Inhibition of cell proliferation by the interferon-inducible 204 ... - Nature

Oncogene (1998) 16, 1543 ± 1551  1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00

Inhibition of cell proliferation by the interferon-inducible 204 gene, a member of the I® 200 cluster M Lembo1, C Sacchi1, C Zappador1, G Bellomo2, M Gaboli3,5, PP Pandol®3, M Gariglio2 and S Landolfo1,4 1

Department of Public Health and Microbiology, Medical School of Torino, Italy; 2Department of Medical Sciences, Medical School of Novara, 3Department of Human Genetics, Memorial Sloan-Kettering Cancer Center, New York 10021, USA; 4Center of Immunogenetics and Experimental Oncology, CNR, Via Santena 9, 10126 ± Torino, Italy.

The role of the IFN-inducible p204 as growth regulator was investigated by transfecting an expression vector constitutively expressing p204 into several cell lines. Like pRB and p107, p204 is a potent growth inhibitor in sensitive cells, as demonstrated by the cell focus assay. Since stable transfectants of sensitive lines constitutively overexpressing p204 could not be established in vitro, we inserted the 204 cDNA into a vector bearing an heavymetal-inducible promoter. Here we show that proliferation of B6MEF ®broblasts lacking endogenous p204 is strongly inhibited by transient p204 expression in the nucleus. p204 delays G1 progression into the S-phase and cells accumulate with a DNA content equivalent to cells arrested in late G1. Moreover, the role of p204 in the control of cell growth in vivo was investigated by generating transgenic mice in which the I® 204 gene was constitutively expressed in all tissues. To this end, expression vectors bearing the 204 cDNA under the control of the SV40 viral promoter were constructed. The overexpression of the p204 transgene achieved by injecting fertilized mouse eggs with these vectors was compatible with embryo development up to the four-cell stage in an in vitro follow-up of 4.5 days. However, no viable animals with an intact copy of the transgene were obtained, suggesting that high and constitutive levels of p204 expression can impair normal embryo development. These ®ndings indicate that p204 plays a negative role in growth regulation and provide new information about the molecular mechanisms exploited by IFNs to inhibit cell proliferation. Keywords: interferon; interferon-inducible genes; p204; cell cycle; growth inhibition;

Introduction Initiation and control of the molecular pathways leading to cell proliferation are mediated by both positive and negative-acting extracellular factors that interact with speci®c cell surface receptors (Pardee, 1989; Pagano and Draetta, 1991). One example of negative-acting factor is the Interferon System (IFNs), a family of secreted proteins that exert antiviral, cell growth regulatory and immunomodulatory eects on Correspondence: S Landolfo 5 On leave of absence from Department of Medical Sciences, Medical School of Novara Received 17 April 1997; revised 22 October 1997; accepted 22 October 1997

target cells (Pestka et al., 1987; Kerr and Stark, 1992; Kimchi, 1992; Landolfo et al., 1995; Friesen et al., 1981). When synchronized mouse or human ®broblasts are stimulated from quiescence to growth by serum addition, treatment with IFN-a/b causes marked prolongation of the G1-phase, a reduced rate of entry into the S-phase, and lengthening of the S- and G2phases (Balkwill and Taylor-Papadimitriou, 1978; Lin et al., 1986; Sen and Lengyel, 1992). Exponentially growing cultures of Daudi Burkitt's lymphoma or M1 myeloid cells exposed to IFN-a accumulate in a state in which they carry a 2 n DNA content, indicating that IFN-a leads to a reversible G0-like arrest (Einat et al., 1985; Tiefenbrun et al., 1996). A set of IFN-induced proteins appear to participate in the IFN-dependent control of cell-proliferation (Revel and Chebath, 1986), but the mechanisms and molecular eectors responsible for growth inhibition by IFNs remain largely unknown. Among the several genes induced by IFNs (430), the mouse I® 200 genes (Lengyel et al., 1995) and their human homologs, MNDA (myeloid nuclear differentiation antigen) (Burrus et al., 1992) and IF116 (Trapani et al., 1992; Dawson and Trapani, 1995), appear to be transcriptionally activated both in vivo and in vitro. The I® 200 family is located on murine chromosome 1 and consists of a cluster of at least six genes, some of which cross-hybridize (Choubey et al., 1989). The 202 gene, the best de®ned member of this family, encodes a 52 kDa phosphoprotein that increases 20 ± 30-fold in cultured cells in response to IFN and translocates after a few hours into the nucleus (Choubey and Lengyel, 1993). Here, p202 binds to both pRB (Retinoblastoma gene product) and the transcription factors E2F, AP-1, c-Fos, c-Jun and NFkB, and inhibits their activity (Min et al., 1996). The 204 protein is a 72 kDa phosphoprotein that increases several fold upon treatment with IFN-a/b and then translocates into the nucleus (Choubey and Lengyel, 1992). However, nothing has been discovered about the proteins bound and the biochemical pathways regulated by p204 once translocated into the nucleus. We have previously demonstrated that both injection in vivo of an IFN-inducer, the synthetic dsRNA poly rI : rC, and IFN-a/b treatment in vitro augment expression of the I® 202 and 204 genes in various strains of mice, with the sole exception of the C57BL/6 strain (Gariglio et al., 1992). This lack of inducibility is limited to the 202 and 204 genes, since another member of the I® 200 family, the p203, is expressed normally in C57BL/6 cell lines stimulated with IFNs (unpublished observations). A few poorly successful attempts have

p204 overexpression inhibits cell growth M Lembo et al

1544

been made to set up stable transfectants constitutively expressing I® 200 genes and thus examine their biological activity. The 202 transfectants either express low p202 levels (Choubey et al., 1996) or lose the transfected gene after some time (Lembo et al., 1995). The exact role of the I® 200 genes as regulators of cell proliferation remains thus unclear. This paper investigates the role of the 204 gene in cell proliferation. A cell focus assay was ®rst used to compare the number of clones growing after 204 cDNA transfection in several cell lines. The 204 cDNA was then inserted into a vector under the control of a heavy-metal-inducible promoter and stably transfected into a C57BL/6-derived cell line, B6MEF, that represents a suitable model to study the function of the p202 and p204 since does not express the endogenous counterpart (Gariglio et al., 1994, 1996). Generation of high and constitutive expression of p204 in the transgenic mouse and its compatibility with embryo development and survival were also investigated. The results obtained provide direct evidence that overexpression of p204 inhibits both in vivo and in vitro cell growth by accumulating transfected cells at the G1S border of the cell cycle and that p204 itself is a negative regulator of normal cell growth.

Results Transfection of a p204 expression vector inhibits cell growth A common feature of growth-suppressor genes, such as p53 (Mercer et al., 1990), pRB (Qin et al., 1992) and p107 (Zhu et al., 1993) is their ability to inhibit cell proliferation when overexpressed in sensitive cell lines. Such inhibition is both cell-type and cell-cycle-stagedependent. As an initial functional test to determine whether overexpression of p204, a member of the I® 200 family, also inhibits cell proliferation, several cell lines were transfected with a p204 expression vector or a control vector and assayed for growth suppression response by a transient cell focus assay. Two weeks after transfection, equal numbers of cells were plated onto 100-mm tissue culture dishes and propagated for 2 weeks in the presence of G418. As shown in Table 1, transfection of the pRcRSV control plasmid generated a conspicuous number of cell foci in all lines. In contrast, transfection of NIH3T3, B6MEF and B/ cMEF with the pRcRSV204 dramatically reduced the number to 8, 4 and 3 respectively. Interestingly, transfection of 115/14 and TS/A with pRcRSV or

pRcRSV204 gave about the same number of foci (161 vs 175 and 91 vs 105 respectively). Consistent with these ®ndings, Western blotting analysis of cell extracts isolated from neomycin-resistant clones of both growth-suppressible and non-growth-suppressible cell lines revealed that the p204 was only detectable in the non-growth-suppressible lines (data not shown). Taken as a whole, these results indicate that the p204 functions as a growth suppressor in some cell lines, but not in others. Establishment and characterization of stably-transfected clones with an inducible 204-expression vector As demonstrated in the transient cell focus assay, it was impossible to obtain stable transfectants constitutively expressing the p204 from NIH3T3, B6MEF and B/cMEF cell lines. To circumvent this obstacle, we constructed an inducible 204 gene vector (pMT204) in which the heavy-metal inducible promoter was linked to 204 coding sequence. This vector or the control vector lacking the 204 sequence (pMT) were transfected into B6MEF cells. After G418 selection, the resulting clones were examined to determine the presence of the p204 transgene and protein expression on the addition of Cd ions. The presence of the 204 cDNA was monitored by PCR ampli®cation of genomic DNA. The choice of the two primers allowed us to discriminate between ampli®cation of the transgene and that of the endogenous 204 gene (data not shown). Western MW KDa

1

2

3

4

5

6

117 —

82 —

49 — 29 —

Actin Table 1 Analysis of the eects of p204 overexpression on cell growth by a cell focus assay Cell line

pRcRSV

NIH3T3 B6MEF B/cMEF 115 TSA

102 46 59 161 91

a

Foci with at least 2 mm in diameter

No of clonesa pRcRSV204 8 4 3 175 105

Figure 1 Western blot analysis of cell extracts from B6MEF untransfected or transfected with pMT204. Equal amounts of total cell extracts were separated by gel electrophoresis and blotted onto nitrocellulose. The presence of p204 was detected with anity-puri®ed rabbit polyclonal antibody. The membrane was then incubated with goat anti-rabbit Ig horseradish peroxidase conjugate as secondary antibody and visualized with an ECL kit (Amersham). Actin immunodetection with a monoclonal antibody (Boehringer) was performed as internal control. Lane 1: untreated Neo.1 cells; lane 2: Cd-treated Neo.1 cells; lane 3: untreated I204.1 cells; lane 4: Cd-treated I204.1 cells; lane 5: untreated I204.2 cells; lane 6: Cd-treated I204.2 cells


blotting analysis was performed on 12 independent neo-resistant cell clones, and two clones, I204.1 and I204.2, expressing low levels of p204 in the absence of Cd were selected. As shown in Figure 1, addition of Cd signi®cantly increased the expression of p204 after 18 h compared to the basal levels due to the leaky activity of the MT promoter. As expected, no p204 was detected in pMT transfected cells (Neo.1) even after Cd addition (Figure 1, lanes 1 and 2). Actin immunodetection, used as internal control, showed that its steady-state level was not aected. p204 was also localized by immuno¯uorescence analysis. Its staining pattern varied with the IFN treatment. Untreated NIH3T3 cells showed a diuse nuclear and cytoplasmic 204 antigen reactivity (Figure 2a), whereas in those treated with IFN-a p204 was predominantly detected in the nucleus, giving raise to a bright staining of the nuclei (Figure 2b). Untreated B6MEF stably transfected with pMT204 displayed weak staining, presumably due to basal expression produced by leaky transcription from the MT promoter (Figure 2c). By contrast, a bright nuclear ¯uorescence was observed upon Cd induction, suggesting that the exogenous p204 was properly localized (Figure 2d). This pattern does not reproduce the

punctate staining described by Choubey and Lengyel (1992), nor allow any speculation about the localization of p204 in the nucleolus. However, these dierences can be attributed to the use of dierent polyclonal antibodies and cell lines, and the examination of cell preparations by confocal laser scanner microscopy instead of traditional ¯uorescence microscopy. Selective induction of p204 impairs cell proliferation Previous studies have suggested that I® 200 gene products aect cell proliferation (Choubey et al., 1996; Lembo et al., 1995). However, they employed transfected clones that express relatively low p202 levels, or those that express high levels, but soon lose the transgene. Exponentially growing I204 clones and Neo.1 cells cultured in 10% DS were stimulated with 2 mM Cd and viable cells were counted every 24 h. The behavior of all 12 clones was closely similar, with respect to the proliferation rate. For simplicity, two clones I204.1 and I204.2 are presented. In the absence of Cd, the growth pattern after 72 h in all three lines was similar (I204.1 : 3.56105; I204.2 : 3.86105; Neo.1 : 4.66105), whereas in its presence the number

Figure 2 Subcellular localization of the 204 protein by indirect immuno¯uorescence microscopy. NIH3T3 or I204.1 cells were grown on coverslips and left untreated or treated with IFN-a (kindly provided by M Brunda, Homan-La Roche) or Cd respectively, for 24 h prior to ®xation. The ®xed cells were immunostained with immunoanity-puri®ed anti-204 antibodies and the signal was detected using FITC-conjugated goat anti-rabbit IgG (Sigma). (a) untreated NIH3T3; (b) IFN-a-treated NIH3T3; (c) untreated I204.1 cells; Cd-treated-I204.1 cells

1545


1546

a

Figure 3 Growth rates of the Neo.1 and I204 clones grown in the absence (open symbols) or presence (full symbols) of 2 mM Cd. Cells were seeded at equal densities in DMEM supplemented with 10% DCS. Twenty-four hours after plating, indicated as time 0, 2 mM Cd was added and maintained for 72 h. Data are means of triplicate determinations with s.d

of I204.1 and I204.2 cells was signi®cantly lower (9.26104 and 8.16104), indicating that Cd-induction of p204 suppressed cell growth (Figure 3). As expected, the Neo.1 growth rate again increased to 4.46105 cells.

b

1

p204 is a cell-cycle-stage-speci®c growth suppressor To monitor cell cycle regulation, the nuclear DNA content of I204.1, I204.2 and Neo.1 clones exposed to Cd for 18 h was evaluated after ¯uorescent PI staining. Flow cytometric pro®les revealed that Cd treatment altered the DNA content of pMT204-transfected cells from an asynchronous growing population of cells in various phases of the cell cycle to one in which approximately 61 and 65% of Cd-treated I204.1 and I204.2 cells respectively, displayed a 2 n DNA content (Figure 4a, lower panel), indicating a G1/S delay in cell cycle progression. In contrast, Cd treatment had no eect on the DNA content of the Neo.1 cells (Figure 4a, upper panel), since their growth pro®les in the absence or presence of Cd did not vary signi®cantly. Cell cycle progression was further analysed by phosphorylation of the retinoblastoma gene product (pRB). It is known that the phosphorylation status of pRB oscillates during the cell cycle being hypophosphorylated in G1-phase and gradually hyperphosphorylated when the cells advance through S-phase into G2-phase (Buchkovich et al., 1989; Beijersbergen and Bernards, 1996). In its hypophosphorylated form pRB binds to the transcription factor E2F and blocks transcription. We therefore examined pRB phosphorylation by Western blot analysis after Cd treatment. Figure 4b shows that, in exponentially growing Neo and I204 clones, a major portion of pRB appears to be the slow-migrating hyperphosphorylated form (indicated as ppRB) and, with p204 overexpression, an almost complete conversion to the rapidly migrating hypophosphorylated form takes places. Therefore, the hypophosphorylated status of pRB in Cd treated I204 cells is another indication that p204 induction arrests cells before S-phase and activates pRB as part of its antiproliferative activity (Resnitzky et al., 1992). [3H]thy uptake by I204.1, I204.2 and Neo.1 clones stimulated with Cd was also evaluated. Time course analysis of this uptake revealed that Cd gradually inhibited DNA synthesis in I204.1 and I204.2 cells after 24 h. The magnitude of the response began to level out by 48 h (Figure 4c). The asynchronous cells required

2

3

4

5

6

— ppRb — pRb

c

Figure 4 (a) Eect of p204 expression on cell cycle phase distribution. Exponentially growing Neo.1 and I204 cells were left untreated (upper panel) or treated with 2 mM Cd (lower panel) for 18 h and then harvested for ¯ow cytometry (see Materials and methods) to determine the cell cycle pro®le. (b) Immunoblot of extracted proteins with anti-pRB antibodies. Total cell extracts from both Neo.1 and I204 clones, untreated or treated with 2 mM Cd for 18 h, were separated by gel electrophoresis and blotted onto nitrocellulose. Western blot analysis was performed with anti-pRB antibodies (ppRB, hyperphosphorylated pRB). Lane 1: untreated I204.1 cells; lane 2: Cd-treated I204.1 cells; lane 3: untreated I204.2 cells; lane 4: Cd-treated I204.2 cells; lane 5: untreated Neo.1 cells; lane 6: Cd-treated Neo.1 cells. (c) Inhibition of DNA synthesis by p204 expression. Rates of DNA synthesis were measured by [3H]thymidine incorporation in cells cultured in 10% DCS. Cells left untreated or treated at time 0 with 2 mM Cd were pulse-labeled with 1 mCi of [3H]thy 12 h before harvesting at the indicated times. The data, expressed as c.p.m.6103, represent the means with standard deviations (+s.d.) from sextuplicate wells

approximately 48 h of Cd treatment for maximum inhibition of [3H]thy uptake. DNA synthesis of Neo.1 cells was not impaired by Cd, indicating that the eect observed is speci®c for the p204 transfected cells.


Taken as a whole, these results demonstrate that p204 expression generates a cell cycle arrest similar to that previously reported for p107 (Zhu et al., 1993). p204 overexpression suppresses generation of transgenic mice Two constructs were generated to constitutively overexpress p204 gene in transgenic mice (Figure 5a, see Materials and methods): (i) pSVK3-204, which consists of the 204 cDNA under the control of the SV40 early promoter, followed by the SV40 polyadenylation site; (ii) pSG5-204-IRES-lacZ, which has the rabbit b-globin second intron, positioned between the promoter and 204 cDNA, since intron sequences can increase the transcriptional eciency and the mRNA stability in transgenic mice (Brinster et al., 1988; Palmiter et al., 1991). In this construct, we also cloned an IRES derived from the encephalomyocarditis virus (Ghattas et al., 1991) at the 3' of the 204 cDNA, followed by the lacZ coding sequence and the SV40 terminator (Figure 5a). The IRES provides an independent translation start site allowing concomitant translation of the 204 and the lacZ genes from the resulting dicistronic mRNA. Both constructs were transfected into NIH3T3 cells, and transient 204 mRNA and p204 expression were con®rmed by Northern blot and Western blot analysis, while b-galactosidase activity from the pSG5-204-IRES-LACZ was tested by a colorimetric assay. Finally, a 3.7 kb HaeII fragment from pSVK3-204 and 9.8 kb SalI fragment from the pSG5-204-IRES-LACZ were puri®ed and injected into fertilized eggs for transgene production according to

a

standard procedures. Integration of the transgene and its intactness were investigated by Southern blot on tail DNA from the litter. The pSVK3-204 construct was injected into 138 two-cell embryos and 26 animals (approximately 19%) were born following their transfer into recipient mothers (Table 2). Southern blot analysis identi®ed one transgenic animal, but further characterization of the transgene revealed that the integrated fragment was rearranged. Northern and Western blot analysis con®rmed that p204 was not expressed in this transgenic line (data not shown). In parallel, the pSG5-204-IRES-LACZ construct was injected into 279 two-cell embryos and 37 animals (approximately 13%) were born. Once again, none were transgenic. Injection of the empty pSG5 construct, as control of DNA toxicity, gave rise to a normal yield of transgenic animals (Table 2). In view of this failure to get transgenic animals constitutively expressing p204, eggs injected with pSG5-204-IRES-LACZ construct in vitro were followed for 4.5 days and then analysed for bgalactosidase activity. Seventy-eight eggs were injected with the SalI-SalI fragment of pSG5-204-IRES-LACZ fragment and 56 fertilized eggs were injected with the linearized pSG5 vector as control. Four days after injection, oocytes were ®xed and stained as described. Forty-eight oocytes injected with the pSG5-204-IRESLACZ (approximately 62%) were positive for bgalactosidase activity (Figure 5b). LacZ expression was observed up to the four-cell stage of development, indicating that the pSG5-204-IRES-LACZ transgene could be correctly integrated and expressed. This experiment rules out a possible nonspeci®c toxic effect of the injected DNA and suggests that constitutive expression of the I® 204 gene impairs the subsequent growth and/or development of the transgenic embryos. Discussion Transfection of the p204 expression plasmid into NIH3T3, B6MEF and B/cMEF cells resulted in a limited number of clones, as observed in a cell focus assay. These results demonstrate that p204 overexpression strikingly inhibits cell proliferation. It seemed unlikely that this arrest was attributable to nonspeci®c toxic eects, in as much as there was no signi®cant reduction in clone number after transfection into 115 or TS/A cell lines. Moreover, clones expressing the p204 transgene could not be found from transfected NIH3T3, B6MEF or B/cMEF cells, whereas 115 and TS/A colonies overexpressing p204 were easily detected, and stable clones, expressing p204 at high levels, could be readily established (data not shown).

b

pS G5

pSG5-204-IRES-LACZ

Figure 5 (a) Schematic representation of the pSVK3-204 and pSG5-204-IRES-LACZ vectors. Both vectors are shown in their linear form as they were prepared for egg injection. The probes utilized for the screening of transgenic mice by Southern blot analysis are also indicated. (b) Fertilized eggs, injected with pSG5 and pSG5-204-IRES-LACZ vectors respectively, were cultured in vitro for 4.5 days and stained for b-galactosidase activity. All the embryos injected with pSG5-204-IRES-LACZ are positive for bgalactosidase activity and therefore for the p204 protein. Eggs injected with both constructs reach the four-cell stage

Table 2 p204 overexpression suppresses generation of transgenic mice Construct injected pSVK3-204 pSG5-204IRES-LACZ pSG5

Number of Number of Number of transferred animals transgenic embryos born animals

% of transgenic animals

138 279

26 37

1 0

0.7 0

98

23

8

8.1

1547


1548

Like other growth-suppressor genes, therefore, p204 does not suppress all lines. A common feature of p53 (Baker et al., 1990; Diller et al., 1990) and pRB (Mercer et al., 1990; Qin et al., 1992), two well-characterized tumor suppressor gene products (Marshall, 1991), is their ability to inhibit cell proliferation when overexpressed in sensitive lines. Such inhibition is both cell-type and cell-cycle-stage dependent. Similarly, p107 depresses cell growth when constitutively transfected into Saos-2, but not U2os cells, and arrests sensitive cells in G1 (Zhu et al., 1993). When overexpressed, p204 aects cell cycle progression of sensitive cells at the G1/S border. These conclusions are drawn from the results of ¯ow cytometry, RB phosphorylation status, and DNA synthesis analysis. The percentage of p204-transfected cells that accumulates in G1 signi®cantly increases upon addition of Cd, and DNA synthesis is still detectable, though signi®cantly decreased. Taken together, our results indicate that p204 is a negative cell growth regulator whose mechanisms are similar to those of p107, namely reduction of S-phase cells and a corresponding increase in G1-phase cells. The G2+M phase does not appear to be aected. High expression of IRF1, another IFNinducible gene, aects the proliferation of transfected cells, but apparently controls dierent biochemical pathways, since it does not speci®cally block any single phase of the cell cycle (Yamada et al., 1991; Kircho et al., 1993). In vivo, overexpression of the p204 transgene driven by the SV40 promoter achieved by injecting fertilized mouse eggs, was compatible with embryo development to the four-cell stage. However, no viable animals with an intact copy of the transgene were obtained, suggesting that high constitutive levels of p204 expression impair normal embryo development. p204 belongs to the I® 200 gene family (Lengyel, 1993). Another member of this family, namely p202, is a nuclear phosphoprotein with a putative consensus sequence (TPKK) for phosphorylation by cyclindependent kinases. It binds to the retinoblastoma tumor suppressor protein (pRb), both in vitro and in vivo, and to the transcription factors AP-1, c-Fos and c-Jun, NF-kB p50 and p65 (Choubey et al., 1996; Min et al., 1996). Transfection studies, though nonconclusive because stably transfected cells do not tolerate high p202 levels, suggest that p202 may be critical in normal cell division. Less information has been accumulated about p204 (Choubey and Lengyel, 1992). It is structurally related to p202, but contains other amino acid motifs. It localizes in the nucleus upon IFN-treatment, but does not bind to the transcription factors recognized by p202. The metabolic pathways exploited by p204 to exert its antiproliferative activity would thus seem to be dierent from those of p202. Stable transfectants expressing high levels of other IFN-inducible proteins have been described. Constitutive expression of a transfected 2',5'-oligoadenylate synthetase cDNA impaired replication of encephalomyocarditis virus (EMCV) and mengo viruses (Chebath et al., 1987). On its transfection into murine cells, the constitutive expression of wild-type human ds-RNA dependent PKR cDNA conferred a partial resistance to EMCV infection (Proud, 1995). A decreased rate of cellular protein synthesis in these

transfectants was also observed (Meurs et al., 1993). Constitutive expression of IFN-inducible human Mx-A or murine Mx-1 in mouse NIH3T3 cells conferred resistance to in¯uenza virus as well as to vesicular stomatitis virus (VSV) (Pavlovic et al., 1992; Staeheli et al., 1986). By contrast, when Cd-treated I204.1 clones were infected with EMCV, mengo virus or VSV, normal virus replication was observed despite elevated levels of p204 expression (data not shown). These observations have some interesting implications. The ®rst is that whereas transfected cells can tolerate high levels of 2'-5' Oase, PKR and Mx proteins, high levels of p204 lead to cell growth arrest and do not allow the establishment of long-term transfectants. Secondly, p204 (and probably the other members of the I® 200 family), does not appear to be involved in induction of the antiviral state, at least against the RNA viruses so far tested. Thirdly, IFNinduced metabolic pathways responsible for growth control and inhibition of virus replication overlap to a large extent, but may partially dier. Growth inhibition by IFNs is a well-established phenomenon (Evinger et al., 1981a; De Maeyer et al., 1988; Sokawa et al., 1977). In some studies employing ®broblasts, epithelial or hematopoietic cell lines, IFN treatment culminated in the accumulation of nondividing cells with 2 n DNA content distributed either in G0/G1 or the late G1/S border of the cell cycle, whereas in other similar studies IFNs were shown to aect both G1 and S+G2, in either synchronous or asynchronous cells stimulated to growth (Evinger et al., 1981b). It is clear, therefore, that IFN control dierent `checkpoints', depending on the cell type and growth regulatory conditions. Our present ®ndings showing that constitutive expression of p204 is incompatible with cell growth, and that its transient expression accumulates transfected cells with a 2 n DNA content at the G1/S border, thus inhibiting cell growth, provide a further illustration of the molecular mechanisms exploited by IFN-a to regulate progression throughout the dierent phases of the cell cycle.

Material and methods Cells B6MEF and B/cMEF are embryonic ®broblast lines derived from C57BL/6 and Balb/c mice respectively and immortalized through several passages in culture. Both lines were cultured in Dulbecco's modi®ed Eagle's Medium (DMEM) supplemented with 10% fetal calf serum (FCS) (Gibco/BRL). NIH3T3 cells from the American Type Culture Collection (ATCC) were cultured in the same medium supplemented with 10% donor calf serum (DCS) (Gibco/BRL). The 115/14 (115) cell line is derived from NIH3T3 cells transformed with c-Ha-ras activated by gene ampli®cation, and was kindly provided by Marco Pierotti, Milan. TS/A is a cell line established from the ®rst in vivo transplant of a moderately dierentiated mammary adenocarcinoma that arose spontaneously in a 20-monthold multiparous BALB/c mouse, and was kindly provided by Mirella Giovarelli, Turin. These lines were cultured in DMEM supplemented with 10% FCS. Stable transfected clones were selected and maintained in the same medium containing G418 500 mg/ml (Gibco/BRL). In the metal ion induction experiments, CdCl2 (Cd) was


added at a ®nal concentration of 2 mM to the medium supplemented with dialyzed serum. Plasmids, transfections and DNA ampli®cation (PCR) pRcRSV204 was constructed by ligation of the blunt-ended 2060 bp EcoRI-XbaI fragment from pSVK3204, containing the entire 204 coding region, into the blunt-ended HindIII site of the expression vector pRcRSV (Invitrogen). pcDNA3MT204 (pMT204) was constructed by ligation of the blunt-ended EcoRI-XbaI fragment of the 204 cDNA into the blunt-ended HindIII site of pcDNA3MT. In the original pcDNA3 plasmid (Invitrogen), the cytomegalovirus (CMV) promoter (NruI-HindIII fragment) has been replaced by the metallothionein promoter derived from plasmid pMTneo (Grignani et al., 1990). For construction of pcDNA3MT (pMT), this promoter was excised from pMTneo by EcoRI and HindIII digestion, ®lled in and inserted into pcDNA3, digested with NruI and HindIII and ®lled in. DNA transfections were performed by the calcium phosphate precipitation technique modi®ed according to Jordan et al. (1996). Brie¯y, 56105 cells were transfected with 10 mg of the indicated plasmid and split 1 : 20 48 h later in 100 mm dishes. Stable transfectants were selected by the addition of 500 mg/ml G418 (Gibco/BRL). Fragments from the 204 cDNA were ampli®ed with the Hybaid thermal cycler, using cloned Taq polymerase (Dynazyme). The ampli®cation protocol employed 35 cycles at 928C for 1 min, 568C for 1 min, 728C for 1 min, with a ®nal extension of 10 min at 728C. Oligonucleotides were selected to discriminate ampli®ed products of the transfected cDNA from the endogenous 204 gene. The coding primer was positioned in the MT promoter of pcDNA3MT and the noncoding primer in the 204 cDNA. Thus, the predicted size of the ampli®ed fragment from the transfected cDNA was 1340 bp. Immunoblotting Cells were washed twice with phosphate-buered saline (PBS), disrupted in cold 3% SDS-lysis buer (0.125 M TrisHCl, pH 6.8, 3% SDS (Sodium Dodecyl Sulfate), 10% glycerol, 10 mM DTT (Dithiothreitol)) with the addition of 1 M PMSF (Phenylmethylsulfonyl Fluoride), 1 mg/ml pepstatin, 10 mg/ml leupeptin and 10 mg/ml aprotinin, and brie¯y sonicated. Insoluble material was removed by centrifugation. Protein concentration was determined by the Bio-Rad Dc Protein Assay (Bio-Rad Laboratories). Proteins separated on SDS-8.5% polyacrylamide gel were transferred onto PVDF membrane (Amersham) by electroblotting in transfer buer (25 mM Tris-HCl, pH 8.3, 150 mM glycine, 20% v/v methanol). Membranes were blocked in blocking solution (10 mM Tris-HCl, pH 7.5, 0.1 M NaCl, 0.1% Tween 20, 5% (wt/vol) non-fat dry milk) and incubated with anity-puri®ed polyclonal antibody (diluted 1 : 2000) generated in rabbit using a GST-204 (amino acids 42 ± 205) fusion protein as immunogen. Goat anti-rabbit IgG-horseradish-peroxidase conjugate was used as second antibody at a dilution of 1 : 4000 and detected by ECL (Amersham). Monoclonal anti-actin antibodies (Boehringer) were used as internal control. For pRB detection, immunoblots were incubated with monoclonal anti-pRB antibodies (G3-245; PharMingen, San Diego) (Resnitzky et al., 1992).

washed with PBS containing 1% bovine serum albumin (BSA). Antigen localization was determined after incubation of permeabilized cells with immunoanity-puri®ed 204 antibodies diluted in PBS with 10% DCS for 1 h at room temperature. The antibodies to the 204 protein were immunoanity-puri®ed by passing the protein-A-puri®ed polyclonal antibody raised in rabbit as described above through a column in which the antigen had been coupled to tresyl-activated sepharose 4B (Pharmacia) as suggested by the suppliers. The antibodies were eluted from the column at alkaline pH (11.5), immediately neutralized and stored at 7208C. After washing with PBS containing 1% BSA and 0.05% Tween-20, the cells were incubated with FITCconjugated goat anti-rabbit antibody in PBS containing 1% BSA for 1 h. They were then stained for DNA with 0.5 mg/ ml of Propidium iodide (PI) and mounted with mounting medium (90% glycerol, 2.5 gr/l DABCO (1,4-Diazabicyclo[2.2.2] octane) in PBS). Fluorescence images were examined with a laser scanner confocal microscope (BioRad MRC 600, equipped with a Nikon PlanApo 60/1.40 oil objective). Five to 7 focal frames were taken along the z axis at 1 mm intervals and then merged to obtain a reconstructed image. Cell viability assay Cell growth was evaluated by the trypan blue exclusion method. Exponentially growing cells (16104) were seeded into 2 cm2 wells of a 24-well plate containing medium plus 10% serum. At 24 h after plating (indicated as time 0), CdCl2 was added at a ®nal concentration of 2 mM. At the indicated time points, cells from triplicate wells were trypsinized combined with any ¯oating cells, pelleted and counted with a hemacytometer. More than 300 cells were counted for each variable. Assay of DNA synthesis by [3H]thymidine incorporation Sextuplicate samples of exponentially growing cells (56103/well) in 96-well microtiter plates were treated with Cd (2 mM) at time 0, then pulse-labeled with 1 mCi/well of [3H]thymidine (Amersham) 12h before harvesting. At the indicated times, cells were washed three times with ice-cold 10% trichloroacetic acid, and lysed with 300 ml of 0.5 N NaOH. Lysates (100 ml) were transferred directly into vials containing liquid scintillation cocktail (Pico¯uor 30, Packard), and radioactivity was quantitated by scintillation counting. Cell focus assay To assay the eect of 204 expression, NIH3T3, B6MEF, B/ cMEF, 115 and TSA cells were transfected with 10 mg of the pRcRSV204 expression vector, which constitutively expresses 204, or with 10 mg of the parental vector, without 204 sequence. Eighteen hours later, they were washed twice with PBS, propagated with fresh medium containing G418 (500 mg/ml) and grown under G418 selection for 2 weeks. Single-cell suspensions were made by trypsinization, and 16103 cells were plated onto 100 mm tissue culture dishes. The transfected cells were cultured for 2 weeks in medium supplemented with G418 (500 mg/ml). They were then washed twice with PBS, and stained with 10% formalin0.5% crystal violet, and the number of foci with a diameter of at least 2 mm were counted.

Immuno¯uorescence microscopy

Flow cytometry analysis of DNA content

Cells were grown on coverlips and ®xed at room temperature for 15 min with freshly prepared 1% paraformaldehyde in PBS, washed with PBS, permeabilized for 20 min on ice with 0.2% Triton X-100 in PBS (vol/vol), and

DNA staining was performed as previously described (Barbiero et al., 1995). Cells were harvested by trypsinization, washed with ice-cold PBS, and ®xed in 70% ice-cold ethanol for at least 30 min. After centrifugation, cells were

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incubated at room temperature in the presence of DNasefree RNase (type 1-A) and propidium iodide (PI) at a ®nal concentration of 0.4 and 0.18 mg/ml PBS respectively. Fluorescence was measured with a FACScan ¯ow cytometer (Becton & Dickinson, Mountain View, CA) equipped with a 488 nm argon laser. Two ®lters were used to collect the red ¯uorescence due to PI staining, one transmitting at 585 nm with a bandwidth of 42 nm (FL2), the other transmitting above 620 nm (FL3). FL2 and FL3 were registered on a linear and on a log scale respectively. Approximately 104 cells were analysed for each sample at a ¯ow rate of about 200 cells/s. The percentages of cells within the G1, S and G2/M phases of the cell cycle were determined by analysis with the CellFit computer program provided by Becton & Dickinson. Generation of transgenic mice The pSVK3-204 expression vector was prepared by subcloning the 2073 bp EcoRI-TthIII Klenow ®lled fragment of 204 cDNA (Choubey et al., 1989) into the unique SmaI site of pSVK3 vector (Pharmacia). The pSG5204-IRES-LACZ expression vector was prepared in two subcloning steps: ®rst the 2094 bp EcoRI-BamHI fragment from pSVK3-204 containing the 204 cDNA was cloned into the EcoRI-BamHI sites of pSG5 (Pharmacia) to generate the pSG5-204 expression vector; subsequently, a 620 bp BamHI-BamHI fragment containing the encephalomyocarditis virus internal ribosome entry site (IRES) was cloned upstream from a 3100 BamHI-BamHI fragment encoding the b-galactosidase enzyme (derived from the MoTEL/1 retroviral vector kindly provided by Dr M Sadelain), and then into the BamHI site of pSG5-204. Correct insertion of the subclonal fragments was analysed by restriction enzyme mapping and sequencing. Prior to injection, the pSVK3-204 and pSG5-204-IRES-LACZ vectors were digested with the HaeII and the SalI restriction enzymes respectively to release the inserts from the plasmid sequences. The 3.7 kb and 9.8 kb fragments were separated by agarose gel electrophoresis, electroeluted, puri®ed according to a standard procedure and resuspended into 5 mM Tris-HCl, 0.5 mM EDTA injection buer (Hogan et al., 1994). (C57BL/66CBA/J) females were superovulated and mated with (C57BL/66CBA/J) males. Fertilized eggs were recovered and DNA was microinjected into pronuclei according to a standard procedure (Hogan et al., 1994). The only transgenic line obtained (see results section) was propagated by mating with (C57BL/66CBA/J) F1 mice.

DNA and RNA analysis Identi®cation of the transgenic animals and analysis of the integration and intactness of the transgene was carried out by Southern blot. In order to identify the pSVK3-204 transgenic mice, DNA extracted from tails was digested with EcoRI and hybridized with the 1118 bp BB and the 438 bp SV40 probes, both derived from the pSVK3 vector (see Figure 5a). For the identi®cation of pSG5-204-IRESLACZ transgenic mice, DNA extracted from tails was digested with BamHI or EcoRI enzymes and hybridized with the 3 kb LACZ probe, and with the 438 bp SV40 probe derived from the pSG5 vector (see Figure 5a). The expression of the pSVK3-204 vector in the only transgenic line obtained was analysed by Northern blot (Sambrook et al., 1989) with mRNA extracted from various tissues according to standard procedures (Chomczynski and Sacchi, 1987). X-galactoside staining Injected oocytes cultured in M16 medium were rinsed in PBS and then transferred in the ®xative medium (1% glutaraldehyde in PBS containing 1% serum) for 5 min. They were then rinsed three times (2 min each) by successive transfer in PBS containing 1% serum, and transferred into the histochemical reaction mixture consisting of 1 mg/ml 4-chloro-5-bromo-3-indolyl-b-galactoside (X-Gal), 4 mM K4Fe(CN)63H2O, 4 mM K3Fe(CN)6 and 2 mM MgCl2 in PBS, incubated at 378C in a humidi®ed chamber and scored for positive blue staining after 20 h.

Acknowledgements We are grateful to Giuseppe Barbiero, Department of Experimental Medicine and Oncology, Turin, for performing ¯ow cytometry analysis. We thank Vera Soares and the Transgenic Facility, MSKCC; Michel Sadelain for the IRES-Lac-Z cassette. This work was supported by grants from the Italian National Research Council (CNR, PF `ACRO.') and the Associazione Italiana per la Ricerca sul Cancro (AIRC) to SL and from the Memorial SloanKettering Cancer Center to PPP Mirella Gaboli was partially supported by Fondazione `A. Bossolasco', Fondazione `Blance¯or Boncompagni-Bildt' and AIRC fellowships.

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