No abstract

Genetic instability is thought to be responsible for the numerous genotypic changes that occur during neoplastic transformation and metastatic progression. To explore the role of genetic instability at the level of point mutations during mammary tumor development and malignant progression, we combined transgenic mouse models of mutagenesis detection and oncogenesis. Bitransgenic mice were generated that carried both a bacteriophage A transgene to assay mutagenesis and a polyomavirus middle T oncogene, mammary gland-targeted expression of which led to metastatic mammary adenocarcinomas. We developed a novel assay for the detection of mutations in the A transgene that selects for phage containing forward mutations only in the A cII gene, using an hfl-bacterial host. In addition to the relative ease of direct selection, the sensitivity of this assay for both spontaneous and chemically induced mutations was comparable to the widely used mutational target gene, A lacI, making the cII assay an attractive alternative for mutant phage recovery for any A-based mouse mutagenesis assay system. The frequencies of A cII mutants were not significantly different in normal mammary epithelium, primary mammary adenocarcinomas, and pulmonary metastases. The cII mutational spectra in these tissues consisted mostly of G/C -> A/T transitions, a large fraction ofwhich occurred at CpG dinucleotides. These data suggest that, in this middle T oncogene model of mammary tumor progression, a significant increase in mutagenesis is not required for tumor development or for metastatic progression.

A large number of insect species have been screened for the presence of the retrotransposable elements RI and R2. These elements integrate independently at specific sites in the 28S rRNA genes. Genomic blots indicated that 43 of 47 insect species from nine orders contained insertions, ranging in frequency from a few percent to >50% of the 28S genes. Sequence analysis of these insertions from 8 species revealed 22 elements, 21 of which corresponded to RI or R2 elements.Surprisingly, many species appeared to contain highly divergent copies of RI and R2 elements. For example, a parasitic wasp contained at least four families of RI elements; the Japanese beetle contained at least five families of R2 elements. The presence of these retrotransposable elements throughout Insecta and the observation that single species can harbor divergent families within its rRNA-encoding DNA loci present interesting questions concerning the age of these elements and the possibility of cross-species transfer.Transposable elements probably exist in the genome of every eukaryote (1). Among the most abundant types of these elements are the retrotransposable elements that, like retroviruses, move by means of an RNA intermediate (2). Retrotransposable elements can be divided into two major classes (3, 4). One class is similar to the retroviruses in that they contain long terminal repeats (LTRs) and their encoded proteins have amino acid similarity to those of the retroviruses. The second class, termed Line 1-like or the non-LTR retrotransposable elements, lacks any type ofterminal repeat and has lower levels of amino acid similarity to the retroviruses.RI and R2 (formerly called type I and type II insertions) are non-LTR retrotransposable elements, each found at a precise location in a fraction of the 28S rRNA genes ofBombyx mori and several dipteran species (5-12). The insertion sites for RI and R2 are 74 base pairs (bp) apart in a highly conserved region of the 28S gene, the large subunit rRNA gene of eukaryotes (Fig. 1A). The presence of either RI or R2 within an rRNA-encoding DNA (rDNA) unit inactivates that unit (13)(14)(15). Only a few copies of RI and R2 are located outside the rDNA units and they appear to be nonfunctional (6,16,17). The high insertion specificity of R2 elements can be explained by an encoded endonuclease activity specific to its 28S gene insertion site (18). Individual copies of RI and R2 have the same 3' end but may be truncated at their 5' end, a feature common to other non-LTR retrotransposable elements (6,7,11,17).Although transposable elements as a group are widespread, it has been difficult to study the distribution of a particular element across broad taxonomic groups due to their dispersed genomic locations and rapid sequence divergence that limits their detection by DNA hybridization. Here we have taken advantage of the remarkable insertion specificities ofRI and R2 to determine their distribution in species throughout the class Insecta. We present evidence that RI and R2 are present in the rRNA ge...

T-cell acute lymphoblastic leukemias and lymphomas (T-ALL) are aggressive hematologic cancers frequently associated with activating mutations in NOTCH1. Early studies identified NOTCH1 as an attractive therapeutic target for the treatment of T-ALL through the use of γ-secretase inhibitors (GSIs). Here, we characterized the interaction between PF-03084014, a clinically-relevant GSI, and dexamethasone in preclinical models of glucocorticoid-resistant T-ALL. Combination treatment of the GSI PF-03084014 with glucocorticoids induced a synergistic antileukemic effect in human T-ALL cell lines and primary human T-ALL patient samples. Mechanistically PF-03084014 plus glucocorticoid treatment induced increased transcriptional upregulation of the glucocorticoid receptor and glucocorticoid target genes. Treatment with PF-03084014 and glucocorticoids in combination was highly efficacious in vivo, with enhanced reduction of tumor burden in a xenograft model of T-ALL. Finally, glucocorticoid treatment effectively reversed PF-03084014-induced gastrointestinal toxicity via inhibition of goblet cell metaplasia. These results warrant the analysis of PF-03084014 and glucocorticoids in combination for the treatment of glucocorticoid-resistant T-ALL.