The tuberous sclerosis-2 (Tsc-2) gene product, tuberin, functions as a renal tumor suppressor. Treatment of Eker (Tsc-2 EK/+) rats and primary renal epithelial cells derived from Tsc-2 EK/+ rats (QTRRE cells) with 2,3,5-tris-(glutathion-S-yl) hydroquinone (TGHQ) results in loss of heterozygosity at the Tsc-2 locus in kidney tumors and QTRRE cells. QTRRE cells are carcinogenic in athymic nude mice. Analysis of kidney tumors formed in Tsc-2 EK/+ + rats following 8-months of TGHQ treatment reveals increases in B-Raf, Raf-1, pERK, cyclin D1, p27Kip1, 4EBP1, p-4EBP1(Thr70), p-4EBP1(Ser65), and p-4EBP1(Thr37/46) protein expression. These data establish the involvement of mTOR and MAPK signaling cascades in tuberin null tumors. Similar increases in 4EBP1 and p4EBP1 are observed in renal tumor QTRRE-xenografts in nude mice. Concomitant with increases in expression of these proteins in TGHQ-induced renal tumors, similar changes are observed in QTRRE cells, which also exhibit high ERK, B-Raf and Raf-1 kinase activity; and increased expression of cyclin D1, p27, p-4EBP1 (Thr70), p-4EBP1 (Ser65), and p-4EBP1 (Thr37/46). Manipulation of the Raf/MEK/ERK kinase cascade in QTRRE cells, with kinase inhibitors and siRNA, indicates that Raf-1/MEK/ERK participates in crosstalk with 4EBP1 to regulate translation of cyclin D1.Cyclin D1 and p27 protein levels are increased in the cytoplasm in our RCC models. In normal HK-2 cells, p27 and cyclin D1 are localized to the nucleus. Due to the instability of the cyclin D1-CDK4 complex, p27 interaction is necessary for cyclin D1-CDK4 complex assembly and stabilization in the nucleus. Manipulation of p27 protein levels in QTRRE cells with phosphodiesterase inhibitors, dibutyryl cAMP, and the proteosome inhibitor MG132, all result in a parallel increase in p27 and cyclin D1. Furthermore, p27 siRNA and sorafenib treatment both cause a decrease in p27 and cyclin D1. Further manipulation of cAMP, Rap1B, and B-Raf proteins, revealed that cAMP/PKA/Rap1B/B-Raf activation and B-Raf//ERK MAPK inhibition both modulate p27 expression and compartmental localization in tuberous sclerosis renal cancer. Phosphodiesterase inhibitors play a role in regulating the expression, degradation, and cytoplasmic localization of p27. Therefore, cytoplasmic p27-cyclin D1 mislocalization and stabilization may have an oncogenic role in the cytosol and play a crucial role in tumor formation.

Studies have shown that long-term opioid agonist (such as morphine) treatment produces antinociceptive tolerance and increased pain sensitivity (hyperalgesia and/or allodynia), limiting the clinical efficacy of morphine. Prolonged opiate administration also upregulates spinal pain neurotransmitter (such as calcitonin gene-related peptide (CGRP)) levels and enhances evoked CGRP release in the dorsal horn of rats. It was suggested that augmented spinal pain neurotransmission may contribute to paradoxical pain sensitization and antinociceptive tolerance. The cellular signal transduction pathways involved in sustained opioid mediated augmentation of spinal pain neurotransmitter are not fully clarified.Sustained morphine treatment was shown to augment the concentrations of inflammatory mediators, such as PGE2 in the spinal cord. Studies have shown that PGE2 stimulates cAMP formation and CGRP release by activation of Gs protein-coupled prostaglandin receptor types in primary sensory neurons. Interestingly, it was found earlier that sustained opioid agonist treatment leads to a Raf-1-dependent sensitization of adenylyl cyclase(s) (AC superactivation), augmenting forskolin-stimulated cAMP formation upon opioid withdrawal (cAMP overshoot). It is well demonstrated that cAMP activates cAMP-dependent protein kinase (PKA), which plays an important role in the modulation of presynaptic neurotransmitter release. Therefore, in this study, we investigate the physiological role of Raf-1 mediated AC superactivation and subsequent PKA activation in A. sustained morphine-mediated augmentation of basal or evoked pain neurotransmitter release in vitro, in cultured primary sensory neurons, and B. in vivo, in sustained morphine mediated paradoxical pain sensitization and antinociceptive tolerance in rats.Our data demonstrates that A. sustained morphine treatment augments both basal and capsaicin-evoked CGRP release from isolated primary sensory neurons in a PKA- and Raf-1- dependent manner. B. sustained morphine treatment- augments of PGE2-evoked CGRP release from these cells. C. selective knockdown of spinal PKA or Raf-1 protein levels by intrathecal PKA- or Raf-1-specific siRNA pretreatment completely attenuates sustained morphine-mediated thermal hyperalgesia, tactile allodynia and greatly reduces antinociceptive tolerance in rats.In conclusion, we suggest that Raf-1-mediated AC superactivation may have a crucial trigger role in sustained morphine-mediated compensatory adaptations in the nervous system. Thus, we expect that pharmacological attenuation of Raf-1-mediated AC superactivation may improve the clinical treatment of chronic and neuropathic pain.

The RbµE2F transcriptional regulatory pathway plays a critical role in the cell cycle. Rb is inactivated through multiple waves of phosphorylation, mediated mainly by cyclin D and cyclin E associated kinases. Once Rb is inactivated, cells can enter Sµphase. Collectively, three Rb family members and ten E2F proteins coordinate every additional stage of the cell cycle, from quiescence to mitosis. However the RbµE2F pathway is frequently altered in cancer. Aside from cell proliferation, the RbµE2F pathway regulates other essential cellular processes including apoptosis, cell differentiation, angiogenesis and DNA damage repair pathways, but its role in invasion and cancer progression is less clear. We demonstrate here that matrix metalloproteinases genes (MMPs), which regulate the invasion, migration and collagen degradation activities of cancer cells during metastasis are transcriptionally regulated by the RbµE2F pathway. Unlike E2F target genes involved in cell proliferation, which are solely regulated by the E2F activators (E2F1µ3), additional E2F family members can regulate MMP9, MMP14, and MMP15. While we had previously shown that Rafµ1 kinase physically interacts with Rb, and that disruption of this interaction with a small molecule inhibitor of the RbµRafµ1 interaction (RRDµ251) can inhibit cell proliferation, angiogenesis, and growth of tumors in mouse models, we now show RRDµ251 inhibits the expression of MMPs and the biological functions mediated by MMPs as well—including invasion, migration, and collagen degradation. RRDµ251 also inhibits metastatic foci development in a tail vein lung colonization model in mice. These results suggest that E2F transcription factors may play a role in promoting metastasis through regulation of MMP genes. Conversely, another MMP gene connected to metastasis, MMP2, is transcriptionally repressed by E2F1 in lung cancer cells through a p53µKAP1µHDAC1µmediated mechanism. However, E2F1 cannot repress the MMP2 promoter in cells that are lacking any component of this complex, such as p53 mutant breast cancer cells. Therefore the role of the RbµE2F pathway in MMP transcription and metastasis is cell type dependent. In addition to growth factors, nicotine can also induce cell proliferation, angiogenesis, EMT, and progression of lung cancer. In our studies, nicotine induced invasion, collagen degradation, and transcription of MMP2, MMP9, MMP14, and MMP15 required nAChRs, and multiple E2F family members. Our studies also show that nicotine not only promotes tumor growth in vivo through the nAChRµE2F pathway—it also results in metastasis to the liver and brain. Taken together, these studies link the RbµE2F pathway to the regulation of many facets of cancer.

During pregnancy, 4 - 7% of women suffer from major depressive disorder. When antidepressive treatment is needed, selective serotonin reuptake inhibitors (SSRIs) are the most commonly used. Although severe complications from SSRI treatment are rare, association with a number of adverse pregnancy and fetal outcomes has been found. Also, antenatal depression per se has been shown to affect pregnancy outcomes. The overall aim of this thesis was to examine the effects of SSRIs on human placenta and embryo. In the first study, gene expression was investigated in placenta from depressed, SSRI-treated and healthy pregnant women, using microarray analysis. Antenatal depression and SSRI treatment induced alterations in gene expression, but only 20 genes in common were noted. Validation with qRT-PCR showed that six out of seven selected genes were altered in SSRI-treated women compared with controls, and two genes were altered between depressed women and controls. In study two, the protein levels in placenta from depressed, SSRI-treated and healthy pregnant women were investigated, focusing on the NGF signaling pathway. NGF, phosphorylated Raf-1, ROCK2 and phosphorylated ROCK2, were altered in both SSRI-treated and depressed women, although the proteins were regulated differently in the two groups. In the third study, human embryos were treated with fluoxetine. Embryo development and protein expression were studied. Fluoxetine had some effect on the timing of embryo developmental stages. Also, several proteins were uniquely found in fluoxetine-treated embryos compared with untreated embryos. Fluoxetine also altered the levels of proteins secreted from the embryo. In the fourth study, the human neuroblastoma cell line SH-SY5Y/TrkA was treated with TPA and NGF. The activation of Raf-1 was investigated and the involvement of Ras and PKC was studied. Both NGF and TPA activated Raf-1, but to a different extent and via different pathways. The NGF-induced activation of Raf-1 was mediated via Ras, while TPA induced signaling via PKC. In conclusion, SSRI treatment and antenatal depression influence placental gene and protein expression. These findings may affect placental development and function, which in turn could affect fetal development. Also, direct exposure of embryos to fluoxetine has some effects on embryo development and protein expression, which may affect the development of the fetus.