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Biomedical subjects

H G Drexler

Publications and source records attributed to H G Drexler.

At least 19 recordsLinked to original sources
PubMed1995

The protein kinase C activator Bryostatin-1 induces the rapid release of TNF alpha from MONO-MAC-6 cells.

Bryostatin-1 is a natural activator of protein kinase C and currently examined in phase I trials as anticancer agent. We found that Bryostatin-1 induced tumor necrosis factor alpha (TNF alpha) expression in the human cell line MONO-MAC-6. Using Northern blot analysis and a bioassay for the detection of the cytokine we observed that Bryo alone was sufficient to transiently induce mRNA synthesis and the rapid release of TNF alpha into the culture medium. However, the combination of Bryo with lipopolysaccharide resulted in a strong synergistic increase of TNF alpha secretion. The biologic activity of the secreted TNF alpha was amenable to inhibition by anti-TNF alpha antibodies. Blockade of the lipopolysaccharide receptor CD14 or inhibition of protein kinase C implied that both, CD14 and protein kinase C, are involved individually in signal transduction pathways leading to TNF alpha secretion from MONO-MAC-6 cells. The results demonstrate that Bryostatin-1 is able to induce TNF alpha secretion in human monocytes via a protein kinase C-dependent and CD14-independent pathway and by a mechanism which is most likely based on a strong increase of the TNF alpha mRNA level.

Antibodies, Blocking↗
PubMed1995

Isoenzyme analysis as a rapid method for the examination of the species identity of cell cultures.

One of the major problems in cell culturing is the misidentification or cross-contamination of authentic continuous cell lines. We applied a rapid and efficient isoelectric focusing (IEF) technique for the routine analysis to detect interspecies contamination of cell cultures and for the identification of unknown animal cell lines. The method is based on the isoelectric separation of a specific set of intracellular enzymes which can be used to distinguish between cell lines of human, murine, or other mammalian origin. By means of preformed agarose gels, standardized conditions and equipment, this technique is especially applicable for routine work and allows the analysis of a large number of unknown samples with reproducible results. One hundred seventy-seven cell lines which have been sent to the Department of Human and Animal Cell Cultures at the DSM (Deutsche Sammlung von Mikroorganismen and Zellkulturen) were analyzed for species authentication; only three cell lines were found not to be of the presumed species. Our study strongly emphasizes standardized IEF as an efficient and rapid method for routinely monitoring the authenticity of cell lines.

Animals↗
PubMed1995

Leukemia cell lines: in vitro models for the study of acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) serves as a paradigm in clinical and biological leukemia research. Firstly, APL represents a model for the new therapeutic approach of differentiation therapy, taking advantage of the ability of APL cells to respond to retinoic acid treatment by terminal differentiation. Secondly, the 15;17 chromosomal translocation specific for APL leads at the molecular genetic level to a chimeric gene fusing the PML and RAR alpha genes and appears to be an instrumental, if not actually the causative event, in the neoplastic process. These unique characteristics of an otherwise rather rare disease have recently attracted intense research interest. As in other types of leukemia where continuous cell lines are powerful research tools, studies using APL-derived cell lines have contributed a large body of relevant data in efforts to unravel the pathobiology and leukemogenesis of APL. Three cell lines have been reported to be derived from patients with APL: HL-60, NB-4 and PL-21. Both HL-60 and PL-21 lack t(15;17) while NB-4 carries this cytogenetic hallmark pathognomonic for APL. Morphological and immunophenotypical examinations of the cell lines do not permit a clear assignment to any stage of myelomonocytic differentiation. Some additional data, such as expression of myeloperoxidase, monocyte-specific esterase and annexin VIII, together with the cytogenetic and molecular biological information, suggest that NB-4 is the only genuine promyelocytic leukemia cell line, whereas HL-60 may represent a discrete stage of differentiation between the late myeloblasts and the promyelocyte; PL-21 has distinct features associated with monocytic cells. These cell lines provide unique in vitro model systems for studying the cellular and molecular events involved in the proliferation and differentiation of normal and leukemic myelomonocytic cells.

Cell Differentiation↗
PubMed1995

Expression of bcl-2 mRNA and protein in leukemia-lymphoma cell lines.

The expression of the proto-oncogene bcl-2 was examined in a panel of 75 continuous human leukemia-lymphoma cell lines originated from different hematopoietic cell types. The presence of the bcl-2 protein, as evidenced by Western blotting, and its mRNA, as determined by Northern blotting, were not restricted to cells with the chromosomal translocation t(14;18)(q32;q21), but were also detected in a large number of cell lines without t(14;18). The amount of the bcl-2 protein and mRNA in the cell lines with t(14;18) was in the same order of magnitude as in other bcl-2 expressing cell lines of the same lineage, but without the translocation. Bcl-2 was found in all types of hematopoietic cell lines which were assigned to the following lineages based on their phenotypical characteristics: pre-B, B, plasma, T, myeloid, monocytic, erythroid-megakaryocytic and Hodgkin's lymphoma derived cell lines. The levels of accumulated mRNA and protein corresponded fairly well in most of the cell lines examined. Our results suggest the notion that bcl-2 expression is widely present in hematopoietic cell lines without restriction to single lineages and, in fact, clearly independent of the chromosomal aberration t(14;18). It is conceivable that bcl-2 expression is a common feature in established hematopoietic cell lines and may contribute to their unlimited growth in vitro.

Blotting, Northern↗
PubMed1995

IL-4R alpha and gamma chain expression in LPS- and IL-4-stimulated MONO-MAC-6 cells.

Short-term stimulation of peripheral blood monocytes (PBMo) and cells of the monocytic cell line MONO-MAC-6 with lipopoly-saccharide (LPS) induces high tumor necrosis factor (TNF)alpha mRNA levels. In contrast to the results obtained with primary cells, this effect could not be inhibited by preincubating the cell line with recombinant human interleukin-4 (rh IL-4). This deficiency in response to the cytokine was not caused by a general unresponsiveness of MONO-MAC-6 cells to IL-4. Thus, the expression of the monocyte-associated differentiation markers CD14 and monocyte-specific esterase (MSE), upregulated by long-term stimulation with LPS, could be decreased by IL-4. Long-term LPS treatment apparently induced IL-4 responsiveness of the cell line. While IL-4R alpha mRNA was upregulated about 3-fold, this positive effect was not apparent at the cell surface protein level. In contrast to the constitutive alpha chain expression, the IL-4R gamma chain expression could not be detected with a specific mAb nor by Northern blot analysis. However, reverse transcriptase polymerase chain reaction (RT-PCR) demonstrated the presence of low-level IL-4R gamma chain mRNA in the cell line. We suggest that the low reactivity of the cells to IL-4 might be correlated with the low expression of the gamma chain.

Antibodies, Monoclonal↗
PubMed1995

Expression of FLT3 receptor and FLT3-ligand in human leukemia-lymphoma cell lines.

The FLT3 gene encodes a receptor tyrosine kinase that is closely related to two well-known receptors, KIT and FMS, that regulate with their respective ligands, stem cell factor (SCF) and macrophage colony-stimulating factor (M-CSF), proliferation and differentiation of hematopoietic cells. The ligand for FLT3, FL, is active in both soluble and membrane-bound forms. We examined expression of FL and FLT3 mRNA in a panel of some 110 continuous human leukemia-lymphoma cell lines from all major hematopoietic cell lineages by Northern blot analysis. FLT3 mRNA is expressed primarily in pre-B cell lines, myeloid and monocytic cell lines whereas FL mRNA was detected in most cell lines from all cell lineages. Analysis of FLT3 receptor protein expression examined with a specific anti-FLT3 monoclonal antibody and flow cytometry in 17 cell lines confirmed the results obtained at the mRNA level. Forty of 110 cell lines displayed both receptor and ligand mRNA suggesting a possible autocrine or intracrine stimulation. In normal hematopoietic cells expression of FLT3 was reported to be associated with CD34 positivity, a cell surface marker of immature and precursor cells. No correlation between FLT3 and CD34 expression was found in the cell lines analyzed. These studies served to illustrate further the importance of the FL-FLT3 ligand-receptor system in the regulation of hematopoietic cells.

Gene Expression↗
PubMed1995

Recurrent chromosomal translocations and fusion genes in leukemia-lymphoma cell lines.

The occurrence of defined chromosomal translocations in specific subtypes of leukemia and lymphoma strongly suggests that these structural alterations play an important role in the process of tumorigenesis. Translocations may activate nearby cellular genes involved in the control of proliferation or differentiation. One mechanism by which genes become activated or overexpressed is by their juxtaposition with the genes coding for the lymphoid antigen receptors (immunoglobulins or T cell receptors). The majority of the translocated genes appear to encode transcription factors, the expression of which might become deregulated due to the influence of regulatory elements in the antigen receptor loci. A second general type of chromosomal translocation by which proto-oncogenes can be activated results in the synthesis of fusion genes. Here, chimera proteins containing portions of two different proteins are generated following exon-exon juxtaposition of two genes lying at the sites of translocation. The list of such tumor-associated fusion genes generated by specific translocations has greatly increased over the last years. A wide variety of leukemia-lymphoma cell lines have been established to act both as genetic resources and as model systems for the different disease types and stages providing an inexhaustible source of replicate material. For many translocations, DNA spanning the breakpoint was isolated by using cell lines carrying the particular abnormality. The cloning of the genes disrupted by the translocations has led to the availability of probes useful for diagnosis and monitoring of patients, eg with polymerase chain reaction (PCR) assays. Here, we review the cytogenetic and molecular data of chromosomal aberrations associated with leukemia-lymphoma and indicate a panel of human cell lines carrying such specific chromosomal abnormalities.

Cloning, Molecular↗
PubMed1994

Chromatid aberration dose responses and dispersal in human G2 lymphocytes treated with bleomycin: comparison with equivalent X-irradiation reveals formation of a novel class of heavily damaged cells.

We describe the dose responses and dispersal of chromatid (ct) aberrations in human peripheral blood lymphocytes, treated with a 5-min pulse of bleomycin (BLM) in doses ranging from 0.78 to 200 micrograms/ml during the G2 phase of the cell cycle. Damage was assessed in cells fixed at the time of peak damage 1 h after treatment. Both ct breaks and the percentages of damaged cells rose according to log BLM dose above 6.3 micrograms/ml only. Below this dose all endpoints exhibited flat responses suggestive of thresholding. A dose of 100 micrograms/ml produced similar amounts and distribution of ct breakage per cell (B/c) as a previously studied X-ray dose of 0.8 Gy, permitting future direct cytogenetic comparisons between clastogens. Within the scorable range (0-29 B/c) the dispersal of ct breakage after BLM treatment resembled that after equivalent X-irradiation; but BLM treatment alone resulted in the formation of heavily damaged cells (HDC) defined as with > or = 30 B/c, representing a cytogenetic endpoint of DNA damage reminiscent of apoptosis. At the dose producing equivalent chromatid breakage, BLM produced 7.4 times fewer exchanges than X-rays in G2.

Bleomycin↗
PubMed1994

c-kit expression in human megakaryoblastic leukemia cell lines.

A panel of 164 continuous human leukemia-lymphoma cell lines was analyzed for expression of c-kit using Northern blotting and reverse transcriptase-polymerase chain reaction (RT-PCR). The c-kit transcripts were detectable in cell lines assigned to the myeloid (in 7 of 29 by Northern blotting and in 4 of 8 by RT-PCR), monocytic (in 1 of 24 by Northern blotting and in 3 of 6 by RT-PCR), erythroid (in 6 of 8 by Northern blotting and in 5 of 5 by RT-PCR), and megakaryoblastic (in 10 of 10 by Northern blotting) lineages, c-kit expression was not seen by Northern blotting or RT-PCR analysis in any of the 93 lymphoid leukemia, myeloma, or lymphoma cell lines. Treatment of four megakaryoblastic cell lines with protein kinase C activators (phorbol ester 12-O-tetradecanoylphorbol 13-acetate and Bryostatin 1) led to terminal differentiation as assessed by morphologic alterations, changes in the surface marker profile, and growth arrest. These effects were associated with enhanced c-kit mRNA expression. Exposure to all-trans retinoic acid down-regulated c-kit mRNA levels, while simultaneously causing morphologic alterations in all four cell lines. Stimulation with growth factors (interleukin-3, granulocyte macrophage-colony stimulating factor, and insulin-like growth factors I and II), used to assess any role of c-kit in proliferative processes, did not lead to significant upregulation or downregulation of c-kit expression. The finding of constitutive and high expression of c-kit mRNA in all megakaryoblastic leukemia cell lines and its modulation by various reagents might further contribute to the understanding of megakaryopoietic proliferation, differentiation, and leukemogenesis.

Apoptosis↗
PubMed1994

Different biological effects of the two protein kinase C activators bryostatin-1 and TPA on human carcinoma cell lines.

Bryostatin 1 (Bryo) is a naturally occurring macrocyclic lactone with antineoplastic activity. Like the phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA) it directly activates the calcium- and phospholipid-dependent protein kinase C (PKC), thus generating a number of different cellular responses. We investigated the effects of Bryo and TPA on DNA synthesis, proliferation, viability and c-myc protooncogene expression of the human carcinoma cell lines COLO-320, MEL-HO, and KB-3-1. TPA inhibited [3H]-thymidine incorporation in all three cell lines in a dose-dependent manner, whereas Bryo only inhibited the DNA synthesis in MEL-HO, but not in KB-3-1 and COLO-320 cells. Within the concentration ranges used, TPA and Bryo were found to have a low toxicity. Counting of the cells confirmed the observed inhibition of cell proliferation. However, the enzymatic conversion of MTT, applied as a colorimetric proliferation assay, was not significantly affected by both biomodulators. Time-course experiments revealed a rapid onset of the inhibitory effect on DNA synthesis. Bryo was further able to antagonize the TPA-mediated effects on proliferation suggesting an (at least partially) different mode of action of these PKC activators. Incubation of MEL-HO and COLO-320 cells with either of the two biomodulators resulted in a rapid and strong increase of c-myc mRNA. The present study emphasizes Bryo as an interesting, natural substance for the study of PKC-mediated biological effects.

Adenocarcinoma↗
PubMed1994

Treatment of mycoplasma contamination in a large panel of cell cultures.

Mycoplasmal contamination remains a significant impediment to the culture of eukaryotic cells. For certain cultures, attempts to eliminate the infection are feasible alternatives to the normally recommended disposal of the contaminated culture. Here, three antibiotic regimens for mycoplasmal decontamination were compared in a large panel of naturally infected cultures: a 1-wk treatment with the fluoroquinolone mycoplasma removal agent (MRA), a 2-wk treatment with the fluoroquinolone ciprofloxacin, and three rounds of a sequential 1-wk treatment with BM-Cyclin containing tiamulin and minocyclin. These antibiotic treatments had a high efficiency of permanent cure: MRA 69%, ciprofloxacin 75%, BM-Cyclin 87%. Resistance to mycoplasma eradication was observed in some cell cultures: BM-Cyclin 0%, MRA 20%, ciprofloxacin 20%. Nearly all resistant contaminants that could be identified belonged to the species Mycoplasma arginini and M. orale. Detrimental effects of the antibiotics were seen in the form of culture death caused by cytotoxicity (in 5 to 13% of the cultures). Alterations of the cellular phenotypic features or selective clonal outgrowth might represent further untoward side effects of exposure to these antibiotics. Overall, antibiotic decontamination of mycoplasmas is an efficient, inexpensive, reliable, and simple method: 150/200 (75%) chronically and heavily contaminated cultures were cured and 50/200 (25%) cultures could not be cleansed and were either lost or remained infected. It is concluded that eukaryotic cell cultures containing mycoplasmas are amenable to antibiotic treatment and that a cure rate of three-quarters is a reasonable expectation.

Cells, Cultured↗
PubMed1994

Leukemia cell lines: in vitro models for the study of chronic myeloid leukemia.

The clinical importance of CML lies in its poor responsiveness to chemotherapy which has proved highly effective in treating ALL. The scientific importance of CML resides in its role as a cancer prototype, permitting the identification of genes centrally involved in both neoplastic change and normal cellular differentiation. One of these genes, the fusion gene BCR/ABL resulting from the balanced translocation (9;22) has received wide attention owing to its intimate involvement in CML. Although a tremendous amount of data have been recently discovered about BCR/ABL, its exact role in leukemogenesis and normal hematopoiesis remains obscure. The study of CML cell lines has already been of considerable help in understanding the molecular events associated with the Ph chromosome [4]. Further advances are likely to be forthcoming, particularly at the molecular genetic, but also at the protein level. CML cell lines may offer an excellent means of addressing many issues as continuous cell lines represent an inexhaustible source of identical cell material that, in addition, can be made available to other researchers around the world. This overview on the thus far reported CML-derived cell lines supports the hypothesis that in some specimens of CML the target cells in which Ph translocation arises are not necessarily lineage-restricted committed progenitor cells, but are in fact in some (or all?) cases precommitted bipotential or multipotential progenitor or stem cells retaining the potential for differentiation in diverse hematopoietic directions [26]. In conclusion, established tumor cell lines with their unique phenotypic and karyotypic features have been extremely useful models for investigation of the molecular and biological characteristics of CML. Considerable progress in understanding the molecular and cell biology of CML has been achieved. Further advances in the knowledge of CML are expected to accrue with the productive use of these powerful research tools for many important unresolved issues. By so doing, these discoveries might open new avenues that promise to move clinicians closer to the goal of the prevention or cure of CML in all patients.

Adult↗
PubMed1994

Effective treatment of mycoplasma contamination in cell lines with enrofloxacin (Baytril).

Continuous cell lines are frequently contaminated with microorganisms, mycoplasmas being the most prominent and cumbersome. In our experience, of the 300 cell lines examined more than one third was infected with mycoplasmas. Mycoplasma contamination can affect virtually every parameter and functional activity of a cultured cell. An alternative to the recommended disposal of infected cultures is an attempt to eliminate the contaminants. Adding antibiotics with strong activity against mycoplasmas to the culture medium is a simple, inexpensive and efficient decontamination method. Here, we studied the effectiveness of the new antibiotic enrofloxacin (Baytril) developed specifically for use against mycoplasmas. Baytril is a new synthetic agent from the group of quinolone derivatives that are DNA gyrase inhibitors. Thirty-two chronically infected cell lines (27 human leukemia-lymphoma cell lines) were treated with Baytril in a prospective study in direct comparison with three other well-established anti-mycoplasma regimens, the antibiotics BM-Cyclin, Ciprobay and MRA (Mycoplasma Removal Agent). Mycoplasmas were detected by DNA staining, agar colony growth, DNA-RNA hybridization, polymerase chain reaction, and monoclonal antibody staining. Treatment with Baytril eliminated the contaminants in 30/32 cultures (94%). The cure rates for Ciprobay, BM-Cyclin and MRA were 91%, 81%, and 75%, respectively. The IC50 values of Baytril for cell lines varied over a wide range depending on the type of hematopoietic cell lineage with T- and B-cell lines being more sensitive targets. Baytril-treated cell lines remained mycoplasma-negative over a 12-week antibiotic-free culture period. Low levels of mycoplasma infection were shown not to persist by repeat testing after growth without antibiotics. A retrospective analysis of anti-mycoplasma treatments with BM-Cyclin, Ciprobay, MRA or Baytril showed that 265/351 cultures (75%) were immediately cured of mycoplasma; however, all of the remaining, mycoplasma-positive cultures harboring mycoplasms resistant to the first antibiotic could be cleaned up by a second round with a different antibiotic. Baytril is an efficient anti-mycoplasma antibiotic and based on its high cure rate might be the treatment of first choice.

Anti-Bacterial Agents↗
PubMed1994

IL-4 inhibits the LPS-induced expression of CD14 and monocyte-specific esterase mRNA in MONO-MAC-6 cells.

The human MONO-MAC-6 cell line expresses the monocyte-associated differentiation markers CD14 and monocyte-specific esterase (MSE) and can be stimulated by lipopolysaccharide (LPS) to produce high mRNA levels of monocyte-related cytokines. This similarity to human peripheral blood monocytes (PBMo) renders this cell line a promising model for studies of monocyte activation and differentiation. Interleukin-4 (IL-4) is known to act antagonistically to LPS during the activation process of PBMo, inhibiting the production of cytokines. Therefore, this study was designed to compare the effects of IL-4 and LPS on the expression of monocytic markers and tumor necrosis factor alpha (TNF alpha) mRNA on PBMo and the MONO-MAC-6 cell line. IL-4 inhibited the LPS-induced expression of TNF alpha mRNA in PBMo and downregulated the LPS receptor CD14 but it had no influence on MONO-MAC-6 cells regarding these parameters. However, upregulation of CD14 and MSE mRNA expression in the cell line by a 2-day incubation with LPS were inhibited by IL-4. This response to IL-4 after long-term treatment with LPS was seemingly contradictory to the missing reduction of TNF alpha mRNA expression after short-term incubation with LPS. Obviously long-term treatment with LPS made the cells responsive to IL-4. The increase in responsiveness was not due to IL-4 receptor (IL-4R) upregulation, as LPS did not influence the constitutive expression of the IL-4R.

Antigens, CD↗
PubMed1994

Expression and regulation of CD30 ligand and CD30 in human leukemia-lymphoma cell lines.

The CD30 antigen was originally described as a specific surface marker for Hodgkin's lymphoma. Recent work established CD30 as a member of the tumor necrosis factor/nerve growth factor receptor superfamily whose ligand (CD30L) has also been cloned and expressed; CD30L is active as membrane-bound type II glycoprotein. Here, CD30L mRNA expression was studied in a panel of 102 continuous human leukemia-lymphoma cell lines and was found only in four Burkitt lymphoma, one Burkit-type acute lymphoblastic leukemia and one non-Hodgkin's lymphoma (NHL) cell line. The product of CD30L mRNA is expressed as a membrane protein on the surface of these malignant B-cell lines. Treatment of these cell lines with soluble CD27L, phorbol ester or staphylococcus aureus Cowan antigen resulted in the enhancement of cell surface CD30L protein expression. CD30L mRNA was not detected in normal unstimulated peripheral blood (PB) monocytes, monocyte-derived macrophages, or T-cells, but was detected in primary granulocytes; exposure to activating reagents induced and upregulated CD30L transcription in these different PB populations. While CD40 and CD30L surface protein expression on PB monocytes could be enhanced or induced by treatment with gamma-interferon, these cells remained negative for CD30, both at the mRNA and at the protein level. Similarly, PB monocyte-derived macrophages and granulocytes remained negative for CD30 mRNA and protein expression, regardless of stimulation. Only activated T-cells expressed CD30 mRNA and surface protein. CD30L-transfected cells and cells constitutively expressing CD30L delivered a similar stimulus for proliferation of the CD30+ Hodgkin's disease (HD)-derived cell line HDLM-2, but inhibited proliferation of the CD30+ large cell anaplastic lymphoma cell line KARPAS-299. These data provide strong evidence for the involvement in growth regulation of recombinant and natural CD30L through its interaction with the CD30 receptor. Collectively, these data suggest that the CD30L-CD30 interaction has potent biological activity and might play a critical role in the immune response and pathogenesis of HD and some NHL, in particular Burkitt lymphomas.

Antigens, CD↗
PubMed1994

Characterization of the monocyte-specific esterase (MSE) gene.

Carboxylic esterases are widely distributed in hematopoietic cells. Monocytes express the esterase isoenzyme (termed 'monocyte-specific esterase', MSE) that can be inhibited by NaF in the alpha-naphthyl acetate cytochemical staining. We examined the expression of MSE in normal cells and primary and cultured leukemia-lymphoma cells. The MSE protein was demonstrated by isoelectric focusing (IEF); MSE mRNA expression was investigated by Northern blotting and reverse transcriptase-polymerase chain reaction (RT-PCR). The following samples were positive for MSE protein and Northern mRNA expression: 20/24 monocytic, 4/32 myeloid, and 1/20 erythroid-megakaryocytic leukemia cell lines, but none of the 112 lymphoid leukemia or lymphoma cell lines; of the normal purified cell populations only the monocytes were positive whereas, T, B cells, and granulocytes were negative; of primary acute (myelo) monocytic leukemia cells (CD14-positive, FAB M4/M5 morphology) 14/20 were Northern mRNA and 11/14 IEF protein positive. RT-PCR revealed MSE expression in 29/49 Northern-negative lymphoid leukemia-lymphoma cell lines. The RT-PCR signals in monocytic cell lines were on average 50-fold stronger than the mostly weak trace expression in lymphoid specimens. On treatment with various biomodulators, only all-trans retinoic acid significantly upregulated MSE message and protein levels but could not induce new MSE expression in several leukemia cell lines; lipopolysaccharide and interferon-gamma increased MSE expression in normal monocytes. Analysis of DNA methylation with sensitive restriction enzymes showed no apparent regulation of gene expression by differential methylation; the MSE gene is evolutionarily conserved among mammalian species; the half-life of the human MSE transcripts was about 5-6 h. The extent of MSE expression varied greatly among different monocytic leukemia samples. However, the MSE overexpression in a significant number of specimens was not associated with gene amplification, gross structural rearrangements or point mutations within the cDNA region. Taken together, the results suggest that MSE expression is not absolutely specific for, but strongly associated with cells of the monocytic lineage; MSE is either not expressed at all or expressed at much lower levels in cells from other lineages. The biological significance, if any, of rare MSE messages in lymphoid cells detectable only by the hypersensitive RT-PCR remains unclear. Further studies on the regulation of this gene and on the physiological function of the enzyme will no doubt be informative with respect to its striking overexpression in some malignant cells and to a possible role in the pathobiology of monocytic leukemias.

Base Sequence↗
PubMed1994

Characterization and expression of tartrate-resistant acid phosphatase (TRAP) in hematopoietic cells.

Acid phosphatase (AcP, EC 3.1.3.2) is represented by a number of enzymes that can be differentiated according to structural and immunological properties, tissue distribution, subcellular location and other features; these AcP isoenzymes share similar catalytic activity toward phosphoesters in an acidic medium. Classically, AcPs have been divided into four types according to their sensitivity to tartrate and to their origin: erythrocytic, lysosomal, prostatic AcP, and an AcP enzyme that was first identified in hairy cell leukemia (HCL). This latter AcP was termed isoenzyme 5 (based on its electrophoretic mobility) or human type 5, tartrate-resistant acid phosphatase (TRAP). Differences in various physicochemical properties, lack of amino acid sequence similarity and different chromosomal locations of the respective genes showed that the four AcP isoenzymes are not related. The biochemical properties of TRAP are unique: resistance to inhibition by tartrate, but inhibition by molybdate; glycoprotein of 30-40 kDa occurring as two similar isoforms with different carbohydrate content, each composed of dissimilar subunits of 16 and 23 kDa in disulfide linkage; active at acid pH (optimum at 5-6) with basic pI (8.5-9.0); presence of an iron active site giving the purified protein a purple color. The TRAPs of different human sources (HCL spleen, osteoclastoma, Gaucher's spleen, placenta) have an 85-94% homology in their amino acid sequences. Full-length TRAP cDNAs (1.4 kb) have been cloned from human placenta and Gaucher's spleen. Variations in TRAP structure appear to result from post-translational modifications and not from the existence of a multigene family as only a single TRAP gene and a single mRNA species have been reported. This notion of a single TRAP gene is supported by the substantial sequence homology found among the various TRAPs from human tissues and from animal sources (e.g. bovine spleen and bone; rat spleen, bone and epidermis; pig uterus). The latter enzyme preparations of animal origin have been described for many years as the purple acid phosphatase (PAP). However, the high degree of sequence homology indicated that TRAP and PAP enzymes represent a single entity belonging to the class of metalloproteins. The human TRAP gene was assigned to chromosome 15 and to chromosome 19 by two groups. TRAP protein is localized in lysosomes or similar organelles and is not secreted. The serum level of TRAP was found to be increased during physiological bone growth, in Gaucher's disease, and in malignancies metastasized to bone (resulting from increased osteoclastic activity).(ABSTRACT TRUNCATED AT 400 WORDS)

Acid Phosphatase↗