Using an inducible transcription system which allows the regulated expression of C/EBP isoforms in tissue culture cells, we have found that the ectopic expression of C/EBP␣, at a level comparable to that found in normal liver tissue, has a pronounced antimitogenic effect in mouse L cells and NIH 3T3 cells. The inhibition of cell division by C/EBP␣ in mouse cells cannot be reversed by simian virus 40 T antigen, by oncogenic ras, or by adenovirus E1a protein. When expressed in thymidine kinase-deficient L cells or 3T3 cells, C/EBP␣ is detected in a protein complex which binds to the E2F binding sites found in the promoters of the genes for E2F-1 and dihydrofolate reductase (DHFR). Bacterially expressed C/EBP␣ has no affinity for these E2F sites, but when recombinant C/EBP␣ is added to nuclear extracts from mouse fibroblasts, a new E2F binding activity appears, which contains the C/EBP␣ protein. Using an E2F-DP1-responsive promoter linked to a reporter gene, it can be shown that C/EBP␣ directly inhibits the induction of this promoter by E2F-DP1 in transienttransfection assays. Furthermore, C/EBP␣ can be shown to inhibit the S-phase induction of the E2F and DHFR promoters in permanent cell lines. These findings delineate a straightforward mechanism for C/EBP␣-mediated cell growth arrest through repression of E2F-DP-mediated S-phase transcription.

Cell cycling and protein synthesis are both key physiological tasks for cancer cells. Here we present a model for how the elongation phase of protein synthesis, governed by elongation factor 2 and elongation factor 2 kinase, both modulates and responds to cell cycling. Within this framework we also discuss survivin, a protein with both pro-mitotic and anti-apoptotic roles whose persistence in the cell is tied to protein synthesis due to its short half-life. Finally, we provide a brief overview of efforts of cancer researchers to target EF2 and EF2 kinase.

We have transferred a mutant hamster gene coding for an altered dihydrofolate reductase to wild-type cultured mouse cells by using total genomic DNA from methotrexate-resistant Chinese hamster ovary A29 cells as donor. By demonstrating the presence of hamster gene sequences in transformants we have provided direct evidence for gene transfer. Transformants selected for increased resistance to methotrexate contain increased amounts of the newly transferred gene. We have used this mutant dhfr gene to introduce the Escherichia coli antibiotic resistance plasmid pBR322 into animal cells. Amplification of the dhfr sequences results in amplification of the pBR322 sequences as well. The use of this gene may allow the introduction and amplification of virtually any genetic element in various new cellular environments. The ability to transfer purified genes into cultured cells provides a unique opportunity to study the function and physical state of exogenous genes in new cellular environments. The development of systems for DNA transfer in animal cells originated with the lytic transfection of cells by using purified viral DNA (1, 2) and progressed to the stable transfer of viral transforming functions to appropriate recipient cells (3). Subsequently, viral genes from the herpesviruses coding for the biochemically selectable marker thymidine kinase (TK) (4-6) were transferred to enzyme-deficient mutant cells. Restriction fragments of herpes simplex virus type 1 encoding TK were isolated (6) and subsequently cloned into bacterial plasmids (7). Through the use of this selectable marker, virtually any gene can now be introduced into recipient cells (8, 9); however, these cells must be tk-mutants. Other potential selection systems are available, and several laboratories have recently demonstrated the DNA-mediated transfer of cellular genes coding for selectable markers such as TK (10), adenine phosphoribosyltransferase (11) and hypoxanthine phosphoribosyltransferase (12,13 Transformation and Selection. Ltk-aprF cells and NIH 3T3 cells were transformed with genomic DNA by the calcium phosphate coprecipitation method (2) as described (11). All DNAs were sterilized by ethanol precipitation and resuspended in 1 mM Tris-HCl/1 mM EDTA, pH 7.9. For tk+ transformation, cells were exposed to hypoxanthine/aminopterin/thymidine selective medium as described (10). Transformants resistant to Mtx were select~d in growth medium containing either 0.1 or 0.2 jqg of Mtx per ml with the same feeding schedule as for tk selection. Afte 2-3 weeks, colonies were isolated from individual dishes with cloning cylinders to ensure that each transformant arose from an independent event. In transformation with ligated DNAs, no more than 1 ,ug of pBR322 DNA was added to 106 cells per dish because higherThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.3567Abbreviations: DHFR, dihydrofolat...

A procedure is presented for the purification of alpha2u globulin, a protein synthesized in the liver, secreted into the serum, and excreted in the urine of male rats. The steady-state levels of alpha2u globulin in the serum and liver cytosol fraction of adult male rats have been determined using a radial immunodiffusion assay. A cell-free protein synthesizing system, easily prepared from commercial wheat germ, has been used to identify and quantitate the mRNA coding for alpha2u globulin. Poly(A)-containing RNA isolated from male rat liver directs the synthesis in the wheat germ translational system of a protein which is precipitated by rabbit anti-alpha2u globulin and migrates with authentic alpha2u globulin on sodium dodecyl sulfate-polyacrylamide gels. Poly(A)-containing RNA from the livers of female rats or from the kidneys of male rats, tissues which synthesize no alpha2u globulin, do not direct the synthesis of alpha2u globulin in the wheat germ system. Thyroidectomized male rats had no detectable alpha2u globulin in their sera or liver cytosols, and the livers from these thyroidectomized males were found to contain no translatable mRNA for alpha2u globulin, as measured in the wheat germ system. Administration of L-thyroxine or triiodo-L-thyronine to hyroidectomized males resulted in the synthesis of alpha2u globulin, as measured by increased levels of this protein in sera and liver cytosols. This increase in alpha2u globulin synthesis following thyroid hormone treatment was accompanied by a parallel increase in the functional level of the hepatic mRNA coding for this protein. Treatment of thyroidectomized males with a variety of androgens failed to stimulate alpha2u globulin synthesis, and no alpha2u globulin mRNA could be detected in the livers from these androgen-treated thyroidectomized males. These findings indicate that thyroid hormones influence alpha2u globulin synthesis in male rat liver by acting pretranslationally, possibly by modulating gene transcription, and rule out the possibility of an indirect androgen-mediated effect of thyroid hormones in modulating alpha2u globulin biosynthesis.

Two different genes coding for the hormonally regulated rat liver protein alpha 2u globulin were introduced into mouse Ltk- cells through co-transfection with the HSV-1 thymidine kinase gene. Three to ten copies of the alpha 2u globulin genes were detected several tk+ clones, over 50% of which could be induced with dexamethasone, the produce alha 2u globulin mRNA and protein. This suggests that the information necessary for hormonal response is contained in the DNA fragment used for transfer.

One of insulin's many biological effects is the increased transcription of AP-1-regulated genes. cJun is the principal component of the AP-1 transcription complex, which is regulated by the newly discovered members of the MAPK superfamily referred to as cJun NH2-terminal kinases (JNKs) or stress-activated protein kinases (SAPKs). We show that insulin stimulates a dose- and time-dependent increase in JNK activity in Rat 1 fibroblasts overexpressing human insulin receptors (Rat 1 HIR cells). Using two different polyclonal anti-JNK antibodies, JNK activity was measured after immunoprecipitation from whole cell extracts by phosphorylation of GSTcJun(1-79). Peak activation occurred 15 min after insulin addition, resulting in a 2.5-fold increase in GSTcJun(1-79) phosphorylation over unstimulated controls. Maximal JNK activation correlated with the onset of AP-1 DNA binding activity. Both insulin-stimulated JNK activity and insulin-induced AP-1 transcriptional activity were found to be Ras-dependent. These data suggest that in Rat 1 cells, JNK activation may play a role in insulin-regulated AP-1 transcriptional activity leading to a mitogenic response.

a2,-Globulin is a rat protein of as yet unknown function whose synthesis can be induced by glucocorticoids and several other hormones. Induction by glucocorticoids is a secondary response to the hormone: protein synthesis is required before the hormone can exert its stimulatory effect on oL2.-globulin transcription. We have used the linker-scanning mutagenesis procedure, followed by transfer of the mutant genes into mouse L-cells for analysis of their phenotype, to determine sequences within a cloned a2u-globulin promoter that are required for its regulation by glucocorticoids. Mutations between positions -115 and -160 abolish or greatly reduce the inducibility of a2.-globulin by the hormone. Mutations just upstream from this region, between positions -177 and -220, have an opposite effect; they increase induction two-to fourfold.Steroid hormones are important regulators of gene expression in many organisms. In general, these hormones exert their effects at the transcriptional level, inducing or repressing the synthesis of specific mRNAs (reviewed in references 1, 61). These effects can be either direct or indirect. A direct or primary response is characterized by insensitivity of mRNA induction to inhibitors of protein synthesis and by a rapid increase in the transcription of the inducible gene (62). In the best-studied example of a primary response, the induction of mouse mammary tumor virus (MMTV) transcription by glucocorticoids, the hormone receptor complex binds to specific DNA sequences within the provirus and, by a still unknown mechanism, stimulates transcription from the viral promoter (reviewed in references 47, 48).Indirect or secondary responses to steroid hormones require protein synthesis for transcription of the induced RNA and usually shows a lag between administration of the hormone and initiation of the response. The classical example of such a response is the induction by ecdysterone of the late puffs on the chromosomes of Drosophila salivary glands (reviewed in reference 46). The late puffs, unlike the early puffs, do not appear if protein synthesis is inhibited when the glands are first treated with the hormone. It seems likely that during the lag period a regulatory protein(s) is synthesized in response to the hormone. This protein is required for the expression of the secondary response (2,13 Mutagenesis. An outline of the mutagenesis procedure is shown in Fig. 3. A library of deletions, extending into the promoter from the a2,-globulin structural gene (3' deletions), was made by Bal 31 nuclease digestion from the SalI site of plasmid p91FHS. A 10-jig portion of plasmid DNA was digested with SalI. The DNA was precipitated with ethanol and dissolved in 300 jil of buffer containing 12 mM CaCl2, 12 mM MgCl2, 20 mM Tris hydrochloride (pH 8), 1 mM EDTA, 0.6 M NaCl, and 3 U of Bal 31 nuclease and incubated at 15°C. After incubation for 5, 10, and 15 min, 100-jil samples of the mixture were added to a tube containing 50 ,ul of phenol-CHCl3 (1:1) and 10 jil of 0.25 M EDTA, and the tube was vortex...

A procedure is presented for the purification of specific mRNAs, which exploits the ability of antibodies prepared against a native protein to bind to the nascent polypeptide on the polysome. Rather than precipitating these soluble antibody-polysome complexes with anti-antibody, which can lead to nonspecific trapping of polysomes, we have linked the anti-antibody to an insoluble matrix. Thus, the antibody-polysome complex binds to the anti-antibody support and nonspecific polysomes can easily be removed by several washes. We have found para-aminobenzyl cellulose (PAB cellulose), to be a suitable matrix for this purpose. This support can bind large quantities of anti-antibody and it displayed no detectable nonspecific affinity for polysomes or RNA. Using this procedure, we have obtained an apparently homogeneous preparation of ovalbumin mRNA.