Priming Effect with G-CSF Enhances In Vitro Apoptosis by Treatment with Ara-C and VP-16 in Leukemic Cell Line.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4333-4333
Author(s):  
Jun-ichi Kitagawa ◽  
Takeshi Hara ◽  
Hisashi Tsurumi ◽  
Nobuhiro Kanemura ◽  
Masahito Shimizu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Priming Effect ◽  
Low Dose ◽  
Combined Treatment ◽  
Annexin V ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Significant Difference

Abstract Introduction: We have recently reported that the effectiveness of low dose Ara-C, VP-16 and G-CSF (AVG therapy) for elderly AML patients who were ineligible for intensive chemotherapy (Hematol Oncol, in press). G-CSF has been reported to potentiate in vitro anti-leukemic effect of Ara-C. The mechanism of the potentiation is assumed to recruit quiescent G0 leukemic cells into cell cycle. We hypothesized that the enhanced cytotoxicity was due to the apoptosis by the effect of the priming of G-CSF, and the effect was depended on the cell cycle. In order to afford proof of this hypothesis, we assayed proliferation, apoptosis, and cell cycle in leukemic cell lines. Materials: Ara-C, VP-16, G-CSF was provided by Nippon Shinyaku, Nihonkayaku, Chugai pharmacy, respectively, Tokyo, Japan. 32D and HL-60 were obtained from RIKEN Bioresource Center Cell Bank (Ibaragi, Japan), Ba/F3 was generous gifts from Dr. Kume, Jichi medical school, Tochigi, Japan. Methods: 5 x 105/ml HL60, 32D and Ba/F3 were cultured with various concentrations of Ara-C and/or VP-16 in the presence or absence of G-CSF 50ng/ml for 3 days. At the end of the culture, cell proliferation and viability were determined by using the trypan blue. The Annexin V-binding capacity of treated cells was examined by flow cytometry using ANNEXIN V-FITC APOPTOSIS DETECTION KIT I purchased from BD Pharmingen™. Cell cycle analysis was done with BrdU Flow KIT purchased from BD Pharmingen™. The incorporated BrdU was stained with specific anti-BrdU fluorescent antibodies, and the levels of cell-associated BrdU are then measured by flow cytometory. Result: Ara-C and VP-16 inhibited proliferation and decreased viability of leukemic cell lines dose-dependently. Half killing concentration (IC50) was redused in combination of Ara-C and VP-16 than Ara-C or VP-16 alone. In G-CSF dependent cell line (32D), IC50 was redeced in the presence of G-CSF than absence of G-CSF at G-CSF, and there was no significant difference between with and without G-CSF in G-CSF independent cell lines (HL-60, Ba/F3) (p<0.05). In combined treatment of low dose Ara-C (10−7M) and VP-16 (10−7M), the percentage of apoptotic cells were increased to 20.67% from 13.04% by addition of G-CSF in 32D, and there was no significant differencebetween with and without G-CSF in HL-60 and Ba/F3 (p<0.05). At combined treatment of low dose Ara-C and VP-16, the percentage of G0/G1 phase cells were decreased to 43.94% from 35.63% and S phase cells were increased to 29.50% from 24.05% in 32D by addition of G-CSF, and there was no significant difference between with and without G-CSF in HL-60 and Ba/F3 (p<0.05). Discussion: We first showed a combination effect of Ara-C and VP-16. Next we demonstrated that the potentiation of the cytotoxicity was mediated through the mechanism of apoptosis, and apoptosis played an important role for eradicating leukemic cells by low dose Ara-C and VP-16. And G-CSF recruited cells G0/G1 phase into S phase in G-CSF dependent cells by addition of G-CSF. These results suggest that priming effect of G-CSF significantly potentiate the cytotoxicity mediated by AVG chemotherapy. Conclusion: The priming effect of G-CSF might be admitted at least of a part in AML cells.

Apoptosis of leukemic cells accompanies reduction in intracellular pH after targeted inhibition of the Na+/H+exchanger

Blood ◽  
2000 ◽  
Vol 95 (4) ◽  
pp. 1427-1434 ◽  
Author(s):  
Ivan N. Rich ◽  
Diana Worthington-White ◽  
Oliver A. Garden ◽  
Philip Musk
Keyword(s):  
Cell Cycle ◽  
Intracellular Ph ◽  
Annexin V ◽  
Leukemic Cell ◽  
Differential Sensitivity ◽  
Leukemic Cells ◽  
Hematopoietic Tissue ◽  
Leukemic Cell Lines ◽  
Targeted Inhibition

The Na+/H+ exchanger isoform 1 (NHE1) is primarily responsible for the regulation of intracellular pH (pHi). It is a ubiquitous, amiloride-sensitive, growth factor–activatable exchanger whose role has been implicated in cell-cycle regulation, apoptosis, and neoplasia. Here we demonstrate that leukemic cell lines and peripheral blood from primary patient leukemic samples exhibit a constitutively and statistically higher pHi than normal hematopoietic tissue. We then show that a direct correlation exists between pHi and cell-cycle status of normal hematopoietic and leukemic cells. Advantage was taken of this relationship by treating leukemic cells with the Na+/H+ exchanger inhibitor, 5-(N, N-hexamethylene)-amiloride (HMA), which decreases the pHiand induces apoptosis. By incubating patient leukemic cells in vitro with pharmacologic doses of HMA for up to 5 hours, we show, using flow cytometry and fluorescent ratio imaging microscopy, that when the pHi decreases, apoptosis—measured by annexin-V and TUNEL methodologies—rapidly increases so that more than 90% of the leukemic cells are killed. The differential sensitivity exhibited between normal and leukemic cells allows consideration of NHE1 inhibitors as potential antileukemic agents.

Differential expression of a novel proline-rich homeobox gene (Prh) in human hematolymphopoietic cells

Blood ◽  
1995 ◽  
Vol 85 (5) ◽  
pp. 1237-1245 ◽  
Author(s):  
G Manfioletti ◽  
V Gattei ◽  
E Buratti ◽  
A Rustighi ◽  
A De Iuliis ◽  
...  
Keyword(s):  
Cell Lines ◽  
Constitutive Expression ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Maturation Stage ◽  
Lymphoid Tissues ◽  
Specific Cell ◽  
B Cell Differentiation ◽  
Leukemic Cell Lines

Proline-rich homeobox (Prh) is a novel human homeobox-containing gene recently isolated from the CD34+ cell line KG-1A, and whose expression appears mainly restricted to hematopoietic tissues. To define the pattern of Prh expression within the human hematopoietic system, we have analyzed its constitutive expression in purified cells obtained from normal hematopoietic tissues, its levels of transcription in a number of leukemia/lymphoma cell lines representing different lineages and stages of hematolymphopoietic differentiation, and its regulation during in vitro maturation of human leukemic cell lines. Prh transcripts were not detected in leukemic cells of T-lymphoid lineage, irrespective of their maturation stage, and in resting or activated normal T cells from peripheral blood and lymphoid tissues. In contrast, high levels of Prh expression were shown in cells representing early stages of B lymphoid maturation, being maintained up to the level of circulating and tissue mature B cells. Terminal B-cell differentiation appeared to be conversely associated with the deactivation of the gene, since preplasmacytic and plasmocytoma cell lines were found not to express Prh mRNA. Prh transcripts were also shown in human cell lines of early myelomonocytic, erythromegakaryocytic, and preosteoclast phenotypes. Prh expression was lost upon in vitro differentiation of leukemic cell lines into mature monocyte-macrophages and megakaryocytes, whereas it was maintained or upregulated after induction of maturation to granulocytes and osteoclasts. Accordingly, circulating normal monocytes did not display Prh mRNA, which was conversely detected at high levels in purified normal granulocytes. Our data, which show that the acquisition of the differentiated phenotype is associated to Prh downregulation in certain hematopoietic cells but not in others, also suggest that a dysregulated expression of this gene might contribute to the process of leukemogenesis within specific cell lineages.

Pioglitazone Inhibits the Growth of Human Leukemic Cell Lines and Primary Leukemic Cells in Vitro.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4493-4493 ◽  
Author(s):  
Yoshihiro Hatta ◽  
Minoru Saiki ◽  
Yuko Enomoto ◽  
Shin Aizawa ◽  
Umihiko Sawada ◽  
...  
Keyword(s):  
Cell Lines ◽  
Colony Formation ◽  
Mononuclear Cells ◽  
Hematopoietic Cells ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Peroxisome Proliferator ◽  
Leukemic Cell Lines ◽  
Ppar Γ

Abstract Troglitazone and pioglitazone are one of thiazolidinediones that are high affinity ligand for the nuclear receptor called peroxisome proliferator-activated receptor gamma (PPAR-γ). Troglitazone is a potent inhibitor of clonogenic growth of acute myeloid leukemia cells when combined with a retinoid. However, the effect of pioglitazone to neoplastic cells and normal hematopoietic cells has not been studied yet. Adult T-cell leukemia (ATL), prevalent in western Japan, is a highly aggressive malignancy of mature T lymphocyte. Therefore, we studied antitumor effect of pioglitazone against leukemic cells including ATL as well as normal hematopoietic cells. With 300 μM of pioglitazone, colony formation of ATL cell lines (MT1, MT2, F6T, OKM3T, and Su9T01) was completely inhibited. Colony formation of HUT102, another ATL cell line, was 12 % compared to untreated control. Clonogenic cells of other leukemic cell lines (K562, HL60, U937, HEL, CEM, and NALM1) was also inhibited to 0–30% of control. Colony formation of primary leukemic cells from 5 AML patients was decreased to 15 %. However, normal hematopoietic cells were weakly inhibited with 300 μM pioglitazone; 77 % of CFU-GM, 70 % of CFU-E, and 33 % of BFU-E survived. Cell cycle analysis showed that pioglitazone decreased the ratio of G2/M phase in HL60 cells, suggesting the inhibition of cell division. By Western blotting, PPAR-γ protein level was similar in all leukemic cells and normal bone marrow mononuclear cells. Taken together, pioglitazone effectively eliminate leukemic cells and could be used as an antitumor agent in vivo.

A Novel Multiparameter Flow Cytometric Cytotoxicity Assay Detects p53 Deletion as the Major Cause of In Vitro Resistance to Fludarabine in B-Cell Chronic Lymphocytic Leukemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4470-4470
Author(s):  
James Z. Huang ◽  
Antony C. Bakke ◽  
Guang Fan ◽  
Rita Braziel ◽  
Ken M. Gatter ◽  
...  
Keyword(s):  
Drug Sensitivity ◽  
Drug Exposure ◽  
Leukemic Cell ◽  
Chemotherapeutic Agents ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Multiparameter Flow Cytometry ◽  
Diagnostic Strategy ◽  
Significant Difference

Abstract Individual patients with B-CLL demonstrate variable responses to standard induction and salvage therapeutic regimens. It would be highly desirable to develop a predictable and reproducible laboratory diagnostic strategy that guides the selection of appropriate drugs and/or regimens based on the drug sensitivity and resistance profiles of leukemic cells for individual patients. As a first step towards this goal, a study was designed to investigate the differences of in vitro drug sensitivity profiles of leukemic cells with different cytogenetic abnormalities from CLL patients. CLL cells from 43 patients were incubated in vitro with four commonly used chemotherapeutic agents (fludarabine, chlorambucil, cladribine or prednisolone) individually or in combination. Multiparameter flow cytometry was utilized to determine the decrease in leukemic cell viability after drug exposure. Both fresh and cryopreserved samples were assessed and were found to be equivalent for assay, regardless of the percentage of B-CLL cells or the degree of spontaneous apoptosis. The highest in vitro resistance to fludarabine, was seen in all seven cases of B-CLL cells with deletions of p53, a cytogenetic abnormality associated with poor clinical outcome. Interestingly, in vitro response to chlorambucil and prednisolone was seen some CLL cases with p53 deletion and correlated with clinical response to these drugs. In CLL cases without p53 deletion, a marked variability in vitro drug sensitivity CLL cells was observed but no significant difference was detected among cases with normal cytogenetics (n=13), ATM deletion (n=4), trisomy 12 (n=3), or 13q deletion (n=7). Our findings provide direct evidence of cellular resistance to fludarabine in CLL associated with p53 deletion, confirming prior clinical observations. In vitro drug sensitivity assay may prove useful in guiding choices for therapy for CLL patients based on the drug sensitivity profile of leukemic cells in individuals.

Improved Therapy of Multiple Myeloma by Combining Milatuzumab (Anti-CD74 Humanized mAb) and Bortezomib: In Vitro and In Vivo Results.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1176-1176
Author(s):  
Rhona Stein ◽  
David M. Goldenberg
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Leukemic Cells ◽  
Tumor Type ◽  
Term Survival ◽  
Significant Difference ◽  
Highly Effective ◽  
Ic50 Values

Abstract Background: The humanized anti-CD74 monoclonal antibody, milatuzumab (hLL1, or IMMU-115; Immunomedics, Inc, Morris Plains, NJ), is in clinical evaluation for therapy of multiple myeloma (MM) after preclinical evidence of activity in this tumor type (Stein et al, Blood2004;104:3705). Here we examine the ability of milatuzumab to increase the efficacy of drugs in MM cell lines. Methods: MTT cytotoxicity assays were performed on a panel of MM cell lines, including CAG, KMS11, KMS12-PE, and MC/CAR, to examine the effects of bortezomib, doxorubicin (dox), and dexamethasone (dex) alone and combined with milatuzumab or milatuzumab + crosslinking 2nd Ab (goat anti-human IgG, GAH). In vivo studies used a CAG-SCID mouse model of disseminated disease. Results: Without drugs, crosslinked milatuzumab, but not milatuzumab alone, yielded significant anti-proliferative effects on the four MM cell lines. In combination studies, crosslinked milatuzumab produced significant reductions in the IC50 values of the anti-MM drugs. For example, in CAG, milatuzumab+GAH decreased the IC50 values 58%, 78%, and 98% for bortezomib, dox, and dex, respectively (P=0.0034, 0.0073, and 0.078, respectively). In vivo, milatuzumab at 100 μg/injection, 2x weekly for 4 weeks, starting 1 day after injection of CAG cells, more than doubled the median survival time (MST) from 42 days in untreated CAG-bearing SCID mice to 103 days. Combination therapy with milatuzumab and bortezomib or dox was compared to milatuzumab alone, with treatments initiated 5 days after injection of CAG cells. Bortezomib alone (1.0 mg/kg) increased MST from 33 to 44 days (P=0.0021 vs. untreated). Treatment with milatuzumab alone (100 μg/mouse) increased the MST to 73 days (P<0.0001 vs. untreated). When bortezomib and milatuzumab treatments were combined, the MST increased to 93 days (P=0.0441 vs. milatuzumab and P=0.0065 vs. bortezomib). Thus, the combination of milatuzumab and bortezomib increased survival significantly compared to either single treatment. Given alone, dox yielded little or no effect on survival compared with untreated animals, and there was no significant difference between milatuzumab monotherapy and milatuzumab plus doxorubicin in this model. In contrast, a milatuzumabdox immunoconjugate was found to be a highly effective therapeutic agent, with all mice achieving long-term survival. The inhibition of the NF-κB survival pathway of B-leukemic cells by milatuzumab supports its complementary effects when combined with drugs having different mechanisms of action, such as bortezomib. Conclusions: The therapeutic efficacies of bortezomib, dox, and dex are enhanced in vitro in MM cell lines when given in combination with milatuzumab. In vivo, milatuzumab alone or especially in combination with bortezomib is highly effective in MM. (Supported in part by USPHS grant P01CA103985 from the NCI, and grants from the Thomas and Agnes Carvel Foundation and the Walter and Louise Sutcliffe Foundation.)

Role of Erythropoietin Receptor in t(12;21) Positive Acute Lymphoblastic Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2792-2792
Author(s):  
Renate Panzer-Gruemayer ◽  
Gerd Krapf ◽  
Dominik Beck ◽  
Gerhard Fuka ◽  
Christian Bieglmayer ◽  
...  
Keyword(s):  
Cell Lines ◽  
Fusion Gene ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cell ◽  
Erythropoietin Receptor ◽  
Molecular Signature ◽  
Leukemic Cells ◽  
Drug Induced ◽  
Induced Apoptosis

Abstract The chromosomal translocation t(12;21)(p13;q22) resulting in the TEL/AML1 (also known as ETV6/ RUNX1) fusion gene is the most frequent translocation in childhood B cell precursor (BCP) ALL. This type of ALL is characterized by a unique molecular signature, which includes the overexpression of the gene for the erythropoietin receptor (EpoR). So far, it is not known what causes the overexpression of the EpoR gene or whether it has any effect on the t(12;21) positive leukemia. We therefore aimed to evaluate potential mechanisms responsible for the upregulation of the EpoR in t(12;21) leukemias and to find out whether signalling via this receptor affects survival or proliferation of leukemic cells. In addition, we planned to explore signalling pathways linked to the respective effects and to elucidate relevant mechanisms that might be essential for cell survival. We first excluded the possibility that the EpoR expression is upregulated as a consequence of high Epo levels in the plasma that are induced by the patients’ low hemoglobin (Hb) levels. While Hb levels from patients with t(12;21)+ ALL were significantly lower compared to those with other subtypes of BCP ALL (median, 6,15g/dL and 7,9g/dL, respectively; p<0.001 Wilcoxon 2- sample test), which correlated with high Epo levels in the plasma, the extent of EpoR mRNA expression of leukemic cells was independent of the respective amount of Epo in the individual patient’s plasma. Next, the influence of Epo on t(12;21) + leukemic cell lines was evaluated and revealed a consistent time and dose dependent increase in proliferation (Epo concentrations 10, 50, 100U/ml for 72 hours) determined by 3H-Thymidine incorporation. This effect was abrogated upon addition of a blocking anti-EpoR antibody thereby confirming the specificity of EpoR signalling. Since Epo may have apoptosis-modulating potential in EpoR expressing malignant cells, we tested its influence on drug-induced apoptosis. For this purpose IC50 concentrations of drugs that are commonly used for the treatment of children with BCP ALL were used. A reduction of glucocorticoid (GC)-induced apoptosis by Epo was demonstrated in t(12;21)+ cell lines while no effect was seen in combination with other drugs or in t(12;21) negative cell lines. Preliminary data indicate that NF-kappa B as well as PI3K/Akt pathways are triggered by Epo, implying that they play a role in this rescue mechanism. Given that cell lines may have intrinsic changes, we are presently evaluating whether the observed results can also be reproduced in primary leukemic cells. In support of this assumption are results in a limited number of primary t(12;21)+ leukemias showing a superior survival (MTT assay) and reduced apoptosis rate to GC when cultured in the presence of Epo. These findings are in contrast to those in t(12;21) negative BCP ALLs. In conclusion, our data indicate that overexpression of EpoR in t(12;21) positive leukemias is not induced by low Hb, a feature that is generally observed in patients with this type of leukemia. Binding of Epo to its receptor in vitro leads to enhanced survival and negatively affects the sensitivity to GCs. Whether these findings have any implications on the treatment and care of patients with t(12;21)+ leukemia needs to be addressed in further studies. Financial support: OENB10720, FWF P17551-B14 and GENAU-CHILD Projekt GZ200.136/1 - VI/1/2005 to RPG.

Targeting Aminopeptidases by Tosedostat (TST) (CHR2797), Alone and with LBH589, Induces Significant Cytotoxicity Against Human Multiple Myeloma (MM) Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1847-1847
Author(s):  
Chirag Acharya ◽  
Mike Y Zhong ◽  
Daniel Tannenbaum ◽  
Michelle Chen ◽  
Matt Ma ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Caspase 3 ◽  
Combined Treatment ◽  
Single Agent ◽  
Annexin V ◽  
Novel Approach ◽  
Human Multiple Myeloma ◽  
Induced Apoptosis ◽  
G1 Cells

Abstract Abstract 1847 Aminopeptidases (AP) are necessary for the growth and development of malignant cells and have a selectively important role in the maintenance of intracellular amino acid (AA) levels in neoplastic cells. CHR2797 is a novel, low nanomolar inhibitor of the M1 family of AP, a group of metalloenzymes containing a central Zn2+ ion. CHR2797 has antiproliferative and apoptotic effects against MM in vitro by inducing the AA deprivation response (AADR). TST, an oral, chronically administered agent with a good safety profile has demonstrated activity in patients with relapsed/refractory AML and is currently under study as part of combination therapy for untreated elderly patients with AML. At the epigenetic regulatory level, Zn-dependent histone deacetylase (HDAC) cause the deacetylation of histone and non-histone cellular proteins which are critical for gene expression, inducing apoptosis and cell cycle arrest in cancer cells. LBH589 (Panobinostat) is an established pan-HDAC inhibitor with potent in vitro anti-cancer activity in many hematological malignancies. The clinical efficacy of Panobinostat is currently being studied in several Phase II/III clinical trials with particular promise seen in the treatment of MM. Here we examined the potential therapeutic effect of CHR2797, alone and with LBH589, against MM cells. Using MTS and CTG assays, CHR2797, at clinically achievable concentrations, decreased survival and proliferation in MM1S and IL-6-dependent ANBL6 cells, in the presence or absence of bone marrow stromal cells following 72 hours incubation. CHR2797 induces apoptosis in MM cells via activation of Caspase 3/7 and 9 but not Caspase 8. Significantly, CHR2797 (10 μM) induced apoptosis in patient MM cells, as seen by % of annexin V and PI from 22 + 1.5% to 39 + 2.3% after 48h incubation. Combined treatment with CHR2797 and LBH589 in MM cells (MM1S, ANBL6, and INA6) further reduced cell viability following 72 hour incubation when compared with CHR2797 treatment alone, as determined by CTG viability luminescent assay. Both drugs together also augmented growth inhibitory effects when compared with single agent alone, after 72 hours incubation followed by MTS assay. Importantly, the combination of both drugs increased caspase 3/7- & 9-mediated apoptosis than CHR2797 alone in these MM cells following 24h-treatment. Cell cycle analysis (CHR2797 at 1μM; LBH589 at 1 nM) showed an increased growth arrest in G0/G1 cells in MM1R cells treated with both drugs versus CHR2797 alone after 24 hours: 68.5±3.3% versus 36±2.5%. Furthermore, CHR2797 inhibited anti-apoptotic protein Mcl-1 in MM1R and U266 MM cells by immunoblottings. Combined treatment with CHR2797 and LBH589 further blocked Mcl-1 when compared with either treatment alone after 24 hours incubation. Together, these results show that the combination of CHR2797 and LBH589 enhanced anti-myeloma effects when compared with either drug alone. This combination, which also has the potential of being without overlapping clinical toxicities, provides a promising novel approach to anti-myeloma therapy. Disclosures: Singer: Cell Therapeutics, Inc: Employment, Equity Ownership. Richardson:Novartis: Membership on an entity's Board of Directors or advisory committees.

Thymic Stromal Lymphopoeitin Signaling Contributes To Increased Risk Of Relapse Of Pediatric Acute Lympoblastic Leukemia Through Enhanced Survival Of Leukemic Cells That Overexpress Thymic Stromal Lymphopoeitin Receptor

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2543-2543
Author(s):  
Christopher D Chien ◽  
Matthew Kreitman ◽  
Haiying Qin ◽  
Terry J Fry
Keyword(s):  
Bone Marrow ◽  
Annexin V ◽  
Thymic Stromal Lymphopoietin ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
Early Stages ◽  
Increased Risk ◽  
Significant Difference ◽  
Risk Of Relapse

Abstract Pediatric acute lymphoblastic leukemia (ALL) is the most common childhood malignancy. Although the cure rate for this disease is greater than 85%, ALL remains the number one cause of cancer-related deaths in children due to relapse of ALL. Therefore, there is a great need to identify new therapies for patients who have recurrent disease. Recently, a subset of pediatric ALL patients whose leukemic cells express high levels of thymic stromal lymphopoietin receptor (TSLPR/CRLF2) have been shown to have an increased risk of relapse and shorter disease free and poorer overall survival. Overexpression of TLSPR occurs in 8% of unscreened pediatric precursor B ALL and occurs by genomic rearrangement of the TSLPR gene, which fuses the unmutated TSLPR gene to altered transcriptional control or by other yet to be described means. The mechanism by which Thymic Stromal Lymphopoietin (TLSP) signaling contributes to increased risk of relapse is unknown. Studies have shown aberrant signaling in high TSLPR expressing ALL patient derived cell lines relying heavily on in vitro experiments. As no pre-clinical model of high TSLPR ALL has been published, we created a model of high TSLPR expressing leukemia to study TSLPR overexpressing leukemia progression. We have created a high TSLPR expressing leukemia cell line through retroviral transduction of a transplantable syngeneic mouse leukemia model in which the leukemic progression can be studied with physiologic levels of TSLP. This high TSLPR leukemia has levels of expression of TSLPR comparable to what is found on human leukemia that overexpress TSLPR. The TSLPR is functional in these cells and we see increased phosphorylation of STAT5 protein in response to IL-7 or TSLP stimulation. When we introduce the leukemia into mice and look at disease progression, we observed an 8 fold difference in the numbers of cells in the bone marrow 5 days after intravenous injection corresponding to an early stage of leukemia progression (Figure 1. high TSLPR 1.61%+/-0.95 vs. low TSLPR 0.20%+/-0.16). Interestingly we find no significant difference in long term survival of mice injected with either low or high TSLPR leukemia lines. The increased numbers of leukemic cells in the bone marrow at early stages of leukemic progression could be due to an increased rate of proliferation or better survival/engraftment. Low and high TSLPR expressing cells show no significant difference in growth rate in vitro or in vivo in dye dilution assays (Figure 2) suggesting that the increase in leukemic cells in the bone marrow is through enhanced survival. To test this, we treated low and high TSLPR leukemia lines with the steroid dexamethasone in the absence or presence of TSLP. We found that the addition of TSLP significantly reduced the Annexin V positive relative to cells not treated with TSLP in the high TSLP expressing leukemia cells, while in low TSLPR expressing cells we observed no decrease in Annexin V positive cells (Figure 3). This suggests that high TSLPR expression sensitizes leukemia cells to TSLP in the leukemia microenvironment. To confirm that this is the case we have found by gene expression analysis that we can detect TSLP in mouse bone marrow. We hypothesize that therapies targeting the TSLP signaling axis in ALL would decrease the risk of relapse. To test this hypothesis we have generated TSLPR-Fc conjugates to block TSLP signaling. We plan on using these reagents to block TSLP signaling to see if we can reverse the increased amounts of leukemia we find in mice at early stages of leukemic progression as well as the eliminate the survival advantage provided by TSLP to high TSLPR expressing leukemic cells in response to chemotherapeutic agents. Disclosures: No relevant conflicts of interest to declare.

Chemoprevention Against Hepatocellular Carcinoma of Cornus officinalis in vitro

2004 ◽  
Vol 32 (05) ◽  
pp. 717-725 ◽  
Author(s):  
Jung-San Chang ◽  
Lien-Chai Chiang ◽  
Fen-Fang Hsu ◽  
Chun-Ching Lin
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Lines ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Xtt Assay ◽  
Leukemic Cell Lines ◽  
Cornus Officinalis ◽  
Anti Oxidant ◽  
Dose Dependent

The water extracts of Cornus officinalis Sieb. et Zuce against hepatocellular carcinoma (HCC) was studied for its chemopreventive potential. Three HCC cell lines (HepG2, SK-Hep1 and PLC/PRF/5) and three leukemic cell lines (U937, K562 and Raji) were tested with XTT assay. Extracts of C. officinalis inhibited all these HCC cells and leukemic cells at a concentration of 100 μg/ml (P<0.05) and was dose-dependent (P<0.0001). P53 (P<0.0001) and Ras (P=0.001) significantly affected its activity against HCC. Extracts of C. officinalis also possessed the anti-oxidant activity through free radicals scavenging activity at a concentration of 50 μg/ml (P<0.05). In summary, our experiment implied that C. officinalis might be a candidate for chemopreventive agent against HCC through the antioxidant and anti-neoplastic effects.

scholarly journals Sertad1 Antagonizes iASPP Function By Hindering Its Entrance into Nuclei to Interact with P53 in Leukemic Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5201-5201
Author(s):  
Shaowei Qiu ◽  
Jing Yu ◽  
Tengteng Yu ◽  
Haiyan Xing ◽  
Na An ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Leukemic Cell ◽  
K562 Cells ◽  
Expression Level ◽  
Leukemic Cells ◽  
Immunoblotting Analysis ◽  
Chemotherapy Drugs

Abstract Introduction: As the important suprressor of P53, iASPP was found to be overexpressed in leukemia, and functioned as oncogene that inhibited apoptosis of leukemia cells. Sertad1 is identified as one of the proteins that can bind with iASPP in our previous study by two-hybrid screen. Sertad1 is highly expressed in carcinomas from pancreatic, lung and ovarian tissues, which considered Sertad1 as an oncoprotein. In this study, our findings revealed that Sertad1 could interact with iASPP in the cytoplasm near nuclear membrane, which could block iASPP to enter into nucleus to interact with P53, and inhibited the function of iASPP eventually. Methods: Co-immunoprecipitation and fluorescence confocal microscopic imaging were used to confirm the interaction between iASPP and Sertad1, the exact binding domains and the subcellular colocalization.The plasmids of iASPP and Sertad1 were transfected alone or co-transfected into K562 cells, the stable subclones that highly expressed iASPP, Sertad1 or both of them were then established by limiting dilution and named as K562-iASPPhi, K562-Sertad1hi, and K562-Douhi, respectively. The cell proliferation, cell cycle and apoptosis of above subclones were investigated by flow cytometry. Further, silence of the above two proteins was performed to confirm their functions. Immunoblotting analysis and immunofluorescence were performed to explore the possible mechanisms of difference between the biological functions of the above subclones. Results: Sertad1 expression level varied in leukemic cell lines and AML patients irrespectively of iASPP and P53. Interaction between iASPP and Sertad1 did exist in 293 cell and leukemic cells, both iASPP and Sertad1 scattered in the cytoplasm and nucleus, and their colocalizations were mainly in the cytoplasm, which encircled the nucleus. iASPP binds directly to Sertad1 through its PHD-bromo domain, C-terminal domain and Cyclin-A domain in a reduced order, and Serta domain failed to bind to iASPP. Overexpression of iASPP in K562 cells (iASPPhi) could result in the increased cell proliferation, cell cycle arrest in G2/M phase and resistance to apoptosis induced by chemotherapy drugs. While overexpression of iASPP and Sertad1 at the same time (Douhi) could slow down the cell proliferation, lead the cells more vulnerable to the chemotherapy drugs. As figure showed, in K562-Douhi cells, both iASPP and Sertad1 were obviously located in the cytoplasm, which encircled the nuclei, the subcellular colocalization was nearly outside the nuclei. The immunoblotting analysis further supported the conclusions. The resistance of iASPP to chemotherapeutic drug was accompanied by Puma protein expression in a p53-independent manner. By knocking down the expersssion of iASPP and Sertad separately, we found that iASPP is dispensable for maintenance of anti-apoptotic function and Sertad1 is indispensable for cell cycle in leukemic cells. Conclusions: In normal situation, the protein iASPP and Sertad1 scatter in the nucleus and cytoplasm, mainly in the cytoplasm. As convinced by our study, iASPP was overexpressed in the leukemia cell lines and primary AML patients, it could function as oncogene through its binding with P53 protein in the nucleus, inhibit the function of P53. When iASPPhi cells were exposed to apoptosis stimuli, Puma protein could play an important role in this process, irrespective of the expression level of P53. But when iASPP and Sertad1 were both overexpressed in the leukemic cells, Sertad1 could tether iASPP outside the nucleus mainly through its PHD-bromo domain, prevent it from inhibiting P53 function, suppress the leukemic cell growth and stimulate cell apoptosis by rescuing the P53 eventually. Our data provided a new insight to overcome iASPP protein, namely through its binding partners, when the similar proteins or drugs that can tether iASPP outside the nucleus such as Sertad1 are transfected into the leukemic cells, it may restore p53 function to eliminate the leukemic cells. Figure 1 Figure 1. Disclosures Wang: Novartis: Consultancy; Bristol Myers Squibb: Consultancy.

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