EXPERIMENTAL

CELL

RESEARCH

203, 449-455 (1992)

Induction of Nuclear Lamins A/C during in Vitro-Induced Differentiation of F9 and PI9 Embryonal Carcinoma Cells ELENAMATTIA,~'~ E. C. Skater

Institute

WOUTER D. HOFF,~JANDENBLAA~WEN,ALEXANDRAM.L.MEIJNE,~ NICOSTUURMAN,~ANDJOSVANRENSWOUDE' for Biochemical

Research,

Lamin B is the major constituent of the nuclear lamina of undifferentiated mouse embryonal carcinoma cells. The full complement of the three major lamins A, B, and C, found in somatic mammalian cells, is acquired in vitro by certain after induction of differentiation drugs. In this study we have examined the time course of lamin AIC expression in the two embryonal carcinoma cell lines F9 and P19. We show here that lamins A/C are detectable in these cell lines, at the mRNA level and at the protein level, after 3 days of growth in media containing retinoic acid or retinoic acid + 3-isobutyl-lmethylxanthine. The data reported here indicate that the expression of lamins A/C is mainly regulated at the transcriptional level and occurs when the cells, by morphological and functional criteria, have differentiated along their developmental pathway. o lssz ACTdemic

Press,

of Amsterdam,

University

Inc.

INTRODUCTION The nuclear lamina consists of a reticular meshwork (lo-20 nm thick) of proteins, lining the inner side of the nuclear membrane (for review see [l]). In most mammalian somatic cells the lamina is composed of three major proteins, lamins A, B, and C (64,67, and 72 kDa, respectively), which are closely related to cytoplasmic ’ Present address: Microbiology Institute, Faculty of Medicine and Surgery, University of Roma “La Sapiensa,” Piazzale A. Moro 5, 00185 Rome, Italy. Fax: 39-6-49914641. *To whom correspondence and reprint requests should be addressed. ‘Present address: Dept. of Microbiology and Biotechnology Center, University of Amsterdam, Nieuwe Achtergracht 127, 1018 WS Amsterdam, The Netherlands. 4 Present address: The Netherlands Cancer Institute, Division of Cell Biology, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. ’ Present address: Department of Pharmacological Sciences, Health Sciences Center, SUNY at Stony Brook, Stony Brook, NY 11734.8651. ’ Present address: Department of Experimental Medicine, Faculty of Medicine and Surgery, University of Roma “La Sapienza,” Viale Regina Elena 324, 00161 Rome, Italy.

Amsterdam

1000 HD,

The Netherlands

intermediate filament proteins [Z, 31. Functionally, the nuclear lamina probably provides nuclear envelope stability, anchorage of nuclear pore complexes and intermediate filaments, and contributes to the organization of chromatin in the interphase nucleus. At mitosis, the lamins are phosphorylated and the nuclear lamina transiently disassembles to allow envelope breakdown and chromosome segregation [4-61. Lamins A and C become soluble while lamin B remains associated with vesicles formed by the nuclear membrane [4, 71. At the end of mitosis the membrane-associated lamin B probably directs the reconstitution of the nuclear envelope around the daughter chromosomes while lamins A and C would interact with chromatin [8-lo]. Several studies on early embryonic development and of stem cell proliferation have shown that lamin B is constitutively expressed in all cells while lamins A and C appear only at particular stages of development and differentiation [ll-131. To gain insight into the role played by these two lamins, we have studied the time course of their appearance in mouse embryonal carcinoma cells (EC) after induction of in vitro differentiation by retinoic acid (RA) and 3isobutyl-I-methylxanthine (MIX). In order to evaluate the possibility that lamin A and C expression is instrumental in the differentiation of these cells, we have compared the expression of these two lamins with that of specific differentiation markers. Using Northern blot analysis as well as immunofluorescence and immunoblotting techniques, we show here that two EC cell lines, P19 and F9, express lamins A and C after about 3 days of culture under differentiation-promoting conditions. Our data indicate that the synthesis of lamins A and C is not an “early event” in the differentiation process but that it takes place when the cells, as judged by morphological and functional criteria, have acquired the characteristics of endoderm-like cells along their developmental pathway. In this study we provide evidence that the expression of lamins A and C is mainly regulated at the transcriptional level. MATERIALS

AND METHODS

Cell culture. P19 EC [14], P19 MES-1 [15], P19 EPI-7, and Pl9 END-2 [16] cells were cultured in gelatinized tissue culture flasks in a

449

0014.4827192

$5.00

Copyright 0 lVY‘2 by Academic Press, Inc. All rights of reproduction in any form reserved.

450

MATTIA

1:l mixture of DMEM and Ham’s F12 media supplemented with 7.5% (v/v) fetal bovine serum, buffered with sodium carbonate in a 5% CO, atmosphere at 37°C. F9 EC cells (a gift from Dr. B. Terrana, Sclavo Research Center, Siena, Italy) and FS-AC cl9 cells [17] were cultured in 90% (v/v) DMEM supplemented with 10% (v/v) fetal calf serum. To induce differentiation, F9 and P19 embryonal carcinoma cells were plated on plastic culture dishes or on glass slides to give a cell density of approximately 5-10 X 103/cm2 and 20 h later, all-trans-retinoic acid (RA, Sigma Chemical Co.) was added to both cell lines to a final concentration of 1O-6 A4 from a lo-* M stock solution in dimethyl sulfoxide (DMSO). F9 cells received MIX (Sigma Chemical Co.) at a final concentration of 0.1 mM, from a 0.1 M stock solution in DMSO, in combination with RA. Antibodies. Anti-stage-specific embryonal antigen 1 (antiSSEAl), a monoclonal antibody provided by Dr. Solter (Wistar Institute, Philadelphia, PA), was used as a marker of the undifferentiated stage [ 181 at a dilution of 1:500. An antiserum against collagen IV was kindly donated by Dr. S. Garbisa (University of Padova, Italy) and used at a 1:lOO dilution. Lamins A and C were detected by a mouse IgM monoclonal antibody produced by the hybridoma cell line 41CC4 obtained from Dr. W. J. van Venrooij (Catholic University of Nijmegen, The Netherlands). This antibody, used in our experiments in a 1:lOOO dilution of the hybridoma supernatant, has been previously characterized [19]. An immune serum obtained from rabbits according to the procedure described by Aebi et al. [3] was used to recognize lamins A, B, and C. Fluorescein-conjugated goat anti-mouse and goat anti-rabbit IgG were purchased from Nordic and used at a 1:lOO dilution. On immunoblots, positive bands were identified with alkalinephosphatase-conjugated goat anti-rabbit IgG. Fluorescence microscopy. Cells grown on glass coverslips were fixed in methanol for 5 min at -20°C. Fixed cells were incubated with “first” antibodies for 45 min, rinsed in phosphate-buffered saline, and then incubated with FITC-conjugated secondary antibodies for 45 min at room temperature. Subsequently, cells were rinsed and counterstained with Hoechst Dye 33258 (0.2 pg/ml) for 5 min at room temperature to reveal DNA. The coverslips were mounted with 0.1% (w/v) p-phenylenediamine in 10% (v/v) phosphate-buffered saline, 90% (v/v) glycerol, pH 8 [ZO]. All specimens were studied using phase contrast and immunofluorescence microscopy and photographed on Kodak Tri-X Pan film, using a Leitz Orthoplan fluorescence microscope. Northern blot analysis. Embryonal carcinoma cells exposed for different lengths of time to RA, as well as differentiated cell lines, were harvested by trypsinization and centrifuged for 5 min at SOOgat 4°C. Samples of approximately 2-4 X 10s cells were lysed in 8 ml of a buffer consisting of 10 mM Tris, pH 7.5, 2 mM MgCl,, 3 mM CaCl,, 3 mM dithiothreitol (DTT), 0.05% (v/v) Nonidet P-40, 10 mM vanadyl ribonucleoside complex, 0.1 mM phenylmethylsulfonyl fluoride, and 100 kallicrein-activating units of aprotinin/ml. After gently vortexing the mixture a few times, isotonicity was restored by addition of 2 ml of a buffer containing 10 mM Tris, pH 7.5, 2 M sucrose, 5 mM MgCl,, 3 mM DTT. The suspension was mixed thoroughly and then centrifuged for 10 min at 1OOOgat 4°C. Nuclei were separated from the cytoplasm and processed for immunoblot analysis (see below). RNA was isolated from the cytoplasm by the guanidinium/cesium chloride method [21] after addition of a 6 M guanidinium isothiocyanate solution to a final concentration of 4 M. RNA concentration and purity were assessed by measurement of optical densities at 260 and 280 nm. For each sample, equal amounts of total RNA were electrophoresed on 1% agarose gels containing 0.66 M formaldehyde and transferred by capillary blotting to nitrocellulose filters. A l-kb BamHI-Hind111 fragment from the 3’ end of lamin C cDNA was used to detect lamin A and C mRNAs [22]. A collagen IV mouse cDNA clone, kindly provided by Dr. M. Kurkinen [23], was linearized by EcoRI. A PstI fragment (1250 bp) of the hamster actin gene cloned in pBR322 [24] was

ET AL. used as an actin probe. All probes were 32P labeled by random priming [25]. Prehybridization and hybridization procedures were performed as previously reported [26]. The washed filters were exposed to Fuji X-ray films at -70°C for periods of time varying from a few hours to 1 week. Zmmunoblot analysis. Nuclei were purified by centrifugation through a sucrose cushion consisting of 2 M sucrose, 10 mM Tris, pH 7.5,3.5 mM MgCl,, 3 mM DTT, for 1 hat 72,000g [27]. Nuclear matrices were isolated from undifferentiated, RA-treated and differentiated cells according to Kaufmann et al. [28]. The assay described by Peterson [29] was used to determine protein concentration. For each sample, 50 pg of nuclear matrix proteins was subjected to electrophoresis in an 8% SDS-polyacrylamide gel according to Laemmli [30]. Proteins were transferred to nitrocellulose membranes by electroblotting [31] and then probed first with the anti-lamin A and C monoclonal antibody 41CC4 and, subsequently, with the rabbit immune serum that recognizes lamins A, B, and C. The immunocomplexes formed were identified using alkaline-phosphatase-conjugated goat anti-rabbit IgG. To this end, the blots probed with the murine monoclonal 41CC4 were pretreated with rabbit anti-mouse antibodies.

RESULTS

Evaluation of in Vitro-Induced Cells by Light Microscopy

Differentiation

of EC

In order to look at the appearance of lamins A and C, P19 and F9 embryonal carcinoma cells were grown on coverslips and induced to differentiate by the addition of RA or RA plus MIX, respectively, to the culture medium. As a positive control for differentiation, END-2 and FS-AC cl9 cell lines, stable derivatives of the former ones, were used. At different times after the addition of the inducer(s), cells were fixed. The differentiation process was monitored by examining the morphological changes by phase contrast microscopy and the presence of differentiation stage-specific antigens by immunofluorescence microscopy. As shown in Fig. la F9 stem cells have a round shape and a high nuclearlcytoplasmic ratio and grow adherent to each other, forming aggregates. Three days after the addition of RA and MIX (Fig. le) the morphology of these cells has dramatically changed; they appear more flat, the contours are irregular, and the cytoplasmic compartment has increased with respect to the nuclear compartment. At 5 days of differentiation (Fig. li) these alterations become even more evident with long cytoplasmic extensions that create large intercellular spaces. Cells fixed in the course of differentiation were double-stained for immunofluorescence with the 41CC4 monoclonal antibody that recognizes lamins A and C. As differentiation markers we used and scored the presence/absence of SSEAl and collagen IV antigens, the first being expressed only in undifferentiated stem cells [18], while the second is characteristic of endoderm-like cells, its synthesis taking place during RA-induced differentiation of mouse teratocarcinoma stem cells [32]. Figure lb shows that nearly 90% of F9 EC cells are peripherally

RA-INDUCED

Ph Con

SSEA

EXPRESSION

OF LAMINS

A/C

IN EC CELLS

451

I

FIG. 1. Phase microscopy and immunofluorescence microscopy Day 0, (e-h) Day 3, (i-n) Day 5, (o-r) FSAC Cl9 cells. Cells fixed on after incubation with antibodies against the embryonic cell surface monoclonal anti-lamin A and C antibody 41CC4 (fourth column). antibodies (see Materials and Methods). Magnification: X760.

of F9 EC cells at different times after the addition of RA + MIX. (a-d) coverslips were observed by phase contrast microscopy (first column) or antigen SSEAl (second column), collagen IV (third column), and the The immunocomplexes formed were stained with FITC-labeled second

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MATTIA

stained by the anti-SSEAl antibody. The fluorescence signal is rapidly lost during the 24 h after the addition of RA and MIX (data not shown). Cells examined 3 days after the induction of differentiation appear homogeneously negative for the SSEAl antigen (Fig. If), much like the differentiated FS-AC cl9 cells. The secreted, extracellular matrix glycoprotein collagen IV is expressed at a very low level in F9 stem cells [32]. Accordingly, when stained with anti-collagen IV antibodies, these cells show no or very little fluorescence in the cytoplasm (Fig. lc). Three days after the addition of the differentiation inducers, the cytoplasmic area has become brightly fluorescent (Fig. lg) and by 5 days discrete spots, presumably representing transport vesicles on their way to the cell membrane, are observed (Fig. lm). A similar spotted pattern is seen in FS-AC cl9 cells stained with anti-collagen IV antibodies (Fig. lq). When F9 stem cells are tested for the presence of lamins A and C, about 90% are negative (Fig. Id). The remaining lo%, probably representing a spontaneously differentiated subpopulation, show very weak perinuclear labeling. One day and 2 days after induction of differentiation, no substantial changes are observed, neither in the level of fluorescence, nor in the percentage of positive cells (data not shown). By 3 days of treatment with RA and MIX, a large percentage of the cell population shows a perinuclear fluorescent ring that indicates the synthesis of lamins A and C (Fig. lh). The intensity of the signal and the number of positive cells increase with the time of the treatment (Fig. In), approaching the level of lamin A and C expression detected in the differentiated derivative FS-AC cl9 cells (Fig. Ir). Results similar to those described for F9 cells were obtained in RA-mediated differentiation of P19 EC cells. Lamin A and C mRNA levels during in Vitro-Induced Differentiation of EC Cells To study the time-course of lamin A and C expression during in vitro-induced differentiation of mouse embryonal carcinoma cells, we measured the levels of lamin A and C mRNAs in P19 EC and F9 EC cells before and after exposure to retinoic acid. As a control for differentiation, lamin A and C mRNAs were also detected in END-2, EPI-7, and MES-1 cells, three clonal differentiated derivatives of P19 EC cells in which the presence of lamins A and C has been previously reported [33]. Equal amounts of total RNA, extracted from P19 EC cells exposed to retinoic acid for different periods of time and from differentiated cell lines, were electrophoresed and blotted onto nitrocellulose paper, according to the procedures described under Materials and Methods. Because of the large degree of identity between lamin

ET AL.

12345 * -3kb

-A

-2.lkb

a

-C

b

FIG. 2. Northern blot analysis of mRNA from differentiated cell lines and from P19 EC cells induced to differentiate by RA. (a) 20 ag of cytoplasmic mRNA from END-2, EPI-7, and MES-1 cells. (b) 20 pg of cytoplasmic mRNA isolated from samples of P19 EC cells grown in culture medium alone (lane 1) or in medium plus RA for 1 day (lane 2), 3 days (lane 3), 5 days (lane 4), and 7 days (lane 5). Purified mRNA samples were electrophoresed on a 0.66 M formaldehyde gel, blotted on nitrocellulose, and probed with a2P-labeled lamin C cDNA (see Materials and Methods).

A and lamin C sequences (lamin A possesses an additional stretch of amino acids at the carboxy-terminal domain), we used 32P-labeled lamin C cDNA in our experiments to light both lamin A and lamin C mRNAs. Figure 2a shows the results of hybridization of this probe to total RNA isolated from the differentiated cell lines. Two bands of about 3 and 2.1 kb are detected, corresponding, respectively, to lamin A and lamin C mRNAs. While these mRNAs are expressed (to a different extent) in the differentiated clones of P19 EC cells, their specific signals are totally absent in undifferentiated P19 EC cells (Fig. 2b). During the course of the differentiation process, lamin A and C mRNA bands appear as soon as 3 days after the cells have been exposed to retinoic acid (Fig. 2b, lane 3). The relative amounts of lamin A and C mRNAs increase as a function of time of exposure to the inducer, while the lamin A/C ratio seems to remain constant. Results similar to those reported above for P19 cells were obtained when F9 EC cells were induced to differentiate by RA + MIX, and their lamin A and C content was tested by Northern blot analysis. Figure 3 shows that the specific bands are absent in samples from cells exposed to the inducers for 1 or 2 days. However, positive signals are detected in cells exposed to the differentiation inducers for 3 days (lane 4). In order to compare the time course of lamin A/C mRNA synthesis with the expression of specific genes that characterize the differentiation process, blots were rehybridized with a collagen IV probe. Figure 3 shows that the collagen IV mRNA signal increases dramatically during the time frame of the experiment, starting between 2 and 3 days after exposure of F9 EC cells to RA + MIX. To check for variations in the amount of RNA transferred onto nitrocellulose, blots were also hybridized with an actin probe. The levels of actin mRNA appeared constant, confirming the specific

RA-INDUCED

EXPRESSION

OF LAMINS

A/C

453

IN EC CELLS

changes with the synthesis of lamin A and lamin C types in addition to lamin B. During the differentiation process the lamin A to lamin C ratio appears to remain constant while the total amount of these two proteins increases with the time of exposure of the cells to RA. Data similar to those reported in Fig. 4 for P19 EC cells were obtained from nuclear matrices of differentiating F9 EC cells (data not shown).

12345

DISCUSSION

c .c v (21

FIG. 3. Northern blot analysis of mRNA from F9 EC cells induced to differentiate by RA & MIX. Cytoplasmic mRNA was isolated from F9 EC cells at Time 0 (lane l), or 1 day (lane 2), 2 days (lane 3), 3 days (lane 4), and 5 days (lane 5) after the addition of RA and MIX to the culture medium (see Materials and Methods). 20 pg of each sample was electrophoresed, blotted, and probed as described for Fig. 2.

increments mRNAs.

observed

for lamin

A/C and collagen

Immunological Determination of Lamin during in Vitro-Induced Differentiation

IV

A and Lamin C of EC Cells

The synthesis of lamin A and C proteins during RAinduced differentiation of P19 and F9 EC cells was followed on immunoblots probed with a monoclonal antibody that recognizes lamin A and lamin C. To enrich for lamins, nuclear matrices were isolated from the cells. Although the intensity of the bands corresponding to lamin A and lamin C was rather weak, the signal could be detected in both cell lines about 3 days after exposure to RA (data not shown). To increase the level of detection of these lamins, the blots were reprobed with an immune serum that reacts with lamins A and C as well as with lamin B. Figure 4 shows the results obtained from nuclear matrices of differentiating P19 cells. The band corresponding to lamin B is predominant in all samples and its intensity does not change with the progress of differentiation. In agreement with data previously reported, the nuclear lamina of undifferentiated P19 EC cells is essentially composed of lamin B type [ 12,33,34]. The faint signals that identify some lamin A and C in control samples might account for a small percentage of spontaneously differentiated cells. The results of the experiment illustrated in Fig. 4 demonstrate that between 3 and 4 days after the exposure of these cells to RA, the complement of the nuclear lamina

Lamin A/C expression has been studied in a number of different cell systems induced to differentiate in uitro [33-391. These studies, essentially conducted at the protein level, have not unequivocally asserted: (i) at which time in the course of the differentiation process lamin A and C expression is switched on; (ii) at which level lamin A and C expression is controlled, and (iii) if the expression of lamin A and C is instrumental to the differentiation process. In the present study, we have induced two mouse embryonal carcinoma cell lines, F9 and P19, to differentiate in vitro by the addition of RA plus or minus MIX. By culturing the cells for up to 7 days in the presence of the inducer(s), we demonstrated that: (i) Lamin A and lamin C are expressed after 3 days of growth under differentiating conditions; the expression of lamin A and C genes was detected by the appearance of the specific mRNAs on Northern blots and of the corresponding proteins on immunoblots. (ii) In P19 and F9 EC cells, the control of lamin A and lamin C expression occurs, at least in part, at the transcriptional level. The variations in the synthesis of the proteins detected by immunoblot and immunofluorescence correlate well with the variations in the levels of the specific mRNAs, indicating that the newly synthesized lamin A and C mRNAs are rapidly translated. (iii) The expression of lamin A and C takes place

1

2

3

4 -A -6 -c

FIG. 4. Immunoblot analysis of lamins during RA-induced differentiation of P19 EC cells. Nuclear matrices were isolated from P19 cells at Time 0 (lane 1) and after 1 day (lane 2), 3 days (lane 3), and 7 days (lane 4) of growth in the presence of RA. Samples of 50 pg each were electrophoresed and blotted on nitrocellulose as described under Materials and Methods. The blots were probed with a rabbit immune serum directed against lamins A, B, and C, followed by alkaline-phosphatase-conjugated goat anti-rabbit IgG.

454

MATTIA

when the cells have already differentiated to endodermlike cells, as judged by morphological and functional (production of collagen IV) criteria. Our results are in agreement with those provided by Paulin-Levasseur and colleagues [37] who, by Northern blot analysis, found an increment of lamin A and lamin C mRNAs in HL60 cells induced to differentiate in vitro along the machrophage or granulocytic pathway. Conflicting results, however, have been obtained in the past with respect to the expression of lamins A and C during in uitro differentiation of mouse embryonal carcinoma cells. Using immunoblotting techniques, the presence of lamin A and C proteins was detected by Stewart and Burke in the nuclei of differentiated derivatives of EC cells [ 121 and later by Lebel and co-workers in the nuclei by RA [ 341. of F9 EC cells during in vitro differentiation Worman and co-workers, in contrast, did not detect the expression of lamins A and C at the protein as well as at the mRNA level, after exposing F9 EC cells for 48 h to RA + CAMP [36]. In the light of the data reported in this work, it is conceivable that Worman and colleagues failed to detect lamin A and lamin C expression in differentiating F9 cells because they expected an activation of lamin A/C genes shortly after the exposure of the cells to the differentiating agents. In fact, by culturing P19 and F9 cells in the presence of RA or RA + MIX for up to 7 days, we have been able to show that the expression of lamin A and C in these cells occurs after about 3 days from the addition of the differentiation inducers. Moreover, our data comparing the time course of the appearance of the mRNA for lamins A and C with that of a differentiation marker like collagen IV further support the concept that lamin A and C expression is a late event in the differentiation process and, therefore, not instrumental to it. In this respect, our results are consistent with those of Rober et al. [38] who found, in hemopoietic cells that were differentiating in vitro, an increase in the percentage of lamin A/C-positive cells parallel to the increment of the differentiated derivatives that could be identified. Taken together, these data indicate that the acquisition of lamins A and C during differentiation takes place when the cells have already been confined to the developmental program to which they are primed. Some lamin A and lamin C protein was detectable on immunoblots of F9 and P19 EC cells before the addition of the inducers. This is probably due to a small percentage of cells that have spontaneously differentiat,ed in culture and similar data have been reported for other cell systems [37]. The appearance of lamins A and C at a particular stage of development poses a question about the role played by these polypeptides in the architectural and/or functional organization of the cell nucleus. Recent studies have provided evidence for in vitro assembly of lamin A and lamin C on the surface of mitotic chromosomes

ET AL.

[40, 411. These findings, which imply different chromatin binding functions for lamin B on one hand and lamins A and C on the other, suggest that the developmental regulation of the latter might be related to the chromatin organization of the differentiated cells. A detailed analysis of the molecular events that accompany or follow the switching on of lamin A and lamin C expression in early embryonic development would help to clarify the role played by each type of lamin in the nuclei of the differentiated cells. The authors thank Roe1 van Driet and Luitzen de Jong for valuable discussions during the course of this work and the preparation of the manuscript. We are grateful to Sergio Ferraro for quality photographic assistance.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

22. 23. 24.

Gerace, L., and Burke, B. (1988) Annu. Reu. Cell Biol. 4, 33% 374. Gerace, L., Blum, A., and Blobel, G. (1978) J. Cell Biol. 79,546566. Aebi, V., Cohn, J., and Buhle, L. Y. (1986) Nature 323,560-564. Gerace, L., and Blobel, G. (1980) Cell 19,277-287. Ottaviano, Y., and Gerace, L. (1985) J. Biol. Chem. 260, 624632. Burke, B., and Gerace, L. (1986) Cell 44, 639-652. Stick, R., Angres, B., Lehner, C. F., and Nigg, E. A. (1988) J. Cell Biol. 107, 397-406. Lohka, M., and Maller, J. (1985) J. Cell Biol. 101, 518-523. Miake-Lye, R., and Kirschner, M. W. (1985) Cell 41, 165-175. Newport, J. (1987) Cell 48, 205-217. Lehner, C. F., Stick, R., Eppenberger, H. M., and Nigg, E. A. (1987) J. Cell Biol. 105, 577-587. Stewart, C., and Burke, B. (1987) Cell 51, 383-392. Rober, R. A., Weber, K., and Osborn, M. (1989) Development 105, 365-378. McBurney, M. M., and Rogers, B. J. (1982) Deu. Biol. 89,503508. Mummery, C. L., Feijen, A., Moolenaar, W. H., van den Brink, C. E., and de Laat, S. W. (1986) Exp. Cell Res. 165,229-242. Mummery, C. L., Feijen, A., van der Saag, P. T., van den Brink, C. E., and de Laat, S. W. (1985) Deu. Biol. 109, 402-410. Howe, C. C., and Sotter, D. (1981) Deu. Biol. 84, 239-243. Sotter, D., and Knowles, B. B. (1978) Proc. N&l. Acad. Sci. USA 75, 5565-5569. Burke, B., Tooze, J., and Warren, G. (1983) EMBO J. 2, 361367. Krenik, K. D., Kephart, G. M., Offord, K. P., Dunnette, S. L., and Gleich, G. J. (1989) J. Immunol. Methods 117, 91-97. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. McKeon, F. D., Kirschner, M. W., and Caput, D. (1986) Nature 3 19, 463-468. Kurkinen, M., Barlow, D. P., Helfmann, D. M., Williams, J. G., and Hogan, L. M. (1983) Nucleic Acids Res., 6199-6209. Dodemont, H. J., Soriano, P., Quax, W. J., Ramaekers, F., Len-

RA-INDUCED stra, J. A., Groenen, M. A. M., Bernardi, (1982)EMBOJ. 1,167-171. 25. 26. 27. 28. 29. 30. 31. 32.

EXPRESSION

A/C

IN EC CELLS

455

H.

33.

Stuurman, N., van Driel, R., De Jong, L., Meijne, A. M. L., and van Renswoude, J. (1989) Exp. Cell Res. 180,460-466.

Feinberg, A. P., and Vogelstein, B. (1983) Anal. Biochem. 132, 6-13. Mattia, E., den Blaauwen, J., and van Renswoude, J. (1990) Biothem. J. 267,553-555. Rao, K., Harford, J. B., Rouault, T., MC Clelland, A., Ruddle, F. H., and Klausner, R. D. (1986) Mol. Cell. Biol. 6, 236-240. Kaufmann, S. H., Gibson, W., and Shaper, J. H. (1983) J. Biol. Chem. 258, 2710-2719. Peterson, G. L. (1977) Anal. Biochem. 83, 346-356. Laemmli, U. K. (1970) Nature 227,680-685. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76,4350-4354. Schindler, J., Matthaei, K., and Scerman, M. (1981) Proc. Natl. Acad. Sci. USA 78,1077-1080.

34.

Lebel, S., Lampron, C., Royal, A., and Raymond, Cell Biol. 105, 1099-1104.

35.

Guilly, M. N., Bensussan, A., Bourge, J. F., Borners, M., and Courvalin, J. C. (1987) EMBO J. 12,3795-3799. Worman, H. J., Yuan, J., Blobel, G., and Georgatos, S. D. (1988) Proc. Natl. Acad. Sci. USA 85, 8531-8534. Paulin-Levasseur, M., Giese, G., Scherbarth, A., and Traub, P. (1989) Eur. J. Cell Biol. 50, 453-461. Rober, R., Gieseler, R. K. H., Peters, J. H., Weber, K., and Osborn, M. (1990) Exp. Cell Res. 190, 185-194. Guilly, M. N., Kolb, J. P., Gosti, F., Godeau, F., and Courvalin, J. C. (1990) Exp. Cell Res. 189, 145-147. Burke, B. (1990) Exp. Cell Res. 186, 169-176. Glass, J. R., and Gerace, L. (1990) J. Cell Biol. 111, 1047-1057.

Received June 29, 1992

G., and Bloemendal,

OF LAMINS

36. 37. 38. 39. 40. 41.

Y. (1987) J.