Anti-transforming growth factor (TGF)-beta antibodies inhibit breast cancer cell tumorigenicity and increase mouse spleen natural killer cell activity. Implications for a possible role of tumor cell/host TGF-beta interactions in human breast cancer progression.

TGF-beta effects on angiogenesis, stroma formation, and immune function suggest its possible involvement in tumor progression. This hypothesis was tested using the 2G7 IgG2b, which neutralizes TGF-beta 1, -beta 2, and -beta 3, and the MDA-231 human breast cancer cell line. Inoculation of these cells in athymic mice decreases mouse spleen natural killer (NK) cell activity. Intraperitoneal injections of 2G7 starting 1 d after intraperitoneal inoculation of tumor cells suppressed intraabdominal tumor and lung metastases, whereas the nonneutralizing anti-TGF-beta 12H5 IgG2a had no effect. 2G7 transiently inhibited growth of established MDA-231 subcutaneous tumors. Histologically, both 2G7-treated and control tumors were identical. Intraperitoneal administration of 2G7 resulted in a marked increase in mouse spleen NK cell activity. 2G7 did not inhibit MDA-231 primary tumor or metastases formation, nor did it stimulate NK cell-mediated cytotoxicity in beige NK-deficient nude mice. Finally, serum-free conditioned medium from MDA-231 cells inhibited the NK cell activity of human blood lymphocytes. This inhibition was blocked by the neutralizing anti-TGF-beta 2G7 antibody but not by a nonspecific IgG2. These data support a possible role for tumor cell TGF-beta in the progression of mammary carcinomas by suppressing host immune surveillance.

Bacterial toxins affect early events of T lymphocyte activation.

The effects of pertussis toxin and cholera toxin on early events of T lymphocyte activation were examined in the T lymphocyte cell line, Jurkat. Pertussis toxin treatment of these T cells increased inositol phosphates production and led to increases in intracellular free calcium concentration. These effects were produced by the isolated B (binding) subunit of pertussis toxin, alone. Inositol phosphates production resulting from perturbation of the T cell antigen receptor-CD3 complex by MAb was not affected by pertussis toxin treatment but was markedly inhibited by cholera toxin. This effect of cholera toxin paralleled elevations in cAMP content. However, forskolin, in concentrations equipotent for cAMP production, was a weaker inhibitor of inositol phosphates production. Cholera toxin inhibition of inositol phosphates production did not result from inhibition of baseline incorporation of inositol into phosphoinositide substrates of phospholipase C. These studies underline the complexity of toxin effects on cellular systems and suggest that other approaches will be required to implicate guanine nucleotide-binding regulatory proteins in control of the early events of T lymphocyte activation. However, the data presented here provide a molecular basis for the clinical observations of lymphocytosis and the in vitro observations of lymphocyte mitogenesis after pertussis toxin stimulation.

Growth inhibition by retinol of a human breast carcinoma cell line in vitro and in athymic mice.

Although the anticarcinogenic and antiproliferative effects of vitamin A (retinol) have been extensively studied in vitro, there are few data regarding the response of human tumor cells to this agent in vivo. We have studied the effects of retinol on the human breast carcinoma cell line, MDA-MB-231. Initial in vitro studies on monolayer cultures demonstrated a retinol-induced growth inhibition that was reversible as well as time and dose dependent. A similar dose-dependent decrease in tumor cell growth was shown in vivo when BALB/c-nu/nu (athymic) mice were inoculated s.c. with MDA-MB-231 cells and given graded nontoxic doses of retinol intragastrically for 3 weeks. Tumor cells were also inhibited from lung colonization as "artificial" metastatic lesions when injected i.v. into athymic mice following retinol treatment. Spleen cells from these mice were assayed for natural killer cells as determined by their cytotoxic activity on 51Cr-labeled target cells. There was no change in natural killer activity with any dose of retinol. We conclude that retinol has a dose-dependent antiproliferative effect on human breast carcinoma in vivo as well as in vitro. Further, the retinol-induced tumor inhibition seen in T-cell-deficient mice does not appear to be due to enhancement of host immunity and thus may be solely a direct effect of retinol on the tumor.

Potential utility of serum neuron-specific enolase levels in small cell carcinoma of the lung.

To assess the value of neuron-specific enolase (NSE) as a possible biomarker of small cell lung cancer, serum levels were determined by radioimmunoassay in 93 newly diagnosed untreated patients and were compared to the NSE levels of 20 healthy adult controls [9.6 +/- 0.7 (S.E.) ng/ml]. Serum NSE was elevated (greater than 20 ng/ml) in 73% of all patients including 23 of 39 (59%) with limited-stage disease and 45 of 54 (83%) with extensive-stage disease. The mean serum NSE was significantly higher in extensive-stage disease (94.5 +/- 13.8 ng/ml) compared to the mean value for limited-stage disease (33.7 +/- 4.7 ng/ml) (p less than 0.001). NSE was elevated in all patients with three or more sites of metastatic disease. Serial NSE determinations were obtained on 57 small cell lung cancer patients. NSE levels fell in 40 of 50 (80%) of patients responding to treatment, increased in 5 of 7 (71%) of patients with progressive disease, and increased in 30 of 35 (86%) of patients who relapsed. A persistent rise in serum NSE occurred as many as 12 weeks before the clinical recognition of relapse in 15 of 23 (65%) of patients for whom adequate serial NSE data were available. These findings indicate that serum NSE may be a useful marker for staging, monitoring treatment, and predicting relapse in patients with small cell lung cancer.

Inhibition of HER2/neu (erbB-2) and mitogen-activated protein kinases enhances tamoxifen action against HER2-overexpressing, tamoxifen-resistant breast cancer cells.

HER2/neu (erbB-2) overexpression has been causally associated with tamoxifen resistance in human breast cancer cells. Forced expression of HER2 in MCF-7 breast cancer cells resulted in mitogen-activated protein kinase (MAPK) hyperactivity and tamoxifen resistance. Inhibition of HER2 and MAPKs with AG1478 and U0126, respectively, as well as dominant-negative MEK-1/2 constructs restored the inhibitory effect of tamoxifen on estrogen receptor (ER)-mediated transcription and cell proliferation. Both AG1478 and U0126 also restored the tamoxifen-mediated association of ER with nuclear receptor corepressor (N-CoR) in the antiestrogen-resistant MCF-7 cells. Treatment with a combination of tamoxifen and a HER2 kinase inhibitor reduced tumor MAPK activity and markedly prevented growth of HER2-overexpressing MCF-7 xenografts in athymic mice. Thus, blockade of HER2 and MAPK signaling may enhance tamoxifen action and abrogate antiestrogen resistance in human breast cancer.

Natural killer cell activity during murine Schistosomiasis mansoni.

Natural killer cell (NK) activity of spleen cells from CBA/J and C57BL/6 mice infected with Schistosoma mansoni was assayed using 51Cr-labeled YAC-1 cells as target cells. No significant difference in NK activity was detected between infected and age-matched control spleen cells from 1 to 4 wk after infection. The NK-mediated cytotoxic abilities of both infected and age-matched control mice fell in parallel, as expected, on an age-related basis. However, on a cell-to-cell basis the splenic NK activity of mice infected from 8 to 18 wk was significantly less than that of age-matched control mice. Meanwhile, because of the splenomegaly associated with patent infection, the total number of nucleated splenocytes in chronically infected mice increased to fourfold that of age-matched control mice. Therefore, the total available NK activity per spleen was increased by chronic infection. The NK activities of spleen cells from age-matched control mice and those infected for 18 wk could be augmented by in vivo administration of polyinosinic-polycytidylic acid, indicating the continued responsiveness of NK cells from chronically infected mice to activation. Spleen cell NK activity in age-matched control and infected C57BL/6 mice exhibited a pattern similar to that of CBA/J mice.

Immune response to chemically induced tumours: correlation of responding cell class with in vivo inhibition of tumour growth.

Lymphoid cells stimulated by soluble tumour antigens in the MCA-induced murine fibrosarcoma system have been identified by subclass and protective capacity in adoptive syngeneic hosts. Lymph-node or spleen cells taken at weekly intervals after inoculation of syngeneic chemically induced fibrosarcomas were enriched by 3 methods in T, B, and "null" cell subclasses, and assayed for proliferative kinetics in response to soluble membrane antigens. The stimulated subpopulations were found to be heterogeneous, their composition varying with time and tumour burden. Initial proliferative responses after tumour inoculation were limited to the T-enriched subpopulation. Later during tumour growth, T, B and null cell fractions were vigorously and equally stimulated by tumour antigen. The ability of the same T, B or null-cell subpopulations to inhibit tumour growth was measured in adoptive hosts by a modified Winn assay. Only the T-cell subpopulation responding to tumour antigen in vitro effectively and consistently retarded tumour growth in vivo. In contrast to the shared specificities on syngeneic tumours identified by the proliferative assay, tumour-growth inhibition was limited to the specific tumour borne by the cell donor.

Mannose 6-, fructose 1-, and fructose 6-phosphates inhibit human natural cell-mediated cytotoxicity.

In vitro human natural cell-mediated cytotoxicity (NCMC) to K-562, Molt-4, and F-265 cells is inhibited in a dose-dependent manner by mannose 6-phosphate, fructose 1-phosphate and fructose 6-phosphate. This inhibition is not observed with mannose, glucose, fucose, glucose 6-phosphate, mannose 1-phosphate, galactose 1-phosphate, or galactose 6-phosphate. Preincubation of the effector cells, obtained from fresh whole blood, with mannose-6-phosphate, fructose-1-phosphate, or fructose-6-phosphate did not inhibit cytotoxicity, which indicated that these hexose phosphates are not nonspecifically toxic towards the effector lymphocytes. Mannose-6-phosphate and the stereochemically similar fructose-1-phosphate are more potent inhibitors than fructose-6-phosphate in terms of concentration required and time of onset of effect. Inhibition of cytotoxicity by mannose-6-phosphate varied with target cell type: F-265 is protected at much lower concentrations of mannose-6-phosphate (less than 1 mM) than is either Molt-4 or K-562. The inhibition of NCMC is also observed with the inhibitors of lysosomal function, NH4Cl, and chloroquine. The presence of a functional mannose-6-phosphate receptor on target cells was demonstrated: (i) Gelonin, a seed protein that inactivates the eukaryotic ribosome but is nontoxic to intact cells, was covalently linked to monophosphopentamannose, and this conjugate ws toxic to both K-562 and F-265 target cells, the latter being by far the more sensitive; and (ii) chloroquine, NH4Cl, and mannose-6-phosphate all inhibited the toxicity of gelonin-monophosphopentamannose. These results suggest either that a cytolytic lymphokine contains a hexose phosphate residue and may be taken up by target cells through the lysosomal/mannose 6-phosphate pathway or that such a residue is involved in target cell-effector cell recognition.

Soluble tumor antigen-induced lymphocyte proliferation: effects of serum from normal and tumor-bearing mice.

The effect of serum from mice bearing methylcholanthrene-induced fibrosarcomas on the lympho-proliferative responses of their spleen and lymph-node cells to 3 M KCl-solubilized tumor antigens has been investigated. Low concentrations of serum (less than or equal to 2.5%) collected throughout the course of tumor growth were inhibitory. Normal sera collected from age-matched control mice were often slightly inhibitory, but to a significantly lesser extent at identical serum concentrations. The inhibitory effect of tumor-bearing serum was not limited to homologous tumors, but was also observed when cross-reacting syngeneic tumor antigens or serum from animals bearing other syngeneic tumors were added to the assay mixture. At the low concentrations used, tumor-bearing sera inhibited only tumor-antigen-induced proliferation, and not proliferation induced by T cell mitogens. The inhibitory effects of tumor-bearing sera on lympho-proliferative responses to soluble tumor antigens appear to be similar to the inhibition seen as "serum blocking" in other assays of tumor immunity.

Effects of orally administered retinol on natural killer cell activity in wild type BALB/c and congenitally athymic BALB/c mice.

Retinoids have been shown to inhibit the growth and development of neoplastic cells in many systems. One mechanism of action may be through activation of the immune system, specifically natural killer (NK) cell activity. The effect of retinol on NK cell cytotoxicity was examined in three groups of mice: BALB/c (wild-type), BALB/c nu/nu (athymic), and BALB/c nu/nu previously injected with human tumor cells. In untreated mice, NK activity was highest in athymic mice without tumors and lowest in wild-type mice, although serum and liver retinol concentrations were identical in all three groups. In mice fed graded, nontoxic doses of retinol daily for 3 weeks, serum retinol levels in all three groups exhibited a sharp peak and decline following daily bolus retinol administration. Retinol stores in the livers showed a dose-dependent increase in all treated animals. However, NK cell activity, differed for each group. Athymic mice without tumors exhibited no change in NK activity as a result of retinol treatment. Athymic mice with tumors had NK levels that tended to increase with increasing retinol doses, but these changes were not statistically significant. Wild-type mice, on the other hand, demonstrated significantly higher NK levels after treatment with retinol doses of 300 and 600 micrograms/day. In subsequent time course experiments, there was a peak in NK activity 1 h following bolus retinol administration similar to the peak seen in serum retinol concentrations, suggesting either an acute activation or recruitment of cytotoxic cells. Retinol thus appears to increase NK activity in wild-type BALB/c mice, and this activity may be an important component of its antineoplastic activity.

T mitogens trigger LPS responsiveness in mouse thymus cells.

Mouse thymus cells are essentially unresponsive to lipopolysaccharide (LPS) stimulation. However, when cultured with minimally mitogenic levels of concanavalin A or submitogenic ratios of mitomycin-treated allogeneic spleen cells, in combination with LPS, they demonstrate levels of DNA synthesis de novo or greater than those induced by the T cell mitogen alone. Dose-response kinetics were characteristic of LPS. The subpopulation containing the LPS responsive cells was of low net buoyant density. Neither phytohemagglutinin nor pokeweed mitogen acted synergistically with LPS in this model to trigger thymus cells. The data suggest that LPS triggering may involve interaction with a T cell subpopulation.

Ability of delayed-type hypersensitivity reactions to distinguish tumor-associated antigens and histocompatibility antigens in soluble extracts from murine fibrosarcomas.

The footpad swelling (FPS) test for delayed-type hypersensitivity in the mouse was evaluated for its ability to measure both tumor-associated antigens (TAA) and histocompatibility (H) antigens solubilized from methylcholanthrene (MCA) induced fibrosarcomas of C57B1/6 (B6) mice. Tests for TAA were performed in mice immune to syngeneic tumors while H-antigens were assayed in mice immunized with skin allografts. FPS was most intense in B6 mice challenged with TAA from the immunizing B6 tumor, but also occurred in response to cross-reactive TAA solubilized from another B6 fibrosarcoma. Tests for tumor-associated H-antigens in allograft immune mice were strongly positive in response to donor/recipient H-antigen differences and proved sensitive to shared third-party H-antigen differences. Comparison of soluble antigens from the same tumor maintained in vitro and in vivo revealed that, while both TAA and H-antigens could be detected in preparations from the in vivo tumor line, only TAA and not H-antigens could be detected by RPS in extracts prepared from the in vitro tumor line. These experiments have demonstrated that the mouse FPS test can distinguish both TAA and H-antigen specificities persent in the same complex mixture of tumor-cell antigens.

Phase I study of MVE-2 evaluating toxicity and biologic response modification capability.

A total of 21 patients were treated in a phase I trial using the biological response modifier MVE-2, a low molecular weight component of pyran copolymer. All patients received weekly IV MVE-2 infused over 2 h. Proteinuria, sometimes of nephrotic proportions, was the dose limiting toxicity, and was seen with increasing incidence as the cumulative dose of MVE-2 exceeded 2500 mg. Other toxicity with MVE-2 was minimal. Biologic response modification at tolerable doses was inconsistent, although several assays, particularly natural cell-mediated cytotoxicity, indicated enhanced activity at higher dosages of MVE-2. No objective tumor responses were observed. MVE-2 is not useful as a biological response modifier using our initial method of administration, since the dose limiting toxicity occurred at lower levels than were necessary to induce consistent biologic response modification. Following completion of the phase I study, we administered MVE-2 by 30-min infusion to 8 additional patients and did not detect proteinuria, in spite of large cumulative doses. It is possible that alternate schedules of MVE-2 administration could minimize proteinuria and allow the administration of dosages necessary for immunologic modification.

Evidence for a positive role of transforming growth factor-beta in human breast cancer cell tumorigenesis.

To determine the biological role of transforming growth factor-beta (TGF-beta) in mammary carcinomas in vivo, estrogen-dependent MCF-7 cells were transfected with a mouse TGF-beta 1 cDNA. Growth characteristics in culture were not altered in the transfected cells. However, the MCF-7/TGF-beta 1 cells formed tumors in ovariectomized athymic mice in the absence of estrogen supplementation. Daily injections of human recombinant TGF-beta 1 supported tumor formation by wild-type MCF-7 cells in castrated nude mice in the absence of exogenous estradiol. In another approach to the same question, the effect of anti-TGF-beta antibodies on tumor formation by estrogen-independent MDA-231 cells was examined. The 2G7 IgG2b (2G7) antibody, which neutralizes TGF-beta 1, -beta 2, and -beta 3, blocked the formation of MDA-231 tumors at the injection site and lung metastases in nude mice. Inoculation of MDA-231 cells inhibited, while injection of 2G7 increased mouse spleen natural killer (NK) activity. 2G7 did not inhibit MDA-231 tumors and metastases in NK-deficient animals. Finally, medium conditioned by MDA-231 cells inhibited lymphocyte-mediated NK activity; this inhibition was abrogated by 2G7, but not by a control IgG2. These data support a positive role for tumor cell TGF-beta in the maintenance and/or progression of mammary carcinoma cells in an intact host.

Specific antigen stimulated lymphocyte proliferation in osteosarcoma.

A lymphocyte proliferation assay (LPA) for cellular immune responses to osteosarcoma antigens is described and applied to an examination of peripheral blood lymphocytes (PBL) taken from osteosarcoma patients. The antigen preparations were derived from 3 M KC1 solubilized osteosarcoma, taken from a limited number of patients. Lymphocytes from most tumor-bearing patients were stimulated to significant proliferation when cultured in normal human serum. Such stimulation was observed whether or not the lymphoid cells were preincubated 24 hours at 37 degrees C prior to addition of antigen. Patients whose lesion had been resected and who were without evidence of disease for 5-70 months had diminished proliferative responses. Lymphocytes from normal subjects, from patients having other types of sarcoma, and patients having carcinomas rarely responded to the soluble osteosarcoma antigens. When responsive PBL taken from tumor-bearing patients were cultured in autologous serum, the proliferative responses were abrogated or blocked. Serial assays made in the course of bearing this tumor under a variety of therapeutic regimens, including an immunotherapy protocol, suggest that the LPA may be useful in monitoring clinical progress of the disease and possibly in other immunotherapy protocols for osteosarcoma.

Human recombinant interleukin 2-activated sheep lymphocytes lyse sheep pulmonary microvascular endothelial cells.

Administration of lymphokine-activated killer (LAK) cells in combination with interleukin 2 (IL-2) has been effective in reducing tumor mass in humans, but has been accompanied by significant toxicity. We used a chronic awake sheep model to investigate the cause of the vascular leak syndrome associated with IL-2 administration. Sheep repeatedly infused with human recombinant IL-2 (hrIL-2) developed mild pulmonary hypertension, systemic hypotension, acidemia, hypoxemia, and increased flow of protein rich lung lymph. We hypothesized that LAK cells may damage lung endothelium in vivo and cause increased lung vascular permeability. Sheep peripheral blood and lung lymph lymphocytes incubated in vitro with hrIL-2 generated cytotoxic activity for human K-562 cells and sheep pulmonary microvascular endothelial cells. In addition, cytotoxic effector cells were isolated from the peripheral blood of a sheep which had received hrIL-2. These observations suggest that LAK cells possess the ability to damage endothelial cells and may contribute to an increased pulmonary vascular permeability observed following hrIL-2 infusion in sheep.

Pleiotropic activities of human interferons are mediated by multiple response pathways.

Among the pleiotropic effects of human interferon are the inhibition of viral replication, the activation of natural killer cells, and the inhibition of cellular growth. Oxyphenbutazone, a nonsteroidal antiinflammatory agent, is a potent inhibitor of the antiviral activity of human alpha and beta interferons as determined by cytopathic effect and vesicular stomatitis virus synthesis and release in human foreskin fibroblasts. The inhibition of interferon activity is dose dependent with maximal inhibition at 25-50 microM and minimal inhibition at 1 microM. In contrast, oxyphenbutazone at concentrations as high as 100 microM has no effect on the activation of natural killer cells by human interferon. Similarly, oxyphenbutazone has no inhibitory effect on interferon-induced antigrowth activity in the human breast carcinoma cell line MDA-MB-231. This cell line is sensitive to oxyphenbutazone inhibition of interferon-induced antiviral activity in vitro. In another human cell line, the vulvar carcinoma A431, oxyphenbutazone apparently augments the antigrowth activity of interferon. Although oxyphenbutazone inhibits the fatty acid cyclooxygenase enzyme in these systems, other inhibitors of cyclooxygenase fail to inactivate the antiviral activity of human interferon. Thus, oxyphenbutazone appears to inhibit the interferon antiviral cascade at a site distinct from prostaglandin biosynthesis. Moreover, the failure to inhibit natural killer cell activation or cellular antigrowth effects of human interferon suggests a pathway different from that associated with the antiviral effect of human interferon.

Blockade of the epidermal growth factor receptor tyrosine kinase suppresses tumorigenesis in MMTV/Neu + MMTV/TGF-alpha bigenic mice.

Overexpression of ErbB-2/Neu has been causally associated with mammary epithelial transformation. Here we report that blockade of the epidermal growth factor receptor (EGFR) kinase with AG-1478 markedly delays breast tumor formation in mouse mammary tumor virus (MMTV)/Neu + MMTV/transforming growth factor alpha bigenic mice. This delay was associated with inhibition of EGFR and Neu signaling, reduction of cyclin-dependent kinase 2 (Cdk2) and mitogen-activated protein kinase (MAPK) activities and cyclin D1, and an increase in the levels of the Cdk inhibitor p27(Kip1). In addition, BrdUrd incorporation into tumor cell nuclei was prevented with no signs of tumor cell apoptosis. These observations prompted us to investigate the stability of p27. Recombinant p27 was degraded rapidly in vitro by untreated but not by AG-1478-treated tumor lysates. Proteasome depletion of the tumor lysates, addition of the specific MEK1/2 inhibitor U-0126, or a T187A mutation in recombinant p27 all prevented p27 degradation. Cdk2 and MAPK precipitates from untreated tumor lysates phosphorylated recombinant wild-type p27 but not the T187A mutant in vitro. Cdk2 and MAPK precipitates from AG-1478-treated tumors were unable to phosphorylate p27 in vitro. These data suggest that increased signaling by ErbB receptors up-regulates MAPK activity, which, in turn, phosphorylates and destabilizes p27, thus contributing to dysregulated cell cycle progression.