Pisosterol induces interphase arrest in HL60 cells with C-MYC amplification

The leukaemia cell line HL60 is widely used in studies of the cell cycle, apoptosis and adhesion mechanisms in cancer cells. One marked characteristic of HL60 cells is the C-MYC proto-oncogene amplification, resulting in the formation of homogeneously staining regions (HSRs) at 8p24. We conducted a fluorescence in situ hybridization study in an HL60 cell line, using a locus-specific probe for C-MYC, before and after treatment with pisosterol (at 0.5, 1.0 and 1.8 μg/mL), a triterpene isolated from the fungus Pisolithus tinctorius. Before treatment, 87.5% of the cells showed HSRs. After treatment, no effects were detected at lower concentrations of pisosterol (0.5 and 1.0 μg/mL). However, at 1.8 μg/mL only 15% of the cells presented HSRs, and 39.5% presented few fluorescent signals (3 or 4 alleles), suggesting that pisosterol probably blocks the cells with HSRs at interphase. This result is particularly interesting because cells that do not show a high degree of C-MYC gene amplification have a less aggressive and invasive behaviour and are easy targets for chemotherapy. Therefore, further studies are needed to examine the use of pisosterol in combination with conventional anti-cancer therapy.

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Studies on an adherent variant of the human promyelomonocytic HL60 leukaemia cell line that constitutively expresses 25-hydroxyvitamin D3-24-hydroxylase activity which is inhibited by 1α,25-dihydroxyvitamin D3

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0070185 ◽

1991 ◽

Vol 7 (3) ◽

pp. 185-195 ◽

Cited By ~ 5

Author(s):

M. E. Hayes ◽

D. Bayley ◽

E. B. Mawer

Keyword(s):

Cell Proliferation ◽

Cell Line ◽

Constitutive Expression ◽

Hl60 Cell ◽

Dependent Manner ◽

Hydroxylase Activity ◽

Leukaemia Cell ◽

Leukaemia Cell Line ◽

Hl60 Cells ◽

Dose Dependent

ABSTRACT The constitutive expression of 25-hydroxyvitamin D3-24-hydroxylase (25-(OH)D3-24-hydroxylase) activity has been studied in an adherent variant (Ad-HL60) of the human promyelomonocytic leukaemia cell line HL60. The Ad-HL60 cells have a more differentiated phenotype than the non-adherent cells from which they were derived, and synthesized 1.88±0.07 (±s.e.m.) pmol 24,25-(OH)2D3/h per 106 cells following culture in RPMI-1640 medium containing <0.02 nm 1α,25-(OH)2D3. They also synthesized 1.66±0.05 pmol 24,25-(OH)2D3/h per 106 cells following culture in 1α,25-(OH)2D3-free medium supplemented with 1 g bovine serum albumin/l instead of 10% serum. In contrast, non-adherent HL60 cells required exposure to 10–100 nm 1α,25-(OH)2D3 to induce equivalent 24,25-(OH)2D3 synthesis. The 25-(OH)D3-24-hydroxylase expressed by Ad-HL60 cells had an apparent Michaelis constant of 1 μm and maximal rate of 20 pmol/h per 106 cells with substrate concentrations from 0.012 to 1.2 μm/incubation (5–500ng/ml). Furthermore, 24,25-(OH)2D3 synthesis was inhibited in a dose-dependent manner by ketoconazole (0.01–10 μm), suggesting that the enzyme is cytochrome P-450 dependent. Ad-HL60 cells expressed approximately 3500 specific receptors for 1α,25-(OH)2D3/cell with a dissociation constant of 40 pm. Following exposure to 0.1–100 nm 1α,25-(OH)2D3, Ad-HL60 cell proliferation was significantly inhibited compared with controls grown in medium containing <0.02 nm 1α,25-(OH)2D3 for 96h. Expression of 25-(OH)D3-24-hydroxylase was also inhibited in a dose- and time-dependent manner; however, expression of non-specific esterase was not induced. Both of these findings are contrary to those previously demonstrated for non-adherent HL60 cells, whereas the dose-dependent inhibition of cell proliferation by 1α,25-(OH)2D3 occurs in both adherent and non-adherent phenotypes. These observations on Ad-HL60 cells represent the first description of a cell type in which 1α,25-(OH)2D3 appears to inhibit 25-(OH)D3-24-hydroxylase activity. The Ad-HL60 cells also constitutively metabolized 1α,25-(OH)2D3 in a manner consistent with formation of 1α,24,25-(OH)3D3 without previous exposure to 1α,25-(OH)2D3. In contrast, many other cell types, including non-adherent HL60 cells, require exposure to 1α,25-(OH)2D3 to induce metabolism of 1α,25-(OH)2D3 to 1α,24,25-(OH)3D3, a reaction that represents the initial step for catabolism of 1α,25-(OH)2D3 to calcitroic acid.

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Down-Regulation of Interleukin-8 Gene Expression in HL60 Cell Line by Human Kunitz-Type Trypsin Inhibitor

Biochemical and Biophysical Research Communications ◽

10.1006/bbrc.1995.1131 ◽

1995 ◽

Vol 206 (3) ◽

pp. 927-934 ◽

Cited By ~ 37

Author(s):

K. Maehara ◽

N. Kanayama ◽

A. Halim ◽

E. Elmaradny ◽

T. Oda ◽

...

Keyword(s):

Gene Expression ◽

Cell Line ◽

Trypsin Inhibitor ◽

Interleukin 8 ◽

Hl60 Cell ◽

Down Regulation ◽

Kunitz Type ◽

Hl60 Cell Line

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Effect of bixin on DNA damage and cell death induced by doxorubicin in HL60 cell line

Human & Experimental Toxicology ◽

10.1177/0960327116630352 ◽

2016 ◽

Vol 35 (12) ◽

pp. 1319-1327 ◽

Cited By ~ 7

Author(s):

GC Santos ◽

MR Almeida ◽

LMG Antunes ◽

MLP Bianchi

Keyword(s):

Dna Damage ◽

Hl60 Cell ◽

Red Pigment ◽

Combined Treatments ◽

Therapeutic Approaches ◽

Cytotoxic Effects ◽

Hl60 Cells ◽

Apoptotic Effect ◽

Induction Of Apoptosis ◽

Hl60 Cell Line

Bixin is a natural red pigment extracted from annatto. Although it is widely used as a coloring agent in food, there are few studies about the effect of this carotenoid on DNA. This study aimed to investigate the effects of bixin on cytotoxicity and genotoxicity induced by doxorubicin in HL60 cells. At concentrations above 0.3 μg/mL, bixin demonstrated cytotoxic effects in HL60 cells. Furthermore, this carotenoid was neither mutagenic nor genotoxic to HL60 cells and reduced the DNA damage induced by doxorubicin. Bixin and doxorubicin showed no apoptotic effect in HL60 cells, but the simultaneous combined treatments showed an increase in the percentage of apoptotic cells. In conclusion, our results showed that bixin modulates the cytotoxicity of doxorubicin via induction of apoptosis. The results of this study provide more knowledge about the toxic effects of anticancer treatments and how the natural compounds can be useful on these therapeutic approaches.

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Effect of Second Mitochondria-Derived Activator of Caspases (Smac) Mimetic Compounds on Leukemic Cell Lines.

Blood ◽

10.1182/blood.v112.11.1609.1609 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1609-1609

Author(s):

Federica Servida ◽

Francesco Onida ◽

Domenico Delia ◽

Cinzia Scavullo ◽

Daniele Lecis ◽

...

Keyword(s):

Cell Line ◽

Cell Lines ◽

Cytotoxic Effect ◽

Leukemic Cell ◽

Hl60 Cell ◽

Caspase 9 ◽

Leukemic Cell Lines ◽

Smac Mimetics ◽

Smac Mimetic ◽

Hl60 Cell Line

Abstract The apoptotic process and its dysfunctions have become the focus of extensive pharmaceutical research in solid and hematopoietic tumors as well as neurodegenerative diseases. The X-Inhibitor of Apoptosis Protein (XIAP) binds caspase 9, 3 and 7, preventing their activation and, consequently, apoptosis. The Smac/DIABLO protein, released from mitochondria, binds XIAP as a dimer on the same caspase 9 (BIR3 domain) binding site. Similarly, the Smac protein interferes with the XIAP binding site for caspases 3 and 7, thus promoting both the extrinsic and intrinsic apoptotic paths. The thin balance of this binding equilibrium is altered in various tumors, including leukemia, where XIAP is overexpressed and a caspase-dependent resistance to enter apoptosis is usually observed. Thus, XIAP inhibition via Smac mimetics’ binding is at the same time a characterized protein-protein interaction, and a validated mechanism for intervention in cancer therapy. We tested 56 Smac mimetic compounds (designed by CISI - Center for biomolecular Interdisciplinary Studies and Industrial applications of the Milan University) for their in vitro capacity to bind to the XIAP BIR3 domain. We also evaluated the ability of the Smac mimetic compounds to inhibit the growth of the human leukemia HL60, K562 and Jurkat cell lines (derived from patients with promyelocyitic leukemia, blastic phase-CML and T acute lymphoblastic leukemia, respectively). Nine compounds which were shown to be active, were further investigated for their effect on cell cycle (by DNA staining with propidium iodide and cytofluorimetric analysis) and for possible synergistic effect in combination with other chemotherapeutic drugs (Cytarabine, Etoposide and Idarubicine). The same compounds were also tested on normal CD34+ hematopoietic progenitor cells. The cytotoxicity was evaluated after 72 hours treatment with Smac mimetic compounds by a colorimetric assay for the quantification of cell proliferation and viability based on the cleavage of the WST-8 tetrazolium salt by mitochondrial dehydrogenases. The effect of Smac mimetic compounds on CD34+ cells enriched from mobilized peripheral blood was assessed as the capability of inhibiting the myeloid colony growth (CFU-GM). The data were expressed as mean percentage of 3 replicates normalized to the untreated control. Overall, a strong correlation between the binding affinity to the XIAP BIR3 domain and the cytotoxic effect on the leukemic cell lines was observed. The more promising compounds showed IC50 ranging from 0,3 to 1 microM on the HL60 cell line. The Jurkat and K562 cell lines were less sensitive, with IC50 ranging from 11,8 microM to more than 50 microM. However, in the K562 cell line, the combined treatment unveiled synergistic effect with Cytarabine and Etoposide (R Kern index = 1,4 and 1,5 respectively). No cytotoxic effect was observed on normal controls at doses up to 80 microM. A consistent sub G1 apoptotic peak (up to 53% of apoptotic cells) was observed in the HL60 cell line after 48 hrs treatment, thus suggesting a strong activation of the apoptotic process. All together, our data suggest that Smac mimetics may have a promising therapeutic potential as a new class of anticancer drugs in hematopoietic malignancies. Further experiments are currently ongoing to confirm the effectiveness of these compounds also on primary cells from leukemia patients, both as single agents and in combination with conventional drugs. In particular, due to their ability to enhance pro-apoptotic effect, Smac mimetic compounds may allow to overcome resistance of cancer cells to standard chemotherapy.

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C-myc containing episomes in the HL60 cell line are replaced by double minutes (DMs) as the passage number increases

Cancer Genetics and Cytogenetics ◽

10.1016/0165-4608(89)90283-5 ◽

1989 ◽

Vol 41 (2) ◽

pp. 236-237

Keyword(s):

Cell Line ◽

Hl60 Cell ◽

Double Minutes ◽

Passage Number ◽

Hl60 Cell Line

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KIF20A, highly expressed in immature hematopoietic cells, supports the growth of HL60 cell line

International Journal of Hematology ◽

10.1007/s12185-018-2527-y ◽

2018 ◽

Vol 108 (6) ◽

pp. 607-614 ◽

Cited By ~ 7

Author(s):

Hiroyuki Morita ◽

Akihito Matsuoka ◽

Jun-ichiro Kida ◽

Hiroyuki Tabata ◽

Kaoru Tohyama ◽

...

Keyword(s):

Cell Line ◽

Hematopoietic Cells ◽

Hl60 Cell ◽

Hl60 Cell Line

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ADP-ribosylation of the GTP-binding protein RhoA blocks cytoplasmic division in human myelomonocytic cells

Biochemical Journal ◽

10.1042/bj3080853 ◽

1995 ◽

Vol 308 (3) ◽

pp. 853-858 ◽

Cited By ~ 11

Author(s):

M Aepfelbacher ◽

M Essler ◽

K Luber De Quintana ◽

P C Weber

Keyword(s):

Growth Regulation ◽

Hl60 Cell ◽

Nuclear Staining ◽

Dependent Manner ◽

Rho Proteins ◽

Hl60 Cells ◽

Adp Ribosylation ◽

Gtp Binding ◽

C3 Exoenzyme

To test the role of Rho GTP-binding proteins in growth regulation of human myelomonocytic tumour cells we used recombinant C3 exoenzyme of Clostridium botulinum to specifically ADP-ribosylate and inactivate Rho proteins in situ. In homogenates of HL60 cells, the C3 exoenzyme [32P]ADP-ribosylated one protein that was identified as RhoA by immunoblot and two-dimensional gel electrophoresis. [32P]ADP ribosylation of RhoA in HL60 homogenates in vitro was reduced to 10-20% when cells in culture were pretreated with C3 exoenzyme (10 micrograms, 24 h), indicating that 80-90% of RhoA could be ADP-ribosylated in situ. The C3 exoenzyme inhibited HL60 cell proliferation by up to 80% and the degree of growth inhibition correlated with the amount of in situ ADP-ribosylated RhoA in a time- and dose-dependent manner. Cell cycle analysis demonstrated that the C3 exoenzyme-treated HL60 cells accumulated in mitosis, and nuclear staining revealed binucleated cells. These findings suggest that RhoA has a key role in human myelomonocytic tumour cell growth by regulating cytoplasmic division.

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