3- Roba, J., et at.: Continental Pharma report (to be published) 4. Roncucci, R., et al.: ibid. 5. Peeters, H., et al.: Nature (submitted) 6. Gillot, P.: unpublished data 7. Finger, K.F., Page, J.G., FeIler, D.R. : Biochem. Pharmacol. 15, tO23 (t966) 8. Blaton, V., Vandamme, D., Peeters, H. : F E B S Letters 44, 185 (t974) 9. Duncombe, W.G.: Biochem. J. 83, 6p (1962); 88, 7 (1963); Clin.. Chim. Acta 8, 122 (1964) t0. Muller, D.W., Allen, D.D.: Soc. Exp. Biol. Med. 136(3), 7tt (t971) ; Andersson, R., et al.: Atherosclerosis 18, 399 (1973); Blackard, ~vV.G., Kokatnur, M.G.: J. Atheroscler. Res. 8, 189 (1968) t l . Dole, V.P.: J. Clin. Invest., 35, 150 (t956)

Induced Cell-Cycle Synchrony of Hepatocytes after Partial Hepatectomy H. M. Rabes, G. Iseler, and H. V. Tuczek Institute of Pathology, University of Munich DNA synthesis of hepatocytes in rat liver after two-thirds hepatectomy commences in a topical sequence with a rather low degree of syrtchrony and variable rate of influx into S phase. The maximum number of DNA-synthesizing hepatocytes is limited [11. These are disadvantages for quantitative biochemical studies, but they can be overcome by a chemically induced accumulation of hepatocytes near the G 1 - S boundary, effected by continuous administration of hydroxyurea (HU) ~2, 3] following partial hepatectomy. After release from the HU block, hepatocytes enter DNA synthesis with a high degree of synchrony E4]. I t is not yet known whether the unbalanced growth [5] due to prolonged inhibition at the G 1 - S boundary influences proliferative activity after release from K U block. Male Wistar rats (AF/Han, 220--240 g) were two-thirds hepatectomized [6] and received a continuous i.v. infusion of HU (t.25 • t0 -a M/kg/h) from t4 to 40 h after operation. After HU termination, incorporation of 3H-thymidine (3H-TdR, 0.8 ~Ci/g, spec. act. 5 Ci/mmoI, i.p., Radiochemical Centre, Amersham) into hepatocytes was estimated by autoradiography 60 min after ~H-TdR injection (paraffin sections 4 ~m, Ilford K 2, 21 days exposure). Nearly all hepatocytes are 3H-TdR labeled, thus indicating a simultaneous start of DNA synthesis after HU treatment irrespective of lobular localization (Fig. I a). After about 9 h first hepatocellular mitoses are seen. However, synchrony of mitoses is low, either due to the different duration of G~ as demonstrated in various parts of the Iobule Ill, or due to failure of cells to start mitosis at all after the synchronizing procedure. By means of a continuous infusion of the metaphasearresting vincristine [71 (t .7 • 10-~ g/kg/h, Vincristin, Lilly, GieBen) for near-maximum time interval of ts + tG 2, i.e. about t 1 h after termination of HU infusion, it can be shown t h a t a high percentage of hepatocytes is accumulated in metaphase (Fig. I b). The reversibility of the arrest is dose-dependent. Prolonged inhibition by HU of DNA synthesis after partial hepatectomy, which results in a highly synchronized start of DNA synthesis after release from the HU block, does not interfere with the progress of hepatocytes through the ceil-

142

Fig. 1. (a) Autoradiogram of rat liver 45 h after partial hepatectomy, 60 min after injection of 8H-thymidine, following a continuous i.v. infusion of hydroxyurea from t4 to 40 h after liver resection. H&E, t 0 0 • (b)Metaphase accumulation of hepatocytes 5t h after partial hepatectomy following a continuous i.v. infusion of hydroxyurea from 14 to 40 h, and vincristir~e from 40 to 51 h after liver resection. H&E, 250 •

cycle phases S, G~, and M. The value of this new synchronized model of a differentiated cell system iu vivo for studies of growth regulation and carcinogenesis is discussed elsewhere

D, 4, 8;. Supported by grants from the Deutsche Forschungsgemeinschaft. Received January 27, t975 1. Rabes, H. M., in: Progress in Liver Diseases, Vol. 5 (eds. H. Popper and F. Schaffner). New York-London: Grune and Stratton (in press) 2. Schwartz, H. S., et at.: Cancer Res. 25, 1867 (1965) 3. Rajewsky, M. F.: Exp. Cell Res. 60, 269 (t970) 4. Rabes, H. M., etal.: Experientia 30, 11t6 (t974) 5- Rueckert, R. R., Mneller, G. C.: Cancer Res. 20, 1584 (1960) 6. ttiggins, G. M., Anderson, R. M.: Arch. Path. 12, t86 (t93t) 7. Noble, R. L., etal.: Ann. N.Y. Acad. Sci. 76, 882 (1958) 8. Rabes, H. M., et al.: Experientia (submitted)

Naturwissenschaften 62 (t975)

9 by Springer-Verlag 1975

Induced cell-cycle synchrony of hepatocytes after partial hepatectomy.

3- Roba, J., et at.: Continental Pharma report (to be published) 4. Roncucci, R., et al.: ibid. 5. Peeters, H., et al.: Nature (submitted) 6. Gillot,...
319KB Sizes 0 Downloads 0 Views