The Function Of Rhob In CML Cells And Primitive Hematopoietic Cells

Part â…  Suppressive effect of RhoB on CML cells proliferationObjective：To study the effect of RhoB on the proliferation of chronic myeloidleukemia （CML） cells.Methods:（1） Wild type RhoB and its lipid modification variants were amplifiedwith PCR;（2） Lentiviral vectors were constructed to overexpress RhoB and its variantsin CML cells;（3） Effect on the growth of CML cell and the alteration of cell cycle wereanalyzed by FACS.Results:（1） Overexpression of RhoB in MEG-01and CD34+cells from CMLpatients reduced their proliferation.（2） The lipid modification deficient variantRhoBC193G could not inhibit the growth of CML cells.（3） Both farnesylated （F） andgeranylgeranylated （GG） RhoB （RhoB-F and RhoB-GG） decreased the proliferationability of CML cells.（4） RhoB, RhoB-F and RhoB-GG transduced CML cells residedmore at G2/M phase compared with control or RhoBC193G transducted cells.Conclusion: RhoB required lipid modification to suppress the growth of CML cells,which was correlated with the arrest of cells at G₂/M. Part â…¡Forced expression of dominant negative form of RhoBimpaired long-term engraftment of primitive hematopoietic cellsObjective：To study the effect of RhoB on the proliferation and engraftment ofmouse primitive hematopoietic cells.Methods:（1）The dominant-negative form of Rhob （RhobT19N）was constructed bysite-directed mutation.（2） RhobT19N was transduced into mouse primitive hematopoietic cells by lentiviruses and the proliferation of these cells was analyzed bycolony-forming cell assay.（3）Effect of RhoBT19N on the engraftment of mouseprimitive hematopoietic cells was detected by using transplantation model in vivo.（4）The expression of c-Kit on RhobT19N transduced primitive hematopoietic cells wasanalyzed by using FACS.（5）Effect of RhoBT19N on migration and adhesion ofRhobT19N transduced primitive hematopoietic cells was assessed by using transwellprogenitor migration assay and α4β1and α5β1integrin-mediated adhesion assay,respectively.Results:（1） Forced expression of RhobT19N reduced the colony formationabilities of primitive hematopoietic cells in vitro.（2） Forced expression of DN RhoBreduced c-Kit expression.（3） Forced expression of RhobT19N impairs both short-termand long-term engraftment of hematopoietic stem/progenitor cells.（4） Dominantnegative form of RhoB enhanced migration and reduced adhesion of primitivehematopoietic cells.Conclusion: RhoB activity is essential for the proliferation and engraftment ofmouse primitive hematopoietic cells.