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Differential Responses of Proliferating versus Quiescent Cells to Adriamycin
Experimental Cell Research 250, 131–141 (1999) Article ID excr.1999.4551, available online at http://www.idealibrary.com on
Differential Responses of Proliferating versus Quiescent Cells to Adriamycin Wai Yi Siu, Talha Arooz, and Randy Y. C. Poon 1 Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong
The relative sensitivity of proliferating and quiescent cells to DNA-damaging agents is a key factor for cancer chemotherapy. Here we undertook a reevaluation of the way that proliferating and quiescent cells differ in their responses and fate to adriamycin-induced damage. Distinct types of assays that measure membrane integrity, metabolic activity, cell size, DNA content, and the ability to proliferate were used to compare growing and quiescent Swiss3T3 fibroblasts after adriamycin treatment. We found that immediately after adriamycin treatment of growing cells, p53 and p21 Cip1/Waf1 were induced but the cells remained viable. In contrast, less p53 and p21 Cip1/Waf1 were induced in quiescent cells after adriamycin treatment, but the cells were more prone to immediate cell death, possibly involving apoptosis. Adriamycin induced a G2/M cell cycle arrest in growing cells and a concomitant increase in cell size. In contrast, adriamycin induced an increase in sub-G1 DNA content in quiescent cells and a decrease in cell size. In contrast to the short-term responses, adriamycin-treated quiescent cells have a better long-term survival and proliferation potential than adriamycin-treated growing cells in colony formation assays. These data suggest that proliferating and resting cells are remarkably different in their short-term and long-term responses to adriamycin. © 1999 Academic Press Key Words: apoptosis; cell cycle; cell death; DNA damage; quiescence.
INTRODUCTION
Proper control of the eukaryotic cell cycle requires several feedback controls, ensuring that each stage of the cell cycle is completed before the next stage is initiated [1]. Deregulation of this kind of checkpoint control may allow cell cycle progression to become insensitive to external signals and DNA damage, resulting in increased genome instability [2]. The eukaryotic cell cycle is driven by the cyclin1 To whom correspondence and reprint requests should be addressed. Fax: [852]-2358-1552. E-mail: [email protected].
dependent kinase (CDK) 2 family. The kinase activity of CDK is tightly regulated by an intricate system of phosphorylation and protein–protein interactions [3]. By definition, the activation of CDKs is dependent on the association with a cyclin subunit. CDKs are also regulated by phosphorylation: the activity of the cyclinCDK holoenzyme is increased by phosphorylation of Thr161 and inhibited by phosphorylation of Thr14/ Tyr15. Thr161 can be phosphorylated by the CDKactivating kinase (CAK) [4] and dephosphorylated by the CDK-interacting phosphatase KAP [5]. Thr14/ Tyr15 can be phosphorylated by the Wee1 and Myt1 protein kinases [6, 7] and dephosphorylated by the members of the Cdc25 protein phosphatase family [7]. The activity of CDKs is negatively regulated by binding to CDK inhibitors, which include the p21 family (p21 Cip1/Waf1, p27 Kip1, and p57 Kip2) and the p16 INK4A family (p16 INK4A, p15 INK4B, p18 INK4C, and p19 INK4D) [8]. The activities of some CDKs are also regulated by folding factors [9]. Following DNA damage, two strategies are generally adopted by cells to ensure that mutations are not passed on to the daughter cells. On the one hand, the cell cycle can be arrested transiently to allow time for DNA repair or be permanently arrested and enter a senescent state [10]; on the other hand, the cells with damaged DNA can be eliminated by apoptosis [11] . Both cell cycle arrest and apoptosis after DNA damage are dependent on p53, whose level and activity are increased following DNA damage [12]. Once activated, p53 then induces the expression of p21 Cip1/Waf1, which in turn may be responsible for the cell cycle arrest. In normal human fibroblasts, p21 Cip1/Waf1 is responsible for the inhibition of cyclin A/E–Cdk2 and partly for cyclin D–Cdk4 after DNA damage, but clearly is not responsible for the inhibition of cyclin A/B–Cdc2 [13, 14]. The mitotic cyclin A/B–Cdc2 complexes, in contrast, are inhibited by Thr14/Tyr15 phosphorylation after DNA damage [15]. Many cells reversibly exit the cell cycle and enter a quiescent state (G0) when they are starved of growth 2 Abbreviations used: CDK, cyclin-dependent kinase; UV, ultraviolet light.
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factors or under contact inhibition conditions. While many of the anticancer drugs are reported to be more active against proliferating cells than against quiescent cells [16], there are still many conflicting reports on whether there are any differences between quiescent and proliferating cells after DNA damage in terms of cytotoxicity and recovery. The question is important in cancer therapy because many common human carcinomas (e.g., colon carcinoma and non-small-cell lung carcinomas) are refractory to chemotherapy, and it is believed that one key factor may be because many solid tumors are likely to contain a high proportion of quiescent cells [17]. Here we set out to investigate the responses of growing and quiescent cells upon treatment with adriamycin. Adriamycin (doxorubicin) is a potent anthracycline that is one of the most widely used clinical cancer chemotherapeutic drugs. One of the mechanisms by which adriamycin causes DNA damage is by targeting topoisomerase II; intercalation may position the adriamycin for complex formation with topoisomerase II [18, 19]. Other possible actions of adriamycin including oxidative effects have also been suggested [20]. Here several distinct assays were used to analyze the cell cycle distribution, p53 and p21 Cip1/Waf1 expression, shortterm cytotoxicity, and long-term recovery when growing or resting Swiss3T3 fibroblasts were treated with adriamycin. We found that quiescent cells were more sensitive than growing cells immediately after adriamycin treatment. However, the long-term proliferation potential of quiescent cells is better than that of growing cells after adriamycin treatment. MATERIALS AND METHODS Cell culture. Mouse Swiss3T3 fibroblasts were obtained from American Type Culture Collection (Rockville, MD). Mouse NIH3T3 fibroblasts were gifts from Tony Hunter. PG13 (a gift from Liang Cao) is a retrovirus packaging cell line derived from mouse TK-NIH/ 3T3 fibroblasts (based on the Gibbon ape leukemia virus) [21]. HeLa cells (human cervical carcinoma cells) were gifts from Hermann Bujard [22]. Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) calf serum (for HeLa cells) or 10% (v/v) fetal bovine serum (for other cells) in a humidified incubator at 37°C, in 10% CO 2. Quiescent cells were obtained by incubating the cells in serum-free DMEM for 48 h. Cell-free extracts were prepared as described [23]. The protein concentration of cell lysates was measured with the bicinchoninic acid protein assay system (Pierce, Rockford, IL) using bovine serum albumin as a standard. DNA damage was induced by addition of the indicated dose of adriamycin (also called doxorubicin) (Calbiochem, La Jolla, CA) to the medium for the indicated time or by irradiation with 15 J/m 2 of UV-C as described [13]. Trypan blue cell counting. Cells were trypsinized, collected by centrifugation, and resuspended in medium. Cell suspensions (0.5 ml) were mixed with 0.1 ml of 0.4% trypan blue stain (Sigma). Total cells and viable cells (cells that excluded blue dye) were counted with a hemocytometer under a light microscope. MTT assay. The indicated number of cells was plated onto 96well plates with 200 ml of medium. After treatment, 20 ml of 10 mg/ml
MTT was added to each well. After incubation at 37°C for 4 h, the supernatant was removed and 200 ml of a solution containing 10% SDS and 0.04 N HCl was added to dissolve the water-insoluble formazan salt. After incubation at 37°C for 60 min, the OD 650 nm– OD 570 nm was measured with an ELISA reader (Dynatech Laboratories). The cytotoxicity index was expressed as (1 2 treatment OD/ control OD) 3 100. Colony formation assay. The indicated number of growing or quiescent cells was seeded onto tissue culture plates. The cells were treated with the indicated dose of adriamycin for 3 h. The cells were washed with phosphate-buffered saline and incubated with fresh medium containing serum. The cells were allowed to grow for another 2 weeks. Colonies were fixed with methanol:acetic acid (2:1, v/v) and visualized by staining with 2% (w/v) crystal violet. TUNEL (TdT-mediated dUTP nick end labeling) assay. Cells were grown on poly-L-lysine-treated coverslips in six-well tissue culture plates. TUNEL assays were performed using the ApopTag fluorescein kit (Oncor) according to the manufacturer’s instructions. The nuclei were counterstained with Hoechst 33258. Flow cytometry analysis. Cells were trypsinized and washed with phosphate-buffered saline. The cells were then fixed in ice-cold 70% ethanol and stained with a solution containing 40 mg/ml propidium iodide and 40 mg/ml RNaseA at 37°C for 30 min. Cell cycle distribution (for 10,000 cells) was analyzed using a FACSort machine (Becton–Dickinson). Antibodies and immunological methods. Monoclonal antibody 421 against p53 and rat monoclonal antibodies YL1/2 against mammalian tubulin were gifts from Dr. Julian Gannon and Dr. Tim Hunt (ICRF, South Mimms, UK). Anti-p21 Cip1/Waf1 antibody was a purified polyclonal antibody raised against the C-terminal peptide of p21 Cip1/Waf1 (Santa Cruz Biotechnology, sc-397). Anti-p27 Kip1 polyclonal antibody was as described [23]. Immunoprecipitation and immunoblotting were performed as described [13].
RESULTS
Assessment of the metabolic activities in growing and quiescent cells after adriamycin treatment. The mouse Swiss3T3 fibroblasts, of which the reversible exit of the cell cycle into G0 when starved of serum is very well defined, was used as a model of proliferating and quiescent cells for this study. To have an initial idea of the cytotoxicity of adriamycin on Swiss3T3 cells, we used MTT assays (also called succinate dehydrogenase inhibition tests) to measure the metabolic activity of the cells. The tetrazolium salt MTT is reduced to a colored, waterinsoluble formazan salt only by metabolically active (hence viable) cells [24, 25]. Figure 1A shows the cytotoxicity index of cells seeded at different densities and treated with different doses of adriamycin. The cytotoxicity index was normalized against cells seeded at the same density but not treated with adriamycin. Based on this result, we used 0.2–0.4 mg/ml of adriamycin and 120,000 cells per well for other experiments in this work to be within approximately linear range of the assay. The MTT assay has not been used typically to follow changes in viability over time. We modified the assay by treating the same number of cells with buffer control or various doses of adriamycin and harvested the cells at different time points. For a particular time point, the cytotoxicity index after adriamycin treat-
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FIG. 1. Evaluation of cytotoxicity by MTT assay. (A) Adriamycin-induced cytotoxicity of Swiss3T3 cells at different densities The indicated number of Swiss3T3 cells was seeded onto 96-well plates, and the cells were then treated with either buffer or different doses of adriamycin for 36 h. The cytotoxicity index was then measured by MTT assay as described under Materials and Methods. (B) Time course of adriamycin-induced cytotoxicity in Swiss3T3 cells. Swiss3T3 cells were seeded onto 96-well plates (12,000 cells per well), and the cells were then treated with either buffer or the indicated dosage of adriamycin. Cells were processed for MTT assay at different time points. The cytotoxicity index for each data point is expressed as the percentage of the untreated control for that time point. (C) Time course of adriamycin-induced cytotoxicity in HeLa cells. This experiment was exactly as in B, except HeLa cells (12,000 per well) were used instead of Swiss3T3 cells.
ment was normalized against that of the control cells. A progressive increase in cytotoxicity was seen with increasing dosage of adriamycin and time (Fig. 1B). A similar pattern of cytotoxicity was seen with other cell lines after adriamycin treatment; the MTT assay for human cervical carcinoma HeLa cells is shown for comparison in Fig. 1C. To study the effect of adriamycin on
quiescent (G0) cells, Swiss3T3 cells were grown in medium in the absence of serum for 48 h. The G0 cells were then treated with adriamycin and MTT assays were performed as before (Fig. 2). The cytotoxicity index in this case was normalized against that of the untreated G0 cells for each time point. It is important to note the meaning of these MTT
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FIG. 2. Evaluation of cytotoxicity of quiescent Swiss3T3 cells by MTT assay. Swiss3T3 cells were incubated in medium without serum for 48 h. Cells were then seeded onto 96-well plates (12,000 cells per well) and treated with either buffer or the indicated dosage of adriamycin. Cells were processed for MTT assay at different time points as described under Materials and Methods. The cytotoxicity index for each data point is expressed as the percentage of the untreated control for that time point.
measurements. A change in MTT cleavage activity represents a change in the cell number as well as in their viability. Hence, for proliferating cells the decrease in MTT cleavage activity after adriamycin treatment can be due to cell cycle arrest as well as a decrease in viability. For G0 cells, however, the decrease in MTT cleavage activity in adriamycin-treated cells in comparison to untreated cells was due to the decrease in viability alone because the G0 cells were already arrested. Comparison of viability in growing and quiescent cells after adriamycin treatment by vital stain. To have a more meaningful comparison between growing and quiescent cells, we directly measured the number of viable cells versus dead cells using the classical vital stain trypan blue. The reactivity of trypan blue is based on the fact that the chromopore is negatively charged and does not interact with the cell unless the membrane is damaged; hence, cells that exclude the dye are regarded as viable [26, 27].
Growing and quiescent Swiss3T3 cells were treated with different doses of adriamycin and harvested at different time points for trypan blue staining. Figure 3A shows that treatment of growing cells with adriamycin completely abolished proliferation. However, viability remained relatively high even with the highest dosage used (Fig. 3B), indicating that the cells were arrested but remained viable for up to 48 h. In comparison, Figs. 3C and 3D show that serum-starved Swiss3T3 cells did not proliferate. The viability of quiescent cells decreased slightly during the experiment even in the absence of adriamycin treatment. In the presence of adriamycin, however, viability decreased dramatically in serum-starved cells. In a variation of the above experiment, when serumstarved cells were stimulated by addition of serum, the cell number increased as the cells reentered the cell cycle (Fig. 3E). This indicates that most of the quiescent cells retained the ability to proliferate. In contrast, the cell number did not increase when adriamycin was added together with serum. Similar to the serum-starved cells shown in Fig. 3D, the viability of serum-stimulated cells was significantly reduced after addition of adriamycin (Fig. 3F). Taken together, these data indicate that immediately following adriamycin treatment (48 h represented one to two cell cycle times), cell proliferation is inhibited, and serumstarved cells are more sensitive (in terms of viability) to adriamycin than growing cells. Cell cycle analysis of growing and quiescent cells after adriamycin treatment. We next investigated the cell cycle distribution of growing and serum-starved cells after adriamycin treatment. Growing Swiss3T3 cells were either untreated or treated with adriamycin and harvested 24 and 48 h later for flow cytometry analysis. Figure 4 shows that adriamycin caused mainly a G2/M cell cycle arrest in growing cells. The majority of the cells were at G2/M at 48 h after addition of adriamycin. For serum-starved cells, nearly all the cells contained only G1 DNA content. As expected, the serumstarved cells remained in G0/G1 after treatment with adriamycin. However, a significant population of cells exhibited a sub-G1 DNA content in the presence of adriamycin (Fig. 4A). The sub-G1 DNA population was more marked at 48 h than at 24 h after adriamycin treatment. Although cells containing sub-G1 DNA content were also detected in untreated serum-starved
FIG. 3. Measurement of the viability of Swiss3T3 cells after adriamycin treatment by trypan blue counting. Semiconfluent growing Swiss3T3 cells (A and B), serum-starved Swiss3T3 cells (C and D), and serum-starved Swiss3T3 cells that were released into the cell cycle by addition of serum to the medium at t 5 0 (E and F) were treated with the indicated doses of adriamycin (E, untreated; l, 0.2 mg/ml; h, 0.4 mg/ml; ‚, 0.8 mg/ml). At different time points, cells were harvested for trypan blue counting as described under Materials and Methods. The cells that excluded trypan blue staining (viable cells) (A, C, and E) and the percentage of viable cells to trypan blue-stained dead cells (B, D, and F) are shown.
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cells at 48 h, the proportion of these cells was far more pronounced in the adriamycin-treated samples. Since the presence of sub-G1 DNA content is one of the characteristics of apoptotic cells, these data suggest that a large proportion of quiescent cells may undergo apoptosis after adriamycin treatment. In contrast, there is no evidence of significant apoptosis in growing cells following addition of adriamycin. These speculations are consistent with the trypan blue assay described above, which showed a higher cytotoxicity in quiescent cells compared with growing cells after adriamycin treatment. Significant changes in the size of the cells were observed after adriamycin treatment. We observed by light-scattering data from flow cytometry (Fig. 4B) that growing cells, but not quiescent cells, underwent a considerable increase in size after treatment with adriamycin. In contrast, an increased proportion of smaller cells was detected when quiescent cells were treated with adriamycin (Fig. 4C), suggesting that the cells may be entering apoptosis. To see in more detail whether the Swiss3T3 cells were undergoing apoptosis after addition of adriamycin, TUNEL assays were performed on growing and G0 cells that are untreated or treated with adriamycin (Fig. 4D). We found that there were relatively little apoptotic cells in a growing or quiescent population or when growing cells were treated with adriamycin. In contrast, the proportion of apoptotic cells increased when quiescent cells were treated with adriamycin as indicated by the TUNEL assay. Induction of p53 and p21 Cip1/Waf1 in adriamycintreated cells. Given that p53 and p21 Cip1/Waf1 are involved in cell cycle arrest and apoptosis following DNA damage, we next investigated the levels of p53 and p21 Cip1/Waf1 in growing and resting cells after adriamycin treatment (Fig. 5). When growing Swiss3T3 cells were treated with adriamycin, induction of p53 and p21 Cip1/Waf1 was detected by immunoblotting with specific antibodies (lanes 4 and 5). Treatment with another DNA-damaging agent, UV irradiation, also induced p53 and p21 Cip1/Waf1 (lanes 6 and 7). In contrast, p53 and p21 Cip1/Waf1 remained at basal levels in untreated cells throughout the experiment (lanes 1–3). In contrast to proliferating cells, relatively little induction of p53 and p21 Cip1/Waf1 was seen when serumstarved cells were treated with adriamycin or UV (Fig. 5). Nevertheless, the level of p53 was clearly higher than that in untreated serum-starved cells. Immunoblotting with anti-p27 Kip1 antibody showed that the protein level of p27 Kip1 was slightly higher in quiescent cells, as described previously [23]. The same samples were immunoblotted with a monoclonal antibody against tubulin to indicate similar loadings (we believe the decrease in tubulin level in G0 cells after treatment
with adriamycin or UV was likely due to the increase in cell death described above). At 24 h after addition of adriamycin, the total level of p53 was actually less than that at 6 h. One possibility is that some cells had repaired their DNA and the level of p53 returned to basal level; another possibility is that some cells may be dying by 24 h (but still have not manifested as cell membrane damage for trypan blue assay detection).
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FIG. 4. Analysis of adriamycin-treated Swiss3T3 cells by flow cytometry. (A) Cell cycle analysis by flow cytometry. Growing or quiescent Swiss3T3 cells were either untreated or treated with 0.4 mg/ml of adriamycin as indicated. The cells were harvested for flow cytometry analysis at 24 and 48 h as described under Materials and Methods. The positions of G1 and G2 DNA contents are indicated. The populations containing sub-G1 DNA content are indicated by arrows. (B) Cell size of proliferating cells. Proliferating Swiss3T3 cells were treated with buffer (left-hand panel) or 0.2 mg/ml of adriamycin (right-hand panel) and incubated for 24 h. The cell size and fluorescence were analyzed by flow cytometry. (C) Cell size of quiescent cells. Quiescent Swiss3T3 cells were treated with buffer (left-hand panel) or 0.2 mg/ml of adriamycin (right-hand panel) and incubated for 24 h. The cell size and fluorescence were analyzed by flow cytometry. (D) TUNEL assays. Growing or quiescent Swiss3T3 cells were either untreated or treated with 0.4 mg/ml of adriamycin as indicated. The cells were harvested for TUNEL assay at 24 h as described under Materials and Methods. The same magnification and exposure time were used for all slides. Apoptotic cells were labeled with fluorescein (green) and the nuclei were counterstained with Hoechst 33258 (blue).
Attempts to investigate the relative sensitivity of 3T3 cells that lack p53/p21 Cip1/Waf1 response after DNA damage were hampered for the following reasons. It is well known that the p53 genes in mouse fibroblasts are readily mutated, especially in later passages. We found that another 3T3 cell line used in our laboratory, NIH3T3, also exhibited p53 and p21 Cip1/Waf1 induction following DNA damage (Fig. 6A). On the other hand, PG13 cells, which were originally derived from NIH3T3, did not show p53 induction after treatment with adriamycin under similar conditions (Fig. 6B). To obtain quiescent cells, PG13 cells were incubated in medium without serum for 48 h. However, unlike Swiss3T3 cells (or NIH3T3 cells; data not shown), PG13 cells did not appear to enter quiescence because the cell number continued to increase as shown by trypan blue assay (Fig. 6C). Addition of adriamycin to serum-starved PG13 cells halted further cell proliferation. Consistent with the fact that PG13 cells cannot be completely arrested in G0 by serum starvation, the viability of the cells did not decrease as much as that in
quiescent Swiss3T3 cells after adriamycin treatment. We have also tested p53 2/2 mouse embryonic fibroblasts, but they also cannot be arrested in G0 by serum starvation (unpublished data). Differential recovery of growing and resting cells after adriamycin treatment. Given that quiescent cells are more sensitive than growing cells to adriamycin in the short term, it is hence essential to investigate the longer term survival of these cells. Growing or serumstarved Swiss3T3 cells were incubated with adriamycin, and the cells were then washed and allowed to grow in medium containing serum. The cells that were viable and were able to continue to proliferate formed visible colonies after about 2 weeks. Despite the fact that the shortterm viability of quiescent cells was lower than that of growing cells, these colony formation assays indicated the opposite relationship for the long-term proliferation potential. Over a wide range of adriamycin dosages tested, a higher proportion of quiescent cells than growing cells were able to form colonies (Fig. 7).
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FIG. 5. Expression of p53 and p21 Cip1/Waf1 following adriamycin treatment. Growing (lanes 1–7) or quiescent (lanes 8 –14) Swiss3T3 cells were untreated (lanes 1–3 and 8 –10) or treated with 0.4 mg/ml of adriamycin (lanes 4 and 5 and 11 and 12) or 15 J/m 2 of UV radiation (lanes 6 and 7 and 13 and 14). Cells were harvested at 0 h (lanes 1 and 8), 6 h (lanes 2, 4, 6, 9, 11, and 13), and 24 h (lanes 3, 5, 7, 10, 12, and 14) after treatment. Cell-free extracts were prepared, and 10 mg of each was applied onto 17.5% SDS–PAGE followed by immunoblotting with antibodies against p53, p21 Cip1/Waf1, p27 Kip1, and tubulin as indicated.
DISCUSSION
How proliferating cells and quiescent cells differ in their response to anticancer drugs like adriamycin is an important question in cancer therapy. Most of the normal cells in the body are in quiescent or differentiated states, but most cancer cells are actively proliferating. The goal of chemotherapy is to remove the cancer cells while sparing the normal cells. Here we evaluate the cytotoxicity of adriamycin on growing and quiescent cells by a number of methods that measure cellular membrane integrity, metabolic function, cell size, DNA content, and colony formation. Methods that measured viability over a shorter time scale (up to 48 h) show that quiescent cells are more sensitive to adriamycin than growing cells, but methods that measured survival/reproduction over the longer term show that growing cells are more sensitive to adriamycin than quiescent cells. Given that there are many assays that indicate viability, it is vital to consider exactly the meaning of the assays. MTT assays provide an indication of the metabolic activity of the cell population. A change in MTT cleavage activity represents a change in the cell number as well as in their viability. Hence, for proliferating cells the decrease in MTT cleavage activity after adriamycin treatment can be due to cell cycle arrest as well as a decrease in viability. For G0 cells, in contrast, the decrease in MTT cleavage activity in adriamycintreated cells in comparison to untreated cells was due
to the decrease in viability alone because G0 cells were already arrested. Hence, MTT assays can be used to compare growing or G0 cells in the presence or absence of adriamycin treatment, but are not useful for comparing between growing and G0 cells. The trypan blue exclusion assay is a more direct approach to measure the number of viable and dead cells. It should be noted that floating cells (mostly nonviable cells) were excluded in the trypan blue assay described here; hence, the viability is likely to be a slight overestimation. Furthermore, membrane damage may not be an immediate event in dead cells; hence, this would also lead to an overestimation of viability [27]. In the experiments described here, floating cells represent only a small proportion of the total cell population. We believe the high cytotoxicity observed in G0 cells or stimulated G0 cells after adriamycin treatment (Fig. 3) would be slightly accentuated if floating cells were included in the assay, because there were more floating cells in adriamycin-treated G0 cells than in any other samples (data not shown). Colony formation assay is probably the ultimate viability assay for many purposes. However, as can be seen here, colony formation assay measures the final survival and reproductive capacity of the cells, but the relative rate of cytotoxicity cannot be measured by colony formation assay. Moreover, it is possible that after adriamycin treatment some cells entered a prolong state of arrest, but nevertheless remained viable; these cells will not be scored in colony formation assays. It should also be noted that for colony formation assay, the colonies formed from growing and quiescent cells were expressed as percentages of the respectively untreated cells. One potential problem is that the plating efficiencies of growing and quiescent cells are very different. The plating efficiency is the percentage of the number of colonies formed to the original number of cell plates in the untreated samples. We found that the typical plating efficiencies of growing and G0 cells were about 75 and 25%, respectively. This is consistent with the fact that the viability of serum-starved cells decreased even in the absence of adriamycin (Fig. 3), and some cells probably lost the ability to reenter the cell cycle after serum starvation. A more precise conclusion is that of the cells that would form colonies: higher percentages actually did so from G0 cells than from proliferating cells after treatment with adriamycin. Previous studies that show that quiescent cells are less sensitive to adriamycin than growing cells also relied on colony formation assays [17, 28]. It should be noted that for other types of DNA damages like X-ray, G0 cells had been reported to be more sensitive than growing cells [29]. Flow cytometry analysis shown here indicates that a substantial population of G0 cells contained sub-G1 DNA content when treated with adriamycin (Fig. 4), suggesting that apoptosis may be involved. Both the
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FIG. 6. Responses to adriamycin in different mouse fibroblasts. (A) p53 expression in Swiss3T3 and NIH3T3. Growing NIH3T3 cells (lanes 1–3) or Swiss3T3 cells (lanes 4 – 6) were either untreated (lanes 1, 2, 4, and 5) or treated with 0.4 mg/ml of adriamycin (lanes 3 and 6). Cells were harvested at t 5 0 (lanes 1 and 4) and t 5 6 (lanes 2, 3, 5, and 6). Cell-free extracts were prepared, and 10 mg of each was applied onto 17.5% SDS–PAGE followed by immunoblotting with antibodies against p53 (top) and p21 Cip1/Waf1 (bottom). (B) p53 expression in PG13 cells. Growing PG13 cells (lanes 1 and 2) or Swiss3T3 cells (lanes 3 and 4) were either untreated (lanes 1 and 3) or treated with 0.4 mg/ml of adriamycin (lanes 2 and 4). Cells were harvested at 6 h after treatment. Cell-free extracts were prepared, and 10 mg of each was applied onto 17.5% SDS–PAGE followed by immunoblotting with antibodies against p53. (C) Serum-starved PG13 cells were treated with the indicated doses of adriamycin (E, untreated; h, 0.4 mg/ml). At different time points, cells were harvested for trypan blue counting as described under Materials and Methods. The cells that excluded trypan blue staining (viable cells) are shown in the left-hand panel and the percentage of viable cells to trypan blue-stained dead cells is shown in the right-hand panel.
TUNEL assays and the decrease in cell size as indicated by flow cytometry also suggest the increase in apoptosis in G0 cells after adriamycin treatment. In growing cells, no apoptosis was seen after adriamycin. At the same time, there was significant cell swelling in growing cells after adriamycin treatment, suggesting that many of the cells may be entering necrosis. Hence, it is possible that distinct mechanisms may be involved in adriamycin-induced cell death in growing and G0 cells. However, the increase in cell size after adriamycin treatment may also be due to “unbalanced growth,” when cell division is precluded but cells continue to grow in size [30]. The normal cell cycle-arresting point
of adriamycin is the G2 phase (see Fig. 4); the fact that G0 cells are not in G2 is likely to have a pronounced effect on the response to adriamycin. The tumor suppressor p53 clearly plays an essential role in the G1/S DNA damage checkpoint. Furthermore, p53 plays an important role in apoptosis following DNA damage, although not all apoptosis requires the function of p53. One question is whether cell death in G0 after DNA damage also involves p53. Only very modest increases in p53 levels were detected after G0 cells were treated with adriamycin (Fig. 5), but it is possible that G0 cells are more sensitive to slight increases in p53 than growing cells. To test this possibil-
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FIG. 7. Colony formation assay of Swiss3T3 cells following adriamycin treatment. Known numbers of growing or quiescent Swiss3T3 cells were plated and incubated with the indicated concentration of adriamycin for 3 h. The cells were washed and incubated in medium containing serum for another 2 weeks. The number of colonies formed was counted after staining with crystal violet. Survival of growing and quiescent cells is expressed as percentage of the respective untreated cells. This is representational of one of several experiments with different densities of cells plated.
ity, we need to be able to conditionally express p53 in growing and G0 cells, but expression of exogenous proteins in G0 cells is technically more difficult. The fact that G0 cells have a weaker p53/p21 Cip1/Waf1 response than growing cells (Fig. 5) may underlie the higher sensitivity of G0 cells to immediate killing than growing cells. It has been reported that in G0 cells UV irradiation induced p53 DNA-binding activity, but not its accumulation, whereas both events took place in G1/S- and S-phase cells [31]. It is also possible that growing cells mainly cause cell cycle arrest (at least for the short term), but induction of p53 in G0 cells may cause immediate cell death. We attempted to use PG13 cells, which do not have p53 induction after adriamycin treatment, to study the effect of p53 on cytotoxicity in G0 cells. However, PG13 cells, as with so many p53negative transformed cells, do not arrest in G0 upon serum starvation (Fig. 6). Moreover, it is likely that PG13 cells contain other mutations apart from that of p53. We have also tested p53 2/2 mouse embryonic fibroblasts, but they also cannot be arrested in G0 by serum starvation (unpublished data). Immediately after adriamycin treatment, why are quiescent cells more sensitive than growing cells? Further, why do quiescent cells appear to be less sensitive than growing cells in terms of reproductive potential? It is still not completely clear when the cells undergo cell cycle arrest and when they undergo apoptosis after
DNA damage. One possibility is that after DNA damage, actively growing cells predominantly enter cell cycle arrest (at G2 phase; see Fig. 4); the cells may then undergo cell death if the DNA damage is severe or not repairable. Furthermore, DNA damage-arrested cells may never recover to proliferate, but remain in a prolong arrest state [32]. Quiescent cells, on the other hand, have already exited the cell cycle, and the cells may immediately undergo cell death upon adriamycin treatment since the initial cell cycle arrest mechanism after DNA damage is bypassed. It is not immediately apparent why quiescent cells are also affected by adriamycin, since the main target of adriamycin is likely to be topoisomerase II and quiescent cells have little DNA synthesis activity. It is possible that there are some basal DNA synthesis or DNA repair activities that involve topoisomerase II in G0 cells. Another possibility is that apart from targeting topoisomerase II, adriamycin may have other effects such as oxidation to which G0 cells are sensitive. It would be interesting in the future to analyze the differential responses of proliferating and quiescent cells described here with other kinds of DNA-damaging agents. We are grateful to Hermann Bujard, Liang Cao, Julian Gannon, Tim Hunt, and Tony Hunter for comments and reagents. We thank members of the Poon lab for invaluable help and discussion. This work was supported in part by Research Grants Council Grant HKUST6188/97M and IDTC Grant AF/178/97 to R.Y.C.P..
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Differential Responses of Proliferating versus Quiescent Cells to Adriamycin Wai Yi Siu, Talha Arooz, and Randy Y. C. Poon 1 Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong
The relative sensitivity of proliferating and quiescent cells to DNA-damaging agents is a key factor for cancer chemotherapy. Here we undertook a reevaluation of the way that proliferating and quiescent cells differ in their responses and fate to adriamycin-induced damage. Distinct types of assays that measure membrane integrity, metabolic activity, cell size, DNA content, and the ability to proliferate were used to compare growing and quiescent Swiss3T3 fibroblasts after adriamycin treatment. We found that immediately after adriamycin treatment of growing cells, p53 and p21 Cip1/Waf1 were induced but the cells remained viable. In contrast, less p53 and p21 Cip1/Waf1 were induced in quiescent cells after adriamycin treatment, but the cells were more prone to immediate cell death, possibly involving apoptosis. Adriamycin induced a G2/M cell cycle arrest in growing cells and a concomitant increase in cell size. In contrast, adriamycin induced an increase in sub-G1 DNA content in quiescent cells and a decrease in cell size. In contrast to the short-term responses, adriamycin-treated quiescent cells have a better long-term survival and proliferation potential than adriamycin-treated growing cells in colony formation assays. These data suggest that proliferating and resting cells are remarkably different in their short-term and long-term responses to adriamycin. © 1999 Academic Press Key Words: apoptosis; cell cycle; cell death; DNA damage; quiescence.
INTRODUCTION
Proper control of the eukaryotic cell cycle requires several feedback controls, ensuring that each stage of the cell cycle is completed before the next stage is initiated [1]. Deregulation of this kind of checkpoint control may allow cell cycle progression to become insensitive to external signals and DNA damage, resulting in increased genome instability [2]. The eukaryotic cell cycle is driven by the cyclin1 To whom correspondence and reprint requests should be addressed. Fax: [852]-2358-1552. E-mail: [email protected].
dependent kinase (CDK) 2 family. The kinase activity of CDK is tightly regulated by an intricate system of phosphorylation and protein–protein interactions [3]. By definition, the activation of CDKs is dependent on the association with a cyclin subunit. CDKs are also regulated by phosphorylation: the activity of the cyclinCDK holoenzyme is increased by phosphorylation of Thr161 and inhibited by phosphorylation of Thr14/ Tyr15. Thr161 can be phosphorylated by the CDKactivating kinase (CAK) [4] and dephosphorylated by the CDK-interacting phosphatase KAP [5]. Thr14/ Tyr15 can be phosphorylated by the Wee1 and Myt1 protein kinases [6, 7] and dephosphorylated by the members of the Cdc25 protein phosphatase family [7]. The activity of CDKs is negatively regulated by binding to CDK inhibitors, which include the p21 family (p21 Cip1/Waf1, p27 Kip1, and p57 Kip2) and the p16 INK4A family (p16 INK4A, p15 INK4B, p18 INK4C, and p19 INK4D) [8]. The activities of some CDKs are also regulated by folding factors [9]. Following DNA damage, two strategies are generally adopted by cells to ensure that mutations are not passed on to the daughter cells. On the one hand, the cell cycle can be arrested transiently to allow time for DNA repair or be permanently arrested and enter a senescent state [10]; on the other hand, the cells with damaged DNA can be eliminated by apoptosis [11] . Both cell cycle arrest and apoptosis after DNA damage are dependent on p53, whose level and activity are increased following DNA damage [12]. Once activated, p53 then induces the expression of p21 Cip1/Waf1, which in turn may be responsible for the cell cycle arrest. In normal human fibroblasts, p21 Cip1/Waf1 is responsible for the inhibition of cyclin A/E–Cdk2 and partly for cyclin D–Cdk4 after DNA damage, but clearly is not responsible for the inhibition of cyclin A/B–Cdc2 [13, 14]. The mitotic cyclin A/B–Cdc2 complexes, in contrast, are inhibited by Thr14/Tyr15 phosphorylation after DNA damage [15]. Many cells reversibly exit the cell cycle and enter a quiescent state (G0) when they are starved of growth 2 Abbreviations used: CDK, cyclin-dependent kinase; UV, ultraviolet light.
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factors or under contact inhibition conditions. While many of the anticancer drugs are reported to be more active against proliferating cells than against quiescent cells [16], there are still many conflicting reports on whether there are any differences between quiescent and proliferating cells after DNA damage in terms of cytotoxicity and recovery. The question is important in cancer therapy because many common human carcinomas (e.g., colon carcinoma and non-small-cell lung carcinomas) are refractory to chemotherapy, and it is believed that one key factor may be because many solid tumors are likely to contain a high proportion of quiescent cells [17]. Here we set out to investigate the responses of growing and quiescent cells upon treatment with adriamycin. Adriamycin (doxorubicin) is a potent anthracycline that is one of the most widely used clinical cancer chemotherapeutic drugs. One of the mechanisms by which adriamycin causes DNA damage is by targeting topoisomerase II; intercalation may position the adriamycin for complex formation with topoisomerase II [18, 19]. Other possible actions of adriamycin including oxidative effects have also been suggested [20]. Here several distinct assays were used to analyze the cell cycle distribution, p53 and p21 Cip1/Waf1 expression, shortterm cytotoxicity, and long-term recovery when growing or resting Swiss3T3 fibroblasts were treated with adriamycin. We found that quiescent cells were more sensitive than growing cells immediately after adriamycin treatment. However, the long-term proliferation potential of quiescent cells is better than that of growing cells after adriamycin treatment. MATERIALS AND METHODS Cell culture. Mouse Swiss3T3 fibroblasts were obtained from American Type Culture Collection (Rockville, MD). Mouse NIH3T3 fibroblasts were gifts from Tony Hunter. PG13 (a gift from Liang Cao) is a retrovirus packaging cell line derived from mouse TK-NIH/ 3T3 fibroblasts (based on the Gibbon ape leukemia virus) [21]. HeLa cells (human cervical carcinoma cells) were gifts from Hermann Bujard [22]. Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) calf serum (for HeLa cells) or 10% (v/v) fetal bovine serum (for other cells) in a humidified incubator at 37°C, in 10% CO 2. Quiescent cells were obtained by incubating the cells in serum-free DMEM for 48 h. Cell-free extracts were prepared as described [23]. The protein concentration of cell lysates was measured with the bicinchoninic acid protein assay system (Pierce, Rockford, IL) using bovine serum albumin as a standard. DNA damage was induced by addition of the indicated dose of adriamycin (also called doxorubicin) (Calbiochem, La Jolla, CA) to the medium for the indicated time or by irradiation with 15 J/m 2 of UV-C as described [13]. Trypan blue cell counting. Cells were trypsinized, collected by centrifugation, and resuspended in medium. Cell suspensions (0.5 ml) were mixed with 0.1 ml of 0.4% trypan blue stain (Sigma). Total cells and viable cells (cells that excluded blue dye) were counted with a hemocytometer under a light microscope. MTT assay. The indicated number of cells was plated onto 96well plates with 200 ml of medium. After treatment, 20 ml of 10 mg/ml
MTT was added to each well. After incubation at 37°C for 4 h, the supernatant was removed and 200 ml of a solution containing 10% SDS and 0.04 N HCl was added to dissolve the water-insoluble formazan salt. After incubation at 37°C for 60 min, the OD 650 nm– OD 570 nm was measured with an ELISA reader (Dynatech Laboratories). The cytotoxicity index was expressed as (1 2 treatment OD/ control OD) 3 100. Colony formation assay. The indicated number of growing or quiescent cells was seeded onto tissue culture plates. The cells were treated with the indicated dose of adriamycin for 3 h. The cells were washed with phosphate-buffered saline and incubated with fresh medium containing serum. The cells were allowed to grow for another 2 weeks. Colonies were fixed with methanol:acetic acid (2:1, v/v) and visualized by staining with 2% (w/v) crystal violet. TUNEL (TdT-mediated dUTP nick end labeling) assay. Cells were grown on poly-L-lysine-treated coverslips in six-well tissue culture plates. TUNEL assays were performed using the ApopTag fluorescein kit (Oncor) according to the manufacturer’s instructions. The nuclei were counterstained with Hoechst 33258. Flow cytometry analysis. Cells were trypsinized and washed with phosphate-buffered saline. The cells were then fixed in ice-cold 70% ethanol and stained with a solution containing 40 mg/ml propidium iodide and 40 mg/ml RNaseA at 37°C for 30 min. Cell cycle distribution (for 10,000 cells) was analyzed using a FACSort machine (Becton–Dickinson). Antibodies and immunological methods. Monoclonal antibody 421 against p53 and rat monoclonal antibodies YL1/2 against mammalian tubulin were gifts from Dr. Julian Gannon and Dr. Tim Hunt (ICRF, South Mimms, UK). Anti-p21 Cip1/Waf1 antibody was a purified polyclonal antibody raised against the C-terminal peptide of p21 Cip1/Waf1 (Santa Cruz Biotechnology, sc-397). Anti-p27 Kip1 polyclonal antibody was as described [23]. Immunoprecipitation and immunoblotting were performed as described [13].
RESULTS
Assessment of the metabolic activities in growing and quiescent cells after adriamycin treatment. The mouse Swiss3T3 fibroblasts, of which the reversible exit of the cell cycle into G0 when starved of serum is very well defined, was used as a model of proliferating and quiescent cells for this study. To have an initial idea of the cytotoxicity of adriamycin on Swiss3T3 cells, we used MTT assays (also called succinate dehydrogenase inhibition tests) to measure the metabolic activity of the cells. The tetrazolium salt MTT is reduced to a colored, waterinsoluble formazan salt only by metabolically active (hence viable) cells [24, 25]. Figure 1A shows the cytotoxicity index of cells seeded at different densities and treated with different doses of adriamycin. The cytotoxicity index was normalized against cells seeded at the same density but not treated with adriamycin. Based on this result, we used 0.2–0.4 mg/ml of adriamycin and 120,000 cells per well for other experiments in this work to be within approximately linear range of the assay. The MTT assay has not been used typically to follow changes in viability over time. We modified the assay by treating the same number of cells with buffer control or various doses of adriamycin and harvested the cells at different time points. For a particular time point, the cytotoxicity index after adriamycin treat-
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FIG. 1. Evaluation of cytotoxicity by MTT assay. (A) Adriamycin-induced cytotoxicity of Swiss3T3 cells at different densities The indicated number of Swiss3T3 cells was seeded onto 96-well plates, and the cells were then treated with either buffer or different doses of adriamycin for 36 h. The cytotoxicity index was then measured by MTT assay as described under Materials and Methods. (B) Time course of adriamycin-induced cytotoxicity in Swiss3T3 cells. Swiss3T3 cells were seeded onto 96-well plates (12,000 cells per well), and the cells were then treated with either buffer or the indicated dosage of adriamycin. Cells were processed for MTT assay at different time points. The cytotoxicity index for each data point is expressed as the percentage of the untreated control for that time point. (C) Time course of adriamycin-induced cytotoxicity in HeLa cells. This experiment was exactly as in B, except HeLa cells (12,000 per well) were used instead of Swiss3T3 cells.
ment was normalized against that of the control cells. A progressive increase in cytotoxicity was seen with increasing dosage of adriamycin and time (Fig. 1B). A similar pattern of cytotoxicity was seen with other cell lines after adriamycin treatment; the MTT assay for human cervical carcinoma HeLa cells is shown for comparison in Fig. 1C. To study the effect of adriamycin on
quiescent (G0) cells, Swiss3T3 cells were grown in medium in the absence of serum for 48 h. The G0 cells were then treated with adriamycin and MTT assays were performed as before (Fig. 2). The cytotoxicity index in this case was normalized against that of the untreated G0 cells for each time point. It is important to note the meaning of these MTT
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FIG. 2. Evaluation of cytotoxicity of quiescent Swiss3T3 cells by MTT assay. Swiss3T3 cells were incubated in medium without serum for 48 h. Cells were then seeded onto 96-well plates (12,000 cells per well) and treated with either buffer or the indicated dosage of adriamycin. Cells were processed for MTT assay at different time points as described under Materials and Methods. The cytotoxicity index for each data point is expressed as the percentage of the untreated control for that time point.
measurements. A change in MTT cleavage activity represents a change in the cell number as well as in their viability. Hence, for proliferating cells the decrease in MTT cleavage activity after adriamycin treatment can be due to cell cycle arrest as well as a decrease in viability. For G0 cells, however, the decrease in MTT cleavage activity in adriamycin-treated cells in comparison to untreated cells was due to the decrease in viability alone because the G0 cells were already arrested. Comparison of viability in growing and quiescent cells after adriamycin treatment by vital stain. To have a more meaningful comparison between growing and quiescent cells, we directly measured the number of viable cells versus dead cells using the classical vital stain trypan blue. The reactivity of trypan blue is based on the fact that the chromopore is negatively charged and does not interact with the cell unless the membrane is damaged; hence, cells that exclude the dye are regarded as viable [26, 27].
Growing and quiescent Swiss3T3 cells were treated with different doses of adriamycin and harvested at different time points for trypan blue staining. Figure 3A shows that treatment of growing cells with adriamycin completely abolished proliferation. However, viability remained relatively high even with the highest dosage used (Fig. 3B), indicating that the cells were arrested but remained viable for up to 48 h. In comparison, Figs. 3C and 3D show that serum-starved Swiss3T3 cells did not proliferate. The viability of quiescent cells decreased slightly during the experiment even in the absence of adriamycin treatment. In the presence of adriamycin, however, viability decreased dramatically in serum-starved cells. In a variation of the above experiment, when serumstarved cells were stimulated by addition of serum, the cell number increased as the cells reentered the cell cycle (Fig. 3E). This indicates that most of the quiescent cells retained the ability to proliferate. In contrast, the cell number did not increase when adriamycin was added together with serum. Similar to the serum-starved cells shown in Fig. 3D, the viability of serum-stimulated cells was significantly reduced after addition of adriamycin (Fig. 3F). Taken together, these data indicate that immediately following adriamycin treatment (48 h represented one to two cell cycle times), cell proliferation is inhibited, and serumstarved cells are more sensitive (in terms of viability) to adriamycin than growing cells. Cell cycle analysis of growing and quiescent cells after adriamycin treatment. We next investigated the cell cycle distribution of growing and serum-starved cells after adriamycin treatment. Growing Swiss3T3 cells were either untreated or treated with adriamycin and harvested 24 and 48 h later for flow cytometry analysis. Figure 4 shows that adriamycin caused mainly a G2/M cell cycle arrest in growing cells. The majority of the cells were at G2/M at 48 h after addition of adriamycin. For serum-starved cells, nearly all the cells contained only G1 DNA content. As expected, the serumstarved cells remained in G0/G1 after treatment with adriamycin. However, a significant population of cells exhibited a sub-G1 DNA content in the presence of adriamycin (Fig. 4A). The sub-G1 DNA population was more marked at 48 h than at 24 h after adriamycin treatment. Although cells containing sub-G1 DNA content were also detected in untreated serum-starved
FIG. 3. Measurement of the viability of Swiss3T3 cells after adriamycin treatment by trypan blue counting. Semiconfluent growing Swiss3T3 cells (A and B), serum-starved Swiss3T3 cells (C and D), and serum-starved Swiss3T3 cells that were released into the cell cycle by addition of serum to the medium at t 5 0 (E and F) were treated with the indicated doses of adriamycin (E, untreated; l, 0.2 mg/ml; h, 0.4 mg/ml; ‚, 0.8 mg/ml). At different time points, cells were harvested for trypan blue counting as described under Materials and Methods. The cells that excluded trypan blue staining (viable cells) (A, C, and E) and the percentage of viable cells to trypan blue-stained dead cells (B, D, and F) are shown.
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cells at 48 h, the proportion of these cells was far more pronounced in the adriamycin-treated samples. Since the presence of sub-G1 DNA content is one of the characteristics of apoptotic cells, these data suggest that a large proportion of quiescent cells may undergo apoptosis after adriamycin treatment. In contrast, there is no evidence of significant apoptosis in growing cells following addition of adriamycin. These speculations are consistent with the trypan blue assay described above, which showed a higher cytotoxicity in quiescent cells compared with growing cells after adriamycin treatment. Significant changes in the size of the cells were observed after adriamycin treatment. We observed by light-scattering data from flow cytometry (Fig. 4B) that growing cells, but not quiescent cells, underwent a considerable increase in size after treatment with adriamycin. In contrast, an increased proportion of smaller cells was detected when quiescent cells were treated with adriamycin (Fig. 4C), suggesting that the cells may be entering apoptosis. To see in more detail whether the Swiss3T3 cells were undergoing apoptosis after addition of adriamycin, TUNEL assays were performed on growing and G0 cells that are untreated or treated with adriamycin (Fig. 4D). We found that there were relatively little apoptotic cells in a growing or quiescent population or when growing cells were treated with adriamycin. In contrast, the proportion of apoptotic cells increased when quiescent cells were treated with adriamycin as indicated by the TUNEL assay. Induction of p53 and p21 Cip1/Waf1 in adriamycintreated cells. Given that p53 and p21 Cip1/Waf1 are involved in cell cycle arrest and apoptosis following DNA damage, we next investigated the levels of p53 and p21 Cip1/Waf1 in growing and resting cells after adriamycin treatment (Fig. 5). When growing Swiss3T3 cells were treated with adriamycin, induction of p53 and p21 Cip1/Waf1 was detected by immunoblotting with specific antibodies (lanes 4 and 5). Treatment with another DNA-damaging agent, UV irradiation, also induced p53 and p21 Cip1/Waf1 (lanes 6 and 7). In contrast, p53 and p21 Cip1/Waf1 remained at basal levels in untreated cells throughout the experiment (lanes 1–3). In contrast to proliferating cells, relatively little induction of p53 and p21 Cip1/Waf1 was seen when serumstarved cells were treated with adriamycin or UV (Fig. 5). Nevertheless, the level of p53 was clearly higher than that in untreated serum-starved cells. Immunoblotting with anti-p27 Kip1 antibody showed that the protein level of p27 Kip1 was slightly higher in quiescent cells, as described previously [23]. The same samples were immunoblotted with a monoclonal antibody against tubulin to indicate similar loadings (we believe the decrease in tubulin level in G0 cells after treatment
with adriamycin or UV was likely due to the increase in cell death described above). At 24 h after addition of adriamycin, the total level of p53 was actually less than that at 6 h. One possibility is that some cells had repaired their DNA and the level of p53 returned to basal level; another possibility is that some cells may be dying by 24 h (but still have not manifested as cell membrane damage for trypan blue assay detection).
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FIG. 4. Analysis of adriamycin-treated Swiss3T3 cells by flow cytometry. (A) Cell cycle analysis by flow cytometry. Growing or quiescent Swiss3T3 cells were either untreated or treated with 0.4 mg/ml of adriamycin as indicated. The cells were harvested for flow cytometry analysis at 24 and 48 h as described under Materials and Methods. The positions of G1 and G2 DNA contents are indicated. The populations containing sub-G1 DNA content are indicated by arrows. (B) Cell size of proliferating cells. Proliferating Swiss3T3 cells were treated with buffer (left-hand panel) or 0.2 mg/ml of adriamycin (right-hand panel) and incubated for 24 h. The cell size and fluorescence were analyzed by flow cytometry. (C) Cell size of quiescent cells. Quiescent Swiss3T3 cells were treated with buffer (left-hand panel) or 0.2 mg/ml of adriamycin (right-hand panel) and incubated for 24 h. The cell size and fluorescence were analyzed by flow cytometry. (D) TUNEL assays. Growing or quiescent Swiss3T3 cells were either untreated or treated with 0.4 mg/ml of adriamycin as indicated. The cells were harvested for TUNEL assay at 24 h as described under Materials and Methods. The same magnification and exposure time were used for all slides. Apoptotic cells were labeled with fluorescein (green) and the nuclei were counterstained with Hoechst 33258 (blue).
Attempts to investigate the relative sensitivity of 3T3 cells that lack p53/p21 Cip1/Waf1 response after DNA damage were hampered for the following reasons. It is well known that the p53 genes in mouse fibroblasts are readily mutated, especially in later passages. We found that another 3T3 cell line used in our laboratory, NIH3T3, also exhibited p53 and p21 Cip1/Waf1 induction following DNA damage (Fig. 6A). On the other hand, PG13 cells, which were originally derived from NIH3T3, did not show p53 induction after treatment with adriamycin under similar conditions (Fig. 6B). To obtain quiescent cells, PG13 cells were incubated in medium without serum for 48 h. However, unlike Swiss3T3 cells (or NIH3T3 cells; data not shown), PG13 cells did not appear to enter quiescence because the cell number continued to increase as shown by trypan blue assay (Fig. 6C). Addition of adriamycin to serum-starved PG13 cells halted further cell proliferation. Consistent with the fact that PG13 cells cannot be completely arrested in G0 by serum starvation, the viability of the cells did not decrease as much as that in
quiescent Swiss3T3 cells after adriamycin treatment. We have also tested p53 2/2 mouse embryonic fibroblasts, but they also cannot be arrested in G0 by serum starvation (unpublished data). Differential recovery of growing and resting cells after adriamycin treatment. Given that quiescent cells are more sensitive than growing cells to adriamycin in the short term, it is hence essential to investigate the longer term survival of these cells. Growing or serumstarved Swiss3T3 cells were incubated with adriamycin, and the cells were then washed and allowed to grow in medium containing serum. The cells that were viable and were able to continue to proliferate formed visible colonies after about 2 weeks. Despite the fact that the shortterm viability of quiescent cells was lower than that of growing cells, these colony formation assays indicated the opposite relationship for the long-term proliferation potential. Over a wide range of adriamycin dosages tested, a higher proportion of quiescent cells than growing cells were able to form colonies (Fig. 7).
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FIG. 5. Expression of p53 and p21 Cip1/Waf1 following adriamycin treatment. Growing (lanes 1–7) or quiescent (lanes 8 –14) Swiss3T3 cells were untreated (lanes 1–3 and 8 –10) or treated with 0.4 mg/ml of adriamycin (lanes 4 and 5 and 11 and 12) or 15 J/m 2 of UV radiation (lanes 6 and 7 and 13 and 14). Cells were harvested at 0 h (lanes 1 and 8), 6 h (lanes 2, 4, 6, 9, 11, and 13), and 24 h (lanes 3, 5, 7, 10, 12, and 14) after treatment. Cell-free extracts were prepared, and 10 mg of each was applied onto 17.5% SDS–PAGE followed by immunoblotting with antibodies against p53, p21 Cip1/Waf1, p27 Kip1, and tubulin as indicated.
DISCUSSION
How proliferating cells and quiescent cells differ in their response to anticancer drugs like adriamycin is an important question in cancer therapy. Most of the normal cells in the body are in quiescent or differentiated states, but most cancer cells are actively proliferating. The goal of chemotherapy is to remove the cancer cells while sparing the normal cells. Here we evaluate the cytotoxicity of adriamycin on growing and quiescent cells by a number of methods that measure cellular membrane integrity, metabolic function, cell size, DNA content, and colony formation. Methods that measured viability over a shorter time scale (up to 48 h) show that quiescent cells are more sensitive to adriamycin than growing cells, but methods that measured survival/reproduction over the longer term show that growing cells are more sensitive to adriamycin than quiescent cells. Given that there are many assays that indicate viability, it is vital to consider exactly the meaning of the assays. MTT assays provide an indication of the metabolic activity of the cell population. A change in MTT cleavage activity represents a change in the cell number as well as in their viability. Hence, for proliferating cells the decrease in MTT cleavage activity after adriamycin treatment can be due to cell cycle arrest as well as a decrease in viability. For G0 cells, in contrast, the decrease in MTT cleavage activity in adriamycintreated cells in comparison to untreated cells was due
to the decrease in viability alone because G0 cells were already arrested. Hence, MTT assays can be used to compare growing or G0 cells in the presence or absence of adriamycin treatment, but are not useful for comparing between growing and G0 cells. The trypan blue exclusion assay is a more direct approach to measure the number of viable and dead cells. It should be noted that floating cells (mostly nonviable cells) were excluded in the trypan blue assay described here; hence, the viability is likely to be a slight overestimation. Furthermore, membrane damage may not be an immediate event in dead cells; hence, this would also lead to an overestimation of viability [27]. In the experiments described here, floating cells represent only a small proportion of the total cell population. We believe the high cytotoxicity observed in G0 cells or stimulated G0 cells after adriamycin treatment (Fig. 3) would be slightly accentuated if floating cells were included in the assay, because there were more floating cells in adriamycin-treated G0 cells than in any other samples (data not shown). Colony formation assay is probably the ultimate viability assay for many purposes. However, as can be seen here, colony formation assay measures the final survival and reproductive capacity of the cells, but the relative rate of cytotoxicity cannot be measured by colony formation assay. Moreover, it is possible that after adriamycin treatment some cells entered a prolong state of arrest, but nevertheless remained viable; these cells will not be scored in colony formation assays. It should also be noted that for colony formation assay, the colonies formed from growing and quiescent cells were expressed as percentages of the respectively untreated cells. One potential problem is that the plating efficiencies of growing and quiescent cells are very different. The plating efficiency is the percentage of the number of colonies formed to the original number of cell plates in the untreated samples. We found that the typical plating efficiencies of growing and G0 cells were about 75 and 25%, respectively. This is consistent with the fact that the viability of serum-starved cells decreased even in the absence of adriamycin (Fig. 3), and some cells probably lost the ability to reenter the cell cycle after serum starvation. A more precise conclusion is that of the cells that would form colonies: higher percentages actually did so from G0 cells than from proliferating cells after treatment with adriamycin. Previous studies that show that quiescent cells are less sensitive to adriamycin than growing cells also relied on colony formation assays [17, 28]. It should be noted that for other types of DNA damages like X-ray, G0 cells had been reported to be more sensitive than growing cells [29]. Flow cytometry analysis shown here indicates that a substantial population of G0 cells contained sub-G1 DNA content when treated with adriamycin (Fig. 4), suggesting that apoptosis may be involved. Both the
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FIG. 6. Responses to adriamycin in different mouse fibroblasts. (A) p53 expression in Swiss3T3 and NIH3T3. Growing NIH3T3 cells (lanes 1–3) or Swiss3T3 cells (lanes 4 – 6) were either untreated (lanes 1, 2, 4, and 5) or treated with 0.4 mg/ml of adriamycin (lanes 3 and 6). Cells were harvested at t 5 0 (lanes 1 and 4) and t 5 6 (lanes 2, 3, 5, and 6). Cell-free extracts were prepared, and 10 mg of each was applied onto 17.5% SDS–PAGE followed by immunoblotting with antibodies against p53 (top) and p21 Cip1/Waf1 (bottom). (B) p53 expression in PG13 cells. Growing PG13 cells (lanes 1 and 2) or Swiss3T3 cells (lanes 3 and 4) were either untreated (lanes 1 and 3) or treated with 0.4 mg/ml of adriamycin (lanes 2 and 4). Cells were harvested at 6 h after treatment. Cell-free extracts were prepared, and 10 mg of each was applied onto 17.5% SDS–PAGE followed by immunoblotting with antibodies against p53. (C) Serum-starved PG13 cells were treated with the indicated doses of adriamycin (E, untreated; h, 0.4 mg/ml). At different time points, cells were harvested for trypan blue counting as described under Materials and Methods. The cells that excluded trypan blue staining (viable cells) are shown in the left-hand panel and the percentage of viable cells to trypan blue-stained dead cells is shown in the right-hand panel.
TUNEL assays and the decrease in cell size as indicated by flow cytometry also suggest the increase in apoptosis in G0 cells after adriamycin treatment. In growing cells, no apoptosis was seen after adriamycin. At the same time, there was significant cell swelling in growing cells after adriamycin treatment, suggesting that many of the cells may be entering necrosis. Hence, it is possible that distinct mechanisms may be involved in adriamycin-induced cell death in growing and G0 cells. However, the increase in cell size after adriamycin treatment may also be due to “unbalanced growth,” when cell division is precluded but cells continue to grow in size [30]. The normal cell cycle-arresting point
of adriamycin is the G2 phase (see Fig. 4); the fact that G0 cells are not in G2 is likely to have a pronounced effect on the response to adriamycin. The tumor suppressor p53 clearly plays an essential role in the G1/S DNA damage checkpoint. Furthermore, p53 plays an important role in apoptosis following DNA damage, although not all apoptosis requires the function of p53. One question is whether cell death in G0 after DNA damage also involves p53. Only very modest increases in p53 levels were detected after G0 cells were treated with adriamycin (Fig. 5), but it is possible that G0 cells are more sensitive to slight increases in p53 than growing cells. To test this possibil-
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FIG. 7. Colony formation assay of Swiss3T3 cells following adriamycin treatment. Known numbers of growing or quiescent Swiss3T3 cells were plated and incubated with the indicated concentration of adriamycin for 3 h. The cells were washed and incubated in medium containing serum for another 2 weeks. The number of colonies formed was counted after staining with crystal violet. Survival of growing and quiescent cells is expressed as percentage of the respective untreated cells. This is representational of one of several experiments with different densities of cells plated.
ity, we need to be able to conditionally express p53 in growing and G0 cells, but expression of exogenous proteins in G0 cells is technically more difficult. The fact that G0 cells have a weaker p53/p21 Cip1/Waf1 response than growing cells (Fig. 5) may underlie the higher sensitivity of G0 cells to immediate killing than growing cells. It has been reported that in G0 cells UV irradiation induced p53 DNA-binding activity, but not its accumulation, whereas both events took place in G1/S- and S-phase cells [31]. It is also possible that growing cells mainly cause cell cycle arrest (at least for the short term), but induction of p53 in G0 cells may cause immediate cell death. We attempted to use PG13 cells, which do not have p53 induction after adriamycin treatment, to study the effect of p53 on cytotoxicity in G0 cells. However, PG13 cells, as with so many p53negative transformed cells, do not arrest in G0 upon serum starvation (Fig. 6). Moreover, it is likely that PG13 cells contain other mutations apart from that of p53. We have also tested p53 2/2 mouse embryonic fibroblasts, but they also cannot be arrested in G0 by serum starvation (unpublished data). Immediately after adriamycin treatment, why are quiescent cells more sensitive than growing cells? Further, why do quiescent cells appear to be less sensitive than growing cells in terms of reproductive potential? It is still not completely clear when the cells undergo cell cycle arrest and when they undergo apoptosis after
DNA damage. One possibility is that after DNA damage, actively growing cells predominantly enter cell cycle arrest (at G2 phase; see Fig. 4); the cells may then undergo cell death if the DNA damage is severe or not repairable. Furthermore, DNA damage-arrested cells may never recover to proliferate, but remain in a prolong arrest state [32]. Quiescent cells, on the other hand, have already exited the cell cycle, and the cells may immediately undergo cell death upon adriamycin treatment since the initial cell cycle arrest mechanism after DNA damage is bypassed. It is not immediately apparent why quiescent cells are also affected by adriamycin, since the main target of adriamycin is likely to be topoisomerase II and quiescent cells have little DNA synthesis activity. It is possible that there are some basal DNA synthesis or DNA repair activities that involve topoisomerase II in G0 cells. Another possibility is that apart from targeting topoisomerase II, adriamycin may have other effects such as oxidation to which G0 cells are sensitive. It would be interesting in the future to analyze the differential responses of proliferating and quiescent cells described here with other kinds of DNA-damaging agents. We are grateful to Hermann Bujard, Liang Cao, Julian Gannon, Tim Hunt, and Tony Hunter for comments and reagents. We thank members of the Poon lab for invaluable help and discussion. This work was supported in part by Research Grants Council Grant HKUST6188/97M and IDTC Grant AF/178/97 to R.Y.C.P..
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