KM3 Cells Research Articles

Nuclear Translocation of βII PKC Isoenzyme in Phorbol Ester-Stimulated KM-3 Pre-B Human Leukemic Cells.

Members of the protein kinase C (PKC) family play a key role in regulating cell growth and differentiation in response to several stimuli, including hormones, neurotransmitters, and growth factors. The different properties and substrate specificity of the PKC isoforms are not fully understood, and they are assumed to have specific functions in intracellular signaling. In lymphoid cells, the effects of PMA and Ca2+ ionophore, singly or in combination, on activation and expression of Ca2+-dependent PKC at the level of protein and messenger RNA have been examine. Starting from these observation and the possibility the differential isoenzyme expression might contribute to the differences in phorbol ester sensitivity of lymphoid cells, it seemed worthwhile to investigate the expression and the modulation of PKC isoforms in KM-3 cells, a human pre-B cell line, upon treatment with phorbol 12-myristate 13-acetate (PMA). Using multiparametric analysis we detected three PKC isoforms in the KM-3 cell line: α, βII, and ζ. PMA treatment causes an intranuclear translocation of the βII isoform, via the nuclear pore complex, associated with the interchromatinic regions. These data suggest that the βII isoenzyme may play a strategic role in signal transduction and regulation of specific gene expression in B lymphocytes.

An Effective, Direct Immunomagnetic Procedure for Purging Acute Lymphoblastic Leukemia Cells from Human Bone Marrow.

The AB4 monoclonal antibody, which recognizes an HLA-DR epitope, was found to bind to a high percentage of malignant blast cells in samples obtained from 27 patients with ALL. These included 11 of 11 cases with c-ALL, 3 of 7 with pre-pre-B, and 8 of 9 cases with pre-B ALL. AB4 was used together with anti CD10 and anti CD19 antibodies and super-paramagnetic particles for developing a direct immunomagnetic procedure for purging human bone marrow of leukemic cells. In model experiments with KM3 cells admixed to mononuclear bone marrow cells, the individual antibodies each removed 2.8-3.1 logs and 3.6-4.1 logs of tumor cells with one and two purging cycles, respectively. In comparison, the efficacy of a mixture of the three antibodies was 4.4 logs with one treatment cycle, and > 5 logs with repeated treatments. Whereas the use of a commercially available anti-HLA-DR antibody resulted in a 90% reduction in the survival of CFU-GMs and normal blast colonies, AB4 had only a moderate effect on the progenitor cells (46% and 30% reduction). In conjunction with autologous transplantation, bone marrow from a patient was purged with the antibody mixture and 50% of the CFU-GMs and 47% of the CD34+ cells remained after treatment. The patient showed a normal engraftment, reaching a level of 0.5 x 109/l neutrophils by day 20 and 20 x 109/l platelets by day 30. It is concluded that the antibody cocktail may safely and effectively be used for rapid autograft purging in patients with c-ALL, and also in phenotypically selected cases with other subtypes of ALL.

A cell surface antigen (BAL) defined by a mouse monoclonal antibody inducing apoptosis in a human lymphocytic leukemia cell line.

The lack of apoptosis or programmed cell death in human tumor cells has been suggested to be one factor allowing uncontrolled growth of neoplasms. We have developed a mouse monoclonal antibody (MAb) that induces programmed cell death in a human acute leukemia cell line (KM-3) of the pre B-cell type. Stable, antibody-producing hybridomas were produced by fusing mouse myeloma cells to spleen cells from mice immunized with viable KM-3 cells. Incubation of KM-3 cells with the MAb (designated anti-BAL) resulted in growth inhibition and subsequent cell death within 2-3 days. Anti-BAL required cross-linking with a rabbit anti-mouse antibody to induce DNA fragmentation typical of apoptosis. Immunoblotting experiments with anti-BAL identified a 37-kDa protein, apparently different from any previously described apoptosis-related surface antigen. Strongest expression of the antigen was generally found on cells of lymphoid or myeloid origin. However, several other cell types such as fibroblasts and endothelial cells were also stained by anti-BAL in flow cytometry but less intensively. Despite the apparent presence of this cell surface-bound 37-kDa antigen on several normal and malignant cell types, anti-BAL induced cell death only in human malignant cell lines expressing a more immature phenotype.

Human protein NEFA, a novel DNA binding/EF-hand/leucine zipper protein. Molecular cloning and sequence analysis of the cDNA, isolation and characterization of the protein.

The cDNA libraries constructed from the human acute lymphoblastic leukemia cell line KM3 in the expression vector lambda gt11, were screened with the anti-CALLA (common acute lymphoblastic leukemia antigen) mAb (monoclonal antibody) J5. The selected J5-positive clone I containing a partial cDNA insert was isolated and sequenced. For completing the cDNA sequence the cDNA libraries were further screened by hybridization with the DIG (digoxigenin)-labelled DNA probe derived from clone I, the 5'-end region was analysed by 5'-RACE (rapid amplification of cDNA ends) using a sequence specific primer. In total a 1639 bp cDNA sequence was determined. The cDNA sequence contains a 1260 bp open reading frame and the untranslated 3'- and 5'-end sides. The 420 residue amino acid sequence, deduced from the cDNA sequence, unexpectedly differs fundamentally from CALLA (CD10) although clones I and II were J5-positive in immuno screening. The mature protein corresponding to the cDNA was isolated and characterized from the KM3 cells using polyclonal antisera raised against the in vitro expressed polypeptide from clone I. The protein is expressed on plasma membrane, in cytosol and is secreted into culture medium, its relative molecular mass was determined to be 55 kDa on SDS-PAGE. The deduced amino acid sequence from cDNA was confirmed by peptide sequences. The new protein contains a basic amino acid rich putative DNA binding domain (b) with a potential nuclear targeting signal, two helix-loop-helix (HLH) motif regions, concurrently EF-hand motifs, an acidic amino acid rich region (a) between the EF-hands, and a leucine zipper (Z) motif. This DNA binding protein therefore is characterized by a linked motif "b/HLH/a/HLH/Z". The protein was designated NEFA: DNA binding/EF-hand/acidic amino acid rich region.

Interleukin 4 inhibits the proliferation and promotes the maturation of human leukemic early B cells.

The effects of interleukin 4 (IL-4) on human leukemic precursor B-cell lines were investigated. Recombinant IL-4 (rIL-4) was added to three acute lymphoblastic leukemia-derived pre B-cell lines: Reh, Km3 and Nalm-6. Our results show that rIL-4 significantly decreases continuous proliferation of Reh and Km3 cells while Nalm-6 cells have a limited response in this respect. This rIL-4 effect is dose-dependent and can be neutralized by anti-IL-4 monoclonal antibody (mAb). Furthermore, rIL-4 down-regulated IL-3-induced proliferation of Reh cells. Phenotypic analysis of rIL-4-treated cells points to significant induction of surface marker maturation of leukemic cells by this cytokine. Together, these in vitro data suggest that IL-4: 1) inhibits the proliferation and 2) promotes the differentiation of certain human leukemic B-cell precursors.

Limited proteolysis and differential down-regulation of protein kinase C subspecies in KM3 cells.

Protein kinase C (PKC) is known to play crucial roles in signal transduction. This enzyme was originally found as an undefined protein kinase, which could be activated by limited proteolysis by a Ca2(+)-dependent neutral protease, calpain. PKC was thought to be a single entity for many years, but recent, molecular cloning and biochemical analysis has shown that this enzyme consists of multiple subspecies with closely related structures. The present studies were undertaken to explore the susceptibility of PKC subspecies to proteolysis by calpain in KM3 cells, a pre-B, pre-T cell line. The PKC from KM3 cells was resolved into two subspecies, type II (mainly beta II) and type III (alpha), upon hydroxyapatite column chromatography. This cell line was also shown to contain calpain I and calpain II, which show different sensitivities to Ca2+. Both type II and type III PKC were hydrolysed by either calpain I or calpain II, but type II PKC was cleaved more effectively than type III. The simultaneous presence of phospholipid and diacylglycerol enhanced the cleavage of these PKC subspecies by calpain I. When these cells were treated with phorbol ester, the rates of translocation and down-regulation of these PKC subspecies were different; type II PKC was translocated and depleted from the cell more rapidly than type III enzyme. The results presented herein suggest different roles of the PKC subspecies in the cellular responses to external stimuli.

Alfred P. Sloan Jr. prize. Studies and prospectives of the protein kinase C family for cellular regulation

Alfred P. Sloan Jr. prize. Studies and prospectives of the protein kinase C family for cellular regulation

Differential down-regulation of protein kinase C subspecies in KM3 cells

The down-regulation of protein kinase C (PKC) subspecies in KM3 cells (a pre-B, pre-T cell line) has been examined. The PKC from KM3 cells was resolved into two subspecies, type II (mainly βII) and type III (α), upon hydroxyapatite column chromatography. Biochemical and immunocytochemical analysis revealed that, when these cells were treated with 12- O-tetradecanoylphorbol 13-acetate (TPA), the time course of down-regulation of the PKC subspecies was different; type II PKC was translocated and depleted from the cell more quickly than type III enzyme. The results suggest that each PKC subspecies plays a different role in the cellular response to TPA and probably to other external stimuli.

Effects of methotrexate on purine and pyrimidine metabolism and cell-kinetic parameters in human malignant lymphoblasts of different lineages

MOLT-4 (T−), RAJI (B−), and KM-3 (non-B-non-T-, common ALL) malignant lymphoblasts demonstrated significant differences in their activities of purine de novo synthesis (PDNS) and purine salvage pathway and in their cell-kinetic parameters. Incubations with concentrations of methotrexate (0.02 and 0.2 μM), which can be maintained during many hours in the oral maintenance therapy of acute lymphoblastic leukemia, indicated large differences between the three cell lines with respect to the inhibition of PDNS, depencling on the concentration of methotrexate (MTX) and on the activities of the two pathways. These dose- and cell line-dependent differences corresponded to the perturbations of cell-kinetics and purine and pyrimidine (deoxy)ribonucleotide pools in the three cell lines. Exposure of MOLT-4 cells to 0.02 μM MTX resulted in an incomplete inhibition of DNA synthesis in early S phase, as shown by DNA-flow cytometry and increase of dCTP levels, which recovered spontaneously after 48 hr. Almost no impairment of RNA synthesis occurred (unbalanced growth). In RAJI cells, exposed to 0.02 μM MTX, DNA synthesis was delayed in the S phase, not arrested, and RNA synthesis was not impaired, also indicating an unbalanced growth pattern, which, however, did not recover in time. KM-3 cells were arrested in G1 phase and subsequently in early S phase after incubation with 0.02 μM MTX, and perturbations of ribonucleotides indicated a complete inhibition of RNA synthesis, resulting in a balanced growth pattern. Cytotoxicity was more pronounced in KM-3 cells. The reliability of the soft agar colony forming assay after low dose MTX treatment is discussed. Exposure of MOLT-4 and KM-3 cells to 0.2 μM MTX resulted in a complete inhibition of DNA synthesis, with cessation of cell progression through all parts of the cell cycle and arrest in G1 phase. RAJI cells showed an increasing accumulation of cells in G1 phase without complete cessation of cell cycle progression. Perturbations of ribonucleotide pools suggested an inhibition of RNA synthesis in all cell lines, indicating a balanced growth pattern in KM-3 cells and MOLT-4 cells. Increased levels of ribonucleotides after prolonged exposure to 0.2 μM MTX were due to reutilization of purine and pyrimidine precursors from nucleic acid breakdown consuming PRPP, whereas consumption of ribo-nucleotides for RNA synthesis was absent. The perturbations of deoxyribonucleotide pools after exposure to 0.02 and 0.2 μM MTX were in agreement with the changes in DNA-flow cytometry. Increased levels of dCTP after exposure to 0.02 μM MTX correlated with accumulation of cells in early S phase. A consplcuous phenomenon was the absence of decrease in dGTP pools in RAJI cells, in which cytotoxicity was low. Cytotoxicity was correlated with the degree of PDNS inhibition and a severe decrease of both dGTP and dTTP pools. The differences between the T−, B−, and non-B-non-T-, common ALL cell lines with respect to the parameters studied may explain the different responses to treatment of patients with corresponding lymphoid leukemias and lymphomas and suggest possibilities for different treatment strategies.

Purine de novo synthesis as the basis of synergism of methotrexate and 6-mercaptopurine in human malignant lymphoblasts of different lineages

Methotrexate (MTX) causes an inhibition of purine de novo synthesis (PDNS), resulting in increased intracellular availability of 5-phosphoribosyl-1-pyrophosphate (PRPP) in human malignant lymphoblasts with an active PDNS. Normal bone marrow cells and peripheral blood lymphocytes lack this capacity. The increased levels of PRPP can be used for enhanced incorporation of 6-mercaptopurine (6MP), indicating a potential time-, sequence- and dose-dependent synergism of both drugs. The effects of 0.02 μM and 0.2 μM MTX on the PDNS of MOLT-4 (T-), RAJI (B-) and KM-3 (non-B-non-T-) human malignant lymphoblasts were studied with respect to PRPP levels, aminoimidazolecarboxamide ribonucleosidemonophosphate (AICAR) levels and the incorporation of labeled glycine into purine metabolites. These results were correlated with the activity of the PDNS (labeled glycine incorporation) and the purine salvage pathway (labeled hypoxanthine incorporation) in untreated cells. Inhibition of PDNS by 0.02 μM MTX was complete in KM-3 cells with a moderately active PDNS and salvage pathway. RAJI cells, with a relatively low PDNS and high salvage pathway, demonstrated an incomplete, but increasing inhibition of PDNS, whereas inhibition of PDNS in MOLT-4 cells with both pathways active was minimal and recovered in time. Treatment with 0.2 μM MTX resulted in a complete inhibition of PDNS in all cell lines. After treatment with MTX an enhanced incorporation of labeled hypoxanthine and 6MP was noticed, confirming the potential rescue from MTX cytotoxicity by hypoxanthine and a potential synergism of MTX and 6MP on cytotoxicity. The enhanced incorporation of 6MP was more obvious in RAJI and KM-3 cells in comparison with MOLT-4 cells. These data demonstrate the important role of both the activities of the PDNS and the purine salvage pathway in malignant lymphoblasts of different subclasses with respect to the synergism of MTX and 6MP.

Utilization of a Colony Assay to Assess the Variables Influencing Elimination of Leukemic Cells From Human Bone Marrow With Monoclonal Antibodies and Complement

We have previously used a chromium-release assay to demonstrate that the cocktail of monoclonal antibodies BA-1, BA-2, BA-3, and complement can effectively lyse human leukemic cells in the presence of excess bone marrow. Using a leukemic cell colony assay, we have reinvestigated the variables influencing lysis of human leukemic cells (KM-3, HPB-NULL, NALM-6) in bone marrow using BA-1, BA-2, BA-3, and complement. Specific variables addressed included the concentration of excess bone marrow cells, the number of treatments, the presence or absence of DNase during the treatment, the combination of antibodies, and the sensitivity of different leukemic cell lines to lysis. Using the colony assay, the BA-1,2,3 cocktail was shown to be more effective than any single antibody or combination of two antibodies. We also determined that the concentration of excess bone marrow cells and number of treatments had a direct bearing on leukemic cell lysis. Although two cycles of treatment were significantly superior to one cycle, three cycles were not significantly superior to two cycles. Inclusion of DNase (10 micrograms/mL) was a critical adjunct that eliminated clumping and facilitated plating cells in the colony assay. Finally, we could show that striking differences existed in the sensitivity of the leukemic cell lines to lysis with the BA-1,2,3 cocktail and complement. NALM-6 cells were the most sensitive (approximately four logs of kill), and KM-3 cells were the most resistant (less than two logs of kill). Our results strongly support the utility of sensitive leukemic cell colony assays in the analysis of marrow treatment variables in autologous bone marrow transplantation.

Utilization of a colony assay to assess the variables influencing elimination of leukemic cells from human bone marrow with monoclonal antibodies and complement

We have previously used a chromium-release assay to demonstrate that the cocktail of monoclonal antibodies BA-1, BA-2, BA-3, and complement can effectively lyse human leukemic cells in the presence of excess bone marrow. Using a leukemic cell colony assay, we have reinvestigated the variables influencing lysis of human leukemic cells (KM-3, HPB- NULL, NALM-6) in bone marrow using BA-1, BA-2, BA-3, and complement. Specific variables addressed included the concentration of excess bone marrow cells, the number of treatments, the presence or absence of DNase during the treatment, the combination of antibodies, and the sensitivity of different leukemic cell lines to lysis. Using the colony assay, the BA-1,2,3 cocktail was shown to be more effective than any single antibody or combination of two antibodies. We also determined that the concentration of excess bone marrow cells and number of treatments had a direct bearing on leukemic cell lysis. Although two cycles of treatment were significantly superior to one cycle, three cycles were not significantly superior to two cycles. Inclusion of DNase (10 micrograms/mL) was a critical adjunct that eliminated clumping and facilitated plating cells in the colony assay. Finally, we could show that striking differences existed in the sensitivity of the leukemic cell lines to lysis with the BA-1,2,3 cocktail and complement. NALM-6 cells were the most sensitive (approximately four logs of kill), and KM-3 cells were the most resistant (less than two logs of kill). Our results strongly support the utility of sensitive leukemic cell colony assays in the analysis of marrow treatment variables in autologous bone marrow transplantation.

Molecular cloning of human terminal deoxynucleotidyltransferase.

A cDNA of the human terminal deoxynucleotidyltransferase (TdT; "terminal transferase," EC 2.7.7.31) was isolated from a human lymphoblastoid cell cDNA library in lambda gt 11 by using immunological procedures. Four inserts containing 723 to 939 base pairs were recloned in pBR322 for hybridization and preliminary sequence studies. mRNA selected by hybridization to recombinant DNA was translated to a 58-kDa peptide that specifically immunoprecipitated with rabbit antibodies to calf terminal transferase and mouse monoclonal antibody to human terminal transferase. Blot hybridization of total poly(A)+ RNA from KM3 (TdT+) cells with nick-translated pBR322 recombinant DNA detected a message of about 2000 nucleotides, sufficient to code for the 580 amino acids in the protein. mRNA from terminal transferase- cells gave no signal in hybrid selection or RNA blot hybridization. The complete sequence of the 939-base-pair insert sequence was obtained from deletions cloned in pUC8. The DNA sequence contains an open reading frame coding for 238 amino acids, about 40% of the protein. Three peptides isolated by HPLC from tryptic digests of succinylated 58-kDa calf thymus terminal transferase were sequenced, providing 20, 18, and 22 residues of peptide sequence. A search of the translated sequence of the 939-base-pair insert shows three regions beginning after arginine that have greater than 90% homology with the sequence determined from the calf thymus terminal transferase peptides. These results provide unambiguous evidence that the human terminal transferase sequence has been cloned.

Functional compartmentation of DNA precursors in human leukaemoblastoid cell lines.

Summary. Human leukaemoblastoid cell lines were incubated with [3H]t hymidine and used to study the utilization for DNA synthesis and degradation of thymine nucleotides labelled via the salvage pathway. Three types of experiments were performed.(1) Cells were continuously labelled with exogenous [3H]thymidine at 3 7°C to compare the incorporation of label into DNA with the specific radioactivities of thymine nucleotides separated by chromatography on polyethyleneimine‐cellulose. These studies demonstrated whether one or more functional pool of thymine nucleotides was present.(2) Intracellular thymine nucleotide pools were labelled with [3H]thymidine at 13°C, and the cells were subsequently incubated in unlabelled medium at 37°C in order to quantify the percentage of thymine nucleotides incorporated into DNA. The results showed that 18%, 75%, 75% and 91% of intracellular thymine nucleotides were incorporated into DNA during the chase, in Raji (B lymphoid), Nalm‐6 (pre‐B cell), HL60 (myeloid) and KM3 (c‐ALL) cells respectively, whereas Molt4 (Thy‐ALL) cells incorporated 100%.(3) In order to quantify the activity of the degradative pathway for thymine nucleotides in these cell lines, cells were labelled as described in (2) in the presence of cytosine arabinoside (an inhibitor of DNA synthesis). 100%, 90%, 65%, 38% and 20% of labelled thymine nucleotides were degraded in Raji, HL60, Nalm‐6, KM3 and Molt‐4 respectively.These results confirm our previous conclusion (Taheri et al, 1981a, b) that thymine nucleotides are functionally compartmentalized in human cells. If these results with cell lines can be extrapolated to apply to de novo leukaemia cases, they suggest that the degree of compartmentation differs widely between different leukaemia cell types. Thy‐ALL cells utilized all their labelled thymine nucleotides for DNA synthesis, whereas B‐ALL, cells utilized only 18% of their labelled thymine nucleotide nucleotides. Thy‐ALL cells have the smallest degradative thymine pool and degradative capacity whereas B‐ALL cells possess the largest degradative pool and capacity. AML, pre‐B‐ALL, c‐ALL cell lines show intermediate results. If these results reflect those of the normal bone marrow cells from which each of the leukaemias arise, they suggest that with progressive B cell development in the bone marrow, degradative ability for DNA precursors increases and the efficiency of utilization of DNA precursors for DNA synthesis decreases.