Author Correction: Non-professional phagocytosis: a general feature of normal tissue cells.

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Non-professional phagocytosis: a general feature of normal tissue cells.

Non-professional phagocytosis by cancer cells has been described for decades. Recently, non-professional phagocytosis by normal tissue cells has been reported, which prompted us to take a closer look at this phenomenon. Non-professional phagocytosis was studied by staining cultured cells with live-cell staining dyes or by staining paraffin-embedded tissues by immunohistochemistry. Here, we report that each of 21 normal tissue cell lines from seven different organs was capable of phagocytosis, including ex vivo cell cultures examined before the 3rd passage as well as the primary and virus-transformed cell lines. We extended our analysis to an in vivo setting, and we found the occurrence of non-professional phagocytosis in healthy skin biopsies immediately after resection. Using dystrophin immunohistochemistry for membrane staining, human post-infarction myocardial tissue was assessed. We found prominent signs of non-professional phagocytosis at the transition zone of healthy and infarcted myocardia. Taken together, our findings suggest that non-professional phagocytosis is a general feature of normal tissue cells.

Cytotoxic effect of Efavirenz in BxPC-3 pancreatic cancer cells is based on oxidative stress and is synergistic with ionizing radiation.

The non-nucleoside reverse transcriptase inhibitor (NNRTI) Efavirenz is frequently used in human immunodeficiency virus treatment, but also efficient against cancer in mouse models. Its radiosensitizing effect makes it a promising drug for combination with radiotherapy. The efficacy of Efavirenz combined with irradiation was assessed with immunostaining of DNA-damage markers and colony formation assays in BxPC-3 pancreatic cancer cells. Gene expression and protein phosphorylation of the Efavirenz-sensitive BxPC-3 cells was compared to the resistant primary fibroblasts SBL-5. Oxidative stress, mitochondrial damage and cell death were studied with live-cell microscopy and flow cytometry. Combined Efavirenz and radiation significantly increased the number of γH2AX and phospho-ataxia telangiectasia mutated foci. Efavirenz and ionizing radiation had a synergistic effect using the clonogenic survival assay. Efavirenz selectively induced cell death in the BxPC-3 cells. The differing gene expression of cell cycle and apoptotic regulator genes in both cell cultures after Efavirenz treatment match with this selective effect against cancer cells. In the phosphoprotein array, an early phosphorylation of extracellular signal-related kinase 1/2 and p38 mitogen-activated protein kinase was notably detected in the cancer cells. The phosphorylation of AKT decreased in the cancer cells whereas it increased in the fibroblasts. Oxidative stress and mitochondrial membrane depolarization appeared in the cancer cells immediately after Efavirenz treatment, but not in the fibroblasts. Efavirenz has an anti-cancer effect against pancreatic cancer mainly by the induction of oxidative stress. The antitumor potential of Efavirenz and radiotherapy are additive.

Prognostic Value of Homotypic Cell Internalization by Nonprofessional Phagocytic Cancer Cells.

BACKGROUND: In this study, we investigated the prognostic role of homotypic tumor cell cannibalism in different cancer types. METHODS: The phenomenon of one cell being internalized into another, which we refer to as "cell-in-cell event," was assessed in 416 cases from five head and neck cancer cohorts, as well as one anal and one rectal cancer cohort. The samples were processed into tissue microarrays and immunohistochemically stained for E-cadherin and cleaved caspase-3 to visualize cell membranes and apoptotic cell death. RESULTS: Cell-in-cell events were found in all of the cohorts. The frequency ranged from 0.7 to 17.3 cell-in-cell events per mm(2). Hardly any apoptotic cells were found within the cell-in-cell structures, although apoptotic cell rates were about 1.6 to two times as high as cell-in-cell rates of the same tissue sample. High numbers of cell-in-cell events showed adverse effects on patients' survival in the head and neck and in the rectal cancer cohorts. In multivariate analysis, high frequency was an adverse prognostic factor for overall survival in patients with head and neck cancer (p = 0.008). CONCLUSION: Cell-in-cell events were found to predict patient outcomes in various types of cancer better than apoptosis and proliferation and might therefore be used to guide treatment strategies.

Clearance of primary necrotic cells by non-professional phagocytes.

BACKGROUND INFORMATION: Homotypic internalisation of tumour cells has frequently been observed in tumour tissue sections. Events of non-professional phagocytosis, however, may also occur in normal tissue if the number of dying cells exceeds the phagocytic capacity of professional phagocytes such as macrophages and dendritic cells. The aim of this study was to investigate the molecular background of non-professional phagocytosis of primary necrotic cells by neighbouring tumour cells and normal skin fibroblasts. RESULTS: We demonstrate that homotypic and heterotypic uptake of necrotic cells is a feature common to various cell types. Investigating critical stimuli of necrotic cell clearance we found that non-professional phagocytes require cytoskeleton rearrangement, recognition of phosphatidylserine and GTPase activity of dynamin II, which is normally engaged in endocytosis. Additionally, we have observed an accumulation of adhesion molecule E-cadherin, phosphorylated actin-linker protein ezrin, lysosomal-associated membrane protein 1 and microtubule-associated protein 1 light chain 3 at the site of engulfment. Loss of membrane integrity and an increase in the intracellular level of heat-shock protein 70 in the necrotic cells have also been observed. CONCLUSIONS: Our results shed light on the mechanism of necrotic cell removal by tumour cells and normal skin fibroblasts in vitro. It is reasonable to assume that this process has a physiological relevance in inflammation and autoimmune disease in normal tissue as well as in tumours regarding immune cell infiltration. We conclude that necrotic cell clearance by non-professional phagocytes contributes to the phagocytic activity by macrophages and that this process may prevent release of proinflammatory damage-associated molecular pattern molecules.

Distinct increased outliers among 136 rectal cancer patients assessed by γH2AX.

BACKGROUND: In recent years attention has focused on γH2AX as a very sensitive double strand break indicator. It has been suggested that γH2AX might be able to predict individual radiosensitivity. Our aim was to study the induction and repair of DNA double strand breaks labelled by γH2AX in a large cohort. METHODS: In a prospective study lymphocytes of 136 rectal cancer (RC) patients and 59 healthy individuals were ex vivo irradiated (IR) and initial DNA damage was compared to remaining DNA damage after 2 Gy and 24 hours repair time and preexisting DNA damage in unirradiated lymphocytes. Lymphocytes were immunostained with anti-γH2AX antibodies and microscopic images with an extended depth of field were acquired. γH2AX foci counting was performed using a semi-automatic image analysis software. RESULTS: Distinct increased values of preexisting and remaining γH2AX foci in the group of RC patients were found compared to the healthy individuals. Additionally there are clear differences within the groups and there are outliers in about 12% of the RC patients after ex vivo IR. CONCLUSIONS: The γH2AX assay has the capability to identify a group of outliers which are most probably patients with increased radiosensitivity having the highest risk of suffering radiotherapy-related late sequelae.

PML-nuclear bodies decrease with age and their stress response is impaired in aged individuals.

BACKGROUND: Promyelocytic leukemia nuclear bodies (PML-NBs) have been depicted as structures which are involved in processing cell damages and DNA double-strand break repairs. The study was designed to evaluate differences in patients' PML-NBs response to stress factors like a cancerous disease and ionizing radiation exposure dependent on age. METHODS: In order to clarify the role of PML-NBs in the aging process, we examined peripheral blood monocytes of 134 cancer patients and 41 healthy individuals between 22 and 92 years of age, both before and after in vitro irradiation. Additionally, we analyzed the samples of the cancer patients after in vivo irradiation. Cells were immunostained and about 1600 cells per individual were analyzed for the presence of PML- and γH2AX foci. RESULTS: The number of existing PML-NBs per nucleus declined with age, while the number of γH2AX foci increased with age. There was a non-significant trend that in vivo irradiation increased the number of PML-NBs in cells of young study participants, while in older individuals PML-NBs tended to decrease. It can be assumed that PML-NBs decrease in number during the process of aging. CONCLUSION: The findings suggest that there is a dysfunctional PML-NBs stress response in aged cells.

Cytotoxic effect of efavirenz is selective against cancer cells and associated with the cannabinoid system.

BACKGROUND: Recently, a regression of precancerous lesions in HIV-1-infected patients after initiation of HAART was reported. Nonnucleoside reverse transcriptase inhibitors (NNRTIs) as efavirenz (EFV) might be mediators of this effect, as they are known to have a cytotoxic effect on tumour cells. A potential mechanism involved in this effect may be the activation of the cannabinoid receptor to mediate tumour toxicity. METHODS: Several tumour-derived and fibroblast cell lines were studied. Cytotoxicity of EFV was evaluated by Annexin-Pi staining. The expression of the cannabinoid receptors CB1, CB2 and GPR55 was analysed by western blot, quantitative reverse transcriptase (qRT-PCR) and fluorescence activated cell sorting. The influence of the cannabinoid agonists and antagonists on the effects of EFV was investigated. Furthermore, the effect of EFV on the phosphorylation state of the growth factors Erk, Akt and the tumour suppressor protein p53 was tested. RESULTS: EFV revealed a selective cytotoxic effect on several tumour cell lines, whereas primary fibroblasts were not affected. The cytotoxic effect was associated with the expression of CB1. The combination of EFV with cannabinoid agonists showed an increase in toxicity. The phosphorylation state of Erk and Akt was not affected by EFV, whereas p53 showed an increased phosphorylation. CONCLUSION: EFV has a selective cytotoxic effect on several tumour cells. Furthermore, EFV led to an activating phosphorylation of the tumour suppressor protein p53 going in line with earlier reports that EFV may be antitumourigenic and a potential cytostatic drug. The observed synergistic effect with cannabinoid agonists implicates an involvement of the cannabinoid system.