USP7 inhibitors suppress tumour neoangiogenesis and promote synergy with immune checkpoint inhibitors by downregulating fibroblast VEGF.

BACKGROUND: Understanding how to modulate the microenvironment of tumors that are resistant to immune checkpoint inhibitors represents a major challenge in oncology.Here we investigate the ability of USP7 inhibitors to reprogram the tumor microenvironment (TME) by inhibiting secretion of vascular endothelial growth factor (VEGF) from fibroblasts. METHODS: To understand the role played by USP7 in the TME, we systematically evaluated the effects of potent, selective USP7 inhibitors on co-cultures comprising components of the TME, using human primary cells. We also evaluated the effects of USP7 inhibition on tumor growth inhibition in syngeneic models when dosed in combination with immune checkpoint inhibitors (ICIs). RESULTS: Abrogation of VEGF secretion from fibroblasts in response to USP7 inhibition resulted in inhibition of tumor neoangiogenesis and increased tumor recruitment of CD8-positive T-lymphocytes, leading to significantly improved sensitivity to immune checkpoint inhibitors. In syngeneic models, treatment with USP7 inhibitors led to striking tumor responses resulting in significantly improved survival. CONCLUSIONS: USP7-mediated reprograming of the TME is not linked to its previously characterized role in modulating MDM2 but does require p53 and UHRF1 in addition to the well-characterized VEGF transcription factor, HIF-1α. This represents a function of USP7 that is unique to fibroblasts, and which is not observed in cancer cells or other components of the TME. Given the potential for USP7 inhibitors to transform "immune desert" tumors into "immune responsive" tumors, this paves the way for a novel therapeutic strategy combining USP7 inhibitors with immune checkpoint inhibitors (ICIs).

LINC01638 sustains human mesenchymal stem cell self-renewal and competency for osteogenic cell fate.

The skeleton forms from multipotent human mesenchymal stem cells (hMSCs) competent to commit to specific lineages. Long noncoding RNAs (lncRNAs) have been identified as key epigenetic regulators of tissue development. However, regulation of osteogenesis by lncRNAs as mediators of commitment to the bone phenotype is largely unexplored. We focused on LINC01638, which is highly expressed in hMSCs and has been studied in cancers, but not in regulating osteogenesis. We demonstrated that LINC01638 promotes initiation of the osteoblast phenotype. Our findings reveal that LINC01638 is present at low levels during the induction of osteoblast differentiation. CRISPRi knockdown of LINC01638 in MSCs prevents osteogenesis and alkaline phosphatase expression, inhibiting osteoblast differentiation. This resulted in decreased MSC growth rate, accompanied by double-strand breaks, DNA damage, and cell senescence. Transcriptome profiling of control and LINC01638-depleted hMSCs identified > 2000 differentially expressed mRNAs related to cell cycle, cell division, spindle formation, DNA repair, and osteogenesis. Using ChIRP-qPCR, molecular mechanisms of chromatin interactions revealed the LINC01638 locus (Chr 22) includes many lncRNAs and bone-related genes. These novel findings identify the obligatory role for LINC01638 to sustain MSC pluripotency regulating osteoblast commitment and growth, as well as for physiological remodeling of bone tissue.

LINC01638 Sustains Human Mesenchymal Stem Cell Self-Renewal and Competency for Osteogenic Cell Fate.

The skeleton forms from multipotent human mesenchymal stem cells (hMSCs) competent to commit to specific lineages. Long noncoding RNAs (lncRNAs) have been identified as key epigenetic regulators of tissue development. However, regulation of osteogenesis by lncRNAs as mediators of commitment to the bone phenotype is largely unexplored. We focused on LINC01638, which is highly expressed in hMSCs and has been studied in cancers, but not in regulating osteogenesis. We demonstrated that LINC01638 promotes initiation of the osteoblast phenotype. Our findings reveal that LINC01638 is present at low levels during the induction of osteoblast differentiation. CRISPRi knockdown of LINC01638 in MSCs prevents osteogenesis and alkaline phosphatase expression, inhibiting osteoblast differentiation. This resulted in decreased MSC cell growth rate, accompanied by double-strand breaks, DNA damage, and cell senescence. Transcriptome profiling of control and LINC01638-depleted hMSCs identified > 2,000 differentially expressed mRNAs related to cell cycle, cell division, spindle formation, DNA repair, and osteogenesis. Using ChIRP-qPCR, molecular mechanisms of chromatin interactions revealed the LINC01638 locus (Chr 22) includes many lncRNAs and bone-related genes. These novel findings identify the obligatory role for LINC01638 to sustain MSC pluripotency regulating osteoblast commitment and growth, as well as for physiological remodeling of bone tissue.

Identification and development of a subtype-selective allosteric AKT inhibitor suitable for clinical development.

The serine/threonine protein kinase AKT plays a pivotal role within the PI3K pathway in regulating cellular proliferation and apoptotic cellular functions, and AKT hyper-activation via gene amplification and/or mutation has been implicated in multiple human malignancies. There are 3 AKT isoenzymes (AKT1-3) which mediate critical, non-redundant functions. We present the discovery and development of ALM301, a novel, allosteric, sub-type selective inhibitor of AKT1/2. ALM301 binds in an allosteric pocket created by the combined movement of the PH domain and the catalytic domain, resulting in a DFG out conformation. ALM301 was shown to be highly selective against a panel of over 450 kinases and potently inhibited cellular proliferation. These effects were particularly pronounced in MCF-7 cells containing a PI3KCA mutation. Subsequent cellular downstream pathway analysis in this sensitive cell line revealed potent inhibition of pAKT signalling up to 48 h post dosing. ALM301 treatment was well tolerated in an MCF-7 xenograft model and led to a dose-dependent reduction in tumour growth. Enhanced efficacy was observed in combination with tamoxifen. In summary, ALM301 is a highly specific AKT 1/2 inhibitor with an excellent pharmacological profile suitable for further clinical development.

Epigenetic and transcriptome responsiveness to ER modulation by tissue selective estrogen complexes in breast epithelial and breast cancer cells.

Selective estrogen receptor modulators (SERMs), including the SERM/SERD bazedoxifene (BZA), are used to treat postmenopausal osteoporosis and may reduce breast cancer (BCa) risk. One of the most persistent unresolved questions regarding menopausal hormone therapy is compromised control of proliferation and phenotype because of short- or long-term administration of mixed-function estrogen receptor (ER) ligands. To gain insight into epigenetic effectors of the transcriptomes of hormone and BZA-treated BCa cells, we evaluated a panel of histone modifications. The impact of short-term hormone treatment and BZA on gene expression and genome-wide epigenetic profiles was examined in ERαneg mammary epithelial cells (MCF10A) and ERα+ luminal breast cancer cells (MCF7). We tested individual components and combinations of 17ß-estradiol (E2), estrogen compounds (EC10) and BZA. RNA-seq for gene expression and ChIP-seq for active (H3K4me3, H3K4ac, H3K27ac) and repressive (H3K27me3) histone modifications were performed. Our results show that the combination of BZA with E2 or EC10 reduces estrogen-mediated patterns of histone modifications and gene expression in MCF-7ERα+ cells. In contrast, BZA has minimal effects on these parameters in MCF10A mammary epithelial cells. BZA-induced changes in histone modifications in MCF7 cells are characterized by altered H3K4ac patterns, with changes at distal enhancers of ERα-target genes and at promoters of non-ERα bound proliferation-related genes. Notably, the ERα target gene GREB1 is the most sensitive to BZA treatment. Our findings provide direct mechanistic-based evidence that BZA induces epigenetic changes in E2 and EC10 mediated control of ERα regulatory programs to target distinctive proliferation gene pathways that restrain the potential for breast cancer development.

LncMIR181A1HG is a novel chromatin-bound epigenetic suppressor of early stage osteogenic lineage commitment.

Bone formation requires osteogenic differentiation of multipotent mesenchymal stromal cells (MSCs) and lineage progression of committed osteoblast precursors. Osteogenic phenotype commitment is epigenetically controlled by genomic (chromatin) and non-genomic (non-coding RNA) mechanisms. Control of osteogenesis by long non-coding RNAs remains a largely unexplored molecular frontier. Here, we performed comprehensive transcriptome analysis at early stages of osteogenic cell fate determination in human MSCs, focusing on expression of lncRNAs. We identified a chromatin-bound lncRNA (MIR181A1HG) that is highly expressed in self-renewing MSCs. MIR181A1HG is down-regulated when MSCs become osteogenic lineage committed and is retained during adipogenic differentiation, suggesting lineage-related molecular functions. Consistent with a key role in human MSC proliferation and survival, we demonstrate that knockdown of MIR181A1HG in the absence of osteogenic stimuli impedes cell cycle progression. Loss of MIR181A1HG enhances differentiation into osteo-chondroprogenitors that produce multiple extracellular matrix proteins. RNA-seq analysis shows that loss of chromatin-bound MIR181A1HG alters expression and BMP2 responsiveness of skeletal gene networks (e.g., SOX5 and DLX5). We propose that MIR181A1HG is a novel epigenetic regulator of early stages of mesenchymal lineage commitment towards osteo-chondroprogenitors. This discovery permits consideration of MIR181A1HG and its associated regulatory pathways as targets for promoting new bone formation in skeletal disorders.

Regulation of osteogenesis by long noncoding RNAs: An epigenetic mechanism contributing to bone formation.

Long noncoding RNAs (lncRNAs) have recently emerged as novel regulators of lineage commitment, differentiation, development, viability, and disease progression. Few studies have examined their role in osteogenesis; however, given their critical and wide-ranging roles in other tissues, lncRNAs are most likely vital regulators of osteogenesis. In this study, we extensively characterized lncRNA expression in mesenchymal cells during commitment and differentiation to the osteoblast lineage using a whole transcriptome sequencing approach (RNA-Seq). Using mouse primary mesenchymal stromal cells (mMSC), we identified 1438 annotated lncRNAs expressed during MSC differentiation, 462 of which are differentially expressed. We performed guilt-by-association analysis using lncRNA and mRNA expression profiles to identify lncRNAs influencing MSC commitment and differentiation. These findings open novel dimensions for exploring lncRNAs in regulating normal bone formation and in skeletal disorders.

Identification and Structure-Guided Development of Pyrimidinone Based USP7 Inhibitors.

Ubiquitin specific protease 7 (USP7, HAUSP) has become an attractive target in drug discovery due to the role it plays in modulating Mdm2 levels and consequently p53. Increasing interest in USP7 is emerging due to its potential involvement in oncogenic pathways as well as possible roles in both metabolic and immune disorders in addition to viral infections. Potent, novel, and selective inhibitors of USP7 have been developed using both rational and structure-guided design enabled by high-resolution cocrystallography. Initial hits were identified via fragment-based screening, scaffold-hopping, and hybridization exercises. Two distinct subseries are described along with associated structure-activity relationship trends, as are initial efforts aimed at developing compounds suitable for in vivo experiments. Overall, these discoveries will enable further research into the wider biological role of USP7.

Selective expression of long non-coding RNAs in a breast cancer cell progression model.

Long non-coding RNAs (lncRNAs) are acknowledged as regulators of cancer biology and pathology. Our goal was to perform a stringent profiling of breast cancer cell lines that represent disease progression. We used the MCF-10 series, which includes the normal-like MCF-10A, HRAS-transformed MCF-10AT1 (pre-malignant), and MCF-10CA1a (malignant) cells, to perform transcriptome wide sequencing. From these data, we have identified 346 lncRNAs with dysregulated expression across the progression series. By comparing lncRNAs from these datasets to those from an additional set of cell lines that represent different disease stages and subtypes, MCF-7 (early stage, luminal), and MDA-MB-231 (late stage, basal), 61 lncRNAs that are associated with breast cancer progression were identified. Querying breast cancer patient data from The Cancer Genome Atlas, we selected a lncRNA, IGF-like family member 2 antisense RNA 1 (IGFL2-AS1), of potential clinical relevance for functional characterization. Among the 61 lncRNAs, IGFL2-AS1 was the most significantly decreased. Our results indicate that this lncRNA plays a role in downregulating its nearest neighbor, IGFL1, and affects migration of breast cancer cells. Furthermore, the lncRNAs we identified provide a valuable resource to mechanistically and clinically understand the contribution of lncRNAs in breast cancer progression.

Discovery and characterization of highly potent and selective allosteric USP7 inhibitors.

Given the importance of ubiquitin-specific protease 7 (USP7) in oncogenic pathways, identification of USP7 inhibitors has attracted considerable interest. Despite substantial efforts, however, the development of validated deubiquitinase (DUB) inhibitors that exhibit drug-like properties and a well-defined mechanism of action has proven particularly challenging. In this article, we describe the identification, optimization and detailed characterization of highly potent (IC50 < 10 nM), selective USP7 inhibitors together with their less active, enantiomeric counterparts. We also disclose, for the first time, co-crystal structures of a human DUB enzyme complexed with small-molecule inhibitors, which reveal a previously undisclosed allosteric binding site. Finally, we report the identification of cancer cell lines hypersensitive to USP7 inhibition (EC50 < 30 nM) and demonstrate equal or superior activity in these cell models compared to clinically relevant MDM2 antagonists. Overall, these findings demonstrate the tractability and druggability of DUBs, and provide important tools for additional target validation studies.

Intranuclear and higher-order chromatin organization of the major histone gene cluster in breast cancer.

Alterations in nuclear morphology are common in cancer progression. However, the degree to which gross morphological abnormalities translate into compromised higher-order chromatin organization is poorly understood. To explore the functional links between gene expression and chromatin structure in breast cancer, we performed RNA-seq gene expression analysis on the basal breast cancer progression model based on human MCF10A cells. Positional gene enrichment identified the major histone gene cluster at chromosome 6p22 as one of the most significantly upregulated (and not amplified) clusters of genes from the normal-like MCF10A to premalignant MCF10AT1 and metastatic MCF10CA1a cells. This cluster is subdivided into three sub-clusters of histone genes that are organized into hierarchical topologically associating domains (TADs). Interestingly, the sub-clusters of histone genes are located at TAD boundaries and interact more frequently with each other than the regions in-between them, suggesting that the histone sub-clusters form an active chromatin hub. The anchor sites of loops within this hub are occupied by CTCF, a known chromatin organizer. These histone genes are transcribed and processed at a specific sub-nuclear microenvironment termed the major histone locus body (HLB). While the overall chromatin structure of the major HLB is maintained across breast cancer progression, we detected alterations in its structure that may relate to gene expression. Importantly, breast tumor specimens also exhibit a coordinate pattern of upregulation across the major histone gene cluster. Our results provide a novel insight into the connection between the higher-order chromatin organization of the major HLB and its regulation during breast cancer progression.

The impact of paliperidone palmitate long-acting injection on hospital admissions in a mental health setting.

We screened 216 patients in a retrospective observational investigator-initiated study, of whom 45.37% patients (n=98) were retained after the inclusion criteria were applied. These patients had been prescribed paliperidone palmitate long-acting injection (PPLAI) with diagnoses of schizophrenia, schizoaffective disorder and bipolar affective disorder. We investigated whether PPLAI has an effect on the frequency and length of admissions to mental health inpatient units, the number of contacts with Crisis Resolution Home Treatment Teams (CRHTT) and frequency of home visits by the CRHTT per patient, over 6 years, split using a 'mirror image' method. In total, 85% of patients continued PPLAI for 1 year, 60% for 2 years and 47% for 3 years. In the sample of patients who continued with PPLAI for 3 years (n=48), the mean number of hospital admissions decreased from 1.03 to 0.35 per patient (P=0.001). The mean number of bed days decreased significantly from 53 to 24 bed days (P=0.0001). The median values showed similar significant differences. There were numerical differences but nonsignificant results between CRHTT's number of contacts and length of episodes across the study period. Our results indicate that PPLAI has been shown to be effective in the reduction of hospital admissions, relapse rates and length of inpatient stay.

Interactions between sanitizers and packaging gas compositions and their effects on the safety and quality of fresh-cut onions (Allium cepa L.).

Onions are one of the most widely utilized vegetables worldwide, with demand for fresh-cut onions steadily increasing. Due to heightened safety concerns and consumer demand, the implications of sanitizing and packaging on fresh-cut onion safety and quality need to be better understood. The objective of this study was to investigate the effect of produce sanitizers, in-package atmospheres, and their interactions on the growth of Salmonella Typhimurium, mesophilic aerobic bacteria, yeast and mold, and the physico-chemical quality of diced onions to determine the best sanitizer and in-package atmosphere combination for both safety and quality. Diced onions were inoculated or not with S. Typhimurium, sanitized in sodium hypochlorite, peroxyacetic acid, or liquid chlorine dioxide, and then packaged in either polylactic acid bags containing superatmospheric O2, elevated CO2/reduced O2, or air, or in polyethylene terephthalate snap-fit containers. Throughout 14 days of storage at 7 °C, packaged diced onions were assessed for their safety (S. Typhimurium), and quality (mesophilic aerobic bacteria, yeasts and molds, physico-chemical analyses, and descriptive and consumer acceptance sensory panels). While sanitizer affected (P<0.05) fewer parameters (S. Typhimurium, mesophiles, yeasts and molds, headspace CO2, weight loss, and pH), in-package atmosphere had a significant (P<0.05) effect on all parameters evaluated. Two-way interactions between sanitizer and atmosphere that affected S. Typhimurium and pH were identified whereas 3-way interactions (sanitizer, atmosphere and time) were only observed for headspace CO2. Sodium hypochlorite and elevated CO2/reduced O2 was the best sanitizer and in-package atmosphere combination for enhancing the safety and quality of packaged diced onions. In addition, this combination led to diced onions acceptable for purchase after 2 weeks of storage by trained and consumer panels.

Transcriptional regulation of inducible nitric oxide synthase gene therapy: targeting early stage and advanced prostate cancer.

BACKGROUND: Using the tumour type specific human osteocalcin (hOC) promoter, we have previously reported strong promoter activation in hormone independent prostate cancer cells in vitro. In the present study, we present a comparative study of the tissue specific promoter prostate specific membrane antigen (PSMA), and the tumour-type specific hOC promoter driving the inducible nitric oxide synthase (iNOS) transgene using both in vitro and in vivo models. METHODS: In vitro cytotoxicity was assessed by clonogenic assay. Quantification of nitric oxide expression was determined by the Griess test. In vivo anti-tumour efficacy was determined by tumour growth delay following direct intra-tumoural injection of the constructs into PC3 xenografts. In addition, tumours were dissected post mortem and examined for morphological differences as well as changes in apoptotic protein expression. RESULTS: PSMA/iNOS produced cytotoxicity in both androgen dependant and independent cell lines. Nitric oxide quantification confirmed that increased cytotoxicity was directly associated with nitric oxide production. Tumour growth delays were observed in all groups treated with the iNOS-expressing constructs ranging from 10.7 days for the hOC/iNOS single dose treatment group to a maximum of 52.2 days for the hOC/iNOS multiple dose group. Intra-tumoural assessment of iNOS and cleaved poly (ADP-ribose) polymerase protein expression demonstrated a significant up-regulation of both proteins, indicating cytotoxicity mediated through the intrinsic apoptotic pathway. CONCLUSIONS: Highly significant tumour growth delay coupled with no detrimental side-effects were observed following treatment with the PSMA/iNOS and hOC/iNOS constructs. We consider that these findings provide a basis for the development of systemically delivered PSMA/iNOS or hOC/iNOS targeting early stage and advanced prostate cancer.

Functional characterization of the atopy-associated gene PHF11.

BACKGROUND: Polymorphisms in the plant homeodomain finger protein 11 gene (PHF11) are associated with increased total serum IgE levels, asthma, and severe atopic dermatitis (AD) in children. Although PHF11 includes a plant homeodomain, a motif often found in transcriptional regulators, the function of PHF11 has not been investigated. OBJECTIVE: We sought to test (1) whether PHF11 regulates the transcription of genes involved in allergic disorders and (2) whether polymorphisms in PHF11 predict changes in the expression or function of this gene. METHODS: Microarray analysis was used to examine the expression of PHF11 in different immune cell subsets, and the function of PHF11 was tested by using small interfering RNA-induced knockdown or overexpression of PHF11 in primary CD4+ T cells or Jurkat T cells. Genotype-dependent effects on PHF11 expression were tested by using an allele-specific gene expression, and the transcriptional activity of PHF11 was determined by using luciferase hybrid gene reporter assays and in vitro DNA-binding electromobility shift assays. RESULTS: PHF11 expression was higher in T(H)1 cells relative to that in T(H)2 cells, and knockdown of PHF11 expression reduced expression of the T(H)1-type cytokines IFN-gamma and IL-2. The G-allele of a 3' untranslated region polymorphism associated with AD was correlated with reduced abundance of PHF11 RNA in T(H)1 cells, as well as an increase in a PHF11 isoform lacking exon II. Evidence was also found for a physical and functional interaction between PHF11 and the p65 subunit of nuclear factor kappaB. CONCLUSION: PHF11 is a regulator of T(H)1-type cytokine gene expression. The reduction in PHF11 expression seen with an AD-associated genotype could contribute to the strong T(H)2 responses that characterize many allergic individuals.

Genetic association studies between the T cell immunoglobulin mucin (TIM) gene locus and childhood atopic dermatitis.

BACKGROUND: The T cell immunoglobulin mucin (TIM) gene family is involved in T cell proliferation and differentiation and has been implicated in allergic disease. We have tested whether TIM gene polymorphisms are associated with atopic dermatitis (AD) in an Australian cohort. METHODS: Transmission disequilibrium testing of 15 single nucleotide polymorphisms across TIM-1, TIM-3 and TIM-4 in 93 Caucasian families, and of a tri-allelic (0, 15 and 18 base pairs) TIM-1 insertion polymorphism in 123 Caucasian and Asian families, was carried out in proband-parent trio families. RESULTS: Transmission of the 18-base pair variant of this insertion was significantly under-represented in the childhood AD cohort (p = 0.02), which is in agreement with a previous study on asthma in an African-American cohort. We also found a novel association between AD and the major haplotype of TIM-4 (p = 0.016). There was no evidence for an association between AD and TIM-3. CONCLUSIONS: In addition to confirming the importance of genetic variation in TIM-1, our results also suggest that genetic variants in the ligand for TIM-1, TIM-4, also contribute to the presentation of AD and related disorders.

Effect of the militarily-relevant heavy metals, depleted uranium and heavy metal tungsten-alloy on gene expression in human liver carcinoma cells (HepG2).

Depleted uranium (DU) and heavy-metal tungsten alloys (HMTAs) are dense heavy-metals used primarily in military applications. Chemically similar to natural uranium, but depleted of the higher activity 235U and 234U isotopes, DU is a low specific activity, high-density heavy metal. In contrast, the non-radioactive HMTAs are composed of a mixture of tungsten (91-93%), nickel (3-5%), and cobalt (2-4%) particles. The use of DU and HMTAs in military munitions could result in their internalization in humans. Limited data exist however, regarding the long-term health effects of internalized DU and HMTAs in humans. Both DU and HMTAs possess a tumorigenic transforming potential and are genotoxic and mutagenic in vitro. Using insoluble DU-UO2 and a reconstituted mixture of tungsten, nickel, cobalt (rWNiCo), we tested their ability to induce stress genes in thirteen different recombinant cell lines generated from human liver carcinoma cells (HepG2). The commercially available CAT-Tox (L) cellular assay consists of a panel of cell lines stably transfected with reporter genes consisting of a coding sequence for chloramphenicol acetyl transferase (CAT) under transcriptional control by mammalian stress gene regulatory sequences. DU, (5-50 microg/ml) produced a complex profile of activity demonstrating significant dose-dependent induction of the hMTIIA FOS, p53RE, Gadd153, Gadd45, NFkappaBRE, CRE, HSP70, RARE, and GRP78 promoters. The rWNiCo mixture (5-50 microg/ml) showed dose-related induction of the GSTYA, hMTIIA, p53RE, FOS, NFkappaBRE, HSP70, and CRE promoters. An examination of the pure metals, tungsten (W), nickel (Ni), and cobalt (Co), comprising the rWNiCo mixture, demonstrated that each metal exhibited a similar pattern of gene induction, but at a significantly decreased magnitude than that of the rWNiCo mixture. These data showed a synergistic activation of gene expression by the metals in the rWNiCo mixture. Our data show for the first time that DU and rWNiCo can activate gene expression through several signal transduction pathways that may be involved in the toxicity and tumorigenicity of both DU and HMTAs.

Genomic instability in human osteoblast cells after exposure to depleted uranium: delayed lethality and micronuclei formation.

It is known that radiation can induce a transmissible persistent destabilization of the genome. We have established an in vitro cellular model using HOS cells to investigate whether genomic instability plays a role in depleted uranium (DU)-induced effects. Transmissible genomic instability, manifested in the progeny of cells exposed to ionizing radiation, has been characterized by de novo chromosomal aberrations, gene mutations, and an enhanced death rate. Cell lethality and micronuclei formation were measured at various times after exposure to DU, Ni, or gamma radiation. Following a prompt, concentration-dependent acute response for both endpoints, there was de novo genomic instability in progeny cells. Delayed reproductive death was observed for many generations (36 days, 30 population doublings) following exposure to DU, Ni, or gamma radiation. While DU stimulated delayed production of micronuclei up to 36 days after exposure, levels in cells exposed to gamma-radiation or Ni returned to normal after 12 days. There was also a persistent increase in micronuclei in all clones isolated from cells that had been exposed to nontoxic concentrations of DU. While clones isolated from gamma-irradiated cells (at doses equitoxic to metal exposure) generally demonstrated an increase in micronuclei, most clonal progeny of Ni-exposed cells did not. These studies demonstrate that DU exposure in vitro results in genomic instability manifested as delayed reproductive death and micronuclei formation.

Depleted uranium-catalyzed oxidative DNA damage: absence of significant alpha particle decay.

Depleted uranium (DU) is a dense heavy metal used primarily in military applications. Published data from our laboratory have demonstrated that DU exposure in vitro to immortalized human osteoblast cells (HOS) is both neoplastically transforming and genotoxic. DU possesses both a radiological (alpha particle) and a chemical (metal) component. Since DU has a low-specific activity in comparison to natural uranium, it is not considered to be a significant radiological hazard. In the current study we demonstrate that DU can generate oxidative DNA damage and can also catalyze reactions that induce hydroxyl radicals in the absence of significant alpha particle decay. Experiments were conducted under conditions in which chemical generation of hydroxyl radicals was calculated to exceed the radiolytic generation by one million-fold. The data showed that markers of oxidative DNA base damage, thymine glycol and 8-deoxyguanosine could be induced from DU-catalyzed reactions of hydrogen peroxide and ascorbate similarly to those occurring in the presence of iron catalysts. DU was 6-fold more efficient than iron at catalyzing the oxidation of ascorbate at pH 7. These data not only demonstrate that DU at pH 7 can induced oxidative DNA damage in the absence of significant alpha particle decay, but also suggest that DU can induce carcinogenic lesions, e.g. oxidative DNA lesions, through interaction with a cellular oxygen species.

Potential late health effects of depleted uranium and tungsten used in armor-piercing munitions: comparison of neoplastic transformation and genotoxicity with the known carcinogen nickel.

Limited data exist to permit an accurate assessment of risks for carcinogenesis and mutagenesis from embedded fragments or inhaled particulates of depleted uranium (DU). Ongoing studies have been designed to provide information about the carcinogenic potential of DU using in vitro and in vivo assessments of morphological transformation as well as cytogenetic, mutagenic, and oncogenic effects. For comparison, we also examined tungsten alloys used in military projectiles and the known carcinogen nickel. Quantitative and qualitative in vitro transformation studies were done to assess the carcinogenic potential of radiation and chemical hazards. Using a human osteosarcoma cell model, we demonstrated that soluble and insoluble DU compounds can transform cells to the tumorigenic phenotype, as characterized by morphological, biochemical, and oncogenic changes consistent with tumor cell behavior. Tungsten alloys and nickel were also shown to be neoplastic transforming agents, although at a frequency less than that of DU. Sister chromatid exchange, micronuclei, and alkaline filter elution assays showed DU and tungsten alloys were genotoxic. Exposure to a nontoxic, nontransforming dose of DU induced a small but statistically significant increase in the number of dicentrics formed in cells. These results suggest that long-term exposure to DU or tungsten alloys could be critical to the development of neoplastic disease in humans and that additional studies are needed.