Immortalization of Rat Embryo Fibroblasts by a 3′-Untranslated Region

EXPERIMENTAL CELL RESEARCH ARTICLE NO.

240, 252–262 (1998)

EX983937

Immortalization of Rat Embryo Fibroblasts by a 3 *-Untranslated Region Andrew J. Powell,*,1 Philip B. Gates,* Diana Wylie,* Cristiana P. Velloso,* Jeremy P. Brockes,†,2 and Parmjit S. Jat†,2 *Ludwig Institute for Cancer Research, Courtauld Building, 91 Riding House Street, London W1P 8BT, United Kingdom; and †Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WC1E 6BT, United Kingdom

We have exploited a cross-species expression screen to search for cellular immortalizing activities. A newt blastemal cDNA expression library was transfected into rat embryo fibroblasts and immortal cell lines were selected. This identified a 1-kb cDNA fragment which has a low representation in the cDNA library and is derived from the 3*-UTR of an a-glucosidaserelated mRNA. Expression of this sequence in rat embryo fibroblasts has shown that it is active in promoting colony formation and immortalization. It is also able to cooperate with an immortalization-defective deletion mutant of SV40 T antigen, indicating that it can exert its growth-stimulatory activity in the pathway activated by a viral immortalizing oncogene. This is the first example of an immortalizing activity mediated by an RNA sequence, and further analysis of its mechanism should provide new insights into senescence and immortalization. q 1998 Academic Press

INTRODUCTION

Most animal cells have a restricted proliferative potential both in vivo and in vitro [1, 2]. When cell types from embryos or adults are cultured, they cease proliferation after a finite number of divisions [3, 4]. Such senescent cells cannot be induced to divide by the addition of fresh growth medium. They do not die, but remain metabolically active and retain some responsiveness to mitogens, as assayed by expression of certain immediate-early genes [5]. It has been suggested that regulation of the finite life span comprises two components: first, a counting mechanism that counts the number of divisions; and second, a process of entry into the postmitotic state [6, 7]. As yet the molecular basis for entry into senescence is poorly understood. A sub-group of viral and cellular oncogenes is able to overcome the finite life span of rodent cells [8]. Cells 1 Present address: Gene Function Unit, Glaxo Wellcome Research and Development, Gunnels Wood Road, Stevenage, SG1 2NY, U.K. 2 To whom correspondence and reprint requests may be addressed. Fax: 44 171 878 4040.

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0014-4827/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.

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which do not undergo senescence are said to be immortal, since they have acquired an indefinitely long life span. Immortal cells remain dependent on the presence of mitogens (although they can have a reduced requirement for them) and cannot overgrow a confluent monolayer. They cannot form tumors in nude mice and are thus distinct from fully transformed malignant cells. Serial cultivation of rodent embryo fibroblasts sometimes results in the production of spontaneously immortal cell lines [9, 10]. The cellular lesions which enable these cells to continue proliferating are poorly defined, although alterations in the gene for the negativegrowth regulator p53 have been observed in certain spontaneously immortal cell lines [11, 12], while increased transcription of the c-myc cellular proto-oncogene has also been observed in other mouse cell lines [5]. In contrast, normal human and avian cells rarely undergo spontaneous immortalization, and thus the frequency at which cells escape senescence appears to be dependent upon the donor species [13]. Serial transfer of genetic material has proven to be a powerful approach to identify the activated oncogenes responsible for some tumor phenotypes [14]. For example, Weinberg and colleagues showed that transfection of genomic DNA from T24 bladder carcinoma cells induces transformation of NIH3T3 cells and used serial transfer to isolate the mutated ras gene, the first cellular oncogene to be isolated [15]. Since the cellular lesions that result in spontaneous immortalization of rodent cells have not yet been fully defined, we attempted to use an expression screen to search for cellular immortalizing activities. We screened a eukaryotic cDNA expression library for cDNAs able to immortalize rat embryo fibroblasts (REF). The source of RNA for preparation of this library was newt limb blastemal cells. These cells may represent an interesting source for studies on in vitro life span because they can be serially passaged for hundreds of generations without detectable crisis or senescence [16, 17]. We have isolated a cDNA fragment which is able to overcome the finite life span of REF. This fragment will also cooperate with an immortalization-defective deletion mutant of SV40 large T antigen to immortalize REF, indicating that it can exert its growth-stimulatory activity in the path-

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way activated by this viral immortalizing oncogene. It is derived from the 3*-untranslated region (UTR) of an a-glucosidase-related mRNA and as such is the first example of a noncoding RNA sequence with immortalizing activity. MATERIALS AND METHODS Cells and cell culture. REF were prepared from 13-day-old Sprague–Dawley rat embryos and maintained at 37 or 337C in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), 2 mM glutamine, 100 units/ml penicillin, and 100 mg/ml streptomycin. Newt hindlimb blastemal mesenchyme (B1H1) cells were cultured and passaged as described previously [17]. All media and components were obtained from GibcoBRL. cDNA library construction, electroporation, and isolation of stable cell lines. Total RNA was extracted from newt limb blastemal tissue at early or mid-bud stages (amputations were carried out as described in [18, 19]). A cDNA library was constructed from this RNA, following poly(A) selection, by Clontech Laboratories Inc., using the phage vector lDR2. This vector has within it an Epstein–Barr virusderived plasmid vector (pDR2) which allows expression of the cloned cDNA in mammalian cells [20]. Plasmid DNA for electroporations was prepared from the library according to the manufacturer’s instructions. REF were passaged 24 h before electroporation to ensure logarithmic growth. Then 107 cells were electroporated in Hepes-buffered saline at 260 V/960 mF using a Bio-Rad Laboratories’ Gene Pulser. Cotransfections were carried out using a 20:1 molar ratio of nonselectable DNA (either carrier plasmid DNA or plasmids expressing SV40 T antigen mutants) to selectable DNA (pDR2–cDNA library clone) in a total of 50 mg DNA. Forty-eight hours after electroporation, cells were passaged and selected in 100 mg/ml of hygromycin B (Calbiochem-Novabiochem Ltd., [21]). All cultures were maintained for 2–5 weeks at 337C and the growth medium was changed every 3–4 days. Wherever possible, colonies were isolated, expanded into cell lines, and analyzed. The plates were stained with 2% (w/v) methylene blue in 50% (v/v) ethanol and the colonies were counted. Recovery of cDNA inserts from established cell lines by PCR amplification. Genomic DNA was prepared from confluent cell monolayers by standard procedures [22]. One hundred nanograms of genomic DNA was amplified by PCR, using two nested sets of primers which flank the cDNA cloning site within the lDR2 vector. The first round of amplification was done using the primers Eu (5*-ACGCCATTTGACCATTCACCACAT-3*, located 11 nucleotides downstream of the RSV LTR) and Ed (5*-ATGTGCTGCAAGGCGATTAAGTTG3*, located 17 nucleotides upstream of the SV40 poly(A) site). Amplification was performed with 0.5 mg of each primer, 2.5 units of Taq DNA polymerase (Promega Corp.) in 10 mM Tris–HCl, pH 8.4, 50 mM KCl, 0.1% (w/v) gelatin, 0.05% (v/v) Nonidet-P40, 0.05% (v/v) Tween 20, 0.2 mM dNTPs (Pharmacia Biotech), and 2 mM MgCl2 and comprised 30 cycles of denaturation at 957C for 30 s, annealing at 707C for 1 min, and polymerization at 727C for 3 min. Then 1 ml of this first-round amplification product was used as template in the second round of PCR amplification using primers Iu (5*-GCACCTCCAAGCTTGTCGAGGAAC-3*, located 6 nucleotides downstream of Eu) and Id (5*-TGCCAAGCTTGCATGCCTGCAGGT-3*, located 45 nucleotides upstream of Ed), but still corresponding to vector sequences on either side of the cDNA cloning site. The PCR conditions were the same for both rounds of amplification. DNA sequencing. Dideoxy-sequencing was carried out using either a Pharmacia T7 sequencing kit or a USB Sequenase sequencing kit according to the manufacturer’s instructions and fractionated on an 8% polyacrylamide 8.3 M urea gel in 11 TBE (90 mM Tris–borate

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and 1 mM EDTA) on an IBI STS45 vertical slab gel sequencing apparatus at 60 W for 3–6 h. Automated sequencing was carried out using the PRISM Ready Reaction DyeDeoxy Terminator Cycle Sequencing kit (Perkin–Elmer Corp.) and analyzed on an Applied Biosystems 373A DNA Sequencer System according to the manufacturer’s instructions. Screening of cDNA libraries. lDR2 newt forelimb blastema and lgt11 newt forelimb blastema libraries were screened as described previously [23]. RNase protections. The primers 3920 (5*-TGTTTGGGCAAGCAGTCTTC-3*) and 3921 (5*-GCTGTGATTAACCCTCTGAC-3*) were used to amplify 203 or 225-bp fragments of newt cDNA from the pDR2-L2 and L3 clones, respectively. These fragments were then inserted into pBluescript and used as templates for preparing riboprobes for RNase protection assays. Sense L2 and L3 riboprobes were prepared from templates linearized by HindIII digestion using T7 RNA polymerase, while antisense riboprobes were prepared from templates linearized by EcoRI digestion using T3 RNA polymerase. Riboprobes were prepared using an RNA transcription kit (Stratagene) and 60 mCi [a-32P]UTP at 440 Ci/mmol per reaction. Probes were purified using Sephadex G-50 spin columns. Twenty micrograms of total RNA was hybridized overnight at 457C with 1 1 106 cpm/reaction of the appropriate probe in 80% formamide [24]. Samples were then treated with ribonuclease A and ribonuclease T1, after which the reactions were terminated by digestion with proteinase K, extracted with phenol:chloroform:iso-amyl alcohol (24:24:1), and ethanol precipitated. The protected fragments were fractionated on an 8 M urea–8% polyacrylamide gel.

RESULTS

Expression Screen of a Newt Limb Blastema Expression Library for cDNAs Able to Immortalize REF The newt limb blastema cDNA expression library was transfected into secondary REF in order to identify immortalizing activities (Fig. 1). For transfection the lDR2-cDNA library was converted to a pDR2-cDNA plasmid library by infecting Escherichia coli AM1, a strain which catalyzes recombination between the two bacteriophage P1-derived loxP sites present within the vector. This results in the production of pDR2 plasmids carrying the cDNA inserts [25] and a hygr gene that encodes resistance to hygromycin B [21]. Expression of the cDNA in mammalian cells is driven from the Rous sarcoma virus (RSV) LTR [26] and transcripts are polyadenylated by the SV40 polyadenylation signal (27). Expression of the hygr gene is driven by the herpes simplex virus thymidine kinase (HSV tk) promoter and transcripts are polyadenylated by the HSV tk polyadenylation signal. These two transcription units give rise to convergent transcripts. Plasmid DNA was prepared from a random single-phage plaque with a 2-kb cDNA insert and was transfected as a negative control. Stable transfectants were isolated and serially passaged to derive cell lines. No cell lines were established from control transfections using the randomly isolated 2-kb cDNA. This is in accordance with many other transfection experiments which we have carried out using a variety of control DNAs as part of our studies

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FIG. 1. Schematic diagram depicting the cross-species expression screen. Multiple electroporations of 1.5–2.5 mg of library DNA into 107 secondary REF were done until approximately 2 1 106 stable transfectants were screened. Control transfections were performed with DNA derived from a single plaque, isolated at random from the lDR2 library. Two days after electroporation, cells were passaged and stable transfectants were isolated in medium containing 100 mg/ml hygromycin B for 2–3 weeks. Hygromycin B was then removed and colonies were allowed to form. Colonies were isolated and expanded into cell lines. Genomic DNA prepared from these cell lines was analyzed by PCR for the presence of cDNA inserts. Since the stability of mRNAs derived from newt cDNAs was unknown at temperatures above the normal growth temperature of newt cells (257C), the transfected REF and the established cell lines were incubated at a temperature lower than normal (337C). We have subsequently found that this is not necessary to assay the activity of the sequence we have isolated.

on immortalization by SV40 large T antigen (A.J.P. and P.S.J., unpublished data). In contrast the blastemal cDNA library yielded 43 lines, thus suggesting that certain sequences were able to stimulate the sustained proliferation of rodent cells (Table 1). Genomic DNA was prepared from these cell lines at passage 3 and

used as a template for nested PCR with primers flanking the cDNA cloning site. Sixteen of the cell lines were found to contain one or more cDNA inserts (Table 1). If expression of these inserts is required to maintain growth, then they should be retained upon serial passaging. Genomic DNA prepared from these cell lines

TABLE 1 Expression Screen of Newt Limb Blastema cDNA Expression Library for Immortalizing Activities Number of cell lines which yielded PCR inserts at passage

DNA

Total amount of pDR2 DNA transfected

Number of transfections carried out

Total number of colonies obtained (Average number/mg pDR2 DNA)

Number of cell lines derived

3

6

pDR2-S pDR2-library

4.0 mg 40.0 mg

2 20

23 (5.75) 260 (6.00)

0 43

— 16

— 4

Note. Secondary REF were transfected (107 cells per electroporation) with indicated amount of the pDR2 cDNA library or pDR2-S (split over the indicated number of electroporations) using pBluescript as carrier DNA (to a total of 50 mg DNA per electroporation). Electroporated cells were plated onto four 15-cm plates, selected in medium containing 100 mg/ml hygromycin B, and incubated at 337C. Colonies were picked after 4–5 weeks and expanded into cell lines wherever possible. Genomic DNA was prepared from these cell lines at p3 and p6 and used as template for PCR amplification, using primers from either side of the cDNA insertion site within the pDR2 vector. The indicated number of cell lines gave PCR inserts at p3 and p6. In an extensive series of experiments not reported here we have been unable to isolate any stable cell lines after transfection of control DNA.

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at passage 6 was amplified and four of the cell lines were found to retain a cDNA insert. The other cell lines failed to retain the cDNA insert upon passaging, suggesting either that the cDNA was not required for the immortal phenotype in these cell lines or that the DNA sequence recognized by one of the PCR primers was lost. None of the cell lines that were isolated expanded very rapidly but readily gave rise to variant cells that were morphologically distinguishable and readily overgrew the original culture. To determine whether any of the cDNA inserts were related, Southern blots of the PCR fragments were hybridized with each PCR product. The 1-kb PCR product amplified from cell line h4 cross-hybridized to the 1-kb insert amplified from cell line d21, suggesting that these two cell lines had been immortalized by related cDNAs. These two cell lines were isolated from independent transfections and retained their inserts during serial passaging. They also had the same orientation within the vector and had similar 5* and 3* ends; the structure of these two inserts is shown in Fig. 2A. Sequence analysis of the h4 and d21 cDNA inserts indicated that they were essentially identical (the few mismatches may be accounted for by errors during the PCR amplification). The other two cDNA inserts did not cross-hybridize to each other. Isolation and Characterization of Corresponding cDNA Clones from the Newt Blastema Library The PCR product from the h4 cell line was used to screen the lDR2 library. Three positive plaques were isolated from a screen of approximately 1.25 1 106 clones, indicating that cDNAs related to h4 were rare in this library. Two of the three positive clones isolated from the library contained 1.3-kb cDNA inserts (pDR2L2 and pDR2-L3; the structures are shown in Fig. 2A), each of which had a poly(A) tail. The position of the poly(A) tail indicated that the L2 cDNA was in an orientation which would produce sense RNA from the RSV promoter, whereas the L3 cDNA and the cDNAs amplified from the h4 and d21 cell lines would produce antisense RNA (Fig. 2A). Furthermore, neither L2 or L3 contained a significant open reading frame. The third clone, pDR2-L4, was also longer than the cDNA insert originally amplified from the two cell lines, corresponding to a 1.9-kb cDNA with a 3* end and poly(A) tail similar to both L2 and L3, but with an extra 600 bp at the 5* end (data not shown). pDR2-L4 also had no significant open reading frame. The sequence of clone L3 is shown in Fig. 3. The sequences of the common regions between L3 and L4 cDNAs differ from those of L2 and the original 1-kb cDNAs amplified from the cell lines in that neither L3 nor L4 has the 22-bp deletion that is 340 bp into the 1.0-kb common region (Fig. 3).

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FIG. 2. Activity of newt cDNA clones in immortalization of secondary REF. (A) Screening of the newt blastema cDNA library with the h4 cDNA fragment identified two 1.3-kb cDNA clones. Their abundance in the library and orientation with respect to the pDR2 RSV LTR and their poly(A) (pA) tails are shown. Clone pDR2-L2 expresses sense RNA, whereas pDR2-L3 expresses antisense RNA. The position of the 22-bp deletion within the L2 clone as well as in the h4/d21 inserts is also indicated. (B) The library clones (1.5 mg) were transfected into secondary REF using pBluescript (48.5 mg) as carrier DNA. Transfected REF were selected in the presence of 100 mg/ml hygromycin B and cultured at 337C for 4 or 5 weeks. Colonies were isolated and, wherever possible, expanded into cell lines. Plates were stained with methylene blue and colonies were counted. The average number of colonies formed from two experiments is shown as a percentage of the number of colonies obtained 2 weeks after transfection of a plasmid expressing wild-type SV40 T antigen. The numbers at the top of the graph indicate the number of cell lines derived from the number of colonies isolated. Both cDNAs gave rise to a significant number of colonies following transfection into REF and some of these colonies were able to expand to yield cell lines. N.D., not done.

Transfection of Library Clones L2 and L3 into Secondary REF Increases Their Life Span The library clones L2 and L3 were transfected into secondary REF and stable transfectants were isolated. The number of colonies formed after 4–5 weeks of incubation at 337C and the number of cell lines derived are shown as a percentage of the number of colonies

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The colonies isolated following transfection of either pDR2-L2 or pDR2-L3 gave some cell lines, with pDR2L2 yielding more than pDR2-L3 (7 of 22 and 3 of 17, respectively). These lines grew slowly, at rates that were comparable to those found for the original h4 and d21 lines, and were found to contain the newt cDNA inserts after analysis by PCR amplification after passage 4 (6 out of 7 for L2 and 2 out of 3 for L3). These cells were not continually passaged because of their slow growth rate and due to the appearance of variant cells which had a much higher growth rate. The L3 cDNA Clone Cooperates with an Immortalization-Defective Mutant of SV40 Large T Antigen

FIG. 3. DNA sequence of the cDNA insert in pDR2-L3. This is shown as expressed from the RSV LTR, but note that this is antisense with respect to the newt mRNA, as predicted by the position of the poly(A) tail. The underlined sequence corresponds to the 951-bp h4/ d21 cDNA insert. The 22 bp lacked by pDR2-L2 clone and h4/d21 is also indicated, as are the restriction sites flanking the cDNA cloning site in the pDR2 vector. The presence of an internal BamHI site and the presence of the poly(A) tail (in the antisense orientation) are also indicated.

obtained following transfection of a plasmid encoding wild-type SV40 large T antigen (Fig. 2B). Plasmids pDR2-L2 and pDR2-L3 yielded a similar number of colonies, while pDR2-S (randomly selected 2-kb cDNA) yielded only one colony, demonstrating that the immortalization activity of L2 and L3 was not simply due to integration of the expression vector causing an activation or inactivation of a cellular gene. It should be noted that this comparison of L2 with L3 cannot be used to deduce the orientation dependence of the activity, since transfectants were found to express both sense and antisense transcripts in comparable amounts due to the pDR2 vector containing two convergent transcription units (see below).

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To determine whether the newt sequences could act in the same pathway as a known immortalizing oncogene, the library clones were cotransfected with several immortalization-defective mutants of SV40 large T antigen. T antigen requires regions within both its amino and its carboxy termini for efficient immortalization of rodent fibroblasts [28–34]. Mutant T128-708 encodes a T antigen lacking the amino-terminal 127 aa [35]; dl1135 has a 10-aa deletion within the conserved region 1 (CR1)-like domain within the amino terminus (aa 17–27), which disrupts a function required for transformation of C3H10T1/2, REF52, primary baby rat kidney, and CREF cells [28, 36]. We have recently found that dl1135 is also unable to immortalize REF but was able to maintain immortalization in conditionally immortal REF cell lines [6, 37] that are dependent upon T antigen for their continued growth (A.J.P. and P.S.J., unpublished data). Mutant 5041 has two point mutations within the carboxy terminus, at aa 426 and 430, which abolish the ability to complex with the negative growth regulator p53 (K. W. Peden and J. M. Pipas, unpublished data). This mutant is unable to immortalize REF or to maintain immortalization in T antigendependent cell lines but can cooperate with dl1135 to immortalize REF (A.J.P. and P.S.J., unpublished data). The results of these cotransfections are shown in Fig. 4; it is important to note that the results presented are for colony formation within 2–3 weeks rather than 4– 5 weeks as shown in Table 1 and Fig. 2. The efficiency of colony formation is expressed as a percentage of the number of colonies that form after transfection of wildtype T antigen (Fig. 4A). In some cases the number of cell lines that were derived by expansion of the colonies is also shown. Transfection of each of the T antigen mutants alone yielded few colonies, with the highest, dl1135, yielding only 6.3% of the number of colonies obtained with wild-type T antigen, and only one of these colonies could be expanded to yield a cell line. The library vector (pDR2) was also unable to yield immortal cell lines. Cotransfection of either of the L2 or

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FIG. 4. Clones L2 and L3 are able to immortalize secondary REF in cooperation with immortalization-defective mutants of SV40 T antigen. (A) 1.5 mg of the indicated DR2 clones and 48.5 mg of T antigen mutants or control pBluescript were cotransfected into secondary REF. Transfected REF were cultured at 377C in the presence of 100–125 mg/ml hygromycin B for 2 weeks. Plates were stained with methylene blue and colonies were counted. The average number of colonies formed from at least three independent experiments is shown as a percentage of the number of colonies obtained with wild-type T antigen. The numbers at the top of the graph indicate the number of cell lines derived compared with the number of colonies isolated. Both the L2 and the L3 clones are able to cooperate with dl1135 to immortalize REF. This is more efficient with the L3 clone. N.D., not done. (B) Representative plates obtained following transfection into REF of pDR2 alone, pDR2-L3 alone, dl1135 plus pDR2, and dl1135 plus pDR2-L3 were stained with methylene blue after 2 weeks in culture. The cotransfection of dl1135 plus pDR2-L3 yields an increased number of colonies, which grow more rapidly than either the dl1135 alone or the pDR2-L3 alone.

L3 clones with mutant 5041 also failed to immortalize within 2–3 weeks, yielding colonies at efficiencies relative to that of wild-type T antigen of 5.3 and 2.0%, respectively. In contrast both L2 and L3 clones produced colonies upon cotransfection with dl1135 and these colonies were obtained within 2–3 weeks (it should be noted that colony formation after transfection of L2 or L3 alone required 4–5 weeks). Colony formation was more efficient when L3 was cotransfected with dl1135, rather than L2 (colony formation for L3 is illustrated by the stained dishes shown in Fig. 4B), and gave an efficiency of 61.4% compared to wildtype T antigen yielding 21 cell lines from the 34 colonies isolated. Clone L2 yielded colonies at an efficiency of 26.8%, and 9 of the 24 colonies expanded to give cell lines. Moreover, the colonies isolated after cotransfection expanded much more rapidly and were phenotypically stable in comparison to those isolated with L2 or L3 alone. While cotransfection of L2 with mutant T128708 failed to immortalize REF, some colonies were formed, and cell lines were obtained, following cotransfection of L3 with T128-708. This occurred at a decreased efficiency (14.4%) compared to cotransfection

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of L3 with dl1135, and fewer cell lines were derived (4 cell lines from 11 colonies isolated). This is consistent with our observation that T128-708 is less active than dl1135 in maintaining the growth of a T antigen-dependent REF cell line (A.J.P. and P.S.J., unpublished data). These data indicate that the newt sequences can cooperate with certain mutants of T antigen that are defective for immortalization and hence may be able to act via the T antigen pathway. Analysis of Expression of the Newt cDNA Fragments in the Immortal Cell Lines To determine whether the cell lines isolated after cotransfection of dl1135 with either L2 or L3 expressed RNA complementary to the cDNA, total cellular RNA was analyzed by RNase protection. Antisense and sense riboprobes were prepared from 203-bp (L2) and 225-bp (L3) PCR fragments which span the 22-bp deletion of L2 (shown in Fig. 6). RNA from each of two cell lines derived from the cotransfections was analyzed. The results of these RNase protections are shown in Figs. 5A (the L2 cell lines) and 5B (the L3 cell lines).

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FIG. 5. Analysis of expression of the newt sequence in the REF cell lines and in newt cells. (A) Sense (S) and antisense (A) riboprobes were prepared from a 203-bp fragment of the L2 cDNA and used to protect 10 mg RNA from each of the indicated cells or cell lines. The positions of the unprotected and 203-bp protected riboprobes are indicated. Both representative REF cell lines, established following the cotransfection of dl1135 with pDR2-L2, expressed sense and antisense RNA from the L2 cDNA. (B) Sense (S) and antisense (A) riboprobes were prepared from a 225-bp fragment of the L3 cDNA and used to protect 20 mg RNA from each of the indicated cells or cell lines. The positions of the unprotected and 225-bp protected riboprobes are indicated (an overexposure is shown to demonstrate the presence of a protected RNA of the correct size in lane 4). Both of the representative REF cell lines, established following cotransfection of dl1135 with pDR2-L3, expressed sense and antisense RNA from the L3 cDNA. To ensure that equal amounts of RNA were analyzed for each sample, RNase protection assays were carried out in parallel using a riboprobe which protects a fragment of the rat g-actin RNA [68].

Protected fragments were observed with both the sense and the antisense L2 and L3 riboprobes in hybridizations to RNA from the L2 cell lines (Fig. 5A, lanes 3– 6) and the L3 cell lines (Fig. 5B, lanes 3–6), respectively. This indicates that all four cell lines expressed both antisense and sense RNA from the transfected newt cDNA clones, irrespective of the orientation of the cDNA with respect to the RSV LTR. No protection was

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detected following hybridization of any of the riboprobes to control RNA prepared from secondary REF (lanes 7 and 8) or tRNA (lanes 11 and 12). The protected fragment was detected when the antisense riboprobe prepared from L2 was hybridized to RNA from newt hindlimb blastemal cells (B1H1 cells; [17]). It was not detected after hybridization with the sense riboprobe, indicating that only the sense RNA

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FIG. 6. Schematic diagram of the newt 7.5-kb mRNA. The 3210-bp coding region is represented by the open box and the positions of the ATG and stop codons are indicated. Distances from the poly(A) tail are indicated in kilobases. Also shown are the two 1.3-kb cDNA clones used in the REF immortalization assays, the original 1-kb h4/d21 cDNA insert, and the DNA fragments used to prepare riboprobes for RNase protection assays. The positions of internal BamHI and XbaI sites are also indicated.

is detectable. In addition the antisense L3 riboprobe yielded a protected fragment in hybridizations with RNA from newt forelimb blastema tissue, while the L3 sense riboprobe did not, indicating that only the sense RNA is expressed in blastemal cells in vivo. The Immortalizing cDNA Fragments Originate from the 3*-UTR of an a-Glucosidase-Related cDNA Northern blot analysis of poly(A)-selected RNA from newt forelimb and hindlimb tissue indicated that the mRNA from which the L2 and L3 cDNA fragments are derived is approximately 7.5 kb (data not shown). The sequence of the full-length newt cDNA was assembled by isolating adjacent cDNA fragments from the lDR2 newt limb blastema library and a lgt11 forelimb blastema library [23]; see Materials and Methods). The fulllength cDNA has a 3210-bp open reading frame, starting at an ATG which is 560 bp from the 5* end of the cDNA, and encodes a protein of 1070 aa (Fig. 6). The predicted amino acid sequence of the protein has 83% similarity and 70% identity to the protein encoded by the open reading frame of an a-glucosidase-related cDNA cloned from the human cell line KG-1 (KIAA0088; [38]). DISCUSSION

We have used a cross-species expression screen to search for cellular cDNAs that are able to immortalize REF. This assay required that REF were stably transfected to hygromycin resistance, grew into a colony, and subsequently expanded into a cell line, while retaining a cDNA insert. Only a few independent transfection events satisfied these criteria (Table 1), and it was striking that two of them contained essentially identical cDNA fragments which were rare in the original library. Two clones obtained after screening the library with the h4 fragment yielded colonies upon

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transfection into REF, although these grew very slowly in comparison to those obtained with SV40 T antigen. It has been shown that T antigen has a number of independent functional domains that are required for immortalization of secondary REF. Therefore we cotransfected L2 or L3 with several T antigen mutants, including dl1135 and 5041, and assayed for immortalization of secondary REF. While neither cDNA cooperated with mutant 5041, both were able to cooperate with dl1135 to immortalize REF and yielded immortal lines that expanded rapidly. Thus the cDNAs can function in the pathway activated by this viral immortalizing gene. SV40 T antigen has been extensively studied to identify the activities that are required to immortalize cells. T antigen binds to and sequesters pRB, p107, p130, the RB family of pocket proteins, and p53 [29, 39–42]. Recently the amino-terminal domain of T antigen has been shown to share functional homology with the J domain of the Dna J family of molecular chaperones [36, 43] and this is also required for immortalization, dense focus formation, and tumorigenesis of the choroid plexus in transgenic mice [44]. In addition to the HPDK/R motif, the J box which is present in all Dna J proteins, the amino terminus also has homology (amino acids 17 to 32) to the CR1 domain of adenovirus E1A [45]. Deletion of this region in dl1135 affects the transformation properties of T antigen and its activity as a J domain, since it removes a portion of the predicted a-1 helix of this domain [36]. Since dl1135 T antigen is able to bind to p53 and the RB family of pocket proteins as well as p300 and p400 [46], it has been proposed that in the absence of a functional J domain, the interactions with the pocket proteins are not productive and this cannot be complemented in trans [36]. In contrast, Rundell and colleagues have shown that mutation of residues 19 and 28, which also affect J domain function and the ability of T antigen to produce foci in human diploid fibroblasts, can be

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complemented in trans by wild-type small t antigen [47]. dl1135 T antigen can also complement adenovirus E1A CR2 mutants, defective for binding to the RB family of pocket proteins, to immortalize baby rat kidney cells [45]. We have found that dl1135 T antigen is able to maintain the growth of conditionally immortal REF cell lines derived by immortalization with the thermolabile tsA58 T antigen and to immortalize secondary REF in cooperation with 5041 (A.J.P. and P.S.J., unpublished data). Here we have shown that both L2 and L3 can cooperate with dl1135, suggesting that they can act in the pathway utilized by T antigen. Since it has been postulated that the deletion in dl1135 prevents T antigen from interacting productively with the family of pocket proteins, it will be very interesting to identify the downstream targets of L2 and L3 and to determine if they ensure a productive interaction between the dl1135 T antigen and the RB family of pocket proteins or act downstream. It will also be interesting to determine if L2 and L3 can cooperate with dl1135 T antigen to produce choroid plexus tumors in mice. It is clear that future studies must address the question of whether the h4 sequence plays a role in the immortal phenotype of limb blastemal cells, although this is difficult to address in the newt system. Even though only the sense transcript was detected in RNA derived from normal limb tissue, it may nonetheless be informative to examine the expression of h4 by in situ hybridization of tissue sections during development and regeneration. Further experiments are required not only to evaluate the possible role of h4 in cell life span, but to assay for any acute activity in stimulating the proliferation of blastemal cells or promoting their formation by dedifferentiation, for example from cultured myotubes [4, 48, 49]. This is the first example of a noncoding RNA sequence with immortalizing activity, and it poses a significant challenge to understanding the mechanism of action. This mechanism is unlikely to be due to the integration of vector sequences resulting in the activation or inactivation of cellular genes because neither the randomly selected 2-kb control cDNA (pDR2-S in Table 1 and Fig. 2B) nor the pDR2 library vector yields immortal cell lines. It is possible, although most unlikely, that the 3*-UTR sequence leads to targeted integration. Recent work has shown that 3*-UTRs may be active in a variety of cellular processes [50]. Some of these appear to involve regulation of gene expression at the posttranscriptional level by controlling the transport of mRNA to the cytoplasm, its stability, or its translation [51–59]. These generally involve action of the 3*-UTR in cis, but similar activities might involve titration of regulatory factors in trans as is the case for the fem3 3*-UTR in Caenorhabditis elegans [60]. There are a variety of examples where antisense 3*-UTRs may act in trans to regulate expression [61–

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63], and if the target mRNA was involved in growth suppression, then the antisense could promote growth, in principle. In support of this possibility, it has recently been shown that the 3*-UTR of prohibitin may function as a trans-acting regulatory RNA whose tumor-suppressor activity has been inactivated by mutation in Group B cells [64]. A related expression strategy has been used to identify the 3*-UTR of a-tropomyosin as a sequence that was able to induce differentiation of a myogenic cell line (NMU2; [65]), suppress formation of rhabdomyosarcomas in mice, and suppress growth in immortal mouse fibroblasts [66]. This sequence can also induce trans-differentiation of cultured chick embryo fibroblasts into muscle cells [67]. These were the first demonstrations that 3*-UTRs could act in trans as regulators of growth and differentiation in the absence of coding sequences. Although our 3*-UTR fragment does not have extensive homology to the a-tropomyosin 3*UTR and does not contain any conserved elements such as those identified within other 3*-UTRs ([52]; L. Duret, personal communication), such comparisons will be further aided by definition of the elements in this sequence that are required for promoting growth. In conclusion we have used a cross-species expression screen to isolate a 1-kb cDNA fragment which has a low representation in the original cDNA library and is derived from the 3*-UTR of an a-glucosidase-related mRNA. Despite uncertainties about the mechanism, we have shown that this 3*-UTR sequence is able to promote the growth of REF and can cooperate with a viral immortalizing gene. New insights into senescence and immortalization should come from further studies on its mechanism of action in rodent cells, together with an analysis of its significance for the life span of newt blastemal cells. We are grateful to A. Gann, A. Kobrna, P. Bates, K. Moore, G. R. Mazars, E. Tanaka, and Z. Ikram for helpful discussions and to R. Patient for his comments on the manuscript. We thank J. Pipas, K. W. Peden, and M. J. Tevethia for T antigen mutants and L. Duret for sequence analysis.

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Received September 9, 1997 Revised version received December 9, 1997

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