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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online August 8, 2000 as doi:10.1096/fj.99-1066fje. |
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Department of Immunology and Oncology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Cantoblanco, 28049 Madrid, Spain
2Correspondence: Department of Immunology and Oncology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Campus Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain. E-mail: imerida{at}cnb.uam.es
SPECIFIC AIMS
Theobjective of our study was to examine the relationship between the lipid second messengers (LSMs) diacylglycerol (DAG) and ceramide and the levels of the lipids phosphatidylcholine (PtdCho) and sphingomyelin (SM), their potential precursors, in lymphocytes cultured in three distinct conditions to favor apoptosis, cell arrest, and proliferation. To achieve this, we chose the interleukin 2 (IL-2)-dependent human T lymphocyte cell line Kit 225 as the experimental model.
PRINCIPAL FINDINGS
To study changes in the generation of lipid mediators and the metabolism of their precursors, we defined three Kit 225 cell culture conditions. Depending on the culture conditions used, the cells entered into one of three distinct states (apoptosis, cell arrest, and proliferation).
1. Cell cycle analysis of Kit 225 cells cultured in different
conditions
Cells cultured in these three different conditions were analyzed
by staining the cellular DNA with propidium iodide to determine their
cell cycle status. When the cells were cultured in basal medium, they
entered rapidly into apoptosis, which was evident after 24 h (31%
of cells in sub-G0/G1) and increased accordingly with time (at 48 h, 47% of cells in sub-G0/G1; at 72 h, 62% of cells in
sub-G0/G1). Cells cultured in complete medium (basal medium + serum)
entered into a state of cell arrest that led to their accumulation in
G0/G1 (53% at 24 h), which was maintained until the last period
of time measured (77% at 72 h). Under these conditions, minimal
cell death was observed. Kit 225 cells do not undergo apoptosis in the
absence of IL-2, but this cytokine is absolutely essential for the
cells to proliferate. Accordingly, when the cells were maintained in
complete medium supplemented with IL-2, the cell cycle analysis
resembled that seen in their routine culture (24 h 32% in S+G2/M,
48 h 43% in S+G2/M, 72 h 58% in S+G2/M). These results
indicated that the cells provide a suitable model for studying
signaling events evoked by IL-2.
2. The intracellular levels of DAG and ceramide rise depending on
cell culture conditions
On the basis of the results from the cell cycle analyses, the next
step was to determine the intracellular levels of DAG and ceramide in
the cell states described above at identical time points. Figure 1
indicates that the levels of intracellular ceramide increased
(approximately fourfold at 72 h) with time when cells were
cultured in basal medium. This was accompanied by minor changes in DAG
content (a decrease to approximately 80% at 72 h compared to time
0). In contrast, the DAG content of cells grown in complete medium
+IL-2 increased with time (approximately fourfold at 72 h). Under these conditions, no changes were observed in the ceramide
content of the cells. In resting cells the intracellular levels of both
DAG and ceramide remained stationary throughout the entire period
examined.
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3. The PtdCho/SM ratio in Kit 225 cells changes depending on cell
culture conditions
We then decided to address the question of whether the levels of
the possible precursors of DAG and ceramide (PtdCho and SM,
respectively) correlated with the changes in the accumulations of
intracellular DAG and ceramide. To achieve this, we used in
vivo metabolic labeling with [methyl-3H]choline,
which readily labeled the PtdCho and SM pools in the cells
(approximately a 6/1 ratio of PtdCho/SM). When the cells were incubated
in basal medium, no change in their PtdCho content was observed. In
contrast, their SM level increased with time. Cells that had been
incubated in complete medium exhibited a minor decrease in their PtdCho
level, whereas their SM level remained constant. The level of PtdCho in
proliferating cells increased with time. No change in the level of SM
was seen in such cells. Figure 2
represents the changes in the PtdCho/SM ratio from cells cultured in
the above conditions and collected at the same time points as those
indicated in Fig. 1
. The results indicate that this ratio increased
with time while cells were proliferating, and by 72 h it was 180%
of that measured at time 0. In contrast, this ratio dramatically
dropped in the cells undergoing apoptosis to approximately 20% of
control at 72 h. In cells that accumulated in G0/G1, this ratio
was almost constant (a slight decrease, 25%, was observed).
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4. Sphingomyelin synthase activity in membranes isolated from Kit
225 cells
In membrane extracts of cultured Kit 225 cells, we found a low
level of sphingomyelin synthase (SMS) activity measured by the
in vitro conversion of the radiolabeled head group of
PtdCho to ceramide yielding radiolabeled SM. Our results also indicated
that SMS activity did not change significantly under proliferative
conditions. In contrast, the activity increased in membrane extracts
from cells that had started their apoptotic program (228% of control
at 72 h).
CONCLUSIONS AND SIGNIFICANCE
Control of lymphocyte cell survival and proliferation by IL-2 is critical for both the immune response and for the prevention of autoimmune and infectious diseases. In this study we have extended previous observation by demonstrating that the ratio DAG/ceramide is far greater in proliferating cells than that in a population of apoptotic cells. These results reinforce the concept that DAG is a positive intracellular mitogenic agent and is opposed by endogenous ceramide, underlying the importance of the balance between DAG and ceramide in the control of cell fate. We also demonstrate that the PtdCho/SM ratio changes with cell status, as does the DAG/ceramide ratio. Therefore, not only lipid mediators but also their potential precursors increase in the different situations.
Future studies will be necessary to address the molecular characterization of various enzymes implicated in lipid metabolism, which, as suggested by this and other studies, could be extremely relevant for correct cellular homeostasis. Other important points worthy of investigation will be the identification of the sites at which the LSMs are generated and their compositional analysis. Finally, the identification of potential targets of long-term generated lipid mediators will allow the dissection of the pathways that control proliferation, differentiation, and cell death.
In the light of the observations presented here, we can reinterpret results obtained more than 25 years ago that focused on the phospholipid composition of lymphocytes (normal and various lymphomas) in distinct stages of transformation. By comparing the grade of cell tumoricity with the percentage of PtdCho vs. total phospholipid, as cell transformation increased, so too did the percentage of PtdCho. SM, on the other hand, strongly showed the opposite effect: as tumoricity increases, the percentage of SM decreases. This would suggest that actively growing cells require a continuous supply of mitogenic LSMs and that the balance is tipped in favor of the synthesis of phospholipids able to provide such a demand. In cases where there is uncontrolled cell growth (such as in tumors, etc.), the level of mitogenic LSMs may be exaggerated above that found in normal cells. Conversely, a shutdown of the biosynthesis of such phospholipids is the result in slow-growing, arrested, and apoptotic cells.
In combination with others, the observations found here lead us to the
concept of the LSMs as ‘cellular lipostats’ in which the relative
concentrations of individual LSMs reflect the state of the cell. In
this case, the tightly regulated balance between mitogenic lipids (DAG
and PtdCho) and anti-mitogenic lipids (ceramide and SM) would be the
indicator of the cellular state (see Fig. 3
). A high number in the ratio of mitogenic to anti-mitogenic lipids
reflects a proliferative state; a low number reflects an apoptotic
state and an intermediate number a quiescent state. Recent evidence
indicates that this balance acts not only as an indicator of the
cellular state, but also as an active component in the decision of the
cellular fate.
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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.99-1066fje 
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