p21WAF1 modulates drug-induced apoptosis and cell cycle arrest in B-cell precursor acute lymphoblastic leukemia

p21^WAF1 is a well-characterized mediator of cell cycle arrest and may also modulate chemotherapy-induced cell death. The role of p21^WAF1 in drug-induced cell cycle arrest and apoptosis of acute lymphoblastic leukemia (ALL) cells was investigated using p53-functional patient-derived xenografts (PDXs), in which p21^WAF1 was epigenetically silenced in T-cell ALL (T-ALL), but not in B-cell precursor (BCP)-ALL PDXs. Upon exposure to diverse cytotoxic drugs, T-ALL PDX cells exhibited markedly increased caspase-3/7 activity and phosphatidylserine (PS) externalization on the plasma membrane compared with BCP-ALL cells. Despite dramatic differences in apoptotic characteristics between T-ALL and BCP-ALL PDXs, both ALL subtypes exhibited similar cell death kinetics and were equally sensitive to p53-inducing drugs in vitro, although T-ALL PDXs were significantly more sensitive to the histone deacetylase inhibitor vorinostat. Transient siRNA suppression of p21^WAF1 in the BCP-ALL 697 cell line resulted in a moderate depletion of the cell fraction in G1 phase and marked increase in PS externalization following exposure to etoposide. Furthermore, stable lentiviral p21^WAF1 silencing in the BCP-ALL Nalm-6 cell line accelerated PS externalization and cell death following exposure to etoposide and vorinostat, supporting previous findings. Finally, the Sp1 inhibitor, terameprocol, inhibited p21^WAF1 expression in Nalm-6 cells exposed to vorinostat and also partially augmented vorinostat-induced cell death. Taken together, these findings demonstrate that p21^WAF1 regulates the early stages of drug-induced apoptosis in ALL cells and significantly modulates their sensitivity to vorinostat.     

CD90 and CD110 correlate with cancer stem cell potentials in human T-acute lymphoblastic leukemia cells

Although cancer stem cells (CSCs) have been recently identified in myeloid leukemia, published data on lymphoid malignancy have been sparse. T-acute lymphoblastic leukemia (T-ALL) is characterized by the abnormal proliferation of T-cell precursors and is generally aggressive. As CD34 is the only positive-selection marker for CSCs in T-ALL, we performed extensive analysis of CD markers in T-ALL cell lines. We found that some of the tested lines consisted of heterogeneous populations of cells with various levels of surface marker expression. In particular, a small subpopulation of CD90 (Thy-1) and CD110 (c-Mpl) were shown to correlate with stem cell properties both in vitro and in transplantation experiments. As these markers are expressed on hematopoietic stem cells, our results suggest that stem cell-like population are enriched in CD90+/CD110+ fraction and they are useful positive-selection markers for the isolation of CSCs in some cases of T-ALL.     

The human pre-B cell line Nalm-6 is highly proficient in gene targeting by homologous recombination

Gene targeting provides a powerful means for analyzing gene function, as exemplified by knockout mouse studies and recent work with the highly recombinogenic chicken DT40 B-lymphocyte line. In human cultured cells, however, the low frequency of gene targeting is a serious barrier to efficiently generate knockout clones. Moreover, commonly used human cell lines are karyotypically abnormal or unstable. Here, we show using promoterless targeting constructs that Nalm-6, a human pre-B ALL cell line, is highly proficient for gene targeting by homologous recombination. Indeed, the efficiency of TP53 gene targeting in Nalm-6 appears nearly two orders of magnitude higher than that in HCT116, a colon cancer cell line popularly used for gene targeting. Expression analysis revealed a lack of MSH2 expression in this cell line. As Nalm-6 has a stable neardiploid karyotype with normal p53 status, our results underscore the usefulness of Nalm-6 for gene knockout studies in humans.     

Sulforaphane Induces Cell Cycle Arrest and Apoptosis in Acute Lymphoblastic Leukemia Cells

Acute lymphoblastic leukemia (ALL) is the most common hematological cancer in children. Although risk-adaptive therapy, CNS-directed chemotherapy, and supportive care have improved the survival of ALL patients, disease relapse is still the leading cause of cancer-related death in children. Therefore, new drugs are needed as frontline treatments in high-risk disease and as salvage agents in relapsed ALL. In this study, we report that purified sulforaphane, a natural isothiocyanate found in cruciferous vegetables, has anti-leukemic properties in a broad range of ALL cell lines and primary lymphoblasts from pediatric T-ALL and pre-B ALL patients. The treatment of ALL leukemic cells with sulforaphane resulted in dose-dependent apoptosis and G2/M cell cycle arrest, which was associated with the activation of caspases (3, 8, and 9), inactivation of PARP, p53-independent upregulation of p21^CIP1/WAF1, and inhibition of the Cdc2/Cyclin B1 complex. Interestingly, sulforaphane also inhibited the AKT and mTOR survival pathways in most of the tested cell lines by lowering the levels of both total and phosphorylated proteins. Finally, the administration of sulforaphane to the ALL xenograft models resulted in a reduction of tumor burden, particularly following oral administration, suggesting a potential role as an adjunctive agent to improve the therapeutic response in high-risk ALL patients with activated AKT signaling.     

Rearrangements of the T cell receptor delta gene in T acute lymphoblastic leukemia cells are distinct from those occurring in B lineage acute lymphoblastic leukemia and preferentially involve one V delta gene segment

Rearrangement of the TCR-delta gene was studied using J delta, C delta, and V delta probes in 61 cases of acute lymphoblastic leukemia (ALL) and several cases of chronic lymphoid neoplasms to define the specificity and the diversity of rearrangements occurring at the delta locus. TCR-delta rearrangements or deletions were found in all T (33 cases) and B lineage (28 cases) ALL but not in any case of B cell chronic proliferations (13 cases). The restriction patterns of rearrangement were clearly distinct between T and B ALL and use of one V delta probe showed that rearrangement of the V delta IDP2 gene segment which is also productively rearranged in the Peer cell line, occurred frequently in T-ALL but never in B lineage ALL. Studies of WT31 and delta TCS1 antibody reactivity showed that at least 4 of 13 CD3+ T-ALL cases expressed the delta protein. CD4 and/or CD8 Ag expression were observed in some of the gamma delta expressing T-ALL. These data show that particular TCR-delta gene rearrangements occur in neoplastic early B cells and that the combinatorial diversity of TCR-delta rearrangements in T cells is higher than initially expected. In addition this study shows that an important proportion of CD3 positive T-ALL cases express the gamma delta heterodimer.     

Anti-cancer activity of carbamate derivatives of melampomagnolide B

Melampomagnolide B (MMB) is a natural sesquiterpene structurally related to parthenolide (PTL). We have shown that MMB exhibits anti-leukemic properties similar to PTL. Unlike PTL, the presence of a primary hydroxyl group in the MMB molecule allows the opportunity for examining the biological activity of a variety of conjugated analogs of MMB. We have now synthesized a series of carbamate analogs of MMB and evaluated these derivatives for anti-cancer activity against a panel of sixty human cancer cell lines. Analogs 6a and 6e exhibited promising anti-leukemic activity against human leukemia cell line CCRF-CEM with GI₅₀ values of 680 and 620 nM, respectively. Analog 6a also showed GI₅₀ values of 1.98 and 1.38 μM respectively, against RPMI-8226 and SR leukemia cell lines and GI₅₀ values of 460 and 570 nM against MDA-MB-435 melanoma and MDA-MB-468 breast cancer cell lines, respectively. Analog 6e had GI₅₀ values of 650 and 900 nM against HOP-92 non-small cell lung and RXF 393 renal cancer cell lines.     

Toddaculin, a natural coumarin from Toddalia asiatica, induces differentiation and apoptosis in U-937 leukemic cells

Chemotherapeutics represent the main approach for the treatment of leukemia. However, the occurrence of adverse side effects and the complete lack of effectiveness in some cases make it necessary to develop new drugs. As part of our screening program to evaluate the potential chemotherapeutic effect of natural coumarins, we investigated the anti-leukemic activities of a series of six prenylated coumarins isolated from the stem bark of Toddalia asiatica (Rutaceae). Among these, 6-(3-methyl-2-butenyl)-5,7-dimethoxycoumarin (toddaculin) displayed the most potent cytotoxic and anti-proliferative effects in U-937 cells. To determine whether these effects resulted from induction of cell death or differentiation, we further evaluated the expression of several apoptosis and maturation markers. Interestingly, while toddaculin at 250 μM was able to induce apoptosis in U-937 cells, involving decreased phosphorylation levels of ERK and Akt, 50 μM toddaculin exerted differentiating effects, inducing both the capacity of U-937 cells to reduce NBT and the expression of differentiation markers CD88 and CD11b, but no change in p-Akt or p-ERK levels. Taken together, these findings indicate that toddaculin displays a dual effect as a cell differentiating agent and apoptosis inducer in U-937 cells, suggesting it may serve as a pharmacological prototype for the development of novel anti-leukemic agents.     

Relationship between the ability to support differentiation of osteoclast-like cells and adipogenesis in murine stromal cells derived from bone marrow

In vitro osteoclast differentiation is supported by stromal cells. In order to isolate a stromal cell line that can support osteoclast differentiation, 22 cell lines were cloned from mouse bone marrow. One of these clones, TMS-14, is a line of preadipocytes that supports osteoclast-like cell formation without any bone resorbing factors; and another, TMS-12, is a line of preosteoblasts that supports osteoclast-like cell formation with bone resorbing factors such as prostaglandin E(2)(PGE(2)). The difference of these two lines for osteoclast formation was not related with their abilities of PGE(2)production, but with the expression of osteoclast differentiation factor (ODF, also called OPGL, RANKL, and TRANCE), which detected with RT-PCR, in both cell lines. In TMS-14 cells, ODF mRNA was detected with or without PGE(2). In TMS-12 cells, ODF expression was detected in the PGE(2)-treated cells alone. When TMS-14 cells were induced to undergo adipogenic differentiation in response to treatment with thiazolidinedione, a ligand and activator of peroxisome proliferator-activated receptor gamma (PPARgamma), the ability of TMS-14 cells to support osteoclast-like cell formation was prevented in the presence or absence of 1,25(OH)(2)D(3). The gene expression of ODF in TMS-14 cells was also inhibited by treatment with thiazolidinedione. These results suggest that adipogenesis in bone marrow cells is related to the ability to support osteoclast differentiation. This is the first report of a cloned stromal cell line that can support osteoclastogenesis without the treatment with any osteotropic factors. Furthermore, this murine clonal preadipose cell line may be useful for studying senescence-dependent osteoporosis.     

Histone deacetylase inhibitors synergistically potentiate death receptor 4-mediated apoptotic cell death of human T-cell acute lymphoblastic leukemia cells

Cell-death signaling through the pro-apoptotic tumor necrosis factor-related apoptosis inducing ligand (TRAIL) receptors, death receptor 4 (DR4) and DR5, has shown tumor-selective apoptotic activity. Here, we examine susceptibility of various leukemia cell lines (HL-60, U937, K562, CCRF-CEM, CEM-CM3, and THP-1) to an anti-DR4 agonistic monoclonal antibody (mAb), AY4, in comparison with TRAIL. While most of the leukemia cell lines were intrinsically resistant to AY4 or TRAIL alone, the two T-cell acute lymphoblastic leukemia (T-ALL) lines, CEM-CM3 and CCRF-CEM cells, underwent synergistic caspase-dependent apoptotic cell death by combination of AY4 or TRAIL with a histone deacetylase inhibitor (HDACI), either suberoylanilide hydroxamic acid (SAHA) or valproic acid (VPA). All of the combined treatments synergistically downregulated several anti-apoptotic proteins (c-FLIP, Bcl-2, Bcl-X_L, XIAP, and survivin) without significant changing the expression levels of pro-apoptotic proteins (Bax and Bak) or the receptors (DR4 and DR5). Downregulation of c-FLIP to activate caspase-8 was a critical step for the synergistic apoptosis through both extrinsic and intrinsic apoptotic pathways. Our results demonstrate that the HDACIs have synergistic effects on DR4-specific mAb AY4-mediated cell death in the T-ALL cells with comparable competence to those exerted by TRAIL, providing a new strategy for the targeted treatment of human T-ALL cells.     

Isolation and Comparative Characteristics of Mesenchymal Stem-Cell Lines Derived from Foreskin of Two Donors of Similar Age

Two new nonimmortalized human cell lines FRSN-1 and FRSN-2 were established from foreskin of two similarly aged donors (2.5 years). Growth characteristics and differentiation potential of these cell lines studied on the sixth passage confirmed their status as mesenchymal stem cells (MSCs). A number of characteristics have been analyzed during long-term cultivation up to the 26th passage. The dynamics of the process of replicative senescence defined by the activity of β-galactosidase differed between these lines. However, at the 26th passage, the process of replicative senescence was equally enhanced in both lines. The plating efficiency markedly differed between the lines on the sixth passage. In FRSN-1, it was higher than in FRSN-2. The plating efficiency substantially dropped to the 26th passage in FRSN-1 and was lost in FRSN-2 line. Growth curves showed active proliferation of these lines at the 6th passage. The average doubling time did not differ between the lines and was 36.9 and 39.0 h, respectively. Analysis of growth curves on the 26th passage revealed a decline in proliferative activity and increase in average doubling time of cell populations in both lines, more in FRSN-2 than in FRSN-1 lines. The patterns of growth curves differed in these lines. Morphological analysis revealed increased cell size and spreading typical for the phase of the replicative senescence. Numerical and structural karyotypic analysis at the sixth passage showed that both lines have normal karyotype 46, XY. We did not discover interline differences in the frequency of chromosomal aberrations. To determine the status of these cell lines, comparative analysis of the surface markers was performed using flow cytometry. It was revealed that cells of both lines expressed surface antigens characteristic of human MSCs: CD44, CD73, CD90, CD105, and HLA-ABC and did not express CD34, CD45 and HLADR. Cells of both lines displayed SSEA-4 and SOX2, markers of human embryonic stem cells (ESCs). Expression of SSEA-4 was also detected at the 26th passage in both lines. FRSN-1 and FRSN-2 cells expressed the markers of early ESC differentiation into three germ layers. The ability of these cell lines to differentiate into osteogenic, chondrogenic, and adipogenic lineages was shown on the sixth passage. Both lines exhibited substantially reduced adipogenic potential on the 20th passage. These data indicate that in contrast to growth characteristics the adipogenic differentiation potential changes even with an average degree of replicative senescence. It appears that the cell replicative senescence contributed to the change in MSC differentiation potential. Overall, the results demonstrate that cell lines derived from different donors are distinguished in growth characteristics and pattern of replicative senescence. The disparity is due to a direct genetic influence and indirectly by different microenvironment in their donor organisms before cell isolation.     

Inhibition of Akt potentiates 2-DG-induced apoptosis via downregulation of UPR in acute lymphoblastic leukemia

The ability to pair the regulation of metabolism and cellular energetics with oncogenes and tumor suppressor genes provides cancer cells with a growth and survival advantage over normal cells. We investigated the mechanism of cell death induced by 2-deoxy-D-glucose (2-DG), a sugar analog with dual activity of inhibiting glycolysis and N-linked glycosylation, in acute lymphoblastic leukemia (ALL). We found that, unlike most other cancer phenotypes in which 2-DG only inhibits cell proliferation under normoxic conditions, ALL lymphoblasts undergo apoptosis. Bp-ALL cell lines and primary cells exhibited sensitivity to 2-DG, whereas T-ALL cells were relatively resistant, revealing phenotypic differences within ALL subtypes. Cotreatment with D-mannose, a sugar essential for N-linked glycosylation, rescues 2-DG-treated ALL cells, indicating that inhibition of N-linked glycosylation and induction of ER stress and the unfolded protein response (UPR) is the predominant mechanism of 2-DG's cytotoxicity in ALL. 2-DG-treated ALL cells exhibit upregulation of P-AMPK, P-Akt, and induction of ER stress/UPR markers (IRE1α, GRP78, P-eIF2α, and CHOP), which correlate with PARP cleavage and apoptosis. In addition, we find that pharmacologic and genetic Akt inhibition upregulates P-AMPK, downregulates UPR, and sensitizes ALL cells to remarkably low doses of 2-DG (0.5 mmol/L), inducing 85% cell death and overcoming the relative resistance of T-ALL. In contrast, AMPK knockdown rescues ALL cells by upregulating the prosurvival UPR signaling. Therefore, 2-DG induces ALL cell death under normoxia by inducing ER stress, and AKT and AMPK, traditionally thought to operate predominantly on the glycolytic pathway, differentially regulate UPR activity to determine cell death or survival.     

Disappearance of terminal deoxynucleotidyltransferase in human malignant T-lymphoblasts treated with a human alpha interferon preparation

Human T-lymphoblastoid cell lines RPMI 8402, MOLT-3, and CCRF-CEM were treated with interferon (IFN) to determine if the treatment would result in the disappearance of cellular terminaldeoxynucleotidyltransferase (TdT), a possible differentiation marker for T-lymphocytes. Incubation of RPMI 8402 cells in the presence of IFN preparation caused a decrease in the number of TdT-positive cells and in TdT activity of the cell extract. The inhibition of cell multiplication was dose dependent. The anticellular effect of IFN preparation was cytostatic, not cytocidal. The IFN preparation modified neither the TdT content nor proliferation of MOLT-3 and CCRF-CEM cell lines. The effects of IFN preparation thus varied with the cell line.     

Growth factor-dependent differentiation along the myeloid and lymphoid lineages in an immature acute T lymphocytic leukemia

Bone marrow cells from a child with an immature (CD2+, CD5+, CD7+) acute T lymphocytic leukemia (T-ALL) were cultured in the presence and absence of human rIL-2, IL-3, or granulocyte-macrophage (GM)-CSF. Cells cultured without growth factors failed to divide and those initiated in the presence of IL-2 or GM-CSF underwent maturation and terminal T lymphoid or myelomonocytic differentiation, respectively. In contrast, a permanent growth factor-dependent cell line, designated TALL-103/3, was established upon culture in IL-3. The TALL-103/3 cells gradually lost the T cell-specific markers and acquired a myeloid phenotype (CD15+, CD33+). Switching of the IL-3-dependent cells at an early passage to medium containing only human rIL-2 resulted in the establishment of a subline, named TALL-103/2, with a T lymphoid phenotype (CD3+, CD8+, TCR-gamma delta +, CD7+). The TALL-103/2 cells strictly require IL-2 for growth, are irreversibly committed to the lymphoid lineage, and cannot survive in the presence of any other hemopoietic growth factor tested so far. In contrast, the IL-3-dependent TALL-103/3 cells could be adapted to grow in synthetic (serum-free) medium also in the presence of either GM-CSF or IL-5, in which they retain a myeloid phenotype. Interestingly, after 18 mo in culture in IL-3, the TALL-103/3 cells can still be phenotypically converted to the lymphoid lineage upon addition of IL-2, thus maintaining its bipotentiality. Despite the marked phenotypic differences, the TALL-103/2 and TALL-103/3 cell lines show the same karyotypes with multiple abnormalities present in the primary malignant clone and have identical rearrangements of the TCR-gamma and -delta loci, thus confirming their derivation from a common precursor cell. Together, these findings indicate that the phenotype of immature T-ALL cells can be drastically modified by the presence of specific hemopoietic growth factors in the environment, leading to either lymphoid or myeloid lineage commitment while leaving their karyotype and genotype intact.     

6-Shogaol induces apoptosis in acute lymphoblastic leukaemia cells by targeting p53 signalling pathway and generation of reactive oxygen species

Combination therapies, using medicinal herbs, are broadly recommended to attenuate the chemotherapy adverse effects. Based on our previous findings considering the anti-leukaemic effects of ginger extract on acute lymphoblastic leukaemia (ALL) cells, the present study was aimed to investigate the anti-cancer role of this pharmaceutical plant on ALL mice models. Moreover, we worked towards identifying the most anti-leukaemic derivative of ginger and the mechanism through which it may exert its cytotoxic impact. In vivo experiments were performed using five groups of six C57BL/6 nude mice, and the anti-leukaemic activity of ginger extract alone or in combination with methotrexate (MTX) was examined. Results showed increased survival rate and reduced damages in mice brain and liver tissues. Subsequently, MTT assay demonstrated synergistic growth inhibitory effect of 6-shogaol (6Sh) and MTX on ALL cell lines and patients primary cells. Eventually, the molecular anti-neoplastic mechanism of 6Sh was evaluated using Bioinformatics. Flow cytometry illustrated 6Sh-mediated apoptosis in Nalm-6 cells confirmed by Western blotting and RT-PCR assays. Further analyses exhibited the generation of reactive oxygen species (ROS) through 6Sh. The current study revealed the in vivo novel anti-leukaemic role of ginger extract, promoted by MTX. Moreover, 6-shogaol was introduced as the major player of ginger cytotoxicity through inducing p53 activity and ROS generation.