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Antitumor activity of a novel histone deacetylase inhibitor (S)-HDAC42 in oral
squamous cell carcinoma
Li-Yuan Bai
a,c, Chang-Fang Chiu
a,b, Shiow-Lin Pan
f, Aaron M. Sargeant
g, Tzong-Ming Shieh
d,
Ying-Chu Wang
e, Jing-Ru Weng
e,⇑aDivision of Hematology and Oncology, Department of Internal Medicine, China Medical University Hospital, Taichung 40402, Taiwan bCancer Center, China Medical University Hospital, Taichung 40402, Taiwan
c
College of Medicine, China Medical University, Taichung 40402, Taiwan
d
Department of Dental Hygiene, China Medical University, Taichung 40402, Taiwan
e
Department of Biological Science and Technology, China Medical University, Taichung 40402, Taiwan
f
Division of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Zhunan 35053, Taiwan
g
Charles River Laboratories, Preclinical Services, Spencerville, OH 45887, USA
a r t i c l e
i n f o
Article history:
Received 8 February 2011
Received in revised form 27 July 2011 Accepted 28 July 2011
Available online 23 August 2011 Keywords:
Oral cancer (S)-HDAC42
Histone deacetylase inhibitor Akt
NF-jB
s u m m a r y
The aberrant regulation of epigenetic systems including histone acetylation contributes to inappropriate gene expression in cancer cells. In this study, we investigated the antitumor effects of the novel histone deacetylase inhibitor (S)-HDAC42 in oral squamous cell carcinoma (OSCC) cells. The antiproliferative effect of (S)-HDAC42 was multifold higher than that of suberoylanilide hydroxamic acid in a panel of oral squamous carcinoma cell lines examined. (S)-HDAC42 mediated caspase-dependent apoptosis by target-ing multiple signaltarget-ing pathways relevant to cell cycle progression and survival. We demonstrated that (S)-HDAC42 downregulated the levels of phospho-Akt, cyclin D1, and cyclin-dependent kinase 6, accom-panied by increased p27 and p21 expression. In addition, (S)-HDAC42 suppressed NF-jB signaling by blocking tumor necrosis factor-a-induced nuclear translocation, and activated reactive oxygen species generation. Finally, (S)-HDAC42 exhibited high potency in suppressing OSCC tumor growth in a Ca922 xenograft nude mouse model. Together, these findings underscore the translational value of (S)-HDAC42 in fostering new therapeutic strategies for OSCC.
Ó 2011 Elsevier Ltd. All rights reserved.
Introduction
Squamous oral cell carcinoma (OSCC) is the most commonly diagnosed oral cancer. Although the etiologies underlying the development of OSCC are not fully understood, tobacco use, alco-hol, and betel quid chewing are major risk factors. The main treat-ment modalities include radical surgery followed by chemoradiation, and definitive chemoradiation. However, the prognosis is poor for relapsed and refractory disease or for patients with metastatic disease even after therapeutic inventions with chemotherapeutic or targeted agents.1This predicament highlights
the necessity to develop novel therapeutic strategies for patients with advanced OSCC.
Aberrant epigenetic regulation has been shown to lead to inap-propriate gene expression, a key event in the pathogenesis of many types of cancer.2,3Accordingly, compounds modulating epigenetic machinery represent a key area of focus in cancer therapeutic development. For example, histone deacetylase (HDAC) inhibitors
have been shown to suppress cell proliferation,4induce
apopto-sis,5,6autophagy,7or senescence8in different cancer cells. (S)-HDAC42 is a phenylbutyrate-based HDAC inhibitor with high antitumor efficacy against various types of cancers.9–13
Evi-dence suggests that (S)-HDAC42 mediates antitumor effects through both histone acetylation-dependent and -independent mechanisms.10,14From a mechanistic perspective, this broad
spec-trum of antitumor mechanisms underlies the high potency of (S)-HDAC42 in suppressing cancer cell growth in vitro and in vivo.9–13 In the present study, we report the in vitro and in vivo antitumor efficacy of (S)-HDAC42 in OSCC cells through the interference of multiple signaling pathways relevant to cell cycle progression and survival, including those regulated by Akt, nuclear factor-kap-pa B (NF-
j
B), and reactive oxygen species (ROS).Materials and methods Cells and culture conditions
The OSCC cell lines Ca922, SAS, and HSC-3 were obtained from Japanese Collection of Research Bioresources (Tokyo, Japan). Ca922
1368-8375/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.oraloncology.2011.07.031
⇑Corresponding author. Address: 91 Hsueh-Shih Road, Taichung 404, Taiwan. Tel.: +886 4 22053366x2511; fax: +886 4 22071507.
E-mail address:columnster@gmail.com(J.-R. Weng).
Contents lists available atSciVerse ScienceDirect
Oral Oncology
cells were cultured in MEM (Invitrogen, Carlsbad, CA); SAS and HSC-3 cells were cultured in DMEM (Invitrogen). All culture med-ium were supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco, Grand Island, NY) and penicillin (100 U/ml)/ streptomycin (100
l
g/ml) (Invitrogen). Normal human oral kerati-nocytes (NHOK) were kindly provided by Dr. Yuan-Chien Chen (China Medical University Hospital) and maintained in the kerati-nocyte serum-free medium (Gibco, Grand Island, NY). All cell types were cultured at 37 °C in an atmosphere of 5% CO2.Reagents
(S)-HDAC42 [(S)-(+)-N-hydroxy-4-(3-methyl-2-phenylbutyryla-mino) benzamide] and suberoylanilide hydroxamic acid (SAHA) were synthesized as previously reported,10 and the identity and
purity were confirmed by nuclear magnetic resonance and mass spectrometry.
MTT assay
Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays in six replicates. After drug treatment, MTT (0.5 mg/ml) was added to each well and cells were incubated at 37 °C for 2 h. Reduced MTT was dissolved in DMSO (200
l
L/well), and the absorbance was measured at 570 nm. TUNEL assayCells (5 105) were cultured in 6-well plates in medium con-taining 5% FBS with or without drug treatment. Apoptotic cells were stained using the APO-Brdu™ TUNEL assay kit (Invitrogen) for 30 min at 37 °C, fixed for 30 min in 70% ethanol, and analyzed by fluorescence microscopy.
Flow cytometry
For assessment of apoptosis, cells were stained with Annexin V-FITC and propidium iodide according to the vendor’s protocols (BD Pharmingen, San Diego). Caspase-3 activation was assessed using a FITC rabbit anti-active caspase-3 kit (BD Pharmingen) according the manufacturer’s protocol. ROS production was detected using the fluorescence probe 5-(and-6)-carboxy-20,70 -dichloro-dihydro-fluorescein diacetate (carboxy-DCFDA). Data were analyzed by ModFitLT V3.0 software program.
Immunoblotting
Cell lysates were prepared using RIPA buffer (150 mM NaCl, 50 mM Tris PH 8.0, 1% NP40, 0.5% sodium deoxycholate and 0.1% sodium dodecyl sulfate) in the presence of a cocktail of protease inhibitors (Sigma–Aldrich, St. Louis, MO) and phosphatase inhibi-tors (Calbiochem, Gibbstown, NJ). Antibodies against various pro-teins were obtained from the following sources: p-Akt (Ser473), p-Akt (Thr308) (Santa Cruz Biotechnology, Santa Cruz, CA),
a
-tubu-lin, poly-ADP-ribose polymerase (PARP), HDAC1, HDAC4, p-Ij
Ba
, Ij
Ba
, Akt, NF-j
B, caspase-9, p27, cyclin D1, CDK6, GAPDH (Cell sig-naling; Beverly, MA), procaspase-8, p21 (Abcam Inc., Cambrige, MA), acetyl-histone H3, and HDAC3 (Upstate, Temecula, CA). The goat anti-rabbit IgG-horseradish peroxidase (HRP) conjugates and goat anti-mouse IgG-HRP conjugates were purchased from Perkin-Elmer life Sciences, Inc. (Boston, MA).Confocal imaging of NF-
j
B nuclear localizationCells (1 105/3 mL) were plated on cover slips in each well of six-well plates, incubated in medium with or without (S)-HDAC42
for 48 h, followed by 10 ng/mL tumor necrosis factor-
a
(TNF-a
) for 30 min. The cells were then fixed in 2% paraformaldehyde for 30 min at room temperature, and permeabilized with 0.1% Triton X-100 for 20 min. After blocking with 1% bovine serum albumin (BSA), cells were incubated with a rabbit anti-human NF-j
B anti-body overnight at 4 °C, followed by anti-rabbit IgG, washed, and subjected to confocal microscopy.In vivo efficacy of (S)-HDAC42 in an OSCC xenograft tumor model Twelve female nude mice of 5–7 weeks of age were obtained from the National Laboratory Animal Center (Taipei, Taiwan). Ca922 cells were cultured in MEM supplemented with 10% heat-inactivated FBS. Each mouse was inoculated subcutaneously with 1 107 Ca922 cells in 0.1 ml phosphate-buffered saline. Tumor diameter was measured twice weekly using calipers and the tumor volume was calculated using a standard formula: width2 length 0.52. Body weights of the mice were measured once weekly. When the mean tumor volume reached 60 mm3, mice were randomized into two groups (n = 6). Mice in the treatment group received (S)-HDAC42 orally at a dose of 25 mg/kg per day, and those in the control group received the methylcellulose/Tween 80 vehicle. All mice received treatments by gavage (10
l
L/g body weight) daily till reaching the endpoint. The criteria for endpoint included death, body weight loss more than 30% or tumor size more than 1200 mm3. The in vivo experiment protocol wasFigure 1 The cytotoxic effect of (S)-HDAC42 and suberoylanilide hydroxamic acid (SAHA) in oral cancer cells and normal human oral keratinocytes (NHOK). Antiproliferative effect of (S)-HDAC42 and SAHA in three different oral cancer cell lines (A). Cells were exposed to (S)-HDAC42 or SAHA at the indicated concentra-tions for 48 h, and cell viability was assessed by MTT assay (n = 6). NHOK were relatively insensitive to (S)-HDAC42 (B). TUNEL assay in Ca922 cells treated with (S)-HDAC42 for 48 h (C). The green color in TUNEL stain denotes DNA fragmenta-tion. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
approved by the Animal Ethical Review Committee at National Tai-wan University.
Statistical analysis
Experiments were performed at least in triplicate and results are represented as means ± standard deviation (SD) except indi-cated otherwise. For evaluating caspase-3 activation and ROS pro-duction, the Student’s t-test was used. In the in vivo study, a Log rank test was used for assessing the effect of (S)-HDAC42 on tumor growth and for analyzing the difference of time to endpoint. A Stu-dent’s t-test was also used to compare the difference in body weight with regards to day on treatment. In all experiments, a p va-lue less than 0.05 was considered statistically significant. Results
(S)-HDAC42 mediates greater cytotoxicity than SAHA in OSCC cell lines The antiproliferative effect of (S)-HDAC42 and SAHA was as-sessed using MTT assays in three OSCC cell lines, HSC-3, SAS and Ca922. Cells (2 105/mL) were exposed to (S)-HDAC42 and SAHA
at the concentration range of 0.1–1 and 1–5
l
M, respectively, both of which showed a dose-dependent decrease in cell viability. Rela-tive to SAHA, (S)-HDAC42 was consistently more potent in these OSCC cell lines (Fig. 1A). The IC50values of (S)-HDAC42 and SAHA to suppress the viability of Ca922, SAS, and HSC-3 cells at 48 h of treatment were 0.16, 0.29, and 0.79l
M, respectively, and 0.72, 1.12, and 3.25l
M, respectively. The IC50values at 24 h were higher than those at 48 h (data not shown). Therefore, we treated cells with DMSO or (S)-HDAC42 for 48 h in the following experiments. Importantly, normal human oral keratinocytes were much less sensitive to (S)-HDAC42 (Fig. 1B). TUNEL assays indicated a greater extent of DNA fragmentation in Ca922 cells in response to (S)-HDAC42 vis-à-vis vehicle control (Fig. 1C), suggesting the role of apoptosis in (S)-HDAC42-mediated cytotoxicity.(S)-HDAC42 mediates apoptotic death
We obtained several lines of evidence that (S)-HDAC42-medi-ated cell death was attributable to apoptosis. As shown, flow cytometry indicated a dose-dependent effect of (S)-HDAC42 on increasing the proportion of apoptotic cells (defined as annexin V+ cells) in all three cell lines (Fig. 2A). Furthermore, Western blot
Figure 2 HDAC42 induced apoptosis and caspase activation. Annexin V-FITC/propodium iodide analysis of apoptosis in three oral cancer cell lines treated with (S)-HDAC42 for 48 h (A). Western blotting analysis of PARP, procaspase-8 and caspase-9 in cells treated with (S)-(S)-HDAC42 for 48 h (B). (S)-(S)-HDAC42 induced a dose-dependent increase of activated caspase-3 (C, n = 3).
analysis showed that (S)-HDAC42 induced PARP cleavage and cas-pase-8 and -9 activation in a dose-dependent manner (Fig. 2B). To further confirm the involvement of caspase activation in (S)-HDAC42-mediated apoptosis, cells treated with indicated concen-trations of (S)-HDAC42 or DMSO were analyzed for activated cas-pase-3 expression by flow cytometry (Fig. 2C). Treatment with (S)-HDAC42 for 48 h induced incremental levels of activated cas-pase-3, from 1.8% in the control group to 5.6%, 10.5%, 21.8%, and 30.9% in Ca922 cells treated with 0.15, 0.2, 0.25, and 0.3
l
M of (S)-HDAC42, respectively (p = 0.013, 0.021, 0.034, and 0.025 com-pared to the control group). The activated caspase-3 was 6.8%, 10.4%, 22.1%, 43.0%, and 73.4% in SAS treated with 0.15, 0.2, 0.3, 0.5, and 1.0l
M of (S)-HDAC42, respectively (p = 0.006, 0.023, 0.024, 0.007, and 0.0002 compared to the control group). For HSC-3, the activated caspase-3 was 10.9%, 13.7%, 17.5%, and 19.1% in cells treated with 1.0, 1.5, 2.0 and 2.5l
M of (S)-HDAC42, respectively (p = 0.023, 0.014, 0.012 and 0.010 compared to the control group). These results support that (S)-HDAC42 induces cas-pase-dependent apoptosis in Ca922 cells.Effects of (S)-HDAC42 on markers related to HDAC inhibition and the activation/expression status of Akt and NF-
j
BTo demonstrate the activity of (S)-HDAC42 on HDAC inhibition, oral cancer cells (2 105/mL) were treated with (S)-HDAC42 for 48 h. HDAC inhibition was evidenced by increased acetylation
lev-els of histone H3 in a concentration-dependent manner (Fig. 3A). It is noteworthy that (S)-HDAC42 not only affected the activity, but also caused dose-dependent downregulation of the expression of HDAC1, HDAC3 and HDAC4 in Ca922, the most sensitive cells, and to a lesser extent, HDAC1 and HDAC4 in SAS cells.
To investigate other mechanisms that might underlie the apop-totic effect of (S)-HDAC42, (S)-HDAC42-treated oral cancer cell ly-sates were also blotted with antibodies against signaling proteins involved in the regulation of cell survival and cell cycle progres-sion. (S)-HDAC42 dose-dependently reduced the phosphorylation levels of Akt (Fig. 3B). The upregulation of the CDK inhibitors p21 and p27 has been reported to be a hallmark feature of HDAC inhi-bition.9,15 As shown, (S)-HDAC42 induced a dose-dependent
in-crease in the expression of p27 and p21. Moreover, our data indicate the unique ability of (S)-HDAC42 in suppressing the expression of other cell cycle-regulatory proteins, including CDK6 and cyclin D1.
As NF-
j
B signaling is a key pathway targeted by HDAC inhibi-tors in several types of cancer cells,9,16,17we analyzed the spatialchange of NF-
j
B by confocal microscopy in drug-treated Ca922, SAS, and HSC-3 cells (Fig. 3C). NF-j
B, located in the cytoplasm of cells treated with DMSO, redistributed to the nuclei when the cells were treated with 10 ng/mL of TNF-a
. Treatment with (S)-HDAC42 at a concentration of 0.17, 0.1, and 1.5l
M in Ca922, SAS and HSC-3 cells for 48 h prohibited the activation and subsequent nuclear translocation of NF-j
B.Figure 3 Effects of (S)-HDAC42 on HDAC inhibition, Akt and NF-jB pathway proteins, and cell cycle-related proteins in oral cancer cells treated with (S)-HDAC42 for 48 h (A and B). Confocal microscopic examination of the spatial change of NF-jB in cells treated with TNF-awith or without (S)-HDAC42 (C). TNF-a(10 ng/mL) for 30 min induced activation and nuclear translocation of NF-jB which was inhibited by pretreatment of (S)-HDAC42 for 48 h. The concentration of (S)-HDAC42 used was 0.17, 0.1 and 1.5lM for Ca922, SAS and HSC-3, respectively.
(S)-HDAC42 increases ROS production
Increased ROS has been reported to be responsible for the activ-ity of some anticancer drugs.18Thus we examined the level of ROS
in oral cancer cells treated with (S)-HDAC42 using a flow cytome-try. (S)-HDAC42 significantly increased ROS generation by 55%, 50%, and 137% compared to the control group in Ca922, SAS, and HSC-3, respectively (⁄
p = 0.0185, ⁄⁄
p = 0.032, and ⁄⁄⁄
p = 0.0006, Fig. 4). Co-treatment with an antioxidant N-acetylcysteine either prohibited or decreased the (S)-HDAC42-mediated increase in ROS production.
(S)-HDAC42 slows growth of Ca922 xenografts and prolongs the survival of tumor-bearing athymic nude mice
As shown inFig. 5A, (S)-HDAC42 significantly inhibited Ca922 xenograft tumor growth by 65% on day 20 compared to vehicle-treated mice (p < 0.05). The mean tumor volume on day 20 was 383 and 1090 mm3 for (S)-HDAC42- and vehicle-treated mice, respectively. In addition to inhibiting growth of xenograft tumors, (S)-HDAC42 significantly prolonged the survival time of mice (Fig. 5B). The median time to endpoint was 19 and 31 days for vehicle and compound-treated groups, respectively. Although the body weight loss of mice treated with (S)-HDAC42 did not reach the criteria for early sacrifice (30% decrease), it was statistically sig-nificant compared with that of mice of the control group beginning on day 7 of treatment (p = 0.02) (Fig. 5C).
Discussion
Epigenetic changes have been shown to play a key role in oral carcinogenesis through the dysregulation of the expression of oncogenes and/or tumor suppressor genes.19,20For example, the
HDAC inhibitor FR901228 caused the reactivation of the mapsin tumor suppressor gene expression.21In addition, HDAC inhibitors have also been shown to prevent radiation-induced oral mucositis and to inhibit chemical-induced oral carcinogenesis in a hamster model,22which was correlated with the suppression of oncomiRs
and Rad51 overexpression, the upregulation of differentiation markers and the decrease of intracellular HDAC activity and oxida-tive stress. These in vitro and in vivo data suggest the translational potential of HDAC inhibitors in oral cancer treatment. In our study, (S)-HDAC42 reduced oral tumor growth in a xenograft mouse mod-el and prolonged the median survival of tumor-bearing mice in the absence of limiting toxicity.
In addition to inhibiting HDACs, (S)-HDAC42 also caused the downregulation of Akt phosphorylation and NF-
j
B activity, and modulated the expression levels of several cell cycle-regulatoryproteins in OSCC cells. It has been demonstrated that Akt inhibition could induce the mesenchymal-to-epithelial reverting transition through the downregulation of Snail and Twist in OSCC, suggesting Akt as a target for controlling cancer metastasis.23NF-
j
B is anotherimportant target for anti-oral cancer therapy since, compared with normal epithelial cells, oral cancer cells usually express higher con-stitutive NF-
j
B levels which contribute to a malignant pheno-type.24,25NF-j
B also regulates the expression of many genes thatare important for cell protection from radiation and chemotherapy, anti-apoptosis, cell survival, tumorigenesis, and cancer metasta-sis.26,27By down regulating the NF-
j
B pathway, bortezomib andTrichostatin A have been shown to effectively inhibit the cell growth of head and neck squamous cell carcinoma.28 The con-certed actions of acetylation-dependent and acetylation-indepen-dent effects of (S)-HDAC42, which were reported in other cancer cells previously,9,12,22are considered to be important for the anti-tumor activity of (S)-HDAC42 in oral cancer.
Increased ROS is implicated in inflammation, cardiovascular disease, stroke, aging process, and cell apoptosis.29Recent cumula-Figure 4 (S)-HDAC42 induced reactive oxygen species (ROS) generation. Oral
cancer cells (2 105
/3 mL) were treated with DMSO or (S)-HDAC42 with or without N-acetylcysteine (NAC) for 48 h. The ROS production in each condition is expressed as percentage of that in the control group.⁄
p = 0.0185,⁄⁄
p = 0.032 and⁄⁄⁄
p = 0.0006, when compared with the control group.
Figure 5 Effect of (S)-HDAC42 on the growth of Ca922 xenograft and on the survival time of tumor-bearing nude mice. Nude mice were treated with (S)-HDAC42 or vehicle orally once daily (six mice per group) and tumor diameter was measured twice weekly. Growth curve of Ca922 xenograft tumors (A). Survival time to endpoint of xenograft study (B). The median time to endpoint was 19 and 31 days for vehicle- and (S)-HDAC42-treated groups, respectively. Body weight change of mice (C).
tive evidences implicate ROS generation as an effective strategy in antitumor therapy as well.18 The HDAC inhibitor LAQ-824 was
shown to induce increased ROS, mitochondrial injury and apopto-sis in human leukemia cells (blocked by N-acetylcysteine).30
Be-sides the direct cytotoxicity, LAQ-824 accentuated fludarabine lethality by ROS generation and modulation of DNA repair pro-cesses in leukemia cells.31PDX101, another HDAC inhibitor,
poten-tiated a bortezomib-induced anti-myeloma effect by inducing oxidative stress and ROS-mediated DNA damage.32 Not reported in previous studies of HDAC42, our study showed that (S)-HDAC42 increases ROS generation which could be rescued by co-treatment with N-acetylcysteine in Ca922 and SAS cells. (S)-HDAC42 induced the highest ROS production in HSC-3 cells which was partially rescued by N-acetylcysteine. This induction of ROS is noteworthy because it may represent one of the antitumor mech-anisms of HDAC inhibitors.
Data from previous studies suggest that (S)-HDAC42 has the fol-lowing advantages in addition to the aforementioned pleiotropic effect. First, it has a lower IC50 than SAHA in suppressing the growth of tumor cells.9,10This higher potency is also exemplified
in the present oral cancer study. Second, mice have been shown to tolerate the therapeutic range of (S)-HDAC42 well without overt signs of toxicity.10–13,33The therapeutic efficacy of (S)-HDAC42 is
demonstrated in the present study by the significant delay in tu-mor growth and prolonging of the time to endpoint of the xeno-graft experiment. The median time to tumor size greater than 1200 mm3was 19 and 31 days for vehicle and (S)-HDAC42-treated mice, respectively.
Preclinical changes of (S)-HDAC42 with seemingly acceptable risk: benefit ratios have included reversible testicular and thymic atrophy and leukopenia; however, mortality and significant effects on body weight after repeated oral administration in the previous studies were lacking.10,12Accordingly, the loss of body weight in the present study was unexpected, and the real impact of (S)-HDAC42 on body weight should be clarified in future studies. Importantly, a contributing effect of body weight loss on delayed growth of the OSCC xenograft tumors, in addition to the pleiotropic anticancer activity of (S)-HDAC42, could not be completely ruled out.
In conclusion, (S)-HDAC42 has promising antitumor activities against oral cancer cells in vivo and in vitro. It induces apoptosis in oral cancer cells through HDAC inhibition, downregulation of Akt and NF-
j
B signaling and modulation of cell cycle-related pro-teins. The xenograft mouse model shows its effect on slowing the growth of oral cancer. Further studies are warranted to validate the effect of (S)-HDAC42 in oral cancer clinically.Role of the funding source
The sponsors have no role in study design, collection, analysis and interpretation of data; in the writing of the manuscript; and in the decision to submit the manuscript.
Conflict of interest statement
The authors declare no competing financial interests. Acknowledgements
This work was supported in part by Grants from the Taiwan Department of Health, China Medical University Hospital Cancer Research of Excellence (DOH100-TD-C-111-005), National Science Council Grant (NSC 99-2320-B-039-007-MY2) and China Medical University (CMU95-304, CMU97-101, CMU97-082, DMR-97-017, DMR-97-021).
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