Ubiquitin-mediated proteolysis controls intracellular levels of various cell cycle regulatory proteins, and its inhibition has been shown to induce apoptosis in proliferating cells. In the present study, we examined induction of apo-ptosis in oral squamous cell carcinoma (OSCC)cells by treatment with specific proteasome inhibitors,carbobenzoxy-l-leucyl-l-leucyl-l-norvalinal and lactacystin. In all three OSCC cell lines examined, apoptotic changes such as apo-ptotic body formation and DNA fragmentation were observed at various degrees after 24 h of the carbobenzoxy-l-leucyl-l-leucyl-l-norvalinal or lactacystin treatment. HSC2 cells showed the most prominent apoptotic changes among the cell lines examined and demonstrated the highest level of accumulation of p27Kip1 protein after the treatment with proteasome inhibitor. Reduced expressions of cyclin D1 and phospho pRb were also observed after the treatment with proteasome inhibitor. Moreover, 12 h of treatment with the proteasome inhibitor inhibited cdk2/cyclin E kinase activity and increased the ratio of the cell cycle population at the G1 phase. The proteasome inhibitor led to inhibition of cell cycle progression. In addition, activation of CPP32 and reduced expression of Bcl-2 were observed. Because apo-ptosis induced by the proteasome inhibitor was inhibited by treatment with antisense p27Kip1oligonucleotide, accumulation of the p27Kip1 protein might play an important role in the apoptosis induced by proteasome inhibitor. The present results suggest that inhibition of proteasome function may be used as a possible target of novel therapy for OSCC.
Abnormalities in cell cycle regulators allow uncontrolled cell growth and division and may participate in carcinogenesis (1, 2). Multiple cyclins and CDKs3 are positive regulators to progress the cell cycle. Cyclin/CDK complexes are activated by phosphorylation by the CDK-activating kinase, whereas cyclin/CDK complexes are negatively regulated by a number of CDK inhibitors, including p27Kip1(3, 4, 5, 6, 7, 8, 9, 10). The activity of CDKs is also regulated by phosphorylation that is controlled by the antagonistic action of wee1 kinase and CDC25 phosphatases (11). Cyclin D-cdk4/cdk6 and cyclin E-cdk2 complexes phosphorylated pRb during the G1-S transition, and this phosphorylation caused the inactivation of the growth-inhibitory function of pRb.
Ubiquitin-mediated proteolysis is involved in the turnover of various cell cycle-regulatory proteins, including the tumor suppressor protein p53 and various cyclins as well as the CDK inhibitor p27Kip1 (12, 13, 14, 15, 16). Accumulating evidence suggests that the ubiquitin-proteasome pathway is a major pathway of proteolysis in eukaryotic cells and plays important roles in many physiological functions by the degradation of various oncoproteins or transcriptional regulators (c-Myc, c-Fos, c-Jun, and nuclear factor-κB/IκB) and various cell cycle-regulatory proteins (12, 13, 14, 15, 16). Some of these proteins are related to the regulation of apoptosis, and it is known that ubiquitin-mediated proteolysis is involved in the regulation of apoptosis in mammalian cells (12, 14). In proteolytic events during apoptosis,activation of the proteolytic enzyme is a characteristic feature of apoptosis. In particular, cystein proteases of the caspase family are known to be as central components of proteolytic machinery (17). Apoptosis induced by proteasome inhibitor is attributable to activation of CPP32, a member of the caspase family of cystein proteases, and appears to occur independently from interleukin-1β converting enzyme activity (18).
The proteasome inhibitors are capable of inducing apoptosis in proliferating cells but not in quiescent, differentiated cells (18, 19, 20, 21). p53-dependent apoptosis was induced by proteasome inhibitors in mammalian cells (22, 23). Besides, in human leukemic HL60 cells, apoptosis induced by inhibition of proteasome-mediated proteolysis is revealed to be accompanied by an increase in the concentration of p27Kip1(18).
In the present study, we treated OSCC cells with LLnV or lactacystin,which are specific proteasome inhibitors, to examine the induction of apoptosis by inhibition of the proteasomal function. We also investigated the expression of various cell cycle-regulatory and apoptosis-related proteins after the treatment of proteasome inhibitors and discussed possible mechanisms of apoptosis induced by the proteasome inhibitors.
MATERIALS AND METHODS
Three OSCC cell lines (HSC2, HSC3, and Ho-1-U-1), normal oral epithelial cells, and gingival fibroblasts were examined. All of the OSCC cell lines were provided by the Japanese Cancer Research Resources Bank. They were routinely maintained in RPMI 1640 (Nissui Co., Tokyo,Japan), supplemented with 10% fetal bovine serum (Boehringer Mannheim K. K., Australia), under conditions of 5%CO2 in air at 37°C. Normal oral epithelial cells and gingival fibroblasts were obtained from oral mucosa or gingival tissues using standard explant techniques (24). These tissues were obtained undergoing routine dental surgery in the Department of Oral Surgery, Hiroshima University Dental Hospital. Normal oral epithelial cells were routinely maintained in Keratinocyte-SFM (Life Technologies, Inc., Grand Island, NY), and gingival fibroblasts were maintained in DMEM supplement with 10% fetal bovine serum. Only cells between passages three and five were used in this study.
Treatment with Proteasome Inhibitor.
We used LLnV and lactacystin, which are specific proteasome inhibitors. LLnV inhibits the chymotrypsin-like activity of the proteasome, and lactacystin targets the catalytic β-subunit of the proteasome. LLnV was obtained from the Peptide Institute (Osaka, Japan). Lactacystin was obtained from the Kyowa Medix Co. (Tokyo, Japan). The compounds were dissolved in an amount of DMSO. LLnV and lactacystin were added to the OSCC cells at the final concentration of 50 and 10μ m, respectively.
Cells were fixed in 2.5% glutaraldehyde, postfixed in 1% osmium tetroxide, and embedded in epoxy resin. Thin sections were stained in uranyl acetate and lead citrate and examined under a Hitachi H500H transmission electron microscope.
Determination of DNA Fragmentation.
For qualitative analysis of DNA fragmentation, cells were harvested at the indicated times by centrifugation and lysed by the addition of 100μl of lysis buffer consisting of 10 mm Tris-HCl (pH 7.4),10 mm EDTA, and 0.1% of Triton X-100. They were incubated by addition of RNase A and proteinase K (37°C for 60 min). After centrifugation, the soluble DNA fragments released into the supernatant were precipitated by the addition of 0.5 volume of 7.5 mammonium acetate and 2.5 volumes of ethanol. DNA pellets were dissolved in TE and loaded onto a 2.0% agarose gel and separated at 100 V for 45 min. DNA fragments were visualized after staining with ethidium bromide by transillumination with UV light.
Cells were plated onto a 24-well plate (Sumilon; Sumitomo Bakelite Co.,Tokyo, Japan) and allowed to attach for 24 h. The culture medium was then replaced with medium that contained 50 μm LLnV or 10 μm lactacystin. For a control, cells were incubated in the medium containing the vehicle, 50 μm DMSO. Trypsinized cells were counted by cell counter (Coulter Z1, Coulter,FL) at 6, 12, and 24 h after treatment. Viable cells, as detected by the trypan blue dye exclusion test, were also counted at 6, 12, and 24 h after treatment.
Flow Cytometric Analysis.
Cell cycle distribution was determined by DNA content analysis after propidium iodide staining using Cycle Test Plus DNA reagent kit (Becton Dickinson, San Jose, CA). Cells were cultured as described above and fixed in 70% ethanol and stored at 4°C before analysis. Flow cytometric determination of DNA content was analyzed by a FACScalibur(Becton Dickinson) flow cytometer. For each sample, 20,000 events were stored. The fractions of the cells in G0-G1, S, and G2-M phases were analyzed using CELLQuest, a cell cycle analysis software.
Western Blot Analysis.
We examined the expression of cell cycle-regulatory and apoptosis-related proteins in OSCC cell lines treated with LLnV by Western blot analysis. The cells were lysed in a buffer containing 50 mm Tris-HCl (pH 7.4), 125 mm NaCl, 0.1% (v/v)NP40 (Sigma Chemical Co., St. Louis, MO), 5 mm EDTA, 0.1 m NaF, 10 μg/ml leupeptin (Sigma), 0.1 μg/ml trypsin inhibitor (Sigma), 0.1 μg/ml aprotinin (Sigma), and 50 μg/ml phenylmethylsulfonyl fluoride (Wako, Osaka, Japan). The protein concentration was determined by Bradford protein assay (Bio-Rad,Richmond, CA) using BSA (Sigma) as a standard. Fifty μg of protein were solubilized in Laemmli’s sample buffer by boiling and subjected to 10% SDS-PAGE, followed by electroblotting onto a nitrocellulose filter (Schleicher & Schuell, Dasse, Germany). The filter was blocked 1 h at 4°C with PBS buffer (137 mm NaCl, 8.1 mmNa2HPO4·12H2O,2.68 mm KCl, and 1.47 mmKH2PO4) containing 5%nonfat dry milk powder. Western blot analysis was performed using an antihuman p27Kip1, cdk4, Rb, and CPP32 mouse monoclonal antibody (Transduction Laboratories, Lexington, Kentucky),antihuman p53 and Bcl-2 mouse monoclonal antibody (Dako, Copenhagen,Denmark), antihuman cyclin D and p21 mouse monoclonal antibody,antihuman cdk2, and cyclin E rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA), and anti-phospho Rb (Ser-780) rabbit polyclonal antibody (MBL, Nagoya, Japan). Primary antibodies were added to PBS containing 5% nonfat dry milk powder and incubated for 60 min at room temperature. Incubation with a secondary peroxidase-coupled goat antimouse antibody was performed under the same conditions. For detection of the immunocomplex, the ECL Western blotting detection system (Amersham, Aylesbury, United Kingdom) was used. Anti-actin mouse monoclonal antibody (C4; Boehringer Mannheim, Australia) was used for normalization of Western blot analysis.
Protein Kinase Assay.
For cdk2/cyclin E-associated kinase activity, 100 μg of the lysate were precleared in lysis buffer with 40 μl of 1:1 slurry of protein A-agarose (Oncogene Research Products, Cambridge, MA) for 30 min at 4°C. Samples were incubated with 1 μg of anti-cdk2 or cyclin E rabbit polyclonal antibody (Santa Cruz Biotechnology) for 3 h at 4°C and subsequently centrifuged. The precipitates were washed three times with lysis buffer and three times with kinase buffer [50 mm Tris-HCl (pH 7.4), 10 mmMgCl2, and 1 mm DTT]. The precipitates were then suspended in 35 μl of kinase buffer containing 6 μg of histone H1 (Sigma; type III-S), followed by a 5-min preincubation at 30°C. Subsequently, 5 μl of a 60 μm[γ-32P]ATP solution (3 μCi) were added, and the kinase reaction was carried out at 30°C for 10 min. The reaction was stopped by adding 20 μl of 4× Laemmli’s sample buffer and boiling. The samples were subjected to 10% SDS-PAGE, followed by autoradiography.
CPP32/Caspase 3 Activity.
CPP32/caspase 3 activity was determined using CPP32/Caspase-3 Colorimetric Protease assay kit (MBL, Nagoya, Japan). The assay is based on spectrophotometric detection of the chromophore pNA after cleavage from the labeled substrate DEVD-pNA. The pNA light emission can be quantified using a microtiter plate reader at 405 nm.
Antisense Oligonucleotide and Cell Viability.
The oligonucleotides were purchased from Greiner Japan (Tokyo, Japan). The antisense oligonucleotide sequence was p27Kip1 antisense (5′-GACACTCTGACGTTTGACAT-3′),which was complementary to the region around the initiation codon of p27Kip1 (25, 26). We used p27Kip1 sense (5′-CTGTGAGAGTG CAAACTGTA-3′)oligonucleotides as a control. The oligonucleotides (100μ m) and lipofectin (Life Technologies, Inc., Rockville,MD) were incubated at 37°C for 15 min. The oligonucleotide-lipofectin mixture was diluted with serum-free medium and added to the cells,giving a final concentration of 1.0 μm. Western blot analysis was performed to investigate whether antisense effectively inhibits the expression of p27Kip1 protein. After 4 days, we treated these cells with LLnV, and cell viability was determined by trypan blue exclusion.
Apoptosis Induced by Proteasome Inhibitor in OSCC Cells but not Normal Oral Epithelial Cells.
All OSCC cells treated with LLnV or lactacystin showed varying degrees of apoptotic changes. At 12 h after the proteasome inhibitor treatment, the cells showed membrane blebbing and cytoplasmic shrinkage(Fig. 1, A–D). Apoptotic body formation was observed at the ultrastructural level (Fig. 1,E). Oligonucleosomal laddering was also demonstrated in the cells at 24 h after treatment with proteasome inhibitors (Fig. 1,F). These apoptotic changes induced by LLnV or lactacystin occurred most prominently in HSC2 cells. There were no morphological changes and laddering in the cells treated with DMSO. Apoptotic change was not shown in most of normal oral epithelial cells and gingival fibroblasts at 24 h after LLnV treatment (Fig. 1 G). At 24 h after LLnV treatment, cell viability was 76 and 92% in normal oral epithelial cells and gingival fibroblasts, respectively, in comparison with 18% in HSC2 cells.
Effects of Proteasome Inhibitor on Growth and Cell Cycle of HSC2 Cells.
Because HSC2 cells showed the most remarkable apoptotic changes with the LLnV or lactacystin treatment among the OSCC cells examined, we used mainly HSC2 cells in the following analyses. LLnV and lactacystin showed time-dependent inhibitory effects on the growth of HSC2 cells,as seen in Fig. 2 A. After 24 h of the LLnV treatment, >80% cells died and detached from the culture dish. DMSO did not show inhibitory effects. We used LLnV in the following analyses mainly because LLnV treatment brought about apoptosis more remarkably than lactacystin.
Flow cytometric analysis is shown in Fig. 2 B. The ratios of the cell cycle populations of nontreated cells at G0-G1, S, and G2-M phases were about 53, 27, and 20%,respectively. After 12 h treatment with LLnV, the ratios of the cell cycle populations at G0-G1, S, and G2-M phases were about 78, 12, and 10%,respectively. The decrease of G1 cells and increase of apoptotic cells became prominent at 24 h after LLnV treatment.
Accumulation of p27Kip1 Protein by Treatment with Proteasome Inhibitor.
Proteasome inhibitors have been reported to induce apoptosis in the p53-dependent pathway (22, 23). Another report showed that the inhibition of proteasomal activity is accompanied by accumulation of p27Kip1 protein (18). Therefore,we examined expression of p53 and p27Kip1 after the LLnV treatment. No change of p53 protein level was observed, but expression of p27Kip1 protein increased in all OSCC cells (Fig. 3 A). We compared the signal intensity of p27Kip1expression before and after the LLnV treatment by densitometric scanning. The relative expression levels of signal intensity at 12 h after the LLnV treatment were 7.9, 2.9, and 1.7 in HSC2, HSC3, and Ho-1-U-1 cells, respectively. The highest level of accumulation of p27Kip1 protein was detected in HSC2 cells.
Expression of Cell Cycle Regulators after Treatment with Proteasome Inhibitor in HSC2 Cells.
After incubation with LLnV, HSC2 cells demonstrated accumulation of p27Kip1 protein at 12 h (Fig. 3,B). Expressions of cyclin D1 was reduced with time. Expression of p53 was not detected before or after incubation with LLnV. Expressions of p21, cdk2, cyclin E, and cdk4 also did not change with the LLnV treatment (Fig. 3 B).
Because accumulation of p27Kip1 and reduced expression of cyclin D1 were observed with the LLnV treatment, we examined cdk2 kinase activity, cyclin E-associated kinase activity, and expression of phospho pRb (Ser-780) to elucidate the functional effects of LLnV on the cell cycle (Fig. 4). After cell extracts were immunoprecipitated with an antibody against cdk2 or cyclin E, cdk2 kinase activity and cyclin E-associated kinase activity were measured by using histone H1 as a phosphorylation substrate. Expression of phospho pRb (Ser-780) was examined by using anti-phospho pRb (Ser-780), which reacts with only phosphorylated pRb at the site of Ser-780. This site is phosphorylated by cdk4/cyclin D1 but not by cdk2/cyclin E/A (27). LLnV treatment inhibited cdk2 kinase activity and cyclin E-associated kinase activity in HSC2 cells. The level of p27Kip1 in the immunoprecipitates was determined by Western blot analysis. The amount of cyclin E-associated p27Kip1 increased at 12 and 24 h after LLnV treatment (Fig. 4,A). Expression of phospho pRb also disappeared by 12 h of LLnV treatment, but pRb did not change expression level (Fig. 4 B).
Expression of Apoptosis-related Proteins after Treatment with Proteasome Inhibitor in HSC2 Cells.
The LLnV treatment also influenced expression of the apoptosis-related proteins, Bcl-2 and CPP32. CPP32 precursor protein was expressed at 0 h, but it declined after treatment (Fig. 5,A). CPP32/caspase 3 activity was also measured. CPP32/caspase 3 activity increased time dependently(Fig. 5,B). Expression of Bcl-2 was reduced time dependently(Fig. 5 A).
Effect of Antisense p27Kip1 Oligonucleotide on HSC2 Cells.
To determine whether accumulation of p27Kip1plays an important role in apoptosis induced by proteasome inhibitor,we used antisense p27Kip1 oligonucleotide to block expression of p27Kip1. We also used p27Kip1 sense oligonucleotides as a control. After 12 h of LLnV treatment, accumulation of p27Kip1 protein was observed in HSC2 cells (Fig. 3,A). Accumulation of p27Kip1 protein was also observed in HSC2 cells with p27Kip1sense oligonucleotide treatment but not in HSC2 cells with p27Kip1 antisense oligonucleotide treatment after 12 h of LLnV treatment (Fig. 6,A). Treatment with antisense p27Kip1 oligonucleotide blocked the expression of p27Kip1. After 24 h of LLnV treatment, cell viability was ∼20% in HSC2 cells and HSC2 cells with p27Kip1 sense oligonucleotide treatment but∼50% in HSC2 cells with p27Kip1 antisense oligonucleotide treatment (Fig. 6, B and C).Treatment with p27Kip1 antisense oligonucleotide inhibited the apoptosis induced by LLnV both time and dose dependently(Fig. 6, B and C). In Fig. 6, B and C, an unexpected protective influence of the sense oligonucleotides was shown by 24 h treatment with 10 or 25μ m LLnV but not by 50μ m LLnV. We suggest that sense oligonucleotides may influence to a lesser degree in proteasome inhibitor treatment. However, sense oligonucleotides did not influence 50μ m LLnV used in all experiments of the present study. To examine whether p27Kip1 is upstream or downstream of CPP32/caspase 3 or Bcl-2, we examined the expression of CPP32/caspase 3 and Bcl-2 in antisense p27Kip1-treated cells. CPP32 expression was detected, but Bcl-2 expression was not detected in HSC2 cells with p27Kip1 antisense oligonucleotide treatment after 12 h of LLnV treatment (Fig. 6 A).
This is the first report demonstrating the induction of apoptosis in the cells forming a solid tumor, OSCC cells, by treatment with proteasome inhibitors. These findings are consistent with reported findings that proteasome inhibitors are capable of inducing apoptosis in proliferating cells (18, 19, 20, 21). We observed the accumulation of p27Kip1 protein and apoptosis in all OSCC cell lines by proteasome inhibitor treatment. Furthermore, the most pronounced apoptotic changes were observed in the HSC2 cells that showed the highest level of accumulation of p27Kip1 protein among the OSCC cells examined(Fig. 3,A). Reduced expression of p27Kip1 has been shown recently in various carcinomas and suggested to be brought about by increased proteasome-mediated degradation rather than altered gene expression (28, 29, 30, 31, 32, 33). We have also found reduced expression of p27Kip1 in OSCC (34). Therefore, we assumed that inhibition of proteasome-mediated degradation by proteasome inhibitors might induce accumulation of p27Kip1 protein in OSCC cells. The present results clearly demonstrated that treatment with proteasome inhibitors induced accumulation of p27Kip1 protein after apoptotic changes in OSCC cells. Therefore, we suggest that accumulation of p27Kip1 protein may play an important part in apoptosis. This hypothesis is supported by the findings that treatment with antisense p27Kip1oligonucleotide inhibited apoptosis induced by proteasome inhibitors in HSC2 cells (Fig. 6).
These findings are consistent with recent reports that overexpression of p27Kip1 protein leads to apoptosis in various cancer cell lines (35, 36). However, details of the mechanism of apoptosis induced by overexpression of p27Kip1 protein are still unclear.
Both cdk2 kinase activity and cyclin E-associated kinase activity were reduced after treatment with proteasome inhibitor (Fig. 4). Accumulation of p27Kip1 protein may directly inhibit cdk2/cyclin E-associated kinase activity, which may cause cell cycle arrest at the G1 phase. Expressions of cyclin D1 and phospho pRb (Ser-780) were also reduced by the treatment with proteasome inhibitor (Fig. 3 B and 4). Cyclin D1 turnover is governed by ubiquitination and proteasomal degradation,which are positively regulated by cyclin D1 phosphorylation on threonine 286, but it does not depend on cdk4 catalytic activity (37). The reduced expression of cyclin D1 may bring about loss of phospho pRb (Ser-780) expression, because the site of Ser-780 in phospho pRb is only phosphorylated by cdk4/cyclin D1 (27). We conceive that the events after LLnV treatment may result in inhibition of cyclin D1 transcription, and effects on proteolysis are minor. Moreover, flow cytometric analysis shows that cell cycle population at the G1 phase increased at 12 h after LLnV treatment. Therefore, we suggest that the accumulation of p27Kip1 protein and the reduction of cyclin D1 protein by treatment with proteasome inhibitor brought about cell cycle arrest at the G1 phase.
Human T-cell leukemia cells, proliferating fibroblastic cells, and pheochromocytoma cells, which have a wild-type p53 protein, were reported to show the accumulation of p53 protein after the proteasome inhibitors treatment (22, 23). All OSCC cells used in the present study have a p53 mutation (38, 39), and they did not express p53 protein (Fig. 3,A). Because mutations in the p53 gene increase the stability of the p53 protein,p53 overexpression has been accepted as an indicator of possible p53 mutations. In our previous study, other OSCC cells with a p53 mutation in codon 248 overexpressed p53 protein by Western blot analysis (40). In HSC2 cells, point mutation appeared at the boundary between exon 6 and intron 6, and this point mutation would lead to the synthesis of aberrant p53 protein, terminating at the stop codon of nucleotides 65–67 of exon 7 (38). In HSC3, a 4-bp insertion was noted between codon 305 and 306 and contained a stop codon in the reading frame (38). We conceive that point mutations in these cells could not increase the stability of the p53 protein. In the present study, proteasome inhibitor treatment did not bring about p53 accumulation in these cells (Fig. 3 A). This result is supported by the report that mutant type p53 proteins were not affected by proteasome inhibition (41). We supposed that apoptosis of OSCC cells was induced by a p53-independent pathway.
A precursor of CPP32 protein, a member of the caspase family, was found to be processed by a granzyme and became an active form (42). We observed the activation of CPP32/caspase 3 in HSC2 cells treated with proteasome inhibitor (Fig. 5). This result is the same as reported findings that proteasome inhibitors enhanced CPP32-like activity (18, 43, 44). Drexler (18) suggests that inhibition of CPP32 activation may also be accomplished by the proteasome through constant degradation or processing of a protein, such as blocking the cell cycle progression located upstream of CPP32, which is critical for CPP32. Activation of CPP32 is thought to be necessary for proteasome inhibitor-induced apoptosis and plays an important role in this apoptosis signaling pathway. Besides, it is also known that Bcl-2 protein blocks the activation of CPP32 in mammalian cells (45, 46). It has been reported recently that apoptosis induced by the defects in the ubiquitin-activating enzyme E1 was blocked by overexpression of Bcl-2 (47). The reduction of Bcl-2 protein induced by the treatment with proteasome inhibitor is considered to act favorably for CPP32-related apoptosis of HSC2. Moreover, CPP32 expression was detected and Bcl-2 was not detected in HSC2 cells with p27Kip1 antisense oligonucleotide treatment after 12 h of LLnV treatment (Fig. 6 A). Treatment with p27Kip1 antisense oligonucleotide inhibited CPP32 activity slightly. This finding suggests that p27 may function upstream of CPP32 protein but not of Bcl-2.
We examined whether proteasome inhibitors induced apoptosis in the normal oral epithelial cells and gingival fibroblasts (Fig. 1 G). Apoptosis was not induced in the most of these cells by the LLnV treatment, which is quite different from the findings in OSCC cells. It is interesting that the proteasome inhibitor was capable of inducing apoptosis in cancer cells but not in normal cells. Although further studies must be done to determine the difference between normal cells and OSCC cells, treatments with proteasome inhibitor may be used as a novel therapy for OSCC.
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Supported in part by Grant 07457429 from the Ministry of Education, Science, Sports and Culture, Japan, and Tsuchiya Foundation, Hiroshima, Japan.
The abbreviations used are: CDK,cyclin-dependent kinase; Rb, retinoblastoma; pRb, Rb protein; OSCC,oral squamous cell carcinoma; LLnV,carbobenzoxy-l-leucyl-l-leucyl-l-norvalinal; pNA, p-nitroanilide.
We thank Dr. Wataru Yasui and Eiichi Tahara (First Department of Pathology, Hiroshima University, School of Medicine) for valuable discussions.