Human Leukemia Jurkat Cells Research Articles

Suppression of Postmitochondrial Signaling and Delayed Response to UV‐induced Nuclear Apoptosis in HeLa Cells

Activation of postmitochondrial pathways by UV irradiation was examined using mouse lymphoma 3SB and human leukemic Jurkat cells and two human carcinoma cell lines (HeLa and MCF‐7). Exposure of 3SB and Jurkat cells resulted in large amounts of cytochrome c and apoptosis‐inducing factor (AIF) being released into the cytosol, and a clear laddering pattern of DNA fragments was observed within 3 h of incubation after irradiation. Simultaneously, activation of caspase‐9 and its downstream caspases was detected. HeLa and MCF‐7 cells also showed extensive release of mitochondrial factors and caspase‐9 activation at 4 to 6 h after exposure, but apoptotic nuclear changes appeared much later. Compared with 3SB and Jurkat cells, these carcinoma cell lines exhibited reduced activation of caspase‐9‐like proteolytic activity by UV radiation, and levels of caspase‐3‐like activity in HeLa cells were extremely low, similar to those in caspase‐3‐deficient MCF‐7 cells. These results suggest that the delayed response to UV‐induced nuclear apoptosis in HeLa cells is due to a reduced activation of the caspase cascade downstream of cytochrome c release and suppression of caspase‐3 activity.

Sphingosine 1-phosphate antagonizes apoptosis of human leukemia cells by inhibiting release of cytochrome c and Smac/DIABLO from mitochondria

AbstractSphingosine 1-phosphate (S-1P) has been implicated as a second messenger preventing apoptosis by counteracting activation of executioner caspases. Here it is reported that S-1P prevents apoptosis and executioner caspase-3 activation by inhibiting the translocation of cytochrome c and Smac/DIABLO from mitochondria to the cytosol induced by anti-Fas, tumor necrosis factor-α (TNF-α), serum deprivation, and cell-permeable ceramides in the human acute leukemia Jurkat, U937, and HL-60 cell lines. Furthermore, the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate, which stimulates sphingosine kinase, the enzyme responsible for S-1P production, also inhibits cytochrome c and Smac/DIABLO release. In contrast, dimethylsphingosine (DMS), a specific inhibitor of sphingosine kinase, sensitizes cells to cytochrome c and Smac/DIABLO release triggered by anti-Fas, TNF-α, serum deprivation, or ceramide. DMS-induced mitochondrial apoptogenic factor leakage can likewise be overcome by S-1P cotreatment. Hence, S-1P, likely generated through a protein kinase C– mediated activation of sphingosine kinase, inhibits the apoptotic cascade upstream of the release of the mitochondrial apoptogenic factors, cytochrome c, and Smac/DIABLO in human acute leukemia cells.

A Fas-associated Death Domain Protein-dependent Mechanism Mediates the Apoptotic Action of Non-steroidal Anti-inflammatory Drugs in the Human Leukemic Jurkat Cell Line

Non-steroidal anti-inflammatory drugs (NSAIDs) are inhibitors of cyclooxygenase-1 and -2 and are useful for prevention and cure of cancers, especially colon and rectal cancers. The NSAIDs indomethacin and sulindac sulfide have been shown to induce apoptosis of colon epithelial cancer cells by a Bax-dependent mechanism that involves mitochondria-mediated activation of a caspase-9-dependent pathway. In this report, we demonstrate that indomethacin and sulindac sulfide induce apoptosis of human leukemic Jurkat cells by a mechanism that requires the Fas-associated Death Domain Protein-mediated activation of a caspase-8-dependent pathway. Therefore, NSAIDs induce apoptosis by different mechanisms depending on the cell type.

Prooxidant activity of melatonin promotes fas-induced cell death in human leukemic Jurkat cells

The antioxidant activity of melatonin (MEL) has been considered to constitute part of its physiological as well as pharmacological effects. However, as described herein we found a profound prooxidant activity of micro- to millimolar concentrations of MEL in the human leukemic Jurkat cell line. This prooxidant effect was increased in glutathione-depleted cells and counteracted by antioxidants. As a consequence MEL promoted fas-induced cell death. These data therefore indicate that MEL may be a modulator of the cellular redox status, but does not necessarily act as an intracellular antioxidant.

Inhibition of Nitric-Oxide-Mediated Apoptosis in Jurkat Leukemia Cells despite Cytochrome c Release

We have recently shown that nitric-oxide (NO)-induced apoptosis in Jurkat human leukemia cells requires degradation of mitochondria phospholipid cardiolipin, cytochrome c release, and activation of caspase-9 and caspase-3. Moreover, an inhibitor of lipid peroxidation, Trolox, suppressed apoptosis in Jurkat cells induced by NO donor glycerol trinitrate. Here we demonstrate that this antiapoptotic effect of Trolox occurred despite massive release of the mitochondrial protein cytochrome c into the cytosol and mitochondrial damage. Incubation with Trolox caused a profound reduction of intracellular ATP concentration in Jurkat cells treated by NO. Trolox prevented cardiolipin degradation and caused its accumulation in Jurkat cells. Furthermore, Trolox markedly downregulated the NO-mediated activation of caspase-9 and caspase-3. Caspase-9 is known to be activated by released cytochrome c and together with caspase-3 is considered the most proximal to mitochondria. Our results suggest that the targets of the antiapoptotic effect of Trolox are located downstream of the mitochondria and that caspase activation and subsequent apoptosis could be blocked even in the presence of cytochrome c released from the mitochondria.

Antileukemic drugs increase death receptor 5 levels and enhance Apo-2L–induced apoptosis of human acute leukemia cells

AbstractIn present studies, treatment with tumor necrosis factor (TNF)–related apoptosis inducing ligand (TRAIL, also known as Apo-2 ligand [Apo-2L]) is shown to induce apoptosis of the human acute leukemia HL-60, U937, and Jurkat cells in a dose-dependent manner, with the maximum effect seen following treatment of Jurkat cells with 0.25 μg/mL of Apo-2L (95.0% ± 3.5% of apoptotic cells). Susceptibility of these acute leukemia cell types, which are known to lack p53wt function, did not appear to correlate with the levels of the apoptosis-signaling death receptors (DRs) of Apo-2L, ie, DR4 and DR5; decoy receptors (DcR1 and 2); FLAME-1 (cFLIP); or proteins in the inhibitors of apoptosis proteins (IAP) family. Apo-2L–induced apoptosis was associated with the processing of caspase-8, Bid, and the cytosolic accumulation of cytochrome c as well as the processing of caspase-9 and caspase-3. Apo-2L–induced apoptosis was significantly inhibited in HL-60 cells that overexpressed Bcl-2 or Bcl-xL. Cotreatment with either a caspase-8 or a caspase-9 inhibitor suppressed Apo-2L–induced apoptosis. Treatment of human leukemic cells with etoposide, Ara-C, or doxorubicin increased DR5 but not DR4, Fas, DcR1, DcR2, Fas ligand, or Apo-2L levels. Importantly, sequential treatment of HL-60 cells with etoposide, Ara-C, or doxorubicin followed by Apo-2L induced significantly more apoptosis than treatment with Apo-2L, etoposide, doxorubicin, or Ara-C alone, or cotreatment with Apo-2L and the antileukemic drugs, or treatment with the reverse sequence of Apo-2L followed by one of the antileukemic drugs. These findings indicate that treatment with etoposide, Ara-C, or doxorubicin up-regulates DR5 levels in a p53-independent manner and sensitizes human acute leukemia cells to Apo-2L–induced apoptosis.

Antileukemic drugs increase death receptor 5 levels and enhance Apo-2L–induced apoptosis of human acute leukemia cells

In present studies, treatment with tumor necrosis factor (TNF)–related apoptosis inducing ligand (TRAIL, also known as Apo-2 ligand [Apo-2L]) is shown to induce apoptosis of the human acute leukemia HL-60, U937, and Jurkat cells in a dose-dependent manner, with the maximum effect seen following treatment of Jurkat cells with 0.25 μg/mL of Apo-2L (95.0% ± 3.5% of apoptotic cells). Susceptibility of these acute leukemia cell types, which are known to lack p53wt function, did not appear to correlate with the levels of the apoptosis-signaling death receptors (DRs) of Apo-2L, ie, DR4 and DR5; decoy receptors (DcR1 and 2); FLAME-1 (cFLIP); or proteins in the inhibitors of apoptosis proteins (IAP) family. Apo-2L–induced apoptosis was associated with the processing of caspase-8, Bid, and the cytosolic accumulation of cytochrome c as well as the processing of caspase-9 and caspase-3. Apo-2L–induced apoptosis was significantly inhibited in HL-60 cells that overexpressed Bcl-2 or Bcl-xL. Cotreatment with either a caspase-8 or a caspase-9 inhibitor suppressed Apo-2L–induced apoptosis. Treatment of human leukemic cells with etoposide, Ara-C, or doxorubicin increased DR5 but not DR4, Fas, DcR1, DcR2, Fas ligand, or Apo-2L levels. Importantly, sequential treatment of HL-60 cells with etoposide, Ara-C, or doxorubicin followed by Apo-2L induced significantly more apoptosis than treatment with Apo-2L, etoposide, doxorubicin, or Ara-C alone, or cotreatment with Apo-2L and the antileukemic drugs, or treatment with the reverse sequence of Apo-2L followed by one of the antileukemic drugs. These findings indicate that treatment with etoposide, Ara-C, or doxorubicin up-regulates DR5 levels in a p53-independent manner and sensitizes human acute leukemia cells to Apo-2L–induced apoptosis.

Glutathione is a factor of resistance of Jurkat leukemia cells to nitric oxide-mediated apoptosis.

We have previously reported that nitric oxide (NO) stimulates apoptosis in different human neoplastic lymphoid cell lines through mitochondrial damage (including degradation of cardiolipin, a major mitochondrial lipid) followed by activation of caspases. Here we demonstrate that Jurkat human leukemia cells which survive after 24 h treatment with NO form subpopulations with higher and lower cardiolipin content (designated as NAO(high) and NAO(low), respectively). Sorted NAO(high) cells were found to survive in culture whereas sorted NAO(low) cells died. Moreover, NAO(high) cells acquired an increased resistance to the exposure to NO donors which remained unchanged during long-term culture. These cells showed a similar cardiolipin content and expressed the same level of anti-apoptotic proteins Bcl-2 and Bcl-x(L) as APO-S unsorted cells but contained significantly higher concentration of the antioxidant glutathione. Depletion of glutathione in these cells with buthionine-sulfoximine (BSO) correlated with a significant stimulation of NO-mediated apoptosis whereas the exposure of NO-sensitive APO-S cells to the glutathione precursor N-acetylcysteine (NAC) resulted in a substantial suppression of this effect. Our data suggest a complex mechanism of the resistence to NO-induced apoptosis in Jurkat human leukemia cells in which glutathione plays an important role.

Identification of high affinity binding sites for inhibin on ovine pituitary cells in culture.

The aim of this study was to identify and characterize binding sites for inhibin in primary cultures of ovine anterior pituitary cells. Recombinant human 31-kDa inhibin A was iodinated by an optimized lactoperoxidase procedure. Fractionation of the labeled protein by gel filtration chromatography on Sephadex G-100 in 0.1 M HCl yielded two immunoactive peak regions, the second of which was bioactive as assessed by in vitro bioassay, with a ratio of bioactivity/immunoactivity of 0.62-0.77 and an iodine incorporation ratio of 1.7-2.0 mol 125I/mol inhibin. The specific binding of purified [125I]inhibin to cultured ovine pituitary cells varied with time, temperature, and cell number. Displacement of the tracer by unlabeled inhibin, as assessed by Scatchard analysis, revealed two binding sites with average Kd values of 0.28 and 3.9 nM and with approximately 250 and 3100 binding sites/anterior pituitary cell, respectively. There was little cross-reaction between inhibin and activin A (<2%), transforming growth factor-beta (<0.2%), or follistatin (<<0.1%). Examination of cell lines that were not expected to have inhibin receptors showed that there was no specific binding of inhibin to human leukemia (Jurkat) cells, whereas the binding to human embryonic kidney (293) cells was displaced by both inhibin and activin with a similar degree of cross-reaction, which suggests binding to an activin receptor. It is concluded that inhibin-binding sites with high affinity and specificity have been identified on ovine pituitary cells, consistent with both inhibin action on the pituitary and the presence of the putative inhibin receptor.

Different Moieties of Tautomycin Involved in Protein Phosphatase Inhibition and Induction of Apoptosis

The effects of tautomycin and its derivatives on protein phosphatases PP1 and PP2A and their apoptosis-inducing activity toward human leukemia Jurkat cells were examined, and the relationship between chemical structure and function was discussed. Among the compounds we examined, tautomycin was the most potent inhibitor and the most effective inducer of apoptosis. It inhibited PP1 and PP2A enzymatic activity concentration-dependently with ic 50 values of 20 and 75 pM, respectively, in the presence of 0.01% Brij-35, and an lc 50 value of 1 μM. Esterification of the anhydride moiety of tautomycin markedly increased the ic 50 for the protein phosphatases. The C 1′-C 7′ fragment of tautomycin had no inhibitory effect, but the fragment containing the C 22-C 26 moiety was inhibitory. These results suggest that the C 22-C 26 moiety is essential for inhibition of protein phosphatase activity and that the anhydride moiety enhances the inhibition. However, the esterification of the anhydride did not decrease, nor did the inclusion of the C 22-C 26 moiety increase the apoptosis-inducing activity. On the other hand, the C 1-C 18 moiety of tautomycin was essential for induction of apoptosis, and the conformation and the arrangement of functionalities of the C 18-C 26 carbon chain affected the apoptosis activity. However, modification of C 1-C 18, C 1-C 21, or C 1-C 26 compounds had little effect on phosphatase inhibitory activity. Our results strongly suggest that different moieties of tautomycin are involved in protein phosphatase inhibition and induction of apoptosis.

Induction of CD38/NADase and Its Monoclonal Antibody-Induced Tyrosine Phosphorylation in Human Leukemia Cell Lines

We previously reported that CD38 characterized as an ecto-enzyme of NAD+glycohydrolase (NADase) was specifically induced by retinoic acid (RA) in human promyelocytic leukemia HL-60 cells and that anti-CD38 monoclonal antibody (mAb) induced tyrosine phosphorylation of cellular proteins in the RA-differentiated cells. In the present study, we found that CD38/NADase was induced in human monocytic leukemia THP-1 cells not only by RA but also by dibutyryl cAMP, which had no effect on the induction of CD38 in HL-60 cells. Similarly in the RA-differentiated HL-60 cells, tyrosine phosphorylation by anti-CD38 mAb was also observed in the CD38-expressing THP-1 cells, regardless of whether CD38 was induced by RA or dibutyryl cAMP. Such tyrosine phosphorylation was, however, not observed in human lymphoblastic leukemia Jurkat cells which had been treated by RA to produce CD38. Thus, the induction of CD38/NADase appeared to be not limited for RA-dependent differentiation and not always linked to protein-tyrosine phosphorylation in human leukemic cell lines.