Manfred F. Rajewsky

Synchronisation in vivo: Kinetics of a malignant cell system following temporary inhibition of DNA synthesis with hydroxyurea☆

Abstract As part of an experimental approach to the synchronisation of proliferating cells in vivo, the population kinetics of a transplantable rat carcinoma (BICR/M1R) were studied following temporary inhibition of DNA synthesis with hydroxyurea (HU). The cell cycle parameters of the asynchronous BICR/M1R carcinoma were determined using the labelled mitoses technique and an optimizing computer program. As required for effective synchronisation, DNA synthesis was almost completely and instantaneously blocked by injection of an appropriate dose of HU, whereas the short time constant for the clearance of the agent from the system permitted a rapid reversal of the block. Single blocks of 5 h and 10 h duration were applied as well as blocks repeated at intervals. The resulting synchronizing effects are reflected by the short term and long term kinetic response of the system. Furthermore, a separation of subpopulations of cells accumulated at the G1-S boundary and blocked in S was observed as a result of the block. There was no evidence for cytotoxic effects after a block duration of 5 h. However, significant inactivation of cells occurred when the block was extended to 10 h. This is mainly ascribed to excessive dissociation of macromolecular syntheses, but may to some extent also be due to direct effects of the inhibitor on S cells. The kinetic and metabolic effects of temporary inhibition of DNA synthesis as well as the significance of the results for the synchronisation of cell populations in vivo are discussed.

Zellproliferation in normalen und malignen Geweben:3H-Thymidin-Einbau in vitro unter Standardbedingungen

ZusammenfassungInfolge der Einführung radioaktiv markierter DNA-Vorstufen haben sich die experimentellen Möglichkeiten zur Messung der Zellproliferation normaler und maligner Gewebe verbessert. Besonders auch Probleme des Wachstums menschlicher Tumoren können jetzt mit mehr Aussicht auf Erfolg bearbeitet werden. In diesem Zusammenhang wird über die Charakteristika einer standardisierten In-vitro-Technik zum Einbau von3H-Thymidin in Explantate aus verschiedenen Geweben von Ratte und Maus berichtet. Mit Hilfe von Autoradiographie und Flüssigkeitsszmtillationszählung wurden die Kinetik des3H-Thymidin-Einbaus und die Verteilung der DNA-synthetisierenden Zellen in den Explantaten in Abhängigkeit vom O2-Partialdruck analysiert. Bei den untersuchten Geweben waren die gemessenen Einbauwerte (relative3H-Aktivität pro mg Gewebe) den entsprechenden nach In-vivo-Pulsmarkierung autoradiographisch bestimmten Thymidin-Markierungsindices in guter Näherung proportional. Es wird die Möglichkeit diskutiert, mit Hilfe der verwendeten Methode die mittleren Thymidin-Markierungsindices von Geweben mit unbekannter proliferativer Aktivität (Tumoren) abzuschätzen.SummaryThe introduction of the use of labelled DNA precursors into cell proliferation research has resulted in better techniques for the measurement of cell population kinetics in normal and malignant tissues. There is now an increased chance of being able to successfully deal with the problems of the growth of human tumours. In this context, the characteristics of a standardised in vitro technique for the measurement of3H-thymidine incorporation into expiants of various tissues of rats and mice are described. With the aid of autoradiography and liquid scintillation counting, the kinetics of3H-thymidine incorporation were analysed as was also the distribution of labelled cells in the explants as a function of O2 partial pressure. The relative tritium activity per mg wet weight is proportional to the autoradiographic labelling index for the tissues which were investigated. The applicability of the standardised in vitro technique for the prediction of thymidine labelling indices of tissues with unknown proliferative activities (tumours) is discussed.Infolge der Einfuhrung radioaktiv markierter DNA-Vorstufen haben sich die experimentellen Moglichkeiten zur Messung der Zellproliferation normaler und maligner Gewebe verbessert. Besonders auch Probleme des Wachstums menschlicher Tumoren konnen jetzt mit mehr Aussicht auf Erfolg bearbeitet werden. In diesem Zusammenhang wird uber die Charakteristika einer standardisierten In-vitro-Technik zum Einbau von3H-Thymidin in Explantate aus verschiedenen Geweben von Ratte und Maus berichtet. Mit Hilfe von Autoradiographie und Flussigkeitsszmtillationszahlung wurden die Kinetik des3H-Thymidin-Einbaus und die Verteilung der DNA-synthetisierenden Zellen in den Explantaten in Abhangigkeit vom O2-Partialdruck analysiert. Bei den untersuchten Geweben waren die gemessenen Einbauwerte (relative3H-Aktivitat pro mg Gewebe) den entsprechenden nach In-vivo-Pulsmarkierung autoradiographisch bestimmten Thymidin-Markierungsindices in guter Naherung proportional. Es wird die Moglichkeit diskutiert, mit Hilfe der verwendeten Methode die mittleren Thymidin-Markierungsindices von Geweben mit unbekannter proliferativer Aktivitat (Tumoren) abzuschatzen.

Immuno-slot-blot: A highly sensitive immunoassay for the quantitation of carcinogen-modified nucleosides in DNA

SummaryWe have established a highly sensitive immuno-slot-blot (ISB) procedure that can be routinely applied for detection and quantitation of any heat- or alkali-stable structural DNA modification (caused by carcinogens or mutagens, for example) for which a specific (monoclonal) antibody (MAB) is available. The essential step in this assay is the immobilization on nitrocellulose filters of the structurally modified DNA in its single-stranded form. The immobilized DNA is first reacted with an MAB specifically directed against a particular modified DNA component (e.g., an alkyldeoxynucleoside), and thereafter with a second antibody directed against the first one. The second antibody can be either labeled with 125I or linked to an enzyme complex capable of eliciting a color reaction with a suitable substrate. The sensitivity of the ISB is demonstrated for two different alkyldeoxynucleosides, O6-ethyldeoxyguanosine (O6-EtdGuo) and O4-ethyldeoxythymidine (O4-EtdThd), both of which are produced in cellular DNA exposed to the alkylating N-nitroso carcinogen N-ethyl-N-nitrosourea and both of which represent DNA lesions miscoding during DNA replication and transcription. Using anti-(O6-EtdGuo) and anti-(O4-EtdThd) MABs, respectively, O6-EtdGuo and O4-EtdThd are detected at levels as low as ≧0.3×10-15mol O6-EtdGuo/3 μg DNA (O6-EtdGuo/deoxyguanosine molar ratio in DNA, ≧2×10-7) and ≧0.1×10-15 mol of O4-EtdThd/3 μg DNA (O4-EtdThd/deoxythymidine molar ratio in DNA, ≧4×10-8).

In vitro studies of cell proliferation in tumours—II: Characteristics of a standardised in vitro system for the measurement of 3H-thymidine incorporation into tissue explants

A standardized in vitro system for the measurement of 3H-thymidine incorporation into tissue explants is described, and by its use a good correlation is demonstrated between relative tritium activity per mg wet wt and autoradiographic thymidine labelling index (L.I.). The value of this technique as a method of determining overall L.I.s is discussed, especially as regards tissues with a low L.I. or with a highly inhomogeneous distribution of DNA synthesizing nuclei, as often occurs in tumours. The importance of the oxygen partial pressure in the incubated explants for the process of DNA synthesis and hence for the demonstration of 3H-labelled nuclei is considered both theoretically and on the basis of experimental data. Results of measurements on the kinetics of 3H-thymidine incorporation in the in vitro system are presented.

In vitro differentiation of neural progenitor cells from prenatal rat brain: Common cell surface glycoprotein on three glial cell subsets

Glial progenitor cell differentiation and cell lineage relationships during brain development are complex hierarchical processes depending on genetic programming, cell‐cell interactions, and microenvironmental factors. The identification of precursor cell‐specific antigens provides a tool for the study of both normal development and deviations from lineage‐specific differentiation associated with malignant transformation. Monoclonal antibody (mAb) RB13‐6 recognizes a 130‐kDa cell surface glycoprotein (gp130RB13‐6) expressed by a subset of 9OAcGD3‐positive glial precursor cells scattered in the rat neuroepithelium on prenatal day (PRD) 13. During prenatal development the fraction of gp130RB13‐6‐positive fetal brain cells (FBC) decreased from about 18% (PRD 14) to about 1.5% (PRD 22), coinciding with increasing fractions of more mature cell types, as indicated by the elevated expression of p24RB21‐15, another cell surface determinant specified by mAb RB21‐15 (Kindler‐Röhrborn et al.; Differentiation 30:53–60, 1985) and other neural cell type‐specific markers. Accordingly, gp130RB13‐6‐positive precursor cells were localized in the ventricular zones throughout brain development. Concomitant with their formation and in the adult rat brain, ependymal layers lining the ventricular surface, choroid plexus, and the leptomeninges were intensely labeled by anti‐gp130RB13‐6 mAb. As visualized by confocal laser scanning microscopy of FBC cultures from PRD 13, gp130RB13‐6 was coexpressed with the RC1 antigen by progenitor cells morphologically resembling radial glia cells. In addition, a very small subpopulation of astrocytes coexpressing gp130RB13‐6 and glial fibrillary acidic protein (GFAP; <5%) occurred 3 days after seeding. Primary FBC cultures from PRD 18 contained an increased subset of astrocytes coexpressing gp130RB13‐6 and GFAP (∼25% of all gp130RB13‐6 expressing cells), apparently generated from gp130RB13‐6‐positive precursors. Corresponding to in vivo conditions, ciliated ependymal cells but also microglial cells/macrophages and leptomeningeal cells showed strong expression of gp130RB13‐6 in culture. We thus present a new glycoprotein on the cell surfaces of a glial progenitor cell subset for further studies of cell lineage relationships between radial glia cells, astrocytes, and ependymal cells. J. Neurosci. Res. 48:95–111, 1997.

NEURAL CELL SURFACE DIFFERENTIATION ANTIGEN GP130RB13-6 INDUCES FIBROBLASTS AND GLIOMA CELLS TO EXPRESS ASTROGLIAL PROTEINS AND INVASIVE PROPERTIES

Transient expression of the differentiation and tumor cell surface antigen gp130RB13‐6 characterizes a subset of rat glial progenitor cells susceptible to ethylnitrosourea‐induced neurooncogenesis. gp130RB13‐6 is as a member of an emerging protein family of ecto‐phosphodiesterases/nucleotide pyrophosphatases that includes PC‐1 and the tumor cell motility factor autotaxin. We have investigated the potential role of gp130RB13‐6 in glial differentiation by transfection of three cell lines of different origin that do not express endogenous gp130RB13‐6 (NIH‐3T3 mouse fibroblasts; C6 and BT7Ca rat glioma cells) with the cDNA encoding gp130RB13‐6. The effect of gp130RB13‐6 expression was analyzed in terms of overall cell morphology, the expression of glial cell‐specific marker proteins, and invasiveness. Transfectant sublines, consisting of 100% gp130RB13‐6‐positive cells, exhibited an altered, bipolar morphology. Fascicular aggregates of fibroblastoid cells subsequently developed into mesh‐like patterns. Contrary to the parental NIH‐3T3 and BT7Ca cells, the transfectant cells invaded into collagen type I. As shown by immunofluorescence staining of the transfectant sublines as well as of primary cultures composed of gp130RB13‐6‐positive and ‐negative cells, expression of gp130RB13‐6 induced coexpression of proteins typical for glial cells and their precursors, i.e., glial fibrillary acidic protein, the low affinity nerve growth factor receptor, and the neural proteins Thy‐1, Ran‐2, and S‐100. In accordance with its expression in the immature rat nervous system, gp130RB13‐6 may thus have a significant role in the glial differentiation program and its subversion in neurooncogenesis.—Deissler, H., Blass‐Kampmann, S., Bruyneel, E., Mareel, M., Rajewsky, M. F. Neural cell surface differentiation antigen gp130RB13‐6 induces fibroblasts and glioma cells to express astroglial proteins and invasive properties. FASEB J. 13, 657–666 (1999)

Role of DNA repair in carcinogen-induced ras mutation.

In this contribution we discuss the gene- and cell type-specific repair of miscoding DNA alkylation products as a risk parameter in both mutation induction and malignant transformation by N-nitroso carcinogens. Upon exposure to N-nitroso compounds such as N-methyl-N-nitrosourea (MeNU) or N-ethyl-N-nitrosourea (EtNU), about a dozen different alkylation products are formed in cellular DNA. Among these are O(6)-methylguanine (O(6)-MeGua) and O(6)-ethylguanine (O(6)-EtGua), respectively, which differ only by one CH(2) group in their alkyl residue and, when unrepaired, cause G:C-->A:T transition mutations by anomalous base pairing during DNA replication. We have analyzed the global and gene-specific repair of O(6)-MeGua and O(6)-EtGua in target cell DNA, ras gene mutation frequencies, and tumor incidence, in the model of mammary carcinogenesis induced in 50-day-old female Sprague-Dawley rats by a single application of MeNU or EtNU. Both carcinogens induce histologically indistinguishable mammary adenocarcinomas at high yield. In the target mammary epithelia, O(6)-MeGua is repaired at similar slow rates in both transcriptionally active genes (Ha-ras, beta-actin), silent genes (lgE heavy chain), and in bulk DNA, by the one-step repair protein O(6)-alkylguanine-DNA alkyltransferase (MGMT; low level of expression in the target cells). The slow repair of O(6)-MeGua translates into a high frequency of mutations at the central position of Ha-ras codon 12 (GGA) in MeNU-induced tumors. O(6)-EtGua, however, is removed approximately 20 times faster than O(6)-MeGua selectively from transcribed genes via an MGMT independent, as yet uncharacterized excision mechanism. Accordingly, no Ha-ras codon 12 mutations are found in the EtNU-induced mammary tumors. Neither MeNU- nor EtNU-induced tumors exhibit mutations at codons 13 and 61 of Ha-ras or at codons 12, 13 and 61 of Ki-ras. While a moderate surplus MGMT activity of the target cells - contributed by a bacterial MGMT transgene (ada) - significantly counteracts mammary tumorigenesis in MeNU-exposed rats, this is not the case in the EtNU-treated animals. Differential repair of structurally distinct DNA lesions in transcribed or (temporarily) silent genes thus determines the probability of mutation and, together with cell type-specific and interindividual differences in DNA repair capacity, influences carcinogenic risk.

Untersuchungen zur Synchronisation in vivo: Temporäre Inhibition der DNA-Synthese durch Hydroxyharnstoff in normalen und malignen Säugerzellsystemen

The synchronous passage of proliferating cells through defined phases of the cell cycle is a prerequisite for the study of a number of problems associated with carcinogenesis and cancer therapy. It is particularly required for investigations of the differential sensitivity of mammalian cells in specific phases of the cell cycle to agents capable of initiating the process of malignant transformation, or causing cell death. The present study is concerned with the in vivo synchronisation of different rat tissues (embryo; liver; spleen; transplantable BICR/M1R tumor) by temporary specific inhibition of DNA synthesis with hydroxyurea (HU). In the cell systems investigated, HU inhibited DNA synthesis rapidly and almost completely. On the other hand, the short half-life (t1/2) of the inhibitor in the organism permitted a termination of blocking periods without delay, as required for effective synchronisation. Following single or multiple doses of HU, the t1/2 values for the HU concentration in BICR/M1R tumor tissue and rat blood were nearly identical. t1/2 in rat and human blood exceeded the corresponding value for the mouse (13 min) by factors of about 2 and 8, respectively. In the rat cell systems investigated, DNA synthesis resumed when the HU concentration decreased below a level of 1–5×10−5 moles/103 g (exception: rat embryo; ∼2×104 moles/103 g). The inhibitory effect of a specific blood concentration of HU on cellular DNA synthesis after in vivo administration of the inhibitor can be measured by the reduction of 3H-thymidine incorporation in reference cells exposed to the respective blood plasma samples in vitro. Cytotoxic effects of HU, which are often confined to cells blocked in S, were particularly evident in cells of the lymphatic type. The BICR/M1R tumor served as a model cell system for the analysis of the kinetics of cell proliferation after single and multiple blocks of varying duration. The results show that partial synchronisation of proliferating cells in vivo can be obtained by temporary inhibition of DNA synthesis under controlled conditions. Die Bearbeitung einer Reihe von Problemstellungen der experimentellen und klinischen Krebsforschung setzt die Möglichkeit einer Synchronisation proliferierender Zellsysteme in vivo voraus. Dies gilt z. B. für die Frage, ob bei Säugerzellen als Funktion ihrer Position im Zellcyclus Empfindlichkeitsunterschiede vorhanden sind, und zwar sowohl hinsichtlich der Auslösbarkeit des Prozesses der malignen Transformation durch Cancerogene, als auch in bezug auf die Inaktivierbarkeit maligner Zellen durch cytocide Agentien oder ionisierende Strahlung. In der vorliegenden Arbeit wird über Untersuchungen zur in vivo-Synchronisation verschiedener Gewebe (Embryo; Leber; Milz; transplantabler BICR/M1R-Tumor) der Ratte durch temporäre Blockade der DNA-Synthese mit Hydroxyharnstoff (HU) berichtet. HU inhibiert die DNA-Synthese in vivo spezifisch, rasch und nahezu vollständig. Das rasche Absinken der HU-Konzentration im Organismus unter den zur Hemmung der DNA-Synthese erforderlichen Schwellenwert gestattet eine hinreichend verzögerungsfreie Beendigung von DNA-Syntheseblocks, wie sie für eine effektive Synchronisation erforderlich ist. Nach ein- oder mehrmaliger Pulsapplikation von HU sind die Halbwertszeiten (t1/2) für die HU-Konzentration in BICR/M1R-Tumorgewebe und Blut annähernd gleich. Die t1/2-Werte im Blut von Maus (13 min), Ratte und Mensch verhalten sich wie etwa 1∶2∶8. In den gemessenen Zellsystemen der Ratte erfolgte die Aufhebung der DNA-Syntheseblocks bei Unterschreiten einer HU-Konzentration von 1–5×10−5 Mol/103 g (Ausnahme: Rattenembryo, ∼2×10−4 Mol/103 g). Die Inhibitorwirkung einer bestimmten, im Blut gemessenen HU-Konzentration kann mit Hilfe des 3H-Thymidineinbaus durch Inkubation entsprechender Blutplasmaproben mit Referenzzellen in vitro bestimmt werden. Cytotoxische Effekte von HU, die wahrscheinlich vorwiegend auf blockierte S-Zellen beschränkt sind, waren besonders deutlich bei Zellen vom lymphatischen Typ. Als Modellsystem für die Analyse der Proliferationskinetik nach ein- und mehrmaligen DNA-Syntheseblocks von verschiedener Dauer diente der BICR/M1R-Tumor. Die Ergebnisse zeigen, daß durch Anwendung eines Inhibitors der DNA-Synthese vom Typ des HU unter kontrollierten Bedingungen eine partielle Synchronisation proliferierender Zellen in vivo erreicht werden kann.

Nigericin enhances mafosfamide cytotoxicity at low extracellular pH

SummaryThe cytotoxicity of many alkylating anticancer drugs is increased at reduced intracellular pH (pHi). The therapeutic index of such agents could therefore be improved by lowering pHi in the target cells prior to their application. We have previously demonstrated that the formation of lactic acid can be selectively enhanced in malignant tissues via glucose-mediated stimulation of tumor cell glycolysis. However, the resulting reduction in pHi is partly compensated by the extrusion of H+ equivalents into the extracellular space, with pHi remaining closer to the physiological value than extracellular pH (pHe). For full exploitation of the proton-mediated increase in the cytotoxicity of alkylating agents, pHi should therefore be equilibrated with pHe in lactic acid-producing cells. In the present study we investigated the question as to whether nigericin, an H+/K+ antiporter enabling the entry into cells of H+ ions at low pHe, can be used to enhance the cytotoxic effect of mafosfamide (MAFO; a precursor of “activated” cyclophosphamide) on cultured M1R rat mammary carcinoma cells. At pHe 7.4, the cytotoxic effect of combined treatment with MAFO and nigericin was not superior to treatment with MAFO alone. At acidic pHe, however, MAFO cytotoxicity was potentiated by nigericin as indicated by the colony-forming capacity of M1R cells. For example, at pHe 6.2 (corresponding to the approximate mean “aggregated pH” in actively glycolyzing tumors), the colonyforming fraction of cells treated with a combination of MAFO and nigericin was 3×10−5 that of controls, as compared with a value of 5×10−2 found for cells exposed to MAFO alone. These results suggest that agents counteracting cellular mechanisms that control pHi may be candidate compounds for investigations aimed at the enhancement of alkylating drug cytotoxicity following glucosemediated pH reduction in malignant tumors in vivo.

Mutagenicities of N-nitrosodimethylamine and N-nitrosodiethylamine in Drosophila and their relationship to the levels of O-alkyl adducts in DNA

N-Nitrosodialkylamines are potent carcinogens in experimental animals. Previously, we reported that the mutagenicity of N-nitrosodimethylamine (NDMA) was 10 times higher than that of N-nitrosodiethylamine (NDEA) in the Drosophila wing spot test. To find out how to explain this difference, we have measured the levels of O-alkylated bases in the DNA of exposed Drosophila larvae. Third instar larvae were fed for 3 or 6 h with NDMA or NDEA. Part of the treated larvae were grown to adult flies to score their wings for the presence of mutant spots. From the remaining larvae, DNA was isolated and digested to deoxyribonucleosides, and the digest fractionated by high-performance liquid chromatography (HPLC). The amounts of specific alkyldeoxyribonucleosides present in the fractions were quantified by a radioimmunoassay (RIA) using monoclonal antibodies. Dose-dependent O6-methylguanine, O6-ethylguanine and O4-ethylthymine formations were found to be correlated with the induction frequencies of mutant wing spots. At the same exposure dose, the values of O6-alkylde- oxyguanosine/106 deoxyguanosine were similar for NDMA and NDEA: on feeding 20 micromol/1.5 ml feeding solution, the values for NDMA were 4.0 with 3 h and 18.5 with 6 h of exposure; with 20 micromol NDEA, the corresponding values were 5.4 with 3 h and 14.6 with 6 h of exposure. The wing spot frequencies were very different; however, with NDMA, the total numbers of spots/wing were 3.5 (3 h) and 15 (6 h), and with NDEA 0.8 (3 h) and 0.9 (6 h). Similar discrepancies exist as well between the mutagenicities and the alkylation rates observed for O4-alkylthymidines. These results suggest that the difference between the mutagenic potencies of NDMA and NDEA cannot be explained by the amounts of O-alkyl adducts formed. Different mechanisms are considered by which NDMA and NDEA may produce the genetic effects observed.