Depression of RNA synthesis in the prematurely condensed chromatin of pulverized HeLa cells

Experimental Cell Research69 (1971) 172-376

DEPRESSION OF RNA SYNTHESIS IN THE PREMATURELY CONDENSED CHROMATIN PULVERIZED HeLa CELLS

OF

S. STENMAN Department

of Pathology

III,

University

of Helsinki,

Helsinki,

Finland

SUMMARY Chromosome pulverization is a premature condensation of interphase chromatin (PCC) in fused cells, apparently related to the presence of metaphase chromosomes in the same cytoplasm. The RNA synthesis in such chromatin in fused cells produced by treatment with Sendai virus was investigated. When 3H-uridine was administered before cell fusion, all PCC cells had incorporated the isotope, but when uridine was administered after virus treatment, the incorporation in PCC cells was strongly reduced. Fusion as such, or the virus infection, were excluded as a cause of the inhibition of RNA synthesis. The phenomenon appears analogous to the inhibition of RNA synthesis occurring during the metaphase coiling of chromosomes or in heterochromatin.

Chromosome pulverization is a premature that depression of RNA synthesis is caused condensation of interphase chromatin [l, 11, also by the fusion-induced premature con231 first described in human cells infected densation of interphase chromatin. with measles virus [15]. By the mechanism of virus-induced cell fusion [16, 19, 241 a metaMATERIAL AND METHODS phase and one, or several, interphase cell(s) form a syncytium in which the interphase HeLa cells were cultured in plastic Petri dishes (15 x 60 mm, C. A. Greiner u. Sohne, Niirtingen, nucleus is “pulverized” while the metaphase BRD) in Eagle basal medium containing double chromosomes remain unaltered [6, 15, 181. concentration of amino acids, 10 % calf serum, penicillin (100 III/ml) and streptomycin (50 pug/ml). In Morphologically the pulverized chromatin coverslip cultures 21 x 25 mm coverslips were placed varies greatly [2] and the condensed chro- at the bottom of a dish. Cell fusion was effected by adding 1 ml of undiluted (4 000 HAU/ml) beta mosomes can have a shape ranging from compropiolactone inactivated, non-infectious [19] Sendai pletely irregular, paired or unpaired, small virus suspension to a cell monolayer for 15 min at Culture medium was then added, and cells fragments of chromatin to a prophase- or 37°C. were harvested after 1 h of virus treatment and a prometaphase-like condition [2, 11, 20, 211. 30 min Colcemid (6 x 1O-s M; Ciba) block. Chromosome preparations were made from hypotonically The chromosome condensation of metatreated cells by conventional flame drying, and stained phase chromosomes [12, 17, 251 and the con- with Giemsa. For labelling experiments, 3H-uridine (10.0 Ci/mM, densation of heterochromatin during interNew England Nuclear Corp., Frankfurt/M) was used. phase [5, 10, 131 are associated with a depres- Before autoradiography 100 cells from each culture were recorded slides were destained in acetic sion or inhibition of DNA dependent RNA --:rl -r~ e-n+.:AThe rriztl, T‘-r.Aolr AD rn Qtrinninn film

Depression

Table I. Incorporation

of 3H-uridine in syncytia of ~~e~ap~ase and prematurely condensed chromosomes Time of labelling (min) 70 66) 30 10

PCC Iabelled

PCC unlabelled

n

%

n

%

102 89 47 21

100 86 52 23

0 14 44 71

14 48 77

Virus treatment 1 h. Concentration of W-uridine 3 &i/ml in the experiment where a 10 min treatment was done; in the others 2 &i/ml. radiograms of coverslip cultures were made after fixation in glacial acetic acid and methanol (1 : 3). The totai incorporation of SH-uridine into a cell culture was calculated by scintillation counting of the TCA-precipitabie material in a Packard Tri Carb liquid scintillation counter.

RNA synthesisin KC

Table 1 shows the frequency of PCC cells synthesizing RNA as detected by autoradiography of conventional chromosome preparations: Virus treatment lasted 1 h and uridine was given to the cultures at different times before harvest, as indicated in table 1. The uridine which was given before the cells were fused by virus treatment was incorporated into the RNA of all PCC cells. When the time of labeiling was reduced, however, unlabelled PCC cells became frequent, especially when the labelling was done during the last 10 min only before harvest, when 77 % of the cells did not incorporate uridine. In the chromosome preparations many syncytial cells contained metaphase chromosomes, prematurely condensed chromosomes (“‘pulverized”), and unaffected interphase nuclei which had not undergone PCC. In every instance such interphase nuclei continued to synthesize RNA even when no syn-

of RNA synthesis in pulverhed

cells

373

thesis could be detected in the PCC in the same cell (fig. 1) Fourhundred syncytial cells containing only interphase nuclei were scored from autoradiograms of coverslip cultures, There was no inhibition of these ntlclei as compared with non-fused interphase cells in the same cultures (fig, 2). etaphase cells were wea labelled -uridine was given for -90 mill before harvest, but unlabelled if uridine was given for lo-30 min before harvest. Single prophase cells were unlabelled snly in the experiment in which uridme was given during the last 10 min in culture. Various mor~ho~og~ca~ types of PCC

The morphology of the prematureIy condensed chromatin depends on which phase in the cell cycle the interphase nucleus occupies at the moment of cell fnsion [ll, 20, Z]. During G2 the chromatin condenses into homogeneously thick strands resembling regular prophase or prometaphase chrome-. somes. uring the beginning of G 1 the prematurely condensed chromosomes are also strands, but thinner than during 62, and Table

2. Akmber of syncytia, ,~~~ta~~~l~g metaphase chromosomesand n~or~~~~~g~~a~~y different kinds of prematurely condense chromosomes,incorporating ~~-~r~di~e Time of labelling (min) Morphology of PCC

Pro- or prometaphase-like Labelled Unlabelled Intermediate Labelled Unlabelled Pulverized Labelled Unlabeiled Virus treatment @X/ml.

1 h.

Concentration

90

68

30

9 0

18 I

5 5

35 a

14 6

6 22

53 2

5’7 7

38 17

W-cridlne

2

of

374

S. Stenman

Fig. 1. Autoradiogram of a syncytial cell containing an interphase nucleus, prematurely condensed interphase chromatin (PCC), and normal metaphase chromosomes. Only the interphase nucleus has incorporated 3Huridine. Fig. 2. Autoradiogram of a syncytial cell containing two normal interphase nuclei and a single interphase cell. The RNA synthesis is not repressed in the binucleate cell.

always eroded. These two types are pooled under the title “pro- and prometaphase-like PCC” in table 2. At the end of Gl and at the beginning of S the chromatin condenses into fragments of varying length connected by thin strands (“Intermediate PCC” in table 2). In the S phase the PCC is irregularly condensed, with small clumps resembling minute fragments of metaphase chromatids (“Pulverized PCC” in table 2). The labelling patterns of these different kinds of PCC are presented in table 2. 3M-uridine was given for varying periods prior to harvest. It is evident that the RNA synthesis was inhibited earlier in the morphologically “intermediate” type of PCC than in the pulverized or the pro- or prometaphase-like PCC. The significance of this observation, however, is not clear. Exptl

Cell Res 69

Total RNA synthesis The effect of virus treatment on random cell cultures is shown in table 3. Each figure is the mean of two cultures. The general depression caused by the virus treatment is most severe during the first 30 min, but thereafter the cells partly recover and the rate of RNA synthesis increasescontinuously until harvest. This is the opposite to the repression of RNA synthesis in PCC cells, which became more severe as the virus treatment continued. DISCUSSION The function of the nucleus in a eukaryote cell is greatly influenced by the cytoplasm. In the egg cell all the information needed for the development of the fertilized egg is

Depression Table 3. Incorporation of YH-uridine in HeLa ceil cultures treated with beta propioiactone inncticated SeEdai virus Time of labeiling (tin)

Counts per minute Virus-treated

Control’

70 (1 .Q&i/ml) * 30 12.3,LGiinl)I 10 (3.5 &ihl)

1 668 (28 %)” I 594 (41 %) 1 447 (53 %j

5 976 3 898 2 784

a Phosphate buffered saline used instead of virus. * Cnncent:ation cf W-uridine. ’ Per cent of control. Virus treatment 1 h. present in the cytoplasm [8], and the cytoplasm of somatic cells can also direct the function of the nucleus [3]. The Sendai virusinduced cell fusion has offered an excellent tool in the investigation of this nuclearcytoplasmic relationship. Chromosome pulverization is a dramatic example of such an influence of the cytoplasm on the nucleus. By virus-induced cell fusion an interphase nucleus is introduced into the cytoplasm of a metaphase cell. Within some minutes of fusion, the interphase nuclear membrane is broken down [I] and the interphase chromatin starts to condense although it is not yet functionally nor structurally mature to enter mitosis. This premature condensation of the chrolmosomes occurs already :5 min after cell fusion [2]. We have discovered in the present work that the condensation is associated with a depression or a complete inhibition of the RNA synthesis of the cell. This is analogous to the decrease in RNA synthesis during mitosis [12, 251 and in heterochromatin [IQ, 131: chromosome condensation leads to depression of RNA synthesis. In our experiment the cells had recovered from the general repression. of RNA synthesis, caused by the virus treatment and manipulation of the cultures, when the repressicn in PCC was most extensive, namely during the last IO-30 min in culture after 1 h virus

of

KVA synthesis in pulverized ce!i~

3 75

treatment. This suggests that the repression of PGC is not an effect of the virus itself, In syncytia containing only interphase nuclei NA synthesis continued in all the individual nuclei, indicating that the cell fusion process did got cause a repression by itself either. Even in syncytia, which in addkion to interphase nuclei contained meeapbase 3r prematurely condensed interphase chmmosomes, the intact nuclei synthesized (fig. 1). Thus the inhibition of RNA syn in PCC is most probably a result of the altered state of interphase chromatin caused by cytoplasmic factors in Thus far activation of been reported in heterokaryons where an ‘“inactive” nucleus has been included in ar. active cytoplasm, for instance chicken eryihrocytes in HeLa ccl! cytoplasm 14: 91~ To ous knowledge, the present report seems ‘ro be the first instance of a reversed relationship, and demonstrates an inactivation of nuclear RNA synthesis by ir,teraction of factors in the cytoplasm of a somatic ceii. The mechanism involved may be analogous to the depression of WA synthesis in melaphase cells, although “heterochromatiration” is not ruled out. The RNA synthesis is depressed Iikewise in somatic cell nuclei transplanted into the cytoplasm of egg cells [8], but the nuclear membrafie remains intact in these nuclei. suggesting another mechanism: probably related to the regulation of gene function. Other such examples are those already numerous cases in which a differentiated function of a cell has been ‘“shut off” in hybrids with other undifferentiated tissue c&are cells, such as Ephrussi’s [73 melanoma-IL ce!i hybrid where melanin production of the former was curtailed, or Mohit’s [14j mouse myeloma cell which stopped producing gamma globulin after TusioD with a lymphoma cel’l.

376

S. Stenman

The author wishes to express his appreciation to Eero Saksela, M.D., for his invaluable advice and critical reading of the manuscript. Sincere thanks are also extended to Professor Kari Cantell who kindly supplied the Sendai virus preparation, and to Karl-Johan Tbtterman, cand. med. for his skilled technical assistance. This study has been supported by Finska I&aresallskapet and the Oskar dflund Foundation.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Exptl

Aula, P, Hereditas 65 (1970) 163. Aula. P & Saksela. E. Hereditas 55 (1966) 362. ’ Bolund, L, Thesis,‘Stbckholm (1971). Bolund, L, Ringertz, N R & Harris, H, J cell sci 4 (1969) 71. Brown, S W, Science 151 (1966) 417. Cantell, K, Saksela, E & Aula, P, Ann med exptl biol Fenn 44 (1966) 225. Davidson, R, Ephrussi, B & Yamanato, K, Proc natl acad sci US 56 (1966) 1437. Gurdon, J B, Problems in biology. RNA in development (ed E W Hanly) p. 217. University of Utah Press, Salt Lake City (1970). Harris, H, Nature 206 (1965) 583. Hsu, T C, Exptl cell res 27 (1962) 332. Johnson, R T & Rao, P N, Nature 226 (1970) 717.

Cell Res 69

12. King, D W & Barnhisel, M L, J cell bio133 (1967) 265. 13. Littau. V C. Allfrev. V G. Frenster. J H & Mirskv. A, Prdc natI acadsci US 52 (1964) 93. I’ 14. Mohit. B & Fan. K. Science 171 (1971) 75. 15. Nichols, W W, &an, A, Aula, P & Norrby, E, Hereditas 54 (1965) 101. 16. Norrby, E, Levan, A & Nichols, W W, Exptl cell res 41 (1965) 483. 17. Prescott, D M & Bender, M A, Exptl cell res 26 (1962) 260. 18. Saksela, E, Aula, P & Cantell, K, Ann med exptl biol Fenn 43 (1965) 132. 19. Saksela, E & Cantell, K, Stand j clin lab invest, suppl. 108 (1969) 46. 20. Sandberg, A A, Aya, T, Ikeuchi, T & Weinfeld, H, J natl cancer inst 45 (1970) 615. 21. Stenman, S, Exptl cell res 58 (1969) 461. 22. - Acta path microbial Stand section A 79 (1971) 212. 23. Stenman, S & Saksela, E, Hereditas 62 (1969) 323. 24. Takagi, N, Aya, T, Kato, H & Sandberg, AA, J natl cancer inst 43 (1969) 335. 25. Taylor, J H, Ann NY acad sci 90 (1960) 409.

Received July 9, 1971 Revised version received August 26, 1971