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PubMed1991

"Differentiation induction" culture of human leukemic myeloid cells stimulates high production of macrophage differentiation inducing factor.

A suitable procedure for the production of human monokines was defined as 'differentiation-induction' culture. Human monocytic leukemia THP-1 cells were well-differentiated from nonfunctional promonocytes into macrophage-like cells by the induction with a combination of mezerein, retinoic acid, and a Mycoplasma fermentans extract. The differentiated THP-1 cells secreted a high amount of macrophage differentiation-inducing factor (DIF) activity and concomitantly produced other known monokines, such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1 alpha (IL-1 alpha) and interleukin-1 beta (IL-1 beta), into the medium. These results suggest that other novel human monokines may also be found in the conditioned medium of THP-1 cells induced by the 'differentiation-induction' culture conditions defined in this study. Macrophage DIF was purified to homogeneity and NH2-terminal amino acid sequence analysis revealed that macrophage DIF is very similar or identical to human leukemia inhibitory factor (LIF). The cDNA encoding human LIF was isolated using the polymerase chain reaction, and a clone producing 3.7 micrograms/10(6) cells day recombinant LIF was selected from Chinese hamster ovary (CHO) cells which were transfected with the LIF cDNA. The recombinant LIF production in CHO cells was quantified using MTT reduction assay with M1 cells.

Amino Acid Sequence↗
PubMed1992

Type beta transforming growth factors promote interleukin-3 (IL-3)-dependent differentiation of human basophils but inhibit IL-3-dependent differentiation of human eosinophils.

Basophils and eosinophils share a common differentiation pathway. Factors regulating terminal commitment toward one cell type, however, have so far not been defined. Interleukin-3 (IL-3) is a potent differentiation factor for both human eosinophils and basophils. In the present study, the effects of various recombinant human (rh) growth regulators on IL-3-dependent growth of eosinophils and basophils were studied in a bone marrow (BM) suspension culture system (normal donors, n = 13). We found that type beta transforming growth factors (TGFs) lead to a significant increase in the absolute numbers of basophils in BM cultures grown in the presence of IL-3 (day 14 of culture; IL-3: 133 +/- 20 v IL-3 + TGF-beta 1: 231 +/- 28 x 10(3)/mL [P less than .01]) and to an increase in the total histamine values (IL-3: 72.6 +/- 22.2 v IL-3 + TGF-beta 1: 142.9 +/- 37.3 ng/mL [P less than .015]) compared with rhIL-3 alone. In contrast, type beta TGFs were found to inhibit the IL-3-dependent growth of eosinophils (IL-3: 170.4 +/- 37.2 v IL-3 + TGF-beta 1: 16.7 +/- 5.2 x 10(3)/mL [P less than .01]) and formation of eosinophil cationic protein in the same culture system. The effect of TGF-beta 1 (and TGF-beta 2) on IL-3-dependent differentiation of basophils and eosinophils was dose- and time-dependent (maximum effects observed with 1 to 10 ng/mL of rhTGF-beta 1 or TGF-beta 2) and could be neutralized by an antibody specific for TGF-beta 1. In contrast to the TGFs, interferon-alpha (IFN-alpha) and IFN-gamma were found to downregulate IL-3-dependent formation of both basophils (IL-3: 167 +/- 33 v IL-3 + IFN-alpha: 67 +/- 25 v IL-3 + IFN-gamma: 65 +/- 33 x 10(3)/mL [P less than .01]) and eosinophils (IL-3: 239 +/- 5 v IL-3 + IFN-alpha: 81 +/- 4 v IL-3 + IFN-gamma: 67 +/- 17 x 10(3)/mL [P less than .05]) in our culture system. Type beta TGFs as well as the IFNs failed to directly induce differentiation of human basophils or eosinophils in the absence of other growth factors. Together, these results show that type beta TGFs and IFNs are potent regulators of cytokine-dependent growth and differentiation of human allergic effector cells.

Antibodies, Monoclonal↗
PubMed2006

[Screening differentially expressed genes in human bone marrow stromal cells at defined stage of differentiation.].

To screen differentially expressed genes involved in osteogenic differentiation of human bone marrow stromal cells (BMSCs) at defined stages, subtractive cDNA library was established by means of suppression subtractive hybridization. The BMSCs cultured for 12 and 21 d were used as driver and tester, respectively. A subtract library was successfully constructed and five positive clones were selected from the library. Sequencing analysis and homology comparison showed that the five clones differentially expressed in BMSCs cultured for 21 d were at least 90% homologous with the known genes in human GenBank. It was interestingly found that the osteogenic BMSCs cultured for 21 d differentially expressed decorin and Bax inhibitor 1. RT-PCR was performed to confirm the differentially expressed genes. The results showed that the expression of Bax inhibitor 1 was significantly higher in the cells of 21-day than that of 12-day, while the expression of decorin was only detected in the cells of 21-day.

Apoptosis Regulatory Proteins↗
PubMed1990

Effect of AML serum on normal human myeloid differentiation and hexamethylene bisacetamide-induced granulocytic differentiation of the human HL-60 promyelocytic leukaemic cell line.

The effect of serum from 32 AML patients on the normal human myeloid differentiation and the hexamethylene-bisacetamide induced granulocytic differentiation of HL-60 promyelocytic leukaemic cell line was studied. Nonadherent normal mononuclear marrow cells were cultured in vitro at a concentration of 5 x 10(5) cells/ml for 6 days with each of the 32 AML sera. Ten normal human AB sera were used as control. The results showed an inhibitory activity on both morphological and functional differentiation of normal human myeloid immature marrow cells by 29 out of the 32 AML sera tested. These 29 AML sera were added to cultures of HL-60 (2.5 x 10(5)/ml) leukaemia cell line which incorporated 2 mM hexamethylene-bisacetamide for 6 days. The results showed no significant inhibition of hexamethylene-bisacetamide induced granulocytic differentiation by any of the 29 AML sera. The efficacy of hexamethylene-bisacetamide in inducing differentiation in the presence of inhibitory factors suggests a possible role in the treatment of AML patients.

Acetamides↗
PubMed1990

Neuroendocrine differentiation in "poorly differentiated" colon carcinomas.

The diagnosis of "poorly differentiated" carcinoma was made in 47 of 683 colon cancers on the basis of conventional light microscopy which showed poorly defined glands, solid architecture or variable admixtures thereof. Samples from 44 of these 47 tumors were assessed by immunohistochemical analysis for the presence of neuroendocrine (NE) antigens. Paraffin sections were immunostained with antibodies to NSE, chromogranin, serotonin, VIP, substance P and somatostatin. Additional sections were also stained with monoclonal antibody (Mab) A-80 that recognizes a glycoprotein related to exocrine (EX) differentiation. Based on our findings, the tumors were phenotypically reclassified as follows: I) pure EX (n = 8), II) pure NE (n = 4), III) mixed EX-NE carcinomas (n = 23), and IV) predominantly EX carcinomas with occasional NE cells (n = 9). Survival among groups II and III appeared to be less than group I and survival in group IV was significantly less than group I. Survival among the four pure NE (group II) and 11 predominantly NE mixed carcinomas (group III) taken together was significantly less than the pure EX carcinomas. This study indicates: 1) The incidence of NE differentiation in tumors of the colon and rectum is higher than previously believed. 2) The poorly differentiated colon carcinomas comprise four distinct groups: pure EX, pure NE, mixed EX-NE carcinomas, and predominantly EX carcinomas with a NE cell subpopulation. 3) The presence of NE differentiation or of a NE cell subpopulation in colon carcinoma appears to be associated with a poorer prognosis.

Aged↗
PubMed1975

Xenopus laevis cement gland as an experimental model for embryonic differentiation. I. In vitro stimulation of differentiation by ammonium chloride.

Ectoblastic cells explanted from the animal pole of young Xenopus laevis gastrulae have been cultured in vitro. When these cells were cultured for five days in standard salt solutions they formed atypical epidermis. When they were first submitted for 6 h to Holtfreter solution containing ammonium chloride and then transferred for five days in standard Barth's solution they underwent differentiation into typical cement gland tissue. The optimum concentration of ammonium chloride was 10 mM. Below and above this concentration the resulting cement glands had a smaller volume. The optimum duration for the initial stimulation with 10 mM ammonium chloride in Holtfreter solution was 6 h. Shorter stimulation times produced only small cement glands. Longer initial incubations in ammonium chloride resulted in progressive dissociation of the explants. To obtain the best differentiation into cement gland it appeared that the pH of the ammonium chloride solution should be between 7-5 and 7-7. When the stimulation is performed under these optimum conditions the cement gland measured after five days of culture accounts for 80-90 percent of the explanted tissue. This means that all or almost all of the competent superficial layer of the ectoblastic cells underwent differentiation into cement gland. No other differentiated tissue was observed in the explants. The cement gland is a very simple organ containing only one single cell type. The gland obtained under the described in vitro conditions is therefore proposed as an experimental model for biochemical studies on early embryonic differentiation.

Ammonium Chloride↗
PubMed1989

Age of differentiation determines rat retinal germinal cell phenotype: induction of differentiation by dissociation.

We are interested in the mechanisms that control cell phenotype during the development of the CNS. Since different neuronal types arise at different times during neurogenesis in the retina, we predicted that the factors that determine cell type must be developmentally regulated as well. To test this hypothesis, we induced retinal germinal cells to differentiate at different ages by dissociating the retina into single cells and culturing them on a variety of substrates. Prior to dissociation, the S-phase germinal cells were labeled with 3H-thymidine so that their fate could be specifically followed. We found that our culture conditions promoted the differentiation of the majority of the germinal cells and that these cells differentiated into different neuronal types depending on the age of the animal from which the retina had been taken; embryonic day 14 germinal cells differentiated primarily into ganglion cells, and never produced rods, while germinal cells from postnatal day 1 retina differentiated into rods, but not ganglion cells. These results are consistent with the hypothesis that temporally regulated factors determine cell phenotype during the development of the retina.

Animals↗
PubMed1989

Differential expression of protein kinase C isozymes and erythroleukemia cell differentiation.

Hexamethylene bisacetamide (HMBA) and other polar/apolar chemical agents are potent inducers of erythroid differentiation in murine erythroleukemia cells (MELC), as well as other transformed cell lines. Although the mechanism of action of HMBA is not yet known, evidence has been obtained that protein kinase C (PKC) plays a role in this process. In this study we provide further evidence that establishes this relationship. MELC contain two principal PKC activities, PKC beta and PKC alpha. MELC variants, selected for resistance to vincristine (VC), which display acceleration of their rates of induced differentiation, are enriched in PKC beta activity. When MELC are exposed to HMBA there is a fall in PKC activity, largely accounted for by a decline in PKC beta. This decline in PKC activity is faster in the VC-resistant, rapidly differentiating MELC. We previously demonstrated that VC-resistant MELC are resistant to the inhibition of differentiation by the phorbol ester, phorbol 12-myristate 13-acetate (PMA). In both VC-sensitive and -resistant MELC, PMA causes rapid membrane translocation and then a decline in PKC activity, accompanied by a generation of a Ca2+- and phospholipid-independent protein kinase activity. In VC/PMA-resistant variants, this Ca2+/phospholipid-independent protein kinase activity persists considerably longer than in the VC-sensitive variants. This correlates with the resistance to PMA and provides additional evidence for a role for the Ca2+/phospholipid-independent protein kinase activity during induced differentiation.

Acetamides↗
PubMed1986

Endocrine and tumor differentiation markers in poorly differentiated small-cell carcinoids of the cervix and vagina.

Immunoreactivity for endocrine peptides (serotonin, gastrin, somatostatin, insulin, corticotropin, calcitonin, neurotensin, vasoactive intestinal peptide, and bombesin), cytoskeletal proteins (high and low molecular weight keratins), and tumor differentiation markers (chromogranin, neuron-specific enolase, carcinoembryonic antigen, S100 protein, and Grimelius stain) was sought on nine cervical and one vaginal poorly differentiated small-cell carcinoids. Dense-core secretory granules were ultrastructurally identified in all cases (seven of ten) in which tissue was available for electron microscopy. Immunoreactivity for endocrine secretory products was rarely noted, and only in a minority cell population (serotonin in two of ten). The majority of the tumors exhibited immunoreactivity for low molecular weight keratin (AE1/AE3 in eight of ten; CAM 5.2 in seven of nine), and three of ten tumors focally expressed high molecular weight keratin. Among the markers of neuroendocrine differentiation, neurospecific enolase was more frequently expressed (ten of ten) than chromogranin (five of ten) or argyrophilia (three of ten). Carcinoembryonic antigen was present in eight of ten tumors. S100 protein was absent in all cases. In summary, poorly differentiated small-cell carcinoids of the lower female genital tract, similarly to other small-cell endocrine tumors, occasionally exhibit focal glandular and squamoid differentiation, and only relatively infrequently or focally express immunohistochemically detectable endocrine secretory products, chromogranin, and argyrophilia.

Antigens, Neoplasm↗
PubMed1977

The effects of neuraminidase and galactose oxidase on murine lymphocytes. I. Evidence for the differential delivery of signal(s) leading to cell proliferation and the differentiation of cytotoxic T cells.

The sequential treatment of normal C57BL/6 mouse spleen cell populations with neuraminidase (NA) and galactose oxidase (GO) resulted in cell proliferation, but not in the differentiation of cytotoxic T cells. In contrast, C57BL/6 spleen cells derived from animals primed 5 to 8 months earlier with alloantigen (P815 mastocytoma cells of the DBA/2 strain) both proliferated and demonstrated T cell-mediated cytotoxicity after NAGO stimulation. T cells differentiating into cytotoxic cells after NAGO treatment demonstrated properties similar to alloantigen-specific 'memory' T cells. These were: 1) cytotoxicity developed only from 'primed' cell populations, 2) cytotoxicity developed within 24 hr after NAGO treatment, 3) DNA synthesis was not required for the differentiation of cytotoxic cells during the first 24 hr of culture but both DNA synthesis and cell proliferation were required for the cytotoxicity developing after 24 hr, and 4) all cytotoxicity induced by NAGO showed specificity for the priming alloantigen. It was found, furthermore, that cytotoxicity could be induced at much lower GO concentrations than needed for increased DNA synthesis. We interpret this finding as an indication that NAGO can differentially deliver two 'signals' to T lymphocytes: one leading to cell proliferation, the other causing the differentiation of memory T cells into cytotoxic effectors.

Animals↗
PubMed1987

Bipotential cell differentiation of KU-812: evidence of a hybrid cell line that differentiates into basophils and macrophage-like cells.

KU-812-F is a subclone of KU-812, which has cytological features similar to the parent clone, and which carries the Philadelphia chromosome. We studied the effects of various chemical agents and serum-free culture (SF-C) condition on its differentiation. The KU-812-F subclone differentiated into macrophage-like cells with phorbol myristate acetate (PMA), but not with other agents. Unexpectedly, under SF-C, KU-812-F cells differentiated into mature basophil-like cells, and the histamine content of cell lysates increased in proportion to maturation. The addition of the condensed supernatant of SF-C promoted the cloning efficiency of KU-812-F cells in semisolid SF-C, whereas the cloning efficiency was reduced in SF-C alone. KU-812-F cells expressed myelomonocytic antigens. Additionally, My4 was induced with PMA and SF-C, but less often. HLA-DR was not expressed in any culture conditions, despite blastic morphology of KU-812-F cells or macrophage-like cells induced with PMA. The IgE receptor could not be demonstrated under any conditions. Correspondingly, immature and mature KU-812-F cells did not respond to anti-IgE or concanavalin A (con A), while histamine release was induced with PMA. In conclusion, KU-812-F belongs to the myeloid cell lineage and is at least a bipotential cell that can differentiate into basophils and macrophage-like cells. Although the functions appear to be dissimilar to those of normal basophils, the KU-812-F cell line may be a good model for basophil differentiation. Moreover, KU-812-F cells may provide new insights because they grow in semisolid culture by an autostimulating mechanism.

Basophils↗
PubMed1982

Effect of differentiation on the adenylate cyclase system of 3T3-C2 and 3T3-L1 cells. Determination of choleragen substrates in differentiating 3T3-L1 and nondifferentiating 3T3-C2 cells.

3T3-L1 preadipocytes, when treated with 3-isobutyl-1-methylxanthine, dexamethasone, and insulin, differentiate into cells with the morphological and biochemical properties of adipocytes; the closely related 3T3-C2 cells, under identical conditions, exhibit a low frequency of adipocyte conversion. During differentiation, 3T3-L1 preadipocytes acquire an increased responsiveness to certain agonists (e.g. isoproterenol and adrenocorticotropic hormone) that influence lipolysis and lipogenesis through activation of adenylate cyclase, whereas 3T3-C2 cells do not. It has been suggested that changes in hormone responsiveness of 3T3-L1 cells during differentiation result from increased amounts of the guanyl nucleotide-binding protein of adenylate cyclase, as demonstrated by choleragen-catalyzed [32P]ADP ribosylation of 42 and 49-50-kilodalton particulate peptides. Particulate fractions from nondifferentiating 3T3-C2 cells, like those from 3T3-L1 cells, contained choleragen substrates of 42 and 46-47 (doublet) kilodaltons. Incubation of intact 3T3-L1 or 3T3-C2 cells with choleragen prior to preparation of particulate fractions prevented the subsequent in vitro choleragen-dependent [32P]ADP ribosylation of only these peptides. Increased incorporation of radioactivity into both the 42 and 46-47-kilodalton peptides was observed during differentiation of 3T3-L1 cells. However, a similar increase was also observed in nondifferentiating 3T3-C2 cells subjected to the differentiation protocol. Therefore, increased hormone responsiveness of 3T3-L1 adipocytes cannot be explained solely on the basis of increased labeling, and perhaps increased amounts, of the guanyl nucleotide-binding protein.

Adenosine Diphosphate Ribose↗
PubMed1983

Differential control by platelet factors of squamous differentiation in normal and malignant human bronchial epithelial cells.

Recently, we developed a nutritionally optimal medium for rapid clonal growth (greater than 1 population doubling/day) of normal human bronchial epithelial (NHBE) cells. Adding fetal bovine or adult human blood-derived serum to this medium depresses the clonal growth rate of NHBE cells in a dose-dependent fashion. In contrast, 10 representative lines of human lung carcinomas either replicate poorly or fail to grow at all when inoculated at clonal density in serum-free medium, and their rates of multiplication increase in direct proportion to the amount of blood-divided serum added to the optimized medium. Thus, the growth factor requirements of these lung carcinoma cell lines are significantly different from those of their normal counterparts. Blood-divided serum reduces the clonal growth rate of NHBE cells by specifically inducing the normal cells, but not lung carcinoma cells, to undergo squamous differentiation. The differentiation-inducing activity was found in platelet lysates. In addition, a growth-inhibiting activity that did not induce squamous differentiation of NHBE cells was also identified in partially purified commercial preparations of platelet-derived growth factor. This observation was in marked contrast to results using human bronchial fibroblasts and human lung carcinoma cell lines; the growth rate of the former was significantly stimulated by commercial preparations of platelet-derived growth factor, whereas the growth rates of the tumor cell lines were unaffected. These results indicate that an aberration in the cellular differentiation as assayed in vitro is positively correlated with cancer and suggests that decreased responsiveness to inducer(s) of differentiation may be a major aspect of bronchial cell carcinogenesis.

Bronchi↗
PubMed1984

Leukemic cell differentiation in vivo and in vitro: arrest of proliferation parallels the differentiation induced by the antileukemic drug Harringtonine.

A variety of chemical agents have been shown to induce differentiation in in vitro cultured neoplastic cell lines. We noted that blast cells in the peripheral blood of acute nonlymphoid leukemia patients treated with the drug Harringtonine appeared to undergo morphological changes that suggested differentiation. In view of the relatively minimal myelotoxicity of Harringtonine, we hypothesized that harringtonine was acting by differentiation-induction, which concomitantly arrested cell division. We tested this hypothesis using two different experimental approaches. First, the promyelocytic leukemic cell line, HL-60, was cultured with Harringtonine and shown to differentiate into a cell, which, by functional cell surface marker and morphological criteria, closely resembled normal monocytes. Furthermore, these changes were accompanied, and indeed slightly preceded by, loss of proliferative capacity. Second, to prove that the leukemic blasts were the cells undergoing the changes observed in vivo, freshly isolated leukemia cell populations were cultured with Harringtonine, and morphological changes paralleling those seen in the patients were observed. Thus, the antileukemic effect of Harringtonine appeared to be due to diversion of the proliferating blast cells into a differentiation pathway, which, as in normal myeloid cells, resulted in the arrest of proliferation.

Acute Disease↗
PubMed1982

Effects of conditioned medium from non-differentiating or differentiated mouse myeloid leukemia cells on formation of macrophage-granulocyte colonies of normal mouse bone marrow cells.

The effects of conditioned media (CM) of various clones of mouse myeloid leukemia cells (M1) on colony formation of normal mouse bone marrow cells were examined under various conditions. The CM of sensitive M1 cells, which could be induced to differentiate into macrophage-like and granulocyte-like cells by various factors stimulating the differentiation (D-factor), slightly inhibited colony formation of normal bone marrow cells by colony stimulating factor (GM-CSF). The CM of resistant M1 cells, which were resistant to the induction of differentiation even with high concentrations of the D-factor, significantly inhibited the colony formation of bone marrow cells by GM-CSF. The inhibitory activity of the CM from resistant cells decreased on treatment of the cells with a low concentration of actinomycin D, which could sensitize the resistant cells to induction of differentiation. The CM of differentiated sensitive M1 cells induced by dexamethasone stimulated the colony formation of bone marrow cells without exogenously added GM-CSF. Most of the GM-CSF activity in the CM was separated from the D-factor of M1 cells by Sephadex G-75 gel filtration.

Animals↗
PubMed1982

Human B cell differentiation. III. Enhancing effect of monoclonal anti-immunoglobulin D antibody on pokeweed mitogen-induced plasma cell differentiation.

The effects of monoclonal anti-delta antibodies on pokeweed mitogen (PWM) responses of blood mononuclear cells (MNC) were studied. Treatment with anti-delta antibody enhanced both B cell proliferation and plasma cell differentiation, which are T cell-dependent responses. The anti-delta enhancement of plasma cell differentiation, predominantly of IgM plasma cells, was surprising because PWM-responsive subpopulations of B cells have been shown to lack IgD and their plasma cell differentiation is easily and selectively suppressed by anti-mu, -gamma and -alpha antibodies. Treatment of MNC with monoclonal anti-delta antibody enhanced the number of IgM plasma cells induced by PWM stimulation by approximately threefold. The degree of enhancement was dependent upon the concentration of anti-delta antibody, and the F(ab')2 fragments were effective. Maximal enhancement was obtained either when MNC were preincubated with anti-delta antibody for 1 day before PWM stimulation or when anti-delta antibody was added with PWM at the beginning of 7-day cultures. Anti-delta antibody had little or no effect when added 1 to 3 days after the initiation of PWM stimulated cultures. Anti-delta treatment overnight induced a population of small IgM+IgD+ B cells to enlarge and converted them from poor to good PWM responders. The results are discussed in the context of a model which proposed that differentiation of both immature and preactivated mature IgD- cells can be inhibited by signals generated via surface immunoglobulin cross-linkage, whereas this stimulus enhances differentiation of the intermediate IgD+IgM+ B cells.

Antibodies, Anti-Idiotypic↗
PubMed1994

The SCL transcription factor and differential regulation of macrophage differentiation by LIF, OSM and IL-6.

The SCL gene is a member of the helix-loop-helix family of transcription factors. First identified because of its involvement in a rare chromosome translocation in human T cell acute lymphoblastic leukemia, it is now recognized to be involved in up to 25% of T cell leukemias. Normally within the hemopoietic system the gene is expressed in progenitor cells, erythroid cells, mast cells and megakaryocytes. During macrophage differentiation the level of SCL mRNA and protein becomes undetectable. To examine this further, SCL was over expressed in murine M1 cells. This resulted in perturbation of macrophage differentiation induced by leukemia inhibitory factor (LIF) and oncostatin-M (OSM) but not interleukin (IL)-6. Moreover the perturbation of LIF-induced differentiation applied to some components of macrophage differentiation but not others. This suggests that signaling through the gp130 homodimer (as occurs with IL-6) does not utilize an SCL-inhibitable pathway. In contrast the LIF receptor/gp130 heterodimer does utilize an SCL-inhibitable pathway for some elements of macrophage differentiation (e.g., lysozyme induction) but not others (e.g., M-CSF receptor induction).

Animals↗
PubMed1995

Differentiation of P19 EC cells leads to differential modulation of cyclin-dependent kinase activities and to changes in the cell cycle profile.

The retinoblastoma gene product (pRb) is essential for normal embryonic development. Phosphorylation of pRb by cyclin dependent kinases (cdk's) is believed to be crucial for the regulation of its function. In this report we have studied the regulation of pRb and cdk's during in vitro differentiation of P19 embryonal carcinoma (EC) cells, as a model for early developmental processes. During EC cell differentiation, the synthesis of pRb is strongly induced. In addition, the phosphorylation state of induced pRb is modulated, yielding mainly underphosphorylated pRb. Concomitantly, the pRb kinases cdk2 and cdk4 are differentially regulated: cdk2 kinase activity is impaired, whereas cdk4 kinase activity is stimulated, due to an induction of cyclins D1 and D2. Furthermore, the DNA binding activity of E2F transcription factors is strongly impaired during differentiation and the number of cells in G1 is increased. Thus, P19 EC cell differentiation is accompanied by changes in cdk-activities, pRb regulation and E2F DNA-binding, resulting in the generation of cell types with an altered cell cycle profile.

Adenovirus E1A Proteins↗