Time-course of DNA synthesis in human leukocyte cultures

Leukocytes

culture

609

and in the cell nests in the posterior lobe of the human pituitary gland. This study was performed with rabbit antiserum specific for human chorionic gonadotropin which was shown to cross react in gel diffusion analysis with purified human pituitary luteinizing hormone and to be capable of neutralizing its biological activity. The author is pleased to acknowledge Fai-Fong Fong.

the technical

assistance

of Miss

Ida

REFERENCES 1. BAHN, R. C., Ross, G. T. and SCIIMIT, R. W., J. Histochem. Cytol. 6, 387 (1958). 2. BARRNETT, R. J., LODMAN, A. J., MCALLASTER, K. J. and SIPERSTEIN, E. R., Endocrinology 59, 398 (1956). 3. EDIMART, E. W., SPICER, S. S. and BATES, R. W., J. Histochem. Cutol. 11. 365 (1963). 4. HALMI, N. S. and DE GROOTE, J. W., J. Clin. Endocrinol. 21, 732-(1961). 5. HYMER, W. C. and MCSHAN, W. H., J. Cell Biot. 17, 67 (1963). 6. LEZNOFF, A., FISHMAN, J., GOODFRIEND, L., McGAR~, E‘., BE&, J. and ROSE, B., Proc. Sot. Exptl Biol. Med. 104, 232 (1960). 7. MIDGLEY, A. R., JR. and PIERCE, G. B., JR., J. Expff Med. 115, 289 (1962). 8. MIDGLEY, A. R., JR., PIERCE, G. B., .JR. and WEIGLE, W. O., Proc. Sot. Exptf Biol. Med. 108.85 (1961). 9. MOUDGAL, N. R. and LI, C. H., Nature 191, 192 (1961). 10. PIERCE, G. B., JR. and MIDGLEY, A. R., JR., Am. J. Pathol. 43, 153 (1963). 11. PURVES, H. D. and GRIESBACH, W. E., Endocrinology 56, 374 (1955). 12. SWANSON, H. E. and EZRIN, C., J. Clin. Endocrinof. 20, 952 (1960). 13. WIDE, L., Roos, P. and GEMZELL, C. A., Acfu Endocrinof. 37, 445 (1961).

TIME-COURSE

OF DNA

SYNTHESIS

IN

HUMAN

LEUKOCYTE

CULTURES A. MICHALOWSKI Institute

of Oncology, Received

September

Warsaw,

Poland

20, 1963

THE widespread application of the short-term culture of human white blood cells to medical genetics and a more theoretical interest in the action of phytohaemagglutinin (PHA) on leukocytes in vitro justifies the need of a thorough investigation of the changes to which mature leukocytes are subject in the culture. The present communication is concerned with the rate of deoxyribonucleic acid (DNA) synthesis as a function of time in the population of human white blood cells cultured with the use of PHA according to a modification [3] of the original method of Moorhead et al.

[S].

A healthy 33-year-old man served as a blood donor throughout this investigation. Several 10 ml aliquots of blood were aspirated from the cubital vein into syringes containing 0.3 ml of heparin Leo (4 mg/ml 0.9 per cent NaCl) in each of 12 separate 40 - 631817

Experimental

Cell Research

32

A. Michalowski

610 experiments. Cartney’s in 0.85 per The plasma 1 : 4 with

The contents of each syringe were transferred into one separate Macbottle with 0.5 ml 6 per cent dextran (Glaxo, mol. wt. 70,000-80,000) cent NaCl. After quick gentle mixing the bottles were kept at + 1°C for 1 hr. layer with leukocytes suspended in it was aspirated afterwards and diluted TC 199 culture medium (Glaxo). Finally, PHA P (Difco, batch 456657)

1

I I 48 lime

jhours)

I I

! 72

I I

Fig. I.-Incorporation of 14C-2-Thymidine into human leukocytes maintained in the PHA-containing medium. The mean value of the activity, standard deviation and the number of bottles assayed at each time-point are given. l , the results obtained from individual bottles.

rehydrated with 5 ml of sterile distilled water, diluted 1 : 9 with 0.9 per cent NaCl was added in the proportion of 0.2 ml per 10 ml of the culture suspension. Three ml aliquots of the final suspension were kept in a water-jacket type incubator at 37°C in a number of screw-cap bottles. 14C-2-Thymidine (Amersham), 0.3 PC/ml culture, specific activity 16 mc/mM, was added at various times during the incubation to measure the rate of DNA synthesis. After 2 hr of additional incubation the cells were washed and their activity measured as described previously [5]. In control experiments in which PHA was not added to the culture suspension the mean activity of the cell sediment at the time between 24 and 84 hr of incubation was 13 counts/min and did not exceed 25 counts/min. At the beginning of the culture, the incorporation of thymidine into the leukocytes maintained in the medium containing PHA is very low (Fig. 1). This incorporation may be due to the presence of a small percentage of cells capable of DNA synthesis which are normally found in peripheral blood [2]. (Contamination of the cellular material with the labelled thymidine may account for its radioactivity as well.) The very low rate of thymidine incorporation remains unchanged for approximately 30 hr of the culture. We assume that this initial period involves two consecutive Experimental

Cell Research

32

Leukocytes

culture

processes: (1) dedifferentiation of mature leukocytes, which leads to the appearance of cells that are potentially capable of the synthesis of DNA, and (2) the next step, in which the changed cells are preparing themselves for the synthesis of DNA (presynthetic gap-G,). The duration of these processes taken separately is hardly to be established as there are no sharp and reliable criteria for the accomplishment of

Fig. 2.-Mitotic index of human leukocytes.

in the

PHA-treated

culture

L-

70 lime (hours)

!

BD

the dedifferentiation. Furthermore, they come to an end after approximately 30 hr of culture in only a small fraction of the whole cell population. For the majority of the remaining cells the dedifferentiation and G, take more time, as may be judged from the further steady increase in the number of cells incorporating thymidinethe percentage of cells that have the capacity to synthesize DNA reaches its maximum after about 60 hr of the culture (4, 71. At the time between 30 and 60 hr of the culture, the rate of thymidine incorporation into the cells increases steadily, whereas the cell number remains almost unchanged. Thus, as opposed to a growing asynchronous population of cells in which the rate of the synthesis of DNA per unit of weight and the labelling index do not change with time, leukocytes in the culture constitute about that time a population partly synchronized in respect of DNA synthesis. In such a case the steepness of the ascending portion of the curve illustrating the rate of the synthesis of DNA reflects the degree of synchronization. There seems to be some drop in the rate of the incorporation of thymidine after about 66 hr of the culture, followed by another rise at 72 hr. This may be related to the morphological inhomogeneity of the cells synthesizing DNA. It is well known that in the culture of white blood cells lymphocytes as well as the “large cells” that characterize it constitute at least two distinct types of cells that become labelled on the addition of tritiated thymidine [4,6]. The cells of the various kinds may contribute to the DNA synthesis of the whole population at different times. One can assume that when some of the cells of one type enter their post-synthetic (GB) period in about the 60th hr of the culture, the bulk of the cells of the other type just approaches the synthetic (S) period to bring the second peak of DNA synthesis at 72 hr. An experiment was carried out to find whether the morphologic inhomogeneity of the cells synthesizing DNA expresses itself likewise in the mitotic index of the culture. Colcemid (Ciba, 0.006 mg/ml culture) was added to the cell suspension for Experimental

Cell Research

32

R. G. Kessel and H. W. Beams

612

2 hr at different times of incubation and smears were then prepared. The number of mitoses was determined in 12,000 cells of two culture bottles for each time (Fig. 2). The presence of two peaks in the 65th hr and 77th hr respectively makes the curve similar to that of the incorporation of thymidine. On comparing the two curves it is possible to estimate the modal duration of G, at approximately 5 hr for the two different cell types supposed. Similarly, Bender and Prescott found that G, lasts shorter than 6 hr in their culture of human white blood cells [l]. I wish to express my sincere thanks to Dr. J. 11’. Byron of Paterson Research Laboratories, Christie Hospital and Holt Radium Institute, Manchester, for his helpful suggestions and discussion preceding the present work. REFERENCES 1. 2. 3.

4. 5. 6. 7. 8.

BENDER, M. A. and PRESCOTT, D. M., Exptl Cell Res. 27, 221 (1962). BOND, V. P, CRONKITE, E. P., FLIEDNER, T. M. and SCHORK, P., Science 128, 202 (1958). BYRON, J. W., Personal communication, 1962. COOPER, E. H., BARKHAX, P. and HALE, A. J., Bit. J. Haemutol. 9, 101 (1963). JASISSKA, J. and MICHALOWSKI, A.. Nature 196, 1326 (1962). LIMA-DE-FARIA, A. and REITALU, .J., J. Cell Biol. 16, 315 (1963). MACKINSEY, A. A., STOHLMAX, F. and BRECHER, G., Blood 19, 349 (1962). MOORHEAD,,P. S., NOWELL, P. C., MELLMAN, W. .J., BATTIPS, D. M. and HUNGERFORD, D. A.,

Exptl

Cell Res. 20, 613 (1960).

NUCLEOLAR

EXTRUSION

IN

R. G. KESSEL Marine

Biological Laboratory, State University

OOCYTES and

September

BRIAREUS

H. W. BEAMS

Woods Hole, Mass. of Iowa, Iowa City,

Received

OF THYONE

and Department Iowa, U.S.A.

of

Zoology,

30, 1963

THE

extrusion of nucleolar material into the cytoplasm has been observed using tissue culture techniques in living cells under a variety of environmental conditions [l, 5, 6, 71. Likewise the extrusion of nucleolar material into the cytoplasm, as interpreted in fixed and stained preparations has been described in so many different cases, especially during oogenesis, that it can hardly be doubted that it is of rather general occurrence in this instance as well (cf. [4, lo]). However, using light microscope preparations, the manner in which the nucleolar substances may pass through the nuclear envelope has been more difficult to establish (cf. [9]). Only a few electron Figs. 1-6.-All of nucleoli (N, x 15,500; Fig,

Experimental

electron micrographs of young oocytes nucleus; NL, nucleolus; EN, extruded 3 x 7,500; Figs. 4, 6 x 11,000.

Cell

Research

32

of Thyone showing nucleolar material;

stages in the extrusion 0, ooplasm). Figs. 1, 2, 5