Heinrich Zankl

Inhibition of cyclin-dependent kinase 1 (CDK1) by indirubin derivatives in human tumour cells

The bisindole indirubin has been described, more than 30 years ago, as being clinically active in the treatment of human chronic myelocytic leukaemia. However, the underlying mechanism of action has remained unclear. We have reported previously that indirubin and its analogues are potent and selective inhibitors of cyclin-dependent kinases (CDK). In this study, we investigated the influence of indirubin and derivatives on CDK1/cyclin B kinase in human tumour cells at concentrations known to induce growth inhibition. Cells of the mammary carcinoma cell line MCF-7, synchronized by serum deprivation, after serum repletion stay arrested in the G1/G0phase of the cell cycle in the presence of 2 μM indirubin-3′-monoxime. At higher drug concentrations (≥ 5 μM) an increase of the cell population in the G2/M phase is additionally observed. Cells synchronized in G2/M phase by nocodazole remain arrested in the G2/M phase after release, in the presence of indirubin-3′-monoxime (≥5 μM). After 24 h treatment with 10 μM indirubin-3′-monoxime a sub-G2 peak appears, indicative for the onset of apoptotic cell death. Treatment of MCF-7 cells with growth inhibitory concentrations of indirubin-3′-monoxime induces dose-dependent inhibition of the CDK1 activity in the cell. After 24 h treatment, a strong decrease of the CDK1 protein level along with a reduction of cyclin B in complex with CDK1 is observed. Taken together, the results of this study strongly suggest that inhibition of CDK activity in human tumour cells is a major mechanism by which indirubin derivatives exert their potent antitumour efficacy.

Induction of apoptosis by an inhibitor of cAMP-specific PDE in malignant murine carcinoma cells overexpressing PDE activity in comparison to their nonmalignant counterparts

In order to study potential changes in phosphodiesterase (PDE) activity associated with malignant transformation, normal primary keratinocytes and cells corresponding to different stages of epidermal tumor development in mouse skin were analyzed with respect to their 3′,5′-cyclic adenosine monophosphate (cAMP) hydrolyzing activity. Expression of cAMP-specific PDE-4, intracellular cAMP content, and the sensitivity to the growth inhibitory effect of the PDE-4-specific inhibitor 7-benzylamino-6-chloro-2 piperazino-4-pyrrolidino-pteridine (DC-TA-46) were studied in the two papilloma cell lines, MSCP6 and 308, and in the highly malignant carcinoma cell line CarB. No significant difference in soluble PDE activity and in intracellular cAMP was found in the two papilloma cell lines when compared to primary keratinocytes. In contrast, the spindle-cell carcinoma cell line CarB exhibited significantly higher PDE activity, concomitant with the lowest cAMP level. In all cell lines and also in the primary keratinocytes, rolipram-sensitive PDE-4 activity accounted for the major cAMP-hydrolyzing activity. In primary keratinocytes and in MSCP6 cells, the PDE-4 inhibitor DC-TA-46 induced at best marginal growth inhibition, whereas cell growth of 308 cells was markedly affected at concentrations >2 μM. The carcinoma cell line CarB showed the highest sensitivity to DC-TA-46 (IC50=0.8±0.3 μM). Treatment of CarB cells with DC-TA-46 strongly inhibits intracellular PDE activity, resulting in a marked and long-lasting rise of cAMP. After 24 h of treatment, arrest in the G0/G1 phase of the cell cycle is induced. Treatment with concentrations >2 μM of this highly effective PDE inhibitor results in induction of apoptotic cell death, as detected by fluorescence microscopy, flow cytometry, and ELISA-based determination of fragmented DNA in intact cells.

Double Minutes and c-MYC Amplification in Acute Myelogenous Leukemia: Are They Prognostic Factors?

A case of acute myelogenous leukemia (AML) with double minutes (dmin) and X chromosome loss is presented. Using comparative genomic hybridization (CGH), a region of high-level DNA amplification was detected at 8q24, the locus of the c-MYC proto-oncogene. Fluorescence in situ hybridization (FISH) with a DNA probe specific for the human c-MYC gene confirmed the extrachromosomal amplification of this proto-oncogene in the dmin of the leukemic cells. During the course of the disease, three relapses occurred; two complete remissions could be achieved by treatment with various chemotherapy regimens. The patients survival time of 25 months was considerably longer than in most reported cases of AML with extrachromosomal c-MYC amplification. Therefore, the present case challenges the view that the occurrence of dmin in AML is generally an indication of poor prognosis.

Exhaustive physical exercise increases frequency of micronuclei

Micronucleus (MN) tests were performed on PHA-stimulated blood lymphocytes of six healthy volunteers before and after two exhaustive sprints, causing lactate concentrations in the peripheral blood between 9.6 and 12.4 mmol/l. The number of micronuclei was significantly increased after 24-48 h in all six subjects. The mean of the total group increased from 37 MN per 3000 binucleated cells before exercise to 56 and 55 MN 24 and 48 h after exercise, respectively. These differences were also significant. These results indicate that exhaustive physical exercise causes severe mutations at the chromosome level in blood lymphocytes.

Genotoxic effects of the α, β-unsaturated aldehydes 2-trans-butenal,2-trans-hexenal and 2-trans, 6-cis-rmnonadienal

The genotoxic effects of the 2-alkenals crotonaldehyde, 2-trans-hexenal and 2-trans-6-cis-nonadienal were studied by cytogenetic methods, analyzing frequencies of sister-chromatid-exchanges, numerical and structural chromosome aberrations and micronucleus induction in human blood lymphocytes and cells of the permanent Namalva line. Crotonaldehyde and hexenal were tested in concentrations of 5 microM to 250 microM and nonadienal from 5 microM to 70 microM. Significant dose-related increases of sister-chromatid-exchanges and micronuclei were found for all three compounds. Structural chromosomal aberrations were significantly increased only by crotonaldehyde, but not by hexenal and nonadienal. In contrast numerical chromosome aberrations were not induced by crotonaldehyde whereas hexenal and nonadienal were potent inducers of aneuploidy. The micronuclei were classified by using a centromere-specific DNA probe in a fluorescence in situ hybridization assay. Hexenal and nonadienal increased the percentage of centromere-positive micronuclei, nonadienal being considerably more potent than hexenal. From these results it was concluded that crotonaldehyde acts more as a clastogen whereas hexenal and nonadienal preferentially show aneugenic effects.

Genotoxic effects of 2-trans-hexenal in human buccal mucosa cells in vivo

In 7 non-smoking healthy volunteers, the number of micronuclei (MN) was determined in exfoliated buccal mucosa cells before and after rinsing the mouth with an aqueous 10 ppm solution of 2-trans-hexenal during 3 consecutive days. All individuals showed at least a doubling of the MN frequency during one of the next 4 days. An increase of the mean group MN frequency was observed on the fourth day, becoming significant between the sixth and the seventh day. During the next 2 days, the MN frequency dropped down to nearly the control level. In a second study, 7 other volunteers were examined before and after eating 3-6 bananas per day over a period of 3 days. The bananas contained about 35 ppm of hexenal. Six of the 7 individuals showed at least a doubling of the MN frequency during one of the next 6 days. An increase in the mean MN counts was also observed, but the difference to the control value become non-significant during the test period. The results show for the first time that the flavoring constituent 2-trans-hexenal, which is present in many human foods exerts genotoxic effects on human buccal mucosa cells in vivo.

Comparison of spontaneous and idoxuridine-induced micronuclei by chromosome painting.

Fluorescence in situ hybridisation (FISH) technique with chromosome specific library (CSL) DNA probes for all human chromosomes were used to study about 9000 micronuclei (MN) in normal and idoxuridine (IUdR)-treated lymphocyte cultures of female and male donors. In addition, MN rates and structural chromosome aberrations were scored in Giemsa-stained chromosome spreads of these cultures. IUdR treatment (40 microg/ml) induced on the average a 12-fold increase of the MN rate. Metaphase analysis revealed no distinct increase of chromosome breaks but a preferential decondensation at chromosome 9q12 (28-79%) and to a lower extend at 1q12 (8-21%). Application of FISH technique with CSL probes to one male and one female untreated proband showed that all human chromosomes except chromosome 12 (and to a striking high frequency chromosomes 9, X and Y) occurred in spontaneous MN. In cultures containing IUdR, the chromosomal spectrum found in MN was reduced to 10 chromosomes in the male and 13 in the female proband. Eight chromosomes (2, 6, 12, 13, 14, 15, 17 and 18) did not occur in MN of both probands. On the contrary chromosomes 1 and especially 9 were found much more frequently in the MN of IUdR-treated cultures than in MN of control cultures. DAPI-staining revealed heterochromatin signals in most of the IUdR-induced MN. In an additional study, spontaneous and IUdR-induced MN were investigated in lymphocytes of another female donor using CSL probes only for chromosomes 1, 6, 9, 15, 16 and X. The results confirmed the previous finding that chromosomes 1 and 9 occur very often in MN after IUdR-treatment. The results indicate that decondensation of heterochromatic regions on chromosomes 1 and 9 caused by IUdR treatment strongly correlates with MN formation by these chromosomes.

Mutagenic and cytotoxic effectiveness of zinc dimethyl and zinc diisononyldithiocarbamate in human lymphocyte cultures.

The mutagenic and cytotoxic effectiveness of the vulcanisation accelerators zinc dimethyldithiocarbamate (ZDMC; ziram) and zinc diisononyldithiocarbamate (ZDINDC; arbestab Z) was tested in lymphocyte cultures of five healthy probands. ZDMC and ZDINDC (c=0.1, 1.0 and 10.0microg/ml) were studied in lymphocyte cultures without external metabolic activation. Additionally, incubation of the compounds (c=10.0microg/ml) was performed in the presence of liver microsomes from aroclor-induced rats (1 and 2h, 1 and 2mg microsomal protein). Genotoxicity testing was performed by analysis of chromosomal aberrations (CA), sister chromatid exchanges (SCEs) and micronuclei (MN). For evaluation of antiproliferative effects, mitotic index (MI) and cell cycle kinetics (CCK) were determined. In contrast to earlier investigations we found no significantly increased mutagenic or cytotoxic activity of ZDMC; ZDINDC also was inactive under these conditions.

Cocultivation studies with cells of patients bearing fragile X chromosomes.

SummaryFourteen cocultivation studies were carried out with cells of four patients with fragile X, one obligate and two possible female heterozygotes, two female controls, and a rabbit. In all cocultivations the number of fragile X chromosomes was sharply reduced in the patient cells. The strongest effect was caused by the animal cells. A distinct difference between the two controls in the reducing ability was observed. No such difference was found between the obligate and possible heterozygotes on the one hand and the controls on the other. To test the influence of the residual serum in the mixed blood cultures, the serum of a patients blood sample was replaced by the serum of a control. The frequency of fragile X chromosomes was not decreased by this procedure. Therefore a soluble factor is supposed to exist which is produced by normal or heterozygote cells in culture and which reduces the expression of fragile sites in patient cells.

Cytogenetic studies on the nucleolar organizer region (NOR) activity in meningioma cells with normal and hypodiploid karyotypes

The association pattern and NOR silver staining of the acrocentric chromosomes was studied in normal and hypodiploid cell lines of four meningioma patients. In three cases the hypodiploid cells showed a more increased association frequency or NOR silver staining or both than the normal ones, indicating a compensatory mechanism between the NORs. Chromosomes #14 seemed to be more frequently involved in this phenomenon than other acrocentric chromosomes. From the silver staining results it was concluded that in meningioma cells, chromosomes #22 which bear active as well as inactive NORs were missing. The compensation between the NORs was probably only activated if an active NOR was lost.