1 Department of Medicine, Winthrop-University Hospital, Mineola, New York 11501; Departments of 2 Pathology and 3 Geriatric Services, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854; and 4 Department of Human Morphology, American University of Beirut, Beirut, Lebanon
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ABSTRACT |
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We have previously demonstrated that a plasma natriuretic factor is present in Alzheimer's disease (AD), but not in multi-infarct dementia (MID) or normal controls (C). We postulated that the natriuretic factor might induce the increased cytosolic calcium reported in AD by inhibiting the sodium-calcium antiporter, thereby activating the apoptotic pathway. To test for a factor in AD plasma that induces apoptosis, we exposed nonconfluent cultured LLC-PK1 cells to plasma from AD, MID, and C for 2 h and performed a terminal transferase-dUTP-nick-end labeling (TUNEL) assay. The plasma from AD increased apoptosis nearly fourfold compared with MID and C. The effect was dose dependent and the peak effect was attained after a 2-h exposure. Additionally, apoptotic morphology was detected by electron microscopy, and internucleosomal DNA cleavage was found. We inhibited apoptosis by removing calcium from the medium, inhibiting protein synthesis with cycloheximide, alternately boiling or freezing and thawing the plasma, and digesting a partially purified fraction with trypsin. Heating AD plasma to 56°C did not deactivate the apoptotic factor. These results demonstrate the presence of an apoptotic factor in the plasma of patients with AD.
LLC-PK1 cells; apoptosis; terminal transferase-dUTP-nick-end labeling; natriuretic factor
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INTRODUCTION |
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THE UNITED STATES HAS AN AGING population that is
expected to result in an increase in the incidence of Alzheimer's
disease (AD). One study showed that 20-50% of the population over
the age of 75 had probable AD based on neurological and
neurophysiological examination, and that the incidence increased with
age (6). Definitive diagnosis of individuals with probable AD is made
at autopsy by the identification of -amyloid plaques and tau protein tangles in the brain (10, 23). The development of biochemical, cell
biological, or genetic tests for detection of AD before the onset of
clinical symptoms is an important step toward the development of
preventive therapies.
Both biochemical and genetic evidence have shown that AD has a complex
pathology involving multiple proteins found in the brain (reviewed in
Ref. 28). The role of these proteins and possible interactions between
them in AD are not well understood, although genetic mutations
associated with early onset of AD increase the rate of deposition of
A42 (42 amino acid residue COOH-terminal fragment of amyloid plaque
protein). One current model of AD states that apoptosis is
induced in neuronal cells, with the resulting loss of cells leading to dementia.
The evidence for increased apoptosis in the brains of patients with AD
has recently been reviewed (4, 12). More terminal transferase-dUTP-nick-end labeling (TUNEL)-positive cells are present
in AD brains than in controls; many of these have apoptotic morphology,
which is the classic definition of apoptosis (14). Consistent with a
role for apoptosis are changes in the expression of death-related genes
and the observation that antibodies specific to the products of
apoptotic proteolysis indicate cleavage by caspases in AD brains.
Furthermore, numerous investigators have found that many of the
proteins implicated in AD can induce apoptosis in cultured cells. In a
transgenic mouse model of AD that expresses -amyloid protein in
neurons (16), internucleosomal DNA breaks have been found to occur in
the brain.
Previous work from our laboratory made use of an in vivo assay to identify a natriuretic factor(s) present in the plasma of individuals with AD (20-22). To facilitate isolation of the factor, it was necessary to develop an in vitro method to enhance reproducibility and sensitivity of the bioassay, shorten the time of study, use less plasma volume, and increase the number of plasmas or fractions of plasma that could be tested. We postulated that the natriuretic potential of the plasma factor might affect sodium-dependent cellular functions, such as sodium/calcium exchange, resulting in an increase in cytosolic calcium and initiation of apoptosis (20-22). LLC-PK1 cells were selected for our apoptotic assay because of their differentiated phenotype as the renal proximal tubule, which is the exclusive or predominant site at which urate and lithium are transported and the major site of action for the plasma natriuretic factor (18, 21). The present studies describe an apoptotic factor found in the plasma of patients with AD using our TUNEL assay.
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METHODS |
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Patients were randomly recruited at the Division of Geriatric
Psychiatry, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, based on their willingness to participate in the study. All subjects were examined by a Board-certified geriatric
neuropsychiatrist who established the diagnosis of dementia. The
bioassay was performed at Winthrop University Hospital. The protocol
for these studies was approved by the respective institutional review
boards of both institutions. Consent from demented patients was
obtained from their legal guardians in all cases. Seventeen AD subjects
met National Institute of Neurological and Communicative Disorders and
Stroke-Alzheimer's Disease and Related Disorders Association criteria
for probable AD (25) and 11 multi-infarct dementia (MID) subjects met
DSM-IIIR criteria for the diagnosis of MID and had Hachinski
Ischemia Scale scores greater than 7 (2, 11). Nine
subjects of the same age and gender distribution served as normal
controls (C). In addition to the routine testing, all patients received
either a computed tomography (CT) scan or magnetic resonance imaging of
the brain and a Mini-Mental State Examination (MMSE) score
(7). Heparinized whole blood from all subjects was
centrifuged with a clinical centrifuge within 30 min after collection.
The plasma was then transferred to a plastic container and stored at
70°C. All samples were stored at
70°C until time
of bioassay, except during overnight shipping on dry ice. Bioassays of
plasma samples were performed in a double-blind manner.
Cell culture and protocol. LLC-PK1, a pig kidney epithelial cell line (18) (kindly supplied by Dr. Julia Lever, University of Texas, Houston), was plated into eight-well Permanox plastic chamber slides (NUNC, Naperville, IL) at a density of 103 cells/well. The cells were cultured at 37°C in 5% CO2 in humidified incubators and grown for 3 days to 70-80% confluency in 300 µl/well of DMEM-F12 supplemented with 10% fetal calf serum, 7.5% sodium bicarbonate, 15 mM HEPES, 200 mM L-glutamate, 100 U penicillin, and 0.1 µg/ml streptomycin (Life Technologies, Gaithersburg, MD). The culture fluid was then removed and cells were exposed to 60 µl of plasma from C, AD, or MID diluted in fresh DMEM-F12 media for 2 h at 37°C in 5% humidified CO2. Before analysis, all plasma samples were dialyzed against 1,000× vol of 20 mmol sodium phosphate, pH 7.1, with three changes of buffer to reduce the possible effects of trace medications. Samples were then subjected to low-speed centrifugation to remove particulates and fats. The cells were then rinsed in PBS and fixed in 4% formaldehyde in PBS for 10 min, permeabilized with 0.5% Triton X-100 (Sigma Chemical, St. Louis, MO) for 5 min, and washed in four changes of distilled water. We obtained a positive control by exposing cells to 0.6 mM H2O2 diluted in DMEM-F12 for 2 h.
Apoptosis assay. Nuclear DNA fragmentation consistent with apoptosis was determined by the TUNEL method (8). We used the ApopDetek cell death assay kit (Enzo, Farmingdale, NY) utilizing terminal deoxynucleotidyl transferase to incorporate Bio-16-dUTP onto the 3'-OH termini in the DNA of apoptotic cells, subsequent binding with strepavidin-horseradish peroxidase, and visualization after conversion of the substrate and chromagen (hydrogen peroxide and aminoethylcarbazole) into a localized brick red precipitate. A blue counterstain was also used. Slides were then observed using a Nikon Optiphot microscope (Nikon, Melville, NY) for morphologically irregular and condensed nuclei containing dark red precipitate, indicating TUNEL-positive cells. Five to six random fields totaling ~1,000-1,500 cells were counted per slide. The apoptotic index (AI), defined as the percentage of cells undergoing apoptosis, is calculated by dividing the number of positive nuclei by the total number of nuclei counted multiplied by 100.
Dose- and time-response studies. We performed the TUNEL assay in LLC-PK1 cells that were exposed to different dilutions of AD and C plasma at different intervals in time. AD and C plasma were diluted with DMEM-F12 (1:100, 1:20, 1:10, 1:5, 1:3, and 1:2) and added to 70-80% confluent LLC-PK1 cells for 2 h. Conversely, AD and C plasma were diluted with DMEM-F12 (1:5) and exposed to LLC-PK1 cells for 2, 4, 6, and 8 h. Fresh medium and C or AD plasma were added every 2 h. TUNEL assays were performed to determine AI at 2, 4, 6, and 8 h for AD plasma and at 8 h for C plasma. The selection of a 2-h exposure in the dilution studies and a 1:5 dilution of plasma in the time-response studies was based on the maximum AI noted in the respective studies without the addition of fresh medium and plasma.
Electron microscopy. LLC-PK1 cells were plated at 103 cells per 35-mm plastic petri dish, exposed to AD or C plasma at 1:5 dilution in DMEM-F12 for 2 h, and fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate, pH 7.2, for 1 h at 4°C. The cells were then postfixed in 1% buffered osmium tetroxide, dehydrated in a graded series of ethanol, and embedded in LX112 (Ladd Research Industries, Burlington, VT). En face and cross-sectional thin sections were stained with uranyl acetate and lead citrate and examined on a Zeiss EM10 transmission electron microscope. AI was determined from three blocks representing different areas from each sample and examined for morphologically apoptotic cells (i.e., condensed, blackened nuclei). At least 300 cells were counted per condition, and the mean number of apoptotic cells ± SE was quantified.
DNA ladder assay. The DNA ladder was observed using a modification of the procedure described by Eastman (5). LLC-PK1 cells (106) were seeded into T75 flasks (Falcon) containing 10 ml of DMEM-F12 and 10% fetal calf serum supplemented with 0.12% NaHCO3, 5 mM glutamine, 15 mM HEPES, and 1% penicillin-streptomycin. Cells were allowed to attach overnight at 37°C in 5% humidified CO2. Five milliliters of medium were withdrawn and 0.5 ml of test plasma were added. Cells in the medium and adherent cells (0.05% trypsin in 0.53 mM EDTA, 3 min, 37°C; GIBCO) were harvested on days 2, 3, 4, and 5 by centrifugation at 142 g for 3 min at room temperature. The cell pellet was warmed to 50°C for 2-3 min and resuspended in 2% Sea Plaque agarose (FMC, Rockland, ME) in 0.125 M EDTA, pH 7.4, and dispensed into a precooled 4°C mold. The agarose plugs were incubated at 50°C for 2 h in 0.5 M EDTA, pH 8.0, 1% sarcosine (Sigma), and 1 mg/ml of proteinase K (Boehringer Mannheim). Plugs were then incubated at 37°C for 30 min in 10× volume of 10 mM Tris · HCl, pH 7.5, and 50 mM EDTA. The buffer was exchanged with TE (10 mM Tris · HCl, pH 7.5, and 1 mM EDTA); RNase A, previously boiled for 15 min (29), was added to a final concentration of 250 µg/ml, and the plugs were incubated an additional 30 min at 37°C. DNA in the plugs was subjected to electrophoresis through a 2% SeaKem (FMC) agarose gel in TAE buffer (40 mM Tris-acetate, 2 mM EDTA, pH 8.5) (29) at 2 V/cm for 14 h and visualized as described (5).
Partial protein purification and heating of plasma. Pooled AD and C plasma were dialyzed in a 10-kDa cutoff membrane in 20 mM phosphate buffer, pH 7.1, and centrifuged at 13,000 g for 15 min. The clear supernatant was loaded onto a 10-ml Affi-Gel Blue Gel affinity column (Bio-Rad, Hercules, CA) to bind and eliminate albumin from plasma. The column was then washed with wash buffer until protein was not detectable, then sequentially eluted with 25 ml of 0.5 M NaCl and 10 ml of 2 M NaCl in buffer. Protein concentration was monitored by ultraviolet absorbance at 280 nm. The three protein fractions (load and wash, 0.5 M NaCl and 2 M NaCl) were dialyzed in a 10-kDa cutoff membrane, concentrated over a bed of PEG 8000 in a 1-kDa cutoff membrane, and dialyzed in a 10-kDa cutoff membrane against 10 mM sodium phosphate buffer, pH 7.1. Cultured LLC-PK1 cells were then exposed to 30-100 µg of the three pooled protein fractions for 2 h at 37°C and a TUNEL assay was performed.
In separate experiments, AD and C plasma were heat treated at 56°C for 30 min. In some experiments, plasma was boiled at 100°C for 5 min and the denatured protein aggregates were removed by sedimentation at 1,000 g for 1 min before being tested by TUNEL assay as noted above. In separate experiments, AD plasma was alternately frozen atIsoelectric focusing. The active fraction from the Affi-Gel Blue Gel run (0.5 M NaCl) was further fractionated by isoelectric focusing (IEF) using a Rotofor (Bio-Rad) after dialysis against 10 mM sodium phosphate buffer, pH 7.1. This active fraction was run at a pH gradient of 3-10 at a constant power of 15 W at 4°C for 4 h using Bio-Lyte ampholyte 3/10. Fractions were pooled according to their protein profile and assayed for apoptotic activity. The fractions with the highest AI were pooled, dialyzed, and refractionated by IEF using a narrower pH gradient of 4-6 at the same settings, utilizing Bio-Lyte ampholyte 4/6 and 3/10, 80:20%, respectively (Bio-Rad).
Effect of protein synthesis on apoptotic activity. LLC-PK1 cells were exposed to AD and C plasma in the absence and presence of cycloheximide (0.2-200 µM; Sigma). AI was measured in these cells by TUNEL assay.
Effect of calcium depletion on factor activity. The TUNEL assay was performed in the usual manner except for the substitution of DMEM-F12 with calcium-free DMEM, supplemented with dialyzed 10% fetal calf serum (Life Technologies) and 0.6 mM EGTA to chelate calcium. Dialyzed AD and C plasma were then added to the calcium-free media for 2 h at 37°C and a TUNEL assay was performed.
Contribution of known apoptotic
inducers.
To test the possibility that the apoptotic factor in AD plasma was
-amyloid (31, 33), tumor necrosis factor-
(TNF-
), or
interleukin-1
(19), the TUNEL assay was repeated as described above
after a 2-h incubation at 37°C with 0.10-50 µM
-amyloid (Peninsula Laboratories, Belmont, CA) or 5 pM to 3 nM TNF-
(Quantikine, Minneapolis, MN). To eliminate the
possibility that a protease in AD plasma was responsible for the
apoptotic activity (13, 32, 34), we studied the effect of a
broad-spectrum protease inhibitor cocktail (Boehringer Mannheim) on AI
using the TUNEL assay. LLC-PK1
cells were incubated with AD plasma with and without the inhibitor
cocktail and assayed as described. This cocktail inhibits a large
spectrum of serine, cysteine, and metalloproteases, as well as
calpains. It consists of aprotenin, leupeptin, EDTA, and Pefabloc.
Statistical analysis. All TUNEL assays were performed in triplicate and the data was expressed as means ± SE. An unpaired Student's t-test was used to compare one set of experiments with the other; P < 0.05 was deemed significant. A multivariate analysis was used to determine whether any medications taken by the patients might affect the results and whether there was a correlation between AI and MMSE scores.
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RESULTS |
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Electron microscopy of LLC-PK1
cells after incubation with AD plasma illustrates the distinct
difference of apoptotic and non-apoptotic cells. Apoptotic cells have
condensed, black nuclei, and some cells are noted to be shrunken and
engulfed by neighboring cells, as shown in Fig.
1, A and
B. A histogram showing
quantification of AI determined by electron microscopy of
LLC-PK1 cells exposed to a 1:5
dilution of the indicated plasmas for 2 h is shown in Fig.
1C.
LLC-PK1 cells incubated for 2 h in
medium plus a 1:5 dilution of AD, MID, or C plasma resulted in AI
values of 14.3 ± 1.8%, 3.3 ± 0.33%, and 4.3 ± 0.33%,
respectively. Light microscopic views of these cells, which had been
labeled in situ by the TUNEL method, are depicted in Fig.
2.
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Table 1 summarizes the results of the
exposure of LLC-PK1 cells to AD,
MID, and C plasma. There was nearly a fourfold increase in AI in
LLC-PK1 cells that were exposed to
AD plasma (25.6 ± 8.8%) compared with C (6.0 ± 2.4%;
P < 0.001) and MID (6.5 ± 2.3%; P < 0.001). There was no
significant difference in AI between C and MID
(P > 0.05). As shown in Fig.
3B, AI
values increased linearly up to 8 h of incubation, with replenishment
of media and plasma every 2 h. Incubation for 2, 4, 6, and 8 h at a 1:5 dilution produced AI values for AD plasma of 18.1 ± 1.4%, 41.9 ± 2.2%, 63.9 ± 4.5%, and 79.9 ± 2.9%,
respectively, and the control AI at 8 h was 6.8 ± 0.8%. Diluting
AD plasma in a range of 1:2 to 1:100 revealed a maximum AI of 12.4 ± 0.2% at a 1:5 dilution of plasma with medium (Fig.
3A). We observed AI values of 3.44 ± 0.13%, 8.3 ± 0.09%, 9.38 ± 0.16%, 12.4 ± 0.15%,
12.1 ± 0.17%, and 9.24 ± 0.11% at dilutions of 1:100, 1:20,
1:10, 1:5, 1:3, and 1:2, respectively. Controls ranged from 3.16 ± 0.09% to 3.71 ± 0.08%. There was a significant
reduction in AI at 1:2 compared with 1:3 and 1:5 dilutions
(P < 0.001 for both), but there was no statistical difference between 1:3 and 1:5 dilutions of plasma. All
plasma samples were dialyzed before the TUNEL assay to reduce possible
effects of trace medications. Furthermore, there was no statistical
correlation between AI and the medications the patients had been taking
at the time of study or the MMSE scores in AD.
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Figure 4 shows internucleosomal DNA
cleavage in LLC-PK1 cells that had
been exposed to AD plasma for intervals of 2, 3, 4, and 5 days. Maximum DNA fragmentation was 4 days after exposure to AD plasma and showed the characteristic 180-bp spacing. C plasma did
not exhibit a ladder, even after incubation for 5 days.
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As noted in Table 2, elimination of calcium
from the incubating medium, fetal bovine serum, and plasma or
incubation with 200 µM cycloheximide resulted in inhibition of
apoptosis by AD plasma, suggesting that apoptosis in the system is
dependent on the level of extracellular calcium and protein synthesis.
There was no inhibition of apoptosis at the lower concentrations of cycloheximide. In a separate group of experiments, heating AD and C
plasma at 56°C for 30 min, which deactivated the complement, did
not alter the apoptotic activity (Table 2). However, boiling the AD
plasma at 100°C for 5 min resulted in AI that was no different from
control plasma. Moreover, freezing and thawing the plasma from
70°C to room temperature at least three times decreased apoptotic activity (Table 2). These data suggest that the apoptotic factor in AD plasma is a protein.
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To exclude the possibility that the factor is -amyloid, we incubated
cells with 0.1, 10, and 50 µM of
-amyloid dissolved in media. No
detectable apoptotic activity was observed even at 50 µM (AI was 6.1 ± 3.1%). To eliminate the possibility that TNF-
might be the
apoptotic factor, we quantitated C and AD plasma for the presence of
TNF-
by ELISA (Quantikine). The levels of TNF-
in C and AD plasma
were less than the lowest level of detection by the ELISA kit (0.3 pM).
We achieved a standard curve with ELISA with TNF-
standards and
blocked the reaction utilizing TNF-
antibody. We also tested the
effect of TNF-
on LLC-PK1 cells at 5, 50, 500, and 3,000 pM for 2 h and found that doses as high as 50 pM yielded background levels of apoptosis (AI was 6%). On a Western
blot, both the C and AD plasma resulted in no signal, whereas a
positive control of 100 ng TNF-
yielded a bright positive signal. A
similar situation occurred with interleukin-1
. Both C and AD plasma
had undetectable levels of interleukin-1
by ELISA.
The protein in AD plasma was fractionated on an Affi-Gel Blue Gel
column. The highest AI of 21% was found in the 0.5 M NaCl eluate. No
activity was found in the load and wash fraction and only a modest
activity was noted in the 2 M NaCl eluate (AI of 6% vs. 9%,
respectively). The 2 M NaCl eluate was mainly composed of albumin. The
partially purified factor in the 0.5 M NaCl eluate was dialyzed
overnight in a 10-kDa cutoff membrane at 4°C against 10 mM sodium
phosphate buffer, pH 7.1, to remove salt. IEF was performed on this
protein fraction at a pH gradient of 3-10 (Fig. 5A).
Fractions within clearly defined protein peaks were pooled and dialyzed
to remove ampholyte, then TUNEL assay was performed to check for
apoptotic activity. Dialysis with a 10-kDa cutoff membrane demonstrated
the retention of apoptotic activity in the dialysis bag, suggesting
that the size of the protein exceeded 10 kDa. The highest AI of 29.4%
was noted in fraction 2 of the pooled samples. An IEF repeated only on
this active fraction by respreading on a narrow-range pH gradient of
4-6 (Fig. 5B) yielded an AI of
22% in fraction 2 of this additional purification step. The pI range
of both fractions was 4.7-5.5.
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DISCUSSION |
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We have demonstrated, in the plasma of patients with AD, the presence of a factor(s) that increases apoptosis in cultured LLC-PK1 cells compared with plasma from C and MID. This conclusion is based on three lines of evidence: 1) TUNEL assay, 2) demonstration of a nucleosomal ladder by agarose gel electrophoresis, and 3) electron micrographic analysis showing typical morphology of apoptosis. It is of interest to note that these findings are consistent with our previous studies, which demonstrated the presence of a natriuretic factor(s) in the plasma of patients with AD but not in those of C or MID (22). The present studies, however, do not distinguish whether or not the apoptotic and natriuretic activities found in AD plasma are the result of a single factor or a combination of several factors. There was no correlation between the extent of apoptosis and MMSE scores or degree of dementia.
The degree of apoptosis determined with the TUNEL assay was dose dependent, with a peak at the 1:5 and 1:3 dilutions of AD plasma. The significant reduction in AI at the lowest dilution (1:2) may be due to an inhibitor that diminishes its activity with increasing dilution of plasma. We anticipate resolution of this issue on purification and characterization of the apoptotic factor.
The conclusion that apoptosis results from a factor(s) in AD plasma is based on observed DNA degradation in the nuclei of affected cells. The degree of apoptosis was dose and time dependent, peaking at 120 min when the factor was not replenished after exposure to AD plasma. Moreover, preliminary removal of albumin by Affi-Gel Blue Gel column was an important step in eliminating a large protein that facilitated partial purification of the factor. Subsequent detection of a protein fraction with high apoptotic activity in the 0.5 M NaCl eluate in the Affi-Gel Blue Gel column; multiple dialyses with a 10-kDa cutoff membrane; inactivation of apoptotic activity by boiling, freeze-thawing, or trypsin digestion; and reproducible demonstration of an isoelectric point between 4.7 and 5.5 pH all argue for the presence of a protein in the plasma of patients with AD that induces apoptosis in cultured LLC-PK1 cells. Because factor activity was still present after dialysis and after further purification of plasma by the Affi-Gel Blue Gel affinity column and IEF, it is highly unlikely that any of the medications the patients had been receiving at the time of the study or a spurious contaminant could explain these findings.
The double-stranded endonucleolytic cleavage of DNA occurs at the linker regions of nucleosomes to produce fragments of multiples of ~180 bp (35); this is seen as the apoptotic ladder by agarose gel electrophoresis. This fragmentation of DNA appears coincident with condensation of nuclear chromatin before cell death and is considered a characteristic biochemical feature of apoptosis (14). Demonstration of this repeat pattern was, therefore, used as an indicator of apoptosis. The 3'-OH ends of this cleaved DNA can also serve as the substrate for terminal deoxynucleotidyl transferase TdT, which led to the development of TUNEL (8). This technique results in the labeling of nuclei in situ, before the appearance of the ladder by gel electrophoresis. TUNEL staining of DNA fragments occurs not only in histologically defined apoptotic cells, but also in intact cells during the early stages of apoptosis (8).
The induction of apoptosis in cultured LLC-PK1 cells by a plasma factor in AD follows a characteristic pattern of apoptosis in other systems. Apoptosis was inhibited by the elimination of calcium and by the inhibition of protein synthesis. Our observations are consistent with a role of increased intracellular calcium in the apoptosis of LLC-PK1 cells induced by AD plasma (27).
A role for apoptosis in AD has been suggested and demonstrated in the post mortem brain of AD compared with age-matched controls (3, 17). The increase in cytosolic calcium might be the stimulus that initiates apoptosis (24, 27, 30). However, the relationship of the apoptotic factor to these observations and to the natriuretic factor in AD remains to be resolved.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. K. Maesaka, Dept. of Medicine, Div. of Nephrology and Hypertension, Winthrop-Univ. Hospital, 222 Station Plaza North, Suite 510, Mineola, NY 11501 (E-mail: jmaesaka{at}winthrop.org).
Received 14 October 1997; accepted in final form 11 November 1998.
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