Zugl.: Giessen, Univ., Diss., 2007

Homing of hematopoietic stem cells (HSC) to their bone marrow (BM) niches is crucial to clinical stem cell transplantation. However, the molecular mechanism controlling this process remains not fully understood. In this study, we aimed to explore novel regulators of HSC homing through investigating downstream signals and effector molecules of the stromal cell-derived factor-1 (SDF-1)/CXCR4 axis. We further characterized specific functions of targeted regulators by in vitro and in vivo migration/homing assays on human cord blood (CB) CD34+ hematopoietic stem/progenitor cells. / In summary, we have provided the first transcriptome profile of CB CD34 + cells downstream of the SDF-1/CXCR4 axis. We also reported the first evidence that HSC homing was regulated by the tetraspanin CD9. By comparing the homing-related responses of CD34+ to SDF-1 and CTCE-0214, we identified RGS13 as another potential regulator of HSC homing. It is anticipated that strategies for modulating the expressions and functions of CD9 and RGS13 might improve HSC homing to their hematopoietic niches. / To investigate the transcriptional regulation provided by the SDF-1/CXCR4 axis, we performed the first differential transcriptome profiling of human CB CD34+ cells in response to a short-term exposure of SDF-1, and identified a panel of genes with putative homing functions. We demonstrated that CD9, a member of the tetraspanin family proteins, was expressed in CD34 +CD38-/lo and CD34+CD38+ cells. CD9 levels were enhanced by SDF-1, which simultaneously downregulated CXCR4 membrane expression. Using specific inhibitors and activators, we demonstrated that CD9 expressions were modulated via the CXCR4, G-protein, PKC, PLC, ERK and JAK2 signals. Pretreatment of CD34+ cells with anti-CD9 mAb ALB6 significantly inhibited SDF-1-mediated transendothelial migration and calcium mobilization, whereas adhesion to fibronectin and endothelial cells were enhanced. Infusion of CD34+ cells pretreated with ALB6 significantly impaired their homing to bone marrow and spleen of sublethally irradiated NOD/SCID mice. There also appeared a preferential homing/retaining of untreated CD34+CD9+ cells to these niches. Our results indicate that CD9, as a downstream member of SDF-1/CXCR4 signals might possess specific and important functions in HSC homing. / We first investigated the effects of SDF-1 and its analog, CTCE-0214 (a small cyclized peptide analog of the SDF-1 terminal regions), on homing-related properties (chemotaxis, transwell migration, adhesion and actin polymerization) of CB CD34+ cells. Our results demonstrated that both SDF-1 and CTCE-0214 induced a robust actin polymerization response and improved adhesion of CD34+ cells to fibronectin. Unlike SDF-1, CTCE-0214 did not induce a chemotactic response when added to the lower chamber of the transwell system. Addition of CTCE-0214 to the upper chamber significantly improved migration of CD34+ cells to a SDF-1 gradient, but there was no preferential enhancement in the migration of specific colony-forming unit (CFU) progenitors or the more primitive CD34+CD38 -/lo subpopulation. Pre-exposure of CD34+ cells to CTCE-0214 for 4 hours promoted cell migration, whereas SDF-1 pretreatment retarded migration. To dissect the molecular mechanisms leading to the observed functional differences mediated by SDF-1 and CTCE-0214, we investigated whether the two compounds differentially regulated the expression of several known regulators of HSC migration. Flow cytometric analysis revealed that the cell surface expression of CD26, CD44, CD49d, CD49e and CD164 was not changed by either compounds. Exposure to SDF-1, but not CTCE-0214, decreased membrane expression of CXCR4 on CD34+ cells. Addition of CTCE-0214 to the upper chamber inhibited the SDF-1-induced CXCR4 downregulation in both migrated and non-migrated cell population in the transwell setting. Notably, SDF-1 and CTCE-0214 had an opposite effect on the expression level of regulator of G-protein signaling 13 (RGS13), a negative regulator of chemokine-induced responses. Treatment of CD34+ with SDF-1 for 4 hours resulted in a significant increase in RGS13 expression, whereas CTCE-0214 induced a time-dependent decrease in RGS13 expression. Our results provide the first evidence that SDF-1 and CTCE-0214 differentially regulate migration of CD34 + cells, and we speculate that this might be attributed to their differential regulation of CXCR4 and RGS13 expression. / Leung, Kam Tong. / Adviser: Karen Kwai Har Li. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 146-167). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Chemokines are responsible for the directed migration of leukocyte chemotactic cytokines, coordinating cell movement during inflammation and the transport of hematopoietic cells. In addition to leukocytes, chemokine receptors are also found in neoplastic cells and tumors associated with stromal cells. Among chemokines, and the CXCR4/CXCL12 CCR7/CCL21 systems have been shown the involvement of lymph node metastases or distant metastases in different cancers. Thus, aim of this study was to evaluate the expression of CXCR4, CXCL12, CCR7, CCL21 and Ki-67 in oral squamous cell carcinoma (SCC) and to correlate these markers with clinicopathological indicators, lymph node metastasis and survival. We conducted a survey of reports and paraffin blocks of excisional biopsies of patients with SCC treated at the Hospital Haroldo JuaÃaba (2001-2009). Data on anatomic location of the lesion, sex, age, patient survival, degree of histological differentiation of the tumor, tumor stage and presence or absence of lymph node metastasis, lymphovascular and perineural invasion, nuclear grade and depth of invasion were collected. For immunohistochemical analysis, followed by the technique of streptavidin-biotin-peroxidase using the anti-CXCR4, anti-CXCL12, anti-CCR7, anti-CCL21 and Ki-67 antibody. Histological sections were photomicrographed in 10 fields chosen randomly and measured for the number of labeled tumor cells and determined the percentage of each labeling antibody. The marking of CXCR4 was detected in the cytoplasm and nucleus, CXCL12, CCR7 and CCL21 were only cytoplasmic, their expression was observed in 18 (60%) 8 (22.66%) 16 (53.3%) and 3 (12%) cases, respectively. We found a significant positive association between lymphovascular invasion and immunostaining of CXCR4 (p = 0.007) and CCR7 (P = 0.01) and among these cases metastasis was present in 62.5% and 37.5%, respectively. When in combination with Ki67, we found a significant positive correlation between CXCR4 (p = 0.0086), CXCL12 (p = 0.036) and CCR7 (p = 0:04). Among patients CXCR4 + over 111 months, only 38.4% were alive (p = 0.845), whereas both patients CCR7 + (p = 0.398) as well as CXCR4 +, and CCR7 + (p = 0.441) after 62 months, everyone had already died. We conclude that these chemokines are associated with lymphovascular invasion and cell proliferation, perhaps favoring the development of metastasis and poor prognosis. / As quimiocinas sÃo citocinas quimiotÃticas responsÃveis pela migraÃÃo direcionada de leucÃcitos, coordenando o movimento celular durante a inflamaÃÃo e o transporte de cÃlulas hematopoiÃticas. AlÃm dos leucÃcitos, os receptores de quimiocinas tambÃm sÃo encontrados em cÃlulas neoplÃsicas e em tumores associados com cÃlulas estromais. Dentre as quimiocinas, os sistemas CXCR4/CXCL12 e CCR7/CCL21 tÃm sido demonstrado no envolvimento de metÃstases linfonodais ou à distÃncia em diferentes tipos de cÃncer. Dessa forma, foi objetivo desse trabalho avaliar a expressÃo de CXCR4, CXCL12, CCR7, CCL21 e Ki-67 em carcinoma de cÃlulas escamosas orais (CEC) e correlacionar estes marcadores com indicadores clÃnicopatolÃgicos, metÃstase linfonodal e sobrevida. Realizou-se um levantamento de laudos e blocos parafinados de biopsias excisionais de pacientes portadores de CEC tratados no Hospital Haroldo JuaÃaba (2001 a 2009). Foram coletados dados sobre localizaÃÃo anatÃmica da lesÃo, sexo, idade, sobrevida do paciente, grau de diferenciaÃÃo histopatolÃgica do tumor, estadiamento tumoral e presenÃa ou ausÃncia de metÃstase linfonodal, invasÃo linfovascular e perineural, grau nuclear e profundidade de invasÃo. Para reaÃÃo de imunohistoquÃmica, seguiu-se a tÃcnica da estreptavidina-biotina-peroxidase, utilizando os anticorpos anti-CXCR4, anti-CXCL12, anti-CCR7, anti-CCL21 e Ki-67. As secÃÃes histolÃgicas foram fotomicrografadas em 10 campos escolhidos aleatoriamente e quantificadas quanto ao nÃmero de cÃlulas tumorais marcadas e determinado o percentual de marcaÃÃo de cada anticorpo. A marcaÃÃo de CXCR4 foi detectada em citoplasma e nÃcleo, CXCL12, CCR7 e CCL21 tiveram marcaÃÃo apenas citoplasmÃtica, sendo observada suas expressÃes em 18 (60%), 8 (22,66%), 16 (53,3%) e 3 (12%) casos, respectivamente. Encontrou-se uma associaÃÃo significativa positiva entre a invasÃo linfovascular e a imunomarcaÃÃo do CXCR4 (p=0.007) e CCR7 (p=0.01) e dentre esses casos a metÃstase esteve presente em 62,5% e 37,5%, respectivamente. Quando em associaÃÃo com o Ki67, encontrou-se uma correlaÃÃo positiva significante entre o CXCR4 (p=0.0086), CXCL12 (p=0.036) e CCR7 (p=0.04). Dentre os pacientes CXCR4+, ao longo de 111 meses, apenas 38,4% estavam vivos (p=0.845), ao passo que tanto para pacientes CCR7+ (p = 0.398), quanto CXCR4+ e CCR7+ (p = 0.441), apÃs 62 meses, todos haviam ido a Ãbito. Conclui-se que essas quimiocinas estÃo associadas com a invasÃo linfovascular e proliferaÃÃo celular, talvez favorecendo o desenvolvimento de metÃstases e um pior prognÃstico.