|ognizant Communication Corporation|
AN INTERNATIONAL JOURNAL
INCORPORATING ANTI-CANCER DRUG DESIGN
VOLUME 14, NUMBER 6
Oncology Research, Volume 14, pp. 267-278
0965-0407/04 $20.00 + .00
Copyright © 2004 Cognizant Comm. Corp.
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Structural Insights Into AGC Kinase Inhibition*
Christine Breitenlechner,1 Michael Gaßel,2 Richard Engh,1,3 and Dirk Bossemeyer2
1Abteilung Strukturforschung, Max-Planck-Institut fuer Biochemie,
D-82152 Martinsried, Germany
2Department for Pathochemistry, German Cancer Research Centre, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany
3Department of Medicinal Chemistry, Roche Diagnostics GmbH, D-82372 Penzberg, Germany
The AGC group of protein kinases comprises several targets for small molecule inhibitors of therapeutic significance. Crystal structure data facilitate the design or improvement of selective inhibitory molecules. Cross-selectivity of kinase inhibitors is often observed among closely related enzymes. Usually an obstacle for inhibitor design, cross-selectivity can be useful to obtain structural data from a related kinase, if not available from the original target. Protein kinase A (PKA), a representative of the AGC kinase group, has been cocrystallized with AGC group inhibitors from diverse chemical groups, thus providing structural information about binding modes, selectivity, and cross-selectivity. "Ersatz" kinases were created by mutating the inhibitor binding site of PKA to resemble other related kinases from the AGC group. The cocrystallization of these ersatz kinases with certain AGC group small molecule inhibitors elucidated some aspects of protein kinase inhibitor selectivity in this group of kinases.
Key words: Cancer; Rho-kinase inhibitor; Protein kinase C inhibitor; Crystal structure; Inhibitor selectivity; Surrogate kinase; Mutagenesis
Address correspondence to Dirk Bossemeyer, Department for Pathochemistry, German Cancer Research Centre, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. Tel: +49 6221 423266; Fax: +49 6221 423259; E-mail: D.Bossemeyer@DKFZ.de
*The essentials of this review were presented as an invited lecture at the 3rd International Conference on Inhibitors of Protein Kinases (IPK'2003), Warsaw Poland, June 22-28, 2003. See Biochem. Biophys. Acta 1697(1-2), Special Issue (Proteins and Proteomics), D. Shugar, ed. (2004).
Formation of Mitoxantrone Adducts in Human Tumor Cells: Potentiation by AN-9 and DNA Methylation
Belinda S. Parker,1 Ada Rephaeli,2 Abraham Nudelman,3 Don R. Phillips,1 and Suzanne M. Cutts1
1Department of Biochemistry, La Trobe University, Victoria,
2Felsenstein Medical Research Center, Sackler School of Medicine, Tel Aviv University, Beilinson Campus, Petach Tikva, 49100, Israel
3Chemistry Department, Bar Ilan University, Ramat Gan, 52900, Israel
The ability of mitoxantrone to form DNA adducts was investigated in a series of human tumor cell lines consisting of human cervical cancer (HeLa), human breast cancer (MCF-7), and human neuroblastoma (IMR-32) cells. The mitoxantrone-resistant human promyelocytic leukemia cell line HL60/MX2 was also compared to the parental cell line HL60 in terms of adduct formation in cellular DNA, RNA, and protein. DNA adduct formation detected using [14C]mitoxantrone as a single agent occurred at very low levels but addition of the formaldehyde-releasing prodrug AN-9 (pivaloyloxymethyl butyrate) increased adduct formation considerably in all cell lines tested. Adduct formation increased when increasing ratios of AN-9 were used, and were observed at maximal levels when AN-9 addition was 4 h after the addition of mitoxantrone. However, low levels of adducts were observed when AN-9 addition was 16 h prior to mitoxantrone. The ability of [14C]mitoxantrone to form adducts with DNA, RNA, and protein was assessed in HL60 cells, and DNA was found to be the major substrate for adduct formation. RNA was also shown to be a good substrate while protein adduct levels were consistently very low. In mitoxantrone-resistant HL60/MX2 cells, DNA adduct levels were approximately fourfold lower. To establish the influence of DNA methylation on the ability of mitoxantrone to form adducts in cells, decitabine was used to reduce DNA methylation levels in cells prior to mitoxantrone treatment. This was clearly shown to influence adduct formation, with increasing decitabine levels leading to a decrease in the level of adducts observed in both IMR-32 and MCF-7 cell lines. Collectively, these results suggest that two major factors that influence the extent of mitoxantrone adduct formation in cells are the availability of formaldehyde and the extent of genomic DNA methylation.
Key words: Mitoxantrone; AN-9; DNA methylation; DNA adducts; Decitabine
Address correspondence to S. M Cutts, Department of Biochemistry, La Trobe University, Victoria, 3086, Australia. Tel: +61-03-9479-1517; Fax: +61-03-9479-2467; E-mail: firstname.lastname@example.org.
Minimal Residual Disease in Neuroblastoma: To GAGE or not to GAGE
S. Oltra,1 F. Martínez,1 C. Orellana,1 E. Grau,1 J. M. Fernández,2 A. Cañete,2 and V. Castel2
1Unidad de Genética and 2Unidad de Oncología Pediátrica, Hospital Universitario La Fe, Valencia, Spain
We assessed the utility of GAGE gene expression as a marker of minimal residual disease (MRD) in neuroblastoma. The GAGE gene family shows a high degree of homology (>90%), clustering into two subgroups. GAGE-1, -2, and -8 form one subset, almost identical among themselves, while GAGE-3 to -7 constitute the other subset. The entire GAGE family (GAGE-1-8) was studied by RT-PCR followed by Southern blotting to increase both the sensitivity and specificity of the technique. Surprisingly, expression of GAGE was detected in 59% of peripheral blood samples from normal controls (20/35) as well as in a similar proportion from neuroblastoma patients with localized disease (stages 1 and 2). The study of GAGE-1, -2, and -8 with specific primers lowered this percentage to 28% (10/35), of which only two (6%) showed a high level of expression (directly visualized after RT-PCR). We conclude that GAGE genes can show a variable, usually low level of illegitimate expression in normal blood cells, and therefore their use as MRD markers should be taken with caution.
Key words: Minimal residual disease; Molecular marker; RT-PCR; Micrometastasis
Address correspondence to J. S. Oltra, Unidad de Genética, Hospital Universitario La Fe, Avd. Campanar 21, 46009 Valéncia, Spain. Tel: 0034961973153; Fax: 0034961973153; E-mail: email@example.com
Estimation of Radiation-Induced Interphase Cell Death in Cultures of Human Tumor Material and in Cell Lines
Elaine S. Marshall,1 Bruce C. Baguley,1 John H. L. Matthews,2 Chakiath C. Jose,2 Christopher E. Furneaux,3 James H. F. Shaw,4 James A. Kirker,5 Randall P. Morton,6 Julian B. White,7 Michael L. Rice,7 Richard J. Isaacs,8 Richard Coutts,8 and John R. Whittaker9
1Auckland Cancer Society Research Centre, University of Auckland,
Auckland, New Zealand
Departments of 2Clinical Oncology, 3Neurosurgery, 4Surgery, and 7Urology, Auckland Hospital, Auckland, New Zealand
Departments of 5Histology and 6Otolaryngology, Green Lane Hospital, Auckland, New Zealand
8Palmerston North Hospital, Palmerston North, New Zealand
9Department of Obstetrics and Gynaecology, National Women's Hospital, Auckland, New Zealand
A short-term assay method able to estimate the radiation response of human cancer tissue samples would be of great advantage to the individualization of radiotherapy in cancer patients. However, the effect of radiation on [3H]thymidine incorporation by proliferating cells reflects a composite of cell cycle arrest and induced cell death pathways. Here we consider whether it is feasible to correct for cell cycle effects based on comparison of the effects of radiation and the mitotic inhibitor paclitaxel on [3H]thymidine incorporation. Sixty-two short-term (7-day) cultures of human tumor tissue from 61 patients with melanoma, gynecological cancer, brain cancer, and head and neck cancer, as well as 18 5-day cultures of low passage human tumor cell lines, were irradiated at doses from 2 to 9 Gy, or exposed to paclitaxel (200 nM). [3H]Thymidine incorporation was measured at the end of the incubation. Cell cycle times could be estimated from the paclitaxel data and were 2.7 to 18.6 days for melanomas, 2.5 to >40 days for carcinomas, 3.9 to 39 days for brain tumors, and 1.1 to 3.8 days for cell lines. The effects of radiation on [3H]thymidine incorporation varied widely (0-97% and 0-99% inhibition for 2 and 9 Gy, respectively), and in 23 of the clinical samples, but in none of the cell lines, radiation caused significantly greater inhibition of [3H]thymidine incorporation than paclitaxel (p < 0.05). We argue that that these differences reflect radiation-induced cell loss from G1 phase and/or S phase. Responses of short-term cultures of clinical tumor material to radiation, with appropriate correction for cell cycle effects, might have the potential to provide information on radiation-induced cell death in individual patients.
Key words: Interphase cell death; Apoptosis; Radiosensitivity; Paclitaxel; Human tumors
Address correspondence to Bruce C. Baguley, Auckland Cancer Society Research Centre, University of Auckland, Private Bag 92019, Auckland, New Zealand. Tel: (64-9) 3737 999; Fax: (64-9) 3737 502; E-mail: firstname.lastname@example.org
A Scalable High-Content Cytotoxicity Assay Insensitive to Changes in Mitochondrial Metabolic Activity
Andreas Vogt, Erica N. Kalb, and John S. Lazo
Department of Pharmacology, University of Pittsburgh, Pittsburgh, PA 15261
Quantitative assessments of cell proliferation and cytotoxicity are key components of anticancer drug discovery. Advancements in genomics and combinatorial chemistry have increased the demand for cytotoxicity measurements, and almost all assays for cell proliferation that are compatible with high-throughput screening are single-parameter, homogeneous biochemical assays. Furthermore, the most popular methods currently in use are based on measurements of mitochondrial activity and therefore provide only an indirect measure of cell growth. Here we describe a high-content screening (HCS) methodology that permits the direct quantification of cell numbers as well as multiple measurements of drug activity in individual cells without the ambiguities of previously described homogenous metabolism-based assays. Using two human tumor cell lines, we compared our HCS method with a commonly used homogenous colorimetric assay that detects mitochondrial activity and found the colorimetric assay substantially underrepresented the cytotoxic effects of two mechanistically diverse, clinically used, anticancer drugs: a DNA-damaging agent, bleomycin, and a tubulin stabilizer, paclitaxel. Simultaneous evaluation of cell numbers, mitochondrial mass, and nuclear morphology by HCS provided an explanation for the differential toxicity. Furthermore, we expanded the scope of the HCS assay to include tubulin mass measurements in paclitaxel-treated cells as an example for a specific drug target. The data illustrate the utility of HCS as a powerful analysis tool compatible with the demands of high-throughput screening, and adaptable to include fluorescence-based markers of drug activity.
Key words: Cytotoxicity; High-content screening; Anticancer agents
Address correspondence to John S. Lazo, Department of Pharmacology, Biomedical Science Tower E-1340, University of Pittsburgh, Pittsburgh, PA 15261; Telephone: 412-648-9319; Fax: 412-648-2229; Email: email@example.com
Metabolic Polymoryphisms, Smoking, and Oral Cancer in Puerto Rico
Heng Xie,1 Lifang Hou,2 Peter G. Shields,5 Deborah M. Winn,3 Gloria Gridley,2 Eleuterio Bravo-Otero,6 Scott R. Diehl,7 Elise D. Bowman,4 Linda M. Brown,2 Richard B. Hayes2
1Division of Cancer Treatment and Diagnosis, 2Division
of Cancer Epidemiology and Genetics, 3Division of Cancer Control
and Population Sciences, and 4Laboratory of Human Carcinogenesis,
National Cancer Institute, Bethesda, MD 20892
5Lombardi Cancer Center, Georgetown University Medical Center, Washington, DC 20007
6School of Dentistry, University of Puerto Rico, San Juan, PR 00936-5067
7Center for Pharmacogenomics and Complex Disease Research, New Jersey Dental School, UMDNJ, Newark, NJ 07101-1709
Genetic polymorphisms resulting in variation in metabolism of tobacco carcinogens may influence oral cancer risk. In a population-based case-control study in Puerto Rico, genotypes of CYP1A1, GSTM1, and GSTT1 were determined by a PCR-based method for 132 oral cancer patients and 143 control subjects. Genotype-associated risks were estimated by logistic regression. The null variant of GSTM1 was associated with a marginally significant decrease in oral cancer risk [odds ratio (OR) = 0.6, 95% confidence interval (CI) = 0.3-1.0, and P for trend = 0.09]. Risks increased with increasing cigarette use among subjects with the GSTM1-present genotype (P for trend <0.0001), rising to OR = 9.5, 95% CI = 3.0-30, among the heaviest cigarette users. In contrast, among subjects with the GSTM1-null genotype, risks did not clearly increase with increasing cigarette use (P for trend <0.61; OR = 1.8, 95% CI = 0.6-5.2 among the heaviest tobacco users). The GSTT1-null variant (OR = 1.0, 95% CI = 0.5-1.9) and CYP1A1462Val variant (OR = 0.9, 95% CI = 0.5-1.7) were not associated with the risk. Risks rose with increasing cigarette use in a similar manner for subjects with or without the CYP1A1462Val variant (P for interaction = 0.3) and for subjects with or without the GSTT1-null genotype (P for interaction = 0.4). In conclusion, cigarette use significantly increased the risk of oral cancer in this population. The GSTM1-present genotype was associated with higher tobacco-associated risk for oral cancer among heavy smokers than the null genotype.
Key words: Polymorphism; Enzymes; Smoking; Carcinoma
Address correspondence to Richard B. Hayes, D.D.S., Ph.D., Division of Cancer Epidemiology and Genetics, National Cancer Institute, Executive Plaza South, Rm. 8114, 6120 Executive Blvd., Bethesda, MD 20892-7240. Tel: (301) 496-9093; Fax: (301) 402-1819; E-mail: firstname.lastname@example.org