Summary How cell commitment and differentiation are controlled in the early stages of embryogenesisis a problem that has long fascinated developmental biologists. Retinoic acidinduced differentiation of embryonal carcinoma cells in culture provides a model in which these questions can be explored. Recent work has yielded exciting insights into the central series of molecular changes which drives the commitment of these cells to formation of a new phenotype. Interacting with the key molecules in this central pathway is a variety of transcription factors, many of which show changes in availability and/or activity during differentiation. In various combinations, these modulate the activities of genes involved in both cell proliferation and in the production of extracellular matrix and other proteins characteristic of diflerentiated cells.

Introduction During the earliest phase of development of the mouse embryo, cells maintain identical and coinpletely undifferentiated characteristics, at least until the %cell morula is formed. Subsequent cell divisions produce the trophectoderm and inner cell mass ( E M ) lineages, the latter giving rise to all embryonic and most extraembryonic tissues. At around 4 days of development, a subset of ICM cells differentiates to form the primitive endoderm. Some of these cells begin to migrate onto the trophoblast where they lay down extracellular matrix and assume the characteristics of parietal endoderm (PE) cells ( ' ). The intracellular events driving the commitment of cells to new phenotypes during this complex series of steps has long fascinated devclopmental biologists. A model system in which some of these events can be studied is provided by embryonal carcinoma (EC) cells. A variety of such cell lines has been established in culture from early mouse embryos, the best characterised being the F9 line. In their undifferentiated state, F9 cells have characteristics similar to those of the ICM, with a very low rate of spontaneous differentiation in vitro (reviewed in ref. 1). Addition of retinoic acid (RA) to F9 cells in monolayer culturc, however, induces morphological and biochemical changes over several days. Product cells have many of the characteristics of YE ( 2 ) . The differentiation process can be enhanced by the addition of cyclic AMP, to

which the cells become sensitive soon after differentiation begins (3). For several years, attention has been focussed on F9 cells as an in vitro model in which the ICM-to-extraembiyonic endoderm differentiation pathway can be examined. There has been extensive documentation of changes in gene expression occurring during the process and this has been the starting point for identifying transcription factors whose binding alters during differentiation. More recently, the identification of the retinoic acid receptor (RAR) family, isolation of RAR genes and study of their control mechanisms have cast further light on the differentiation process. Bringing together these elements, it is now possible to propose a model for commitment and control in 1'9 cell differentiation. This modcl is reinarkable for the complexity of the feedback loops and interactions affccting every step of the process, and it is certain that wc are far from understanding its full details. Howcvcr, the model can provide a useful basis for further experimentation to identify some oS the missing links in the pathway, and also to test thc cxtcnt to which the series of events now being clarified in vitro, reflects events in the equivalent differentiation pathway in vivo.

Changes in Gene Expression during F9 Cell Differentiation in vitro Previous reviews have summarised changes in gene expression observed during differentiation of F9 cells following RA treatment (4,5). Three main groups of genes were identified, the first changing in expression within the first 24 hours of RA addition, in some cases via a post-transcriptional mechanism ( 5 ) . The second group, changing transcription during what was tentatively named the primitive endoderm phase, includes genes activated both in the PE pathway for €79 cell differentiation, and in an alternative pathway followed by cells in suspension culture, to produce cells of the visceral endoderm type 5). Examples of this second group include introduced genes under the control of SV40 and pol yorna virus early gene promoters, and genes encoding some componcnts of the extracellular matrix. Altered expression of these genes is evident 48 hours after RA addition. The third group of genes is activated during differentiation only in the PE pathway. This group includcs genes encoding SPARC, tissue plasminogen activator (t-PA) and histocompatibility genes, such as H-2Kb. Activation of these genes commences somewhat later than for group 2 genes, although the precise timing varies with the F9 subline being examined and the culture conditions used (reviewed in reference 5 and summarised in Figure 1). ( ' 3

Changes in Transcription Factor Binding With their well characterised promoter regions, viral genes have provided the ideal starting point for studies on how transcription Factors control gene expression during F9 cell differentiation. Comparison of these viral gcnes with others activated at the same time during differentiation soon led to the realisation that change in any single transcription factor was unlikely to explain the gene expression patterns

laminin A

era 1 kox 1.1

increased expression

collagen IV endo A,B H2 genes SV40, Py

Fig. 1. Time course of differentiation induccd in F9 cells by

addition of retinoic acid. The figure shows time in hours after RA laminin B l addition when transcript levels froin the indicated genes are significantly increased (or decreased). Information [or this figure was obtained from ref. 5 and also refs. decreased 7,8.25,47,48. The sets of genes GROUP 3 GROUP 1 GROUP 2 expression showing altered expression at 0-24, 48 andbeyond 7 2 hours ~ after RA addition are those referred to as Groups 1 to 3, respectively, in the text. Group 3 genes show altered expression only in parietal endodem-type product cells, while expression of the remaining genes is generally affected in both parietal and visceral endoderm differentiation pathways. Exact timings for altered gene expression may vary with cul~ureconditions and cell subline used. spare t-plas. act H-2Kb

C-JIln

I

observed. As reviewed previously ( 5 ) , in closely related EC cell types or under different conditions for induction of differentiation, the order and extent to which different genes are activated varies. This has led to the hypothesis that multiple transcription factors (exerting a mixture of positive and negative effects) are acting together and in different combinations to conlrol expression of different members or the gene set. Table 1 lists some of the transcription factors whose binding to DNA has been found Lo change during F9 cell differcntiation. In many cases, there is no1 yet any information on the consequence\ of altered binding (is transcription activated. suppressed or unaffected'!) or on the cause of altered binding (new protein synthesis, or alteration of pre-existing protein?). Nevcrthcless, it is clear that there is a very major shift

in the population of transcriptioll factors binding to DNA during the process of F9 cell differentiation. Some which appear to play a central role in the differentiation proces? are discussed in more detail below.

The Role of the AP-1 Complex Key studies carried out in the laboratory of Yaniv (h) dcmonstrated that in differentiating F9 cells. activation of the transcriptional enhancer region associated with the pol yorna virus early gene promoter could be largely attributed to increased binding activity of- the factor PEAI. This factor was subsequently shown to have the same DNA binding sequence and to be the mouse equivalent of the huinan tranVectors expressing c-Jun, H key scription factor AP- l((>).

Table 1. Sorne examples of altered transcription factor binding dwiizg F9 cell diffeeventiarion Factor

Kinding Sequcnw

Lcinuing iictmiry dilfci cntiated F9 F9

Ref

Retinoic acid receptoi

GTGAAC or TGACC inverted vr direct repcat

k

+++

11

AP-l(PEA1)'

TGA CIG TCA

-

GGAAAGTCCC

-

++ +++

29

TCIIA factoix (EPB-1, NF-KB) TCllB tactor*

GGAAAGI CCC TGCGGATTCCCCA

+

+++

25,3 I ,42,43

PEA2 (repressor)

TGACTGGCC

++

k

30

ATF family** (CREB, NRB)

TGACGT/A CTCAGGAG

++

*

44,45

NFA3 (repressor)

ATTTGCAT

++

-

46

DRTF 1 (EW

TTTCGCGCTAAA

++

*

38.39

(€I2TFI, KBFI, CTF, NFI)

31

'Alternative names for binding factors are shown in brackets *Part of a family of factors binding to related sequences **A family of factors recognising related DNA sequences. Mcniberq of the family show variable changes in binding during F9 differentiation - the iiiost common patteni is indicated.

component of the AP-I complex, or the viral equivalent, vJun, were shown to induce some aspects of differentiation when they were introduced into F9 cells. In these and in the P1Y EC cell line, ectopic c-jun expression only partially induced expression of differentiation markers such as endoA and t-PA,(7,g)while some other markers were more fully expressed. A dominant negative mutant of c-jun, expressed blocked RA-induced differentiation (9). These ate a key role for the c-Jun component of the AP1 complex. although other meinhers of the complex (JunD. JunB, c-Fos) appear to play little if any primary role in F9 cell differentiation(*). An interesting twist to the story comes from the observation that c-jun expression is positively autoregulated. The gene product binds as a homodimer to an AP-1 recognition sequence in the c-jm gene promoter and activates its own production(*'. A relalively small initial increase in active, DNA-binding AP-1 during cell differentiation can be amplified by this feedback mechanism, and the cells become fixed into a pattern of elevated c-jun expression. This mechanism, which is increasingly being revealed as a feature of the regulation of transcription factor activity@), goes a long way toward explaining the puzzling phenomenon of cell commitment during differentiation.

What Initiates c-jun Expression? Increased transcription of the c - j m gene and elevation of protein levels commences about 24 hours after RA is added to F9 cells. and peaks at around 48 hours@'. This suggests that increased c-jun expression is not a primary response to RA action. What steps precede c-jun activation in determining the course of the differentiation process? What is the event triggering the escalation of c-Jun synthesis during F9 cell differentiation? It appears that there may be two pathways directing this escalation. The first involves binding of RA to its nuclear RA receptor (RAR), triggering a sequence of events culminating in transcriptional activation of c-jun. The second pathway appears to be RAR, (but not RA), independent. Activation of c-jun transcnption via this pathway requires a binding element in the c-jun promoter region called the RERE (RNE I A response element)('*). Activation via RA Receptors Three genes encoding members of the RAR tamily h a w now been isolated. RARa 1s expressed in undifferentiated FY cells, while the p receptor is present only at low leveldl'). Lt has been shown that when RA is added to cells, the activated a receptor binds to B RA recponse element in the promoter of the RARP gene(11x'2),.The result is an increa\e in the exprcssion of RARB, the form of the receptor which is most active in stimulating transcription" '1. Like c-jun, the RARP gene also har a positive autoregulatory loop, whereby the production of RARP is furthcr increased by the presence of RA-bound RARP(13'. The cells quickly proceed to high expression of RARP, which increases in F9 cells 20-fold within 24 hours ofRA addition(' I ) .

In F9 cells, RAKP levelr remain elevated for at least 4 days in the presence ofRA(' I).However, a much briefer KA trcatment (less than 12 hours) is needed for commitment of cells to differentiation(' l). This may be explained by the ten-fold higher affinity of RARP for RA. compared with KAKa. High levels of RA are thus important to triggcr the initial nse in RARP levels, but subsequently the levels of RA present in serum (ca 10-loM)are sufficient to continue the processc7). The link between RAR activation and increased c-Jun appears to be indirect, since no RA-response element has been detected in the c-jun pronioter. It is possiblc that appearance of a new factor, labelled X in Figure 2, is the link between thew two p r o c e s s e ~ ' ~ ~ ' ~ ) .

RAR-independent Activation Kitabayashi and co-workers('O)have recently described an RAR-independent effect of RA and of adenovirus EIA protein on c-jun transcription in F9 cells. Stimulation of c-jun transcription via this pathway occurs 48-60 hrs after the initial stimulus. This is somewhat slower than the effect mediated by RARB and is presumably indirect although independent of new transcription or translation. The sequence element RERE - C(G)C(G)GTGAC(G)NT - in the c-jun promoter is required for transcriptional stimulation; the same element acts as a silcncer in undifferentiated cells, indicating a blocking effect by an inhibitory or ineffective transcription factor binding at the site. In neither of the RAR-dependent or -independent pathways is the direct stimulus to c,jun transcription understood. However, the full escalation of c J u n production requires the AP- 1 binding site in the c,jun promoter, indicating that only a small increase in c-Jim level or activity may be needed, to trigger increased c,jun transcription via autoregulation. lnteraction with other proteins is known to exert a strong influence on the activily or c-Jun as a transcription and autoregulatory factor. It is possible, although not yet proven, that the major stimulus to c-jun transcription in F9 cells may be activation of preformed c-Jun. Baichwal and colleagues described a cell-specific inhibitor ol c-Jun which binds to the c-Jun protein adjacent to its transcriptional activation domain. In cell types containing the inhibitor. expression of the c-src or c-ras oncogenes leads to the release o f c J u n from its inhibitor complex, resulting in increased AP- I activity and stimulation of c-jun expression via autoregulation. This inhibitor may bc prescnt in undiffercntiated F9 cells. An earlier report described the appearance of active AP- 1 (PEA- 1 ) in F9 cclls following activation of cH a rus expression. This resulted in derepression of pol yoma virus gene expression("). Since a milch reduced stimulus was seen by c-Ha ras in differentiated cells, it seems likely that the influcncc of the c-Jun inhibitor is reduced during F9 cell differentiation. Thc inhibitor itself is still to be identified. Lamph and coworkers('g) have shown that the CAMP response element binding protein (CREB) in its unphosphorylated form can render c-Jun inactive, possibly by competition for binding sites on DNA, or by complex formation with the Jun protein('9)).This effect of CREB is lost when the protein is acti-

vated by phosphorylation, and phosphorylated CREB becomes an activator of c-jun expression (18). Interestingly, the REFE sequence element described above(10)bears a close resemblance to that recognised by the ATF family of transcription factors, including CREB (see Table 1). The rise in c-jun transcription and c-Jun DNAbinding activity which appears to be a pivotal event in F9 cell differentiation may thus be regulatcd by a number of intcrlinked mechanisms - a RAR-dependent pathway, possibly involving appearance of an intcrmediate factor as yet unidentified - a RAR-independent pathway, also indirect, and perhaps involving a meniber of the ATF transcription factor family - autoregulation of transcription by c-Jun - modulation of the activity of c-Jun both for binding and autoregulation by one or more factors interacting with it. These may include CREB, or other non CAMP-responsive members of the ATF family, known to form heterodimers with c-Jud20).

A Model for the Differentiation Process A model linking all of these elements is shown in Figure 2. In this model, the process of F9 cell differentiation is charactcrised by at least two different positive feedback loops. These scrve to converl the initial htimulus (a pulse of RA) into a fixed and irreversible program of events which is central to the phenotypic changes observed. There is certainly additional modulation of the process. For cxample, a recent report has described a feedback effect of AP-1 on the

expression of RARP. AP-1 is able to bind to the promoter region of R A R P and act5 synergistically with RARa to activate receptor prod~ction'~,.The synergistic efrecls are observed at very low levels of RA, equivalent to the levels in scmm as described abovd7). In addition, RAR activity is modulated by interaction with 22) and with other members of the RXR receptor cell type-specific co-regulator protein@'. Finally, antagonistic effects between AP-1 and the RARs may soinetimes be seen'*". The outcome of this interaction depends on the ratios of the two protein types; in combination with the transcriptional feedback loops descnbed above, the result I \ presumably very close control of the overall activities of both types of transcription factor.

Other Positively Acting Transcription Factors Produced During F9 Cell Differentiation Table 1 shows that in addition to the cnicial appearance of active AP- 1 complexes. other positively acting tranw-iption factors appear during F9 cell differentiation. Included amongst these is the RA receptor itself. In addition to triggering the pathway leading to activation of AP-1. this factor is able to act directly on some but not all genes activated during the differentiation process. For example, active RARP is able to bind directly to thc promoter of [he laminin B 1 gene('5). Transcription from this gene begins to rise relatively early in the differentiation process, although it does not peak until a much later stage, indicating that factors in addition to the retinoic acid receptor are required for maximal reduced growth

Fig. 2. Proposed pathway for conmitmenl and differentiation of F9 cells during the first 48 hours after RA addition. Clearly identified components of the pathway are shown in black, while hypothetical steps are shown in hatched lines. Shaded circles show DNA-binding factors which have key roles in the control of differentiation. A. In one -.__I) --part of thc differentiation pathway, active E2F factor disappears about 48 hours into differentiation. This may be the major effect responsible for altering the growth characteristics of the cells (see text). Its disap_I____)pearance is due to the formation of larger protein complexes which also include the rctinoblastonia proB. In the lowcr part of the diatein. I gram are proposed two autoregulatory loops controlling transcription tramcriptional of the RARP and c-jun gencs and a (BI activation further feedback control by c-Jun causing activation of the, RARB gene. Linkage between these two loops is uncertain, but may include one or more protein phosphorylation steps, possibly leading to activation of an inhibitor molecule normally suppressing c-jun expression (c.g. CREB protein). A second, RAR-independent pathway stimulating c-Jun production is also shown. Appearance of active c-Jun protein appears to be the major stimulus for activation of genes to produce the characteristic products of differentiated cells, as shown in Figure I, although other subsets of transcription factors also contribute to this activation (Table 1. and discussed in the text).

-b

angmjc.+(@

4+

In F9 cells, it appears that E2F is largely present in an [email protected] has been suggested that different arrangeuncomplexed form due to the absence of suitably underphosments of RA response elements (e.g. as monomers or dimcrs, phorylated RB protein for complex f o r r n a t i ~ n ( ~ ~The .~~,. or with slight divergences from the consensus sequence) can presence of this uncomplexed E2F provides some of the modify the affinity of binding of the receptor, thus determinsame transcriptional effects in F9 cells as it does when ing the amount of active receptor coinplex needed in the cells before transcriptional activation can o c ~ ~ r ( ~ This ~ , ~ ~released - ~ ~ )by. E l A in other cell types. The existence of free E2F thercfore accounts for the reported El A-like activity in these effect could contribute to scquential or diffcrential cells. Around 48 hours after RA addition to F9 cells, the expression of genes during differentiation induced by RA. DRTF complex increases in This is due to entry of At the same time, some changes in transcription factor RE protein into the complex. This change coincides with a activity may be mediated through the RAR-independent slowing in cell growth and loss of transformed characterispathway. For example, when cxccss RARP was expressed in tics such as the ability to form colonies in sort agar. It s e m s F9 cells in the presence of RA, the level of expression of the likcly that removal of free E2F by RB complex formation endo A gene was not correlated with RAR leveld7). In results in reduced transcription of a range of gcnes involved addition, a dominant negative RARol mutant blocked some, in cellular proliferation. although the specific genes involved but not all of the effects of differentiationtz8), indicating in F9 cells have yet to be ~ d e n t i f i e d ( ~ This ~ ~ change ~ ~ ) . would changes in the cells independent of RAR activation. appear to explain the changes in growth properties that accompany morphological differentiation. Repressors of Gene Expression in F9 Cells Several groups have reported repression of the expression of Conclusions and Questions particular genes in F9 cells. Those repressed include the early Intensive studies over the last few years have revealed what genes froin polyoma and SV40 viruses, endoA and B genes is proposed in this review to be the essential process of F9 and genes of thc histocompatibility class I e.g H-2Kbw For many of these genes, sequences involved in repression of cell differentiation induced by RA. This model is outlined in promoter activity and/or potentially repressive transcription Figure 2 and has been discussed above. Synergistic action of factors have been idcntified. As shown in Table 1, one of the c J u n in conjunction with RARa keeps RARP transcription first such factors to be identified was PEA-2. a labile represhigh, even in the presence of declining RA levels, while sor which binds to the polyoma virus promoter/enhancer interactions between c-Jun and RAR proteins control their region adjacent to the binding site for PEA-I (AP-I) and relative activities. A second major event in differentiation, blocks or inhibits PEA-I function(29,30).Additional suppresnot yet clearly linked to the first pathway, is the sequestration sive factors have been found in F9 cclls associated with the of the transcription factor E2F, possibly through altered TCIIA and TCIIB elements of the SV40 early gene enhancer phosphorylation of the retinoblastoma protein. One conseregion(31).Factors binding in these regions recognise similar quence of E2F removal is a reduction in cell proliferative binding sequences in the H-2Kb and endoA gene promotcapacity. ersi31,32). The sizes of DNA-protein complexes seen suggest These ccntral events in F9 differcntiatioii (shown as A and that there may he a family of factors binding to closely R in Figure 2) are accompanied by various other changes related sequences and active on different genes. Many of the whosc interlinkages are currently unknown. What appears to group 2 genes shown in Figure 1 as activated at around 48 be crucial for altered cxpression of particular genes is the balhours into the differentiation process appear to have a strong ance in bindinghctivity of differing sets of transcription facdependence on AP- 1 for stimulation of their transcription. tors (positive and negative). It is clear that difrerentiation is There may be a major factor similarly driving group 3 gene accompanied by widespread alterations in the availability of activation, but this has not yet been identified. Clearly there these factors. The mechanism for this is unclear, but altered is still considerable work required to clarify the net effects of phosphorylation has been proposed as one possibility. different subsets of positively and negatively acting factors Altered phosphorylation could be the major factor linhng binding to the promoter regions of the individual genes putative protein X cffect, modulation of c-Jun DNA binding which show altered expression as F9 cells differentiate. activity, E2F sequestration and the widespread changes in transcription factor activity that have been observed. The model proposcd in Figure 2 attempts to bring together An E l A-Like Activity in F9 Cells? all of the hithcrto unconnected segments of F9 differcnVarious groups have reported the presence in F9 cells of an tiation. If the model is correct, it reveals an elegant and activity resembling that of the adcnovirus protein E l A. This tightly controlled mechanism for fixing cells into a particular activity has been implicated in various properties of F9 cells, progression of changcs once the initial stimulus for differenincluding suppression of viral enhanccr activity and the tiation is received, and provides a foundation for targeting transformed growth phenotype(??’.Within cells, El A itself some of the inissing links in the pathway. The factor linking acts by releasing the transcnption factor E2F (DRTF) from RAR and c-jun gene activation should soon be identified and its complex with the retinoblastoma protein (RR)(74).The the putative receptor-independent pathway clarified. When freed E2F binds to and activates transcription of a variety of this is linked to changes in E2F complex formation, the genes including those involved in cellular proliferation such essential events controlling altered cell phenotype will be as cfos, c-riiyr and c-jun(35.36). understood.

We have recently reported that DINLIF (leukemia inhibitory factor) blocks diffcrentiation of a mutant F9 cell Line, OTF9("), but not of the parent F9 cells. We proposed that OTF9 cells were mutated in one of two pathways for RA-induced F9 differentiation. Since LIF also blocks RAindependent differentiation in embryonic stem cells(41)it is possible that this agent may be targeting the RAR-independent pathway described above in F9 cells. The observation provides a focus for further dissection of the elements of F9 EC cell differentiation in vitm. In addition, it should now be possible to investigate whether the general principles predicted for differentiation of ICM to extracmbryonic endoderm from these in v i m studies reflect the pattern of events seen during embryo development in vivo. If the general picture described here reflects the in vivo situation. then differentiation of inner cell mass to extra embryonic endoderm may be one of the first processes of mammalian development to be understood in detail.

Acknowledgements The author is grateful for the comments and suggestions of Drs. M. Frommer and P. Molloy during the preparation of this review. Work in the author's laboratory has been supported by thc Australian Meat & Livestock Research and Development Committee. References 1 Hogan, B.L.M., Rarlow, D.P. and Tilly, R. ( I 983). F9 (eratocarcinoma cells ar B model for the differentlntion of parietal a n d \iscerol endodenn in the mouse embryo. CnrrcerSurv. 2. 115-140. 2 Strickland, S. and Mahdavi, V. (1978). The inductioii of differentiation in teratocarcinonia cells by relinoic acid. (.'el/ 15, 393-403. 3 Strickland, S., Smith, K.K. and Marotti, K.R. (1980).Ihrnional induction or differentiation in teratocarcinoma slemcells. C d / ,21.347-355. 4Sleigh,M.J. (1985). Virusexpression as aprobeof aregulatory events inearly mouse emhryogencsis. Trends Gener. I, 17-21. 5 Sleigh, M.J. (1989). Gene cxpiession and diffeiciitiation in F9 mouse embryonal cnrcinoma cell\. Birrhein. Sor. Syni/i. 55, 1 - 1 2. 6 Kryszke, M.H., Piette, J. and Yaniv, M.(1987). Induction of a factor that hinds to thc polyoma rims A enhancer on differentiation of embr?.onal carcinoma cells. Nurut-e 328,254-256. 7 de Groot, R. P., Kruyt, F. A.E., van der Saag, P. T. and Kruijer, W.(1990). Cctopic expression ol' c-jun lead\ to ddlerentiation of PI 5 emhryoiial carcinoma cells. EMBO J. 9. 1831-1837. 8 Yang-Yen. H-F., Chin, R. and Karin, M. (19901. Elcvation of AP- I activity during F9 cell differentiation is due to increasedcjun transcription. NetvBid. 2,351-361. 9 Verma, I.M.. Ransone, LJ., lnoue, J-I., Kerr, L.D., Ofir, R., Dwarki, V.J. And Hunter, D. (1591). Nuclear oncoprotcins as transcription factors. J. Cell Bioclzeni Suppl. 15B,89. 10 Kitabayashi, l., Kawakami, Z., Chin, R., Ozawa, K., Matsuoka, T., Toyoshima, S., Umesono, K., Evans, R.M., Gachelin, G. and Yoknyama, K. ( 1992). Transciiptional regulation of the c-jun gene by retinoic acid and El A during dillerenliation ofF9 cells. EMBO J. 11. 167-175. 11 Hu. L. and Gudas, IJ. (1 550). Cyclic AMP analogs and rctinoic acid influence the expression of retinoic acid receptor u,0, dnd y trtKNAs in F9 teratocarcinoma cells. Mol. CellBiol. 10,391-396. 1 2 deThe, H., Vivanco-Ruiz, M-dM., Tiollais, P., Stunnenberp, H. and Dejean, A. (lY90). Identification of a ietinoic acid responsive element in the retinoic acid receptor 0 gene. iVotirre343, 177-180. 13 Sucov, H.M.. Ylurakami, K.K. and Evans, R.M. (1950). Characterization of an autoregulated response element in Itie mouse retinoic acid rcccptor type p gene. Proc. R'utlAcud. Sci. USA 87.5392-5396. 14 Griep, A.E. and Deluca, H.F. (1986) Stodiea on the relalion of DNA synthesis to retinoic acid-induced diffcrentiation of F9 teratocarcinoma cells. E.xp. Cell Kes. 164, 223-231. 15 deGrool, K.P., Pals, C. and Kruijer. W.(1991). Transcriptional control of c-jitn by rctiiioic acid. Nuci. AcidsIks.19. 1585-1SY I 16 Baichwal. V.R., Park, A. and Tjian, R. (1991). v-Src and EJ Ras alleviatc repression of c-Jun by a ccll-specific inhibitor. Notiri-r352, 165-1hX.

17 Wasyljk, C., Imler, J.L., Perez-Mntul, J. and Wasylyk, R. (I987). The c-Hu-ru., oncogene and a tumor promoter acthate the polyoma virus enhancer. Cell 48. 525-

534. 18 I,ainph, W.W., Dwarki, V.J., Ofir. R., Montminy, M. and Verma, I.hI. (1990). Negative and positive regulation by tianscription factor CAMP rcsponse elementbinding protein is modulated by phosphorylation. Proc. Noti A w d . Sci USA. 87,43204324. 19 I'amph, W.W. (1991). Cross-coupliiig of Ap-l irndintncellularhormone icceptori. Cancer CeZls3, 183-185. 20 Brindle, P.K. and Montmiiiy, M.R. (1992). The CREE Family of transcription aclivntors. Curr. Bid. 2, 199-204. 21 Mangelsdorf, D.J., Borgmeyer, U., Heyman, R.A., Yang, Zhou, J., Ong, E.S., Oro, A.E., Kakizuka, A. and Evans, R.M. (1992). Charactenzation of three RXR genes that mediate the action of 9 - c retiiioic ~~ acid. Gens f k v . 6, 329-344 22 Kliewer, S.A., Umesono, K., MangelsdiBrf, D.J. and Evans, R.M. (1992). Retiooid X rcccptor interacr? wil h nuclear receptors in retinoic acid, thyroid hormone and bitamin I13 \ignalling. Nazure 355.446-449. 23 Glass, C.K., Dcvary, O.V. and Rosenfeld, M.G. (1991)). Mulople cell typespecific proteins differentially regulate targel sequence recognition by the u retinoic acid rxsptor. Ccli 63,729-738. 24 Yang-Yen, H-F., %hang, X., Graupncr, G., Tzukcnnan, M., Sakamoto, B.. Karin, M. and PRhl, M. (1991). Antagonism between rctinoic acid reccptors and AP1: implications for tumor promotion and inflammation. A h , B i d 3, 1206-12119. 25 Vasios, G., Mader, S., Gold, J.D., Leid, M., I,utz, Y.. Gaub, M-P., Chambon, P. and Gudas, 1,. (1991). The late retinoic acid induction oflaminin B 1 gcne transcription involves RAR binding to the responsive clement. EMBO .I.IO, 1149.1 158. 26 Vasios, G.W., Gold, J.D., Petkovich, M.,Chambon, P.and Gudas, L.J. (19S9). A retinoic acitl-responsive element is present in the 5' flanking region ofthe laniinin B 1 gene. Proc. NatlAcad. Sci USA 86.3099-9103. 27 Umesono, K., Murakami, K.K., Thompwn, C.C. and Evans, R.M. (1991). Direct repeats as selective response elements for the thyroid hormone, retinoic acid, andvitamin Di receptors. Ceii65,1255-1266. 28 EFpeseth, A S . , Murphy, S.P. and I h n e y , E. (1989). Retinoic acid receptnr expression vector inhibits differentiation of F9 embryonal carcinoma cella. Geties Dev. 3,1647-1656. 29 Martin, M.E., Piette, .I., Yaniv. M., Tang, W-J and Folk, W-.R. (1988). Activation of the polyomavirus enhancer by ii murine activator protein 1 (AP-I) honiolog and two contiguous proteins. Proc. .VurZAcnd. Sci. USA 85,5839-5843. 30 Wasylyk, R., Imler, J.I,., Chattnn, B., Scliata, C. and Wasylyk, C. (1988). Negative and positive factors determine the actiwty of Ihe rdyoma VIIW cnhanccr u domain in undifferentiated and differentiated cell types. Proc l\'ulat/Acad. Sci. LEA 85. 7952-7956. 31 Macchi, M., Uornert, J-M., Dsvidsun. I., Kanno, M., Rosales, R., Vigneron, M., Xiao, J-H,,Fromental, C. and Chambon, P. (1989). The SV40 'I'C-II(KH) enhanson binds ubiquitous and cell type spccifically inducible nuclear proteins from lymphoid and non-lymphoidcell linen. Et4lRO.I. 8.4215-4227. 32 Onclcrcq, R., Lavenu, A. and Cremisi, C. (lYX9). Pleiotrophic derepression of devclopmentally regulated cellular and viral genes by c - m y proto-oncogene products m undifferentiated embryonal carcinoma cells. Nucl. Acids Res. 17,735-753. 33 Bandara, T,.R. and La Thangue, N.B. (1991). .4denovirus E l a prevents the retinoblastoma gene product Cram complexing wilh a cellular tranicription factor. Nulirr-e 351,494-497. 34 Chellappan, S.P., Heihert, S., Mudryj, M., Horowitz, J.M. and Nevins, J.R. (1991). The E2F transcriphur factor is A cellular taigct for thc RB protein. CeU 65, 1053-1061. 35 Hiebert, S.W., Chellappan, S.P., Horowitz, J.M. and Nevins. J.R. (1992) The interaction ofRB aithE2Fcoincides with an inh~bilionof the transcriptional activity of E2F GrmcJ.Dcv. 6,177-185. 36 De Caprio, J.A., Furukawa, Y., Ajchenhaum. F., Griffin, J.D. and L i ~ n p t o n , D.M. [ 1992). The retiiioblastoma-susceptibility gene product becomes phosphorylated in multiple stages during cell cycle entry and progression. Proc N d Al'ad. Sci. US;Z 89, 1795-1798. 37 Wagner, S. and Green, M.R. (1991). A transcriptlnnal tryst. Nuture 352, 189-190. 38 Shivj, M.K.K. and La Thangue, N.B. (1391). Multicomponent diflrentiationregulated transcnption factors i n F9 embryoiial carcinoina stem cells. Mol. Cell B d . 11, 1686-1605. 39 Reichel, R., Kovesdi, I and Ncvins, J.R. (1987). Developmenlai control of a promoter-specific factor that is also rcgulatcd by thc E I A gene product. C ' d / 48, 501 506. 40 Brown, G.S., Brown, M.A., Hilton, D.. Gough, N.M. and Sleigh, M.J. (1992). Inhibitjun of differentiation in a inuriiie F9 embryonal carcinoma cell rubline by leukemia inhibilory factoi-(LIF). Cruivrh Factorx, in press 41 Hilton, D.J. and Gough, N.M. (1951). Leukemia inhibitory factor: a biological pcrspectivc. J. Cell. Biochem. 46,21-26. 42 Kanno, M., Fromental. C., Staub, A., Kuffenach, F., Davidson, I. and C h a m h n , P. (lY89). Thc SV40 TC-II(kB) and the related H-2Kh enhmsons exhibit different cell type specific and inducible protoenhancer activities, but Ihe SV40 core sequence and thc AP-2 binding site have no enhanson propcrties. EMBO J. 8, 42054214. 43 Baldw+n, AS., .Ir. and Sharp, P.A. (1987). Binding of a nuclear k t o r to a

regulatory sequence in the promoter of thc mouse H-2Kh class 1 major histocompatibility gene. Mol. CelZHinZ. 7, 305-313. 44 Taysios, P.T. and La Thangue, N.B. (1990). A multiplicity of differentiationregulated ATF site-binding activities in embryonal carcinoma cells with distinct sequence and promoter specificities. New B i d . 2, 1123-1 134.

induction of laininin BI . collagen IV(a1). andothcl-diffcrentiation-specific niRNAs by retinoic acid in F9 teratocarcinoma cell\. J. C ' d l I'hy.siol. 136,305 31 1 48 Hosler,B.A.,LaKosd,C;.J.,(;rippo, J.P.andGudas,L.J. (IYX9).Expressiooof REX-1. a gene containing zinc tingcr motifs. is rapidly rcduccd by retinoic acid in F9 reratocarcinoma cells. Moi. Cdl. Aiol. 9. 5623-5629.

45 Flanagan, J.R., Murata, M., Burke, PA., Shiraynshi, Y., Appella, E.. Sharp, P.A. end Ozato, K. (1991). negative regulslion of (he major histocomp;itibility complex class i promoter in embryonal carcinoma cells. PTOC.Narl Acud. Sci. USA 83, 31 45.3 149. 46 Lenardo, MJ., Staudt, L., Robhins, P., Kuang. A., Mulligan, R.C. and Baltimore, D. (1989). Repression of the TgH enhancer i n leralocarcinoma cells associated with a novel octatner factor. Science 243.544-546. 47 Wang, S-Y.and Gudaq, L.J. (1988). Protem synthesis inhibitora prevent the

' Engineering, Merilyn J. Sleigh is at the CSIRO Division of Biomolecular PO Box 184,North Rydc, NSW 21 13, Australia.

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THE NEW YORK ACADEMY OF SCIENCES

Human Gene Therapy June 26 to 30,1993 Hyatt Regency Washington, On Capitol Hill, Washington, DC Somatic cell gene therapy is a rapidly developing, new therapeutic approach for the treatment of human disease. This new therapeutic approach promises to open new avenues in which human disease may be approached from a genctic levcl. By definition, somatic cell gene therapy is the transfer of defined genetic material into particular target cells of a patient, thereby altering the genotype and in most cases, the phenotype of those cells for the purpose of treating a particular disease state. The first clinical trials involving genc transfer and gene therapy are ongoing. Many diverse clinical applications of gene therapy are presently under extensive review before final approval and implementation. The purpose and scope of this confcrence is to describe recent advances in the applications of gene therapy for the treatment of human disease. The conference will include discussion of: - Dilferent gene therapy strategies - Advances in gene delivery systems with special attention directed at the advantages and disadvantsges of viralmediatcd genc delivery systems, liposomes and receptor-mediated gene delivery systems - Advances in construction of artificial genes

-Target cells in gene therapy -

Applications of human gene therapy in hereditary disease, neoplastic disease and viral disease

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Ethical, regulatory and medical issues in gene therapy and public policy

There will be contributed poster sessions in conjunction with this conference and these will form an integral part of the program. The deadline for submission of poster abstracts is March 15,1993. The entire ahstract, including title, authorts), and affiliations, musL be typed single-space and contained within a rectangle that measures 5" x 4+" (wxl). (Abstract form is not necessary.) Abstracts should be sent to: Dr. B. E. Huber, Wellcome Research Laboratories, 3030 Cornwallis Rd., Research Triangle Park, NC 27709, USA. For further informat~oncontact' ConferenceDepartment, New York ALademy of Sciciices 2 Edst 63rdStreet,New York, NY 10021, USA. Phone (212) 838-0210 Fax (212) 888-2894 Cable NYACSCI ~ ___

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