Browsing by Author "Stabler, Sally P."

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    Evidence Favoring a Positive Feedback Loop for Physiologic Auto Upregulation of hnRNP-E1 during Prolonged Folate Deficiency in Human Placental Cells
    (Oxford University Press, 2017-04) Tang, Ying-Sheng; Khan, Rehana A.; Xiao, Suhong; Hansen, Deborah K.; Stabler, Sally P.; Kusumanchi, Praveen; Jayaram, Hiremagalur N.; Antony, Aśok C.; Medicine, School of Medicine
    Background: Previously, we determined that heterogeneous nuclear ribonucleoprotein E1 (hnRNP-E1) functions as an intracellular physiologic sensor of folate deficiency. In this model, l-homocysteine, which accumulates intracellularly in proportion to the extent of folate deficiency, covalently binds to and thereby activates homocysteinylated hnRNP-E1 to interact with folate receptor-α mRNA; this high-affinity interaction triggers the translational upregulation of cell surface folate receptors, which enables cells to optimize folate uptake from the external milieu. However, integral to this model is the need for ongoing generation of hnRNP-E1 to replenish homocysteinylated hnRNP-E1 that is degraded.Objective: We searched for an interrelated physiologic mechanism that could also maintain the steady-state concentration of hnRNP-E1 during prolonged folate deficiency.Methods: A novel RNA-protein interaction was functionally characterized by using molecular and biochemical approaches in vitro and in vivo.Results: l-homocysteine triggered a dose-dependent high-affinity interaction between hnRNP-E1 and a 25-nucleotide cis element within the 5'-untranslated region of hnRNP-E1 mRNA; this led to a proportionate increase in these RNA-protein complexes, and translation of hnRNP-E1 both in vitro and within placental cells. Targeted perturbation of this RNA-protein interaction either by specific 25-nucleotide antisense oligonucleotides or mutation within this cis element or by small interfering RNA to hnRNP-E1 mRNA significantly reduced cellular biosynthesis of hnRNP-E1. Conversely, transfection of hnRNP-E1 mutant proteins that mimicked homocysteinylated hnRNP-E1 stimulated both cellular hnRNP-E1 and folate receptor biosynthesis. In addition, ferrous sulfate heptahydrate [iron(II)], which also binds hnRNP-E1, significantly perturbed this l-homocysteine-triggered RNA-protein interaction in a dose-dependent manner. Finally, folate deficiency induced dual upregulation of hnRNP-E1 and folate receptors in cultured human cells and tumor xenografts, and more selectively in various fetal tissues of folate-deficient dams.Conclusions: This novel positive feedback loop amplifies hnRNP-E1 during prolonged folate deficiency and thereby maximizes upregulation of folate receptors in order to restore folate homeostasis toward normalcy in placental cells. It will also functionally impact several other mRNAs of the nutrition-sensitive, folate-responsive posttranscriptional RNA operon that is orchestrated by homocysteinylated hnRNP-E1.
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    Folate Deficiency Facilitates Genomic Integration of Human Papillomavirus Type 16 DNA In Vivo in a Novel Mouse Model for Rapid Oncogenic Transformation of Human Keratinocytes
    (Oxford, 2018) Xiao, Suhong; Tang, Ying-Sheng; Kusumanchi, Praveen; Stabler, Sally P.; Zhang, Ying; Antony, Aśok C.; Medicine, School of Medicine
    Background Epidemiologic and in vitro studies suggest independent linkages between poor folate and/or vitamin B-12 nutrition, genomic human papillomavirus (HPV) type 16 viral integration, and cancer. However, there is no direct evidence in vivo to support the causative role of poor folate nutrition in HPV16 integration into the cellular genome. Objective We tested the hypothesis that folate deficiency enables the integration of HPV16 into the genome of HPV16-harboring keratinocytes, and could thereby influence earlier transformation of these cells to cancer in an animal model. Methods HPV16-harboring human keratinocytes [(HPV16)BC-1-Ep/SL] were differentiated into 3-dimensional HPV16-organotypic rafts under either folate-replete or folate-deficient conditions in vitro. These were then subcutaneously implanted in severely immunocompromised female Beige Nude XID (Hsd: NIHS-LystbgFoxn1nuBtkxid) mice (4–6 wk old, 16–18 g) fed either a folate-replete diet (1200 nmol folate/kg diet) or a progressively folate-deficient diet (600 or 400 nmol folate/kg diet) for 2 mo prior to raft-implantation surgery, and indefinitely thereafter. The tumors that subsequently developed were characterized for onset, pattern of growth, morphology, HPV16 oncogene expression, and HPV16-genomic integration. Results All HPV16-organotypic rafts developed in either folate-replete or physiologic low-folate media in vitro and subsequently implanted in folate-replete mice eventually transformed into aggressive malignancies within weeks. When compared to HPV16-high folate-organotypic raft-derived tumors from mice fed either a 1200 or 600 nmol folate/kg diet, those raft-derived cancers that developed in mice fed a 400 nmol folate/kg diet expressed significantly more HPV16 E6 (1.8-fold more) and E7 (2.8-fold more) oncogenic proteins (P = 0.001), and revealed significantly more HPV16-integration sites in genomic DNA (2-fold more), either directly into, or in the vicinity of, cellular genes (P < 0.05). Conclusions This unprecedented animal model for the consistent rapid transformation of differentiated (HPV16)BC-1-Ep/SL-derived organotypic raft-keratinocytes to cancer in Beige Nude XID mice confirms that dietary folate deficiency can profoundly influence and modulate events leading to HPV16-induced carcinogenesis, and facilitates genomic integration of HPV16 DNA in vivo.