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Cell Growth & Differentiation Vol. 10, 155-162, March 1999
© 1999 American Association for Cancer Research

Dose-dependent Effects of DNA-damaging Agents on p53-mediated Cell Cycle Arrest1

Daniel Chang, Feng Chen, Fenfen Zhang, Bruce C. McKay and Mats Ljungman2

Department of Radiation Oncology, Division of Cancer Biology, University of Michigan Comprehensive Cancer Center, Ann Arbor, Michigan 48109-0582


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
We examined the dose-dependent effects of DNA-damaging agents on G1 arrest in isogenic human cell lines differing in their p53 status. As expected, 5 or 20 Gy of ionizing radiation induced a p53-dependent G1 arrest. In contrast, UV light or actinomycin D induced a modest G1 arrest that was p53-dependent only at lower doses. At higher doses, cells were arrested in G1 in a p53-independent manner coinciding with inhibition of RNA synthesis and abolished cyclin E expression. Interestingly, expression of cyclin E was enhanced after exposure to moderate doses of UV light and actinomycin D, and this enhancement was suppressed by wild-type p53. We propose that agents inducing transcription-blocking DNA lesions will at higher doses inhibit the progression of cells into S phase by a p53-independent mechanism involving the attenuation of E2F-mediated transcription of genes, such as cyclin E.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The progression of cells through the cell cycle is governed by the precise activation and inactivation of cyclin proteins and Cdks3 (1) . The transition from the G1 phase into the S phase of the cell cycle requires the activities of Cdk4/Cdk6 in association with D cyclins and Cdk2/cyclin E. It is thought that these G1 Cdks phosphorylate the Rb protein, which leads to the activation of genes whose products are required for the progression into S phase as well as for the replication of DNA (2, 3, 4, 5) . The Rb protein was recently shown to be recruited to target promoters by transcription factor E2F-1 and to repress these target genes by interaction with a histone deacetylase (5, 6, 7, 8) . Phosphorylation of Rb is thought to disrupt the interaction with E2F-1, which results in the induction of expression of genes containing E2F-1 binding sites.

The tumor suppressor protein p53 has been shown to mediate a G1 arrest in cells exposed to IR and certain other DNA-damaging agents (9) . After the induction of DNA damage, 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 p21WAF1. The expression of p21WAF1 results in the inhibition of Rb phosphorylation, and, thus, the subsequent expression of E2F-dependent genes is blocked (12) . The evidence for the important roles of both p53 and p21WAF1 in the IR-induced G1 cell cycle arrest is deduced from studies using cells defective in either p53 (9 , 13) or p21WAF1 (14 , 15) .

Certain DNA-damaging agents such as UV light and cisplatin will at high doses inhibit mRNA synthesis (16, 17, 18) . In accordance with an affect on transcription, recent studies have shown that the expression of p21WAF1 is reduced or delayed at higher doses of UV light (19, 20, 21) and cisplatin (18) . Thus, p53 may be unable to transactivate the p21WAF1 gene because of the presence of transcription-blocking lesions in the p21WAF1 gene, and, thus, the ability to arrest the cells in G1 should be attenuated. However, proficient transcription is also required for the G1-S transition by activating E2F-responding genes. We, therefore, hypothesized that arrest in 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-dependent effect of IR, UV light, and actinomycin D on cell cycle distribution of two RKO cell lines that differed in their p53 status.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Induction of p53 and p21WAF1 Is Attenuated in HPV16 E6-expressing Cells.
In this study, the human colon cancer cell line RKO and a derived cell line expressing the HPV16 E6 protein (22 , 23) were used to study the role of p53 in cell cycle control after exposure to moderate and high doses of the DNA-damaging agents IR and UV light and of the RNA synthesis inhibitor actinomycin D. Expression of the E6 protein has been shown to cause rapid degradation of the p53 protein even after exposure to DNA-damaging agents (22 , 24 , 25) . The doses chosen for IR and UV have been shown to give similar toxicity in these cells (26) . The doses of actinomycin D were chosen because these doses affect p53 and p21WAF1 expression to a similar extent as the doses chosen for UV light (Ref. 18 ; also see Fig. 1Citation ).



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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 actinomycin D.

 
We confirm that the expression of p53 was almost abolished in the RKO-E6 cells, whereas p53 readily accumulates in the parental cells after exposure to DNA-damaging agents (Fig. 1Citation ; Ref. 23 ). Furthermore, expression of p21WAF1 was significantly reduced in the RKO-E6 cells compared with the parental cells (Fig. 1B)Citation . A small increase in p21WAF1 protein level was observed in the RKO-E6 cells after exposure to IR and lower doses of UV and actinomycin D. However, the induced levels of p21WAF1 were below the baseline level of p21WAF1 in RKO cells. We conclude that the expression of the HPV16 E6 protein in RKO-E6 cells results in a near p53-null phenotype.

Abolished Induction of p21WAF1 after Exposure to High Doses of UV Light and Actinomycin D.
Significant induction of p21WAF1 was observed in RKO cells after exposure to IR and moderate doses of UV light and actinomycin D (Fig. 1B)Citation . However, after exposure to higher doses of UV light and actinomycin D, the expression of p21WAF1 was abolished. It has been shown previously (27) using Northern blot that the expression of p21WAF1 RNA is induced in RKO cells after 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 RNA (27) , whereas IR stimulates p21WAF1 mRNA levels in cells (28) .

To explore whether the loss of p21WAF1 in the RKO cells was due to the inhibition of general transcription, we analyzed the incorporation of [3H]uridine into nascent RNA and poly(A)RNA. It was found that 30 J/m2 of UV light and 200 nM actinomycin D reduced nascent RNA synthesis to below 25% (Table 1)Citation . In contrast, 20 Gy of IR had only marginal effect on total RNA synthesis, and poly(A)RNA synthesis was reduced to about 55%. We conclude that the loss of p21WAF1 expression after exposure to high doses of UV or actinomycin D correlated with a severe inhibition of RNA synthesis, which is in agreement with previous results (18) .


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Table 1 Nascent total RNA and full-length mRNA synthesis in RKO cells 6 h after exposure to UV light, IR, or addition of actinomycin D

 
p53-dependent G1 Arrest after Low and High Doses of IR.
The cell cycle distribution at 6 or 24 h after exposure to IR was determined by incubating the cells with 30 µM BrdUrd for 15 min to label cells synthesizing DNA. Cells were fixed, incubated with an anti-BrdUrd antibody, and stained with PI. The cell samples were then analyzed using flow cytometry.

As expected, 24 h after exposure to 5 Gy of IR, the parental RKO cells arrested in the G1 and G2-M phases of the cell cycle with very few cells traversing the S phase (Fig. 2)Citation . The RKO-E6 cells were found to arrest in the G2-M phase; but, in contrast to the parental RKO cells, a significant number of RKO-E6 cells were found in the S phase. Twenty-four h after irradiation with 20 Gy, all of the cell samples were essentially devoid of S-phase cells. Although parental cells showed evidence of a G1 arrest, the RKO-E6 cells did not show a G1 phase population. Instead, virtually all of the RKO-E6 cells were found occupying the G2-M window. These results suggest that radiation-induced G1 arrest but not G2-M arrest is dependent on wild-type p53 function. It has been recently shown that the arrest in G2 but not in M-phase is dependent on p53 (29 , 30) .



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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.

 
Interestingly, we did not observe any signs of a G1 arrest 6 h after irradiation with 5 Gy (Fig. 2)Citation . In fact, more cells appeared in the S phase at this time than was observed in control cells, and these cells incorporated BrdUrd at rates similar to control cells (Table 2)Citation , which indicated that the irradiation had not induced an S-phase arrest. This may reflect an increase in E2F-1 expression shortly after exposure to IR (31) . Six h after exposure to 20 Gy of IR, there was a "clearing out" of parental RKO cells in transition from G1 to S phase (lower left portion of window D in Fig. 2Citation ). This was not evident for the RKO-E6 cells. The mean FITC signal decreased in the later stages of the S phase, perhaps as a result of attenuated replication of damaged templates leading to an apparent overall decrease in DNA synthesis (Table 2)Citation .


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Table 2 Relative amount of DNA synthesis in treated cells compared with untreated control cells

 
Although the arrest of cells from entering S phase was only marginally apparent at 6 h after irradiation, a marked accumulation of cells in G2-M was observed, which suggested that the G2-M checkpoint had been activated. Thus, it appears that the activation of the G2-M checkpoint is a faster event than the activation of the G1 arrest in these cells.

Role for p53 in G1 Arrest after Exposure to Moderate but not High Doses of UV Light or Actinomycin D.
We next compared the effect of moderate or high doses of UV light and actinomycin D on cell cycle distribution in the RKO and RKO-E6 cell lines. As can be seen in Fig. 3Citation , the distribution of cells in the cell cycle was not significantly different between the two cell lines 6 h after irradiation with 10 J/m2. Interestingly, there was a higher percentage of cells in S phase in the irradiated sample at this time point compared with the unirradiated control sample. However, the mean intensity of the FITC signal, reflecting the amount of BrdUrd incorporated per cell during the 15 min labeling period, was significantly lower in the irradiated cells, which suggested that the irradiated cells were synthesizing DNA at a slower rate (Table 2)Citation . This reduced rate of DNA synthesis after UV irradiation was most likely a result of the presence of UV lesions in the DNA template (32) . Therefore, the increased percentage of cells in S phase 6 h after irradiation with 10 J/m2 may be the result of a slower progression through the S phase of the cell cycle, which leads to an accumulation of cells in S phase. Alternatively, UV light may induce a signal-stimulating S-phase entry.



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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. 2Citation .

 
The cell cycle distribution 24 h after irradiation with 10 J/m2 (Fig. 3)Citation or treatment with 20 nM actinomycin D (Fig. 4)Citation was found to differ depending on the p53 status of the cells. We observed that, compared with RKO-E6 cells, fewer parental RKO cells occupied the S phase at this time, which suggested that a p53-dependent G1 arrest had been activated. The RKO-E6 cells entering S phase accumulated in an early stage of the S phase. Furthermore, a significant proportion of RKO-E6 cells with S-phase DNA content did not incorporate BrdUrd after exposure to 10 J/m2 suggesting that these cells were arrested in S phase (see box F in Fig. 3Citation ). The low value for DNA synthesis in the RKO-E6 cells at 24 h after exposure to 10 J/m2 of UV light (Table 2)Citation does not reflect the induction of a G1 arrest. Rather, cells were entering S phase at a rate that was even higher than for untreated control cells, but they were unable to complete the progression through S phase (see box F in Fig. 3Citation ). The finding that S-phase cells harboring wild-type p53 may be more efficiently synthesizing DNA after exposure to moderate doses of UV light is in agreement with a previous report (32) and may reflect p53-mediated enhancement of nucleotide excision repair, clearing the DNA template from DNA polymerase-blocking lesions (33, 34, 35, 36) .



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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. 2Citation .

 
We observed a severe depletion of the fraction of S-phase cells in samples from both the p53 wild-type and the E6-expressing cell lines 24 h after exposure to high doses of UV light (30 J/m2) or actinomycin D (200 nM; Figs. 3Citation and 4Citation ; Table 2Citation ). We conclude that in contrast to moderate doses of UV light and actinomycin D, high doses of these agents delayed the entry into S phase of the cell cycle by a mechanism that did not depend on p53. Furthermore, the G1 arrest observed at higher doses was not dependent on p21WAF1 because, at these doses, the cells were unable to express p21WAF1 (Fig. 1)Citation .

Attenuated Expression of Cyclin E after Exposure to High Doses of UV and Actinomycin D.
One potential mechanism for the p53-independent induction of G1 arrest after exposure to high doses of UV light and actinomycin D may be that the expression of genes required for the transition from the G1 to the S phase was abrogated. To explore this possibility, we analyzed the expression of cyclin E protein in the two cell lines after exposure to the DNA-damaging agents. Expression of cyclin E, which normally occurs in the late G1 phase and is mediated by the E2F-1 transcription factor (4 , 5) , was significantly reduced in both cell lines after exposure to high doses of UV light or actinomycin D (Fig. 5)Citation . Our results are in agreement with a model in which high doses of DNA-damaging agents induce a p53-independent G1 arrest by interfering with the transcription of S phase-promoting genes such as cyclin E.



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Fig. 5. Expression of cyclin E was greatly reduced in both cell lines after exposure to high doses of UV light and actinomycin D, whereas expression was greatly increased in the RKO-E6 cells at moderate doses. Cells were irradiated or treated with actinomycin D for 24 h followed by Western blot using anti-cyclin E antibodies.

 
Moderate Doses of UV Light or Actinomycin D Induced High Levels of Cyclin E Protein Which Was Antagonized by Wild-Type p53.
A slight increase in cyclin E expression was observed in the parental RKO cells after exposure to moderate doses of UV light and actinomycin D (Fig. 5)Citation . However, the same treatment led to a much higher induction of cyclin E in the RKO-E6 cells. Thus, moderate doses of UV light and actinomycin D but not IR strongly stimulated cyclin-E expression, and this stimulation was suppressed by wild-type p53. Perhaps this finding explains the cell cycle effects shown in Figs. 3Citation and 4Citation in which the accumulation of RKO-E6 cells in early S phase 24 h after exposure to 10 J/m2 or 20 nM actinomycin D may be due to the increased expression of cyclin E.


    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
It is thought that agents that induce the p53 response will arrest cells in the G1 phase of the cell cycle by increased expression of the p21WAF1 gene (10) . This p53-dependent arrest is readily seen in cells exposed to IR (12 , 14 , 15) , whereas studies using UV light have yielded contradictory results (37, 38, 39, 40, 41, 42, 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 efficient despite high induced levels of p53 (19, 20, 21) .

In this study, we explored the role of p53 in the induction of G1 arrest after moderate or high doses of the DNA-damaging agents IR and UV light and the RNA synthesis inhibitor actinomycin D. As expected, cell cycle analysis revealed that cells arrested in the G1 phase of the cell cycle in a p53-dependent manner after exposure to moderate or high doses of IR (Fig. 2)Citation . This G1 arrest correlated with increased expression of the Cdk-inhibitor p21WAF1 (Fig. 1)Citation . In contrast to IR, we found dose-dependent effects of UV light and actinomycin D on p53-mediated G1 arrest. Lower doses of these agents resulted in a modest p53-dependent block of cells from entering S phase. This G1 arrest, which was evident at 24 h but not at 6 h after irradiation or the start of actinomycin D treatment, correlated to increased p21WAF1 expression. At higher doses of UV light and actinomycin D, cells from both cell lines were blocked from entering S phase. This G1 arrest did not coincide with increased expression of p21WAF1. On the contrary, p21WAF1 protein expression was severely attenuated at these doses, which may be a reflection of severe impairment of transcription at these doses (Table 1)Citation . These results suggest that the delay in the entry into S phase by 30 J/m2 of UV light or 200 nM actinomycin D was not mediated by the p21WAF1 protein and was not dependent on wild-type p53 function.

The seemingly contradictory results of reduced expression of p21WAF1 and induced delay of entry into S phase after exposure to high doses of UV light or actinomycin D may be reconciled by the effect that these agents have on general transcription. Because the cell cycle transition from the G1 phase to the S phase of the cell cycle is stimulated by transcription of E2F-regulated genes (4) , we investigated the effect these agents may have on the expression of cyclin E that is induced by E2F-1. Because IR is a poor inhibitor of transcription (Refs. 18 , 44 , 45 ; Table 1Citation ), the expression of S phase-promoting genes would not be expected to be attenuated by IR directly. However, UV light and actinomycin D are potent inhibitors of RNA synthesis (18 , 46) and would be expected to interfere with the unfolding of the genetic program required for the G1-to-S-phase transition. In support of this argument we found that IR caused no measurable inhibition of cyclin E expression even after high doses (Fig. 5)Citation . However, exposure of the cells to high doses of either UV light or actinomycin D significantly reduced cyclin E levels. Thus, we propose that the p53- and p21WAF1-independent block of S-phase entry after high doses of UV light and actinomycin D is caused by severe inhibition of general transcription leading to the inability of the cells to express cyclin E and other S phase-promoting proteins (Fig. 6)Citation .



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Fig. 6. Model for G1 arrest after moderate or high doses of IR, UV light, or actinomycin D. After exposure to p53-inducing agents that only partially 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 UV light or actinomycin D, at which transcription is severely suppressed, cells arrest in G1 in a p53-independent manner. We propose that this G1 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 transition.

 
In contrast to the inhibition of cyclin E expression after exposure to high doses of UV light and actinomycin D, moderate doses of these agents caused a marked increase in cyclin E expression especially in the E6-expressing cells (Fig. 5)Citation . This induced expression of cyclin E may explain the "mitogenic" effect observed in our cell cycle studies in which the entry into S phase was greatly increased by these agents in the RKO-E6 cells (Figs. 3Citation and 4)Citation . The mitogenic signal induced by these agents was apparently masked in the p53 wild-type cells, perhaps by high levels of p21WAF1 expression at these doses (Fig. 1)Citation . We propose that, in addition to orchestrating a G1 arrest after exposure of cells to DNA-damaging agents, p53 may also play a role in reducing the proliferative signal induced by UV light and actinomycin D. This suppression may be the result of reduced E2F-1 activity by p21WAF1-mediated inhibition of phosphorylation of the Rb protein (Fig. 6)Citation .

What is the mechanism for the induction of cyclin E expression in the RKO-E6 cells after exposure to moderate doses of UV light and actinomycin D? Because transcription of the cyclin E gene is induced by the transcription factor E2F-1 (4 , 5) , it is possible that these agents may stimulate the release of E2F-1 by phosphorylation of the Rb protein. Indeed, exposure of cells to IR (23) , UV light (47) , or actinomycin D (48) leads to dephosphorylation of Rb. However, we did not in our study observe any induction of cyclin E by IR despite the potential up-regulation of E2F-1 expression (31) . Furthermore, IR, UV light, and actinomycin D have been shown to cause a decrease in the level of Rb in exposed cells (49) . Another possibility for the induction of cyclin E in the RKO-E6 cells at moderate doses of UV light or actinomycin 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 mechanism. Additional studies are required to elucidate the potential 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 induction.

In summary, this study points out that the type and the dose of different DNA-damaging agents will differentially affect 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 caused accumulation of p53 but not p21WAF1, presumably because of inhibition of general transcription. Despite the lack of p21WAF1 expression at high doses of UV light or actinomycin D, exposed cells were inhibited from entering S phase in a p53- and p21WAF1-independent manner. We propose that this arrest is caused by a general blockage of RNA polymerase II, which cripples the unfolding of the genetic program required for entering S phase.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cell lines, Cell Culture, and Irradiation.
In this study, we used two 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 by Dr. Albert Fornace, Jr. (National Cancer Institute, NIH, Bethesda, MD, via Dr. Ted Lawrence, University of Michigan). The parental cell line, RKO, harbors an endogenous wild-type p53 (52) . The RKO-E6 cell line was 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 with the cellular protein E6AP to target the p53 protein for proteosome-mediated degradation by ubiqutinating the p53 protein (24 , 53) .

Cells were grown in monolayers in MEM (Life Technologies, Inc.) supplemented with 10% fetal bovine serum, 1x vitamins, 1x amino acids, 1x antibiotics. Subconfluent cells, seeded 2 days before the experiments, were irradiated on ice with IR (cobalt source with a dose rate of about 2 Gy/min), or were irradiated at room temperature with a germicidal UV light (254 nm). The fluence of the UV light source (Philips) was measured before each experiment with a UVX radiometer (UVP, Inc.). Actinomycin D was dissolved in DMSO at 1 mM concentrations and diluted in media before the experiments.

Western Blot.
Western blot was performed as described previously (17) using 12% SDS-PAGE for p53 Westerns and 15% SDS-PAGE for p21 Westerns. Detection of p53 and p21WAF1 proteins were performed using different anti-p53 (Ab-2), anti-p21WAF1, and anti-cyclin E (Oncogene Sciences), horseradish-peroxidase-conjugated secondary antibodies, enhanced chemiluminescence (SuperSignal CL-HRP Substrate System, Pierce), and X-ray film. To assess whether equal amounts of protein were loaded in each lane, the nylon membranes were stained with Coomassie blue after the completion of the film exposure.

Measurements of Nascent RNA Synthesis.
Cells were seeded and incubated in 185 Bq/ml [14C]thymidine for 2 days before the experiments. Cells were then irradiated or treated with actinomycin D. After incubation for 5.5 h, nascent RNA was pulse-labeled for 30 min in 0.5 ml of medium containing 7.4 x 105 Bq (20 µCi) of [3H]uridine. After rinsing three times with ice-cold PBS, the cells were scraped off the plates with a cell 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 scintillation counter.

Total nascent RNA synthesis was assessed by counting the activity of TCA insoluble material that did not bind to the poly(dT) magnetic beads. Equal volumes of sample and 10% ice-cold TCA were mixed and put on ice for 30 min; the TCA insoluble material was then collected on filters (GF/A, Whatman). The precipitates on the filters were washed with 5x 1 ml of 5% TCA, 5x 1 ml distilled water, and 2x 1 ml of 95% ethanol, and then dried for 10 min under a heating lamp. The dried filters were then counted in a scintillation counter. Relative total RNA synthesis and poly(A)RNA synthesis was then determined by calculating the ratio of 3H:14C for each sample and comparing it with the ratio in an untreated control sample.

BrdUrd Incubation and Cell Fixation.
Cells were pulse-labeled with 30 mM BrdUrd (Sigma) at 37°C for 15 min. The plates were then rinsed with PBS followed by the collection of the cells by scraping them with a rubber policeman in 2 ml of PBS. After centrifugation, the cells were fixed by adding ice-cold 70% ethanol dropwise under vortexing.

Denaturation, FITC-PI Staining, and Flow Cytometry.
The procedure was performed essentially as described previously (54) . Cells, which had been fixed for at least 30 min, were centrifuged at 1100 rpm (224 x g) for 7 min, washed with PBS, and spun as before. The cells were then suspended in 1 ml of a solution of 10 mM sodium acetate (pH 5.2), 10 mg/ml RNase A, and 10 mM Tris-HCl (pH 7.4) that had previously been boiled for 15 min and then cooled. The samples were incubated at 37°C for 30 min. Next, 1 ml of PBS was added and cells were spun as before, resuspended in 1 ml of 0.1 M HCl and 0.7% Triton X-100 (Sigma), and incubated on ice for 10 min. PBS (1 ml) was then added and cells were spun as before. The pellets were resuspended in 1 ml of sterile distilled deionized water, incubated at 97°C for 15 min, and then cooled on ice for 15 min, after which 1 ml of 0.5% Tween 20 (Bio-Rad) in PBS was added. Cells were again pelleted and suspended in 100 µl of HBT and transferred to microfuge tubes. One hundred µl of 1:20 dilution of antihuman BrdUrd (PharMingen) in HBT (12.5 µg/ml) was added, and cells were incubated at room temperature for 30 min. Then 1 ml of HBT was added and cells were spun at 3200 rpm for 2 min. The pellets were resuspended in 150 ml of 1:15 dilution of antimouse IgG FITC conjugate (Sigma Immuno Chemicals) and incubated for 30 min at room temperature. Then 1 ml of HBT was added, and cells were spun at 830 x g for 2 min. Tubes were wrapped in aluminum foil, and cells were finally suspended in 0.5 ml of 18 µg/ml PI and 40 µg/ml RNase A and incubated at 4°C for at least 0.5 h. The cell samples were then analyzed for BrdUrd content (FITC) and DNA content (PI) using flow cytometry (Coulter Elite ESP Cell Sorter).


    Acknowledgments
 
We thank Al Fornace and Ted Lawrence for their kind gifts of the RKO cell lines used in this study. We thank the Flow Cytometry Core at the University of Michigan for their excellent technical assistance.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by a grant from the University of Michigan Comprehensive Cancer Center’s Institutional Grant from the American Cancer Society. Back

2 To whom requests for reprints should be addressed, at Department of Radiation Oncology, Division of Cancer Biology, University of Michigan Comprehensive Cancer Center, 4306 CCGC, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0582. E-mail: ljungman{at}umich.edu Back

3 The abbreviations used are: Cdk, cyclin-dependent kinase; Rb, retinoblastoma; IR, ionizing radiation; HBT, 0.5% Tween 20 and 5% fetal bovine serum in PBS; PI, propidium iodide; TCA, trichloroacetic acid. Back

Received for publication 5/27/98. Revision received 1/ 4/99. Accepted for publication 1/ 6/99.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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