Resistance of hypoxic cells to ionizing radiation is influenced by homologous recombination status

PURPOSE: To determine the role of DNA repair in hypoxic radioresistance. METHODS AND MATERIALS: Chinese hamster cell lines with mutations in homologous recombination (XRCC2, XRCC3, BRAC2, RAD51C) or nonhomologous end-joining (DNA-PKcs) genes were irradiated under normoxic (20% oxygen) and hypoxic (<0.1% oxygen) conditions, and the oxygen enhancement ratio (OER) was calculated. In addition, Fanconi anemia fibroblasts (complementation groups C and G) were compared with fibroblasts from nonsyndrome patients. RAD51 foci were studied using immunofluorescence. RESULTS: All hamster cell lines deficient in homologous recombination showed a decrease in OER (1.5-2.0 vs. 2.6-3.0 for wild-types). In contrast, the OER for the DNA-PKcs-deficient line was comparable to wild-type controls. The two Fanconi anemia cell strains also showed a significant reduction in OER. The OER for RAD51 foci formation at late times after irradiation was considerably lower than that for survival in wild-type cells. CONCLUSION: Homologous recombination plays an important role in determining hypoxic cell radiosensitivity. Lower OERs have also been reported in cells deficient in XPF and ERCC1, which, similar to homologous recombination genes, are known to play a role in cross-link repair. Because Fanconi anemia cells are also sensitive to cross-linking agents, this strengthens the notion that the capacity to repair cross-links determines hypoxic radiosensitivity.     

Deregulation of cap-dependent mRNA translation increases tumour radiosensitivity through reduction of the hypoxic fraction

Background and purpose

Tumour hypoxia is an important limiting factor in the successful treatment of cancer. Adaptation to hypoxia includes inhibition of mTOR, causing scavenging of eukaryotic initiation factor 4E (eIF4E), the rate-limiting factor for cap-dependent translation. The aim of this study was to determine the effect of preventing mTOR-dependent translation inhibition on hypoxic cell survival and tumour sensitivity towards irradiation.

Material and methods

The effect of eIF4E-overexpression on cell proliferation, hypoxia-tolerance, and radiation sensitivity was assessed using isogenic, inducible U373 and HCT116 cells.

Results

We found that eIF4E-overexpression significantly enhanced proliferation of cells under normal conditions, but not during hypoxia, caused by increased cell death during hypoxia. Furthermore, eIF4E-overexpression stimulated overall rates of tumour growth, but resulted in selective loss of hypoxic cells in established tumours and increased levels of necrosis. This markedly increased overall tumour sensitivity to irradiation.

Conclusions

Our results demonstrate that hypoxia induced inhibition of translational control through regulation of eIF4E is an important mediator of hypoxia tolerance and radioresistance of tumours. These data also demonstrate that deregulation of metabolic pathways such as mTOR can influence the proliferation and survival of tumour cells experiencing metabolic stress in opposite ways of nutrient replete cells.     

Zebularine-induced apoptosis in Calu-6 lung cancer cells is influenced by ROS and GSH level changes

Zebularine (Zeb) is a DNA methyltransferase (DNMT) inhibitor that has various biological properties including anti-cancer effect. In the present study, we evaluated the effects of Zeb on the growth and death of Calu-6 lung cancer cells in relation to reactive oxygen species (ROS) and glutathione (GSH) levels. Zeb inhibited the growth of Calu-6 cells with an IC₅₀ of approximately 150 μM at 72 h in a dose-dependent manner. Zeb induced an S phase arrest of the cell cycle and apoptosis in Calu-6 cells. Pan-caspase inhibitor (Z-VAD) and caspase-8 inhibitor (Z-IETD) significantly rescued some cells from Zeb-induced Calu-6 cell death. In relation to ROS and GSH levels, O₂ ^?? level was significantly increased in Zeb-treated Calu-6 cells and caspase inhibitors reduced O₂ ^?? level in these cells. Zeb induced GSH depletion in HeLa cells, which was attenuated by caspase inhibitors. L-buthionine sulfoximine (BSO), a GSH synthesis inhibitor, intensified the apoptotic cell death, ROS level, and GSH depletion in Zeb-treated Calu-6 cells. In addition, BSO increased Bax protein and decreased Bcl-2 protein in Zeb-treated Calu-6 cells. In conclusion, Zeb inhibited the growth of Calu-6 lung cancer cells via cell cycle arrest and caspase-dependent apoptosis and its cell death was influenced by ROS and GSH level changes.     

Phosphorylation of eIF2alpha is required for mRNA translation inhibition and survival during moderate hypoxia.

BACKGROUND AND PURPOSE: Human tumors are characterized by temporal fluctuations in oxygen tension. The biological pathways that respond to the dynamic tumor microenvironment represent potential molecular targets for cancer therapy. Anoxic conditions result in eIF2alpha dependent inhibition of overall mRNA translation, differential gene expression, hypoxia tolerance and tumor growth. The signaling pathway which governs eIF2alpha phosphorylation has therefore emerged as a potential molecular target. In this study, we investigated the role of eIF2alpha in regulating mRNA translation and hypoxia tolerance during moderate hypoxia. Since other molecular pathways that regulate protein synthesis are frequently mutated in cancer, we also assessed mRNA translation in a panel of cell lines from different origins. MATERIALS AND METHODS: Immortalized human fibroblast, transformed mouse embryo fibroblasts (MEFs) and cells from six cancer cell lines were exposed to 0.2% or 0.0% oxygen. We assayed global mRNA translation efficiency by polysome analysis, as well as proliferation and clonogenic survival. The role of eIF2alpha was assessed in MEFs harboring a homozygous inactivating mutation (S51A) as well as in U373-MG cells overexpressing GADD34 (C-term) under a tetracycline-dependent promoter. The involvement of eIF4E regulation was investigated in HeLa cells stably expressing a short hairpin RNA (shRNA) targeting 4E-BP1. RESULTS: All cells investigated inhibited mRNA translation severely in response to anoxia and modestly in response to hypoxia. Two independent genetic cell models demonstrated that inhibition of mRNA translation in response to moderate hypoxia was dependent on eIF2alpha phosphorylation. Disruption of eIF2alpha phosphorylation caused sensitivity to hypoxia and anoxia. CONCLUSIONS: Disruption of eIF2alpha phosphorylation is a potential target for hypoxia-directed molecular cancer therapy.     

Regulation of gene expression by tumor promoters. III. Complementation of the developmental program in myeloid leukemic cells by regulating mRNA production and mRNA translation

Regulation of gene expression has been analysed in different clones of mouse myeloid leukemic cells treated with the tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate (TPA), the macrophage- and granulocyte-inducing protein MGI, and combined treatment with TPA and MGI. Two-dimensional gel electrophoresis was used to measure changes in the rate of synthesis of specific proteins and in the amount of corresponding translatable mRNAs assayed in the reticulocyte cell-free translation system. TPA induced different subsets of differentiation-associated protein changes in the different clones and the degree of response to TPA was not necessarily related to the degree of response to MGI. It is shown that TPA can induce protein changes either by inducing the synthesis of new mRNA, by increasing or decreasing the amount of pre-existing mRNA, or by modulating the translation of a constant amount of mRNA. Combined treatment with TPA and MGI resulted in an enhancement of protein changes induced by MGI or TPA alone and induced differentiation-associated protein changes not induced by either compound separately in differentiation-defective clones. This complementation of gene expression appeared to be due to each compound inducing functions not induced by the other, so that the combined treatment resulted in new gene expression. Complementation also occurred at the level both of mRNA production and of mRNA translation. It is suggested that the ability of TPA to regulate gene expression at the level of mRNA production and mRNA translation and to complement changes in gene expression induced by other compounds such as MGI are important functions for its role as a tumor promoter.     

Hypoxia induced expression of endogenous markers in vitro is highly influenced by pH.

BACKGROUND: Genes such as carbonic anhydrase IX (Ca9), glucose transporter 1 (Glut1), lactate dehydrogenase A (LDH-A), osteopontin (OPN) and lysyl oxidase (LOX) have been suggested as hypoxic markers, but inconsistent results suggest that factors other than oxygen influence their expression. The current study is a detailed investigation using a range of pH values from 6.3 to 7.5 in two human cell lines to establish the pH dependency of hypoxia induced gene expression. METHODS: Human tumour cell lines (uterine cervix squamous cell carcinoma (SiHa) and pharyngeal squamous cell carcinoma [FaDu(DD)]) were used. Hypoxia was induced by gassing cells in airtight chambers with various oxygen concentrations (21%, 1%, 0.1%, 0.01% and 0%) for up to 24h. The media were titrated to a range of pH values (7.5, 7.0, 6.7, 6.5 and 6.3). Gene expression was determined by real-time PCR. RESULTS: In both SiHa and FaDu(DD) cells Ca9 and LOX reached the highest level of expression at 1% oxygen. In FaDu(DD) cells, a pH of 6.5 had a medium suppression effect on the hypoxia induced expression of Ca9. pH 6.3 resulted in severe suppression of expression for Ca9 and LOX in both SiHa and FaDu(DD). Glut1 and LDH-A had a similar expression pattern to each other, with a maximum expression at 0.01% oxygen, in both cell lines. For these genes pH 6.5 and 6.3 changed the expression pattern in SiHa cells. OPN was up regulated at low oxygen in SiHa cells, but was not induced by hypoxia in FaDu(DD) cells. CONCLUSION: As tumour hypoxia occurs in a deprived microenvironment, other environmental factors, for example low pH, might interact with the effect of low oxygen concentration on gene expression. This study shows that pH in two cell lines has a profound influence on the oxygen dependent induction of certain endogenous hypoxic markers.     

The myelotoxicity of carboplatin is influenced by the time of its administration

Previous studies have demonstrated that there are circadian rhythms in susceptibility to a range of commonly used cytotoxic drugs. In this study we have compared the pharmacokinetics and myelotoxicity of carboplatin administered at 18.00 and 06.00 in random order in patients with advanced ovarian carcinoma. Carboplatin treatment at 06.00 is associated with significantly greater thrombocytopenia than at 18.00 (platelet nadir 95,000 versus 180,000, p less than 0.05). There was no pharmacokinetic difference in the patients' handling of ultrafilterable platinum therefore it is possible that there is an intrinsic rhythm of susceptibility of bone marrow to carboplatin.     

Signaling control of mRNA translation in cancer pathogenesis

The regulation of translation and the control of ribosome biogenesis are essential cellular processes whose impact on cell growth and proliferation is manifested at a number of specific levels. Disruption in one or more of the steps that control protein biosynthesis has been associated with alterations in the regulation of cell growth and cell cycle progression. Consistent with this, tumor suppressors and proto-oncogenes have been found to act on these functions and may therefore regulate malignant progression by affecting the protein synthetic machinery. Although many studies have correlated deregulation of protein biosynthesis with cancer, it remains to be established whether this process is necessary and/or sufficient for neoplastic transformation and metastasis.     

Metastatic pattern in colorectal cancer is strongly influenced by histological subtype

BackgroundClinical studies regarding colorectal cancer (CRC) have suggested differences in metastatic patterns between mucinous adenocarcinoma (MC), signet-ring cell carcinoma (SRCC) and the more common adenocarcinoma (AC). The current study systematically evaluates metastatic patterns of different histological subtypes in CRC patients and analyzes metastatic disease upon primary tumor localization.Patients and methodsA nationwide retrospective review of pathological records of 5817 patients diagnosed with CRC who underwent an autopsy between 1991 and 2010 was performed. Patients were selected from the Dutch pathology registry (PALGA). To substantiate clinical relevance, metastatic patterns were compared with the prospective randomized multicenter Total Mesorectal Excision (TME) trial, which investigated efficacy of preoperative radiotherapy in rectal cancer patients.ResultsIn the autopsy study, 1675 patients had metastatic disease. MC and SRCC patients more frequently had metastatic disease (33.9% and 61.2% versus 27.6%; P < 0.0001) and had metastases at multiple sites more often compared with AC patients (58.6% and 70.7% versus 49.9%; P = 0.001). AC predominantly metastasized to the liver, and MC and SRCC more frequently had peritoneal metastases. Metastatic patterns were also related to the primary tumor site, with a high rate of abdominal metastases in colon cancer patients, whereas rectal cancer patients more often had metastases at extra-abdominal sites. Results from the TME trial confirmed findings in rectal cancer patients from the autopsy study.ConclusionThere are profound differences in metastatic patterns between histological subtypes and the localization of the primary tumor in CRC. Findings from this study should encourage to take these factors into account for follow-up strategies and future studies.     

Preclinical and post-treatment changes in the HCC-associated serum proteome

SELDI-based proteomic profiling of body fluids is currently in widespread use for cancer biomarker discovery. We have successfully used this technology for the diagnosis of hepatocellular carcinoma (HCC) in hepatitis C patients and now report its application to serial serum samples from 37 hepatitis C patients before development of HCC, with HCC and following radiofrequency ablation of the tumour. As with alpha-fetoprotein, an accepted biomarker for HCC, we hypothesised that HCC-associated proteomic features would 'return to normal' following successful treatment and the primary aim of our study was to test this hypothesis. Several SELDI peaks that changed significantly during HCC development were detected but they did not reverse following treatment. These data may be interpreted to suggest that the characteristic SELDI profile is not linearly related to tumour burden but may result from the progression of underlying liver disease or from the emergence of precancerous lesions. beta2-Microglobulin, a protein previously reported to be markedly elevated in patients with HCV related HCC, was also the most significantly HCC associated proteomic feature (m/z 11720) in this study.     

The anti-tumor effect of recombinant interferon alpha or gamma is influenced by tumor location

The in vivo anti-tumor effects of a recombinant human hybrid interferon alpha, rHuIFN-alpha A/D, and recombinant murine interferon gamma (rMuIFN-gamma) were evaluated against experimental hepatic metastases and s.c. tumor growth of the murine reticulum cell sarcoma M5076. The 2 interferons were equally active in preventing experimental hepatic metastases. However, the interferons differed in their relative ability to influence the growth of the same tumor when treatment was initiated following injection of tumor cells. Greater efficacy was obtained in the treatment of metastatic foci in the liver with rHuIFN-alpha A/D, while rMuIFN-gamma was more active in the therapy of an s.c. growing M5076 tumor. These results demonstrate that the same tumor growing at different sites can have different relative sensitivities to IFN-alpha and IFN-gamma.     

Malignant transformation in a defined genetic background: proteome changes displayed by 2D-PAGE

Background  

Cancer arises from normal cells through the stepwise accumulation of genetic alterations. Cancer development can be studied by direct genetic manipulation within experimental models of tumorigenesis. Thereby, confusion by the genetic heterogeneity of patients can be circumvented. Moreover, identification of the critical changes that convert a pre-malignant cell into a metastatic, therapy resistant tumor cell, however, is one necessary step to develop effective and selective anti-cancer drugs. Thus, for the current study a cell culture model for malignant transformation was used: Primary human fibroblasts of the BJ strain were sequentially transduced with retroviral vectors encoding the genes for hTERT (cell line BJ-T), simian virus 40 early region (SV40 ER, cell line BJ-TE) and H-Ras V12 (cell line BJ-TER).     

Megakaryocytic differentiation of human progenitor cells is negatively influenced by direct contact with stroma.

The recently defined ligand for the Mpl receptor, thrombopoietin (TPO), has been found to be the principal regulatory cytokine of megakaryocytopoiesis. Furthermore, it has been hypothesized that direct interaction between stroma or stroma-derived extracellular matrix (ECM) and human progenitor cells (HPC) may modulate megakaryocytopoiesis. For that purpose, we studied megakaryocytic development of HPC, obtained from patients with hematological malignancies in complete remission or solid tumors without bone marrow involvement, under the influence of megakaryocyte growth and development factor (MGDF, a pegylated, truncated molecule related to the endogenous Mpl ligand). The megakaryocytic development of HPC cultured in contact with a stromal layer (contact cultures) was compared with cultures in which HPC were grown separated from a stromal layer by a microporous membrane (non-contact cultures). A significantly lower number of megakaryocytes (CD41- and CD61-positive cells) was obtained from contact cultures compared to non-contact cultures. Furthermore, the expression of CD42b was higher in non-contact cultures, as compared to contact cultures, indicating an increase in megakaryocyte maturation in non-contact cultures. In contrast, no difference in clonogenic capacity was observed (CFU-GM, BFU-E, CFU-Mk). The possibility that direct contact between HPC and stroma induces the production of a soluble inhibitory cytokine as an explanation for the diminished megakaryocytic development in contact cultures, was excluded by performing transwell experiments in which HPC were cultured in direct contact and in a transwell simultaneously. The percentage of megakaryocytes raised from HPC present in the transwell did not decrease, irrespective of the presence of HPC simultaneous below the transwell in direct contact with stroma. It is concluded that both megakaryocytic development out of HPC and megakaryocytic differentiation is reduced in contact cultures, as compared to non-contact cultures. This is due to modulation by adhesive interactions with stroma, stroma-derived ECM or cytokines bound to the membrane of stromal cells, rather than resulting from the production of diffusible cytokines by stromal cells.