Concentration of progenitor cells collected from bone marrow fluid using a continuous flow cell separator.

In ABO major incompatibility on bone marrow transplantation (BMT), red cells must be removed from collected marrow fluid to prevent hemolysis. We report the concentration of progenitor cells collected using a continuous flow cell separator (Cobe Spectra). The average volume of concentrated bone marrow was 132 +/- 47 ml and that of red cells included was 5.1 +/- 2.4 ml. The red cell removal rate was 97.6%. The recovery rate was 40.6% for total nuclear cells, 77.9% for mononuclear cells, 100% for CD34+ cells, and 93.9% for colony forming unit granulocyte-macrophage. Eighteen patients undergoing allogeneic BMT showed no signs of fever or hemolysis during concentrated marrow fluid transfusion. The recovery rate of progenitor cells was high, indicating sufficient recovery of hemopoiesis. This technique is applicable in ABO-incompatible BMT and in frozen-storage stem cells.

Successful graft-versus-leukemia effect of second bone marrow transplantation on relapsed leukemia cutis that was refractory to intensive chemotherapy and donor lymphocyte transfusions in a patient with acute monocytic leukemia.

We report a patient with acute monocytic leukemia (AMoL; M5) who received a second bone marrow transplantation (BMT) with graft-versus-leukemia (GVL) effect on relapsed leukemia cutis, which had been refractory to intensive chemotherapy and donor lymphocyte transfusions (DLTs). A 21-year-old woman was diagnosed with AMoL and achieved complete remission after intensive chemotherapy. The patient received a nonmanipulated allogeneic BMT from her HLA-identical father. Skin tumors developed in her upper extremities, chest, and thigh 11 months after BMT. Leukemia cutis was confirmed by skin biopsy. There was no evidence of relapse in bone marrow. The patient received several courses of chemotherapy and DLTs for the skin relapse, but the skin tumors persisted. The patient then received a second BMT from the same donor. On day 80, grade II acute graft-versus-host disease developed, and the remaining skin tumors were eradicated on day 98, most probably because of GVL effect.

Characterization of imidazole as a DNA denaturant by using TGGE of PCR products from a random pool of DNA.

Perpendicular temperature gradient gel electrophoresis (TGGE) profiles were analyzed for PCR products from a random pool of DNA [60 nts random region flanked by two primer (20 nts) sites]. Besides a normal transition profile of a homoduplex, unique mobility transition profiles of two kinds of heteroduplex with a big internal loop were observed, representing the successive helix-coil transitions of the DNAs. As the appearance of the heteroduplex band is an estimator of the complexity of a random pool, it will be applicable to monitor the extent of the selection process in the in vitro selection method. When imidazole was added to the electrophoretic buffer, the transition pattern shifted to the low temperature side. At a concentration of 1 M, imidazole lowered the melting temperature (Tm) of DNA by 13+/-2 degrees C for all the three chain separation transitions observed. Thus imidazole is a stronger denaturant than urea, at least at dilute concentration. Dependence of Tm on concentration of imidazole and the mobility change suggested that imidazole binds to nucleotide in the single-stranded state.

Philadelphia chromosome-negative cells with trisomy 8 after busulfan and interferon treatment of Ph1-positive chronic myelogenous leukemia.

A 48-year-old Japanese man with Philadelphia chromosome (Ph1)-positive chronic myelogenous leukemia (CML) was treated with busulfan followed by interferon-alpha (IFN-alpha). Ten months after IFN-alpha treatment, Ph1(-) cells with trisomy 8 were detected by the conventional banding technique and fluorescence in situ hybridization (FISH) analysis. Add(Y)(q12) was also found in Ph1(-) cells with trisomy 8. Although Ph1(+) cells disappeared after the treatment with IFN-alpha, Ph1(-) cells with trisomy 8 did not. We summarize four previous case reports of Ph1(+) CML developing Ph1(-) cells with trisomy 8. All four patients had received busulfan and IFN-alpha. These drugs may be related to the ontogenesis of Ph1(-) cells with trisomy 8, but the significance of Ph1(-) cells with trisomy 8 is not known, and further observation is needed.

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. The normal counterpart fused to the MLL locus was proved to be derived from chromosome 22q11(AF-22) by somatic cell hybrids analysis and FISH. By FISH, AF-22 was localized to the region more centromeric to the BCR gene.

Deletion mapping on chromosome 1p in well-differentiated gastric cancer.

To define the region on the short arm of chromosome 1 that is thought to include one or more tumour-suppressor genes for gastric cancers, we carried out loss of heterozygosity (LOH) studies in 26 gastric adenocarcinomas, using three restriction fragment length polymorphism (RFLP) markers and nine microsatellite markers. All tumours were informative with at least one locus; three revealed replication errors (RERs) at multiple microsatellite loci, and interstitial or telomeric allelic deletions were observed in 12 cases. Deletion mapping of these tumours defined a commonly deleted region between two loci, D1S201 and D1S197, that are 13 cM apart. As two loci within the commonly deleted region, D1S57 (pYNZ2) and D1S62 (pTHI54), were mapped respectively to 1p35 and 1p34.3 by fluorescence in situ hybridisation, we conclude that a locus likely to contain a tumour-suppressor gene for gastric cancer is located within a 13 cM region encompassing two chromosomal bands.

PTPN13, a fas-associated protein tyrosine phosphatase, is located on the long arm of chromosome 4 at band q21.3.

PTPN13 is a protein tyrosine phosphatase that associates with the C-terminal negative regulatory domain in the Fas (APO-1/CD95) receptor. The PTPN13 protein contains six GLGF repeats that have been found in the rat postsynaptic density protein (PSD-95) and the Drosophila tumor suppressor protein, lethal-(1)-disc-large-1 (dlg-1). The localization of the PTPN13 gene to human chromosome 4q21.3 was determined by both FISH and PCR analysis of somatic cell hybrids. This 4q21.3 chromosomal region contains a gene for autosomal dominant polycystic kidney disease as well as the region frequently deleted in liver and ovarian cancers, suggesting that PTPN13 is a candidate for one of the putative tumor suppressor genes on the long arm of chromosome 4.

Chromosomal loci of 50 human keratinocyte cDNAs assigned by fluorescence in situ hybridization.

The chromosomal loci of expressed genes provide useful information for a candidate gene approach to the genes responsible for genetic diseases. A large set of randomly isolated cDNAs catalogued by partial sequencing can serve as a resource for accessing and isolating these disease genes. Using fluorescence in situ hybridization, we examined the chromosomal loci of 217 human keratinocyte-derived cDNAs, with independent novel sequence tags at the 3' end region. Among them, we determined the loci of 50 cDNAs. Single-pass sequencing of these from the 5' ends indicated that 39 cDNAs still can be produced for new genes. These cDNAs with identified chromosomal loci are powerful tools that can be used to help elucidate the genes responsible for hereditary skin disorders.

Clonal origin of Philadelphia chromosome negative cells with trisomy 8 appearing during the course of alpha-interferon therapy for Ph positive chronic myelocytic leukemia.

The transient appearance of a Philadelphia chromosome (Ph) negative clone with trisomy 8 was found in the bone marrow cells from a patient with Ph positive CML during the course of alpha-interferon (IFN) therapy. To determine whether this clone was derived from a Ph positive clone or from some other cell lineage, we performed molecular cytogenetic studies on bone marrow cells from the patient by fluorescence in situ hybridization (FISH). No fusion of BCR-ABL could be detected in cells with trisomy 8, clearly indicating that the Ph negative trisomy 8 clone was not derived from the Ph positive CML population.

cDNA cloning and chromosomal localization of the human ciliary neurotrophic factor gene.

Full-length cDNA for human ciliary neurotrophic factor (CNTF) was isolated from a human sciatic nerve cDNA library. Sequence analysis revealed that the longest cDNA was comprised of a 48-bp 5'-untranslated region, a 600-bp coding region and a 1207-bp 3'-untranslated region containing four ATTTA pentamer motifs and a polyadenylation sequence. The transcription starting point was assigned at 81 bp upstream of the initiation methionine by 5' RACE analysis. Using the cDNA and genomic DNA fragment including the entire intron region as mixed probes, the human CNTF gene was localized to the long arm of chromosome 11 at region q12 by fluorescence in situ hybridization.

High-resolution cytogenetic mapping of the short arm of chromosome 1 with newly isolated 411 cosmid markers by fluorescence in situ hybridization: the precise order of 18 markers on 1p36.1 on prophase chromosomes and "stretched" DNAs.

A high-resolution cytogenetic map of the short arm of chromosome 1 with newly isolated 411 cosmid markers was constructed by fluorescence in situ hybridization (FISH). These markers were scattered throughout chromosome 1p, but they were preferentially concentrated on R-band dominant regions such as 1p36, 1p34, 1p32, 1p22, and 1p13. Among these markers, 197 were localized on chromosome band 1p36, a region frequently deleted in neuroblastoma. Of these, 18 were precisely ordered on 1p36.1 by multicolor FISH of prophase chromosomes and "stretched" DNAs as follows: 1pter-163-41-11-1-226-586-568-614-631-665-451-199-190-561-241-74-1 76-652-1cen. The high-density map of chromosome 1p constructed here can provide useful landmarks for constructing a contig map of the short arm of chromosome 1 with YACs and cosmid clones and will expedite the identification of breakpoints and/or tumor suppressor gene(s) associated with several types of malignant tumors that frequently exhibit chromosomal aberrations or deletions of chromosome 1p.

Precise ordering of 26 cosmid markers on chromosome region 3p23-->p21.3 by two-color FISH on human prophase chromosomes and stretched DNAs.

To construct a detailed cytogenetic map of human chromosome region 3p23-->p21.3, we determined the order of 26 cosmid markers (cCI 3 series) previously localized within this region by fluorescence in situ hybridization (FISH). Two-color pairwise FISH analysis of prophase chromosomes provided the order of these markers as follows: pter - 245 (D3S647) - 872 (D3S1018) - 818 (D3S996) - 905 (D3S1022) - 515 (D3S685) - 1195 (D3S1125) - 718 (D3S935) - 911 (D3S1025) - 878 (D3S1020) - 717 (D3S934) - 401 (D3S664) - [708 (D3S926)/524 (D3S686)] - 848 (D3S1011) - 771 (D3S966) - 917 (D3S1029) - 533 (D3S688) - 470 (D3S676) - 940 (D3S1037) - 785 (D3S974) - 810 (D3S988) - 9 (D3S643) - 382 (D3S660) - 769 (D3S965) - 792 (D3S978) - 604 (D3S705) - cen. The two-color signals of 524 (D3S686) and 708 (D3S926) were visualized as an overlapping pattern on prophase chromosomes, and, further, the string signals also overlapped on stretched DNAs, allowing us to determine their precise order as pter - D3S926 - D3S686 - cen. The precise order of 26 DNA markers on 3p23-->p21.3 can provide useful information for the positional cloning of tumor suppressor gene(s) and cancer breakpoint(s) encompassed in this region.

Assignment of the human protein tyrosine phosphatase, receptor-type, zeta (PTPRZ) gene to chromosome band 7q31.3.

Human protein tyrosine phosphatase, receptor-type, zeta (PTPRZ; also denoted HPTP zeta or RPTP beta) has a large extracellular region with the N-terminal carbonic anhydrase-like domain and a cytoplasmic region with two tandemly located protein tyrosine phosphatase domains. One of the characteristics of PTPRZ is its preferential expression in the central nervous system. We localized the human PTPRZ gene to chromosome band 7q31.3 by somatic cell hybrid mapping and fluorescence in situ hybridization.

Human arylhydrocarbon receptor: functional expression and chromosomal assignment to 7p21.

We isolated the human arylhydrocarbon receptor (AhR) cDNA from a human lung cDNA library, by using mouse AhR cDNA as a labeled probe. The nucleotide sequence of cloned human AhR cDNA is identical to the previously reported human AhR sequence [Dolwick et al. (1993), Mol. Pharmacol. 44, 911-917] from cell line HepG2. The overall amino acid identity with mouse AhR from cell line Hepa-1 is 72.5%. The human AhR expressed either in COS-7 cells or in a reticulocyte lysate in vitro translation system showed specific dioxin-binding activity and Arnt-dependent DNA-binding activity. Chromosomal localization of the AhR gene was determined to be chromosome 7p21 by fluorescent in situ hybridization and DNA blot hybridization using 23 human x mouse or Chinese hamster hybrid cell DNAs.

Molecular cloning and analysis of the human Tec protein-tyrosine kinase.

Mouse Tec is a non-receptor type protein-tyrosine kinase and is highly expressed in many hematopoietic cell lines. To investigate the roles of the Tec kinase in the human hematopoietic system, we isolated cDNAs encoding the human Tec kinase. The human tec cDNAs can encode a peptide of 631 amino acid residues with a calculated molecular mass of 73,624. The predicted human Tec protein is highly homologous to those of the members of the Tec family including mouse Tec type IV (94% homology), mouse Tsk/Itk (60%), and human Btk (57%). The homology between human Tec and other members of the Tec family can be observed not only in the Src homology 3 (SH3), SH2, and kinase domains, but also in the N-terminal unique domain. Northern blot analysis demonstrated that the major transcripts of tec could be detected at 2.6 kb and 3.6 kb in a wide range of human hematopoietic cell lines including myeloid, B-, and T-cell lineages. Interestingly, high expression of the tec gene could be detected in all of the three patients examined with myelodysplastic syndrome. The human tec gene was mapped by fluorescence in situ hybridization (FISH) to chromosome 4p12.

Molecular cloning of a novel non-receptor tyrosine kinase, HYL (hematopoietic consensus tyrosine-lacking kinase).

We identified a novel non-receptor tyrosine kinase from a human megakaryoblastic cell line, UT-7, by means of a PCR-based cloning method. The HYL gene contained a SH2 and SH3 domain and a tyrosine kinase catalytic domain. The deduced amino acid sequence of the protein encoded by this gene was most homologous to CSK (c-src kinase). This gene and CSK shared some unique structural properties such as the absence of a myristylation signal and phosphorylation sites of tyrosine residues corresponding to tyrosines 416 and 527 of chicken p60c-src. Unlike CSK, the SH3 domain of HYL was unique since the ALYDY motif was absent. Northern blot analysis revealed a 2.2 kb transcript in various myeloid cell lines but not in adult tissues except for the brain and the lung, whereas CSK mRNA was ubiquitously expressed. The expression of HYL was upregulated when these myeloid cells were differentiated by induction with phorbol myristate acetate. We named this gene, hematopoietic consensus tyrosine-lacking kinase, HYL. The HYL gene was assigned to chromosome 19 at band p13. It is suggested that HYL plays a significant role in the signal transduction of hematopoietic cells.

Chromosomal localization of the protein tyrosine phosphatase G1 gene and characterization of the aberrant transcripts in human colon cancer cells.

We have recently described the isolation of the human PTPG1 gene which encodes a member of intracellular protein tyrosine phosphatases that may be candidates for tumor suppressor genes. In order to investigate the abnormality of the PTPG1 transcript in various human cancer cell lines, we have analyzed the consensus catalytic region of PTPG1 cDNA, using the reverse transcription polymerase chain reaction. In a colorectal carcinoma cell line, DLD-1, we found three aberrant transcripts. Sequencing analysis revealed that one had a missense point mutation and the remainders contained 77 bp and 173 bp deletions, respectively. These alterations might directly affect their phosphatase activities. Our findings provide the first evidence for the aberrant transcripts of the protein tyrosine phosphatase in human cancer cells, and suggest that the aberration of PTPG1 gene might be involved in the tumorigenesis. Moreover, the human PTPG1 gene is localized on chromosome 7q11.23, a region with frequent abnormalities implicated in some human cancers.

Molecular cloning of a human transmembrane-type protein tyrosine phosphatase and its expression in gastrointestinal cancers.

To determine the expression of various protein-tyrosine phosphatases (PTPs) in human gastric cancers, cDNAs encoding conserved PTP domains were amplified by reverse transcriptase polymerase chain reaction from KATO-III cell mRNA and sequenced. Among 72 polymerase chain reaction clones, one of the cDNA sequences encoded a novel potential PTP (stomach cancer-associated PTP, SAP-1). The full length (3.9 kilobases) of the SAP-1 cDNA was further isolated from the KATO-III cell cDNA library and the WiDr cell cDNA library. The predicted amino acid sequence of the SAP-1 cDNA showed that mature SAP-1 consisted of 1093 amino acids and a transmembrane-type PTP, which possessed a single PTP-conserved domain in the cytoplasmic region. The extracellular region of SAP-1 consisted of eight fibronectin type III-like structure repeats and contained multiple N-glycosylation sites. These data suggest that SAP-1 is structurally similar to HPTP beta and that SAP-1 and HPTP beta represent a subfamily of transmembrane-type PTPs. SAP-1 was mainly expressed in brain and liver and at a lower level in heart and stomach as a 4.2-kilobase mRNA, but it was not detected in pancreas or colon. In contrast, among cancer cell lines tested, SAP-1 was highly expressed in pancreatic and colorectal cancer cells. The bacterially expressed SAP-1 fusion protein had tyrosine-specific phosphatase activity. Immunoblotting with anti-SAP-1 antibody showed that SAP-1 is a 200-kDa protein. In addition, transient transfection of SAP-1 cDNA to COS cells resulted in the predominant expression of a 200-kDa protein recognized by anti-SAP-1 antibody. SAP-1 is mapped to chromosome 19 region q13.4 and might be related to carcinoembryonic antigen mapped to 19q13.2.