Species differences and mechanism of action of A3 adenosine receptor allosteric modulators.

Activity of the A3 adenosine receptor (AR) allosteric modulators LUF6000 (2-cyclohexyl-N-(3,4-dichlorophenyl)-1H-imidazo [4,5-c]quinolin-4-amine) and LUF6096 (N-{2-[(3,4-dichlorophenyl)amino]quinolin-4-yl}cyclohexanecarbox-amide) was compared at four A3AR species homologs used in preclinical drug development. In guanosine 5'-[γ-[35S]thio]triphosphate ([35S]GTPγS) binding assays with cell membranes isolated from human embryonic kidney cells stably expressing recombinant A3ARs, both modulators substantially enhanced agonist efficacy at human, dog, and rabbit A3ARs but provided only weak activity at mouse A3ARs. For human, dog, and rabbit, both modulators increased the maximal efficacy of the A3AR agonist 2-chloro-N 6-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide as well as adenosine > 2-fold, while slightly reducing potency in human and dog. Based on results from N 6-(4-amino-3-[125I]iodobenzyl)adenosine-5'-N-methylcarboxamide ([125I]I-AB-MECA) binding assays, we hypothesize that potency reduction is explained by an allosterically induced slowing in orthosteric ligand binding kinetics that reduces the rate of formation of ligand-receptor complexes. Mutation of four amino acid residues of the human A3AR to the murine sequence identified the extracellular loop 1 (EL1) region as being important in selectively controlling the allosteric actions of LUF6096 on [125I]I-AB-MECA binding kinetics. Homology modeling suggested interaction between species-variable EL1 and agonist-contacting EL2. These results indicate that A3AR allostery is species-dependent and provide mechanistic insights into this therapeutically promising class of agents.

The ATP binding site of the chromatin remodeling homolog Lsh is required for nucleosome density and de novo DNA methylation at repeat sequences.

Lsh, a chromatin remodeling protein of the SNF2 family, is critical for normal heterochromatin structure. In particular, DNA methylation at repeat elements, a hallmark of heterochromatin, is greatly reduced in Lsh(-/-) (KO) cells. Here, we examined the presumed nucleosome remodeling activity of Lsh on chromatin in the context of DNA methylation. We found that dynamic CG methylation was dependent on Lsh in embryonic stem cells. Moreover, we demonstrate that ATP function is critical for de novo methylation at repeat sequences. The ATP binding site of Lsh is in part required to promote stable association of the DNA methyltransferase 3b with the repeat locus. By performing nucleosome occupancy assays, we found distinct nucleosome occupancy in KO ES cells compared to WT ES cells after differentiation. Nucleosome density was restored to wild-type level by re-expressing wild-type Lsh but not the ATP mutant in KO ES cells. Our results suggest that ATP-dependent nucleosome remodeling is the primary molecular function of Lsh, which may promote de novo methylation in differentiating ES cells.

Patterns of Recurrence in Patients with Stage IIIB/C Cutaneous Melanoma of the Head and Neck Following Surgery With and Without Adjuvant Radiation Therapy: Is Isolated Regional Recurrence Salvageable?

BACKGROUND: Understanding recurrence patterns is vital for guiding treatment. This study describes recurrence patterns for patients with stage IIIB/C head and neck melanoma (HNM) after therapeutic lymph node dissection (TLND) ± adjuvant radiation therapy (RT). We also report outcomes for salvage therapy for patients with isolated regional relapse. METHODS AND MATERIALS: A single-institution prospective database of 173 patients with American Joint Committee on Cancer (AJCC) stage IIIB/C HNM undergoing TLND between 1997 and 2012 was retrospectively reviewed. Timing and patterns of recurrence were reviewed. Univariable and multivariable analyses were undertaken using the Kaplan-Meier and Cox regression methods to determine factors predictive of recurrence. Median follow-up was 32 months. RESULTS: Adjuvant RT was administered to 66/173 (38 %) patients. Patients selected for RT had a higher AJCC stage and had more extracapsular invasion. The 5-year distant, cervical nodal and in-transit recurrence rates were 38, 10, and 13 %, respectively, following surgery alone compared with 60, 17, and 31 %, respectively, for the adjuvant RT group. The head and neck regional 5-year recurrence rate (combining in-basin nodal and in-transit) was 23 % for the entire cohort. Isolated cervical recurrence occurred in 19 patients: 17/19 underwent salvage surgery (10/17 patients received RT after salvage surgery) and 2/19 had RT alone. However, distant recurrence occurred in 12/19 salvage patients, with most occurring within 12 months, while 4/19 were disease free. CONCLUSIONS: Using a selective approach for adjuvant RT, isolated cervical recurrence after TLND is uncommon. Isolated cervical recurrence can be salvaged effectively with further local therapy; however, distant disease frequently follows.

Patterns of care for ductal carcinoma in situ of the breast: Queensland's experience over a decade.

OBJECTIVES: To review management of ductal carcinoma in situ (DCIS) of the breast in Queensland, with reference to breast conserving surgery (BCS) and adjuvant radiation therapy (RT). In addition, we examined the incidence of invasive breast cancer recurrence and factors predictive of invasive recurrence. MATERIALS AND METHODS: A retrospective review of the Queensland Oncology Repository identified women with resected DCIS (TisN0) ± adjuvant RT between 2003 and 2012. Time to invasive breast cancer recurrence was analysed using the Kaplan Meier method. Median follow-up was 4.9 years. RESULTS: 3038 women had surgery. 940 (31%) had mastectomy and 2098 (69%) underwent BCS. Of 2098 women having BCS, 1100 (52%) received BCS alone and 998(48%) received adjuvant RT. The use of RT significantly increased over the decade from 25% to 62% (p=<0.001). Clinicopathological factors associated with RT use on multivariate analysis included age ≤70, higher socioeconomic status, larger tumour size, higher nuclear grade and surgical margins ≤5 mm. Invasive breast cancer recurrence at 5 years was 1.7% [95% CI 1.0-3.0] in RT group versus 2.8% [95% CI 2.1-3.8] in BCS alone group. Factors associated with increased risk of invasive recurrence on multivariate analysis were age <40 and surgical margins ≤2 mm. CONCLUSION: The use of adjuvant RT in Queensland significantly increased between 2003 and 2012. Selection of patients for RT was based on clinicopathological factors associated with higher recurrence risk. Although longer follow-up is required, the selective use of radiation therapy after BCS is associated with a low rate of invasive breast cancer recurrence at 5 years.

Effect of high fat diet on paternal sperm histone distribution and male offspring liver gene expression.

Several studies have described phenotypic changes in the offspring of mice exposed to a variety of environmental factors, including diet, toxins, and stress; however, the molecular pathways involved in these changes remain unclear. Using a high fat diet (HFD)-induced obesity mouse model, we examined liver gene expression in male offspring and analyzed chromatin of paternal spermatozoa. We found that the hepatic mRNA level of 7 genes (out of 20 evaluated) was significantly altered in HFD male offspring compared to control mice, suggesting that phenotypic changes in the offspring depend on parental diet. We examined 7 imprinted loci in spermatozoa DNA from HFD-treated and control fathers by bisulfite sequencing, but did not detect changes in DNA methylation associated with HFD. Using chromatin immunoprecipitation followed by high-throughput sequencing, we found differential histone H3-occupancy at genes involved in the regulation of embryogenesis and differential H3K4me1-enrichment at transcription regulatory genes in HFD fathers vs. control mice. These results suggest that dietary exposure can modulate histone composition at regulatory genes implicated in developmental processes.