Adaptive homeostasis and the p53 isoform network.

All living organisms have developed processes to sense and address environmental changes to maintain a stable internal state (homeostasis). When activated, the p53 tumour suppressor maintains cell and organ integrity and functions in response to homeostasis disruptors (stresses) such as infection, metabolic alterations and cellular damage. Thus, p53 plays a fundamental physiological role in maintaining organismal homeostasis. The TP53 gene encodes a network of proteins (p53 isoforms) with similar and distinct biochemical functions. The p53 network carries out multiple biological activities enabling cooperation between individual cells required for long-term survival of multicellular organisms (animals) in response to an ever-changing environment caused by mutation, infection, metabolic alteration or damage. In this review, we suggest that the p53 network has evolved as an adaptive response to pathogen infections and other environmental selection pressures.

A pilot study using unique targeted testing of the urogenital microbiome has potential as a predictive test during IVF for implantation outcome.

PURPOSE: This pilot study aimed to develop a methodology characterising the urogenital microbiome as a predictive test in the IVF workup. METHODS: Using unique custom qPCRs, we tested for the presence of specific microbial species from vaginal samples and First Catch Urines from the male. The test panel included a range of potential urogenital pathogens, STIs, 'favourable bacteria' (Lactobacillus spp.) and 'unfavourable bacteria' (anaerobes) reported to influence implantation rates. We tested couples attending Fertility Associates, Christchurch, New Zealand for their first round of IVF. RESULTS: We found that some microbial species affected implantation. The qPCR result was interpreted qualitatively using the Z proportionality test. Samples from women at the time of Embryo Transfer who did not achieve implantation had significantly higher percent of samples that were positive for Prevotella bivia and Staphylococcus aureus compared to women who did achieve implantation. DISCUSSION: The results provide evidence that most other microbial species chosen for testing had little functional effect on implantation rates. The addition of further microbial targets (yet to be determined) could be combined in this predictive test for vaginal preparedness on the day of embryo transfer. This methodology has a substantial advantage of being affordable and easily performed in any routine molecular laboratory. This methodology is most suitable as a foundation on which to develop a timely test of microbiome profiling. Using the indicators detected to have a significant influence, these results can be extrapolated. CONCLUSION: Using a rapid antigen test, a woman can self-sample prior to embryo transfer and obtain an indication of microbial species present which could influence implantation outcome.

Increased Expression of the Δ133p53ß Isoform Enhances Brain Metastasis.

The Δ133p53ß isoform is increased in many primary tumors and has many tumor-promoting properties that contribute to increased proliferation, migration and inflammation. Here we investigated whether Δ133p53ß contributed to some of the most aggressive tumors that had metastasized to the brain. Δ133p53ß mRNA expression was measured in lung, breast, melanoma, colorectal metastases and, where available, the matched primary tumor. The presence of Δ133p53ß expression was associated with the time for the primary tumor to metastasize and overall survival once the tumor was detected in the brain. Δ133p53ß was present in over 50% of lung, breast, melanoma and colorectal metastases to the brain. It was also increased in the brain metastases compared with the matched primary tumor. Brain metastases with Δ133p53ß expressed were associated with a reduced time for the primary tumor to metastasize to the brain compared with tumors with no Δ133p53ß expression. In-vitro-based analyses in Δ133p53ß-expressing cells showed increased cancer-promoting proteins on the cell surface and increased downstream p-AKT and p-MAPK signaling. Δ133p53ß-expressing cells also invaded more readily across a mock blood-brain barrier. Together these data suggested that Δ133p53ß contributes to brain metastases by making cells more likely to invade the brain.

The epithelial sodium channel has a role in breast cancer cell proliferation.

PURPOSE: Breast cancer is the most common cancer affecting women worldwide with half a million associated deaths annually. Despite a huge global effort, the pathways of breast cancer progression are not fully elucidated. Ion channels have recently emerged as novel regulators of cancer cell proliferation and metastasis. The epithelial sodium channel, ENaC, made up of α, ß and γ subunits is well known for its role in Na+ reabsorption in epithelia, but a number of novel roles for ENaC have been described, including potential roles in cancer. A role for ENaC in breast cancer, however, has yet to be described. Therefore, the effects of ENaC level and activity on breast cancer proliferation were investigated. METHODS: Through the publicly available SCAN-B dataset associations between αENaC mRNA expression and breast cancer subtypes, proliferation markers and epithelial-mesenchymal transition markers (EMT) were assessed. αENaC expression, through overexpression or siRNA-mediated knockdown, and activity, through the ENaC-specific inhibitor amiloride, were altered in MCF7, T47D, BT549, and MDAMB231 breast cancer cells. MTT and EdU cell proliferation assays were used to determine the effect of these manipulations on breast cancer cell proliferation. RESULTS: High αENaC mRNA expression was associated with less aggressive and less proliferative breast cancer subtypes and with reduced expression of proliferation markers. Decreased αENaC expression or activity, in the mesenchymal breast cancer cell lines BT549 and MDAMB231, increased breast cancer cell proliferation. Conversely, increased αENaC expression decreased breast cancer cell proliferation. CONCLUSION: αENaC expression is associated with a poor prognosis in breast cancer and is a novel regulator of breast cancer cell proliferation. Taken together, these results identify ENaC as a potential future therapeutic target.

Elevation of the TP53 isoform Δ133p53ß in glioblastomas: an alternative to mutant p53 in promoting tumor development.

As tumor protein 53 (p53) isoforms have tumor-promoting, migration, and inflammatory properties, this study investigated whether p53 isoforms contributed to glioblastoma progression. The expression levels of full-length TP53α (TAp53α) and six TP53 isoforms were quantitated by RT-qPCR in 89 glioblastomas and correlated with TP53 mutation status, tumor-associated macrophage content, and various immune cell markers. Elevated levels of Δ133p53ß mRNA characterised glioblastomas with increased CD163-positive macrophages and wild-type TP53. In situ-based analyses found Δ133p53ß expression localised to malignant cells in areas with increased hypoxia, and in cells with the monocyte chemoattractant protein C-C motif chemokine ligand 2 (CCL2) expressed. Tumors with increased Δ133p53ß had increased numbers of cells positive for macrophage colony-stimulating factor 1 receptor (CSF1R) and programmed death ligand 1 (PDL1). In addition, cells expressing a murine 'mimic' of Δ133p53 (Δ122p53) were resistant to temozolomide treatment and oxidative stress. Our findings suggest that elevated Δ133p53ß is an alternative pathway to TP53 mutation in glioblastoma that aids tumor progression by promoting an immunosuppressive and chemoresistant environment. Adding Δ133p53ß to a TP53 signature along with TP53 mutation status will better predict treatment resistance in glioblastoma. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

A mouse model of the Δ133p53 isoform: roles in cancer progression and inflammation.

This review paper outlines studies on the Δ122p53 mouse, a model of the human Δ133p53 isoform, together with studies in other model organisms, cell culture, and where available, clinical investigations. In general, these studies imply that, in contrast to the canonical p53 tumor suppressor, Δ133p53 family members have oncogenic capability. Δ122p53 is multi-functional, conferring survival and proliferative advantages on cells, promoting invasion, metastasis and vascularization, as does Δ133p53. Cancers with high levels of Δ133p53 often have poor prognosis. Δ122p53 mediates its effects through the JAK-STAT and RhoA-ROCK signaling pathways. We propose that Δ133p53 isoforms have evolved as inflammatory signaling molecules to deal with the consequent tissue damage of p53 activation. However, if sustained expression of the isoforms occur, pathologies may result.

Telomere profiles and tumor-associated macrophages with different immune signatures affect prognosis in glioblastoma.

Telomere maintenance is a hallmark of cancer and likely to be targeted in future treatments. In glioblastoma established methods of identifying telomerase and alternative lengthening of telomeres leave a significant proportion of tumors with no defined telomere maintenance mechanism. This study investigated the composition of these tumors using RNA-Seq. Glioblastomas with an indeterminate telomere maintenance mechanism had an increased immune signature compared with alternative lengthening of telomeres and telomerase-positive tumors. Immunohistochemistry for CD163 confirmed that the majority (80%) of tumors with an indeterminate telomere maintenance mechanism had a high presence of tumor-associated macrophages. The RNA-Seq and immunostaining data separated tumors with no defined telomere maintenance mechanism into three subgroups: alternative lengthening of telomeres like tumors with a high presence of tumor-associated macrophages and telomerase like tumors with a high presence of tumor-associated macrophages. The third subgroup had no increase in tumor-associated macrophages and may represent a distinct category. The presence of tumor-associated macrophages conferred a worse prognosis with reduced patient survival times (alternative lengthening of telomeres with and without macrophages P=0.0004, and telomerase with and without macrophages P=0.013). The immune signatures obtained from RNA-Seq were significantly different between telomere maintenance mechanisms. Alternative lengthening of telomeres like tumors with macrophages had increased expression of interferon-induced proteins with tetratricopeptide repeats (IFIT1-3). Telomerase-positive tumors with macrophages had increased expression of macrophage receptor with collagenous structure (MARCO), CXCL12 and sushi-repeat containing protein x-linked 2 (SRPX2). Telomerase-positive tumors with macrophages were also associated with a reduced frequency of total/near total resections (44% vs >76% for all other subtypes, P=0.014). In summary, different immune signatures are found among telomere maintenance mechanism-based subgroups in glioblastoma. The reduced extent of surgical resection of telomerase-positive tumors with macrophages suggests that some tumor-associated macrophages are more unfavorable.

A clinicopathological study of episomal papillomavirus infection of the human placenta and pregnancy complications.

Viral infections are known to adversely affect pregnancy, but scant attention has been given to human papilloma virus (HPV) infection. We aimed to determine the molecular and histopathological features of placental HPV infection, in association with pregnancy complications including fetal growth restriction, pre-maturity, pre-eclampsia, and diabetes. Three hundred and thirty-nine placentae were selected based on the presence or absence of pregnancy complications. Five independent methods were used to identify HPV in the placenta, namely, immunohistochemistry for L1 viral capsid, in situ hybridization to high-risk HPV DNA, PCR, western blotting, and transmission electron microscopy. Pregnancy complications and uterine cervical smear screening results were correlated with placental HPV histopathology. In this study, which was deliberately biased towards complications, HPV was found in the decidua of 75% of placentae (253/339) and was statistically associated with histological acute chorioamnionitis (P<0.05). In 14% (35/253) of the HPV positive cases, HPV L1 immunoreactivity also occurred in the villous trophoblast where it was associated with a lymphohistiocytic villitis (HPV-LHV), and was exclusively of high-risk HPV type. HPV-LHV significantly associated with fetal growth restriction, preterm delivery, and pre-eclampsia (all P<0.05). All cases of pre-eclampsia (20/20) in our cohort had high-risk placental HPV. A further 55 cases (22%, 55/253) of HPV positive placentae had minimal villous trophoblast HPV L1 immunoreactivity, but a sclerosing pauci-immune villitis, statistically associated with diabetes (49.1%, 27/55, P<0.05). For women with placental HPV, 33% (69/207) had an HPV-related positive smear result before pregnancy compared with (9.4% 8/85) of women with HPV-negative placentae (P=0.0001). Our findings support further investigations to determine if vaccination of women and men will improve pregnancy outcomes.

Increased paired box transcription factor 8 has a survival function in glioma.

BACKGROUND: The molecular basis to overcome therapeutic resistance to treat glioblastoma remains unclear. The anti-apoptotic b cell lymphoma 2 (BCL2) gene is associated with treatment resistance, and is transactivated by the paired box transcription factor 8 (PAX8). In earlier studies, we demonstrated that increased PAX8 expression in glioma cell lines was associated with the expression of telomerase. In this current study, we more extensively explored a role for PAX8 in gliomagenesis. METHODS: PAX8 expression was measured in 156 gliomas including telomerase-negative tumours, those with the alternative lengthening of telomeres (ALT) mechanism or with a non-defined telomere maintenance mechanism (NDTMM), using immunohistochemistry and quantitative PCR. We also tested the affect of PAX8 knockdown using siRNA in cell lines on cell survival and BCL2 expression. RESULTS: Seventy-two percent of glioblastomas were PAX8-positive (80% telomerase, 73% NDTMM, and 44% ALT). The majority of the low-grade gliomas and normal brain cells were PAX8-negative. The suppression of PAX8 was associated with a reduction in both cell growth and BCL2, suggesting that a reduction in PAX8 expression would sensitise tumours to cell death. CONCLUSIONS: PAX8 is increased in the majority of glioblastomas and promoted cell survival. Because PAX8 is absent in normal brain tissue, it may be a promising therapeutic target pathway for treating aggressive gliomas.

Omeprazole-induced acute interstitial nephritis: a possible Th1-Th17-mediated injury?

BACKGROUND: Omeprazole is an important cause of drug-induced acute interstitial nephritis (AIN). How omeprazole induces injury is unknown. METHODS AND RESULTS: Detailed clinical assessment of 25 biopsy-proven cases of omeprazole-induced AIN showed that all patients presented with impaired renal function, sterile pyuria with varying amounts of proteinuria but no eosinophiluria and no systemic symptoms to suggest a vasculitis. Histological analyses were characteristic of an acute tubulitis with an inflammatory cellular infiltrate. Using modified Banff scheme criteria, mild tubulitis (t1) was present in 56% of cases, a moderate tubulitis (t2) in 24% of cases, and a severe tubulitis in 20% of cases. Most (78%) of cases had mononuclear cell infiltrates, no significant eosinophilic infiltrates were found, and glomeruli were not involved. Immunostaining for CD4, CD8, IL-17A, IL-17F, Foxp3 and T-bet (T cell subsets), CD20 and CD163 defined the cellular infiltrates. The predominant inflammatory cells were CD4+ lymphocytic aggregates (77% of cases), combined with co-staining of CD4 IL and 17A/F in 44-48% of all cases, suggesting a Th17-mediated inflammatory process. T-bet+ cell infiltrates were present to a lesser degree, suggesting additional Th1 involvement. How omeprazole induces this inflammatory response is unclear, but may include direct effects by IL-17 expressing CD4+ cells on renal tubular cells. CONCLUSION: This large biopsy series of omeprazole-induced AIN demonstrates the features of acute tubulitis, with significant interstitial infiltrates consistent with immunopathological Th17 and Th1 processes.

Combining TP53 mutation and isoform has the potential to improve clinical practice.

The clinical importance of assessing and combining data on TP53 mutations and isoforms is discussed in this article. It gives a succinct overview of the structural makeup and key biological roles of the isoforms. It then provides a comprehensive summary of the roles that p53 isoforms play in cancer development, therapy response and resistance. The review provides a summary of studies demonstrating the role of p53 isoforms as potential prognostic indicators. It further provides evidence on how the presence of TP53 mutations may affect one or more of these activities and the association of p53 isoforms with clinicopathological data in various tumour types. The review gives insight into the present diagnostic hurdles for identifying TP53 isoforms and makes recommendations to improve their evaluation. In conclusion, this review offers suggestions for enhancing the identification and integration of TP53 isoforms in conjunction with mutation data within the clinical context.

Hyperproliferation, cancer, and inflammation in mice expressing a Δ133p53-like isoform.

The p53 protein is a pivotal tumor suppressor that is frequently mutated in many human cancers, although precisely how p53 prevents tumors is still unclear. To add to its complexity, several isoforms of human p53 have now been reported. The Δ133p53 isoform is generated from an alternative transcription initiation site in intron 4 of the p53 gene (Tp53) and lacks the N-terminus. Elevated levels of Δ133p53 have been observed in a variety of tumors. To explore the functions of Δ133p53, we created a mouse expressing an N-terminal deletion mutant of p53 (Δ122p53) that corresponds to Δ133p53. Δ122p53 mice show decreased survival and a different and more aggressive tumor spectrum compared with p53 null mice, implying that Δ122p53 is a dominant oncogene. Consistent with this, Δ122p53 also confers a marked proliferative advantage on cells and reduced apoptosis. In addition to tumor development, Δ122p53 mice show a profound proinflammatory phenotype having increased serum concentrations of interleukin-6 and other proinflammatory cytokines and lymphocyte aggregates in the lung and liver as well as other pathologies. Based on these observations, we propose that human Δ133p53 also functions to promote cell proliferation and inflammation, one or both of which contribute to tumor development.

The alternative lengthening of telomeres pathway may operate in non-neoplastic human cells.

The alternative lengthening of telomeres (ALT) mechanism represents an alternative to the enzyme telomerase in the maintenance of mammalian telomeres in 25-60% of sarcomas and a minority of carcinomas (about 5-15%). ALT-positive cells are distinguished by long and heterogeneous telomere length distributions by terminal restriction fragment (TRF) Southern blotting. Another diagnostic marker of ALT is discrete nuclear co-localized signals of telomeric DNA and the promyelocytic leukaemia protein (PML), referred to as ALT-associated PML bodies (APBs). Recently, we detected smaller sized co-localized PML and telomere DNA (APB-like) bodies in endothelial cells adjacent to astrocytoma tumour cells in situ. In this study, we examined a wide variety of non-neoplastic tissues, and report that co-localized signals of PML and telomere DNA are present in endothelial, stromal, and some epithelial cells. Co-localized signals of PML and telomere DNA showed an increased frequency in non-neoplastic cells with DNA damage. These results suggest that a mechanism similar to that in ALT-positive tumours also operates in non-neoplastic cells, which may be activated by DNA damage.

Activated CD90/Thy-1 fibroblasts co-express the Δ133p53ß isoform and are associated with highly inflamed rheumatoid arthritis.

BACKGROUND: The p53 isoform Δ133p53ß is known to be associated with cancers driven by inflammation. Many of the features associated with the development of inflammation in rheumatoid arthritis (RA) parallel those evident in cancer progression. However, the role of this isoform in RA has not yet been explored. The aim of this study was to determine whether Δ133p53ß is driving aggressive disease in RA. METHODS: Using RA patient synovia, we carried out RT-qPCR and RNAScope-ISH to determine both protein and mRNA levels of Δ133p53 and p53. We also used IHC to determine the location and type of cells with elevated levels of Δ133p53ß. Plasma cytokines were also measured using a BioPlex cytokine panel and data analysed by the Milliplex Analyst software. RESULTS: Elevated levels of pro-inflammatory plasma cytokines were associated with synovia from RA patients displaying extensive tissue inflammation, increased immune cell infiltration and the highest levels of Δ133TP53 and TP53ß mRNA. Located in perivascular regions of synovial sub-lining and surrounding ectopic lymphoid structures (ELS) were a subset of cells with high levels of CD90, a marker of 'activated fibroblasts' together with elevated levels of Δ133p53ß. CONCLUSIONS: Induction of Δ133p53ß in CD90+ synovial fibroblasts leads to an increase in cytokine and chemokine expression and the recruitment of proinflammatory cells into the synovial joint, creating a persistently inflamed environment. Our results show that dysregulated expression of Δ133p53ß could represent one of the early triggers in the immunopathogenesis of RA and actively perpetuates chronic synovial inflammation. Therefore, Δ133p53ß could be used as a biomarker to identify RA patients more likely to develop aggressive disease who might benefit from targeted therapy to cytokines such as IL-6.

Reduction of lithium induced interstitial fibrosis on co-administration with amiloride.

Long-term administration of lithium is associated with chronic interstitial fibrosis that is partially reduced with exposure to amiloride. We examined potential pathways of how amiloride may reduce interstitial fibrosis. Amiloride was administered to a rat model of lithium induced interstitial fibrosis over a long term (6 months), as well as for short terms of 14 and 28 days. Kidney cortical tissue was subjected to RNA sequencing and microRNA expression analysis. Gene expression changes of interest were confirmed using immunohistochemistry on kidney tissue. Pathways identified by RNA sequencing of kidney tissue were related to 'promoting inflammation' for lithium and 'reducing inflammation' for amiloride. Validation of candidate genes found amiloride reduced inflammatory components induced by lithium including NF-κB/p65Ser536 and activated pAKTSer473, and increased p53 mediated regulatory function through increased p21 in damaged tubular epithelial cells. Amiloride also reduced the amount of Notch1 positive PDGFrß pericytes and infiltrating CD3 cells in the interstitium. Thus, amiloride attenuates a multitude of pro-inflammatory components induced by lithium. This suggests amiloride could be repurposed as a possible anti-inflammatory, anti-fibrotic agent to prevent or reduce the development of chronic interstitial fibrosis.

Harnessing Ultrasound for Targeting Drug Delivery to the Brain and Breaching the Blood-Brain Tumour Barrier.

Despite significant advances in developing drugs to treat brain tumours, achieving therapeutic concentrations of the drug at the tumour site remains a major challenge due to the presence of the blood-brain barrier (BBB). Several strategies have evolved to enhance brain delivery of chemotherapeutic agents to treat tumours; however, most approaches have several limitations which hinder their clinical utility. Promising studies indicate that ultrasound can penetrate the skull to target specific brain regions and transiently open the BBB, safely and reversibly, with a high degree of spatial and temporal specificity. In this review, we initially describe the basics of therapeutic ultrasound, then detail ultrasound-based drug delivery strategies to the brain and the mechanisms by which ultrasound can improve brain tumour therapy. We review pre-clinical and clinical findings from ultrasound-mediated BBB opening and drug delivery studies and outline current therapeutic ultrasound devices and technologies designed for this purpose.

Increased Ascorbate Content of Glioblastoma Is Associated With a Suppressed Hypoxic Response and Improved Patient Survival.

Glioblastoma multiforme is a challenging disease with limited treatment options and poor survival. Glioblastoma tumours are characterised by hypoxia that activates the hypoxia inducible factor (HIF) pathway and controls a myriad of genes that drive cancer progression. HIF transcription factors are regulated at the post-translation level via HIF-hydroxylases. These hydroxylases require oxygen and 2-oxoglutarate as substrates, and ferrous iron and ascorbate as cofactors. In this retrospective observational study, we aimed to determine whether ascorbate played a role in the hypoxic response of glioblastoma, and whether this affected patient outcome. We measured the ascorbate content and members of the HIF-pathway of clinical glioblastoma samples, and assessed their association with clinicopathological features and patient survival. In 37 samples (37 patients), median ascorbate content was 7.6 µg ascorbate/100 mg tissue, range 0.8 - 20.4 µg ascorbate/100 mg tissue. In tumours with above median ascorbate content, HIF-pathway activity as a whole was significantly suppressed (p = 0.005), and several members of the pathway showed decreased expression (carbonic anhydrase-9 and glucose transporter-1, both p < 0.01). Patients with either lower tumour HIF-pathway activity or higher tumour ascorbate content survived significantly longer than patients with higher HIF-pathway or lower ascorbate levels (p = 0.011, p = 0.043, respectively). Median survival for the low HIF-pathway score group was 362 days compared to 203 days for the high HIF-pathway score group, and median survival for the above median ascorbate group was 390 days, compared to the below median ascorbate group with 219 days. The apparent survival advantage associated with higher tumour ascorbate was more prominent for the first 8 months following surgery. These associations are promising, suggesting an important role for ascorbate-regulated HIF-pathway activity in glioblastoma that may impact on patient survival.

Ascorbate content of clinical glioma tissues is related to tumour grade and to global levels of 5-hydroxymethyl cytosine.

Gliomas are incurable brain cancers with poor prognosis, with epigenetic dysregulation being a distinctive feature. 5-hydroxymethylcytosine (5-hmC), an intermediate generated in the demethylation of 5-methylcytosine, is present at reduced levels in glioma tissue compared with normal brain, and that higher levels of 5-hmC are associated with improved patient survival. DNA demethylation is enzymatically driven by the ten-eleven translocation (TET) dioxygenases that require ascorbate as an essential cofactor. There is limited data on ascorbate in gliomas and the relationship between ascorbate and 5-hmC in gliomas has never been reported. Clinical glioma samples (11 low-grade, 26 high-grade) were analysed for ascorbate, global DNA methylation and hydroxymethylation, and methylation status of the O-6-methylguanine-DNA methyltransferase (MGMT) promoter. Low-grade gliomas contained significantly higher levels of ascorbate than high-grade gliomas (p = 0.026). Levels of 5-hmC were significantly higher in low-grade than high-grade glioma (p = 0.0013). There was a strong association between higher ascorbate and higher 5-hmC (p = 0.004). Gliomas with unmethylated and methylated MGMT promoters had similar ascorbate levels (p = 0.96). One mechanism by which epigenetic modifications could occur is through ascorbate-mediated optimisation of TET activity in gliomas. These findings open the door to clinical intervention trials in patients with glioma to provide both mechanistic information and potential avenues for adjuvant ascorbate therapy.

Pilocytic astrocytomas have telomere-associated promyelocytic leukemia bodies without alternatively lengthened telomeres.

Telomere maintenance by either telomerase activity or the recombination-mediated alternative lengthening of telomeres (ALT) mechanism is a hallmark of cancer. Tumors that use ALT as their telomere maintenance mechanism are characterized by long telomeres of great heterogeneity in length and by specific nuclear structures of co-localized promyelocytic leukemia protein and telomere DNA, called ALT-associated promyelocytic leukemia bodies (APBs). Recent advances have revealed a direct role for APBs in telomere recombination in ALT-positive cells. In this study, we investigated the possibility that APBs could occur before the long 'alternatively' lengthened telomeres arise, particularly in low-grade tumors. We measured APBs, telomere length, and telomerase activity in 64 astrocytomas inclusive of grade 1-4 tumors. Almost all grade 1-3 tumors (93%) were APB-positive using published criteria. Grade 2-3 APB-positive tumors also had long telomeres and were confirmed as ALT positive. However, grade 1 tumors lacked long telomeres and were therefore classified as ALT negative, but positive for telomere-associated promyelocytic leukemia bodies (TPB). This is the first report of a TPB-positive but ALT-negative tumor, and suggests that low-grade tumors have the foundation for recombinational telomere repair, as in ALT. Further work is warranted to characterize the TPB-positive phenotype in other early malignancies, as well as to determine whether TPBs predispose to telomere maintenance by ALT.

Identification and validation of DNA methylation changes in pre-eclampsia.

INTRODUCTION: Pre-eclampsia (PE) is a dangerous placental condition that can lead to premature labour, seizures and death of mother and infant. Several studies have identified altered placental DNA methylation in PE; however, there is widespread inconsistency between studies and most findings have not been replicated. This study aimed to identify and validate consistent differences in methylation across multiple PE cohorts. METHODS: Seven publicly available 450K methylation array datasets were analysed to identify consistent differentially methylated positions (DMPs) in PE. DMPs were identified based on methylation difference (≥10%) and significance (p-value ≤ 1 × 10-7). Targeted deep bisulfite sequencing was then performed to validate a subset of DMPs in an additional independent PE cohort. RESULTS: Stringent analysis of the seven 450K datasets identified 25 DMPs (associated with 11 genes) in only one dataset. Using more relaxed criteria confirmed 19 of the stringent 25 DMPs in at least four of the remaining six datasets. Targeted deep bisulfite sequencing of eight DMPs (associated with three genes; CMIP, ST3GAL1 and DAPK3) in an independent PE cohort validated two DMPs in the CMIP gene. Seven additional CpG sites in CMIP were found to be significantly differentially methylated in PE. DISCUSSION: The identification and validation of significant differential methylation in CMIP suggests that the altered DNA methylation of this gene may be associated with the pathogenesis of PE, and may have the potential to serve as diagnostic biomarkers for this dangerous condition of pregnancy.