Multiple molecular forms of human lactoferrin. Identification of a class of lactoferrins that possess ribonuclease activity and lack iron-binding capacity.

Lactoferrin (Lf), the major iron-binding component of milk, also a major constituent of the specific granules of neutrophils involved in antimicrobial activity and a glycoprotein thought to play a role in regulatory functions in the hematopoietic system as well as other physiologic activities, is shown to occur in three isoforms. One, Lf-alpha, binds iron; the other two, Lf-beta and Lf-gamma, express potent RNase activity, but do not bind iron. The three isoforms are very similar or identical in Mr, pI, partial proteolytic peptide patterns, NH2-terminal amino acid sequence, and reactivity with mAbs and polyclonal antisera against the RNase and Lf, respectively. The finding of structurally similar but enzymatically distinct forms of Lf may be related to the diverse functions of the molecule.

Spontaneous regression of Friend virus-induced erythroleukemia. I. The role of the helper murine leukemia virus component.

The RFV strain of the Friend virus complex induces an erythroleukemia that spontaneously regresses. The tropism of regressing Friend virus complex (RFV), which is conferred by its helper MuLV component, MuLV-RF, is different from that of the conventional virus strain, CFV. RFV is NB-tropic and CFV is N-tropic. Passage of nonregressing CFV through Fv-1 incompatible Swiss/ICR mice changed the tropism of CFV from N to NB and resulted in a virus strain which induced erythroleukemia that regressed. Passage of NB-tropic CFV back through Fv-1 compatible mice maintained NB-tropism and regression. Altering the quantity or type of helper MuLV in RFV complex by addition of Ri-MuLV inhibited regression in proportion to the amount of added Ri-MuLV. These studies indicate a relationship between a change in virus tropism to NB by passage in certain hosts (e.g., Swiss/ICR mice) and the ability of Friend virus to induce erythroleukemia that spontaneously regresses. MuLV-RF isolated from the RFV complex induced lymphocytic leukemia in newborn mice which regressed and caused the regression of CFV-induced erythroleukemia. MuLV-RF is NB-tropic, contains no spleen focus-forming virus (SFFV) activity and helps SFFV form spleen foci in genetically restrictive mice. Pseudotype viruses were prepared, consisting of MuLV-RF, or other MuLV's, and SFFV derived from FV-B. The pseudotype viruses each acquired the tropism of the MuLV used in rescue. The pseudotype prepared with MuLV-RF or another NB-tropic MuLV-F, but not the virus obtained by rescue with N-tropic MuLV-F, induced erythroleukemia that spontaneously regressed. These studies demonstrate that the ability of RFV to induce erythroleukemia that spontaneously regresses is due to its helper MuLV component.

Absence of fibronectin and presence of plasminogen activator in both normal and malignant human mammary epithelial cells in culture.

Primary monolayer cultures of normal and malignant human mammary epithelial cells were tested for fibronectin by indirect immunofluorescence using antisera specific for fibronectin. This protein was not detectable on either the normal or malignant epithelial cells. Similar results were obtained for normal and malignant mouse mammary epithelial cell cultures. Control normal and transformed fibroblasts exhibited the expected result: the normal cells were positive and the transformed cells were negative. With the use of supernatant fluids from the same cultures or an agar-overlay assay on viable cells, high levels of plasminogen-dependent fibrinolytic activity were detectable in both the normal and malignant mammary cells. Thus, two characteristics that distinguish normal from transformed fibroblasts do not serve as markers of malignancy in mammary epithelial/carcinoma systems.

Spontaneous regression of friend virus induced leukemia: coinfection with regressing and conventional strains of virus.

Mixtures of friend virus (CFV) and the regressing strain of friend virus (RFV) induce leukemia which regresses. The dominance of the regressing phenotype is solely a function of a threshold dose of RFV. The minimum amount of RFV which induced regression of CFV leukemia is below the titer for induction of friend disease, but does correlate ith the titer of lymphocytic leukemia (helper) activity in the these stocks.

Effect of the extracellular matrix on plasminogen activator isozyme activities of human mammary epithelial cells in culture.

Multiple molecular forms of plasminogen activator were detected in normal human mammary epithelial cells in culture. Cells derived from (normal) breast mammoplasty specimens and grown on the surface of collagen gels exhibited three major classes of plasminogen activator isozymes (Mr = 100,000 [100K], 75,000 [75K], and 55,000 [55K]). The activity of the 100K and 75K isozymes was greatly reduced when the cells were grown on conventional tissue-culture-grade plastic surfaces. MCF-7, a human mammary carcinoma cell line, exhibited predominantly or exclusively the 55K isozyme, irrespective of the cell growth substratum. The activity of the 55K isozyme was more than twofold higher for MCF-7 cells grown on collagen gels than for cells grown on plastic. Progesterone, diethylstilbestrol, and estrogen stimulated the activity of the 55K isozyme of MCF-7 cells, but only when the cells were grown on a plastic surface. The plasminogen activator activities of the normal human mammary epithelial cells were not stimulated by these hormones, irrespective of the growth substratum. These results show that the expression of plasminogen activator isozymes by human mammary epithelial cells is subject to modulation by the extracellular matrix. Normal and malignant cells may differ in their responsiveness to these effects.

Stimulation of normal erythropoiesis in vitro by macrophages from erythroleukemic mice.

The erythroleukemia induced in mice by the Friend virus complex is caused to regress by macrophages. To determine whether the effect of macrophages on leukemia is due to their role in the regulation of erythropoiesis, investigators examined the influence of macrophages from normal and leukemic mice on in vitro colony formation by erythroid progenitor cells [colony-forming units of erythroid progenitor cells (CFU-E)]. Plasma clot cultures of CFU-E were grown over monolayers of macrophages separated from the CFU-E cultures by a layer of agar. Macrophages from progressors (those leukemic mice that will not undergo leukemia regression) significantly stimulated CFU-E, whereas macrophages from regressors (those that will undergo leukemia regression) had no effect on colony formation. Monolayers of normal resident macrophages did not affect in vitro erythropoiesis, while less mature macrophages (peripheral blood mononuclear cells) or nonspecifically activated macrophages (exudate induced) in monolayer stimulated CFU-E. Stimulation by macrophages from leukemic mice was dependent on the presence of virus-producing cells. CFU-E from erythroleukemic mice were resistant to stimulation by leukemic macrophages. These results demonstrated that macrophages from progressively leukemic mice influenced normal colony formation of CFU-E in vitro and that these effects could be related to the ability of macrophages to cause leukemia regression.

Effects of thymectomy and antithymocyte serum on spontaneous regression of Friend virus-induced erythroleukemia.

To evaluate the role of immune response in regression of leukemia, we studied the effect of immunosuppression on the spontaneous regression of a leukemia induced by a specific strain of Friend murine leukemia virus complex (RFV). Thymectomy of newborn but not adult outbred Swiss mice markedly inhibited regression. The effect of antithymocyte serum (ATS) on regression depended on the timing of ATS treatment. Regression was markedly inhibited in leukemic mice given ATS just before the start of regression. During leukemia development, ATS treatment but not thymectomy potentiated splenomegaly and delayed the start of regression. Both ATS treatment and neonatal thymectomy increased mortality as a function of the decrease in disease regression. Treatment with normal rabbit serum also inhibited regression but, when given during leukemia development, affected neither the splenomegalic response to RFV nor the number of deaths. The data demonstrated that an intact immune system was required for leukemia regression and suggested that some thymus-dependent parameter of immune response was a major factor in regression.

Presence of a mouse mammary tumor virus-related antigen in human breast carcinoma cells and its absence from normal mammary epithelial cells.

Antigen(s) related to the major external glycoprotein (gp52) of mouse mammary tumor virus was detected in the human breast cancer cell line MCF-7. No such antigenic determinants were detectable in normal human mammary epithelial cells.

Acute hemorrhagic necrosis of tumors induced by interleukin-1 alpha: effects independent of tumor necrosis factor.

Tumor necrosis factor (TNF), a protein synthesized in response to the endotoxin bacterial lipopolysaccharide (LPS), is the classical mediator of acute hemorrhagic necrosis of tumors. We have demonstrated that interleukin-1 alpha (IL-1 alpha), with a spectrum of activities very similar to those of TNF, also causes acute hemorrhagic necrosis of tumors. Both TNF and IL-1 induce a cascade of events including the synthesis or release of each other. The present studies were thus undertaken to determine whether the hemorrhagic necrosis induced in tumors by IL-1 alpha is due to TNF. Kinetic parameters of IL-1 alpha-induced hemorrhage were similar to those observed with recombinant murine TNF-alpha (TNF-alpha) or LPS in RIF-1 fibrosarcomas in C3H/HeN (endotoxin-sensitive) mice. However, the amount of TNF found in the sera or tumors of animals treated with LPS was more than 20-fold higher than in mice treated with IL-1 alpha, and LPS induced similar degrees of hemorrhagic necrosis, which was measured by determining the packed volume of red blood cells by 59Fe labeling. A low but significantly hemorrhagic dose of IL-1 alpha induced no detectable TNF in tumors. Pretreatment with 250 micrograms of neutralizing antibody to TNF had no effect on IL-1 alpha-induced hemorrhage, whereas TNF-alpha- and LPS-induced hemorrhagic effects were significantly reduced. These results demonstrate an important antitumor activity of IL-1 alpha that appears to be independent of TNF.

Growth of normal and malignant human mammary epithelial cells in culture.

Normal and malignant human mammary epithelial cells were placed in culture. Normal cells were recovered from late-lactation milk and breast fluids, and malignant cells were isolated from primary breast tumors by collagenase digestion. The concentration of cells obtained from breast fluid samples was inversely proportional to the volume of fluid secreted. Most of these cells adhered rapidly to the substrate, did not replicate, displayed Fc receptor-dependent phagocytic activity, and were thus identified as macrophages. The remaining cells grew out into large islands comprised of one or two distinct morphologic types of mammary epithelial cells. Optimum growth of these cells was obtained in medium buffered to pH 6.8, and the epidermal growth factor markedly prolonged the exponential growth phase of the cells. Two morphologically distinct populations of epithelial cells were also observed in cultures established from individual breast tumors. Growth of the malignant cells was relatively unaffected by the pH of the culture medium, and the cells were unresponsive to exogenously added hormones. Overgrowth of malignant epithelial cells in primary cultures by stromal fibroblasts was retarded by replacement of standard growth medium with fresh medium containing a serum substitute; growth of the malignant epithelial cells was unaffected. A feeder layer of mitomycin C-treated human fibroblasts increased the plating efficiency of both normal and malignant cells in primary culture and also facilitated passage of these cells to secondary and tertiary cultures.

Spontaneous regression of Friend murine leukemia virus-induced erythroleukemia. IV. Effects of radiation and athymia on leukemia regression in mice.

The spontaneous regression of the erythroleukemia induced by the regressing Friend murine leukemia virus (F-MuLV) complex was inhibited by irradiation of the animals prior to F-MuLV inoculation. This inhibition was proportional to the dose of radiation used. Treatment of the mice with the bone-seeking isotope 89Sr also inhibited erythroleukemia regression, which implicates the same effector mechanisms involved in the resistance to F-MuLV or F-MuLV-induced immunosuprression. Erythroleukemias induced in athymic nude mice by the regressing F-MuLV complex exhibited higher rates of lethality than did the leukemias in heterozygous or homozygous thymus gland-containing controls. These data suggested the involvement of the immune system in erythroleukemia regression and the specific participation of thymus cells and an 89Sr-susceptible function, perhaps marrow-dependent cells, in the process of regression.

Spontaneous regression of Friend virus-induced erythroleukemia. II. regression of Friend murine leukemia virus-induced lymphocytic leukemia.

We characterized several aspects of spontaneous regression of lymphocytic leukemia in mice. The disease, induced by the helper murine leukemia virus (MuLV) component obtained from the regressing Friend virus complex (RFV), was characterized by spleen and lymph node enlargement, thymus involvement, and anemia. Leukemia regression occurred in about 25% of infected mice and resulted in the return of lymphoid organs to near-normal weight and normal histology and the recovery from anemia. A tenfold to 1,000-fold decrease in virus titer was seen in those mice in which leukemia regressed when compared to leukemic animals, although infectious virus was still recoverable from apparently normal spleens. The sera of mice in which leukemia regressed contained potent virus-neutralizing activity that was associated mainly with immunoglobulins. These studies firmly supported the evidence that the regressing phenotype of RFV was due to its helper MuLV component (MuLV-RF).

Metalloporphyrin enhancement of magnetic resonance imaging of human tumor xenografts in nude mice.

Paramagnetic metalloporphyrins were examined for their in vivo bio-distribution and their ability to enhance nuclear magnetic resonance imaging of human tumor xenografts in nude mice. The metalloporphyrins tested were: manganese tetrasodium-meso-tetra(4-sulfonatophenyl)-porphine (MnTPPS); manganese meso-tetra-4-pyridylporphine; and gadolinium meso-tetra-4-pyridylporphine. All exhibited high molar relaxivities in aqueous solution. In vivo, at a dose of 2 mg/mouse, MnTPPS depressed the longitudinal relaxation time, T1, significantly in the kidney and less in lung and blood. Manganese meso-tetra-4-pyridylporphine depressed T1 in the kidney, lung and liver, while gadolinium meso-tetra-4-pyridylporphine caused large T1 depressions in the blood, liver, brain and tumor, probably due to dissociation of the metalloporphyrin and binding of Gd to plasma or tissue proteins. At a dose of 10 mg/mouse, MnTPPS caused marked T1 depressions of all tissues tested within 5 min of inoculation, but 48-72 h later, T1 values of normal tissues had returned to near normal, while those of the tumors remained significantly depressed. MnTPPS was able to significantly enhance the intensity of nuclear magnetic resonance images of MX-1 and ZR-75 human breast tumors and CX-1 and LS174T human colon tumor xenografts in nude mice. The results demonstrate that paramagnetic metalloporphyrins, because of their high relaxivities and retention in tumors, have the potential for use as tumor-selective contrast agents for nuclear magnetic resonance imaging.

Induction of leukemia regression in mice by immunotherapeutic transfer of T-lymphocytes.

The erythroleukemia induced in susceptible mice by Friend virus (FV) is a progressive, lethal disease. A variant strain of Friend virus (regressing FV) produces a histopathologically identical leukemia except that the disease spontaneously regresses in 50% of leukemic mice. Normal T-cell and macrophage function are required for regression to occur and in animals that are going to regress, specifically reactive T-cells are found in the spleen. Passive transfer of sensitized T-cells from regressing FV immunized or regressed mice caused regression of the conventional lethal leukemia induced by FV. To expand the effector cell populations, characterize them and improve their therapeutic efficacy, sensitized T-cells were cultured in vitro. The T-cells, isolated from regressed or immunized mice, were grown and expanded in vitro with interleukin 2 and antigen (mitomycin C treated regressing FV-infected cell lines). The T-cells demonstrated high levels of in vitro cytotoxicity against FV antigens but exhibited no blastogenic response to the same antigens. When fully FV-induced leukemic mice (14 days post virus inoculation; spleen weight, greater than 0.75 g) were given one injection of 5 X 10(6) in vitro cultured T-cells and no other treatment the mice experienced permanent regressions of their disease. From T-cell cultures depleted of specific cell populations with monoclonal antisera, helper Lyt-1+ cells were shown to be responsible for permanent regressions (cures), whereas cytotoxic Lyt-2+ cells caused temporary leukemia remissions. This model thus provides an experimental system of highly effective passive cellular immunotherapy against an autochthonous, fully developed leukemia, requiring no adjunctive treatment for activity.

Human lactoferrin inhibits growth of solid tumors and development of experimental metastases in mice.

The antitumor effects of the multifunctional iron-binding glycoprotein, lactoferrin (Lf), were investigated. Lf inhibited growth in mice of transplantable solid tumors induced by v-ras transformed fibroblasts and a methylcholanthrene-induced fibrosarcoma. Lf also substantially reduced lung colonization (experimental metastasis) by B16-F10 melanoma cells in syngeneic mice. Iron-saturated and apo-Lf exhibited comparable levels of tumor inhibition and antimetastatic activity. Transferrin, a related iron-binding protein, had no effect on lung colonization. In the B16-F10 system, elimination of natural killer cell activity by pretreatment of mice with anti-asialo GM1 antibody abrogated the effects of Lf, whereas inhibition of macrophage function with silica did not. The results demonstrate a novel activity for Lf and suggest a potentially important role for this molecule in the primary defense against tumorigenesis.

Prognostic value of estrogen receptor determinations in patients with breast cancer.

The estrogen receptor content of human breast cancer specimens is related to the degree of differentiation (grade) of the tumor. In addition, patients with estrogen receptor-positive tumors experience fewer recurrences and remain disease free for a longer priod of time than do patients with receptor-negative tumors. The presence of estrogen receptor is not correlated with lymph node infiltration.

Growth of human mammary epithelial cells on collagen gel surfaces.

We have developed a method for sustained growth of human mammary epithelial cells in monolayer cultures. Epithelial organoids derived from solid breast tissues were grown on the surface of thin (approximately 1 mm) collagen gel layers in an enriched growth medium supplemented with hormones, growth factors, fetal calf serum, and horse serum. To transfer the cultures, the collagen layers were dislodged and digested with collagenase. The monolayers of cells released into suspension were then dissociated into single cells using trypsin-ethylene-diaminetetraacetate. Dissociated single cells were repleted with 75 to 95% efficiency onto collagen layers or tissue culture plastic surfaces. The dissociated cells could also be cryopreserved and reactivated with greater than 80% plating efficiency on collagen layers. Normal human mammary epithelial cells grown under these conditions progressed through 12 to 15 population doublings. The population-doubling times for normal and malignant mammary cells on collagen layers were 34 and 65 hr, respectively. After reaching confluence, cells in some cultures, derived from either normal or malignant tissues, penetrated the gel surface and grew into the collagen. Within the gels, the cells became organized into three-dimensional tubular structures. The use of collagen layers eliminates a major problem in growth of human mammary epithelial cells in culture, difficulty in efficient dissociation, and cell transfer from monolayers.

Mechanism of macrophage reversal of Friend erythroleukemia: macrophage regulation of normal and leukemic erythropoiesis.

The acute erythroleukemia induced in mice by the anemia-inducing strain of the Friend virus complex is caused to regress by normal macrophages. We examined the possibility that reversal of leukemia is related to a macrophage regulatory function in erythropoiesis. We found that the ability of macrophages to induce leukemia regression correlates with nonimmunological, in vivo suppression of normal and susceptible leukemic erythroid progenitors. The macrophage effect on erythropoiesis appears to be due to changes in a humoral regulator, related to but independent of erythropoietin. The results suggest a novel regulatory system for erythropoiesis, operative in vivo, and involving macrophages as accessory or suppressor cells. This regulation appears to be disrupted in erythroleukemic mice, but can be restored, and the disease can be made to regress by treatment with normal macrophages.

Potentiation of mitomycin C and porfiromycin antitumor activity in solid tumor models by recombinant human interleukin 1 alpha.

The time- and dose-dependent effects of recombinant human interleukin 1 alpha (IL-1 alpha) on the antitumor activity of mitomycin C (MMC) and porfiromycin (PORF) were studied in RIF-1 and Panc02 solid tumor model systems. IL-1 alpha produced dose-dependent sensitization of clonogenic RIF-1 tumor cells to MMC in vivo. IL-1 alpha chemosensitization was highly schedule dependent, and the most efficacious schedules produced dose-modifying factors of 3.6 and 5.1 for MMC and PORF, respectively. More than additive clonogenic cell kill after IL-1 alpha-chemotherapy combinations reflected increased cellular sensitivity to MMC and PORF. The combinations also produced marked decreases in the yield of viable tumor cells, suggesting that the bioreductive drugs may have also potentiated the microvascular injury and ischemia produced by IL-1 alpha. Dexamethasone inhibited and ketoconazole, an inhibitor of corticosterone biosynthesis, enhanced IL-1 alpha-mediated chemosensitization in these models. IL-1 alpha mediated chemosensitization to MMC, and PORF was also demonstrated by tumor growth inhibition in the RIF-1 model and increased survival of mice in the spontaneously metastasizing Panc02 system. Chemosensitization of bone marrow spleen colony-forming units was not seen. IL-1 alpha (1000 units/ml) had no effect on MMC and PORF cytotoxicity in RIF-1 and PORF cell lines in vitro. The results indicate that the tumor-specific IL-1 alpha-induced pathophysiologies can sensitize solid tumors to agents which are preferentially activated, retained, and cytotoxic to cells under hypoxic conditions. Our results suggest that strategies combining bioreductively activated hypoxic cell cytotoxins and biological agents might offer efficacious alternatives or adjuvants to conventional combination approaches.

Antitumor effects of recombinant human interleukin 1 alpha in RIF-1 and Panc02 solid tumors.

The antitumor effects of recombinant human interleukin 1 alpha (IL-1) were determined in RIF-1 and Panc02 murine solid tumors. Acute tumor hemorrhage was observed in both models as early as 3 h after a single 25 micrograms/kg IL-1 treatment and was quantitated by the intratumor accumulation of 59Fe-labeled erythrocytes (RBC). The IL-1-mediated hemorrhagic response was maximal 6-12 h after treatment and greater in Panc02 tumors than in RIF-1 tumors. Hemorrhagic responses to RIF-1 tumors growing in athymic nude mice were similar to those seen for RIF-1 tumors in C3H/HeJ mice. This acute vascular injury was accompanied by progressive edema in tumors but not in skin or muscle. In RIF-1 tumors, the extracell water volume at 12 h after IL-1 (395 microI/g) was nearly twice that in untreated controls (215 microI/g). IL-1 also produced marked reductions in tumor blood flow as early as 1 h after treatment. Maximal blood flow restriction was seen at 6 h after IL-1. Although restricted blood flow was observed in tumors for up to 48 h, IL-1 effects on muscle, liver, and skin blood flow were transient with recovery by 12 h after treatment. IL-1 (up to 0.4 ng/ml for 72 h) was not toxic to RIF-1 tissue culture cells in vitro, but 0.2 ng/ml IL-1, for 20 h, reduced the clonogenicity of RIF-1 cells in primary explant cultures by approximately 50%. In vivo, the clonogenic cellularity of RIF-1 tumors was reduced by 70%, 24 h after a single 25 micrograms/kg treatment. Increased clonogenic cell proliferation was observed at 24 h, and rapid repopulation of the clonogenic cell population was seen by 48 h. Although IL-1 transiently slowed the growth of RIF-1 tumors, no significant regrowth delay was observed. In Panc02 tumors, cell proliferation was also inhibited after IL-1. Recovery, however, was delayed and occurred more slowly than in RIF-1 tumors. Significant growth inhibition and regrowth delay (5 days) was observed in Panc02 tumors after a single IL-1 treatment. The results of these studies show that IL-1 has significant effects on the pathophysiology of both RIF-1 and Panc02 tumors in vivo. Further, our results indicate that these effects may be mediated through the activation of a non T-cell, adherent cell population residing in the tumor at the time of IL-1 treatment.