PI3K/Akt pathway regulates retinoic acid‐induced Hox gene expression in F9 cells

Retinoic acid (RA), the most potent natural form of vitamin A, is a key morphogen in vertebrate development and a potent regulator of both adult and embryonic cell differentiation. Specifically, RA regulates clustered Hox gene expression during embryogenesis and is required to establish the anteroposterior body plan. The PI3K/Akt pathway was also reported to play an essential role in the process of RA‐induced cell differentiation. Therefore, we tested whether the PI3K/Akt pathway is involved in RA‐induced Hox gene expression in a F9 murine embryonic teratocarcinoma cells. To examine the effect of PI3K/Akt signaling on RA‐induced initiation of collinear expression of Hox genes, F9 cells were treated with RA in the presence or absence of PI3K inhibitor LY294002, and time‐course gene expression profiles for all 39 Hox genes located in four different clusters—Hoxa, Hoxb, Hoxc, and Hoxd—were analyzed. Collinear expression of Hoxa and ‐b cluster genes was initiated earlier than that of the ‐c and ‐d clusters upon RA treatment. When LY294002 was applied along with RA, collinear expression induced by RA was delayed, suggesting that the PI3K/Akt signaling pathway somehow regulates RA‐induced collinear expression of Hox genes in F9 cells. The initiation of Hox collinear expression by RA and the delayed expression following LY294002 in F9 cells would provide a good model system to decipher the yet to be answered de novo collinear expression of Hox genes during gastrulation, which make the gastrulating cells to remember their positional address along the AP body axis in the developing embryo.     

24p3 in differentiation of myeloid cells

24p3 is a secreted lipocalin that has been variously related to apoptosis, proliferation, and the neutrophil lineage of blood cells. We have investigated the expression of 24p3 mRNA and protein in myeloid cell lines induced to differentiate by insulin-like growth factor 1 (IGF-1) and the granulocytic-colony simulating factor (G-CSF). Both these growth factors, which cause myeloid cells to differentiate into granulocytes, induced a marked increase in the expression of both 24p3 protein and mRNA. The mRNA especially appeared early after the cells were induced with either IGF-1 or G-CSF, at a time when the cells were still proliferating and are morphologically undifferentiated. 24p3 can be considered an early marker of granulocytic differentiation.     

Mutation ofp53Is Not a Prerequisite for Immortalization of Human Fibroblasts by SV40 T Antigen

Thein vitrolife span of human cells is under genetic control and limited. Immortalized cells, however, can be obtained at a low frequency following expression of the SV40 T antigen gene though the steps that lead to immortality are not well understood. p53 has been implicated in cell cycle regulation and evidence suggests it may have a role in controlling life span in rodent and human cells. In this study, we investigated whether allelic loss or mutation ofp53was an essential step during SV40 immortalization leading to the appearance of immortal cell lines. The gross structure of thep53gene was examined in a primary fibroblast cell strain (1BR.3) and two SV40-immortalized derivatives, 1BRMT1 and 1BRgn2. There was no evidence for allelic loss of thep53gene during immortalization. The primary cells and the immortal derivatives all expressed authenticp53mRNAs, though the immortal cell lines had higher levels of expression. Sequence analysis of exons 5–8 did not detect mutations associated with the immortal phenotype. These data are consistent with SV40 immortalization being independent of genetic changes inp53.     

Conditionally Immortalized Neural Cell Lines: Potential Models for the Study of Neural Cell Function

Studies on primary cell cultures have contributed significantly to our understanding of neural cell function. Nevertheless, for many studies the value of these primary cell cultures has been limited by the time the cultures survivein vitro,the quantity of cellular material available for analysis, and the need to prepare the cells on a regular basis from fresh tissue. Techniques for immortalizing cells have existed for some time, but the repertoire of immortalizing genes has grown significantly. This has expanded our ability to generate useful cell lines of specific neural types that are better models of thein vivophenotype than previously. The constitutive expression of oncogenes keeps cells in a proliferative state that could lead to the loss of differentiated gene expression and function. An appealing improvement of immortalization methodology is the use of temperature-sensitive oncogenes that generate cell lines that can proliferate at a permissive temperature and “differentiate” at a nonpermissive temperature. The proliferation of such conditionally immortalized cell lines can be suppressed simply by increasing the temperature. Cell lines maintained at the nonpermissive temperature can enter into a stage in which they express differentiated properties of the cell. The potential ability of conditionally immortalized neural cell lines to accurately reflect theirin vivofunction has now been demonstrated on several occasions through transplantation experiments. In this report, the generation of these cell lines is described along with a discussion of their potential applications in neurobiology.     

A novel immortalization vector for the establishment of penaeid shrimp cell lines

Cell immortalization technology based on gene transfer has been successfully used to generate cell lines from a wide variety of cell types. The inability to stably introduce and express foreign genes has hampered application of this strategy in shrimp cells. We report here the use of replication-defective pantropic retrovirus to achieve a novel immortalization vector in which simian virus 40 large T antigen (SV40T) gene is expressed from Moloney murine leukemia virus (MoMLV) promoter. Data confirmed the presence of transferred SV40T gene and its stable mRNA expression in transduced lymphoid cells of Penaeus chinensis. The transduced cells showed a higher growth rate and a longer replication life-span compared with their untransduced counterparts. These results indicate the pantropic retrovirus-based immortalization-inducing gene delivery system is a potential tool for establishing cell lines from shrimp. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Immortalization of normal human mammary epithelial cells in two steps by direct targeting of senescence barriers does not require gross genomic alterations

Telomerase reactivation and immortalization are critical for human carcinoma progression. However, little is known about the mechanisms controlling this crucial step, due in part to the paucity of experimentally tractable model systems that can examine human epithelial cell immortalization as it might occur in vivo. We achieved efficient non-clonal immortalization of normal human mammary epithelial cells (HMEC) by directly targeting the 2 main senescence barriers encountered by cultured HMEC. The stress-associated stasis barrier was bypassed using shRNA to p16^INK4; replicative senescence due to critically shortened telomeres was bypassed in post-stasis HMEC by c-MYC transduction. Thus, 2 pathologically relevant oncogenic agents are sufficient to immortally transform normal HMEC. The resultant non-clonal immortalized lines exhibited normal karyotypes. Most human carcinomas contain genomically unstable cells, with widespread instability first observed in vivo in pre-malignant stages; in vitro, instability is seen as finite cells with critically shortened telomeres approach replicative senescence. Our results support our hypotheses that: (1) telomere-dysfunction induced genomic instability in pre-malignant finite cells may generate the errors required for telomerase reactivation and immortalization, as well as many additional “passenger” errors carried forward into resulting carcinomas; (2) genomic instability during cancer progression is needed to generate errors that overcome tumor suppressive barriers, but not required per se; bypassing the senescence barriers by direct targeting eliminated a need for genomic errors to generate immortalization. Achieving efficient HMEC immortalization, in the absence of “passenger” genomic errors, should facilitate examination of telomerase regulation during human carcinoma progression, and exploration of agents that could prevent immortalization.     

MLL-ENL cooperates with SCF to transform primary avian multipotent cells

The MLL gene is targeted by chromosomal translocations, which give rise to heterologous MLL fusion proteins and are associated with distinct types of acute lymphoid and myeloid leukaemia. To determine how MLL fusion proteins alter the proliferation and/or differentiation of primary haematopoietic progenitors, we introduced the MLL-AF9 and MLL-ENL fusion proteins into primary chicken bone marrow cells. Both fusion proteins caused the sustained outgrowth of immature haematopoietic cells, which was strictly dependent on stem cell factor (SCF). The renewing cells have a long in vitro lifespan exceeding the Hayflick limit of avian cells. Analysis of clonal cultures identified the renewing cells as immature, multipotent progenitors, expressing erythroid, myeloid, lymphoid and stem cell surface markers. Employing a two-step commitment/differentiation protocol involving the controlled withdrawal of SCF, the MLL-ENL-transformed progenitors could be induced to terminal erythroid or myeloid differentiation. Finally, in cooperation with the weakly leukaemogenic receptor tyrosine kinase v-Sea, the MLL-ENL fusion protein gave rise to multilineage leukaemia in chicks, suggesting that other activated, receptor tyrosine kinases can substitute for ligand-activated c-Kit in vivo.     

IFI 16 gene encodes a nuclear protein whose expression is induced by interferons in human myeloid leukaemia cell lines

We have characterized the induction of mRNA and protein products of the human IFI 16 gene in response to IFN-γ, IFN-α, and IFN-β₂ (IL-6). We demonstrate that the IFI 16 gene product is a novel nucleoprotein expressed in association with the differentiation of myeloid precursor cell lines. In Northern blots, IFI 16 mRNA was increased ～25-fold above barely detectable levels in unstimulated promyelocytic HL-60 cells, in response to IFN-γ. Other myeloid cell lines, U937 and K562, also demonstrated a marked IFN-γ-inducibility of IFI 16 mRNA. However, all three cell lines were far less responsive to IFN-α, and there was no response to IL-6. By comparison, a panel of T and B cell lines demonstrated high constitutive expression of IFI 16 mRNA that was not regulated by these cytokines. Culture of HL-60 cells in medium containing dimethylsulfoxide, retinoic acid, and 1,25 dihydroxyvitamin D₃, agents that stimulate the differentiation of HL-60 along myeloid pathways, also caused the induction of IFI 16 mRNA. To characterize the protein product of IFI 16, a monoclonal antibody was raised against a recombinant bacterial protein comprising the amino terminal 159 amino acids of IFI 16 fused to glutathione S-transferase. The antibody, designated 1G7, was used in Western blotting to demonstrate the strong induction of a cluster of proteins of 85–95 kDa in the nuclear extracts of IFN-γ-treated HL-60. The nuclear localization of IFI 16 antigen was confirmed by immunohistochemical staining of HL-60 cells treated with IFN-γ, dimethylsulfoxide, and retinoic acid. IFI 16 was also detected in the nuclei of monocytes, neutrophils, and lymphocytes in normal peripheral blood. Database comparisons of the IFI 16 amino acid sequence revealed 51% identity with the recently cloned myeloid cell nuclear differentiation antigen (MNDA), and extensive similarity to protein products of the Gene 200 cluster of IFN-inducible genes, Ifi 202 and Ifi 204. The amino terminal domain of IFI 16 encodes a putative nuclear localization signal, ¹²⁴PGAQKRKK, which is strongly conserved in MNDA and 204. Nuclear IFI 16 was able to bind double-stranded DNA in vitro and exhibited a similar elution profile from DNA-cellulose as previously observed for MNDA and 204. Therefore, IFI 16 and MNDA are members of a novel family of human DNA-binding proteins whose expression is associated with myeloid cell differentiation induced by cytokines and chemical agents.     

Identification of key genes responsible for cytokine-induced erythroid and myeloid differentiation and switching of hematopoietic stem cells by RAGE

We utilized a unique culture system to analyze the expression patterns of gene, protein, and cell surface antigen, and the biological process of the related genes in erythroid and myeloid differentiation and switching of hematopoietic stem cells （HSCs） in response to cytokine alterations. Gene-specific fragments （266） identified from five populations of cytokine-stimulated HSCs were categorized into three groups： （1） expressed specifically in a single cell population; （2） expressed in two cell populations, and （3） expressed in three or more populations. Of 145 defined cDNAs, three （2%） were novel genes. Protein two-dimensional gel electrophoresis and flow cytometry analyses showed overlapped and distinguished protein expression profiles in the cell populations studied. Biological process mapping of mRNAs expressed in erythroid and myeloid lineages indicated that mRNAs shared by both lineages attended ＇core processes,＇ whereas genes specifically expressed in either lineage alone were related to specific processes or cellular maturation. Data from this study support the hypothesis that committed HSCs （El4 or G14） cells can still be redirected to develop into myeloid or erythroid cells when erythropoietin （EPO） is replaced with granulocyte-colony stimulating factor （G-CSF） under erythroid-cultured condition or G-CSF with EPO in myeloid-cultured environment, respectively. Our results suggest that genes or proteins co-expressed in erythroid and myeloid lineages may be essential for the lineage maintenance and switching in hematopoiesis.     

Expression of the Evi-1 zinc finger gene in 32Dc13 myeloid cells blocks granulocytic differentiation in response to granulocyte colony-stimulating factor.

Expression of the Evi-1 gene is frequently activated in murine myeloid leukemias by retroviral insertions immediately 5' or 90 kb 5' of the gene. The Evi-1 gene product is a nuclear, DNA-binding zinc finger protein of 145 kDa. On the basis of the properties of the myeloid cell lines in which the Evi-1 gene is activated, it has been hypothesized that its expression blocks normal differentiation. To explore this proposed role, we have constructed a retrovirus vector containing the gene and examined its effects on an interleukin-3-dependent myeloid cell line that differentiates in response to granulocyte colony-stimulating factor (G-CSF). Expression of the Evi-1 gene in these cells did not alter the normal growth factor requirements of the cells. However, expression of the Evi-1 gene blocked the ability of the cells to express myeloperoxidase and to terminally differentiate to granulocytes in response to G-CSF. This effect was not due to altered expression of the G-CSF receptor or to changes in the initial responses of the cells to G-CSF. These results support the hypothesis that the inappropriate expression of the Evi-1 gene in myeloid cells interferes with the ability of the cells to terminally differentiate.     

G-CSF/anti-G-CSF antibody complexes drive the potent recovery and expansion of CD11b<Superscript>+</Superscript>Gr-1<Superscript>+</Superscript> myeloid cells without compromising CD8<Superscript>+</Superscript> T cell immune responses

Background

Administration of recombinant G-CSF following cytoreductive therapy enhances the recovery of myeloid cells, minimizing the risk of opportunistic infection. Free G-CSF, however, is expensive, exhibits a short half-life, and has poor biological activity in vivo.

Methods

We evaluated whether the biological activity of G-CSF could be improved by pre-association with anti-G-CSF mAb prior to injection into mice.

Results

We find that the efficacy of G-CSF therapy can be enhanced more than 100-fold by pre-association of G-CSF with an anti-G-CSF monoclonal antibody (mAb). Compared with G-CSF alone, administration of G-CSF/anti-G-CSF mAb complexes induced the potent expansion of CD11b⁺Gr-1⁺ myeloid cells in mice with or without concomitant cytoreductive treatment including radiation or chemotherapy. Despite driving the dramatic expansion of myeloid cells, in vivo antigen-specific CD8⁺ T cell immune responses were not compromised. Furthermore, injection of G-CSF/anti-G-CSF mAb complexes heightened protective immunity to bacterial infection. As a measure of clinical value, we also found that antibody complexes improved G-CSF biological activity much more significantly than pegylation.

Conclusions

Our findings provide the first evidence that antibody cytokine complexes can effectively expand myeloid cells, and furthermore, that G-CSF/anti-G-CSF mAb complexes may provide an improved method for the administration of recombinant G-CSF.