Rb and FZR1/Cdh1 determine CDK4/6-cyclin D requirement in C. elegans and human cancer cells.

Cyclin-dependent kinases 4 and 6 (CDK4/6) in complex with D-type cyclins promote cell cycle entry. Most human cancers contain overactive CDK4/6-cyclin D, and CDK4/6-specific inhibitors are promising anti-cancer therapeutics. Here, we investigate the critical functions of CDK4/6-cyclin D kinases, starting from an unbiased screen in the nematode Caenorhabditis elegans.

Caenorhabditis elegans cyclin D/CDK4 and cyclin E/CDK2 induce distinct cell cycle re-entry programs in differentiated muscle cells.

Cell proliferation and differentiation are regulated in a highly coordinated and inverse manner during development and tissue homeostasis. Terminal differentiation usually coincides with cell cycle exit and is thought to engage stable transcriptional repression of cell cycle genes. Here, we examine the robustness of the post-mitotic state, using Caenorhabditis elegans muscle cells as a model.

C. elegans MCM-4 is a general DNA replication and checkpoint component with an epidermis-specific requirement for growth and viability.

DNA replication and its connection to M phase restraint are studied extensively at the level of single cells but rarely in the context of a developing animal. C. elegans lin-6 mutants lack DNA synthesis in postembryonic somatic cell lineages, while entry into mitosis continues.

Functional conservation of the human EXT1 tumor suppressor gene and its Drosophila homolog tout velu.

Heparan sulfate proteoglycans play a vital role in signaling of various growth factors in both Drosophila and vertebrates. In Drosophila, mutations in the tout velu (ttv) gene, a homolog of the mammalian EXT1 tumor suppressor gene, leads to abrogation of glycosaminoglycan (GAG) biosynthesis. This impairs distribution and signaling activities of various morphogens such as Hedgehog (Hh), Wingless (Wg), and Decapentaplegic (Dpp).

Coordination of cell proliferation and differentiation: finding a GEM in the root?

How cells acquire specific fates in conjunction with cell division is a major developmental question. In a recent issue of Nature, Caro and colleagues describe the Arabidopsis protein GEM, which interacts both with DNA-replication and transcriptional regulators (Caro et al., 2007).

Quantitative analysis of Hedgehog gradient formation using an inducible expression system.

Background: The Hedgehog (Hh) family of secreted growth factors are morphogens that act in development to direct growth and patterning. Mutations in human Hh and other Hh pathway components have been linked to human diseases. Analysis of Hh distribution during development indicates that cholesterol modification and receptor mediated endocytosis affect the range of Hh signaling and the cellular localization of Hh.

Rasp, a putative transmembrane acyltransferase, is required for Hedgehog signaling.

Members of the Hedgehog (Hh) family encode secreted molecules that act as potent organizers during vertebrate and invertebrate development. Post-translational modification regulates both the range and efficacy of Hh protein. One such modification is the acylation of the N-terminal cysteine of Hh.

Nijmegen breakage syndrome. The International Nijmegen Breakage Syndrome Study Group.

Background: Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder. NBS-1, the gene defective in NBS, is located on chromosome 8q21 and has recently been cloned. The gene product, nibrin, is a novel protein, which is member of the hMre11/hRad50 protein complex, suggesting that the gene is involved in DNA double strand break repair.

Hedgehog movement is regulated through tout velu-dependent synthesis of a heparan sulfate proteoglycan.

Hedgehog (Hh) molecules play critical roles during development as a morphogen, and therefore their distribution must be regulated. Hh proteins undergo several modifications that tether them to the membrane. We have previously identified tout velu (ttv), a homolog of the mammalian EXT tumor suppressor gene family, as a gene required for movement of Hh.

Tout-velu is a Drosophila homologue of the putative tumour suppressor EXT-1 and is needed for Hh diffusion.

Hedgehog (Hh) proteins act through both short-range and long-range signalling to pattern tissues during invertebrate and vertebrate development. The mechanisms allowing Hedgehog to diffuse over a long distance and to exert its long-range effects are not understood. Here we identify a new Drosophila gene, named tout-velu, that is required for diffusion of Hedgehog.

Requirement of Drosophila NF1 for activation of adenylyl cyclase by PACAP38-like neuropeptides.

The human neurofibromatosis type 1 (NF1) tumor suppressor protein functions as a Ras-specific guanosine triphosphatase-activating protein, but the identity of Ras- mediated pathways modulated by NF1 remains unknown. A study of Drosophila NF1 mutants revealed that NF1 is essential for the cellular response to the neuropeptide PACAP38 (pituitary adenylyl cyclase-activating polypeptide) at the neuromuscular junction. The peptide induced a 100-fold enhancement of potassium currents by activating the Ras-Raf and adenylyl cyclase-adenosine 3',5'-monophosphate (cAMP) pathways.

Rescue of a Drosophila NF1 mutant phenotype by protein kinase A.

The neurofibromatosis type 1 (NF1) tumor suppressor protein is thought to restrict cell proliferation by functioning as a Ras-specific guanosine triphosphatase-activating protein. However, Drosophila homozygous for null mutations of an NF1 homolog showed no obvious signs of perturbed Ras1-mediated signaling. Loss of NF1 resulted in a reduction in size of larvae, pupae, and adults.

A functional assay for heterozygous mutations in the GTPase activating protein related domain of the neurofibromatosis type 1 gene.

The GTPase-activating protein related domain of the human neurofibromatosis type 1 protein (NF1GRD) can down-regulate RAS in Saccharomyces cerevisiae. Using a technique termed the FASAY method, for Functional Analysis of Separated Alleles in Yeast, we designed a rapid method for detection of heterozygous NF1GRD loss-of-function mutations. In our method, PCR amplified NF1GRD cDNA is directly cloned into a centromeric vector by homologous recombination in a cdc25 temperature-sensitive mutant strain expressing human Ha-ras.

Large E1B proteins of adenovirus types 5 and 12 have different effects on p53 and distinct roles in cell transformation.

The formation of complexes between oncoproteins of DNA tumor viruses and the cellular protein p53 is thought to result in inactivation of the growth suppressor function of p53. In cells transformed by nononcogenic human adenovirus type 5 (Ad5), the 55-kDa protein encoded by E1B forms a stable complex with p53 and sequesters it in the cytoplasm. However, the homologous 54-kDa protein of highly oncogenic Ad12 does not detectably associate with p53.

The expression of two P-glycoprotein (pgp) genes in transgenic Caenorhabditis elegans is confined to intestinal cells.

P-glycoproteins can cause multidrug resistance in mammalian tumor cells by active extrusion of cytotoxic drugs. The natural function of these evolutionarily conserved, membrane-bound ATP binding transport proteins is unknown. In mammals, P-glycoproteins are abundantly present in organs associated with the digestive tract.

Neurofibromatosis type 1 gene mutations in neuroblastoma.

The introduction of human chromosome 17 suppresses the tumourigenicity of a neuroblastoma cell line in the absence of any effects on in vitro growth and the neurofibromatosis type 1 (NF1) gene may be responsible. Here we report that 4 out of 10 human neuroblastoma lines express little or no neurofibromin and that two of these lines show evidence of NF1 mutations, providing further proof that NF1 mutations occur in tumours that are not commonly found in NF1 patients. We also show that NF1 deficient neuroblastomas show only moderately elevated ras-GTP levels, in contrast to NF1 tumour cells, indicating that neurofibromin contributes differently to the negative regulation of ras in different cell types.

The P-glycoprotein gene family of Caenorhabditis elegans. Cloning and characterization of genomic and complementary DNA sequences.

P-glycoproteins, encoded by families of evolutionarily conserved genes, can confer a multidrug-resistant phenotype to mammalian tumor cells. To obtain more information on their functions in normal cells we have cloned genomic and complementary DNA sequences of four P-glycoprotein gene homologs of the genetically well-characterized nematode Caenorhabditis elegans, termed pgp-1, pgp-2, pgp-3 and pgp-4, respectively. The genes were physically mapped on chromosome IV (pgp-1), I (pgp-2) and X (pgp-3 and pgp-4).