Chloroquine diverts ACTH from a regulated to a constitutive secretory pathway in AtT-20 cells

AtT-20 cells, a mouse pituitary line, externalize a viral membrane glycoprotein and the precursor of ACTH constitutively, that is, rapidly without storage or regulation. They also have a regulated pathway in which they cleave the precursor to mature hormones, ACTH and beta-endorphin, store them in secretory granules and discharge them only in the presence of a secretagogue. An analogy exists for newly synthesized lysosomal enzymes which are either delivered to the lysosome or secreted from the cell. Targeting to the lysosomes may require a low pH step, since chloroquine causes the enzymes to be secreted from the cell. Here we show that chloroquine (200 microM) also appears to block the storage of newly synthesized ACTH in secretory granules and instead diverts it to the outside of the cell via the constitutive pathway. Chloroquine has no effect on the constitutive pathway and does not block the exocytosis of pre-packaged ACTH. Thus like lysosomal enzymes, peptide hormones are not sent to their correct destinations in the presence of chloroquine, but are diverted instead to a constitutive pathway that is chloroquine-insensitive.     

Therapeutic approaches to protein-misfolding diseases

Several sporadic and genetic diseases are caused by protein misfolding. These include cystic fibrosis and other devastating diseases of childhood as well as Alzheimer's, Parkinson's and other debilitating maladies of the elderly. A unified view of the molecular and cellular pathogenesis of these conditions has led to the search for chemical chaperones that can slow, arrest or revert disease progression. Molecules are now emerging that link our biophysical insights with our therapeutic aspirations.     

Ribosomal mutations cause p53-mediated dark skin and pleiotropic effects

Mutations in genes encoding ribosomal proteins cause the Minute phenotype in Drosophila and mice, and Diamond-Blackfan syndrome in humans. Here we report two mouse dark skin (Dsk) loci caused by mutations in Rps19 (ribosomal protein S19) and Rps20 (ribosomal protein S20). We identify a common pathophysiologic program in which p53 stabilization stimulates Kit ligand expression, and, consequently, epidermal melanocytosis via a paracrine mechanism. Accumulation of p53 also causes reduced body size and erythrocyte count. These results provide a mechanistic explanation for the diverse collection of phenotypes that accompany reduced dosage of genes encoding ribosomal proteins, and have implications for understanding normal human variation and human disease.     

Functional analysis of secreted and transmembrane proteins critical to mouse development.

We describe the successful application of a modified gene-trap approach, the secretory trap, to systematically analyze the functions in vivo of large numbers of genes encoding secreted and membrane proteins. Secretory-trap insertions in embryonic stem cells can be transmitted to the germ line of mice with high efficiency and effectively mutate the target gene. Of 60 insertions analyzed in mice, one-third cause recessive lethal phenotypes affecting various stages of embryonic and postnatal development. Thus, secretory-trap mutagenesis can be used for a genome-wide functional analysis of cell signaling pathways that are critical for normal mammalian development and physiology.     

Germline mutations in the ribonuclease L gene in families showing linkage with HPC1.

Although prostate cancer is the most common non-cutaneous malignancy diagnosed in men in the United States, little is known about inherited factors that influence its genetic predisposition. Here we report that germline mutations in the gene encoding 2'-5'-oligoadenylate(2-5A)-dependent RNase L (RNASEL) segregate in prostate cancer families that show linkage to the HPC1 (hereditary prostate cancer 1) region at 1q24-25 (ref. 9). We identified RNASEL by a positional cloning/candidate gene method, and show that a nonsense mutation and a mutation in an initiation codon of RNASEL segregate independently in two HPC1-linked families. Inactive RNASEL alleles are present at a low frequency in the general population. RNASEL regulates cell proliferation and apoptosis through the interferon-regulated 2-5A pathway and has been suggested to be a candidate tumor suppressor gene. We found that microdissected tumors with a germline mutation showed loss of heterozygosity and loss of RNase L protein, and that RNASEL activity was reduced in lymphoblasts from heterozyogous individuals compared with family members who were homozygous with respect to the wildtype allele. Thus, germline mutations in RNASEL may be of diagnostic value, and the 2-5A pathway might provide opportunities for developing therapies for those with prostate cancer.