Active RHOA favors retention of human hematopoietic stem/progenitor cells in their niche
© Jaganathan et al.; licensee BioMed Central Ltd. 2013
Received: 17 August 2013
Accepted: 10 September 2013
Published: 11 September 2013
Hematopoietic stem/progenitor cells (HSPCs) maintain the hematopoietic system by balancing their self-renewal and differentiation events. Hematopoietic stem cells also migrate to various sites and interact with their specific microenvironment to maintain the integrity of the system. Rho GTPases have been found to control the migration of hematopoietic cells and other cell types. Although the role of RAC1, RAC2 and CDC42 has been studied, the role of RHOA in human hematopoietic stem cells is unclear.
By utilizing constitutively active and dominant negative RHOA, we show that RHOA negatively regulates both in vitro and in vivo migration and dominant negative RHOA significantly increased the migration potential of human HSC/HPCs. Active RHOA expression favors the retention of hematopoietic stem/progenitor cells in the niche rather than migration and was found to lock the cells in the G0 cell cycle phase thereby affecting their long-term self-renewal potential.
The current study demonstrates that down-regulation of RHOA might be used to facilitate the migration and homing of hematopoietic stem cells without affecting their long-term repopulating ability. This might be of interest especially for increasing the homing of ex vivo expanded HSPC.
Hematopoietic stem cells maintain the hematopoietic system through their proliferation, differentiation and migration from their niche. These processes are regulated at several levels and dysregulation was found to lead to pathological conditions. Of many factors that were found to control the migration, proliferation and the self-renewal capacity of stem cells, Rho GTPases also play an essential role. Rho GTPases belong to the Ras superfamily of GTPases. They are involved in the migration of different cell types, tumour cells and also in the differentiation of mesenchymal stromal cells [1–7]. Rho GTPases act as molecular switches that cycle between active GTP-bound state and inactive GDP-bound form. This conversion is tightly regulated by Guanine-nucleotide Exchange factor (GEFs) and GTPase-activating proteins (GAPs) [8, 9]. Dysregulation of Rho GTPases was associated with neutrophil dysfunction, leukemia, and Fanconi anemia [10–12].
Enhanced activity of Rho GTPases was found to be associated with diverse hematologic abnormalities and malignancies [13–16]. Rac and Cdc42 have been found to be up-regulated in human AML [17, 18]. Rac1, one of the members of the RhoGTPase family was shown to regulate the homing of hematopoietic stem cells through CXCR4/SDF1 signalling . Rac1 was also found to be important for homing and engraftment of HSC into the BM niche and deficiency of Rac1 resulted in low engraftment rates . Hematopoiesis was also found to be affected in Rac1 deficient embryo which is correlated with the impaired response to CXCL12 stimulation . Hematopoietic specific Rac2 was found to be important for HSPC migration and adhesion and lack of Rac2 resulted in increased number of circulating HSPC. This suggests that Rac2 is essential for stem cell adhesion to its niche . Furthermore, Rac2 was found to control these activities via the modulation of Rac1 and Cdc42 . In addition, Rac2 mutation in hematopoietic cells resulted in severe neutrophilia [23, 24] and decreased Cdc42 resulted in defective homing in Fanconi anemia . Cdc42 was also found to regulate HSC migration and their retention in the hematopoietic stem cell niche. Mice lacking Cdc42 showed increased number of circulating HSC, rapidly cycling cells with deficiency in directional migration and adhesion . Cdc42 activity also drastically affected the actin organization and cell adhesion. The increase in cycling of the cells was found to be mediated by p21CIP1 and c-Myc .
Although the role of Rac1, Rac2 and Cdc42 in hematopoiesis is well studied, function of RhoA is still not clearly understood. Our current study and other reports indicate that RhoA function seems to be cell type specific and species specific. In mice, inhibition of RhoA by dominant negative RhoAN19 reduced the migration of HSPCs but increased their engraftment . However, the role of RhoA on human hematopoietic stem cell function, migration, and engraftment is still unclear.
Our studies in human cells show that RHOA inhibition increased the chemotactic migration of HSPCs without affecting their engraftment potential. We report here for the first time that RHOA expression favors retention of HSPCs in the niche rather than inducing their migration. Furthermore, inhibition of RHOA significantly increased the in vivo BM migration and homing of human HSPCs in mice without affecting their engraftment levels.
Isolation of lineage depleted cord blood mononuclear cells (HSPCs)
Umbilical cord blood was obtained after informed consent from the Royal London Hospital, London, UK in accordance with the local Research Ethics Committees guidelines. The mononuclear cells were separated by density gradient centrifugation and enriched for progenitor cells using human progenitor enrichment cocktail and Stem Sep column (Stem Cell Technologies, Vancouver, Canada). The resulting lineage depleted mononuclear cells (CBLin-) termed as Hematopoietic stem/Progenitor cells (HSPCs) were used for further experiments.
Quantitative real-time PCR was performed on sub-fractions of the hematopoietic cells to quantify the transcript level of Rho GTPases RHOA. Total cellular RNA was extracted using RNeasy kit (Qiagen, Crawley, UK) and reverse transcribed into cDNA with Superscript III reverse transcriptase (Invitrogen). Real-time PCR was performed with SYBR-Green (ABI Biosystems, Carlsbad, USA) in an ABI 7900HT (ABI Biosystems) real-time PCR machine. The specificity of the product was verified in a 2% agarose gel. The primers used were: RHOA forward 5′-CTGGTGATTGTTGGTGATGG-3′ and RHOA reverse 5′-GCGATCATAATCTTCCTGCC-3′ and GAPDH forward 5′- GGGAAGGTGAAGGTCGGAGT-3′ and GAPDH reverse 5′- GGGTCATTGATGGCAACAATA-3′.
The lentiviral vectors used for the study were based on pHRcPPT SIEW Sin vector with IRES regulating eGFP reporter gene. The vector contains SFFV (Spleen Focus Forming Virus)-LTR promoter and WPRE (Woodchuck Hepatitis Virus) element for post-transcriptional processing. RHOA constitutively active (RHOAV14) and dominant negative (RHOAN19) sequences were cloned from pBluescript vectors (kindly provided by Dr. Michael Way, Cancer Research UK, London, UK) by PCR using the primers F: 5′-GCGCGGATCCATGGCTGCCATCCGGAA-3′; R: 5′-GCGCGGATCCTCACAAGACAAGGCAAC-3′. The sequences were cloned into Topo Cloning vector (Invitrogen, Paisley, UK) and subcloned subsequently into SIEW by BamHI digestion. The orientation and the presence of mutation were confirmed by DNA sequencing. Lentiviral vector with only IRES GFP was used as experimental control.
Lentiviral production and concentration
Lentiviral particles were generated by transfecting the transfer plasmid into 293 T cells with the packaging plasmids pCMVR8.94 and envelope pMD.G as described previously . Viral supernatants were collected 48 and 72 hr after transfection and concentrated by ultracentrifugation.
Lentiviral transduction of lineage depleted cord blood mononuclear cells
Freshly isolated or frozen lineage depleted mononuclear cells were stimulated for 8 hr with cytokines hFlt3L (50 ng/ml), hSCF (50 ng/ml), hIL-6 (10 ng/ml) and hTPO (20 ng/ml). After stimulation, transduction of HSPC cells were performed by the addition of the lentivirus particles containing control, RHOAV14 and RHOAN19 at a multiplicity of infection (M.O.I) of 80 in the presence of polybrene (4 μg/ml). Sixteen hours after transduction, the cells were washed and used for further experiments.
Liquid culture, LTC-IC and CFU-C assay
CFU-C assay was performed for cells transduced with RHOA constructs in methylcellulose medium (Methocult H4434, Stem Cell Tech, Vancouver, Canada). Briefly, 1 × 103 cells were seeded in 35 mm culture dishes and incubated at 37°C, 5% CO2. GFP positive cell aggregates of more than 50 cells were counted as colonies at 14 days in an inverted fluorescent microscope (Leica, Switzerland) according to the colony morphology. Long-term culture-initiating cell assay (LTC-IC) was performed by plating 1 × 104 transduced cells on a monolayer of irradiated M2-10B4 cells and half-media replaced every 7 days. At the end of 5 weeks, the cells were collected, plated in methylcellulose medium for CFU-C assay and scored after 14 days. Liquid culture was performed to maintain the cells in progenitor stage by seeding the cells in serum free medium (Stem Cell Tech) containing hSCF (300 ng/ml), hFlt3L (300 ng/ml) and hTPO (20 ng/ml)and fresh media was added every 2–3 days.
In vitro transwell migration assay
Cord blood lineage depleted cells (HSPCs) were transduced with control, constitutively active RHOA (RHOAV14) or dominant negative RHOA (RHOAN19) and cultured in serum free medium supplemented with hSCF (300 ng/ml), hFLT3L (300 ng/ml) and hTPO (20 ng/ml) for 7 days. Cytokines were added every 2–3 days. 100,000 cells were seeded in the transwell chambers (5 μm pore size) coated with fibronectin. SDF1α was added to the lower well (125 ng/ml) and the cells were allowed to migrate for 4 hours. The migrated cells in the lower well was collected and enumerated by flow cytometry (LSR II, Becton Dickinson) with the counting beads (Molecular Probes).
Phenotype and cell cycle analysis
The transduced cells were identified by their expression of the reporter gene eGFP. Cell surface markers expression was determined by staining the cells with fluorescent conjugated antibodies and analyzed by flow cytometry. Cell cycle analysis was performed by fixing the cells with 2% paraformaldehyde and permeabilised with 0.1% Triton X-100. The cells were stained with anti-Ki67 conjugated with Alexa647 (Molecular Probes) and resuspended in 2% FCS containing DAPI (4′, 6-diamidino-2-phenylindole) and analyzed by flow cytometry.
Short-term homing experiment
RHOA transduced HSPC cells were expanded in serum free media containing stem cell factor (SCF, 300 ng/ml), FMS likle tyrose kinase ligand (FLT3L, 300 ng/ml) and thrombopoietin (TPO, 20 ng/ml) for one week with addition of growth factors every second day. The cells were washed and 0.5 × 105 transduced cells were injected intravenously in NOD/SCID/β2 mice. Bone marrow homing was analysed 24 hours post-injection by staining the bone marrow cells with antibodies against human CD45.
In vivo migration and xeno-transplantation assay
All in vivo experiments were performed in accordance with the UK Home Office regulations and Cancer Research UK guidelines. NOD/SCID/β2 microglobulin null mice were purchased from Charles River Laboratories, UK. Mice aged 8–12 weeks were irradiated sub-lethally with an irradiation dose of 375 cGy from a 137Cs source. For migration experiments, 0.1 × 105 transduced HSPCs cells were injected intra-bone into one of the hind limbs of the mice. Bones from each limb was collected separately after 7–8 weeks and analyzed for human cell engraftment by staining with antibodies against human CD45. For engraftment experiments, mice were intravenously injected with 0.5 – 1 × 105 transduced HSPCs. The animals were sacrificed 12 weeks after transplantation, femurs and tibiae were collected and the cells were flushed and analyzed for human engraftment by flow cytometry.
Statistical analysis was performed using One-way ANOVA test (SPSS software) and statistical significance in in vivo experiments were assessed using generalized linear models based on the negative binomial distribution (R software).
Reduced RHOA levels in HSPC population
Dominant negative RHOA increases HSPC migration
To analyze the effect of RHOA on hematopoietic cells, HSPCs were lentivirally transduced with constitutively active RHOA (RHOAV14), dominant negative RHOA (RHOAN19) and the control vector. The lentivirus used has an SFFV promoter expressing EGFP driven by IRES (Figure 1B). RHOA genes were cloned between the SFFV promoter and the IRES sequence. We found that RHOAV14 expression increased the RHOA active level whereas RHOAN19 expression effectively reduced the active RHOA level in the transduced cells.
Active RHOA favors retention in niche
Active RHOA decreases HSPC proliferation
Taken together, these findings suggest that RHOA negatively regulates in vitro chemotactic and in vivo migration of human HSPCs and RHOA seems to favor the retention of HSPC in the niche rather than their migration. Active RHOA significantly reduced the proliferation of the HSPC in vivo.
Hematopoietic stem cell proliferation, self-renewal, differentiation and migration are controlled by various factors and stimuli. HSC migration to the bone marrow and its differentiation is essential to improve the HSC engraftment during transplantation therapy. Earlier studies have found that Rho GTPases, Rac 1, Rac 2, and Cdc42 control various aspects of migration, differentiation and self-renewal capabilities of murine HSPC but the role of RHOA in human hematopoiesis has not been investigated [19, 21, 22]. To understand the role of RHOA in human HSPCs, we modulated the active RHOA level in HSPCs through constitutively active and dominant negative forms. Transcript analysis of endogenous RHOA expression in the stem and progenitor cell population showed that RHOA was expressed in both stem enriched (CD34+CD38-) and progenitor enriched (CD34+CD38+) populations. In the present study, we show that decreased RHOA activity through dominant negative RHOA expression resulted in significantly increased in vitro migration through SDF1α stimuli and in in vivo migration and homing. We found a 2-fold increase in the migration of HSPC from the injected bone to the non-injected bone in xeno-transplantation assay. As previously reported in human HSPCs by Bug et al. and in MSC by Jaganathan et al., our present study suggests that decreased RHOA activity is essential for migration in human HSPCs both in vitro and in vivo. Our observations are in contrast with the results reported recently, where RhoAN19 expression resulted in the decrease of SDF1α induced migration in murine HSPCs . In addition, the increase in RHOA level through RHOAV14 expression resulted in an increase in the retention of HSPCs in BM niche and a decreased migration to the contra-lateral non-injected bone in an in vivo migration assay although no difference in the engraftment percentage was seen. Our data goes along with an earlier report suggesting that migration and engraftment of HSPCs occurs as separate events in vivo and thus might not always correlate together as suggested .
The cell cycle control by Rho GTPases has been documented well in hematopoietic progenitor cells, cancer cells and fibroblasts [27, 31–34]. In murine hematopoietic cells, RhoAN19 expression resulted in decreased levels of P21Kip1/Waf1 and a higher number of cells in S phase . Although we did not find any changes in the cell cycle profile when RHOAN19 was expressed in human HSPCs under cytokine induction, there was a significant increase in the number of colonies obtained in short-term and long-term colony forming assays, which shows that RHOA controls the proliferation of these cells. In addition, RHOAV14 expression in human HSPCs locked the cells in G0 phase of the cell cycle even under cytokine stimulation and reduced their long-term but not short-term colony forming ability or in vivo repopulating ability. Nevertheless, despite the non-significant effect on the 7 to 10 weeks long-term repopulation in vivo, we observed a significant decrease in the percentage of CD34+cells in the mice transplanted with RHOAV14, providing some evidence that RHOA negatively regulates the HSPC self-renewal but does not inhibit the progenitor cell proliferation. Studies using secondary transplantation should be performed to further confirm these data.
In conclusion, we show that an active form of RHOA in human HSPCs reduces the in vitro and in vivo migration, bone marrow homing and lock the cells in G0 stage of the cell cycle, favoring the retention of the cells in the niche. In contrast RHOAN19 expression resulted in an increase in migration and in the short-term and long-term colony forming abilities without altering the cell cycle or the repopulating ability of these cells. Thus, down-regulation of RHOA might be used to facilitate the migration, homing of hematopoietic stem cells without affecting their long-term repopulating ability. This might be of interest especially for increasing the homing of ex vivo expanded HSPC.
Thanks to Oscar Quintana Bustamante for help with in vivo experiments. BGJ is currently affiliated with Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati, Assam, India. This work was supported by Cancer Research UK (DB) and partially supported by Department of Biotechnology (DBT), India (BGJ).
- Banyard J, Anand-Apte B, Zetter BR SM: Motility and invasion are differentially modulated by Rho family GTPases. Oncogene. 2000, 19 (4): 580-591. 10.1038/sj.onc.1203338.View ArticlePubMedGoogle Scholar
- Bug G, Rossmanith T, Henschler R, Kunz-Schughart LA, Schroder B, Kampfmann M, Kreutz M, Hoelzer D, Ottmann OG: Rho family small GTPases control migration of hematopoietic progenitor cells into multicellular spheroids of bone marrow stroma cells. J Leukoc Biol. 2002, 72 (4): 837-845.PubMedGoogle Scholar
- Jaganathan BG, Ruester B, Dressel L, Stein S, Grez M, Seifried E, Henschler R: Rho inhibition induces migration of mesenchymal stromal cells. Stem Cells. 2007, 25 (8): 1966-1974. 10.1634/stemcells.2007-0167.View ArticlePubMedGoogle Scholar
- Millan J, Williams L, Ridley AJ: An in vitro model to study the role of endothelial rho GTPases during leukocyte transendothelial migration. Methods Enzymol. 2006, 406: 643-655.View ArticlePubMedGoogle Scholar
- Parri M, Chiarugi P: Rac and Rho GTPases in cancer cell motility control. Cell Commun Signal. 2010, 8: 23-10.1186/1478-811X-8-23.PubMed CentralView ArticlePubMedGoogle Scholar
- Sordella R, Jiang W, Chen GC, Curto M, Settleman J: Modulation of Rho GTPase signaling regulates a switch between adipogenesis and myogenesis. Cell. 2003, 113 (2): 147-158. 10.1016/S0092-8674(03)00271-X.View ArticlePubMedGoogle Scholar
- Xu Y, Wagner DR, Bekerman E, Chiou M, James AW, Carter D, Longaker MT: Connective tissue growth factor in regulation of RhoA mediated cytoskeletal tension associated osteogenesis of mouse adipose-derived stromal cells. PLoS One. 2010, 5 (6): e11279-10.1371/journal.pone.0011279.PubMed CentralView ArticlePubMedGoogle Scholar
- Bishop AL, Hall A: Rho GTPases and their effector proteins. Biochem J. 2000, 348 (Pt 2): 241-255.PubMed CentralView ArticlePubMedGoogle Scholar
- Hall A, Nobes CD: Rho GTPases: molecular switches that control the organization and dynamics of the actin cytoskeleton. Philos Trans R Soc Lond B Biol Sci. 2000, 355 (1399): 965-970. 10.1098/rstb.2000.0632.PubMed CentralView ArticlePubMedGoogle Scholar
- Barabe F, Kennedy JA, Hope KJ, Dick JE: Modeling the initiation and progression of human acute leukemia in mice. Science. 2007, 316 (5824): 600-604. 10.1126/science.1139851.View ArticlePubMedGoogle Scholar
- Kasper B, Tidow N, Grothues D, Welte K: Differential expression and regulation of GTPases (RhoA and Rac2) and GDIs (LyGDI and RhoGDI) in neutrophils from patients with severe congenital neutropenia. Blood. 2000, 95 (9): 2947-2953.PubMedGoogle Scholar
- Mulloy JC, Cancelas JA, Filippi MD, Kalfa TA, Guo F, Zheng Y: Rho GTPases in hematopoiesis and hemopathies. Blood. 2009, 115 (5): 936-947.View ArticlePubMedGoogle Scholar
- Gu Y, Jia B, Yang FC, D’Souza M, Harris CE, Derrow CW, Zheng Y, Williams DA: Biochemical and biological characterization of a human Rac2 GTPase mutant associated with phagocytic immunodeficiency. J Biol Chem. 2001, 276 (19): 15929-15938. 10.1074/jbc.M010445200.View ArticlePubMedGoogle Scholar
- Pasqualucci L, Neumeister P, Goossens T, Nanjangud G, Chaganti RS, Kuppers R, Dalla-Favera R: Hypermutation of multiple proto-oncogenes in B-cell diffuse large-cell lymphomas. Nature. 2001, 412 (6844): 341-346. 10.1038/35085588.View ArticlePubMedGoogle Scholar
- Reuther GW, Lambert QT, Booden MA, Wennerberg K, Becknell B, Marcucci G, Sondek J, Caligiuri MA, Der CJ: Leukemia-associated Rho guanine nucleotide exchange factor, a Dbl family protein found mutated in leukemia, causes transformation by activation of RhoA. J Biol Chem. 2001, 276 (29): 27145-27151. 10.1074/jbc.M103565200.View ArticlePubMedGoogle Scholar
- Sanchez-Aguilera A, Rattmann I, Drew DZ, Muller LU, Summey V, Lucas DM, Byrd JC, Croce CM, Gu Y, Cancelas JA, Johnston P, Moritz T, Williams DA: Involvement of RhoH GTPase in the development of B-cell chronic lymphocytic leukemia. Leukemia. 2009, 24 (1): 97-104.View ArticlePubMedGoogle Scholar
- Bojesen SE, Ammerpohl O, Weinhausl A, Haas OA, Mettal H, Bohle RM, Borkhardt A, Fuchs U: Characterisation of the GRAF gene promoter and its methylation in patients with acute myeloid leukaemia and myelodysplastic syndrome. Br J Cancer. 2006, 94 (2): 323-332. 10.1038/sj.bjc.6602939.PubMed CentralView ArticlePubMedGoogle Scholar
- Somervaille TC, Cleary ML: Identification and characterization of leukemia stem cells in murine MLL-AF9 acute myeloid leukemia. Cancer Cell. 2006, 10 (4): 257-268. 10.1016/j.ccr.2006.08.020.View ArticlePubMedGoogle Scholar
- Gu Y, Filippi MD, Cancelas JA, Siefring JE, Williams EP, Jasti AC, Harris CE, Lee AW, Prabhakar R, Atkinson SJ, Kwiatkowski DJ, Williams DA: Hematopoietic cell regulation by Rac1 and Rac2 guanosine triphosphatases. Science. 2003, 302 (5644): 445-449. 10.1126/science.1088485.View ArticlePubMedGoogle Scholar
- Ghiaur G, Ferkowicz MJ, Milsom MD, Bailey J, Witte D, Cancelas JA, Yoder MC, Williams DA: Rac1 is essential for intraembryonic hematopoiesis and for the initial seeding of fetal liver with definitive hematopoietic progenitor cells. Blood. 2008, 111 (7): 3313-3321. 10.1182/blood-2007-08-110114.PubMed CentralView ArticlePubMedGoogle Scholar
- Yang FC, Atkinson SJ, Gu Y, Borneo JB, Roberts AW, Zheng Y, Pennington J, Williams DA: Rac and Cdc42 GTPases control hematopoietic stem cell shape, adhesion, migration, and mobilization. Proc Natl Acad Sci USA. 2001, 98 (10): 5614-5618. 10.1073/pnas.101546898.PubMed CentralView ArticlePubMedGoogle Scholar
- Jansen M, Yang FC, Cancelas JA, Bailey JR, Williams DA: Rac2-deficient hematopoietic stem cells show defective interaction with the hematopoietic microenvironment and long-term engraftment failure. Stem Cells. 2005, 23 (3): 335-346. 10.1634/stemcells.2004-0216.View ArticlePubMedGoogle Scholar
- Ambruso DR, Knall C, Abell AN, Panepinto J, Kurkchubasche A, Thurman G, Gonzalez-Aller C, Hiester A, deBoer M, Harbeck RJ, Oyer R, Johnson GL Roos D: Human neutrophil immunodeficiency syndrome is associated with an inhibitory Rac2 mutation. Proc Natl Acad Sci USA. 2000, 97 (9): 4654-4659. 10.1073/pnas.080074897.PubMed CentralView ArticlePubMedGoogle Scholar
- Kurkchubasche AG, Panepinto JA, Tracy TF, Thurman GW, Ambruso DR: Clinical features of a human Rac2 mutation: a complex neutrophil dysfunction disease. J Pediatr. 2001, 139 (1): 141-147. 10.1067/mpd.2001.114718.View ArticlePubMedGoogle Scholar
- Zhang X, Shang X, Guo F, Murphy K, Kirby M, Kelly P, Reeves L, Smith FO, Williams DA, Zheng Y, Pang Q: Defective homing is associated with altered Cdc42 activity in cells from patients with Fanconi anemia group A. Blood. 2008, 112 (5): 1683-1686. 10.1182/blood-2008-03-147090.PubMed CentralView ArticlePubMedGoogle Scholar
- Yang L, Wang L, Geiger H, Cancelas JA, Mo J, Zheng Y: Rho GTPase Cdc42 coordinates hematopoietic stem cell quiescence and niche interaction in the bone marrow. Proc Natl Acad Sci USA. 2007, 104 (12): 5091-5096. 10.1073/pnas.0610819104.PubMed CentralView ArticlePubMedGoogle Scholar
- Ghiaur G, Lee A, Bailey J, Cancelas JA, Zheng Y, Williams DA: Inhibition of RhoA GTPase activity enhances hematopoietic stem and progenitor cell proliferation and engraftment. Blood. 2006, 108 (6): 2087-2094. 10.1182/blood-2006-02-001560.View ArticlePubMedGoogle Scholar
- Suwa H, Ohshio G, Imamura T, Watanabe G, Arii S, Imamura M, Narumiya S, Hiai H, Fukumoto M: Overexpression of the rhoC gene correlates with progression of ductal adenocarcinoma of the pancreas. Br J Cancer. 1998, 77 (1): 147-152. 10.1038/bjc.1998.23.PubMed CentralView ArticlePubMedGoogle Scholar
- Cancelas JA, Lee AW, Prabhakar R, Stringer KF, Zheng Y, Williams DA: Rac GTPases differentially integrate signals regulating hematopoietic stem cell localization. Nat Med. 2005, 11 (8): 886-891. 10.1038/nm1274.View ArticlePubMedGoogle Scholar
- Gazitt Y: Homing and mobilization of hematopoietic stem cells and hematopoietic cancer cells are mirror image processes, utilizing similar signaling pathways and occurring concurrently: circulating cancer cells constitute an ideal target for concurrent treatment with chemotherapy and antilineage-specific antibodies. Leukemia. 2004, 18 (1): 1-10. 10.1038/sj.leu.2403173.View ArticlePubMedGoogle Scholar
- Gottig S, Mobest D, Ruster B, Grace B, Winter S, Seifried E, Gille J, Wieland T, Henschler R: Role of the monomeric GTPase Rho in hematopoietic progenitor cell migration and transplantation. Eur J Immunol. 2006, 36 (1): 180-189. 10.1002/eji.200525607.View ArticlePubMedGoogle Scholar
- Olson MF, Paterson HF, Marshall CJ: Signals from Ras and Rho GTPases interact to regulate expression of p21Waf1/Cip1. Nature. 1998, 394 (6690): 295-299. 10.1038/28425.View ArticlePubMedGoogle Scholar
- Roovers K, Klein EA, Castagnino P, Assoian RK: Nuclear translocation of LIM kinase mediates Rho-Rho kinase regulation of cyclin D1 expression. Dev Cell. 2003, 5 (2): 273-284. 10.1016/S1534-5807(03)00206-5.View ArticlePubMedGoogle Scholar
- Welsh CF, Roovers K, Villanueva J, Liu Y, Schwartz MA, Assoian RK: Timing of cyclin D1 expression within G1 phase is controlled by Rho. Nat Cell Biol. 2001, 3 (11): 950-957. 10.1038/ncb1101-950.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.