Apoptotic volume decrease and the incredible shrinking cell.
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Animals↗
Biomedical subjects
C D Bortner
Publications and source records attributed to C D Bortner.
Differential involvement of initiator caspases in apoptotic volume decrease and potassium efflux during Fas- and UV-induced cell death.
Caspase activation and apoptotic volume decrease are fundamental features of programmed cell death; however, the relationship between these components is not well understood. Here we provide biochemical and genetic evidence for the differential involvement of initiator caspases in the apoptotic volume decrease during both intrinsic and extrinsic activation of apoptosis. Apoptosis induction in Jurkat T lymphocytes by Fas receptor engagement (intrinsic) or ultraviolet (UV)-C radiation (extrinsic) triggered the loss of cell volume, which was restricted to cells with diminished intracellular K(+) ions. These characteristics kinetically coincided with the proteolytic processing and activation of both initiator and effector caspases. Although the polycaspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone completely inhibited the Fas-mediated apoptotic volume decrease and K(+) efflux, it was much less effective in preventing these processes during UV-induced cell death under conditions whereby caspase activities and DNA degradation were blocked. To define the roles of specific initiator caspases, we utilized Jurkat cells genetically deficient in caspase-8 or stably transfected with a dominant-negative mutant of caspase-9. The results show that the activation of caspase-8, but not caspase-9, is necessary for Fas-induced apoptosis. Conversely, caspase-9, but not caspase-8, is important for UV-mediated shrunken morphology and apoptosis progression. Together, these findings indicate that cell shrinkage and K(+) efflux during apoptosis are tightly coupled, but are differentially regulated by either caspase-8 or caspase-9 depending on specific pathways of cell death.
Amino Acid Chloromethyl Ketones↗
Modification of alternative splicing of Bcl-x pre-mRNA in prostate and breast cancer cells. analysis of apoptosis and cell death.
There is ample evidence that deregulation of apoptosis results in the development, progression, and/or maintenance of cancer. Since many apoptotic regulatory genes (e.g. bcl-x) code for alternatively spliced protein variants with opposing functions, the manipulation of alternative splicing presents a unique way of regulating the apoptotic response. Here we have targeted oligonucleotides antisense to the 5'-splice site of bcl-x(L), an anti-apoptotic gene that is overexpressed in various cancers, and shifted the splicing pattern of Bcl-x pre-mRNA from Bcl-x(L) to Bcl-x(S), a pro-apoptotic splice variant. This approach induced significant apoptosis in PC-3 prostate cancer cells. In contrast, the same oligonucleotide treatment elicited a much weaker apoptotic response in MCF-7 breast cancer cells. Moreover, although the shift in Bcl-x pre-mRNA splicing inhibited colony formation in both cell lines, this effect was much less pronounced in MCF-7 cells. These differences in responses to oligonucleotide treatment were analyzed in the context of expression of Bcl-x(L), Bcl-x(S), and Bcl-2 proteins. The results indicate that despite the presence of Bcl-x pre-mRNA in a number of cell types, the effects of modification of its splicing by antisense oligonucleotides vary depending on the expression profile of the treated cells.
Alternative Splicing↗
Flow cytometric analysis of cell shrinkage and monovalent ions during apoptosis.
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Animals↗
Age-dependent skin tumorigenesis and transgene expression in the Tg.AC (v-Ha-ras) transgenic mouse.
Transgenic Tg.AC (v-Ha-ras ) mice develop skin tumors in response to specific carcinogens and tumor promoters. The Tg.AC mouse carries the coding sequence of v-Ha ras, linked to a zeta-globin promoter and an SV40 polyadenylation signal sequence. The transgene confers on these mice the property of genetically initiated skin. This study examines the age-dependent sensitivity of the incidence of skin papillomas in Tg.AC mice exposed to topically applied 12-O:-tetradecanoylphorbol-13-acetate (TPA) treatment, full thickness skin wounding or UV radiation. Skin tumor incidence and multiplicity were strongly age-dependent, increasing with increasing age of the animal when first treated at 5, 10, 21 or 32 weeks of age. Furthermore, the temporal induction of transgene expression in keratinocytes isolated from TPA-treated mouse skin was also influenced by the age of the mice. Transgene expression was seen as early as 14 days after the start of TPA treatment in mice that were 10-32 weeks of age, but was not detected in similarly treated 5-week old mice. When isolated keratinocytes were fractionated by density gradient centrifugation the highest transgene expression was found in the denser basal keratinocytes. Transgene expression could be detected in the denser keratinocyte fraction as early as 9 days from start of TPA treatment in 32-week old mice. Using flow cytometry, a positive correlation was observed between expression of the v-Ha-ras transgene and enriched expression of the cell surface protein beta1-integrin, a putative marker of epidermal stem cells. This result suggests that, in the Tg.AC mouse, an age-dependent sensitivity to tumor promotion and the correlated induction of transgene expression are related to changes in cellular development in the follicular compartment of the skin.
Age Factors↗
Glucocorticoid-induced plasma membrane depolarization during thymocyte apoptosis: association with cell shrinkage and degradation of the Na(+)/K(+)-adenosine triphosphatase.
Multiple signaling pathways are known to induce apoptosis in thymocytes through mechanisms that include the loss of mitochondrial membrane potential, cell shrinkage, caspase activation, and DNA degradation but little is known about the consequences of apoptosis on the properties of the plasma membrane. We have previously shown that apoptotic signals, including survival factor withdrawal and glucocorticoids, induce plasma membrane depolarization during rat thymocyte apoptosis, but the mechanisms involved in this process are unknown. We report here that inhibition of the Na(+)/K(+)-adenosine triphosphatase (Na(+)/K(+)-ATPase) with ouabain similarly depolarized control thymocytes and enhanced glucocorticoid-induced membrane depolarization, suggesting a link between Na(+)/K(+)-ATPase and plasma membrane depolarization of thymocytes. To determine whether repression of Na(+)/K(+)-ATPase levels within cells can account for the loss of plasma membrane potential, we assessed protein levels of the Na(+)/K(+)-ATPase in apoptotic thymocytes. Spontaneously dying thymocytes had decreased levels of both catalytic and regulatory subunits of Na(+)/K(+)-ATPase, and glucocorticoid treatment enhanced the loss of Na(+)/K(+)-ATPase protein. The pan caspase inhibitor (z-VAD) blocked both cellular depolarization and repression of Na(+)/K(+)-ATPase in both spontaneously dying and glucocorticoid-treated thymocytes; however, specific inhibitors of caspase 8, 9, and caspase 3 did not. Interestingly, glucocorticoid treatment simultaneously induced cell shrinkage and depolarization. Furthermore, depolarization and the loss of Na(+)/K(+)-ATPase protein were limited to the shrunken population of cells. The data indicate an important role for Na(+)/K(+)-ATPase in both spontaneous and glucocorticoid-induced apoptosis of rat thymocytes.
Amino Acid Chloromethyl Ketones↗
Plasma membrane depolarization without repolarization is an early molecular event in anti-Fas-induced apoptosis.
The movement of intracellular monovalent cations has previously been shown to play a critical role in events leading to the characteristics associated with apoptosis. A loss of intracellular potassium and sodium occurs during apoptotic cell shrinkage establishing an intracellular environment favorable for nuclease activity and caspase activation. We have now investigated the potential movement of monovalent ions in Jurkat cells that occur prior to cell shrinkage following the induction of apoptosis. A rapid increase in intracellular sodium occurs early after apoptotic stimuli suggesting that the normal negative plasma membrane potential may change during cell death. We report here that diverse apoptotic stimuli caused a rapid cellular depolarization of Jurkat T-cells that occurs prior to and after cell shrinkage. In addition to the early increase in intracellular Na(+), (86)Rb(+) studies reveal a rapid inhibition of K(+) uptake in response to anti-Fas. These effects on Na(+) and K(+) ions were accounted for by the inactivation of the Na(+)/K(+)-ATPase protein and its activity. Furthermore, ouabain, a cardiac glycoside inhibitor of the Na(+)/K(+)-ATPase, potentiated anti-Fas-induced apoptosis. Finally, activation of an anti-apoptotic signal, i.e. protein kinase C, prevented both cellular depolarization in response to anti-Fas and all downstream characteristics associated with apoptosis. Thus cellular depolarization is an important early event in anti-Fas-induced apoptosis, and the inability of cells to repolarize via inhibition of the Na(+)/K(+)-ATPase is a likely regulatory component of the death process.
Antibodies↗
A selective requirement for elevated calcium in DNA degradation, but not early events in anti-Fas-induced apoptosis.
Jurkat cells undergo apoptosis in response to anti-Fas antibody through a caspase-dependent death cascade in which calcium signaling has been implicated. We have now evaluated the role of calcium during this death cascade at the single cell level in real time utilizing flow cytometric analysis and confocal microscopy. Fluo-3 and propidium iodide were employed to evaluate calcium fluxes and to discriminate between viable and non-viable cells, respectively. Anti-Fas treatment of Jurkat cells resulted in a sustained increase in intracellular calcium commencing between 1 and 2 h after treatment and persisting until subsequent loss of cell membrane integrity. The significance of this rise in calcium was evaluated by buffering intracellular calcium with BAPTA and/or removing calcium from the extracellular medium and monitoring the effects of these manipulations on calcium signaling and components of the apoptotic process. Complete inhibition of the anti-Fas induced rise in intracellular calcium required both chelation of [Ca(2+)](i) and removal of extracellular calcium. Interestingly, this condition did not abrogate several events in Fas-induced apoptosis including cell shrinkage, mitochondrial depolarization, annexin binding, caspase activation, and nuclear poly(A)DP-ribose polymerase cleavage. Furthermore, calcium-free conditions in the absence of anti-Fas antibody weakly induced these apoptotic components. In marked contrast, calcium depletion did not induce DNA degradation in control cells, and inhibited apoptotic DNA degradation in response to anti-Fas. These data support the concept that the rise in intracellular calcium is not a necessary component for the early signal transduction pathways in anti-Fas-induced apoptosis in Jurkat cells, but rather is necessary for the final degradation of chromatin via nuclease activation.
Apoptosis↗
Protein kinase C (PKC) inhibits fas receptor-induced apoptosis through modulation of the loss of K+ and cell shrinkage. A role for PKC upstream of caspases.
Cell shrinkage and loss of intracellular K(+) are early requisite features for the activation of effector caspases and apoptotic nucleases in Fas receptor-mediated apoptosis of Jurkat cells, although the mechanisms responsible for both process remain unclear (Bortner, C. D., Hughes, F. M., Jr., and Cidlowski, J. A. (1997) J. Biol. Chem. 272, 32436-32442). We have now investigated the role of protein kinase C (PKC)-dependent signaling in the regulation of Fas-induced cell shrinkage and loss of K(+) during apoptosis. Anti-Fas induced cell shrinkage was blocked during PKC stimulation by the phorbol ester 12-O-tetradecanoylphorbol-3-acetate (PMA) and by bryostatin-1. Conversely, inhibition of PKC with Gö6976, enhanced the anti-Fas-mediated loss of cell volume. Analyses of mitochondrial membrane potential and DNA fragmentation revealed that the PKC-mediated effect observed in cell volume is propagated to these late features of apoptosis. Flow cytometric analyses and (86)Rb efflux experiments revealed that a primary effect of PKC appears to be on the modulation of Fas-induced K(+) efflux, since both PMA and bryostatin-1 inhibited extrusion of K(+) that occurs during Fas-mediated cell death, and Gö6976 exacerbated the effect of anti-Fas. Interestingly, high extracellular K(+) significantly blocked the effect of anti-Fas alone or anti-Fas combined with Gö6976, suggesting an underlying effect of PKC on K(+) loss. Western blot analyses showed the caspase-dependent proteolysis of PKC isotypes delta, epsilon, and theta in whole cell extracts from anti-Fas treated Jurkat T cells. However, stimulation of PKC by PMA or bryostatin-1 prevented this isotypic-specific PKC cleavage during apoptosis, providing further evidence that PKC itself exerts an upstream signal in apoptosis and controls the caspase-dependent proteolytic degradation of PKC isotypes. Finally, we show that PMA or bryostatin-1 prevents the activation of caspase-3 and caspase-8. Thus, this study shows that the protective effect that PKC stimulation exerts in the Fas-mediated apoptotic pathway occurs at a site upstream of caspases-3 and -8.
Antineoplastic Agents↗
Cell volume regulation in immune cell apoptosis.
The loss of cell volume is an early and fundamental feature of programmed cell death or apoptosis; however, the mechanisms responsible for cell shrinkage during apoptosis are poorly understood. The loss of cell volume is not a passive component of the apoptotic process, and a number of experimental findings from different laboratories highlight the importance of this process as an early and necessary regulatory event in the signaling of the death cascade. Additionally, the loss of intracellular ions, particularly potassium, has been shown to play a primary role in cell shrinkage, caspase activation, and nuclease activity during apoptosis. Thus, an understanding of the role that ion channels and plasma membrane transporters play in cellular signaling during apoptosis may have important physiological implications for immune cells, especially lymphocyte function. Furthermore, this knowledge may also have an impact on the design of therapeutic strategies for a variety of diseases of the immune system in which apoptosis plays a central role, such as oncogenic processes or immune system disorders. The present review summarizes our appreciation of the mechanisms underlying the early loss of cell volume during apoptosis and their association with downstream events in lymphocyte apoptosis.
Animals↗
Volume regulation and ion transport during apoptosis.
The loss of cell volume is a defining characteristic of programmed cell death, which separates apoptosis from necrosis. Assessment of cell volume during apoptosis can easily be accomplished by flow cytometry, by examining changes in the light-scattering properties of living cells that are directly proportional to cell size and density. Flow cytometry quantitates only relative changes in cell size; however, when used in conjunction with Coulter (Hialeah, FL) counter analysis, more precise cell volume data can be obtained. Flow cytometric analysis of multiple characteristics during apoptosis, such as changes in intracellular ion concentrations, membrane integrity, and DNA content, along with changes in cell size, permits sorting of various subpopulations of apoptotic cells. Below, we describe methods for rapid examination of cell volume and ion changes during apoptosis.
Animals↗
28S ribosome degradation in lymphoid cell apoptosis: evidence for caspase and Bcl-2-dependent and -independent pathways.
Apoptosis, a physiological form of cell death, is characterized by the activation of a program that kills cells and recycles their constituents. We have used thymoma cell lines to examine the role of Bcl-2 and caspases in ribosomal destruction during apoptosis. Glucocorticoid- and calcium ionophore (A23187)-induced apoptosis of S49 Neo cells resulted in both 28S rRNA and DNA degradation. Interestingly, anisomycin, a potent protein synthesis inhibitor, also induced 28S rRNA and DNA fragmentation suggesting that the responsible nucleases are present in the viable cells and become activated during apoptosis. The anti-apoptotic protein, Bcl-2, inhibited both glucocorticoid- and anisomycin-induced DNA and 28S rRNA degradation but could not protect against A23187-induced nucleic acid degradation. We next examined the role of caspase activation in the generation of 28S rRNA degradation through the use of ZVAD, a general caspase inhibitor. Under conditions where ZVAD substantially decreased 28S rRNA degradation induced by glucocorticoid or anisomycin, no decrease was observed when A23187 was used to induce apoptosis. Surprisingly, RNA degradation, like DNA degradation, occurs exclusively in shrunken lymphocytes but not those with normal cell volume despite equivalent exposure of the cells to the apoptotic signals. Together, these findings indicate the ribosome is a specific target for death effectors during apoptosis and that a caspase/Bcl-2-independent pathway exists to activate its destruction.
Amino Acid Chloromethyl Ketones↗
The cell and molecular biology of apoptosis in T-lymphocytes.
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Apoptosis↗
Caspase independent/dependent regulation of K(+), cell shrinkage, and mitochondrial membrane potential during lymphocyte apoptosis.
The loss of cell volume is a fundamental feature of apoptosis. We have previously shown that DNA degradation and caspase activity occur only in cells which have shrunken as a result of potassium and sodium efflux (Bortner, C. D., Hughes, F. M., Jr., and Cidlowski, J. A. (1997) J. Biol. Chem. 272, 32436-32442). Furthermore, maintaining a normal intracellular potassium concentration represses the cell death process by inhibiting the activity of apoptotic nucleases and suppressing the activation of effector caspases (Hughes, F. M., Jr., Bortner, C. D. Purdy, G. D., and Cidlowski, J. A. (1997) J. Biol. Chem. 272, 30567-30576). We have now investigated the relationship between cell shrinkage, ion efflux, and changes in the mitochondrial membrane potential, in addition to the role of caspases in these apoptotic events. Treatment of Jurkat cells with a series of inducers which act via distinct signal transduction pathways, resulted in all of the cell death characteristics including loss of cell viability, cell shrinkage, K(+) efflux, altered mitochondrial membrane potential, and DNA fragmentation. Interestingly, only cells which shrunk had a loss of mitochondrial membrane potential and the other apoptotic characteristics. Treatment of Jurkat cells with an anti-Fas antibody in the presence of the general caspase inhibitor z-VAD, abrogated these features. In contrast, when Jurkat cells were treated with either the calcium ionophore A23187 or thapsigargin, z-VAD failed to prevent cell shrinkage, K(+) efflux, or changes in the mitochondrial membrane potential, while effectively inhibiting DNA degradation. Treatment of Jurkat cells with various apoptotic agents in the presence of either the caspase-3 inhibitor DEVD, or the caspase-8 inhibitor IETD also blocked DNA degradation, but failed to prevent other characteristics of apoptosis. Together these data suggest that the cell shrinkage, K(+) efflux, and changes in the mitochondrial membrane potential are tightly coupled, but occur independent of DNA degradation, and can be largely caspase independent depending on the particular signal transduction pathway.
Antibodies↗
A necessary role for reduced intracellular potassium during the DNA degradation phase of apoptosis.
Apoptosis is a highly organized mechanism that allows specific cells to die in a controlled manner. Apoptosis can be induced by a variety of agents in a great number of cell types, but cell shrinkage and discrete chromatin cleavage remain common components of apoptosis that is manifested by constitutive enzymes. Despite intensive investigation, the actual event or events that signal this cell death process have not been completely identified. Because of their ability to effect a great number of cellular enzymes simultaneously, we have hypothesized that ions in high concentration (150 mM) in living cells may act as a repressor of apoptotic effectors. In this manuscript we review data that shows that DNA degradation only occurs in the shrunken population of lymphocytes treated with glucocorticoid, shrunken cells loose K+ and become hypotonic, and physiological concentrations on K+ inhibit nuclease activity irrespective of proteins. Together, these data suggest that the reduction in potassium levels appears necessary for the DNA degradation that occurs during apoptosis.
Apoptosis↗
A necessary role for cell shrinkage in apoptosis.
The loss of cell volume is a fundamental and universal characteristic of programmed cell death. However, what was once thought to be a passive, secondary feature of the cell death process has now become an area of research interest. Recent studies have integrated cell volume regulation and the movement of ions with the activation of apoptosis. A dramatic reduction of potassium and sodium concentration has been shown to occur in apoptotic cells that exhibit a shrunken morphology. Furthermore, maintaining the normal physiological intracellular concentration of monovalent ions, particularly potassium, inhibits the activation and activity of the death cascades. Thus, the role ions play during apoptosis is more extensive than just facilitation of the loss of cell volume. In this article, we will review the concepts of cell volume regulation and the loss of volume during apoptosis. Additionally, we will underscore our current understanding of ion movement as it relates to the activation of the cell death process.
Animals↗
A primary role for K+ and Na+ efflux in the activation of apoptosis.
Cell shrinkage is a major characteristic of apoptosis, but the mechanism and role of this process in cell death are poorly understood. The primary factor that controls volume regulation in all cells is ions, and thus we have examined the movement of ions at the single cell level in lymphocytes during apoptosis. Activation of the death program with several stimuli that act through independent pathways to stimulate apoptosis results in a synchronous shift of cells from a normal cell size to a shrunken cell size. Only the shrunken cells exhibit DNA fragmentation and an approximate 4-fold elevation of caspase-3-like activity. Analysis of K+ and Na+ ion content of individual cells by flow cytometry revealed that the intracellular ionic strength of apoptotic cells decreased substantially from their non-shrunken counterparts. Additionally, we show apoptosis is enhanced under conditions where the intracellular K+ concentration is diminished and that apoptosis is inhibited when K+ efflux is prevented. These data show that the efflux of ions, primarily potassium, plays a necessary and perhaps a pivotal role in the cell death program.
Animals↗
Intracellular K+ suppresses the activation of apoptosis in lymphocytes.
Little is known about the mechanisms of suppression of apoptosis. We have addressed the novel possibility that the level of intracellular K+ regulates the apoptotic process by controlling the activity of death enzymes. We show that K+, at normal intracellular levels, inhibits both apoptotic DNA fragmentation and caspase-3(CPP32)-like protease activation, suggesting that intracellular K+ loss must occur early during apoptosis. Direct measurement of K+ by inductively coupled plasma/mass spectrometry and flow cytometry indicates a major decrease in intracellular K+ concentration in the apoptotic cell. Flow cytometric analysis revealed that caspase and nuclease activity were restricted to the subpopulation of cells with reduced K+. Disruption of the natural K+ electrochemical gradient suppressed the activity of both caspase and nuclease independent of the mode of activation of the apoptotic inducing agent, demonstrating that a decrease in intracellular K+ concentration is a necessary, early event in programmed cell death.
Animals↗