Sequential Treatment with Cytarabine and Decitabine Has an Increased Anti-Leukemia Effect Compared to Cytarabine Alone in Xenograft Models of Childhood Acute Myeloid Leukemia

<div><p>The current interest in epigenetic priming is underpinned by the belief that remodelling of the epigenetic landscape will sensitise tumours to subsequent therapy. In this pre-clinical study, paediatric AML cells expanded in culture and primary AML xenografts were treated with decitabine, a DNA demethylating agent, and cytarabine, a frontline cytotoxic agent used in the treatment of AML, either alone or in combination. Sequential treatment with decitabine and cytarabine was found to be more effective in reducing tumour burden than treatment with cytarabine alone suggesting that the sequential delivery of these agents may a have real clinical advantage in the treatment of paediatric AML. However we found no evidence to suggest that this outcome was dependent on priming with a hypomethylating agent, as the benefits observed were independent of the order in which these drugs were administered.</p></div

Arginine Methyltransferases Are Regulated by Epstein-Barr Virus in B Cells and Are Differentially Expressed in Hodgkin’s Lymphoma

Although there is increasing evidence that aberrant expression of those enzymes which control protein arginine methylation contribute to carcinogenesis, their de-regulation by oncogenic viruses in primary cells has yet to be reported. We first show that the protein arginine methyltransferases, CARM1, PRMT1 and PRMT5 are strongly expressed in Hodgkin Reed-Sternberg (HRS) cells, and up-regulated in Hodgkin&#039;s lymphoma (HL) cell lines. Given that Epstein-Barr virus (EBV) can be detected in approximately 50% of primary HL, we next examined how EBV infection of germinal centre (GC) B cells, the presumptive precursors of HRS cells, modulated the expression of these proteins. EBV infection of GC B cells was followed by the up-regulation of CARM1, PRMT1 and PRMT5, and by the down-regulation of the arginine deiminase, PADI4. Latent membrane protein 1 (LMP1), the major EBV transforming gene was shown to induce PRMT1 in GC B cells and in a stably transfected B cell line. The recent development of compounds which inhibit PRMT-mediated reactions provides a compelling case for continuing to dissect the contribution of virus induced changes in these proteins to lymphomagenesis

Pyrosequencing analysis of 4 tumour suppressor genes following DAC treatment.

<p>Changes in methylation status of a CpG site for 4 representative candidate genes (CSMD1, RARB, CADM1 and CDH13), following treatment with DAC at a range of concentrations in 8 primary AML patient cultures. Cells were treated at 0, 24, 48, 72 and 96 h, DNA methylation was measured at 120 h. All experiments were performed in duplicate.</p

Q-PCR analysis of candidate genes identified from the expression arrays.

<p>Changes in the expression of 5 genes which were concordantly de-regulated in all three xenografts following sequential treatment with either Ara-C followed by DAC (ILF3 and MARCKS) or DAC followed by Ara-C (FLT3, CTBP1 and MLL5). Assays were carried out in triplicate.</p

The <i>in vitro</i> effects of DAC and Ara-C in primary paediatric AML.

<p>A). Changes in viability following treatment with DAC or Ara-C at a range of concentrations in 8 primary AML patient samples. B) Changes in proliferation following treatment with DAC or Ara-C at the IC50 dose in 8 primary AML patient cultures. Cells were treated at 0, 24, 48, 72 and 96 h, both cell viability and proliferation were measured at 120 h. All experiments were performed in triplicate.</p

Summary of methylation changes following treatment with different drug regimens.

<p>For each xenograft, the number of hypomethylation and hypermethylation changes as well as the median change in beta value following treatment with each drug regimen. Differentially methylated CpG sites were identified using Genome Studio; results were filtered to retain CpG in which the change in beta values between PBS and drug treated samples was >±0.2.</p

The in vivo effects of DAC and Ara-C in primary paediatric AML xenografts.

<p>A and B) Cells isolated from BM of three xenografts following 5 or 10 days of treatment were stained for human CD45 and CD33 and for mouse CD45; the proportion of human cells which were CD45⁺CD33⁺ from each treatment group were compared to Ara-C treatment alone in the BM. C) Cells isolated from the spleen which were human CD45⁺CD33⁺ compared to PBS (* P <0.001).</p

The <i>in vitro</i> effects of DAC and Ara-C in combination in primary paediatric AML.

<p><b>A</b>) The percentage of viable cells following treatment with each drug regimen compared to PBS controls in 5 primary AML patient cultures (* P<0.01). All experiments were performed in triplicate. <b>B</b>) The percentage of cells in each phase of cell cycle following treatment with each drug regimen, for a representative primary AML sample (AML-7). All experiments were performed in duplicate.</p

Evidence of disrupted high-risk human papillomavirus DNA in morphologically normal cervices of older women

High-risk human papillomavirus (HR-HPV) causes nearly 100% of cervical carcinoma. However, it remains unclear whether HPV can establish a latent infection, one which may be responsible for the second peak in incidence of cervical carcinoma seen in older women. Therefore, using Ventana in situ hybridisation (ISH), quantitative PCR assays and biomarkers of productive and transforming viral infection, we set out to provide the first robust estimate of the prevalence and characteristics of HPV genomes in FFPE tissue from the cervices of 99 women undergoing hysterectomy for reasons unrelated to epithelial abnormality. Our ISH assay detected HR-HPV in 42% of our study population. The majority of ISH positive samples also tested HPV16 positive using sensitive PCR based assays and were more likely to have a history of preceding cytological abnormality. Analysis of subsets of this population revealed HR-HPV to be transcriptionally inactive as there was no evidence of a productive or transforming infection. Critically, the E2 gene was always disrupted in those HPV16 positive cases which were assessed. These findings point to a reservoir of transcriptionally silent, disrupted HPV16 DNA in morphologically normal cervices, re-expression of which could explain the increase in incidence of cervical cancer observed in later life