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ARE CELLS RESCUED FROM ‘LOW DENSITY DEATH’ BY CO-OPERATION BETWEEN PHOSPHOLIPASES C AND D?
Cell Biology International 2000, Vol. 24, No. 2, 121–123 doi:10.1006/cbir.1999.0458, available online at http://www.idealibrary.com on
HYPOTHESIS
ARE CELLS RESCUED FROM ‘LOW DENSITY DEATH’ BY CO-OPERATION BETWEEN PHOSPHOLIPASES C AND D? MORTEN RASMUSSEN* and LEIF RASMUSSEN Institute of Medical Biology, Department of Cell Biology, Odense University, DK-5230 Odense M, Denmark Received 8 April 1999; accepted 20 August 1999
Cells of the ciliate Tetrahymena thermophila die when transferred at low density to a lipid-free nutritionally complete medium. This death is prevented and they will start to proliferate if protein kinase C is activated and this activation is sustained. We propose that this takes place in two stages. Firstly, the phospholipase C pathway beginning with and specific for phosphatidylinositol leads to the formation of diacylglycerol and inositol tris-phosphate. Diacylglycerol activates protein kinase C, and inositol tris-phosphate via Ca2+ phospholipase D (PLD). Secondly, the protein kinase C response can now be sustained by diacylglycerol produced by phospholipase D, using phosphatidylcholine and phosphatidylserine as substrates. Should this switching from PI-specific phospholipase C (PLC) to phospholipase D fail, then the cell will die 2000 Academic Press in the course of milliseconds during the minutes following inoculation. K: cell signalling; phospholipases C and D; cell death; phosphatidylinositol; diacylglycerol; Tetrahymena; protein kinase C.
INTRODUCTION
OBSERVATIONS
Some of the first reactions which came to light in the field of cell signalling were the conversion of phosphatidylinositol (PI) via phosphatidylinositol bis-phosphate (PIP2) to inositol tris-phosphate (IP3) and diacylglycerol (DAG). These compounds activate protein kinase C (PKC), but a question remains unanswered: what happens when the cell runs short of PI? It clearly has to continue generating DAG. We have uncovered some clues that may provide answers to these questions based upon our work with living ciliates. These clues may also shed light on the well-known phenomenon of the low survival frequencies of cells of all types—not just ciliates—when cultured from very low initial densities in minimal nutritional media. We know of no other experiments that have focused on these enigmas.
Cells transferred to nutrient medium at low initial densities have a high risk of dying before exiting their lag period (Freshney, 1987). The ciliate Tetrahymena thermophila is no exception when subcultured in a lipid-free, synthetic nutrient medium (Christensen et al., 1995; for review, see Christensen et al., 1997). Recently, we established that the transition from normal swimming to death occurs in a single cell within milliseconds in the interval between 4 and 30 min after transfer to the new medium at low cell density (Rasmussen and Rasmussen, 2000). Our group previously reported that moribund cells were rescued when PKC was activated by phorbol esters (Straarup et al., 1997). Recently, we also noted that pure phospholipids added to the lipid-free medium lead to activation of cell multiplication, and that PLD was involved in the rescue process (Rasmussen and Rasmussen, 1999, 2000). Previous observations are relevant to our hypothesis. Firstly, Christensen et al. (1996)
*To whom correspondence [email protected] 1065–6995/00/020121+03 $35.00/0
should
be
addressed.
E-mail:
2000 Academic Press
122
Cell Biology International, Vol. 24, No. 2, 2000
PLC cascade –
IP3 4
+
phosphatidylcholine (PC) and phosphatidylserine (PS) as precursors.
PC/PS
PI
3
Ca2+
6
1
DAG +
2
PPH 7
PLD +
BENEFITS
PA 8
PKC 5
Fig. 1. Tetrahymena thermophila cells need a sustained activation of protein kinase C in order to exit the lag phase after subcultivation. This activation seems to occur in two stages. First there is a production of diacylglycerol (DAG) from phosphatidylinositol (PI) via phosphatidylinositol bisphosphate (PIP2) and this in turn activates the protein kinase C cascade (PKC) (reactions 1 and 2). The PI-specific phospholipase C (PLC) cascade also produces inositol trisphosphate (IP3) which activates phospholipase D, PLD, (reactions 4, 5 and 8). From phosphatidylcholine (PC) and phosphatidylserine (PS) the enzyme PLD produces phosphatidate (PA) which is converted to DAG, (reactions 6, 7 and 2). The upregulated PKC activity feedback inhibits PLC (reaction 3). In this way the PLC cascade works as an enzymatic ‘kick starter’ for PKC activation. The shift in lipolysis from phosphatidylinositol (PI) to phosphatidylcholine (PC) and phosphatidylserine (PS) seems to be crucial for entry into the proliferative mode.
reported that human recombinant insulin down to nanomolar concentrations prevented Tetrahymena at low initial densities from dying. Secondly, Kovacs et al. (1996, 1997) demonstrated that micromolar concentrations of insulin increased the activity of PLD in T. pyriformis. Connections between insulin effects on cell survival and PLD have therefore been established in Tetrahymena. HYPOTHESIS We propose a scheme combining old and new results on cell death and multiplication following subcultivation. The sources of DAG for activation of PKC are central in our hypothesis. Our scheme consists of two loops (Fig. 1, left and right). In the first loop (reactions 1–3), the PI-specific PLC cascade leads to formation of DAG which activates PKC. PKC feedback in turn inhibits PLC (Kikkawa et al., 1989). Reactions 4 and 5 relay the signal from PLC to PLD via an increase in the intracellular calcium level, upregulating calciumsensitive PLD activity. In the second loop (reactions 6, 7, 2, 8) a feed-forward loop is formed, upregulating PLD and producing DAG by using
There are three benefits for the cell in this shift between the two phospholipases, the PI-specific PLC and the PLD: (1) The cellular PI pool used by the PLC amounts to only a few per cent of the total phospholipids, whereas the PC+PS pool used by PLD accounts for approximately 60%; thus the cell switches from a sparse to an abundant source for DAG. (2) PI hydrolysis (via PIP2) involves several pathways, such as Ca2+ and DAG signalling, whereas PC/PS hydrolysis (via PA) leads to DAG alone. (3) The PLC loop leads to a rapid activation of PKC via PLD and to a subsequent inactivation of itself, in the manner of an enzymatic ‘kick starter’. MECHANISM OF CELL DEATH AND ITS PREVENTION Our experiments indicate that the PI-specific PLC is centrally involved in ‘low density death’. Both addition of sonicated PI (the precursor to the PLC cascade) to the cells or partial inhibition of PLC prevent the cells from dying (Rasmussen and Rasmussen, 2000). That means that both stimulation and inhibition of the same enzyme gives the same result. If nothing is done, the cells invariably die within minutes. Thus, the fate of the cells can be controlled by simple means. Our cells can undergo programmed apoptoticlike cell death when treated with staurosporine (Christensen et al., 1998). This involves chromatin condensation and membrane blebbing and requires in excess of 12 h. The fast—indeed explosive— death we have observed is morphologically distinct from both necrosis and apoptosis (Rasmussen and Rasmussen, 2000). The underlying mechanisms are also different from those behind necrosis and apoptosis and can be reversed by simple means, for example by addition of (phospho)lipids (Christensen and Rasmussen, 1992; Schousboe and Rasmussen, 1994; Rasmussen and Rasmussen, 2000). The active lipids included PI, PC, PS, PE (phosphatidylethanolamine), Peth (phosphatidylethanol), asolectin, and monoacyl- and diacylglycerol.
Cell Biology International, Vol. 24, No. 2, 2000
REMARKS ON EXPERIMENTAL CONDITIONS Many lipids activate the mechanisms we are dealing with here. Our observations can therefore be made with only nutritionally complete, but lipid-free, media and have to our knowledge yet to be carried out in other cell systems, such as mammalian cell cultures. Rich complex media are often used for growing cells, and nutritional lipids can often activate both the enzyme systems and hence proliferation, and thus obscure the basic mechanisms. Furthermore, cell-produced growth factors activate these mechanisms (Christensen and Rasmussen, 1992; for a review, see Christensen et al., 1997). We therefore carried out our experiments with cultures in lipid-free media at very low initial cell densities to exclude these effects. Starving, low-density cultured Tetrahymena cells survive for 70 h, 10-fold longer than fed cells (Christensen et al., 1995). It is intuitively paradoxical that free access to all the necessary nutrients for cell growth actually speeds up processes leading to ‘low density cell death’ (Christensen et al., 1997). We suggest that the cells under lipid-free conditions may be wearing themselves out in an attempt to survive the lag phase. The observed ‘low density cell death’ is thus reminiscent of incidental death due to lack of control of lipolysis, rather than activation of mechanisms with the single function of leading to cell death.
CONCLUSIONS Tetrahymena thermophila cells need sustained activation of PKC in order to proliferate. After transfer to new medium at low cell density the PI-specific PLC seems to provide the necessary IP3 and DAG from phosphatidylinositol. Apparently, this activates PLD, which takes over and provides DAG from phosphatidylcholine and phosphatidylserine. When this transition is not made, the single cell dies suddenly and explosively within a matter of minutes after transfer to new medium. It is so far unknown whether the same enzymes or similar mechanisms are involved when cells undergo ‘low density death’ (albeit mammalian cells).
123
ACKNOWLEDGEMENTS It is a pleasure to thank Drs Hans Boye, Odense, and Denys Wheatley, Aberdeen, for their comments in this study. REFERENCES C ST, R L, 1992. Evidence for growth factors, which control cell multiplication in Tetrahymena thermophila. Acta Protozool 31: 215–219. C ST, W DN, R MI, R L, 1995. Mechanisms controlling death, survival and proliferation in a model unicellular eukaryotic cell system, Tetrahymena thermophila. Cell Death & Differentiation 2: 301–308. C ST, Q H, K K, R L, 1996. Cell death, survival and proliferation in Tetrahymena thermophila. Effects of insulin, sodium nitroprusside, 8-bromo cyclic GMP, N-methyl-L-arginine and methylene blue. Cell Biol Internatl 20: 653–666. C ST, R L, L V, W DN, 1997. Signaling in unicellular eukaryotes. Regulation of cell survival, proliferation, differentiation, mating, chemosensory behavior and programmed cell death. Internatl Rev Cytol 177: 181–253. C ST, C J, S EM, K K, W DN, R L, 1998. Staurosporine-induced cell death in Tetrahymena thermophila has mixed characteristics of both apoptotic and autophagic degeneration. Cell Biol Internatl 22: 591–598. F RI, 1987. Culture of Animal Cells: A Manual of Basic Techniques. New York, Liss. K U, K A, N Y, 1989. The protein kinase family: heterogeneity and its implications. Ann Rev Biochem 58: 31–44. K P, C G, I Y, N Y, 1996. Effect of insulin on the phospholipase D activity of untreated and insulin pretreated (hormonally imprinted) Tetrahymena. Biochem Biophys Res Com 222: 359–361. K P, C G, N S, N Y, 1997. Phospholipase D activity in Tetrahymena pyriformis GL. Cell Biochem Funct 15: 53–60. R M, R L, 1999. Phospholipase D in Tetrahymena. Activity and significance. Comp Biochem Physiol 124: 467–473. R M, R L, 2000. Survival/death in the early lag phase of Tetrahymena—the roles of phospholipases C and D. Cell Biochem. Funct (In press). S P, R L, 1994. Survival of Tetrahymena thermophila at low initial cell densities. Effects of lipids and long chain alcohols. J Euk Microbiol 41: 195–199. S EM, S P, H HQ, K K, H EK, R L, C ST, 1997. Effects of protein kinase C activators and staurosporine on protein kinase activity, cell survival and proliferation in Tetrahymena thermophila. Microbios 91: 181–190.
HYPOTHESIS
ARE CELLS RESCUED FROM ‘LOW DENSITY DEATH’ BY CO-OPERATION BETWEEN PHOSPHOLIPASES C AND D? MORTEN RASMUSSEN* and LEIF RASMUSSEN Institute of Medical Biology, Department of Cell Biology, Odense University, DK-5230 Odense M, Denmark Received 8 April 1999; accepted 20 August 1999
Cells of the ciliate Tetrahymena thermophila die when transferred at low density to a lipid-free nutritionally complete medium. This death is prevented and they will start to proliferate if protein kinase C is activated and this activation is sustained. We propose that this takes place in two stages. Firstly, the phospholipase C pathway beginning with and specific for phosphatidylinositol leads to the formation of diacylglycerol and inositol tris-phosphate. Diacylglycerol activates protein kinase C, and inositol tris-phosphate via Ca2+ phospholipase D (PLD). Secondly, the protein kinase C response can now be sustained by diacylglycerol produced by phospholipase D, using phosphatidylcholine and phosphatidylserine as substrates. Should this switching from PI-specific phospholipase C (PLC) to phospholipase D fail, then the cell will die 2000 Academic Press in the course of milliseconds during the minutes following inoculation. K: cell signalling; phospholipases C and D; cell death; phosphatidylinositol; diacylglycerol; Tetrahymena; protein kinase C.
INTRODUCTION
OBSERVATIONS
Some of the first reactions which came to light in the field of cell signalling were the conversion of phosphatidylinositol (PI) via phosphatidylinositol bis-phosphate (PIP2) to inositol tris-phosphate (IP3) and diacylglycerol (DAG). These compounds activate protein kinase C (PKC), but a question remains unanswered: what happens when the cell runs short of PI? It clearly has to continue generating DAG. We have uncovered some clues that may provide answers to these questions based upon our work with living ciliates. These clues may also shed light on the well-known phenomenon of the low survival frequencies of cells of all types—not just ciliates—when cultured from very low initial densities in minimal nutritional media. We know of no other experiments that have focused on these enigmas.
Cells transferred to nutrient medium at low initial densities have a high risk of dying before exiting their lag period (Freshney, 1987). The ciliate Tetrahymena thermophila is no exception when subcultured in a lipid-free, synthetic nutrient medium (Christensen et al., 1995; for review, see Christensen et al., 1997). Recently, we established that the transition from normal swimming to death occurs in a single cell within milliseconds in the interval between 4 and 30 min after transfer to the new medium at low cell density (Rasmussen and Rasmussen, 2000). Our group previously reported that moribund cells were rescued when PKC was activated by phorbol esters (Straarup et al., 1997). Recently, we also noted that pure phospholipids added to the lipid-free medium lead to activation of cell multiplication, and that PLD was involved in the rescue process (Rasmussen and Rasmussen, 1999, 2000). Previous observations are relevant to our hypothesis. Firstly, Christensen et al. (1996)
*To whom correspondence [email protected] 1065–6995/00/020121+03 $35.00/0
should
be
addressed.
E-mail:
2000 Academic Press
122
Cell Biology International, Vol. 24, No. 2, 2000
PLC cascade –
IP3 4
+
phosphatidylcholine (PC) and phosphatidylserine (PS) as precursors.
PC/PS
PI
3
Ca2+
6
1
DAG +
2
PPH 7
PLD +
BENEFITS
PA 8
PKC 5
Fig. 1. Tetrahymena thermophila cells need a sustained activation of protein kinase C in order to exit the lag phase after subcultivation. This activation seems to occur in two stages. First there is a production of diacylglycerol (DAG) from phosphatidylinositol (PI) via phosphatidylinositol bisphosphate (PIP2) and this in turn activates the protein kinase C cascade (PKC) (reactions 1 and 2). The PI-specific phospholipase C (PLC) cascade also produces inositol trisphosphate (IP3) which activates phospholipase D, PLD, (reactions 4, 5 and 8). From phosphatidylcholine (PC) and phosphatidylserine (PS) the enzyme PLD produces phosphatidate (PA) which is converted to DAG, (reactions 6, 7 and 2). The upregulated PKC activity feedback inhibits PLC (reaction 3). In this way the PLC cascade works as an enzymatic ‘kick starter’ for PKC activation. The shift in lipolysis from phosphatidylinositol (PI) to phosphatidylcholine (PC) and phosphatidylserine (PS) seems to be crucial for entry into the proliferative mode.
reported that human recombinant insulin down to nanomolar concentrations prevented Tetrahymena at low initial densities from dying. Secondly, Kovacs et al. (1996, 1997) demonstrated that micromolar concentrations of insulin increased the activity of PLD in T. pyriformis. Connections between insulin effects on cell survival and PLD have therefore been established in Tetrahymena. HYPOTHESIS We propose a scheme combining old and new results on cell death and multiplication following subcultivation. The sources of DAG for activation of PKC are central in our hypothesis. Our scheme consists of two loops (Fig. 1, left and right). In the first loop (reactions 1–3), the PI-specific PLC cascade leads to formation of DAG which activates PKC. PKC feedback in turn inhibits PLC (Kikkawa et al., 1989). Reactions 4 and 5 relay the signal from PLC to PLD via an increase in the intracellular calcium level, upregulating calciumsensitive PLD activity. In the second loop (reactions 6, 7, 2, 8) a feed-forward loop is formed, upregulating PLD and producing DAG by using
There are three benefits for the cell in this shift between the two phospholipases, the PI-specific PLC and the PLD: (1) The cellular PI pool used by the PLC amounts to only a few per cent of the total phospholipids, whereas the PC+PS pool used by PLD accounts for approximately 60%; thus the cell switches from a sparse to an abundant source for DAG. (2) PI hydrolysis (via PIP2) involves several pathways, such as Ca2+ and DAG signalling, whereas PC/PS hydrolysis (via PA) leads to DAG alone. (3) The PLC loop leads to a rapid activation of PKC via PLD and to a subsequent inactivation of itself, in the manner of an enzymatic ‘kick starter’. MECHANISM OF CELL DEATH AND ITS PREVENTION Our experiments indicate that the PI-specific PLC is centrally involved in ‘low density death’. Both addition of sonicated PI (the precursor to the PLC cascade) to the cells or partial inhibition of PLC prevent the cells from dying (Rasmussen and Rasmussen, 2000). That means that both stimulation and inhibition of the same enzyme gives the same result. If nothing is done, the cells invariably die within minutes. Thus, the fate of the cells can be controlled by simple means. Our cells can undergo programmed apoptoticlike cell death when treated with staurosporine (Christensen et al., 1998). This involves chromatin condensation and membrane blebbing and requires in excess of 12 h. The fast—indeed explosive— death we have observed is morphologically distinct from both necrosis and apoptosis (Rasmussen and Rasmussen, 2000). The underlying mechanisms are also different from those behind necrosis and apoptosis and can be reversed by simple means, for example by addition of (phospho)lipids (Christensen and Rasmussen, 1992; Schousboe and Rasmussen, 1994; Rasmussen and Rasmussen, 2000). The active lipids included PI, PC, PS, PE (phosphatidylethanolamine), Peth (phosphatidylethanol), asolectin, and monoacyl- and diacylglycerol.
Cell Biology International, Vol. 24, No. 2, 2000
REMARKS ON EXPERIMENTAL CONDITIONS Many lipids activate the mechanisms we are dealing with here. Our observations can therefore be made with only nutritionally complete, but lipid-free, media and have to our knowledge yet to be carried out in other cell systems, such as mammalian cell cultures. Rich complex media are often used for growing cells, and nutritional lipids can often activate both the enzyme systems and hence proliferation, and thus obscure the basic mechanisms. Furthermore, cell-produced growth factors activate these mechanisms (Christensen and Rasmussen, 1992; for a review, see Christensen et al., 1997). We therefore carried out our experiments with cultures in lipid-free media at very low initial cell densities to exclude these effects. Starving, low-density cultured Tetrahymena cells survive for 70 h, 10-fold longer than fed cells (Christensen et al., 1995). It is intuitively paradoxical that free access to all the necessary nutrients for cell growth actually speeds up processes leading to ‘low density cell death’ (Christensen et al., 1997). We suggest that the cells under lipid-free conditions may be wearing themselves out in an attempt to survive the lag phase. The observed ‘low density cell death’ is thus reminiscent of incidental death due to lack of control of lipolysis, rather than activation of mechanisms with the single function of leading to cell death.
CONCLUSIONS Tetrahymena thermophila cells need sustained activation of PKC in order to proliferate. After transfer to new medium at low cell density the PI-specific PLC seems to provide the necessary IP3 and DAG from phosphatidylinositol. Apparently, this activates PLD, which takes over and provides DAG from phosphatidylcholine and phosphatidylserine. When this transition is not made, the single cell dies suddenly and explosively within a matter of minutes after transfer to new medium. It is so far unknown whether the same enzymes or similar mechanisms are involved when cells undergo ‘low density death’ (albeit mammalian cells).
123
ACKNOWLEDGEMENTS It is a pleasure to thank Drs Hans Boye, Odense, and Denys Wheatley, Aberdeen, for their comments in this study. REFERENCES C ST, R L, 1992. Evidence for growth factors, which control cell multiplication in Tetrahymena thermophila. Acta Protozool 31: 215–219. C ST, W DN, R MI, R L, 1995. Mechanisms controlling death, survival and proliferation in a model unicellular eukaryotic cell system, Tetrahymena thermophila. Cell Death & Differentiation 2: 301–308. C ST, Q H, K K, R L, 1996. Cell death, survival and proliferation in Tetrahymena thermophila. Effects of insulin, sodium nitroprusside, 8-bromo cyclic GMP, N-methyl-L-arginine and methylene blue. Cell Biol Internatl 20: 653–666. C ST, R L, L V, W DN, 1997. Signaling in unicellular eukaryotes. Regulation of cell survival, proliferation, differentiation, mating, chemosensory behavior and programmed cell death. Internatl Rev Cytol 177: 181–253. C ST, C J, S EM, K K, W DN, R L, 1998. Staurosporine-induced cell death in Tetrahymena thermophila has mixed characteristics of both apoptotic and autophagic degeneration. Cell Biol Internatl 22: 591–598. F RI, 1987. Culture of Animal Cells: A Manual of Basic Techniques. New York, Liss. K U, K A, N Y, 1989. The protein kinase family: heterogeneity and its implications. Ann Rev Biochem 58: 31–44. K P, C G, I Y, N Y, 1996. Effect of insulin on the phospholipase D activity of untreated and insulin pretreated (hormonally imprinted) Tetrahymena. Biochem Biophys Res Com 222: 359–361. K P, C G, N S, N Y, 1997. Phospholipase D activity in Tetrahymena pyriformis GL. Cell Biochem Funct 15: 53–60. R M, R L, 1999. Phospholipase D in Tetrahymena. Activity and significance. Comp Biochem Physiol 124: 467–473. R M, R L, 2000. Survival/death in the early lag phase of Tetrahymena—the roles of phospholipases C and D. Cell Biochem. Funct (In press). S P, R L, 1994. Survival of Tetrahymena thermophila at low initial cell densities. Effects of lipids and long chain alcohols. J Euk Microbiol 41: 195–199. S EM, S P, H HQ, K K, H EK, R L, C ST, 1997. Effects of protein kinase C activators and staurosporine on protein kinase activity, cell survival and proliferation in Tetrahymena thermophila. Microbios 91: 181–190.