The Sp/KLF family contains at least twenty identi®ed members which include Sp1-4 and numerous kru È ppel-like factors. Members of the family bind with varying af®nities to sequences designated as`Sp1 sites' (e.g., GC-boxes, CACCC-boxes, and basic transcription elements). Family members have different transcriptional properties and can modulate each other's activity by a variety of mechanisms. Since cells can express multiple family members, Sp/KLF factors are likely to make up a transcriptional network through which gene expression can be ®ne-tuned. Sp1 site'-dependent transcription can be growth-regulated, and the activity, expression, and/or post-translational modi®cation of multiple family members is altered with cell growth. Furthermore, Sp/KLF factors are involved in many growth-related signal transduction pathways and their overexpression can have positive or negative effects on proliferation. In addition to growth control, Sp/KLF factors have been implicated in apoptosis and angiogenesis; thus, the family is involved in several aspects of tumorigenesis. Consistent with a role in cancer, Sp/ KLF factors interact with oncogenes and tumor suppressors, they can be oncogenic themselves, and altered expression of family members has been detected in tumors. Effects of changes in Sp/KLF factors are context-dependent and can appear contradictory. Since these factors act within a network, this diversity of effects may arise from differences in the expression pro®le of family members in various cells. Thus, it is likely that the properties of the overall network of Sp/KLF factors play a determining role in regulation of cell growth and tumor progression.

Nonsense-mediated mRNA decay (NMD), also called mRNA surveillance, is an important pathway used by all organisms that have been tested to degrade mRNAs that prematurely terminate translation and, as a consequence, eliminate the production of aberrant proteins that could be potentially harmful. In mammalian cells, NMD appears to involve splicing-dependent alterations to mRNA as well as ribosome-associated components of the translational apparatus. To date, human (h) Upf1 protein (p) (hUpf1p), a group 1 RNA helicase named after its Saccharomyces cerevisiae orthologue that functions in both translation termination and NMD, has been the only factor shown to be required for NMD in mammalian cells. Here, we describe human orthologues to S. cerevisiae Upf2p and S. cerevisiae Upf3p (Caenorhabditis elegans SMG-4) based on limited amino acid similarities. The existence of these orthologues provides evidence for a higher degree of evolutionary conservation of NMD than previously appreciated. Interestingly, human orthologues to S. cerevisiae Upf3p (C. elegans SMG-4) derive from two genes, one of which is X-linked and both of which generate multiple isoforms due to alternative pre-mRNA splicing. We demonstrate using immunoprecipitations of epitope-tagged proteins transiently produced in HeLa cells that hUpf2p interacts with hUpf1p, hUpf3p-X, and hUpf3p, and we define the domains required for the interactions. Furthermore, we find by using indirect immunofluorescence that hUpf1p is detected only in the cytoplasm, hUpf2p is detected primarily in the cytoplasm, and hUpf3p-X localizes primarily to nuclei. The finding that hUpf3p-X is a shuttling protein provides additional indication that NMD has both nuclear and cytoplasmic components.The biogenesis of functionally mature mRNAs in mammalian cells is remarkably involved and inherently subject to inefficiencies and inaccuracies that result in the generation of abnormal translational reading frames. Mammalian mRNAs are transcribed initially as precursors, most of which contain multiple introns that must be removed by the process of premRNA splicing. If transcription initiates incorrectly or an intron either fails to be removed or is removed using one or more abnormal splice sites, then product mRNA has the potential to harbor a premature termination codon (PTC) that could derive from an upstream reading frame, a retained intron, or a shift in the reading frame.In order to cope with the generation of PTCs and their potential to result in deleterious proteins that function in new or dominant-negative ways, mammalian cells have evolved a pathway called nonsense-mediated mRNA decay (NMD) or mRNA surveillance (reviewed in references 20, 28, 30, 31, and 32). This pathway surveys all translated mRNAs, whether they be normal or defective, in order to degrade those that prematurely terminate translation more than 50 to 55 nucleotides (nt) upstream of the final exon-exon junction (7,8,41,43,44,48,49)-a feature of most PTCs but not most normal termination codons (34). These and other data i...

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waf1/cip1 and p27 kip1 . Reversal of these cell cycle regulatory effects was coincident with activator-induced down-regulation of PKC ␣, ␦, and ⑀. Differential down-regulation of individual PKC isozymes revealed that PKC ␣ in particular is sufficient to mediate cell cycle arrest by PKC agonists in this system. Taken together, the data implicate PKC ␣ in negative regulation of intestinal epithelial cell growth both in vitro and in situ via pathways which involve modulation of Cip/Kip family cyclin-dependent kinase inhibitors and the retinoblastoma growth suppressor protein.

Botulinum neurotoxin (BoNY) type A, a causative agent of botulism, is a di-chain protein (molecular weight 140,000) from Clostridium botulinum, and the most neurotoxic substance known. Some cases of sudden infant cot deaths have been attributed to such a neuroparalytic condition. BoNT inhibits irreversibly the release of acetylcholine from peripheral nerves in a highly selective manner. Hence, it is potentially an invaluable probe for studying the mechanism of transmitter release. Here we demonstrate specific labelling of murine motor nerve terminals with neurotoxic, 125I-labelled BoNT (type A) by autoradiography. We observed saturable, temperature-sensitive binding of BoNT to sites which reside solely on the nerve terminal membrane; these were distributed on all unmyelinated areas, at an average density of 150-500 per micron2 of membrane. The binding was mediated by the larger subunit of the toxin and was inhibited partially by tetanus toxin, another microbial protein. No specific binding was detectable on any other cell types examined, including noradrenergic terminals. Following binding, internalization of radioactivity was observed; this process was energy-dependent as it could be prevented totally by azide or dinitrophenol (DNP). This direct demonstration of separable steps, including highly selective binding and acceptor-mediated internalization, is reconcilable with the unique potency and the multiphasic inhibitory action of BoNT on transmitter release, as shown electrophysiologically.

Members of the protein kinase C (PKC) family of signal transduction molecules have been widely implicated in regulation of cell growth and differentiation, although the underlying molecular mechanisms involved remain poorly defined. Using combined in vitro and in vivo intestinal epithelial model systems, we demonstrate that PKC signaling can trigger a coordinated program of molecular events leading to cell cycle withdrawal into G0. PKC activation in the IEC-18 intestinal crypt cell line resulted in rapid downregulation of D-type cyclins and differential induction of p21waf1/cip1 and p27kip1, thus targeting all of the major G1/S cyclin-dependent kinase complexes. These events were associated with coordinated alterations in expression and phosphorylation of the pocket proteins p107, pRb, and p130 that drive cells to exit the cell cycle into G0 as indicated by concomitant downregulation of the DNA licensing factor cdc6. Manipulation of PKC isozyme levels in IEC-18 cells demonstrated that PKCα alone can trigger hallmark events of cell cycle withdrawal in intestinal epithelial cells. Notably, analysis of the developmental control of cell cycle regulatory molecules along the crypt–villus axis revealed that PKCα activation is appropriately positioned within intestinal crypts to trigger this program of cell cycle exit–specific events in situ. Together, these data point to PKCα as a key regulator of cell cycle withdrawal in the intestinal epithelium.

Abstract. The mechanisms underlying control of cell growth and differentiation in epithelial tissues are poorly understood. Protein kinase C (PKC) isozymes, members of a large family of serine/threonine ldnases of fundamental importance in signal transduction, have been increasingly implicated in the regulation of cell growth, differentiation, and function. Using the rat intestinal epithelium as a model system, we have examined PKC-specific activity as well as individual PKC isozyme expression and distribution (i.e., activation status) in epithelial cells in situ. Increased PKC activity was detected in differentiating and functional cells relative to immature proliferating crypt cells. Immunofluorescence and Western blot analysis using a panel of isozyme-specific antibodies revealed that PKC or, BII, 6, ¢, and ~" are expressed in rat intestinal epithelial cells and exhibit distinct subcellular distribution patterns along the crypt-villus unit. The combined morphological and biochemical approach used permitred analysis of the activation status of specific PKC isozymes at the individual cell level. These studies showed that marked changes in membrane association and level of expression for PKC a, BII, 6, and ~" occur as cells cease division in the mid-crypt region and begin differentiation. Additional changes in PKC activation status are observed with acquisition of mature function on the villus. These studies clearly demonstrate naturally occurring alterations in PKC isozyme activation status at the individual cell level within the context of a developing tissue.Direct activation of PKC in an immature intestinal crypt cell line was shown to result in growth inhibition and coincident translocation of PKC tx from the cytosolic to the particulate subcellular fraction, paralleling observations made in situ and providing further support for a role of intestinal PKC isozymes in post-mitotic events. PKC isozymes were also found to be tightly associated with cytoskeletal elements, suggesting participation in control of the structural organization of the enterocyte. Taken together, the results presented strongly suggest an involvement of PKC isoforms in cellular processes related to growth cessation, differentiation, and function of intestinal epithelial cells in situ.

EMT is associated with poor survival in surgically resected pancreatic adenocarcinoma. A correlation between activated Erk and fibronectin was identified that may open avenues for targeted therapy for this subgroup.