A constitutively expressed, truncated umuDC operon regulates the recA-dependent DNA damage induction of a gene in Acinetobacter baylyi strain ADP1

In response to environmentally caused DNA damage, SOS genes are up-regulated due to RecA-mediated relief of LexA repression. In Escherichia coli, the SOS umuDC operon is required for DNA damage checkpoint functions and for replicating damaged DNA in the error-prone process called SOS mutagenesis. In the model soil bacterium Acinetobacter baylyi strain ADP1, however, the content, regulation, and function of the umuDC operon are unusual. The umuC gene is incomplete, and a remnant of an ISEhe3-like transposase has replaced the middle 57% of the umuC coding region. The umuD open reading frame is intact, but it is 1.5 times the size of other umuD genes and has an extra 5' region that lacks homology to known umuD genes. Analysis of a umuD::lacZ fusion showed that umuD was expressed at very high levels in both the absence and presence of mitomycin C and that this expression was not affected in a recA-deficient background. The umuD mutation did not affect the growth rate or survival after UV-induced DNA damage. However, the UmuD-like protein found in ADP1 (UmuDAb) was required for induction of an adjacent DNA damage-inducible gene, ddrR. The umuD mutation specifically reduced the DNA damage induction of the RecA-dependent DNA damage-inducible ddrR locus by 83% (from 12.9-fold to 2.3-fold induction), but it did not affect the 33.9-fold induction of benA, an unrelated benzoate degradation gene. These data suggest that the response of the ADP1 umuDC operon to DNA damage is unusual and that UmuDAb specifically regulates the expression of at least one DNA damage-inducible gene.     

Rapid and preferential activation of the c-jun gene during the mammalian UV response.

Exposure of mammalian cells to DNA-damaging agents leads to activation of a genetic response known as the UV response. Because several previously identified UV-inducible genes contain AP-1 binding sites within their promoters, we investigated the induction of AP-1 activity by DNA-damaging agents. We found that expression of both c-jun and c-fos, which encode proteins that participate in formation of the AP-1 complex, is rapidly induced by two different DNA-damaging agents: UV and H2O2. Interestingly, the c-jun gene is far more responsive to UV than any other immediate-early gene that was examined, including c-fos. Other jun and fos genes were only marginally affected by UV or H2O2. Furthermore, UV is a much more efficient inducer of c-jun than phorbol esters, the standard inducers of c-jun expression. This preferential response of the c-jun gene is mediated by its 5' control region and requires the TPA response element, suggesting that this element also serves as an early target for the signal transduction pathway elicited by DNA damage. Both UV and H2O2 lead to a long-lasting increase in AP-1 binding activity, suggesting that AP-1 may mediate the induction of other damage-inducible genes such as human collagenase.     

A Constitutively Expressed, Truncated umuDC Operon Regulates the recA-Dependent DNA Damage Induction of a Gene in Acinetobacter baylyi Strain ADP1

In response to environmentally caused DNA damage, SOS genes are up-regulated due to RecA-mediated relief of LexA repression. In Escherichia coli, the SOS umuDC operon is required for DNA damage checkpoint functions and for replicating damaged DNA in the error-prone process called SOS mutagenesis. In the model soil bacterium Acinetobacter baylyi strain ADP1, however, the content, regulation, and function of the umuDC operon are unusual. The umuC gene is incomplete, and a remnant of an ISEhe3-like transposase has replaced the middle 57% of the umuC coding region. The umuD open reading frame is intact, but it is 1.5 times the size of other umuD genes and has an extra 5′ region that lacks homology to known umuD genes. Analysis of a umuD::lacZ fusion showed that umuD was expressed at very high levels in both the absence and presence of mitomycin C and that this expression was not affected in a recA-deficient background. The umuD mutation did not affect the growth rate or survival after UV-induced DNA damage. However, the UmuD-like protein found in ADP1 (UmuDAb) was required for induction of an adjacent DNA damage-inducible gene, ddrR. The umuD mutation specifically reduced the DNA damage induction of the RecA-dependent DNA damage-inducible ddrR locus by 83% (from 12.9-fold to 2.3-fold induction), but it did not affect the 33.9-fold induction of benA, an unrelated benzoate degradation gene. These data suggest that the response of the ADP1 umuDC operon to DNA damage is unusual and that UmuDAb specifically regulates the expression of at least one DNA damage-inducible gene.     

c-myc gene expression is stimulated by agents that activate protein kinase C and does not account for the mitogenic effect of PDGF

The role of the phosphoinositide turnover-protein kinase C pathway in mediating PDGF-stimulated c-myc expression and cell proliferation was studied. Both direct activators of kinase C (e.g. phorbol ester analogues) and hormones that activate kinase C via receptor-mediated phosphoinositide turnover (e.g. PDGF, bradykinin, or vasopressin) elicited a rapid increase in c-myc mRNA expression. Desensitization of the kinase C pathway by prolonged exposure to phorbol abolished the induction of c-myc by subsequent phorbol challenge and attenuated c-myc induction by PDGF and bradykinin, but did not affect PDGF-stimulated mitogenesis. Bradykinin and phorbol esters stimulated the same magnitude of c-myc expression as PDGF but elicited less than one-tenth the PDGF-induced mitogenic response. We conclude that stimulation of c-myc expression is a common response to a diverse group of agents that elicit phosphoinositide turnover and activate protein kinase C, and that neither activation of protein kinase C nor enhanced c-myc expression is sufficient for the mitogenic action of PDGF.     

The activation of protein kinase G by daphnane, ingenane and tigliane diterpenoid esters

The activation of protein kinase C by daphnane, ingenane and tigliane diterpenoid eaters. In this review, the mechanism of action of phorbol esters and related diterpenes is described. These compounds have been shown to stimulate a Ca^{2 +} and phospholipid dependent protein kinase, termed kinase C. Phorbol esters activate protein kinase C by substituting for the natural effector, the second messenger, diacylglycerol. The various known protein substrates of this enzyme are described. Many of these substrates are involved in regulation of protein synthesis, DNA expression, cell transformation etc. This provides the explanation for the tumour promotion effects of some phorbol esters. Evidence for the biochemical mechanisms of action of phorbol esters that have other biological effects are also described. Recent evidence from our laboratories indicates that phorbol esters with limited biological effects, e.g. inflammatory but not tumour promoting, also act through this protein kinase. These phorbol esters appear to stimulate the phosphorylation of a different range of substrate proteins in vivo.     

Induction of apoptosis and stress response in ovarian carcinoma cell lines treated with ST1926, an atypical retinoid

To understand the molecular mechanisms mediating apoptosis induction by a novel atypical retinoid, ST1926, the cellular response to drug treatment was investigated in IGROV-1 ovarian carcinoma cells carrying wild-type p53 and a cisplatin-resistant p53 mutant subline (IGROV-1/Pt1). Despite a similar extent of drug-induced DNA strand breaks, the level of apoptosis was substantially higher in p53 wild-type cells. p53 activation and early upregulation of p53-target genes were consistent with p53-dependent apoptosis in IGROV-1 cells. Stress-activated protein kinases were activated in both cell lines in response to ST1926. This event and activation of AP-1 were more pronounced in IGROV-1/Pt1 cells, in which the modulation of DNA repair-associated genes suggests an increased ability to repair DNA damage. Inhibition of JNK or p38 stimulated ST1926-induced apoptosis only in IGROV-1 cells, whereas inhibition of ERKs enhanced apoptosis in both the cell lines. Such a pattern of cellular response and modulation of genes implicated in DNA damage response supports that the genotoxic stress is a critical event mediating drug-induced apoptosis. The results are consistent with apoptosis induction through p53-dependent and -independent pathways, regulated by MAP kinases, which likely play a protective role.     

Hallmarks for senescence in carcinogenesis: novel signaling players

Cellular senescence is a potent anti-cancer mechanism controlled by tumor suppressor genes, particularly p53 and pRb, which is characterized by the irreversible loss of proliferation. Senescence induced by DNA damage, oncogenic stimulation, or excessive mitogenic input, serves as a barrier that counteracts cancer progression. Emerging evidence in cellular and in in vivo models revealed the involvement of additional signaling players in senescence, including PML, CK2, Bcl-2, PI3K effectors such as Rheb, Rho small GTPases, and cytokines. Recent studies have also implicated protein kinase C (PKC) isozymes as modulators of senescence phenotypes and showed that phorbol esters, widely used PKC activators, can induce senescence in a number of cancer cells. These novel findings suggest a complex array of cross-talks between senescence pathways and may have significant implications in cancer therapy.     

Lex marks the spot: the virulent side of SOS and a closer look at the LexA regulon

The SOS response that responds to DNA damage induces many genes that are under LexA repression. A detailed examination of LexA regulons using genome-wide techniques has recently been undertaken in both Escherichia coli and Bacillus subtilis. These extensive and elegant studies have now charted the extent of the LexA regulons, uncovered many new genes, and exposed a limited overlap in the LexA regulon between the two bacteria. As more bacterial genomes are analysed, more curiosities in LexA regulons arise. Several notable examples include the discovery of a LexA-like protein, HdiR, in Lactococcus lactis, organisms with two lexA genes, and small DNA damage-inducible cassettes under LexA control. In the cyanobacterium Synechocystis, genetic and microarray studies demonstrated that a LexA paralogue exerts control over an entirely different set of carbon-controlled genes and is crucial to cells facing carbon starvation. An examination of SOS induction evoked by common therapeutic drugs has shed new light on unsuspected consequences of drug exposure. Certain antibiotics, most notably fluoroquinolones such as ciprofloxacin, can induce an SOS response and can modulate the spread of virulence factors and drug resistance. SOS induction by beta-lactams in E. coli triggers a novel form of antibiotic defence that involves cell wall stress and signal transduction by the DpiAB two-component system. In this review, we provide an overview of these new directions in SOS and LexA research with emphasis on a few themes: identification of genes under LexA control, the identification of new endogenous triggers, and antibiotic-induced SOS response and its consequences.     

Evolution of Natural Transformation: Testing the DNA Repair Hypothesis in Bacillus Subtilis and Haemophilus Influenzae

The hypothesis that the primary function of bacterial transformation is DNA repair was tested in the naturally transformable bacteria Bacillus subtilis and Haemophilus influenzae by determining whether competence for transformation is regulated by DNA damage. Accordingly, DNA damage was induced by mitomycin C and by ultraviolet radiation at doses that efficiently induced a known damage-inducible gene fusion, and the ability of the damaged cultures to transform was monitored. Experiments were carried out both under conditions where cells do not normally become competent and under competence-inducing conditions. No induction or enhancement of competence by damage was seen in either organism. These experiments strongly suggest that the regulation of competence does not involve a response to DNA damage, and thus that explanations other than DNA repair must be sought for the evolutionary functions of natural transformation systems.     

Induction of angiogenesis in vitro by vanadate, an inhibitor of phosphotyrosine phosphatases

We have previously shown that capillary endothelial cells grown on the surface of three-dimensional collagen gels can be induced to invade the underlying fibrillar matrix and to form capillary-like tubular structures in response to tumor-promoting phorbol esters or the angiogenic agent fibroblast growth factor (FGF). Since both phorbol esters and FGF stimulate phosphorylation of tyrosine residues, we treated endothelial cells with vanadate, an inhibitor of phosphotyrosine-specific phosphatases, to determine whether this agent could induce the expression of an angiogenic phenotype in these cells. We show here that vanadate stimulates endothelial cells to invade collagen matrices and to organize into characteristic tubules resembling those induced by FGF or phorbol esters. We have further observed that vanadate concomitantly stimulates endothelial cells to produce plasminogen activators (PAs), proteolytic enzymes which are induced by phorbol esters and FGF, and which have been implicated in the neovascular response; this stimulation can be accounted for by an increase in the levels of urokinase-type PA and tissue type PA mRNA. These results suggest a role for tyrosine phosphorylation in the regulation of the angiogenic phenotype in capillary endothelial cells.