Expression of coproporphyrinogen oxidase and synthesis of hemoglobin in human erythroleukemia K562 cells

Abstract

Coproporphyrinogen oxidase (CPOX), the sixth enzyme in the heme-biosynthetic pathway, catalyzes oxidative decarboxylation of coproporphyrinogen to protoporphyrinogen and is located in the intermembrane space of mitochondria. To clarify the importance of CPOX in the regulation of heme biosynthesis in erythroid cells, we established human erythroleukemia K562 cells stably expressing mouse CPOX. The CPOX cDNA-transfected cells had sevenfold higher CPOX activity than cells transfected with vector only. Expression of ferrochelatase and heme content in the transfected cells increased slightly compared with the control. When K562 cells overexpressing CPOX were treated with delta-aminolevulinic acid (ALA), most became benzidine-positive without induction of the expression of CPOX or ferrochelatase, and the heme content was about twofold higher than that in ALA-treated control cells. Increases in cellular heme concomitant with a marked induction of the expression of heme-biosynthetic enzymes, including CPOX, ferrochelatase and erythroid-specific delta-aminolevulinic acid synthase, as well as of alpha-globin synthesis, were observed when cells were treated with transforming growth factor (TGF)beta 1. These increases in the transfected cells were twice those in control cells, indicating that overexpression of CPOX enhanced induction of the differentiation of K562 cells mediated by TGF beta 1 or ALA. Conversely, the transfection of antisense oligonucleotide to human CPOX mRNA into untreated and TGF beta 1-treated K562 cells led to a decrease in heme production compared with sense oligonucleotide-transfected cells. These results suggest that CPOX plays an important role in the regulation of heme biosynthesis during erythroid differentiation.

Methotrexate used in combination with aminolaevulinic acid for photodynamic killing of prostate cancer cells

“…For example, murine erythroleukaemia MEL cells induced to differentiate by dimethylsulphoxide (DMSO), showed a large induction in mRNA for CPO within 10 h (Kohno et al, 1993). Likewise, human erythroleukaemia K562 cells undergoing differentiation in the presence of TGF-b, also displayed increased CPO expression (Taketani et al, 2001). In erythroid cells, differentiation-inducing agents induced several of the haeme-synthetic enzymes simultaneously (Taketani et al, 1995), but even under such circumstances, CPO may be rate-limiting and therefore important in the regulation of haeme and PpIX production (Conder et al, 1991).…”

A critical role for the co-repressor N-CoR in erythroid differentiation and heme synthesis

“…In erythrocytes, the enzyme ALA-S2 controls the rate-limiting step of the heme synthesis from glycine to 5-aminolevulinic acid [24]. Previous studies have shown that ALA-S2 is often induced during erythroid differentiation [25][26][27][28], we thus analyzed whether Ara-C also induces the expression of ALA-S2 in K562 cells. K562 cells were subjected to Ara-C-and hemin-induced erythroid differentiation for 3 days and the levels of ALA-S2 mRNA were then determined by RT-PCR.…”

Involvement of ABC7 in the biosynthesis of heme in erythroid cells: interaction of ABC7 with ferrochelatase

“…22 For the selection, G418 at a final concentration of 500 g/mL was added to the culture medium. After 7 days, the G418-resistant cells were diluted, seeded in a 96-well tissue-culture plate, and cultured in the medium containing 500 g/mL G418.…”

“…Immunoblotting was done with antiferrochelatase, anti-c-Myc-tag, anticatalase, and antithioredoxin antibodies as the primary antibodies. 6,22 Immunoprecipitation was performed as described 23 using antiferrochelatase or anti-c-Myc (9E10) antibodies.…”