Vol. 10, 155–162, March 1999 Cell Growth & Differentiation 155
Dose-dependent Effects of DNA-damaging Agents on p53-
mediated Cell Cycle Arrest1
Daniel Chang, Feng Chen, Fenfen Zhang, The tumor suppressor protein p53 has been shown to
Bruce C. McKay, and Mats Ljungman2 mediate a G1 arrest in cells exposed to IR and certain other
Department of Radiation Oncology, Division of Cancer Biology, DNA-damaging agents (9). After the induction of DNA dam-
University of Michigan Comprehensive Cancer Center, Ann Arbor, age, the level and activity of p53 in exposed cells is increased
resulting in p53-mediated transactivation of target genes (10,
11). One of the target genes encodes the Cdk-inhibitor
Abstract p21WAF1. The expression of p21WAF1 results in the inhibition
We examined the dose-dependent effects of DNA- of Rb phosphorylation, and, thus, the subsequent expression
damaging agents on G1 arrest in isogenic human cell of E2F-dependent genes is blocked (12). The evidence for
lines differing in their p53 status. As expected, 5 or 20 the important roles of both p53 and p21WAF1 in the IR-
Gy of ionizing radiation induced a p53-dependent G1 induced G1 cell cycle arrest is deduced from studies using
arrest. In contrast, UV light or actinomycin D induced a cells defective in either p53 (9, 13) or p21WAF1 (14, 15).
modest G1 arrest that was p53-dependent only at lower Certain DNA-damaging agents such as UV light and cis-
doses. At higher doses, cells were arrested in G1 in a platin will at high doses inhibit mRNA synthesis (16 –18). In
p53-independent manner coinciding with inhibition of accordance with an affect on transcription, recent studies
RNA synthesis and abolished cyclin E expression. have shown that the expression of p21WAF1 is reduced or
Interestingly, expression of cyclin E was enhanced delayed at higher doses of UV light (19 –21) and cisplatin (18).
after exposure to moderate doses of UV light and Thus, p53 may be unable to transactivate the p21WAF1 gene
actinomycin D, and this enhancement was suppressed because of the presence of transcription-blocking lesions in
by wild-type p53. We propose that agents inducing the p21WAF1 gene, and, thus, the ability to arrest the cells in
transcription-blocking DNA lesions will at higher doses G1 should be attenuated. However, proficient transcription is
inhibit the progression of cells into S phase by a p53- also required for the G1-S transition by activating E2F-re-
independent mechanism involving the attenuation of sponding genes. We, therefore, hypothesized that arrest in
E2F-mediated transcription of genes, such as cyclin E. the G1 phase of the cell cycle could be accomplished in a
p53-independent manner by the inhibition of transcription. In
this study, we tested this hypothesis by studying the dose-
Introduction dependent effect of IR, UV light, and actinomycin D on cell
The progression of cells through the cell cycle is governed by cycle distribution of two RKO cell lines that differed in their
the precise activation and inactivation of cyclin proteins and p53 status.
Cdks3 (1). The transition from the G1 phase into the S phase
of the cell cycle requires the activities of Cdk4/Cdk6 in as- Results
sociation with D cyclins and Cdk2/cyclin E. It is thought that
Induction of p53 and p21WAF1 Is Attenuated in HPV16
these G1 Cdks phosphorylate the Rb protein, which leads to
E6-expressing Cells. In this study, the human colon cancer
the activation of genes whose products are required for the
cell line RKO and a derived cell line expressing the HPV16 E6
progression into S phase as well as for the replication of DNA
protein (22, 23) were used to study the role of p53 in cell
(2–5). The Rb protein was recently shown to be recruited to
cycle control after exposure to moderate and high doses of
target promoters by transcription factor E2F-1 and to repress
the DNA-damaging agents IR and UV light and of the RNA
these target genes by interaction with a histone deacetylase
synthesis inhibitor actinomycin D. Expression of the E6 pro-
(5– 8). Phosphorylation of Rb is thought to disrupt the inter-
tein has been shown to cause rapid degradation of the p53
action with E2F-1, which results in the induction of expres-
protein even after exposure to DNA-damaging agents (22,
sion of genes containing E2F-1 binding sites.
24, 25). The doses chosen for IR and UV have been shown to
give similar toxicity in these cells (26). The doses of actino-
mycin D were chosen because these doses affect p53 and
Received 5/27/98; revised 1/4/99; accepted 1/6/99.
The costs of publication of this article were defrayed in part by the p21WAF1 expression to a similar extent as the doses chosen
payment of page charges. This article must therefore be hereby marked for UV light (Ref. 18; also see Fig. 1).
advertisement in accordance with 18 U.S.C. Section 1734 solely to indi- We confirm that the expression of p53 was almost abol-
cate this fact.
Supported by a grant from the University of Michigan Comprehensive ished in the RKO-E6 cells, whereas p53 readily accumulates
Cancer Center’s Institutional Grant from the American Cancer Society. in the parental cells after exposure to DNA-damaging agents
To whom requests for reprints should be addressed, at Department of
Radiation Oncology, Division of Cancer Biology, University of Michigan
(Fig. 1; Ref. 23). Furthermore, expression of p21WAF1 was
Comprehensive Cancer Center, 4306 CCGC, 1500 East Medical Center significantly reduced in the RKO-E6 cells compared with the
Drive, Ann Arbor, MI 48109-0582. E-mail: firstname.lastname@example.org.
parental cells (Fig. 1B). A small increase in p21WAF1 protein
The abbreviations used are: Cdk, cyclin-dependent kinase; Rb, retino-
blastoma; IR, ionizing radiation; HBT, 0.5% Tween 20 and 5% fetal bovine level was observed in the RKO-E6 cells after exposure to IR
serum in PBS; PI, propidium iodide; TCA, trichloroacetic acid. and lower doses of UV and actinomycin D. However, the
156 DNA Damage and Role of p53 in G1 Arrest
Fig. 1. The induction of p53 and
p21WAF1 protein accumulation
was attenuated in the RKO-E6
cells. Western blots showing the
protein levels of (A) p53 and (B)
p21WAF1 in RKO and RKO-E6
cells 24 h after exposure to IR,
UV light, or the addition of acti-
induced levels of p21WAF1 were below the baseline level of Table 1 Nascent total RNA and full-length mRNA synthesis in RKO
p21WAF1 in RKO cells. We conclude that the expression of cells 6 h after exposure to UV light, IR, or addition of actinomycin D
the HPV16 E6 protein in RKO-E6 cells results in a near The values represent the mean of 2– 4 biological experiments SE.
p53-null phenotype. Relative total Relative mRNA
Abolished Induction of p21WAF1 after Exposure to High RNA synthesis synthesis
Doses of UV Light and Actinomycin D. Significant induc- Control 100 100
tion of p21WAF1 was observed in RKO cells after exposure to IR, 5 Gy 90 3 77 6
IR and moderate doses of UV light and actinomycin D (Fig. IR, 20 Gy 90 3 54 10
UV, 10 J/m2 58 8 77 27
1B). However, after exposure to higher doses of UV light and UV, 30 J/m2 25 9 14 7
actinomycin D, the expression of p21WAF1 was abolished. It actD,a 20 nM 37 2 27 4
has been shown previously (27) using Northern blot that the actD, 200 nM 9 2 5 1
expression of p21WAF1 RNA is induced in RKO cells after a
actD, actinomycin D.
exposure to 10 J/m2 but inhibited after exposure to 30 J/m2.
Furthermore, actinomycin D treatment has been shown at
higher doses to effectively reduce expression of p21WAF1
p53-dependent G1 Arrest after Low and High Doses of
RNA (27), whereas IR stimulates p21WAF1 mRNA levels in
IR. The cell cycle distribution at 6 or 24 h after exposure to
cells (28). IR was determined by incubating the cells with 30 M BrdUrd
To explore whether the loss of p21WAF1 in the RKO cells for 15 min to label cells synthesizing DNA. Cells were fixed,
was due to the inhibition of general transcription, we ana- incubated with an anti-BrdUrd antibody, and stained with PI.
lyzed the incorporation of [3H]uridine into nascent RNA and The cell samples were then analyzed using flow cytometry.
poly(A)RNA. It was found that 30 J/m2 of UV light and 200 nM As expected, 24 h after exposure to 5 Gy of IR, the parental
actinomycin D reduced nascent RNA synthesis to below RKO cells arrested in the G1 and G2-M phases of the cell
25% (Table 1). In contrast, 20 Gy of IR had only marginal cycle with very few cells traversing the S phase (Fig. 2). The
effect on total RNA synthesis, and poly(A)RNA synthesis was RKO-E6 cells were found to arrest in the G2-M phase; but, in
reduced to about 55%. We conclude that the loss of p21WAF1 contrast to the parental RKO cells, a significant number of
expression after exposure to high doses of UV or actinomy- RKO-E6 cells were found in the S phase. Twenty-four h after
cin D correlated with a severe inhibition of RNA synthesis, irradiation with 20 Gy, all of the cell samples were essentially
which is in agreement with previous results (18). devoid of S-phase cells. Although parental cells showed
Cell Growth & Differentiation 157
Fig. 2. RKO cells exposed to IR arrested in the G1 phase of the cell cycle in a p53-dependent manner. The distribution of cells in the cell cycle after
exposure to 5 or 20 Gy of IR was analyzed 6 or 24 h after irradiation by pulse-labeling with BrdUrd for 15 min, followed by FITC and PI staining. The PI
staining intensity (i.e., DNA content) is expressed on the X-axis while FITC intensity (i.e., BrdUrd incorporation) is expressed on the Y-axis. The position of
cells in G1, S phase, and G2-M are indicated in the top left panel.
Table 2 Relative amount of DNA synthesis in treated cells compared with untreated control cells
DNA synthesis was determined from multiplying the number of cells incorporating BrdUrd with the mean FITC signal in the BrdUrd positive windows (the
H windows in Fig. 2–5).
IR UV light Actinomycin D
Cells 5 Gy 20 Gy 10 J/m 30 J/m 20 nM 200 nM
0 6h 24 h 6h 24 h 0 6h 24 h 6h 24 h 0 6h 24 h 6h 24 h
RKO 100 197 36 50 4 100 73 92 102 54 100 213 29 13 5
RKO-E6 100 235 151 75 16 100 75 65 26 13 100 176 67 11 6
evidence of a G1 arrest, the RKO-E6 cells did not show a G1 mulation of cells in G2-M was observed, which suggested
phase population. Instead, virtually all of the RKO-E6 cells that the G2-M checkpoint had been activated. Thus, it ap-
were found occupying the G2-M window. These results sug- pears that the activation of the G2-M checkpoint is a faster
gest that radiation-induced G1 arrest but not G2-M arrest is event than the activation of the G1 arrest in these cells.
dependent on wild-type p53 function. It has been recently Role for p53 in G1 Arrest after Exposure to Moderate
shown that the arrest in G2 but not in M-phase is dependent but not High Doses of UV Light or Actinomycin D. We
on p53 (29, 30). next compared the effect of moderate or high doses of UV
Interestingly, we did not observe any signs of a G1 arrest light and actinomycin D on cell cycle distribution in the RKO
6 h after irradiation with 5 Gy (Fig. 2). In fact, more cells and RKO-E6 cell lines. As can be seen in Fig. 3, the distri-
appeared in the S phase at this time than was observed in bution of cells in the cell cycle was not significantly different
control cells, and these cells incorporated BrdUrd at rates between the two cell lines 6 h after irradiation with 10 J/m2.
similar to control cells (Table 2), which indicated that the Interestingly, there was a higher percentage of cells in S
irradiation had not induced an S-phase arrest. This may phase in the irradiated sample at this time point compared
reflect an increase in E2F-1 expression shortly after exposure with the unirradiated control sample. However, the mean
to IR (31). Six h after exposure to 20 Gy of IR, there was a intensity of the FITC signal, reflecting the amount of BrdUrd
“clearing out” of parental RKO cells in transition from G1 to S incorporated per cell during the 15 min labeling period, was
phase (lower left portion of window D in Fig. 2). This was not significantly lower in the irradiated cells, which suggested
evident for the RKO-E6 cells. The mean FITC signal de- that the irradiated cells were synthesizing DNA at a slower
creased in the later stages of the S phase, perhaps as a result rate (Table 2). This reduced rate of DNA synthesis after UV
of attenuated replication of damaged templates leading to an irradiation was most likely a result of the presence of UV
apparent overall decrease in DNA synthesis (Table 2). lesions in the DNA template (32). Therefore, the increased
Although the arrest of cells from entering S phase was only percentage of cells in S phase 6 h after irradiation with 10
marginally apparent at 6 h after irradiation, a marked accu- J/m2 may be the result of a slower progression through the
158 DNA Damage and Role of p53 in G1 Arrest
Fig. 3. A modest G1 arrest after UV irradiation was p53-dependent after exposure to 10 J/m2 but not after 30 J/m2. The two cell lines were exposed to
UV light followed by BrdUrd pulse-labeling for 15 min at 6 or 24 h after irradiation. Cell cycle analysis was performed as described in legend to Fig. 2.
Fig. 4. G1 arrest after exposure to actinomycin D was p53-dependent at moderate doses but not after high doses. The two cell lines were exposed to 20
or 200 nm of actinomycin D followed by BrdUrd pulse-labeling for 15 min in the presence of the drug at either 6 or 24 h after addition of the drug. Cell cycle
analysis was performed as described in legend to Fig. 2.
S phase of the cell cycle, which leads to an accumulation of vated. The RKO-E6 cells entering S phase accumulated in an
cells in S phase. Alternatively, UV light may induce a signal- early stage of the S phase. Furthermore, a significant pro-
stimulating S-phase entry. portion of RKO-E6 cells with S-phase DNA content did not
The cell cycle distribution 24 h after irradiation with 10 incorporate BrdUrd after exposure to 10 J/m2 suggesting
J/m2 (Fig. 3) or treatment with 20 nM actinomycin D (Fig. 4) that these cells were arrested in S phase (see box F in Fig. 3).
was found to differ depending on the p53 status of the cells. The low value for DNA synthesis in the RKO-E6 cells at 24 h
We observed that, compared with RKO-E6 cells, fewer pa- after exposure to 10 J/m2 of UV light (Table 2) does not
rental RKO cells occupied the S phase at this time, which reflect the induction of a G1 arrest. Rather, cells were enter-
suggested that a p53-dependent G1 arrest had been acti- ing S phase at a rate that was even higher than for untreated
Cell Growth & Differentiation 159
or 20 nM actinomycin D may be due to the increased expres-
sion of cyclin E.
It is thought that agents that induce the p53 response will
arrest cells in the G1 phase of the cell cycle by increased
Fig. 5. Expression of cyclin E was greatly reduced in both cell lines after expression of the p21WAF1 gene (10). This p53-dependent
exposure to high doses of UV light and actinomycin D, whereas expres-
sion was greatly increased in the RKO-E6 cells at moderate doses. Cells arrest is readily seen in cells exposed to IR (12, 14, 15),
were irradiated or treated with actinomycin D for 24 h followed by Western whereas studies using UV light have yielded contradictory
blot using anti-cyclin E antibodies. results (37– 43). Because UV light, but to a lesser extent IR,
inhibits RNA synthesis by inducing transcription-blocking
DNA lesions, transactivation of the p21WAF1 gene may not be
control cells, but they were unable to complete the progres- efficient despite high induced levels of p53 (19 –21).
sion through S phase (see box F in Fig. 3). The finding that In this study, we explored the role of p53 in the induction
S-phase cells harboring wild-type p53 may be more effi- of G1 arrest after moderate or high doses of the DNA-dam-
ciently synthesizing DNA after exposure to moderate doses aging agents IR and UV light and the RNA synthesis inhibitor
of UV light is in agreement with a previous report (32) and actinomycin D. As expected, cell cycle analysis revealed that
may reflect p53-mediated enhancement of nucleotide exci- cells arrested in the G1 phase of the cell cycle in a p53-
sion repair, clearing the DNA template from DNA polymer- dependent manner after exposure to moderate or high doses
ase-blocking lesions (33–36). of IR (Fig. 2). This G1 arrest correlated with increased ex-
We observed a severe depletion of the fraction of S-phase pression of the Cdk-inhibitor p21WAF1 (Fig. 1). In contrast to
cells in samples from both the p53 wild-type and the E6- IR, we found dose-dependent effects of UV light and acti-
expressing cell lines 24 h after exposure to high doses of UV nomycin D on p53-mediated G1 arrest. Lower doses of these
light (30 J/m2) or actinomycin D (200 nM; Figs. 3 and 4; Table agents resulted in a modest p53-dependent block of cells
2). We conclude that in contrast to moderate doses of UV from entering S phase. This G1 arrest, which was evident at
light and actinomycin D, high doses of these agents delayed 24 h but not at 6 h after irradiation or the start of actinomycin
the entry into S phase of the cell cycle by a mechanism that D treatment, correlated to increased p21WAF1 expression. At
did not depend on p53. Furthermore, the G1 arrest observed higher doses of UV light and actinomycin D, cells from both
at higher doses was not dependent on p21WAF1 because, cell lines were blocked from entering S phase. This G1 arrest
at these doses, the cells were unable to express p21WAF1 did not coincide with increased expression of p21WAF1. On
(Fig. 1). the contrary, p21WAF1 protein expression was severely at-
Attenuated Expression of Cyclin E after Exposure to tenuated at these doses, which may be a reflection of severe
High Doses of UV and Actinomycin D. One potential impairment of transcription at these doses (Table 1). These
mechanism for the p53-independent induction of G1 arrest results suggest that the delay in the entry into S phase by 30
after exposure to high doses of UV light and actinomycin D J/m2 of UV light or 200 nM actinomycin D was not mediated
may be that the expression of genes required for the transi- by the p21WAF1 protein and was not dependent on wild-type
tion from the G1 to the S phase was abrogated. To explore p53 function.
this possibility, we analyzed the expression of cyclin E pro- The seemingly contradictory results of reduced expression
tein in the two cell lines after exposure to the DNA-damaging of p21WAF1 and induced delay of entry into S phase after
agents. Expression of cyclin E, which normally occurs in the exposure to high doses of UV light or actinomycin D may be
late G1 phase and is mediated by the E2F-1 transcription reconciled by the effect that these agents have on general
factor (4, 5), was significantly reduced in both cell lines after transcription. Because the cell cycle transition from the G1
exposure to high doses of UV light or actinomycin D (Fig. 5). phase to the S phase of the cell cycle is stimulated by
Our results are in agreement with a model in which high transcription of E2F-regulated genes (4), we investigated the
doses of DNA-damaging agents induce a p53-independent effect these agents may have on the expression of cyclin E
G1 arrest by interfering with the transcription of S phase- that is induced by E2F-1. Because IR is a poor inhibitor of
promoting genes such as cyclin E. transcription (Refs. 18, 44, 45; Table 1), the expression of S
Moderate Doses of UV Light or Actinomycin D Induced phase-promoting genes would not be expected to be atten-
High Levels of Cyclin E Protein Which Was Antagonized uated by IR directly. However, UV light and actinomycin D
by Wild-Type p53. A slight increase in cyclin E expression are potent inhibitors of RNA synthesis (18, 46) and would be
was observed in the parental RKO cells after exposure to expected to interfere with the unfolding of the genetic pro-
moderate doses of UV light and actinomycin D (Fig. 5). gram required for the G1-to-S-phase transition. In support of
However, the same treatment led to a much higher induction this argument we found that IR caused no measurable inhi-
of cyclin E in the RKO-E6 cells. Thus, moderate doses of UV bition of cyclin E expression even after high doses (Fig. 5).
light and actinomycin D but not IR strongly stimulated cy- However, exposure of the cells to high doses of either UV
clin-E expression, and this stimulation was suppressed by light or actinomycin D significantly reduced cyclin E levels.
wild-type p53. Perhaps this finding explains the cell cycle Thus, we propose that the p53- and p21WAF1-independent
effects shown in Figs. 3 and 4 in which the accumulation of block of S-phase entry after high doses of UV light and
RKO-E6 cells in early S phase 24 h after exposure to 10 J/m2 actinomycin D is caused by severe inhibition of general tran-
160 DNA Damage and Role of p53 in G1 Arrest
D is that these agents induce Myc and Ras, which have been
shown to induce the accumulation of cyclin E as well as
E2F-1 (50). Indeed, low-to-moderate doses of UV light have
been shown to induce expression of both c-H-ras and c-myc
(51). Finally, it is possible that the induced levels of cyclin E
is not the cause but rather the result of increased proliferation
and S-phase entry caused by some other inducible mecha-
nism. Additional studies are required to elucidate the poten-
tial roles of E2F-1, Myc, and Ras in the induction of cyclin E
after moderate doses of DNA-damaging and RNA-synthesis-
inhibiting agents and the role of p53 in masking this induc-
In summary, this study points out that the type and the
dose of different DNA-damaging agents will differentially af-
fect cell cycle progression of exposed cells. Although IR
induced the accumulation of both p53 and p21WAF1 even at
high doses, equitoxic doses of UV light or actinomycin D
Fig. 6. Model for G1 arrest after moderate or high doses of IR, UV light, caused accumulation of p53 but not p21WAF1, presumably
or actinomycin D. After exposure to p53-inducing agents that only partially because of inhibition of general transcription. Despite the
inhibit RNA synthesis (i.e., IR or moderate doses of UV light or actinomycin
D), G1 arrest is dependent on functional p53. However, at higher doses of lack of p21WAF1 expression at high doses of UV light or
UV light or actinomycin D, at which transcription is severely suppressed, actinomycin D, exposed cells were inhibited from entering S
cells arrest in G1 in a p53-independent manner. We propose that this G1
phase in a p53- and p21WAF1-independent manner. We pro-
arrest is caused by the direct interference of cyclin E expression and other
E2F-1-transactivated gene products required for the G1 to S phase tran- pose that this arrest is caused by a general blockage of RNA
sition. polymerase II, which cripples the unfolding of the genetic
program required for entering S phase.
scription leading to the inability of the cells to express cyclin Materials and Methods
E and other S phase-promoting proteins (Fig. 6). Cell lines, Cell Culture, and Irradiation. In this study, we used two
In contrast to the inhibition of cyclin E expression after different cell lines derived from the human colon cancer cell line RKO with
different functional status of p53. These cells were generously given to us
exposure to high doses of UV light and actinomycin D, mod- by Dr. Albert Fornace, Jr. (National Cancer Institute, NIH, Bethesda, MD,
erate doses of these agents caused a marked increase in via Dr. Ted Lawrence, University of Michigan). The parental cell line, RKO,
cyclin E expression especially in the E6-expressing cells (Fig. harbors an endogenous wild-type p53 (52). The RKO-E6 cell line was
5). This induced expression of cyclin E may explain the derived from RKO cells transfected with the vector pCMV-E6 containing
the HPV 16 E6 gene (22, 23). The E6 protein has been shown to interact
“mitogenic” effect observed in our cell cycle studies in which
with the cellular protein E6AP to target the p53 protein for proteosome-
the entry into S phase was greatly increased by these agents mediated degradation by ubiqutinating the p53 protein (24, 53).
in the RKO-E6 cells (Figs. 3 and 4). The mitogenic signal Cells were grown in monolayers in MEM (Life Technologies, Inc.) sup-
induced by these agents was apparently masked in the p53 plemented with 10% fetal bovine serum, 1 vitamins, 1 amino acids, 1
wild-type cells, perhaps by high levels of p21WAF1 expression antibiotics. Subconfluent cells, seeded 2 days before the experiments,
were irradiated on ice with IR (cobalt source with a dose rate of about 2
at these doses (Fig. 1). We propose that, in addition to Gy/min), or were irradiated at room temperature with a germicidal UV light
orchestrating a G1 arrest after exposure of cells to DNA- (254 nm). The fluence of the UV light source (Philips) was measured before
damaging agents, p53 may also play a role in reducing the each experiment with a UVX radiometer (UVP, Inc.). Actinomycin D was
proliferative signal induced by UV light and actinomycin D. dissolved in DMSO at 1 mM concentrations and diluted in media before the
This suppression may be the result of reduced E2F-1 activity
Western Blot. Western blot was performed as described previously
by p21WAF1-mediated inhibition of phosphorylation of the Rb (17) using 12% SDS-PAGE for p53 Westerns and 15% SDS-PAGE for p21
protein (Fig. 6). Westerns. Detection of p53 and p21WAF1 proteins were performed using
What is the mechanism for the induction of cyclin E ex- different anti-p53 (Ab-2), anti-p21WAF1, and anti-cyclin E (Oncogene Sci-
pression in the RKO-E6 cells after exposure to moderate ences), horseradish-peroxidase-conjugated secondary antibodies, en-
hanced chemiluminescence (SuperSignal CL-HRP Substrate System,
doses of UV light and actinomycin D? Because transcription Pierce), and X-ray film. To assess whether equal amounts of protein were
of the cyclin E gene is induced by the transcription factor loaded in each lane, the nylon membranes were stained with Coomassie
E2F-1 (4, 5), it is possible that these agents may stimulate the blue after the completion of the film exposure.
release of E2F-1 by phosphorylation of the Rb protein. In- Measurements of Nascent RNA Synthesis. Cells were seeded and
incubated in 185 Bq/ml [14C]thymidine for 2 days before the experiments.
deed, exposure of cells to IR (23), UV light (47), or actinomy-
Cells were then irradiated or treated with actinomycin D. After incubation
cin D (48) leads to dephosphorylation of Rb. However, we did for 5.5 h, nascent RNA was pulse-labeled for 30 min in 0.5 ml of medium
not in our study observe any induction of cyclin E by IR containing 7.4 105 Bq (20 Ci) of [3H]uridine. After rinsing three
despite the potential up-regulation of E2F-1 expression (31). times with ice-cold PBS, the cells were scraped off the plates with a cell
Furthermore, IR, UV light, and actinomycin D have been scraper. Poly(A)mRNA was isolated from cell lysates using the Straight A’s
mRNA isolation system (Novagen) and 3H activity was assessed using a
shown to cause a decrease in the level of Rb in exposed cells scintillation counter.
(49). Another possibility for the induction of cyclin E in the Total nascent RNA synthesis was assessed by counting the activity of
RKO-E6 cells at moderate doses of UV light or actinomycin TCA insoluble material that did not bind to the poly(dT) magnetic beads.
Cell Growth & Differentiation 161
Equal volumes of sample and 10% ice-cold TCA were mixed and put on 9. Kastan, M. B., Onyekwere, O., Sidransky, D., Vogelstein, B., and Craig,
ice for 30 min; the TCA insoluble material was then collected on filters R. W. Participation of p53 protein in the cellular response to DNA damage.
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ml of 5% TCA, 5 1 ml distilled water, and 2 1 ml of 95% ethanol, and 10. Levine, A. J. p53, the cellular gatekeeper for growth and division. Cell,
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counted in a scintillation counter. Relative total RNA synthesis and pol-
11. Giaccia, A., and Kastan, M. The complexity of p53 modulation:
y(A)RNA synthesis was then determined by calculating the ratio of 3H:14C
emerging patterns from divergent signals. Genes Dev., 12: 2973–2983,
for each sample and comparing it with the ratio in an untreated control
BrdUrd Incubation and Cell Fixation. Cells were pulse-labeled with 12. Dulic, V., Kaufmann, W. K., Wilson, S. J., Tlsty, T. D., Lees, E., Harper,
30 mM BrdUrd (Sigma) at 37°C for 15 min. The plates were then rinsed with J. W., Elledge, S. J., and Reed, S. I. p53-dependent inhibition of cyclin-
PBS followed by the collection of the cells by scraping them with a rubber dependent kinase activities in human fibroblasts during radiation-induced
policeman in 2 ml of PBS. After centrifugation, the cells were fixed by G1 arrest. Cell, 76: 1013–1023, 1994.
adding ice-cold 70% ethanol dropwise under vortexing. 13. Kuerbitz, S., Plunkett, B., Walsh, V., and Kastan, M. Wild-type p53 is
Denaturation, FITC-PI Staining, and Flow Cytometry. The proce- a cell cycle checkpoint determinant following irradiation. Proc. Natl. Acad.
dure was performed essentially as described previously (54). Cells, which Sci. USA, 89: 7491–7495, 1992.
had been fixed for at least 30 min, were centrifuged at 1100 rpm (224 14. Deng, C. X., Zhang, P. M., Harper, J. W., Elledge, S. J., and Leder, P.
g) for 7 min, washed with PBS, and spun as before. The cells were then Mice lacking p21CIP1/WAF1 undergo normal development, but are defec-
suspended in 1 ml of a solution of 10 mM sodium acetate (pH 5.2), 10 tive in G1 checkpoint control. Cell, 82: 675– 684, 1995.
mg/ml RNase A, and 10 mM Tris-HCl (pH 7.4) that had previously been
15. Waldman, T., Kinzler, K. W., and Vogelstein, B. p21 is necessary for
boiled for 15 min and then cooled. The samples were incubated at 37°C
the p53-mediated G1 arrest in human cancer cells. Cancer Res., 55:
for 30 min. Next, 1 ml of PBS was added and cells were spun as before,
resuspended in 1 ml of 0.1 N HCl and 0.7% Triton X-100 (Sigma), and
incubated on ice for 10 min. PBS (1 ml) was then added and cells were 16. Mayne, L., and Lehmann, A. Failure of RNA synthesis to recover after
spun as before. The pellets were resuspended in 1 ml of sterile distilled UV-irradiation: an early defect in cells from individuals with Cockayne’s
deionized water, incubated at 97°C for 15 min, and then cooled on ice for syndrome and xeroderma pigmentosum. Cancer Res., 42: 1473–1478,
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Cells were again pelleted and suspended in 100 l of HBT and transferred 17. Ljungman, M., and Zhang, F. Blockage of RNA polymerase as a
to microfuge tubes. One hundred l of 1:20 dilution of antihuman BrdUrd possible trigger for UV light-induced apoptosis. Oncogene, 13: 823– 831,
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room temperature for 30 min. Then 1 ml of HBT was added and cells were 18. Ljungman, M., Zhang, F., Chen, F., Rainbow, A., and McKay, B.
spun at 3200 rpm for 2 min. The pellets were resuspended in 150 ml of Inhibition of RNA polymerase II as a trigger for the p53 response. Onco-
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19. Lu, X., Burbidge, S. A., Griffin, S., and Smith, H. M. Discordance
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Acknowledgments damage induced by ultraviolet light inhibits p21waf1 and bax expression:
We thank Al Fornace and Ted Lawrence for their kind gifts of the RKO cell implications for DNA repair, UV sensitivity and the induction of apoptosis.
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