Kathrin Zaugg

Carnitine palmitoyltransferase 1C promotes cell survival and tumor growth under conditions of metabolic stress

Tumor cells gain a survival/growth advantage by adapting their metabolism to respond to environmental stress, a process known as metabolic transformation. The best-known aspect of metabolic transformation is the Warburg effect, whereby cancer cells up-regulate glycolysis under aerobic conditions. However, other mechanisms mediating metabolic transformation remain undefined. Here we report that carnitine palmitoyltransferase 1C (CPT1C), a brain-specific metabolic enzyme, may participate in metabolic transformation. CPT1C expression correlates inversely with mammalian target of rapamycin (mTOR) pathway activation, contributes to rapamycin resistance in murine primary tumors, and is frequently up-regulated in human lung tumors. Tumor cells constitutively expressing CPT1C show increased fatty acid (FA) oxidation, ATP production, and resistance to glucose deprivation or hypoxia. Conversely, cancer cells lacking CPT1C produce less ATP and are more sensitive to metabolic stress. CPT1C depletion via siRNA suppresses xenograft tumor growth and metformin responsiveness in vivo. CPT1C can be induced by hypoxia or glucose deprivation and is regulated by AMPKα. Cpt1c-deficient murine embryonic stem (ES) cells show sensitivity to hypoxia and glucose deprivation and altered FA homeostasis. Our results indicate that cells can use a novel mechanism involving CPT1C and FA metabolism to protect against metabolic stress. CPT1C may thus be a new therapeutic target for the treatment of hypoxic tumors.

A Role for the Deubiquitinating Enzyme USP28 in Control of the DNA-Damage Response

The Chk2-p53-PUMA pathway is a major regulator of DNA-damage-induced apoptosis in response to double-strand breaks in vivo. Through analysis of 53BP1 complexes we have discovered a new ubiquitin protease, USP28, which regulates this pathway. Using a human cell line that faithfully recapitulated the Chk2-p53-PUMA pathway, we show that USP28 is required to stabilize Chk2 and 53BP1 in response to DNA damage. In this cell line, both USP28 and Chk2 are required for DNA-damage-induced apoptosis, and they accomplish this in part through regulation of the p53 induction of proapoptotic genes like PUMA. Our studies implicate DNA-damage-induced ubiquitination and deubiquitination as a major regulator of the DNA-damage response for Chk2, 53BP1, and a number of other proteins in the DNA-damage checkpoint pathway, including several mediators, such as Mdc1, Claspin, and TopBP1.

Trigger-dependent gene expression profiles in cardiac preconditioning: evidence for distinct genetic programs in ischemic and anesthetic preconditioning.

BackgroundDNA chips facilitate genomic-wide exploration of gene expression. The authors hypothesized that ischemic (IPC) and anesthetic preconditioning (APC) would differentially modulate gene expression in hearts. MethodsAffymetrix rat U34A gene chips were used to explore the transcriptional response to IPC and APC, sustained ischemia (110 min) without reperfusion, and time-matched perfusion in isolated rat hearts. IPC was induced by three cycles of 5 min of ischemia, and APC was induced by 1.5 minimum alveolar concentration isoflurane (110 min). For each heart, a separate chip was used for hybridization. Data were analyzed for significant ≥ 2.0-fold changes in gene expression. Microarray results were confirmed by quantitative real-time reverse-transcription polymerase chain reaction. ResultsOf the 8,799 genes represented on U34A, 217 transcripts in the APC group, 234 in the IPC group, and 29 in the ischemia group displayed significant ≥ 2.0-fold up-regulation in messenger RNA levels, and 185 transcripts in the APC group, 55 in the IPC group, and 49 in the ischemia group displayed significant ≥ 2.0-fold down-regulation. Many of these transcripts were unknown genes. A high number of commonly regulated genes were found in IPC and APC (39 up-regulated, 17 down-regulated). Genes commonly regulated included those associated with cell defense (heat shock protein 10, aldose reductase, Bcl-xS). Conversely, a pool of protective and antiprotective genes was differentially regulated in APC versus IPC (heat shock protein 27/70, programmed cell death 8), suggesting trigger-dependent transcriptome variability. ConclusionsThe novel microarray technology provides evidence for distinct cardioprotective phenotypes in IPC and APC. The observed transcriptional changes raise the possibility of a second window of protection by volatile anesthetics. The authors’ molecular portraits are the first global genomic comparison between IPC and APC.

Metastatic bone pain: treatment options with an emphasis on bisphosphonates

IntroductionOne of the key targets for metastatic cancer cells is the skeleton. Once metastatic cells are established within the bone matrix, skeletal integrity becomes increasingly compromised. Bone lesions lead to various complications, including bone pain, fractures and spinal cord compression.Mechanisms of bone painBone pain is debilitating and affects quality of life of the patient. In addition, it increases the use of health care resources. Many patients with metastatic bone disease experience substantial bone pain despite state-of-the-art systemic analgesic treatment. Incident pain is the predominant pain syndrome.Treatment options for bone painTypically, this syndrome requires moderate baseline analgesia with increased on-demand doses. Other techniques for treating bone pain, including radiation therapy, neuraxial application of analgesics, nerve blocks and local stabilisation procedures, should be considered. In addition, therapy with bisphosphonates targeting bone-specific pain is an important strategy. This review discusses the various management options for bone pain arising from metastatic bone disease.

Cross-talk between Chk1 and Chk2 in double-mutant thymocytes

Chk1 is a checkpoint kinase and an important regulator of mammalian cell division. Because null mutation of Chk1 in mice is embryonic lethal, we used the Cre-loxP system and the Lck promoter to generate conditional mutant mice in which Chk1 was deleted only in the T lineage. In the absence of Chk1, the transition of CD4−CD8− double-negative (DN) thymocytes to CD4+CD8+ double-positive (DP) cells was blocked due to an increase in apoptosis at the DN2 and DN3 stages. Strikingly, loss of Chk1 activated the checkpoint kinase Chk2 as well as the tumor suppressor p53 in these thymocytes. However, the developmental defects caused by Chk1 deletion were not rescued by p53 inactivation. Significantly, even though Chk1 deletion is highly lethal in proliferating tissues, we succeeded in using in vivo methods to generate Chk1/Chk2 double-knockout T cells. Analysis of these T cells revealed an interesting interaction between Chk1 and Chk2 functions that partially rescued the apoptosis of the double-mutant cells. Thus, Chk1 is both critical for the survival of proliferating cells and engages in cross-talk with the Chk2 checkpoint kinase pathway. These factors have implications for the targeting of Chk1 as an anticancer therapy.

Effect of high dose per pulse flattening filter-free beams on cancer cell survival.

PURPOSE To investigate if there is a statistically significant difference in cancer cell survival using a high dose per pulse flattening filter-free (FFF) beam compared to a standard flattened beam. MATERIAL AND METHODS To validate the radiobiological effect of the flattened and FFF beam, two glioblastoma cell lines were treated with either 5 or 10 Gy using different dose rates. Dose verification was performed and colony formation assays were carried out. To compare the predictability of our data, radiobiological models were included. RESULTS The results presented here demonstrate that irradiation of glioblastoma cell lines using the FFF beam is more efficient in reducing clonogenic cell survival than the standard flattened beam, an effect which becomes more significant the higher the single dose. Interestingly, in our experimental setting, the radiobiological effect of the FFF beam is dependent on dose per pulse rather than on delivery time. The used radiobiological models are able to describe the observed dose rate dependency between 6 and 24 Gy/min. CONCLUSION The results presented here show that dose per pulse might become a crucial factor which influences cancer cell survival. Using high dose rates, currently used radiobiological models as well as molecular mechanisms involved urgently need to be re-examined.

Key targets for the execution of radiation-induced tumor cell apoptosis: the role of p53 and caspases

In many human hematologic and solid malignancies, intrinsic or acquired treatment resistance remains a major obstacle for successful cancer therapy. The molecular understanding of how tumor cells respond to chemotherapy and ionizing radiation is rapidly evolving. Induction of programmed cell death, apoptosis, is one important strategy for successful cancer therapy. This has been shown convincingly for oncogene-transformed normal cells as well as tumor cells of lymphoid origin. However, the relevance of apoptosis in solid human malignancies is less clear. Loss of apoptosis might be linked to specific mutations in the often tissue-specific apoptotic pathways due to aberrations in the stress-related signal transduction cascades. Restoration of a dysfunctional apoptotic program in cancer tissue where apoptosis has been identified as an important mechanism for tissue homeostasis is one rational approach for innovative cancer therapy. In this review, we focus on the relevance of the tumor suppressor p53 for apoptosis-induction and successful cancer therapy outlining the importance of an intact caspase machinery for apoptosis execution. Strategies are discussed to overcome treatment resistance and a high apoptotic threshold in human malignancies where apoptosis is the dominant mode of cell death and the status of p53 is an important determinant for apoptosis induction.

Depletion of the novel p53-target gene carnitine palmitoyltransferase 1C delays tumor growth in the neurofibromatosis type I tumor model

Despite the prominent pro-apoptotic role of p53, this protein has also been shown to promote cell survival in response to metabolic stress. However, the specific mechanism by which p53 protects cells from metabolic stress-induced death is unknown. Earlier we reported that carnitine palmitoyltransferase 1C (CPT1C), a brain-specific member of a family of mitochondria-associated enzymes that have a central role in fatty acid metabolism promotes cell survival and tumor growth. Unlike other members of the CPT family, the subcellular localization of CPT1C and its cellular function remains elusive. Here, we report that CPT1C is a novel p53-target gene with a bona fide p53-responsive element within the first intron. CPT1C is upregulated in vitro and in vivo in a p53-dependent manner. Interestingly, expression of CPT1C is induced by metabolic stress factors such as hypoxia and glucose deprivation in a p53 and AMP activated kinase-dependent manner. Furthermore, in a murine tumor model, depletion of Cpt1c leads to delayed tumor development and a striking increase in survival. Taken together, our results indicate that p53 protects cells from metabolic stress via induction of CPT1C and that CPT1C may have a crucial role in carcinogenesis. CPT1C may therefore represent an exciting new therapeutic target for the treatment of hypoxic and otherwise treatment-resistant tumors.

Ckap2 Regulates Aneuploidy, Cell Cycling, and Cell Death in a p53-Dependent Manner

We used DNA microarray screening to identify Ckap2 (cytoskeleton associated protein 2) as a novel p53 target gene in a mouse erythroleukemia cell line. DNA damage induces human and mouse CKAP2 expression in a p53-dependent manner and p53 activates the Ckap2 promoter. Overexpressed Ckap2 colocalizes with and stabilizes microtubules. In p53-null cells, overexpression of Ckap2 induces tetraploidy with aberrant centrosome numbers, suggesting disturbed mitosis and cytokinesis. In p53-competent cells, Ckap2 does not induce tetraploidy but activates p53-mediated cell cycle arrest and apoptosis. Our data suggest the existence of a functional positive feedback loop in which Ckap2 activates the G1 tetraploidy checkpoint and prevents aneuploidy.

Stem cell-like human endothelial progenitors show enhanced colony-forming capacity after brief sevoflurane exposure: preconditioning of angiogenic cells by volatile anesthetics.

BACKGROUND: Endothelial progenitor cells play a pivotal role in tissue repair, and thus are used for cell replacement therapies in “regenerative medicine.” We tested whether the anesthetic sevoflurane would modulate growth or mobilization of these angiogenic cells. METHODS: In an in vitro model, mononuclear cells isolated from peripheral blood of healthy donors were preconditioned with sevoflurane (3 times 30 min at 2 vol% interspersed by 30 min of air). Colony-forming units were determined after 9 days in culture and compared with time-matched untreated control. Using magnetic cell sorting, CD133+/CD34+ endothelial progenitors were enriched from human umbilical cord blood, and vascular endothelial growth factor (VEGF), VEGFR2 (KDR), granulocyte colony-stimulating factor (G-CSF), STAT3, c-kit, and CXCR4 expressions were determined in sevoflurane-treated and untreated cells by real-time reverse transcriptase polymerase chain reaction. In a volunteer study with crossover design, we tested whether sevoflurane inhalation (<1 vol% end-tidal concentration) would mobilize endothelial progenitor cells from the bone marrow niche into the circulation using flow cytometry of peripheral blood samples. VEGF and G-CSF plasma levels were also measured. RESULTS: In vitro sevoflurane exposure of mononuclear cells enhanced colony-forming capacity and increased VEGF mRNA levels in CD133+/CD34+ cord blood cells (P = 0.017). Sevoflurane inhalation in healthy volunteers did not alter the number of CD133+/CD34+ or KDR+/CD34+ endothelial progenitors in the circulation, but increased the number of colony-forming units (P = 0.034), whereas VEGF and G-CSF plasma levels remained unchanged. CONCLUSIONS: Sevoflurane preconditioning promotes growth and proliferation of stem cell-like human endothelial progenitors. Hence, it may be used to promote perioperative vascular healing and to support cell replacement therapies.