ATP requirement for acidic resistance in Escherichia coli.

ATP participates in many cellular metabolic processes as a major substrate to supply energy. Many systems for acidic resistance (AR) under extremely acidic conditions have been reported, but the role of ATP has not been examined. To clarify whether or not ATP is necessary for the AR in Escherichia coli, the AR of mutants deficient in genes for ATP biosynthesis was investigated in this study. The deletion of purA or purB, each of which encodes enzymes to produce AMP from inosinate (IMP), markedly decreased the AR. The content of ATP in these mutants decreased rapidly at pH 2.5 compared to that of the wild type. The AR was again decreased significantly by the mutation of adk, which encoded an enzyme to produce ADP from AMP. The DNA damage in the purA and purB mutants was higher than that in the wild type. These results demonstrated that metabolic processes that require ATP participate in survival under extremely acidic conditions, and that one such system is the ATP-dependent DNA repair system.

The role of OmpC and OmpF in acidic resistance in Escherichia coli.

The roles of OmpC and OmpF in acidic resistance (AR) were examined. When ompC and ompF were deleted, AR was decreased. The decreased level of AR seen in the mutant that was deficient in ompC and ompF was elevated by the addition of glutamate, but not by the addition of arginine or lysine. The expression levels of adiA and cadB were diminished by the deletion of ompC and ompF, and the conversion of arginine to agmatine and lysine to cadaverine by intact cells were reduced in the mutant. The expression of gadA/gadB was not affected by the deletion of ompC and ompF. These results suggest that the transport of arginine, lysine, and their decarboxylated products through OmpC and/or OmpF is essential for the survival of Escherichia coli cells under extremely acidic conditions.

Expression and activity of Kdp under acidic conditions in Escherichia coli.

Escherichia coli has three major K(+) uptake systems, Trk, Kup, and Kdp, which have been studied extensively at near neutral pH. However, the function of these transporters under acidic conditions is not well understood, although growth and survival under acidic conditions are important for bacterial pathogenesis. In this study, we examined the expression and activity of Kdp under acidic conditions and found that the transport activity of Kdp is decreased at low pH and that the expression of kdp is regulated by the internal K(+) concentration in a pH-independent manner. Consequently, the low activity of Kdp was compensated for by the induction of its elevated expression by low K(+) accumulation via Kdp at acidic pH.

HU participates in expression of a specific set of genes required for growth and survival at acidic pH in Escherichia coli.

The major histone-like Escherichia coli protein, HU, is composed of alpha and beta subunits respectively encoded by hupA and hupB in Escherichia coli. A mutant deficient in both hupA and hupB grew at a slightly slower rate than the wild type at pH 7.5. Growth of the mutant diminished with a decrease in pH, and no growth was observed at pH 4.6. Mutants of either hupA or hupB grew at all pH levels tested. The arginine-dependent survival at pH 2.5 was diminished approximately 60-fold by the deletion of both hupA and hupB, whereas the survival was slightly affected by the deletion of either hupA or hupB. The mRNA levels of adiA and adiC, which respectively encode arginine decarboxylase and arginine/agmatine antiporter, were low in the mutant deficient in both hupA and hupB. The deletion of both hupA and hupB had little effect on survival at pH 2.5 in the presence of glutamate or lysine, and expression of the genes for glutamate and lysine decarboxylases was not impaired by the deletion of the HU genes. These results suggest that HU regulates expression of the specific set of genes required for growth and survival in acidic environments.

Extracellular acidic environments induce phosphorylation of ZAP-70 in Jurkat T cells.

In solid tumor and inflammation loci, low pH conditions have been observed as a consequence of either a lack of sufficient vascularization or excess activity of tumor cells, and T cells have been reported to infiltrate tumors and inflammation sites. However, it remains unclear how extracellular acidic environments affect immune cell function. A previous report proposed that a different signal transduction cascade might occur under low pH conditions in Jurkat T cells (Fukamachi T, Saito H, Kakegawa T, Kobayashi H. Different proteins are phosphorylated under acidic environments in Jurkat cells. Immunol Lett 2002;82:155-8). In this study, we investigated the protein phosphotyrosine level in Jurkat and Jurkat mutant cells under different pH conditions. The ZAP-70 phosphorylation level increased under acidic environments. P38 MAPK was more activated at acidic pH. The level of active p38 was low in mutant P116 deficient in ZAP-70, and interestingly the level remained consistently low at all pH values tested. The activation of ERK was not stimulated at low pH. These results suggest that extracellular low pH stimulates or enhances TCR signaling via ZAP-70 and p38.

Different proteins are phosphorylated under acidic environments in Jurkat cells.

In order to test the ability of immune cells to grow under acidic conditions, Jurkat cells were cultured in medium at pH 6.3. At this acidic pH, the cells required approximately 2 days per generation, while the generation time was approximately 1 day for cells grown in near neutral medium. Western blot analysis using anti-phosphotyrosine antibody (4G10) showed that protein phosphorylation was affected by external pH. For example, the level of a 66-kDa phosphoprotein increased dramatically in acidic medium, whereas the amount of a 34-kDa phosphoprotein decreased. These results suggest that Jurkat cells use different signal pathways under acidic environments.

Expression of acidosis-dependent genes in human cancer nests.

Previous studies investigating cancer cells cultured at acidic pH have shown that the expression level of ~700 genes were more than two-fold higher than those of the cells cultured in alkaline medium at pH 7.5. The aim of the present study was to confirm whether these acidosis-induced genes are expressed in human cancer tissues. Therefore, 7 genes were selected from our previous study, which encoded interleukin 32 (IL-32), lysosomal H+ transporting ATPase, V0 subunit d2 (ATP6V0D2), tumor necrosis factor receptor superfamily, member 9 (TNFRSF9), amphiregulin, schwannoma-derived growth factor (AREG), v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (ErbB3), PRR5-ARHGAP8 (LOC553158) and dimethylglycine dehydrogenase (DMGDH), and their expression was examined in human clinical specimens from patients with cancer. In addition, the expression of the gene encoding manganese superoxide dismutase (MnSOD) was examined. The specimens from patients with colon, stomach and renal cancer showed increased MnSOD, IL-32, and TNFRSF9 transcripts compared to those from non-tumorous regions of the same patients. Notably, an elevated expression of ATP6V0D2 was found in the specimens from patients with stomach cancer, whereas the expression was decreased in those from patients with colon and renal cancer. The expression of LOC553158 was upregulated in colon and stomach cancer specimens. These results indicate that the investigation of gene expression under acidic conditions is useful for the development of novel cancer markers and/or chemotherapeutic targets.

Acidic environments enhance the inhibitory effect of statins on proliferation of synovial cells.

Many previous studies in animal models and clinical investigations have suggested that statins are useful chemotherapeutics against rheumatoid arthritis, whereas in vitro experiments using synovial cell lines showed no significant effect of statins on cell proliferation until now. Since synovial fluid in rheumatoid joint knee was found to be acidic, we examined the effect of statins on human synovial sarcoma cell line SW982 cells in acidic medium. Statins suppressed the proliferation of SW982 cells at pH6.7, while the suppression was very weak in pH7.5 medium. It was shown that the suppression was caused by the decrease in geranylgeranyl diphosphate, suggesting that a geranylgeranylated protein(s) has an essential role in cell proliferation of SW982 cells under acidic conditions. Our present data clearly implied that statins had high efficacy against SW982 cells in acidic medium whose pH is close to that of rheumatoid arthritis loci in patients. These results lead us to anticipate that screening of chemicals having high therapeutic efficacy in acidic medium promotes the development of new microenvironment-dependent medicines for chemotherapies against rheumatoid arthritis.

Gene Expressions for Signal Transduction under Acidic Conditions.

Although it is now well known that some diseased areas, such as cancer nests, inflammation loci, and infarction areas, are acidified, little is known about cellular signal transduction, gene expression, and cellular functions under acidic conditions. Our group showed that different signal proteins were activated under acidic conditions compared with those observed in a typical medium of around pH 7.4 that has been used until now. Investigations of gene expression under acidic conditions may be crucial to our understanding of signal transduction in acidic diseased areas. In this study, we investigated gene expression in mesothelioma cells cultured at an acidic pH using a DNA microarray technique. After 24 h culture at pH 6.7, expressions of 379 genes were increased more than twofold compared with those in cells cultured at pH 7.5. Genes encoding receptors, signal proteins including transcription factors, and cytokines including growth factors numbered 35, 32, and 17 among the 379 genes, respectively. Since the functions of 78 genes are unknown, it can be argued that cells may have other genes for signaling under acidic conditions. The expressions of 37 of the 379 genes were observed to increase after as little as 2 h. After 24 h culture at pH 6.7, expressions of 412 genes were repressed more than twofold compared with those in cells cultured at pH 7.5, and the 412 genes contained 35, 76, and 7 genes encoding receptors, signal proteins including transcription factors, and cytokines including growth factors, respectively. These results suggest that the signal pathways in acidic diseased areas are different, at least in part, from those examined with cells cultured at a pH of around 7.4.

Zoledronic acid produces combinatory anti-tumor effects with cisplatin on mesothelioma by increasing p53 expression levels.

We examined anti-tumor effects of zoledronic acid (ZOL), one of the bisphosphonates agents clinically used for preventing loss of bone mass, on human mesothelioma cells bearing the wild-type p53 gene. ZOL-treated cells showed activation of caspase-3/7, -8 and -9, and increased sub-G1 phase fractions. A combinatory use of ZOL and cisplatin (CDDP), one of the first-line anti-cancer agents for mesothelioma, synergistically or additively produced the cytotoxicity on mesothelioma cells. Moreover, the combination achieved greater anti-tumor effects on mesothelioma developed in the pleural cavity than administration of either ZOL or CDDP alone. ZOL-treated cells as well as CDDP-treated cells induced p53 phosphorylation at Ser 15, a marker of p53 activation, and up-regulated p53 protein expression levels. Down-regulation of p53 levels with siRNA however did not influence the ZOL-mediated cytotoxicity but negated the combinatory effects by ZOL and CDDP. In addition, ZOL treatments augmented cytotoxicity of adenoviruses expressing the p53 gene on mesothelioma. These data demonstrated that ZOL-mediated augmentation of p53, which was not linked with ZOL-induced cytotoxicity, played a role in the combinatory effects with a p53 up-regulating agent, and suggests a possible clinical use of ZOL to mesothelioma with anti-cancer agents.

Respiration and the F1Fo-ATPase enhance survival under acidic conditions in Escherichia coli.

Besides amino acid decarboxylation, the ADP biosynthetic pathway was reported to enhance survival under extremely acidic conditions in Escherichia coli (Sun et al., J. Bacteriol. 193∶ 3072-3077, 2011). E. coli has two pathways for ATP synthesis from ADP: glycolysis and oxidative phosphorylation. We found in this study that the deletion of the F(1)Fo-ATPase, which catalyzes the synthesis of ATP from ADP and inorganic phosphate using the electro-chemical gradient of protons generated by respiration in E. coli, decreased the survival at pH 2.5. A mutant deficient in hemA encoding the glutamyl tRNA reductase, which synthesizes glutamate 1-semialdehyde also showed the decreased survival of E. coli at pH 2.5. Glutamate 1-semialdehyde is a precursor of heme synthesis that is an essential component of the respiratory chain. The ATP content decreased rapidly at pH 2.5 in these mutants as compared with that of their parent strain. The internal pH was lowered by the deletion of these genes at pH 2.5. These results suggest that respiration and the F(1)Fo-ATPase are still working at pH 2.5 to enhance the survival under such extremely acidic conditions.

Zoledronic acid produces antitumor effects on mesothelioma through apoptosis and S-phase arrest in p53-independent and Ras prenylation-independent manners.

INTRODUCTION: We examined whether zoledronic acid (ZOL), the third generation of bisphosphonates, produced cytotoxic effects on human mesothelioma cells in vitro and in vivo, and investigated a possible involvement of p53, Ras, and extracellular signal-regulated kinase1/2 (ERK1/2) pathways. METHODS: Cytotoxicity and cell cycles were assessed with a colorimetric assay and flow cytometry, respectively. Expression levels of apoptosis-linked proteins and prenylation of small guanine-nucleotide-binding regulatory proteins were tested with p53-small interfering RNA, an ERK kinase1/2-inhibitor, and prenyl alcohols. The antitumor activity was examined in an orthotopic animal model. RESULTS: ZOL treatments suppressed growth of mesothelioma cells bearing the wild-type p53 gene through apoptosis induction accompanied by activation of caspases, or S-phase arrest by up-regulated cyclin A and B1. ZOL induced p53 phosphorylation and subsequent activation of the downstream pathways. Down-regulated p53 expression with the small interfering RNA, however, showed that both apoptosis and S-phase arrest were irrelevant to the p53 activation. Geranylgeranyl but not farnesyl pyrophosphate inhibited ZOL-induced apoptosis and S-phase arrest, and the geranylgeraniol supplement decreased ZOL-mediated Rap1A but not Ras unprenylation. Inhibition of ERK1/2 pathways suppressed ZOL-induced apoptosis but not S-phase arrest. We further demonstrated that ZOL, administrated intrapleurally, inhibited the tumor growth in the pleural cavity. CONCLUSIONS: These data indicate that ZOL induces apoptosis or S-phase arrest, both of which are independent of p53 activation and Ras unprenylation, and suggest that ZOL is a possible therapeutic agent to mesothelioma partly through non-Ras- and ERK1/2-mediated pathways.

E1B-55 kDa-defective adenoviruses activate p53 in mesothelioma and enhance cytotoxicity of anticancer agents.

INTRODUCTION: Genetic characterization of malignant mesothelioma shows a homozygous deletion of the INK4A/ARF locus, which results in inactivation of the p53 pathways. METHODS: We examined possible antitumor effects of adenoviruses with a deletion of the E1B-55kD gene (Ad-delE1B55) on mesothelioma and investigated combinatory actions with the first-line chemotherapeutic agents. RESULTS: Ad-delE1B55 produced cytotoxicity on mesothelioma cells, which was associated with p53 phosphorylation, pRb dephosphorylation, and cleavage of caspases. Ad-delE1B55-infected cells displayed hyperploidy at the cell-cycle analysis and showed enlarged nuclear configurations. Combination of Ad-delE1B55 plus cisplatin or pemetrexed produced antitumor effects in vitro. Furthermore, Ad-delE1B55 and cisplatin showed combinatory effects in an orthotopic animal model. CONCLUSIONS: Cell death caused by Ad-delE1B55 is attributable to cell-cycle arrest at M-phase checkpoint followed by activated apoptotic pathways, and combination of the first-line chemotherapeutic agents and the oncolytic adenovirus is a potential therapeutic for mesothelioma.

Tumor specific low pH environments enhance the cytotoxicity of lovastatin and cantharidin.

In tumor cell masses, the extracellular pH decreases below 6.5. The effect of external acidic pH on the efficacy of 24 chemical compounds including molecular-targeted inhibitors and anti-tumor reagents was investigated in human cancer cells. Lovastatin showed no cytotoxicity in mesothelioma or pancreatic carcinoma cells at concentrations up to 10 µM and pH around 7.4, but 10 µM lovastatin decreased the survival of these cells below 40% at acidic pH. Lovastatin inhibits HMG-CoA reductase, resulting in a decrease in the levels of cholesterol and prenylated proteins. An inhibitor of the former pathway showed pH-independent cytotoxic activity, whereas an inhibitor of the latter pathway had stronger activity at acidic pH. The inhibitory efficacy of cantharidin also increased at acidic pH. On the other hands, no pH dependency or slightly impaired efficacy at low pH conditions was observed in other 20 reagents, and especially, the activity of aphidicolin was suppressed under acidic conditions. These results suggested that screening under acidic conditions would be useful for developing new chemotherapeutic reagents.

Catalytic asymmetric total synthesis of tangutorine.

The first enantioselective total synthesis of tangutorine has been achieved, wherein a Pd-catalyzed asymmetric allylic amination using a chiral diaminophosphine oxide (DIAPHOX) preligand was the key step.

Requirement of an IkappaB-beta COOH terminal region protein for acidic-adaptation in CHO cells.

We previously reported that an IkappaB-beta COOH terminal region protein (designated CTIB) was essential for the proliferation of CHO cells under acidic stress (Lao et al., 2005. J Cell Physiol 203(1):186-192). In order to investigate the mechanisms underlying the requirement of CTIB for acidic adaptation, CTIB was silenced with an RNAi technique in CHO cells. CTIB silencing resulted in those cells completely failing to proliferate and maintain intracellular pH (pHi) homeostasis at an extracellular pH (pHe) of 6.3. An increased activation of p38 MAP kinase was induced by CTIB silencing at the low pH value. CTIB was only present in the cytoplasm and co-immunoprecipitation of the cytoplasmic fraction revealed that the loss of CTIB led to a loss of p65 in the immunoprecipitate complex. CTIB silencing reduced both the decrease in p65 and the increase in p50 in the nucleus when the cells were incubated at pHe 6.3. In cells with CTIB silenced, the transcriptions of p65, p105, and IL1-beta were suppressed, and decreases in both the transcription and activity of MnSOD were observed at pHe 6.3. Suppression of these genes suggested a suppressed NF-kappaB activity since p105, IL1-beta, and MnSOD were target genes of NF-kappaB. Our data demonstrated that CTIB functioned to prevent the over-accumulation of p65 in the nucleus, ensuring the appropriate composition of the NF-kappaB complex in the nucleus to respond to stimuli under acidic conditions.

Prolonged transcriptional silencing and CpG methylation induced by siRNAs targeted to the HIV-1 promoter region.

In addition to the degradation of homologous RNAs through the RNA interference (RNAi) pathway, small interfering RNAs (siRNAs) can in some systems induce cytosine methylation and transcriptional silencing of homologous promoters. Targeting of HIV-1 by RNAi results in transient suppression of the virus through degradation of viral transcripts. In an effort to prolong the suppressive effect of siRNAs on productive HIV-1 infection, we targeted conserved tandem NF-kappaB binding motifs in the viral LTR. A 21-nucleotide-RNA duplex induced marked and durable (at least 30 days) suppression of productive HIV-1 infection in chronically infected Magic-5 cells. This suppression is associated with CpG methylation within the 5'LTR and marked reduction of HIV-1 transcription in nuclear run-on assays. We then assessed three additional siRNAs targeting other sites within the HIV-1 promoter region. These siRNAs suppressed HIV-1 infection to different extents and the degree of suppression correlated with the extent of de novo methylation of CpG motifs within the HIV-1 promoter region. These findings indicate that HIV-1 can be silenced by an RNA-directed mechanism that suppresses transcription and induces CpG methylation. In addition to providing evidence that this RNA-directed DNA methylation is active in mammalian cells, this is the first report of prolonged suppression of HIV-1 infection induced by siRNA.

An IkappaB-beta COOH terminal region protein is essential for the proliferation of CHO cells under acidic stress.

CHO-K1 cells were able to proliferate and maintain pHi homeostasis at pH 6.3. A novel acidic sensitive mutant, AS-5B, which proliferated at pH 7.4 but failed to either proliferate or maintain pHi homeostasis at pH 6.3, was derived from CHO-K1 using a replica method. The acidic-sensitivity of AS-5B was not due to deficiencies in sodium proton exchangers, HCO3- (co)transporters or H+-ATPases. A cDNA clone encoding a COOH terminal region of IkappaB-beta conferred partial acidic-resistance on AS-5B, and the encoded protein was present in CHO-K1, but was nearly absent from AS-5B. Our data demonstrated that the expression of this small protein was essential for the proliferation of CHO cells under acidic stress.