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Experimental Therapeutics |
Pharmaceutical Research Institute, Kyowa Hakko Kogyo Co., Ltd., Shimotogari, Nagaizumi-cho, Sunto-gun, Shizuoka 411-8731 Japan [K. S., T. A., M. S., T. T., S. A.], and Medical Research Council Toxicology Unit, University of Leicester, Leicester, LE1 9HN United Kingdom [C. C., A. G.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although the PKC enzyme family has been considered to be a major target of UCN-01, it is unclear how PKC inhibition can cause anticancer activity of the drug, and the most important target for UCN-01 is still obscure. Cell cycle analysis revealed that UCN-01 induces G1 phase accumulation in a wide spectrum of cancer cell lines (5 , 9 , 14 , 15) , suggesting that this event is one of the major consequences of treatment of human cancer cells with UCN-01. This feature is rarely observed with traditional cytotoxic anticancer agents. We have also shown that the UCN-01-induced G1 phase accumulation is mediated via direct and indirect inhibition of Rb kinase(s), such as CDK2 (16) .
Furthermore, studies from the National Cancer Institute group (6 , 17 , 18) and our laboratories (14) suggest that induction of apoptosis is intrinsically linked to the cytotoxic effect of UCN-01. UCN-01 induced apoptosis both in leukemia and colon carcinoma cells, the latter being relativity resistant to apoptosis induction by conventional cytotoxic agents, such as camptothecin (17) .
The aim of this study was to improve our understanding of the mechanism of antineoplastic action of UCN-01. Its special objective was to explore the intriguing association between apoptosis induction, G1 phase accumulation, and Rb kinase inhibition, which determines the cellular response toward this drug using a human A549 lung adenocarcinoma cell line with acquired resistance against UCN-01 (A549/UCN cells). When these cells were derived and compared with their wild-type counterparts, they were shown to possess down-regulated PKC-
, -
, and -
and to express the multidrug resistance-associated protein, but they lacked cross-resistance against STP and its PKC-specific analogue CGP 41251 (19)
. Specifically, we wished to compare A549/UCN and A549 cells in terms of: (a) their sensitivity to the growth-arresting properties of selected anticancer drugs with differing mechanisms of cytotoxicity; (b) their susceptibility toward induction of apoptosis and toward G1 phase accumulation elicited by UCN-01; and (c) their susceptibility toward UCN-01-induced changes in expression of proteins involved in apoptosis and G1 and S phase checkpoint functions.
UCN-01 is one of the first representatives of a relatively new generation of kinase-directed signal transduction modulators that are currently progressing into clinical trials. Therefore, the characterization of cellular resistance mechanisms operative against this drug was thought to help predict some determinants of clinical resistance that might eventually complicate therapy using such kinase-directed agents.
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MATERIALS AND METHODS |
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Antibodies were purchased from the following suppliers: anti-p21, p27, CDK4, CDK6, and bax polyclonal antibodies and anticyclin B1 monoclonal antibody from Santa Cruz Biotechnology (Santa Cruz, CA); anti-Rb and cyclin E monoclonal antibodies from PharMingen (San Diego, CA); anticyclin D1 monoclonal antibody from IBL (Gunma, Japan); anti-PARP monoclonal antibody from Enzyme Systems Products (Dublin, CA); anti-bcl-2 monoclonal antibody from Genosys (Cambridgeshire, United Kingdom); anti-bcl-xL polyclonal antibody from Transduction Laboratories (Lexington, KY); anti-bak monoclonal antibody from Calbiochem (Cambridge, MA); anticyclin A and CDK2 monoclonal antibodies were prepared as described previously (16) .
Cell Culture and Assessment of Inhibition of Cell Growth.
A549/UCN cells were derived from their parent A549 cells, as described previously (19)
. Cells were maintained in Ham’s F-12 medium supplemented with 10% fetal bovine serum, 100 IU of penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine (Life Technologies Inc., Grand Island, NY) in an atmosphere of 5% CO2. A549/UCN cells were maintained in the presence of 200 nM UCN-01. In growth inhibition experiments, cells were incubated in culture medium for 24 h in 96-well microwell plates (Nunc, Roskilde, Denmark) before incubation with drug for 72 h. Cell viability was determined by the 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT; Sigma Chemical Co.) assay (20)
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Cell Cycle Analysis.
Cells were incubated in the culture medium for 24 h in Falcon 3003 plastic dishes (Becton Dickinson, Lincoln Park, NJ) before incubation with UCN-01 for 24 h. The cells were harvested by trypsin-EDTA treatment, fixed with ice-cold ethanol (70%), and stored at 4°C. For flow cytometric analysis, the RNA was hydrolyzed using 250 µg/ml RNase A (type 1-A; Sigma Chemical Co.) at 37°C for 30 min, and the cells were stained with propidium iodide (PI; Sigma Chemical Co.) for 20 min. DNA content of cells was analyzed using an EPICS ELITE flow cytometer (Coulter Electronics, Hialeah, FL). Cell cycle distribution was calculated by a Multicycle program (Phoenix Flow Systems, Inc., San Diego, CA).
Assessment of Apoptosis.
For the TUNEL assay, cells were processed according to the manufacturer’s instructions using ApopTag Direct Kit (Oncor, Inc., Gaithersburg, MD; Ref. 21
). Briefly, cells were fixed in 1% formaldehyde solution for 15 min on ice, washed in PBS, suspended in 70% ethanol, and stored at -20°C. After washing in PBS, the cells were resuspended first in equilibration buffer for 15 min at room temperature, then in TdT reaction buffer (50 µl) at 37°C for 30 min. After stopping the TdT reaction, the cells were incubated in PBS (1 ml) containing 10 µg/ml PI and 10 µg/ml RNase A for 15 min at room temperature in the dark. Bivariate analysis of apoptosis (green fluorescence) and DNA content (PI) was performed flow cytometrically. The resulting bivariate plots allow detection of apoptotic events initiated within the cell cycle. Using the control specimen to define normal levels of green fluorescence (i.e., basal levels of apoptosis), the R1 region was set. Cells with fluorescence within the R1 region were considered apoptotic. Data from 20,000 cells were collected and analyzed using the Multi2D program (Phoenix Flow Systems).
Western Blotting.
Western blotting was performed as described previously (16)
. Cells exposed to drug were harvested by trypsin-EDTA treatment, washed with PBS, and stored at -80°C. They were lysed in lysis buffer [50 mM HEPES/NaOH (pH 7.4), 150 mM NaCl, 0.1% Triton X-100, 50 mM sodium fluoride, 80 mM glycerophosphate, 0.1 mM sodium o-vanadate, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, and 1 µg/ml leupeptin] for 20 min at 4°C. The cell lysate was centrifuged (14,000 rpm) for 10 min at 4°C, and its protein content was determined using a protein assay kit (Bio-Rad Laboratories). Equal amounts of protein were heated in SDS-sample buffer for 5 min at 95°C and subjected to SDS-PAGE. Protein was transferred to a p-membrane (ATTO, Tokyo, Japan), probed with primary antibodies, followed by secondary antibodies conjugated with horseradish peroxidase (Amersham Life Sciences, Buckinghamshire, United Kingdom), and detected by enhanced chemiluminescence (Amersham Life Sciences).
CDK2 Kinase Assay.
CDK2 kinase assay was performed, as described previously (16)
. Cell lysate was prepared as described above. Equal amounts of protein (300 µg) of the cell lysate were added to protein A-Sepharose CL-4B (20%, v/v; Pharmacia, Uppsala, Sweden) preassociated with anti-CDK2 antibody and mixed gently for 2 h at 4°C. CDK2-immunoprecipitate was washed with lysis buffer twice and wash buffer [50 mM HEPES/NaOH (pH 7.4), 10 mM MgCl2, and 1 mM DTT] two times, and incubated with kinase buffer (40 µl) [50 mM HEPES/NaOH (pH 7.4), 10 mM MgCl2, 1 mM DTT, 16 µg of histone H1 (Boehringer Mannheim, Mannheim, Germany), 50 µM ATP, and 2.5 µCi [
-32P]ATP (5000 Ci/mmol; Amersham Life Sciences)] for 10 min at 30°C. Each sample was mixed with 3 x SDS-sample buffer (30 µl) to stop the reaction, heated for 5 min at 95°C, and subjected to SDS-PAGE. Gel was dried, stained with Coomassie Brilliant Blue, and analyzed by a BAS2000 image analyzer (Fuji photo film, Tokyo, Japan).
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RESULTS |
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, in comparison with wild-type A549 cells, A549/UCN cells showed 3.6- and 2.6-fold resistance against UCN-01 and CGP 41251, respectively, but lacked cross-resistance against STP and UCN-02. Dose-response curves of UCN-01 showed that acquired resistance to UCN-01 was more pronounced at higher concentrations [i.e., at 300 nM UCN-01 the percentage decrease in cell number was 90% for A549, but only 55% for A549/UCN cells (data not shown)]. Dose-response curves of CGP 41251 also showed that resistance against CGP 41251 was weak because at higher concentrations both cell lines were sensitive to the drug, A549/UCN cells even more so than A549 cells at concentrations of above 3 µM CGP 41251 (data not shown). To characterize A549/UCN cells further, their potential sensitivity to the following clinically used anticancer drugs was investigated: adriamycin representing DNA-intercalating topoisomerase II poisons; cisplatin and mitomycin C representing DNA intrastrand cross-linking agents; and Taxol and navelbine representing mitotic poisons. A549/UCN cells were not cross-resistant against adriamycin, Taxol, or navelbine. However, they showed 4.4- and 14-fold cross-resistance against mitomycin C and cisplatin at IC50 levels, respectively (Table 1)
. A549/UCN cells were also investigated in terms of their sensitivity to flavopiridol, a CDK inhibitor currently under clinical scrutiny (22)
, because UCN-01 has also been shown to inhibit CDK2 in a direct and indirect manner (15
, 16)
. A549/UCN cells were not resistant against flavopiridol (Table 1)
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shows that UCN-01 at 0.08 µM induced only a slight accumulation of A549 cells in G1 phase, whereas the drug at higher concentrations blocked cells in the S and G2M phases, but not in G1 phase. The percentages of cells in G1 phase were 43%, 47%, 34%, and 22% at 0, 0.08, 0.24, and 0.4 µM of UCN-01, respectively (Fig. 1)
. In contrast, in A549/UCN cells, UCN-01 induced a clearly concentration-dependent increase in the G1 phase population (Fig. 1)
. The percentages of cells in G1 phase were 43%, 58%, 66%, and 67% at 0, 0.08, 0.24, and 0.4 µM of UCN-01, respectively (Fig. 1)
. In A549 cells, UCN-01 elicited a concentration-dependent increase in TUNEL-positive cells and in the sub-G1 cell population, which was also positive in the TUNEL assay (Fig. 2)
. Furthermore, an apparent concentration-dependent cleavage of PARP protein assessed by the appearance of an 85-kDa protein was also observed after UCN-01 treatment in these cells (Fig. 5)
. Morphological changes such as cell shrinkage, chromatin condensation, and nuclear fragmentation in A549 cells after treatment with UCN-01 supported the inference made as to induction of apoptosis (data not shown). In sharp contrast, the drug induced hardly any apoptosis in A549/UCN cells at concentrations as high as 0.4 µM, as adjudged by TUNEL assay (Fig. 2)
, PARP cleavage assay (Fig. 5)
, and assessment of morphological indicators (data not shown). These results suggest that attenuation of apoptosis induction and augmentation of G1 phase checkpoint function might be pivotal determinants of the acquired resistance of A549/UCN cells against UCN-01.
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. This result suggests that A549/UCN cells display selective resistance against UCN-01-induced apoptosis, which is consistent with the sensitivity pattern of these cells to the growth-arresting properties of UCN-01, Taxol, and STP (Table 1)
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. In contrast, expression of Rb and S and G2M cyclins A and B1 and CDK2 was down-regulated, whereas levels of CDK4 and CDK6 proteins were only slightly decreased (Fig. 4A)
. The apoptosis-regulatory proteins, the levels of which were measured, were the antiapoptotic molecules bcl-2 and bcl-xL and the proapoptotic species bak, bax, and PARP. Whereas wild-type A549 cells expressed bcl-xL but not bcl-2, A549/UCN cells expressed bcl-2 with only traces of bcl-xL (Fig. 4B)
. A549/UCN cells displayed slight up-regulation of expression of bak and bax proteins (Fig. 4B)
. PARP, an important substrate for the death-inducing enzyme caspase 3, was apparently down-regulated in A549/UCN cells (Fig. 5)
. Because PKC family proteins were reported to play a role as an upstream regulator of Raf-1 protein (23)
, we have examined the expression level of Raf-1 protein in A549/UCN and A549 cells. There was no difference in Raf-1 expression level between both cell lines (data not shown).
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). Cells were incubated with UCN-01 (0.08, 0.24, and 0.4 µM) for 24 h and analyzed by Western blotting. Although UCN-01 did not affect the level of p21 in A549 cells, it induced its expression in A549/UCN cells (Fig. 5)
, a phenomenon that has previously been reported in other cell types (16)
. UCN-01 did not affect the expression level of p27 in either A549 or A549/UCN cells (Fig. 5)
, which differs from its effects reported previously (16)
. UCN-01 hardly affected the levels of cyclins D1, E, and B1 in either cell line (data not shown). The expression level of cyclin A was decreased slightly by UCN-01 in A549 cells; in contrast, in A549/UCN cells, it was markedly reduced to undetectable levels (Fig. 5)
. In both cell types, UCN-01 induced a decrease in Rb protein levels and the emergence of a Rb species with faster mobility, indicating dephosphorylation of Rb (Fig. 5)
. In A549/UCN cells, UCN-01 induced the disappearance of a faster migrating band of CDK2, which indicates dephosphorylation of the CDK2 protein (Fig. 5)
. This result suggests that the dephosphorylation of CDK2 might play an important role in the G1 phase accumulation induced by UCN-01. Although the expression levels of bcl-2 were not affected by UCN-01, bcl-xL protein levels were slightly decreased by UCN-01 in both cell lines, although its basal expression level was much higher in A549 cells than in A549/UCN cells. UCN-01 also did not cause a shift in mobility of the bcl-2 and bcl-xL proteins in these cells, an event that has been reported for mitotic poisons and interpreted as a marker of M phase arrest and/or apoptosis induction (24
, 25)
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Susceptibility of A549/UCN Cells Toward CDK2 Kinase Activity.
Because the results of Fig. 5
have suggested that dephosphorylation of CDK2 might play an important role in G1 phase accumulation induced by UCN-01, we further compared the effect of UCN-01 on CDK2 kinase activity in A549 and A549/UCN cells. Cells were incubated with UCN-01 (0.08, 0.24, and 0.4 µM) for 24 h, and CDK2 activity was determined after immunoprecipitation with anti-CDK2 monoclonal antibody using histone H1 as a substrate. Although there was little change on CDK2 activities in A549 cells treated with UCN-01, CDK2 activity of A549/UCN cells was inhibited by UCN-01 in a concentration-dependent manner (Fig. 6)
. This result is consistent with the mobility shift of CDK2 and cyclin A protein level (Fig. 5)
, suggesting that inhibition of CDK2 activity might play an important role in the G1 phase accumulation induced by UCN-01.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The determination of expression levels of G1 checkpoint proteins (i.e., of negative regulators such as the CDK inhibitors p21, p27, and Rb, as well as of positive regulators such as cyclins and CDKs) allowed further inferences to be made as to the mechanism of resistance against UCN-01 operative in A549/UCN cells. In the resistant cells, p21 and p27, as well as G1 cyclins D1 and E, were unambiguously clearly up-regulated, suggesting that these cells are characterized by a compromised balance of cell cycle-inhibitory and -accelerating molecules. Because p21 and p27 proteins are thought to play an important role as G1 checkpoint proteins (27) , it is conceivable that the reinforcement of the G1 checkpoint function renders cells resistant against UCN-01. It has previously been suggested that dephosphorylation of both Rb and CDK2 proteins is important for the G1-phase accumulation induced by UCN-01 (16) . However, in the study described here, UCN-01 induced the dephosphorylation of Rb protein in both A549 cells and their UCN-01 resistant counterparts, suggesting that a step downstream of Rb protein dephosphorylation might be responsible for G1 phase accumulation. The fact that the phosphorylated form of CDK2 was down-regulated by UCN-01 only in A549/UCN cells but not in wild-type A549 cells suggests that CDK2 dephosphorylation plays a more important role than Rb dephosphorylation in the G1 phase accumulation induced by UCN-01. Because UCN-01 has been shown to inhibit CDK2 activity in several cell lines (15 , 16) , the results presented here suggest that a CDK2 substrate other than Rb might be crucial for cell cycle blockage and/or apoptosis induced by UCN-01. Alternatively, the effects of UCN-01 on CDK2 in A549/UCN cells may be the consequence rather than the cause of the altered cell cycle arrest phenotype in the resistant cells. It is also conceivable that a protein upstream of CDK2, such as CDK-activating kinase, might be inhibited by UCN-01, thus engendering the down-regulation of CDK2 activity.
As to apoptosis-regulatory proteins, although A549 cells expressed bcl-xL but not bcl-2, in accordance with a previous study (28 , 29) , A549/UCN cells expressed bcl-2 but not bcl-xL. This difference suggests that bcl-2 might be an important determinant of UCN-01 sensitivity. It has been proposed that overexpression of bcl-2 can be responsible for resistance against apoptosis inducers (30 , 31) , a notion that prompted us to examine the susceptibility of UCN-01-resistant A549 cells toward apoptosis induction by Taxol and STP. A549/UCN cells were even more sensitive than A549 cells to apoptosis induction by both drugs, which is consistent with the pattern of susceptibility toward growth inhibitory potency, suggesting that the acquired resistance against UCN-01 in A549/UCN cells is a feature selective to UCN-01, at least among the drugs tested here.
Recently, p21 was reported to regulate negatively the induction of apoptosis, and the protein was shown to be a substrate of caspase 3, an important executioner of apoptosis (32, 33, 34) . p21 protein was clearly up-regulated by UCN-01 in A549/UCN cells but not in A549 cells, which harbor the wild-type p53 protein. These data are consistent with the notion that UCN-01 induces p21 via a p53-independent mechanism (16) . p27, which has been shown to be associated with cytotoxic drug resistance in cells under anchorage-independent growth conditions (35 , 36) , was also up-regulated in A549/UCN cells. Taken together, the overexpression of p21 and p27 in A549/UCN cells might be an important determinant of acquired resistance against UCN-01, however, the mechanism of their overexpression remains to be elucidated.
The pattern of cross-resistance against a variety of anticancer agents observed in A549/UCN cells is in accordance with alterations in the checkpoint governing G1 rather than that regulating G2M. The cells were not cross-resistant against Taxol and navelbine, which are M phase-acting drugs, or adriamycin, which arrests cells selectively at the G2 checkpoint (37) . Unexpectedly, A549/UCN cells exhibited cross-resistance against DNA cross-linking agents such as mitomycin C and cisplatin, both of which are thought to act on the G1-S phase, as well as the G2 phase, in p53 wild-type cells, such as A549. This cross-resistance can be interpreted to indicate that reinforcement of the G1 checkpoint also protects cells from the lethal consequences of DNA-damaging agents. Alternatively, UCN-01, cisplatin and mitomycin C could contribute directly to DNA damage leading to an increase in availability of DNA present in replication forks. Thus, susceptibility to apoptosis could arise from S phase arrest.
In conclusion, the results described above suggest that acquired resistance of A549/UCN cells against UCN-01 is mediated through the attenuation of apoptosis induction, which in turn might be the corollary of the enforcement of G1 checkpoint proteins, as well as antiapoptotic proteins. The results also suggest that the susceptibility of cells toward G1 phase accumulation elicited by UCN-01 can be inversely correlated to susceptibility to apoptosis induction and cellular sensitivity toward the drug. UCN-01 is one of several new kinase-directed antisignaling drugs that are currently entering clinical evaluation as anticancer drugs. Should any of these agents be eventually included into the oncologist’s chemotherapeutic armamentarium, it is prudent to be aware of the possibility that they may elicit clinical resistance as a consequence of the ability to alter processes that regulate the cell cycle machinery and apoptosis in a complicated fashion analogous to that described here for UCN-01.
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FOOTNOTES |
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1 To whom requests for reprints should be addressed, at Kyowa Hakko Kogyo Co., Ltd., Shimotogari, Nagaizumi-cho, Sunto-gun, Shizuoka 411-8731, Japan. 
2 The abbreviations used are: UCN-01, 7-hydroxystaurosporine; CDK, cyclin-dependent kinase; PKC, protein kinase C; Rb, retinoblastoma; STP, staurosporine; PARP, poly(ADP-ribose) polymerase; TdT, terminal deoxynucleotidyl transferase; TUNEL, TdT-mediated nick end labeling; PI, propidium iodide.
Received 3/18/99. Accepted 7/ 6/99.
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