Inducers of leukemic cell differentiation cause down-regulation of RB gene expression.

Expression of the retinoblastoma (RB) tumor suppressor gene during cell differentiation induced by dimethyl sulfoxide or sodium butyrate was studied in HL-60 human promyelocytic leukemia cells. As cells progressed through the cell cycle, the amount of RB protein per cell increased with homeostasis maintained, so that the amount of RB protein relative to the total cell mass remained almost constant. Dimethyl sulfoxide was used to induce these promyelocytic leukemia cells to undergo terminal differentiation into mature myeloid cells. There was an early reduction in the RB protein expressed per cell. The reduction in expression was similar for cells in all cell cycle phases. There was also progressively reduced expression at later times as cells terminally differentiated. This was compared to the case in which sodium butyrate was used to induce the differentiation of HL-60 cells into mature monocytic cells. An early reduction in RB protein expression per cell also occurred. It occurred for cells in all cell cycle phases as well. Thus, the induced differentiation of HL-60 cells along either the myeloid or the monocytic differentiation lineage involves an early reduction in RB expression, which is common to both pathways. The reduction anteceded proliferative arrest or differentiation. In both cases, the final, resulting G0-differentiated cells had less RB protein per cell than the proliferating, immature, leukemic precursor cells.     

Coupled down-regulation of the RB retinoblastoma and c-myc genes antecedes cell differentiation: possible role of RB as a "status quo" gene.

The ability of the well known morphogen, retinoic acid (RA), as well as 1,25-dihydroxy-vitamin D3 (VD), whose receptor complex binds a DNA consensus sequence related to that of the retinoic acid receptor, to regulate expression of the retinoblastoma (RB) tumor suppressor gene in a context of induced cell differentiation was characterized. HL-60 human promyelocytic leukemia cells were induced to undergo myeloid or monocytic terminal cell differentiation by these agents. To investigate the potential coupling between down-regulation of RB and c-myc oncogene expression with cell differentiation, dose response relationships for the induced down-regulation of RB and c-myc expression were compared with each other and with induced cell differentiation. The total amount of RB protein per cell increased as cells advanced through the cell cycle, but the amount of RB protein relative to the total cell mass remained approximately constant. Treated with RA or VD, an early progressive decrease in cellular content of the RB protein occurred in all cell cycle phases well before any cell cycle modulation or phenotypic differentiation. For a differentiation-defective variant HL-60 cell line, failure to differentiate was preceded by a failure to down-regulate cellular levels of the RB protein. In dose response experiments, progressively increasing RA or VD concentrations caused progressively greater reductions in RB as well as c-myc expression with an increasing fraction of cells terminally differentiating. For both RA and VD, the dose response relationships for reductions in RB and c-myc expression were similar suggesting that their down-regulation may be coupled. These observations are consistent with a model whereby RB expression acts as a cellular brake to sustain a developmentally ordained state of differentiation (i.e., preserve the "status quo"); and the down-regulation of heterogeneously distributed RB protein per cell below a threshold is part of the metabolic cascade culminating in terminal cell differentiation. Thus, RB may have a role in this developmental context.     

C-FMS dependent HL-60 cell differentiation and regulation of RB gene expression

The dependence of induced myelomonocytic cell differentiation, and regulation of the RB tumor suppressor gene during this process, on the c-fms gene product, the CSF-1 lymphokine receptor, was determined in HL-60 promyelocytic leukemia cells. Adding a monoclonal antibody with specificity for the c-fms gene product to cells treated with various inducers of myelomonocytic or macrophage differentiation, including retinoic acid and 1,25-dihydroxy vitamin D3, inhibited the rate of differentiation. During the period of inducer treatment usually preceding onset of differentiation, longer periods of antibody exposure caused greater inhibition of differentiation. In a stable HL-60 transfectant overexpressing the CSF-1 receptor at the cell surface due to a constitutively driven c-fms trans gene, the rate of differentiation was enhanced compared to the wild type cell, consistent with a positive regulatory role for the CSF-1 receptor. The anti-fms antibody caused much less inhibition of differentiation in the transfectants than in wild type cells, consistent with a larger number of receptors causing reduced sensitivity. During the induced metabolic cascade leading to differentiation, the typical early down regulation of RB gene expression was inhibited by the antibody. The antibody itself caused an increase in RB expression per cell, which offset the decrease normally caused by differentiation inducers (1,25-dihydroxy vitamin D3 and retinoic acid). The changes in RB expression preceded changes in the RB protein to the hypophosphorylated state. Most of the RB protein in proliferating cells was phosphorylated and no significant accumulation of hypophosphorylated RB protein occurred until after onset of GO arrest. Thus the metabolic cascade leading to myelomonocytic differentiation of HL-60 cells appears to be driver by a function of the c-fms protein. Inhibiting that process by attacking this receptor impedes differentiation and also compromises the early down regulation of RB tumor suppressor gene expression which normally precedes differentiation. These findings provide additional support for a potential role for down regulating RB expression in promoting cell differentiation, and suggest the possibility that RB may be either a target or intermediate mediator of CSF-1 actions. © 1993 Wiley-Liss, Inc.     

Different nucleolar antigen expression in resting and proliferating human lymphocytes as studied by fluorescence microscopy and flow cytometry

The aim of this study was to investigate the use of a nucleolar antigen to discriminate between proliferating and resting cells. Antinucleolar antibodies (Si87) were obtained from a scleroderma patient. The specificity of immunostaining was verified and morphological changes in nucleoli were monitored using a fluorescence microscope. Fluorescence of propidium iodide-stained DNA and nucleolar immunofluorescence were measured by flow cytometry. Following phytohaemagglutinin stimulation the number of nucleoli of normal human peripheral blood lymphocytes increased about 3-fold, accompanied by enlargement of nucleolar size. Simultaneously a mean increase in total immunofluorescence per cell by a factor of three was detected. The method developed and applied here allows a discrimination between resting and proliferating human lymphocytes on the basis of their nucleolar antigen content.     

Different nucleolar antigen expression in resting and proliferating human lymphocytes as studied by fluorescence microscopy and flow cytometry

Abstract. The aim of this study was to investigate the use of a nucleolar antigen to discriminate between proliferating and resting cells. Antinucleolar antibodies (Si87) were obtained from a scleroderma patient. The specificity of immunostaining was verified and morphological changes in nucleoli were monitored using a fluorescence microscope. Fluorescence of propidium iodide-stained DNA and nucleolar immunofluorescence were measured by flow cytometry.
Following phytohaemagglutinin stimulation the number of nucleoli of normal human peripheral blood lymphocytes increased about 3-fold, accompanied by enlargement of nucleolar size. Simultaneously a mean increase in total immunofluorescence per cell by a factor of three was detected. The method developed and applied here allows a discrimination between resting and proliferating human lumphocytes on the basis of their nucleolar antigen content.     

Phosphorylation of the retinoblastoma gene product is modulated during the cell cycle and cellular differentiation

Introduction of an exogenous retinoblastoma (RB) gene in RB-deficient retinoblastoma or osteosarcoma cells has been shown to suppress their neoplastic phenotype. In experiments designed to explore the potential mechanism of RB tumor suppression, we report here that the phosphorylation state of RB protein is modulated during normal cellular events. In resting cells, RB protein is present in its least phosphorylated form; in rapidly proliferating cells, RB protein is highly phosphorylated. Maximal phosphorylation is associated with S phase of the cell cycle. Induction of differentiation in several human leukemia cell lines by treatment with phorbol ester or retinoic acid leads to dephosphorylation of RB. Time course studies indicate that RB dephosphorylation precedes the total arrest of cell growth during differentiation. These observations strongly suggest that the function of RB protein is modulated by a phosphorylation/dephosphorylation mechanism during cell proliferation and differentiation.     

The role of serine hydroxymethyltransferase in cell proliferation: DNA synthesis from serine following mitogenic stimulation of lymphocytes

It is shown that the time-course of incorporation of radioactivity from [3-¹⁴C]serine into nucleic acids parallels DNA synthesis following mitogenic stimulation of human peripheral blood lymphocytes by phytohaemagglutinin (PHA). The activity of serine hydroxymethyltransferase was elevated about four-fold in PHA-stimulated lymphocytes compared to that in unstimulated control ceils. It is suggested that lymphocytes, in common with other proliferating cell systems:, may synthesize serine de novo for utilization in pathways of nucleotide biosynthesis following mitogenic stim--ulation.     

Interleukin-2-triggered Raf-1 expression, phosphorylation, and associated kinase activity increase through G1 and S in CD3-stimulated primary human T cells.

To gain further insight into the role of Raf-1 in normal cell growth, c-raf-1 mRNA expression, Raf-1 protein production, and Raf-1-associated kinase activity in normal human T cells were analyzed. In contrast to the constitutive expression of Raf-1 in continuously proliferating cell lines, c-raf-1 mRNA and Raf-1 protein levels were barely detectable in freshly isolated G0 T lymphocytes. Previous work with fibroblasts has suggested that Raf-1 plays a signaling role in the G0-G1 phase transition. In T cells, triggering via the T-cell antigen receptor (TCR)-CD3 complex (TCR/CD3) resulted in an approximately fourfold increase in c-raf-1 mRNA. In addition, the promotion of G1 progression by interleukin 2 (IL-2) was associated with a 5- to 10-fold immediate/early induction of c-raf-1 mRNA, resulting in up to a 12-fold increase in Raf-1 protein expression. TCR/CD3 activation did not alter the phosphorylation state of Raf-1, whereas interleukin 2 receptor stimulation resulted in a rapid increase in the phosphorylation state of a subpopulation of Raf-1 molecules progressively increasing throughout G1. These findings were complemented by assays for Raf-1-associated kinase activity which revealed a gradual accumulation of serine and threonine autokinase activity in Raf-1 immunoprecipitates during G1, which remained elevated throughout DNA replication.     

A hemopoietic specific gene encoding a small GTP binding protein is overexpressed during T cell activation

We have isolated, from a human B cell line cDNA library, a cDNA (Gx) encoding a small G protein identical to rac 2, a member of the ras superfamily. Gx/rac 2 gene is expressed as a unique mRNA of 1,7 Kb in peripheral blood lymphocytes, in purified B and T cells, in thymus as well as in several B and T cell lines. It is not expressed in many other tissues analysed including liver, brain, lung, heart and kidney. Upon in vitro stimulation with phytohemagglutinin A, peripheral blood lymphocytes show a clear increase of the Gx/rac 2 mRNA after 6 hours; a 30-50 fold accumulation is reached at 24 hours and persists thereafter. Purified T lymphocytes exhibit a similar increase in Gx/rac 2 mRNA expression upon mitogenic stimulation. Therefore, the expression of the Gx/rac 2 gene appears to be restricted to cells of the hemopoietic lineage and to be strongly stimulated during T cell activation. Gx/rac 2 protein must fulfill a specific role in activated T cells that could provide a new model for studying the function of small G proteins.     

Calmodulin in lymphocyte mitogenic stimulation and in lymphoid cell line growth

Calmodulin levels are elevated three- to fourfold in the dividing cells, resulting from the lectin-induced stimulation of fresh human lymphocytes. This increase in calmodulin appears to be related mainly to progression into S phase and supports the hypothesis that calmodulin might be crucial in regulating the progression of lymphoblasts through their division cycle. Calmodulin levels are higher in a lymphoid cell line derived from human acute lymphoblastic leukemia blood cells than in a lymphoid cell line derived from normal human blood cells, suggesting that calmodulin could be an important mediator of the leukemogenetic process.     

Roles of histone acetylation modification in basal and inducible expression of hsp26 gene in D. melanogaster

Src-suppressed C kinase substrate (SSeCKS) plays a role in membrane-cytoskeletal remodeling to regulate mitogenesis, cell differentiation, and motility. Previous study showed that lipopolysaccharide (LPS) induced a selective and strong expression of SSeCKS in the vascular endothelial cells of lung. Here we show that LPS stimulation elevated expression of SSeCKS mRNA and protein in Rat pulmonary microvascular endothelial cell (RPMVEC). LPS potentiated SSeCKS phosphorylation in a time- and dose-dependent manner, and partly induced translocation of SSeCKS from the cytosol to the membrane after LPS challenge. The PKC inhibitor, Calphostin C, significantly decreased LPS-induced phosphorylation of SSeCKS, inhibited SSeCKS translocation and actin cytoskeleton reorganization after LPS challenge, suggesting that PKC may play a role in LPS-induced SSeCKS translocation and actin rearrangement. We conclude that SSeCKS is located downstream of PKC and that SSeCKS and PKC are both necessary for LPS-induced stress fiber formation. Chun Cheng and Haiou Liu are contributed equally to this work.     

A cytophotometric and flow cytofluorometric approach to the differentiation of T lymphocytes in the thymus.

By cytophotometric and flow cytofluorometric DNA and protein determinations two main proliferating subpopulations of thymus lymphocytes with a different percentage of cells in the S phase could be distinguished. One subpopulation had a very low protein content, was cortisone sensitive and located in the cortex. Cells with comparable low protein contents were not found amongst lymphocytes of the peripheral blood. The other lymphocyte subpopulation had a higher protein content, was cortisone resistant and situated in the cortex around a group of epithelial cells and in the medulla. The protein content of these thymus lymphocytes appeared to be comparable to that of the peripheral blood lymphocytes. On the basis of the protein content per cell, it is possible to identify and isolate the more often described major subpopulation of cortisone sensitive thymus lymphocytes remaining and dying in the thymus, and the minor cortisone resistant subpopulation of thymus lymphocytes which is the source of the peripheral T lymphocyte.     

The NF-kappa B cascade is important in Bcl-xL expression and for the anti-apoptotic effects of the CD28 receptor in primary human CD4+ lymphocytes

We explored the role of the NF-kappa B pathway in the survival of primary human CD4+ T lymphocytes during CD28 costimulation. Transduction of proliferating CD4+ T cells with a tetracycline-regulated retrovirus encoding for a dominant-interfering, degradation-resistant I-kappaBalpha (inhibitor of kappa B alpha factor) mutant induced apoptosis. Using DNA arrays, we show that Bcl-xL features as a prominent anti-apoptotic member among a number of early CD28-inducible genes. A 1.2-kb segment of the proximal Bcl-xL promoter, linked to a luciferase reporter, responded to CD3/CD28 stimulation in Jurkat cells. Mutation of an NF-kappa B site around -840 decreased, while ectopic expression of I-kappa B kinase-beta (IKK beta) enhanced reporter gene activity. Na+-salicylate and cyclopentenone PGs, direct inhibitors of IKK beta, interfered in the activation of the Bcl-xL promoter and induced apoptosis in CD28-costimulated CD4+ T cells. Moreover, salicylate blocked nuclear localization of NF-kappa B factors that bind to the NF-kappa B binding site in the Bcl-xL promoter, as well as the expression of Bcl-xL protein. HuT-78, a lymphoblastoid T cell line with constitutive NF-kappa B activity, contained elevated levels of Bcl-xL protein and, similar to proliferating CD4+ T cells, was resistant to apoptotic stimuli such as anti-Fas and TNF-alpha. In contrast, the same stimuli readily induced apoptosis in a Jurkat T cell clone with no detectable Bcl-xL expression. Jurkat BMS2 cells also differed from HuT-78 in collapse of mitochondrial membrane potential and superoxide generation in the mitochondrium. Taken together, these data demonstrate that CD3/CD28-induced activation of IKK beta and expression of Bcl-xL promote the survival of primary human CD4+ T lymphocytes.     

Presence of a reversible inhibitor for human lymphoid cell DNA synthesisin the extracts of human peripheral lymphocytes.

An endogenous inhibitor of human lymphocyte DNA synthesis contained in extracts of purified human peripheral lymphocytes is described. It was found that the peripheral lymphocyte extract inhibits the DNA synthesis of phytohemagglutinin (PHA) stimulated human peripheral lymphocytes, lymphocytes in mixed lymphocyte culture, and human lymphoid cells in a long-term culture (PGLC-33H). This extract did not inhibit the DNA synthesis of nonlymphoid cells including HeLa and human embryonic lung. The effects of the inhibitor were reversible and noncytotoxic. Initial characterization showed the inhibitor to be thermolabile, DNase resistant, trypsin sensitive, and stable in a pH range 5.4–8.4. It appears that the inhibitor contained in the purified human peripheral lymphocyte extract is similar to a previously described inhibitor extracted from a human lymphoid cell line (PGLC-33H). Quantitation of the inhibitor in various lymphoid cell populations showed the amount of inhibitor per cell to be higher in resting peripheral lymphocytes than in PHA stimulated peripheral lymphocytes or human lymphoid cells in long-term culture (PGLC-33H). This data suggest that the inhibitor described may play a regulatory role in lymphocyte metabolism.     

In vitro generation of tumour-specific lymphocyte reactivity to colonic carcinoma cells

Summary Purified tumour cells and normal mucosa cells from fresh human colorectal cancer resection specimens, and T-cell-enriched autologous peripheral blood lymphocytes, were mixed in short-term (6 day) mixed lymphocytetumour cell (MLTC) microcultures. Lymphocyte stimulation was measured by ³H-thymidine uptake, and a stimulation index (SI=[lymphocytes vs tumour cells (cpm)–tumour cells (cpm)]/[lymphocytes (cpm)])>3 was regarded as significant. Significant lymphocyte reactivity was found in 10/15 patients with colon carcinoma. However, 1 patient with autologous tumour reactivity, also showed significant stimulation against autologous normal mucosa cells, suggesting tumour-associated reactivity. Maximum stimulation occurred most frequently at a lymphocyte:tumour cell ratio of 2:1 and with nylon wool-passaged lymphocytes.Project was supported by a grant from the Cancer Research CampaignSupported by New South Wales Cancer Council Fellowship     

A cytophotometric and flow cytofluorometric approach to the differentiation of T lymphocytes in the thymus

Summary By cytophotometric and flow cytofluorometric DNA and protein determinations two main proliferating subpopulations of thymus lymphocytes with a different percentage of cells in the S phase could be distinguished. One subpopulation had a very low protein content, was cortisone sensitive and located in the cortex. Cells with comparable low protein contents were not found amongst lymphocytes of the peripheral blood. The other lymphocyte subpopulation had a higher protein content, was cortisone resistant and situated in the cortex around a group of epithelial cells and in the medulla. The protein content of these thymus lymphocytes appeared to be comparable to that of the peripheral blood lymphocytes. On the basis of the protein content per cell, it is possible to identify and isolate the more often described major subpopulation of cortisone sensitive thymus lymphocytes remaining and dying in the thymus, and the minor cortisone resistant subpopulation of thymus lymphocytes which is the source of the peripheral T lymphocyte.