Analysis of Events Associated .With Cell Cycle
`Arrest at G2 Phase and .Cell Death
`Induced by Cisplatin
`
`Christine M. Sorenson, Michael A. Barry, Alan Eastman*
`
`DNA is the accepted target for cisplatin, but recent evidence
`has shed doubt on DNA synthesis as the critical proces.s.
`Ll210/0 cells incubated for 2 hours with cisplatin progres.s to
`the G2 phase of the cell cycle and are arrested there for several
`days. They then either progres.s in the cell cycle or die. In cells
`that eventually die, total transcription, polyadenylated
`[poly(AtJ RNA synthesis, and protein synthesis were mark(cid:173)
`edly inhibited only after 48 hours. Nicotinamide adenine
`dinucleotide (NAD) and adenosine triphosphate (ATP) levels
`decreased after 3 days. Cell membrane integrity was lost after
`4 days. These results demonstrate that cells can be lethally
`damaged, yet continue to undergo apparently normal meta(cid:173)
`bolic activities for several days. In a previous study, DNA
`double-strand breaks were detected after 1 day. We now
`show that by 2 days, breaks are visible as fragmentation in the
`nucleosome spacer regions of chromatin. This type of damage
`is consistent with cell death occurring by the proces.s of
`apoptosis. Cell shrinkage and morphology were also consis(cid:173)
`tent with this type of cell death. The slow cell death reported
`here appears to occur at the GifM transition and may involve
`events that normally occur at this stage of the cell cycle. These
`results demonstrate the importance or DNA degradation as
`an early and po~ibly essential step in cell death. [J Natl
`Cancer Inst 82:749-755, 1990]
`
`Cisplatin has been shown to be an effective antineoplastic
`agent in the treatment of a variety of tumors(/). DNA has been
`implicated as the critical target for cytotoxicity (2). Most of the
`damage is due to DNA- intrastrand cross-links: DNA-interstrand
`and DNA-protein cross-links represent less than I% of the total
`platination of DNA (3). The relative contribution of each lesion to
`toxicity is still in contention.
`Inhibition of DNA synthesis has been observed in a variety of
`cells following platination (4-6). DNA synthesis has also been
`reported to be more sensitive to cisplatin than either RNA or
`protein synthesis (2). Thus inhibition of DNA synthesis has been
`logically envisioned as the critical step in toxicity. We have
`previously questioned this assumption (7,8). Rather than being
`arrested in the S phase of the cell cycle, as would be expected if
`DNA synthesis were inhibited, cells were arrested in the G2 phase
`before dying. Although slowed DNA synthesis may occur during
`progression to the G2 phase, this did not correlate with toxicity. In
`Chinese hamster ovary cells either proficient or deficient for
`DNA repair, and therefore exhibiting marlcedly different sensitiv-
`
`Vol. 82, No. 9, May 2, 1990
`
`ities to cisplatin, we observed that any inhibition of DNA
`synthesis was related to the applied drug concentration and not to
`the degree of toxicity . Very sensitive cells progressed at a normal
`rate to the G2 phase and subsequently died. ln contrast, more
`resistant cells could tolerate higher drug concentrations that did
`slow DNA synthesis.
`These observations led to the two questions addressed here:
`why are cells arrested in the G2 phase, and by what mechanism do
`they subsequently die? It should be emphasized that, although G2
`arrest appears to be a prerequisite for cell death (except at very
`high drug concentrations), all such arrested cells do not die. At
`minimally toxic concentrations of cisplatin, cells may eventually
`bypass the block and return to normal cycling. Hence, there are
`two possible fates for a cell arrested in the G2 phase: survival or
`death. It is important to determine how a cell regulates the
`outcome.
`We initially hypothesized that cells incubated with cisplatin
`were arrested in the G2 phase by an inhibition of transcription,
`specifically, 3!1 inability to produce the full-length messenger
`RNA (mRNA) needed for passage to mitosis. Earlier studies had
`investigated only the quantity, rather than the quality, of RNA
`s.ynthesis (4-6). We therefore investigated inhibition of transcrip(cid:173)
`tion and other parameters that might be involved in cell death.
`Previously, we had shown DNA double-strand breaks to be the
`earliest change detected in cells destined to die (7) . Here we
`demonstrate that these breaks occur in the nucleosome spacer
`region of chromatin DNA, giving rise to "nucleosome ladders" by
`gel electrophoresis. Such breaks are characteristic of cells dying
`by the mechanism of apoptosis (9).
`
`Materials and Methods
`Cell Culture
`Ll210/0 cells were maintained in an exponential suspension
`culture at 37 °C in a humidified atmosphere of 5% COr95% air
`
`Received November 13, 1989; revised January 25, 1990; accepted February 2,
`1990.
`Supponed by Public Health Service grant CA-36039 from the National Cancer
`Institute, National Institutes of Health, Oeparuncn.1 of Health and Human
`Services.
`C. M. Sorenson, McArdle Laboratory for Cancer Rcsean:h, University of
`Wisconsin, Madison, WI.
`M. A. Barry, A. Eastman, Department of Pharmacology and Toxicology,
`Dartmouth Medical School, Hanover, NH.
`•co"upondtnce 10: Alan Easun.an, Ph.D., Department of Phannacology,
`Dartmouth Medical School, Hanover, NH 03756.
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`in McCoy's 5a (modified) medium (GIBCO Laboratories, Grand
`Island, NY) supplemented with 15% calf serum, penicillin,
`streptomycin, and amphotericin B (Fungizone). In all experi(cid:173)
`ments, L 1210/0 cells were incubated in various concentrations of
`cisplatin (Bristol Laboratories, Syracuse, NY) for 2 hours at
`37 °C. Cell size was detennined on a Coulter Channelyzer 256.
`
`DNA Synthesis
`At each time point, I 06 cells were resuspended in 2 mL of fresh
`medium containing 0.5 µ.Ci of [3H]thymidine (6.7 Ci/mmol; ·
`New England Nuclear Corp., Boston, MA) and incubated for 30
`minutes to produce radiolabeled DNA. The cells were then
`centrifuged, rinsed twice with cold phosphate-buffered saline
`(PBS), and resuspended in 100 µ.L of PBS, and 100 µL of a
`solution of salmon spenn DNA [500 µg/mL in 20 mM edetic acid
`(EDTA)] was added. To each sample was added 5 mL of 10%
`ice-cold trichloroacetic acid (TCA). This was incubated on ice for
`15 minutes, and the precipitate was collected by filtration through
`2.4-cm-cliameter Whatman glass microfiber filters (GF/C; What(cid:173)
`man International Ltd ., Maidstone, England). The filters were
`rinsed with 10% TCA followed by ethanol, solubilized with 0.5
`mL of NCS tissue solubilizer (Amersham Corp., Arlington
`Heights, IL) for 30 minutes at 37 °C, and neutralized, and the
`radioactivity was detennined with a Beckman scintillation
`counter.
`
`Total RNA Synthesis
`Fresh medium containing 0.5 µCi of [3H]uridine (28.5 Ci/
`mmol; New England Nuclear Corp.) in 2 mL was added to 106
`cells and incubated for 30 minutes at 37 °C to produce radiola(cid:173)
`beled RNA. The cells were then centrifuged, rinsed twice with
`cold PBS containing 200 µM uridine, and resuspended in I 00 µL
`of PBS containing 200 µ.M uridine, and 100 µ.L of salmon spenn
`DNA (500 µg/mL) was added . TCA was added, the precipitate
`was collected, and the radioactivity was assayed as described
`above.
`
`Poly(Ar RNA Synthesis
`A total of 2 x 106 cells were resuspended in 4 mL of fresh
`medium containing 2 µCi of [3H]uridine and incubated at 37 °C
`for 2 hours. The cells were centrifuged and rinsed twice with cold
`PBS containing 200 µ.M uridine. Polyadenylated [poly(A)+](cid:173)
`RNA was isolated by a modification of the method of Badley
`et al. (10). Unlabeled Ll210/0poly(A)+ RNA (80 ng) was added
`to the equilibrated oligodeoxythymidylate [oligo(dDkellulose
`in batch fashion and incubated at room temperature for 15
`minutes. The Ll210/0 cell lysate was added, and the incubation
`was continued for an additional 45 minutes. The oligo(dT)-cel(cid:173)
`lulose was briefly centrifuged, the supernatant removed, and
`binding buffer added. This sequence was repeated until the
`absorbance at 260 nm (A260) of the supernatant was less than
`0.05. The poly(A)+ RNA waseluled wilh 1-rnLaliquotsofsterile
`water and collected by centrifugation, and the radioactivity in
`each fraction was determined.
`
`Protein Synthesis
`A total of 106 L1210/0 cells were resuspended in 2 mL of
`methionine-free McCoy's medium containing l µCi ofTran3sS-
`
`label ([3sS]methionine-cysteine; ICN Biomedicals, Inc., Costa
`Mesa, CA) and incubated at 37 °C for l hour. The cells were then
`centrifuged, rinsed twice with cold PBS containing 200 µM
`methionine, and resuspended in I 00 µL of PBS containing 200
`µM methionine. TCA was added, the precipitate was collected,
`and radioactivity was assayed as described above.
`ATP and NAD Levels
`At each time point, 107 cells were harvested and rinsed with
`Hanks' balanced salt solution containing I mM sodium phos(cid:173)
`phate, l mM potassium phosphate, and , as phosphatase inhibi(cid:173)
`tors, 5 mM sodium fluoride and 5 mM sodium glycerol phosphate
`(1 /).The cell pellet was then resuspended in 100 µL of 0.4 M
`perchloric acid containing phosphatase inhibitors and incubated
`on ice for 30 minutes. The cell lysate was spun for 10 minutes at
`4 °C, and 16 µ.L of potassium bicarbonate was added to neutralize
`the supernatant. Samples were analyzed by anion exchange
`high-pressure liquid chromatography on a Whatman Partisil 10
`SAX column ( 12). Quantitation was performed by comparison of
`peak heights with a standard curve obtained from injection of
`known amounts of nicotinamide adenine dinucleotide (NAD) or
`adenosine triphosphate (ATP).
`
`DNA Degradation
`
`Cells were analyzed by a gel electrophoresis method adapted
`from Eckhardt(/J). A 125-mL volumeof2% agarose in TBE(89
`mMTris, 89 mM boric acid, 2.5 mM EDTA; pH 8.0) was poured
`into a minigel support with the comb. After the gel solidified, the
`portion of the gel above the comb (2 cm) was removed . This space
`was filled with 16 mL of 0.8% agarose-2% sodium dodecyl
`sulfate (SDS) in TBE to which l .25 mg of proteinase K per mL
`was added after the agarose had cooled below 50 °C. Each pellet
`of I 06 cells was suspended in 15 µL of sample buff er (I 0 mg of
`ribonucleaselmL, 15% Ficoll 70, 0.01% bromophenol blue in
`TBE), and the suspended pellet was transferred to a well. Cell
`lysis began in the sample buffer and was completed during
`electrophoresis at 20 V for l hour. DNA fragments were then
`separated by electrophoresis for 3 hours at 90 V. Molecular
`weight standards (Msp I-digested pBR322; New England Bio(cid:173)
`labs, Beverly, MA) were subjected to electrophoresis in adjacent
`lanes.
`Following electrophoresis, the gel was rinsed in distilled water
`and incubated overnight at room temperature with gentle shaking
`in 100 mL of TE (I 0 mM Tris, l mM EDT A; pH 8. 0) containing
`2 mg of ribonuclease A. This procedure removed the SDS and
`contaminating RNA that had comigrated with the smaller nu(cid:173)
`cleosome fragments. The gel was rinsed in distilled water and
`stained for 30 minutes in 100 mL of water containing 50 µg of
`ethidium bromide. The gel was rinsed , destained in water for 4
`hours, and photographed in ultraviolet light.
`The approximate amount of DNA detected in these gels was
`assessed by laser densitometry of the photographic negatives.
`These values were compared with a standard curve produced by
`electrophoresis of a known amounl of sheared DNA under
`identical conditions. Minimally detectable degradation repre(cid:173)
`sented about 50 ng, while the maximum degradation detected in
`these studies was about 500 ng. The majority of DNA always
`remained in the well and could not be quantitated by densitome(cid:173)
`try. The total DNA applied to each well was about l 0-20 µg; the
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`latter value reflects cells with twice the nonnal content of DNA as
`a result of arrest in the G2 phase.
`~~ing El~~on Micr<!5C0py
`Electron microscopy was perfonned on Ll210/0 cells at vari(cid:173)
`ous· time intervals following incubation with cisplatin. Approxi(cid:173)
`mately 107 cells were pelleted and resuspended in I mL of 2%
`glyceraldehyde in 0.1 M cacodylate buffer (pH 7 .4) and stored at
`4 °C. The cells were centrifuged. with a cytocentrifuge at 500 rpm
`for 30 minutes onto a round coverslip coated with 0.1 % poly(cid:173)
`L-lysine. The coverslip was postfixed with I% osmium tetroJtide
`in 0.1 M phosphate buffer (pH 7.4), dehydrated through an
`ascending ethanol series, and critical-point-dried with Freon 13.
`The coverslips were mounted on specimen stubs, sputter-coated
`with gold, and examined in a Philips 515 scanning electron
`microscope.
`
`Results
`Cell Survival
`
`(>ti] THYMIDINE
`
`PH) URIOINE
`
`(J's) METHIONINE
`
`INCtJSA TlOH TIME (Daya)
`
`Figure 1. Inhibition of macromolecular synthesis in Ll210/0 ceUs al various
`times following 2 hr of incubation wilh cisplatin concentrations of0.12 (O), 0.25
`(• ), 0.5 (.c.), I( • }· 2 (O), and 4 (• ) µg/mL. Incorporation of [3H]thymidine,
`[ 3HJuridine, and [ ~S]methionine into TCA-precipitable mat.erial represents
`DNA, RNA, and protein synthesis, respectively.
`
`The various parameters by which the toxicity of cisplatin has
`been measured in L1 210/0 cells have been discussed previously
`(7). Following 2 hours of drug treatment, the concentration
`inhibiting growth by 50% over a 3-day period was 0. 7 µg/mL .
`However, this value does not adequately assess cells that are
`arrested in the G2 phase for several days and then recover. A more
`accurate assessment of toxicity was obtained by measuring trypan
`blue dye exclusion for up to 14 days after drug treatment. The
`time to maJtimum loss of membrane integrity was 6-8 days. By
`this criterion, a cisplatin concentration of 0. 7 µg/mL killed less
`than 10% of the cells. Approximately 50% of the cells were killed
`at 2 µg/mL. This was supported by flow cytometry. At 0.7
`µg/mL, the cells were arrested maJtimally in the G2 phase on day
`1 and then recovered over the following 2 days, with no apparent
`cell disintegration. At 2 µg/mL, more than 80% of the cells were
`arrested in the G2 phase by 2 days; after 4-Q days, approximately
`half of the cells were observed as debris. Higher concentrations of
`cisplatin led to virtually complete disintegration of cells by 6
`days. Therefore, drug concentrations of less than 2 µg/mL are
`considered minimally toxic, 2 µ.g/mL represents an intennediate
`tollicity, and higher concentrations are significantly toxic.
`
`Inhibition of Macromolecular Synthesis
`
`We investigated several parameters to elucidate the sequence
`of events occurring in cells destined to die following incubation
`with cisplatin. The timing of such events could then be compared
`with the maximal G2 arrest on day 2 and the loss of membrane
`integrity around day 6. DNA, RNA, and protein synthesis studies
`were perf ormea to determine when inhibition of these processes
`occurs following platination.
`At various time intervals after drug treatment, cells were
`incubated with [3H]thymidine, [3H]uridine, or [3.5S]methionine,
`and incorporation was assayed as radioactivity in the acid(cid:173)
`precipitable fraction (fig. I). A significant inhibition of DNA
`synthesis was observed even at the earliest time point of 12 hours.
`At minimally toxic drug concentrations, DNA synthesis returned
`to near-control levels by 2 days; intermediate concentrations
`required 5 days. The inhibition of DNA synthesis between days 1
`
`and 5 correlated with arrest in the G2 phase. At toxic drug
`concentrations, DNA synthesis was permanently suppressed by
`2 days.
`A significant inhibition of total RNA synthesis was not ob(cid:173)
`served until 2 days following incubation with cisplatin. At
`minimally toxic drug concentrations, recovery of total RNA
`synthesis was noted by 4 days. This corresponded to the transient
`· arrest in the G2 phase of the cell cycle, followed by the re(cid:173)
`emergence of a cycling population. Total RNA synthesis contin(cid:173)
`ued to decrease after 2 days at toxic drug concentrations and was
`permanently suppressed by 3 days.
`The trend of inhibition observed with newly synthesized
`protein was similar to that observed with total newly synthesized
`RNA . A significant inhibition of protein synthesis was observed
`at 2 days. This decrease in synthesis was continuous at toxic
`concentrations, while a transient decrease was observed at mini(cid:173)
`mally toxic concentrations.
`We hypothesized that cells might be arrested in the G2 phase
`due to truncation of newly synthesized mRNA required for
`mitosis. However, no reduction in the amount of new poly(A)+
`RNA was observed until 2 days (fig. 2). This was after a
`significant G2 arrest had occurred (7). The panem of inhibition of
`m.RNA synthesis closely followed that observed with total RNA.
`At minimally toxic drug concentrations, only a slight decrease of
`poly(A)+ RNA synthesis was observed during the 4-day experi(cid:173)
`ment. At toxic drug concentrations, a continual decrease in newly
`synthesized poly(A)+ RNA was noted.
`DNA Degradation
`We had previously observed that fonnation of DNA double(cid:173)
`strand breaks appeared to be the earliest change detected in cells
`destined to die (7). We pursued this observation further by using
`gel ~lectrophoresis to resolve nucleosome fragments (fig. 3). This
`modified technique permi!S rapid and sensitive detection of DNA
`degradation but results in some blurring of the smaller bands as
`compared with techniques that require an initial purification of
`the DNA. The limit of detection on these gels is about 50 ng of
`
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`140
`
`INCUBATION TIME (DAYS)
`
`z
`Q
`~
`c( a:
`Figure 2. Inhibition o~
`a: = 110
`Q. 0
`oc
`of
`synthesis
`or
`in 08
`poly(A)+ RNA
`Ll210/0 cells at vari-
`~o 80
`ous times following 2
`~1111
`c;--
`hr of incubation with
`the indicated concen-
`ir
`:>
`trations of cisplatin.
`......
`:c
`..,
`
`•O
`
`20
`
`L...J
`
`• o 2s JJQ/mt
`
`1 µg/ml
`
`4 µg/ml
`
`e
`0, 0 5
`3
`z
`0
`i==
`<( a:
`1-z
`~ 2
`z
`0
`(.)
`
`•
`
`(!)
`:::>
`a:
`0
`
`0
`
`Figure 4. Changes in
`size of LI 21 OIO cells at
`various times follow(cid:173)
`ing 2 hr of incubation
`with the indicated con(cid:173)
`centrations of cispla(cid:173)
`tin .
`
`CELL VOLUME (p Iller)
`
`size increased markedly at 2 days. This correlated with the
`passage of cells into the G2 arrest. By 4 days at the higher drug
`concentrations, a significant proportion of the cells had shrunk to
`become smaller in size than the control cells. This was particu(cid:173)
`larly noticeable in the cells incubated with the most toxic
`concentration of cisplatin.
`
`'
`3
`2
`INCUBATION TIME COays)
`
`DNA, or about 0.5% of that added to the wells. At 1 day, little if
`any fragmentation was observed at any drug concentration. By 2
`days, however, nucleosome fragments were observed following
`toxic drug concentrations, and the intensity increased at later
`times. At these drug concentrations, a characteristic nucleosorne
`ladder consisting of multimers of approximately 180 base pairs
`was observed. At intermediate concentrations, this ladder be(cid:173)
`came most pronounced at 4 days. At minimally toxic drug
`concentrations, no nucleosome fragments were observed. Hence,
`the appearance of nucleosome fragments correlated with cell
`death and occurred several days before loss of trypan blue dye
`exclusion.
`Cell Size
`Since it has been reported that shrinkage occurs in cells dying
`by apoptosis (9), we measured cell size following incubation with
`cisplatin (fig. 4). At minimally toxic concentrations of cisplatin,
`there was only a slight increase in cell size, but this was reversed
`by day 3. However, at higher drug concentrations, the distribu(cid:173)
`tion of cell size became more heterogeneous, and the mean cell
`
`I day
`
`2 deya
`
`3 d•ys
`
`4 deys
`
`o~-N'lllfCDOci-N"if'CD od-N'CCDO~-C'lot•CO J'Q/ml
`
`bo
`
`-MO
`
`- 360
`
`- 1so
`
`Figure 3. DNA degradation in Ll210/0 cells following 2 hr of incubation with
`cisplatin. Cells were harvested after 1-4 days, and DNA degradation was
`analyzed by gel electrophoresis. The molecular weights were obtained from
`adjacent lanes containing Msp I-digested pBR322 DNA standards.
`
`Figure 5. Scanning electron micrographs of Ll 210/0 cells at various times
`following 2 hr of incubation with 8 µg of cisplatin per rnL. A: control; B: 1 day;
`C: 2 days; D: 3 days; E: 4 days; F: 6 days. The white bars at the bottom of each
`picture represent 10 µm.
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`ATP
`
`0
`
`0
`INCUBATION TIME (Daya)
`
`~
`
`Figure 6. Changes in NAO and ATP levels in Ll210/0 ceUs at various times
`following 2 hr of incubation with the indicated concentrations of cisplatin.
`
`Scanning Electron Microscopy
`Another factor that has been associated with apoptosis is
`changes on the cell surface, such as convolution and formation of
`blebs. L 1210/0 cells were incubated with 8 µg of cisplatin per mL
`(toxic concentration) for 2 hours and incubated posttreatment for
`up to 6 days, and electron microscopy was performed (fig. 5).
`Undamaged cells showed characteristic roughened surfaces.
`However, following incubation with cisplatin, marked changes
`occurred; most pronounced was the porous appearance at 4-6
`days and occasionally for cells at earlier time points. This
`corresponds to the timing of loss of trypan blue dye exclusion in
`these cells (7). Prior to this change, some cells were seen to have
`blebs on their surf aces. Such blebs are usually very transient, as
`the observable changes in apoptosis can occur in less than 15
`minutes (9). Accordingly, very few cells would be expected to
`show this phenomenon simultaneously.
`
`ATP and NAD Levels
`
`Increased poly(ADP-ribosyl)ation is commonly associated
`with DNA strand breaks (14). (ADP = adenosine diphosphate.)
`NAO serves as a substrate in this reaction, thus causing NAO
`pools to decrease. ATP levels subsequently decline. Alterna(cid:173)
`tively, loss of NAO and ATP could precede DNA degradation,
`indicating loss of osmoregulation as an early step in cell death. At
`minimally toxic drug concentrations, no significant decrease in
`ATP or NAO levels was observed during the 5-day experiment
`(fig. 6). However, at toxic drug concentrations, ATP and NAD
`levels dramatically decreased at 3 days. Because these changes
`occurred after DNA degradation, they were a result of, and not a
`contributor to, the damage.
`
`Discussion
`To date, very linle is known about the processes involved in
`cell death . The general consensus suggests inhibition of DNA
`synthesis as the critical step in cisplatin-induced cytotoxicity. Our
`earlier work had questioned this dogma (7,8). We therefore
`sought an alternative explanation. In this report we have at(cid:173)
`tempted to profile the events that occurred as a result of cisplatin
`
`treatment. In particular, we have investigated events that may be
`associated with drug-induced G2 arrest and the subsequent deg(cid:173)
`radation of DNA that appears to be an essential step in cell death.
`_. -We initially hyp<>thesized.that cells are arrested in.the 0 2.phase.
`due to an inhibition of transcription, speciticaJly, an inability to
`-produce the full-length mRNA needed for passage to ·mitosis; ··- -· ~
`First, we compared the effect of drug treatment on DNA, RNA,
`and protein synthesis. Consistent with previous reports (2), we
`observed that DNA synthesis was the most sensitive to drug
`treatment in that it was the first suppressed. Since the cells still
`survived at the lower concentrations, suppression of DNA syn(cid:173)
`thesis did not predict cell death. These curves show an initial
`suppression of DNA synthesis during the S phase. This is
`followed by recovery, reflecting passage into the G2 phase, and a
`subsequent arrest there. Recovery of DNA synthesis at 3-5 days
`represents passage through the next cell cycle. At the highest
`concentrations, the cells were dying, and no recovery was
`observed. In contrast, neither RNA synthesis nor protein synthe(cid:173)
`sis was suppressed until about 2 days after drug treatment.
`These experiments, as well as those previously reported (2),
`did not take into consideration that the quality of transcription
`could be altered; that is, transcription could tenninate on reaching
`an adduct in DNA, but then reinitiate from the beginning. The
`result could be the same quantity of RNA produced, but with
`much of it incomplete. We therefore measured the quantity of
`newly synthesized poly(A)+ RNA as an indicator of completion
`of transcription. Again, we observed no significant suppression
`of synthesis until 2 days after drug treatment. Therefore, 0 2 arrest
`cannot be attributed to detectable changes in transcription.
`A recent explanation for drug-induced 0 2 arrest comes from
`the yeast RAD9 mutant (/5). Cells with this phenotype were
`unable to be arrested in the G2 phase following introduction of
`DNA damage; they continued to cycle and died. The RAD9 gene
`product was demonstrated to be essential for arrest of cell division
`following DNA damage. It is possible that RAD9 is involved in
`the surveillance mechanism previously hypothesized by Tobey
`(16).
`The question still remains as to the cause of a cell 's demise
`following incubation with cisplatin. There are two known mech(cid:173)
`anisms of cell death: necrosis and apoptosis (9). The latter
`pathway, also known as programmed cell death, occurs during
`metamorphosis, differentiation, and general cell turnover. Apo(cid:173)
`ptosis is also the cause of death in thymocytes exposed to
`glucocorticoids (/7) or x rays (18). One of the characteristics of
`apoptosis is chromatin condensation associated with DNA degra(cid:173)
`dation, giving rise to internucleosomal cleavage products. The
`endonuclease involved is of nonlysosomaJ origin, because the
`membranes remain intact. ln addition, new protein synthesis is
`required . In the LI 210/0 cells studied here, we detected the
`formation of nucleosome ladders at toxic concentrations within
`2 days of drug treatment. The intensity of the nucleosome ladder
`increased with time and drug concentration. At nontoxic drug
`concentrations, no such degradation was observed. It is probable
`that the DNA degradation previously observed as DNA double(cid:173)
`strand breaks by neutral elution at 24 hours (7) progresses and by
`48 hours is observable as intemucleosomal cleavage products.
`These events occurred long before loss of membrane integrity.
`The cells did not begin to take up trypa.ri blue until 4 days after
`drug treatment (7).
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`Several other characteristics of apoptosis have been investi(cid:173)
`gated. Following incubation of Ll210/0 cells with cisplatin, we
`observed an initial increase in cell size associated with arrest in
`the G2 phase (fig. 4). At minimally toxic concentrations, the cell
`size had recovered by 4 days, but at toxic concentrations, a
`significant proportion of the cells demonstrated a reduction in
`volume to less than control cells. Cell shrinkage is often associ(cid:173)
`ated with apoptosis, but the increased size has not previously been
`reported. This increase may not have been observed before for
`several reasons. First, many of the observations have been made
`in whole tissues, in which apoptotic cells are typically quite
`isolated and infrequent (9). Because of this relative infrequence,
`it would be diffic~lt to observe cells undergoing early stages of
`apoptosis, particularly since such swollen cells might not be
`recognized as abnonnal. Second, work in thymocytes shows a
`much more rapid induction of apoptosis in response to dexa(cid:173)
`methasone (17,19). Generally, in vitro studies have not been
`performed under conditions that would pennit detection of de(cid:173)
`layed cell death. We also detected occasional cell surf ace blebs by
`scanning electron microscopy (fig. 5). The holes subsequently
`observed correlated with the timing of trypan blue uptake. The
`holes probably resulted from the bursting of the blebs.
`We also monitored NAD and ATP levels following incubation
`of the cells with cisplatin. NAD is a substrate for poly(ADP(cid:173)
`ribosyl)ation of proteins. This protein modification is stimulated
`by DNA strand breaks and generally suppresses protein activity
`(20). Extensive or unrepaired DNA strand breaks reportedly
`cause poly(ADP-ribose) polymerase to be continuously active.
`This causes depletion of NAD and, subsequently, depletion of
`ATP during attempts to replenish the NAD pool. Such alterations
`are thought to account for the rapid cell death ~hat occurs before
`DNA repair takes place (20,21). However, we were able to detect
`significant decreases in NAD and ATP only at toxic concentra(cid:173)
`tions and 3 days following drug treatment. This was 1 day after
`the detection of DNA double-strand breaks. These data suggest
`either that significant poly(ADP-ribosyl)ation was a delayed
`response or that these breaks did not stimulate poly(ADP(cid:173)
`ribosyl)ation. An endonuclease potentially involved in apoptosis
`is itself inhibited by poly(ADP-ribosyl)ation (22). Reduced NAD
`could therefore lead to its activation. However, since the changes
`we observed occurred after DNA degradation, they were an effect
`of, and not a contributor to, the damage.
`The results presented here affinn the importance of DNA
`degradation as an early and presumably essential step in cell
`death. The inhibition of transcription and protein synthesis
`occurred at about the same time as DNA degradation, so it is not
`possible to confirm which is cause or effect. Loss of NAD and
`ATP occurred later, presumably as a consequence of these
`changes. These results clearly demonstrate that cells can be
`lethally damaged, yet continue to undergo apparently normal
`metabolic activities for several days. These results are also
`different from those reporting rapid cell death. However, similar
`events appear to occur in both types of cell death; the difference is
`presumably in the signal transduction pathway. The slow cell
`death reported here appears to occur at the G2/M transition and
`may therefore involve events that normally occur at this stage of
`the cell cycle. This,/~· ~onsistent with the idea that apoptosis is
`related to chromatin1Condensation.
`Tremendous progress has been made recently in understanding
`
`the events essential for passage of cells into mitosis. The major
`component appears to be the kinase coded for by the cdc2 gene
`(histone Hl kinase) that is activated and inactivated by specific
`phosphorylations and by a variety of associated proteins (23,24).
`One essential protein is cyclin B, the only protein whose synthesis
`is required for passage into mitosis (14). Two regulators of
`histone Hl kinase are defined in yeast by the cdc25 and weel
`genes; the former activates the kinase, while the latter suppresses
`it. Mutants with an imbalance in these genes can undergo a
`"mitotic catastrophe" reminiscent of premature chromatin con(cid:173)
`densation (25). It seems highly probable that these events are
`involved as part of the signal transduction pathway during cell
`death by apoptosis. The events reported in this paper are not
`restricted to DNA-damaging agents, as many other toxic agents
`induce the same events (26). Therefore, there will probably be
`multiple steps within the pathway at which drug effects can be
`manifested.
`
`References
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`produced by anticancer platinum complexes. Pharmacol Tuer 34: 155-166,
`1987
`(4) HAIDER HC, ROSENBERG 8: Inhibitory effects of anti-tumor platinum
`compound on DNA, RNA and protein synlhcsis in mammalian cells in vitro.
`Int J Cancer 6:207-216, 1970
`(5) HOWLE JA, GALE GR: Cis-Dichlorodiammineplatinum(U): Persistent and
`selective inhibition of deoxyribonucleic acid synthesis in vivo. Biochem
`Phannacol 19:2757-2762, 1970
`(6) SALLES B, 8lTTOUR JL, MACQUET JP: cis-Pt(NH3)2Cl2 and rrans(cid:173)
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`Biochem Biophys Res Commun 112:555-563, 1983
`(7) SORENSON CM , EASTMA