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Changes in nucleoside transport of HL-60 human promyelocytic cells during N,N-dimethylformamide induced
Abstract:
The rate of nucleoside transport decreased profoundly in human promyelocytic leukemia HL-60 cells after myeloid differentiation was induced by 5-6 days of exposure to 0.8% N,N-dimethylformamide (DMF). The facilitated diffusion of 100 microM radiolabeled adenosine and 2'-deoxyadenosine, measured by rapid transport assays, decreased 10- to 20-fold. The transport of 2 microM coformycin or 2'-deoxycoformycin, which is mediated by the same mechanism and was monitored by the adenosine deaminase titration assay, decreased 29-fold. The reduction in nucleoside transport capacity after DMF treatment was confirmed by a 19-fold decrease in the number of specific binding sites per cell (from 24-30 X 10(4) to 1.2-1.7 X 10(4)) for [3H]-6-p-nitrobenzylthioinosine, a nucleoside transport inhibitor. The binding affinity of 6-p-nitrobenzylthioinosine was not altered significantly and nucleoside transport remained sensitive to the transport inhibitors, 6-p-nitrobenzylthioinosine, dipyridamole, and dilazep after DMF-induced maturation. Time-dependence studies showed that the rate of 100 microM deoxyadenosine transport was unchanged for the first 24 h of exposure to DMF but fell to about 36% of control rates at 24-26 h and then gradually decreased further to about 4-5% of control rates after 5-6 days. In contrast, transport rates of the purine bases were reduced only 2- to 3-fold in HL-60 cells after 5 days of DMF treatment. The rates of adenosine and deoxyadenosine transport were unchanged or reduced by no more than 2-fold after 5-6 days of exposure to 0.8% DMF in the following human tumor cell lines that are not inducible with DMF: ARH-77 (multiple myeloma), KG-1 (acute myelogenous), and K-562 (chronic myelogenous). Thus, changes in nucleoside transport may serve as an early, membrane-associated marker of differentiation of the HL-60 cell line.
Insights
Myeloid differentiation in HL-60 cells, induced by N,N-dimethylformamide (DMF), profoundly reduces nucleoside transport rates. This decrease in nucleoside transporter function is linked to fewer binding sites and may serve as a differentiation marker.
Area of Science:
- Cellular Biology
- Molecular Pharmacology
- Cancer Research
Background:
- Nucleoside transport is crucial for cellular metabolism and proliferation.
- HL-60 cells are a human promyelocytic leukemia cell line commonly used to study myeloid differentiation.
- N,N-dimethylformamide (DMF) is a known inducer of myeloid differentiation in HL-60 cells.
Purpose of the Study:
- To investigate the effect of DMF-induced myeloid differentiation on nucleoside transport in HL-60 cells.
- To determine if changes in nucleoside transport can serve as a marker for HL-60 cell differentiation.
Main Methods:
- HL-60 cells were exposed to 0.8% N,N-dimethylformamide (DMF) for 5-6 days to induce myeloid differentiation.
- Nucleoside transport rates for radiolabeled adenosine and 2'-deoxyadenosine were measured using rapid transport assays.
- The transport of coformycin and 2'-deoxycoformycin was assessed via adenosine deaminase titration assays.
- The number of specific binding sites for the nucleoside transport inhibitor [3H]-6-p-nitrobenzylthioinosine was quantified.
- Transport rates of purine bases and nucleoside transport in other cell lines were also evaluated.
Main Results:
- DMF-induced differentiation caused a 10- to 20-fold decrease in adenosine and 2'-deoxyadenosine transport.
- Coformycin and 2'-deoxycoformycin transport decreased by 29-fold, indicating a reduction in transporter function.
- The number of nucleoside transporter binding sites decreased by 19-fold, while binding affinity remained unchanged.
- Purine base transport was reduced only 2- to 3-fold, and nucleoside transport was largely unaffected in non-inducible cell lines.
Conclusions:
- Myeloid differentiation in HL-60 cells significantly impairs nucleoside transport capacity.
- The observed reduction in nucleoside transport is primarily due to a decrease in the number of transporter binding sites.
- Changes in nucleoside transport serve as an early, membrane-associated marker of HL-60 cell differentiation.