Covalent attachment of myristic acid to pp6o0-sc, the transforming protein of Rous sarcoma virus, was studied in a cell-free system. Using a synthetic peptide containing the first 11 amino acids of the mature pp60 V-src polypeptide sequence as a substrate, we probed lysates from a variety of cells and tissues for N-myristyl transferase (NMT) activity. Nearly every eucaryotic cell type tested contained NMT, including avian, mammalian, insect, and plant cells. Since NMT activity was detected in rabbit reticulocyte lysates, we took advantage of the translational capability of these lysates to determine the precise point during translation at which myristate is attached to pp6o0-src. src mRNA, transcribed from cloned v-src DNA, was translated in reticulocyte lysates which had been depleted of endogenous myristate. Addition of [3H]myristate to lysates 10 min after the start of synchronized translation resulted in a dramatic decrease in the incorporation of radiolabeled myristate into pp60v-src polypeptide chains. These results imply that although myristate can be attached posttranslationally to synthetic peptide substrates, myristylation in vivo is apparently a very early cotranslational event which occurs before the first 100 amino acids of the nascent polypeptide chain are polymerized.Covalent attachment of the 14-carbon fatty acid, myristic acid, has been shown to occur on a wide variety of viral and cellular proteins (11,18,22,23,35,41). One of the bestcharacterized myristylated proteins is pp6O-srC, the transforming protein of Rous sarcoma virus (RSV; 6, 14). Studies of numerous point and deletion mutants of pp6O-src which lack the myristate attachment site have established that myristylation is a necessary event for directing pp60v-sr to the plasma membrane. Mutant pp6O-src molecules which are not myristylated remain in the cytoplasm and do not transform cells (10, 15).The structural requirements for myristylation of a polypeptide chain have recently been elucidated. Myristate is attached through an amide linkage with a glycine residue at the amino terminus of the acceptor protein (7,24,34) by N-myristyl transferase (NMT). An amino-terminal glycine is essential but not sufficient for myristylation, and a serine or threonine residue is present at position 5 in nearly all myristylated proteins (39, 40). Additional sequence determinants are important, since deletions within the first 10 amino acids of the mature pp6v-vsrc polypeptide sequence abolish myristylation of the resultant protein (26,27). However, no clear consensus sequence seems to exist among all of the known myristylated proteins.Myristylation of both viral and cellular proteins has been shown to be an early event during protein biosynthesis. Treatment of cells with cycloheximide, an inhibitor of protein synthesis, results in immediate cessation of myristylation (5,18,20,22). Moreover, nascent polypeptide chains which are covalently associated with tRNA are already myristylated (44), implying that myristylation occurs cotranslationally. Exactly when t...
BackgroundLeukemia initiating cells (LIC) contribute to therapeutic resistance through acquisition of mutations in signaling pathways, such as NOTCH1, that promote self-renewal and survival within supportive niches. Activating mutations in NOTCH1 occur commonly in T cell acute lymphoblastic leukemia (T-ALL) and have been implicated in therapeutic resistance. However, the cell type and context specific consequences of NOTCH1 activation, its role in human LIC regeneration, and sensitivity to NOTCH1 inhibition in hematopoietic microenvironments had not been elucidated.Methodology and Principal FindingsWe established humanized bioluminescent T-ALL LIC mouse models transplanted with pediatric T-ALL samples that were sequenced for NOTCH1 and other common T-ALL mutations. In this study, CD34+ cells from NOTCH1Mutated T-ALL samples had higher leukemic engraftment and serial transplantation capacity than NOTCH1Wild-type CD34+ cells in hematopoietic niches, suggesting that self-renewing LIC were enriched within the NOTCH1Mutated CD34+ fraction. Humanized NOTCH1 monoclonal antibody treatment reduced LIC survival and self-renewal in NOTCH1Mutated T-ALL LIC-engrafted mice and resulted in depletion of CD34+CD2+CD7+ cells that harbor serial transplantation capacity.ConclusionsThese results reveal a functional hierarchy within the LIC population based on NOTCH1 activation, which renders LIC susceptible to targeted NOTCH1 inhibition and highlights the utility of NOTCH1 antibody targeting as a key component of malignant stem cell eradication strategies.
Linear forms of simian virus 40 (SV40) DNA, when added to transfection mixtures containing circular SV40 and +X174 RFI DNAs, enhanced the frequency of SV40/+X174 recombination, as measured by infectious center in situ plaque hybridization in monkey BSC-1 cells. The sequences required for the enhancement of recombination by linear DNA reside within the SV40 replication origin/regulatory region (nucleotides 5,171 to 5,243/0 to 128). Linearization of +X174 RFI DNA did not increase the recombination frequency. The SV40/+X174 recombinant structures arising from transfections supplemented with linear forms of origin-containing SV40 DNA contained 4X174 DNA sequences interspersed within tandem head-to-tail repeats derived from the recombination-enhancing linear DNA. Evidence is presented that the tandem repeats are not formed by homologous recombination and that linear forms of SV40 DNA must compete with circular SV40 DNA for the available T antigen to enhance recombination. We propose that the enhancement of recombination by linear SV40 DNA results from the entry of that DNA into a rolling circle type of replication pathway which generates highly recombinogenic intermediates.The pathways and underlying mechanisms responsible for recombination in animal cells are poorly understood. Integrative recombination by DNA tumor viruses such as simian virus 40 (SV40), polyoma virus, and adenovirus does not generally involve specific sites in the viral or cellular genome, and extensive base sequence homology is not required (reviewed in reference 69), although a possible role for very short regions of homology has not been excluded (11). Because the molecular analysis of viral integration sites in transformed cells is feasible only many cell generations after the initial cell-virus recombination event, we and others have devised short-term extrachromosomal recombination systems, based either upon the cell-mediated excision of infectious SV40 DNA from transfected plasmid constructs (65,73,74) or upon intermolecular recombination between cotransfected SV40 and foreign DNAs (1,24,31,75,76). Some of these studies have led to the realization that, in contrast to lower eucaryotes, nonhomologous recombination in higher eucaryotes is prevalent.Previously, we have shown that DEAE-dextran-mediated cotransfection of monkey BSC-1 cells with circular SV40 and 4X174 RFI DNAs generates recombinant virus which replicates in the presence of wild-type SV40 (76). The frequency of recombination was measured, in terms of the proportion of SV40-replicating cells which generate SV40/40X174 hybrid virions, by an infectious center in situ plaque hybridization procedure (24). Only recombinants which retain a functional SV40 origin of replication and are of a size which can be encapsidated into SV40 virions are scored by this assay. The number of recombinant-producing cells (10-3 with respect to the total cell population and 10-2 with respect to the successfully transfected subpopulation) is unaffected by the level of gross nucleotide sequence homology bet...
Background Optimizing the therapeutic ratio for radiation therapy (RT) in head and neck squamous cell carcinoma (HNSCC) is uniquely challenging owing to high rates of early and late toxicity involving nearby organs at risk. These toxicities have a profound impact on treatment compliance and quality of life. Emerging evidence suggests that RT dose alone cannot fully account for the variable severity of RT-related adverse events (rtAEs) observed in HNSCC patients. Next-generation sequencing has become an increasingly valuable tool with widespread use in the oncology field and is being robustly explored for predicting rtAEs beyond dosimetric data. Methods Patients who had Foundation Medicine sequencing data and received RT for primary or locally recurrent HNSCC were selected for this study. Early and late toxicity data were collected and reported based on Common Terminology Criteria for Adverse Events version 5.0. Dosimetric parameters were collected for pertinent structures. Results A total of HNSCC 37 patients were analyzed in this study. Genetic alterations in BRCA2, ERBB3, NOTCH1 and CCND1 were all associated with higher mean grade of toxicity with BRCA2 alteration implicated in all toxicity parameters evaluated including mucositis, early dysphagia, xerostomia and to a lesser extent, late dysphagia. Interestingly, patients who exhibited alterations in both BRCA2 and ERBB3 experienced a twofold or greater increase in early dysphagia, early xerostomia and late dysphagia compared to ERBB3 alteration alone. Furthermore, several gene alterations were associated with improved toxicity outcomes. Within an RT supersensitive patient subset, alterations were found in TNFAIP3, HNF1A, SPTA1 and CASP8. All of these alterations were not found in the RT insensitive patient subset. We found 17 gene alterations in the RT insensitive patient subset that were not found in the RT supersensitive patient subset. Conclusion Despite consistent RT dosimetric parameters, patients with HNSCC experience heterogeneous patterns of rtAEs. Identifying factors associated with toxicity outcomes offers a new avenue for personalized precision RT therapy and prophylactic management. Here, next-generation sequencing in a population of HNSCC patients correlates several genetic alterations with severity of rtAEs. Further analysis is urgently needed to identify genetic patterns associated with rtAEs in order to reduce harmful outcomes in this challenging population.
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