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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online May 18, 2001 as doi:10.1096/fj.00-0738fje. |
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Brain Research Institute,
* Pharmacological Institute and
Department of Surgery, University of Vienna, 1090 Vienna, Austria; and
Howard Hughes Medical Institute Laboratories, Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA
2Correspondence: Brain Research Institute, University of Vienna, Division of Biochemistry and Molecular Biology, Spitalgasse 4, A-1090 Vienna, Austria. E-mail: christian.pifl{at}univie.ac.at
SPECIFIC AIMS
The aim of our study was to determine the mechanism and site of action of the cytotoxic potential of the catecholamine transmitters dopamine (DA) and norepinephrine (NE). We established SK-N-MC neuroblastoma cell lines stably transfected with the cDNAs of the human DA transporter (DAT) or NE transporter and studied the effects of DA and NE in cells with and without catecholamine uptake.
PRINCIPAL FINDINGS
1. Low micromolar concentrations of DA or NE (1–10 µM) inhibit
cell growth
Exposure of cells expressing DA uptake at a maximal initial rate
(Vmax) of 18 to 24
pmol/min·105 cells to 10 µM
DA in the culture medium had a profound effect on cell growth: instead
of the normal time-dependent increase, there was a gradual loss of
viable cells (Fig. 1A
). The effect of DA was related to the level of DAT
expression (Fig. 1B
) and could be prevented by the transport
blocker mazindol; in this low micromolar range, DA had no effect in
parental cells lacking the DAT (Fig. 1C
). NE, which is a
substrate of the DAT, had an effect slightly stronger than DA on growth
of our DAT-expressing cells (Fig. 1D
, E
). The
noncatecholamine substrate of DAT 1-methyl-4-phenylpyridinium
(MPP+) was, in contrast, less effective than the
two catecholamines (Fig. 1D
, E
).
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We could rule out the possibility that transporter activation and the necessary higher energy demand per se elicited cell growth inhibition. Neither DA or NE receptor blockers nor cell-permeable blockers of intracellular signaling pathways modified the catecholamine effect.
2. Oxidative stress is not involved in catecholamine inhibition of
cell growth
None of the known experimentally effective antioxidants prevented
the cell loss induced by DA in the DAT-expressing cells. Nor did we
find any accumulation of intracellular oxidants. We used the inhibition
of aconitase activity and the fluorescence of hydroethidine as a
measure of intracellular superoxide radicals and the fluorescence of
dichlorofluorescein as an indicator of hydrogen peroxide. Whereas the
toxic transporter substrate MPP+ produced effects
indicative of oxidative stress in our cells, intracellular accumulation
of DA or NE did not. Thus, a major contribution of oxidative stress to
the catecholamine effects studied can be excluded.
3. Catecholamines arrest cell cycle progression and induce
apoptosis
Since cell growth and cell death are intimately connected to cell
cycle events, we determined the effect of DA and NE on cell cycle by
flow cytometry based on DNA content. In cells without DA uptake,
neither 10 µM DA nor 10 µM NE added for 24 h to the culture
medium altered the cell cycle progression. However, in cells with DA
uptake (Fig. 2
), the share of the Go/G1
population of cells was significantly increased (from 50% to
80%; after 6 h of exposure, to nearly 60%). The profound
effects of the catecholamines on cell cycle distribution was not
observed after 10 µM MPP+. Deferoxamine, known
to block cellular proliferation before the G1/S,
arrested the progression in the
G0/G1 phase of the cell
cycle, as expected, in cells with and without DAT.
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In DAT-expressing cells, low micromolar levels of DA or NA and 100 µM deferoxamine in the medium for 24 h elicited the pattern of DNA laddering characteristic of apoptosis. Flow cytometric measurements using the fluorescent dye YO-PRO-1 showed the share of apoptotic cells to be doubled relative to controls.
4. The catecholamine-induced inhibition of cell growth is
antagonized by iron (III)
As shown above, in DAT-expressing cells the effect of DA and NE
had striking parallels to the action of deferoxamine:
G1 arrest with a similar time course and
induction of apoptosis. In addition, deferoxamine showed an
antiproliferative effect, with the growth inhibition being additive to
that of DA. As deferoxamine is an effective iron chelator, interaction
with cellular iron was investigated. 90 µM
FeCl3 antagonized not only the effect of 100 µM
deferoxamine, but also the effect of 3 µM DA on cell growth [% of
control cells ±SE (n): 55 ±6 (4),
vehicle; 79 ±1 (4), FeCl3;
P<0,05]; the effect of 3 µM MPP+
on cell numbers, however, was not influenced. Furthermore, 90 µM
FeCl3 antagonized the arrest of the cell cycle
elicited by 1 µM catecholamines. This iron concentration reduced the
intracellular DA levels by 18 ± 3% (after 6 h of incubation
with 3 µM DA; n=3); the initial DA uptake rate was reduced
by 10 ± 2%.
CONCLUSIONS AND SIGNIFICANCE
The effect of catecholamines in the low micromolar range on cell growth and viability clearly had an intracellular site of action: 1) It was absent in parental cells; 2) the susceptibility of cells transfected with the transporter was related to the initial rate of uptake and prevented by uptake blockade (mazindol); and 3) continuous transporter activation (entailing increased energy demand) was not involved. Oxidative stress is commonly considered to be crucial for DA cytotoxicity. This notion is based on the protective action of antioxidants. In fact, in our neuroblastoma cells without DA uptake, cytotoxicity of high micromolar catecholamines was blocked by antioxidants. However, for low micromolar concentrations of DA or NE acting in DAT-expressing neuroblastoma cells, we could rule out oxidative stress as a key mechanism.
Our study was performed in dividing cells with an active cell cycle. The effect of DA was primarily antiproliferative in nature, with the sum of necrotic and apoptotic cells representing only a minor portion of the DA-induced cell loss. DA and NE induced an apparent arrest in the G1 phase, whereas MPP+ (a structurally unrelated cytotoxic DAT substrate) was without effect. Thus, the effect on the distribution of cells within the cycle phases was specific for catecholamines and not shared by every substrate of the DAT.
In view of the known property of catecholamines to bind iron and other metals, iron chelation as a likely mechanism suggested itself. Intracellular iron is essential for cell growth, and iron-chelating compounds have been shown to interfere with the cell cycle by inhibition of ribonucleotide reductase. In our cells expressing the DAT, the antiproliferative action of DA was prevented by addition of FeCl3 to the medium; the effect was specific in that the action of MPP+ on cell numbers was not affected. Furthermore, addition of FeCl3 antagonized the arrest of the cell cycle elicited by the catecholamines. This implicates iron chelation as the mechanism of catecholamine-induced antiproliferation. Another explanation might be that iron (III) destabilized DA in the medium by accelerating its autoxidation. However, intracellular levels of the catecholamines determined in the absence or presence of 90 µM FeCl3 make it unlikely that accelerated decomposition of extracellular DA is a major factor in the protective action of iron(III).
Binding of intracellular iron as a mechanism of catecholamine-induced growth inhibition is greatly supported by the similar time course of cell cycle arrest at G1 and growth inhibition induced by the known iron chelator deferoxamine. The finding that at submaximal concentrations the effect of deferoxamine was only additive to that of DA, being antagonized by addition of FeCl3, favors an identical site of action of these two otherwise quite different compounds.
The cell cycle effect of DA, NE, and deferoxamine was accompanied by apoptosis as revealed by DNA laddering and flow cytometry. Interfering with the cell cycle has been demonstrated to be a potent trigger of apoptosis. G1 arrest and apoptosis are often interrelated. Thus, in agreement with our observations, data in the literature show that apoptosis of proliferating neuronal cells after growth factor withdrawal correlated with arrest in G1.
The most obvious relevance of our observations lies in aspects of neurodevelopment and neuroprotection. Regulation of the cell division cycle apparently has an effect on neuronal differentiation. It is believed that the final division of neuronal progenitor, especially events during the G1 phase of the cycle, influences to some degree its final differentiation.
We made our observations in actively dividing neuroblastoma
cells. Could our findings also be relevant for adult neurons? Agents
that block the G1/S transition, including the
iron chelator deferoxamine, have been shown to suppress the apoptosis
of differentiated PC12 cells and sympathetic neurons. These findings
are in line with the suggestion that, although antiproliferative agents
will cause apoptosis in cycling cells, by preventing re-entry into an
abortive cycle they will promote survival in terminally differentiated
cells, such as neurons. The parallels between deferoxamine and
catecholamines in our proliferating neuroblastoma cells in terms of
growth inhibition, G1 arrest, and induction of
apoptosis suggest that, similar to deferoxamine, catecholamines might
be neuroprotective in mature NE and DA neurons (Fig. 3
). The possibility of neuroprotection stands in contrast to the
current hypotheses implicating DA in the etiology of nigral cell
death in Parkinson’s disease.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0738fje ; to cite this
article, use FASEB J. (May 18, 2001) 10.1096/fj.00-0738fje 
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