Targeting of an interrupted polypurine:polypyrimidine sequence in mammalian cells by a triplex-forming oligonucleotide containing a novel base analogue.

The DNA triple helix consists of a third strand of nucleic acid lying in the major groove of an intact DNA duplex. The most stable triplexes form on polypurine:polypyrimidine sequences, and pyrimidine interruptions in the purine strand are destabilizing. Sequence stringency is imparted by specific Hoogsteen hydrogen bonds between third strand bases and the purine bases in the duplex. Appropriate base and sugar modifications of triple helix-forming oligonucleotides (TFOs) confer chromosome targeting activity in living cells. However, broad utilization of TFOs as gene targeting reagents in mammalian cells has been limited by the requirement for homopurine target sequences. Although there have been a number of base analogues described that appear to be promising as candidates for triplex target expansion, none has been examined in a biological system. We have employed a postsynthetic strategy to prepare a collection of TFOs with base analogues at a defined position. Following assessment of affinity for a triplex target with a single C:G inversion, TFOs with a second generation of analogues were synthesized. One of these, TFO-5a, with 2'-OMe-guanidinylethyl-5-methylcytosine at the position corresponding to the C:G interruption in the target sequence, was further modified to confer bioactivity. The activity of this TFO, linked to psoralen, was measured in a mammalian cell line that was engineered by directed sequence conversion to carry a triplex target with a single C:G interruption. TFO-5a was active against this target and inactive against the corresponding target with an uninterrupted polypurine:polypyrimidine sequence.

DNA repair and mutagenesis in Werner syndrome.

Werner syndrome (WS) is the hallmark premature aging syndrome in which the patients appear much older than their actual chronological age. The disorder is associated with significantly increased genome instability and with transcriptional deficiencies. There has been some uncertainty about whether WS cells are defective in DNA repair. We thus examined repair in vitro in nuclear and mitochondrial DNA. Whereas cellular studies so far do not show significant DNA repair deficiencies, biochemical studies with the Werner protein clearly indicate that it plays a role in DNA repair.

Coordinate action of the helicase and 3' to 5' exonuclease of Werner syndrome protein.

Werner syndrome is a human disorder characterized by premature aging, genomic instability, and abnormal telomere metabolism. The Werner syndrome protein (WRN) is the only known member of the RecQ DNA helicase family that contains a 3' --> 5'-exonuclease. However, it is not known whether both activities coordinate in a biological pathway. Here, we describe DNA structures, forked duplexes containing telomeric repeats, that are substrates for the simultaneous action of both WRN activities. We used these substrates to study the interactions between the WRN helicase and exonuclease on a single DNA molecule. WRN helicase unwinds at the forked end of the substrate, whereas the WRN exonuclease acts at the blunt end. Progression of the WRN exonuclease is inhibited by the action of WRN helicase converting duplex DNA to single strand DNA on forks of various duplex lengths. The WRN helicase and exonuclease act in concert to remove a DNA strand from a long forked duplex that is not completely unwound by the helicase. We analyzed the simultaneous action of WRN activities on the long forked duplex in the presence of the WRN protein partners, replication protein A (RPA), and the Ku70/80 heterodimer. RPA stimulated the WRN helicase, whereas Ku stimulated the WRN exonuclease. In the presence of both RPA and Ku, the WRN helicase activity dominated the exonuclease activity.

Targeted gene knockout by 2'-O-aminoethyl modified triplex forming oligonucleotides.

Triplex forming oligonucleotides (TFOs) are of interest because of their potential for facile gene targeting. However, the failure of TFOs to bind target sequences at physiological pH and Mg(2+) concentration has limited their biological applications. Recently, pyrimidine TFOs with 2'-O-aminoethyl (AE) substitutions were shown to have enhanced kinetics and stability of triplex formation (Cuenoud, B., Casset, F., Husken, D., Natt, F., Wolf, R. M., Altmann, K. H., Martin, P., and Moser H. E. (1998) Angew. Chem. Int. Ed. 37, 1288--1291). We have prepared psoralen-linked TFOs with varying amounts of the AE-modified residues, and have characterized them in biochemical assays in vitro, and in stability and HPRT gene knockout assays in vivo. The AE TFOs showed higher affinity for the target in vitro than a TFO with uniform 2'-OMe substitution, with relatively little loss of affinity when the assay was performed in reduced Mg(2+). Once formed they were also more stable in "physiological" buffer, with the greatest affinity and stability displayed by the TFO with all but one residue in the AE format. However, TFOs with lesser amounts of the AE modification formed the most stable triplexes in vivo, and showed the highest HPRT gene knockout activity. We conclude that the AE modification can enhance the biological activity of pyrimidine TFOs, but that extensive substitution is deleterious.

Expression of ATM in ataxia telangiectasia fibroblasts rescues defects in DNA double-strand break repair in nuclear extracts.

Ataxia telangiectasia (A-T) is a human genetic disorder characterized by progressive cerebellar degeneration, hypersensitivity to ionizing radiation (IR), immunodeficiency, and high cancer risk. At the cellular level, IR sensitivity and increased frequency of spontaneous and IR-induced chromosomal breakage and rearrangements are the hallmarks of A-T. The ATM gene, mutated in this syndrome, has been cloned and codes for a protein sharing homology with DNA-PKcs, a protein kinase involved in DNA double-strand break (DSB) repair and DNA damage responses. The characteristics of the A-T cellular phenotypes and ATM gene suggest that ATM may play a role similar to that of DNA-PKcs in DSB repair and that there is a primary DNA repair defect in A-T cells. In the current study, the function of ATM in DNA DSB repair was evaluated in an in vitro system using two plasmids, carrying either an EcoRI-induced DSB within the lacZalpha gene or various endonuclease-induced DSB in the SupF suppressor tRNA gene. We found that the DSB repair efficiency in A-T nuclear extracts was comparable to, if not higher than, that in normal nuclear extracts. However, the repair fidelity in A-T nuclear extracts was decreased when repairing DSB with short 5' and 3' overhangs (<4 base pairs (bp)) or blunt ends, but not 5' 4-bp overhangs. Sequencing of the mutant plasmids revealed that deletions involving 1-6 nucleotide microhomologies were the major class of mutations in both A-T and normal extracts. However, the size of the deletions in plasmids from A-T nuclear extracts was larger than that from normal nuclear extracts. Expression of the ATM protein in A-T cells corrected the defect in DSB repair in A-T nuclear extracts. These results suggest that ATM plays a role in maintaining genomic stability by preventing the repair of DSB from an error-prone pathway.

Unwinding of a DNA triple helix by the Werner and Bloom syndrome helicases.

Bloom syndrome and Werner syndrome are genome instability disorders, which result from mutations in two different genes encoding helicases. Both enzymes are members of the RecQ family of helicases, have a 3' --> 5' polarity, and require a 3' single strand tail. In addition to their activity in unwinding duplex substrates, recent studies show that the two enzymes are able to unwind G2 and G4 tetraplexes, prompting speculation that failure to resolve these structures in Bloom syndrome and Werner syndrome cells may contribute to genome instability. The triple helix is another alternate DNA structure that can be formed by sequences that are widely distributed throughout the human genome. Here we show that purified Bloom and Werner helicases can unwind a DNA triple helix. The reactions are dependent on nucleoside triphosphate hydrolysis and require a free 3' tail attached to the third strand. The two enzymes unwound triplexes without requirement for a duplex extension that would form a fork at the junction of the tail and the triplex. In contrast, a duplex formed by the third strand and a complement to the triplex region was a poor substrate for both enzymes. However, the same duplex was readily unwound when a noncomplementary 5' tail was added to form a forked structure. It seems likely that structural features of the triplex mimic those of a fork and thus support efficient unwinding by the two helicases.

Stability of DNA triplexes on shuttle vector plasmids in the replication pool in mammalian cells.

Triple helix-forming oligonucleotides may be useful as gene-targeting reagents in vivo, for applications such as gene knockout. One important property of these complexes is their often remarkable stability, as demonstrated in solution and in cells following transfection. Although encouraging, these measurements do not necessarily report triplex stability in cellular compartments that support DNA functions such as replication and mutagenesis. We have devised a shuttle vector plasmid assay that reports the stability of triplexes on DNA that undergoes replication and mutagenesis. The assay is based on plasmids with novel variant supF tRNA genes containing embedded sequences for triplex formation and psoralen cross-linking. Triple helix-forming oligonucleotides were linked to psoralen and used to form triplexes on the plasmids. At various times after introduction into cells, the psoralen was activated by exposure to long wave ultraviolet light (UVA). After time for replication and mutagenesis, progeny plasmids were recovered and the frequency of plasmids with mutations in the supF gene determined. Site-specific mutagenesis by psoralen cross-links was dependent on precise placement of the psoralen by the triple helix-forming oligonucleotide at the time of UVA treatment. The results indicated that both pyrimidine and purine motif triplexes were much less stable on replicated DNA than on DNA in vitro or in total transfected DNA. Incubation of cells with amidoanthraquinone-based triplex stabilizing compounds enhanced the stability of the pyrimidine triplex.

Increased spontaneous mutation frequency in human cells expressing the phage PBS2-encoded inhibitor of uracil-DNA glycosylase.

The Ugi protein inhibitor of uracil-DNA glycosylase encoded by bacteriophage PBS2 inactivates human uracil-DNA glycosylases (UDG) by forming a tight enzyme:inhibitor complex. To create human cells that are impaired for UDG activity, the human glioma U251 cell line was engineered to produce active Ugi protein. In vitro assays of crude cell extracts from several Ugi-expressing clonal lines showed UDG inactivation under standard assay conditions as compared to control cells, and four of these UDG defective cell lines were characterized for their ability to conduct in vivo uracil-DNA repair. Whereas transfected plasmid DNA containing either a U:G mispair or U:A base pairs was efficiently repaired in the control lines, uracil-DNA repair was not evident in the lines producing Ugi. Experiments using a shuttle vector to detect mutations in a target gene showed that Ugi-expressing cells exhibited a 3-fold higher overall spontaneous mutation frequency compared to control cells, due to increased C:G to T:A base pair substitutions. The growth rate and cell cycle distribution of Ugi-expressing cells did not differ appreciably from their parental cell counterpart. Further in vitro examination revealed that a thymine DNA glycosylase (TDG) previously shown to mediate Ugi-insensitive excision of uracil bases from DNA was not detected in the parental U251 cells. However, a Ugi-insensitive UDG activity of unknown origin that recognizes U:G mispairs and to a lesser extent U:A base pairs in duplex DNA, but which was inactive toward uracil residues in single-stranded DNA, was detected under assay conditions previously shown to be efficient for detecting TDG.

Mutation spectra in supF: approaches to elucidating sequence context effects.

Shuttle vectors carrying the supF suppressor tRNA gene were originally developed for mutagenesis experiments in primate and human cells. Since then, the supF gene has been used as a mutation reporter in other mammalian cells, yeast, Escherichia coli, and transgenic mice. The widespread use of the vector for studies of many DNA reactive agents has produced a large database of mutation spectra. These provide primary information on the kinds and distribution of mutations provoked by many agents and, in many instances, allow comparisons between related agents or the same agent in different cell backgrounds. In this review we will discuss some of these data with a primary focus on the interpretation of UV mutation spectra. We will also describe our development and application of custom supF marker genes as an approach to studying the effect of sequence context on mutation hotspots and cold spots. Our studies suggest that C-C photoproducts are not mutagenic in certain sequence contexts in which T-C photoproducts are mutation hotspots. In addition, we have found several examples of sequence context effects acting as much as 80 bases away from the site of mutation. We will consider some of the problems raised by these studies and the possible resolution of some of them offered by the newly discovered family of damage bypass DNA polymerases.

Triple-helix formation induces recombination in mammalian cells via a nucleotide excision repair-dependent pathway.

The ability to stimulate recombination in a site-specific manner in mammalian cells may provide a useful tool for gene knockout and a valuable strategy for gene therapy. We previously demonstrated that psoralen adducts targeted by triple-helix-forming oligonucleotides (TFOs) could induce recombination between tandem repeats of a supF reporter gene in a simian virus 40 vector in monkey COS cells. Based on work showing that triple helices, even in the absence of associated psoralen adducts, are able to provoke DNA repair and cause mutations, we asked whether intermolecular triplexes could stimulate recombination. Here, we report that triple-helix formation itself is capable of promoting recombination and that this effect is dependent on a functional nucleotide excision repair (NER) pathway. Transfection of COS cells carrying the dual supF vector with a purine-rich TFO, AG30, designed to bind as a third strand to a region between the two mutant supF genes yielded recombinants at a frequency of 0.37%, fivefold above background, whereas a scrambled sequence control oligomer was ineffective. In human cells deficient in the NER factor XPA, the ability of AG30 to induce recombination was eliminated, but it was restored in a corrected subline expressing the XPA cDNA. In comparison, the ability of triplex-directed psoralen cross-links to induce recombination was only partially reduced in XPA-deficient cells, suggesting that NER is not the only pathway that can metabolize targeted psoralen photoadducts into recombinagenic intermediates. Interestingly, the triplex-induced recombination was unaffected in cells deficient in DNA mismatch repair, challenging our previous model of a heteroduplex intermediate and supporting a model based on end joining. This work demonstrates that oligonucleotide-mediated triplex formation can be recombinagenic, providing the basis for a potential strategy to direct genome modification by using high-affinity DNA binding ligands.

Targeted correction of an episomal gene in mammalian cells by a short DNA fragment tethered to a triplex-forming oligonucleotide.

Triplex-forming oligonucleotides (TFOs) can bind to polypurine/polypyrimidine regions in DNA in a sequence-specific manner and provoke DNA repair. We have coupled a TFO to a short donor fragment of DNA that shares homology to a selected gene as a strategy to mediate gene targeting and correction. In this bifunctional oligonucleotide, the TFO domain is designed to bind the target gene and stimulate repair and recombination, with the donor domain positioned for recombination and information transfer. A series of these tethered donor-TFO (TD-TFO) molecules with donor domains of 40-44 nucleotides and TFO domains in both the purine and pyrimidine triplex motifs were tested for their ability to mediate either gene correction or mutation of a supF reporter gene contained in a SV40 shuttle vector in mammalian cells. In vitro binding assays revealed that the attachment of the donor domain via a flexible linker did not significantly alter the binding affinity of the TFO domain for the polypurine site in the supF target DNA, with equilibrium dissociation constants in the 10(-8) M range. Experiments in which the target vector and the linked TD-TFOs were pre-incubated in vitro and co-transfected into cells led to conversion frequencies approaching 1%, 4-fold greater than with the two domains unlinked. When cells that had been previously transfected with the SV40 vector were electroporated with the TD-TFOs, frequencies of base pair-specific gene correction were seen in the range of 0.04%, up to 50-fold over background and at least 3-fold over either domain alone or in unlinked combinations. Sequence conversion by the TD-TFOs was achieved using either single- or double-stranded donor domains and either triplex motif. Substitution of either domain in the TD-TFO with control sequences yielded reagents with diminished activity, as did mixtures of unlinked TFO and donor DNA segments. The boost in activity provided by the attached TFO domain was reduced in cells deficient in the nucleotide excision repair factor XPA but was restored in a subclone of these cells expressing XPA cDNA, suggesting a role for nucleotide excision repair in the pathway of triple helix-stimulated gene conversion. The ability to correct or mutate a specific target site in mammalian cells using the TD-TFO strategy may provide a useful tool for research and possibly for therapeutic applications.

Triplex formation by oligonucleotides containing 5-(1-propynyl)-2'-deoxyuridine: decreased magnesium dependence and improved intracellular gene targeting.

Oligonucleotides capable of sequence-specific triple helix formation have been proposed as DNA binding ligands useful for modulation of gene expression and for directed genome modification. However, the effectiveness of such triplex-forming oligonucleotides (TFOs) depends on their ability to bind to their target sites within cells, and this can be limited under physiologic conditions. In particular, triplex formation in the pyrimidine motif is favored by unphysiologically low pH and high magnesium concentrations. To address these limitations, a series of pyrimidine TFOs were tested for third-strand binding under a variety of conditions. Those containing 5-(1-propynyl)-2'-deoxyuridine (pdU) and 5-methyl-2'-deoxycytidine (5meC) showed superior binding characteristics at neutral pH and at low magnesium concentrations, as determined by gel mobility shift assays and thermal dissociation profiles. Over a range of Mg2+ concentrations, pdU-modified TFOs formed more stable triplexes than did TFOs containing 2'-deoxythymidine. At 1 mM Mg2+, a DeltaTm of 30 degreesC was observed for pdU- versus T-containing 15-mers (of generic sequence 5' TTTTCTTTTTTCTTTTCT 3') binding to the cognate A:T bp rich site, indicating that pdU-containing TFOs are capable of substantial binding even at physiologically low Mg2+ concentrations. In addition, the pdU-containing TFOs were superior in gene targeting experiments in mammalian cells, yielding 4-fold higher mutation frequencies in a shuttle vector-based mutagenesis assay designed to detect mutations induced by third-strand-directed psoralen adducts. These results suggest the utility of the pdU substitution in the pyrimidine motif for triplex-based gene targeting experiments.

Targeted gene knockout mediated by triple helix forming oligonucleotides.

Triple helix forming oligonucleotides (TFOs) recognize and bind sequences in duplex DNA and have received considerable attention because of their potential for targeting specific genomic sites. TFOs can deliver DNA reactive reagents to specific sequences in purified chromosomal DNA (ref. 4) and nuclei. However, chromosome targeting in viable cells has not been demonstrated, and in vitro experiments indicate that chromatin structure is incompatible with triplex formation. We have prepared modified TFOs, linked to the DNA-crosslinking reagent psoralen, directed at a site in the Hprt gene. We show that stable Hprt-deficient clones can be recovered following introduction of the TFOs into viable cells and photoactivation of the psoralen. Analysis of 282 clones indicated that 85% contained mutations in the triplex target region. We observed mainly deletions and some insertions. These data indicate that appropriately constructed TFOs can find chromosomal targets, and suggest that the chromatin structure in the target region is more dynamic than predicted by the in vitro experiments.

Aliphatic and alicyclic diols induce melanogenesis in cultured cells and guinea pig skin.

We have found that several aliphatic and alicyclic diols induce melanogenesis in cultured S91 mouse melanoma cells and normal human epidermal melanocytes (NHEM). In addition, these compounds induce melanogenesis when applied to guinea pig skin, with transfer of melanin to keratinocytes and formation of "supranuclear caps," as occurs in naturally pigmented skin. The relative order of potency of some of these diols in NHEM is 5-norbornene-2,2-dimethanol > 3,3-dimethyl-1,2-butanediol > cis-1,2-cyclopentanediol > 2,3-dimethyl-2,3-butanediol > 1,2-propanediol. Following treatment with these diols or 3-isobutyl-1-methylxanthine, melanin and tyrosinase activity are increased within S91 cells and NHEM; however, for cultured NHEM, the largest increases of melanin and tyrosinase occur in an extracellular particulate fraction, shown by electron microscopy to consist almost entirely of stage III and IV melanosomes. These results indicate that cultured NHEM treated with diols export melanosomes in a fashion that is commensurate with natural melanogenic processes. In contrast, S91 mouse melanoma cells exhibit aberrant melanosomal trafficking, in accordance with the known defect in myosin-V mediated melanosomal transport. Both S91 cells and NHEM exhibit morphologic changes and growth arrest indicative of differentiation following treatment with diols. The diols described in this report are candidates for use as cosmeceutical tanning agents.

Recombination induced by triple-helix-targeted DNA damage in mammalian cells.

Gene therapy has been hindered by the low frequency of homologous recombination in mammalian cells. To stimulate recombination, we investigated the use of triple-helix-forming oligonucleotides (TFOs) to target DNA damage to a selected site within cells. By treating cells with TFOs linked to psoralen, recombination was induced within a simian virus 40 vector carrying two mutant copies of the supF tRNA reporter gene. Gene conversion events, as well as mutations at the target site, were also observed. The variety of products suggests that multiple cellular pathways can act on the targeted damage, and data showing that the triple helix can influence these pathways are presented. The ability to specifically induce recombination or gene conversion within mammalian cells by using TFOs may provide a new research tool and may eventually lead to novel applications in gene therapy.

Single nucleotide positions have proximal and distal influence on UV mutation hotspots and coldspots.

Base substitution mutation frequency is influenced by the sequence context surrounding lesions in the DNA. We have been studying ultraviolet mutagenesis in human repair-deficient cells in the supF marker gene carried in a shuttle vector plasmid. There are prominent hotspots, on opposite strands, at the 5' TC sites in the eight base palindrome 5' CTTCGAAG. Recently, we developed a reporter system which permits sequence manipulation in the vicinity of mutational hotspots. We have used the system to characterize the influence of individual positions in the palindrome on the frequency of mutagenesis at the two UV hotspots. In this paper we have determined the contribution of bases at the second and third positions in the palindrome. Changes in bases that were in the primer template duplex when the replication complex encountered the photoproducts at one of the hotspot sites significantly increased or decreased the probability of mutations at the site. We also observed modulation of hotspot activity at other sites as a function of single base changes as much as 80 bases away from the hotspots. In these instances, the site of the changed base was in the unreplicated template ahead of the primer terminus when the polymerase encountered the relevant photoproduct. Our results indicate that sequence context has both proximal and distal consequences for mutagenesis.

Mutagenesis by third-strand-directed psoralen adducts in repair-deficient human cells: high frequency and altered spectrum in a xeroderma pigmentosum variant.

Psoralen-conjugated triple-helix-forming oligonucleotides have been used to generate site-specific mutations within mammalian cells. To investigate factors influencing the efficiency of oligonucleotide-mediated gene targeting, the processing of third-strand-directed psoralen adducts was compared in normal and repair-deficient human cells. An unusually high mutation frequency and an altered mutation pattern were seen in xeroderma pigmentosum variant (XPV) cells compared with normal, xeroderma pigmentosum group A (XPA), and Fanconi anemia cells. In XPV, targeted mutations were produced in the supF reporter gene carried in a simian virus 40 vector at a frequency of 30%, 3-fold above that in normal or Fanconi anemia cells and 6-fold above that in XPA. The mutations generated by targeted psoralen crosslinks and monoadducts in the XPV cells formed a pattern distinct from that in the other three cell lines, with mutations occurring not just at the damaged site but also at adjacent base pairs. Hence, the XPV cells may have an abnormality in trans-lesion bypass synthesis during repair and/or replication, implicating a DNA polymerase or an accessory factor as a basis of the defect in XPV. These results may help to elucidate the repair deficiency in XPV, and they raise the possibility that genetic manipulation via triplex-targeted mutagenesis may be enhanced by modulation of the XPV-associated activity in normal cells.

Mutagenesis in mammalian cells induced by triple helix formation and transcription-coupled repair.

When mammalian cells were treated with triplex-forming oligonucleotides of sufficient binding affinity, mutations were specifically induced in a simian virus 40 vector contained within the cells. Triplex-induced mutagenesis was not detected in xeroderma pigmentosum group A cells nor in Cockayne's syndrome group B cells, indicating a requirement for excision repair and for transcription-coupled repair, respectively, in the process. Triplex formation was also found to stimulate DNA repair synthesis in human cell extracts, in a pattern correlating with the inhibition of transcription in such extracts. These findings may have implications for therapeutic applications of triplex DNA and raise the possibility that naturally occurring triple helices are a source of genetic instability.

The influence of single base changes on UV mutational activity at two translocated hotspots.

Mutation hotspots have been a staple of mutation spectra since the introduction of fine structure mutation mapping almost 40 years ago. It has been well established that sequence context is an important determinant of mutational activity at mutagen induced hotspots and coldspots. However, our understanding of the sequence effectors of base substitution hotspots is quite limited. This is because manipulation of the sequence about a hotspot site in a marker gene is restricted by the need to maintain a functional marker. In this work, we describe a generalizable system for studying sequence context effects on mutagenesis. We have prepared a variant of the supF tRNA gene (a marker used by us in previous studies) in which an eight-base palindrome, the site of two UV hotspots in the interior of the gene, was copied into the acceptor stem and pre-tRNA region. The variant tRNA was active. The UV mutation spectrum of this variant showed that the new copy of the palindrome generated two hotspots which were as intense as the original sites in the interior of the gene. Variant genes were constructed with all possible bases at the first position in the palindrome in the pre-tRNA sequence, which does not affect tRNA function. The mutation analysis showed that activity at one of the hotspots could be reduced or enhanced by the changes, while activity at the other site was not significantly affected. The base changes did not influence the frequency of cyclobutane dimer or (6-4) photoproduct formation at the two hotspot sites. Thus, the changes in mutational activity were due to the influence of sequence context on the efficiency of mutation formation at the sites of UV lesions.

IL-3 and ribavirin induce differentiation and growth suppression during long-term treatment of a megakaryocytic leukemia cell line.

Differentiation of the megakaryocytic leukemia cells, CMK, was induced by long-term (12 day) treatment with the combination of IL-3 and the nucleoside analogue ribavirin (RV), which reduces cellular GTP levels. In a previous report we demonstrated the induction of early messages and antigens, as well as the formation of giant polyploid cells in the cultures (Majumdar et al., 1994, J. Cell. Physiol., 160:29-39). Here we show high level induction of messages for the late markers, Platelet Factor 4, GMP140 (P-Selectin), thrombospondin, and beta thromboglobulin. The induced cells are also positive for these antigens by immunocytochemical analysis. The high level message induction resulted from synergy between the inducers. Pretreatment of the cells with IL-3 could accelerate the rise in message seen with the inducer combination. The increase in differentiation markers was accompanied by a reduction of the proliferative capacity of the cells. Riboguanosine, which has anti differentiation activity, blocked the induction of early and late antigens by the inducer combination, and also by IL-3 acting alone, but did not block the reduction in proliferative competence. In this model of megakaryocytic differentiation IL-3 treatment yields an initial stimulation of growth followed by growth suppression, and is the principal driver of the differentiation process. RV functions primarily as a stimulator of message and protein expression in synergy with IL-3.