Receptor binding and mitogenic effects of insulin and insulinlike growth factors I and II for human myeloid leukemic cells

Insulin and insulinlike growth factors I and II (IGF-I and IGF-II) influence mesodermal cell proliferation and differentiation. As multiple growth factors are involved in hemopoietic cell proliferation and differentiation, we assessed the receptor binding and mitogenic effects of these peptides on a panel of mesodermally derived human myeloid leukemic cell lines. The promyelocytic cell line HL60 had the highest level of specific binding for these 125I-labeled ligands, with lower binding to the less differentiated myeloblast cell line KG1 and undifferentiated blast variants of these cell lines (HL60blast, KG1a). Insulin binding affinity and receptor numbers were reduced significantly by chemically induced granulocytic differentiation of HL60 cells and was unchanged following induced monocytic differentiation. No substantial alteration in IGF-I or -II binding occurred with induced HL60 cell differentiation. Insulin and IGF-I demonstrated cross competition for receptor binding and down-regulated their homologous receptors without detectable cross modulation of the heterologous receptors on HL60 cells. IGF-I and insulin increased HL60 cell proliferation, as assessed by 3H-thymidine uptake, IGF-I greater than insulin. IGF-I binding and mitogenic effects were blocked by the monoclonal anti-IGF-I receptor antibody IR3, indicating that IGF-I-induced proliferative effects were mediated via its homologous receptor. In contrast, insulin binding and mitogenesis displayed blocking by both anti-IGI-I and anti-insulin receptor antibodies, indicating mediation of its activity through both receptors. These data demonstrate specific binding and mitogenic interactions between insulin, IGFs, and hemopoietic cells which are associated with their state of differentiation.     

Insulin-like growth factor-binding protein enhancement of insulin-like growth factor-i (IGF-I)–mediated DNA synthesis and IGF-I binding in a human breast carcinoma cell line

The insulin-like growth factors (IGFs) are potent mitogens for malignant cell proliferation. The majority of secreted IGFs are bound to specific IGF-binding proteins (IGFBPs) that are secreted by a large number of cells. These proteins may either inhibit or enhance IGF actions. Breast carcinoma cells secrete a variety of IGFBPs. We have previously demonstrated that retinoic acid (RA) inhibition of IGF-l– stimulated MCF-7 cell proliferation is associated with increased IGFBP-3 levels in the conditioned media. We therefore investigated the effect of recombinant IGFBP-3 as well as IGFBP-2, -4 and -5 on IGF-l stimulation of DNA synthesis and IGF-I binding in the MCF-7 human breast carcinoma cell line. IGFBP-2 and -3 enhanced IGF-l stimulation of DNA synthesis in MCF-7 cells while IGFBP-4 and -5 had no effect. Transfection of MCF-7 cells with an IGFBP-3 expression vector resulted in the enhanced secretion of IGFBP-3 with an accompanying increase in IGF-l binding as well as increased cell proliferation upon treatment of the cells with IGF-l. IGF-l preincubation of MCF-7 cells transfected with control pSVneo plasmids results in cells refractory to further IGF-l stimulation of thymidine incorporation while IGF-l continues to stimulate [³H]-thymidine incorporation in IGFBP-3–transfected MCF-7 cells, suggesting that IGFBP-3 protects the cells from IGF-l–mediated down regulation of its receptor. Therefore, IGFBP-3 secreted by MCF-7 cells can enhance IGF-l stimulation of DNA synthesis, increase IGF-l binding to these cells, and prevent IGF-l–induced desensitization of its own receptor, suggesting that IGFBP-3 plays a significant role in IGF-l–mediated breast carcinoma proliferation. © 1994 Wiley-Liss, Inc.     

Demonstration of insulin-like growth factor (IGF-I and -II) receptors and binding protein in human breast cancer cell lines

The insulin like growth factors (IGFs), potent mitogens for a variety of normal and transformed cells, have been reported to be secreted by several human breast cancer cell lines (BC). We have investigated the binding characteristics of IGF-I and -II in four human BC: MCF-7, T-47D, MDA 231 and Hs578T. Binding studies in microsomal membrane preparations detected high specific binding for both IGF in all four BC studied. Cross-linking with 125I-IGF-I, followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reduced conditions, revealed the presence of an alpha subunit of apparent Mr = 130,000 in MCF-7, T-47D and MDA 213 cells. When 125I-IGF-II was cross-linked, a major band of apparent Mr = 260,000 was seen in all BC. This band was inhibited by IGF-II, but not by insulin. Cross-linking of 125I-IGF-I to conditioned media from BC demonstrated the presence of three binding proteins of apparent Mr = 45,000, 36,000 and 29,000 in all BC but T-47D, in which the 36,000 band was not seen. These data demonstrate that BC possess classical receptors for both IGF-I and -II and, furthermore, that BC produce specific binding proteins for these growth factors.     

Insulin-like growth factor I (IGF-I) receptors and IGF-I action in oligodendrocytes from rat brains.

Oligodendrocyte progenitor cells were prepared by mechanical dissociation of 1-day-old rat brain cultures. These cells undergo proliferation and differentiation into oligodendrocytes as demonstrated by the expression of proliferation and differentiation-related specific antigens. We have used this unique culture system to characterize insulin-like growth factor I (IGF-I) receptors and their action in the central nervous system (CNS). 125I-IGF-I specifically binds to these cultures with high affinity. Competition-inhibition data suggest that IGF-I is most potent in competing for 125I-IGF-I binding, followed by IGF-II and insulin. Scatchard analyses of the binding data indicate a curvilinear plot with a Kd for high affinity of 0.2 nM, and a Bmax of 247 fmol/mg, and a Kd for low affinity of 3.2 nM and Bmax of 1213 fmol/mg protein. Covalent cross-linking followed by SDS-PAGE analysis demonstrated a radioactive band of Mr 135,000 which corresponds to the alpha subunit of the IGF-I receptor. Solution hybridization/RNase protection assay produced a single protected band corresponding to IGF-I receptor messenger RNA, further confirming the presence of these receptors. Incubation of progenitor cells with IGF-I resulted in a time- and concentration-dependent increase in [3H]thymidine incorporation and cell numbers. This effect appears to be mediated by IGF-I receptors since IGF-II and insulin were proportionately less potent. In addition to its effect on proliferation, IGF-I also increased the number of 4E7- and GC-antigen positive cells. These observations indicate that oligodendrocytes in primary culture express specific IGF-I receptors and that the interaction of IGF-I with these receptors results in the proliferation as well as differentiation of oligodendrocytes.     

Characterization of a chimeric monoclonal antibody against the insulin-like growth factor-I receptor

The insulin-like growth factors (IGFs) signaling system has been shown to play important roles in neoplasia. The IGF receptor type 1 (IGF-IR) is overexpressed in many types of solid and hematopoietic malignancies, and there is substantial experimental and clinical evidence that targeting IGF-IR is a promising therapeutic strategy against cancer. It has been previously reported that a mouse monoclonal antibody (mAb), 4G11, blocked IGF-I binding to IGF-IR and downregulated the IGF-IR in MCF-7 cells. We cloned this antibody, constructed a human-mouse chimeric antibody, designated m590, and characterized it. The chimeric IgG1 m590 bound to cell-associated IGF-IR on NWT c43 stably transfected cells and MCF-7 breast cancer cells as efficiently as the parental murine antibody. Using purified IGF-IR extracellular domains, we found that both the chimeric m590 and the parental 4G11 antibodies bind to conformational epitopes on IGF-IR. Neither of these antibodies bound to the insulin receptor (IR) ectodomain. Furthermore, IgG1 m590 blocked the binding of IGF-I and IGF-II to IGF-IR, and inhibited both IGF-I and IGF-II induced phosphorylation of IGF-IR in MCF-7 cells. These results suggest that m590 could be an useful antibody in diagnosis and treatment of cancer, as well as a research tool.     

Characterization of a chimeric monoclonal antibody against the insulin-like growth factor-I receptor

The insulin-like growth factors (IGFs) signaling system has been shown to play important roles in neoplasia. The IGF receptor type 1 (IGF-IR) is overexpressed in many types of solid and hematopoietic malignancies, and there is substantial experimental and clinical evidence that targeting IGF-IR is a promising therapeutic strategy against cancer. It has been previously reported that a mouse monoclonal antibody (mAb), 4G11, blocked IGF-I binding to IGF-IR and downregulated the IGF-IR in MCF-7 cells. We cloned this antibody, constructed a human-mouse chimeric antibody, designated m590, and characterized it. The chimeric IgG1 m590 bound to cell-associated IGF-IR on NWT c43 stably transfected cells and MCF-7 breast cancer cells as efficiently as the parental murine antibody. Using purified IGF-IR extracellular domains, we found that both the chimeric m590 and the parental 4G11 antibodies bind to conformational epitopes on IGF-IR. Neither of these antibodies bound to the insulin receptor (IR) ectodomain. Furthermore, IgG1 m590 blocked the binding of IGF-I and IGF-II to IGF-IR, and inhibited both IGF-I and IGF-II induced phosphorylation of IGF-IR in MCF-7 cells. These results suggest that m590 could be an useful antibody in diagnosis and treatment of cancer, as well as a research tool.     

A panel of monoclonal antibodies for the type I insulin-like growth factor receptor. Epitope mapping, effects on ligand binding, and biological activity.

We obtained 20 mouse monoclonal antibodies specific for human type I insulin-like growth factor (IGF) receptors, using transfected cells expressing high levels of receptors (IGF-1R/3T3 cells) as immunogen. The antibodies immunoprecipitated receptor.125I-IGF-I complexes and biosynthetically labeled receptors from IGF-1R/3T3 cells but did not react with human insulin receptors or rat type I IGF receptors. Several antibodies stimulated DNA synthesis in IGF-1R/3T3 cells, but the maximum stimulation was only 25% of that produced by IGF-I. The antibodies fell into seven groups recognizing distinct epitopes and with different effects on receptor function. All the antibodies reacted with the extracellular portion of the receptor, and epitopes were localized to specific domains by investigating their reaction with a series of chimeric IGF/insulin receptor constructs. Binding of IGF-I was inhibited up to 90% by antibody 24-60 reacting in the region 184-283, and by antibody 24-57 reacting in the region 440-586. IGF-I binding was stimulated up to 2.5-fold by antibodies 4-52 and 16-13 reacting in the region 62-184, and by antibody 26-3 reacting downstream of 283. The latter two groups of antibodies also dramatically stimulated insulin binding to intact IGF-1R/3T3 cells (by up to 50-fold), and potentiated insulin stimulation of DNA synthesis. Scatchard analysis indicated that in the presence of these antibodies, the affinity of the type I IGF receptor for insulin was comparable with that of the insulin receptor. These data indicate that regions both within and outside the cysteine-rich domain of the receptor alpha-subunit are important in determining the affinity and specificity of ligand binding. These antibodies promise to be valuable tools in resolving issues of IGF-I receptor heterogeneity and in studying the structure and function of classical type I receptors and insulin/IGF receptor hybrids.     

Characterization of Insulin-Like Growth Factor Binding to Human Granulosa Cells Obtained During in Vitro Fertilizationd

Abstract

Insulin and IGF-I affect in vitro ovarian stromal and follicular cell function in several species. We previously characterized insulin receptors on human granulosa cells obtained from in vitro fertilization procedures but were unable to demonstrate specific binding of IGF-I.

Following modification of the assay conditions, we now report specific, high affinity IGF-1 binding sites on human granulosa cells. Substitution of equimolar concentrations of sucrose for sodium chloride in the buffer solution increased binding of IGF but not insulin in equilibrium assays. Maximal specific IGF-I binding was 2.69 ± 0.30%/10⁵ cells (SEM, n=9) with half-maximal inhibition of binding at 2 ng/ml IGF-I. Unlabeled insulin recognized the type I IGF receptor with low affinity. An IGF-I receptor monoclonal antibody (αIR-3) inhibited ¹²⁵I-IGF-I but not ¹²⁵I-insulin binding. Affinity crosslinking followed by SDS/PAGE under reducing conditions revealed IGF-I binding at a molecular weight compatible with the αsubunit of the type I IGF receptor and with a pattern of inhibition by various ligands that paralleled the equilibrium binding assays.

IGF-I receptors are present on freshly isolated human ovarian granulosa cells obtained following pharmacologic stimulation with gonadotrophin according to the protocols of in vitro fertilization. The biologic function of these receptors currently is being investigated.     

Regulation of breast cancer growth by insulin-like growth factors

The IGFs may be important autocrine, paracrine or endocrine growth factors for human breast cancer. IGF-I and II stimulate growth of cultured human breast cancer cells. IGF-I is slightly more potent, paralleling its higher affinity for the IGF-I receptor. Antibody blockade of the IGF-I receptor inhibits growth stimulation induced by both IGFs, suggesting that this receptor mediates the growth effects of both peptides. However, IGF-I receptor blockade does not inhibit estrogen (E₂)-induced growth suggesting that secreted IGFs are not the major mediators of E₂ action. Several breast cancer cell lines express IGF-II mRNA by both Northern analysis and RNase protection assay. IGF-II activity is found in conditioned medium by radioimmuno and radioreceptor assay, after removal of somatomedin binding proteins (BP) which are secreted in abundance. IGF-I is undetectable. BPs of 25 and 40 K predominate in ER-negative cell lines while BPs of 36 K predominate in ER-positive cells. Blockade of the IGF-I receptor inhibits anchorage-independent and monolayer growth in serum of a panel of breast cancer cell lines. Growth of one line (MDA-231) was also inhibited in vivo by receptor antibody treatment of nude mice. The antibody had no effect on growth of MCF-7 tumors. These data suggest the IGFs are important regulators of breast cancer cell proliferation and that antagonism of this pathway may offer a new treatment strategy.     

The insulin-like growth factor II (IGF-II)/mannose 6-phosphate receptor mediates IGF-II-induced motility in human rhabdomyosarcoma cells.

Insulin-like growth factor-II (IGF-II) is an autocrine growth and motility factor for human rhabdomyosarcoma. It interacts with three different receptors: the IGF-I, the IGF-II, and the insulin receptor. A specific function of the IGF-II receptor in mediating IGF-II responses has not been defined. In this report we investigate the mechanism of IGF-II-mediated motility in rhabdomyosarcoma cells. We demonstrate that IGF-II and [Leu27]IGF-II, an analog selective for the IGF-II receptor, stimulate motility at concentrations in which they interact only with their own receptor. An antibody that blocks the IGF-I receptor does not inhibit either peptide activity, while an antibody specific for the IGF-II receptor suppresses the IGF-II-induced motility. This antibody does not interfere with rhabdomyosarcoma cell proliferation. We conclude that in rhabdomyosarcoma cells IGF-II stimulates two different responses mediated by distinct receptors: 1) a mitogenic response through the type I receptor and 2) a motility response through the type II receptor.     

Insulin-like growth factors modulate the growth inhibitory effects of retinoic acid on MCF-7 breast cancer cells

Retinoids are currently being tested for the treatment and prevention of several human cancers, including breast cancer. However, the anti-cancer and growth inhibitory mechanisms of retinoids are not well understood. All-trans retinoic acid (RA) inhibits the growth of the estrogen receptor-positive (ER+) breast cancer cell line, MCF-7, in a reversible and dose-dependent manner. In contrast, insulin-like growth factors (IGF-I,IGF-II) and insulin are potent stimulators of the proliferation of MCF-7 and several other breast cancer cell lines. Pharmacologic doses of RA (≤10^?6M) completely inhibit IGF-I-stimulated MCF-7 cell growth. Published data suggest that the growth inhibitory action of RA on IGF-stimulated cell growth is linear and dose-dependent, similar to RA inhibition of unstimulated or estradiol-stimulated MCF-7 cell growth. Surprisingly, we have found that IGF-I or insulin-stimulated cell growth is increased to a maximum of 132% and 127%, respectively, by cotreatment with 10^?7 M RA, and that 10^?9–10^?7 M RA increase cell proliferation compared to IGF-I or insulin alone. MCF-7 cells that stably overexpress IGF-II are also resistant to the growth inhibitory effects of 10^?9–10^?7 M RA. Treatment with the IGF-I receptor blocking antibody, αIR-3, restores RA-induced growth inhibition of IGF-I-treated or IGF-II-overexpressing MCF-7 cells, indicating that the IGF-I receptor is mediating these effects. IGFs cannot reverse all RA effects since the altered cell culture morphology of RA-treated cells is similar in growth-inhibited cultures and in IGF-II expressing clones that are resistant to RA-induced growth inhibition. These results indicate that RA action on MCF-7 cells is biphasic in the presence of IGF-I or insulin with 10^?9–10^?7 M RA enhancing cell proliferation and ≥ 10^?6M RA causing growth inhibition. As IGF-I and IGF-II ligands are frequently detectable in breast tumor tissues, their potential for modulation of RA effects should be considered when evaluating retinoids for use in in vivo experimental studies and for clinical purposes. Additionally, the therapeutic use of inhibitors of IGF action in combination with RA is suggested by these studies. © 1995 Wiley-Liss Inc.     

Autocrine and paracrine growth regulation of human breast cancer

Previous work from our laboratory has demonstrated that human breast cancer (BC) cells in culture can be stimulated by physiologic concentrations of estrogen. In an effort to further understand this process, we have examined the biochemical and biological properties of proteins secreted by human BC cells in vitro. We have developed a defined medium system which simultaneously allows the collection of factors secreted by the BC cells, facilitates their purification and allows for an unequivocal assay of their effect on other BC cells. By both biochemical and radioimmunoassay procedures, MCF-7 cells secrete large quantities of IGF-I-like activity. The cells contain receptors for IGF-I and are stimulated by physiologic concentrations of IGF-I. Multiple additional peaks of growth stimulatory activity can be obtained by partial purification of conditioned media from human BC cells by sequential dialysis, acid extraction and Biogel P60 chromatography. These peaks are induced up to 200-fold by physiologic concentrations of estrogen. Several of these peaks cross-react in a radioreceptor assay with EGF and are thus candidates for transforming growth factors. Monoclonal antibodies (MCA) have been prepared which react with secreted proteins from the MCF-7 cells. One of these MCAs binds to material from MCF-7 and ZR-75-1 hormone-dependent BC cells only when these two lines are treated with estrogen but reacts with conditioned medium from several other hormone-independent cell lines in the absence of estrogen stimulation. This MCA is currently undergoing further characterization and evaluation of its biological potency. We conclude that with estrogen stimulation, hormone-dependent human BC cells secrete peptides which when partially purified can replace estrogen as a mitogen. Their role as autocrine or paracrine growth factors and their effects on surrounding nonneoplastic stroma may suggest a means of interfering with tumor proliferation.     

Expression and characterization of a functional human insulin-like growth factor I receptor

Stable transfectants of Chinese hamster ovary (CHO) cells were developed that expressed the protein encoded by a human insulin-like growth factor I (IGF-I) receptor cDNA. The transfected cells expressed approximately 25,000 high affinity receptors for IGF-I (apparent Kd of 1.5 X 10(-9) M), whereas the parental CHO cells expressed only 5,000 receptors per cell (apparent Kd of 1.3 X 10(-9) M). A monoclonal antibody specific for the human IGF-I receptor inhibited IGF-I binding to the expressed receptor and immunoprecipitated polypeptides of apparent Mr values approximately 135,000 and 95,000 from metabolically labeled lysates of the transfected cells but not control cells. The expressed receptor was also capable of binding IGF-II with high affinity (Kd approximately 3 nM) and weakly recognized insulin (with about 1% the potency of IGF-I). The human IGF-I receptor expressed in these cells was capable of IGF-I-stimulated autophosphorylation and phosphorylation of endogenous substrates in the intact cell. This receptor also mediated IGF-I-stimulated glucose uptake, glycogen synthesis, and DNA synthesis. The extent of these responses was comparable to the stimulation by insulin of the same biological responses in CHO cells expressing the human insulin receptor. These results indicate that the isolated cDNA encodes a functional IGF-I receptor and that there are no inherent differences in the abilities of the insulin and IGF-I receptors to mediate rapid and long term biological responses when expressed in the same cell type. The high affinity of this receptor for IGF-II also suggests that it may be important in mediating biological responses to IGF-II as well as IGF-I.     

Characterization of the receptor for insulin-like growth factor II in bone cells

We have previously shown that insulin-like growth factor II (IGF-II) is produced by bone cells and that IGF-II stimulates cell proliferation and collagen synthesis in bone cells. We now extend these in vitro findings by demonstrating specific IGF-II binding to bone cells derived from newborn mouse calvaria and embryonic chick calvaria. The kinetics of [125I] IGF-II binding in embryonic chick calvaria cells showed time and temperature dependence. Scatchard analysis of [125I]IGF-II binding to chick calvaria cells showed an apparent Kd of 1.4 x 10(-10) M, with a calculated receptor site concentration of 40,000/cell. The specificity characteristics showed that IGF-II was significantly more potent than IGF-I or insulin in displacing IGF-II tracer. Competition for binding of [125I]IGF-II by unlabeled IGF-II showed a dose-dependent displacement between 0.5 and 25 ng/ml. Fifty percent displacement of [125I]IGF-II binding to chick and mouse calvarial cells was achieved at 1-2 ng/ml; 90% of specific binding of [125I]IGF-II was displaceable in the presence of 125 ng/ml of unlabeled IGF-II. IGF-I showed less than 5% cross reactivity for displacement of [125I]IGF-II binding to chick and mouse bone cells. Type II receptor inhibitory antibodies, R-II-PAB1 inhibited the binding of [125I]IGF-II to mouse bone cells and H-35 rat hepatoma cells (which contain type II but not type I receptors) in a dose-dependent manner. R-II-PAB1 also inhibited basal cell proliferation as well as IGF-II-, IGF-I-, and fibroblast growth factor (FGF)-induced cell proliferation in mouse bone cells. In chick calvaria bone cells and TE89 human osteosarcoma cells, R-II-PABI inhibited neither binding of [125I]IGF-II nor IGF-II-induced cell proliferation. These results together with our findings that IGF-II increased chick bone cell proliferation in the presence of maximal doses of IGF-I suggest that at least part of the mitogenic action of IGF-II is mediated through type II rather than type I receptors in bone cells.     

Influence of luteinizing hormone-releasing hormone agonists on human mammary carcinoma cell lines and their xenografts

The specific binding of luteinizing hormone-releasing hormone (LH-RH) agonist in estradiol-dependent MCF-7 and estradiol-independent MDA-MB-231 human breast cancer cells has been studied using [3H]Ovurelin [(D-3H-Phe6),des-Gly10-LH-RH- ethylamide]. The results of Scatchard analyses suggest the presence of a single class of receptor sites, both in cell suspensions and membrane fractions. Evaluation of these peptide receptors appears to reflect additional characteristics of biological behaviour of these human breast cancer cells. The synthetic LH-RH agonist Ovurelin [(D-Phe6),des-Gly10-LH-RH-ethylamide] can directly interfere (25-30%) with the proliferation of MDA-MB-231 human breast cancer cells in culture. The inhibitory effect of Ovurelin in vitro was negligible in the MCF-7 cell line. In the in vivo experiments the treated immunosuppressed mice bearing either MCF-7 or MDA-MB-231 xenografts responded to the high-dose LH-RH analogue Zoladex depot and Decapeptyl depot therapy. Since the MDA-MB-231 tumour was found to be ER-negative it seems possible that the regression of this xenograft results from the direct antitumor action of the LH-RH agonist.     

IGF-I affects glycosaminoglycan/proteoglycan synthesis in breast cancer cells through tyrosine kinase-dependent and -independent pathways

The insulin-like growth factor I (IGF-I) has been implicated in breast cancer development acting through insulin-like growth factor I receptor (IGF-IR), but also through estrogen receptor (ER). The effect of IGF on proteoglycan (PG) synthesis by two human breast cancer epithelial cell lines, the ER-positive MCF-7 and the ER-negative BT-20, was studied alone and in combination with genistein. Both cell lines synthesise hyaluronan (HA), matrix secreted and cell membrane-associated galactosaminoglycan containing proteoglycans (GalAGPGs) and heparan sulphate proteoglycans (HSPGs) in variable amounts. IGF-I affects the synthesis of PGs by BT-20 cells by decreasing the amounts of HA and secreted GalAGPGs and HSPGs and upregulates the expression of cell membrane-associated GalAGPGs and HSPGs. IGF-I exerts this effect on BT-20 cells acting mainly through receptors with protein tyrosine kinase activity (PTK). In contrast, IGF-I stimulates the synthesis of secreted GalAGPGs and HSPGs by MCF-7 cells, exhibiting only a slight suppression on synthesis of cell-associated GalAGPGs and HSPGs. The regulatory effect of IGF-I on PGs distribution in MCF-7 cells is mediated through a mix of pathways, which involves both receptors with PTK activity and PTK-independent signalling. It is suggested that the effects of IGF-I on the synthesis and distribution of PGs by epithelial breast cancer cells also depend on the presence or the absence of ER. The result of the IGF-I action is the balanced biosynthesis between the matrix and cell-associated PGs in both cell lines, approaching a common biosynthetic phenotype.     

High performance liquid chromatography analysis of 1,25-dihydroxyvitamin D3 receptor in malignant cells. Correlation of effects on cell proliferation and receptor concentration

Malignant cells were assayed for 1,25(OH)2D3 receptors and for the effects of 1,25(OH)2D3 on cell proliferation. The established lines studied were human promyelocytic leukemia (HL-60), T-cell lymphocytic leukemias (Molt-4, RPMI-8402, CEM), mouse leukemia (L1210), breast cancers (HT-39 and MCF-7) and a glioma (C-6) cultures. A TSK 3000 SW (0.75 X 60 cm) HPLC size exclusion column was used to characterize specific 1,25(OH)2D3 binding. We show for the first time that this column is capable of resolving the 3.2-3.5S 1,25(OH)2D3 mammalian receptor (Rs = 32 A) from the 5.5-6.0S form of the mammalian serum 25(OH)D3 transport receptor (Rs = 40 A). The molecular size of the 1,25(OH)2D3 receptors from these cancer cell lines was identical to that from rabbit intestine. HT-39, HL-60, MCF-7, Molt-4, C-6, RPMI-8402 and L1210 cells demonstrated specific 1,25(OH)2[3H]D3 binding (120, 90, 80, 45, 30 and 18 fmoles of sites/mg protein, respectively). Receptors were not detected in the CEM line. 1,25(OH)2D3 inhibited cell proliferation of HT-39, HL-60, MCF-7 and Molt-4 cells by 20% to 70%. In contrast, mouse leukemia (L1210) cells were stimulated to proliferate by this hormone. Proliferation of RPMI and CEM cells was not affected by 1,25(OH)2D3. We demonstrate that size-exclusion HPLC of 1,25(OH)2D3 binding proteins from mammalian intestine and cancer cells provided a rapid method for identification of specific 1,25(OH)2D3 receptors. Furthermore, in the cells studied, the presence and concentration of 1,25(OH)2D3 receptors qualitatively predicted the potency of this hormone to alter cell proliferation. We believe this assay will be useful for rapid analysis of human tumor receptor concentrations.     

Recycling of tumor necrosis factor-alpha receptor in MCF-7 cells

Kinetics of regulation of membrane receptors for tumor necrosis factor-alpha (TNF) in human breast adenocarcinoma MCF-7 cells was investigated. When MCF-7 cells were incubated with radioiodinated human recombinant TNF, they bound TNF specifically and accumulated it intracellularly. Preincubation of cells with native TNF up to 1 x 10(-9) M for 12 h stimulated specific binding of TNF, indicating that concentrations of membrane receptors for TNF were regulated by the ligand. Accumulation of radioactivity in cells incubated with [125I]TNF proceeded at a constant rate for up to 24 h. Kinetics of binding and internalization of TNF were similar in the presence and absence of protein synthesis for at least 1 h, suggesting that the TNF receptor was either replenished from an intracellular pool of receptors or was recycled (reutilized) during the course of TNF internalization. Data were analyzed kinetically by fitting equations of compartmental models of ligand-cell interactions with and without the term for receptor recycling. Fits were obtained only for the model with receptor recycling; absence of the term for receptor recycling resulted in physically impossible best-fit parameter values. Analysis of the best-fit parameters indicated that both internalization and recycling of the receptor were stimulated by the ligand.     

Characterization of monoclonal antibodies to the IGF-I receptor that inhibit IGF-I binding to human cells

A mouse monoclonal antibody directed against the human placental IGF-I receptor is shown to completely inhibit IGF-I binding to both human placental membranes and circulating human mononuclear leukocytes. Insulin binding is unaffected. Studies with 125I-labelled anti-receptor antibody indicate that the antibody reacts with an epitope distinct from the IGF-I binding site. These antibodies will be powerful probes of IGF-I binding and action in human cells.