TREX1, originally designated DNase III, was isolated as a major nuclear DNA-specific 335 exonuclease that is widely distributed in both proliferating and nonproliferating mammalian tissues. The cognate cDNA shows homology to the editing subunit of the Escherichia coli replicative DNA polymerase III holoenzyme and encodes an exonuclease which was able to serve a DNA-editing function in vitro, promoting rejoining of a 3 mismatched residue in a reconstituted DNA base excision repair system. Here we report the generation of genetargeted Trex1؊/؊ mice. The null mice are viable and do not show the increase in spontaneous mutation frequency or cancer incidence that would be predicted if Trex1 served an obligatory role of editing mismatched 3 termini generated during DNA repair or DNA replication in vivo. Unexpectedly, Trex1 ؊/؊ mice exhibit a dramatically reduced survival and develop inflammatory myocarditis leading to progressive, often dilated, cardiomyopathy and circulatory failure.Two distinct nuclear exonucleases account for the major part of the total exonucleolytic activity on DNA observed in mammalian cell extracts (24, 25). They were identified as a 3Ј35Ј exonuclease acting preferentially on single-stranded DNA and a 5Ј33Ј exonuclease specific for double-stranded DNA that could remove a single-stranded 5Ј overhang as an oligonucleotide. These nuclear enzymes were designated DNase III and DNase IV, as they are distinct from the pancreatic and macrophage lysosomal DNA endonucleases DNase I and DNase II. DNase IV was later renamed flap endonuclease 1 (FEN1) (23), and its main function is processing displaced 5Ј single strands that arise during lagging-strand DNA replication, as well as during DNA repair, recombination, and triplet repeat expansion. The elimination of Fen1 activity leads to early embryonic lethality in mice, consistent with an essential role of the enzyme in DNA replication (20). In contrast, DNase III is expressed at similar levels in nonproliferating and proliferating tissues; it was isolated as the major nuclear 3Ј35Ј DNA exonuclease from the adult rabbit liver (14) and also from the calf thymus and human myoblasts, where it was designated TREX1 (37, 38).The human TREX1/DNase III cDNA (14, 29) shares amino acid sequence homology with the Escherichia coli DnaQ/MutD editing subunit of the replicative DNA polymerase III holoenzyme, which can increase the fidelity of an exonuclease-deficient mammalian DNA polymerase in vitro (36). The TREX1/ DNase III cDNA encodes a nonprocessive 3Ј35Ј DNA-specific exonuclease, with a preference for single-stranded DNA or mispaired 3Ј termini; like the native protein isolated from mammalian cells, it forms homodimers (14, 29, 30). Since two of the major mammalian nuclear DNA polymerases, Pol␣ and Pol␤, do not have an intrinsic 3Ј exonuclease function, it was proposed that TREX1/DNase III may serve to edit mismatched deoxyribonucleotides during lagging-strand DNA synthesis or gap filling in DNA base excision repair, which are conducted by Pol␣ and Pol␤, respectively...

We therefore believe that FLIM has a potential future clinical role in imaging BCCs for rapid and noninvasive tumour delineation and as an aid to determine adequate excision margins with best preservation of normal tissue.

Lung cancer is the commonest cancer killer. Small cell lung cancer (SCLC) is initially chemosensitive, but rapidly relapses in a chemoresistant form with an overall survival of <5%. Consequently, novel therapies are urgently required and will likely arise from an improved understanding of the disease biology. Our previous work showed that fibroblast growth factor-2 induces proliferation and chemoresistance in SCLC cells. Here, we show that the selective fibroblast growth factor receptor (FGFR) inhibitor PD173074 blocks H-510 and H-69 SCLC proliferation and clonogenic growth in a dose-dependent fashion and prevents FGF-2-induced chemoresistance. These effects correlate with the inhibition of both FGFR1 and FGFR2 transphosphorylation. We then determined the efficacy of daily oral administration of PD173074 for 28 days in two human SCLC models. In the H-510 xenograft, tumor growth was impaired similar to that seen with single-agent cisplatin administration, increasing median survival compared with control sham-treated animals. Crucially, the effect of cisplatin was significantly potentiated by coadministration of PD173074. More dramatically, in H-69 xenografts, PD173074 induced complete responses lasting >6 months in 50% of mice. These effects were not a consequence of disrupted tumor vasculature but instead correlated with increased apoptosis (caspase 3 and cytokeratin 18 cleavage) in excised tumors. Moreover, in vivo imaging with 3′-deoxy-3′-

To investigate the role of signaling by the small GTPase Ral, we have generated mice deficient for RalGDS, a guanine nucleotide exchange factor that activates Ral. We show that RalGDS is dispensable for mouse development but plays a substantial role in Ras-induced oncogenesis. Lack of RalGDS results in reduced tumor incidence, size, and progression to malignancy in multistage skin carcinogenesis, and reduced transformation by Ras in tissue culture. RalGDS does not appear to participate in the regulation of cell proliferation, but instead controls survival of transformed cells. Experiments performed in cells isolated from skin tumors suggest that RalGDS mediates cell survival through the activation of the JNK/SAPK pathway. These studies identify RalGDS as a key component in Ras-dependent carcinogenesis in vivo.

Fluorescence lifetime imaging (FLIM) depends on the fluorescence decay differences between tissues to generate image contrast. In the present study FLIM has been applied to fixed (but unstained) breast cancer tissues to demonstrate the feasibility of this approach for histopathological assessment. As the FLIM method relies on natural autofluorescence, it may be possible to circumvent tissue processing altogether and so FLIM has the potential to be a powerful new method of in vivo tissue imaging via an endoscopic or per-operative approach in a variety of organs, as well as a research tool for in vivo animal models of disease. Unstained, alcohol-fixed tissue samples from 13 patients were stimulated by laser pulses at 415 nm. The temporal decay of the autofluorescence was imaged over a period of 2 ns after cessation of the pulse. The decay rate at each image pixel was calculated as the 'lifetime' factor tau. A tissue classification scheme was used to define regions in each image. The average lifetimes of different tissue regions were compared. A total of 167 tissue regions were measured. Within individual fields, stroma had a larger tau (slower decay) than epithelium (p < 0.001). Within individual patients (taking the mean tau of a given tissue type across all fields from each patient), there was a statistically significant difference between benign and malignancy-associated stroma (p < 0.05). Also, benign collagen had a longer tau than benign epithelium (p < 0.05). Multivariate analysis showed a significant difference between benign stroma, malignancy-associated stroma, blood vessels, and malignant epithelium (p < 0.05). Statistically significant differences between benign and malignancy-associated stroma were obtained even with small patient numbers, indicating that lifetime-based instruments can be developed for real-time diagnostic imaging with microscopic resolution.

No abstract

Tissue destruction characterizes infection with Mycobacterium tuberculosis (Mtb). Type I collagen provides the lung's tensile strength, is extremely resistant to degradation, but is cleaved by matrix metalloproteinase (MMP)-1. Fibroblasts potentially secrete quantitatively more MMP-1 than other lung cells. We investigated mechanisms regulating Mtb-induced collagenolytic activity in fibroblasts in vitro and in patients. Lung fibroblasts were stimulated with conditioned media from Mtb-infected monocytes (CoMTb). CoMTb induced sustained increased MMP-1 (74 versus 16 ng/ml) and decreased tissue inhibitor of metalloproteinase (TIMP)-1 (8.6 versus 22.3 ng/ml) protein secretion. CoMTb induced a 2.7-fold increase in MMP-1 promoter activation and a 2.5-fold reduction in TIMP-1 promoter activation at 24 hours (P = 0.01). Consistent with this, TIMP-1 did not co-localize with fibroblasts in patient granulomas. MMP-1 up-regulation and TIMP-1 down-regulation were p38 (but not extracellular signal-regulated kinase or c-Jun N-terminal kinase) mitogen-activated protein kinase-dependent. STAT3 phosphorylation was detected in fibroblasts in vitro and in tuberculous granulomas. STAT3 inhibition reduced fibroblast MMP-1 secretion by 60% (P = 0.046). Deletion of the MMP-1 promoter NF-κB-binding site abrogated promoter induction in response to CoMTb. TNF-α, IL-1β, or Oncostatin M inhibition in CoMTb decreased MMP-1 secretion by 65, 63, and 25%, respectively. This cytokine cocktail activated the same signaling pathways in fibroblasts and induced MMP-1 secretion similar to that induced by CoMTb. This study demonstrates in a cellular model and in patients with tuberculosis that in addition to p38 and NF-κB, STAT3 has a key role in driving fibroblast-dependent unopposed MMP-1 production that may be key in tissue destruction in patients.

The autofluorescence of biological tissue can be exploited for the detection and diagnosis of disease but, to date, its complex nature and relatively weak signal levels have impeded its widespread application in biology and medicine. We present here a portable instrument designed for the in situ simultaneous measurement of autofluorescence emission spectra and temporal decay profiles, permitting the analysis of complex fluorescence signals. This hyperspectral fluorescence lifetime probe utilizes two ultrafast lasers operating at 355 and 440 nm that can excite autofluorescence from many different biomolecules present in skin tissue including keratin, collagen, nicotinamide adenine dinucleotide (phosphate), and flavins. The instrument incorporates an optical fiber probe to provide sample illumination and fluorescence collection over a millimeter-sized area. We present a description of the system, including spectral and temporal characterizations, and report the preliminary application of this instrument to a study of recently resected (<2 h) ex vivo skin lesions, illustrating its potential for skin cancer detection and diagnosis.