Recombinant Origins of Pathogenic and Nonpathogenic Mouse Gammaretroviruses with Polytropic Host Range.

Ecotropic, xenotropic, and polytropic mouse leukemia viruses (E-, X-, and P-MLVs) exist in mice as infectious viruses and endogenous retroviruses (ERVs) inserted into mouse chromosomes. All three MLV subgroups are linked to leukemogenesis, which involves generation of recombinants with polytropic host range. Although P-MLVs are deemed to be the proximal agents of disease induction, few biologically characterized infectious P-MLVs have been sequenced for comparative analysis. We analyzed the complete genomes of 16 naturally occurring infectious P-MLVs, 12 of which were typed for pathogenic potential. We sought to identify ERV progenitors, recombinational hot spots, and segments that are always replaced, never replaced, or linked to pathogenesis or host range. Each P-MLV has an E-MLV backbone with P- or X-ERV replacements that together cover 100% of the recombinant genomes, with different substitution patterns for X- and P-ERVs. Two segments are always replaced, both coding for envelope (Env) protein segments: the N terminus of the surface subunit and the cytoplasmic tail R peptide. Viral gag gene replacements are influenced by host restriction genes Fv1 and Apobec3 Pathogenic potential maps to the env transmembrane subunit segment encoding the N-heptad repeat (HR1). Molecular dynamics simulations identified three novel interdomain salt bridges in the lymphomagenic virus HR1 that could affect structural stability, entry or sensitivity to host immune responses. The long terminal repeats of lymphomagenic P-MLVs are differentially altered by recombinations, duplications, or mutations. This analysis of the naturally occurring, sometimes pathogenic P-MLV recombinants defines the limits and extent of intersubgroup recombination and identifies specific sequence changes linked to pathogenesis and host interactions.IMPORTANCE During virus-induced leukemogenesis, ecotropic mouse leukemia viruses (MLVs) recombine with nonecotropic endogenous retroviruses (ERVs) to produce polytropic MLVs (P-MLVs). Analysis of 16 P-MLV genomes identified two segments consistently replaced: one at the envelope N terminus that alters receptor choice and one in the R peptide at the envelope C terminus, which is removed during virus assembly. Genome-wide analysis shows that nonecotropic replacements in the progenitor ecotropic MLV genome are more extensive than previously appreciated, covering 100% of the genome; contributions from xenotropic and polytropic ERVs differentially alter the regions responsible for receptor determination or subject to APOBEC3 and Fv1 restriction. All pathogenic viruses had modifications in the regulatory elements in their long terminal repeats and differed in a helical segment of envelope involved in entry and targeted by the host immune system. Virus-induced leukemogenesis thus involves generation of complex recombinants, and specific replacements are linked to pathogenesis and host restrictions.

Sequence Diversity, Intersubgroup Relationships, and Origins of the Mouse Leukemia Gammaretroviruses of Laboratory and Wild Mice.

UNLABELLED: Mouse leukemia viruses (MLVs) are found in the common inbred strains of laboratory mice and in the house mouse subspecies ofMus musculus Receptor usage and envelope (env) sequence variation define three MLV host range subgroups in laboratory mice: ecotropic, polytropic, and xenotropic MLVs (E-, P-, and X-MLVs, respectively). These exogenous MLVs derive from endogenous retroviruses (ERVs) that were acquired by the wild mouse progenitors of laboratory mice about 1 million years ago. We analyzed the genomes of seven MLVs isolated from Eurasian and American wild mice and three previously sequenced MLVs to describe their relationships and identify their possible ERV progenitors. The phylogenetic tree based on the receptor-determining regions ofenvproduced expected host range clusters, but these clusters are not maintained in trees generated from other virus regions. Colinear alignments of the viral genomes identified segmental homologies to ERVs of different host range subgroups. Six MLVs show close relationships to a small xenotropic ERV subgroup largely confined to the inbred mouse Y chromosome.envvariations define three E-MLV subtypes, one of which carries duplications of various sizes, sequences, and locations in the proline-rich region ofenv Outside theenvregion, all E-MLVs are related to different nonecotropic MLVs. These results document the diversity in gammaretroviruses isolated from globally distributedMussubspecies, provide insight into their origins and relationships, and indicate that recombination has had an important role in the evolution of these mutagenic and pathogenic agents. IMPORTANCE: Laboratory mice carry mouse leukemia viruses (MLVs) of three host range groups which were acquired from their wild mouse progenitors. We sequenced the complete genomes of seven infectious MLVs isolated from geographically separated Eurasian and American wild mice and compared them with endogenous germ line retroviruses (ERVs) acquired early in house mouse evolution. We did this because the laboratory mouse viruses derive directly from specific ERVs or arise by recombination between different ERVs. The six distinctively different wild mouse viruses appear to be recombinants, often involving different host range subgroups, and most are related to a distinctive, largely Y-chromosome-linked MLV ERV subtype. MLVs with ecotropic host ranges show the greatest variability with extensive inter- and intrasubtype envelope differences and with homologies to other host range subgroups outside the envelope. The sequence diversity among these wild mouse isolates helps define their relationships and origins and emphasizes the importance of recombination in their evolution.

Phase I study of TP300 in patients with advanced solid tumors with pharmacokinetic, pharmacogenetic and pharmacodynamic analyses.

BACKGROUND: A Phase I dose escalation first in man study assessed maximum tolerated dose (MTD), dose-limiting toxicity (DLT) and recommended Phase II dose of TP300, a water soluble prodrug of the Topo-1 inhibitor TP3076, and active metabolite, TP3011. METHODS: Eligible patients with refractory advanced solid tumors, adequate performance status, haematologic, renal, and hepatic function. TP300 was given as a 1-hour i.v. infusion 3-weekly and pharmacokinetic (PK) profiles of TP300, TP3076 and TP3011 were analysed. Polymorphisms in CYP2D6, AOX1 and UGT1A1 were studied and DNA strand-breaks measured in peripheral blood mononuclear cells (PBMCs). RESULTS: 32 patients received TP300 at 1, 2, 4, 6, 8, 10, 12 mg/m(2). MTD was 10 mg/m(2); DLTs at 12 (2/4 patients) and 10 mg/m(2) (3/12) included thrombocytopenia and febrile neutropenia; diarrhoea was uncommon. Six patients (five had received irinotecan), had stable disease for 1.5-5 months. TP3076 showed dose proportionality in AUC and Cmax from 1-10 mg/m(2). Genetic polymorphisms had no apparent influence on exposure. DNA strand-breaks were detected after TP300 infusion. CONCLUSIONS: TP300 had predictable hematologic toxicity, and diarrhoea was uncommon. AUC at MTD is substantially greater than for SN38. TP3076 and TP3011 are equi-potent with SN38, suggesting a PK advantage. TRIAL REGISTRATION: EU-CTR2006-001345-33.

Characterization of the human SNM1A and SNM1B/Apollo DNA repair exonucleases.

Human SNM1A and SNM1B/Apollo have both been implicated in the repair of DNA interstrand cross-links (ICLs) by cellular studies, and SNM1B is also required for telomere protection. Here, we describe studies on the biochemical characterization of the SNM1A and SNM1B proteins. The results reveal some fundamental differences in the mechanisms of the two proteins. Both SNM1A and SNM1B digest double-stranded and single-stranded DNA with a 5'-to-3' directionality in a reaction that is stimulated by divalent cations, and both nucleases are inhibited by the zinc chelator o-phenanthroline. We find that SNM1A has greater affinity for single-stranded DNA over double-stranded DNA that is not observed with SNM1B. Although both proteins demonstrate a low level of processivity on low molecular weight DNA oligonucleotide substrates, when presented with high molecular weight DNA, SNM1A alone is rendered much more active, being capable of digesting kilobase-long stretches of DNA. Both proteins can digest past ICLs induced by the non-distorting minor groove cross-linking agent SJG-136, albeit with SNM1A showing a greater capacity to achieve this. This is consistent with the proposal that SNM1A and SNM1B might exhibit some redundancy in ICL repair. Together, our work establishes differences in the substrate selectivities of SNM1A and SNM1B that are likely to be relevant to their in vivo roles and which might be exploited in the development of selective inhibitors.

Evodiamine, a dual catalytic inhibitor of type I and II topoisomerases, exhibits enhanced inhibition against camptothecin resistant cells.

DNA topoisomerases are nuclear enzymes that are the targets for several anticancer drugs. In this study we investigated the antiproliferative activity against human leukaemia cell lines and the effects on topoisomerase I and II of evodiamine, which is a quinazolinocarboline alkaloid isolated from the fruit of a traditional Chinese medicinal plant, Evodia rutaecarpa. We report here the anti-proliferative activity against human leukaemia cells K562, THP-1, CCRF-CEM and CCRF-CEM/C1 and the inhibitory mechanism on human topoisomerases I and II, important anti-cancer drugs targets, of evodiamine. Evodiamine failed to trap [Topo-DNA] complexes and induce any detectable DNA damage in cells, was unable to bind or intercalate DNA, and arrested cells in the G(2)/M phase. The results suggest evodiamine is a dual catalytic inhibitor of topoisomerases I and II, with IC(50) of 60.74 and 78.81 µM, respectively. The improved toxicity towards camptothecin resistant cells further supports its inhibitory mechanism which is different from camptothecin, and its therapeutic potential.

Human SNM1A and XPF-ERCC1 collaborate to initiate DNA interstrand cross-link repair.

One of the major DNA interstrand cross-link (ICL) repair pathways in mammalian cells is coupled to replication, but the mechanistic roles of the critical factors involved remain largely elusive. Here, we show that purified human SNM1A (hSNM1A), which exhibits a 5'-3' exonuclease activity, can load from a single DNA nick and digest past an ICL on its substrate strand. hSNM1A-depleted cells are ICL-sensitive and accumulate replication-associated DNA double-strand breaks (DSBs), akin to ERCC1-depleted cells. These DSBs are Mus81-induced, indicating that replication fork cleavage by Mus81 results from the failure of the hSNM1A- and XPF-ERCC1-dependent ICL repair pathway. Our results reveal how collaboration between hSNM1A and XPF-ERCC1 is necessary to initiate ICL repair in replicating human cells.

Measurement of DNA damage in individual cells using the Single Cell Gel Electrophoresis (Comet) assay.

The Single Cell Gel Electrophoresis (Comet) assay is a simple, versatile and sensitive method for measuring DNA damage in individual cells, allowing the determination of heterogeneity of response within a cell population. The basic alkaline technique described is for the determination of DNA strand break damage and its repair at a single cell level. Specific modifications to the method use a lower pH ('neutral' assay), or allow the measurement of DNA interstrand cross-links. It can be further adapted to, for example, study specific DNA repair mechanisms, be combined with fluorescent in situ hybridisation, or incorporate lesion specific enzymes.

SG2285, a novel C2-aryl-substituted pyrrolobenzodiazepine dimer prodrug that cross-links DNA and exerts highly potent antitumor activity.

The pyrrolobenzodiazepines (PBD) are naturally occurring antitumor antibiotics, and a PBD dimer (SJG-136, SG2000) is in phase II trials. Many potent PBDs contain a C2-endo-exo unsaturated motif associated with the pyrrolo C-ring. The novel compound SG2202 is a PBD dimer containing this motif. SG2285 is a water-soluble prodrug of SG2202 in which two bisulfite groups inactivate the PBD N10-C11 imines. Once the bisulfites are eliminated, the imine moieties can bind covalently in the DNA minor groove, forming an interstrand cross-link. The mean in vitro cytotoxic potency of SG2285 against human tumor cell lines is GI(50) 20 pmol/L. SG2285 is highly efficient at producing DNA interstrand cross-links in cells, but they form more slowly than those produced by SG2202. Cellular sensitivity to SG2285 was primarily dependent on ERCC1 and homologous recombination repair. In primary B-cell chronic lymphocytic leukemia samples, the mean LD(50) was significantly lower than in normal age-matched B and T lymphocytes. Antitumor activity was shown in several human tumor xenograft models, including ovarian, non-small cell lung, prostate, pancreatic, and melanoma, with cures obtained in the latter model with a single dose. Further, in an advanced-stage colon model, SG2285 administered either as a single dose, or in two repeat dose schedules, was superior to irinotecan. Our findings define SG2285 as a highly active cytotoxic compound with antitumor properties desirable for further development.

Anaplastic plasmacytomas: relationships to normal memory B cells and plasma cell neoplasms of immunodeficient and autoimmune mice.

Anaplastic plasmacytomas (APCTs) from NFS.V(+) congenic mice and pristane-induced plasmacytic PCTs from BALB/c mice were previously shown to be histologically and molecularly distinct subsets of plasma cell neoplasms (PCNs). Here we extended these comparisons, contrasting primary APCTs and PCTs by gene expression profiling in relation to the expression profiles of normal naïve, germinal centre, and memory B cells and plasma cells. We also sequenced immunoglobulin genes from APCT and APCT-derived cell lines and defined surface phenotypes and chromosomal features of the cell lines by flow cytometry and by spectral karyotyping and fluorescence in situ hybridization. The results indicate that APCTs share many features with normal memory cells and the plasma cell-related neoplasms (PLs) of FASL-deficient mice, suggesting that APCTs and PLs are related and that both derive from memory B cells. Published in 2010 by John Wiley & Sons, Ltd.

Measurement of DNA interstrand crosslinking in individual cells using the Single Cell Gel Electrophoresis (Comet) assay.

The Single Cell Gel Electrophoresis (Comet) assay, originally developed to allow visualisation of DNA strand break damage in individual cells, has been adapted to measure DNA interstrand cross-links. DNA interstrand cross-links are formed in cells by a number of commonly used cancer chemotherapy agents and are considered to be the critical lesion formed by such agents. This technique allows the analysis of DNA interstrand cross-link formation and repair at a single cell level, requires few cells, allows the determination of heterogeneity of response within a cell population and is sensitive enough to measure DNA interstrand cross-links at pharmacologically relevant doses. The method can be applied to any in vitro or in vivo application where a single cell suspension can be obtained. The method has also become invaluable in studies using human tissue and can be used as a method for pharmacodynamic analysis in early clinical trials.

Measurement of DNA interstrand crosslinking in naked DNA using gel-based methods.

Bifunctional DNA damaging agents continue to be the mainstay in various chemotherapeutic regimens used in the clinic. DNA interstrand crosslinks are considered to be the critical cytotoxic lesions for the biological activity of such agents. Gel-based electrophoretic assays can efficiently separate denatured single-stranded DNA from double-stranded, covalently-linked DNA resulting from the presence of an interstrand crosslink. The methods described here offer a simple way for the assessment of crosslinking efficiencies of bifunctional agents in both long fragments of DNA (e.g. 1-5 kb) and short oligonucleotide DNA duplexes. As the repair of interstrand crosslinks is a key determinant of cellular and clinical chemosensitivity, these methods can be useful for the characterization and isolation of site-directed adducted substrates for use in subsequent biochemical analysis of cellular recognition and DNA repair processes.

Expression of infectious murine leukemia viruses by RAW264.7 cells, a potential complication for studies with a widely used mouse macrophage cell line.

The mouse macrophage-like cell line RAW264.7, the most commonly used mouse macrophage cell line in medical research, was originally reported to be free of replication-competent murine leukemia virus (MuLV) despite its origin in a tumor induced by Abelson MuLV containing Moloney MuLV as helper virus. As currently available, however, we find that it produces significant levels of ecotropic MuLV with the biologic features of the Moloney isolate and also MuLV of the polytropic or MCF class. Newborn mice developed lymphoma following inoculation with the MuLV mixture expressed by these cells. These findings should be considered in interpretation of increasingly widespread use of these cells for propagation of other viruses, studies of biological responses to virus infection and use in RNA interference and cell signalling studies.

Functionalised cyclopentadienyl titanium compounds as potential anticancer drugs.

A number of new ionic titanocene compounds have been isolated and characterised, which exhibit excellent cytotoxicity against different human tumour cell lines including a defined cisplatin resistant cell line. A range of biological assays have been carried out to determine levels of cytotoxicity and levels of DNA interstrand crosslinking.

Novel nitrogen mustard-armed combi-molecules for the selective targeting of epidermal growth factor receptor overexperessing solid tumors: discovery of an unusual structure-activity relationship.

To enhance the potency of "combi-molecules", we designed 6a-d and 18 to release an inhibitor of EGFR TK and a bifunctional alkylator. The combi-molecules blocked EGFR TK with potency increasing with the basicity of the mustard moiety. They selectively killed cells transfected with EGFR and were potent against the DU145 prostate cancer cells. Combi-molecule 6a blocked EGFR phosphorylation in an irreversible manner, induced DNA-cross-links, and arrested the cells in mid-S.

Anaplastic, plasmablastic, and plasmacytic plasmacytomas of mice: relationships to human plasma cell neoplasms and late-stage differentiation of normal B cells.

We have compared histologic features and gene expression profiles of newly identified plasmacytomas from NFS.V(+) congenic mice with plasmacytomas of IL6 transgenic, Fasl mutant, and SJL-beta2M(-/-) mice. NFS.V(+) tumors comprised an overlapping morphologic spectrum of high-grade/anaplastic, intermediate-grade/plasmablastic, and low-grade/plasmacytic cases with similarities to subsets of human multiple myeloma and plasmacytoma. Microarray and immunohistochemical analyses of genes expressed by the most prevalent tumors, plasmablastic plasmacytomas, showed them to be most closely related to immunoblastic lymphomas, less so to plasmacytomas of Fasl mutant and SJL mice, and least to plasmacytic plasmacytomas of IL6 transgenic mice. Plasmablastic tumors seemed to develop in an inflammatory environment associated with gene signatures of T cells, natural killer cells, and macrophages not seen with plasmacytic plasmacytomas. Plasmablastic plasmacytomas from NFS.V(+) and SJL-beta2M(-/-) mice did not have structural alterations in Myc or T(12;15) translocations and did not express Myc at high levels, regular features of transgenic and pristane-induced plasmacytomas. These findings imply that, as for human multiple myeloma, Myc-independent routes of transformation contribute to the pathogenesis of these tumors. These findings suggest that plasma cell neoplasms of mice and humans exhibit similar degrees of complexity. Mouse plasmacytomas, previously considered to be homogeneous, may thus be as diverse as their human counterparts with respect to oncogenic mechanisms of plasma cell transformation. Selecting specific types of mouse plasmacytomas that relate most closely to subtypes of human multiple myeloma may provide new opportunities for preclinical testing of drugs for treatment of the human disease.

Modulation of topoisomerase IIalpha expression by a DNA sequence-specific polyamide.

Topoisomerase IIalpha (topo IIalpha) is an important target for several chemotherapeutic agents, including etoposide and doxorubicin. Confluent cells express low levels of topo IIalpha and are resistant to etoposide treatment. Repression of transcription in confluent cells is mediated by binding of the transcription factor NF-Y to inverted CCAAT motifs within the topo IIalpha promoter. To block the repressive binding of NF-Y, a polyamide (JH-37) was designed to bind to the flanking regions of selected CCAAT sites within the topo IIalpha promoter. Electrophoretic mobility shift assays and DNase I footprinting assays showed occupancy of the inverted CCAAT sites by JH-37. Chromatin immunoprecipitation assays confirmed in vivo inhibition of NF-Y binding to the topo IIalpha promoter. Following incubation of confluent NIH3T3 cells with JH-37, increased expression of topo IIalpha mRNA and protein was detectable. This correlated both with increased DNA double-strand breaks as shown by comet assay and decreased cell viability following exposure to etoposide. Polyamides can modulate gene expression and chemosensitivity of cancer cells.

Expression of the cyclin-dependent kinase inhibitor p27 and its deregulation in mouse B cell lymphomas.

CDKN1B (p27) regulates cell-cycle progression at the G1-S transition by suppressing the cyclin E/CDK2 kinase complex. In normal lymphocytes and most human B cell non-Hodgkin lymphomas (NHL), there is an inverse correlation between proliferative activity and expression of p27; however, a subset of NHL with high mitotic indices expresses p27, which is inactive due to sequestration in nuclear protein complexes or due to cytoplasmic retention. Our studies of mouse B cell NHL also identified cases with high proliferative activity and high levels of p27 at a surprisingly high frequency. Here, p27 was complexed with D-type cyclins 1 and 3 and with the COPS9 protein, JAB1. In addition, we found cytoplasmic sequestration following phosphorylation by activated AKT.

Histologic and molecular characterizations of megakaryocytic leukemia in mice.

Six cases of megakaryocytic leukemia (MKL) were identified and analyzed for morphology and molecular features. MKL were composed of megakaryocyte lineage cells ranging from immature to quite mature cells. VWF, GATA1 and RUNX1 were strongly expressed in megakaryocytes in both normal spleen and MKL as analyzed by immunohistochemistry (IHC). Altered expression of Meis1, Pbx1 and Psen2 and Lef1 in MKL detected with oligonucleotide microarrays was confirmed by qPCR and IHC. This is the first report of spontaneous MKL in mice, defining VWF as a biomarker for diagnosis and suggesting possible involvement of a series of genes in disease pathogenesis.

High-throughput retroviral tagging for identification of genes involved in initiation and progression of mouse splenic marginal zone lymphomas.

Human B-cell lymphomas are frequently associated with specific genetic changes caused by chromosomal translocations that activate proto-oncogenes. For lymphomas of mice expressing murine leukemia virus, mutagenic proviral insertions are thought to play a similar role. Here we report studies designed to determine whether specific retroviral integration sites might be associated with a specific subset of mouse B-cell lymphomas and if the genes associated with these sites are regularly altered in expression. We studied splenic marginal zone lymphomas (MZL) of NFS.V(+) mice that are unusual in exhibiting frequent progression from low to high grade, potentially allowing assignment of cancer genes to processes of initiation and progression. We used inverse PCR to clone and analyze 212 retroviral integration sites from 43 MZL at different stages of progression. Sixty-two marked common integration sites and included 31 that had been marked previously. Among the new common integration sites, seven were unique to MZL. Using microarrays and real-time quantitative PCR analysis, we defined differential patterns of gene expression in association with disease progression for Gfi1, Sox4, Brca2, Snf1lk, Nfkb1, Pou2af1, Prdm1, Stat6, and Blnk. Heightened expression of Gfi1 distinguishes MZL from other lymphoma types. The combined use of proviral tagging and analyses of gene expression thus provides a powerful approach to understanding of genes that collaborate in tumorigenesis.