Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0023418 (leukemia)
 93,477 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cocultivation of a clonal factor-dependent hematopoietic cell line (FDC-P1JL26) with an irradiated bone marrow stromal cell line (D2XRII) significantly increased the frequency of isolation of factor-independent subclones. Eight out of nine factor-independent subclonal lines showed expression of IL-3, GM-CSF or both cytokine mRNAs by reverse-transcription polymerase chain reaction (RT-PCR) and seven of these expressed biologically active GM-CSF or IL-3. In three cell lines that synthesized biologically active IL-3 (FIJ1, FIJ4D and FIJ10D) insertion of an IAP sequence into the IL-3 gene was detected by PCR analysis and the insertions were confirmed by DNA sequence analysis of PCR or RT-PCR fragments. In the four cell lines in which no IL-3 expression was detected no IAP insertions were detected. Rearrangements of the GM-CSF gene were detected in three factor-independent cell lines and an insertion of an IAP into the GM-CSF gene was confirmed by DNA sequence analysis of PCR fragments. In contrast to results with IL-3, insertion of an IAP into the GM-CSF gene did not correlate with GM-CSF expression. In one cell line that contained an IAP insertion into the GM-CSF gene, no GM-CSF was detected by biological assay nor by RT-PCR. Retrotransposition of IAPs may be responsible for the emergence of factor-independent cells in our cocultivation system and other IAP insertions may prove to be responsible for the factor-independent phenotype seen in the non-autocrine factor-independent cell line, FI7CL2.
Leukemia 1998 Jan
PMID:The role of intracisternal A-type particles in the evolution of factor-independent murine hematopoietic cell lines. 943 15

Although transduction with amphotropic murine leukemia virus (MLV) vectors has been optimized successfully for hematopoietic differentiated progenitors, gene transfer to early hematopoietic cells (stem cells) is still highly restricted. A similar restriction to gene transfer was observed in the mouse stem cell line FDC-Pmix compared with transfer in the more mature myeloid precursor cell line FDC-P1 and the human erythroleukemia cell line K562. Gene transfer was not improved when the vector was pseudotyped with gp70SU of the 10A1 strain of MLV, which uses the receptor of the gibbon ape leukemia virus (Pit1), in addition to the amphotropic receptor (Pit2). Although 10A1 and amphotropic gp70SU bound to FDC-P1, K562, and fibroblasts, no binding to FDC-Pmix cells was detected. This indicates that FDC-Pmix cells lack functional Pit2 and Pit1 receptors. Pseudotyping with the vesicular stomatitis virus G protein improved transduction efficiency in FDC-Pmix stem cells by 2 orders of magnitude, to fibroblast levels, confirming a block to retroviral infection at the receptor level.
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PMID:Entry of amphotropic and 10A1 pseudotyped murine retroviruses is restricted in hematopoietic stem cell lines. 944 44

Hematopoietic stem cells (HSC) are an important target for retroviral gene transfer. However, transduction efficiency in these HSC is extremely low compared to fibroblasts or more mature hematopoietic cells. This infection block was analyzed in the HSC line FDC-Pmix. The infection frequency with the amphotropic murine leukemia virus (MLV-A) is more than 100-fold lower in FDC-Pmix cells as compared to fibroblasts. Pseudotyping with the env of the 10A1 strain (MLV-10A1), which uses both the amphotropic receptor (Pit-2) and the receptor for gibbon ape leukemia virus (Pit-1), did not improve the infection efficiency. Vectors pseudotyped with VSV G protein were found to overcome the infection block in FDC-Pmix, confirming that the block is at the level of virus binding and possibly penetration. Accordingly, we could not detect virus binding of MLV-A or MLV-10A1 to FDC-Pmix cell lines. Northern blot analysis was performed to detect whether the defect is at the level of transcription. Surprisingly, similar levels of Pit-2 receptor transcripts were detected in all cell types. The overexpression of rat Pit-2 DNA in CHO but not in FDC-Pmix cells improved amphotropic infection frequency after introducing rat Pit-2 DNA into the cells. Taken together these results show that the inefficient infection of FDC-Pmix is due to a lack of functional receptors. Either the receptor protein is incorrectly processed in these cells or a cofactor is missing in FDC-Pmix cells that is necessary for efficient binding and/or penetration.
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PMID:Lack of functional Pit-1 and Pit-2 expression on hematopoietic stem cell lines. 958 96

The Bcr/Abl tyrosine kinase that is expressed from the Philadelphia chromosome protects leukemia cells from apoptosis caused by removal of growth factors or by cytotoxic agents and ionizing irradiation. This resistance to apoptosis is associated with a Bcr/Abl-mediated G2/M delay. Therefore, inhibiting Bcr/Abl signaling pathways should block the ability of the Bcr/Abl kinase to protect cells from apoptosis. The monoterpenes, limonene and perillyl alcohol (POH) are new anticancer agents that selectively induce apoptosis in neoplastic cells of a variety of rodent carcinoma models. Since the potential antitumor activities of monoterpenes overlap with signaling pathways affected by the Bcr/Abl kinase, POH and limonene were tested for antileukemia activity. POH, but not limonene selectively induced G0/G1 arrest followed by apoptosis in Bcr/Abl-transformed, but not nontransformed FDC.P1 and 32D myeloid cell lines. In contrast to their greater sensitivity to POH, Bcr/Abl-transformed cells were more resistant than nontransformed cells to several chemotherapy agents and ionizing irradiation. Since in Bcr/Abl-transformed cells, POH induces apoptosis associated with G0/G1 arrest, POH must activate an apoptotic pathway that is not protected by the Bcr/Abl-induced G2/M delay. Monoterpenes may represent novel agents for treating Ph+ leukemias.
Leukemia 1999 Oct
PMID:Perillyl alcohol selectively induces G0/G1 arrest and apoptosis in Bcr/Abl-transformed myeloid cell lines. 1051 60

Mcl-1, a member of the Bcl-2 family, has been identified as an inhibitor of apoptosis induced by anticancer agents and radiation in myeloblastic leukemia cells. The molecular mechanism underlying this phenomenon, however, is not yet understood. In the present study, we report that hyperpolarization of the membrane potential is required for prevention of mcl-1 mediated cell death in murine myeloblastic FDC-P1 cells. In cells transfected with mcl-1, the membrane potential, measured by the whole-cell patch clamp, was hyperpolarized more than -30 mV compared with control cells. The membrane potential was repolarized by increased extracellular K(+) concentration (56 mV per 10-fold change in K(+) concentration). Using the cell-attached patch-clamp technique, K(+) channel activity was 1.7 times higher in mcl-1 transfected cells (NP(o) = 22.7 +/- 3. 3%) than control cells (NP(o) = 13.2 +/- 1.9%). Viabilities of control and mcl-1 transfected cells after treatment with the cytotoxin etoposide (20 microgram/ml), were 37.9 +/- 3.9% and 78.2 +/- 2.0%, respectively. Suppression of K(+) channel activity by 4-aminopyridine (4-AP) before etoposide treatment significantly reduced the viability of mcl-1 transfected cells to 49.0 +/- 4.6%. These results indicate that as part of the prevention of cell death, mcl-1 causes a hyperpolarization of membrane potential through activation of K(+) channel activity.
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PMID:Protection from cell death by mcl-1 is mediated by membrane hyperpolarization induced by K(+) channel activation. 1055 59

Despite the fact that RAF-1 lies immediately downstream of p21RAS in the MAP kinase-signalling cascade, recent evidence in non-haematopoietic environments suggest that RAS and RAF can transduce signals through alternative pathways specific to a particular cell type. Since mutational activation of RAS occurs at high frequency in human leukaemia, we have investigated the contribution of signalling from mutant RAF in mediating the transforming effects of the N-RAS oncogene in the growth factor-dependent cell line, FDC-P1. Independent activation of N-RAS extended the period of exponential growth leading to an increased saturating density under optimal growth conditions. Under conditions of growth factor withdrawal, cells expressing mutant RAS, but not control cells, demonstrated protection against apoptotic death. Although RAF promoted cell proliferation in a similar manner to that observed in FDCP-RAS cells, expression of mutant RAF was not as effective at protecting these cells against apoptotic death following growth factor withdrawal. The results suggest that RAS utilises RAF-dependent signals in promoting the proliferation of FDC-P1 cells but the anti-apoptotic effects of this oncogene are mediated through a RAF- and BCL-2-independent pathway.
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PMID:Alternative effects of RAS and RAF oncogenes on the proliferation and apoptosis of factor-dependent FDC-P1 cells. 1063 45

In this study, the abilities of constitutive and conditional forms of the three Raf kinases to abrogate the cytokine dependency of FDC-P1 cells were examined. The constitutively active forms (delta) of all three Raf kinases were fused to the hormone-binding domain of the estrogen receptor (ER), rendering their activities conditionally dependent upon exogenous beta-estradiol. The vast majority of deltaRaf:ER-infected FDC-P1 cells remained cytokine-dependent; however, cells were obtained at low frequency in which expression of deltaRaf:ER abrogated cytokine dependency. Isoform specific differences between the Raf kinases were observed as cytokine-independent cells were obtained more frequently from deltaA-Raf:ER than either deltaRaf-1:ER or deltaB-Raf:ER infected cells. To determine whether the regulatory phosphorylation sites in the Raf proteins were necessary for abrogation of cytokine dependency, they were changed by site-directed mutagenesis. Substitution with phenylalanine eliminated the transforming ability of the deltaB-Raf:ER and deltaRaf-1:ER kinases. However, a similar substitution in A-Raf did not extinguish its transforming activity. The activated Raf proteins induced essential downstream MEK1 activity as treatment with the MEK1 inhibitor, PD98059, suppressed Raf-mediated growth. Activated MAP kinases (ERK1 and ERK2) were detected in deltaRaf:ER-transformed cells, and their presence was dependent upon a functional MEK1 protein. The cytokine-independent phenotype required the continued activity of the deltaRaf:ER proteins as removal of beta-estradiol caused the cells to stop growing and undergo apoptosis. The Raf-responsive cells were found to express autocrine growth factors, which promoted their growth. Constitutive activation of the Raf-1 oncogene resulted in malignant transformation as cytokine-independent FDC-P1 cells infected with a retrovirus encoding an activated Raf-1 protein formed tumors upon injection of immunocompromised mice. In summary, Raf kinases can abrogate cytokine dependency, prevent apoptosis and induce the tumorigenicity of a certain subpopulation of FDC-P1 cells by a MEK1-dependent mechanism.
Leukemia 2000 Apr
PMID:Differential abilities of the Raf family of protein kinases to abrogate cytokine dependency and prevent apoptosis in murine hematopoietic cells by a MEK1-dependent mechanism. 1076 50

Flt3-ligand (Flt3-L) is an early acting costimulatory cytokine that has been shown to possess antitumor properties in murine solid tumor models. Flt3-L is a trans-membrane protein (tm) but can be proteolytically cleaved to a soluble form, which is also biologically active. In this study, the antitumor effect of both soluble and tmFlt3-L was evaluated in a mouse leukemia model. To mimic the multiorgan involvement characteristic of human leukemia, a factor-dependent cell line FDC.P1 was made leukemogenic by transfection with the human BCR/ABL gene. The resulting cell line, AW, expresses BCR/ABL RNA and protein. It maintains a similar in vitro growth rate as the parent cell line, but unlike the parent cell line, AW cells are factor independent and tumorigenic. Growth of FDC.P1 and AW cells are unaffected by the addition of soluble human Flt3-L to the culture medium. Also, AW growth is unaltered after transduction with a retroviral vector expressing the tm isoform of human Flt3-L (AW/tmFlt3-L). When 10(5) AW cells were i.v. injected into syngeneic DBA/2 mice, fatal leukemia developed in nine of nine (100%) mice within 4-6 weeks with involvement of the blood, bone marrow, spleen, and thymus. Systematic administration of soluble human Flt3-L (500 microg/kg/day) for 10 days protected mice from leukemia, with 11 of 17 mice tumor free at week 8 (64.7%) The tm isoform of Flt3-L also was protective. When 10(4) AW/tmFlt3-L cells were injected i.v. into mice, only 35.7% (5 of 14) developed leukemia versus 100% in control groups. Adoptive transfer of immunity was also demonstrated; T cells obtained from tumor-free animals conferred protection to 87% (seven of eight) naive mice challenged with AW cells. These results demonstrate that both soluble and membrane-bound human Flt3-L has antitumor activity in this leukemia model.
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PMID:Antileukemic activity of Flt3 ligand in murine leukemia. 1076 77

The Raf oncoprotein plays critical roles in the transmission of mitogenic signals from cytokine receptors to the nucleus. There are three Raf family members: A-Raf, B-Raf and Raf-1. Conditionally active forms of the Raf proteins were created by ligating N-terminal truncated activated forms to the estrogen-receptor (ER) hormone-binding domain resulting in beta-estradiol-inducible constructs. We introduced these chimeric deltaRaf:ER oncoproteins into the murine FDC-P1 hematopoietic cell line. Two different types of cells were recovered after drug selection in medium containing either cytokine or beta-estradiol: (1) cytokine-dependent cells that expressed the deltaRaf:ER oncoproteins; and (2) Raf-responsive cells that grew in response to the deltaRaf:ER oncoprotein. Depending upon the particular deltaRaf:ER oncoprotein, cytokine-dependent cells were recovered 10(3) to 10(5) times more frequently than Raf-responsive cells. To determine whether BCL2 could synergize with the deltaRaf:ER oncoproteins and increase the frequency of cytokine-independent cells, cytokine-dependent deltaRaf:ER-expressing cells were infected with either a BCL2 containing retrovirus or an empty retroviral vector. BCL2 overexpression, by itself, did not relieve cytokine dependency of the parental cell line. However, BCL2 overexpression increased the frequency of Raf-responsive cells approximately five- to 100-fold. Cytokine-dependent deltaRaf:ER-infected cells entered the G1 phase of the cell cycle after cytokine withdrawal and entered S phase only after cytokine addition. Raf-responsive deltaRaf:ER cells entered the G1 phase of the cell cycle after estrogen deprivation and re-entered the cell cycle after addition of either IL-3 or the estrogen receptor antagonist tamoxifen which activates the deltaRaf:ER constructs. Expression of the BCL2 oncoprotein often delayed the exit from the S and G2/M phases demonstrating the protective effects BCL2 provided to these Raf and BCL2 infected cells. The deltaRaf:ER cells expressed the deltaRaf:ER proteins and downstream MEK and ERK activities after beta-estradiol treatment. Raf-responsive cells that were also infected with BCL2 expressed higher levels of BCL2 than the cells that were not infected with BCL2. Thus BCL2 can synergize with the activated Raf in the abrogation of cytokine dependency of certain hematopoietic cells. These cells will be useful in furthering our understanding of the roles of the Raf and BCL2 oncoproteins in hematopoietic cell growth, cell cycle progression and prevention of apoptosis.
Leukemia 2000 Jun
PMID:Synergy between Raf and BCL2 in abrogating the cytokine dependency of hematopoietic cells. 1086 73

The MEK1 oncoprotein plays a critical role in Ras/Raf/MEK/MAPK-mediated transmission of mitogenic signals from cell surface receptors to the nucleus. In order to examine this pathway's role in leukemic transformation, a conditionally active (beta-estradiol-inducible) form of the MEK1 protein was created by ligating a cDNA encoding an N-terminal truncated form of MEK1 to the hormone-binding domain of the estrogen receptor (ER). We introduced this chimeric deltaMEK1:ER oncoprotein into cytokine-dependent human TF-1 and murine FDC-P1 hematopoietic cell lines. Two different types of cells were recovered after drug selection in medium containing either cytokine or beta-estradiol: (1) cells that expressed the deltaMEK1:ER oncoprotein but remained cytokine-dependent and (2) MEK1-responsive cells that grew in response to deltaMEK1:ER activation. Cytokine-dependent cells were recovered 10(2) to 10(4) times more frequently than MEK1-responsive cells depending upon the particular cell line. To determine whether BCL2 overexpression could synergize with the deltaMEK1:ER oncoprotein in relieving cytokine dependence, the cytokine-dependent deltaMEK1:ER-expressing cells were infected with a BCL2-containing retrovirus, and the frequency of MEK1-responsive cells determined. BCL2 overexpression, by itself, did not relieve cytokine dependency of the parental cells, however, it did increase the frequency at which MEK1-responsive cells were recovered approximately 10-fold. DeltaMEK1:ER+BCL2 cells remained viable for at least 3 days after estradiol deprivation, whereas viability was readily lost upon withdrawal of beta-estradiol in the MEK1-responsive cells which lacked BCL2 overexpression. The MAP kinases, ERK1 and ERK2 were activated in response to deltaMEK1:ER stimulation in both deltaMEK1:ER and deltaMEK1:ER+BCL2 cells. As compared to the cytokine-dependent deltaMEK1:ER and BCL2 infected cells, MEK1-responsive BCL2 infected cells expressed higher levels of BCL2. While both MEK1-responsive deltaMEK1:ER and deltaMEK1:ER+BCL2 infected cells expressed cDNAs encoding the autocrine cytokine GM-CSF, more GM-CSF cDNAs and bioactivity were detected in the MEK1-responsive deltaMEK1:ER+BCL2 cells than in the MEK1-responsive cells lacking BCL2 or cytokine-dependent cells. These conditionally transformed cells will be useful in furthering our understanding of the roles MEK1 and BCL2 play in the prevention of apoptosis in hematopoietic cells.
Leukemia 2000 Jun
PMID:Combined effects of aberrant MEK1 activity and BCL2 overexpression on relieving the cytokine dependency of human and murine hematopoietic cells. 1086 74


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