ABSTRACT
Hypothemycin, a resorcylic acid lactone antibiotic, was identified as active in a screen for inhibitors of T cell activation. It was found to inhibit the proliferation of mouse and human T cells stimulated with anti-CD3 mAb + PMA and of human PBMC stimulated with anti-CD3 mAb alone. This inhibition was partially reversed by exogenous IL-2 indicating that it is not due to non-specific toxicity. Hypothemycin potently suppressed the production of IL-2 (IC50: 9 nM) but affected IL-2-induced proliferation to a lesser extent (IC50: 194 nM). Hypothemycin also inhibited IL-6, IL-10, IFN-gamma and TNF-alpha production. By contrast, it markedly enhanced the production of IL-4, IL-5 and IL-13. These effects were seen both at the mRNA and protein secretion levels. Analysis of the effect of hypothemycin on CD69 induction suggested that it disrupts calcineurin-independent rather than calcineurin-dependent signaling. Furthermore, hypothemycin was able to inhibit the phosphorylation of ERK1/2 induced by PMA treatment of T cells. Therefore, hypothemycin represents an inhibitor of T cell activation with a novel mode of action and unique modulatory activity on cytokine production.
Subject(s)
Antineoplastic Agents/pharmacology , Cytokines/drug effects , Immunosuppressive Agents/pharmacology , Lymphocyte Activation/drug effects , RNA, Messenger/drug effects , T-Lymphocytes/drug effects , Animals , Cytokines/metabolism , Estrogens, Non-Steroidal/pharmacology , Humans , Mice , RNA, Messenger/metabolism , T-Lymphocytes/metabolism , Tacrolimus/pharmacology , Zearalenone/pharmacologyABSTRACT
The synthesis of C32-O-arylethyl ether derivatives of ascomycin that possess equivalent immunosuppressant activity but reduced toxicity, compared to FK-506, is described.
Subject(s)
Immunosuppressive Agents/chemical synthesis , Immunosuppressive Agents/pharmacology , Macrolides/chemical synthesis , Macrolides/pharmacology , Tacrolimus/analogs & derivatives , Administration, Oral , Animals , Calcineurin Inhibitors , Drug Evaluation, Preclinical , Hypothermia/chemically induced , Immunophilins/metabolism , Immunosuppressive Agents/metabolism , Immunosuppressive Agents/toxicity , Inhibitory Concentration 50 , Injections, Intravenous , Kidney Diseases/chemically induced , Male , Mice , Mice, Inbred BALB C , Rats , Rats, Sprague-Dawley , Structure-Activity Relationship , T-Lymphocytes/drug effects , Tacrolimus/chemistry , Tacrolimus/pharmacology , Tacrolimus/toxicity , Tacrolimus Binding Proteins , Toxicity TestsABSTRACT
Cytokine production upon T cell activation results from the integration of multiple signaling pathways from TCR/CD3 and from costimulatory molecules such as CD28. Among these pathways, the possible role of p38 mitogen activated protein kinase (MAPK) is the least understood. Here, we used a highly specific p38 MAPK inhibitor, the SB203580 compound, to examine the role of this enzyme in the induction of various cytokines in human T cells stimulated with anti-CD3 and anti-CD28 mAb together or in combination with PMA. Cytokine induction was monitored by ELISA and at the mRNA level. While SB203580 had little effect on IL-2 production and proliferation, it significantly reduced the production of several other cytokines. The secretion of IL-4, IL-5, IL-13, and TNF-alpha was inhibited by 20-50% with modes of T cell activation involving the CD28 pathway, whereas their mRNA expression was little affected. In contrast, IFN-gamma induction via CD28/PMA or CD3/CD28, but not CD3/PMA, was markedly diminished both at the protein and at the mRNA levels. Most interestingly, SB203580 also suppressed IL-10 secretion and mRNA induction via CD28-dependent activation by 75-85% (IC50 approximately 0.2 microM). Subset analysis suggested that this inhibition did not reflect a differential effect on T cell subsets. Therefore, p38 MAPK activity appears to contribute to cytokine production, mostly via CD28-dependent signaling. Moreover, IL-10 seems to rely more on this activity than other cytokines for its induction in T cells.
Subject(s)
CD28 Antigens/physiology , Calcium-Calmodulin-Dependent Protein Kinases/physiology , Cytokines/biosynthesis , Interleukin-10/biosynthesis , Mitogen-Activated Protein Kinases , T-Lymphocytes/metabolism , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Cells, Cultured , Cytokines/genetics , Dose-Response Relationship, Drug , Humans , Imidazoles/pharmacology , Interferon-gamma/biosynthesis , Interleukin-2/biosynthesis , Lymphocyte Activation/drug effects , Pyridines/pharmacology , RNA, Messenger/analysis , p38 Mitogen-Activated Protein KinasesABSTRACT
BACKGROUND: Tacrolimus (FK506) has potent immunosuppressive properties reflecting its ability to block the transcription of lymphokine genes in activated T cells through formation of a complex with FK506 binding protein-12, which inhibits the phosphatase activity of calcineurin. The clinical usefulness of tacrolimus is limited, however, by severe adverse effects, including neurotoxicity and nephrotoxicity. Although this toxicity, like immunosuppression, appears mechanistically related to the calcineurin inhibitory action of the drug, a large chemistry effort has been devoted to search for tacrolimus analogs with reduced toxicity but preserved immunosuppressive activity that might have enhanced therapeutic utility. METHODS: Here, we report on the identification of such an analog, which was synthetically derived from ascomycin (ASC), the C21 ethyl analog of tacrolimus, by introducing an indole group at the C32 position. The profile of biological activity of indolyl-ASC was characterized in rodent models of immunosuppression and toxicity. RESULTS: Indolyl-ASC was found to exhibit an immunosuppressive potency equivalent to that of tacrolimus in T-cell activation in vitro and in murine transplant models, even though indolyl-ASC bound about 10 times less to intracellular FK506 binding protein-12 than tacrolimus or ASC. Further evaluation of indolyl-ASC revealed that it is threefold less potent than tacrolimus in inducing hypothermia, a response that may reflect neurotoxicity, and in causing gastrointestinal transit alterations in mice. Moreover, indolyl-ASC was at least twofold less nephrotoxic than tacrolimus upon 3-week oral treatment in rats. CONCLUSIONS: Altogether, these data indicate a modest but definite improvement in the therapeutic index for indolyl-ASC compared with tacrolimus in rodent models.
Subject(s)
Immunosuppressive Agents/pharmacology , Tacrolimus/analogs & derivatives , Animals , Blood Urea Nitrogen , Body Temperature/drug effects , Cell Division/drug effects , Female , Immunosuppressive Agents/toxicity , Ionomycin/pharmacology , Kidney/drug effects , Kidney/pathology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Rats , Rats, Inbred SHR , Rats, Sprague-Dawley , Tacrolimus/pharmacology , Tacrolimus/toxicity , Thyroid Gland/transplantationABSTRACT
A series of C32-O-aralkyl ether derivatives of the FK-506 related macrolide ascomycin have been prepared based on an earlier reported C32-O-cinnamyl ether design. In the present study, the nature of the aryl tethering group was varied in an attempt to improve oral activity. An imidazol-2-yl-methyl tether was found to be superior among those investigated and has resulted in an ascomycin analog, L-733,725, with in vivo immunosuppressive activity comparable to FK-506 but with an improved therapeutic index.
Subject(s)
Imidazoles/chemical synthesis , Immunosuppressive Agents/chemical synthesis , Tacrolimus/analogs & derivatives , Tacrolimus/chemical synthesis , Animals , Biological Availability , Hypothermia/chemically induced , Imidazoles/pharmacology , Imidazoles/toxicity , Immunophilins/metabolism , Immunosuppressive Agents/pharmacology , Immunosuppressive Agents/toxicity , Indicators and Reagents , Kidney/drug effects , Kidney/pathology , Mice , Mice, Inbred BALB C , Molecular Conformation , Molecular Structure , Neurotoxins/toxicity , Rats , Structure-Activity Relationship , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , Tacrolimus/pharmacology , Tacrolimus/therapeutic use , Tacrolimus/toxicity , Tacrolimus Binding ProteinsABSTRACT
FK506 blocks T cell activation by preventing the transcription of lymphokine genes through binding to the intracellular protein FKBP12 and formation of complex that inhibits the phosphatase activity of calcineurin. Beside exerting potent suppressive activity on cellular and humoral immune responses, in vivo treatment with FK506 in rodent models induces thymic alterations characterized by a selective reduction of mature CD4+8- cells. The potential relationship between such thymic alterations and the immunosuppressive and calcineurin inhibitory activities of FK506 has not been defined. Here, we took advantage of the availability of FK506 analogs with different immunosuppressive potencies to address this question. Intravenous daily administration of FK506 in Sprague-Dawley rats for 4 days was found to be sufficient to cause a depletion of CD4+8- thymocytes with an ED50=0.06 mg/kg/day. Under the same conditions, L-683,590 which is 2-3-fold less potent than FK506 in inhibiting T cell activation and calcineurin function gave an ED50=0.17 mg/kg/day. In contrast, the nonimmunosuppressive, calcineurin noninhibitory antagonist L-685,818, failed to deplete the CD4+8- thymocyte subset but could reverse the reducing effect of FK506 on this subset. Another analog, L-688,617, which does not completely inhibit T cell activation in vitro, also behaved as a partial agonist of CD4+8- cell depletion. Therefore, the ability of FK506 analogs to deplete the CD4+8- thymocytes subset correlates with their immunosuppressive and calcineurin inhibitory potencies. This suggests that calcineurin is involved in the intra-thymic maturation processes of CD4+8- T cells. Moreover, the short-term treatment protocol described here provides a rapid and quantitative assay to determine the immunosuppressive potency of FK506-like compounds in vivo
Subject(s)
CD4-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/drug effects , Calmodulin-Binding Proteins/antagonists & inhibitors , Immunosuppressive Agents/pharmacology , Phosphoprotein Phosphatases/antagonists & inhibitors , T-Lymphocyte Subsets/drug effects , Tacrolimus/analogs & derivatives , Animals , CD4-CD8 Ratio/drug effects , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Calcineurin , Dose-Response Relationship, Drug , Male , Rats , Rats, Sprague-Dawley , Receptors, Antigen, T-Cell/biosynthesis , Tacrolimus/pharmacology , Thymus Gland/cytologyABSTRACT
FK-506 inhibits Ca(2+)-dependent transcription of lymphokine genes in T cells, and thereby acts as a powerful immunosuppressant. However, its potential therapeutic applications may be seriously limited by several side effects, including nephrotoxicity and neurotoxicity. At present, it is unclear whether these immunosuppressive and toxic effects result from interference with related biochemical processes. FK-506 is known to interact with FK-binding protein-12 (FKBP-12), an abundant cytosolic protein with cis-trans peptidyl-prolyl isomerase activity (PPIase) activity. Because rapamycin (RAP) similarly binds to FKBP-12, although it acts in a manner different from FK-506, by inhibiting T cell responses to lymphokines, such an interaction with FKBP-12 is not sufficient to mediate immunosuppression. Recently, it was found that the complex of FKBP-12 with FK-506, but not with RAP, inhibits the phosphatase activity of calcineurin. Here, we used L-685,818, the C18-hydroxy, C21-ethyl derivative of FK-506, to explore further the role of FKBP-12 in the immunosuppressive and toxic actions of FK-506. Although L-685,818 bound with high affinity to FKBP-12 and inhibited its PPIase activity, it did not suppress T cell activation, and, when complexed with FKBP-12, did not affect calcineurin phosphatase activity. However, L-685,818 was a potent antagonist of the immunosuppressive activity of both FK-506 and RAP. Moreover, L-685,818 did not induce any toxicity in dogs and rats or in a mouse model of acute FK-506 nephrotoxicity, but it blocked the effect of FK-506 in this model. Therefore, FK-506 toxicity involves the disruption of biochemical mechanisms related to those implicated in T cell activation. Like immunosuppression, this toxicity is not due to the inhibition of the PPIase activity of FKBP-12, but may be linked to the inhibition of the phosphatase activity of calcineurin by the drug FKBP-12 complex.
Subject(s)
Immunosuppressive Agents/pharmacology , Polyenes/antagonists & inhibitors , Tacrolimus/analogs & derivatives , Tacrolimus/toxicity , Amino Acid Isomerases/antagonists & inhibitors , Animals , Calcineurin , Calmodulin-Binding Proteins/antagonists & inhibitors , Calmodulin-Binding Proteins/metabolism , Carrier Proteins/antagonists & inhibitors , Carrier Proteins/metabolism , Female , Lymphocyte Activation , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Peptidylprolyl Isomerase , Phosphoprotein Phosphatases/antagonists & inhibitors , Phosphoprotein Phosphatases/metabolism , Sirolimus , T-Lymphocytes/immunology , Tacrolimus/antagonists & inhibitors , Tacrolimus/pharmacology , Tacrolimus Binding ProteinsABSTRACT
In this report we have approached two questions relating to the mechanism of action of cyclosporin A (CsA). First, we address whether the major cytosolic protein for CsA, cyclophilin, is directly involved in mediating the immunosuppressive activity of this drug, and, in particular, whether inhibition of this protein's peptidyl-prolyl cis-trans isomerase (PPIase) activity results in inhibition of murine T cell activation. Second, we ask whether the nephrotoxicity observed with CsA is related to inhibition of PPIase-dependent pathways in cells other than lymphocytes. Using a series of 61 cyclosporin analogues, we generally found a good correlation between cyclophilin binding and immunosuppressive activity for the majority of analogues analyzed. However, a number of compounds of distinct structural classes were found that could interact with cyclophilin but were much less immunosuppressive than expected. The inability of these analogues to inhibit lymphocyte activation could not be explained by their failure to enter the cell and bind to cyclophilin under the conditions used in the cellular assays. Surprisingly, a nonimmunosuppressive analogue, MeAla-6, which bound well to cyclophilin and was active as a PPIase inhibitor, did not induce renal pathology in vivo. Furthermore, another analogue, MeBm2t, which was immunosuppressive in vitro, possessed little or no activity as a PPIase inhibitor. These findings pose serious questions concerning a direct role of cyclosporin in mediating CsA's immunosuppressive and nephrotoxic activities. In addition, they raise doubts about whether PPIase has a direct function in lymphocyte signal transduction.
Subject(s)
Amino Acid Isomerases/metabolism , Carrier Proteins/metabolism , Cyclosporins/pharmacology , Immunosuppression Therapy , Kidney/pathology , Lymphocyte Activation/drug effects , T-Lymphocytes/immunology , Animals , Cyclosporins/toxicity , In Vitro Techniques , Kidney/drug effects , Kinetics , Mice , Mice, Inbred BALB C , Molecular Structure , Peptidylprolyl Isomerase , Protein Binding , Structure-Activity Relationship , T-Lymphocytes/drug effectsABSTRACT
The structurally related immunosuppressive macrolides FK-506 and rapamycin (RAP) were previously shown to inhibit T cell stimulation through different mechanisms. FK-506 acts similarly to cyclosporin A (CsA) and prevents IL-2 production and IL-2R expression. RAP has little or no effect on these events but markedly impedes the response to IL-2. The present study was initiated to examine the possibility of a complementation between the immunosuppressive actions of RAP and FK-506 or CsA on various murine T cell responses. RAP potentiated the effect of CsA on proliferation and IL-2R expression in T cells stimulated with ionomycin + PMA. However, in the same system, RAP acted as a potent antagonist of FK-506 suppression. RAP also blocked FK-506- but not CsA-mediated inhibition of IL-2 mRNA induction. By using model systems sensitive to inhibition by RAP but not FK-506 we further demonstrated that FK-506 reciprocally behaves as an antagonist of RAP. In one such model, the stimulation of splenic T cells with IL-2 + PMA, FK-506, but not CsA, reversed the suppressive effect of RAP on proliferation. FK-506 also antagonized RAP-mediated inhibition with respect to the induction of Ly-6E Ag expression by IFN in YAC cells. To explore further the competition between the two macrolides at the cellular level, we performed binding experiments with a radiolabeled derivative of FK-506. Both FK-506 and RAP, but not CsA, inhibited the binding of this probe in YAC cells. Taken together, these data demonstrate that FK-506 and RAP antagonize each other's biologic activity and physically interact with a common receptor site(s) in T cells. Moreover, CsA acts at a site distinct from the cellular target(s) of FK-506 or RAP.
Subject(s)
Anti-Bacterial Agents/pharmacology , Immunosuppressive Agents/pharmacology , Lymphocyte Activation/drug effects , T-Lymphocytes/drug effects , Animals , Anti-Bacterial Agents/antagonists & inhibitors , Binding, Competitive , Cyclosporins/pharmacology , Gene Expression/drug effects , In Vitro Techniques , Interleukin-2/genetics , Ionomycin/pharmacology , Mice , Mice, Inbred C57BL , Polyenes/antagonists & inhibitors , Polyenes/pharmacology , Receptors, Interleukin-2/metabolism , Signal Transduction/drug effects , Sirolimus , Tacrolimus , Tetradecanoylphorbol Acetate/pharmacologyABSTRACT
FK-506 and the structurally related macrolide rapamycin (RAP) were investigated in comparison with cyclosporin A (CsA) for their immunosuppressive effects on murine T cells. All three agents suppressed the proliferation of splenic T cells triggered by lectins or antibodies to CD3 and Ly-6C. FK-506 or CsA also inhibited proliferation, IL-2 production, and IL-2R expression in splenic T cells activated with ionomycin + PMA. However, RAP minimally affected IL-2 production and IL-2R expression in these cells, although it reduced proliferation. Similarly, FK-506 and CsA, but not RAP, suppressed IL-2 production by activated DO.11.10 T hybridoma cells. In such a system, as well as in normal T cells stimulated with high ionomycin concentrations, FK-506 and CsA enhanced proliferation, indicating that they both abrogate negative signals associated with T cell activation. On the contrary, RAP diminished the autonomous proliferation of hybridoma cells, whereas FK-506 and CsA had little effect. The proliferative response induced in D10.G4 cells by IL-1 + ionomycin but not that induced by IL-1 + PMA was sensitive to inhibition by FK-506 and CsA. In contrast, RAP inhibited equally well both types of stimulation. Finally, T cell proliferation driven by IL-2 or IL-4 was found to be relatively resistant to FK-506 or CsA but sensitive to RAP. Altogether, these data demonstrate that FK-506 and CsA alter similar calcium-associated events of T cell activation and block T cell proliferation primarily by suppressing lymphokine production. RAP interferes with a different set of events and inhibits T cells by impairing their response to growth-promoting lymphokines.
Subject(s)
Anti-Bacterial Agents/pharmacology , Immunosuppressive Agents/pharmacology , Lymphocyte Activation/drug effects , Animals , Cyclosporins/pharmacology , Hybridomas , Interleukin-2/antagonists & inhibitors , Interleukin-2/biosynthesis , Interleukin-4/antagonists & inhibitors , Interleukin-4/biosynthesis , Ionomycin/pharmacology , Mice , Mice, Inbred C57BL , Polyenes/pharmacology , Receptors, Interleukin-2/metabolism , Sirolimus , Tacrolimus , Tetradecanoylphorbol Acetate/pharmacologyABSTRACT
In order to establish whether CyP is the pharmacologically relevant CsA receptor, the CyP binding v immunosuppressive activity was measured for an extensive, structurally varied group of CsA analogues. Overall, CyP binding was found to parallel immunosuppressive activity. Other than MeAla6-CsA, the few exceptions to the correlation could be ascribed to cellular metabolism. These results strongly implicate CyP or a related protein in the mechanism of action of cyclosporine.
Subject(s)
Carrier Proteins/metabolism , Cyclosporins/metabolism , Immunosuppression Therapy , Immunosuppressive Agents , Lymphocyte Activation/drug effects , Animals , Ethers/pharmacology , In Vitro Techniques , Interleukin-2/metabolism , Ionomycin , Lymphocyte Culture Test, Mixed , Mice , Peptidylprolyl Isomerase , Structure-Activity Relationship , Tetradecanoylphorbol Acetate/pharmacologyABSTRACT
Monoclonal antibodies (mAb) to graft-specific class I or class II major histocompatibility antigens were tested for their ability to enhance the survival of allogeneic skin transplants. Mutant mouse strains were grafted with wild type tissue to restrict the antigenic differences being recognized. For allogeneic recognition of the class I antigen Ld, mutant BALB/c-H-2dm2 (dm2) mice were grafted with wild type BALB/cKh skin, and two dm2 anti-BALB/cKh mAb, 23-10-1 and 30-5-7, were tested for their ability to enhance. The anti-Ld antibody 23-10-1 (IgM) was found not to enhance the survival of BALB/c skin on dm2 mice. 30-5-7, however, an IgG2a antibody of indistinguishable specificity from 23-10-1, prolonged graft survival for approximately 5 days. For recognition of selected Iab determinants, mutant B6.C-H-2bm12 (bm12) mice were grafted with wild type B6/Kh skin, and mAb specific for the serological change(s) in bm12 were tested for their ability to enhance. The anti-Iab antibody 25-9-17 (IgG2a) was found not to enhance B6 grafts on bm12 mice. However, the enhancement seen with 25-9-17 using (C3H X bm12)F1 recipients was extraordinary, such that treated mice had a mean survival time three times that of the controls. Since 25-9-17 is of C3H origin, these results suggest that allotype (or possibly idiotype) compatibility is important in antibody enhancement. Another anti-Iab antibody 28-16-8 (IgM), also of C3H origin, failed to enhance a B6 graft on (C3H X bm 12)F1 mice.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Antibodies, Monoclonal/immunology , Graft Enhancement, Immunologic , Skin Transplantation , Animals , Graft Survival , Histocompatibility Antigens Class II/immunology , Immunoglobulin G/immunology , Immunoglobulin M/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Transplantation, HomologousABSTRACT
The T cell repertoire of B6.C-H-2bm12 mice (an I-A mutant mouse strain) to wild-type Iab antigens was investigated using both secondary proliferative cultures and cloned T cell lines. Because bm12 mice have a gain-loss mutation of their gene encoding the Ia beta-chain polypeptide, bm12 anti-B6 T cell responses are specific for the select component of Iab specificities that was lost as a result of the mutation. Although stimulator cells bearing Iab antigens elicited the strongest responses, Iaq, d, and s antigens also resulted in reproducible stimulations of these bm12 anti-B6-primed T cells. Cloned T cell lines isolated from bm12 anti-b6 cultures revealed similar findings, with most clones recognizing determinants unique for Iab antigens; however, clones showing cross-reactions with Iad and/or q were also selected. Using F1 hybrid responder T cells (mutant x cross-reactive strain), we further dissected this cross-reactivity into several distinct cross-reactive determinants. Because bm12 mice lack the serologically defined Ia differentiation antigen W39, T cell recognition of this determinant was investigated by using bm12 anti-B6-primed cells. Stimulation by Ia.W39+ cells was appreciably better than by Ia.W39- (Xid-defective) cells, suggesting that bm12 T cells recognize an Xid-regulated, W39-like Ia differentiation antigen.
Subject(s)
Genes, MHC Class II , Mice, Mutant Strains/immunology , T-Lymphocytes/immunology , Animals , Clone Cells/immunology , Cross Reactions , Epitopes/immunology , Female , Lymphocyte Activation , Male , Mice , Mice, Inbred C57BL , Mice, Inbred CBASubject(s)
Mammary Neoplasms, Experimental/drug therapy , Neoplasms, Hormone-Dependent/drug therapy , Progesterone/administration & dosage , Animals , Castration , Estradiol/administration & dosage , Female , Mammary Neoplasms, Experimental/blood , Mammary Neoplasms, Experimental/therapy , Neoplasms, Experimental/metabolism , Neoplasms, Hormone-Dependent/blood , Neoplasms, Hormone-Dependent/therapy , Progesterone/blood , Prolactin/metabolism , Rats , Rats, Inbred WFSubject(s)
Prolactin/blood , Animals , Female , Half-Life , Kinetics , Mammary Neoplasms, Experimental/blood , Radioligand Assay , Rats , SheepSubject(s)
Mammary Neoplasms, Experimental/therapy , Ovary/physiology , Prolactin/physiology , Adrenocorticotropic Hormone/pharmacology , Animals , Castration , Female , Growth Hormone/pharmacology , Hydroxyprogesterones/pharmacology , Mammary Neoplasms, Experimental/blood , Neoplasms, Experimental/metabolism , Prolactin/blood , Prolactin/metabolism , Rats , Rats, Inbred WF , Remission, SpontaneousABSTRACT
Growth of the transplantable mammary tumor, MTW9, in W/Fu rats is greatly enhanced by elevated serum prolactin concentrations. This report compares the prolactin binding to tumor membranes in two mammary tumor strains derived from MTW9. Maximum binding to membranes of both tumors occurred at pH 7.6 after incubation for 30 hr at 4 degrees. The binding was inhibited only by polypeptide hormones that possess lactogenic activity. MTW9-P, an ovariectomy-responsive tumor developed in rats maintained on daily perphenazine injections, had 4-fold-higher prolactin binding than had MTW9MtT, an ovariectomy-nonresponsive tumor developed in rats bearing the mammosomatotropic pituitary tumor, MtTW10. Withdrawal of perphenazine from rats bearing MTW9-P caused a fall to normal of plasma prolactin, no tumor regression, and no significant change in prolactin binding. In contrast, resection of MtT resulted in tumor regression, a fall to normal of serum prolactin, and a nearly 3-fold increase in prolactin binding. Scatchard plots of prolactin binding data yield an apparent affinity constant, Ka, of 1.2 X 109 liters/mole for both tumors. The 4-fold-higher prolactin binding in the ovariectomy responsive variant suggests a positive correlation between ovariectomy response and the number of membrane prolactin-binding sites. No correlation between prolactin sensitivity and prolacting binding is apparent.
PIP: Prolactin binding in ovariectomy-responsive and ovariectomy-nonresponsive carcinoma in the Wistar/Furth rat is compared. The time course of binding of prolactin at 4, 24, ad 37 degrees for mammary tumor (MTW9) coimplanted with MtTW10, a mammosomatotropic pituitary tumor (MTW9-MtT) or with MTW9 maintained with daily perphenazine injections (MTW9-P) was measured. Maximum binding to membranes of both tumors occurred at 4 degrees after about 30 hours incubation. The binding was inhibited by polypeptide hormones that possess lactogenic activity. Mammary tumors from animals maintained on perphenazine had a 4-fold greater binding capacity than did tumors from MtT-supported animals. When perphenazine therapy was halted the binding capacity of MTW9-P membranes was unaffected. This result held when MTW9-P animals were ovariectomized. Resection of MtT resulted in tumor regression, a fall to normal of serum prolactin, and a nearly 3-fold increase in prolactin binding. Scatchard plots of prolactin binding data yield an apparent affinity constant, K(a) of 1.2 X 10(9) liters/mole for both tumors.
Subject(s)
Mammary Neoplasms, Experimental/metabolism , Ovary/physiology , Prolactin/metabolism , Receptors, Cell Surface/metabolism , Animals , Binding, Competitive , Castration , Cell Membrane/metabolism , Female , Hydrogen-Ion Concentration , Perphenazine/pharmacology , Placental Lactogen/pharmacology , Prolactin/blood , Rats , Rats, Inbred WF , Temperature , Time FactorsABSTRACT
MTW9, a transplantable mammary tumor in Wistar Furth rats, shows little growth unless host serum prolactin is increased. This study compares the response to ovariectomy of MTW9-MtT, a tumor developed in rats bearing the mammosomatotropic tumor MtTW10 (serum prolactin 500 to 7000 ng/ml) with MTW9-P developed in rats given chronic perphenazine treatment (4 mg/kg/day). Serum prolactin concentrations were 150 to 600 ng/ml in MTW9-P bearing rats. MTW9-MtT does not regress after ovariectomy but does regress after surgical removal (resection) of MtT. Ovariectomy plus MtT resection leads to greater tumor regression than MtT resection alone. MTW9-P does not regress when perphenazine administration is stopped but does regress after ovariectomy, whether or not rats are given perphenazine. Administration of estradiol (10 mug/day) to rats with complete ovariectomy-induced regression of MTW9-P results in regrowth of tumor. These data suggest that MTW9-P may represent a clone of MTW9 with a lower requirement for prolactin.