Factors associated with undercorrected refractive errors in an older population: the Blue Mountains Eye Study.

AIMS: To identify characteristics of people with clinically relevant undercorrected refractive errors. METHODS: The Blue Mountains Eye Study was a population based survey of 3654 Australians aged 49-97 years. Examinations included a standardised refraction and measurement of presenting and best corrected visual acuity. Clinically relevant undercorrected refractive error was defined as improvement of >/=10 letters (2+ lines on the logMAR chart) in subjects with presenting acuity 6/9 or worse. Associations with a range of demographic and ocular variables were explored, adjusting for age and sex, presented as odds ratios (OR) with 95% confidence intervals (CI). RESULTS: Undercorrected refractive error was present in 814/3654 subjects (10.2%). Older age (p <0.001), hyperopia (OR 1.45, CI 1.15 to 1.83), longer interval from last eye examination (p <0.001), past occupation as tradesperson (OR 1.64, 1.13 to 3.29) or labourer (OR 2.00, CI 1.39 to 2.89), receipt of government pension (OR 1.47, CI 1.12 to 1.94), and living alone (OR 1.34, CI 1.05 to 1.72) were all associated with undercorrected refractive error. Past or current use of distance glasses (OR 0.25, CI 0.20 to 0.32) and driving (OR 0.67, CI 0.52 to 0.86) were associated with a lower prevalence. CONCLUSIONS: Increasing age and measures of socioeconomic disadvantage and isolation were found to predict undercorrected refractive error. Given the documented impacts from correctable visual impairment, these findings suggest a need to target education and eye care services.

Transforming growth factor-beta-induced growth inhibition in a Smad4 mutant colon adenoma cell line.

Transforming growth factor-beta (TGF-beta) inhibits growth and induces apoptosis of colon epithelial cells. Binding of TGF-beta to its receptor induces phosphorylation of the Smad proteins Smad2 and Smad3, which then form heteromeric complexes with Smad4, translocate to the nucleus, and activate gene transcription. Smad4 function has been considered an obligate requirement for TGF-beta signaling, and Smad4 mutations present in some cancers have been considered sufficient to inactivate TGF-beta signaling. In this work, we describe studies with a nontransformed human colon epithelial cell line that is mutant for Smad4 but remains growth-inhibited by TGF-beta. The colon cell line VACO-235 has lost one of its Smad4 alleles via a chromosome 18q deletion. The remaining allele bears two missense point mutations located in regions important for Smad4 trimer formation, which is thought necessary for Smad4 function. As expected, pSBE4-BV/Luc, a Smad4-activated transcriptional reporter, was inactive in VACO-235. Nonetheless, VACO-235 demonstrated 80% growth inhibition in response to TGF-beta, as well as retention of some TGF-beta-mediated activation of the p3TP-Lux transcriptional reporter. Transient transfection of the VACO-235 Smad4 mutant allele into a Smad4-null cell line confirmed that this allele is functionally inactive as assayed by both the pSBE4-BV and p3TP-Lux reporters. The simplest explanation of these results is that there is a non-Smad4-dependent pathway for TGF-beta-mediated signaling and growth inhibition in VACO-235 cells.

Mechanisms underlying losses of heterozygosity in human colorectal cancers.

Losses of heterozygosity are the most common molecular genetic alteration observed in human cancers. However, there have been few systematic studies to understand the mechanism(s) responsible for losses of heterozygosity in such tumors. Here we report a detailed investigation of the five chromosomes lost most frequently in human colorectal cancers. A total of 10,216 determinations were made with 88 microsatellite markers, revealing 245 chromosomal loss events. The mechanisms of loss were remarkably chromosome-specific. Some chromosomes displayed complete loss such as that predicted to result from mitotic nondisjunction. However, more than half of the losses were associated with losses of only part of a chromosome rather than a whole chromosome. Surprisingly, these losses were due largely to structural alterations rather than to mitotic recombination, break-induced replication, or gene conversion, suggesting novel mechanisms for the generation of much of the aneuploidy in this common tumor type.

Molecular detection of smad2/smad4 alterations in colorectal tumors.

The signaling pathways mediated by the transforming growth factor-ß (TGF-ß) family of factors are implicated in a wide array of biological processes including cell differentiation and proliferation, determination of cell fate during embryogenesis, cell adhesion and cell death. The recent discovery of the SMAD family of signal transducer proteins as mediators of TGF-ß relaying signals from cell membrane to nucleus has revolutionized the understanding of the molecular basis of these processes (1,2). To date, at least eight homologues of the Smad genes have been identified and shown to be downstream of the serine/threonine kinase receptors Table 1 ). SMADs are molecules of relative mass 42K-60K composed of two regions of homology at the amino and carboxy terminals of the protein. The activation of SMADs by receptors upon TGF-ß binding results in the formation of hetero-oligomeric complexes and translocation to the nucleus where transcription of target genes is effected. However, some of the SMADs apparently inhibit rather than mediate, TGF-ß signaling. These inhibitory SMADs are also induced by TGF-ß stimulation suggesting that there is an intracellular negative-feedback loop. Table 1 Human SMAD Genes and Cancers Gene Map position Affected cancers Reference(s) SMAD1 4q28-31 None 24 SMAD2 18q21 Colon 24 , 26 , 32 SMAD3 15q21-22 None 24 , 33 SMAD4 18q21 Lung, pancreatic,and colon 22-25 , 33 SMAD5 5q31 None 24 , 33 SMAD6 15q21-22 None 24 , 33 SMAD7 18q21 None 24 , 33 , 34 SMAD8/MADH6 13q12-14 None 35.

Mutational inactivation of transforming growth factor beta receptor type II in microsatellite stable colon cancers.

We previously demonstrated that mutational inactivation of transforming growth factor beta type II receptors (RIIs) is very common among the 13% of human colon cancers with microsatellite instability. These mutations principally cluster in the BAT-RII polyadenine sequence repeat. Among microsatellite stable (MSS) colon cancers, we now find that non-BAT-RII point mutations inactivate RII in another 15% of cases, thus doubling the known number of colon cancers in which RII mutations are pathogenetic. Functional analysis confirms that these mutations inactivate RII signaling. Moreover, another 55% of MSS colon cancers demonstrate a transforming growth factor beta signaling blockade distal to RII. The transforming growth factor beta pathway and RII in particular are major targets for inactivation in MSS colon cancers as well as in colon cancers with microsatellite instability.

Homeosis and polyposis: a tale from the mouse.

Homeobox genes play essential roles in specifying the fates of different cell types during embryogenesis. In Drosophila, the homeotic gene caudal is important for the generation of posterior structures. In the mouse, the caudal homologue Cdx2 has been implicated in directing early processes in intestinal morphogenesis and in the maintenance of the differentiated phenotype. A recent study showed that Cdx2 null mutation was embryonically lethal, whereas Cdx2+/- mice developed multiple intestinal polyps in the proximal colon in addition to developmental defects. There are striking phenotypic similarities and differences between Cdx2+/- and other mice predisposed to intestinal neoplasia. The possible role of Cdx2 in human colorectal tumorigenesis is discussed.

Frequency of Smad gene mutations in human cancers.

Much excitement has recently been generated by the discovery of the Smad genes, encoding proteins that transduce signals from the transforming growth factor beta family of cytokines. Here, we report the completion of cloning of the six known human Smads, providing novel sequences for Smad5 and Smad6. Previously, Smad4 and Smad2 were shown to be mutated in human cancers. However, analysis of the other four Smad genes revealed no mutations in a total of 167 tumors, including those from colon, breast, lung, and pancreas. These results suggest that the various Smad genes have different functions and demonstrate that mutations in these four genes do not, in general, account for the widespread resistance to transforming growth factor beta that is found in human tumors.

14-3-3sigma is a p53-regulated inhibitor of G2/M progression.

Exposure of colorectal cancer (CRC) cells to ionizing radiation results in a cell-cycle arrest in G1 and G2. The G1 arrest is due to p53-mediated induction of the cyclin-dependent kinase inhibitor p21WAF1/CIP1/SDI1, but the basis for the G2 arrest is unknown. Through a quantitative analysis of gene expression patterns in CRC cell lines, we have discovered that 14-3-3sigma is strongly induced by gamma irradiation and other DNA-damaging agents. The induction of 14-3-3sigma is mediated by a p53-responsive element located 1.8 kb upstream of its transcription start site. Exogenous introduction of 14-3-3sigma into cycling cells results in a G2 arrest. As the fission yeast 14-3-3 homologs rad24 and rad25 mediate similar checkpoint effects, these results document a molecular mechanism for G2/M control that is conserved throughout eukaryotic evolution and regulated in human cells by p53.

Evaluation of candidate tumour suppressor genes on chromosome 18 in colorectal cancers.

Chromosome deletions are the most common genetic events observed in cancer. These deletions are generally thought to reflect the existence of a tumour suppressor gene within the lost region. However, when the lost region does not precisely coincide with a hereditary cancer locus, identification of the putative tumour suppressor gene (target of the deletion) can be problematic. For example, previous studies have demonstrated that chromosome 18q is lost in over 60% of colorectal as well as in other cancers, but the lost region could not be precisely determined. Here we present a rigorous strategy for mapping and evaluating allelic deletions in sporadic tumours, and apply it to the evaluation of chromosome 18 in colorectal cancers. Using this approach, we define a minimally lost region (MLR) on chromosome 18q21, which contains at least two candidate tumour suppressor genes, DPC4 and DCC. The analysis further suggested genetic heterogeneity, with DPC4 the deletion target in up to a third of the cases and DCC or a neighbouring gene the target in the remaining tumours.

Mad-related genes in the human.

Resistance to the growth inhibitory effects of TGF-beta is common in human cancers. However, the mechanism(s) by which tumour cells become resistant to TGF-beta are generally unknown. We have identified five novel human genes related to a Drosophila gene called Mad which is thought to transduce signals from TGF-beta family members. One of these genes was found to be somatically mutated in two of eighteen colorectal cancers, and three of the other genes were located at chromosomal positions previously suspected to harbor tumour suppressor genes. These data suggest that this gene family may prove to be important in the suppression of neoplasia, imparting the growth inhibitory effects of TGF-beta-like ligands.

Evaluation of the FHIT gene in colorectal cancers.

A variety of studies suggests that tumor suppressor loci on chromosome 3p are important in various forms of human neoplasia. Recently, a chromosome 3p14.2 gene called FHIT was discovered and proposed as a candidate tumor suppressor gene in colorectal and other cancers. We evaluated the FHIT gene in a panel of colorectal cancer cell lines and xenografts, which allowed a comprehensive mutational analysis. A transcript containing the complete coding sequence was found to be expressed at robust levels in 29 of 31 cancers tested. The complete sequence of the coding region of the gene was determined and found to be normal in all 29 of these cases. These studies suggest either that FHIT is inactivated by an unusual mechanism or that it plays a role in relatively few colorectal tumors.

PAK1, a gene that can regulate p53 activity in yeast.

The ability of p53 protein to activate transcription is central to its tumor-suppressor function. We describe a genetic selection in Saccharomyces cerevisiae which was used to isolate a mutant strain defective in p53-mediated transcriptional activation. The defect was partially corrected by overexpression of a yeast gene named PAK1 (p53 activating kinase), which localizes to the left arm of chromosome IX. PAK1 is predicted to encode an 810-aa protein with regions of strong similarity to previously described Ser/Thr-specific protein kinases. PAK1 sequences upstream of the coding region are characteristic of those regulating genes involved in cell cycle control. Expression of PAK1 was associated with an increased specific activity of p53 in DNA-binding assays accompanied by a corresponding increase in transactivation. Thus, PAK1 is the prototype for a class of genes that can regulate the activity of p53 in vivo, and the system described here should be useful in identifying other genes in this class.

p53 tagged sites from human genomic DNA.

The product of the tumor suppressor gene p53 binds to DNA and activates transcription from promoters containing its consensus binding site. This activity has been hypothesized to be responsible for its biological effects. However, the total number and nature of human genomic sites with which p53 can functionally interact is unknown. In this paper, we have used a Saccharomyces cerevisiae-based screen to identify human genomic sequences that activate transcription from an adjacent reporter gene in a p53-dependent manner (p53-tagged sites, PTS). Fifty-seven different PTS were identified, and the total number of such sites in the human genome was predicted to be between 200 and 300. Almost all contained two adjacent copies of the previously defined consensus 5'-PuPuPuC(A/T)(T/A)GPyPyPy-3'. Spacing between the copies was found to be critical for sequence-specific transcriptional activation in vivo. These results further refine the nature of the genomic sequences likely to be most important for p53-mediated tumor suppression.

Sequence-specific transcriptional activation is essential for growth suppression by p53.

Although several biochemical features of p53 have been described, their relationship to tumor suppression remains uncertain. We have compared the ability of p53-derived proteins to act as sequence-specific transcriptional (SST) activators with their ability to suppress tumor cell growth, using an improved growth-suppression assay. Both naturally occurring and in vitro derived mutations that abrogated the SST activity of p53 lost the ability to suppress tumor cell growth. Additionally, the N- and C-terminal ends of p53 were shown to be functionally replaceable with foreign transactivation and dimerization domains, respectively, with concordant preservation of both SST and tumor-suppressive properties. Only the central region of p53, conferring specific DNA binding, was required to suppress growth by such hybrid proteins. The SST activity of p53 thus appeared to be essential for the protein to function as a tumor suppressor.

The multiple roles for ATP in the Escherichia coli UvrABC endonuclease-catalyzed incision reaction.

The biochemical properties of the Escherichia coli UvrA tandem ATPase site mutants in nucleotide excision repair have been studied. In these and earlier studies it was found that ATP binding is required for protein-protein and nucleoprotein association reactions, whereas the dissociation reactions are driven by the hydrolysis of ATP. The self-association of UvrA to form the reactive dimeric species UvrA2 is driven by nucleotide binding, but its dissociation from DNA requires ATP hydrolysis. Similarly, ATP binding drives those allosteric changes in DNA topology during UvrA2-nucleoprotein formation (Oh, E.Y., and Grossman, L. (1986) Nucleic Acids Res. 14, 8557-8571). The manifestation of the UvrB-associated cryptic ATPase requires UvrA and DNA in a helicase-catalyzed supercoiling reaction. The UvrA2B helicase activity requires ATP hydrolysis by the C-terminal ATPase site of UvrA in addition to UvrB. ATP hydrolysis by the C-terminal ATPase site of UvrA also participates in the localization of damaged sites contributing to the formation of damage-specific high affinity nucleoprotein complexes. The levels of complementation to UV survival by the ATPase site mutants of UvrA (Thiagalingam, S., and Grossman, L. (1991) J. Biol. Chem. 266, 11395-11403) correspond to its ability to self-bind and translocate in combination with the UvrB subunit in its search for damaged sites during the preincision mode of nucleotide excision.

Oncoprotein MDM2 conceals the activation domain of tumour suppressor p53.

The tumour-suppressor gene p53 is inactivated in most human malignancies either by missense mutations or by binding to oncogenic proteins. In human soft tissue sarcomas, inactivation apparently results from MDM2 gene amplification. MDM2 is an oncogene product that may function by binding to p53 and inhibiting its ability to activate transcription. Here we show that, when expressed in Saccharomyces cerevisiae, human MDM2 inhibits human p53's ability to stimulate transcription by binding to a region that nearly coincides with the p53 acidic activation domain. The isolated p53 activation domain fused to another DNA-binding protein is also inactivated by MDM2, confirming that MDM2 can inhibit p53 function by concealing the activation domain of p53 from the cellular transcription machinery.

Oncogenic forms of p53 inhibit p53-regulated gene expression.

Mutant forms of the gene encoding the tumor suppressor p53 are found in numerous human malignancies, but the physiologic function of p53 and the effects of mutations on this function are unknown. The p53 protein binds DNA in a sequence-specific manner and thus may regulate gene transcription. Cotransfection experiments showed that wild-type p53 activated the expression of genes adjacent to a p53 DNA binding site. The level of activation correlated with DNA binding in vitro. Oncogenic forms of p53 lost this activity. Moreover, all mutants inhibited the activity of coexpressed wild-type p53, providing a basis for the selection of such mutants during tumorigenesis.

Both ATPase sites of Escherichia coli UvrA have functional roles in nucleotide excision repair.

The roles of the two tandemly arranged putative ATP binding sites of Escherichia coli UvrA in UvrABC endonuclease-mediated excision repair were analyzed by site-directed mutagenesis and biochemical characterization of the representative mutant proteins. Evidence is presented that UvrA has two functional ATPase sites which coincide with the putative ATP binding motifs predicted from its amino acid sequence. The individual ATPase sites can independently hydrolyze ATP. The C-terminal ATPase site has a higher affinity for ATP than the N-terminal site. The invariable lysine residues at the ends of the glycine-rich loops of the consensus Walker type "A" motifs are indispensable for ATP hydrolysis. However, the mutations at these lysine residues do not significantly affect ATP binding. UvrA, with bound ATP, forms the most favored conformation for DNA binding. The initial binding of UvrA to DNA is chiefly at the undamaged sites. In contrast to the wild type UvrA, the ATPase site mutants bind equally to damaged and undamaged sites. Dissociation of tightly bound nucleoprotein complexes from the undamaged sites requires hydrolysis of ATP by the C-terminal ATPase site of UvrA. Thus, both ATP binding and hydrolysis are required for the damage recognition step enabling UvrA to discriminate between damaged and undamaged sites on DNA.

ATPase activity of the UvrA and UvrAB protein complexes of the Escherichia coli UvrABC endonuclease.

We have analyzed the ATPase activity exhibited by the UvrABC DNA repair complex. The UvrA protein is an ATPase whose lack of DNA dependence may be related to the ATP induced monomer-dimer transitions. ATP induced dimerization may be responsible for the enhanced DNA binding activity observed in the presence of ATP. Although the UvrA ATPase is not stimulated by dsDNA, such DNA can modulate the UvrA ATPase activity by decreases in Km and Vm and alterations in the Ki for ADP and ATP-gamma-S. The induction of such changes upon binding to DNA may be necessary for cooperative interactions of UvrA with UvrB that result in a DNA stimulated ATPase for the UvrAB protein complex. The UvrAB ATPase displays unique kinetic profiles that are dependent on the structure of the DNA effector. These kinetic changes correlate with changes in footprinting patterns, the stabilization of protein complexes on DNA damage and with the expression of helicase activity.