The GLI-Kruppel family of human genes.

Previous characterization of GLI, a gene found to be amplified and expressed in a subset of human brain tumors, revealed the presence of five tandem zinc fingers related to those of Krüppel (Kr), a Drosophila segmentation gene of the gap class. We have used the GLI cDNA as a molecular probe to isolate related sequences from the human genome. Partial characterization of six related loci, including sequence determination, expression studies, and chromosome localization, revealed that each locus could encode a separate finger protein. The predicted proteins all had similar H-C links, i.e., a conserved stretch of 9 amino acids connecting the C-terminal histidine of one finger to the N-terminal cysteine of the next. On the basis of amino acid sequence and intron-exon organization, the genes could be placed into one of two subgroups: the GLI subgroup (with the consensus finger amino acid sequence [Y/F]XCX3GCX3[F/Y]X5LX2HX3-4H[T/S]GEKP) or the Kr subgroup (with the consensus finger amino acid sequence [Y/F]XCX2CX3FX5LX2HXRXHTGEKP). Unlike GLI or Kr, most of the newly isolated genes were expressed in many adult tissues. The predicted proteins probably control the expression of other genes and, by analogy with Kr and GLI, may be important in human development, tissue-specific differentiation, or neoplasia.     

A molecular genetic linkage map of mouse chromosome 18 reveals extensive linkage conservation with human chromosomes 5 and 18

An interspecific backcross between C57BL/6J and Mus spretus was used to generate a molecular genetic linkage map of mouse chromosome 18 that includes 23 molecular markers and spans approximately 86% of the estimated length of the chromosome. The Apc, Camk2a, D18Fcr1, D18Fcr2, D18Leh1, D18Leh2, Dcc, Emb-rs3, Fgfa, Fim-2/Csfmr, Gnal, Grl-1, Grp, Hk-1rs1, Ii, Kns, Lmnb, Mbp, Mcc, Mtv-38, Palb, Pdgfrb, and Tpl-2 genes were mapped relative to each other in one interspecific backcross. A second interspecific backcross and a centromere-specific DNA satellite probe were used to determine the distance of the most proximal chromosome 18 marker to the centromere. The interspecific map extends the known regions of linkage homology between mouse chromosome 18 and human chromosomes 5 and 18 and identifies a new homology segment with human chromosome 10p. It also provides molecular access to many regions of mouse chromosome 18 for the first time.     

Molecular Cloning of the N-Terminus of GTBP

Defects in mismatch repair genes cause the genetic instability characteristic of hereditary nonpolyposis colorectal cancer and a subset of sporadic colon tumors. The newest member of the mismatch repair gene family,GTBP, has recently been identified as a partial cDNA. Here, we describe the isolation of its 5′ terminus, allowing definition of the entire coding region. Several polymorphisms within the 5′ end were identified and are presented.     

Mismatch Repair Genes on Chromosomes 2p and 3p Account for a Major Share of Hereditary Nonpolyposis Colorectal Cancer Families Evaluable by Linkage

Two susceptibility loci for hereditary nonpolyposis colo-rectal cancer (HNPCC) have been identified, and each contains a mismatch repair gene: MSH2 on chromosome 2p and MLH1 on chromosome 3p. We studied the involvement of these loci in 13 large HNPCC kindreds originating from three different continents. Six families showed close linkage to the 2p locus, and a heritable mutation of the MSH2 gene was subsequently found in four. The 2p-linked kindreds included a family characterized by the lack of extracolonic manifestations (Lynch I syndrome), as well as two families with cutaneous manifestations typical of the Muir-Torre syndrome. Four families showed evidence for linkage to the 3p locus, and a heritable mutation of the MLH1 gene was later detected in three. One 3p-linked kindred was of Amerindian origin. Of the remaining three families studied for linkage, one showed lod scores compatible with exclusion of both MSH2 and MLH1, while lod scores obtained in the other two families suggested exclusion of one HNPCC locus (MSH2 or MLH1) but were uninformative for markers flanking the other locus. Our results suggest that mismatch repair genes on 2p and 3p account for a major share of HNPCC in kindreds that can be evaluated by linkage analysis.     

Presymptomatic direct detection of adenomatous polyposis coli (APC) gene mutations in familial adenomatous polyposis

The recent identification of the familial adenomatous polyposis (FAP) gene (designated as APC) enables conclusive genetic testing of at-risk family members for the specific mutation in families in which the germline gene mutation has been characterized. Presymptomatic molecular diagnosis of FAP was performed by direct direction of mutations in lymphocyte DNA in four families. Each of the families has a different mutation of the APC gene. Twenty-seven offspring of affected individuals (a priori risk of 50%) were tested. Ten of the 27 had already developed clinical features of FAP. Of the remaining seventeen, two had had a negative colon exam at an early age, and nine had never had colon exams (mean age, 12.1±3.1 SD years). Six children from this group (54%) were found to carry their affected parent's mutation. No change in the conventional FAP colon screening regimen is recommended for these children. In contrast, when direct tests indicate that an individual does not have the FAP mutation, we recommended that screening be decreased. Reduction of uncertainty for at-risk FAP family members is an important benefit of genetic testing.     

Saikosaponin-d,a novel SERCA inhibitor,induces autophagic cell death in apoptosis-defective cells

Autophagy is an important cellular process that controls cells in a normal homeostatic state by recycling nutrients to maintain cellular energy levels for cell survival via the turnover of proteins and damaged organelles. However, persistent activation of autophagy can lead to excessive depletion of cellular organelles and essential proteins, leading to caspase-independent autophagic cell death. As such, inducing cell death through this autophagic mechanism could be an alternative approach to the treatment of cancers. Recently, we have identified a novel autophagic inducer, saikosaponin-d (Ssd), from a medicinal plant that induces autophagy in various types of cancer cells through the formation of autophagosomes as measured by GFP-LC3 puncta formation. By computational virtual docking analysis, biochemical assays and advanced live-cell imaging techniques, Ssd was shown to increase cytosolic calcium level via direct inhibition of sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase pump, leading to autophagy induction through the activation of the Ca²⁺/calmodulin-dependent kinase kinase–AMP-activated protein kinase–mammalian target of rapamycin pathway. In addition, Ssd treatment causes the disruption of calcium homeostasis, which induces endoplasmic reticulum stress as well as the unfolded protein responses pathway. Ssd also proved to be a potent cytotoxic agent in apoptosis-defective or apoptosis-resistant mouse embryonic fibroblast cells, which either lack caspases 3, 7 or 8 or had the Bax-Bak double knockout. These results provide a detailed understanding of the mechanism of action of Ssd, as a novel autophagic inducer, which has the potential of being developed into an anti-cancer agent for targeting apoptosis-resistant cancer cells.     

Distribution of immunoreactive mesotocin and vasotocin in the brain and pituitary of chickens

The distribution of vasotocin and mesotocin in the pituitary and central nervous system in male chickens was determined using radioimmunoassays. Neither peptide was detected in the pineal. Mesotocin, but not vasotocin, was detected in the cerebellum. Both peptides were found in the septal area, archistriatum, paleostriatum, optic lobe, anterior, medial and posterior hypothalamus, midbrain, pons, medulla oblongata, and the anterior and posterior pituitary. Equal amounts of the 2 peptides were present in the septal area, archistriatum and anterior hypothalamus whereas vasotocin was more abundant (2- to 10-fold) in the paleostriatum, optic lobe, midbrain, and pituitary. The amount of mesotocin was about twice that of vasotocin in the medulla oblongata and the medial and posterior hypothalamus. The wide distribution of vasotocin and mesotocin in extrahypothalamic sites in the central nervous system suggests that the peptides may, as in mammals, have a role in a variety of autonomic and endocrine regulatory processes in chickens.     

Genetic instability and darwinian selection in tumours

Genetic instability has long been hypothesized to be a cardinal feature of cancer. Recent work has strengthened the proposal that mutational alterations conferring instability occur early during tumour formation. The ensuing genetic instability drives tumour progression by generating mutations in oncogenes and tumour-suppressor genes. These mutant genes provide cancer cells with a selective growth advantage, thereby leading to the clonal outgrowth of a tumour. Here, we discuss the role of genetic instability in tumour formation and outline future work necessary to substantiate the genetic instability hypothesis.     

Use of isogenic human cancer cells for high-throughput screening and drug discovery

Cell-based screening for novel tumor-specific drugs has been compromised by the lack of appropriate control cells. We describe a strategy for drug screening based on isogenic human cancer cell lines in which key tumorigenic genes have been deleted by targeted homologous recombination. As a test case, a yellow fluorescent protein (YFP) expression vector was introduced into the colon cancer cell line DLD-1, and a blue fluorescent protein (BFP) expression vector was introduced into an isogenic derivative in which the mutant K-Ras allele had been deleted. Co-culture of both cell lines allowed facile screening for compounds with selective toxicity toward the mutant Ras genotype. Among 30,000 compounds screened, a novel cytidine nucleoside analog was identified that displayed selective activity in vitro and inhibited tumor xenografts containing mutant Ras. The present data demonstrate a broadly applicable approach for mining therapeutic agents targeted to the specific genetic alterations responsible for cancer development.