Glucocorticoid receptor expression in receptorless mutants isolated from the human leukemic cell line CEM-C7

The molecular basis for the loss of steroid binding activity in receptorless (r-) glucocorticoid-resistant (dexr) mutants isolated from the glucocorticoid-sensitive (dexs) cell line CEM-C7 was investigated. Although there was little binding of the reversibly associating ligand [3H]dexamethasone in r- mutants, labeling with the covalent affinity ligand [3H] dexamethasone 21-mesylate revealed significant amounts of a 92 kilodalton human glucocorticoid receptor (hGR) protein. Immunoblots of hGR protein in r- and normal cells showed that r- mutants expressed approximately half the amount of immunoreactive hGR protein seen in dexs cells. Comparison of the genomic organization of the hGR genes in normal and mutant cells revealed no discernable differences in the structure, or dosage, indicating that the r- phenotype was not the result of gross deletion or rearrangement of the hGR genes. In addition, r- cells expressed the same 7 kilobase mRNA as normal cells. More importantly, the amount of hGR mRNA expressed in r- cells was never significantly less, and in some cases was greater than, that seen in normal cells, indicating that the decrease in immunoreactive hGR protein seen in r- cells is not the result of loss of hGR mRNA expression. Taken together with the known mutation rate of the hGR gene(s) in these cells, these results suggest that the hGR genes in dexs CEM-C7 cells are allelic and that dexs cells express both a normal hGR protein and one with an altered steroid binding site. Furthermore, they suggest that the r- phenotype is acquired as the result of mutation within the coding region of the originally functional allele, leading to loss of ligand binding and expression of immunoreactive product.     

Metabolism of c-myc gene products: c-myc mRNA and protein expression in the cell cycle.

The presence and synthesis of c-myc protein and mRNA in the cell cycle has been studied. We find that c-myc mRNA is present, at equivalent levels, at all times in the cell cycle with the possible exception of mitosis. Furthermore, we demonstrate that this mRNA is transcribed in both G1 and G2 phases. An analysis of the c-myc protein in vivo shows that de novo synthesis occurs in G1 and G2 and the protein turns over with a half-life of approximately 20-30 min in both phases. Furthermore, the level of c-myc protein rapidly increases in cell populations when they re-initiate the cell cycle, thereafter decreasing as the culture reaches quiescence. The results therefore suggest that expression of c-myc can be rapidly modulated and that it is activated during the G0 to G1 transition, but is expressed thereafter in the cell cycle.     

Proteins encoded by the human c-myc oncogene: differential expression in neoplastic cells.

To examine myc protein products in the wide variety of human tumor cells having alterations of the c-myc locus, we have prepared an antiserum against a synthetic peptide corresponding to the predicted C-terminal sequence of the human c-myc protein. This antiserum (anti-hu-myc 12C) specifically precipitated two proteins of 64 and 67 kilodaltons in quantities ranging from low levels in normal fibroblasts to 10-fold-higher levels in Epstein-Barr virus-immortalized and Burkitt's lymphoma cell lines, to 20- to 60-fold-higher levels in cell lines having amplified c-myc. The p64 and p67 proteins were found to be highly related by partial V8 proteolytic mapping, and both were demonstrated to be encoded by the c-myc oncogene, using hybrid-selected translation of myc-specific RNA. In addition, the p64 protein was specifically precipitated from cells transfected with a translocated c-myc gene. Both p64 and p67 were found to be nuclear phosphoproteins with extremely short half-lives. In tumor cell lines having alterations at the c-myc locus due to amplification or translocation, we observed a significant change in the expression of p64 relative to p67 when compared with normal or Epstein-Bar virus-immortalized cells.     

Retroviral expression of the human IL-2 gene in a murine T cell line results in cell growth autonomy and tumorigenicity.

In mature T lymphocytes (T cells) the regulated expression of the genes for interleukin-2 (IL-2) and its receptor (IL-2R) constitutes an essential part in controlling the cell growth. Evidence has been provided which suggests the involvement of an aberrant function of the IL-2 system in developing T cell neoplasms, particularly the adult T cell leukemia/lymphoma (ATL). As an approach to examine the extent of the IL-2 system contribution to T cell neoplasms, we created the experimental conditions wherein both IL-2 and IL-2R are expressed constitutively in a murine T cell line. We made use of a retroviral vector to infect an IL-2-dependent CTLL-2 line and lead to the expression of human IL-2. Here, we show that the virus-infected cells not only proliferate in vitro in the absence of exogenously supplied IL-2 under certain conditions, but also develop tumors (lymphomas) in nude and syngeneic mice.     

Deoxyadenosine toxicity in an adenosine deaminase-inhibited human CCRF-CEM T-lymphoblastoid cell line causes cell swelling.

The human T-lymphoblastoid cell line CCRF-CEM, pre-treated with 2'-deoxycoformycin, was used as a model for adenosine deaminase deficiency to investigate how 2'-deoxyadenosine exerts its cytotoxic effects. Incubation of these cells with 1 microM or 5 microM deoxyadenosine for 24 and 48 h caused an increase of up to 50% in their modal cell volume as measured by a Coulter Size Distribution Analyzer and this increase in cell volume was accompanied by an increase in their fragility and deformability. The swelling of cells was concomitant with the phosphorylation of deoxyadenosine and its intracellular accumulation as dATP. There was no evidence of osmotic imbalance or of inhibition of the Na+/K(+)-dependent ATPase activity as the intracellular concentrations (and the intracellular:extracellular ratios) of Na+, K+ and Ca2+ were essentially unchanged. Cytochalasin B (20 microM) also caused lymphoblasts to swell over a 6-h period and its effect on cell size was similar to that of either 1 microM or 5 microM deoxyadenosine over 24 or 48 h. Longer time-courses of incubation with cytochalasin B caused severe toxicity leading to the death and lysis of a significant proportion of the cells. Other drugs, such as colchicine, vincristine and vinblastine that are known to affect various components of the cytoskeleton also caused swelling of cells in a concentration- and time-dependent manner but there was no evidence that these effects were additive or synergistic with those of deoxyadenosine. Inhibition of DNA synthesis, either directly by aphidicolin or indirectly by hydroxyurea, was less cytotoxic than the effect caused by deoxyadenosine. We conclude that one of the toxic effects resulting from the excessive phosphorylation of deoxyadenosine and its accumulation as dATP in human T-lymphoblasts is not dependent on inhibition of DNA synthesis but may be caused by the disruption of the cytoskeleton in these cells.     

Melatonin synthesized by Jurkat human leukemic T cell line is implicated in IL-2 production

Human lymphocytes have recently been described as an important physiological source of melatonin (N-acetyl-5-methoxytryptamine), which could be involved in the regulation of the human immune system. On the other hand, stimulation of IL-2 production by exogenous melatonin has been shown in the Jurkat human lymphocytic cell line. Furthermore, both melatonin membrane and nuclear receptors are present in these cells. In this study, we show that the necessary machinery to synthesize melatonin is present and active in resting and stimulated Jurkat cells. Accordingly, we have found that cells synthesize and release melatonin in both conditions. Therefore, we investigated whether endogenous melatonin produced by Jurkat cells was involved in the regulation of IL-2 production. When melatonin membrane and nuclear receptors were blocked using specific antagonists, luzindole and CGP 55644, respectively, we found that IL-2 production decreased, and this drop was reverted by exogenous melatonin. Additionally, PHA activation of Jurkat cells changed the profile of melatonin nuclear receptor mRNA expression. A previous study showed that exogenous melatonin is able to counteract the decrease in IL-2 production caused by prostaglandin E2 (PGE2) in human lymphocytes via its membrane receptor. In our model, when we blocked the melatonin membrane receptor with luzindole, the inhibitory effect of PGE2 on IL-2 production was higher. Therefore, we have demonstrated the physiological role of endogenous melatonin in this cell line. These findings indicate that endogenous melatonin synthesized by human T cells would contribute to regulation of its own IL-2 production, acting as an intracrine, autocrine, and/or paracrine substance.     

Down-regulation of endothelial cell growth inhibitors by enhanced MYCN oncogene expression in human neuroblastoma cells.

Recent evidence indicates that the genetic alterations of the multistage process of malignant transformation appear to activate tumor neovascularization by altering the balance between stimulators and inhibitors of angiogenesis. In the present study, we have attempted to define the effect of enhanced MYCN oncogene expression on the profile of endothelial cell growth modulators in neuroblastoma cells. We report here that conditioned medium of human neuroblastoma cells with normal MYCN expression contains three inhibitors of endothelial cell proliferation, which appear to be novel proteins as judged by their physicochemical, immunological and biological properties. All three inhibitors are diminished or become undetectable upon experimental increase of MYCN expression. Our results suggest that enhanced MYCN expression in human neuroblastoma cells alters the angiogenic balance by down-regulating endothelial cell growth inhibitors but leaving the expression of the stimulators unaffected. These data shed light on the molecular mechanisms linking the genetic changes of malignant transformation with initiation of tumor angiogenesis. Moreover, our observations might explain the poor prognosis of human neuroblastomas following MYCN oncogene amplification through initiation of angiogenesis and subsequent tumor growth and spread.     

Clostridium difficile toxin A induces multinucleation in the human leukemic T cell line JURKAT.

Clostridium difficile toxin A is a cytotoxic enterotoxin known to be active on all mammalian cell lines tested up to now. It induces a disruption of the cytoskeleton, particularly the microfilament system, leading to inhibition of cell proliferation. Here, we describe some effects of toxin A on the leukemic T cell line JURKAT. Cells exposed to the toxin did not divide, as cell numbers remained constant for 3 days in the presence of 0.5 to 1.0 micrograms/ml of the toxin. However, these cells were found to become multinucleated, a phenomenon which was time- and dose-dependent. After treatment for 72 h with 0.5 micrograms/ml toxin A, 95% of the cells were multinucleated and had a considerably increased cell diameter. These effects in JURKAT cells were partially reversible upon removal of the toxin within 12 h after the beginning of toxin exposure, but irreversible after 24 h of toxin treatment. These results suggest a continuing nuclear division in the absence of cytoplasmic division, i.e., an effect of toxin A on contractile ring formation. The JURKAT cell is the first cell type reported to respond to toxin A with multinucleation.     

Presence and expression of a novel variant form of ST2 gene product in human leukemic cell line UT-7/GM.

A novel variant cDNA from the human ST2 gene other than ST2 or ST2L was identified and tentatively named ST2V. Alternative splicing inserts a new exon which leads to a change in the C-terminal portion of ST2, causing it to gain a hydrophobic tail instead of losing the third immunoglobulin-like domain. ST2V is expressed in human leukemic cell line UT-7 and its sublines UT-7/GM, UT-7/EPO, and UT-7/TPO, in addition to human helper T cell line 5C10. The amount of ST2V mRNA is greatly diminished when UT-7/GM cells are induced to differentiate into either erythroblastic or megakaryoblastic phenotypes. The possible roles of the ST2V in growth and differentiation are intriguing.