Proliferation of PHA-stimulated lymphocytes measured by combined autoradiography and sister chromatid differential staining

Preliminary

notes

423

g&C, l’hypothese selon laquelle l’organisa3. Ryser, U, Fakan, S & Braun, R, Exp cell res 78 (1973) 89. teur nucleolaire n’existe pas chez Physarum 4. Lord, A, Nicole, L & Lafontaine, J G, J cell sci 23 polycephalum. L’egale repartition des res(1977) 25. 0 F. Guttes, S & Rusch, H P, Biochim tes nucleolaires tout autour de la masse 5. Nvaaard. bibihys acta 38 (1960) 298. chromosomique, en prometaphase et meta6. Bernhard. W. J ultrastruct res 27 (1969) 250. Burglen, M J; J microsc 21 (1974)‘193. phase, conduirait alors a une distribution a 7. 8. Resultats non publies. 9. Ploton, D & Gontcharoff, M, Compt rend acad sci peu pres Cgale du rDNA dans les noyaux 285 (1977) 1251. tils. 10. Lafontaine, J G & Lord, A, J cell sci 16 (1974) 63. 11. Voet. V M & Braun. R. J mol biol 106(1976) 567. D’autre part, des etudes autoradio12. Rug,‘G & Gontcharoff, M, Compt rend acad sci graphiques realisees apt-esmarquage a l’uri283 (1976) 829. dine [3H] 10 min apt-es metaphase [12] ont 13. Gontcharoff, M & Toublan, B, Biologie cellulaire (1977) 50 a. deja montre une synthese de rRNA dans les 14. 29 De la Torre, C & Clowes, F A L, J cell sci 1I corps prenucleolaires (synthese qui Ctait (1972) 713. C S, Sonenshein, G E & Holt, C E, restee a un niveau non negligeable durant la 15. Newlon, Biochemistry 12 (1973) 2338. mitose [ 131). L’apparition de granules preJuly 25, 1978 ribosomaux a leur niveau confirme la re- Received Accepted September 27, 1978 prise rapide de la transformation des RNP fibrillaires en RNP granulaires. II apparait ainsi que les corps prenucleolaires sont pleinement fonctionnels dans la synthese du Printed in Sweden Copyright @ 1979 by Academic Press. lnc rRNA tres peu de temps apres la telophase All rights of reproduction in any form reserved 0014.4827/79/020423-OS$O?.oO/O et jouent done le role dun nucleole restructure. Ce fait est a her a l’absence de phase Proliferation of PHA-stimulated lymphocytes Gl dans le cycle cellulaire de Physarum measured by combined autoradiography and polycephalum. En effet un tel phenomene sister chromatid differential staining a deja CtCnote chez Zea mays [14] ou cerPETER E. CROSSEN and WILLIAM F. MORGAN, taines cellules ne possedant pas de Gl, Cytogenetics Unit, Cancer Society of New Zealand, presentent un nucleole organise des la telo- Christchurch Hospital, Christchurch, New Zeuland phase. Summary. Lymphocyte proliferation in culture was Enfin il se pose le probleme de la nature studied by combined [3H]TdR incorporation and sister chromatid differential staining. The majority of 1st extrachromosomique du rDNA et de l’ex- division metaphases in a 72 h culture commenced istence des restes nucleolaires, contenant DNA synthesis after 48 h and had a cell cycle of less than 24 h. A small proportion of cells from some probablement le rDNA, Cgalement extra- donors commenced DNA synthesis between 24-30 h chromosomiques. Ces deux constatations and had cell cycle times of up to 48 h. Although many entered DNA synthesis at the same time, they conduisent Q envisager les sous-unites nu- cells showed marked asynchrony in the length of their cell cleolaires et les restes nucleolaires comme cycle, with some completing one, some two and others cell cycles in the 72 h culture period. The time de veritables minichromosomes porteurs du three taken for cells to enter S following stimulation with rDNA (dont la duplication se realise tout au PHA ranged from 24 to 48 h and there was considerable variation between donors in the number of fast long de S et G2 [ 151). and slow responding cells. Phytohemagglutinin (PHA)-stimulated lymI. Guttes, S, Guttes, E & Ellis, R A, J ultrastruct res phocyte cultures are frequently used to as22 (1968) 508. 2. Ryser, U, 2 Zellforsch mikrosk Anat 110 (1970) sess the effects of mutagens and carcino108. gens, both in vivo and in vitro and as tests

References

424

Preliminary

notes

of immunological responsiveness. Since the cultures were washed twice in normal saline and rein fresh medium containing BUdR at a final number of cell cycles undergone between incubated concentration of 10 pg/ml. All cultures were mainexposure to an experimental agent and tained in the dark to avoid photolysis of the BUdRsubstituted DNA f61. Chromosome nreoarations were sampling will influence the result, par- made by the flame technique [7] after hypotonic treatticularly in cytogenetic studies [l], it is es- ment in 0.075 M KCI and fixation in acetic methanol I : 3 as previously described [8]. Slides were first sential that accurate knowledge of lympho- treated for sister chromatid differential staining using cyte proliferation in culture be obtained. the method of Perry & Wolff [9]. Those slides showing differential staining of the chromatids were deTraditional methods for studying lympho- clear stained in 50% acetic acid and stripped with Kodak cyte proliferation have relied on r3H]TdR AR10 stripping film. Following exposure for one week were developed in Kodak Dl9B developer and incorporation followed by either autoradio- they restained in Giemsa (Gurrs R66). One hundred metagraphy or liquid scintillation counting. phases from each culture were analysed for the presence of label. A metaphase was considered labelled if However, Sbromodeoxyuridine (BUdR) in- at least one quarter of the chromosomes showed label corporation combined with sister chromatid that was confined to the chromosomes. The metawere also classified as to whether they were differential staining has provided new in- phases 1st 2nd or 3rd division cells using criteria of differenformation about lymphocyte cell cycle tial Giemsa staining previously described [lo]. The number of labelled cells in each culture was detertimes [2, 31. Using the BUdR-Giemsa tech- mined from counts of 1000 mononuclear cells. A cell nique we have demonstrated that 48 h lym- was considered labelled if it showed three times the ofgrains of an equivalent area of background. phocyte cultures contain 2nd division meta- numbers A second series of cultures from different donors phases, and that there is considerable varia- were labelled continuously with r3H]TdR for the first h after which the [3H]TdR was removed, the cultion among donors in the numbers of lst, 48 tures washed twice in normal saline and then incubated 2nd and 3rd division metaphases in a 72 h in fresh medium containing BUdR for a further 24 h. from these cultures were treated for sister chroculture [4]. Of particular interest is the ori- Slides matid differential staining and autoradiography in the gin of the 1st division metaphases found in same manner as those from the pulse-labelled culcultures from most donors. Are they cells tures. with a long cell cycle or are they cells with a delayed response to PHA? We have com- Results bined sister chromatid differential staining Data from cultures pulse-labelled with r3H]with r3H]TdR incorporation to give a TdR at four hourly intervals between 24 and double labelling technique which answers 34 h and then treated for sister chromatid these questions and provides additional in- differential staining are summarised in table formation about lymphocyte proliferation in 1. Based on previous studies it was envisaged that the majority of cultures laculture. belled between 24-30 h would show a large number of labelled cells. However, we Materials and Methods found considerable variation among donors Peripheral blood samples were obtained from normal donors who were in good health and not receiving in the numbers of labelled lymphocytes and medication. The don&s ages ranged from 16 to 60 it was not until the 30-34 h labelling period years. Following detibrination and gelatine sedimentation [S] the leukocyte-rich serum was cultured for 72 h that all cultures had high numbers of lain Ham’s F10 medium containing PHA 25 PI/ml (Bur- belled lymphocytes. Only nine of the roughs Wellcome, Reagent Grade). Colchicine (Aqua Colchin, Parke Davis) was added to the cultures 4 h donors had labelled metaphases and these prior to harvest at a final concentration of 0.1 wg/ml. were mainly 1st and 2nd division cells. The [3H]TdR (Amersham, spec. act. 5000 mCi/mM) was double labelling technique permits us to deadded to a series of cultures at four hourly intervals from 24 to 34 h at a final concentration of 2 &i/ml. termine when a lymphocyte was synthesFour different donors were studied at each time interval. Following incubation with [3H]TdR for 4 h the izing DNA and how many cell cycles it had E.vp Cdl

Rc,.\ IIN I 197Y)

Preliminary

Table 1. Incidence tween 24-34 h Labelling period (hours) 24-48

28-32

30-34

425

of labelled cells and metaphases in cultures pulsed with [3H]TdR be-

% Labelled cells

Donor 1 2 3 4 5 6 7 8 9 10 II I2 13 14 I5 16

26-30

notes

Labelled metaphases % Labelled metaphases 1st 2nd 3rd 22 0 0 0 0 I 5 0 2 1 0 0 11 5 9 10

29.0 0.0 0.5 0.3 2.1 2.5 8.8 0.8 1.2 6.9 A:: 20.5 12.9 16.0 14.8

completed. In the case of donor 1, labelled 1st division metaphases must have entered S between 24-48 h and taken a further 44 h to reach metaphase. Labelled 2nd and 3rd division metaphases must have also commenced DNA synthesis at the same time, but have considerably shorter cell cycle times-in the region of 22 and 14 h respectively. Unlabelled lst, 2nd and 3rd division metaphases must have commenced DNA synthesis after the removal of the [3H]TdR (28 h) and therefore have shorter cell cycle times. Similar analyses can be made for the

0 1 4 0 0

I 0 0 0 0 0 0 0 0

8 0 I 3 3 4

: 0 1 0 0 0

other donors at the various labelling intervals. Table 2 shows the incidence of unlabelled metaphases in cultures labelled continuously with [3H]TdR for 48 h. Unlabelled metaphases must have commenced DNA synthesis after the removal of the [3H]TdR and therefore taken 48 h or longer to enter the S phase. The vast majority of unlabelled metaphases from all four donors were 1st divisions, indicating that these cells have a delayed response to PHA. Donor 20 had two unlabelled 2nd division cells. These

Table 2. Combined data from cultures labelled with r3H]TdR from 26-30 h and cultures exposed to r3H] TdR for 48 h Cultures labelled from 26-30 h % Labelled cells

% Labelled metaphases

1st

2nd

3rd cycle

Cultures labelled for 48 h % Unlabelled metaphases

11.50 15.40 0.01 0.01

6 10 0 0

1 5 0 0

5 5 0 0

0 0 0 0

19 28 14 69

Labelled metaphases in Donor 17 18 19

20

E.rl, Cdl Res II8 i 1979)

426

Preliminary notes

also must have commenced DNA synthesis after 48 h but were able to complete two cell cycles. There was considerable variation among the four donors in the numbers of lymphocytes entering S after 48 h. Donor 20 had 69% of metaphases which entered S after 48 h whereas donor 19 had only 14%. Separate cultures from these four donors were pulsed with r3H]TdR at 26-30 h and the results are also included in table 2. Again there was considerable variation. Donors 17 and 18 showed a high percentage of labelled lymphocytes, whereas donors 19 and 20 had very few lymphocytes synthesising DNA during this time period. Discussion By combining C3H]TdR incorporation with sister chromatid differential staining the number of lymphocytes synthesizing DNA, the time of DNA synthesis and the number of cell cycles can be ascertained. The combined technique showed that the majority of 1st divisions in a 72 h lymphocyte culture commence DNA synthesis after 48 h and have a cell cycle of 24 h or less. A small number of 1st division metaphases from some donors can, however, commence DNA synthesis between 24-30 h and have a 48 h cell cycle. Using a BUdR/Hoechst sequential staining technique Craig-Holmes & Shaw [3] also found lymphocytes with a 48 h cell cycle. However, there was no mention of inter-donor variation in the number of lymphocytes with a 48 h cycle. The time taken for lymphocytes to enter their first S following stimulation with PHA varied considerably between donors. Twenty-nine per cent of lymphocytes from donor 1 entered S between 24-28 h, whereas the majority of lymphocytes from donor 20 took at least 48 h. Experiments using extended culture times indicate that some lymphocytes may enter their first S between

72 and 100 h [ 11, 121.Since DNA synthesis in a lymphocyte culture is a direct result of stimulation by PHA, the variability can be directly attributed to individual variation in PHA response. From our data it would appear that there are individuals whose lymphocytes have a rapid response to PHA and others whose lymphocytes have a delayed response. Donor 1 can be regarded as a fast responder, with 29% of lymphocytes synthesizing DNA at 24-28 h, whereas donor 20 can be regarded as a slow responder, no DNA synthesis at 2630 h and 69% of metaphases entering S after 48 h. Further information regarding fast and slow responding lymphocytes comes from the experiments where separate cultures from the same donors were pulse-labelled at 26-30 h and continuously labelled for 48 h. Donors 17 and 18 would also be classified as fast responders with 11 and 15% respectively of lymphocytes synthesizing DNA at 26-30 h and low numbers of lymphocytes entering S after 48 h. Donor 19 on the other hand could be classified as an intermediate responder with no lymphocytes synthesizing DNA at 2630 h and a low number commencing S after 48 h. Presumably, the majority of metaphases (86 %) from this donor initiated S between 30-48 h. Although donor 20 also showed no DNA synthesis between 2630 h, he would be classified as a slow responder because of the high number of lymphocytes which commenced S after 48 h. There was also marked asynchrony in the rate of cell division. While a considerable number of lymphocytes entered DNA synthesis at the same time some were able to complete one cell cycle, some two and others three. Whether lymphocytes with different cell cycle times represent different subpopulations as has been suggested [ 131 remains to be elucidated. Lymphocyte cultures are clearly complex

Preliminary

mixtures of cells; some with rapid entry into S (24 h), some with delayed entry (100 h), some with a long cell cycle (48 h) and others with a short cell cycle (12 h). Individual donors show wide variation in the numbers of each type of cell making the precise measurement of lymphocyte proliferation difficult. The combination of r3H]TdR incorporation combined with sister chromatid differential staining should provide further data about lymphocyte proliferation and may provide additional information about cellular proliferation in general. This work was supported by the MRC of New Zealand and the Canterbury and Westland Division of the Cancer Society of New Zealand. We thank Dr P. H. Fitzgerald for his constructive criticism.

References I. Heddle, J A, Evans, H J & Scott, D, Human radiation cytogenetics (ed H J Evans, W M Court Brown & A S McLean) p. 6. North Holland, Amsterdam (1967). 2. Tice, R, Schneider, E L & Rary, J M, Exp cell res 102(1976) 232. 3. Craig-Holmes, A P & Shaw, M W, Exp cell res 99 (1976) 79. 4. Crossen, P E & Morgan, W F, Exp cell res 104 (1977) 453. 5. Coulson, A S & Chalmers, D G, Lancet i (1964) 468. 6. Ikushima, T&Wolff, S, Exp cell res 87 (1974) 15. 7. Scherz, R G, Stain technol37 (1%2) 386. 8. Crossen. P E. Clin genet 3 (1972) 169. 9. Perry, P’& Wolff, S: Nature 251’(1974) 156. IO. Crossen. P E & Morean. W F. Cell immunol 32 (1977) 432. Il. Sot&, L, Exp cell res 78 (1973) 201. 12. Steffen, J A & Stolzmann, W M, Exp cell res 56 (1969) 453. 13. Jasinska, J, Steffen, J A & Michalowski, A, Exp cell res 61 (1970) 337. Received July 25, 1978 Revised version received October 9, 1978 Accepted October I I, 1978

notes

421

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Scanning electron microscopic visualization of a microexudate prepared by the release of cells by urea CHARLES E. SCHWARTZ,‘* LOREN HOFFMAN.* CARL G. HELLEROVIST’ and LEON W. CUNNINGHAM,’ ‘Department of Biochemistry and *Department of Anatomy, Vanderbilt University, Nashville, TN 37232, USA Summary. The scanning electron microscope (SEM) was used to examine the residue remaining after human fibroblasts, permitted to attach to plastic dishes in the absence of serum, were removed with buffered 1 M urea. The ‘urea carpet’ is entirely different from the “substrate-attached material” (SAM) of Culp [4, 5, 6] in that it contains no “footpad” material. Furthermore, the SEM pictures clearly indicate that urea carpet stimulates the adhesion and spreading of newly added tibroblasts.

Freshly trypsinized human fibroblasts, in the absence of serum, attach to plastic and secrete a microexudate carpet to which they then adhere [l]. We have shown [2] that radioactive proline is incorporated into hydroxyproline containing proteins in this microcarpet and have reported [3] that such cells, after 4 h in serum-free medium, could be released by bacterial collagenase. It was also shown that collagenase pretreatment of a microexudate carpet, prepared by the release of cells by buffered 1 M urea (‘urea carpet’) caused up to a 80 % reduction in the rate of adhesion of single cells to this carpet. Brief trypsin treatment had a lesser effect. We now present scanning electron microscopic (SEM) evidence that the urea carpet is markedly different from the “substrate-attached material” (SAM) of Culp [4, 5, 61 in that no “footpads” are left behind when cells are removed by urea. Furthermore, SEM pictures corroborate the results of adhesion studies previously reported [3]. * Present address: Department of Biochemistry, University of Vermont, Burlington, VT 05401, USA. E.rp Cell Rrs I I8 f 1979)