Enhancement of fractionated bone marrow transplantation on hematopoietic cell homing in a mouse allogeneic model.

BACKGROUND: The conventional method of bone mar row transplantation (BMT) requires a large quantity of bone marrow cells (BMC) transplanted by a single injection. However, the homing efficiency of BMC is low, because the emptying of available niches might be a continuous process after stem-cell death after irradiation. In this article, the death character of CD34 stem cells was directly detected, and a novel method of fractionated (Fr) BMT that aimed to better use each niche that was continuously emptied was developed. METHODS: Apoptosis and necrosis were detected using trivariate fluorescence-activated cell sorting after labeling cells with phycoerythrin-CD34-fluorescein isothiocyanate-Annix V-7-aminoactinomycin D. Fr-BMT was conducted in a model of allogeneic BMT, where the total dosage of BMC was divided into four doses, and each dose was administrated for 4 consecutive days at 24-hr intervals after lethal irradiation. RESULTS: CD34 cells underwent a continuous death process after lethal irradiation and presented three death peaks within a week. Fr-BMT decreased the BMC transplanted but increased the accumulatively homed BMC and enhanced the survival rates of recipients. CONCLUSIONS: Fr-BMT is able to enhance hematopoietic cell homing and engraftment, and this method might prove to be a solution to the previous clinical problem that a single unit of umbilical cord blood is not sufficient for engraftment in adults.

Effects of anesthesia-induced modest hypothermia on cellular radiation sensitivity.

To assess the mechanisms of modest hypothermia (MH) and its effects on cellular radiation response, a model of anesthesia-induced modest hypothermia (AIMH) in the adult mice and a model of pure MH in the newborn mice were established. The survival rate of lethally irradiated mice was increased to 72% through AIMH before irradiation. Both apoptosis and necrosis of human fetal bone marrow CD34(+) hematopoietic stem cells cultured under MH were significantly decreased as detected by MTT and flow cytometry, with three-color labeled by PE-CD(34) (+)/ FITC-AnnexinV /7AAD. The survival and proliferation of mouse bone marrow MNC treated with MH after irradiation were also increased. The MH exerted similar protective effects on the leukemia cell lines A20, HL60, K562 to the normal bone marrow cells, but it enhanced the radiation sensitivity of leukemia cell line FBL3 and mouse melanoma B16F10. No effects have been found on the radiation sensitivity of those cells treated with MH before irradiation. The results also showed that MH mediated the effects on radiation sensitivity, in addition to increasing the oxygen tension. These results show different effects of MH on different cells: (i) AIMH exerts a protective effect on the normal hematopoietic stem cells, some leukemia cell lines A20, HL60, K562, and some neoplasma 3LL, LOVO. And MH exhibits a synthetic effect with anesthetic. (ii) MH enhances the radiation sensitivity of another leukemia and neoplasma cell lines FBL3, B16F10 and CT26. Therefore, AIMH has a potential to enhance the effects of radiation-therapy and decrease side effects on some tumors.

[Effects of STAT3 antisense oligodeoxynucleotides on apoptosis and proliferation of mouse melanoma cell line B16].

BACKGROUND & OBJECTIVE: STAT3 protein has been found constitutively activated in a wide variety of human tumor tissues and cell lines. It may be involved in tumorigenesis and development. This study was to observe the activation of STAT3 protein in mouse melanoma cell line B16, human liver cancer cell lines SMMC-7721 and HepG-2, human lung cancer cell line A549, and human cervical cancer cell line HeLa, and to investigate the effects of STAT3 antisense oligodeoxynucleotides (ASODN) on proliferation and apoptosis of B16 cells. METHODS: B16 cells were transfected with STAT3 ASODN or STAT3 sense oligodeoxynucleotides (STAT3 SODN), respectively. The expression and phosphorylation levels of STAT3 protein in all the tumor cells were measured by Western blot. The proliferation of B16 cells was detected by MTT assay. Cell apoptosis was determined by Hoechst33258 staining and Annexin V/PI using flow cytometry. RESULTS: STAT3 was highly expressed and phosphorylated in all the tumor cells. Transfection of STAT3 ASODN suppressed the expression and phosphorylation levels of STAT3 protein in B16 cells. Forty-eight hours after transfection, the proliferation of B16 cells was inhibited the higher the concentration (0-200 nmol/L) of STAT3 ASODN was, the heavier the inhibition was (P<0.01); when transfected with STAT3 SODN over 250 nmol/L, the proliferation of B16 cells was also inhibited (P<0.05). Inhibitory effects appeared 24 h later after transfection of STAT3 ASODN, and became more obvious 48 h later (P<0.01). The early apoptosis rate was significantly higher in 400, 800, 2,000 nmol/L STAT3 ASODN groups and 2,000 nmol/L STAT3 SODN group than in blank control group (8.22%, 9.99%, 16.97%, and 13.31% vs. 5.52%, P<0.01); no significant difference was found among 400 and 800 nmol/L STAT3 SODN groups and blank control group (5.87%, 5.36% and 5.52%, P>0.05). CONCLUSIONS: STAT3 is highly expressed and activated in all the tumor cells detected. STAT3 ASODN can abrogate the activity of STAT3 protein, inhibit proliferation and induce apoptosis of B16 cells, which support that STAT3 may be a potential molecular target for tumor therapy.