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Cancer-related fatigue (CRF) and stress are common symptoms in cancer patients and represent early side effects of cancer treatment which affect the life quality of the patients. CRF may partly depend on disruption of the circadian rhythm. Locomotor activity and corticosterone rhythms are two important circadian outputs which can be used to analyze possible effects on the circadian function during cancer development and treatment. The present study analyzes the relationship between locomotor activity rhythm, corticosterone levels, hepatocellular carcinoma (HCC) development, and radiotherapy treatment in a mouse model. HCC was induced in mice by single injection of diethylnitrosamine (DEN) and chronic treatment of phenobarbital in drinking water. Another group received chronic phenobarbital treatment only. Tumor bearing animals were divided randomly into four groups irradiated at four different Zeitgeber time points. Spontaneous locomotor activity was recorded continuously; serum corticosterone levels and p-ERK immunoreaction in the suprachiasmatic nucleus (SCN) were investigated. Phenobarbital treated mice showed damped corticosterone levels and a less stable 24 hours activity rhythm as well as an increase in activity during the light phase, reminiscent of sleep disruption. The tumor mice showed an increase in corticosterone level during the inactive phase and decreased activity during the dark phase, reminiscent of CRF. After irradiation, corticosterone levels were further increased and locomotor activity rhythms were disrupted. Lowest corticosterone levels were observed after irradiation during the early light phase; thus, this time might be the best to apply radiotherapy in order to minimize side effects.

This study investigates whether a chronotherapeutic treatment of hepatocellular carcinoma (HCC) may improve treatment efficacy and mitigate side effects on healthy liver (HL). HCC was induced in Per2::luc mice which were irradiated at four time points of the day. Proliferation and DNA-double strand breaks were investigated in irradiated and non-irradiated organotypic slice culture (OSC) and ex vivo samples by detection of Ki67 and γ-H2AX. OSC proved useful to determine dose-dependent effects on proliferation and DNA damage but appeared unsuited to test the chronotherapeutic approach. Irradiation of ex vivo samples was most effective at the proliferation peaks in HCC at ZT02 (early inactivity phase) and ZT20 (late activity phase). Irradiation effects on HL were minimal at ZT20. Ex vivo samples revealed disruption in daily variation and down-regulation of all investigated clock genes except Per1 in non-irradiated HCC as compared with HL. Irradiation affected rhythmic clock gene expression in HL and HCC at all ZTs except at ZT20. Irradiation at ZT20 had no effect on total leukocyte numbers. Our results indicate ZT20 as the optimal time point for irradiation of HCC in mice. Translational studies are now needed to evaluate whether the late activity phase is the optimal time point for irradiation of HCC in man.

The homeobox gene HLXB9 encodes for the transcription factor HB9, which is essential for pancreatic as well as motor neuronal development. Beside its physiological expression pattern, aberrant HB9 expression has been observed in several neoplasias. Especially in infant translocation t(7;12) acute myeloid leukemia, aberrant HB9 expression is the only known molecular hallmark and is assumed to be a key factor in leukemic transformation. However, so far, only poor functional data exist addressing the oncogenic potential of HB9 or its influence on hematopoiesis. We investigated the influence of HB9 on cell proliferation and cell cycle in vitro, as well as on hematopoietic stem cell differentiation in vivo using murine and human model systems. In vitro, HB9 expression led to premature senescence in human HT1080 and murine NIH3T3 cells, providing for the first time evidence for an oncogenic potential of HB9. Onset of senescence was characterized by induction of the p53–p21 tumor suppressor network, resulting in growth arrest, accompanied by morphological transformation and expression of senescence-associated β-galactosidase. In vivo, HB9-transduced primary murine hematopoietic stem and progenitor cells underwent a profound differentiation arrest and accumulated at the megakaryocyte/erythrocyte progenitor stage. In line, gene expression analyses revealed de novo expression of erythropoiesis-related genes in human CD34+hematopoietic stem and progenitor cells upon HB9 expression. In summary, the novel findings of HB9-dependent premature senescence and myeloid-biased perturbed hematopoietic differentiation, for the first time shed light on the oncogenic properties of HB9 in translocation t(7;12) acute myeloid leukemia.

The mechanisms of skin aging have not been completely elucidated. Anecdotal data suggests that EGFR inhibition accelerates aging-like skin changes.The objective of the study was to evaluate the clinical characteristics and investigate the cellular and molecular mechanisms underlying skin changes associated with the use of EFGRIs.Patients during prolonged treatment with EGFRIs (3 months) were analyzed for aging-like skin changes. Baseline EGFR expression was compared in young (25 years old) vs. old (65 years old) skin. In addition, the regulation of extracellular matrix, senescence-associated genes, and cell cycle status was measured in primary human keratinocytes treated with erlotinib in vitro.There were progressive signs of skin aging, including xerosis cutis, atrophy, rhytide formation, and/or actinic purpura in 12 patients. Keratinocytes treated with erlotinib in vitro showed a significant down-modulation of hyaluronan synthases (HAS2 and HAS3), whereas senescence-associated genes (p21, p53, IL-6, maspin) were upregulated, along with a G1 cell cycle arrest and stronger SA β-Gal activity. There was significantly decreased baseline expression in EGFR density in aged skin, when compared to young controls.EGFR inhibition results in molecular alterations in keratinocytes that may contribute to the observed skin aging of patients treated with respective targeted agents.

The prorenin receptor (PRR) is highly expressed in podocytes, but its role in the maintenance of podocyte function is unknown. Here we generated podocyte-specific PRR-knockout mice and found that these animals died between 2 to 3 wk after birth. Within 14 d, PRR-knockout mice developed nephrotic syndrome, albuminuria with podocyte foot-process fusion, and cytoskeletal changes. Podocyte-specific PRR deletion also led to disturbed processing of multivesicular bodies and enrichment of autophagosomal (LC3) and lysosomal (LAMP2) markers, indicating a functional block in autophagosome-lysosome fusion and an overload of the proteasomal protein-degradation machinery. In vitro, PRR knockdown and pharmacologic blockade of vacuolar H(+)-ATPases, which associate with the PRR, increased vesicular pH, led to accumulation of LC3-positive and LAMP2-positive vesicles and altered the cytoskeleton. Taken together, these results suggest that the PRR is essential for podocyte function and survival by maintaining autophagy and protein-turnover machinery. Furthermore, PRR contributes to the control of lysosomal pH, which is important for podocyte survival and cytoskeletal integrity.

Proteases of the caspase family are thought to be activated by proteolytic processing of their inactive zymogens. However, although proteolytic cleavage is sufficient for executioner caspases, a different mechanism has been recently proposed for initiator caspases, such as caspase-8, which are believed to be activated by proximity-induced dimerization. According to this model, dimerization rather than proteolytic processing is considered as the critical event for caspase-8 activation. Such a mechanism would suggest that in the absence of a dimerization platform such as the death-inducing signaling complex, caspase-8 proteolytic cleavage would result in an inactive enzyme. As several studies have described caspase-8 cleavage during mitochondrial apoptosis, we now investigated whether caspase-8 becomes indeed catalytically active in this pathway. Using an in vivo affinity labeling approach, we demonstrate that caspase-8 is activated in etoposide-treated cells in vivo in the absence of the receptor-induced death-inducing signaling complex formation. Furthermore, we show that both caspase-3 and -6 are required for the efficient activation of caspase-8. Our data therefore indicate that interchain cleavage of caspase-8 in the mitochondrial pathway is sufficient to produce an active enzyme even in the absence of receptor-driven procaspase-8 dimerization.

Depending on multiple factors DNA damage leads either to cell cycle arrest or apoptosis. One of the main players deciding the fate of a cell is the tumor suppressor p53 that modulates these responses in a transcription-dependent and -independent manner. Over the past few years, however, strong evidence accumulated that p53 engages also powerful pro-survival pathways by transcriptionally activating a multitude of genes whose products efficiently counteract apoptosis. Our review summarizes the current knowledge concerning approximately forty p53-regulated proteins that exert their anti-apoptotic potential by interfering with diverse cellular processes. These activities are surely essential for normal development and maintenance of a healthy organism, but may easily turn into the dark side of the tumor suppressor p53 contributing to tumorigenesis.

Due to their tremendous apoptosis-inducing potential, proteasomal inhibitors (PIs) have recently entered clinical trials. Here we show, however, that various PIs rescued proliferating tumor cells from death receptor-induced apoptosis. This protection correlated with the stabilization of X-linked IAP (XIAP) and c-FLIP and the inhibition of caspase activation. Together with the observation that PIs could not protect cells expressing XIAP or c-FLIP short interfering RNAs (siRNAs) from death receptor-induced apoptosis, our results demonstrate that PIs mediate their protective effect via the stabilization of these antiapoptotic proteins. Furthermore, we show that once these proteins were eliminated, either by long-term treatment with death receptor ligands or by siRNA-mediated suppression, active caspases accumulated to an even larger extent in the presence of PIs. Together, our data support a biphasic role for the proteasome in apoptosis, as they show that its constitutive activity is crucial for the rapid initiation of the death program by eliminating antiapoptotic proteins, whereas at later stages, the proteasome acts in an antiapoptotic manner due to the proteolysis of caspases. Thus, for a successful PI-based tumor therapy, it is crucial to carefully evaluate basal proteasomal activity and the status of antiapoptotic proteins, as their PI-mediated prolonged stability might even cause adverse effects, leading to the survival of a tumor.