Differential regulation of telomerase activity and telomerase RNA during multi-stage tumorigenesis.

Telomeres consist of short repeated sequences that are synthesized by telomerase, a ribonucleo-protein DNA polymerase. Telomerase activity is present in many tumours and not detected in many normal tissues. Telomere shortening in human and mouse tissues and primary cell cultures may be due to the absence of telomerase activity. To determine when telomerase is activated during tumour development and progression, we examined telomerase activity and expression of the recently cloned mouse telomerase RNA component (mTR) in two different transgenic mouse models of multi-stage tumorigenesis. These mouse models allow examination of many independent tumours from genetically identical individuals. These mice reproducibly develop pancreatic islet cell carcinoma and squamous cell carcinoma of the skin. In both carcinoma types, we detected telomerase activity only in late-stage tumours; in contrast, we found mTR levels were upregulated in the early preneoplastic stages, and further increased during progression. Surprisingly, mTR levels did not parallel the amount of telomerase activity detected and a subset of tumours lacked telomerase activity and yet expressed telomerase RNA. Regulation of telomerase activity may therefore be separable from expression of its RNA component. These results clearly demonstrate telomerase is activated in late stages of tumour progression, and show for the first time that the initial up regulation of telomerase RNA is an early event, concurrent with the hyperproliferation elicited by viral oncogenes.

Cationic liposomes target angiogenic endothelial cells in tumors and chronic inflammation in mice.

This study sought to determine whether angiogenic blood vessels in disease models preferentially bind and internalize cationic liposomes injected intravenously. Angiogenesis was examined in pancreatic islet cell tumors of RIP-Tag2 transgenic mice and chronic airway inflammation in Mycoplasma pulmonis-infected C3H/HeNCr mice. For comparison, physiological angiogenesis was examined in normal mouse ovaries. We found that endothelial cells in all models avidly bound and internalized fluorescently labeled cationic liposomes (1,2-dioleoyl-3-trimethylammonium-propane [DOTAP]/cholesterol or dimethyldioctadecyl ammonium bromide [DDAB]/cholesterol) or liposome-DNA complexes. Confocal microscopic measurements showed that angiogenic endothelial cells averaged 15-33-fold more uptake than corresponding normal endothelial cells. Cationic liposome-DNA complexes were also avidly taken up, but anionic, neutral, or sterically stabilized neutral liposomes were not. Electron microscopic analysis showed that 32% of gold-labeled liposomes associated with tumor endothelial cells were adherent to the luminal surface, 53% were internalized into endosomes and multivesicular bodies, and 15% were extravascular 20 min after injection. Our findings indicate that angiogenic endothelial cells in these models avidly bind and internalize cationic liposomes and liposome-DNA complexes but not other types of liposomes. This preferential uptake raises the possibility of using cationic liposomes to target diagnostic or therapeutic agents selectively to angiogenic blood vessels in tumors and sites of chronic inflammation.

A new donor cornea harvesting technique for posterior lamellar keratoplasty.

AIMS: To describe a technique for posterior lamellar keratoplasty donor preparation. METHODS: In an experimental study eight human donor research corneas were mounted onto an artificial anterior chamber and deep stromal pockets dissected. Four corneas were mounted in the standard endothelial side down orientation and dissected using standard instruments (group 1). Another four corneas were mounted endothelial side up and dissected using a flat spatula (group 2). Trephined lamellar graft thickness was assessed by ultrasound pachymetry. The grafts were also analysed using vital staining of the endothelium and standard histological preparation. RESULTS: Achieved posterior graft thickness was 118 (SD 32) microm (group 1) and 92 (23) microm (group 2) (p=0.324). Percentage of devitalised endothelial cells was 0.86% (1.48%) (group 1) and 3.9% (2.9%) (group 2) (p=0.185). The dissections using both harvesting techniques remained in plane and were smooth. CONCLUSIONS: A blunt spatula and endothelium side up orientation on an artificial anterior chamber can be used to create posterior lamellar dissections without compromising endothelial cell number or planarity when compared to standard endothelium side down harvest.

Device for the application of a dynamic biaxially uniform and isotropic strain to a flexible cell culture membrane.

A large number of studies have demonstrated that mechanical perturbation modulates cellular metabolism; however, the systematic characterization of the molecular and cellular transduction mechanisms underlying mechanically induced metabolic modulation has been impeded, in part, by the limitations of the mechanical device. The objective of this investigation was to develop an in vitro experimental system that would provide independent control of the spatial and temporal biaxial strain distribution imposed on a flexible transparent tissue culture membrane that permits attachment, proliferation, and maintenance of the phenotypic expression of cultured embryonic osteoblasts. Such a device would permit a systematic investigation of the cellular response to specific, independently controlled parameters of mechanical deformation. Using a prototype device designed to impose a dynamic sinusoidal spatially isotropic biaxial strain profile, we confirmed experimentally that the strain was biaxially uniform and isotropic (radial = circumferential strain over the entire culture membrane) to within 14% (SD/mean) for the range of the peak strains tested (2.3-9.4%). Additionally, the uniformity was maintained at 1 Hz for at least 5 days of continuous operation. This experimental verification of the theoretically predicted isotropic strain profile suggests that the design principle is sound. Embryonic osteoblasts cultured on the flexible substrate proliferated and exhibited a temporal pattern of phenotypic expression (extracellular matrix accumulation and mineralization) comparable with that observed on polystyrene of tissue culture grade.

Bloom syndrome: multiple retinopathies in a chromosome breakage disorder.

AIM: To describe multiple retinal abnormalities in a patient with Bloom syndrome, including early macular drusen, diabetic retinopathy, and the onset of leukaemic retinopathy. METHODS: Clinical data were collected over 1 year of follow up, and ocular abnormalities in Bloom syndrome were reviewed from the literature. RESULTS: A 39 year old man with a rare autosomal recessive "chromosome breakage" syndrome was followed. A variety of ocular findings have been reported in Bloom syndrome; this patient had hard drusen in both maculae, non-proliferative diabetic retinopathy, and haemorrhagic retinopathy as a herald of acute lymphocytic leukaemia. CONCLUSIONS: Bloom syndrome is a rare disorder of genomic instability, in which a variety of ocular abnormalities have been found. Described here are multiple retinal manifestations arising from characteristic systemic associations of diabetes mellitus and leukaemia, as well as macular hard drusen.

Bilateral diffuse lamellar keratitis following bilateral simultaneous versus sequential laser in situ keratomileusis.

AIM: To determine the difference in the incidence of bilateral diffuse lamellar keratitis (DLK) in patients undergoing simultaneous versus sequential laser in situ keratomileusis (LASIK) as an indication of intrinsic risk for inflammation. METHODS: A retrospective non-comparative case series of 1632 eyes that had undergone bilateral, simultaneous or sequential LASIK between April 1998 and February 2001 at a university based refractive centre by three surgeons. All cases that developed clinically evident DLK were identified and reviewed. In order to identify isolated cases and exclude those caused by environmental factors, when more than one patient in a given session developed DLK, the session was excluded. The main outcome measure was the incidence of unilateral and bilateral isolated, non-epidemic DLK. RESULTS: Of 1632 eyes, 126 eyes (7.7%) of 107 patients developed at least grade 1 DLK. In six operating sessions, DLK was observed in more than one patient per session, and on this basis 13 patients were excluded. 16 of the 94 remaining patients developed DLK in both eyes (17.0%). Six of 41 patients (14.6%) in the simultaneous group, versus 10 of 53 patients (18.9%) in the sequential group developed bilateral DLK (p >0.5). CONCLUSION: In isolated, non-epidemic bilateral DLK, a similar incidence was observed regardless of whether the surgery was simultaneous or sequential, suggesting an underlying intrinsic cause for DLK.

Mouse models for the study of telomerase.

The ends of chromosomes, or telomeres, consist of short repeated sequences that are synthesized by a ribonucleoprotein-DNA polymerase called telomerase. The RNA component of telomerase is essential for enzyme activity. The maintenance of telomere length by telomerase has been proposed to be essential for cellular viability and to play an important role in cellular senescence and immortalization. We are interested in using the mouse as a model system for the study of telomerase. We studied telomerase activity and expression of the mouse telomerase RNA component (mTR) in two different transgenic mouse models of multistage tumorigenesis: models of islet cell carcinoma and squamous cell carcinoma. In both tumour models, telomerase activity was detected only in late-stage tumours, whereas the telomerase RNA was present at higher than normal levels in pre-neoplastic stages and increased further in late-stage tumours. However, the RNA levels did not parallel the amounts of telomerase activity detected, suggesting that regulation of telomerase activity does not correlate with the regulation of its RNA component. These results establish a direct correlation between progression to late-stage tumours and induction of telomerase activity, and suggest that the initial upregulation of telomerase RNA is an early event. To address the role of telomerase during normal mouse development and tumour formation, we have constructed a knockout mouse for the mouse telomerase RNA, mTR-/-. These mice and the cell lines derived from them are telomerase deficient.