mTOR (mammalian target of rapamycin) is the center for cellular activities. It controls many cell activities via inhibiting apoptosis and promoting cell growth. Rheb can activate mTOR signaling pathway and participate in genesis and development of multiple cancers. This study was purposed to explore the underlying role of Rheb in human myeloid leukemia by using the myeloid leukemia cell lines. Two myeloid leukemia cell lines HL-60 and K562 overexpressing Rheb were established with retrovirus containing Rheb. The mRNA and protein expressions of Rheb were determined by Real-Time PCR and Western blot respectively. Cell proliferation rate was examined by CCK-8 assay and apoptosis rate was analyzed using Annexin V and 7-AAD double-staining. The results showed that Rheb was overexpressed in both HL-60 and K562 cell lines. The Rheb overexpression cell lines were successfully established. It is found that overexpression of Rheb could promote cell growth. Furthermore, the overexpression of Rheb could accelerate cells entering into G2/M phase (P < 0.01), while did not affect the apoptosis. It is concluded that Rheb overexpression promotes myeloid leukemia cell proliferation through accelerating cell cycle progression.
Cell Cycle ; Cell Proliferation ; HL-60 Cells ; Humans ; K562 Cells ; Monomeric GTP-Binding Proteins ; metabolism ; Neuropeptides ; metabolism ; Ras Homolog Enriched in Brain Protein ; Signal Transduction

OBJECTIVETo construct recombinant lentiviral vectors carrying Rheb gene and its mutant Rheb'D60K gene, and examine their expression in human liver cancer cells.

METHODSRheb gene was amplified by PCR to construct the recombinant plasmid LV31-Rheb-WT and LV31-Rheb-D60K. HEK-293 FT cells were contransfected with the recombinant lentiviral vector together with a lentiviral package plasmid to produce the lentiviral particles. The expression of PS6 protein was detected in the lentivirus-infected MCF-7 cells. The apoptosis of SK-HEP-1 cells transfected with LV31-Rheb-WT or LV31-Rheb-D60K was observed.

RESULTSThe recombinant LV31-Rheb-WT and LV31-Rheb-D60K vectors were confirmed by PCR and DNA sequencing. Western blotting showed that PS6 protein expression was increased in LV31-Rheb-WT-transfected cells while decreased in LV31-Rheb-D60K-transfected cells. LV31-Rheb-D60K-transfected SK-HEP-1 cells showed more obvious apoptosis after starvation than LV31-Rheb-WT-transfected cells.

CONCLUSIONLentiviral vectors carrying Rheb gene and its mutant has been successfully constructed, which can be useful in further investigation of the role of Rheb gene in cancer cells.

Apoptosis ; genetics ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Genetic Vectors ; genetics ; HEK293 Cells ; Humans ; Lentivirus ; genetics ; metabolism ; Liver Neoplasms ; metabolism ; pathology ; MCF-7 Cells ; Monomeric GTP-Binding Proteins ; biosynthesis ; genetics ; Mutant Proteins ; genetics ; Neuropeptides ; biosynthesis ; genetics ; Ras Homolog Enriched in Brain Protein ; Recombinant Proteins ; biosynthesis ; genetics ; Transfection

OBJECTIVETo investigate the role of Rheb (mTOR activator) in AML development by measuring Rheb expression in bone marrow of adult AML patients and in AML cell line HL-60.

METHODSReal-time PCR assay was used to measure the Rheb mRNA expression in 27 AML patients and 29 ITP patients as control. The relationship between Rheb mRNA expression and age, AML subtype, fusion gene, splenomegaly, hepatomegaly and survival of AML patients was analyzed and compared. In addition, HL-60 cell line over-expressing Rheb was established, and the HL-60 cells and HL-60 cells with overexpression of Rheb were treated with Ara-C of different concentrations, the proliferation level was detected by CCK-8 method, and the IC50 was calculated.

RESULTSThe mRNA level of Rheb in AML patients was similar to that in ITP patients (control). Interestingly, higher expression of Rheb was associated with better survival and was sensitive to Ara-C treatment. However, the expression level of Rheb was not associated with age, AML subtype, fusion gene, and hepatomegaly of patients. Lower expression level of Rheb was associated with splenomegaly. In vitro analysis of HL-60 line indicated that overexpression of Rheb could increased the cell sensitivity to Ara-C treatment (IC50=0.54 µmol/L) and caused HL-60 cell apoptosis.

CONCLUSIONThe lower Rheb expression is a poor prognostic indicator for AML patients, which is associated with AML splenomegaly, the patients and HL-60 cells with low expression of Rheb are insensitive to Ara-C treatment.

Adult ; Apoptosis ; Bone Marrow ; metabolism ; Cytarabine ; pharmacology ; HL-60 Cells ; Humans ; Leukemia, Myeloid, Acute ; genetics ; metabolism ; pathology ; Monomeric GTP-Binding Proteins ; genetics ; metabolism ; Neuropeptides ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Ras Homolog Enriched in Brain Protein ; Real-Time Polymerase Chain Reaction ; Spleen ; pathology