Childhood Brain Tumors: A Review of Strategies to Translate CNS Drug Delivery to Clinical Trials.

Brain and spinal tumors affect 1 in 1000 people by 25 years of age, and have diverse histological, biological, anatomical and dissemination characteristics. A mortality of 30-40% means the majority are cured, although two-thirds have life-long disability, linked to accumulated brain injury that is acquired prior to diagnosis, and after surgery or chemo-radiotherapy. Only four drugs have been licensed globally for brain tumors in 40 years and only one for children. Most new cancer drugs in clinical trials do not cross the blood-brain barrier (BBB). Techniques to enhance brain tumor drug delivery are explored in this review, and cover those that augment penetration of the BBB, and those that bypass the BBB. Developing appropriate delivery techniques could improve patient outcomes by ensuring efficacious drug exposure to tumors (including those that are drug-resistant), reducing systemic toxicities and targeting leptomeningeal metastases. Together, this drug delivery strategy seeks to enhance the efficacy of new drugs and enable re-evaluation of existing drugs that might have previously failed because of inadequate delivery. A literature review of repurposed drugs is reported, and a range of preclinical brain tumor models available for translational development are explored.

Professionalising care into compliance: The challenge for personalised care models.

One of the most basic understandings of nursing is that a nurse is a caregiver for a patient who helps to prevent illness, treat health conditions, and manage the physical needs of patients. Nursing is often presented as a caring profession, which provides patient care driven by ideals of empathy, compassion and kindness. These ideals of care have further been foregrounded through the development and implementation of stress on patient centred care (PCC) and/or person-centred practice (PCP). Although the idealisation of nursing as a caring profession is common, and one certainly seen as integral by nurses and written into the heart of regulatory documentation, we contend that the actual delivery of care is being undercut by the very regulatory climate that strives to professionalise care. As we outline, with specific reference to the context of Australian Nursing, this transformation delivers a commodified, even McDonaldized, model of patient management rather than care. It seems that even with its explicit stress on PCC and PCP, Australian Nursing cannot live up to its own care ideals. Having outlined this problem, the paper then demonstrates the ways in which PCC is thwarted at the coal face of nursing practice and that there must be an institutionalised change to be able to provide genuine patient-centred care.

Empowerment as an alternative to traditional patient advocacy roles.

There has long been acceptance within healthcare that one of the roles that nurses fulfil is to do with patient advocacy. This has historically been positioned as part of the philosophical and inherent requirements of the nursing profession at large and is supported through shared conceptualisations of the nursing profession. Such conceptualisations are communicated to nursing professionals by way of first their education, and second their professional codes, guidelines and standards for practice. The focus on advocacy is further embedded within patient-centred care frameworks and concepts of the nurse-patient therapeutic relationship. Nurses have also been considered ideally placed to undertake the work of patient advocacy due to the 24/7 nature and intimacy of the care provided. What this means is that nurses are under the impression that that they must be an advocate for their patients through their nursing practice. However, for a fundamental concept of nursing, advocacy is poorly defined, and practices commonly associated with advocacy are undercut by the professionalisation of nursing and other constraints. In addition, nursing standards and frameworks of care are being actively reframed around ideas of empowerment which do not necessarily fit well with those of advocacy. This article thus suggests that it is time to recognise that the work of advocacy is no longer representative of what nurses (can) do in practice, and to explicitly reorient conceptualisations of nurse practice around empowerment. This article will further analyse what this may look like in practice.

shRNA-mediated PPARα knockdown in human glioma stem cells reduces in vitro proliferation and inhibits orthotopic xenograft tumour growth.

The overall survival for patients with primary glioblastoma is very poor. Glioblastoma contains a subpopulation of glioma stem cells (GSC) that are responsible for tumour initiation, treatment resistance and recurrence. PPARα is a transcription factor involved in the control of lipid, carbohydrate and amino acid metabolism. We have recently shown that PPARα gene and protein expression is increased in glioblastoma and has independent clinical prognostic significance in multivariate analyses. In this work, we report that PPARα is overexpressed in GSC compared to foetal neural stem cells. To investigate the role of PPARα in GSC, we knocked down its expression using lentiviral transduction with short hairpin RNA (shRNA). Transduced GSC were tagged with luciferase and stereotactically xenografted into the striatum of NOD-SCID mice. Bioluminescent and magnetic resonance imaging showed that knockdown (KD) of PPARα reduced the tumourigenicity of GSC in vivo. PPARα-expressing control GSC xenografts formed invasive histological phenocopies of human glioblastoma, whereas PPARα KD GSC xenografts failed to establish viable intracranial tumours. PPARα KD GSC showed significantly reduced proliferative capacity and clonogenic potential in vitro with an increase in cellular senescence. In addition, PPARα KD resulted in significant downregulation of the stem cell factors c-Myc, nestin and SOX2. This was accompanied by downregulation of the PPARα-target genes and key regulators of fatty acid oxygenation ACOX1 and CPT1A, with no compensatory increase in glycolytic flux. These data establish the aberrant overexpression of PPARα in GSC and demonstrate that this expression functions as an important regulator of tumourigenesis, linking self-renewal and the malignant phenotype in this aggressive cancer stem cell subpopulation. We conclude that targeting GSC PPARα expression may be a therapeutically beneficial strategy with translational potential as an adjuvant treatment. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Combined use of CDK4/6 and mTOR inhibitors induce synergistic growth arrest of diffuse intrinsic pontine glioma cells via mutual downregulation of mTORC1 activity.

BACKGROUND: Diffuse intrinsic pontine glioma (DIPG) is a lethal type of pediatric brain tumor that is resistant to conventional chemotherapies. Palbociclib is a putative novel DIPG treatment that restricts the proliferation of rapidly dividing cancer cells via selective inhibition of cyclin-dependent kinase (CDK) 4 and CDK6. However, implementing palbociclib as a monotherapy for DIPG is unfeasible, as CDK4/6 inhibitor resistance is commonplace and palbociclib does not readily cross the blood-brain barrier (BBB) or persist in the central nervous system. To inhibit the growth of DIPG cells, we aimed to use palbociclib in combination with the rapamycin analog temsirolimus, which is known to ameliorate resistance to CDK4/6 inhibitors and inhibit BBB efflux. MATERIALS AND METHODS: We tested palbociclib and temsirolimus in three patient-derived DIPG cell lines. The expression profiles of key proteins in the CDK4/6 and mammalian target of rapamycin (mTOR) signaling pathways were assessed, respectively, to determine feasibility against DIPG. Moreover, we investigated effects on cell viability and examined in vivo drug toxicity. RESULTS: Immunoblot analyses revealed palbociclib and temsirolimus inhibited CDK4/6 and mTOR signaling through canonical perturbation of phosphorylation of the retinoblastoma (RB) and mTOR proteins, respectively; however, we observed noncanonical downregulation of mTOR by palbociclib. We demonstrated that palbociclib and temsirolimus inhibited cell proliferation in all three DIPG cell lines, acting synergistically in combination to further restrict cell growth. Flow cytometric analyses revealed both drugs caused G1 cell cycle arrest, and clonogenic assays showed irreversible effects on cell proliferation. Palbociclib did not elicit neurotoxicity in primary cultures of normal rat hippocampi or when infused into rat brains. CONCLUSION: These data illustrate the in vitro antiproliferative effects of CDK4/6 and mTOR inhibitors in DIPG cells. Direct infusion of palbociclib into the brain, in combination with systemic delivery of temsirolimus, represents a promising new approach to developing a much-needed treatment for DIPG.

The distribution, clearance, and brainstem toxicity of panobinostat administered by convection-enhanced delivery.

OBJECTIVE The pan-histone deacetylase inhibitor panobinostat has preclinical efficacy against diffuse intrinsic pontine glioma (DIPG), and the oral formulation has entered a Phase I clinical trial. However, panobinostat does not cross the blood-brain barrier in humans. Convection-enhanced delivery (CED) is a novel neurosurgical drug delivery technique that bypasses the blood-brain barrier and is of considerable clinical interest in the treatment of DIPG. METHODS The authors investigated the toxicity, distribution, and clearance of a water-soluble formulation of panobinostat (MTX110) in a small- and large-animal model of CED. Juvenile male Wistar rats (n = 24) received panobinostat administered to the pons by CED at increasing concentrations and findings were compared to those in animals that received vehicle alone (n = 12). Clinical observation continued for 2 weeks. Animals were sacrificed at 72 hours or 2 weeks following treatment, and the brains were subjected to neuropathological analysis. A further 8 animals received panobinostat by CED to the striatum and were sacrificed 0, 2, 6, or 24 hours after infusion, and their brains explanted and snap-frozen. Tissue-drug concentration was determined by liquid chromatography tandem mass spectrometry (LC-MS/MS). Large-animal toxicity was investigated using a clinically relevant MRI-guided translational porcine model of CED in which a drug delivery system designed for humans was used. Panobinostat was administered at 30 µM to the ventral pons of 2 juvenile Large White-Landrace cross pigs. The animals were subjected to clinical and neuropathological analysis, and findings were compared to those obtained in controls after either 1 or 2 weeks. Drug distribution was determined by LC-MS/MS in porcine white and gray matter immediately after CED. RESULTS There were no clinical or neuropathological signs of toxicity up to an infused concentration of 30 µM in both small- and large-animal models. The half-life of panobinostat in rat brain after CED was 2.9 hours, and the drug was observed to be distributed in porcine white and gray matter with a volume infusion/distribution ratio of 2 and 3, respectively. CONCLUSIONS CED of water-soluble panobinostat, up to a concentration of 30 µM, was not toxic and was distributed effectively in normal brain. CED of panobinostat warrants clinical investigation in patients with DIPG.

Evaluation of the quality of RNA extracted from archival FFPE glioblastoma and epilepsy surgical samples for gene expression assays.

AIMS: Histopathological tissue samples are being increasingly used as sources of nucleic acids in molecular pathology translational research. This study investigated the suitability of glioblastoma and control central nervous system (CNS) formalin-fixed paraffin embedded (FFPE) tissue-derived RNA for gene expression analyses. METHODS: Total RNA was extracted from control (temporal lobe resection tissue) and glioblastoma FFPE tissue samples. RNA purity (260/280 ratios) was determined and RNA integrity number (RIN) analysis was performed. RNA was subsequently used for RT-qPCR for two reference genes, 18S and GAPDH. RESULTS: Reference gene expression was equivalent between control and glioblastoma tissue when using RNA extracted from FFPE tissue, which has key implications for biological normalisation for CNS gene expression studies. There was a significant difference between the mean RIN values of control and glioblastoma FFPE tissue. There was no significant correlation between 260/280 or RIN values versus total RNA yield. The age of the tissue blocks did not influence RNA yield, fragmentation or purity. There was no significant correlation between RIN or 260/280 ratios and mean qPCR cycle threshold for either reference gene. CONCLUSIONS: This study showed that routinely available CNS FFPE tissue is suitable for RNA extraction and downstream gene expression studies, even after 60 months of storage. Substantial RNA fragmentation associated with glioblastoma and control FFPE tissue blocks did not preclude downstream RT-qPCR gene expression analyses. Cross validation with both archival and prospectively collated FFPE specimens is required to further demonstrate that CNS tissue blocks can be used in novel translational molecular biomarker studies.

Repurposing the anti-epileptic drug sodium valproate as an adjuvant treatment for diffuse intrinsic pontine glioma.

Targeting epigenetic changes in diffuse intrinsic pontine glioma (DIPG) may provide a novel treatment option for patients. This report demonstrates that sodium valproate, a histone deacetylase inhibitor (HDACi), can increase the cytotoxicity of carboplatin in an additive and synergistic manner in DIPG cells in vitro. Sodium valproate causes a dose-dependent decrease in DIPG cell viability in three independent ex vivo cell lines. Furthermore, sodium valproate caused an increase in acetylation of histone H3. Changes in cell viability were consistent with an induction of apoptosis in DIPG cells in vitro, determined by flow cytometric analysis of Annexin V staining and assessment of apoptotic markers by western blotting. Subsequently, immunofluorescent staining of neuronal and glial markers was used to determine toxicity in normal rat hippocampal cells. Pre-treatment of cells with sodium valproate enhanced the cytotoxic effects of carboplatin, in three DIPG cell lines tested. These results demonstrate that sodium valproate causes increased histone H3 acetylation indicative of HDAC inhibition, which is inversely correlated with a reduction in cell viability. Cell viability is reduced through an induction of apoptosis in DIPG cells. Sodium valproate potentiates carboplatin cytotoxicity and prompts further work to define the mechanism responsible for the synergy between these two drugs and determine in vivo efficacy. These findings support the use of sodium valproate as an adjuvant treatment for DIPG.

The transcription factor PPARα is overexpressed and is associated with a favourable prognosis in IDH-wildtype primary glioblastoma.

AIMS: PPARα agonists are in current clinical use as hypolipidaemic agents and show significant antineoplastic effects in human glioblastoma models. To date however, the expression of PPARα in large-scale glioblastoma datasets has not been examined. We aimed to investigate the expression of the transcription factor PPARα in primary glioblastoma, the relationship between PPARα expression and patients' clinicopathological features and other molecular markers associated with gliomagenesis. METHODS AND RESULTS: With protein immunoblotting techniques and reverse transcription quantitative real-time PCR, PPARα was found to be significantly overexpressed in glioblastoma compared with control brain tissue (P = 0.032 and P = 0.005). PPARA gene expression was found to be enriched in the classical glioblastoma subtype within The Cancer Genome Atlas (TCGA) dataset. Although not associated with overall survival when assessed by immunohistochemistry, cross-validation with the TCGA dataset and multivariate analyses identified PPARA gene expression as an independent prognostic marker for overall survival (P = 0.042). Finally, hierarchical clustering revealed novel, significant associations between high PPARA expression and a putative set of glioblastoma molecular mediators including EMX2, AQP4, and NTRK2. CONCLUSIONS: PPARα is overexpressed in primary glioblastoma and high PPARA expression functions as an independent prognostic marker in the glioblastoma TCGA dataset. Further studies are required to explore genetic associations with high PPARA expression and to analyse the predictive role of PPARα expression in glioblastoma models in response to PPARα agonists.

Nursing assessment of older people who are in hospital: exploring registered nurses' understanding of their assessment skills.

BACKGROUND: Nurses worldwide are expected to take a leading role in caring for older people. Considerable literature dedicated to the range and application of assessment skills used by nurses vary. There is limited knowledge of registered nurses' (RNs) views of their assessment of older adults. AIM: The aim of this project was to explore RNs current perceptions of nursing assessment, and the core skills they identified as necessary. DESIGN: A qualitative descriptive design study was conducted in three inpatient units in one regional hospital in Victoria. METHOD: Date were collected through participant observation of RNs (n = 13) followed by 1:1 semi-structured interviews. Data were analysed thematically. CONCLUSION: This research has illuminated that an ill-defined repertoire of skills was used by RNs when assessing older persons. Skills identified appeared to be based on years of personal-professional experience. Differences were noted between the descriptions nurses gave and what was observed during interactions with older persons.

Two steps forward, one step back: the intricacies of engaging with e-portfolios in nursing undergraduate education.

OBJECTIVES: To share the experience of implementing and refining the use of e-portfolios into an undergraduate nursing degree. DESIGN: This is a reflective piece that explores the hurdles experienced in introducing and maintaining e-portfolio's into higher education, particularly in undergraduate nursing curricula. DATA SOURCES: Nil REVIEW METHODS: Review of the literature, and comparing and contrasting this against the experience of the authors. RESULTS: Hurdles included access to computer and quality internet connections, along with the levels of IT literacy of staff, and the common misconception that all students are IT savvy. What was also evident was how the need for both pedagogical and technical support to both staff and students was paramount in achieving an understanding of the software, and the scope and perspective of e-portfolio pedagogy. With each of these hurdles, staff reluctance to engage in e-portfolio use in the tertiary sector was evident. On reflection of each of these challenges, the authors identified that their experience mirrored other tertiary institutions in that a single hurdle alone is not responsible for fragmented e-portfolio implementation, but a combination of factors. Through exploring recommendations from other tertiary institutions, the authors recognize the limitations within their own working environment that contributed to the pitfalls experienced. CONCLUSION: Despite e-portfolios being introduced into higher education for over a decade, its successful implementation into undergraduate nursing curricula continues to be fraught with pitfalls, creating the sense of moving backwards more so than forwards. As with any new learning tool, careful consideration needs to be given to comprehensive planning, implementation, review and evaluation to either prevent the hurdles identified, or limit their impact on the quality of the portfolio produced and the learning attained from the process.

Problematising autonomy and advocacy in nursing.

Customarily patient advocacy is argued to be an essential part of nursing, and this is reinforced in contemporary nursing codes of conduct, as well as codes of ethics and competency standards governing practice. However, the role of the nurse as an advocate is not clearly understood. Autonomy is a key concept in understanding advocacy, but traditional views of individual autonomy can be argued as being outdated and misguided in nursing. Instead, the feminist perspective of relational autonomy is arguably more relevant within the context of advocacy and nurses' work in clinical healthcare settings. This article serves to highlight and problematise some of the assumptions and influences around the perceived role of the nurse as an advocate for patients in contemporary Western healthcare systems by focusing on key assumptions concerning autonomy inherent in the role of the advocate.

Type VII collagen regulates expression of OATP1B3, promotes front-to-rear polarity and increases structural organisation in 3D spheroid cultures of RDEB tumour keratinocytes.

Type VII collagen is the main component of anchoring fibrils, structures that are integral to basement membrane homeostasis in skin. Mutations in the gene encoding type VII collagen COL7A1 cause recessive dystrophic epidermolysis bullosa (RDEB) an inherited skin blistering condition complicated by frequent aggressive cutaneous squamous cell carcinoma (cSCC). OATP1B3, which is encoded by the gene SLCO1B3, is a member of the OATP (organic anion transporting polypeptide) superfamily responsible for transporting a wide range of endogenous and xenobiotic compounds. OATP1B3 expression is limited to the liver in healthy tissues, but is frequently detected in multiple cancer types and is reported to be associated with differing clinical outcome. The mechanism and functional significance of tumour-specific expression of OATP1B3 has yet to be determined. Here, we identify SLCO1B3 expression in tumour keratinocytes isolated from RDEB and UV-induced cSCC and demonstrate that SLCO1B3 expression and promoter activity are modulated by type VII collagen. We show that reduction of SLCO1B3 expression upon expression of full-length type VII collagen in RDEB cSCC coincides with acquisition of front-to-rear polarity and increased organisation of 3D spheroid cultures. In addition, we show that type VII collagen positively regulates the abundance of markers implicated in cellular polarity, namely ELMO2, PAR3, E-cadherin, B-catenin, ITGA6 and Ln332.

Implied consent and nursing practice: ethical or convenient?

Nursing professionals in a variety of practice settings routinely use implied consent. This form of consent is used in place of or in conjunction with informed or explicit consent. This article looks at one aspect of a qualitative exploratory study conducted in a Day of Surgery Admission unit. This article focuses on the examination of nurses' understandings of implied consent and its use in patient care in nursing practice. Data were collected through one-on-one interviews and analysed using a thematic analysis. Nurses participating in this study revealed that they routinely used implied consent in their nursing practice. This article will look at whether implied consent supports or impedes a patient's autonomy.

Identification and characterization of DSPIa, a novel isoform of human desmoplakin.

Desmoplakin is a ubiquitous component of desmosomes and desmosome-like structures, such as the cardiomyocyte area composita. Two major isoforms, desmoplakin I (DSPI) and desmoplakin II (DSPII) are encoded by alternative mRNA transcripts differentially spliced from the same gene. The resulting proteins are identical in amino acid sequence with the exception that DSPII contains only one third of the central alpha-helical rod domain present in DSPI. Here we describe a novel minor isoform of desmoplakin that is also produced by alternative splicing of the desmoplakin gene and that we name desmoplakin Ia (DSPIa). DSPIa is an alternatively spliced DSPI mRNA with a unique splice donor site that is 90% homologous to and downstream of the DSPII specific donor. The resulting DSPIa mRNA is in-frame and encodes a protein that has a central alpha-helical rod domain of intermediate size and that is 156 amino acids larger than DSPII and 443 amino acids smaller than DSPI. We demonstrate, through recombinant expression and short interfering RNA knockdown, that the DSPIa protein is readily detectable, albeit at substantially lower levels than the dominant isoforms, DSPI and DSPII. DSPIa mRNA has a similar tissue distribution to that of DSPI and of DSPII.

Using the postpartum hospital stay to address mothers' and fathers' smoking: the NEWS study.

OBJECTIVE: The objective of this study was to test the feasibility and acceptability of introducing an intervention to address mothers' and fathers' smoking during the postpartum hospitalization. METHODS: During a 14-month period (February 2005 to April 2006), we assessed the smoking status of both parents of all newborns who were delivered at a hospital child birth center. Parents who were current smokers (1 cigarette, even a puff, in past 30 days) or recent quitters (smoked since 1 month before conception) were eligible for the study. Parents were assigned to intervention or usual care control condition on the basis of day of study enrollment. Smoking outcomes were assessed at 3 months by telephone survey and cotinine confirmation; quitline use was assessed at 3 months by using quitline database. RESULTS: A total of 101 (64%) of 159 eligible parents enrolled in the study (n = 53 control subject, n = 48 intervention), including 72 (71%) current smokers and 29 (29%) recent quitters. All parents in the intervention group received the in-hospital counseling session, 94% had a fax sent to a provider, and 36 (75%) accepted quitline enrollment. In an intention-to-treat analysis that included both current smokers and recent quitters, self-reported 7-day abstinence decreased from 31% to 25% among intervention parents versus 38% to 23% among control subjects (effect size 9.4%; nonsignificant). Among current smokers at baseline who were reached at follow-up (n = 36), self-reported 24-hour quit attempts were higher in the intervention group versus control group (64% vs 18%; P = .005), whereas the cotinine-confirmed 7-day abstinence rates at follow-up were 9% in the intervention group and 3% in the control group (nonsignificant). CONCLUSIONS: Enrolling mothers and fathers into tobacco treatment services during the immediate postpartum hospital stay is feasible and seems to stimulate quit attempts. The birth of an infant presents a teachable moment to reach both parents and to provide cessation assistance.

QTL analyses of lineage-negative mouse bone marrow cells labeled with Sca-1 and c-Kit.

Differences in the number of functionally and/or phenotypically defined bone marrow cells in inbred mouse strains have been exploited to map quantitative trait loci (QTL) that determine the variation in cell frequency. To extend this approach to the differences in the stem/progenitor cell compartment in CBA/H and C57BL/6 mice, we have exploited the resolution of flow cytometry and the power of QTL analyses in 124 F(2) mice to analyze lineage-negative (Lin(-)) bone marrow cells according to the intensity of labeling with Sca-1 and c-Kit. In the Lin(-) Sca-1(+) c-Kit(+) enriched population, six QTL were identified: one significant and five suggestive. Whereas previous in vitro clonogenic, LTC-IC, day 35 CAFC, and flow cytometry each identified different QTL, our approach identified the same or very similar QTL at all three loci (chromosomes 1, 17, and 18) as well as QTL on chromosomes 6 and 10. In silico analyses implicate hematopoietic stem cell homing involving Cxcr4 and Cxcl12 as being the determining pathway. The mapping of the same or very similar QTL in independent studies using different assay(s) suggests a common genetic determinant, and thus reinforces the biological and genetic significance of the QTL. These data also suggest that mouse bone marrow cell subpopulations can be functionally, phenotypically, and genetically defined.

GPR26: a marker for primary glioblastoma?

Glioblastomas are highly malignant brain tumours; they have been described as one of the most deadly human cancers. Two conceptual classifications of the condition exist: primary (de novo), which does not exhibit prior disease and secondary glioblastoma, which develops from a pre-existing glioma. This study investigates whether GPR26 is differentially transcribed in glioblastoma tissue from patients of different ages, in order to define a candidate genetic marker. The transcriptional profile of GPR26 was compared in nine samples: seven glioblastoma tissues and two normal brain tissues using PCR. Despite GPR26 being present in the glioblastoma tissues, it was not transcribed in any of the four cell lines tested. GPR26 transcription ratios were compared between normal and cancerous samples, also age categories <50 and >60 years were compared. Results suggested differential transcription of GPR26, which is significantly less transcribed in tissues from older patients, implied by a p-value of 0.03. This study has identified GPR26 to be a genetic indicator of primary glioblastoma, suggesting that it could be a suppressor of primary glioblastoma development.

DNA methylation during mouse hemopoietic differentiation and radiation-induced leukemia.

OBJECTIVE: To examine DNA methylation in mouse hemopoiesis before and after in vivo exposure to a leukemogenic dose of x-rays, and address whether methylation levels are associated with the relative radiosensitivity of tissues in vivo. METHODS: The methylation status of control CBA/H and C57BL/6 mouse tissues before and after exposure to 3-Gy x-rays, and myeloid and lymphoid leukemias and lymphomas, was assessed by the direct analysis of the 5-methylcytosine (5-(Me)C) content of DNA, and by Southern blot analysis of genomic repeat sequences. RESULTS: The DNA 5-(Me)C content of bone marrow is 15% lower than spleen. Together with the analyses of stem (myeloid) and progenitor (lymphoid) leukemias and lymphomas, we found a trend of increasing methylation during hemopoietic differentiation. Exposure to x-rays induced greater cell death in the hypomethylated bone marrow (>80%) than spleen (50%) in vivo, supporting the observed correlation found between methylation status and radiosensitivity of other high-turnover hierarchical tissues. Furthermore, there was an 8% DNA 5-(Me)C content decrease in bone marrow after in vivo exposure to 3-Gy x-rays, but this was genotype dependent, being observed in AML-susceptible (CBA/H) but not AML-resistant (C57BL/6) inbred mice. CONCLUSION: Together these data suggest that methylation status may be related to the relative radiosensitivity of high-turnover hierarchical tissues such as bone marrow and that radiation-induced DNA hypomethylation has a role in radiation leukemogenesis.

Evidence for clustered tumour suppressor gene loci on mouse chromosomes 2 and 4 in radiation-induced acute myeloid leukaemia.

PURPOSE: To investigate the influence of genetic and epigenetic factors on allelic loss on chromosomes 2 and 4 in mouse radiation-induced acute myeloid leukaemia (r-AML). METHODS: r-AML that arose in (CBA/HxC57BL/6)F1xCBA/H and F1xC57BL/6 mice were screened for transcription factor PU1 (also known as SPI-1) gene mutations and methylation of the paired box gene 5 (Pax5) gene promoter. We have increased the statistical significance of a genetic linkage analysis of affected F1xCBA/H mice to test for linkage to loci implicated directly or indirectly with r-AML-susceptibility. RESULTS: There was a statistically significant difference ( p < 10-4) in the frequency of PU1 gene mutations in F1xCBA/H and F1xC57BL/6 r-AML, implicating a second linked but genotype-dependent myeloid leukaemia suppressor gene on chromosome 2. A suggestive CBA/H r-AML-resistance locus maps within 10 cM of the minimally deleted region on chromosome 4. The Pax5 gene promoter is subject to ongoing subclonal promoter methylation in the r-AML, evidence that Pax5 gene silencing confers a selective advantage during clonal expansion in vivo. CONCLUSIONS: Allelic loss in mouse r-AML and subsequent tumour suppressor gene mutation (PU1) or silencing (Pax5) is strongly influenced by genetic background and/or epigenetic factors, and driven by in vivo clonal selection.