Genetic studies have identified mutations in key regulators of the Wnt͞-catenin pathway in a variety of cancers, most frequently in colon cancers. However, whether the pathway is activated in clinical cancer samples is not easily determined, and therefore it is useful to find markers that could be surrogates to show activation of the Wnt͞-catenin pathway. Gene expression profiles were analyzed in SW620, a colon cancer cell line in which -catenin levels are stabilized as a consequence of truncated adenomatous polyposis coli and were compared with profiles of the same cells transfected with antisense oligodeoxynucleotides. Treatment of cells with -catenin antisense oligodeoxynucleotides resulted in a decrease in the levels of axin2 and human naked cuticle (hnkd) mRNAs. Interestingly, the proteins encoded by both of these mRNAs are known inhibitors of the -catenin pathway. In 30 human cell lines derived from different origins, axin2 and hnkd were expressed only in human colon cancer cell lines that are known to have activating mutations in the Wnt͞-catenin pathway. Further, levels of both axin2 and hnkd mRNA were also found to be elevated in about 65% of laser microdissected cells from human colon tumors compared with laser microdissected cells of normal morphology from the same patient samples. The increased expression of axin2 and hnkd correlated with truncations in adenomatous polyposis coli in the same patient samples. These results reveal that it is possible to detect activation of a carcinogenic pathway in human cancer samples with specific markers.
Transmembrane receptors for hormones, neurotransmitters, light, and odorants mediate their cellular effects by activating heterotrimeric guanine nucleotide-binding proteins (G proteins). Crystal structures have revealed contact surfaces between G protein subunits, but not the surfaces or molecular mechanism through which Galphabetagamma responds to activation by transmembrane receptors. Such a surface was identified from the results of testing 100 mutant alpha subunits of the retinal G protein transducin for their ability to interact with rhodopsin. Sites at which alanine substitutions impaired this interaction mapped to two distinct Galpha surfaces: a betagamma-binding surface and a putative receptor-interacting surface. On the basis of these results a mechanism for receptor-catalyzed exchange of guanosine diphosphate for guanosine triphosphate is proposed.
Multiple myeloma (MM) is the second most common hematologic malignancy. Despite recent treatment advances, it remains incurable. Here, we report that Pim2 kinase expression is highly elevated in MM cells and demonstrate that it is required for MM cell proliferation. Functional interference of Pim2 activity either by short hairpin RNAs or by a potent and selective small-molecule inhibitor leads to significant inhibition of MM cell proliferation. Pim inhibition results in a significant decrease of mammalian target of rapamycin C1 (mTOR-C1) activity, which is critical for cell proliferation. We identify TSC2, a negative regulator of mTOR-C1, as a novel Pim2 substrate and show that Pim2 directly phosphorylates TSC2 on Ser-1798 and relieves the suppression of TSC2 on mTOR-C1. These findings support Pim2 as a promising therapeutic target for MM and define a novel Pim2-TSC2-mTOR-C1 pathway that drives MM proliferation.
Abstract. The major hepatitis B virus (HBV) ~ core protein is a viral structural protein involved in nucleic acid binding. Its coding sequence contains an extension of 29 codons (the "precore" region) at the amino terminus of the protein which is present in a fraction of the viral transcripts. This region is evolutionarily conserved among mammalian and avian HBVs, suggesting it has functional importance, although at least for duck HBV it has been shown to be nonessential for replication of infectious virions.Using in vitro assays for protein translocation across the endoplasmic reticulum membrane, we found that the precore region of the HBV genome encodes a signal sequence. This signal sequence was recognized by signal recognition particle, which targeted the nascent precore protein to the endoplasmic reticulum membrane with efficiencies comparable to those of other mammalian secretory proteins. A 19-amino acid signal peptide was removed by signal peptidase on the lumenal side of the microsomal membrane, generating a protein similar to the HBV major core protein, but containing 10 additional amino acids from the precore region at its amino terminus. Surprisingly, we found that 70-80% of this signal peptidase-cleaved product was localized on the cytoplasmic side of the microsomal vesicles and was not associated with the membranes. We conclude that translocation was aborted by an unknown mechanism, then the protein disengaged from the translocation machinery and was released back into the cytoplasm. Thus, a cytoplasmically disposed protein was created whose amino terminus resulted from signal peptidase cleavage. The remaining 20-30% appeared to be completely translocated into the lumen of the microsomes. A deletion mutant lacking the carboxy-terminal nucleic acid binding domain of the precore protein was similarly partitioned between the lumen of the microsomes and the cytoplasmic compartment, indicating that this highly charged domain is not responsible for the aborted translocation. We discuss the implications of our findings for the protein translocation process and suggest a possible role in the virus life cycle.H UMAN hepatitis B virus (HBV) t is a member of a group of enveloped DNA viruses (hepadna viruses) that use reverse transcription as part of their life cycle (for review see Tiollais et al., 1985;Standring and Rutter, 1986). The HBV virion genome is a circular partially double-stranded DNA of 3.2 kb. Due to its small size, the coding information in the DNA is densely packed into four partially overlapping reading frames. The products of two of these open reading frames are found as the major structural components in the viral particles, the core protein and the J.-H. Ou's present address is
The elastic response of a living cell is affected by its physiological state. This property provides mechanical fingerprints of a cell's dysfunctionality. The softness (kilopascal range) and thickness (2-15 μm) of mammalian cells imply that the force exerted by the probe might be affected by the stiffness of the solid support. This observation makes infinite sample thickness models unsuitable to describe quantitatively the forces and deformations on a cell. Here, we report a general theory to determine the true Young's moduli of a single cell from a force-indentation curve. Analytical expressions are deduced for common geometries such as flat punches, paraboloids, cones, needles, and nanowires. For a given cell and indentation, the influence of the solid support on the measurements is reduced by using sharp and high aspect ratio tips. The theory is validated by finite element simulations.
Purpose: PIM kinases have been shown to act as oncogenes in mice, with each family member being able to drive progression of hematologic cancers. Consistent with this, we found that PIMs are highly expressed in human hematologic cancers and show that each isoform has a distinct expression pattern among disease subtypes. This suggests that inhibitors of all three PIMs would be effective in treating multiple hematologic malignancies.Experimental Design: Pan-PIM inhibitors have proven difficult to develop because PIM2 has a low K m for ATP and, thus, requires a very potent inhibitor to effectively block the kinase activity at the ATP levels in cells. We developed a potent and specific pan-PIM inhibitor, LGB321, which is active on PIM2 in the cellular context.Results:LGB321 is active on PIM2-dependent multiple myeloma cell lines, where it inhibits proliferation, mTOR-C1 signaling and phosphorylation of BAD. Broad cancer cell line profiling of LGB321 demonstrates limited activity in cell lines derived from solid tumors. In contrast, significant activity in cell lines derived from diverse hematological lineages was observed, including acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), multiple myeloma and non-Hodgkin lymphoma (NHL). Furthermore, we demonstrate LGB321 activity in the KG-1 AML xenograft model, in which modulation of pharmacodynamics markers is predictive of efficacy. Finally, we demonstrate that LGB321 synergizes with cytarabine in this model. Conclusions:We have developed a potent and selective pan-PIM inhibitor with single-agent antiproliferative activity and show that it synergizes with cytarabine in an AML xenograft model. Our results strongly support the development of Pan-PIM inhibitors to treat hematologic malignancies.
Immunoglobulin heavy-chain binding protein (BiP, GRP-78) associates tightly in the endoplasmic reticulum (ER) with newly synthesized proteins that are incompletely assembled, have mutant structures, or are incorrectly glycosylated. The function of BiP has been suggested to be to prevent secretion of incorrectly folded or incompletely assembled protein, to promote folding or assembly of proteins, or to solubilize protein aggregates within the ER lumen. Here we examine the interaction of BiP with newly synthesized polypeptides in an in vitro protein translation-translocation system. We find that BiP forms tight complexes with nonglycosylated yeast invertase and incorrectly disulphide-bonded prolactin, but does not associate detectably with either glycosylated invertase or correctly disulphide-bonded prolactin. Moreover, BiP associates detectably only with completed chains of prolactin, not with chains undergoing synthesis. We conclude that BiP recognizes and binds with high affinity in vitro to aberrantly folded or aberrantly glycosylated polypeptides, but not to all nascent chains as they are folding.
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