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

Studies on synchronisation in vivo: Temporary inhibition of DNA synthesis in normal and malignant mammalian cell systems with hydroxyurea

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Summary

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.

Zusammenfassung

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.

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Abbreviations

HU:

Hydroxyharnstoff

PCA:

Perchlorsäure

t C :

mittlere (mediane) Zellcyclusdauer

t G1 :

mittlere Dauer der G1-Periode des Zellcyclus

t S :

mittlere Dauer der S-Periode des Zellcyclus

t G2 :

mittlere Dauer der G2-Periode des Zellcyclus

t M :

mittlere Dauer der Mitose

n:

Gesamtzahl der Zellen einer Zellpopulation

n p :

Anzahl der proliferierenden Zellen einer Zellpopulation

n p /n:

Proliferative Fraktion einer Zellpopulation

n S :

Anzahl der in der S-Periode des Zellcyclus befindlichen Zellen einer Zellpopulation (S-Zellen)

n S* :

Anzahl der 3H-Thymidin einbauenden S-Zellen einer Zellpopulation

n S*/n:

3H-Thymidin-Markierungsindex

n M :

Anzahl der in Mitose befindlichen Zellen einer Zellpopulation

n M* :

Anzahl der in der Mitose befindlichen, 3H-Thymidin-markierten Zellen einer Zellpopulation

n M− :

Anzahl der in Mitose befindlichen, nicht 3H-Thymidin-markierten Zellen einer Zellpopulation

n M /n:

Mitoseindex

n M */n M :

Anteil 3H-Thymidm-markierter Zellen in Mitose an der Gesamtzahl der Zellen in Mitose

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Authors and Affiliations

  1. Max-Planck-Institut für Virusforschung, Abteilung Physikalische Biologie, Tübingen

    M. F. Rajewsky, D. F. Hülser & E. Fabricius

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  1. M. F. Rajewsky
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  2. D. F. Hülser
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  3. E. Fabricius
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Herrn Professor P. Dembowski (†) gewidmet.

Die Arbeit wurde mit dem Gerhard-Domagk-Preis 1970 ausgezeichnet.

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Rajewsky, M.F., Hülser, D.F. & Fabricius, E. Untersuchungen zur Synchronisation in vivo: Temporäre Inhibition der DNA-Synthese durch Hydroxyharnstoff in normalen und malignen Säugerzellsystemen. Z. Krebsforsch. 76, 266–292 (1971). https://doi.org/10.1007/BF00304031

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