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Biomedical subjects

H G Drexler

Publications and source records attributed to H G Drexler.

At least 145 records · Page 8Linked to original sources
PubMed1994

Sensitivity of different methods for the detection of myeloperoxidase in leukemia cells.

We examined the sensitivity of different myeloperoxidase (MPO) detection methods in leukemia cell lines. To this end the MPO-positive acute promyelocytic leukemia cell line NB-4 was diluted into cell populations of the MPO-negative myeloma cell line MM-1 at different ratios. MPO protein was identified by classical cytochemical staining and by a specific anti-MPO monoclonal antibody in an immunofluorescent reaction. Cytochemical staining detected 1% positive cells among 99% negative cells. Careful, but time-consuming observation enabled the detection of positive cells in even higher dilutions. At least a 10-fold increase in sensitivity was achieved with the immunofluorescent method, as brightly fluorescent cells are more amenable for a screening of slides at lower microscopic magnification than the cytochemically visualized cells. MPO mRNA expression was examined in whole cell populations by Northern blotting (maximal sensitivity 1%), a reverse transcriptase-polymerase chain reaction (RT-PCR) amplification assay (sensitivity 0.1%), and by RT-PCR followed by Southern blotting (sensitivity 0.05%). The high sensitivity of PCR-based techniques is offset by the fact that these methods do not allow for the identification and further characterization of the individual, MPO-positive cells. Thus, methods examining bulk populations require homogeneous cell samples in order to avoid false-positivity stemming from a few residual bystander cells. The five different techniques were used to determine the status of MPO expression in 20 randomly chosen leukemia cell lines of myelomonocytic origin. In 11 cell lines (8 positive and 3 negative) all five tests provided concordant results. Three cell lines were Northern-negative, but RT-PCR-positive and MPO protein-positive suggesting that Northern blot analysis is the least sensitive tool. Six cell lines were devoid of MPO protein, at least according to the methods used here, but trace expression of MPO message was documented by PCR. All five techniques have advantages and drawbacks and must be carefully selected in order to obtain useful data. The detection of MPO is of experimental and clinical importance in the distinction of myeloid from lymphoid leukemias, and in the lineage assignment of apparently biphenotypic or unclassifiable cases.

Blotting, Northern↗
PubMed1993

Myeloperoxidase: expression and modulation in a large panel of human leukemia-lymphoma cell lines.

Myeloperoxidase (MPO) is found exclusively in the azurophilic granules (primary lysosomes) of normal myelomonocytic cells. Cytochemical staining for MPO activity is used clinically to distinguish myeloid from lymphoid leukemias. We studied the expression of MPO at the RNA and protein level in 140 continuous human leukemia-lymphoma cell lines using classical cytochemistry, immunofluorescent staining with a specific monoclonal antibody, Northern blot analysis, and a reverse transcription-polymerase chain reaction (RT-PCR) amplification assay. Seventy-eight lymphoid leukemia, myeloma, and lymphoma cell lines were negative; only 3 pre-B-acute lymphoblastic leukemia (ALL) cell lines were MPO-positive. Two of these MPO-positive pre-B-ALL cell lines showed a trace expression after RT-PCR and Southern blotting corresponding to 4% to 6% of the transcripts found in other positive myeloid cell lines. The third pre-B-ALL cell line was positive in Northern blots and cytochemical/immunofluorescent staining; however, only few cells were weakly positive in the latter assay. Although 15 of 59 cell lines assigned to the myeloid, monocytic, megakaryocytic, or erythroid lineages were MPO-positive in Northern blots, those 15 and 13 additional cell lines showed bands of mRNA after RT-PCR. MPO protein was detected in all 16 Northern-positive cell lines; on the other hand, there were 4 cell lines that were protein-positive, but Northern-negative. Differentiation induced by protein kinase C activators 12-O-tetradecanoylphorbol 13-acetate and Bryostatin 1 or by all-trans retinoic acid was associated with a decrease in MPO mRNA in all 7 initially positive cell lines studied, even leading to the complete absence of transcripts, but the enzymatic activity of the differentiated cells was only slightly less than that of unstimulated cells. MPO expression could not be induced in 10 initially negative cell lines. The half-life of MPO mRNA was found to be about 6 hours and was not shortened by prior exposure of the cells to the differentiation-inducing agents. These results confirm that MPO expression is mainly associated with myelomonocytic cells, but also underline the notion that MPO cannot be used as an absolutely lineage-specific marker for the distinction of leukemic cells. MPO can be used as an excellent parameter to characterize the various stages of normal and induced differentiation.

Base Sequence↗
PubMed1993

Specificity and sensitivity of polymerase chain reaction (PCR) in comparison with other methods for the detection of mycoplasma contamination in cell lines.

The polymerase chain reaction (PCR) amplification was used for the detection of mycoplasma contamination in 42 continuous cell lines. Using the microbiological cultivation on agar as the reference method, 29 cell lines were regarded as positive and 13 cell lines as negative. The double-step PCR analysis employed nested primers that anneal to gene sequences coding for the evolutionarily conserved 16 S rRNA of some 25 different mycoplasma species (including the ones most commonly found in cell cultures). In terms of the positivity or negativity of mycoplasma infection the results were identical for the agar assay and PCR amplification. All positive cell lines displayed distinct, unequivocal, objectively discernible bands on agarose gels while the non-infected specimens showed no DNA amplification. A simultaneously performed comparison with four other commonly used detection methods (DNA-RNA hybridization in solution, DAPI DNA fluorescence staining, immunostaining with a monoclonal antibody and an ELISA) showed that PCR produced significantly less false-negative or false-positive results than all the other methods. Furthermore, in dilution experiments, PCR correctly detected the infecting mycoplasmas at the lowest level of 1/10(4) whereas the other assays were less sensitive. It is concluded that double-step PCR employing nested primers is superior to other mycoplasma detection methods in many respects: simplicity and speed, high specificity and extreme sensitivity, objectivity and accuracy.

Animals↗
PubMed1993

The Pearson product-moment correlation coefficient is better suited for identification of DNA fingerprint profiles than band matching algorithms.

A database of DNA fingerprint profiles from permanently established human and animal cell lines was prepared with a computer program originally designed for numerical taxonomy of bacteria. Identifications of cell line DNA profiles were performed, both by the Pearson product-moment correlation coefficient and by band matching. Under the conditions used the Pearson product-moment correlation coefficient was consistently more reliable.

Algorithms↗
PubMed1993

Mycoplasma detection by PCR analysis.

The polymerase chain reaction (PCR) method was used to detect mycoplasma contamination in a panel of 42 continuous cell lines. According to the microbiological cultivation assay on agar, 29 cell lines were chronically infected and 13 cell lines were negative. Sets of outer and inner primers (nested double-step PCR) were applied which anneal to DNA sequences coding for conserved regions of the 16S rRNA. These oligonucleotides allow for the amplification of DNA regions found in at least 25 mycoplasma species (including the ones most commonly found in cell cultures), but do not cross-hybridize with DNA from eukaryotic cells. Mycoplasma-positive cell lines showed distinctive bands in ethidium bromide-stained gels, both after the first round of amplification as well as after the second PCR; all agar-negative cell lines were also unambiguously negative in the PCR assay. Thus, neither false-positive nor false-negative results occurred. Provided that the proper PCR working conditions are scrupulously observed, the PCR amplification has several outstanding advantages: high sensitivity, specificity, reliability, objectivity, speed, and simplicity.

Animals↗
PubMed1993

Effects of dolastatins on human B-lymphocytic leukemia cell lines.

Dolastatins are naturally occurring peptides isolated from marine animals and are known to be potent anti-neoplastic agents. Here, the three compounds dolastatin 10 (Dola 10), dolastatin 15 (Dola 15) and deo-dolastatin 10 (Deo-Dola 10) were added to cultures of two human chronic B-leukemia cell lines, JVM-2 and EHEB. The three dolastatins (Dola 10 > Dola 15 > Deo-Dola 10) inhibited cell proliferation and immunoglobulin production. Decreased cell growth and lack of accumulation of immunoglobulin was not caused by cytotoxicity as cell viability in the cultures remained high. The unchanged surface marker immunoprofiles during treatment and a predominance of treated cells in S and G2/M phase suggested cytostatic rather than directly cytotoxic effects. Exposure to these reagents increased quickly, albeit only short c-myc and bcl-2 mRNA expression. These results indicate that the three dolastatins are potent inhibitors of leukemic B-cell proliferation.

Antigens, Neoplasm↗
PubMed1993

Expression and modulation of annexin VIII in human leukemia-lymphoma cell lines.

Annexin VIII is a calcium- and phospholipid-binding protein with anticoagulant activity. Annexin VIII mRNA was found to be specifically expressed in acute promyelocytic leukemia (APL) cells; it was not found in other types of acute myeloid leukemia (AML) nor in lymphoid malignancies. Using Northern blot analysis we investigated annexin VIII expression in 142 continuous human leukemia and lymphoma cell lines at the mRNA level. While the only APL cell line, NB-4, was indeed positive, other cell lines also displayed annexin VIII mRNA: 4/22 myeloid cell lines, 8/23 monocytic cell lines, 2/8 megakaryoblastic cell lines, 5/26 lymphoma-derived cell lines, 2/10 myeloma cell lines and 1/44 lymphoid leukemia cell lines. The strongest expression was seen in NB-4 and in the Hodgkin's disease derived cell line HDLM-2. Treatment of NB-4 cells with all-trans retinoic acid (ATRA) or the phorbol ester TPA induced terminal differentiation and down-regulated annexin VIII mRNA expression rapidly within a few hours; vitamin D3 was ineffective in this regard; the protein kinase C activator Bryostatin 1 up-regulated the expression. A panel of initially negative cell lines could not be induced by any of these biomodulators to transcribe annexin VIII. The half-life (T1/2) of annexin VIII mRNA was about 3-4 h using actinomycin D as transcription inhibitor. Treatment with ATRA or TPA prior to exposure to actinomycin shortened the T1/2 to 2 h while Bryostatin 1 extended it to 6h. As 21/141 non-APL cell lines were positive, annexin VIII cannot be used as a marker gene for APL cells; however, it might be associated with myelomonocytic or erythro-megakaryoblastic precursor cells. Annexin VIII gene expression might play a unique role in the proliferation and/or differentiation of leukemic cells and could be associated with the particular abnormal hemostasis of some leukemias.

Annexins↗
PubMed1993

Different effects of the two protein kinase C activators bryostatin-1 and TPA on growth and differentiation of human monocytic leukemia cell lines.

The effects of bryostatin-1 (Bryo) and 12-O-tetradecanoyl-phorbol 13-acetate (TPA), both activators of protein kinase C (PKC), on proliferation and differentiation of two monocytic leukemia cell lines, JOSK-I and JOSK-M, were investigated. Treatment with TPA or Bryo inhibited cellular proliferation in a dose-dependent manner. Both drugs induced distinct phenotypic changes associated with monocytic differentiation. Although c-myc mRNA is often found to be down-regulated during biomodulator-triggered in vitro myelomonocytic differentiation, however, here the modulation of c-myc expression was less pronounced. All parameters studied were more prominently altered in TPA- than in Bryo-treated cells, and, were more distinct in JOSK-I than in JOSK-M. Since Bryo was able to antagonize the TPA-mediated effects on proliferation and morphological alterations, an (at least partially) different mode of action of these PKC activators on monocytic cell lines may be suggested.

Adult↗
PubMed1993

Retrovirus tests of human leukemia/lymphoma cell lines at DSM.

Permanently established human cell lines can produce several retroviruses. It is important to routinely test such cell lines for human T cell lymphotropic virus (HTLV) type I and II, and for human immunodeficiency virus (HIV) type 1 and 2 in order to exclude any potential biohazard from cell lines producing human retroviruses. Reverse transcriptase assay, polymerase chain reaction, and dot-blot hybridization of in-vitro amplified DNA with virus-specific probes are used.

Containment of Biohazards↗
PubMed1993

Induction of differentiation of B-cell leukemia cell lines JVM-2 and EHEB by bryostatin 1.

The effects of bryostatin 1 (Bryo 1), a protein kinase C (PKC) activator, on proliferation, differentiation and macromolecular synthesis were investigated in the two cell lines EHEB and JVM-2, established from patients with chronic B-cell leukemia. Treatment with Bryo 1 inhibited the proliferation, DNA and RNA synthesis in a time- and dose-dependent fashion. The cells differentiated along the B-cell pathway to plasmacytoid cells as judged by morphological examination and increased their production and secretion of immunoglobulins. c-myc mRNA expression was induced in both cell lines. The phorbol ester TPA, a pharmacological PKC activator, had similar differentiation-inducing effects. The biomodulators failed to induce significant alterations in the cell surface marker profile. Except for their surface markers, all parameters studied were more strongly altered in JVM-2 than in EHEB cells. JVM-2 was established from a patient with B-prolymphocytic leukemia (PLL), whereas EHEB originated from a case of B-chronic lymphocytic leukemia (CLL). These data support the notion that PLL cells appear to be activated B-cells, in contrast to the rather quiescent CLL cells. Since Bryo 1 lacks tumor-promoting activity, this naturally occurring compound, extracted from marine animals, has a potential role in the therapy of B-cell neoplasms as a differentiating agent.

Antigens, Neoplasm↗
PubMed1993

Recent results on the biology of Hodgkin and Reed-Sternberg cells. II. Continuous cell lines.

The relative scarcity of Hodgkin (H) and Reed-Sternberg (RS) cells within biopsies from cases with Hodgkin's disease (HD) is an impediment to the analysis of the nature and function of these cells. Continuous cell lines as uniform and permanently available sources of cells provide a valid alternative. Development of HD cell lines has proven to be rather difficult when compared with the results on leukemia and Non-Hodgkin lymphoma cells. Only a few cell lines containing cells that resemble in-vivo H-RS cells have been established. Because the in-vitro culture conditions favor the self-propagation of residual normal cells, e.g. Epstein-Barr virus transformed B-lymphoblastoid cells or monocyte/macrophage monolayers, early attempts at culturing HD tissue resulted mainly in the generation of such cell lines. Even for the bona fide HD cell lines it is difficult to prove that the immortalized cells originated from an H-RS cell. These 13 HD cell lines have been extensively characterized in a large variety of aspects. These data have resulted in widely varying conclusions about the nature of the cell lines. It is apparent that all HD cell lines are unique among hematopoietic cell lines and are also different from one another. No conclusive evidence towards the origin of the cells has been obtained for some cell lines, while others could be operationally, albeit not always unequivocally, assigned to the T- or B-cell or monocyte-macrophage lineages. The overall phenotypes are often not concordant with those of normal hematopoietic cells; some cell lines show clearly mixed lineage attributes. The artifactual expansion of non-HRS cells in culture and the acquisition or loss of certain properties during the adaptation to culture systems cannot be excluded. There was also a bias for the establishment of cell lines from cases with advanced clinical stages, nodular sclerosing subtype and pleural effusions. The extensive analysis of a few cell lines has provided a wealth of information useful for the understanding of the biology of H-RS cells. The striking heterogeneity could be reflective of a biologically heterogeneous disease.

Animals↗
PubMed1993

Differentiation and growth modulation of myeloid leukemia cells by the protein kinase C activating agent bryostatin-1.

Bryostatin-1 (Bryo), a macrocyclic lactone of the sea water bryozoan Bugula neritina, is a potent activator of protein kinase C and was found to exhibit antineoplastic activity in several systems. We studied the effect of Bryo on differentiation and growth modulation of human myeloid leukemia cell lines and freshly explanted blood cells from patients with myeloid leukemia. Alterations at the molecular level and phenotypic changes triggered by Bryo were similar, but not identical, to those induced by phorbol esters. Bryo was able to inhibit cellular proliferation as evidenced by [3H]-thymidine uptake and induced morphological changes associated with monocytic differentiation. In studies using continuous cell lines, the glucocorticoid dexamethasone was unable to prevent the Bryo-induced growth inhibition or the induced phenotypic changes. However, in fresh myeloid blood cells dexamethasone attenuated these Bryo-triggered effects. Our own data taken together with reports from the literature reviewed here suggest the following conclusions: (i) Bryo, while lacking tumor promoting activity, is able to induce differentiation in maturation arrested leukemia cells; (ii) it exhibits selective antiproliferative properties in normal or malignant hematopoietic cells and supports growth of multipotent stem cells. These features might qualify Bryostatin-1 as a potential candidate for promising research and possibly for future clinical applications.

Antineoplastic Agents↗
PubMed1993

Multiparameter approach in the identification of cross-contaminated leukemia cell lines.

A common problem in cell culturing is cross-contamination with other cells or misidentification of cells. An effective cell culture quality and identity control is required in order to avoid inter- and intraspecies contamination of cell lines and their further propagation and dissemination. We present evidence that supposedly unrelated cell lines that we received from the original investigators are in fact related to the chronic myeloid leukemia cell line K-562. The sister cell lines SPI-801 and SPI-802 were originally established from a patient with T-cell acute lymphoblastic leukemia and displayed T-cell associated features. However, data from morphological evaluation, immunophenotyping, bcr-abl gene rearrangement analysis, DNA fingerprinting, Northern blot analysis of globin gene expression and esterase isoenzyme analysis clearly established that the three cell lines are related. Cytogenetic examination while not proving the common identity of the cells provided further evidence for the suspected common origin of all three cell lines. Chromosome banding, DNA fingerprinting and bcr-abl genotyping suggested further evolution of these clones during long-term cultivation. Quality and identity control is an essential feature of cell culture technique. Only regular monitoring for purity and integrity of cell lines will significantly reduce the incidence of cell line contamination and misidentification.

Antigens, Neoplasm↗
PubMed1993

Transient expression of the tartrate-resistant acid phosphatase (TRAP) gene in hamster cells: a pilot study.

Tartrate-resistant acid phosphatase (TRAP) became known as the characteristic, albeit not specific, marker enzyme for hairy cell leukemia (HCL). It is also expressed in other types of leukemic cells and in a variety of normal hematopoietic cells under both physiological and artificial conditions. The role of this distinctive enzyme is still unknown. TRAP hydrolyzes several chemical compounds, but its physiological substrate remains to be elucidated. Here, the insertion of a human TRAP cDNA into mammalian expression vector pSBC-2 yielded a construct that encoded enzymatically active TRAP in transfected baby hamster kidney cells (BHK-21). TRAP was expressed transiently, as shown by Northern blot analysis, polyacrylamide gel electrophoresis, isoelectric focusing and cytochemical staining. BHK-21 cells over-expressing the enzyme had a growth rate that was approximately 50% of that observed in control cells. A stable expression could not be achieved. Recent evidence suggested that TRAP might function as a protein tyrosine phosphatase. The effect of TRAP on protein tyrosine phosphorylation was examined by immunoblotting with an anti-phosphotyrosine monoclonal antibody (MoAb). The level of tyrosine phosphorylation was clearly lower in transfected BHK-21-TRAP cells than in the control cultures. Phorbol ester-mediated induction of protein tyrosine phosphorylation could overcome the status of reduced phosphorylation in BHK-21-TRAP cells. Furthermore, upon treatment with 12-O-tetra-decanoylphorphol-13-acetate tyrosine phosphorylation reached similar levels to stimulated control cells, indicating the existence of a functional endogenous phosphorylation network. Thus, TRAP might function as an antagonistic counterpart of cellular protein tyrosine kinases and could be involved in the control of cellular activation, proliferation, and differentiation.

Acid Phosphatase↗
PubMed1993

Acute myeloid leukemias expressing lymphoid-associated antigens: diagnostic incidence and prognostic significance.

A portion of patients with acute myeloid leukemia also display surface antigens associated with lymphoid development (Ly+AML). The incidence of Ly+AML varies considerably between independent studies, both overall and with regard to individual antigens. On average, lymphoid-associated antigen expression is relatively low in AML. The reasons for some striking differences between conflicting reports are not clear, but are most probably due to various technical aspects including several arbitrary parameters. The data accumulated from the literature lead to the following conclusions: (i) use of different reagents against the CD surface antigens, different positive/negative cut-off levels, analysis of fresh or frozen cell material and variable sensitivities of the analytical instruments (expression of lymphoid-associated antigens was commonly weaker than myeloid-associated markers) seriously influence the results; (ii) most antigens (CD1, CD2, CD3, CD5, CD8, CD10, CD19, CD20, CD21, CD22) were expressed on less than 10% of AML cases; (iii) the CD4 and CD7 antigens, also found on normal monocytic and immature myeloid progenitor cells, were detected in 24% and 15% of AML cases, and their expression correlated with FAB M4/M5 and M1/M2 morphology, respectively; (iv) differences between pediatric and adult Ly+AML were restricted to CD4 and CD19 expression being detected more often in childhood cases; (v) there is no cytogenetic anomaly specific for Ly+AML; anomalies exclusively associated with lymphoid malignancies were not seen; aberrations involving 11q23, 14q32, and the 9;22 translocation seem to be increased; (vi) in most studies, expression of lymphoid-associated antigens (with the exception of CD7) on AML blasts lacked prognostic significance; CD7+AML appears to be a particular subset of malignant myeloid progenitors. In summary, these findings suggest that in general, Ly+AML may not represent a biologically distinct form of leukemia as these cases have similar clinical features and respond to therapy in a comparable manner.

Adult↗