The effects of thinning on carbon and nutrient fluxes input into forest floor via litterfall in black pine afforestation sites.

As a component of the biogeochemical cycle, litterfall contributes carbon and nutrients to forest ecosystems by transferring organic material to mineral soil. Litterfall therefore serves as an important indicator for soil fertility and ecosystem health. This study aimed to determine the impact of different levels of thinning (light, moderate, and heavy) on litterfall quantity (needles, branches, bark, cones, and miscellaneous parts) and on the amount of carbon and nutrients entering the ecosystem in black pine afforestation areas. Three levels of low thinning, namely light, moderate, and heavy thinning (15%, 25%, and 35% of breast height area, respectively), were applied as treatments. Additionally, a control plot was included in the experiment. Litterfall samples were collected four times per year (once per season) from 12 treatment plots for three years. In the laboratory, dry weight measurements and analyses of carbon and macro-micro nutrient elements (N, P, K, Ca, Mg, S, Na, Fe, Cu, Zn, and Mn) were performed on litterfall samples taken from the field. Differences between treatments in terms of litterfall and the amount of carbon and nutrient elements entering the ecosystem were evaluated through variance analysis and the Duncan test. According to the findings, the quantity of litterfall input into the forest floor was highest in the control treatment, at 6,543 kg ha-1 year-1 and lowest in the heavy treatment, at 4,378 kg ha-1 year-1, showing a significant variation in litterfall quantity. The input of C to the soil ranged between 2,233 kg ha-1 year-1 and 3,347 kg ha-1 year-1 depending on thinning treatment. Although thinning treatment reduced C input to the soil, there was no significant difference among treatments. This also applied to nutrient elements such as N, P, K, Mg, and S. Needles constituted the majority of litterfall components (60%) and had the highest C density among all components, at 51.2%. The weighted carbon ratio for litterfall was calculated at 50.8%. Considering carbon-focused planning, performing moderate thinning interventions in the study area or similar pine-afforested areas may be a suitable option for maintaining the sustainability and health of the forest.

Nutrient pools and loss due to the removal of harvesting residue in cedar plantations (a case study from Türkiye).

Determining the nutrient stocks and revealing the extent to which these stocks will be affected by the interventions in forest ecosystems are crucial for sustainable forest management. This study aimed to determine the nutrient stock of cedar (Cedrus libani A. Rich.) plantations at different stands with various diameter classes and estimate the nutrient stock to be removed from the forest due to harvesting. Soil and plant samples were collected from 40 plots in Eskisehir and Afyonkarahisar provinces in Turkey. The variation in the nutrient concentrations and stocks of different components of the ecosystem were evaluated by analysis of variance and the decrease via harvesting by regression analysis. The results showed that the concentrations of N, P, K, Mg, S, Fe, Zn, and Mn were highest in the needles, Ca in the bark, Cu in the needles, dead branches, and root. In the large-diameter forest (LDF), dbh=20.0-35.9 cm, the highest P stock was found in the trees, Fe stock in the forest floor, and S stock in the soil and trees. As a result, the forest floor should be protected as it is the crucial component of both the nutrient cycle and the Fe stock in the ecosystem. In LDF, 28.4-37.3% of the nutrient stored in the trees would be removed from the ecosystem in the case of moderate thinning with whole-tree harvesting, while only 5.9-14.1% of the nutrient stock in the case of stem-only harvesting. For these reasons, leaving logging residues after harvesting in the forest would minimize nutrient loss. The study results showed that improved nutrient management in a forest ecosystem will make a significant contribution to the sustainability of forests.

Array-based comparative gene expression analysis of tumor cells with increased apoptosis resistance after hypoxic selection.

Tumor hypoxia is an adverse prognostic factor. In a recent study, we could demonstrate that cyclic hypoxia selects for hypoxia-tolerant tumor cells, which are cross-resistant to other stimuli of mitochondrial death pathways. In contrast, sensitivity of the cells to death-receptor ligands was mainly not affected. The aim of the present study was to further elucidate cellular changes induced by cyclic hypoxia and to identify alterations in gene expression pattern upon hypoxic selection by means of DNA-microarray analysis. Our data reveal that cyclic hypoxia resulted in the selection of cells with resistance to doxorubicine and radiation. Furthermore, hypoxic selection was accompanied by constitutive changes of the gene expression pattern with downregulation of 156 and upregulation of 82 genes. Most of the differentially regulated genes were involved in cellular responses to hypoxia and reoxygenation. While many of the genes that were downregulated upon hypoxic selection represent genes that are usually upregulated by acute hypoxia, the genes that were upregulated represent genes that are involved in stress resistance and anti-apoptotic signalling. Most importantly, hypoxic selection was not associated with changes of single apoptosis relevant genes, but with alterations in gene expression levels of a wide variety of genes indicating a more complex adaptation process.

Loss of p21 disrupts p14 ARF-induced G1 cell cycle arrest but augments p14 ARF-induced apoptosis in human carcinoma cells.

The human INK4a locus encodes two structurally unrelated tumor suppressor proteins, p16 INK4a and p14 ARF (p19 ARF in the mouse), which are frequently inactivated in human cancer. Both the proapoptotic and cell cycle-regulatory functions of p14 ARF were initially proposed to be strictly dependent on a functional p53/mdm-2 tumor suppressor pathway. However, a number of recent reports have implicated p53-independent mechanisms in the regulation of cell cycle arrest and apoptosis induction by p14 ARF. Here, we show that the G1 cell cycle arrest induced by p14 ARF entirely depends on both p53 and p21 in human HCT116 and DU145 carcinoma cells. In contrast, neither loss of p53 nor p21 impaired apoptosis induction by p14 ARF as evidenced by nuclear DNA fragmentation, phosphatidyl serine exposure, and caspase activation, which included caspase-3/7- and caspase-9-like activities. However, lack of functional p21 resulted in the accumulation of cells in G2/M phase of the cell cycle and markedly enhanced p14 ARF-induced apoptosis that was, nevertheless, efficiently inhibited by the cell permeable broad-spectrum caspase inhibitor zVAD-fmk (valyl-alanyl-aspartyl-(O)-methyl)-fluoromethylketone). Thus, loss of cell cycle restriction point control in the absence of p21 may interfere with p14 ARF-induced apoptosis. Finally, these data indicate that the signaling events required for G1 cell cycle arrest and apoptosis induction by p14 ARF dissociate upstream of p53.

Molecular ordering of hypoxia-induced apoptosis: critical involvement of the mitochondrial death pathway in a FADD/caspase-8 independent manner.

Dys-regulated growth and improper angiogenesis commonly lead to areas of hypoxia in human tumors. Hypoxia is known to be associated with a worse outcome since a lack of oxygen interferes with the efficacy of chemotherapy or radiotherapy. In parallel, hypoxia-induced apoptosis may also impose a selection pressure favoring growth of more resistant tumor cells. However, the mechanisms of hypoxia-induced apoptosis and the relative contribution of intrinsic and extrinsic apoptotic pathways are not understood. Therefore, Jurkat cell lines with defined defects in the extrinsic or intrinsic signaling cascades were used to evaluate the role of either pathway for induction of apoptosis under hypoxic conditions. Jurkat cells were incubated in hypoxia and the rate of apoptosis induction was determined by Western blotting, fluorescence microscopy and flow cytometry. Hypoxia-induced apoptosis was not affected by lack of caspase-8 or FADD, whereas overexpression of Bcl-2 or expression of dominant-negative caspase-9 mutant rendered the cells resistant to hypoxia-induced apoptosis. These results suggest that hypoxia-induced apoptosis mainly relies on intrinsic, mitochondrial pathways, whereas extrinsic pathways have no significant implications in this process. Thus, in human tumors, hypoxia will mainly lead to the selection of hypoxia-resistant cells with defects in mitochondrial apoptosis signaling pathways.

Multidomain Bcl-2 homolog Bax but not Bak mediates synergistic induction of apoptosis by TRAIL and 5-FU through the mitochondrial apoptosis pathway.

The death ligand TRAIL synergizes with DNA-damaging therapies such as chemotherapeutic drugs or ionizing irradiation. Here, we show that the synergism of TRAIL and 5-fluorouracil (5-FU) and cross-sensitization between TRAIL and 5-FU for induction of apoptosis, entirely depend on Bax proficiency in human DU145 and HCT116 carcinoma cells. DU145 prostate carcinoma cells that have lost Bax protein expression due to mutation fail to release cytochrome c and to activate caspase-3 and -9 when exposed to TRAIL and 5-FU. In contrast, TRAIL sensitized for 5-FU-induced apoptosis and vice versa upon reconstitution of Bax expression. Isobolographic analyses of ED50 doses for 5-FU at increasing TRAIL concentrations showed a clear synergism of TRAIL and 5-FU in Bax-expressing cells. In contrast, the effect was merely additive in DU145 cells lacking Bax. Notably, both DU145 and HCT116 Bax-deficient cells still express Bak. This indicates that Bak is not sufficient to mediate cross-sensitization and synergism between 5-FU and TRAIL. Stable overexpression of Bak in DU145 sensitized for epirubicin-induced apoptosis but failed to confer synergy between TRAIL and 5-FU. Moreover, we show by the use of EGFP-tagged Bax and Bak that TRAIL and 5-FU synergistically trigger oligomerization and clustering of Bax but not Bak. These data clearly establish distinct roles for Bax and Bak in linking the TRAIL death receptor pathway to the mitochondrial apoptosis signaling cascade and delineate a higher degree of specificity in signaling for cell death by multidomain Bcl-2 homologs.

Adenovirus-mediated overexpression of p14(ARF) induces p53 and Bax-independent apoptosis.

The human INK4a gene locus encodes two structurally unrelated tumor suppressor proteins, p16(INK4a) and p14(ARF), which are frequently inactivated in human cancer. Whereas p16(INK4a) acts through engagement of the Rb-cdk4/6-cyclin D pathway, both the pro-apoptotic and cell cycle-regulatory functions of p14(ARF) were shown to be primarily dependent on the presence of functional p53. Recent reports have also implicated p14(ARF) in p53-independent mechanisms of cell cycle regulation and apoptosis induction, respectively. To further explore the pro-apoptotic function of p14(ARF) in relation to functional cellular p53, we constructed a replication-deficient adenoviral vector for overexpression of p14(ARF) (Ad-p14(ARF)). As expected, Ad-p14(ARF) efficiently induced apoptosis in p53/Rb wild-type U-2OS osteosarcoma cells at low multiplicities of infection. Interestingly, Ad-p14(ARF) also induced apoptosis in both p53-deleted SAOS-2 osteosarcoma cells and HCT116 colon cancer cells with a bi-allelic knock-out of p53 (HCT116-p53(-/-)). Similarly, adenovirus-mediated overexpression of p14(ARF) induced apoptosis in p53/Bax-mutated DU145 prostate cancer cells as well as in HCT116 cells devoid of functional Bax (HCT116-Bax(-/-)). Restoration of Bax expression by retroviral gene transfer in DU145 cells did not further enhance p14(ARF)-triggered cell death. Infection with Ad-p14(ARF) induced activation of mitochondrial permeability shift transition, caspase activation and apoptotic DNA fragmentation irrespective of the presence or absence of either Bax or functional cellular p53. Nevertheless, overexpression of the anti-apoptotic Bcl-2 homolog Bcl-x(L) markedly inhibited p14(ARF)-induced apoptosis. This may indicate that p14(ARF) triggers a so far unknown activator of mitochondrial apoptosis which can be inhibited by Bcl-2 but which acts either independently or downstream of Bax. Taken together, this report demonstrates the participation of signaling pathways apart from the p53/Mdm-2 rheostat and Bax in p14(ARF)-mediated apoptosis.

Cyclic exposure to hypoxia and reoxygenation selects for tumor cells with defects in mitochondrial apoptotic pathways.

The negative influence of hypoxia on the outcome of malignant tumors may be caused by direct oxygen effects, and potentially, the selection of resistant tumor cells under repetitive hypoxia. To evaluate whether cyclic hypoxia selects for resistant cells and to analyze the underlying mechanisms, the influence of cyclic hypoxia on intracellular death pathways was determined in tumor cells. It could be demonstrated that cyclic hypoxia selects for cells with increased resistance against hypoxia-induced apoptosis. These cells exhibited a cross-resistance against paradigmatic triggers of mitochondrial apoptotic pathways (ionizing radiation/etoposide). In contrast, TRAIL-receptor mediated apoptosis remained unaffected. Thus, cyclic hypoxia selects for cells with defects of the mitochondrial rather than receptor-mediated pathways. Selection of p53-defective cells has been described as a consequence of cyclic hypoxia; therefore, we evaluated the impact of hypoxic selection on activation of p21 and downstream mediators of p53-dependent apoptosis. p53 function and protein levels of key mediators of mitochondrial apoptosis remained unaffected by hypoxic selection. However, radiation-induced conformational changes of Bax were reduced after cyclic hypoxia. In summary, it could be demonstrated that hypoxic stress confers a selection pressure on mitochondrial apoptotic pathways and, consecutively, to an increased resistance toward mitochondrial death triggers.

Guardians of cell death: the Bcl-2 family proteins.

Apoptosis is mediated through at least three major pathways that are regulated by (i) the death receptors, (ii) the mitochondria and (iii) the ER (endoplasmic reticulum). In most cells, these pathways are controlled by the Bcl-2 family of proteins that can be divided into anti-apoptotic and pro-apoptotic members. Although the overall amino acid sequence homology between the family members is relatively low, they contain highly conserved domains, referred to as BH (Bcl-2 homology) domains (BH1-4), that are essential for homo- and hetero-complex formation, as well as for their cell-death-inducing capacity. Structural and functional analyses revealed that the pro-apoptotic homologues can be subdivided into the Bax subfamily and the growing BH3-only subfamily. Recent data indicate that BH3-only proteins act as mediators that link various upstream signals, including death receptors and DNA damage signalling, to the mitochondrial and the ER pathway. This review discusses recent structural and functional insights into how these subfamilies promote or inhibit cell-death signals, and how these properties may be utilized for development of apoptosis-promoting small molecules, e.g. in cancer therapy.

Disruption of cell death signaling in cancer: impact on disease prognosis and response to therapy.

Disruption of cell cycle and apoptosis signaling pathways are key events during tumorigenesis, tumor progression and development of resistance against anticancer therapies. Thus, the analysis of functional alterations within these signaling cascades is of utmost importance for the understanding of resistance mechanisms, clinical outcome and risk-adapted treatment strategies. Key signaling pathways involved in the treatment resistance include the p53/p14ARF signaling complex and the mitochondrial apoptosis machinery. Apart from the direct genetic events, these signaling cascades are subject to epigenetic modulations implied by the tumor microenvironment.

p14ARF induces G2 cell cycle arrest in p53- and p21-deficient cells by down-regulating p34cdc2 kinase activity.

The human INK4a gene locus encodes two structurally unrelated tumor suppressor proteins, p16(INK4a) and p14(ARF). Although primarily proposed to require a functional p53.Mdm-2 signaling axis, recently p14(ARF) has been implicated in p53-independent cell cycle regulation. Here we show that p14(ARF) preferentially induces a G(2) arrest in tumor cells lacking functional p53 and/or p21. Expression of p14(ARF) impaired mitotic entry and enforced a primarily cytoplasmic localization of p34(cdc2) that was associated with a decrease in p34(cdc2) kinase activity and reduced p34(cdc2) protein expression. A direct physical interaction between p14(ARF) and p34(cdc2) was, nevertheless, ruled out by lack of co-immunoprecipitation. The p14(ARF)-induced depletion of p34(cdc2) was associated with impaired cdc25C phosphatase expression and a prominent shift to inhibitory Tyr-15-phosphorylation in G(2)-arrested cells lacking either p53, p21, or both. Finally, reconstitution of p34(cdc2) using a constitutively active, phosphorylation-deficient p34(cdc2AF) mutant alleviated this p14(ARF)-induced G(2) arrest, thereby allowing cell cycle progression. Taken together, these data indicate that p14(ARF) arrests cells lacking functional p53/p21 in the G(2) phase of the cell cycle by targeting p34(cdc2) kinase. This may represent an important fail-safe mechanism by which p14(ARF) protects p53/p21-deficient cells from unrestrained proliferation.

Multigene analysis of Rb pathway and apoptosis control in esophageal squamous cell carcinoma identifies patients with good prognosis.

Deregulation of cell-cycle G(1)-restriction point control by disruption of Rb-pathway components is a frequent event in cancer. In concert with the inactivation of cell death pathways, such events not only contribute to tumor development but also determine the intrinsic and acquired resistance to cancer therapy and, ultimately, disease prognosis. We previously observed that the cyclin-dependent kinase inhibitor p16(INK4a) and the proapoptotic Bcl-2 homolog Bax are positive prognostic factors and identify patients with good prognosis in esophageal squamous cell carcinoma (SCC). In the present study, we therefore extend our analysis to additional genes controlling the G(1) restriction point and apoptosis, respectively. This retrospective analysis was performed in a cohort of 53 patients undergoing surgery for esophageal SCC with curative intent, i.e., R0 resection. Protein expression profiles of cyclin D1, p16(INK4a), Rb, p21(CIP/WAF-1), p53, Bax and Bcl-2 were analyzed by immunohistochemistry and compared to p53 mutational status, as determined by SSCP-PCR of exons 5-8. Loss of p16(INK4a), Rb, p21(CIP/WAF-1) or Bax and overexpression of cyclin D1 were associated individually with shorter overall survival, while Bcl-2 expression and p53 mutation were not of prognostic relevance. The longest survival was observed in a subgroup of patients whose tumors bore a combination of favorite genotypes, i.e., low cyclin D1 and high Rb, p21(CIP/WAF-1), p16(INK4a) and Bax protein expression. These results show that multigene analyses based on limited sets of functionally linked genes reliably identify patients with good vs. poor prognosis.