T-leukemia Cells Research Articles

Effect of ABC transporters on HIV-1 infection: inhibition of virus production by the MDR1 transporter.

The MDR1 multidrug transporter P-gp (P-glycoprotein) is an efflux pump that extrudes diverse hydrophobic drugs and peptides from cells. Since the entry of HIV-1 into cells involves an initial interaction of the viral gp41 hydrophobic peptide with the plasma membrane, a potential effect of P-gp on HIV-1 infectivity was explored. Virus production was greatly decreased when P-gp was overexpressed at the surface of a continuous CD4(+) human T-leukemic cell line (12D7) infected with HIV-1(NL4-3), a T-tropic molecular clone of HIV-1. P-gp overexpression did not significantly alter the surface expression or distribution of either the HIV-1 receptor CD4 or the coreceptor CXCR4. Reduction of HIV-1 infectivity in P-gp-expressing cells occurred both during the fusion of viral and plasma membranes and at subsequent step(s) in the HIV-1 life cycle.

Expression of interleukin-4 in apoptotic cells: stimulation of the type-2 cytokine by different toxins in human peripheral blood mononuclear and tumor cells.

Immunological reactivity is regulated by T-cell populations (type-1 and type-2 cells) via cytokine secretion, but their influence on apoptosis remains unclear. Intracellular expression of type-1 (interferon [IFN]-gamma) and type-2 (interleukin [IL]-4) cytokines and apoptosis-related molecules (Apo2. 7, Bcl-2 protein) was studied by flow cytometry in human peripheral blood mononuclear cells (PBMC), myeloma (U-266), monocytic (THP-1), and T-leukemia cells (MOLT-4) in response to toxins, which act on different intracellular targets (actinomycin D, cycloheximide, the mistletoe lectins [ML]-1 and ML-3, brefeldin A, staurosporine). The apoptosis-inducing toxins stimulated intracellular IL-4 expression mainly in PBMC with high expression of the mitochondrial apoptosis marker, Apo2.7, but with decreased level of the anti-apoptotic Bcl-2 protein. Up-regulation of IL-4 coincided with a significant down-regulation of IFN-gamma in CD4(+) and CD8(+) cells. The inhibitor of oxidative phosphorylation, oligomycin, and the caspase inhibitor, z-VAD-fmk, abolished IL-4 expression and DNA fragmentation in the PBMC. Also in the myeloma, monocytic, and T-leukemia cells, IL-4 was mainly observed in the Apo2.7(+) apoptotic cells in response to the toxins. We suggest that the different apoptotic toxins activate a common pathway in which IL-4 production plays a yet unknown intracellular role further downstream during apoptosis.

Defective chemokine production in T-leukemia cell lines and its possible functional role.

Peripheral blood lymphocytes and T-cell clones produced nanogram quantities of the chemokines RANTES, MIP-lα, MIP-lβ, MCP-l, IL-8 and GRO-α as well as the motogenic cytokine HGF. In contrast, various T-leukemia cell lines at different stages of differentiation did not produce the same chemokines/cytokines. In order to study the possible functional importance of the poor chemokine production different T-cell lines were compared with respect to development of motile forms and migration on extracellular matrix components in the absence and presence of various chemokines. RANTES, MIP-1α, MIP-1β, IL-8, GRO-α and lymphotactin did not augment the development of motile forms including the size and appearance of the pseudopodia activity of the T-leukemia cell lines. The T-cell lines migrated spontaneously on/to fibronectin in a Boyden chamber assay system. Chemokines augmented the migration of the T-leukemia cell lines on fibronectin in the Boyden system in a chemotactic fashion with peak responses at 10 to 50 ng/ml. Thus, the production of chemokines is defective, in neoplastic T-lymphocytes. The defective chemokine production does not seem to play any major role for the basic locomotor capacity of the cells but may modulate the responsiveness to exogenous chemokines.

Isolation and Characterization of a Cytotoxic Pentapeptide, κ-casecidin, from Bovine κ-casein Digested with Bovine Trypsin

Bovine κ-casein produced cytotoxic activity toward mouse spleen cells when it was digested with bovine trypsin or chymotrypsin. The cytotoxic activity reached a maximum when the casein was digested with the trypsin at 45°C for 5h and was observed when the spleen cells were cultured for 6h with the digest. A cytotoxic peptide was isolated from a digest prepared from κ-casein treated with trypsin for 5h by a combination of ion-exchange column chromatography, gel filtration and reversed-phase high performance liquid chromatography. The sequence of the isolated peptide was estimated as Phe-Phe-Ser-Asp-Lys, being compatible with sequence 17-21 of bovine κ-casein. A chemically synthesized peptide, Phe-Phe-Ser-Asp-Lys, produced cytotoxic activity toward mouse spleen cells at a dose response manner. On the other hand, the isolated peptide retained cytotoxic activities toward animal cells such as mouse spleen T- and B-lymphocyte-enriched cells, bovine milk cells and Jurkat, Clone E6-1 cells (a cell line from human acute leukemic T-lymphocyte). The mouse lymphocytes cultured with the isolated peptide revealed an internucleosomal DNA fragmentation. The isolated peptide significantly inhibited the proliferative responses of mouse spleen cells stimulated by lipopolysaccharide, pokeweed mitogen, phytohemagglutinin and concanavalin A. Moreover, the isolated peptide showed notable antibacterial activity against Staphylococcus aureus IFO3060.

Molecular mechanisms of prostate cancer cell death upon inhibition of arachidonate 5-lipoxygenase: Involvement of FAS/APO1-mediated signals

It has recently been proposed that doxorubicin (DOX) can induce apoptosis in human T-leukemia cells via the Fas/FasL system in an autocrine/paracrine way. We show here that treatment of Jurkat cells with either anti-Fas antibodies, anthracyclin drugs or actinomycin D induces the activation of CPP32 (caspase-3) and apoptosis. However, DOX treatment did not induce the expression of membrane FasL or the release of soluble FasL and co-incubation with blocking anti-Fas antibodies prevented Fas-induced but not DOX-induced apoptosis. All the morphological and biochemical signs of apoptosis induced by anti-Fas or DOX can be prevented by Z-VAD-fmk, a general caspase inhibitor. DEVD-cho, a specific inhibitor of CPP32-like caspases which completely blocks Fas-mediated apoptosis, prevented drug-induced nuclear apoptosis but not cell death. We conclude that: (i) DOX-induced apoptosis in human T-leukemia/lymphoma is Fas-independent and (ii) caspase-3 is responsible of DOX-induced nuclear apoptosis but other Z-VAD-sensitive caspases are implicated in cell death.

Reduction ofmdrlGene Amplification in Human Multidrug-Resistant LoVo DX Cell Line Is Promoted by Triple Helix-Forming Oligonucleotides

We have demonstrated previously that the GT triplex-forming oligodeoxyribonucleotide (TFO) d(TGTGTTTTTGTTTTGTTGGTTTTGTTT), named TFO ID, targeted to a polypyrimidine-polypurine coding sequence located within human multidrug-resistance mdrl gene, specifically and significantly reduced mdrl mRNA levels in the drug-resistant T-leukemic CEM-VLB100 cell line. In this article, we demonstrate that TFO 1D is effective at inhibiting not only transcription but also replication of mdrl genes, leading to a loss of amplified gene copies in the drug-resistant colon adenocarcinoma LoVo DX cell line. In contrast, TFO ID does not alter replication of the constitutive mdrl gene copy in the corresponding parental sensitive LoVo 109 cell line. A specific reduction in mdrl gene amplification levels was also obtained with the pyrimidine TFO d(CTTTTTCTTTTCTTCCTTTTCTTT), named TFO 24TC, directed against the same polypyrimidine-polypurine sequence of the mdrl gene. We suggest that triple helix-forming oligonucleotides might affect the replication of unstable chromosomal elements as amplicons in actively replicating cells by causing a local impairment of DNA polymerase activity. This study lends support to the notion that TFO may be used to reduce gene amplification aiming to control neoplastic progression in cancer cells bearing amplified oncogenes.

Study on apoptosis of human acute T-lymphocyte leukemia cell line induced by allicin

Objective: To explore the law and role of apoptosis inducing effect of allicin on human acute T-lymphocyte leukemia cell line (6T-CEM).Methods: Terminal deoxylnucleotide transferase directed X-UTP nick and end labeling (TUNEL) assay, DNA gel electrophoresis, light microscopy and revers transcription-polymerase chair reaction (RT-PCR) were used to observe apoptosis of cells.Results: Allicin showed significant inducing effect on apoptosis of 6T-CEM cell when the concentration was 0.1 μg/ml, reaching its peak effect when the concentration was 50 μg/ml, with the apoptosis rate as 36.4%. Allicin (15 μg/ml) acted on 6T-CEM for 2 h and induced obvious cell apoptosis, the highest effect reached after 24 h of action, with the apoptosis rate as 56.9%. Actidione or actinomycin D could induce apoptosis of 6T-CEM, the allicin could not enhance their apoptosis inducing effect. Allicin could cause significant reduction of bcl-2 and bax gene expression of 6T-CEM, especially the bcl-2.Conclusion: Allicin could induce cell apoptosis of 6T-CEM with significant time and dose effect. Mechanism of the effect is through inhibiting the bcl-2 gene expression to reduce bcl-2/bax ratio.

Lineage switch in childhood leukemia with monosomy 7 and reverse of lineage switch in severe combined immunodeficient mice

Morphophenotypic lineage switches occur in a small percentage of those with acute leukemia, and the underlying mechanisms are not clear. In this study, we attempted to induce a lineage switch in acute myelocytic leukemia (AML) with monosomy 7, whose lineage had switched from acute T-lymphocytic leukemia (T-ALL) during chemotherapy, in severe combined immunodeficient (SCID) mice. Although the transplanted myeloid cells were engrafted in SCID mice without cytokine administration, T-ALL developed in SCID mice treated with recombinant human granulocyte-macrophage colony-stimulating factor or recombinant human interleukin 3. Analysis of the nucleotide sequences of the rearranged T-cell receptor γ-chain (TCR-γ) gene revealed that this lineage switch resulted from the selection of the T-lineage subclone in SCID mice, which had expanded at onset. In addition, we found that the T-lineage and myeloid cells belonged to the distinct subclones, which were different in TCR-γ gene rearrangements, but were derived from a common clone with an identical N- ras gene mutation for both subclones. In in vitro cultures, only the myeloid subclone grew; the T-lineage subclone failed to grow even in the presence of recombinant human granulocyte-macrophage colony-stimulating factor or recombinant human interleukin 3. These results suggested that the initial diagnostic T-lymphoid subclone, whose growth was dependent on these cytokines and the hematopoietic microenvironment, emerged from a bipotential T-lymphoid/myeloid leukemic stem cell, and further genetic event(s) induced the myeloid subclone, which grew independently of these cytokines and the microenvironment.

Regulation of Casein Kinase 2 by Direct Interaction with Cell Surface Receptor CD5

The transmembrane protein CD5, expressed on all T cells and the B1 subset of B cells, modulates antigen receptor-mediated activation. We used the yeast two-hybrid system to identify proteins that interact with its cytoplasmic domain and play a role in CD5 proximal signaling events. We found that the beta subunit of the serine/threonine kinase casein kinase 2 (CK2) interacts specifically with the cytoplasmic domain of CD5. Co-immunoprecipitation experiments showed activation-independent association of CK2 with CD5 in human and murine B and T cell lines and murine splenocytes. The interaction of CK2 holoenzyme with CD5 is mediated by the amino terminus of the regulatory subunit beta. CK2 binds and phosphorylates CD5 at the CK2 motifs flanked by Ser459 and Ser461. Cross-linking of CD5 leads to the activation of CD5-associated CK2 in a murine B-lymphoma cell line and a human T-leukemia cell line and is independent of net recruitment of CK2 to CD5. In contrast, CK2 is not activated following cross-linking of the B cell receptor complex or the T cell receptor complex. This direct regulation of CK2 by a cell surface receptor provides a novel pathway for control of cell activation that could play a significant role in regulation of CD5-dependent antigen receptor activation in T and B cells.

T-Cell Receptor Signaling Pathway Exerts a Negative Control on Thrombin-Mediated Increase in [Ca2+]i and p38 MAPK Activation in Jurkat T Cells: Implication of the Tyrosine Kinase p56Lck

Activation of the mitogen-activated protein kinase (Erk) and c-Jun terminal kinase is a well-documented mechanism for the seven transmembrane spanning receptors. We have previously shown that thrombin stimulation of the T-leukemic cell line Jurkat induced a transient increase in [Ca2+]i and tyrosine phosphorylation of several cellular proteins. Here, we have analyzed p42-44 MAPK, JNK and p38 MAPK activation using Jurkat T-cell lines deficient in either the tyrosine kinase p56Lck (JCaM1) or the tyrosine phosphatase CD45 (J45.01). Our results demonstrate that p56Lck and CD45 exert a negative control on thrombin-induced p38 MAPK activation and [Ca2+]i release in Jurkat cells. Thrombin receptor expression was identical on the different cell lines as assessed by FACS analysis. Tyrosine phosphorylation of p38 MAPK was drastically increased after thrombin stimulation of JCaM1 or J45.01 cells, as compared with parental cells (JE6.1). P42-44 MAPK and JNK activity also enhanced after thrombin treatment of JE6.1 and JCaM1 cell lines, whereas basal kinase activity was higher in J45.01 cells and was not further stimulated by thrombin. Thrombin and thrombin receptor agonist peptide-induced [Ca2+]imobilization paralleled p38 MAPK activation in JCaM1 and J45.01 cells. Moreover, reconstitution of J45.01 and JCaM1 cell lines with either CD45 or Lck is accompanied by restoration of a normal thrombin-induced [Ca2+]i response and p38MAPK phosphorylation. These data show that a component of the T-cell receptor signaling pathway exerts a negative control on thrombin-induced responses in Jurkat T cells. Accordingly, we found that thrombin enhanced tyrosine phosphorylation of p56Lck and decreased p56Lck kinase activity in J45.01 cells. Our results are consistent with a negative role for p56Lck on thrombin-induced [Ca2+]i release and p38 MAPK activation in Jurkat T-cell lines.

T-Cell Receptor Signaling Pathway Exerts a Negative Control on Thrombin-Mediated Increase in [Ca2+]i and p38 MAPK Activation in Jurkat T Cells: Implication of the Tyrosine Kinase p56Lck

AbstractActivation of the mitogen-activated protein kinase (Erk) and c-Jun terminal kinase is a well-documented mechanism for the seven transmembrane spanning receptors. We have previously shown that thrombin stimulation of the T-leukemic cell line Jurkat induced a transient increase in [Ca2+]i and tyrosine phosphorylation of several cellular proteins. Here, we have analyzed p42-44 MAPK, JNK and p38 MAPK activation using Jurkat T-cell lines deficient in either the tyrosine kinase p56Lck (JCaM1) or the tyrosine phosphatase CD45 (J45.01). Our results demonstrate that p56Lck and CD45 exert a negative control on thrombin-induced p38 MAPK activation and [Ca2+]i release in Jurkat cells. Thrombin receptor expression was identical on the different cell lines as assessed by FACS analysis. Tyrosine phosphorylation of p38 MAPK was drastically increased after thrombin stimulation of JCaM1 or J45.01 cells, as compared with parental cells (JE6.1). P42-44 MAPK and JNK activity also enhanced after thrombin treatment of JE6.1 and JCaM1 cell lines, whereas basal kinase activity was higher in J45.01 cells and was not further stimulated by thrombin. Thrombin and thrombin receptor agonist peptide-induced [Ca2+]imobilization paralleled p38 MAPK activation in JCaM1 and J45.01 cells. Moreover, reconstitution of J45.01 and JCaM1 cell lines with either CD45 or Lck is accompanied by restoration of a normal thrombin-induced [Ca2+]i response and p38MAPK phosphorylation. These data show that a component of the T-cell receptor signaling pathway exerts a negative control on thrombin-induced responses in Jurkat T cells. Accordingly, we found that thrombin enhanced tyrosine phosphorylation of p56Lck and decreased p56Lck kinase activity in J45.01 cells. Our results are consistent with a negative role for p56Lck on thrombin-induced [Ca2+]i release and p38 MAPK activation in Jurkat T-cell lines.

Expression and regulation of mRNA for distinct isoforms of cAMP-specific PDE-4 in mitogen-stimulated and leukemic human lymphocytes.

We reported previously that the gene for PDE-1B1 is induced in isolated human peripheral blood lymphocytes (HPBL) following mitogenic stimulation (Jiang, X., Li, J., Paskind, M., and Epstein, P.M. [1996] Proc. Natl. Acad. Sci. USA 93, 11,236-11,241). Using reverse transcription-polymerase chain reaction (RT-PCR), we investigated possible changes in the expression of the four genes for cAMP-specific phosphodiesterase (PDE-4A-D) in HPBL under the same conditions. Isolated, quiescent HPBL express mRNA for PDE-4B as the principal transcript. Following mitogenic stimulation with phytohemagglutinin (PHA), mRNA for PDE-4A and PDE-4D are clearly induced. HPBL appear not to express PDE-4C under resting or stimulated conditions. The PHA induced increase in PDE-1B1, PDE-4A, and PDE-4D mRNA is mimicked by incubation of HPBL with dibutyryl cAMP (dBcAMP) and 1-methyl-3-isobutylxanthine (IBMX). The B-lymphoblastoid cell line, RPMI 8392, and the T-leukemic cell line, Molt 4, express PDE-4A mRNA as the most abundant transcript, but incubation with dBcAMP and IBMX induces an increase in the expression of mRNA for PDE-4B in both of these cell lines, and in PDE-4D3 in the RPMI 8392 cell line. These studies demonstrate that expression of mRNA for PDE-1B1 and some of the subtypes of PDE-4 are induced in HPBL following mitogenic stimulation, possibly secondarily to elevation of cAMP induced by the mitogen. As already indicated for PDE-1B1, some of these subtypes of PDE-4 might also provide additional therapeutic targets for treatment of immunoproliferative disorders and immune dysfunction.

In VitroSide-View Imaging Technique and Analysis of Human T-Leukemic Cell Adhesion to ICAM-1 in Shear Flow

The objective of the present study is to apply a novel side-view imaging technique to investigate T-leukemic Jurkat cell adhesion to a surface-immobilized ICAM-1 in shear flow, a ligand for leukocyte LFA-1. Images have revealed that Jurkat cell adhesion on ICAM-1 under flow conditionsin vitrois quasistatic. The cell-substrate contact length steadily increased with time during the initial cell attachment to the ICAM-1-coated surface and subsequently decreased with time as the trailing edge of the cell membrane peeled away from the substrate under the influence of fluid shear forces. Changes in flow shear stresses, cell deformability, or substrate ligand strength resulted in a significant change in the characteristic adhesion binding time and contact length. A 3-D flow field with shear stresses acting on an adherent cell was calculated by using finite element methods based on cell shapes obtained from thein vitroimages. The maximum shear stress acting on an actual cell body was found to be 3–5 times greater than the upstream inlet wall shear stress and was influenced by the extent of cell deformation within the flow channel. Therefore, the application of such a side-view imaging technique has provided a practical assay to study the mechanics of cell-surface adhesion in 3-D. The elongation of cells in shear flow tempers hydrodynamic shear forces on the cell, which affects the transients in cell-surface adhesion.

Doxorubicin-induced apoptosis in human T-cell leukemia is mediated by caspase-3 activation in a Fas-independent way

It has recently been proposed that doxorubicin (DOX) can induce apoptosis in human T-leukemia cells via the Fas/FasL system in an autocrine/paracrine way. We show here that treatment of Jurkat cells with either anti-Fas antibodies, anthracyclin drugs or actinomycin D induces the activation of CPP32 (caspase-3) and apoptosis. However, DOX treatment did not induce the expression of membrane FasL or the release of soluble FasL and co-incubation with blocking anti-Fas antibodies prevented Fas-induced but not DOX-induced apoptosis. All the morphological and biochemical signs of apoptosis induced by anti-Fas or DOX can be prevented by Z-VAD-fmk, a general caspase inhibitor. DEVD-cho, a specific inhibitor of CPP32-like caspases which completely blocks Fas-mediated apoptosis, prevented drug-induced nuclear apoptosis but not cell death. We conclude that: (i) DOX-induced apoptosis in human T-leukemia/lymphoma is Fas-independent and (ii) caspase-3 is responsible of DOX-induced nuclear apoptosis but other Z-VAD-sensitive caspases are implicated in cell death.

Prolactin is an autocrine growth factor for the Jurkat human T-leukemic cell line

Despite convincing evidence of cooperation between IL-2 and endogenous prolactin (PRL) during T cell activation, the individual role of PRL as a T-cell lineage cytokine remains to be defined. We have examined the production and function of PRL on the Jurkat human T-leukemic cell line, which does not constitutively produce IL-2. The majority of Jurkat cells expressed PRL receptor (R) under standard culture conditions, whereas appearance of the α chain of the IL-2-R required PHA–PMA stimulation, as did IL-2 synthesis. Western blotting revealed a predominant band at 23.5 kDa and a weaker band at 25.5 kDa in both Jurkat cell lysates and human (h) pituitary PRL. Metabolic labeling of the cell lysates with 35 S -methionine and immunoprecipitation with an antiserum against hPRL showed that both forms of PRL are actively synthesized by the Jurkat cell line. PRL released in the medium was biologically active in the rat Nb2 lymphoma mitogenic assay. Depletion of medium PRL with two polyclonal anti-hPRL antisera inhibited the growth of Jurkat cells in a dose-dependent manner, as evaluated by cell number and 3 H -TdR uptake. Purified pituitary or recombinant hPRL at a wide range of concentrations had no significant effect on their growth, but reversed the blocking activity of the anti-hPRL antibody. Recombinant IL-2 had no effect on the antibody-induced growth inhibition. Taken as a whole, these results demonstrate that PRL can act as an autocrine T cell growth factor independently of IL-2 and are the first evidence of its involvement in human leukemic growth and possibly in leukemic transformation.

Plasminogen activators play an essential role in extracellular-matrix invasion by lymphoblastic T cells.

Involvement of extravascular sites, in particular infiltration of the central nervous system, is a frequent complication of T-lymphoblastic leukemia and contributes to leukemia-associated morbidity. In this report, we studied the contribution of plasminogen activators to the invasive properties of 7 human T-cell lines in a model of transmigration through an extracellular matrix. The T-cell lines were found to express either urokinase (u-PA) and high levels of u-PA receptor or tissue-type plasminogen activator (t-PA) and low levels of u-PA receptor. The rate of transmigration was consistently higher for u-PA-expressing cells than for t-PA-expressing cells. PA-inhibitor type 1 (PAI-1) was detected in the conditioned medium of one cell line and PAI-2 was detected in cell extracts from 6 lines. The transmigration of 6 out of 7 cell lines was inhibited by trasylol, an inhibitor of plasmin, by an excess of exogenous PAI-1 or PAI-2, and by antibodies to the particular PA type expressed by the cells. Partial inhibition of transmigration by the amino-terminal fragment of u-PA implies that the u-PA receptor contributes to transmigration. Thus, the transmigration of T-leukemia cells through a barrier of extracellular matrix requires PA-dependent proteolysis, which can be provided either by u-PA or t-PA. Specific inhibition of the PA system could provide a means to inhibit tissue invasion by T lymphoblastic cells.

Intracellular calcium signaling by Jurkat T-lymphocytes exposed to a 60 Hz magnetic field.

To explore possible biochemical mechanisms whereby electromagnetic fields of around 0.1 mT might affect immune cells or developing cancer cells, we studied intracellular calcium signaling in the model system Jurkat E6-1 human T-leukemia cells during and following exposure to a 60 Hz magnetic field. Cells were labeled with the intracellular calcium-sensitive fluorescent dye Fluo-3, stimulated with a monoclonal antibody against the cell surface structure CD3 (associated with ligand-stimulated T-cell activation), and analyzed on a FACScan flow-cytometer for increases in intensity of emissions in the range of 515-545 nm. Cells were exposed during or before calcium signal-stimulation to 0.15 mTrms 60 Hz magnetic field. The total DC magnetic field of 78.2 microT was aligned 17.5 degrees off the vertical axis. Experiments used both cells cultured at optimal conditions at 37 degrees C and cells grown under suboptimal conditions of 24 degrees C, lowered external calcium, or lowered anti-CD3 concentration. These experiments demonstrate that intracellular signaling in Jurkat E6-1 was not affected by a 60 Hz magnetic field when culture and calcium signal-stimulation were optimal or suboptimal. These results do not exclude field-induced calcium-related effects further down the calcium signaling pathway, such as on calmodulin or other calcium-sensitive enzymes.

CARCINOGENIC CHROMIUM(VI) INDUCES OXIDATIVE STRESS IN CULTURED HUMAN LEUKEMIC T-LYMPHOCYTES. 1. GENERATIO OF HYDROGEN PEROXIDE DURIN INTRACELLULAR REDUCTION OF CHROMATE

Treatment of human leukemic T-lymphocyte MOLT4 cells with potassium chromate, a chromium (VI) compound, increased the form ation of the oxidized compound dichlorofluorescein (DCF), a highly fluorescent compound, from the parent nonfluorescent compound, 2,7-dichlorofluorescin diacetate (DCF-DA). No such increase in DCF formation was observed when cells were treated with Cr(III) compounds. In cell-free system s, dichlorofluorescin diacetate was also oxidized to DCF by strong oxidants (such as hydrogen peroxide and hydroperoxides) in the presence of peroxidase, suggesting that Cr(VI) treatment increased the intracellular level of these peroxides in MOLT4 cells. The rate of generation of peroxides was found to be both dose and time dependent. These results suggest that accumulation of intracellular peroxides may, at least in part, be associated with chromium-induced genotoxicity and add to the emerging concept that these phenomena may, in part, be mediated via oxidative m echanisms.

Mechanisms of the Carcinogenic Chromium(VI)-induced DNA-Protein Cross-linking and Their Characterization in Cultured Intact Human Cells

DNA-protein complexes (DPCs) were induced in human leukemic T-lymphocyte MOLT4 cells by treatment with potassium chromate. DPCs were isolated by ultracentrifugal sedimentation in the presence of 2% SDS and 5 M urea. The complexes were analyzed by two-dimensional SDS-polyacrylamide gel electrophoresis. Three acidic proteins of 74, 44, and 42 kDa and a basic protein of 51 kDa were primarily complexed to DNA following 25 microM chromate treatment. Higher concentrations of chromate cross-linked many other proteins to DNA. Amino acid sequencing and immunoblotting studies indicated that the acidic 44-kDa protein could be nuclear beta-actin. Lectin and aminoglycoside nucleotidyltransferase were also found to cross-link with DNA by chromate treatment. The composition and stability of the DPCs were studied using nucleases, proteinase K, and disruptive chemicals. Pretreatment of cells with antioxidants inhibited the formation of DPCs, measured as K+-SDS precipitable DPCs, indicating the involvement of oxidative mechanisms. Because chromate causes certain nuclear proteins to form complexes with DNA and the complexes are resistant to treatments such as 2% SDS and 5 M urea, but disruptable under gel electrophoretic conditions, chromium could be used as a cross-linking agent for the identification of other proteins, such as transcription factors, that transiently interact with DNA.

Apoptosis as a mechanism of cell death induced by different chemotherapeutic drugs in human leukemic T-lymphocytes

The involvement of apoptosis in the mechanism of cell death induced by six clinically relevant anticancer drugs [methotrexate (MTX), doxorubicin (ADR), daunorubicin (DNR), vincristine (VCR), 6-mercaptopurine (6MP), and prednisolone (PRD)] in human leukemic T-lymphocytes (CCRF-CEM and Jurkat) was investigated by analysing changes in cell size and morphology, changes in membrane integrity, alterations in [Ca 2+] i and induction of DNA fragmentation. MTX, ADR, and DNR showed pronounced dose- and time-dependent cytotoxic effects on both cell lines, whereas cell viability was not considerably reduced by 6MP or PRD. On the other hand, the cytotoxic activity of VCR was much higher on Jurkat cells than on CEM cells. With the exception of 6MP and PRD, all the other compounds induced extensive chromatin condensation, nuclear fragmentation, plasma membrane blebbing, and formation of apoptotic bodies and fragmentation of DNA in both cell lines. Occurrence of DNA fragmentation always preceded loss of membrane integrity. These observations are consistent with cell death being mediated by apoptosis. Significant increases in [Ca 2+] i were only observed in CEM cells preincubated with MTX or DNR (10 μM). In contrast, MTX as well as VCR induced a reduction in the basal intracellular Ca 2+concentration in Jurkat T-cells. Although the ability to induce changes in [Ca 2+] i correlated with higher cytotoxic potency of the anticancer drugs, a causal relationship between increased [Ca 2+] iand induction of apoptosis could not be clearly established. These results, therefore, suggest no determinant role for Ca 2+ in triggering the process of endonucleolytic cleavage of genomic DNA in these leukemic T-lymphocytes.