Fertility and Pregnancy Outcomes After Conservative Management of Adenocarcinoma In Situ of the Cervix.

OBJECTIVE: The aim of the study was to evaluate the pregnancy outcomes of women who underwent conservative management of adenocarcinoma in situ (AIS). MATERIALS AND METHODS: We conducted a retrospective chart review of patients diagnosed with AIS at a single tertiary institution between January 1, 1991, and December 31, 2019. We collected demographic data, AIS-specific information, and fertility outcomes and performed bivariate analyses to compare demographic characteristics and AIS-specific information between patients with and without hysterectomy after diagnosis. Patients with conservative management who achieved pregnancy were described. RESULTS: Among 87 patients with AIS, 38 (44%) underwent a hysterectomy within 6 months of diagnosis and 49 (56%) underwent conservative management. Six of 19 patients (32%) had residual AIS despite undergoing definitive management after an excisional procedure with negative margins and negative endocervical curettage (ECC). Nine of 19 patients (47%) had residual AIS after an excisional procedure with positive margins and/or a positive ECC. Patients who opted for conservative management were younger (median = 31.6 [interquartile range = 27.4-34.9] vs 38.5 y [32.3-44.8 y], p < .001) and nulligravid. Among patients with conservative management, there were 15 pregnancies and 14 live births (29%). Seven were preterm, although 2 were for medical indications. CONCLUSIONS: Residual AIS in patients with negative margins and ECC leading to definitive hysterectomy (32%) and the rate of preterm birth (36%) were higher than previous reports and nationally reported rates. However, only 1 patient had a preterm birth before 34 weeks. These findings reflect important information for counseling patients who elect for conservative management of AIS.

Policy-influencer perspectives on the development, adoption, and implementation of provincial school-based daily physical activity policies across Canada: A national case study.

OBJECTIVES: School-based daily physical activity (DPA) policies represent a promising intervention for increasing physical activity levels among children. Between 2005 and 2010, five Canadian provinces adopted and implemented DPA policies. This national case study explored facilitators and challenges to developing, adopting and implementing DPA policies from the perspective of key stakeholders ('policy-influencers') in these five provinces. METHODS: Development, adoption and implementation of DPA policies at the provincial level was the phenomenon of interest, with each province constituting a distinct case. Semi-structured interviews were conducted with 15 purposively selected policy-influencers across the five case provinces. Cases were first analyzed separately, and then concurrently in a cross-case comparison. Qualitative content analysis was used to code interviews and develop themes. RESULTS: Four themes related to the development, adoption, and implementation of DPA policies emerged: existing resources and capacity for policy change; top-down policy development/adoption and bottom-up implementation; political will and windows of opportunity; and, ideology and policy change. Each of these themes encompassed facilitators and challenges related to policy processes surrounding development, adoption and implementation of DPA policies. CONCLUSION: These findings can inform development of future health-related polices in schools. Stakeholders can, for instance, remain attuned to the opening of political windows and capitalize on them as an opportunity to advocate for policy change or create communities of practice to enhance coordination among policy stakeholders. Future studies that explore why other jurisdictions have failed to adopt DPA policies might yield novel insights regarding leverage points to support widespread diffusion of DPA policies.

Activation of the hypoxia pathway in breast cancer tissue and patient survival are inversely associated with tumor ascorbate levels.

BACKGROUND: The transcription factor hypoxia inducible factor (HIF) -1 drives tumor growth and metastasis and is associated with poor prognosis in breast cancer. Ascorbate can moderate HIF-1 activity in vitro and is associated with HIF pathway activation in a number of cancer types, but whether tissue ascorbate levels influence the HIF pathway in breast cancer is unknown. In this study we investigated the association between tumor ascorbate levels and HIF-1 activation and patient survival in human breast cancer. METHODS: In a retrospective analysis of human breast cancer tissue, we analysed primary tumor and adjacent uninvolved tissue from 52 women with invasive ductal carcinoma. We measured HIF-1α, HIF-1 gene targets CAIX, BNIP-3 and VEGF, and ascorbate content. Patient clinical outcomes were evaluated against these parameters. RESULTS: HIF-1 pathway proteins were upregulated in tumor tissue and increased HIF-1 activation was associated with higher tumor grade and stage, with increased vascular invasion and necrosis, and with decreased disease-free and disease-specific survival. Grade 1 tumors had higher ascorbate levels than did grade 2 or 3 tumors. Higher ascorbate levels were associated with less tumor necrosis, with lower HIF-1 pathway activity and with increased disease-free and disease-specific survival. CONCLUSIONS: Our findings indicate that there is a direct correlation between intracellular ascorbate levels, activation of the HIF-1 pathway and patient survival in breast cancer. This is consistent with the known capacity of ascorbate to stimulate the activity of the regulatory HIF hydroxylases and suggests that optimisation of tumor ascorbate could have clinical benefit via modulation of the hypoxic response.

Adoption and diffusion of zoning bylaws banning fast food drive-through services across Canadian municipalities.

BACKGROUND: Healthy public policy is an important tool for creating environments that support human health and wellbeing. At the local level, municipal policies, such as zoning bylaws, provide an opportunity for governments to regulate building location and the type of services offered. Across North America, there has been a recent proliferation of municipal bylaws banning fast food drive-through services. Research on the utilization of this policy strategy, including bylaw adopters and adopter characteristics, is limited within the Canadian context. The aim of this study was to identify and characterize Canadian municipalities based on level of policy innovation and nature of their adopted bylaw banning fast food drive-through services. METHODS: A multiple case history methodology was utilized to identify and analyse eligible municipal bylaws, and included development of a chronological timeline and map of adopter municipalities within Canada. Grey literature and policy databases were searched for potential adopters of municipal fast food drive-through service bylaws. Adopters were confirmed through evidence of current municipal bylaws. Geographic diffusion and diffusion of innovations theories provided a contextual framework for analysis of bylaw documents. Analysis included assignment of adopter-types, extent and purpose of bans, and policy learning activities of each adopter municipality. RESULTS: From 2002 to 2016, 27 municipalities were identified as adopters: six innovators and twenty-one early adopters. Mapping revealed parallel geographic diffusion patterns in western and eastern Canada. Twenty-two municipalities adopted a partial ban and five adopted a full ban. Rationales for the drive-through bans included health promotion, environmental concerns from idling, community character and aesthetics, traffic concerns, and walkability. Policy learning, including research and consultation with other municipalities, was performed by nine early adopters. CONCLUSION: This study detailed the adoption of fast food drive-through bylaws across Canada. Understanding the adopter-type characteristics of municipalities and the nature of their bylaws can assist other jurisdictions in similar policy efforts. While the implications for research and practice are evolving and dynamic, fast food drive-through service bans may play a role in promoting healthier food environments. Further research is required to determine the viability of this strategy for health promotion and chronic disease prevention.

Ascorbate availability affects tumor implantation-take rate and increases tumor rejection in Gulo-/- mice.

In solid tumors, HIF1 upregulates the expression of hundreds of genes involved in cell survival, tumor growth, and adaptation to the hypoxic microenvironment. HIF1 stabilization and activity are suppressed by prolyl and asparagine hydroxylases, which require oxygen as a substrate and ascorbate as a cofactor. This has led us to hypothesize that intracellular ascorbate availability could modify the hypoxic HIF1 response and influence tumor growth. In this study, we investigated the effect of variable intracellular ascorbate levels on HIF1 induction in cancer cells in vitro, and on tumor-take rate and growth in the Gulo-/- mouse. These mice depend on dietary ascorbate, and were supplemented with 3,300 mg/L, 330 mg/L, or 33 mg/L ascorbate in their drinking water, resulting in saturating, medium, or low plasma and tissue ascorbate levels, respectively. In Lewis lung carcinoma cells (LL/2) in culture, optimal ascorbate supplementation reduced HIF1 accumulation under physiological but not pathological hypoxia. LL/2, B16-F10 melanoma, or CMT-93 colorectal cancer cells were implanted subcutaneously into Gulo-/- mice at a range of cell inocula. Establishment of B16-F10 tumors in mice supplemented with 3,300 mg/L ascorbate required an increased number of cancer cells to initiate tumor growth compared with the number of cells required in mice on suboptimal ascorbate intake. Elevated ascorbate intake was also associated with decreased tumor ascorbate levels and a reduction in HIF1α expression and transcriptional activity. Following initial growth, all CMT-93 tumors regressed spontaneously, but mice supplemented with 33 mg/L ascorbate had lower plasma ascorbate levels and grew larger tumors than optimally supplemented mice. The data from this study indicate that improved ascorbate intake is consistent with increased intracellular ascorbate levels, reduced HIF1 activity and reduced tumor initiation and growth, and this may be advantageous in the management of cancer.

Pharmacokinetic and anti-cancer properties of high dose ascorbate in solid tumours of ascorbate-dependent mice.

Despite recent evidence for an anti-tumour role for high-dose ascorbate, potential mechanisms of action are still unclear. At mM concentrations that are achieved with high-dose intravenous administration, autoxidation of ascorbate can generate cytotoxic levels of H2O2. Ascorbate is also a required co-factor for the hydroxylases that suppress the transcription factor hypoxia-inducible factor (HIF-1). HIF-1 supports an aggressive tumour phenotype and is associated with poor prognosis, and previous studies have shown that optimizing intracellular ascorbate levels down-regulates HIF-1 activation. In this study we have simultaneously measured ascorbate concentrations and the HIF-1 pathway activity in tumour tissue following high dose ascorbate administration, and have studied tumour growth and physiology. Gulo-/- mice, a model of the human ascorbate dependency condition, were implanted with syngeneic Lewis lung tumours, 1g/kg ascorbate was administered into the peritoneum, and ascorbate concentrations were monitored in plasma, liver and tumours. Ascorbate levels peaked within 30min, and although plasma and liver ascorbate returned to baseline within 16h, tumour levels remained elevated for 48h, possibly reflecting increased stability in the hypoxic tumour environment. The expression of HIF-1 and its target proteins was down-regulated with tumour ascorbate uptake. Elevated tumour ascorbate levels could be maintained with daily administration, and HIF-1 and vascular endothelial growth factor protein levels were reduced in these conditions. Increased tumour ascorbate was associated with slowed tumour growth, reduced tumour microvessel density and decreased hypoxia. Alternate day administration of ascorbate resulted in lower tumour levels and did not consistently decrease HIF-1 pathway activity. Levels of sodium-dependent vitamin C transporters 1 and 2 were not clearly associated with ascorbate accumulation by murine tumour cells in vitro or in vivo. Our results support the suppression of the hypoxic response by ascorbate as a plausible mechanism of action of its anti-tumour activity, and this may be useful in a clinical setting.

A multiple case history and systematic review of adoption, diffusion, implementation and impact of provincial daily physical activity policies in Canadian schools.

BACKGROUND: Few children meet physical activity (PA) recommendations, and are therefore at increased risk for overweight/obesity and adverse health outcomes. To increase children's opportunities for PA, several Canadian provinces have adopted school-based daily PA (DPA) policies. It is not clear why some jurisdictions have adopted DPA policies, and others have not, nor whether these policies have been implemented and have achieved their intended outcomes. The purpose of this study was to understand the processes underlying adoption and diffusion of Canadian DPA policies, and to review evidence regarding their implementation and impact. METHODS: We adopted a multiple case history methodology in which we traced the chronological trajectory of DPA policies among Canadian provinces by compiling timelines detailing key historical events that preceded policy adoption. Publicly available documents posted on the internet were reviewed to characterize adopter innovativeness, describe the content of their DPA policies, and explore the context surrounding policy adoption. Diffusion of Innovations theory provided a conceptual framework for the analyses. A systematic literature search identified studies that had investigated adoption, diffusion, implementation or impact of Canadian DPA policies. RESULTS: Five of Canada's 13 provinces and territories (38.5%) have DPA policies. Although the underlying objectives of the policies are similar, there are clear differences among them and in their various policy trajectories. Adoption and diffusion of DPA policies were structured by the characteristics and capacities of adopters, the nature of their policies, and contextual factors. Limited data suggests implementation of DPA policies was moderate but inconsistent and that Canadian DPA policies have had little to no impact on school-aged children's PA levels or BMI. CONCLUSIONS: This study detailed the history and current status of Canadian DPA policies, highlighting the conditional nature of policy adoption and diffusion, and describing policy and adopter characteristics and political contexts that shaped policy trajectories. An understanding of the conditions associated with successful policy adoption and diffusion can help identify receptive contexts in which to pioneer novel legislative initiatives to increase PA among children. By reviewing evidence regarding policy implementation and impact, this study can also inform amendments to existing, and development of future PA policies.

Restoring physiological levels of ascorbate slows tumor growth and moderates HIF-1 pathway activity in Gulo(-/-) mice.

Hypoxia-inducible factor-1 (HIF-1) governs cellular adaption to the hypoxic microenvironment and is associated with a proliferative, metastatic, and treatment-resistant tumor phenotype. HIF-1 levels and transcriptional activity are regulated by proline and asparagine hydroxylases, which require ascorbate as cofactor. Ascorbate supplementation reduced HIF-1 activation in vitro, but only limited data are available in relevant animal models. There is no information of the effect of physiological levels of ascorbate on HIF activity and tumor growth, which was measured in this study. C57BL/6 Gulo(-/-) mice (a model of the human ascorbate dependency condition) were supplemented with 3300 mg/L, 330 mg/L, or 33 mg/L of ascorbate in their drinking water before and during subcutaneous tumor growth of B16-F10 melanoma or Lewis lung carcinoma (LL/2). Ascorbate levels in tumors increased significantly with elevated ascorbate intake and restoration of wild-type ascorbate levels led to a reduction in growth of B16-F10 (log phase P < 0.001) and LL/2 tumors (lag growth P < 0.001, log phase P < 0.05). Levels of HIF-1α protein in tumors decreased as dietary ascorbate supplementation increased for both tumor models (P < 0.001). Similarly, tumor ascorbate was inversely correlated with levels of the HIF-1 target proteins CA-IX, GLUT-1, and VEGF in both B16-F10 and LL/2 tumors (P < 0.05). The extent of necrosis was similar between ascorbate groups but varied between models (30% for B16-F10 and 21% for LL/2), indicating that ascorbate did not affect tumor hypoxia. Our data support the hypothesis that restoration of optimal intracellular ascorbate levels reduces tumor growth via moderation of HIF-1 pathway activity.

Current limitations of murine models in oncology for ascorbate research.

The role of vitamin C (ascorbate) in cancer prevention, tumor growth, and treatment is of intense public interest. Clinical trial data have been sparse, contradictory, and highly controversial, and robust pre-clinical data are required for progress. This paper reviews pre-clinical models and their limitations with respect to ascorbate research. Most studies have utilized animals able to synthesize ascorbate and thus are not ideal models of the human condition. More recently, genetically modified mouse models have become available; yet, all studies compared healthy and scorbutic mice. The majority of investigations to date concluded that increased ascorbate led to decreased tumor growth, but data on mechanisms and doses are inconclusive. Clinically relevant animal studies are still required to convince a generally sceptical medical audience of the potential worth of ascorbate as an adjunct to therapy.

Gulonolactone Addition to Human Hepatocellular Carcinoma Cells with Gene Transfer of Gulonolactone Oxidase Restores Ascorbate Biosynthesis and Reduces Hypoxia Inducible Factor 1.

Humans are unable to synthesise ascorbate (Vitamin C) due to the lack of a functional gulonolactone oxidase (Gulo), the enzyme that catalyses the final step in the biosynthesis pathway. Ascorbate is a vital micronutrient required for many biological functions, including as a cofactor for metalloenzymes that regulate the transcription factor hypoxia-inducible factor-1 (HIF-1), which governs cell survival under hypoxia. In most animals, ascorbate is made in liver cells. This study aimed to restore ascorbate synthesis to human hepatocellular carcinoma HepG2 cells and determine the effect of internally produced ascorbate on HIF-1 activation. HepG2 cells were gene-modified with a plasmid encoding the mouse Gulo cDNA, tested for genomic incorporation by PCR and ascorbate synthesis by high performance liquid chromatography. Levels of HIF-1 protein were measured using Western blotting. Gulo-modified HepG2 cells showed increased adherence compared to control HepG2 cells. A PCR-positive clone synthesised ascorbate when the Gulo substrate, l-gulono-1,4-lactone, was supplied. Intracellular ascorbate concentrations reached 5% of saturation levels (6 nmol/106 cells). Addition of ascorbate or gulonolactone reduced HIF-1 accumulation in the Gulo clone, but also in parental HepG2 cells. Our data confirm the requirement for a number of factors in addition to Gulo in the ascorbate biosynthesis pathway in human cells.

Primary culture of ovarian surface epithelial cells and ascites-derived ovarian cancer cells from patients.

Our laboratory has refined the technique for isolating primary cultures of normal human ovarian surface epithelial (OSE) cells by combining two different protocols involving the enzymatic and mechanical removal of OSE cells from ovarian biopsies. A simple protocol of obtaining primary epithelial ovarian cancer (EOC) cells from the ascites fluid removed from patients with high-grade ovarian cancer is also described. These methods allow for the direct application of many molecular and cellular analyses of normal versus cancer cells isolated freshly from patients, with the added potential for retrospective analyses of archived cells and tissues. Thus, we have included optional steps for the immediate preparation of ascites-derived EOC cells to be used for subsequent cytological analyses. Initial isolation of OSE or EOC cells can be completed in 1 h, and primary cells are further expanded in culture for several weeks.

Epigenetic regulation of proprotein convertase PACE4 gene expression in human ovarian cancer cells.

Proprotein convertases (PC) are a family of serine endoproteases that play important roles in regulating cell function by converting proproteins to biologically active molecules. Several lines of evidence suggest that overexpression of PCs contributes to tumor formation and progression in various types of cancer. In this study, we examined PC expression in six normal ovarian surface epithelium (OSE) cultures, nine primary ovarian cancer (OC) cultures, and three established OC cell lines (Hey, HeyC2, and OCC-1). Our results show that furin and PC7 expression in OC cells was comparable to that in normal OSE. However, PACE4 expression was greatly reduced in all OC samples studied. PACE4 promoter activity was measured in HeyC2 and OCC-1 cells using transiently transfected luciferase reporter plasmids. Both cell lines supported PACE4 promoter activity, showing that the transcription factors critical for PACE4 expression are present in OC cells. The observation that established OC cell lines have reduced PACE4 expression, but maintained the ability to support PACE4 promoter activity, led to the hypothesis that reduced expression may be due to epigenetic modification of the PACE4 gene, such as DNA methylation and histone deacetylation. Methylation analysis of 79 CpG dinucleotides within the PACE4 promoter and exon I (-196/+340) revealed that the percentage of methylated cytosine nucleotides was 8-9% in normal OSE, but 58-93% in OC cells. Treatment with the demethylating agent 5-aza-2'-deoxycytidine and/or the histone deacetylase inhibitor trichostatin A greatly increased PACE4 expression in OC cells. These data suggest that the reduction of PACE4 expression in OC cells is caused, in part, by DNA hypermethylation and histone deacetylation.