We examined the role of MCP-1, a potent chemotactic and activating factor for macrophages, in perfusion, inflammation, and skeletal muscle regeneration post-ischemic injury. MCP-1-/- or C57Bl/6J control mice [wild-type (WT)] underwent femoral artery excision (FAE). Muscles were collected for histology, assessment of tissue chemokines, and activity measurements of lactate dehydrogenase (LDH) and myeloperoxidase. In MCP-1-/- mice, restoration of perfusion was delayed, and LDH and fiber size, indicators of muscle regeneration, were decreased. Altered inflammation was observed with increased neutrophil accumulation in MCP-1-/- versus WT mice at Days 1 and 3 (P< or =0.003), whereas fewer macrophages were present in MCP-1-/- mice at Day 3. As necrotic tissue was removed in WT mice, macrophages decreased (Day 7). In contrast, macrophage accumulation in MCP-1-/- was increased in association with residual necrotic tissue and impaired muscle regeneration. Consistent with altered inflammation, neutrophil chemotactic factors (keratinocyte-derived chemokine and macrophage inflammatory protein-2) were increased at Day 1 post-FAE. The macrophage chemotactic factor MCP-5 was increased significantly in WT mice at Day 3 compared with MCP-1-/- mice. However, at post-FAE Day 7, MCP-5 was significantly elevated in MCP-1-/- mice versus WT mice. Addition of exogenous MCP-1 did not induce proliferation in murine myoblasts (C2C12 cells) in vitro. MCP-1 is essential for reperfusion and the successful completion of normal skeletal muscle regeneration after ischemic tissue injury. Impaired muscle regeneration in MCP-1-/- mice suggests an important role for macrophages and MCP-1 in tissue reparative processes.
Myelodysplastic syndrome (MDS) is a complex family of pre-leukemic diseases in which hematopoietic stem cell defects lead to abnormal differentiation in one or more blood lineages. Disease progression is associated with increasing genomic instability and a large proportion of patients go on to develop acute myeloid leukemia. Primarily a disease of the elderly, it can also develop following chemotherapy. We have previously reported that CREB binding protein (Crebbp) heterozygous mice have an increased incidence of hematological malignancies, and others have shown that CREBBP is one of the genes altered by chromosomal translocations found in patients suffering from therapy-related MDS. This led us to investigate whether hematopoietic tumor development in Crebbp+/- mice is preceded by a myelodysplastic phase and whether we could uncover molecular mechanisms that might contribute to its development. We report here that Crebbp+/- mice invariably develop myelodysplastic/myeloproliferative neoplasm within 9-12 months of age. They are also hypersensitive to ionizing radiation and show a marked decrease in PARP1 activity after irradiation. In addition, protein levels of XRCC1 and APEX1, key components of base excision repair machinery, are reduced in unirradiated Crebbp+/- cells or upon targeted knock down of CREBBP levels. Our results thus provide validation of a novel myelodysplastic/myeloproliferative neoplasm mouse model and, more importantly, point to defective repair of DNA damage as a contributing factor to the pathogenesis of this currently incurable disease.
Recent studies suggest that PARP1 inhibitors, several of which are currently in clinical trial, may selectively kill BRCA1/2 mutant cancers cells. It is thought that the success of this therapy is based on immitigable lethal DNA damage in the cancer cells resultant from the concurrent loss or inhibition of two DNA damage repair pathways: single-strand break (SSB) repair and homologous recombination repair (HRR). Presumably, inhibition of PARP1 activity obstructs the repair of SSBs and during DNA replication, these lesions cause replication fork collapse and are transformed into substrates for HRR. In fact, several previous studies have indicated a hyper-recombinogenic phenotype in the absence of active PARP1 in vitro or in response to DNA damaging agents. In this study, we demonstrate an increased frequency of spontaneous HRR in vivo in the absence of PARP1 using the pun assay. Furthermore, we found that the HRR events that occur in Parp1 nullizygous mice are associated with a significant increase in large, clonal events, as opposed to the usually more frequent single cell events, suggesting an effect in replicating cells. In conclusion, our data demonstrates that PARP1 inhibits spontaneous HRR events, and supports the model of DNA replication transformation of SSBs into HRR substrates.
Introduction Early detection of lung cancer in high-risk individuals reduces mortality. Low-dose spiral computed tomography (LDCT) is the current standard but suffers from an exceedingly high false-positive rate (>96%) leading to unnecessary and potentially dangerous procedures. We, therefore, set out to develop a simple, noninvasive, and quantitative assay to detect lung cancer. Methods This proof-of-concept study evaluated the sensitivity/specificity of the CyPath Early Lung Cancer Detection Assay to correctly classify LDCT-confirmed cohorts of high-risk control (n = 102) and cancer (n = 26) subjects. Fluorescence intensity parameters of red fluorescent cells (RFCs) from tetra (4-carboxyphe-nyl) porphyrin (TCPP)-labeled lung sputum samples and subjects’ baseline characteristics were assessed for their predictive power by multivariable logistic regression. A receiver operating characteristic curve was constructed to evaluate the sensitivity/specificity of the CyPath assay. Results RFCs were detectable in cancer subjects more often than in high-risk ones (p = 0.015), and their characteristics differed between cohorts. Two independent predictors of cancer were the mean of RFC average fluorescence intensity/area per subject (p < 0.001) and years smoked (p = 0.003). The CyPath-based classifier had an overall accuracy of 81% in the test population; false-positive rate of 40% and negative predictive value of 83%. Conclusions The tetra (4-carboxyphenyl) porphyrin-based CyPath assay correctly classified study participants into cancer or high-risk cohorts with considerable accuracy. Optimizing sputum collection, sample reading, and refining the classifier should improve sensitivity and specificity. The CyPath assay thus has the potential to complement LDCT screening or serve as a stand-alone approach for early lung cancer detection.
Background In vitro cell culture experiments with primary cells have reported that cell proliferation is retarded in the presence of ambient compared to physiological O2 levels. Cancer is primarily a disease of aberrant cell proliferation, therefore, studying cancer cells grown under ambient O2 may be undesirable. To understand better the impact of O2 on the propagation of cancer cells in vitro, we compared the growth potential of a panel of ovarian cancer cell lines under ambient (21%) or physiological (3%) O2.Principal FindingsOur observations demonstrate that similar to primary cells, many cancer cells maintain an inherent sensitivity to O2, but some display insensitivity to changes in O2 concentration. Further analysis revealed an association between defective G2/M cell cycle transition regulation and O2 insensitivity resultant from overexpression of 14-3-3 σ. Targeting 14-3-3 σ overexpression with RNAi restored O2 sensitivity in these cell lines. Additionally, we found that metastatic ovarian tumors frequently overexpress 14-3-3 σ, which in conjunction with phosphorylated RB, results in poor prognosis.ConclusionsCancer cells show differential proliferative sensitivity to changes in O2 concentration. Although a direct link between O2 insensitivity and metastasis was not determined, this investigation showed that an O2 insensitive phenotype in cancer cells to correlate with metastatic tumor progression.
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