A RAC-GEF network critical for early intestinal tumourigenesis.

RAC1 activity is critical for intestinal homeostasis, and is required for hyperproliferation driven by loss of the tumour suppressor gene Apc in the murine intestine. To avoid the impact of direct targeting upon homeostasis, we reasoned that indirect targeting of RAC1 via RAC-GEFs might be effective. Transcriptional profiling of Apc deficient intestinal tissue identified Vav3 and Tiam1 as key targets. Deletion of these indicated that while TIAM1 deficiency could suppress Apc-driven hyperproliferation, it had no impact upon tumourigenesis, while VAV3 deficiency had no effect. Intriguingly, deletion of either gene resulted in upregulation of Vav2, with subsequent targeting of all three (Vav2-/- Vav3-/- Tiam1-/-), profoundly suppressing hyperproliferation, tumourigenesis and RAC1 activity, without impacting normal homeostasis. Critically, the observed RAC-GEF dependency was negated by oncogenic KRAS mutation. Together, these data demonstrate that while targeting RAC-GEF molecules may have therapeutic impact at early stages, this benefit may be lost in late stage disease.

High speed optically sectioned fluorescence lifetime imaging permits study of live cell signaling events.

We present a time domain optically sectioned fluorescence lifetime imaging (FLIM) microscope developed for high-speed live cell imaging. This single photon excited system combines wide field parallel pixel detection with confocal sectioning utilizing spinning Nipkow disc microscopy. It can acquire fluorescence lifetime images of live cells at up to 10 frames per second (fps), permitting high-speed FLIM of cell dynamics and protein interactions with potential for high throughput cell imaging and screening applications. We demonstrate the application of this FLIM microscope to real-time monitoring of changes in lipid order in cell membranes following cholesterol depletion using cyclodextrin and to the activation of the small GTP-ase Ras in live cells using FRET.

Prenatal diagnosis and characterization of an unbalanced whole arm translocation resulting in monosomy for 18p.

Monosomy for the short arm of chromosome 18 is one of the most frequent autosomal deletions observed. While most cases result from terminal deletion of 18p, 16% of cases reported were as a result of an unbalanced whole arm translocation resulting in monosomy 18p. The origin and structure of these derivative chromosomes were reported in only a few cases. We report the prenatal diagnosis and characterization of a new case of monosomy 18p as a result of an unbalanced whole arm translocation. Amniocentesis was performed at 15 weeks of gestation on a 34-year-old woman initially referred for advanced maternal age. Holoprosencephaly was identified by ultrasound at the time of amniocentesis. Karyotype analysis showed an unbalanced whole arm translocation between the long arm of one chromosome 18 and the long arm of one chromosome 22, 45,XX,der(18;22)(q10;q10), in all metaphases. In effect, the fetus had monosomy for 18p. Parental karyotypes were normal, suggesting a de novo origin for the der(18;22). Fluorescence in situ hybridization (FISH) analysis was performed with alpha-satellite probes D18Z1 and D14Z1/D22Z1 to identify the origin of the centromere on the der(18;22). Signal was observed with both probes, indicating that the centromere was composed of alpha-satellite DNA from both constituent chromosomes. Genotyping of the fetus and her parents with chromosome 18p STS marker D18S391 showed only the paternal 187 bp allele was present in the fetus, indicating that it was the maternal chromosome 18 involved in the der(18;22). This case and previous reports show that de novo unbalanced whole arm translocations are more likely to retain alpha-satellite sequences from the two chromosomes involved.

Candidate region for Coffin-Siris syndrome at 7q32-->34.

Coffin-Siris syndrome is characterized by intrauterine growth retardation, mental deficiency, coarse face, hypoplastic fifth fingers and nails, hirsutism, and initial difficulties with feeding. The etiology of this syndrome is unknown. We report on an 11-year-old girl with Coffin-Siris syndrome and a de novo, apparently balanced reciprocal translocation between chromosomes 7 and 22 [t(7;22)(q32;q11.2)]. The 7q breakpoint in our patient is very similar to the breakpoint reported in a previous case [McPherson et al., 1997: Am J Med Genet 71:430-433] with a balanced t(1;7)(q21.3;q34). Together, these patients provide evidence that the region 7q32-->34 is a candidate region for the gene responsible for Coffin-Siris syndrome.

Monosomy 16 as the sole abnormality in myeloid malignancies.

The majority of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) patients reported with chromosome 16 abnormalities had the inv(16)(p13q22) or t(16;16)(p13;q22) rearrangements, which were associated with a favorable prognosis. In contrast, del(16)(q22) was reported less commonly but was associated with a less favorable prognosis. We describe an 80-year-old woman who presented with MDS (refractory anemia). Chromosome analysis from bone marrow aspirate cultures showed monosomy 16 as the sole cytogenetic abnormality. Comparison of this patient with previously reported cases of monosomy 16 showed that this uncommon abnormality was associated with myeloid disorders. Monosomy 16 patients, similar to del(16)(q22) patients, tended to be elderly, presented with MDS or AML, and had a poor prognosis. The similarity in clinical course for del(16)(q22) and monosomy 16 patients suggests that the phenotype in both groups resulted from loss of important gene(s) on 16q, as distinct from the fusion gene product identified in the inv(16) and t(16;16) rearrangements.

Genomic instability induced by ionizing radiation.

Genomic instability is characterized by the increased rate of acquisition of alterations in the mammalian genome. These changes encompass a diverse set of biological end points including karyotypic abnormalities, gene mutation and amplification, cellular transformation, clonal heterogeneity and delayed reproductive cell death. The loss of stability of the genome is becoming accepted as one of the most important aspects of carcinogenesis, and the numerous genetic changes associated with the cancer cell implicate genomic stability as contributing to the neoplastic phenotype. Multiple metabolic pathways govern the accurate duplication and distribution of DNA to progeny cells; other pathways maintain the integrity of the information encoded by DNA and regulate the expression of genes during growth and development. For each of these functions, there is a normal baseline frequency at which errors occur, leading to spontaneous mutations and other genomic anomalies. This review summarizes the current status of knowledge about radiation-induced genomic instability. Those events and processes likely to be involved in the initiation and perpetuation of the unstable phenotype, the potential role of epigenetic factors in influencing the onset of genomic instability, and the delayed effects of cellular exposure to ionizing radiation are discussed.

Natural antimutagenic agents.

Following a brief review of recent discoveries in the field of natural antimutagenic and tumor chemopreventive agents, contemporary findings in the author's laboratories employing the direct acting mutagen, ethyl methanesulfonate, in modified Ames tests and eukaryotic murine FM3A mammary tumor cells modified to be subject to thymidine-less death are described to illustrate the underlying principles. The EMS studies are illustrated with the isolation of the novel antimutagen, plicatin B, from the medicinal plants, Psoralea juncaea and P. plicata. The FM3A studies are carried out with extracts of Styrax asiatica, a plant previously studied extensively with the EMS system. The FM3A findings closely parallel the earlier work with EMS showing that the responsible agents, cinnamic acid, cinnamoyl ricinoleate and cinnamoyl cinnamate are effective both in prokaryotic and eukaryotic tests and that the new FM3A assay system has useful properties for screening and assay of novel antimutagenic agents.

Synthesis and biological evaluation of 4-fluoro-, 7-fluoro-, and 4,7-difluoro-5,6-dihydroxytryptamines.

The 5,6-dihydroxytryptamine (5,6-DHT) derivatives 4-fluoro- and 7-fluoro-5,6-DHTs (26a,b) and 4,7-difluoro-5,6-DHT (26c) were synthesized from 3-fluoroanisole (1) and 1,4-difluoro-2,3-dimethoxybenzene (13), respectively. Efficient methods were developed for the conversion of 1 to 4-fluoro- and 7-fluoro-5,6-bis(benzyloxy)indoles (12a,b, respectively), and 13 to 4,7-difluoro-5,6-[( diphenylmethylene)dioxy]indole (19) via reductive cyclization of 2-nitro-beta-(dialkylamino)styrenes prepared in situ from 2-nitrotoluenes. Indoles 12a,b and 19 were then converted to 26a-c via the corresponding indole-3-acetonitriles. The fluorine-substituted 5,6-DHTs displayed increased phenol acidities, determined spectrophotometrically, and decreased inherent potential to undergo oxidation as determined by cyclic voltammetry. Fluorine substitution did not have a significant adverse effect on the cytotoxic potential as judged from the IC50 values of 117, 125, 135, and 92 microM for 26a,c and 5,6-DHT, respectively, for the inhibition of incorporation of [3H]thymidine into the DNA of neuroblastoma clone N-2a cells in culture. Surprisingly, 26a-c exhibited 32-, 23-, and 13-fold higher affinities, respectively, compared to 5,6-DHT for the serotonergic uptake system of N-2a cells as measured by the ability of 26a-c and 5,6-DHT to antagonize the uptake of [3H]5-HT into the N-2a cells. These desirable chemical and biological properties of 26a-c should make them useful tools for the study of the molecular mechanism of neurodegenerative action of 5,6-DHT.

The FDA report on intraocular lenses.

Clinical studies of intraocular lenses (IOLs) as investigational devices have been regulated in the United States by the Food and Drug Administration (FDA) since 9 February 1978. As of August 1982, data have been collected on more than one million IOLs implanted. During the last 12 months of the study, 409 000 IOLs were implanted. Visual acuity of 20/40 or better at one year after surgery was present in 85% of over 45 000 cases reviewed. Increasing patient age, surgical problems, postoperative complications, and adverse reactions were factors that reduced the visual acuity. The current trend in the USA is for implantation of posterior chamber and anterior chamber IOLs.

The FDA report on intraocular lenses.

Clinical studies of intraocular lenses (IOLs) as investigational devices have been regulated in the United States by the Food and Drug Administration (FDA) since February 9, 1978. As of August 1982, data have been collected on more than one million IOLs implanted. During the last 12 months of the study, 409,000 IOLs were implanted. Visual acuity of 20/40 or better at one year after surgery was present in 85% of over 45,000 cases reviewed. Increasing patient age, surgical problems, postoperative complications, and adverse reactions were factors that reduced the visual acuity. The current trend in the USA is for implantation of the posterior chamber and anterior chamber IOLs.

Measurement of liver blood flow in the rat using an 85Krypton clearance technique.

A method for the measurement of liver blood flow (LBF) in rats has been developed by recording the clearance of 85Kr from the liver after injection of a solution of the gas into the portal vein. This technique provides a measure of tissue perfusion at a cellular level. The animal preparation was maintained in a stable haemodynamic and respiratory state for up to 4-3 h by careful fluid and anaesthetic control. Stability of the model was reflected by relative constancy of the LBF measured throughout the experiment in all of the animals studied. The mean LBF determined in six rats was 2-41+/-(s.d.) 0-50 ml/g/min.