Mutated D4-guanine diphosphate-dissociation inhibitor is found in human leukemic cells and promotes leukemic cell invasion.
OBJECTIVE
Rho GTPase may be involved in human cancer invasion via the augmentation of cell motility and adhesion. We report on two point mutations of the D4-guanine diphosphate (GDP)-dissociation inhibitor (GDI) gene, one of the Rho-GDIs, which were found in a human leukemic cell line, Reh, and the mutated D4-GDI functions as an accelerator of leukemic cell invasion.


METHODS
We investigated the altered activity of GDP dissociation by mutated (mt) D4-GDI and the functions of this mt and wild-type (wt) D4-GDI in invasion. The mice inoculated with wt or mt D4-GDI vector-transfected Raji cells were observed and examined pathologically. Adhesiveness and cell motility of wt or mt D4-GDI vector-transfected Raji cells were examined. Finally, it was examined whether Rho activation was changed by mutation of D4-GDI under the condition of Rho-GDI knockdown.


RESULTS
Two point mutations of the D4-GDI gene were found in Reh cells. The region of mutations is conserved among members of the Rho-GDI family at the amino acid level. D4-GDI with two mutations (V68L and V69A) functioned in a dominant negative manner in the inhibition of GDP dissociation from Rho. Severe combined immune-deficient mice inoculated with Raji cells developed hemiparalysis. The Raji cells were present in bone marrow and peripheral blood, and hepatic invasion was observed in 20% of the mice. Mice inoculated with wt D4-GDI vector-transfected Raji cells (wt D4) showed later paralysis and none developed hepatic invasion. Mice inoculated with mt D4-GDI-transfected Raji cells (mt D4) showed a 5-day reduction in the time to paraplegia and death. In addition, hepatic invasion was evident in 80% of mice transplanted with mt D4 cells. There were no differences in growth rates and amounts of guanine triphosphate (GTP)-bound Rho, cdc42, or Rac among all clones, however, GTP-bound Rho in mt D4 clone with short hairpin RNA (shRNA) vector for Rho-GDI knockdown was increased compared with wt D4 clone with shRNA vector for Rho-GDI knockdown. The mt D4 cells showed an augmentation of adhesiveness and cell motility. On the other hand, wt D4 cells showed a decreased ability of cell motility.


CONCLUSIONS
These results suggest the mutated D4-GDI functions as a dominant negative molecule against the wt D4-GDI and accelerates invasion via regulation of cytoskeletal machinery.
Department of Pathology, Keio University School of Medicine, Tokyo, Japan.
Yuji
Y
Nakata
Kensuke
K
Kondoh
Sachiko
S
Fukushima
Akinori
A
Hashiguchi
Wenlin
W
Du
Mutsumi
M
Hayashi
Jun-ichiroh
J
Fujimoto
Jun-ichi
J
Hata
Taketo
T
Yamada
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002051:Burkitt Lymphoma / Q000473:pathology; D002448:Cell Adhesion; D045744:Cell Line, Tumor / Q000378:metabolism; D002465:Cell Movement; D002648:Child; D017124:Conserved Sequence; D005260:Female; D005799:Genes, Dominant; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000235:genetics* / Q000502:physiology; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006801:Humans; D015458:Leukemia, T-Cell / Q000473:pathology; D051379:Mice; D016513:Mice, SCID; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D009361:Neoplasm Invasiveness / Q000503:physiopathology*; D009363:Neoplasm Proteins / Q000235:genetics* / Q000502:physiology; D010291:Paresis / Q000209:etiology; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000473:pathology*; D016415:Sequence Alignment; D025521:Tumor Suppressor Proteins / Q000235:genetics* / Q000502:physiology; D020741:rho GTP-Binding Proteins / Q000378:metabolism*; D062949:rho Guanine Nucleotide Dissociation Inhibitor beta; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors
C569658:ARHGDIB protein, human; D020730:Guanine Nucleotide Dissociation Inhibitors; D009363:Neoplasm Proteins; D025521:Tumor Suppressor Proteins; D062949:rho Guanine Nucleotide Dissociation Inhibitor beta; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D006153:Guanosine Diphosphate; D020741:rho GTP-Binding Proteins