Modulator of heme biosynthesis induces apoptosis in leukemia cells.
OBJECTIVE
The purpose of this research is the investigation of the possible cause(s) of the dark-cell death phenomenon induced by 1,10-phenanthroline (Oph), a porphyrin biosynthesis modulator.


BACKGROUND
We have previously shown that porphyrin biosynthesis modulators, such as Oph, which is also an iron-chelating agent, enhance protoporphyrin IX (Proto) accumulation in mammalian neoplastic cells treated with delta-aminolevulinic acid (ALA). As a result of the enhanced Proto accumulation, a significant increase in photodynamic damage was observed under illumination. Also tetrapyrrole and heme-biosynthesis modulators have been shown to cause death in treated insect larvae in darkness, a phenomenon referred to as dark-cell death. Dark-cell death was also observed in Oph + ALA-treated transformed mammalian cells.


METHODS
Neoplastic cells were treated with ALA, Oph, and ALA + Oph, and the following cell properties were investigated: growth arrest, membrane permeability, cell survival, nucleosomal cleavage, and cell cycle alterations.


RESULTS
It was observed that Oph but not ALA induced growth arrest, in a T-cell leukemia line (MLA 144) as assessed by reduction in DNA synthesis. Exogenous Proto and isomers of Oph lacking the iron-chelating property of Oph also caused a dose-dependent inhibition of proliferation in MLA 144 cells. Although the plasma membrane of Oph-treated cells remained intact following 3 h of dark-incubation, the cells exhibited DNA internucleosomal cleavage, characteristic of cells undergoing apoptosis. Cell cycle analysis using the DNA intercalating dye propidium iodide (PI) coupled to flow cytometry, indicated that 81 +/- 5.6% of Oph-treated MLA 144 cells were apoptotic, with the majority of the cells arrested in the early S phase. On the other hand, treatment with either ALA or Proto did not alter the cell cycle. Also, using a double-labeling protocol with Hoechst 33342, and PI, and analysis by flow cytometry, Oph-treated cells were found to be 82% apoptotic after 3 h of dark-incubation. Apoptosis was reduced by 75% (p < 0.05) by the cytoplasmic protein synthesis inhibitor cycloheximide.


CONCLUSIONS
These results indicate that in addition to enhancing Proto accumulation, the heme biosynthesis modulator Oph also induces growth arrest and apoptosis in transformed cells in darkness.
Rebeiz
N
N
Laboratory of Immunophysiology, Department of Animal Sciences, University of Illinois at Urbana-Champaign, Illinois, USA. CRebeiz@uiuc.edu
Arkins
S
S
Kelley
K W
KW
Durack
G
G
Rebeiz
C A
CA
D000622:Aminolevulinic Acid / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002453:Cell Cycle / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002463:Cell Membrane Permeability / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004247:DNA / Q000096:biosynthesis; D003624:Darkness; D005434:Flow Cytometry; D006922:Hylobates; D007502:Iron Chelating Agents / Q000494:pharmacology; D007938:Leukemia / Q000378:metabolism; D008027:Light; D008223:Lymphoma / Q000378:metabolism; D016555:Matched-Pair Analysis; D009707:Nucleosomes / Q000187:drug effects; D010618:Phenanthrolines / Q000494:pharmacology; D017319:Photosensitizing Agents / Q000378:metabolism / Q000494:pharmacology*; D011524:Protoporphyrins / Q000378:metabolism / Q000494:pharmacology; D011963:Receptors, GABA-A / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000187:drug effects
D007502:Iron Chelating Agents; D009707:Nucleosomes; D010618:Phenanthrolines; D017319:Photosensitizing Agents; D011524:Protoporphyrins; D011963:Receptors, GABA-A; D000622:Aminolevulinic Acid; D004247:DNA; C028025:protoporphyrin IX; C025205:1,10-phenanthroline