Oral Rehabilitation of Patients Sustaining Orofacial Injuries: The UPenn Initiative.

Tissue injuries in the oral and maxillofacial structures secondary to trauma, warfare, ablative cancer, and benign tumor surgery result in significant losses of speech, masticatory and swallowing functions, aesthetic deformities, and overall psychological stressors and compromise. Optimal oral rehabilitation remains a formidable challenge and an unmet clinical need due to the influence of multiple factors related to the physiologic limitations of tissue repair, the lack of site and function-specific donor tissues and constructs, and an integrated team of multidisciplinary professionals. The advancements in stem cell biology, biomaterial science, and tissue engineering technologies, particularly the 3-dimensional bioprinting technology, together with digital imaging and computer-aided design and manufacturing technologies, have paved the path for personalized/precision regenerative medicine. At the University of Pennsylvania, we have launched the initiative to integrate multidisciplinary health professionals and translational/clinical scientists in medicine, dentistry, stem cell biology, tissue engineering, and regenerative medicine to develop a comprehensive, patient-centered approach for precision and personalized reconstruction, as well as oral rehabilitation of patients sustaining orofacial tissue injuries and defects, especially oral cancer patients.

Biliverdin's regulation of reactive oxygen species signalling leads to potent inhibition of proliferative and angiogenic pathways in head and neck cancer.

BACKGROUND: In this study, we evaluate whether the use of biliverdin (BV), a natural non-toxic antioxidant product of haeme catabolism, can suppress head and neck squamous cell carcinoma (HNSCC) cell proliferation and improve the tumour survival both in vitro and in vivo. Furthermore, we investigate whether this therapeutic outcome relies on BV's potent antioxidant effect on reactive oxygen species (ROS)-mediated signalling. METHODS: Two well-characterised HNSCC cell lines and a mouse model with human HNSCC were used for this study. In vitro, the effect of BV on ROS was assayed. Subsequently, critical regulatory proteins involved in growth, antiapoptotic, and angiogenic pathways were investigated by western blot analysis. In addition, the antiproliferative effect of BV was also evaluated using the clonogenic assay. Moreover, tumour growth inhibition was assessed using a mouse model with HNSCC. RESULTS: Biliverdin treatment resulted in decreased ROS, leading to suppression of proliferation and angiogenesis pathways of HNSCC, significantly decreasing the expression and phosphorylation of oncogenic factors such as epidermal growth factor receptor (EGFR), phosphorylation of Akt, and expression of angiogenic marker and transcription factor, hypoxia-inducible factor1-α (HIF1-α). Furthermore, this downregulation of ROS by BV led to a significant suppression of tumour growth in vivo. CONCLUSIONS: Our study demonstrates the efficacy of a novel therapeutic approach using BV as an antitumour agent against HNSCC through its effect on EGFR/Akt and HIF1-α/angiogenesis signal transduction pathways. Our findings indicate that BV's inhibitory effect on these tumorigenic pathways relies on its antioxidant effect, and may extend its therapeutic potential to other solid cancers.

FRAP reveals that mobility of oestrogen receptor-alpha is ligand- and proteasome-dependent.

Here we report the use of fluorescence recovery after photobleaching (FRAP) to examine the intranuclear dynamics of fluorescent oestrogen receptor-alpha (ER). After bleaching, unliganded ER exhibits high mobility (recovery t1/2 < 1 s). Agonist (oestradiol; E2) or partial antagonist (4-hydroxytamoxifen) slows ER recovery (t1/2 approximately 5-6 s), whereas the pure antagonist (ICI 182,780) and, surprisingly, proteasome inhibitors each immobilize ER to the nuclear matrix. Dual FRAP experiments show that fluorescent ER and SRC-1 exhibit similar dynamics only in the presence of E2. In contrast to reports that several nuclear proteins show uniform dynamics, ER exhibits differential mobility depending upon several factors that are linked to its transcription function.

Molecular disruption of NBS1 with targeted gene delivery enhances chemosensitisation in head and neck cancer.

BACKGROUND: a fibroblast growth factor 2 (FGF2)-targeted adenoviral system can alter viral tropism and allow for improved transduction and reduced systemic toxicity. This study is to investigate if the FGF2-targeted adenoviral mutant Nijmegen breakage syndrome 1 (FGF2-Ad-NBS1) gene transfer can enhance cisplatin chemosensitisation not only by targeting DNA repair, but also through the induction of antiangiogenesis, whereas at the same time reducing toxicities in treating head and neck squamous cell carcinoma (HNSCC). METHODS: the human HNSCC cell line was treated in vitro and in a nude mouse xenograft model. We conducted verification of binding ability of mutant NBS1 and downregulation of MRN complex, evaluation of transduction efficiency and combined antitumour activities. The antiangiogenesis mechanism was also investigated. Finally, we estimated the distribution of adenoviral vector in the liver. RESULTS: the mutant NBS1 protein retains the binding ability and effectively suppresses the expression level of the MRN in infected cells. Transduction efficiency in vitro and cisplatin chemosensitisation were upregulated. The FGF2-Ad-NBS1 also showed detargeting the viral vectors away from the liver. The downregulation of NF-κB expression was supposed to correlate with increased antiangiogenesis. CONCLUSIONS: FGF2-targeted adenoviral system enhances the cisplatin chemosensitisation of mutant NBS1 and may avoid viral-associated liver toxicities.

Estrogen-induced cytodifferentiation of the ovalbumin-secreting glands of the chick oviduct.

The histological, ultrastructural, and biochemical changes occurring during hormone-induced cytodifferentiation of the ovalbumin-secreting glands in the chick oviduct have been studied. Marked perivascular edema is an initial response of the immature oviduct stroma to diethylstilbestrol administration and is accompanied by an interstitial migration of mononuclear cells. Mitotic activity in the immature mucosal epithelium increases within 24 hr, and glands begin to develop on days 2-4 as budlike invaginations into the subepithelial stroma. An immediate intracellular effect of the hormone is aggregation of previously dispersed ribosomes. Ribosomal zones in the nucleolus gain prominence, and there is a progressive development of rough endoplasmic reticulum in the epithelial cells. Extensive profiles of endoplasmic reticulum are present in the gland cells by day 6. Fine apical progranules appear in the epithelial cells on day 2, and ovalbumin can be measured immunochemically by day 3 at about the same time that new species of nuclear RNA have been identified. Ovalbumin granules form within condensing vacuoles in the Golgi zone and begin to be released into the lumina of the gland acini at about day 6 of the treatment.

Primary sequence of ovomucoid messenger RNA as determined from cloned complementary DNA.

Ovomucoid messenger RNA (mRNAom) comprises approximately 8% of the total mRNA in the estrogen-stimulated oviduct. The recombinant plasmid pOM100 contained DNA complementary to the 3' end of mRNAom. DNA complementary to the 5' end of mRNAom was obtained from a partially purified preparation of mRNAom by polymerization by reverse transcriptase in the presence of a restriction fragment primer from pOM100. The complementary DNA mixture was amplified by molecular cloning using poly dG/dC tailing to form recombinant bacterial plasmids. Recombinant plasmids containing ovomucoid DNA sequences were selected by in situ hybridization to 32P-labeled pOM100 fragments. The longest plasmid containing ovomucoid DNA sequences was designated pOM502. The complete DNA sequence of both pOM100 and pOM502 was determined. The two plasmids appear to contain sequences complementary to the entire length of mRNAom. The nucleic acid sequence agrees with the known amino acid sequences for both ovomucoid and its N-terminal signal peptide. Highly homologous sequences occur in two regions that coincide with structural domains of the protein. Comparison of the sequence of mRNAom with that for other eucaryotic mRNAs allowed identification of possible functional regions in the mRNA molecule.

Female steroid hormones and target cell nuclei.

The data discussed herein demonstrate the great variation in target-tissue response that can occur after administration of steroid hormones. The female sex steroids can exert regulatory effects on the synthesis, activity, and possibly even the degradation of tissue enzymes and structural proteins. Each response, nevertheless, appears to be dependent on the synthesis of nuclear RNA. In many instances, the steroid actually promotes a qualitative change in the base composition and sequence of the RNA synthesized by the target cell, implying a specific effect on gene transcription. Most important is our direct quantitative evidence that sex steroids cause a net increase in the intracellular amounts of specific mRNA molecules in target tissues. It thus appears that we are discovering a pattern of steroid hormone action which includes (Fig. 1): (i) uptake of the hormone by the target cell and binding to a specific cytoplasmic receptor protein; (ii) transport of the steroid-receptor complex to the nucleus; (iii) binding of this "active" complex to specific "acceptor" sites on the genome (chromatin DNA and acidic protein); (iv) activation of the transcriptional apparatus resulting in the appearance of new RNA species which includes specific mRNA's; (v) transport of the hormone-induced RNA to the cytoplasm resulting in synthesis of new proteins on cytoplasmic ribosomes; and (vi) the occurrence of the specific steroid-mediated "functional response" characteristic of that particular target tissue. To elucidate fully the mechanism of steroid hormone action we must study the biochemistry of the process by which information held by the steroid hormone-receptor complex is transferred to the nuclear transcription apparatus. If our assumptions are correct, we should ultimately be able to discover how this hormone-receptor complex exerts a specific regulatory effect on nuclear RNA metabolism. Such regulation might be achieved (i) by direct effects on chromatin template leading to increased gene transcription and thus RNA synthesis; (ii) by activation of the polymerase complex itself; (iii) by inhibition of RNA breakdown; or (iv) by intranuclear processing of large precursor molecules so that smaller biologically active sequences are produced, and (v) by transport of RNA from the nucleus to the cytoplasmic sites of cellular protein synthesis.

Steroid hormones: effects on adenyl cyclase activity and adenosine 3',5'-momophosphate in target tissues.

The adenyl cyclases of chick oviduct and rat prostate were not stimulated by estrogen and testosterone, respectively, suggesting that growth and differentiation of these target tissues are not mediated by adenosine 3',5'-monophosphate. Estrogen acutely activated adenyl cyclase in the castrate rat uterus, but this was prevented by administration of DL-propranolol, suggesting that the effect was mediated by catecholamines. Progesterone produced a delayed stimulation of oviduct adenyl cyclase preceding and concomitant with the induction of synthesis of avidin.

Estrogenic induction of ornithine decarboxylase in vivo and in vitro.

Injection of estrogens (17beta-estradiol or diethylstilbestrol) into immature chicks results in a marked (30- to 50-fold) increase in the ornithine decarboxylase activity of oviductal homogenates within a 4-hour period. Similar stimulations were obtained when estrogen was injected into hypophysectomized or castrated rats and the uterus was examined for decarboxylase activity. An elevation of decarboxylase activity was obtained in vitro when oviducts from immature chicks were incubated in the presence of estrogen. These data indicate a direct action of estrogen on oviduct tissue to promote a rapid increase in the activity of a specific enzyme and represent the first example of a completely in vitro enzyme response to estrogen.

Protein synthesis: differential stimulation of cell-specific proteins in epithelial cells of chick oviduct.

Immunofluorescent and radioautographic studies demonstrate that ovalbumin and avidin are cell-specific proteins synthesized by different epithelial cells in the chick oviduct mucosa. The mechanism of the selective induction of ovalbumin synthesis by estrogen and of avidin synthesis by progesterone may be through stimulation of specific target cells by these hormones.

Distribution of RNA transcripts from structural and intervening sequences of the ovalbumin gene.

A study was made of the function of the intervening sequences in the ovalbumin gene, Radioactively labeled DNA probes for the intervening sequences were prepared and RNA's were isolated from whole cells, nuclei, and polysomes of estrogen-stimulated chick oviducts. The concentrations of messenger RNA (mRNA) transcripts from ovalbumin structural sequences (mRNAov) and transcripts corresponding to intervening sequences were then estimated by hybridization to cloned DNA probes. Oviduct tissue contains approximately 58,000 molecules of mRNAov sequences per tubular gland cell and most of these sequences are present in the cytoplasm. In contrast, there are 200 to 300 molecules of RNA per cell which are transcribed from the intervening sequences of the natural ovalbumin gene and almost all of these are found in the nucleus. The difference in distribution of structural and intervening sequence transcripts suggests that, unlike mature mRNA, the intervening sequences are not preferentially transported to cytoplasmic polysomes.

Dopamine activation of an orphan of the steroid receptor superfamily.

The chicken ovalbumin upstream promoter transcription factor (COUP-TF) is a member of the steroid receptor superfamily and participates in the regulation of several genes. While a number of functions have been ascribed to COUP-TF, no ligand or activator molecule has been identified, and thus it is classified as one of a group of orphan receptors. Activation of COUP-TF by physiological concentrations of the neurotransmitter dopamine was observed in transient transfection assays. Treatment of transfected cells with the dopamine receptor agonist alpha-ergocryptine also activated COUP-dependent expression of a reporter gene. COUP-TF that contained a deletion in the COOH-terminal domain was not activated by these compounds. These observations suggest that dopamine may be a physiological activator of COUP-TF.

Estrogen-dependent increase in transfer RNA during differentiation of the chick oviduct.

Estrogenic hormones induce morphologic and biochemical differentiation in the oviduct of the immature chick. Concomitant with the hormone-stimulated tissue growth, there was an increase in 4S RNA, as judged by polyacrylamide-gel electrophoresis, and a corresponding increase in cellular transfer RNA activity, as measured by the amino acid acceptor capacity. This system may be suitable for studying the relation of hormones to transfer RNA in a differentiating tissue.

Sequence and characterization of a coactivator for the steroid hormone receptor superfamily.

A yeast two-hybrid system was used to identify a protein that interacts with and enhances the human progesterone receptor (hPR) transcriptional activity without altering the basal activity of the promoter. Because the protein stimulated transactivation of all the steroid receptors tested, it has been termed steroid receptor coactivator-1 (SRC-1). Coexpression of SRC-1 reversed the ability of the estrogen receptor to squelch activation by hPR. Also, the amino terminal truncated form of SRC-1 acted as a dominant-negative repressor. Together, these results indicate that SRC-1 encodes a coactivator that is required for full transcriptional activity of the steroid receptor superfamily.

Molecular cloning of complementary DNA encoding the avian receptor for vitamin D.

Vitamin D3 receptors are intracellular proteins that mediate the nuclear action of the active metabolite 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Two receptor-specific monoclonal antibodies were used to recover the complementary DNA (cDNA) of this regulatory protein from a chicken intestinal lambda gt11 cDNA expression library. The amino acid sequences that were deduced from this cDNA revealed a highly conserved cysteine-rich region that displayed homology with a domain characteristic of other steroid receptors and with the gag-erbA oncogene product of avian erythroblastosis virus. RNA selected via hybridization with this DNA sequence directed the cell-free synthesis of immunoprecipitable vitamin D3 receptor. Northern blot analysis of polyadenylated RNA with these cDNA probes revealed two vitamin D receptor messenger RNAs (mRNAs) of 2.6 and 3.2 kilobases in receptor-containing chicken tissues and a major cross-hybridizing receptor mRNA species of 4.2 kilobases in mouse 3T6 fibroblasts. The 4.2-kilobase species was substantially increased by prior exposure of 3T6 cells to 1,25(OH)2D3. This cDNA represents perhaps the rarest mRNA cloned to date in eukaryotes, as well as the first receptor sequence described for an authentic vitamin.

Convergent pathways for steroid hormone- and neurotransmitter-induced rat sexual behavior.

Estrogen and progesterone modulate gene expression in rodents by activation of intracellular receptors in the hypothalamus, which regulate neuronal networks that control female sexual behavior. However, the neurotransmitter dopamine has been shown to activate certain steroid receptors in a ligand-independent manner. A dopamine receptor stimulant and a D1 receptor agonist, but not a D2 receptor agonist, mimicked the effects of progesterone in facilitating sexual behavior in female rats. The facilitory effect of the neurotransmitter was blocked by progesterone receptor antagonists, a D1 receptor antagonist, or antisense oligonucleotides to the progesterone receptor. The results suggest that in rodents neurotransmitters may regulate in vivo gene expression and behavior by means of cross-talk with steroid receptors in the brain.

Partial hormone resistance in mice with disruption of the steroid receptor coactivator-1 (SRC-1) gene.

The in vivo biological function of a steroid receptor coactivator was assessed in mice in which the SRC-1 gene was inactivated by gene targeting. Although in both sexes the homozygous mutants were viable and fertile, target organs such as uterus, prostate, testis, and mammary gland exhibited decreased growth and development in response to steroid hormones. Expression of RNA encoding TIF2, a member of the SRC-1 family, was increased in the SRC-1 null mutant, perhaps compensating partially for the loss of SRC-1 function in target tissues. The results indicate that SRC-1 mediates steroid hormone responses in vivo and that loss of its coactivator function results in partial resistance to hormone.

Regulation of progesterone receptor-mediated transcription by phosphorylation.

The progesterone receptor (PR) in the chicken oviduct is a phosphoprotein that regulates gene transcription in the presence of progesterone. Treatment with progesterone in vivo stimulates phosphorylation of the progesterone receptor. With transient transfection assays, the present work has tested whether phosphorylation participates in the regulation of PR-mediated transcription. Treatment with 8-bromo-cyclic adenosine monophosphate (8-Br cAMP), a stimulator of cAMP-dependent protein kinase [protein kinase A (PKA)], mimicked progesterone-dependent, receptor-mediated transcription in the absence of progesterone. Inhibition of PKA blocked hormone action. Treatment with okadaic acid, an inhibitor of protein phosphatases 1 and 2A, stimulated transcription in a manner similar to that of progesterone. These observations suggest that phosphorylation of the PR or other proteins in the transcription complex can modulate PR-mediated transcription in vivo.

Dopaminergic and ligand-independent activation of steroid hormone receptors.

The current view of how steroid hormone receptors affect gene transcription is that these receptors, on binding ligand, change to a state in which they can interact with chromatin and regulate transcription of target genes. Receptor activation is believed to be dependent only on this ligand-binding event. Selected steroid hormone receptors can be activated in a ligand-independent manner by a membrane receptor agonist, the neurotransmitter dopamine. In vitro, dopamine faithfully mimicked the effect of progesterone by causing a translocation of chicken progesterone receptor (cPR) from cytoplasm to nucleus. Dual activation by progesterone and dopamine was dissociable, and a serine residue in the cPR was identified that is not necessary for progesterone-dependent activation of cPR, but is essential for dopamine activation of this receptor.