Download D0301 / E100

Research Program D
Diagnostics and Experimental Therapy
Unit D0301
Toxicology and Chemotherapy
Unit of Toxicology and Chemotherapy (D0301 / E100)
Head: Prof. Dr. med. Martin R. Berger
Tel.: 06221-423310 Fax: 06221-423313 E-mail: [email protected]
Visiting Scientists
Dr. Spiro M. Konstantinov (5-8/01; 9-12/02) Sofia, Bulgaria
Dr. Milka Georgieva (3-5/01, 5-7/02; 12/02 - 2/03) Sofia,
Bulgaria
Prof. Iana Tsoneva (10-11/01; 10-12/02) Sofia, Bulgaria
Graduate Students
Cristina Voß
Michael Conzelmann
Christoph Dieterle
Hassan Adwan
Jan Sänger
Maike Leible
Tobias Bäuerle (9/01 - )
Maya Zaharieva (Erasmus Programm, 6-9/01) Sofia, Bulgaria
Magdalena Kraus (4/02- )
Michael Rodenbach (10/02- )
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Technicians
Birgit Kaiser (part time)
Gerd Braun (2/01 - )
The Unit of Toxicology and Chemotherapy originated in
1993 from the former Division of Carcinogenesis and
Chemotherapy.
The objective of this unit is to better understand mechanisms of toxicity and carcinogenesis of drugs and chemicals and to develop improved methods for diagnosis and
treatment of cancer.
In this context the mechanism of action as well as the
spectrum of anticancer and anti-protozoan activities of
alkylphosphocholines was studied. In addition, colorectal
micro-metastases as well as disseminated tumor cells
(DTC) were identified by using sensitive assays. These
included a PCR-RFLP assay for K-ras mutations in DTC
that were shed from K-ras mutated colon carcinoma, as
well as RT-PCR assays for cytokeratin 20 and
guanylylcyclase C. Finally, models of liver metastasis of
colorectal cancer as well as of bone metastasis of breast
cancer were developed and used for evaluating new
therapies.
Pharmacological characterization of
antineoplastic alkylphosphocholines
M.R. Berger, S.M. Konstantinov, M. Georgieva,
M. Zaharieva
In cooperation with: Prof. Dr. Hansjörg Eibl, Max Planck Institut
für Biophysikalische Chemie, Göttingen; Prof. Dr. Clemens Unger,
Klinik für Tumorbiologie, Freiburg; Dr. Uwe Zillmann, Zentrales
Tierlabor des DKFZ
High anti cancer activity in methylnitrosourea-induced primary rat mammary carcinoma, as first described by our
group (Berger et al., Cancer Treat. Rev., 1987, 14, 307317; Muschiol et al., Lipids, 1987, 22, 930-934) and then
by others (Hilgard et al. Eur. J. Cancer, 1988, 24, 14571461), prompted the clinical use of hexadecylphosphocholine (HPC; INN: miltefosine) in the topical treatment of
skin metastases of locally recurring breast cancer. Corresponding to our pre-clinical results, anti-cancer activity
was observed in patients, leading to remissions or stable
disease in 50% of treated women. Systemic administration
of HPC, however, failed to result in tumor responses since
gastrointestinal toxicity prevented the administration of
sufficiently high dose levels. Nevertheless, the low dose
levels administered caused significant increases in platelet
and leukocyte counts. Alkylphosphocholines have been a
main topic of interest since then and were followed to establish dose/response and structure/activity relationships
as well as new pharmacological effects and the mechanisms of action.
a) Structure activity relationships of
alkylphosphocholines in mammary carcinoma
Studies on the structure/activity relationships of alkylphosphocholines concentrated on the length of the alkyl chain,
the introduction of double bonds into the alkyl chain and
on modifications of the polar phosphocholine head. We
demonstrated that an elongation of the alkyl chain was associated with increased toxicity, but introduction of a
double bond reduced toxicity. A completely different physical property was obtained with an alkyl chain length of 22
carbon atoms since this structure no longer forms micelles
in solution and can be administered i.v. due to the absence of haemolytic properties. Compounds of this chain
length, like erucylphosphocholine, have a superior therapeutic ratio and are considered to be second generation
alkylphosphocholines (Berger et al. Drugs of Today, 1998,
34, 73-81).
b) Structure activity relationships of
alkylphosphocholines in hematopoietic cells
Studies in hematopoietic cells showed that these alkylphosphocholines caused no toxicity in normal bone marrow cells of rats and mice at concentrations used for
therapy. Instead, mild to moderate growth stimulation of
platelets and leukocytes was observed. By contrast, alkylphosphocholines inhibited the growth of a panel of leukemic cell lines with their cytotoxicity pending on the length
of the respective alkyl chain and induced apoptosis. The
DKFZ 2003: Research Report 2001/2002
Research Program D
Diagnostics and Experimental Therapy
Unit D0301
Toxicology and Chemotherapy
efficacy of these drugs was synergistically increased in
combination with gemcitabine and antisense oligonucleotides directed against the gene bcr [1, 2].
[5]. The size of osteolytic lesions in the hind-limb of rats
was detected and followed by X-rays (fig. 1) and CT-scan.
Both models will be used for therapy experiments.
Sensitive detection and quantitation of colon
carcinoma cells
Fig. 1: X-ray image of an
osteolytic metastasis in
rat tibia caused by MDAMB-231 human breast
cancer cells.
M.R. Berger, M. Conzelmann, C. Dieterle, M. Kraus
In cooperation with: Dr. Ulrich Linnemann, Klinikum Nürnberg
Nord, Nürnberg; Dr. Matthias Seelig, Klinikum Ludwigshafen,
Ludwigshafen
The main focus was on studies related to early diagnosis
of colorectal cancer. To that purpose, colorectal cancers
were analyzed for K-ras codon 12 and 13 mutations using
a sensitive PCR-RFLP assay. For patients with K-ras mutated tumors this mutation was used as marker for detecting disseminated tumor cells (DTC) in typical target tissues of colorectal cancer metastasis.
The PCR-RFLP assay used was sensitive enough to detect up to one mutant in 106 wild type cells. Based on this
assay, a K-ras mutation rate of 46% was found the primary
tumor of colorectal cancer patients. The most prevalent
mutation at codon 12 was a G→C transversion, followed
by a G→A transition which resulted in amino acids alanine
and asparagine, respectively, instead of glycine. At codon
13 G→A transitions were found only, resulting in amino
acid asparagin instead of glycine. Concomitantly investigated tissues with potential metastatic spread (lymph
nodes, liver, bone marrow) were found positive for K-ras
mutants even if histologic examinations failed to detect
any metastases. Thus, it was demonstrated that K-ras mutated disseminated tumor cells can be detected earlier
than with conventional diagnostic means by using the
PCR-RFLP assay for tissue samples from critical organs
such as the liver [3,4].
In addition, markers of epithelial cells (cytokeratin 20) and
gastrointestinal cells (guanylycyclase C) were used as surrogate markers for disseminated colorectal cancer cells as
well in order to include patients bearing tumors without Kras mutation.
Publications (* = external co-author)
[1] Georgieva M.C., Konstantinov, S.M., *Topashka-Ancheva, M.,
Berger, M.R.: Combination effects of alkylphosphocholines and
gemcitabine in malignant and normal hematopoietic cells. Cancer
Letters: 182, 163-174 (2002)
[2] Konstantinov, S. M., Georgieva, M. C., *Topashka-Ancheva,
M., *Eibl, H., Berger M. R.: Combination with an antisense oligonucleotide synergistically improves the antileukemic efficacy of
erucylphospho-N,N,N-trimethylpropyl-ammonium in CML cell
lines. Molecular Cancer Therapeutics 1, 877-884 (2002)
[3] Schimanski, C.C.; *Linnemann, U., Berger, M.R.: Sensitive detection of K-ras mutations augments diagnosis of colorectal cancer metastases in the liver. Cancer Res. 59, 5169-5175 (1999)
[4] Schimanski, C.C., *Linnemann, U., *Arbogast, R., Berger,
M.R.: Extended staging results from the detection of isolated tumor cells in the liver of colorectal cancer patients. Oncol. Rep 8:
185-188 (2001)
[5] Wittmer, A., Khazaie, K., Berger, M.R.: Quantitative detection
of lac-Z-transfected CC531 colon carcinoma cells in an orthotopic
rat liver metastasis model. Clin. Exp. Metastas. 17, 369-376
(1999)
Rat models mimicking metastasis
M.R. Berger, H. Adwan, J. Sänger, M. Leible,
T. Bäuerle, M. Rodenbach
In cooperation with: Dr. Matthias Seelig, Klinikum Ludwigshafen,
Ludwigshafen; Prof. Dr. G. Golomb, Hebrew University, Jerusalem, Israel
Rat models of metastatic colon cancer (by transplanting
tumor cells into the liver) and metastatic mammary cancer
(by transplanting tumor cells into the femur and tibia) were
developed and used to investigate the anticancer potential
of certain new drugs and treatment modes (antisense oligonucleotides encapsulated into nanoparticles, locoregional administration, chemoembolisation, antibodies).
The number of rat colon cancer cells growing in the liver
was determined by assaying the activity of β-galactosidase after transfecting the cell line with this marker gene
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