Role of vitamin A/retinoic acid in regulation of embryonic and adult hematopoiesis

Vitamin A is an essential micronutrient throughout life. Its physiologically active metabolite retinoic acid (RA), acting through nuclear retinoic acid receptors (RARs), is a potent regulator of patterning during embryonic development, as well as being necessary for adult tissue homeostasis. Vitamin A deficiency during pregnancy increases risk of maternal night blindness and anemia and may be a cause of congenital malformations. Childhood Vitamin A deficiency can cause xerophthalmia, lower resistance to infection and increased risk of mortality. RA signaling appears to be essential for expression of genes involved in developmental hematopoiesis, regulating the endothelial/blood cells balance in the yolk sac, promoting the hemogenic program in the aorta-gonad-mesonephros area and stimulating eryrthropoiesis in fetal liver by activating the expression of erythropoietin. In adults, RA signaling regulates differentiation of granulocytes and enhances erythropoiesis. Vitamin A may facilitate iron absorption and metabolism to prevent anemia and plays a key role in mucosal immune responses, modulating the function of regulatory T cells. Furthermore, defective RA/RARα signaling is involved in the pathogenesis of acute promyelocytic leukemia due to a failure in differentiation of promyelocytes. This review focuses on the different roles played by vitamin A/RA signaling in physiological and pathological mouse hematopoiesis duddurring both, embryonic and adult life, and the consequences of vitamin A deficiency for the blood system

Regulation of p27kip1 and p57kip2 functions by natural polyphenols

In numerous instances, the fate of a single cell not only represents its peculiar outcome but also contributes to the overall status of an organism. In turn, the cell division cycle and its control strongly influence cell destiny, playing a critical role in targeting it towards a specific phenotype. Several factors participate in the control of growth, and among them, p27Kip1 and p57Kip2, two proteins modulating various transitions of the cell cycle, appear to play key functions. In this review, the major features of p27 and p57 will be described, focusing, in particular, on their recently identified roles not directly correlated with cell cycle modulation. Then, their possible roles as molecular effectors of polyphenols’ activities will be discussed. Polyphenols represent a large family of natural bioactive molecules that have been demonstrated to exhibit promising protective activities against several human diseases. Their use has also been proposed in association with classical therapies for improving their clinical effects and for diminishing their negative side activities. The importance of p27Kip1 and p57Kip2 in polyphenols’ cellular effects will be discussed with the aim of identifying novel therapeutic strategies for the treatment of important human diseases, such as cancers, characterized by an altered control of growth

Identification of Genes Involved in Hematopoietic Stem Cell Differentiation and Leukemia Differentiation and Leukemia

Hematopoiesis is maintained by a proper balance between self renewal and multipotent differentiation of the hematopoietic stem cells (HSC). Acute myelogenous leukemia (AML) is characterized by the blockage in the differentiation of HSC, while self renewal and proliferation is preserved. It is important to understand the mechanisms involved in the inhibition of hematopoietic differentiation and maintenance of the HSC state in order to develop better therapies for AML. In these studies I have explored the role of Hsp90, omega-3 fatty acids and YB-1 in hematopoietic differentiation. EML, a hematopoietic precursor cell line, was used as a model for the hematopoietic system in these studies. My preliminary data showed the activation of Wnt signaling upon inhibition of Hsp90 in EML cells. This data suggested the involvement of Hsp90 in the regulation of Wnt signaling in EML cells. Moreover, my initial data with fatty acid studies indicated that omega-3 fatty acids could affect Wnt signaling in EML cells. Unfortunately, further progression of both these studies was marred by variability in my data. In my latest study, I have identified YB-1 as a marker involved in the maintenance of the hematopoietic stem cell state. YB-1 was found to be highly expressed in the EML cell line and in the mouse bone marrow-derived HSC and myeloid progenitor cells. In addition, YB-1 expression was downregulated during myeloid differentiation in retinoic acid (RA) and granulocyte macrophage colony stimulating factor (GM-CSF) treated EML cells, as well as in the granulocytes derived from mouse bone marrow. Further, abnormal YB-1 expression was observed in myeloid leukemic cell lines. Knockdown of YB-1 expression and arsenic trioxide treatment (As2O3) in erythroleukemic, K562 cell line resulted in apoptosis and inhibition of cell proliferation. Most importantly, these treatments led to the induction of megakaryocytic differentiation in these cells. Overall my data suggests that increased expression of YB-1 in the leukemic cells contributes to the leukemic cell properties by promoting cell proliferation, cell survival and blocking cell differentiation. Thus, YB-1 could be a potential target for therapy in myeloid leukemia

Expression and regulation of c-myb in B-lymphocyte development

B lymphocytes are continually produced in bone marrow from pluripotential hematopoietic stem cells. Lymphopoiesis is characterized by a series of highly regulated genotypic and phenotypic changes resulting in immunocompetent effector cells which express cell surface immunoglobulin. Our laboratory has focused on defining extracellular signals regulating lymphoid progenitor cell survival, proliferation, and differentiation. These studies have demonstrated that pro-B cell survival, proliferation, and differentiation are regulated by interactions with fibroblastic stromal cells in the hematopoietic microenvironment. However, specific molecular mechanisms by which stromal cells regulate B lymphoid development are largely unknown. In an attempt to better understand molecular mechanisms regulating maturation in this lineage, we developed a panel of pro-B cell clones from 14-day murine fetal liver. These pro-B cell clones remain dependent on stromal cells for survival, do not form tumors, and reconstitute B lymphocytes in severe combined immunodeficient (SCID) mice. In vitro, pro-B cell clones continuously proliferate and do not differentiate. We noted that pro B cell lines were characterized by expression of high levels of the oncogene c-myb. Although several laboratories have proposed a role for c-myb in regulation of hematopoiesis, virtually nothing is known about the function of c-myb in normal B lineage cells. To investigate the role of c-myb in the survival, proliferation, and differentiation of B lineage cells, we utilized a stromal cell dependent pro-B cell line that expresses mRNA and protein for c-myb. Experiments utilizing RT-PCR and Western blot analysis reveal that c-myb is regulated in pro-B cells by stromal cells, specifically by stromal cell adhesion contacts. Both DMSO and antisense oligonucleotides were used to downregulate c-myb protein to determine the role this intracellular regulator plays in B lymphocyte development. Our investigations revealed that downregulation of c-myb did not affect pro-B cell survival but did interrupt both pro-B cell proliferation and differentiation. In vivo investigations in mice carrying homozygous mutations of the c-myb gene indicate that lymphopoiesis is severely diminished in embryonic knockout animals. These data suggest a central role for c-myb in proliferation and differentiation of developing B lymphocytes

The expression and regulation of matrilysin (MMP-7) in human colon cancer and leukaemia cell lines

Matrilysin (MMP-7, EC 3.4.24.23) is the smallest member o f the matrix metalloproteinase (MMP) family and has been shown to be overexpressed in various tumours including breast and colon cancers. Matrilysin has also been shown to play an important role in several aspects of tumour biology including growth, progression, invasion and metastasis. With respect to colon cancer, matrilysin is unique in that it is the only MMP expressed exclusively by the malignant epithelia o f colonic adenocarcinomas. These facts combine to make matrilysin a promising therapeutic target. However, in order to develop drugs which specifically inhibit matrilysin it is important to understand how matrilysin gene expression is controlled, something which to date remains poorly understood. We have examined a panel of human colon tumour cell lines and have shown that matrilysin expression can be upregulated by a number of cytokines including EGF, IL-6 and bFGF. Analysis of the matrilysin promoter revealed the presence of a number of potential transcription factor binding sites including three ETS sites. We have shown that EGF treatment increased matrilysin gene expression by activation of PEA3 transcription factors using artificial promoter, western blot and EMSA analysis. ‘Supershift’ EMSA analysis showed that other PEA3 subfamily members such as ERM and ER81 may also be involved which is in agreement with other studies. In addition, we have found that EGF increased cellular levels o f [3-catenin through destabilisation of the E-cadherin/catenin complex which resulted in increased binding to the T c f site within the matrilysin promoter. We also examined the expression and regulation of matrilysin in the K562 and HL-60 myeloid leukaemia cell lines. Results showed that only the K562 cell line expressed matrilysin and in vitro invasion assays showed that the K562 cells were up to 4 times more invasive than the HL- 60 cell line. Matrilysin antibody blocking experiments showed a significant decrease in invasion in the K562 cell line suggesting a role for matrilysin in leukaemia invasion. The MMP and TIMP profiles o f these cell lines were also examined. Our data suggests that EGF plays an important role in the regulation of matrilysin gene expression via a number of new mechanisms. Furthermore, we have shown that matrilysin plays an important role in leukaemia cell line invasion. These findings have identified possible new drug targets that will inhibit matrilysin expression which in turn should lead to decreased tumourigenesis and invasion and metastasis

When the bear lost its tail : targeting the GRK/β-arrestins downstream IGF1R in non-epithelial malignancies

Plasma membrane receptors are highly specialized cell surface structures that receive extracellular information and process them into biological responses. Among them, this thesis focuses on two major receptor families: the G protein-coupled receptors (GPCR) and the receptor tyrosine kinases (RTKs). One of the most studied RTKs, IGF1R has been demonstrated to be essential for cancer development in a wide range of tissues. Recognition of IGF1R signaling hijacking by twisted malignant processes was rapidly shadowed by anti-cancer therapeutics developments, yet with disappointing results in clinical setting. Failures in past anti-IGF1R strategies required bedside to bench shift and re-evaluation of the mechanism controlling IGF1R tumorigenesis. In particular its non-canonical, kinase-independent signaling capabilities, through use of the GRKs/β-arrestins system - prominent controller of GPCR-signaling-, is today acknowledged to orchestrate IGF1R oncogenic power. This thesis aims to explore the GRK/β-arrestin system downstream IGF1R and uncover its targeting potential as a cancer therapeutic strategy in non-epithelial cancers. Study I describes the functional roles of GRK isoform modulation in IGF1R downregulation to develop anti-IGF1R targeting strategies via inhibition of GRK2. Our results establish the potential for clinical applicability of cross-targeting the IGF1R through pharmacological inhibition of GRK2 in Ewing sarcoma by using paroxetine, a commonly prescribed antidepressant. In Study II, we investigate the therapeutic potential of p53 activation through targeting MDM2 in conjunctival melanoma. The use of Nutlin3 to reactivate p53 via inhibition of MDM2 proved more effective than siRNA inhibition of MDM2. This suggests that the additional effect of Nutlin3 on IGF1R degradation is highly beneficial in cancer targeting. This study reveals double-hit IGF1R/p53 targeting strategy as a potent therapy for recurring and metastatic conjunctival melanoma. In study III, we investigate the disruption of the p53/MDM2/IGF1R axis via unbalancing the β-arrestin system to improve treatment response to chemotherapy in malignant melanoma. This study demonstrates novel dual therapeutic strategy in which inhibition of β-arrestin1 signaling or β-arrestin2 hyperactivation can enhance response to chemotherapy. Considering the significance of IGF1R and downstream biased signaling in blood cancer, study IV investigates the individual effect of kinase signaling versus GRK/β-arrestin signaling downstream the receptor in leukemia. Our results uncover both arms of IGF1R signaling as targets for cell proliferation and survival (kinase) and cell differentiation (GRK/β-arrestin) in acute myeloid leukemia. This work establishes the potential of targeting IGF1R kinase and/or C-terminus to induce peripheral differentiation. In summary, the findings described in the present thesis provide new insights for the therapeutic potential of non-canonical IGF1R signaling

The in vitro and in vivo effects of human growth hormone administration on tumor growth of rats bearing a transplantable rat pituitary tumor (7315b)

Abstract The direct effects of human GH and IGF-I on PRL secretion and cell proliferation were studied on PRL secreting rat pituitary tumor 7315b cells in vitro, as well as the effects in vivo of human GH administration on body weight, IGF-I levels and tumor size in rats bearing this transplantable tumor. In the in vitro studies IGF-I levels above 5 nM stimulated PRL release in a dose-dependent manner while GH, in concentrations of 0.23–45 nM, did not affect PRL release. Cell proliferation was stimulated by IGF-I in a dose-dependent manner from 0.5 nM onwards, while GH did not have an effect. The in vivo studies showed that 1 mg GH/rat/day prevented tumor-induced cachexia and normalized the suppressed IGF-I levels without stimulating tumor growth. It is concluded that tumor-induced cachexia can be prevented by exogenous GH administration without an increase in tumor mass, even if a tumor model is used whose cultured tumor cells respond to exposure to IGF-I with a mitotic response

Vitamin D in cancer chemoprevention

Context: There is increasing evidence that Vitamin D (Vit D) and its metabolites, besides their well-known calcium-related functions, may also exert antiproliferative, pro-differentiating, and immune modulatory effects on tumor cells in vitro and may also delay tumor growth in vivo. Objective: The aim of this review is to provide fresh insight into the most recent advances on the role of Vit D and its analogues as chemopreventive drugs in cancer therapy. Methods: A systematic review of experimental and clinical studies on Vit D and cancer was undertaken by using the major electronic health database including ISI Web of Science, Medline, PubMed, Scopus and Google Scholar. Results and conclusion: Experimental and clinical observations suggest that Vit D and its analogues may be effective in preventing the malignant transformation and/or the progression of various types of human tumors including breast cancer, prostate cancer, colorectal cancer, and some hematological malignances. These findings suggest the possibility of the clinical use of these molecules as novel potential chemopreventive and anticancer agent

REGULATION OF MACROAUTOPHAGY BY VITAMIN A/ RETINOIDS

Retinoic acids (RAs) have diverse biologic effects and regulate several cellular functions. Here, we investigated the role of RA on autophagy by studying its effects on autophagosome (AUT) maturation, as well as on upstream regulators of autophagosome biogenesis. Our studies, based on the use of pH-sensitive fluorescent reporter markers, suggest that RA promotes AUT acidification and maturation. By using competitive inhibitors and specific agonists, we demonstrated that this effect is not mediated by the classic Retinoic Acid Receptor (RAR) and Retinoid X Receptors (RXR). RA did not affect the protein expression levels of upstream regulators of autophagy, such as Beclin-1, phospho-mTOR, and phospho-Akt1, but induced redistribution of both endogenous cation-independent mannose-6-phosphate receptor CI-MPR and transiently transfected GFP and RFP full-length CI-MPR fusion proteins from the trans-Golgi region to acidified AUT structures. Those structures were found to be amphisomes (acidified AUTs) and not autophagolysosomes. The critical role of CI-MPR in AUT maturation was further demonstrated by siRNA-mediated silencing of endogenous CI-MPR. Transient CI-MPR knockdown resulted in remarkable accumulation of nonacidified AUTs, a process that could not be reversed with RA.These results suggest that RA induces AUT acidification and maturation by regulating CI-MPR subcellular location, a process critical in the cellular autophagic mechanism