Biol Cell (1990) 69, 205-210 © Elsevier, Paris

205

Original article

In vitro induction of early mouse embryo intracisternal particles particles) in cultured cell lines

Jacqueline Lasneret, Laurent Dianoux, Jo~l Lesser, Jorge Peries, Martine Canivet

UPR A43 CNRS R~trovirus et rdtrotransposons des vert~brds, H6pital Saint-Louis, 75010 Paris Cedex, France (Received 25 January 1990; accepted 4 October 1990)

Summary - The experimental induction of~ particles, retrovirus-like structures corresponding to the small IA particles of the mouse, was studied by electron microscopy in rodent-cultured cell lines. Among the chemicals tested, only IdUr was shown to be an effective inducer, but not cycloheximide, puromycine, deoxy-fluorouracile or 5-azacytine. However, only two mouse-derived cell lines: Ki- BALB and FG 10, among 27 cell lines of mouse, rat and mink origins tested, expressed e particles upon IdUr treatment. Epsilon particles thus respond to chemical inducers very differently in comparison with large IAP. Moreover, the addition of interferon previ- ously shown to attenuate IAP production, had no effect on that of ~ particles.

epsilon-particles / intracisternal A-type particles I in vitro induction / rodent cells / retrotransposons / interferon

I n t r o d u c t i o n

Among murine retrovirus-like particles [9], those located within the cisternae of rough endoplasmic reticulum and called intracisternal A particles (lAP), have been described by numerous authors in mouse tumors [22] and embryo ceils [2, 3, 5 - 7 , 21, 23]. In a study of early mouse em- bryo, Chase and Piko introduced the distinction between large and small A-type particles [8]. The large type cor- responds to classical IAP [12], whereas the small type, which presents a definitively distinct morphology, was later referred to as epsilon (E) particle [26-28]. In fact, these E particles are different from IAP in size and internal struc- ture. Their wheel-like aspect is reminiscent of, but differ- ent from the intracisternal R-type particles (IRP) found in Syrian hamster tumor cells and embryos [1, 19, 20]. The expression of ~ and IA particles in oocytes and early mouse embryos, exhibited opposite patterns of developmental stage dependence, whatever the mouse strain examined. Epsilon particles had never been reported in normal and neoplastic mouse cells. Nevertheless, in a transformed fibroblastic cell line (Ki-BALB) treated with iododeoxyu- ridine (IdUr), we observed the induction of structures iden- tical to this category of particles [14].

The biological nature of the e particles is not yet known. Some data suggest that they may be related to lAP be- cause of their reciprocal regulation in early mouse em- bryos, and their frequent simultaneous presence in the same cell.

In the present article, we present results of experiments performed to study the regulation of e particle production. For this purpose, we attempted to induce these particles in mouse and other rodent cell lines by treatment with IdUr and other chemicals known for their capacity to activate endogenous retroviruses in mouse cells cultured in vitro [10, 16, 17]. In further experiments, we studied the effect o f a known biological regulator o f endogenous retroviruses : interferon (IFN) on the expression of e par- ticles [4, 18].

M a t e r i a l s a n d m e t h o d s

Ce//s

Cells used in this study are described in table I. All lines were grown in Dulbecco's medium supplemented with 1007o heat- inactivated calf fetal serum. Clones were isolated by the end point dilution method as described in [41.

Induction experiments

Cells were treated with the following chemicals: 1, 5, 10, 20 or 40/~g/ml of ldUr, 10 t~g/ml of puromycine, 10 -6 M of dexa- methasone, 10/~g/ml of cycloheximide, 0.8/~g/ml of deoxy- fluorouracil or 2 tag/ml of 5-azacytidine, as indicated for each experiment. These inducers were purchased from Sigma (St Louis, MD, USA). Induction procedures followed a protocol previously described [14, 15]. Briefly, cells were treated one day after seed- ing for 6 to 48 h, depending on the inducer and the experiment. Cultures were then carefully washed and refed with fresh medi- um. Cells were harvested for electron microscopy from 24 to 48 h after the end of the treatment period.

Electron microscopy

Cells were fixed in 2.5070 glutaraldehyde, post-fixed in I°70 os- mic acid and embedded in Epon. Ultra-thin sections were stained with uranyl acetate and lead citrate. At least 200 cellular sections were systematically scanned for each cellular sample in a Philips 301 electron microscope. Care was taken to avoid examining fields close to one another. Virus-like particles were identified according to their morphology, size and localization, as follows: - for lAP : intracisternal location, maturation by budding from the reticulum membrane, diameter of 80-90 nm, 2 electron-dense rings bordered by the inner lamina of the unit membrane and no intermediate layer between the nucleoid and the external envelope; - for e particles: trilaminar unit membrane derived from the reticulum, a central nucleoid composed of a 20-30 nm electron- lucent space surrounded by a thin electron-dense ring, exclusive location in rough endoplasmic reticulum, wheel-like morpholo-

206 j Lasneret et al

gy with radial array of spokes projecting from the central nucleoid to the outer membrane, diameter of 75-80 nm.

IA and ~ particles were quantified by counting the total num- ber of particles in 100 cellular sections and by evaluating the per- centage of positive cells showing at least one particle. Samples were examined blind. Statistical significance was evaluated by analyzing the variance between treated and untreated samples.

Interferon preparation

Semi-purified ~//3 mouse interferon (IFN) prepared on L 129 cells was a gift from E Falcoff (U 196, INSERM, Paris). At the con- centration of IFN employed (200 IU/ml), the preparation did not affect cell DNA or protein synthesis, nor did it alter the growth of cells over a period of 5 d.

R e s u l t s

All experiments which were performed to find inducers of ~ particles other than IdUr were unsuccessful. We were unable to induce e particles with cycloheximide, puromy- cin, deoxyfluorouracil or 5-azacytidine, in any cell line used in our assays (table I). As expected, treatment with IdUr induced e particles in Ki-BALB cells, but among the other cell lines tested, only one of them, FG 10, was indu- cible. Ki-BALB and FG l0 are transformed by Kirsten sar- coma virus (Ki-MSV) and Moloney sarcoma virus (M-MSV) and carry v-Ki-ras and v-mos oncogenes respec- tively.

By electron microscopic observation of untreated Ki- BALB and FG 10 cells, we ascertained the presence of some IAP and the absence of e particles. After IdUr treat- ment, a substantial increase in the number of lAP and an

induction of e particles were observed. The latter were generally found in clusters of 10 or more units (fig l) and were sometimes intermixed with IAP (fig 2). In addition to these structures, type C particles, as could be expected, were present in induced cell lines. Hybrid forms of E and IA or type C particles were occasionally observed (fig 3).

Table II shows the results of a comparative study of Ki- BALB and FG 10 cell inducibility. Several experiments clearly showed that Ki-BALB cells were generally more in- ducible than FG 10 cells. Even in the case where the per- centage of cells carrying at least one particle was nearly the same in both cell lines, the total number of ~ particles per 100 cell sections was significantly higher in Ki-BALB cells.

The percentage of cells showing E particles after induc- tion was always lower than the percentage of those con- taining IAP under the same conditions. It is noteworthy that e particle induction showed wide variability in the different experiments.

After splitting the treated cultures, the e particles dis- appeared, unlike IAP which remained high with respect to untreated cells. Double treatment with either IdUr and 5-azacytidine or IdUr and dexamethasone did not signifi- cantly increase the number of IA and epsilon particles com- pared to IdUr treatment alone.

In order to dispose of a high ~ particle producer cell sub- line, we proceeded to clone the Ki-BALB cell line. We then systematically scanned clones for their ability to produce particles after treatment with IdUr. Table III shows the results obtained with 18 clones. There was a great varia- bility of clones, both in percentage of positive cells and in the number of particles per 100 cell sections. These two parameters ranged, respectively, from 0 to 10.8 and from 0 to 241, without concordance between them. The three

T a b l e I. Different cell lines used for e particle induction assays.

Name o f cell lines Animal strains IdUr treatment

Description Presence o f Increase e particles o f lAP

D 152 Mouse Balb/c KiBALB D245 MSV 85 Balb/Mc~5 D55 Mouse Swiss D55 + MSV 3T3 MSV 3T3 MLV FG 10 EF-AKR Mouse AKR EF-C3H Mouse C3H C3H/McA 5 Mouse C3H EF-129 Mouse 129 PCC 4 PCC 3 PCC3d + PCD I PCD 3 PYS-2 EF-A/He Mouse A/Heston PCC 6 Mouse A/Heston NRK Rat Ki-NRK Rat CCL 64 Mink Mink S+L - Mink Ki-Mink Mink

Fibroblasts Non producing Ki-MSV transformed fibroblasts 3T3 transformed by M.MSV, S+L - 3T3 producing M-MSV transformed fibroblasts Methyl cholantrene transformed fibroblasts Fibroblasts M-MSV primary infected fibroblasts M-MSV chronically infected fibroblasts M-MLV chronically infected fibroblasts 3T3 transformed by M-MSV, S+L - Primary culture of embryo fibroblasts Primary culture of embryo fibroblasts Methyl cholantrene transformed fibroblasts Primary culture of embryofibroblasts Multipotential embryonic carcinoma Multipotential embryonic carcinoma Pluritissular differentiated from PCC 3 Myocardial differentiated teratocarcinoma Fibroblastic differentiated teratocarcinoma Endodermal differentiated teratocarcinoma Primary culture of embryo fibroblasts Nullipotential embryonic carcinoma Normal kidney cells Non producing Ki-MSV transformed NRK Normal lung cells Transformed by M-MSV, S+L - Non producing Ki-MSV transformed fibroblasts

+ + - +

- +

- +

+ +

- +

- +

- +

- +

- +

- +

- +

Induction of ~-particles 207

clones (2, 12 and 13) in which the total number of parti- cles was the highest, had a percentage of positive cells rang- ing f rom 6.5 to 10.8. Clone 20 presented relatively numerous particles, but had a low percentage of positive ceils. Moreover, clones exhibited varying inducibility in later experiments. In any case, biological cloning failed to produce a cell line which was highly and stably induci- ble. It should be noted that for IAP, there was less fluc- tuation in the different clones.

Previous results had been obtained using the same IdUr induction scheme: cells were collected for electron microscopy processing 24 h after the end of a 24-h treat- ment with 40 ffg/ml ldUr. In later experiments, whatever

the cells tested (FG 10, Ki-BALB and Ki-BALB clones), we were unable to establish concentration and time-drug dependence of ~ induction unlike lAP activation [14].

According to our previous data [13], IFN diminished the number of IAP constitutively present in Ki-BALB ceils and blocked the induction of IAP by IdUr. In order to test its effect on e induction, Ki-BALB cells simultaneously treated with IdUr (40 ffg/ml) and IFN (200 IU/ml) , and Ki-BALB cells treated with IdUr alone, were scanned by electron microscopy. Results are shown in table IV : neither the percentage of positive cells nor the number of ~ parti- cles per 100 cell sections were significantly different, un- like the results for lAP.

Fig 1. Electron micrograph. Dramatic increase in endogenous retrovirus-like epsilon particles in IdUr-induced Ki-BALB cell. Mag- nification x 18000.

"~ & " " - " ' ' ~ ' , r '" ~ . ' - ~ " ' " '.. ', , k~ t . .~ . : - ' , , "- ';dL.": '!t~ " : " ~ " ~ ' ~ , i ~"

- - " ,~ - - " / " : ~ ' L ~ ' ~ ~ ' I " - " , ~ . - . a i ~ i E ~ , ~ " ; 1 I " ~ 1 ". ~ - , ~ I I F ! I . ' l ~ " ! . l _ l

. . . . , / . . . . . ,~ . , - ,, . : K C d i L _ ' ~ d J F . , , t l ~. ~ 1 ~ . , , . . ~ ., , . . . , i t ; : . .

, " . ~ ".., ' , . . . . ",.: . o~. ~ . . ,.

Fig 2. Epsilon particle (double arrows) and lAP (arrows) in groups. Magnification x 53 000.

208 .I Lasneret et al

~ , ,~m. . v ,?.- , . ~ " "

e 4 ~ " ~ I , "]'f~'(" r..: . . . . .. " ~

• • .7 . , 6 , 7 ~ . ; . ' . ':

":... ,,. . ~IW~r~ . , .',~ • . ~ . . . . , . , , ~.-" ~...

-. • ~ • " • l r .'g ~ ~ - ";- 4 , . z," • ~:," , , . " , , ~ ~ . : ' . . . . . . . . _ _ : ~ . . . . - . . ~ . ~ " ~' " ~ - " . , ~ " : , ~ -- -~:,~ " ~,'~"

.~ , ~.'.'.'-..~,~;':- ..~.¢.-,: . ",,.~,?,~ .a.~,,... o" . ~.;. . , . . , . : _ , . ~- , , , , , ,

• - " '" - ' ~ ' " "- . . . . • ' - : ~ - .~ - - ' I : " : ~ ' , ~ " ; , ~ : ~ - . . . .

~"~ "" -~-~-:" ~'2~-;~",~I~. .~";~ i'~,~. "".I~W"" ,:,." ~: .... ' " ~ " " " , - g ~ t , , ~ - ~ - r ~ ' , A i ~ ' ~. ,~, ~; / : ' . ~ . . ~ . ~ ~ , . - .,:;'~ ~' ~,.

, ~, :.-'/ > £ ~ ' a ~ l ~ - - , , ~ - . , " "'- ~ ~. F . . . . . ~.af¢."" ." .~:~ • " ~.., , -, ~ : . . ~ . . . . . ~ - ,, ;,.'~. . . . . . .

• , . -r .~ - - .~ , - , : . , . . . ~ • • ' c: . . . . . . . ; ~

• • , . . ~ ,-.,-'~.:" . , ~"~J ~, . - ,_ ., • .

" ': _ _ - % : : • . - : ; ~ ~ "~,0-- " ,t : ' , . ~k"-' ' : : ~ " ~ ~-. . . . . " ~, -

~ ' ~ , ~ 1 n H I ~ ' , ~ . 3 . ' d ~ . ~ , l ~ : , ~ • -'~.)- ~-, ,- : ! _ v , - I " . . . . - I ' , .,. ~ ~ / - ' ~ : ~ . ~ ' ~ , . " ~

Fig 3. Details of different particles and hybrid structures observed in a treated cell. a: Epsilon particle and IAP (× 125000); b: type-C particle (× 125 000); c: hybrid structure between ~ and type-C particle (× 120 000); d: hybrid structure between E particle and lAP ( x 120000).

Table 11. Comparison between IAP and E particle induction by IdUr in KiBALB and FG10 cells.

Table !II. Induction of e-particles in different clones of Ki-BALB cell line. Comparison with IAP activation.

E x p I d U r % o f T o t a l N o o f C l o n e P e r c e n t o f N o o f par t i c l e s p e r t r e a t m e n t ~ p o s i t i v e cells ~ par t i c les p e r 100 n u m b e r ~ p o s i t i v e cel ls b in: 100 cel lular s e c t i o n s ~

cel lular s ec t i ons b in: E 1,4 P ~ IA P E-particles IA P E-particles IA P

K i - B A L B c e l b 1 3.5 47 112 205 0 0 15 0 105 2 6.6 56.5 241 430

1 + 15 55 225 553 4 2.5 59 33 253

2 0 0 26 0 96 5 4.2 37.5 44 148 + 5 55 190 380 7 0 50 0 357

3 0 0 14 0 30 8 0 57.5 0 175 + 4 58 86 625 12 7.5 50.8 235 293

4 0 0 ND 0 ND 13 10.8 55.8 218 312 + 16 63 210 750 14 1.5 50 10 166

5 0 0 20 0 47 15 0 55 0 140 + 4.5 67 87 797 16 4.5 51 147 205

F G 10 c e l ~ 17 5.8 44 39 178 0 0 2 0 15 18 3 52.5 58 157

1 + 6 40 80 120 19 2 47 70 345 0 0 1.5 0 12 20 2.5 55.8 175 217

2 + 5 35 20 132 21 3.7 58.8 4 194 0 0 3 0 23 22 0 47.5 0 144

3 + 4.5 45 90 297 23 2 43 20 135

a Cells were treated with IdUr (40 ~g /ml ) for 24 h and collect- ed for electron microscopy 24 h after treatment, b Values represent the average of results from triplicate cultures

a Ceils were treated with IdUr (40 tzg/ml) for 24 h and collect- ed for electron microscopy 24 h after treatment, b Values represent the average of results from triplicate cultures

Induction of e-particles 209

Discussion

Previous electron microscope studies showed that e parti- cles were found transiently during mouse normal embryo- genesis at the 2-cell stage [27]. They were not observed either in later embryos or in adult tissues. To-date, the presence of these particles has been reported only in Ki- BALB mouse cells induced by IuDr [14]. In this study we show that, after treatment with IdUr, we visualized ~ par- ticles in two mouse derived cell lines. The mouse strains from which the cell lines are issued, BALB/c and Swiss, are not considered to be high producers of e particles dur- ing embryogenesis [27], but rather as intermediate and low producers, respectively. The fact that both these cell lines are transformed by sarcoma viruses could be related to the observation of Yotsuyanagi and Sz6116si concerning the accidental production of ~ particles in certain cases of de- velopmental disorders [26, 28]. Nevertheless, two other cell lines: 3T3 MSV (BALB/c) and D55 MSV (Swiss) trans- formed by M-MSV, did not exhibit particles after IdUr treatment. This was also the case for cell lines derived from mouse embryonic carcinomas.

It should be noted that AKR fibroblasts did not produce e particles after induction, although strain AKR was con- sidered as a high producer in early embryos [27].

Epsilon particle regulation seems to differ from that of IAP. Factors influencing the induction of e particles and the activation of lAP act in a different manner for these two types of particles in cultured cell lines. Under our ex- perimental conditions, ~ particles were only induced in two cell lines, while IAP were activated in numerous cell lines. Moreover, for the former, IdUr was the only effective in- ducer, whereas 5-azacytidine activated IAP, even more ef- ficiently than IdUr [15].

There was a significant relationship between IAP acti- vation and dose or duration of treatment, but e particle induction did not appear to be dependent on these fac- tors. However, for the latter, it should be emphasized that induction was prone to great irregularity. Indeed, in a given experiment, the number of particles fluctuated over a wide range in several samples. If we take into consideration this variability, the effect of IFN on particle induction was not significant, contrarily to IAP activation.

Hybrid forms have already been mentioned in early mouse embryos [27] and in somatic cell hybrids harbor- ing several types of different particles [24, 25]. These mor-

Table IV. Action of IFN on IA and e particles in IdUr-treated Ki-BALB cells.

Treatment % o f positive Total No o f cells d f o r particles per 100

cellular sections a E particles I A P ~ particles IAP

Control cells 0 18+3.5 0 27+ 7.5 IdUr a.b 7.3+0.7 61___6 222__.97 486+43 (IdUr + IFN) a.b 5.6_+0.6 4 6 _ + 4 190+99 140-+10 IdUr a.c 3.8-+0.5 82-+7 81_+60 555_+55 (ldUr + IFN) ~,¢ 3.2_+0.4 15_+5 12_+10 120_+90

a Cells were treated with IdUr (40/zg/ml) for 24 h. Half of them were submitted to IFN (200 IU/ml) 8 h before and during IdUr treatment; b cells were collected 24 h after IdUr treatment; c cells were collected 48 h after IdUr treatment; a values represent the average of results from triplicate cultures.

phological hybrids provide the best illustration of the dis- tinction between e particles and other types of particular structures.

In vivo, the expression of e particles is very stringent and their regulation is co-ordinated with that of IAP. In our in vitro system, the expression of e particles is still strin- gent, but their regulation does not seem to be co-ordinated with that of IAP and is even dissimilar.

The detection of IAP-associated antigens described by Huang and Calarco [11], where e and IA particles are simultaneously present, could be used in IdUr inducible cell lines to further investigate an eventual immunologi- cal relationship between the two types of structures.

Acknowledgments

We are grateful to Y Yotsuyanagi for helpful suggestions. We thank P Bittoun for expert technical assistance, B Boursin for performing the photography, and M Pasquet and MC Poeuf for excellent editorial assistance.

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