of a guinea pig cell line.

We transfected cells of a guinea pig cell line with RCK cDNA inserted in a pIRES1neo expression vector. The overexpression of rck/p54 was confirmed by Western blot and RT-PCR analysis. In two clones expressing rck/p54, the cell growth was highly inhibited; and their anchorage-independent growth, which is an important character of malignant transformation, was not found.

Predominant expression of the src homology 2-containing tyrosine phosphatase protein SHP2 in vascular smooth muscle cells.

src homology 2 (SH2)-containing protein-tyrosine phosphatase SHP2 is known to transduce positive signals from activated receptor protein-tyrosine kinases such as platelet-derived growth factor receptor (PDGFR) beta and insulin receptor. Here, we demonstrate the physiological expression of SHP2 in rats. In northern and western blot analyses, SHP2 expressions were recognized in all tissues, but their expression levels varied significantly among tissues: it is lowest in the liver and kidney.

Molecular cloning and chromosome mapping of rat phospholipase D genes, Pld1a, Pld1b and Pld2.

We have previously obtained three partial rat phospholipase D (PLD) cDNA fragments by a reverse transcriptase-polymerase chain reaction (RT-PCR) method using degenerate primers based on two conserved amino acid sequences in PLDs of human and yeast. The entire coding regions of these genes were isolated and sequenced. The longest clone, Pld1a encodes a 1075 amino acid (aa) protein that was highly similar (89% identity) to human PLD1a, especially in four conserved regions present in other PLDs.

Involvement of ICE family proteases in apoptosis induced by reoxygenation of hypoxic hepatocytes.

Cell death due to reoxygenation after hypoxia was characterized in primary cultured hepatocytes. Fluorescence and electron microscopic analyses of reoxygenated hepatocytes revealed morphological characteristics of apoptosis, including chromatin condensation, nuclear fragmentation, and formation of apoptotic bodies. Few necrotic hepatocytes, defined by loss of plasma membrane integrity, mitochondrial swelling, and formation of large vacuoles, were observed.

Comparative genome mapping of the ataxia-telangiectasia region in mouse, rat, and Syrian hamster.

Chromosomal locations of the Atm (ataxia-telangiectasia (AT)-mutated) and Acat1 (mitochondrial acetoacetyl-CoA thiolase) genes in mouse, rat, and Syrian hamster were determined by direct R-banding FISH. Both genes were colocalized to the C-D band of mouse chromosome 9, the proximal end of q24.1 of rat chromosome 8, and qa4-qa5 of Syrian hamster chromosome 12.

Molecular cloning of the breakpoint of t(11;22) (q23;q11) chromosome translocation in an adult acute myelomonocytic leukaemia.

Southern blot analysis with a cDNA probe of MLL indicated that the breakpoint is in a BamHI 8.3 kb fragment which carries the exon 5-11 of MLL gene in DNA from an adult acute myelomonocytic leukaemia with a t(11;22) (q23;q11) translocation. The structural analysis of the rearranged MLL locus demonstrated that the breakpoint is localized between exon 8 and 9 of MLL locus.

Identification and chromosome mapping of the mouse homologue of the human gene (DDX6) that encodes a putative RNA helicase of the DEAD box protein family.

The human DDX6 gene (alias RCK) at chromosome 11 band q23 was identified through the study of the breakpoint of t(11;14)(q23;q32) translocation in a B-cell lymphoma cell line, RC-K8. DDX6 encodes a DEAD box protein/RNA helicase. Positive mouse genomic and cDNA recombinant clones were obtained by screening mouse B-cell genomic and cDNA libraries with a human DDX6 cDNA probe.

The rck/p54 candidate proto-oncogene product is a 54-kilodalton D-E-A-D box protein differentially expressed in human and mouse tissues.

Expression of the RCK gene, which is a target gene on 11q23 of the t(11;14) (q23;q32) translocation in the B-cell lymphoma cell line RC-K8, was studied by Northern and Western blot analyses. The RCK gene product is a member of the D-E-A-D box protein/RNA helicase family. With the use of Northern blot analysis, a 7.

Cyclic bcl-2 gene expression in human uterine endometrium during menstrual cycle.

To investigate an apoptotic role for bcl-2 in menses, we studied bcl-2 protein production throughout the cycle. Bcl-2 protein was observed in endometrial glandular, stromal and myometrial smooth-muscle cells. Glandular cells expressed bcl-2 at proliferative through early secretory phases but not at late secretory through menstrual phases.

Multiple subcellular localization of bcl-2: detection in nuclear outer membrane, endoplasmic reticulum membrane, and mitochondrial membranes.

The use of biochemical fractionation, immunofluorescence laser-scanning confocal microscopy, and immunoelectron microscopy with mouse anti-human bcl-2 monoclonal antibody to analyze the subcellular localization of the bcl-2 gene product revealed the protein prominently in the nuclear envelope, endoplasmic reticulum membrane, and mitochondrial membranes. Electron microscopy at high magnification more precisely localized bcl-2 to the nuclear outer membrane as confirmed by the biochemical fractionation, as well as to mitochondrial outer and, to a lesser degree, inner membrane. This multisite membrane distribution of bcl-2 suggests an important role for this protein in several different membrane compartments.

Long-range mapping of the 11q23 region involved in chromosome aberrations in human tumors by pulsed-field gel electrophoresis with a yeast artificial chromosome.

We have previously demonstrated that the RCK gene involved in t(11;14)(q23;q32) and the more centromeric MLL/ALL1 gene involved in t(4;11)(q21;q23) and t(11;19)(q23;p13) are localized on different adjacent NotI fragments by using pulsed-field gel electrophoresis (PFGE) analysis with the yeast artificial chromosome (YAC) clone yB22B2. The PFGE analysis using the YACs of YTY17 containing the prophobilinogen deaminase (PBGD), CBL2 and THY1 genes and yB22B2 allowed the following ordering of genes and breakpoints from CD3 to THY1 on 11q23: cent-CD3-ALL/MLL1-RCK-PBGD-CBL2-THY1, and the establishment of a long-range restriction map covering these genes. In addition, we showed that the FLI1 region involved in the t(11;22)(q24;q12) in Ewing's sarcoma was more telomeric region that the THY1 gene by analyzing somatic cell hybrids carrying the 11q- and/or 14q+ chromosome of the t(11;14)(q23;q32) translocation, and by PFGE analysis of the YAC clone YTY17.

Two distinct portions of LTG19/ENL at 19p13 are involved in t(11;19) leukemia.

We previously isolated cDNA clones, MLL-a and MLL-b, derived from the 11q23 breakpoint region and detected gene rearrangements with MLL-b cDNA in infantile leukemia cell lines with 11q23 abnormalities. We also showed chimeric mRNAs between MLL and genes on partner chromosomes such as 4q21 and 19p13. In the present study, we isolated overlapping MLL cDNA clones of 11 kb and demonstrated that MLL-a and MLL-b were derived from the same gene, MLL/ALL-1/HRX.