An essential role for the interferon-inducible, double-stranded RNA-activated protein kinase PKR in the tumor necrosis factor-induced apoptosis in U937 cells.

Tumor necrosis factor alpha (TNF-alpha) is well-characterized for its necrotic action against tumor cells; however, it has been increasingly associated with an apoptosis-inducing potential on target cells. While the signaling events and the actual cytolytic mechanism(s) for both TNF-alpha-induced necrosis and apoptosis remain to be fully elucidated, we report here on (i) the ability of TNF-alpha to induce apoptosis in the promonocytic U937 cells, (ii) the discovery of a cross-talk between the TNF-alpha and the interferon signaling pathways, and (iii) the pivotal role of interferon-inducible, double-stranded RNA-activated protein kinase (PKR) in the induction of apoptosis by TNF-alpha. Our data from microscopy studies, trypan blue exclusion staining, and apoptotic DNA ladder electrophoresis revealed that a subclone derived from U937 and carrying a PKR antisense expression vector was resistant to TNF-alpha-induced apoptosis. Further, TNF-alpha initiated a generalized RNA degradation process in which the participation of PKR was required. Finally, the PKR gene is a candidate "death gene" since overexpression of this gene could bring about apoptosis in U937 cells.

DNA strand annealing is promoted by the yeast Rad52 protein.

The Saccharomyces cerevisiae RAD52 gene plays a pivotal role in genetic recombination. Here we demonstrate that yeast Rad52 is a DNA binding protein. To show that the interaction between Rad52 and DNA is direct and not mediated by other yeast proteins and to facilitate protein purification, a recombinant expression system was developed. The recombinant protein can bind both single- and double-stranded DNA and the addition of either Mg2+ or ATP does not enhance the binding of single-stranded DNA. Furthermore, a DNA binding domain was found in the evolutionary conserved N terminus of the protein. More importantly, we show that the protein stimulates DNA annealing even in the presence of a large excess of nonhomologous DNA. Rad52-promoted annealing follows second-order kinetics and the rate is 3500-fold faster than that of the spontaneous reaction. How this annealing activity relates to the genetic phenotype associated with rad52 mutant cells is discussed.

Chromosomal double-strand break repair in Ku80-deficient cells.

The x-ray sensitive hamster cell line xrs-6 is deficient in DNA double-strand break (DSB) repair and exhibits impaired V(D)J recombination. The molecular defect in this line is in the 80-kDa subunit of the Ku autoantigen, a protein that binds to DNA ends and recruits the DNA-dependent protein kinase to DNA. Using an I-SceI endonuclease expression system, chromosomal DSB repair was examined in xrs-6 and parental CHO-K1 cell lines. A DSB in chromosomal DNA increased the yield of recombinants several thousand-fold above background in both the xrs-6 and CHO-K1 cells, with recombinational repair of DSBs occurring in as many as 1 of 100 cells electroporated with the endonuclease expression vector. Thus, recombinational repair of chromosomal DSBs can occur at substantial levels in mammalian cells and it is not grossly affected in our assay by a deficiency of the Ku autoantigen. Rejoining of broken chromosome ends (end-joining) near the site of the DSB was also examined. In contrast to recombinational repair, end-joining was found to be severely impaired in the xrs-6 cells. Thus, the Ku protein appears to play a critical role in only one of the chromosomal DSB repair pathways.

Overexpression of the transcription factor UBF1 is sufficient to increase ribosomal DNA transcription in neonatal cardiomyocytes: implications for cardiac hypertrophy.

The accelerated protein accumulation characteristic of cardiomyocyte hypertrophy results from increased cellular protein synthetic capacity (elevated ribosome content). The rate limiting step in ribosome accumulation is transcription of the rRNA genes. During neonatal cardiomyocyte hypertrophy induced by norepinephrine or spontaneous contraction, changes in the expression of a ribosomal DNA transcription factor, UBF, correlated with increased rates of ribosome biogenesis. We hypothesized that elevated expression of UBF was part of the mechanism by which these hypertrophic stimuli effected increases in the rate of transcription from the rDNA promoter. In this study, we have examined directly the effect of overexpressing UBF on rDNA transcription in neonatal cardiomyocytes in culture. In control experiments, a novel reporter construct for rDNA transcription (pSMECAT) showed similar increases in activity in response to hypertrophic stimuli (10(-4) M phenylephrine, 10(-7) M endothelin, and spontaneous contraction) as did the endogenous rRNA genes. When contraction-arrested cardiomyocytes were cotransfected with pSMECAT and increasing amounts of a UBF1 expression vector; a dose-dependent (3-5 fold) increase in rDNA transcription was observed. Western blot analysis confirmed that the overexpressed, FLAG-tagged UBF accumulated in the cardiomyocyte nuclei. The observation that overexpression of UBF1 is sufficient to increase rDNA transcription in neonatal cardiomyocytes provides evidence in support of the hypothesis that the regulation of UBF is a key component of the increased ribosome biogenesis and protein accumulation associated with cardiomyocyte hypertrophy.

ATP-dependent uptake of natural product cytotoxic drugs by membrane vesicles establishes MRP as a broad specificity transporter.

MRP is a recently isolated ATP-binding cassette family transporter. We previously reported transfection studies that established that MRP confers multidrug resistance [Kruh, G. D., Chan, A., Myers, K., Gaughan, K., Miki, T. & Aaronson, S. A. (1994) Cancer Res. 54, 1649-1652] and that expression of MRP is associated with enhanced cellular efflux of lipophilic cytotoxic agents [Breuninger, L. M., Paul, S., Gaughan, K., Miki, T., Chan, A., Aaronson, S. A. & Kruh, G. D. (1995) Cancer Res. 55, 5342-5347]. To examine the biochemical mechanism by which MRP confers multidrug resistance, drug uptake experiments were performed using inside-out membrane vesicles prepared from NIH 3T3 cells transfected with an MRP expression vector. ATP-dependent transport was observed for several lipophilic cytotoxic agents including daunorubicin, etoposide, and vincristine, as well as for the glutathione conjugate leukotriene C4 (LTC4). However, only marginally increased uptake was observed for vinblastine and Taxol. Drug uptake was osmotically sensitive and saturable with regard to substrate concentration, with Km values of 6.3 microM, 4.4 microM, 4.2 microM, 35 nM, and 38 microM, for daunorubicin, etoposide, vincristine, LTC4, and ATP, respectively. The broad substrate specificity of MRP was confirmed by the observation that daunorubicin transport was competitively inhibited by reduced and oxidized glutathione, the glutathione conjugates S-(p-azidophenacyl)-glutathione (APA-SG) and S-(2,4-dinitrophenyl)glutathione (DNP-SG), arsenate, and the LTD4 antagonist MK571. This study establishes that MRP pumps unaltered lipophilic cytotoxic drugs, and suggests that this activity is an important mechanism by which the transporter confers multidrug resistance. The present study also indicates that the substrate specificity of MRP is overlapping but distinct from that of P-glycoprotein, and includes both the neutral or mildly cationic natural product cytotoxic drugs and the anionic products of glutathione conjugation. The widespread expression of MRP in tissues, combined with its ability to transport both lipophilic xenobiotics and the products of phase II detoxification, indicates that the transporter represents a widespread and remarkably versatile cellular defense mechanism.

Cloning of a novel receptor expressed in rat prostate and ovary.

We have cloned a novel member of the nuclear receptor superfamily. The cDNA of clone 29 was isolated from a rat prostate cDNA library and it encodes a protein of 485 amino acid residues with a calculated molecular weight of 54.2 kDa. Clone 29 protein is unique in that it is highly homologous to the rat estrogen receptor (ER) protein, particularly in the DNA-binding domain (95%) and in the C-terminal ligand-binding domain (55%). Expression of clone 29 in rat tissues was investigated by in situ hybridization and prominent expression was found in prostate and ovary. In the prostate clone 29 is expressed in the epithelial cells of the secretory alveoli, whereas in the ovary the granuloma cells in primary, secondary, and mature follicles showed expression of clone 29. Saturation ligand-binding analysis of in vitro synthesized clone 29 protein revealed a single binding component for 17beta-estradiol (E2) with high affinity (Kd= 0.6 nM). In ligand-competition experiments the binding affinity decreased in the order E2 > diethylstilbestrol > estriol > estrone > 5alpha-androstane-3beta,17beta-diol >> testosterone = progesterone = corticosterone = 5alpha-androstane-3alpha,17beta-diol. In cotransfection experiments of Chinese hamster ovary cells with a clone 29 expression vector and an estrogen-regulated reporter gene, maximal stimulation (about 3-fold) of reporter gene activity was found during incubation with 10 nM of E2. Neither progesterone, testosterone, dexamethasone, thyroid hormone, all-trans-retinoic acid, nor 5alpha-androstane-3alpha,I7beta-diol could stimulate reporter gene activity, whereas estrone and 5alpha-androstane-3beta,17beta-diol did. We conclude that clone 29 cDNA encodes a novel rat ER, which we suggest be named rat ERbeta to distinguish it from the previously cloned ER (ERalpha) from rat uterus.

Generation of mice with a 200-kb amyloid precursor protein gene deletion by Cre recombinase-mediated site-specific recombination in embryonic stem cells.

Gene disruptions and deletions of up to 20kb have been generated by homologous recombination with appropriate targeting vectors in murine embryonic stem (ES) cells. Because we could not obtain a deletion of about 200 kb in the mouse amyloid precursor protein gene by the classical technique, we employed strategies involving the insertion of loxP sites upstream and downstream of the region to be deleted by homologous recombination and elicited excision of the loxP-flanked region by introduction of a Cre expression vector into the ES cells. In the first approach, the loxP sequences were inserted in two successive steps and after each step, ES cell clones were isolated and characterized. Deletion of the loxP-flanked sequence was accomplished by introducing the cre gene in a third step. In the second approach, ES cells containing the upstream loxP cassette were electroporated simultaneously with the downstream loxP targeting vector and the Cre expression plasmid. ES cells were obtained that gave rise to chimeric mice capable of germ-line transmission of the deleted amyloid precursor protein allele.

Preferential induction of a Th1 immune response and inhibition of specific IgE antibody formation by plasmid DNA immunization.

We compared the antigen-specific antibody isotypes and lymphokine secretion by CD4+ T cells in BALB/c mice immunized intradermally with either Escherichia coli beta-galactosidase (beta-gal) or plasmid DNA (pDNA) encoding beta-gal in a cytomegalovirus-based expression vector (pCMV-LacZ). pCMV-LacZ induced mainly IgG2a, whereas beta-gal in saline or alum induced IgG1 and IgE beta-gal-specific antibodies. In addition, splenic CD4+ T helper (Th) cells isolated from pDNA-immunized mice secreted interferon-gamma but not interleukin (IL)-4 and IL-5, whereas Th cells from beta-gal-injected mice secreted IL-4 and IL-5 but not interferon-gamma after in vitro stimulation with antigen. Together these data demonstrate that pDNA immunization induced a T helper type 1 (Th1) response, whereas protein immunization induced a T helper type 2 (Th2) response to the same antigen. Interestingly, priming of mice with pCMV-LacZ prevented IgE antibody formation to a subsequent i.p. beta-gal in alum injection. This effect was antigen-specific, because priming with pCMV-LacZ did not inhibit IgE anti-ovalbumin antibody formation. Most importantly, intradermal immunization with pCMV-LacZ (but not pCMV-OVA) of beta-gal in alum-primed mice caused a 66-75% reduction of the IgE anti-beta-gal titer in 6 weeks. Also, pCMV-LacZ induced specific IgG2a antibody titers and interferon-gamma secretion by Th cells in the beta-gal in alum-primed mice. The data demonstrate that gene immunization induces a Th1 response that dominates over an ongoing protein-induced Th2 response in an antigen-specific manner. This suggests that immunization with pDNA encoding for allergens may provide a novel type of immunotherapy for allergic diseases.

The open reading frame of bamboo mosaic potexvirus satellite RNA is not essential for its replication and can be replaced with a bacterial gene.

A satellite RNA of 836 nt depends on the bamboo mosaic potexvirus (BaMV) for its replication and encapsulation. The BaMV satellite RNA (satBaMV) contains a single open reading frame encoding a 20-kDa nonstructural protein. A full-length infectious cDNA clone has been generated downstream of the T7 RNA polymerase promoter. To investigate the role of the 20-kDa protein encoded by satBaMV, satBaMV transcripts containing mutations in the open reading frame were tested for their ability to replicate in barley protoplasts and in Chenopodium quinoa using BaMV RNA as a helper genome. Unlike other large satellite RNAs, mutants in the open reading frame did not block their replication, suggesting that the 20-kDa protein is not essential for satBaMV replication. Precise replacement of the open reading frame with sequences encoding chloramphenicol acetyltransferase resulted in high level expression of chloramphenicol acetyltransferase in infected C. quinoa, indicating that satBaMV is potentially useful as a satellite-based expression vector.

Importance of the carboxyl-terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate: sugar phosphotransferase system for phosphoryl donor specificity.

The first protein component of the Escherichia coli phosphoenolpyruvate: sugar phosphotransferase system (PTS) is the 64-kDa protein enzyme I (EI), which can be phosphorylated by phosphoenolpyruvate (PEP) and carry out phosphotransfer to the acceptor heat-stable protein (HPr). The isolated amino-terminal domain (EIN) of E. coli EI is no longer phosphorylated by PEP but retains the ability to participate in reversible phosphotransfer to HPr. An expression vector was constructed for the production of large amounts of EIN, and conditions were developed for maximal expression of the protein. A three-column procedure is described for purification to homogeneity of EIN; a 500-ml culture yields approximately 80 mg of pure protein in about a 75% yield. Intact E. coli EI is effective in phosphotransfer from PEP to HPr from E. coli but not to the HPrs from Bacillus subtilis or Mycoplasma capricolum. Phosphotransfer from EI to enzyme IIAglc (EIIAglc) from E. coli or M. capricolum requires the intermediacy of HPr. The phosphorylated form of EIN is capable of more general phosphotransfer; it will effect phosphotransfer to HPrs from E. coli, B. subtilis, and M. capricolum as well as to EIAglc from E. coli. These studies demonstrate that the carboxyl-terminal domain of EI confers on the protein the capability to accept a phosphoryl group from PEP as well as a discriminator function that allows the intact protein to promote effective phosphoryl transfer only to E. coli HPr.

Regulation of microfilament organization and anchorage-independent growth by tropomyosin 1.

Variants of chemically immortalized Syrian hamster embryo cells that had either retained (supB+) or lost (supB-) the ability to suppress tumorigenicity when hybridized with a fibrosarcoma cell line were subcloned. Both supB cell types are nontumorigenic; however, the supB- but not supB+ cells exhibit conditional anchorage-independent growth. Alterations of actin microfilament organization were observed in supB- but not supB+ cells that corresponded to a significant reduction of the actin-binding protein tropomyosin 1 (TM-1) in subB- cells. To examine the possibility of a direct relationship between TM-1 expression and the subB- phenotype, subB+ cells were transfected with an expression vector containing the TM-1 cDNA in an antisense orientation. The antisense-induced reduction of TM-1 levels in supB+ clones caused a microfilament reorganization and conferred anchorage-independent growth potential that were indistinguishable from those characteristic of supB- cells. These data provide direct evidence that TM-1 regulates both microfilament organization and anchorage-independent growth and suggest that microfilament alterations are sufficient for anchorage-independent growth.

Characterization of Arabidopsis thaliana cDNAs that render yeasts tolerant toward the thiol-oxidizing drug diamide.

Diamide oxidizes cellular thiols and induces oxidative stress. To isolate plant genes which may, when overexpressed, increase tolerance of plants toward oxidative damage, an in vivo diamide tolerance screening in yeasts was used. An Arabidopsis cDNA library in a yeast expression vector was used to transform a yeast strain with intact antioxidant defense. Cells from approximately 10(5) primary transformants were selected for resistance to diamide. Three Arabidopsis cDNAs which confer diamide tolerance were isolated. This drug tolerance was specific and no cross tolerance toward hydroperoxides was found. One cDNA (D3) encodes a polypeptide which has an amino-terminal J domain characteristic of a divergent family of DnaJ chaperones. Another (D18) encodes a putative dTDP-D-glucose 4,6-dehydratase. Surprisingly, the third cDNA (D22) encodes a plant homolog of gamma-glutamyltransferases. It would have been difficult to predict that the expression of those genes would lead to an improved survival under conditions of depletion of cellular thiols. Hence, we suggest that this cloning approach may be a useful contribution to the isolation of plant genes that can help to cope with oxidative stress.

The angiotensin II type 2 (AT2) receptor antagonizes the growth effects of the AT1 receptor: gain-of-function study using gene transfer.

The type 1 angiotensin II (AT1) receptor is well characterized but the type 2 (AT2) receptor remains an enigma. We tested the hypothesis that the AT2 receptor can modulate the growth of vascular smooth muscle cells by transfecting an AT2 receptor expression vector into the balloon-injured rat carotid artery and observed that overexpression of the AT2 receptor attenuated neointimal formation. In cultured smooth muscle cells, AT2 receptor transfection reduced proliferation and inhibited mitogen-activated protein kinase activity. Furthermore, we demonstrated that the AT2 receptor mediated the developmentally regulated decrease in aortic DNA synthesis at the latter stages of gestation. These results suggest that the AT2 receptor exerts an antiproliferative effect, counteracting the growth action of AT1 receptor.

Oligomeric structure of caveolin: implications for caveolae membrane organization.

A 22-kDa protein, caveolin, is localized to the cytoplasmic surface of plasma membrane specializations called caveolae. We have proposed that caveolin may function as a scaffolding protein to organize and concentrate signaling molecules within caveolae. Here, we show that caveolin interacts with itself to form homooligomers. Electron microscopic visualization of these purified caveolin homooligomers demonstrates that they appear as individual spherical particles. By using recombinant expression of caveolin as a glutathione S-transferase fusion protein, we have defined a region of caveolin's cytoplasmic N-terminal domain that mediates these caveolin-caveolin interactions. We suggest that caveolin homooligomers may function to concentrate caveolin-interacting molecules within caveolae. In this regard, it may be useful to think of caveolin homooligomers as "fishing lures" with multiple "hooks" or attachment sites for caveolin-interacting molecules.

Role of glutathione in the export of compounds from cells by the multidrug-resistance-associated protein.

Multidrug-resistance-associated protein (MRP) is a plasma membrane glycoprotein that can confer multidrug resistance (MDR) by lowering intracellular drug concentration. Here we demonstrate that depletion of intracellular glutathione by DL-buthionine (S,R)-sulfoximine results in a complete reversal of resistance to doxorubicin, daunorubicin, vincristine, and VP-16 in lung carcinoma cells transfected with a MRP cDNA expression vector. Glutathione depletion had less effect on MDR in cells transfected with MDR1 cDNA encoding P-glycoprotein and did not increase the passive uptake of daunorubicin by cells, indicating that the decrease of MRP-mediated MDR was not due to nonspecific membrane damage. Glutathione depletion resulted in a decreased efflux of daunorubicin from MRP-transfected cells, but not from MDR1-transfected cells, suggesting that glutathione is specifically required for the export of drugs from cells by MRP. We also show that MRP increases the export of glutathione from the cell and this increased export is further elevated in the presence of arsenite. Our results support the hypothesis that MRP functions as a glutathione S-conjugate carrier.