The limits of the cellular capacity to mediate an estrogen response.

While steroid response is generally restricted by the availability of steroid receptors, the theoretical limits of the response are not known. We have constructed a series of cell lines that stably express the estrogen receptor (ER) at levels up to 5,000,000 ERs per cell and employed these cells to explore the limits of the estrogen response. Several reporter genes with estrogen response elements upstream of the herpes thymidine kinase promoter showed hyperbolic saturation kinetics with increasing ER. Maximum response was 10 times that seen in cell lines with receptor titers comparable to physiological levels. Half-maximal responses required 500,000 receptors per cell, and cells with 5,000,000 ERs showed greater than 90% maximum induction. Estradiol dose-response studies indicated that the receptors are limiting below 500,000 ERs per cell, but at higher ER titers there are spare receptors. In contrast to most reporters, the widely used reporter pA2-CAT, which has 200 base pairs of Xenopus vitellogenin DNA between the response element and the promoter, showed squelching at ER levels beyond 500,000 per cell. Cell lines that expressed ER above this level activated pA2-CAT with a distorted hormone dependence, where saturating ligand concentrations were inhibitory. All reporters displayed squelching when the ER was provided by transient transfection at a level that we judge is 20,000,000 per cell by extrapolation from the behavior of stable cell lines. These findings suggest that saturation of the cellular capacity to mediate an estrogen response and ER-dependent squelching occur at receptor titers well above those encountered in nature. If current models of steroid hormone action are correct, the findings also imply that estrogen response elements are occupied to very small extents under normal conditions.     

Cutaneous malignant melanoma and familial dysplastic nevi: evidence for autosomal dominance and pleiotropy.

Segregation of familial cutaneous melanoma has been shown to be compatible with autosomal dominant transmission with incomplete penetrance. However, the combined phenotype of melanoma and a known melanoma-precursor lesion, the dysplastic nevus (DN), has not previously been found to fit a Mendelian model of inheritance using complex segregation analysis. Employing a life-table and disease-free survival analysis approach, we estimated the lifetime incidence of melanoma in the sibs and offspring of DN-affected individuals to be 46%, consistent with a highly penetrant, autosomal dominant mode of inheritance. To further elucidate the relationship between the two traits, we conducted a linkage analysis between the melanoma locus and a hypothetical DN locus, and obtained a maximum lod score of 3.857 at theta = .08. Furthermore, all families giving evidence for linkage were in the coupling phase and the maximum likelihood estimate of theta was not significantly different from 0 (P = .1). This provides evidence that the DN and melanoma traits may represent pleiotropic effects of a single, highly penetrant gene behaving in an autosomal dominant manner.     

Anti-idiotypic treatment of BALB/c mice induces CRIa-bearing suppressor cells with altered Igh-restricted function

The ABA-specific antibody response of A/J mice (Igh Ie) is dominated by the CRIa idiotype. In contrast, BALB/c mice (Igh Ia) do not produce CRIa-bearing anti-ABA antibodies after antigenic challenge. We have shown previously that treatment with rabbit anti-CRIa (R-anti-CRIa) induces the expression of "CRIa-like" anti-arsonate antibodies in BALB/c mice. In the present report, we demonstrate that R-anti-CRIa treatment enables BALB/c mice to respond to A/J ABA-specific first-order suppressor molecules (TsF1). Manipulated BALB/c also produced CRIa bearing ABA-specific immune response. Thus, R-anti-CRIa treatment induces a change in the characteristic Igh restriction pattern typically seen in this system. These data suggest that Igh restriction in the ABA-specific T suppressor cell pathway is the result of CRIa+ dominance in the T suppressor cell response of A/J mice. The effectiveness of idiotypic manipulation in inducing the expression of a given idiotype at both the B cell and T suppressor cell levels is discussed.     

Syngeneic monoclonal internal image anti-idiotopes as prophylactic vaccines

A syngeneic monoclonal anti-idiotope that behaves as an internal image of the mammalian reovirus type 3 cellular attachment protein (viral hemagglutinin) was used in the syngeneic host for the induction of a prophylactic anti-viral antibody response. These studies were performed without the aid of co-stimulation by viral antigens. The high stringency of this system enables us to define the maximum constraints on the use of anti-idiotopes as anti-viral vaccines. We have used the murine BALB/c monoclonal IgM anti-idiotope 87.92.6 to study the idiotope and antigen specificity, kinetics, dose dependence, adjuvant, carrier, and valency requirements of anti-idiotope-induced anti-viral antibody responses. These studies show that the production of high titer neutralizing antibody requires a lengthy (60 day) immunization protocol, which includes the use of adjuvant and multivalent anti-idiotope, and is dependent on anti-idiotope concentrations of greater than 50 micrograms. When administered in this manner anti-idiotope can stimulate serotype-specific antibody responses across species barriers at levels comparable with those obtained after inoculation with virus. The practical efficacy of these reagents and procedures is documented by the ability of maternal immunization with anti-idiotope to confer complete protection in neonates from a potentially lethal reovirus type 3 viral infection.     

Inhibition of human leukocyte 3-hydroxy-3-methylglutaryl coenzyme A reductase activity by ascorbic acid. An effect mediated by the free radical monodehydroascorbate

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity in microsomes isolated from cultured lymphoid (IM-9) cells or freshly isolated human leukocytes was markedly decreased by either ascorbic acid or its oxidized derivative, dehydroascorbate. Inhibition of IM-9 leukocyte HMG-CoA reductase activity was log linear between 0.01 and 10 mM ascorbic acid (25 and 81% inhibition, respectively) and 0.1 and 10 mM dehydroascorbate (5 and 75% inhibition, respectively). Inhibition was noncompetitive with respect to HMG-CoA (Km = 10.2 microM (RS); ascorbic acid, Ki = 6.4 mM; dehydroascorbate, Ki = 15 mM) and competitive with respect to NADPH (Km = 16.3 microM; acetic acid, Ki = 6.3 mM; dehydroascorbate, Ki = 3.1 mM). Ascorbic acid and dehydroascorbate are interconverted through the free radical intermediate monodehydroascorbate. Reducing agents are required to convert dehydroascorbate to monodehydroascorbate, but prevent formation of the free radical from ascorbate. In microsomes from IM-9 cells, the reducing agent, dithiothreitol, abolished HMG-CoA reductase inhibition by ascorbate but enhanced inhibition by dehydroascorbate. In addition, the concentration of monodehydroascorbate present in ascorbate solutions was directly proportional to the degree of HMG-CoA reductase inhibition by 1.0 mM ascorbate. Fifty per cent inhibition of enzyme activity occurred at a monodehydroascorbate concentration of 14 microM. These data indicate that monodehydroascorbate mediates inhibition of HMG-CoA reductase by both ascorbate and dehydroascorbate. This effect does not appear to be due to free radical-induced membrane lipid modification, however, since both ascorbate and dehydroascorbate inhibited the protease-solubilized, partially purified human liver enzyme. Since inhibition of HMG-CoA reductase occurs at physiological concentrations of ascorbic acid in the human leukocyte (0.2-1.72 mM), this vitamin may be important in the regulation of endogenous cholesterol synthesis in man.     

Monoclonal antibody OKT11A inhibits and recombinant interleukin 2 (IL 2) augments expression of IL 2 receptors at a pretranslational level

The expression of receptors for interleukin 2 (IL 2) represents a critical event regulating the growth of normal T lymphocytes. We investigated the effects of the inhibitory monoclonal antibody OKT11A (anti-sheep erythrocyte receptor) and of purified recombinant IL 2 (rIL 2) on the expression of IL 2 receptors by activated T cells at both the protein and the mRNA levels. Adding OKT11A antibody (0.5 microgram/ml) to phytohemagglutinin (PHA)-stimulated cultures of human peripheral blood mononuclear cells (PBMC) markedly suppressed cellular proliferation (assessed by [3H]thymidine incorporation) and IL 2 receptor expression (determined by immunofluorescence assay by using the anti-IL 2-receptor antibody, anti-Tac). Northern blot analysis performed with the use of a cDNA probe specific for the human IL 2 receptor gene demonstrated that OKT11A antibody also decreased the accumulation of IL 2 receptor mRNA induced by PHA in PBMC. Purified rIL 2 (10 U/ml) alone had little effect on the expression of IL 2 receptors in unstimulated PBMC cultures. In combination with PHA or with PHA plus OKT11A, however, rIL 2 augmented both the expression of IL 2 receptor protein on PBMC and the accumulation of IL 2 receptor mRNA in PBMC. Adding anti-Tac antibody to PBMC cultures to block the interaction of IL 2 with its receptor diminished the accumulation of IL 2 receptor mRNA induced by PHA. Taken together, these data demonstrate that OKT11A antibody inhibits and IL 2 augments expression of IL 2 receptors on PHA-stimulated T cells, at least in part, at a pretranslational level.