Thymineless death in Bacillus megaterium

Wachsman, J. T. (University of Illinois, Urbana), S. Kemp, and L. Hogg. Thymineless death in Bacillus megaterium. J. Bacteriol. 87:1079-1086. 1964.-Strain KM:T(-), a thymine auxotroph of Bacillus megaterium strain KM, rapidly loses the ability to multiply when incubated in the absence of thymine, on an otherwise sufficient medium. At 37 C, there is a lag of approximately 60 min, prior to the onset of exponential death (decrease of 1 decade per 50 min). The extent of the decrease in viable count varies from 4 to 5 decades after 5 hr of starvation. The cells die more slowly at 30 C (decrease of 1 decade per 120 min) after a lag of approximately 90 min. Thymine starvation permits substantial net ribonucleic acid (RNA) and protein synthesis, but only slight deoxyribonucleic acid synthesis. In contrast with the changes occurring at 30 C, thymineless death at 37 C is eventually accompanied by a rapid hydrolysis of RNA and by cell lysis. Chloramphenicol inhibits thymineless death at 37 C. Strain T(-)R(1), a derivative of strain KM:T(-), undergoes a very low rate of thymineless death at 37 C (decrease of 1 decade per 240 min). Neither hydrolysis of RNA nor cell lysis occurs during 8 hr of thymine starvation. Strain KM:T(-)H(-) (doubly auxotrophic for thymidine and histidine) requires histidine for maximal thymineless death at 37 C. Preincubation of this strain on the basal medium supplemented with thymidine alone enables the population to become increasingly immune to subsequent thymineless death.     

Thapsigargin analogues for targeting programmed death of androgen-independent prostate cancer cells.

A number of analogues of thapsigargin, a selective inhibitor of the sarco-endoplasmic reticulum Ca2+-ATPases have been synthesized. In all of the prepared analogues the butanoyl residue at O-8 has been replaced with a residue containing an aromatic amine. The amine can be used as an anchoring point for attaching a peptide group sensitive to the proteolytic enzyme, prostate specific antigen, secreted by prostate cancer cells. Like thapsigargin, the analogues are capable of elevating the cytoplasmic Ca2+ concentration approximately sevenfold when tested at effective cytotoxic doses. The analogues in which the 8-O-butanoyl group has been replaced with 3-(4-aminophenyl)propanoyl or 4-aminocinnamoyl were found potently to induce programmed cell death of the prostate cancer cells.     

Thymineless death inLactobacillus acidophilus R-26

Thymineless death (TLD) was studied inLactobacillus acidophilus R-26. Thymine synthesis was inhibited with 5-fluorouracil (FU) or deoxyadenylate (dAMP) or by the absence of folic acid. In the case of FU, the maximum rate of dying was obtained at concentrations exceeding 0.1 μg/ml. This concentration did not affect the growth of the bacteria in the presence of thymine (4 μg/ml) and uracil (10 μg/ml). At higher FU concentrations up to 10 μg/ml, the course of TLD was unaltered, but the growth of bacteria in complete medium was slower. In the case of dAMP, the same course of TLD was obtained at a concentration of 150 μg/ml. If 1,500 μg dAMP/ml was used, the pre-death lag phase was shortened the rate of dying being unaltered. These concentrations of dAMP retarded the growth of bacteria even in a complete medium. If the thymine synthesis was prevented by the absence of folic acid the rate of dying was much lower than that caused by the presence of FU or dAMP. This was true even if the aminopterin was added. The authors conclude that the folic acid starvation did not inhibit completely the synthesis of thymine.     

Thymineless death inLactobacillus acidophilus R-26

Thymineless death (TLD) as well as deoxyribosideless death (DRLD) can be observed inLactobacillus acidophilus R-26 during growth in media lacking thymine or deoxyriboside respectively. Both phenomena exhibit the same interval of lage period (2–3 h) but the rate of inactivation is 2–3 times faster in TLD. Transfer experiments show that inactivation of bacterial reproduction is accelerated immediately if—DR medium is replaced by—T one. In the opposite case the deceleration of the inactivation rate does not appear immediately but after a 1–2 h lag period, in which no changes in the number of viable bacteria can be observed. Our results suggested that the accumulation of deoxyriboside compounds has no causal role in the inactivation of bacterial reproduction. However, the presence of deoxyribosides can accelerate the process of inactivation.     

Proteomic analysis of androgen-independent growth in low and high passage human LNCaP prostatic adenocarcinoma cells

The present study compared the proteomic characteristics of a low passage number (L-33) and high passage number (H-81) LNCaP cell clone. Marked differences in protein expression were noted in the response of L-33 and H-81 cells to androgens. To investigate if regulation of these proteins was androgen-dependent, expression of the androgen receptor was silenced via small interfering RNA. Consistent with the proteomic data, abrogation of androgen receptor production in H-81 cells resulted in the reversed expression level into L-33 cells compared with non-treated H-81 LNCaP cells. The results clarify the progression into an androgen-independent phenotype.     

Ferruginol suppresses survival signaling pathways in androgen-independent human prostate cancer cells

Ferruginol, a bioactive compound isolated from a Chilean tree (Podocarpaceae), attracts attention as a consequence of its pharmacological properties, which include anti-fungal, anti-bacterial, cardioprotective, anti-oxidative, anti-plasmodial and anti-ulcerogenic actions. Nevertheless, the molecular basis for these actions remains only partly understood and hence we investigated the effects of ferruginol on androgen-independent human prostate cancer cells (PC3), a known model for solid tumor cells with an exceptional resistance to therapy. The results show that ferruginol induces PC3 cell death via activation of caspases as well as apoptosis-inducing factor (AIF) as confirmed by its translocation into the nucleus. In order to clarify the biochemical mechanism responsible for the anti-tumor activity of ferruginol, we analyzed a set of molecular mediators involved in tumor cell survival, progression and aggressiveness. Ferruginol was able to trigger inhibition/downregulation of Ras/PI3K, STAT 3/5, protein tyrosine phosphatase and protein kinases related to cell cycle regulation. Importantly, the toxic effect of ferruginol was dramatically impeded in a more reducing environment, which indicates that at least in part, the anti-tumoral activity of ferruginol might be related to redox status modulation. This study supports further examination of ferruginol as a potential agent for both the prevention and treatment of prostate cancer.     

Thymineless death in Escherichia coli mutants deficient in the RecF recombination pathway

Like recF and recQ mutants studied earlier, two other classes of Escherichia coli mutants defective in the RecF conjugal recombination pathway, recJ and recO, were found to be partially resistant to thymineless death. In contrast, a recN mutant, also belonging to the pathway, was indistinguishable from the wild type with respect to thymineless death.     

Thymineless death in Escherichia coli dnaB mutants and in a dnaB dnaG double mutant.

The interference of dnaB mutations of Escherichia coli with thymineless death is described. All the isogenic Thy- dnaB mutants of E. coli we have tested show a remarkable immunity towards cell death induced by thymine deprivation at the nonpermissive temperature. We have also constructed and tested an isogenic double dnaB dnaG mutant. It loses its viability in the absence of thymine at both permissive and nonpermissive temperatures. The role of the dnaB gene product is discussed.     

Inhibition of Vitamin D3 metabolism enhances VDR signalling in androgen-independent prostate cancer cells

Induction of growth arrest and differentiation by 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) occurs in non-malignant cell types but is often reduced in cancer cells. For example, androgen-independent prostate cancer cells, DU-145 and PC-3, are relatively insensitive to the anti-proliferative action of 1,25-(OH)₂D₃. This appears to be due to increased 1,25-(OH)₂D₃-metabolism, as a result of CYP24 enzyme-induction, which in turn leads to decreased anti-proliferative efficacy. In the in vitro rat kidney mitochondria assay, the 2-(4-hydroxybenzyl)-6-methoxy-3,4-dihydro-2H-naphthalen-1-one (4) was found to be a potent inhibitor of Vitamin D₃ metabolising enzymes (IC₅₀ 3.5 μM), and was shown to be a more potent inhibitor than the broad spectrum P450 inhibitor ketoconazole (IC₅₀ 20 μM). The combination of the inhibitor and 1,25-(OH)₂D₃ caused a greater inhibition of proliferation in DU-145 cells than when treated with both agents alone. Examination of the regulation of VDR target gene mRNA in DU-145 cells revealed that co-treatment of 1,25-(OH)₂D₃ plus inhibitor of Vitamin D₃ metabolising enzymes co-ordinately upregulated CYP24, p21^waf1/cip1 and GADD45.     

Thymineless Mutagenesis in Escherichia coli

To clarify the relationship between thymineless death and thymineless mutagenesis, the induction of arginine revertants of Escherichia coli TAU-bar by thymine starvation was examined in physiological terms. Induced revertants were detectable both on minimal medium lacking arginine and minimal medium supplemented with 1 mug of arginine per ml. Substantial thymineless mutagenesis occurred during the period before the onset of thymineless death. Mutagenesis and loss of viability were observed upon incubation in medium lacking thymine and arginine, and both were inhibited upon incubation in medium lacking thymine and uracil. Mutagenesis also occurred during thymine starvation at 25 C, where there was relatively little loss of viability. At 37 C thymineless mutagenesis did not require complete thymine starvation, and the induction of revertants appeared to be initiated at the same suboptimal thymine concentration at which lethality was first detectable. Mutagenesis was found not to occur preferentially at the growing point of deoxyribonucleic acid replication. These results suggest that thymineless mutagenesis does not involve simply errors in base pairing due to the absence of thymine. The data also suggest that the induction of mutations and thymineless death are due to the same primary event but that mutagenesis is the more sensitive response.     

Survival advantage of AMPK activation to androgen-independent prostate cancer cells during energy stress

Androgen-independent prostate cancer usually develops as a relapse following androgen ablation therapy. Removing androgen systemically causes vascular degeneration and nutrient depletion of the prostate tumor tissue. The fact that the malignancy later evolves to androgen-independence suggests that some cancer cells are able to survive the challenge of energy/nutrient deprivation. AMP-activated protein kinase (AMPK) is an important manager of energy stress. The present study was designed to investigate the role of AMPK in contributing to the survival of the androgen-independent phenotype. Most of the experiments were carried out in the androgen-dependent LNCaP cells and the androgen-independent C4-2 cells. These two cell lines have the same genetic background, since the C4-2 line is derived from the LNCaP line. Glucose deprivation (GD) was instituted to model energy stress encountered by these cells. The key findings are as follows. First, the activation of AMPK by GD was much stronger in C4-2 cells than in LNCaP cells, and the robustness of AMPK activation was correlated favorably with cell viability. Second, the response of AMPK was specific to energy deficiency rather than to amino acid deficiency. The activation of AMPK by GD was functional, as demonstrated by appropriate phosphorylation changes of mTOR and mTOR downstream substrates. Third, blocking AMPK activation by chemical inhibitor or dominant negative AMPK led to increased apoptotic cell death. The observation that similar results were found in other androgen-independent prostate cancer cell lines, including CW22Rv1 abd VCaP, provided further assurance that AMPK is a facilitator on the road to androgen-independence of prostate cancer cells.     

Thymineless death is inhibited by CsrA in Escherichia coli lacking the SOS response

Thymineless death (TLD) is the rapid loss of colony-forming ability in bacterial, yeast and human cells starved for thymine, and is the mechanism of action of common chemotherapeutic drugs. In Escherichia coli, significant loss of viability during TLD requires the SOS replication-stress/DNA-damage response, specifically its role in inducing the inhibitor of cell division, SulA. An independent RecQ- and RecJ-dependent TLD pathway accounts for a similarly large additional component of TLD, and a third SOS- and RecQ/J-independent TLD pathway has also been observed. Although two groups have implicated the SOS-response in TLD, an SOS-deficient mutant strain from an earlier study was found to be sensitive to thymine deprivation. We performed whole-genome resequencing on that SOS-deficient strain and find that, compared with the SOS-proficient control strain, it contains five mutations in addition to the SOS-blocking lexA(Ind⁻) mutation. One of the additional mutations, csrA, confers TLD sensitivity specifically in SOS-defective strains. We find that CsrA, a carbon storage regulator, reduces TLD in SOS- or SulA-defective cells, and that the increased TLD that occurs in csrA⁻ SOS-defective cells is dependent on RecQ. We consider a hypothesis in which the modulation of nucleotide pools by CsrA might inhibit TLD specifically in SOS-deficient (SulA-deficient) cells.     

Selective amino acid restriction targets mitochondria to induce apoptosis of androgen-independent prostate cancer cells

Relative specific amino acid dependency is one of the metabolic abnormalities of cancer cells, and restriction of specific amino acids induces apoptosis of prostate cancer cells. This study shows that restriction of tyrosine and phenylalanine (Tyr/Phe), glutamine (Gln), or methionine (Met), modulates Raf and Akt survival pathways and affects the function of mitochondria in DU145 and PC3, in vitro. These three restrictions inhibit energy production (ATP synthesis) and induce generation of reactive oxygen species (ROS). Restriction of Tyr/Phe or Met in DU145 and Met in PC3 reduces mitochondrial membrane potential (DeltaPsim) and induces caspase-dependent and -independent apoptosis. In DU145, Tyr/Phe or Met restriction reduces activity of Akt, mitochondrial distribution of phosphorylated Raf and apoptosis inducing factor (AIF), and increases mitochondrial distribution of Bak. Mitochondrial Bcl-XL is increased in Tyr/Phe-restricted but decreased in Met-restricted cells. Under Tyr/Phe or Met restriction, reduced mitochondrial Raf does not inactivate the pro-apoptotic function of Bak. Tyr/Phe restriction also inhibits Bcl-2 and Met restriction inhibits Bcl-XL in mitochondria. These comprehensive actions damage the integrity of the mitochondria and induce apoptosis of DU145. In PC3, apoptosis induced by Met restriction was not associated with alterations in intracellular distribution of Raf, Bcl-2 family proteins, or AIF. All of the amino acid restrictions inhibited Akt activity in this cell line. We conclude that specific amino acid restriction differentially interferes with homeostasis/balance between the Raf and Akt survival pathways and with the interaction of Raf and Bcl-2 family proteins in mitochondria to induce apoptosis of DU145 and PC3 cells.