[Biological behavior of stromal cell-derived factor-1 on migration, adhesion and apoptosis in different kinds of AML cell lines].

The study was aimed to investigate the biological behavior of stromal cell-derived factor-1 (SDF-1) in migration, adhesion and apoptosis as well as the related signaling transduction pathways in different kinds of acute myeloid leukemia (AML) cell lines. The expression of surface molecules on AML (KG1a, ML1 and U937) cells were analyzed by flow cytometry. The cell adhesion was detected by MTT assay. The cell migration was checked by transwell assay. Bcl-xl was checked by immunoblotting after activation of phosphionositide-3 kinase (PI3K) in AML cells treated with SDF-1. The results indicated that the expressions of the surface molecules on AML (KG1a, ML1 and U937) cells were different. The list of the expression showed CD34 (KG1a = 95.6%, ML1 = 4.6%, U937 = 4.8%), CD45 (KG1a = 98.3%, U937 = 97.5%, ML1 = 17.8%), CXCR4 (ML1 = 85.4%, U937 = 43.6%, KG1a = 3.8%), ICAM (KG1a = 75.8%, U937 = 41.8% and ML1 = 46.3%). SDF-1 could not upregulate their expression, but could trigger the establishment of polarized morphology of the cells which expressed CXCR4 high. SDF-1 promoted ML1 and U937 cell adhesion to the stroma cells (HS5, HS27), stimulated PI3K in the cells. It was also confirmed that SDF-1 could increase the leukemic cell survival by stimulate this pathway. After addition of wortmaninn or PTX, the cell death increased. It is concluded that the SDF-1 increases the leukemic cell adhesion, migration and survival by stimulating the PI3K pathway. These functions can be depressed by the PI3K inhibitor and also the inhibitor of G protein as well.

[Preliminary study of biological characteristics of myelodysplastic syndromes clonal cells].

OBJECTIVE: To investigate the biological difference of clonal cells between myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). METHOD: Bone marrow (BM) clonal cells (which had cytogenetic markers detected by FISH assay) and blasts were quantitatively analysed in 51 MDS and 11 AML patients. The clonal cell percentage in orthochromatic normoblasts, granulocytes and megakaryocytes were assayed. The biological functions for phagocytosis and oxidation of MDS peripheral blood (PB) neutrophils were compared with that of normal controls. RESULTS: Almost all MDS patients BM had a higher clonal cell percentage (mean 48.2%) than blasts percentage (mean 6.7%) (P < 0.01), but with the subtype of MDS advancing this percentage gap was closing up, and in 11 AML patients no such gap was observed. This gap in MDS patients with + 8 abnormality was smaller than in those with 5q -. In MDS BM, clonal cells were detected in segmented granulocytes (mean 45.9%), orthochromatic normoblasts (mean 46.0%) and mature megakaryocytes (mean 38.0%). In Addition, an approximate amount of clonal cells with the same karyotype abnormality in BM were detected in MDS PB (mean 37.3% in blood vs 48.6% in marrow). Functional analysis showed that the neutrophils in MDS PB could exert nearly normal physiological functions (P > 0.05), but those from AML could not as compared to healthy donors (P < 0.01). CONCLUSION: There is a significant difference in the biological features between MDS and AML clonal cells.

[Abnormal polarization of T lymphocyte induces negative hematopoietic regulation in myelodysplastic syndromes].

OBJECTIVE: To explore polarization of T lymphocyte and its relationship with apoptosis of marrow cells in patients with myelodysplastic syndromes (MDS). METHODS: Measurements of Th1, Th2, Tc1, Tc2 subsets in bone marrows from 34 patients with MDS and 13 normal controls were performed by flow cytometry. INF-gamma and TNF-alpha in marrow serum were determined by ELISA (Enzyme-linked immunosorbent assay). Apoptosis index of marrow cells was detected by TUNEL (TdT-mediated dUTP nick end labeling). Correlations between Th1, Th2, Tc1, Tc2 subsets and INF-gamma, TNF-alpha levels as well as apoptosis index were analyzed, and relationship between TNF-alpha, INF-gamma levels and apoptosis index was also investigated. RESULTS: (1) The percentage of Th1 cells [(10.1 +/- 1.6)%], Tc1 cells [(24.0 +/- 3.6)%] and Tc1/Tc2 ratio (50.0 +/- 11.1) was significantly increased in patients with MDS than in normal controls [(4.0 +/- 0.5)%, (5.8 +/- 0.6)% and 13.4 +/- 2.7, respectively]. Levels of INF-gamma [(58.6 +/- 21.7) microg/L] and TNF-alpha [(15.7 +/- 3.8) microg/L] in marrow serum of MDS patients was markedly elevated compared to normal controls [0 and (0.3 +/- 0.2) microg/L, respectively]. An increased apoptosis index of nucleated cells was observed in MDS patients [(7.8 +/- 1.5)%] as compared to controls [(2.1 +/- 0.3)%, P < 0.05]. The Th1 cell percentage showed a positive correlation with the levels of INF-gamma and TNF-alpha (r = 0.38, P < 0.05 and r = 0.39, P < 0.05, respectively), and with apoptotic index of nucleated marrow cells in MDS patients (r = 0.33, P < 0.05). Furthermore, a positive correlation was observed between INF-gamma, TNF-alpha levels and apoptotic index of marrow cells (r = 0.74, P < 0.01 and r = 0.73, P < 0.01, respectively). (2) Th1, Tc1 cells and Tc1/Tc2 ratio in MDS-RCMD patients was markedly elevated (P < 0.01) but did not in RCMD-RS, RAEB-1 and RAEB-2 patients as compared to normal controls. (3) An elevation in the percentages of Th1, Tcl and Tc1/ Tc2 ratio was detected in patients with IPSS lower-risk but did not in higher-risk group as compared to controls. (4) Increased Th1 and Tc1 percentages and Th1/Th2 and Tc1/Tc2 ratios were observed in RCMD patients with normal karyotype, but did not in those with abnormal karyotype. Conclusions Th1/Th2 and Tc1/Tc2 in bone marrow of MDS patients were unbalanced, polarizing to type I reaction especially in patients with RCMD subtype, IPSS lower-risk and normal karyotype. The increased Th1 cells in bone marrow may account for the increased apoptosis of nucleated marrow cells in MDS, through proapoptotic cytokines such as INF-gamma and TNF-alpha.

Differential effects of bexarotene on intrinsic and extrinsic pathways in TRAIL-induced apoptosis in two myeloid leukemia cell lines.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces programmed cell death (apoptosis) preferentially in tumor cells. However, not all cancer cells are sensitive to TRAIL. We determined whether ligation of the retinoid receptor, RXR, would sensitize cells to TRAIL-mediated apoptosis. The leukemic cell lines KG1a (apoptosis-resistant) and ML-1 (apoptosis-sensitive) were treated with the RXR-specific retinoid bexarotene, TRAIL, or both, and apoptosis was determined. In KG1a cells, bexarotene downregulated FLIP(Long) and activated caspase-8, thereby allowing for TRAIL-triggered apoptosis. Overexpression of FLIP(Long) in ML-1 cells abrogated apoptosis. In unmodified ML-1 cells bexarotene enhanced programmed cell death via truncation of Bid and release of cytochrome C. Blockade of caspase-8 prevented enhancement in both cell lines; blockade of caspase-9 had a significant effect only in ML-1 cells. Thus, the effect of bexarotene on TRAIL-mediated programmed cell death involved proximal events of the extrinsic pathway; however, downstream signals involved the intrinsic pathway in ML-1 but not in KG1a cells. These studies add further information to the regulation of programmed cell death in leukemic cells that have to be considered when designing therapeutic strategies.

[Expression of insulin-like growth factor receptor type I in marrow nucleated cells from hematologic malignancies and its anti-apoptotic effect].

To explore the expression of insulin-like growth factor receptor type I (IGF-IR) and its relationship to apoptosis in hematopoietic cells of MDS and AML marrow, bone marrow nucleated cells from 16 patients with myelodysplastic syndrome (MDS) and 16 patients with acute myeloid leukemia (AML) were collected for analysis, respectively. Another 16 normal donors' marrow samples were taken as controls. Immunocytochemical method (APAAP) and TdT-mediated dUTP nick end labeling (TUNEL) fluorescence were used simultaneously on cytospins of nucleated cells from these patients. Then, the ratios of IGF-IR positive cells and apoptosis cells in all nucleated cells were counted separately. The results showed that (1) there was a higher IGF-IR expression rate (56.8 +/- 14.3)% in nucleated cells of MDS marrow than that in normal marrow (40.4 +/- 9.6)% (P < 0.01). Also IGF-IR positive rate in AML marrow (86.8 +/- 13.8)% was significantly higher than that in normal marrow (P < 0.01). Furthermore, IGF-IR had higher expression in AML marrow when compared to MDS marrow (P < 0.01); (2) apoptosis in nucleated cells of MDS marrow (5.4 +/- 3.0)% was significantly higher than that in normal marrow (1.2 +/- 0.9)% (P < 0.01) and AML marrow (0.3 +/- 0.4)% (P < 0.01), while there was less apoptosis in AML marrow than that in normal marrow (P < 0.01); (3) apoptosis occurred mainly in IGF-IR negative cells (9.0 +/- 4.8)% and less in IGF-IR positive cells (1.4 +/- 2.4)% (P < 0.01). IGF-IR expression showed negative correlation with apoptosis (r = -0.852, P < 0.01); (4) IGF-IR of MDS nucleated cells in RAEB/RAEB-t/CMML expressed higher than that in RA/RAS (64.1 +/- 3.2% vs 53.5 +/- 16.2%) subgroup, although no significant difference was found (P > 0.05); and apoptosis in RAEB/RAEB-t/CMML subgroup was lower than that in RA/RAS cases (3.1 +/- 2.1% vs 6.4 +/- 2.8%) (P < 0.05); (5) IGF-IR positive rate in nucleated cells of MDS and AML marrow showed positive correlation with blast rate (r = 0.677; P < 0.01). It is concluded that there is overexpression of IGF-IR in marrow nucleated cells in MDS and AML cases. And it seems that the overexpression of IGF-IR may suggest some malignant proliferation tendency and suppress cell apoptosis through some mechanism in these malignant hematologic ailments. So, anti-IGF-IR will become a new approach for therapy of MDS and AML.

[Apoptotic characteristics of CD34 positive cells in myelodysplastic syndromes].

OBJECTIVE: To investigate the apoptotic situation of CD(34) positive cells in myelodysplastic syndromes (MDS). METHOD: In 36 MDS patients, immunocytochemical technique was used for the detection of the expression of CD(34) antigen and DNA in situ end labelling (ISEL) (fluorescein) for the apoptotic signals. Fourteen cases of iron deficiency anemias (IDA) were used as controls. RESULTS: (1) CD(34) expression in MDS group was much higher than that in controls (49.2 +/- 38.5 vs 10.2 +/- 9.7, P < 0.01), and MDS cases had an obviously higher apoptotic rate than control did (69.1 +/- 28.2 vs 17.8 +/- 11.2, P < 0.01). (2) Expression of CD(34) was higher in transforming group (P < 0.05) than in non-transforming and post-transforming groups. Apoptotic rates in both non-transforming/transforming group were higher than in post-transforming group (P < 0.02 and < 0.05 respectively). (3) No apoptosis was found in CD(34) positive cells in MDS; (4) Both CD(34) positive cells and apoptotic cells formed into small or large clusters but did not co-distributed in a given area. CONCLUSION: There is overexpression of CD(34) antigen on hematopoietic cells in MDS. High CD(34) expression accompanied high apoptosis coexisted in the process of transformation from MDS to AML. Apoptosis-resistance of these CD(34) positive cells suggested that they came from malignant hematopoietic cell clones.

[Relationship between macrophages and apoptosis in patients with myelodysplastic syndromes].

OBJECTIVE: To observe the relationship between macrophage proliferation and cell apoptosis in patients with myelodysplastic syndromes (MDS). METHODS: A double labelling method of immunohistochemistry (alkaline phosphatase anti-alkaline phosphatase, APAAP) and ISEL (DNA in situ end labelling) was used to detect the positive CD68 expression (macrophages) and apoptosis on cold plastic embedded bone marrow biopsy sections in 30 MDS cases. 12 cases of iron deficient diseases (IDA) were used as the control. RESULTS: (1) The number of CD68 positive cells in MDS were higher than that in controls (29.2 +/- 33.0/mm(2) bone marrow tissue vs 21.2 +/- 16.7/mm(2)) (P > 0.05); (2) The number of apoptotic cells in MDS group was much higher than that in the controls (71.5 +/- 70.9/mm(2) vs 37.3 +/- 23.0/mm(2), P < 0.05); (3) The number of CD68 expression (35.5 +/- 37.0/mm(2)) and apoptosis (90.7 +/- 74.6/mm(2)) in less advanced MDS were much higher than that in advanced MDS group (14.6 +/- 11.7/mm(2) and 26.8 +/- 33.1/mm(2), P < 0.05 and < 0.01 respectively); (4) CD68 expression showed an obvious positive correlation to apoptosis in MDS cases (r = 0.83, P < 0.001); (5) CD68 positive cells did not show location correlation to apoptotic cells; (6) CD 68 positive cells in MDS showed simultaneous apoptosis. CONCLUSIONS: Over-apoptosis existed in MDS. Less advanced group has a higher ratio of apoptosis than in advanced group. The correlation between macrophages and apoptosis indicates the participation of TNFalpha in apoptosis-induction during MDS development.