Induction of HSP70 and polyubiquitin expression associated with plant virus replication

By examining the front of virus invasion in immature pea embryos infected with pea seed-borne mosaic virus (PSbMV), the selective control of different host genes has been observed. From our observations, the early responses to PSbMV replication can be grouped into three classes, inhibited host gene expression, induced host gene expression, and no effect on a normal host function. The expression of two heat-inducible genes encoding HSP70 and polyubiquitin was induced coordinately with the onset of virus replication and the down-regulation of two other genes encoding lipoxygenase and heat shock cognate protein. The down-regulation was part of a general suppression of host gene expression that may be achieved through the degradation of host transcripts. We discuss the possibilities of whether the induction of HSP70 and polyubiquitin genes represents a requirement for the respective protein products by the virus or is merely a consequence of the depletion of other host transcripts. The former is feasible, as the induction of both genes does result in increased HSP70 and ubiquitin accumulation. This also indicates that, in contrast to some animal virus infections, there is not a general inhibition of translation of host mRNAs following PSbMV infection. This selective control of host gene expression was observed in all cell types of the embryo and identifies mechanisms of cellular disruption that could act as triggers for symptom expression.

Survival function of ERK1/2 as IL-3-activated, staurosporine-resistant Bcl2 kinases

Bcl2 phosphorylation at Ser-70 may be required for the full and potent suppression of apoptosis in IL-3-dependent myeloid cells and can result from agonist activation of mitochondrial protein kinase C (PKC). Paradoxically, expression of exogenous Bcl2 can protect parental cells from apoptosis induced by the potent PKC inhibitor, staurosporine (stauro). High concentrations of stauro of up to 1 μM only partially inhibit IL-3-stimulated Bcl2 phosphorylation but completely block PKC-mediated Bcl2 phosphorylation in vitro. These data indicate a role for a stauro-resistant Bcl2 kinase (SRK). We show that aurintricarboxylic acid (ATA), a nonpeptide activator of cellular MEK/mitogen-activated protein kinase (MAPK) kinase, can induce Ser-70 phosphorylation of Bcl2 and support survival of cells expressing wild-type but not the phosphorylation-incompetent S70A mutant Bcl2. A role for a MEK/MAPK as a responsible SRK was implicated because the highly specific MEK/MAPK inhibitor, PD98059, also can only partially inhibit IL-3-induced Bcl2 phosphorylation, whereas the combination of PD98059 and stauro completely blocks phosphorylation and synergistically enhances apoptosis. p44MAPK/extracellular signal-regulated kinase 1 (ERK1) and p42 MAPK/ERK2 are activated by IL-3, colocalize with mitochondrial Bcl2, and can directly phosphorylate Bcl2 on Ser-70 in a stauro-resistant manner both in vitro and in vivo. These findings suggest a role for the ERK1/2 kinases as SRKs. Thus, the SRKs can serve to functionally link the IL-3-stimulated proliferative and survival signaling pathways and, in a novel capacity, may explain how Bcl2 can suppress stauro-induced apoptosis. In addition, although the mechanism of regulation of Bcl2 by phosphorylation is not yet clear, our results indicate that phosphorylation may functionally stabilize the Bcl2-Bax heterodimerization.

The cellular ecology of progressive neoplastic transformation: A clonal analysis

A comparison was made of the competence for neoplastic transformation in three different sublines of NIH 3T3 cells and multiple clonal derivatives of each. Over 90% of the neoplastic foci produced by an uncloned transformed (t-SA′) subline on a confluent background of nontransformed cells were of the dense, multilayered type, but about half of the t-SA′ clones produced only light foci in assays without background. This asymmetry apparently arose from the failure of the light focus formers to register on a background of nontransformed cells. Comparison was made of the capacity for confluence-mediated transformation between uncloned parental cultures and their clonal derivatives by using two nontransformed sublines, one of which was highly sensitive and the other relatively refractory to confluence-mediated transformation. Transformation was more frequent in the clones than in the uncloned parental cultures for both sublines. This was dramatically so in the refractory subline, where the uncloned culture showed no overt sign of transformation in serially repeated assays but increasing numbers of its clones exhibited progressive transformation. The reason for the greater susceptibility of the pure clones is apparently the suppression of transformation among the diverse membership that makes up the uncloned parental culture. Progressive selection toward increasing degrees of transformation in confluent cultures plays a major role in the development of dense focus formers, but direct induction by the constraint of confluence may contribute by heritably damaging cells. In view of our finding of increased susceptibility to transformation in clonal versus uncloned populations, expansion of some clones at the expense of others during the aging process would contribute to the marked increase of cancer with age.

Angiostatin gene transfer: Inhibition of tumor growth in vivo by blockage of endothelial cell proliferation associated with a mitosis arrest

The antitumoral effects that follow the local delivery of the N-terminal fragment of human plasminogen (angiostatin K3) have been studied in two xenograft murine models. Angiostatin delivery was achieved by a defective adenovirus expressing a secretable angiostatin K3 molecule from the cytomegalovirus promoter (AdK3). In in vitro studies, AdK3 selectively inhibited endothelial cell proliferation and disrupted the G2/M transition induced by M-phase-promoting factors. AdK3-infected endothelial cells showed a marked mitosis arrest that correlated with the down-regulation of the M-phase phosphoproteins. A single intratumoral injection of AdK3 into preestablished rat C6 glioma or human MDA-MB-231 breast carcinoma grown in athymic mice was followed by a significant arrest of tumor growth, which was associated with a suppression of neovascularization within and at the vicinity of the tumors. AdK3 therapy also induced a 10-fold increase in apoptotic tumor cells as compared with a control adenovirus. Furthermore, we showed that systemic injection of AdK3 delayed C6 tumor establishment and growth, confirming that angiostatin can function in a paracrin manner. Our data support the concept that targeted antiangiogenesis, using adenovirus-mediated gene transfer, represents a promising alternative strategy for delivering antiangiogenic factors as their bolus injections present unsolved pharmacological problems.

Muscarinic cholinergic regulation of cardiac myocyte ICa-L is absent in mice with targeted disruption of endothelial nitric oxide synthase

Cardiac myocytes have been shown to express constitutively endothelial nitric oxide synthase (eNOS) (nitric oxide synthase 3), the activation of which has been implicated in the regulation of myocyte L-type voltage-sensitive calcium channel current (ICa-L) and myocyte contractile responsiveness to parasympathetic nervous system signaling, although this implication remains controversial. Therefore, we examined the effect of the muscarinic cholinergic agonist carbachol (CCh) on ICa-L and contractile amplitude in isoproterenol (ISO)-prestimulated ventricular myocytes isolated from adult mice, designated eNOSnull mice, with targeted disruption of the eNOS gene. Although both eNOSnull and wild-type (WT) ventricular myocytes exhibited similar increases in ICa-L in response to ISO, there was no measurable suppression of ICa-L by CCh in cells from eNOSnull mice, in contrast to cells from WT mice. These results were reflected in the absence of an effect of CCh on the positive inotropic effect of ISO in eNOSnull myocytes. Also, unlike myocytes from WT animals, eNOSnull myocytes failed to exhibit an increase in cGMP content in response to CCh. Nevertheless, the pharmacologic nitric oxide donors 3-morpholino-sydnonimine and S-nitroso-acetyl-cystein increased cGMP generation and suppressed ISO-augmented ICa-L in eNOSnull cells, suggesting that the signal transduction pathway(s) downstream of eNOS remained intact. Of importance, activation of the acetylcholine-activated K+ channel by CCh was unaffected in atrial and ventricular eNOSnull myocytes. These results confirm the obligatory role of eNOS in coupling muscarinic receptor activation to cGMP-dependent control of ICa-L in cardiac myocytes.

Overexpression of the large subunit of the protein Ku suppresses metallothionein-I induction by heavy metals

Metallothioneins (MT) are involved in the scavenging of the toxic heavy metals and protection of cells from reactive oxygen intermediates. To investigate the potential role of the protein Ku in the expression of MT, we measured the level of MT-I mRNA in the parental rat fibroblast cell line (Rat 1) and the cell lines that stably and constitutively overexpress the small subunit, the large subunit, and the heterodimer of Ku. Treatment with CdS04 or ZnS04 elevated the MT-I mRNA level 20- to 30-fold in the parental cells and the cells (Ku-70) that overproduce the small subunit or those (Ku-7080) overexpressing the heterodimer. By contrast, the cells (Ku-80) overexpressing the large subunit of Ku failed to induce MT-I. In vitro transcription assay showed that the MT-I promoter activity was suppressed selectively in the nuclear extracts from Ku-80 cells. The specificity of the repressor function was shown by the induction of hsp 70, another Cd-inducible gene, in Ku-80 cells. Addition of the nuclear extract from Ku-80 cells at the start of the transcription reaction abolished the MT-l promoter activity in the Rat 1 cell extract. The transcript once formed in Rat 1 nuclear extract was not degraded by further incubation with the extract from Ku-80 cells. The repressor was sensitive to heat. The DNA-binding activities of at least four transcription factors that control the MT-I promoter activity were not affected in Ku-80 cells. These observations have set the stage for further exploration of the mechanisms by which the Ku subunit mediates suppression of MT induction.

The negative feedback actions of progesterone on gonadotropinreleasing hormone secretion are transduced by the classical progesterone receptor

Progesterone (P) powerfully inhibits gonadotropin-releasing hormone (GnRH) secretion in ewes, as in other species, but the neural mechanisms underlying this effect remain poorly understood. Using an estrogen (E)-free ovine model, we investigated the immediate GnRH and luteinizing hormone (LH) response to acute manipulations of circulating P concentrations and whether this response was mediated by the nuclear P receptor. Simultaneous hypophyseal portal and jugular blood samples were collected over 36 hr: 0–12 hr, in the presence of exogenous P (P treatment begun 8 days earlier); 12–24 hr, P implant removed; 24–36 hr, P implant reinserted. P removal caused a significant rapid increase in the GnRH pulse frequency, which was detectable within two pulses (175 min). P insertion suppressed the GnRH pulse frequency even faster: the effect detectable within one pulse (49 min). LH pulsatility was modulated identically. The next two experiments demonstrated that these effects of P are mediated by the nuclear P receptor since intracerebroventricularly infused P suppressed LH release but 3α-hydroxy-5α-pregnan-20-one, which operates through the type A γ-aminobutyric acid receptor, was without effect and pretreatment with the P-receptor antagonist RU486 blocked the ability of P to inhibit LH. Our final study showed that P exerts its acute suppression of GnRH through an E-dependent system because the effects of P on LH secretion, lost after long-term E deprivation, are restored after 2 weeks of E treatment. Thus we demonstrate that P acutely inhibits GnRH through an E-dependent nuclear P-receptor system.

Plasticity of the neoplastic phenotype in vivo is regulated by epigenetic factors

Age of host and transplantation-site microenvironment influence the tumorigenic potential of neoplastically transformed liver epithelial cells. Tumorigenic BAG2-GN6TF rat liver epithelial cells consistently form tumors at ectopic sites, but differentially express tumorigenicity or hepatocytic differentiation in the liver depending on host age and route of cell transplantation into the liver. Direct inoculation into host livers concentrates tumor cells locally, resulting in undifferentiated tumors near the transplantation site in both young (3-month-old) and old (18-month-old) rats. Transplantation-site tumors regress within 1 month in the livers of young rats, but grow progressively in old rats. However, inoculation of cells into the spleen distributes transplanted cells individually throughout the liver, resulting in hepatocytic differentiation by tumor cells with concomitant suppression of their tumorigenicity in young rats. When transplanted into livers of old rats by splenic inoculation, or when young hepatic-transplant recipients are allowed to age, hepatocytic progeny of BAG2-GN6TF cells proliferate to form foci, suggesting that the liver microenvironment of old rats incompletely regulates the proliferation and differentiation of tumor cell-derived hepatocytes. Upon removal from the liver, BAG2-GN6TF-derived hepatocytes revert to an undifferentiated, aggressively tumorigenic phenotype. We posit that the spectrum between normal differentiation and malignant potential of these cells reflects the dynamic interaction of the specific transformation-related genotype of the cells and the characteristics of the tissue microenvironment at the transplantation site. Changes in the tissue milieu, such as those that accompany normal aging, may determine the ability of a genetically aberrant cell to produce a tumor.

Gene transfer of Fas ligand induces tumor regression in vivo

The Fas–Fas ligand (FasL) system plays an important role in the induction of lymphoid apoptosis and has been implicated in the suppression of immune responses. Herein, we report that gene transfer of FasL inhibits tumor cell growth in vivo. Although such inhibition is expected in Fas+ tumor cell lines, marked regression was unexpectedly observed after FasL gene transfer into the CT26 colon carcinoma that does not express Fas. Infection by an adenoviral vector encoding FasL rapidly eliminated tumor masses in the Fas+ Renca tumor by inducing cell death, whereas the elimination of Fas− CT26 cells was mediated by inflammatory cells. Analysis of human malignancies revealed Fas, but not FasL, expression in a majority of tumors and susceptibility to FasL in most Fas+ cell lines. These findings suggest that gene transfer of FasL generates apoptotic responses and induces potent inflammatory reactions that can be used to induce the regression of malignancies.

Bimodal regulation of Na+–Ca2+ exchanger by β-adrenergic signaling pathway in shark ventricular myocytes

In shark heart, the Na+–Ca2+ exchanger serves as a major pathway for both Ca2+ influx and efflux, as there is only rudimentary sarcoplasmic reticulum in these hearts. The modulation of the exchanger by a β-adrenergic agonist in whole-cell clamped ventricular myocytes was compared with that of the Na+–Ca2+ exchanger blocker KB-R7943. Application of 5 μM isoproterenol and 10 μM KB-R7943 suppressed both the inward and the outward Na+–Ca2+ exchanger current (INa−Ca). The isoproterenol effect was mimicked by 10 μM forskolin. Isoproterenol and forskolin shifted the reversal potential (Erev) of INa−Ca by approximately −23 mV and −30 mV, respectively. An equivalent suppression of outward INa−Ca by KB-R7943 to that by isoproterenol produced a significantly smaller shift in Erev of about −4 mV. The ratio of inward to outward exchanger currents was also significantly larger in isoproterenol- than in control- and KB-R7943-treated myocytes. Our data suggest that the larger ratio of inward to outward exchanger currents as well as the larger shift in Erev with isoproterenol results from the enhanced efficacy of Ca2+ efflux via the exchanger. The protein kinase A-mediated bimodal regulation of the exchanger in parallel with phosphorylation of the Ca2+ channel and enhancement of its current may have evolved to satisfy the evolutionary needs for accelerated contraction and relaxation in hearts of animals with vestigial sarcoplasmic Ca2+ release stores.

Polymorphism in the 3′-untranslated region of TNFα mRNA impairs binding of the post-transcriptional regulatory protein HuR to TNFα mRNA

Tumor necrosis factor α (TNFα) acts as a beneficial mediator in the process of host defence. In recent years major interest has focused on the AU-rich elements (AREs) present in the 3′-untranslated region (3′-UTR) of TNFα mRNA as this region plays a pivotal role in post-transcriptional control of TNFα production. Certain stimuli, such as lipopolysaccharides, a component of the Gram-negative bacterial cell wall, have the ability to relinquish the translational suppression of TNFα mRNA imposed by these AREs in macrophages, thereby enabling the efficient production of the TNFα. In this study we show that the polymorphism (GAU trinucleotide insertional mutation) present in the regulatory 3′-UTR of TNFα mRNA of NZW mice results in the hindered binding of RNA-binding proteins, thereby leading to a significantly reduced production of TNFα protein. We also show that the binding of macrophage proteins to the main ARE is also decreased by another trinucleotide (CAU) insertion in the TNFα 3′-UTR. One of the proteins affected by the GAU trinucleotide insertional mutation was identified as HuR, a nucleo-cytoplasmic shuttling protein previously shown to play a prominent role in the stability and translatability of mRNA containing AREs. Since binding of this protein most likely modulates the stability, translational efficiency and transport of TNFα mRNA, these results suggest that mutations in the ARE of TNFα mRNA decrease the production of TNFα protein in macrophages by hindering the binding of HuR to the ARE.

Inactivation of Saccharomyces cerevisiae OGG1 DNA repair gene leads to an increased frequency of mitochondrial mutants

The OGG1 gene encodes a highly conserved DNA glycosylase that repairs oxidized guanines in DNA. We have investigated the in vivo function of the Ogg1 protein in yeast mitochondria. We demonstrate that inactivation of ogg1 leads to at least a 2-fold increase in production of spontaneous mitochondrial mutants compared with wild-type. Using green fluorescent protein (GFP) we show that a GFP–Ogg1 fusion protein is transported to mitochondria. However, deletion of the first 11 amino acids from the N-terminus abolishes the transport of the GFP–Ogg1 fusion protein into the mitochondria. This analysis indicates that the N-terminus of Ogg1 contains the mitochondrial localization signal. We provide evidence that both yeast and human Ogg1 proteins protect the mitochondrial genome from spontaneous, as well as induced, oxidative damage. Genetic analyses revealed that the combined inactivation of OGG1 and OGG2 [encoding an isoform of the Ogg1 protein, also known as endonuclease three-like glycosylase I (Ntg1)] leads to suppression of spontaneously arising mutations in the mitochondrial genome when compared with the ogg1 single mutant or the wild-type. Together, these studies provide in vivo evidence for the repair of oxidative lesions in the mitochondrial genome by human and yeast Ogg1 proteins. Our study also identifies Ogg2 as a suppressor of oxidative mutagenesis in mitochondria.

Twenty-first aminoacyl-tRNA synthetase–suppressor tRNA pairs for possible use in site-specific incorporation of amino acid analogues into proteins in eukaryotes and in eubacteria

Two critical requirements for developing methods for the site-specific incorporation of amino acid analogues into proteins in vivo are (i) a suppressor tRNA that is not aminoacylated by any of the endogenous aminoacyl-tRNA synthetases (aaRSs) and (ii) an aminoacyl-tRNA synthetase that aminoacylates the suppressor tRNA but no other tRNA in the cell. Here we describe two such aaRS–suppressor tRNA pairs, one for use in the yeast Saccharomyces cerevisiae and another for use in Escherichia coli. The “21st synthetase–tRNA pairs” include E. coli glutaminyl-tRNA synthetase (GlnRS) along with an amber suppressor derived from human initiator tRNA, for use in yeast, and mutants of the yeast tyrosyl-tRNA synthetase (TyrRS) along with an amber suppressor derived from E. coli initiator tRNA, for use in E. coli. The suppressor tRNAs are aminoacylated in vivo only in the presence of the heterologous aaRSs, and the aminoacylated tRNAs function efficiently in suppression of amber codons. Plasmids carrying the E. coli GlnRS gene can be stably maintained in yeast. However, plasmids carrying the yeast TyrRS gene could not be stably maintained in E. coli. This lack of stability is most likely due to the fact that the wild-type yeast TyrRS misaminoacylates the E. coli proline tRNA. By using error-prone PCR, we have isolated and characterized three mutants of yeast TyrRS, which can be stably expressed in E. coli. These mutants still aminoacylate the suppressor tRNA essentially quantitatively in vivo but show increased discrimination in vitro for the suppressor tRNA over the E. coli proline tRNA by factors of 2.2- to 6.8-fold.

Genetic disruption of PPARδ decreases the tumorigenicity of human colon cancer cells

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that have been implicated in a variety of biologic processes. The PPARδ isotype was recently proposed as a downstream target of the adenomatous polyposis coli (APC)/β-catenin pathway in colorectal carcinogenesis. To evaluate its role in tumorigenesis, a PPARδ null cell line was created by targeted homologous recombination. When inoculated as xenografts in nude mice, PPARδ −/− cells exhibited a decreased ability to form tumors compared with PPARδ +/− and wild-type controls. These data suggest that suppression of PPARδ expression contributes to the growth-inhibitory effects of the APC tumor suppressor.

Induced fit DNA recognition by a minor groove binding analogue of Hoechst 33258: fluctuations in DNA A tract structure investigated by NMR and molecular dynamics simulations

NMR analysis and molecular dynamics simulations of d(GGTAATTACC)2 and its complex with a tetrahydropyrimidinium analogue of Hoechst 33258 suggest that DNA minor groove recognition in solution involves a combination of conformational selection and induced fit, rather than binding to a preorganised site. Analysis of structural fluctuations in the bound and unbound states suggests that the degree of induced fit observed is primarily a consequence of optimising van der Waals contacts with the walls of the minor groove resulting in groove narrowing through: (i) changes in base step parameters, including increased helical twist and propeller twist; (ii) changes to the sugar–phosphate backbone conformation to engulf the bound ligand; (iii) suppression of bending modes at the TpA steps. In contrast, the geometrical arrangement of hydrogen bond acceptors on the groove floor appears to be relatively insensitive to DNA conformation (helical twist and propeller twist). We suggest that effective recognition of DNA sequences (in this case an A tract structure) appears to depend to a significant extent on the sequence being flexible enough to be able to adopt the geometrically optimal conformation compatible with the various binding interactions, rather than involving ‘lock and key’ recognition.