Flavaglines target primitive leukemia cells and enhance anti-leukemia drug activity.

Identification of agents that target human leukemia stem cells is an important consideration for the development of new therapies. The present study demonstrates that rocaglamide and silvestrol, closely related natural products from the flavagline class of compounds, are able to preferentially kill functionally defined leukemia stem cells, while sparing normal stem and progenitor cells. In addition to efficacy as single agents, flavaglines sensitize leukemia cells to several anticancer compounds, including front-line chemotherapeutic drugs used to treat leukemia patients. Mechanistic studies indicate that flavaglines strongly inhibit protein synthesis, leading to the reduction of short-lived antiapoptotic proteins. Notably though, treatment with flavaglines, alone or in combination with other drugs, yields a much stronger cytotoxic activity toward leukemia cells than the translational inhibitor temsirolimus. These results indicate that the underlying cell death mechanism of flavaglines is more complex than simply inhibiting general protein translation. Global gene expression profiling and cell biological assays identified Myc inhibition and the disruption of mitochondrial integrity to be features of flavaglines, which we propose contribute to their efficacy in targeting leukemia cells. Taken together, these findings indicate that rocaglamide and silvestrol are distinct from clinically available translational inhibitors and represent promising candidates for the treatment of leukemia.

Cloning and characterization of a ninth member of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase family, ppGaNTase-T9.

We have cloned, expressed and characterized the gene encoding a ninth member of the mammalian UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (ppGaNTase) family, termed ppGaNTase-T9. This type II membrane protein consists of a 9-amino acid N-terminal cytoplasmic region, a 20-amino acid hydrophobic/transmembrane region, a 94-amino acid stem region, and a 480-amino acid conserved region. Northern blot analysis revealed that the gene encoding this enzyme is expressed in a broadly distributed manner across many adult tissues. Significant levels of 5- and 4.2-kilobase transcripts were found in rat sublingual gland, testis, small intestine, colon, and ovary, with lesser amounts in heart, brain, spleen, lung, stomach, cervix, and uterus. In situ hybridization to mouse embryos (embryonic day 14.5) revealed significant hybridization in the developing mandible, maxilla, intestine, and mesencephalic ventricle. Constructs expressing this gene transiently in COS7 cells resulted in no detectable transferase activity in vitro against a panel of unmodified peptides, including MUC5AC (GTTPSPVPTTSTTSAP) and EA2 (PTTDSTTPAPTTK). However, when incubated with MUC5AC and EA2 glycopeptides (obtained by the prior action of ppGaNTase-T1), additional incorporation of GalNAc was achieved, resulting in new hydroxyamino acid modification. The activity of this glycopeptide transferase is distinguished from that of ppGaNTase-T7 in that it forms a tetra-glycopeptide species from the MUC5AC tri-glycopeptide substrate, whereas ppGaNTase-T7 forms a hexa-glycopeptide species. This isoform thus represents the second example of a glycopeptide transferase and is distinct from the previously identified form in enzymatic activity as well as expression in embryonic and adult tissues. These findings lend further support to the existence of a hierarchical network of differential enzymatic activity within the diversely regulated ppGaNTase family, which may play a role in the various processes governing development.

Low levels of GRP-Ca expression in transgenic mice.

The GRP-Ca gene of the rat encodes a member of the glutamine/glutamic-acid-rich protein (GRP) family. This gene is expressed in a highly tissue-specific fashion, with transcription being found only in the acinar cells of the submandibular gland (SMG). To begin to define the cis-acting elements governing GRP-Ca expression, we constructed transgenic mice containing the rat GRP-Ca gene plus 9.5 kb of 5' genomic sequence and 1 kb of 3 genomic sequence. Expression of GRP-Ca was detectable in progeny from only 1 of 3 independent founders. Expression levels of the transgenic GRP-Ca were much lower than the native GRP-Ca found in the rat SMG. Furthermore, GRP-Ca in transgenic mice was not tissue-specifically expressed, being found in both the SMG/SLG complex and the liver. These results indicate that the genomic region of GRP-Ca included in these transgenic mice is not sufficient to confer the high levels of tissue-specific expression seen in the rat.

Characterization of a UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase that displays glycopeptide N-acetylgalactosaminyltransferase activity.

We report the cloning, expression, and characterization of a novel member of the mammalian UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (ppGaNTase) family that transfers GalNAc to a GalNAc-containing glycopeptide. Northern blot analysis revealed that the gene encoding this enzyme, termed ppGaNTase-T6, is expressed in a highly tissue-specific manner. Significant levels of transcript were found in rat and mouse sublingual gland, stomach, small intestine, and colon; trace amounts were seen in the ovary, cervix, and uterus. Recombinant constructs were expressed transiently in COS7 cells but demonstrated no transferase activity in vitro against a panel of unmodified peptides, including GTTPSPVPTTSTTSAP (MUC5AC). However, when incubated with the total glycosylated products obtained by action of ppGaNTase-T1 on MUC5AC (mainly GTT(GalNAc)PSPVPTTSTT(GalNAc)SAP), additional incorporation of GalNAc was achieved, resulting in new hydroxyamino acids being modified. The MUC5AC glycopeptide failed to serve as a substrate for ppGaNTase-T6 after modification of the GalNAc residues by periodate oxidation and sodium borohydride reduction, indicating a requirement for the presence of intact GalNAc. This suggests that O-glycosylation of multisite substrates may proceed in a specific hierarchical manner and underscores the potential complexity of the processes that regulate O-glycosylation.

Cloning and expression of a novel, tissue specifically expressed member of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase family.

We report the cloning and expression of the fifth member of the mammalian UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (ppGaNTase) family. Degenerate polymerase chain reaction amplification and hybridization screening of a rat sublingual gland (RSLG) cDNA library were used to identify a novel isoform termed ppGaNTase-T5. Conceptual translation of the cDNA reveals a uniquely long stem region not observed for other members of this enzyme family. Recombinant proteins expressed transiently in COS7 cells displayed transferase activity in vitro. Relative activity and substrate preferences of ppGaNTase-T5 were compared with previously identified isoforms (ppGaNTase-T1, -T3, and -T4); ppGaNTase-T5 and -T4 glycosylated a restricted subset of peptides whereas ppGaNTase-T1 and -T3 glycosylated a broader range of substrates. Northern blot analysis revealed that ppGaNTase-T5 is expressed in a highly tissue-specific manner; abundant expression was seen in the RSLG, with lesser amounts of message in the stomach, small intestine, and colon. Therefore, the pattern of expression of ppGaNTase-T5 is the most restricted of all isoforms examined thus far. The identification of this novel isoform underscores the diversity and complexity of the family of genes controlling O-linked glycosylation.

cDNA cloning and expression of a novel UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase.

The cDNA for a fourth member of the mammalian UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase family, termed ppGaNTase-T4, has been cloned from a murine spleen cDNA library and expressed transiently in COS7 cells as a secreted functional enzyme. Degenerate primers, based upon regions that are conserved among the known mammalian members of the enzyme family (ppGaNTase-T1, -T2, and -T3) and three Caenorhabditis elegans homologues (ppGaNTase-TA, -TB, and -TC), were used in polymerase chain reactions to identify and clone this new isoform. Substrate preferences for recombinant murine ppGaNTase-T1 and ppGaNTase-T4 isozymes were readily distinguished. ppGaNTase-T1 glycosylated a broader range of synthetic peptide substrates; in contrast, the ppGaNTase-T4 preferentially glycosylated a single substrate among the panel of 11 peptides tested. Using Northern blot analysis, a ppGaNTase-T4 message of 5.5 kilobases was detectable in murine embryonic tissues, as well as the adult sublingual gland, stomach, colon, small intestine, lung, cervix, and uterus with lower levels detected in kidney, liver, heart, brain, spleen, and ovary. Thus, the pattern of expression for ppGaNTase-T4 is more restricted than for the three previously reported isoforms of the enzyme. The variation in expression patterns and substrate specificities of the ppGaNTase enzyme family suggests that differential expression of these isoenzymes may be responsible for the cell-specific repertoire of mucin-type oligosaccharides on cell-surface and secreted O-linked glycoproteins.