Activating Akt1 mutations alter DNA double strand break repair and radiosensitivity.

The survival kinase Akt has clinical relevance to radioresistance. However, its contributions to the DNA damage response, DNA double strand break (DSB) repair and apoptosis remain poorly defined and often contradictory. We used a genetic approach to explore the consequences of genetic alterations of Akt1 for the cellular radiation response. While two activation-associated mutants with prominent nuclear access, the phospho-mimicking Akt1-TDSD and the clinically relevant PH-domain mutation Akt1-E17K, accelerated DSB repair and improved survival of irradiated Tramp-C1 murine prostate cancer cells and Akt1-knockout murine embryonic fibroblasts in vitro, the classical constitutively active membrane-targeted myrAkt1 mutant had the opposite effects. Interestingly, DNA-PKcs directly phosphorylated Akt1 at S473 in an in vitro kinase assay but not vice-versa. Pharmacological inhibition of DNA-PKcs or Akt restored radiosensitivity in tumour cells expressing Akt1-E17K or Akt1-TDSD. In conclusion, Akt1-mediated radioresistance depends on its activation state and nuclear localization and is accessible to pharmacologic inhibition.

The additional loss of Bak and not the lack of the protein tyrosine kinase p56/Lck in one JCaM1.6 subclone caused pronounced apoptosis resistance in response to stimuli of the intrinsic pathway.

Ionising radiation, hypoxia, and the cyclooxygenase-2 inhibitor Celecoxib are known agonists of the intrinsic apoptosis pathway that involves mitochondrial damage upstream of caspase activation. Mitochondrial integrity is regulated by the pro-apoptotic Bcl-2 protein family members Bak and Bax. Upstream of the mitochondria, many kinases and phosphatases control the apoptotic response. However, the role of the non-receptor tyrosine kinase p56/Lck during apoptosis is controversial. The present investigation demonstrate the existence of two JCaM1.6 subclones, one expressing and one deficient for Bak. The lack of p56/Lck expression in JCaM1.6 cells per se did hardly affect apoptosis induced by ionising radiation, hypoxia, or Celecoxib. Only the additional loss of Bak expression, as observed in one JCaM1.6 subclone, rendered the cells resistant. siRNA-mediated downregulation of Bak and p56/Lck mimicked the observed effects in the subclones. Earlier experiments performed with the Bak-negative clone might have lead to the wrong assumption that lack of p56/Lck alone, and not the additonal loss of Bak, was responsible for reduced sensitivity towards stimuli of the intrinsic apoptosis pathway.

Bcl-2 mediated inhibition of erucylphosphocholine-induced apoptosis depends on its subcellular localisation.

The synthetic phospholipid derivative erucylphosphocholine (ErPC) is a potent inducer of apoptosis in human tumor cell lines. This membrane-targeted drug induces apoptosis independently from death receptor signaling through a mitochondrial pathway that is inhibited by over-expression of Bcl-2. Within the cell, Bcl-2 resides in membranes of mitochondria, endoplasmic reticulum (ER) and the nucleus. However, the importance of its subcellular localisation in distinct organelles for protection against apoptosis is not completely understood. To investigate the impact of Bcl-2 localised at defined subcellular compartments on its protective effects against ErPC-induced apoptosis, Bcl-2 expression was directed to the outer membrane of the mitochondria or the ER of Jurkat T Lymphoma cells, using Bcl-2 mutants with modified membrane anchors. The mitochondrial insertion sequence of ActA directed Bcl-2 to the mitochondria (Bcl-2/MT), the ER-specific sequence of cytochrome b5 to the ER (Bcl-2/ER). Additionally, Jurkat cells expressing wild-type Bcl-2 (Bcl-2/WT) or a transmembrane domain-lacking mutant (Bcl-2/DeltaTM) were employed. While restricted expression of Bcl-2 either at membranes of the mitochondria or the ER strongly interfered with ErPC-induced mitochondrial damage and apoptosis, cytosolic Bcl-2/DeltaTM exhibited only reduced protection. Thus, membrane localisation of Bcl-2 is a prerequisite for substantial protection against ErPC-induced apoptosis. For efficient long-term inhibition of ErPC-induced apoptosis Bcl-2 had to be present in the membranes of both compartments, the ER and the mitochondria. The finding that ER-targeted Bcl-2 interferes with ErPC-induced mitochondrial damage points to an involvement of the ER in apoptosis signaling upstream of the mitochondria and to a crosstalk between both compartments.

Membrane targeted anticancer drugs: potent inducers of apoptosis and putative radiosensitisers.

In the last two decades, cellular membranes have been identified as novel targets for antineoplastic drugs. Two classes of synthetic phospholipid analogues: the alkyllysophospholipids (ALP) with the prototypical 1-O-Octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (Et-18-OCH3, Edelfosine), as well as the alkylphosphocholines (APC) with the prototypical hexadecylphosphocholine (HePC, Miltefosine), have been identified targeting cellular membranes and exerting potent antineoplastic effects in cell culture and animal models. In contrast to most of the chemotherapeutic agents in clinical use, APC and ALP primarily interfere with cellular membranes without direct interaction with the DNA. They modulate membrane permeability and fluidity, membrane lipid composition, metabolism of phospholipids and proliferation signal transduction. Interestingly, similar to DNA-damaging drugs, ALP and APC induce apoptotic cell death. Furthermore, combination experiments with cytotoxic drugs or radiation revealed a synergistic effect in leukaemic and brain tumour cell lines. These findings together with the observations that ALP and APC selectively kill malignant cells, that they lack bone marrow toxicity and even exert growth stimulatory effects on hematopoietic progenitor cells make ALP and APC a promising tool for novel approaches in cancer chemotherapy. In this contribution, novel findings on the mechanism of action, apoptotic signalling pathways and putative radiosensitising effects of ALP and APC were reviewed, with a special focus on erucylphosphocholine (ErPC), the prototype of the novel intravenously applicable APC derivatives.

A new type of thermoalkalophilic hydrolase of Paucimonas lemoignei with high specificity for amorphous polyesters of short chain-length hydroxyalkanoic acids.

A novel type of hydrolase was purified from culture fluid of Paucimonas (formerly Pseudomonas) lemoignei. Biochemical characterization revealed an unusual substrate specificity of the purified enzyme for amorphous poly((R)-3-hydroxyalkanoates) (PHA) such as native granules of natural poly((R)-3-hydroxybutyrate) (PHB) or poly((R)-3-hydroxyvalerate) (PHV), artificial cholate-coated granules of natural PHB or PHV, atactic poly((R,S)-3-hydroxybutyrate), and oligomers of (R)-3-hydroxybutyrate (3HB) with six or more 3HB units. The enzyme has the unique property to recognize the physical state of the polymeric substrate by discrimination between amorphous PHA (good substrate) and denatured, partially crystalline PHA (no substrate). The pentamers of 3HB or 3HV were identified as the main products of enzymatic hydrolysis of native PHB or PHV, respectively. No activity was found with any denatured PHA, oligomers of (R)-3HB with five or less 3HB units, poly(6-hydroxyhexanoate), substrates of lipases such as tributyrin or triolein, substrates for amidases/nitrilases, DNA, RNA, casein, N-alpha-benzoyl-l-arginine-4-nitranilide, or starch. The purified enzyme (M(r) 36,209) was remarkably stable and active at high temperature (60 degrees C), high pH (up to 12.0), low ionic strength (distilled water), and in solvents (e.g. n-propyl alcohol). The depolymerase contained no essential SH groups or essential disulfide bridges and was insensitive to high concentrations of ionic (SDS) and nonionic (Triton and Tween) detergents. Characterization of the cloned structural gene (phaZ7) and the DNA-deduced amino acid sequence revealed no homologies to any PHB depolymerase or any other sequence of data banks except for a short sequence related to the active site serine of serine hydrolases. A classification of the enzyme into a new family (family 9) of carboxyesterases (Arpigny, J. L., and Jaeger, K.-E. (1999) Biochem. J. 343, 177-183) is suggested.

Mobilization of poly(3-hydroxybutyrate) in Ralstonia eutropha.

Ralstonia eutropha H16 degraded (mobilized) previously accumulated poly(3-hydroxybutyrate) (PHB) in the absence of an exogenous carbon source and used the degradation products for growth and survival. Isolated native PHB granules of mobilized R. eutropha cells released 3-hydroxybutyrate (3HB) at a threefold higher rate than did control granules of nonmobilized bacteria. No 3HB was released by native PHB granules of recombinant Escherichia coli expressing the PHB biosynthetic genes. Native PHB granules isolated from chromosomal knockout mutants of an intracellular PHB (i-PHB) depolymerase gene of R. eutropha H16 and HF210 showed a reduced but not completely eliminated activity of 3HB release and indicated the presence of i-PHB depolymerase isoenzymes.