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Biomedical subjects

J A Cidlowski

Publications and source records attributed to J A Cidlowski.

At least 37 records · Page 2Linked to original sources
PubMed2000

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↗
PubMed2000

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↗
PubMed2000

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↗
PubMed2000

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↗
PubMed2000

Apoptotic nuclease assays.

One of the defining biochemical characteristics of apoptosis is the degradation of chromatin into regularly sized (oligonucleosomal and approximately 30- to 50-kb) fragments. Because destruction of the genome represents a clear commitment to death, considerable interest has focused on this component of apoptosis and numerous assays have been developed to assess the relevant nucleases involved. These assays fall into two major categories: (1) those independent of chromatin structure and (2) those dependent on chromatin structure. The chromatin-independent assays (plasmid degradation assay and radioactive gel assay) examine the ability to degrade naked DNA and are advantageous because of their simplicity and speed and ability to analyze single nucleases or mixtures of nucleases. However, these assays do not mimic the conditions present in normal cells and consequently do not assess the ability of an enzyme to function in apoptosis. In contrast, chromatin structure-dependent assays (nuclear autodigestion and HeLa nuclei assay) present intact chromatin to either endogenous or exogenous enzymes and assess the ability to degrade chromatin in a manner that recapitulates the genomic destruction seen in vivo. Detailed protocols are discussed for both classes of assays. These assays have been instrumental in the identification of several apoptotic nucleases.

Animals↗
PubMed2000

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↗
PubMed2000

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↗
PubMed2000

Glucocorticoid resistance in asthma is associated with elevated in vivo expression of the glucocorticoid receptor beta-isoform.

BACKGROUND: Glucocorticoid-resistant bronchial asthma is characterized by failure of corticosteroids to suppress key asthma-relevant, cell-mediated inflammatory responses in the airways. OBJECTIVE: The mechanism of this phenomenon is not clear but may involve aberrant expression of the beta-isoform of the glucocorticoid receptor. METHODS: We have measured expression of the alpha- and beta-glucocorticoid receptor isoforms in tuberculin-driven cutaneous cell-mediated inflammatory lesions in people with asthma who are glucocorticoid sensitive and resistant after 9 days of therapy with oral prednisolone (40 mg/day) or matching placebo in a random order, crossover design. RESULTS: After placebo therapy, the mean numbers of cells expressing glucocorticoid receptor alpha immunoreactivity in the lesions evoked in glucocorticoid-sensitive and -resistant patients with asthma were statistically equivalent. The numbers of cells expressing glucocorticoid receptor beta were significantly elevated in the patients who were glucocorticoid resistant, resulting in an 8-fold higher ratio of expression of glucocorticoid receptor alpha/glucocorticoid receptor beta in the patients who were glucocorticoid sensitive. Glucocorticoid receptor alpha/glucocorticoid receptors beta were colocalized to the same cells. Oral prednisolone therapy was associated with a significant decrease in the numbers of cells expressing glucocorticoid receptor alpha but not glucocorticoid receptor beta in the subjects who were glucocorticoid sensitive. No significant change was found in the numbers of cells expressing glucocorticoid receptor alpha and glucocorticoid receptor beta in the patients who were glucocorticoid resistant. Prednisolone therapy reduced the ratio of glucocorticoid receptor alpha/glucocorticoid receptor beta expression for the patients who were glucocorticoid sensitive to a level seen in the patients who were glucocorticoid resistant before therapy. CONCLUSION: Because glucocorticoid receptor beta inhibits alpha-glucocorticoid receptor-mediated transactivation of target genes, the increased expression of glucocorticoid receptor beta in inflammatory cells might be a critical mechanism for conferring glucocorticoid resistance.

Adult↗
PubMed2000

Identification of potassium-dependent and -independent components of the apoptotic machinery in mouse ovarian germ cells and granulosa cells.

Recent studies with thymocytes have suggested a critical role for intracellular potassium in the regulation of apoptosis. In this study, we examined the pathways of K(+) regulation during ovarian cell death. In initial studies, fluorographic analysis demonstrated a significant loss of K(+) during apoptosis stimulated by doxorubicin in oocytes and trophic hormone deprivation in granulosa cells. In oocytes, suppression of potassium efflux by potassium-enriched medium prevented condensation, budding, and fragmentation, although it did not block DNA degradation, suggesting the existence of potassium-independent nucleases in oocytes. Culture of granulosa cells in potassium-enriched medium inhibited internucleosomal DNA cleavage, although high-molecular weight DNA cleavage was apparent, suggesting that the nuclease or nucleases responsible for generating 50-kilobase (kb) fragments in these cells is potassium independent. To address this directly, isolated granulosa cell nuclei were stimulated to autodigest their DNA, and internucleosomal, but not large-fragment, cleavage was completely blocked by 150 mM potassium. We next examined whether the proapoptotic caspases are targets for potassium regulation. In cell-free assays, processing of pro-interleukin-1beta and proteolysis of cellular actin by recombinant caspase-1 and caspase-3, respectively, were suppressed by the presence of 150 mM potassium. Other monovalent ions (NaCl, LiCl) exerted a similar effect in these cell-free assays. Thus, in oocytes and granulosa cells, potassium efflux appears to occur early in the cell death program and may regulate a number of apoptotic events including caspase activity and internucleosomal DNA cleavage. However, there also exist novel potassium-independent pathways in both ovarian germ cells and somatic cells that signal certain apoptotic events, such as large-fragment DNA cleavage.

Animals↗
PubMed2000

Delineation of the signaling pathways involved in glucocorticoid-induced and spontaneous apoptosis of rat thymocytes.

In primary rat thymocytes, both glucocorticoids and the withdrawal of in vivo survival factors elicit apoptosis. In this study we wanted to determine whether distinct pathways leading to apoptosis are engaged by these two stimuli. To address this question, we conducted a multiparametric analysis of cell viability, DNA fragmentation, activation of caspase-3-like activity, cell shrinkage, the loss of mitochondrial membrane potential, and externalization of phosphatidylserine in the absence and presence of protein and RNA synthesis. The role of caspase activity was also examined in both glucocorticoid-and survival factor withdrawal-induced cell death. We show that glucocorticoid-induced, but not spontaneous, loss of viability is dependent upon macromolecular synthesis and caspase activity. Furthermore, glucocorticoid-induced phosphatidylserine externalization and cell shrinkage are dependent upon gene regulation and caspase activity, whereas these features manifest independently of gene regulation and caspase activity in spontaneous death. In contrast, the loss of mitochondrial membrane potential was dependent upon macromolecular synthesis only in glucocorticoid-induced death and was independent of caspases in both spontaneous and dexamethasone-induced death. These results suggest that thymocytes can die by a caspase-independent mechanism and that a major difference between glucocorticoid- and survival factor deprivation-induced death is the dependence on gene expression.

Adrenalectomy↗
PubMed2000

Delineation of an antiapoptotic action of glucocorticoids in hepatoma cells: the role of nuclear factor-kappaB.

Glucocorticoids are primarily recognized for their profound anti-inflammatory actions and their ability to induce lymphocyte apoptosis. We report here that, in contrast to their effect on cells of the immune system, glucocorticoids suppress serum deprivation induced apoptosis of rat hepatoma (HTC) cells. Suppression of apoptosis in these cells occurs at physiological concentrations of glucocorticoid and is abrogated by the glucocorticoid antagonist RU486. Although HTC cells also express receptors for progesterone, estrogen, and thyroid hormone, ligands for these receptors fail to rescue these cells from programmed cell death. Because the sensitivity of cells to apoptotic stimuli is often regulated by the ratio of antiapoptotic to proapoptotic Bcl-2 family members, we analyzed the influence of glucocorticoids and induction of apoptosis by serum starvation on the expression of these proteins. Bcl-2, Bcl-xL, Bad, Bak, and Bax levels were not altered by either treatment. Mitochondrial function has recently been implicated as a critical early regulator of apoptosis in many cells including hepatocytes. Dexamethasone treatment blocked a decrease in this potential (delta psi(m)) during serum deprivation induced apoptosis in HTC cells, indicating an action of this hormone upstream of mitochondria. We also show that the induction of apoptosis in HTC cells is associated with a decrease in nuclear factor (NF)-kappaB. Treatment with dexamethasone effectively blocked the loss of nuclear NF-kappaB, suggesting that this hormone acts to suppress apoptosis of HTC cells via regulation of this nuclear transcription factor. This hypothesis was confirmed by transfection experiments that show that expression of a superrepressor of NF-kappaB inhibits the ability of dexamethasone to rescue HTC cells from apoptosis induced by serum deprivation.

Animals↗
PubMed2000

CBP (CREB binding protein) integrates NF-kappaB (nuclear factor-kappaB) and glucocorticoid receptor physical interactions and antagonism.

Nuclear factor-kappaB (NF-kappaB) and the glucocorticoid receptor (GR) are transcription factors with opposing actions in the modulation of immune/inflammatory responses. NF-kappaB induces the expression of proinflammatory genes, while GR suppresses immune function in part by suppressing expression of the same genes. Previously, we demonstrated that physiological antagonism between NF-kappaB and GR is due to a mutual transcriptional antagonism that requires the p65 subunit of NF-kappaB and multiple domains of GR (1). To elucidate the mechanism(s) of NF-kappaB p65 and GR transcriptional antagonism, we analyzed the interactions of wild-type p65 and p65 RHD (rel homology domain, a dominant negative mutant of p65 which lacks a transactivation domain) with GR. We show that p65RHD blocks p65-mediated transactivation, yet does not block the repression of GR transactivation by p65, indicating that transcriptional activity by p65 is not required to repress GR function. Both p65 and p65 RHD physically interact with GR, but only intact p65 represses GR-mediated signaling, implicating the p65 transactivation domain in the transcriptional repression of GR. To further characterize p65-GR interactions, we examined the role of the transcriptional co-integrator CREB binding protein (CBP) in their mutual antagonism. GR-mediated repression of p65 transactivation and p65-mediated repression of GR transactivation, as well as the physical interaction between NF-kappaB and GR, are enhanced by CBP. GR bound to the antagonist RU 486, although transcriptionally inactive, retains the ability to repress p65 transactivation. However, CBP does not physically interact with antagonist-bound GR and does not enhance its repressive effect on p65. These data suggest that CBP functions as an integrator of p65/GR physical interaction, rather than as a limiting cofactor for which p65 and GR compete.

Animals↗
PubMed1999

The dominant negative activity of the human glucocorticoid receptor beta isoform. Specificity and mechanisms of action.

Alternative splicing of the human glucocorticoid receptor gene generates a nonhormone binding splice variant (hGRbeta) that differs from the wild-type receptor (hGRalpha) only at the carboxyl terminus. Previously we have shown that hGRbeta inhibits the transcriptional activity of hGRalpha, which is consistent with reports of elevated hGRbeta expression in patients with generalized and tissue-specific glucocorticoid resistance. The potential role of hGRbeta in the regulation of target cell sensitivity to glucocorticoids prompted us to further evaluate its dominant negative activity in other model systems and to investigate its mode of action. We demonstrate in multiple cell types that hGRbeta inhibits hGRalpha-mediated activation of the mouse mammary tumor virus promoter. In contrast, the ability of the progesterone and androgen receptors to activate this promoter is only weakly affected by hGRbeta. hGRbeta also inhibits hGRalpha-mediated repression of an NF-kappaB-responsive promoter but does not interfere with homologous down-regulation of hGRalpha. We show that hGRbeta can associate with the heat shock protein hsp90 although with lower affinity than hGRalpha. In addition, hGRbeta binds GRE-containing DNA with a greater capacity than hGRalpha in the absence of glucocorticoids. Glucocorticoid treatment enhances hGRalpha, but not hGRbeta, binding to DNA. Moreover, we demonstrate that hGRalpha and hGRbeta can physically associate with each other in a heterodimer. Finally, we show that the dominant negative activity of hGRbeta resides within its unique carboxyl-terminal 15 amino acids. Taken together, our results suggest that formation of transcriptionally impaired hGRalpha-hGRbeta heterodimers is an important component of the mechanism responsible for the dominant negative activity of hGRbeta.

Alternative Splicing↗
PubMed1999

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↗
PubMed1999

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↗
PubMed1999

Immunocytochemical analysis of the glucocorticoid receptor alpha isoform (GRalpha) using GRalpha-specific antibody.

The alpha isoform of the glucocorticoid receptor (GRalpha) binds glucocorticoids and functions as a ligand-dependent transcription factor. Although GRalpha is expressed in almost all tissues and cells, its subcellular distribution is controversial. Many studies have reported that GRalpha translocates from the cytoplasm to the nucleus in a hormone-dependent manner whereas others have concluded that GRalpha is constitutively located in the nucleus. These conflicting data may result from the use of antibodies that do not discriminate GRalpha from a splice variant of the GR gene termed GRbeta. Using a GRbeta-specific antibody, we have recently demonstrated that GRbeta resides in the nucleus of cells independent of glucocorticoid treatment. In the following study we have generated a novel GRalpha-specific antibody (AShGR) in order to assess, unambiguously, the subcellular distribution of GRalpha. AShGR recognizes recombinant GRalpha on Western blots and in immunoprecipitation experiments but does not cross-react with recombinant GRbeta. Endogenous GRalpha is detected by AShGR in a variety of human cell lines including HeLa S3, CEM-C7, HEK-293, MCF-7, Hep G2, and secondary lung epithelial cells. In addition, AShGR detects endogenous rat and mouse GRalpha. Immunocytochemistry was performed with AShGR on COS-I cells transfected with human GRalpha and on HTC rat hepatoma cells expressing endogenous GRalpha. In both systems, GRalpha was found in the cytoplasm of cells in the absence of hormone and in the nucleus after hormone treatment. These studies mark the first time a GRalpha-specific antibody has been employed to examine the expression and subcellular distribution of endogenous GRalpha.

Amino Acid Sequence↗
PubMed1999

Potassium is a critical regulator of apoptotic enzymes in vitro and in vivo.

Apoptosis, or programmed cell death, is a fundamental biological process involved in many physiological and pathological phenomena. This process is predominantly catabolic in which cellular macromolecules are broken down by distinct enzymes to be later recycled in healthy cells. These enzymes are arranged in an elaborate cascade that serves to both propagate and amplify a death signal as well as process bulk macromolecules and inhibit repair systems. One of the best-characterized enzyme systems involved in apoptosis is the activation of a nuclease(s) that degrades the genome into discrete oligonucleosomal fragments, clearly committing a cell to death. Using two different in vitro models, we have shown that K+ directly inhibits this/these nuclease(s) with complete inhibition observed at cellular concentrations of K+ found in non-dying cells (150 mM). These data suggest that K+ concentrations in living cells suppress apoptotic nuclease activity. One of the nucleases involved in apoptotic DNA degradation (NUC18/Cyclophilin) has been purified in our laboratory and found to be inhibited by similar concentrations of K+. Upstream of DNA fragmentation a specific class of proteases, termed caspases, are activated which propagate an apoptotic signal and lead to downstream events such as DNA fragmentation. Using an in vitro model of caspase activation, we also observed that activation of this enzyme is also completely inhibited by normal physiological K+ levels, suggesting that K+ levels in non-dying cells suppress multiple portions of the apoptotic enzyme cascade. These results suggest that K+ concentrations may decrease in cells undergoing apoptosis and both physical and fluorescence techniques document an intracellular K+ concentration of 35 mM in apoptotic cells. This loss of K+ also accounts for changes in cell volume (cell shrinkage) that are universally associated with apoptosis. Measurements of K+ concentration in shrunken and non-shrunken cells demonstrated that only shrunken cells contain a reduced concentration of intracellular K+. Importantly, both the nuclease and caspase activity were found exclusively in this shrunken population providing an absolute correlation between the activity of these enzymes and low K+ levels within a cell. Finally, suppressing K+ efflux in whole cells prevents the activation of these enzymes whereas enhancing the efflux of this ion facilitates enzymatic activity. These results suggest a direct cause and effect relationship between the level of K+ and the activity of apoptotic enzymes. Taken together we have shown a critical and novel mechanism that regulates apoptosis by a direct effect on the apoptotic enzymes.

Animals↗