Thomas Marti

DNA mismatch repair and mutation avoidance pathways.

Unpaired and mispaired bases in DNA can arise by replication errors, spontaneous or induced base modifications, and during recombination. The major pathway for correction of mismatches arising during replication is the MutHLS pathway of Escherichia coli and related pathways in other organisms. MutS initiates repair by binding to the mismatch, and activates together with MutL the MutH endonuclease, which incises at hemimethylated dam sites and thereby mediates strand discrimination. Multiple MutS and MutL homologues exist in eukaryotes, which play different roles in the mismatch repair (MMR) pathway or in recombination. No MutH homologues have been identified in eukaryotes, suggesting that strand discrimination is different to E. coli. Repair can be initiated by the heterodimers MSH2‐MSH6 (MutSα) and MSH2‐MSH3 (MutSβ). Interestingly, MSH3 (and thus MutSβ) is missing in some genomes, as for example in Drosophila, or is present as in Schizosaccharomyces pombe but appears to play no role in MMR. MLH1‐PMS1 (MutLα) is the major MutL homologous heterodimer. Again some, but not all, eukaryotes have additional MutL homologues, which all form a heterodimer with MLH1 and which play a minor role in MMR. Additional factors with a possible function in eukaryotic MMR are PCNA, EXO1, and the DNA polymerases δ and ϵ. MMR‐independent pathways or factors that can process some types of mismatches in DNA are nucleotide‐excision repair (NER), some base excision repair (BER) glycosylases, and the flap endonuclease FEN‐1. A pathway has been identified in Saccharomyces cerevisiae and human that corrects loops with about 16 to several hundreds of unpaired nucleotides. Such large loops cannot be processed by MMR. J. Cell. Physiol. 191: 28–41, 2002.

DNA repair nucleases

Stability of DNA largely depends on accuracy of repair mechanisms, which remove structural anomalies induced by exogenous and endogenous agents or introduced by DNA metabolism, such as replication. Most repair mechanisms include nucleolytic processing of DNA, where nucleases cleave a phosphodiester bond between a deoxyribose and a phosphate residue, thereby producing 5′-terminal phosphate and 3′-terminal hydroxyl groups. Exonucleases hydrolyse nucleotides from either the 5′ or 3′ end of DNA, while endonucleases incise internal sites of DNA. Flap endonucleases cleave DNA flap structures at or near the junction between single-stranded and double-stranded regions. DNA nucleases play a crucial role in mismatch repair, nucleotide excision repair, base excision repair and double-strand break repair. In addition, nucleolytic repair functions are required during replication to remove misincorporated nucleotides, Okazaki fragments and 3′ tails that may be formed after repair of stalled replication forks.

Human agonistic TRAIL receptor antibodies Mapatumumab and Lexatumumab induce apoptosis in malignant mesothelioma and act synergistically with cisplatin.

BackgroundThe incidence of malignant pleural mesothelioma (MPM) is associated with exposure to asbestos, and projections suggest that the yearly number of deaths in Western Europe due to MPM will increase until 2020. Despite progress in chemo- and in multimodality therapy, MPM remains a disease with a poor prognosis. Inducing apoptosis by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or agonistic monoclonal antibodies which target TRAIL-receptor 1 (TRAIL-R1) or TRAIL-R2 has been thought to be a promising cancer therapy.ResultsWe have compared the sensitivity of 13 MPM cell lines or primary cultures to TRAIL and two fully human agonistic monoclonal antibodies directed to TRAIL-R1 (Mapatumumab) and TRAIL-R2 (Lexatumumab) and examined sensitization of the MPM cell lines to cisplatin-induced by the TRAIL-receptor antibodies. We found that sensitivity of MPM cells to TRAIL, Mapatumumab and Lexatumumab varies largely and is independent of TRAIL-receptor expression. TRAIL-R2 contributes more than TRAIL-R1 to death-receptor mediated apoptosis in MPM cells that express both receptors. The combination of cisplatin with Mapatumumab or Lexatumumab synergistically inhibited the cell growth and enhanced apoptotic death. Furthermore, pre-treatment with cisplatin followed by Mapatumumab or Lexatumumab resulted in significant higher cytotoxic effects as compared to the reverse sequence. Combination-induced cell growth inhibition was significantly abrogated by pre-treatment of the cells with the antioxidant N-acetylcysteine.ConclusionOur results suggest that the sequential administration of cisplatin followed by Mapatumumab or Lexatumumab deserves investigation in the treatment of patients with MPM.

Starvation-induced activation of ATM/Chk2/p53 signaling sensitizes cancer cells to cisplatin

BackgroundOptimizing the safety and efficacy of standard chemotherapeutic agents such as cisplatin (CDDP) is of clinical relevance. Serum starvation in vitro and short-term food starvation in vivo both stress cells by the sudden depletion of paracrine growth stimulation.MethodsThe effects of serum starvation on CDDP toxicity were investigated in normal and cancer cells by assessing proliferation, cell cycle distribution and activation of DNA-damage response and of AMPK, and were compared to effects observed in cells grown in serum-containing medium. The effects of short-term food starvation on CDDP chemotherapy were assessed in xenografts-bearing mice and were compared to effects on tumor growth and/or regression determined in mice with no diet alteration.ResultsWe observed that serum starvation in vitro sensitizes cancer cells to CDDP while protecting normal cells. In detail, in normal cells, serum starvation resulted in a complete arrest of cellular proliferation, i.e. depletion of BrdU-incorporation during S-phase and accumulation of the cells in the G0/G1-phase of the cell cycle. Further analysis revealed that proliferation arrest in normal cells is due to p53/p21 activation, which is AMPK-dependent and ATM-independent. In cancer cells, serum starvation also decreased the fraction of S-phase cells but to a minor extent. In contrast to normal cells, serum starvation-induced p53 activation in cancer cells is both AMPK- and ATM-dependent. Combination of CDDP with serum starvation in vitro increased the activation of ATM/Chk2/p53 signaling pathway compared to either treatment alone resulting in an enhanced sensitization of cancer cells to CDDP. Finally, short-term food starvation dramatically increased the sensitivity of human tumor xenografts to cisplatin as indicated not only by a significant growth delay, but also by the induction of complete remission in 60% of the animals bearing mesothelioma xenografts, and in 40% of the animals with lung carcinoma xenografts.ConclusionIn normal cells, serum starvation in vitro induces a cell cycle arrest and protects from CDDP induced toxicity. In contrast, proliferation of cancer cells is only moderately reduced by serum starvation whereas CDDP toxicity is enhanced. The combination of CDDP treatment with short term food starvation improved outcome in vivo. Therefore, starvation has the potential to enhance the therapeutic index of cisplatin-based therapy.

Blocking the epithelial-to-mesenchymal transition pathway abrogates resistance to anti-folate chemotherapy in lung cancer

Anticancer therapies currently used in the clinic often can neither eradicate the tumor nor prevent disease recurrence due to tumor resistance. In this study, we showed that chemoresistance to pemetrexed, a multi-target anti-folate (MTA) chemotherapeutic agent for non-small cell lung cancer (NSCLC), is associated with a stem cell-like phenotype characterized by an enriched stem cell gene signature, augmented aldehyde dehydrogenase activity and greater clonogenic potential. Mechanistically, chemoresistance to MTA requires activation of epithelial-to-mesenchymal transition (EMT) pathway in that an experimentally induced EMT per se promotes chemoresistance in NSCLC and inhibition of EMT signaling by kaempferol renders the otherwise chemoresistant cancer cells susceptible to MTA. Relevant to the clinical setting, human primary NSCLC cells with an elevated EMT signaling feature a significantly enhanced potential to resist MTA, whereas concomitant administration of kaempferol abrogates MTA chemoresistance, regardless of whether it is due to an intrinsic or induced activation of the EMT pathway. Collectively, our findings reveal that a bona fide activation of EMT pathway is required and sufficient for chemoresistance to MTA and that kaempferol potently regresses this chemotherapy refractory phenotype, highlighting the potential of EMT pathway inhibition to enhance chemotherapeutic response of lung cancer.

Amino acid sequence of the heavy chain of porcine plasmin. Comparison of the carbohydrate attachment sites with the human and bovine species

Abstract The amino acid sequence of the heavy chain of porcine plasmin (560 residues) was determined. Selected fragments were produced by cleavage with cyanogen bromide, elastase, Staphylococcus aureus V8 protease, trypsin or clostripain or with combinations thereof. The fragments were aligned with overlapping sequences. Sequence comparison with the human and bovine species indicated that the porcine molecule contains the same structural and functional domains as the two others. In particular, the characteristic arrangement of the disulfide bonds clearly indicates the five kringle structures. The heavy chain of porcine plasmin has a sequence homology of 84% with the bovine and of 78% with the human molecule. The average degree of identity among the kringles is over 80%. In the connecting strands between the kringles the identity decreases drastically (to 42% in the connecting strand between kringles 4 and 5) except for the strand between kringles 2 and 3 (over 80%). Porcine plasminogen contains two carbohydrate moieties. The N-glycosidic site at Asn289 is only partially glycosylated as in the human and bovine species and corresponds to Asn288 (human) and Asn289. (bovine), respectively. The O-glycosidic site is located at Thr249 in the connecting strand between kringles 2 and 3, whereas in the human and bovine molecule it occurs in the connecting strand between kringles 3 and 4, at Thr345 and Ser339, respectively.

Increased PD-L1 expression and IL-6 secretion characterize human lung tumor-derived perivascular-like cells that promote vascular leakage in a perfusable microvasculature model.

Pericytes represent important support cells surrounding microvessels found in solid organs. Emerging evidence points to their involvement in tumor progression and metastasis. Although reported to be present in the human lung, their specific presence and functional orientation within the tumor microenvironment in non-small cell lung cancer (NSCLC) has not yet been adequately studied. Using a multiparameter approach, we prospectively identified, sorted and expanded mesenchymal cells from human primary NSCLC samples based on co-expression of CD73 and CD90 while lacking hematopoietic and endothelial lineage markers (CD45, CD31, CD14 and Gly-A) and the epithelial marker EpCAM. Compared to their normal counterpart, tumor-derived Lineage-EpCAM-CD73+CD90+ cells showed enhanced expression of the immunosuppressive ligand PD-L1, a higher constitutive secretion of IL-6 and increased basal αSMA levels. In an in vitro model of 3D microvessels, both tumor-derived and matched normal Lineage-EpCAM-CD73+CD90+ cells supported the assembly of perfusable vessels. However, tumor-derived Lineage-EpCAM-CD73+CD90+ cells led to the formation of vessels with significantly increased permeability. Together, our data show that perivascular-like cells present in NSCLC retain functional abnormalities in vitro. Perivascular-like cells as an eventual target in NSCLC warrants further investigation.

Toll-like receptor 4 activation attenuates profibrotic response in control lung fibroblasts but not in fibroblasts from patients with IPF

Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease with a median survival of 3 yr. IPF deteriorates upon viral or bacterial lung infection although pulmonary infection (pneumonia) in healthy lungs rarely induces fibrosis. Bacterial lipopolysaccharide (LPS) activates Toll-like receptor 4 (TLR4), initiating proinflammatory pathways. As TLR4 has already been linked to hepatic fibrosis and scleroderma, we now investigated the role of TLR4 in IPF fibroblasts. Lung tissue sections from patients with IPF were analyzed for TLR4 expression. Isolated normal human lung fibroblasts (NL-FB) and IPF fibroblasts (IPF-FB) were exposed to LPS and transforming growth factor-β (TGF-β) before expression analysis of receptors, profibrotic mediators, and cytokines. TLR4 is expressed in fibroblast foci of IPF lungs as well as in primary NL-FB and IPF-FB. As a model for a gram-negative pneumonia in the nonfibrotic lung, NL-FB and IPF-FB were coexposed to LPS and TGF-β. Whereas NL-FB produced significantly less connective tissue growth factor upon costimulation compared with TGF-β stimulation alone, IPF-FB showed significantly increased profibrotic markers compared with control fibroblasts after costimulation. Although levels of antifibrotic prostaglandin E2 were elevated after costimulation, they were not responsible for this effect. However, significant downregulation of TGF-β receptor type 1 in control fibroblasts seems to contribute to the reduced profibrotic response in our in vitro model. Normal and IPF fibroblasts thus differ in their profibrotic response upon LPS-induced TLR4 stimulation.

Environmental changes in Northern Iceland since the Younger Dryas inferred from periglacial slope deposits

The slopes in Northern Iceland show the widespread occurrence of solifluction features, indicative of an active periglacial environment due to annual mean temperatures around 3°C at sea level and seasonal soil frost. In order to reconstruct periods with active and inactive solifluction in the past we excavated 18 solifluction lobes for analysing the sediment sequences. Dating of the sediments was realised mainly by tephrochronology and 14C. The oldest solifluction layer could be dated to the Younger Dryas (YD), just after the deglaciation of Northern Iceland. The early to mid Holocene up to the deposition of Hekla 3 Tephra (~3 ka BP) is characterized by the accumulation of loess and tephra layers, which show no signs of secondary remobilisation or erosion, indicating stable slopes during the mid-Holocene climatic optimum (MCO). After the deposition of Hekla 3 Tephra and especially during the ‘Little Ice Age’ (LIA), solifluction reappeared in the profiles as a probable consequence of Neoglacial cooling. The results fit well with other proxies from Iceland (glacier variations, pollen), from North Atlantic marine cores and from Greenland ice records.

18P Epithelial-to-mesenchymal transition (EMT) is required for resistance to anti-folate chemotherapy in lung cancer.

cells. Prognoscan assessment identified decrease SASH1 mRNA expression lead to a prognostic reduction in patient survival. The depletion of SASH1 in lung cells resulted in a significant increase in cellular proliferation in cancer lung cells. Connectivity mapping predicted the drug Chloropyramine would lead to an increase in SASH1 expression. We demonstrated that Chloropyramine upregulates SASH1 in malignant cell lines. In keeping with this we have demonstrated that Chloropyramine inhibited lung cancer proliferation in vitro. These novel observations support the tumour suppressive role of SASH1 in lung tumourgenesis. Further work is ongoing to understand the function of SASH1 in lung cancer growth. Conclusions: The upregulation of SASH1, either by chemical agents or gene therapy, is a potential novel approach to the management of lung cancer and other solid tumours. Legal entity responsible for the study: Queensland University of Technology Funding: Queensland Health Disclosure: All authors have declared no conflicts of interest.