Blocking ligand occupancy of the αVß3 integrin inhibits the development of nephropathy in diabetic pigs.

Hyperglycemia stimulates secretion of αVß3 ligands from vascular cells, including endothelial cells, resulting in activation of the αVß3 integrin. This study determined whether blocking ligand occupancy of αVß3 would inhibit the development of diabetic nephropathy. Ten diabetic pigs received an F(ab)2 fragment of an antibody directed against the extracellular domain of the ß3-subunit, and 10 received a control IgG F(ab)2 for 18 weeks. Nondiabetic pigs excreted 115 ± 50 µg of protein/mg creatinine compared with control F(ab)2-treated diabetic animals (218 ± 57 µg/mg), whereas diabetic animals treated with the anti-ß3 F(ab)2 excreted 119 ± 55 µg/mg (P < .05). Mesangial volume/glomerular volume increased to 21 ± 2.4% in control-treated diabetic animals compared with 14 ± 2.8% (P < .01) in animals treated with active antibody. Diabetic animals treated with control F(ab)2 had significantly less glomerular podocin staining compared with nondiabetic animals, and this decrease was attenuated by treatment with anti-ß3 F(ab)2. Glomerular basement membrane thickness was increased in the control, F(ab)2-treated diabetic animals (212 ± 14 nm) compared with nondiabetic animals (170 ± 8.8 nm), but it was unchanged (159.9 ± 16.4 nm) in animals receiving anti-ß3 F(ab)2. Podocyte foot process width was greater in control, F(ab)2-treated, animals (502 ± 34 nm) compared with animals treated with the anti-ß3 F(ab)2 (357 ± 47 nm, P < .05). Renal ß3 tyrosine phosphorylation decreased from 13 934 ± 6437 to 6730 ± 1524 (P < .01) scanning units in the anti-ß3-treated group. We conclude that administration of an antibody that inhibits activation of the ß3-subunit of αVß3 that is induced by hyperglycemia attenuates proteinuria and early histologic changes of diabetic nephropathy, suggesting that it may have utility in preventing the progression of this disease complication.

Glucose regulation of thrombospondin and its role in the modulation of smooth muscle cell proliferation.

Smooth muscle cells (SMC) maintained in high glucose are more responsive to IGF-I than those in normal glucose. There is significantly more thrombospondin-1 (TSP-1) in extracellular matrix surrounding SMC grown in 25 mM glucose. In this study we investigated 1) the mechanism by which glucose regulates TSP-1 levels and 2) the mechanism by which TS-1 enhances IGF-I signaling. The addition of TSP-1 to primary SMC was sufficient to enhance IGF-I responsiveness in normal glucose. Reducing TSP-1 protein levels inhibited IGF-I signaling in SMC maintained in high glucose. We determined that TSP-1 protected IAP/CD47 from cleavage and thereby facilitated its association with SHP substrate-1 (SHPS-1). We have shown previously that the hyperglycemia induced protection of IAP from cleavage is an important component of the ability of hyperglycemia to enhance IGF-I signaling. Furthermore we determined that TSP-1 also enhanced phosphorylation of the beta3 subunit of the alphaVbeta3 integrin, another molecular event that we have shown are critical for SMC response to IGF-I in high glucose. Our studies also revealed that the difference in the amount of TSP-1 in the two different glucose conditions was due, at least in part, to a difference in the cellular uptake and degradation of TSP-1.

Chromosomal instability in Barrett's esophagus is related to telomere shortening.

Barrett's esophagus is a useful model for the study of carcinogenesis, as the metaplastic columnar epithelium that replaces squamous esophageal epithelium is at elevated risk for development of adenocarcinoma. We examined telomere length and chromosomal instability (CIN) in Barrett's esophagus biopsies using fluorescence in situ hybridization. To study CIN, we selected centromere and locus-specific arm probes to chromosomes 17/17p (p53), 11/11q (cyclin D1), and 9/9p (p16 INK4A), loci reported to be involved in early stages of Barrett's esophagus neoplasia. Telomere shortening was observed in Barrett's esophagus epithelium at all histologic grades, whereas CIN was highest in biopsies with dysplastic changes; there was, however, considerable heterogeneity between patients in each variable. Alterations on chromosome 17 were strongly correlated with telomere length (r = 0.55; P < 0.0001) and loss of the 17p arm signal was the most common event. CIN on chromosome 11 was also associated with telomere shortening (r =0.3; P = 0.05), although 11q arm gains were most common. On chromosome 9p, arm losses were the most common finding, but chromosome 9 CIN was not strongly correlated with telomere length. We conclude that CIN is related to telomere shortening in Barrett's esophagus but varies by chromosome. Whether instability is manifested as loss or gain seems to be influenced by the chromosomal loci involved. Because telomere shortening and CIN are early events in Barrett's esophagus neoplastic progression and are highly variable among patients, it will be important to determine whether they identify a subset of patients that is at risk for more rapid neoplastic evolution.

Quantitative fluorescence in situ hybridization (QFISH) of telomere lengths in tissue and cells.

Telomeres are repetitive DNA sequences at the end of each chromosome that provide stability and prevent end-to-end chromosome fusions. In order to understand mechanisms responsible for telomere shortening, it is necessary to develop methods for accurate telomere length measurement that can be applied to archival and fresh tissue and cells. This unit describes in situ-based quantitative fluorescence in situ hybridization (QFISH) protocols using a fluorescence-conjugated telomere probe (peptide nucleic acid, PNA) that stains telomeres proportionally to their length. These protocols can be used on formalin-fixed paraffin-embedded tissue, lightly fixed tissue, cells isolated from tissue, cultured cells, and agar-embedded cells. The basic protocol for QFISH staining is modified to achieve excellent QFISH staining for a variety of cell preparations. Image-analysis techniques to quantitate average telomere lengths from tissues and isolated stained cells are also described.

Distinct functions for WRN and TP53 in a shared pathway of cellular response to 1-beta-D-arabinofuranosylcytosine and bleomycin.

Mutations in the WRN or the TP53 genes lead to spontaneous genetic instability, an elevated risk of tumor formation, and sensitivity to compounds that interfere with DNA replication, such as camptothecin and DNA interstrand cross-linking drugs. We investigated the hypothesis that WRN and TP53 are involved in cellular responses to DNA replication-blocking lesions by exposing WRN deficient and TP53 mutant lymphoblastoid cell lines (LCLs) to 1-beta-d-arabinofuranosylcytosine (AraC) and bleomycin. Loss of WRN or TP53 function resulted in induction of apoptosis and lesser proliferative survival in response to AraC and bleomycin. WRN and TP53 operate in a shared DNA damage response pathway, since in cells in which TP53 was inactivated by SV-40 transformation, no difference in AraC and bleomycin sensitivity was found regardless of WRN status. In contrast to TP53 mutant LCLs, WRN-deficient cells showed unaffected cell cycle arrest after AraC and bleomycin exposure, which indicates that WRN is not involved in DNA damage-activated cell cycle arrest. Neither WRN nor TP53 deficiency affected cellular recovery from exposure to AraC and bleomycin, which disagrees with a direct role in repair of these DNA lesions. Our results indicate that WRN and TP53 perform different functions in a shared DNA damage response pathway.

Telomere length assessment in tissue sections by quantitative FISH: image analysis algorithms.

BACKGROUND: Telomeres are tandem repeated DNA sequences at the ends of every chromosome, which cap, stabilize, and prevent chromosome fusions and instability. Telomere regulation is an important mechanism in cellular proliferation and senescence in normal diploid and neoplastic cells. Quantitative methods to assess telomere lengths are essential to understanding how telomere dynamics play a role in these processes. METHODS: Telomere lengths have been conventionally measured using terminal restriction fragment (TRF), quantitative fluorescence in situ hybridization (QFISH), and flow FISH. In this study, we have applied QFISH to measure average telomere lengths in cultured cells and human tissues of the GI tract. Importantly, this method can be used to analyze telomere lengths in sections using confocal microscopy. We describe and compare three image analysis algorithms: a simple pixel histogram calculation of background corrected fluorescence, a telomere spot-finding method, and a background curve subtraction algorithm. RESULTS: Using normal human diploid fibroblasts (NHDF) either dropped on slides or sectioned after agar embedding, similar telomere length shortening is evident with increasing population doubling levels (PDLs), using peptide nucleic acid (PNA) and an N3'-P5'-phosphoamidate (PA) oligonucleotide probe for all three methods. Validation of these in situ telomere quantification methods showed excellent agreement with the commonly used telomere repeat fragment-Southern blot method. Telomere length reductions can also be demonstrated in tissue sections from histologically normal mucosa from patients with chronic ulcerative colitis (with dysplasia or cancer elsewhere in the colon), in colon adenomas, and in mucosal biopsies from patients with Barrett's esophagus. Both on slides and in tissue sections, the telomere spot-finding method has the greatest variability, while intra- and inter-biopsy variability in telomere length assessment using the other methods is relatively low. CONCLUSIONS: Accurate and reproducible telomere length measurements can be made in tissue sections using QFISH and confocal microscopy. The simplest methods proved the most reliable and make these methods readily accessible to many laboratories. The use of these methods will enhance the ability to measure telomere lengths in tissue samples and aid in the understanding of the role of telomere length in aging and disease.

Chromosomal instability in ulcerative colitis is related to telomere shortening.

Ulcerative colitis, a chronic inflammatory disease of the colon, is associated with a high risk of colorectal carcinoma that is thought to develop through genomic instability. We considered that the rapid cell turnover and oxidative injury observed in ulcerative colitis might accelerate telomere shortening, thereby increasing the potential of chromosomal ends to fuse, resulting in cycles of chromatin bridge breakage and fusion and chromosomal instability associated with tumor cell progression. Here we have used quantitative fluorescence in situ hybridization to compare chromosomal aberrations and telomere shortening in non-dysplastic mucosa taken from individuals affected by ulcerative colitis, either with (UC progressors) or without (UC non-progressors) dysplasia or cancer. Losses, but not gains, of chromosomal arms and centromeres are highly correlated with telomere shortening. Chromosomal losses are greater and telomeres are shorter in biopsy samples from UC progressors than in those from UC non-progressors or control individuals without ulcerative colitis. A mechanistic link between telomere shortening and chromosomal instability is supported by a higher frequency of anaphase bridges--an intermediate in the breakage and fusion of chromatin bridges--in UC progressors than in UC non-progressors or control individuals. Our study shows that telomere length is correlated with chromosomal instability in a precursor of human cancer.

Werner syndrome diploid fibroblasts are sensitive to 4-nitroquinoline-N-oxide and 8-methoxypsoralen: implications for the disease phenotype.

The clinical phenotype of Werner Syndrome (WRN) includes features reminiscent of accelerated aging and an increased incidence of sarcomas and other tumors of mesenchymal origin. This syndrome results from mutations in the WRN DNA helicase/exonuclease gene. We found that WRN deficient primary fibroblasts, as well as lymphoblastoid cell lines (LCLs), show reduced proliferative survival in response to 4-nitroquinoline-N-oxide (4NQO) and 8-methoxypsoralen (8MOP), compared with WRN-proficient cells. This is the first demonstration of drug hypersensitivity in primary cells of mesenchymal origin from WRN patients. Notably, 8MOP-induced DNA interstrand crosslinks, but not 8MOP mono-adducts, produced S-phase apoptosis in WRN-deficient LCLs. In contrast, 8MOP did not induce S-phase apoptosis in WRN-deficient diploid fibroblasts, in which drug hypersensitivity was entirely due to reduced cell proliferation. Such reduced proliferation of damaged mesenchymal cells in WRN patients may lead to earlier proliferative senescence. In addition, failure of WRN-deficient mesenchymal cells to undergo apoptosis in response to DNA damage in S-phase may promote genomic instability and could help clarify the increased risk of sarcoma in WRN patients. Because interstrand crosslinks are believed to be repaired through homologous recombination, these results suggest an important role for WRN in recombinational resolution of stalled replication forks.

Flow cytometric analysis of the cell cycle phase specificity of DNA damage induced by radiation, hydrogen peroxide and doxorubicin.

We have optimized a flow cytometric DNA alkaline unwinding assay to increase the sensitivity in detecting low levels of DNA damage (strand breaks and alkali-labile sites) and to permit the measurement of the extent of DNA damage within each cell cycle compartment. The lowest gamma radiation dose that induced detectable DNA damage in each cell cycle phase of HeLa and CEM cells was 10 cGy. The lowest H(2)O(2) concentration that induced detectable DNA damage in each cell cycle phase was 0.5 microM in HeLa cells, and 1-2.5 TmicroM in CEM cells. For both HeLa cells and CEM cells, DNA damage in each cell cycle compartment increased approximately linearly with increasing doses of gamma radiation and H(2)O(2). Although untreated HeLa and CEM cells in S phase consistently exhibited greater DNA unwinding than did G(1) or G(2) cells (presumably due to DNA strand breaks associated with replication forks), there was no difference between the susceptibility of G(0)/G(1), S and G(2)/M phase cells to DNA damage induced by gamma radiation or H(2)O(2), or in the rate of repair of this damage. In each cell cycle phase, the susceptibility to gamma radiation-induced DNA damage was greater in CEM cells than in HeLa cells. In contrast to the lack of cell cycle phase-specific DNA damage induced by exposure to gamma radiation or H(2)O(2), the cancer chemotherapeutic drug doxorubicin (adriamycin) predominantly induced DNA damage in G(2) phase cells.