MDM2 protein overexpression inhibits apoptosis of TF-1 granulocyte-macrophage colony-stimulating factor-dependent acute myeloblastic leukemia cells.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a growth factor for acute myeloblastic leukemia (AML) cells. Murine double minute 2 (MDM2) oncoprotein, a potent inhibitor of wild-type p53 (wtp53), can function both to induce cell proliferation and enhance cell survival, and is frequently overexpressed in leukemias. Therefore, we focused on the importance of MDM2 protein in GM-CSF-dependent versus GM-CSF- independent growth of AML cells. The TF-1 AML cell line, which has both wtp53 and mutant p53 genes, showed GM-CSF-dependent growth; deprivation of GM-CSF resulted in G1 growth arrest and apoptosis. MDM2 mRNA and protein were highly expressed in proliferating TF-1 cells in the presence of GM-CSF and decreased significantly with deprivation of GM-CSF. In contrast, p53 protein increased with GM-CSF deprivation. Ectopic overexpression of MDM2 in TF-1 AML cells conferred resistance to GM-CSF deprivation, and is associated with decreased p53 protein expression. Moreover, a variant of TF-1 cells that grows in a GM-CSF-independent fashion also expressed high levels of MDM2 and low levels of p53. These results suggest that GM-CSF-independent growth of AML cells is associated with overexpression of MDM2 protein and related modulation of p53 expression.

Characterization of p16(INK4A) expression in multiple myeloma and plasma cell leukemia.

Loss of p16(INK4A) (p16) expression is frequently associated with the development of epithelial and lymphoid malignancies. However, the frequency and significance of p16 abnormalities in multiple myeloma (MM) and the more aggressive phase of plasma cell leukemia (PCL) have not been well defined. Accordingly, the goal of this study was to define the expression and function of p16 in fresh samples of MM and PCL. We found that p16 protein was highly expressed in primary MM cells, although it was undetectable in fresh samples of PCL. Additionally, p16 protein was also absent in four of four MM-derived cell lines. To determine the mechanism for p16 underexpression in PCL and MM-derived cell lines, we performed PCR analysis to evaluate both gene deletion and the presence of methylation. Interestingly, the p16 gene was present and methylated in all patient PCL cells and MM cell lines, whereas it was unmethylated in patient MM cells and normal B cells. Furthermore, treatment with the demethylating agent 5-deoxyazacytidine or p16 retrofection restored p16 protein expression and induced G1 growth arrest in patient PCL cells and MM cell lines. These results suggest that inactivation of the p16 gene by methylation may be associated with decreased growth control and the development of PCL in a subset of patients with MM.

The clinical application of an ATP assay to predict the chemosensitivity of gastric cancer.

The chemosensitivity of human xenografts in nude mice and fresh surgical specimens of gastric cancer was evaluated in vitro using the ATP assay and the MTT assay. The in vitro sensitivity of 6 human xenografts was detected by the ATP assay and compared with the in vivo sensitivity of the xenografts in nude mouse. The assay showed a 56.3% true-positive rate and a 85.7% true-negative rate, with 90.0% sensitivity and 46.2% specificity. When 10 surgical specimens obtained from gastric cancer patients were divided into two groups and sensitivities assessed by the ATP and the MTT assays, the overall correlation of both assays was 81.3%. The ATP assay might be useful in evaluating the chemosensitivity of human gastric carcinomas.

A novel pre-B acute lymphoblastic leukemia cell line with chromosomal translocation between p16(INK4A)/p15(INK4B) tumor suppressor and immunoglobulin heavy chain genes: TGFbeta/IL-7 inhibitory signaling mechanism.

p16 INK4A and/or p15 INK4B genes are frequently deleted in leukemias and other cancers. We have established a novel pre-B acute lymphoblastic leukemia (ALL) cell line (JKB2) with a chromosomal translocation between 9p2l and 14q32, on which p16INK4A/p15INK4B and heavy chain immunoglobulin (Ig) genes, respectively, are located. Homozygous deletions of P16INK4A/p15INK4B genes in JKB2 cells were confirmed by polymerase chain reaction, and their protein products were not detectable by Western blotting. Therefore JKB2 is the first example of an immunoglobulin heavy chain translocation associated with deletions of these genes. In JKB2 cells, cyclin-dependent kinase(CDK)4 and CDK6 formed complexes with cyclin D, due to the lack of p16, triggering phosphorylation of retinoblastoma protein (pRB) and continuous cell proliferation. Moreover, the growth of JKB2 cells was partially inhibited by TGF beta or IL-7, accompanied by decreased CDK4 and CDK6 expression, increased p2l and p27 expression, decreased p27 binding to CDK4/CDK6, and increased binding of p27 to CDK2. In addition, IL-7 both inhibited proliferation and induced differentiation of JKB2 cells. These studies suggest that a t(9;14)(p21;q32) chromosomal translocation can result in deletion of both p16 INK4A and p15 INK4B genes in pre-B ALL, and that the JKB2 cell line therefore provides a model for the study of leukemogenesis related to abnormalities in chromosome 9p2l. Moreover, they suggest that TGF-beta can, suppress JKB2 cell growth in a p15-independent mechanism.

Ex vivo expansion of umbilical cord blood hematopoietic progenitor cells by combinations of cytokines.

Ex vivo expansion of hematopoietic progenitor cells in the umbilical cord blood mononuclear cells (CB-MNC) was investigated in liquid culture system with various combinations of cytokines (stem cell factor [SCF], interleukin [IL]-3, IL-6, granulocyte-colony stimulating factor [G-CSF], erythropoietin [EPO], and interferon [INF]-gamma). Non-lineage-committed hematopoietic progenitor cells and lineage committed hematopoietic progenitor cells were represented as CD34+CD38- and CD34+CD38+ subpopulations, respectively. Although absolute CD34+CD38- cell numbers decreased even in the presence of multicytokines, the combinations of SCF plus IL-6 and SCF plus IL-3 plus IL-6 plus IFN-gamma were significantly effective in maintaining CD34+CD38- cells than the other combinations (P < 0.05). After 4 weeks of culture. CD34+CD38- cells disappeared in all combinations of cytokines. Absolute CD34+CD38+ cell numbers increased in the presence of cytokines. Maximal expansion of CD34+CD38+ cells were observed in the combinations of SCF plus IL-3 plus IL-6 plus EPO (19.8 +/- 3.3-fold) and SCF plus IL-3 plus IL-6 plus G-CSF (18.3 +/- 2.6). The combination of SCF plus IL-3 plus IL-6 was also effective to expand CD34+CD38- cells (15.8 +/- 3.9). However, the expansion was transient and they decreased to zero within 3 weeks. In the combinations of SCF plus IL-6 and SCF plus IL-3 plus IL-6 plus INF-gamma, maximal expansion was inferior to the others but CD34+CD38+ cells were maintained more than 4 weeks. These results suggested that the indication of CBT can be expanded into older children by ex vivo augmentation of CB hematopoietic progenitor cells using multi-cytokines.