Nicotine inhibition of apoptosis suggests a role in tumor promotion. SUSAN C. WRIGHT,1. JIAN. ZHONG, HUI ZHNG,. AND JAMES. W. LARIUCE. Palo Alto ...
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Nicotine inhibition promotion SUSAN
-
of apoptosis
C. WRIGHT,1
Palo Alto Institute
“ -
ZHONG,
JIAN
of Molecular
HUI ZHNG,
Medicine,
Mountain
Recent evidence indicates that cell death through apoptosis may be an important mechanism to prevent tumor development. Therefore, agents that inhibit apoptosis may function as tumor promotors. The purpose of this study was to determine the effects of nicotine on the process of apoptosis. The results demonstrate that nicotine inhibits apoptosis induced by diverse stimuli including tumor necrosis factor (TNF), UV light, chemotherapeutic drugs, and calcium ionophore. This phenomenon was observed in normal and transformed cells derived from a variety of species and tissues, including tumor cell types related to tobacco use. The major nicotine metabolite, cotinine, also inhibited apoptosis, whereas N-nitrosodiethylamine, a carcinogen found in tobacco, was without effect. Therefore, nicotine-mediated inhibition of apoptosis may contribute to the pathogenesis of tobacco-related cancer as well as decrease the efficacy of cancer therapies.-Wright, S. C., Zhong, J., Zheng, H., Larrick, J. W. Nicotine inhibition of apoptosis suggests a role in tumor production. FASEB J. 7: 1045-1051; 1993. ABSTRACT
Key Wordr: nicotine
apoptostr
Aovrosis
IS
death
DNA fragmentation
THE
NORMAL
that functions
bryogenesis,
dependent
responses,
immune
tissues,
tumor necrosis factor.
process
PHYSIOLOGICAL
to control and normal
cell populations hormone
withdrawal
tissue homeostasis
em-
supports
the theory
that
Cell
lines
The
human
induction
of
tumors. This hypothesis is supported that expression of oncogenes such as
tumor
suppressor
AND
METHODS
histiocytic
lymphoma,
U937,
as well as 3T3,
WEHI-164,
mechanism to eliminate nascent tumor cells. Thus, the acquisition of resistance to apoptosis would confer a survival
bcl-2 (7) or mutant
MATERIALS
necrotic criteria
immune defense mechanisms (4, 5) or homeostatic controls (6) is an important
advantage to emerging by the recent findings
USA
According to the two-stage model of carcinogenesis, tumors are initiated by exposure of cells to agents that cause mutations which tend to release cells from growth controls. Subsequent exposure to tumor-promoting agents accelerates cancer development, albeit by poorly understood mechanisms. We postulate that inhibition of apoptosis may be one mechanism of tumor promotion. It follows that exposure to agents in the environment that inhibit apoptosis could promote tumor formation, especially if it coincides with or follows exposure to known carcinogens. Although the occurrence of carcinogens in the environment has been widely studied, little is known about the prevalence of agents that inhibit apoptosis. The use of tobacco has been well documented to increase the risk for various cancers, including lung, oral, esophageal, and bladder (13). Of the many components of tobacco, several strong carcinogens have been identified, such as N-nitroso compounds and benzo[a]pyrene (for review see ref 14). However, little is known concerning the presence in tobacco of potential tumor-promoting agents. Nicotine is the major alkaloid in tobacco and is not carcinogenic, although it is addictive. The present study was undertaken to determine if nicotine can inhibit apoptosis, thus implicating it as a potential tumor-promoting agent in tobacco-related carcinogenesis.
(reviewed
apoptotic bodies, cell shrinking, and DNA fragmentation into multiples of 180 base pairs before cell death. In contrast, necrosis is characterized by cell and organelle swelling, loss of membrane integrity, and digestion of DNA into fragments of a continuous spectrum of sizes after cell death.
evidence
W. LARIUCE
CA 94043,
from
(reviewed in ref 3). Hallmarks of apoptosis include cytoplasmic membrane blebbing with release of membrane-bounded
Growing
AND JAMES Vie
a role in tumor
of cell
during
in refs 1 and 2). It can be distinguished from the form of cell death by morphological and biochemical
apoptosis through other tissue-specific
suggests
genes such as p53 (8)
a cell from apoptosis. Moreover, many chemoagents, including drugs (9, 10), radiation (11), and heat treatment (12), appear to kill tumor cells by apoptosis. Therefore, understanding the mechanisms of acquisition
Cos 7, Du-145, FADU, A427, and H661 cell lines were obtained from the KItC. All cell lines were maintained in RPMI 1640 supplemented with 10% FCS, penicillinstreptomycin, and L-glutamine (2 mM). All cell lines were routinely tested for mycoplasma and always found to be negative according to the Mycotect kit (GIBCO, Grand Island, N.Y.). Reagents Purified human rTNF (specific activity = 1 x 10 purchased from R&D Systems, Minneapolis, Nicotine, (- )-nicotine, (+ )-nicotine, cotinine, and N-nitrosodiethylamine were purchased from Louis, Mo.). For all experiments, (±)-nicotine unless otherwise indicated.
U/mg) was Minn. (± )anabasine, Sigma (St. was used
may protect therapeutic
and expression provements
of resistance
in the prevention
0892-6638/93/0007-1045/$01.50.
to apoptosis and
© FASEB
treatment
may lead to imof cancer.
iTo whom reprint requests should be addressed, at: Palo Alto Institute of Molecular Medicine, 2462 Wyandotte St., Mountain View, CA 94043, USA.
1045
RESEARCH Quantitative
COMMUNICATIONS assay
of DNA
The assay we routinely use to quantitate DNA fragmentation is a modification of a previously described assay (11) that was adapted to the 96-well microtiter format. The major advantage of our modified assay is that it facilitates processing of large numbers of samples. This assay was described in detail previously and found to produce results similar to other standard DNA fragmentation assays (15). The assay was set up in triplicate in a flat-bottom, 96-well microtiter plate in RPMI 1640 with 2.5% FCS in a total volume of 0.1 ml. Target cells were suspended at 1 x 106/ml and 0.05 ml was added to each well. Wells for total counts received an additional 0.05 ml of medium, whereas experimental wells received 0.05 ml of the appropriate concentrations of TNF. Plates were usually incubated for 20 h at 37#{176}C but in some ,experiments the incubation time was shortened as indicated. The assay was terminated by addition of 150 il 10 mM EDTA and 0.3% Triton X-100 to each well. Plates were harvested using a Packard harvester and samples were counted using a Packard Matrix 96 beta counter. The percent of DNA release was calculated as follows: total % DNA
release
cpm
-
experimental
cpm x 100
=
total
cpm
Using this technique, the percent of spontaneous DNA release varied from 0 to 5% over a 5 h incubation period and did not exceed 10% release in a 20 h incubation. Correcting for these low levels of spontaneous release did not significantly alter the results, and therefore it was not performed. Standard deviations were always less than 15% of the mean of the triplicate values. Visualization electrophoresis
of DNA
fragmentation
by gel
The second method to analyze DNA fragmentation used agarose gel electrophoresis. Target cells were treated with TNF or the various agents and the DNA was harvested as described previously (15) under conditions similar to cell lysis in the above DNA fragmentation assay. Triton X-100 lyses the cytoplasmic membrane, whereas chelation of divalent cations with EDTA releases nuclear DNA. After lysing the cells, debris was removed by centrifugation at 13,000 x g for 10 s. DNA in the supernatant was ethanol-precipitated after phenol extraction. Equivalent amounts of material from a fixed number of cells were loaded and electrophoresed on a 1.0% agarose slab gel.
RESULTS Nicotine inhibits chemotherapeutic
U93 7 apoptosis drugs
induced
by TNF
or
Our laboratory has been studying the mechanism (or mechanisms) of apoptosis using in vitro model systems with several target cells and apoptosis-inducing agents. Our previous studies demonstrated that tumor necrosis factor-a (TNF)2 induces apoptosis in the human histiocytic lymphoma cell line, U937 (15). Figure 1 demonstrates that nicotine caused a dose-dependent inhibition of TNF-induced digestion of DNA. Nicotine completely abolished apoptosis at a concentration of 1.0 mM and suppressed 60-70% of DNA fragmentation at 0.1 mM. Nicotine was nontoxic in all the experiments reported in this study, as judged by trypan blue
1046
Vol. 7
August
1993
TNF Control
II
fragmentation
TNF+nicothe buM 40
lOOuM
TNF+nicotine
TNF+n,cotine b000uM 30 0
0 E 0 U-
20
10
z a 4
6
TNF Figure
1. Nicotine
inhibits
8
10
apoptosis.
U937
12
(ng/mI)
TNF-induced
target
cells were pretreated for 30 mm with different concentrations of nicotine followed by addition of TNE DNA fragmentation was measured by release of [5H]thymidine-labeled DNA fragments after a 20 h incubation as described in Materials and Methods. Each point represents the mean of triplicate values and all standard deviations (not shown) were less than 15%. This experiment has
been repeated
on four other occasions
with similar
results.
exclusion, and in no case was there any evidence of necrosis, as judged by microscopic examination. Nicotine also inhibited apoptosis of U937 induced by chemotherapeutic drugs, such as vinblastine (VBL), taxol, and doxorubicin (Dox) (Fig. 2A). Nicotine
inhibits
apoptosis
induced
by UV
light
To extend these findings, we studied the effect of nicotine on ultraviolet (UV) light treatment, which rapidly and potently activates apoptosis in U937 and several other tumor cell lines. In this experiment, U937 cells cultured in the presence of nicotine for 30 mm or 2 days before exposure to UV light were highly resistant to apoptosis (Fig. 2B). Nicotine as low as 10 iM caused 80-95% suppression of DNA fragmentation. This finding was confirmed by agarose gel analysis of DNA extracted from normal U937 (Fig. 3, lane 2), and cells exposed to UV light in the presence and absence of nicotine. UV light induced the DNA to fragment into multiples of 180 base pairs, producing the characteristic “ladder” pattern (lane 3). However, no such fragments were visible in extracts from UV-treated cells incubated with 10 M (lane 6) or 100 tM nicotine (lane 7). Nicotine alone had no apparent effect on control U937 cells (lanes 4 and 5). These combined data demonstrate that nicotine inhibits apoptosis induced in U937 cells by diverse stimuli. To explore the generality of the phenomenon, we tested the effects of nicotine on UV light-induced apoptosis in a variety of cell types. The results in Fig. 4 show that nicotine inhibited apoptosis of the nontransformed murine fibroblast,
2Abbreviations: Dox, PMA, phorbol myristate VBL, vinblastine.
The FASEB Journal
doxorubicin; PKC, protein acetate; TNF, tumor necrosis
kinase
C;
factor-a;
WRIGHT
El AL.
RESEARCH A H
1.0
VOL
0.2
0.04
-J1-u-
VOl.
T65d
0.2 04
10 2 0.4
H
Fil
]-
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_____________ 10 +
Taxol
2 0.4
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50 25
Dox
50
+
Dox
25 12.1
_____________ H 20
30
40
50
Although apoptosis
showed that nicotine inhibits of cell types, we wanted to
the above in a wide
studies variety
cell lines
linked
to
inhibits
apoptosis
of normal
thymocytes
Further experiments were performed to determine if this phenomenon was limited to in vitro maintained cell lines. It has been reported that treatment of normal murine thymocytes with the calcium ionophore A23187 induces apoptosis (16). Therefore, we tested the effects of nicotine in this sys-
H
10
of tumor
Nicotine
J-l
H
12.5
Nicotine inhibits apoptosis tobacco-related malignancies
confirm that this also applied to tumor cells assocaited with tobacco-related cancer. Therefore we tested the effects of nicotine on UV light-induced apoptosis of the human pharyngeal carcinoma, FADU, as well as two human lung cancer cell lines, A427 and H661. The results shown in Fig. 5 demonstrate that nicotine dose-dependently suppressed DNA fragmentation in all three cell lines at concentrations ranging from 60 to 500 LM. These findings support the hypothesis that nicotine inhibition of apoptosis may contribute to the development of tobacco-related malignancies.
1.0 +
COMMUNICATIONS
6070
80
tem. The results (Fig. 6) show that nicotine inhibits apoptosis of freshly explanted murine thymocytes. Therefore, nicotine inhibition of DNA fragmentation appears to be a phenomenon of widespread occurrence in both normal and
90
DNA Fragmentation (%)
transformed cells of various ducers of apoptosis.
B
Specificity
UV Control
of nicotine
types
inhibition
responding
to diverse
in-
of apoptosis
To examine the specificity of the nicotine effect, we tested several other tobacco-related compounds. The major nicotine metabolite, cotinine, as well as a related alkaloid, anabasine, also inhibited TNF-induced apoptosis of U937 cells
1234567 2
days
10
20
30
40
50
60
DNA Fragmentation
70
80
90
100
(%)
Figure 2. Nicotine inhibits chemotherapeutic drug and UV lightinduced apoptosis. A) U937 target cells were pretreated with nicotine for 30 mm, then the various drugs were added and DNA fragmentation was measured after 20 h as described in Fig. 1. Each point represents the mean of triplicates +1standard deviation. This experiment has been repeated on two other occasions with similar results. B) U937 cells were treated for the indicated length of time with different concentrations of nicotine. After exposure to UV light at 0.2 J/cm2 in a Stratalinker (Stratagene Inc., La Jolla, Calif.), cells were incubated an additional 2 h and harvested to assess DNA fragmentation as described in Fig. 1.
3T3 cells, as well as the murine fibrosarcoma, WEH1164, the SV4O-transformed monkey kidney Cos-7 line, and the human prostate cancer Du-145 line. Therefore, nicotine dose-dependently inhibits apoptosis in both normal and
transformed ous
cell lines derived
species.
NICOTINE
INHIBITS
APOPTOSIS
from different
tissues and van-
Figure 3. Nicotine inhibits UV light activation of endonuclease. U937 cells were pretreated with nicotine 30 mm before the addition of TNF at 10 ng/ml. The cells were incubated an additional 20 h and the DNA extracted and electrophoresed as described in Materials and Methods. Lane 1, MW markers; lane 2, untreated U937; lane 3, U937 + UV (0.2 J/cm2); lane 4, U937 + nicotine (10 iM); lane 5, U937 + nicotine (100 ELM); lane 6, U937 + nicotine (10 LM) + UV; lane 7, U937 + nicotine (100 tiM) + UV.
1047
RESEARCH COMMUNICATIONS Coil Line
Nicotine
Cell Line
Nicotine M)
(IIM)
H
FADU
‘I,
125
60
1000
30
40 A427
WENI-164
-
1000 200 40
H
500 250
1-l
125
60
Cool
1
1000 200 40
1-I
H
H661
‘I,
250 125
Du-145
60
H
1000
200 40
I 0
I
I
I
10
20
30
I 40
I 50
10
20
30
DNA Fragmentation
40
50
60
Figure 5. Nicotine with tobacco-related
(%)
Figure 4. Nicotine inhibits UV light-induced apoptosis in adherent cell lines of diverse origin. [3H]Thymidine-labeled target cells were pretreated with the indicated concentration of nicotine before exposure to UV light at 0.2 J/cm2. After an additional 20 h incubation, the assay was harvested as described previously for Fig. 1. Each value represents the mean +1- standard deviation from one representative
experiment
of a total
of three.
(Fig. 7A). This is not a nonspecific effect of any component of tobacco because the structurally unrelated compound, N-nitrosodiethylammne, which is a known carcinogen found in tobacco, did not inhibit apoptosis. Furthermore, the inhibition mediated by nicotine is probably not due to the fact that it is a weak base, as alkalinization of cellular compartments by treatment with ammonium sulfate does not inhibit apoptosis (unpublished results). To examine the stereospecificity of the effect, we tested the different stereoisomers of nicotine. The results shown in Fig. 7B demonstrate that both (- )-nicotine and (+ )-nicotine inhibit UV light-induced apoptosis equally as compared to the mixture of isomers used in the present and all the previous experiments. In this experiment, the IC50 value for the three nicotine preparations fell between 50 and 100 tM. These findings differ from the known stereospecific effects of nicotine in the central nervous system (17), which are due at least in part to the higher binding affinity of (- )-nicotine to the nicotinic acetylcholine receptor (18). This suggests that nicotine’s effect on apoptosis may not be receptor-mediated, or it may involve a nonconventional nicotine receptor. To examine this question, we tested the effects of acetyicholine receptor antagonists on UV light-induced DNA fragmentation of both U937 targets (Fig. 8A) and FADU targets (Fig. 8B). The results show that the nicotinic acetyicholine
1048
Vol.
7
August 1993
inhibits
apoptosis
carcinogenesis.
in tumor
I
70
(%)
DNA Fragmentation 0
I 60
cell lines
[3H]Thymidine
associated
labeled
target
cells were pretreated with the indicated concentration of nicotine before exposure to UV light at 0.1 J/cm2. After an additional 20 h incubation, the assay was harvested as described previously for Fig. 1.
receptor antagonists, pentolinium (1.0 mM) and hexamethonium (1.0 mM) did not reverse nicotine inhibition of DNA fragmentation in either cell line. As expected, the muscaninic acetylcholine receptor antagonist, atropine (1.0 mM), was also without effect. These data support the hypothesis that
Nicotine
1.0mM
*23187 (nU)
50 10 2
+
H
I-I
50
H i
10
I
20
L
I
30
40
DNA Fragmentation (%) Figure 6. Nicotine inhibits apoptosis of thymocytes. Munine thymocytes from outbred CD mice were cultured with the indicated concentrations of the calcium ionophore A23187 to induce apoptosis as described previously (16). After 48 h the soluble vs. intact DNA was measured using the diphenylamine reaction, according to a previously described procedure (11). Each point represents the mean of triplicates +1- standard deviation.
The FASEB Journal
WRIGHT
Er
AL.
inhibitor
RESEARCH presses apoptosis blocks apoptosis
A
(NM)
NIcotin.
1000
CotMilns
1000
I-I
-w H
H An.b..ln.
1000
N.nlfro.o. dI.thytwnln.
1000
H I 10
I
20
40
30
DNA Fragmentation
60
(%)
inhIbitor
(MM)
B
H 250 100
::
H
is not induced
COMMUNICATIONS
known. However, the fact that it by stimuli as diverse as TNF, UV
light, and chemotherapeutic drugs suggests that nicotine is not acting on an early signaling event. One possibility is that it interferes with the function of the putative endonuclease responsible for DNA fragmentation. Ongoing studies are aimed at investigating whether nicotine blocks activating signals on directly inhibits endonuclease activity. Nicotine is known to exert its effects on many cell types by binding to nicotinic cholinergic receptors which can be blocked by specific antagonists. However, we were unable to reverse nicotine’s inhibition of apoptosis in U93 7 or the FADU cell lines using the nicotinic cholinergic blockers hexamethonium or pentolinium. Therefore, the nicotine effect may not be receptor-mediated, or it occurs through unconventional nicotine receptors. This possibility is supported by a report describing noncholinergic nicotine receptors expressed on monocytes and neutrophils (19). Further work is necessary to determine if the effects of nicotine on apoptosis are mediated through unconventional nicotine receptors. The fact that relatively high concentrations of nicotine (10-500 tM) are required to inhibit DNA fragmentation raised the possibility that a contaminant present in the nicotine preparation may be responsible for the inhibition. However, the finding that three different nicotine preparations were equally inhibitory, and that the related compounds cotinine and anabasine were also active argues against this explanation. Although we cannot absolutely rule
+/-
Mons
A. 1.1937Target
Ant.onlV
50
H
90
H .500
200
i-I
100 50
i-I
90
200 100
‘H
___H
H
+200
60
P.ntollnlwn Hsxsmstho.tiwn
25 I
I
I
I
I
I
0
10
20
30
40
50
DNA Fragmentation
(%) 10
Figure 7. Specificity of nicotine inhibition of apoptosis. A) U937 target cells were pretreated with the indicated compounds for 30 mm before the addition of TNF (10 ng/ml) and subsequent incubation for 20 h. DNA fragmentation was measured as described in the legend for Fig. 1. All compounds were tested at nontoxic concentrations as indicated by trypan blue exclusion. B) U937 cells were pretreated with the different stereoisomers of nicotine for 30 mm and then exposed to UV light (0.15 J/cm2). The assay was harvested after an additional 2 h incubation.
20
30
DNA Fragmentation
B. FADU Target
.1- NIcotine
(SM)
40
(%)
Antagetist
I-I
-
.500
-
PsntOlkthan
LH
VAvopins
nicotine nicotinic
inhibition of apoptosis cholinergic receptors.
does not involve
I
conventional
0
This study demonstrated that nicotine inhibits apoptosis in normal thymocytes, untransformed fibroblasts, and many tumor cell lines including two lung cancer lines and a pharyngeal carcinoma. The mechanism by which nicotine sup-
NICOTINE INHIBITSAPOPTOSIS
I 20
DNA Fragmentation Figure
DISCUSSION
10
8. Acetylcholine
receptor
antagonists
I
I
I
30
40
50
(%) do not block nicotine
of apoptosis. U937 cells A) or FADU cells B) were pretreated with 1.0 mM of each of the antagonists for 30 mm, and then nicotine was added for an additional 30. The cells were then exposed to UV light and the assay harvested after an additional 2 h incubation. All agents used in this experiment were nontoxic as assessed by trypan blue exclusion. inhibition
1049
RESEARCH COMMUNICATIONS out the possibility that an inhibitory contaminant was present in all five preparations, it seems highly unlikely. Nicotine at millimolar concentrations has been reported to induce new gene expression and differentiation in human myelocytic HL-60 cells (20). However, this phenomenon is not involved in our system as inhibitors of protein synthesis do not reverse nicotine inhibition of UV light or TNFinduced apoptosis (unpublished observations). TNF-induced apoptosis of U937 cells does not require de novo protein synthesis (15), similar to some other models of apoptosis (21). Another possible mechanism of nicotine action is activation of protein kinase C (PKC), which has been reported in nicotine-treated adrenal chromaffin cells (22). We have found that the PKC activator phorbol myristate acetate (PMA) inhibits both TNF and UV light-induced apoptosis in U937 cells (S. C. Wright et al., unpublished observations). This is in accord with other reports that PMA inhibits apoptosis in a variety of model systems (23-28). Further work will be necessary to determine if nicotine-induced elevation of PKC is responsible for the suppression of apoptosis reported here. PMA is a prototype tumor promoter, although the mechanism of promotion is not understood. We postulate that inhibition of apoptosis is at least one mechanism of tumor promotion. Therefore, nicotine may function as a tumor promoter by suppressing apoptosis, as a step in the pathogenesis of tobacco-related cancer. Although the average blood concentration of nicotine in smokers is 0.25 iM (29-31), it is rapidly metabolized to cotinine, which can be found in the blood at concentrations as high as 2.0 ILM (29-31). The fact that blood cell cancers are not strongly associated with tobacco use is in keeping with the observation that serum nicotine concentrations are too low to suppress apoptosis in our studies. Furthermore, it is likely that local nicotine concentrations in the ororespiratory tract will greatly exceed serum levels. Other tissues may contain even higher concentrations of nicotine that would coincide with those found to inhibit apoptosis in cells in culture. For example, nicotine concentrations of 100 iM in hair and 10 M in cervical mucus were reported in female smokers (32, 33). Furthermore, nicotine concentrations as high as 1.0 to 10.0 mM have been reported in the saliva of snuff users (34). Because these concentrations would completely block apoptosis as measured in our studies, nicotine could be a major factor contributing to the higher incidence of oral cancers occurring in smokeless tobacco users (35). The concept that nicotine may promote tumor formation in tissues where it may be found in relatively high concentrations was recently proposed by Maneckjee and Minna (36). This study demonstrated that nicotine could reverse opioidmediated growth inhibition of lung cancer cell lines. Whether this phenomenon may involve interference with apoptosis is not known. The process of apoptosis functions in a variety of defense mechanisms that are thought to be important in immune surveillance against tumor cells. TNF (37-39), natural killer cells (40), and cytotoxic T lymphocytes (41) have all been shown to induce apoptosis in their targets. Furthermore, many chemotherapeutic drugs kill tumor cells by apoptosis, as shown in this report and elsewhere (9, 10). Therefore, exposure to inhibitors of apoptosis, such as nicotine, may promote the emergence of new tumors as well as decrease the efficacy of cancer therapies. A survey of other environmental compounds for tumor-promoting activity through inhibition of apoptosis is warranted.
II
1050
Vol. 7
August
1993
The authors figures. This awarded
by the
thank work
S. Lama for expert assistance was supported in part by
National
Cancer
in preparing the grant CA47669
Institute.
REFERENCES 1. Duvall, E., and Wyllie, A. H. (1986) Death and the ceU. Immunol. Today 7, 115-119 2. Gerschenson, L. E., and Rotello, R. J. (1992) Apoptosis: a different type of cell death. FASEB j 6, 2450-2455 3. Walker, N. I., Harmon, B. V., Gobe, G. C., and Kerr, J. F. R. (1988) Patterns of cell death. Methods Achiev. Exp. Pathol. 13, 18-54 4. Williams, G. T. (1991) Programmed cell death: apoptosis and oncogenesis. Cell 65, 1097-1098 5. Bursch, W., Oberhammer, F., and Schulte-Hermann, R. (1992) Cell death by apoptosis and its protective role against disease. Trends Pharmacol. Sd. 13, 243-251 6. Raff, M. C. (1992) Social controls on cell survival and cell death. Nature (London) 356, 397-400 7. Henderson, S., Rowe, M., Gregory, C., Croom-Carter, D., Wang, F., Longnecker, R., Kieff, F., and Rickison, A. (1991) Induction of bcl-2 expression by Epstein-Barr virus latent membrane protein 1 protects infected B cells from programmed cell death. Cell 65, 1107-1115 8. Yonish, Rouach, E., Resnitzky, D., Lotem, J., Sachs, L., Kimchi, A., and Oren, M. (1991) Wild-type p53 induces apoptosis of myetoid leukaemic cells that is inhibited by interleukin-6. Nature (London) 352, 345-347 9. Kaufmann, S. H. (1989) Induction of endonudeolytic DNA deavage in human acute myelogenous leukemia cells by etoposide, camptothecin, and other cytotoxic anticancer drugs: a cautionary note. Cancer Res. 49, 5870-5878 10. Eastmann, A. (1990) Activation of programmed cell death by anticancer agents: cisplatin as a model system. Cancer Cells 2, 275-280 11. Sellins, K. S., and Cohen, J. J. (1987) Gene induction by. -y-irradiation leads to DNA fragmentation in lymphocytes. j Immunol. 139, 3199-3206 12. Takano, Y. S., Harmon, B. V., and Kerr, J. F. (1991) Apoptosis induced by mild hyperthermia in human and murine tumour cell lines: a study using electron microscopy and DNA gel electrophoresis. j Pathol. 163, 329-336 13. International agency for research on cancer (1986) IARC monographs on the evaluation of carcinogenic risk of chemicals to humans. Tobacco Smoking 38, 199-298 14. Hoffmann, D., and Hecht, S. S. (1985) Nicotine-derived N-nitrosamines and tobacco-related cancer: current status and future directions. Cancer Res. 45, 935-944 15. Wright, S. C., Kumar, P., Tam, A. W, Shen, N., Varma, M., and Larrick, J. W. (1992) Apoptosis and DNA fragmentation precede TNF-induced cytolysis of U937 cells. j Cell. Bioche,n. 48, 344-355 16. Kizaki, H., Tadakuma, T., Idaka, C., Muramatsu, J., and Ishimura, Y. (1989) Activation of a suicide process of thymocytes through DNA fragmentation by calcium ionophores and phorbol esters. j Immunol. 143, 1790-1794 17. Meltzer, L. T., Rosecrans, J. A., Aceto, M. D., and Harris, L. S. (1980) Discriminative stimulus properties of the optical isomers of nicotine. Psychopharmacology 68, 283-286 18. Vincek, W. C., Martin, B. R., Aceto, M. D., Tripathi, H. L., and Hams, L. S. (1981) Synthesis of 4,4-ditritio-( + )-nicotine: comparative binding and distribution studies with the natural enantiomer. j Pharm. Sci. 70, 1292-1293 19. Davies, B. D., Hoss, W., Lin, J. -P., and Lionetti, F. (1982) Evidence for a noncholinergic nicotine receptor on human phagocytic leukocytes. Mol. Cell. Biochem. 44, 23-31 20. Wu, J. M., and Maturana, J. (1990) Induction of monocyte differentiation by nicotine in HL-60 leukemic cells. FASEBJ 4, A1975 (abstr.) 21. Martin, S. J., Lennon, S. V., Bonham, A. M., and Cotter, T G. (1990) Induction of apoptosis (programmed cell death) in human leukemic HL-60 cells by inhibition of RNA or protein synthesis.j Immunol. 145, 1859-1867 22. Tuominen, R. K., McMillan, M. K., Ye, H. Stachowiak, M. K., Hudson, P. M., and Hong, J. 5. (1992) Long term activation of protein kinase C by nicotine in bovine adrenal chromaffin cells. j Neurothem. 58, 1652-1658 23. Kanter, P., Leister, K. J., Tomei, L. D., Wenner, P. A., and Wenner, C. E. (1984) Epidermal growth factor and tumor promoters prevent DNA fragmentation by different mechanisms. Biochem. Biophys. Res. Commun. 118, 392-399
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WRIGHT
Er AL.
RESEARCH COMMUNICATIONS 24. Rodriguez-Thrduchy, G., and Lopez-Rivas, A. (1989) Phorbol esters inhibit apoptosis in IL-2-dependent T lymphocytes. Biochem. Biophys. Res. Commun. 164, 1069-1075 25. McConkey, D. J., Orrenius, S., and Jondal, M. (1990) Agents that elevate CAMP stimulate DNA fragmentation in thymocytes. j Immunol. 145, 1227-1230 26. Lucas, M., Solano, F., and Sanz, A. (1991) Induction of programmed cell death (apoptosis) in mature lymphoctyes. FEBS LeU. 279, 19-20 27. McConkey, D. J., Aguilar-Santelises, M., Hartzell, P., Eriksson, I., Mellstedt, H., Orrenius, S., and Jondal, M. (1991) Induction of DNA fragmentation in chronic B-lymphocytic leukemia cells. j Immunol. 146, 1072-1076 28. McConkey, D. J., Hartzell, P., Jondal, M., and Orrenius, S. (1989) Inhibition of DNA fragmentation in thymocytes and isolated thymocyte nuclei by agents that stimulate protein kinase C. j Biol. Chem. 264, 13399-13402 29. Russell, M. A. H., Wilson, C., Patel, U. A., Feyerabend, C., and Cole, P. V. (1975) Plasma nicotine levels after smoking cigarettes with high, medium and low nicotine yields. Br Med. J. 2, 414-416 30. Benowitz, N. L., Hall, S. M., Herning, R. I., Jacob, P., Jones, T R., and Osman, A. L. (1983) Smokers of low-yield cigarettes do not consume less nicotine. N Engl. j Med. 309, 139-142 31. Hemning, R. I., Jones, T, Benowitz, N. L., and Mines, A. H. (1983) How a cigarette is smoked determined blood nicotine levels. Clin. Pharmacol. Ther. 33, 84-90 32. Haley, N. J., and Hoffmann, D. (1985) Analysis for nicotine and cotinine in hair to determine cigarette smoker status. Clin. Chem. 31, 1598-1600 33. Sasson, I. M., Haley, N.J., Hoffmann, D., Wynder, F. L., Hellberg, D., and Nilsson, S. (1985) Cigarette smoking and neoplasia of the uterine
smoke constituents in cervical mucus. N EngI. j Med. 312, 315-316 Hoffman, D., and Adams, J. D. (1981) Carcinogenic tobacco-specific Nnitrosoamines in snuff and in the saliva of snuff dippers. Cancer Res. 41, 4305-4308 Win, D. M., Blot, W. J., Shy, C. M., et al. (1981) Snuff dipping and oral cancer among women from the southern United States. N EngI. j Med. 304, 745-749 Maneckjee, R., and Minna, J. D. (1990) Opioid and nicotine receptors affect growth regulation of human lung cancer cell lines. Proc. Nail. Acad. &i. USA 87, 3294-3298 Schmid, D. S., Hornung, R., McGrath, K. M., Paul, N., and Ruddle, N. H. (1987) Target cell DNA fragmentation is mediated by lymphotoxin and tumor necrosis factor. Lymphokine Res. 6, 195-202 Dealtry, G. B., Naylor, M. S., Fiers, W., and Balkwill, F. R. (1987) DNA fragmentation and cytotoxicity caused by tumor necrosis factor is enhanced by interferon-v. Eur. j Immunoi 17, 689-693 Laster, S. M., Wood, J. G., and Gooding, L. R. (1988) Tumor necrosis factor can induce both apoptic and necrotic forms of cell lysis. j Immunol. 141, 2629-2634 McConkey, D. J., Chow, S. C., Orrenius, S., and Jondal, M. (1990) NK cell-induced cytotoxicity is dependent on a Ca increase in the target. FASEBJ 4, 2661-2664 Duke, R. C., Chervenak, R., and Cohen, J. J. (1983) Endogenous endonuclease-induced DNA fragmentation: an early event in cellmediated cytolysis. Proc. Nail. Acad. &i. USA 80, 6361-6365 cervix:
34.
35.
36.
37.
38.
39.
40.
41.
Received for publication October 3, 1992. Accepted for publication April 14, 1993.
1993 AMERICAN PHYSIOLOGICAL SOCIETY CONFERENCE PHYSIOLOGY AND PHARMACOLOGY OF MOTOR CONTROL OCTOBER 2-5, 1993 San Diego, California Program Saturday, October 2
Overview
Floyd E. Bloom (Scripps Clinic) Anatomy of Neurotransmitter Systems Chair: Tomas H#{244}kfelt (Karolinska Institute) Sunday, October 3
Neurotransmitlers and Receptors Chair: Floyd E. Bloom Neuropharmacology of Movement Control Chair: Sten Griliner (Karolinska Institute)
Monday, October 4
Neurophysiology of Control of Movement in Mammals Chair: James C. Houk (Northwestern University) Neuropharmacology of Motoneurons Chair: Jack L. Feldman (UCLA) Keynote
Tuesday, October 5
Diseases
Address Gerald M. Edelman
(Scripps
Research
Institute)
of Movement
Chair: Joseph B. Martin (UCSF) Summation Joseph
NICOTINE INHIBITSAPOPTOSIS
B. Martin
1051