Tissue-type plasminogen activator-induced invasion and metastasis of murine melanomas.

The role of tissue-type plasminogen activator (tPA) in the 'spontaneous' as well as 'experimental' metastasis of ocular melanomas in mice was evaluated by transfecting the D5.1G4 murine melanoma cell line that possesses low metastatic activity and low tPA activity with a full length cDNA encoding human tPA. For comparison, a highly metastatic melanoma cell line (Queen's) that constitutively expresses high tPA production, was transfected with a cDNA coding for human plasminogen activator inhibitor type 1 (PAI-1). Unlike non-transfected controls, transfected D5.1G4 melanoma cells expressed high levels of tPA and produced extensive pulmonary metastases following intravenous injection. By contrast, PAI-1 transfected Queen's melanoma cells expressed low tPA activity and displayed significantly reduced metastatic potential compared with nontransfected controls. Moreover, PAI-1 transfected Queen's melanoma cells did not metastasize from the eye while nontransfected parental cells produced extensive spontaneous metastases. Expression of tPA activity in transfected and nontransfected cell lines was completely blocked by an anti-tPA antibody. This antibody significantly inhibited the organ localization and frequency of lung metastases of both Queen's and tPA-transfected D5.1G4 melanomas. This study demonstrates that tPA is involved in the metastasis of murine intraocular melanomas.

Capacity of simian virus 40 T antigen to induce self-tolerance but not immunological privilege in the anterior chamber of the eye.

Transgenic mice bearing the simian virus 40 (SV40) large T oncogene developed progressively growing intraocular tumors and displayed characteristics of immunological tolerance to SV40 T antigen. Transgenic mice failed to mount CTL responses to SV40 T antigen-bearing tumor cell lines derived from the transgenic intraocular tumors. Spleen cells from transgenic hosts were able to prevent the in vivo and in vitro generation of CTL responses by lymphocytes from normal syngeneic FVB/N mice. Adoptive transfer of spleen cells from tolerant transgenic donors temporarily inhibited the immunological rejection of SV40 T antigen-positive tumor cells transplanted to normal syngeneic FVB/N recipients. Thus, introduction of SV40 transforming sequences into the mouse germline induced tolerance to SV40 T antigen. However, in normal FVB/N mice, SV40 T antigen-bearing tumor cells failed to experience immune privilege in the anterior chamber and did not elicit systemic down-regulation of delayed-type hypersensitivity responses that characteristically occur when antigens are introduced into the anterior chamber. The results indicate that within the anterior chamber of the eye, SV40 T antigen-bearing cells are perceived by the host's immune system much differently than are other categories of antigen. Thus, SV40 T antigen effectively induces self-immunological tolerance when its gene is introduced into the host's germline but fails to experience immunological privilege in the anterior chamber of the eye in normal hosts.

Ligand-dependent down-regulation of stably transfected human glucocorticoid receptors is associated with the loss of functional glucocorticoid responsiveness.

The effect of glucocorticoids on the regulation of stably transfected human glucocorticoid receptors has been examined. Exposure of a Chinese hamster ovary-derived cell line containing stably transfected human glucocorticoid receptor genes and glucocorticoid-responsive dihydrofolate reductase genes to 5 nM dexamethasone resulted in a rapid, time-dependent reduction in the level of glucocorticoid receptor protein to 50% of control levels within 5 h of steroid treatment. This decrease in receptor protein was persistent, with a maximal 70% reduction observed even after 4 weeks of dexamethasone treatment. Immunocytochemical analysis of the influence of dexamethasone on stably transfected glucocorticoid receptors revealed efficient translocation of receptors to the nucleus within 1 h of hormone treatment. However, upon longer exposure to dexamethasone (5 h), immunoreactive glucocorticoid receptors were localized primarily to the cytoplasm. By 24 h of treatment, glucocorticoid receptors were absent from the cytoplasm and the nucleus, suggesting that the ligand-induced loss of glucocorticoid receptors may be a cytoplasmic event. The decrease in transfected glucocorticoid receptor protein was largely reflected by similar changes in steady state levels of human glucocorticoid receptor mRNA; however, the effects of hormone on receptor protein levels were more profound than on receptor mRNA. There was an initial rapid reduction in transfected glucocorticoid receptor mRNA to 50% of control levels within 2 h of dexamethasone treatment. This reduction was followed by a transient rise in mRNA expression after 12 h of hormone treatment. With prolonged exposure to dexamethasone (> 12 h) a second, more gradual decline in human glucocorticoid receptor mRNA was observed. This biphasic pattern of glucocorticoid receptor gene expression was not reflected at the level of receptor protein, suggesting that both transcriptional and translational control mechanisms may be involved in ligand-dependent receptor regulation. When cells were removed from dexamethasone after up to 48 h of treatment, glucocorticoid receptor mRNA levels fully recovered within 12 h. Receptor protein recovered only partially during this same time period. Down-regulation of glucocorticoid receptor protein and mRNA levels by dexamethasone in stably transfected cells led to corresponding reductions in the hormone sensitivity to two glucocorticoid-regulated genes: a transiently transfected chloramphenicol acetyltransferase receptor gene and a stably integrated dihydrofolate reductase gene. These results demonstrate that stably transfected human glucocorticoid receptors are subject to ligand-induced down-regulation in a heterologous cell line. Moreover, glucocorticoid receptor autoregulation appears to be a highly conserved mechanism for attenuating cellular responsiveness to hormone.

Methotrexate-induced overexpression of functional glucocorticoid receptors in Chinese hamster ovary cells.

We have used a modified cotransfection and selection strategy to create a series of mammalian cell lines that stably express high levels of intact glucocorticoid receptors. These cell lines were produced by subjecting Chinese hamster ovary (CHO) cells, which had been previously cotransfected with a glucocorticoid-responsive dihydrofolate reductase (DHFR) gene and the human glucocorticoid receptor gene, to growth in increasing concentrations of methotrexate (MTX). By linking the MTX selection process to glucocorticoid receptor function via the DHFR gene, stable cell lines resistant to a range of MTX concentrations (50 nM to 3 microM) were isolated that were strictly dependent upon glucocorticoids for growth. Quantitation of steroid binding capacity in MTX-resistant cells revealed a progressive increase in the number of glucocorticoid receptors as a function of increasing MTX concentration. This increase in receptor content was maximal at the highest level of MTX resistance examined (3 microM MTX) and represented a 25-fold elevation in glucocorticoid receptor number relative to CHO cells expressing only endogenous hamster receptor. The increases in steroid binding obtained after MTX selection were reflected by similar increases in the level of glucocorticoid receptor protein as determined by immunoblot analysis. Examination of glucocorticoid receptor structure by sucrose density gradient centrifugation revealed that oligomeric (9 S) steroid receptor complexes were formed at all levels of receptor expression. Subcellular localization of the glucocorticoid receptor protein by immunocytochemical staining revealed effective nuclear translocation of the overexpressed receptors in MTX-resistant cells. Functional transfection studies using a glucocorticoid-responsive reporter gene indicated that the additional glucocorticoid receptors in CHO cells were competent to activate transcription. To determine the molecular basis for the MTX-induced increases in functional glucocorticoid receptors, steady-state levels of glucocorticoid receptor mRNA were examined. MTX selection produced a 5- to 7-fold increase in transfected glucocorticoid receptor gene expression relative to untreated cells. MTX-resistant cells also expressed increased levels of a putative hamster glucocorticoid receptor mRNA species. Interestingly, the observed increases in receptor gene expression in these cells could not be accounted for by amplification of either the human or the hamster glucocorticoid receptor genes.

Autoregulation of glucocorticoid receptor gene expression.

Glucocorticoid receptors are members of a highly conserved family of steroid receptor proteins, which are ligand-dependent transcription factors. Previous studies have shown that the presence of functional glucocorticoid receptors is a prerequisite for manifestation of cellular responses to hormone. Glucocorticoid receptors undergo down-regulation following treatment with glucocorticoids. To define the molecular mechanisms that are involved in this process we have analyzed the down-regulation of glucocorticoid receptors both in HeLa cells, which contain endogenous receptors, and in cells containing receptors that have been introduced by DNA transfection. Our results show that cells that contain glucocorticoid receptors--either endogenous or transfected--undergo down-regulation of steroid-binding capabilities, as well as reductions in receptor protein and mRNA levels, in a remarkably similar fashion. DNA sequences in the coding region of the human glucocorticoid receptor cDNA appear to be sufficient to account for down-regulation of receptor. This novel finding suggests that unique mechanisms are involved in controlling glucocorticoid receptor homeostasis.

Novel antipeptide antibodies to the human glucocorticoid receptor: recognition of multiple receptor forms in vitro and distinct localization of cytoplasmic and nuclear receptors.

We have synthesized two peptides that correspond to unique regions of the amino-terminus of the human glucocorticoid receptor (GR). Peptides representing amino acids 245-259 and 346-367 (designated 59 and 57, respectively) were chosen on the basis of hydrophobicity/hydrophilicity ratios as well as overall proline content. These peptides were then used as antigens to produce epitope-specific antibodies that recognize and interact with human GR in a variety of physical states. Antiserum directed against each peptide recognizes denatured, [3H] dexamethasone mesylate-labeled GR as well as unliganded receptor on Western blots. In contrast to other antipeptide GR antibodies, these antibodies recognize and form stable complexes with unactivated and molybdate-stabilized forms of the GR, indicating that neither epitope is occluded when the receptor exists in an oligomeric state. Activated, 4S DNA-binding forms of the receptor are also recognized by both antibodies. The interaction of antibodies 59 and 57 with human GR in various states is highly specific based on the observation that preincubation of either antiserum with the appropriate peptide completely precludes the recognition of receptor by antibody. Titration analysis of antisera reveals that an increase in the antibody concentration cause discrete increases in the sedimentation coefficient of GR on sucrose gradients. These shifts occur under high salt conditions and are consistent with the formation of multiple stable antibody-receptor complexes. Interestingly, neither antibody interferes with the ability of the GR to be activated into a DNA-binding form or with the ability of the activated GR to interact with DNA cellulose. Consistent with these observations, both antibodies recognize and form stable complexes with GR when the receptor is associated with DNA fragments that contain specific glucocorticoid-responsive elements. Thus, both antibodies appear to recognize all known forms of the human GR protein. Using immunohistochemical techniques to visualize GR in HeLa S3 cells as well as in Chinese hamster ovary cells that stably express transfected human GR, a cytoplasmic location for receptor is observed in the absence of ligand. In contrast, immunoreactive GR is predominantly nuclear after hormone treatment, further supporting a role for nuclear translocation in GR function.

Stable overproduction of intact glucocorticoid receptors in mammalian cells using a selectable glucocorticoid responsive dihydrofolate reductase gene.

We have generated several mammalian cell lines that stably express high levels of intact glucocorticoid receptor. These cells were created by cotransfecting a glucocorticoid-dependent dihydrofolate reductase (DHFR) gene into DHFR-deficient Chinese hamster ovary (CHO) cells together with a plasmid directing the expression of human glucocorticoid receptor. Using this approach, transfection frequencies indicate that the inclusion of glucocorticoid receptor cDNA increased the efficiency of DHFR transformation greater than 10-fold over nonreceptor control DNA. When a stably cotransfected line (designated MG/hGR) was subjected to short term growth in cytotoxic concentrations of the antifolate methotrexate, these cells strongly resisted growth inhibition when dexamethasone was present in the medium. This effect was steroid specific and was inhibited by the glucocorticoid antagonist RU38486. In an effort to exploit the methotrexate-induced coamplification properties of the DHFR gene as a means of creating cell lines having increased levels of glucocorticoid receptor, MG/hGR cells were chronically exposed to a relatively low concentration of methotrexate (50 nM). After this treatment a resistant line was isolated (MG/hGR/MTX50) that displayed complete dependence on exogenous glucocorticoid for growth. To investigate the molecular basis for the enhanced ability of MG/hGR/MTX50 cells to resist the cytotoxic effects of methotrexate in the presence of dexamethasone, glucocorticoid receptor protein in these cells was characterized and compared to parental CHO cells and methotrexate sensitive MG/hGR cells. Affinity labeling with [3H]dexamethasone mesylate and Western blot analysis with antiglucocorticoid receptor antiserum revealed that nontransfected CHO cells have virtually undetectable levels of glucocorticoid receptor protein whereas cotransfected MG/hGR cells contain at least 3 times more intact monomeric receptor protein of Mr 94,000. Correspondingly, analysis of receptor protein in MG/hGR/MTX50 cells indicated that these cells contain 8 to 10 times more glucocorticoid receptor than nontransfected CHO cells. Scatchard analysis of steroid binding curves revealed that these increases correspond to 6,600, 22,000 and 63,000 dexamethasone binding sites per cell for nontransfected CHO cells, cotransfected MG/hGR cells, and MG/hGR/MTX50 cells, respectively. Sedimentation profiles of native receptor in transfected and methotrexate-resistant cells further support the progressive increase in receptor content and demonstrate that glucocorticoid receptor exists in cotransfected cels as an oligomeric complex under hypotonic conditions (9S complex in the presence of 20 mM sodium molybdate, 7S in the absence of molybdate), which dissociates to a monomeric 4S species in the presence of 0.4 M KCl. These physicochemical properties are indistinguishable from those observed for the endogenous hamster glucocorticoid receptor and suggest that stably transfected human glucocort