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C C Uphoff

Publications and source records attributed to C C Uphoff.

At least 37 records · Page 2Linked to original sources
PubMed1997

Occurrence of TEL-AML1 fusion resulting from (12;21) translocation in human early B-lineage leukemia cell lines.

The recurrent (12;21)(p13;q22) translocation fuses the two genes TEL and AML1 that have previously been cloned from translocation breakpoints in myeloid leukemias. Using mainly reverse transcriptase-polymerase chain reaction (RT-PCR), the TEL-AML1 chimeric transcript has been observed in 22-27% of pediatric patients with acute lymphoblastic leukemia (ALL), in particular in the early B-lineage ALL subtype, making it the most common genetic lesion in these patients. The vast majority of acute myeloid leukemias, other ALL subtypes and even adults with early B-lineage ALL were TEL-AML1-negative. We determined whether the TEL-AML1 fusion gene can also be observed in continuous human leukemia cell lines with an early B-lineage phenotype. Twenty-nine such cell lines established from children (n = 13) or adults (n = 13) with early B-lineage ALL and five cell lines derived from chronic myeloid leukemia in blast crisis or B cell non-Hodgkin's lymphoma were investigated for the occurrence of the TEL-AML1 rearrangement by RT-PCR. While all 13 adult early B-lineage ALL cell lines and the five cell lines from other leukemias or lymphomas were negative, 1/13 pediatric cell lines (cell line REH) was found to be positive for TEL-AML1; though neither reciprocal AML1-TEL, nor normal TEL, mRNA was detectable by RT-PCR in this cell line. These findings agreed with the results of conventional cytogenetic and FISH analysis of REH which was found to carry the der(21) partner only of t(12;21)(p13;q22), probably resulting from a complex translocation, t(4;12;21;16)(q32;p13;q22;q24.3). Hybridization with flanking cosmid clones (179A6 and 148B6), covering exons 1 and 8 respectively of TEL, confirmed a rearrangement accompanying the t(12;21), and showed cryptic deletion of the residual allele resulting from an apparently reciprocal t(5;12)(q31;p13). These findings in REH provide a further example of, and possible cytogenetic mechanism for, the paradigm of TEL-AML1 fusion accompanied by deletion of the residual TEL allele. The low rate of early B-lineage ALL cell lines carrying this translocation contrasts clearly with the relative high frequency of TEL-AML1-positive cases in primary material. It is possible that expression of the fusion product hampers the in vitro growth and establishment in culture of such leukemic cells. Nevertheless, the cell line REH represents a powerful tool for the further molecular characterization of this unique breakpoint and can serve as a positive control in routine PCR reactions.

Adolescent↗
PubMed1997

Two acute monocytic leukemia (AML-M5a) cell lines (MOLM-13 and MOLM-14) with interclonal phenotypic heterogeneity showing MLL-AF9 fusion resulting from an occult chromosome insertion, ins(11;9)(q23;p22p23).

We describe two new human leukemia cell lines, MOLM-13 and MOLM-14, established from the peripheral blood of a patient at relapse of acute monocytic leukemia, FAB M5a, which had evolved from myelodysplastic syndrome (MDS). Both cell lines express monocyte-specific esterase (MSE) and MLL-AF9 fusion mRNA. Gene fusion is associated with a minute chromosomal insertion, ins(11;9)(q23;p22p23). MOLM-13 and MOLM-14 are the first cell lines with, and represent the third reported case of, MLL gene rearrangement arising via chromosomal insertion. Both cell lines carry trisomy 8 which was also present during the MDS phase, as well as the most frequent trisomies associated with t(9;11), ie, +6, +13, +19 variously present in different subclones. Despite having these features in common, differences in antigen expression were noted between the two cell lines: that of MOLM-13 being CD34+, CD13-, CD14-, CD15+, CD33+; whereas MOLM-14 was CD4+, CD13+, CD14+, CD15+, CD33+. Differentiation to macrophage-like morphology could be induced in both cell lines after stimulation with INF-gamma alone, or in combination with TNF-alpha, which treatment also induced or upregulated, expression of certain myelomonocyte-associated antigens, including CD13, CD14, CD15, CD64, CD65 and CD87. Together, these data confirm that both cell lines are likely to be novel in vitro models for studying monocytic differentiation and leukemogenesis.

Adult↗
PubMed1996

Esterase isoenzyme profiles of 255 leukemia-lymphoma cell lines from all hematopoietic cell lineages.

Carboxylic esterase isoenzymes isolated from a panel of well-characterized continuous human leukemia-lymphoma cell lines were separated by isoelectric focusing. Typical isoenzyme patterns designated Mono 1/Mono 2 (for monocyte-associated), My 1/My 2 (for myeloid or myeloma), Lym 1/Lym 2 (for lymphoid) and Und (for undifferentiated) could be reproducibly discerned. The Mono patterns contained one unique isoenzyme encoded by the monocyte-specific esterase gene. This comparative analysis of 255 leukemia-lymphoma cell lines covered the major cell lineage that are affected by hematological neoplasias. The results showed that (except for myelomas) lymphoid-derived malignancies, both leukemias and lymphomas, expressed primarily the Und and Lym esterase isoenzyme profiles. In contrast, myeloid leukemia cells and the related erythroid and megakaryocytic cell lines displayed mainly the My patterns. The Mono patterns were detected predominantly in monocyte-derived leukemias. As the B-lymphocytic hierarchy progresses from pre B-cells via B-cells to plasma cells, number and intensity of the isoenzymes increased as well from the Und pattern to the My isoenzyme profile. Hodgkin's disease and anaplastic large cell lymphoma lines displayed heterogenous isoenzyme profiles consistent with their heterogenous cellular origin. The present study using continuous leukemia-lymphoma cell lines as model systems provides a biochemical characterization of different hematopoietic cell lineages and stages of differentiation.

Carboxylesterase↗
PubMed1996

Differential expression of the ufo/axl oncogene in human leukemia-lymphoma cell lines.

The ufo protein (also termed axl) is a member of a new family of receptor tyrosine kinases and is encoded by a transforming gene that was initially isolated from primary human myeloid leukemia cells by DNA-mediated transformation of NIH/3T3 cells. The ligand, Gas6, a protein S-related molecule lacking any known function yet, has recently been identified. We report the expression pattern of ufo mRNA in a panel of 76 human continuous leukemia-lymphoma cell lines. The gene was not expressed in cell lines derived from lymphoid malignancies (n=28), but transcription was seen in 3/11 myeloid, 0/6 monocytic, 9/13 erythroid and 11/18 megakaryocytic cell lines. Several cell lines were treated with phorbol ester leading to significant upregulation of the ufo message in constitutively positive cells. An apparent ufo mRNA overexpression was not found in any of the positive leukemia cell lines, but was identified in the drug-resistant subclones of the cervix carcinoma cell line HeLa. Southern blot analysis of restriction enzyme-digested genomic DNA did not provide evidence for gene amplification, but the HeLa subclones showed banding patterns suggestive of gene rearrangement. Two main ufo mRNA bands of 3.2 and 5.0 kb were identified; no differences in the half-lives (t1/2 = 2.5 h) of these two mRNA species could be identified. In summary, ufo, representing a novel type of receptor tyrosine kinase, is expressed solely in myeloid and erythro-megakaryocytic leukemias but not in lymphoid malignancies. These and previous data suggest an involvement of the ufo receptor tyrosine kinase in normal and malignant myelopoiesis; however, its exact role, if any, and mode of operation in leukemogenesis remains to be determined.

3T3 Cells↗
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↗
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

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

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

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↗
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

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

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

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

Modulation of gene expression in the acute promyelocytic leukemia cell line NB4.

The human leukemic cell line NB4 was derived from a patient with acute promyelocytic leukemia and is characterized by a specific 15;17 chromosomal translocation. We analyzed the response of NB4 and HL-60 cells to the biomodulators all-transretinoic acid (ATRA), vitamin D3 (Vit D3) and the protein kinase C agonists bryostatin 1 (Bryo 1) and phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). HL-60 cells were used for comparison being arrested at the myeloblastic-promyelocytic stage, but lacking the t(15;17) abnormality. In most experiments Vit D3 was only weakly or not at all effective. The other three reagents effectively slowed or stopped the proliferation of the cells in suspension. Associated with this proliferation arrest was the cell differentiation along the myeloid cell lineages: ATRA modulated morphological features indicative of granulocytic differentiation; Bryo 1 and TPA caused also distinct morphological changes. The inducers up-regulated the expression of CD11b (without changing the surface expression of other markers, e.g. CD13, CD14, CD15, CD33, CD68, HLA-DR) and completely down-regulated the originally strong expression of myeloperoxidase and c-myc at the mRNA level. Thus, ATRA- or protein kinase C activator-induced differentiation involved changes associated with maturational processes. Induction of terminal differentiation of leukemic cells by physiological or pharmacological modulators may be able to control the growth of the malignant cells and has therapeutic implications.

Antigens, CD↗
PubMed1993

MDR1 expression in human leukemia-lymphoma cell lines.

We examined the expression of the mdr1 gene in 90 human tumor cell lines of which 79 were derived from leukemia or lymphoma samples. Using Northern blot analysis and the mdr1.1 probe, only three cell lines, KB-V1 (a positive control), EM-2, A-704, displayed mdr1 mRNA expression. Immunocytochemical APAAP staining of these three and 15 other cell lines with the anti-p170 glycoprotein monoclonal antibody JSB-1 was negative in all but the KB-V1 cells. In contrast to the results seen in freshly explanted neoplastic cells, the mdr1 gene does not seem to be functionally expressed in the majority of unmanipulated and unselected, continuous human tumor cell lines.

ATP Binding Cassette Transporter, Subfamily B, Mem↗