Anti-tumor monoclonal antibodies in conjunction with ß-glucans: a novel anti-cancer immunotherapy.

Monoclonal antibodies (mAbs) have greatly advanced the field of anti-cancer immunotherapy and have made a major impact in clinical medicine. While more mAbs have been approved by the FDA and entered into the clinical therapeutic arena with indications to treat various solid tumors and hematologic malignancies, extensive efforts have also been made to make mAb therapy more effective. Combination therapy of anti-tumor mAbs with chemotherapeutic drugs has been widely used in the clinical patient care. In addition, many immune stimulating agents have been specifically studied for this very purpose. One compound in particular, ß-glucan, has shown very promising and exciting results in pre-clinical animal models and early phase human clinical trials. ß-Glucans are naturally occurring, abundant polysaccharides with different structures that can be extracted and purified from fungi, bacteria, oats and barley. The active components of yeast-derived ß-glucan exert their unique immune stimulating functions by binding specifically to complement receptor 3 (CR3) via lectin-like domain (LLD) and activating CR3 to promote cellular cytotoxicity of iC3b-coated cancer cells. In addition, particulate yeast-derived ß-glucan stimulates both innate and adaptive anti-tumor immune responses. This review covers the anti-cancer mechanisms of anti-tumor mAbs and ß-glucans, the pre-clinical studies done with ß-glucans in conjunction with anti-tumor mAbs in human carcinoma xenograft models, and the preliminary results of human clinical trials with different ß-glucans, as well as those of phase I/II and III studies using the combination of yeast-derived soluble ß-glucan and anti-tumor mAbs.

Alemtuzumab in previously treated chronic lymphocytic leukemia patients who also had received fludarabine.

PURPOSE: This phase II pilot study determined the efficacy and safety of alemtuzumab (Campath-1H; Burroughs Wellcome, United Kingdom) in patients with chronic lymphocytic leukemia (CLL), all of whom had previously received fludarabine and other chemotherapy regimens. PATIENTS AND METHODS: Twenty-four patients were treated with intravenous alemtuzumab at six centers in the United States. The target dose of 30 mg over 2 hours, three times weekly, was administered for up to 16 weeks. Responses were evaluated by an independent panel of experts using 1996 National Cancer Institute-sponsored Working Group criteria. Safety assessments included analysis of lymphocyte subpopulations. Antimicrobial prophylaxis was not mandatory. RESULTS: Eight patients (33%) achieved a major response (all partial remissions), with a median time to response of 3.9 months (range, 1.6 to 5.3 months). The median duration of response was 15.4 months (range, 4.6 to >or= 38.0 months), the median time to disease progression was 19.6 months (range, 7.7 to >or= 42.0 months), and the median survival time was 35.8 months (range, 8.8 to >or= 47.1 months). Acute infusion-related events, mainly grades 1 and 2, were most common and most severe in the first week. Ten patients (eight nonresponders and two responders) experienced major infections on-study. Pneumocystis carinii pneumonia was reported in two patients on-study; neither had received prophylaxis. Median CD4+ and CD8+ counts decreased and then began to increase by the end of the study, with further recovery by 1-month follow-up. One of 53 samples obtained from 10 patients had a low titer of alemtuzumab antibodies. CONCLUSION: Alemtuzumab has significant activity in poor-prognosis, fludarabine-treated CLL patients. However, because of a relatively high incidence of opportunistic infections accompanying profound lymphopenia, future protocols should include mandatory prophylaxis.

Brief-duration high-intensity chemotherapy for patients with small noncleaved-cell lymphoma or FAB L3 acute lymphocytic leukemia: results of cancer and leukemia group B study 9251.

PURPOSE: To define the activity and feasibility of brief-duration high-intensity chemotherapy for adults with small noncleaved, non-Hodgkin's lymphoma (SNC) and the L3 variant of acute lymphocytic leukemia (L3 ALL). PATIENTS AND METHODS: Seventy-five adults with either SNC or L3 ALL (median age, 44 years) were treated with an aggressive regimen that consisted of one cycle of cyclophosphamide and prednisone followed by cycles containing either ifosfamide or cyclophosphamide; high-dose methotrexate, vincristine, dexamethasone, and either doxorubicin or etoposide/cytarabine; or intrathecal triple therapy with prophylactic CNS irradiation. RESULTS: All 24 patients with L3 ALL and the 30 of 51 patients with SNC confirmed by central histologic review were included in this analysis. Forty-three of 54 patients achieved complete response (CR) (18 of 24 with ALL and 25 of 30 with SNC), and 28 are alive and in continuous CR with a median follow-up of 5.1 years. Hematologic toxicity was profound, and nonhematologic toxicity was notable, with 10 of 75 patients treated developing significant neurologic toxicity consisting of transverse myelitis in five patients, CNS toxicity in three, and severe peripheral neuropathy in two. All patients who did not achieve CR died of the disease, and all recurrences occurred within 16 months of the end of treatment. Responses and toxicities were similar in the patients with both lymphoma and leukemia. CONCLUSION: Aggressively delivered chemotherapy is potentially curative in as many as half of patients with SNC and the L3 ALL variant. This treatment regimen had considerable neurologic toxicity and has been modified.

A randomized phase 2 study of PBPC mobilization by stem cell factor and filgrastim in heavily pretreated patients with Hodgkin's disease or non-Hodgkin's lymphoma.

This randomized, controlled study compared the ability to mobilize and collect an optimal target yield of 5 x 10(6) CD34+ cells/kg using stem cell factor (SCF; 20 microg/kg/day) plus filgrastim (G-CSF; 10 microg/kg/day) vs filgrastim alone (10 microg/kg/day) in 102 patients diagnosed with non-Hodgkin's lymphoma (NHL) or Hodgkin's disease (HD), who were prospectively defined as being heavily pretreated. Leukapheresis began on day 5 of cytokine administration and continued daily until the target yield was reached, or until a maximum of five leukaphereses had been performed. Compared with the filgrastim-alone group (n = 54), the SCF plus filgrastim group (n = 48) showed an increase in the proportion of patients reaching the target yield within five leukaphereses (44% vs 17%, P = 0.002); reduction in the number of leukaphereses required to reach the target yield (P = 0.003); reduction in the proportion of patients failing to reach a minimum yield of 1 x 10(6) CD34+ cells/kg to proceed to transplant (16% vs 26%, P = NS); increase in the median yield of CD34+ cells per leukapheresis (0.73 x 10(6)/kg vs 0.48 x 10(6)/kg, P = 0.04); and an increase in the median total CD34+ cells collected within five leukaphereses (3.6 x 10(6)/kg vs 2.4 x 10(6)/kg, P = 0.05). All patients receiving SCF were premedicated (antihistamines and albuterol), and treatment was generally well tolerated. Five patients experienced severe mast cell-mediated reactions, none of which were life-threatening. In this study of heavily pretreated lymphoma patients, SCF plus filgrastim was more effective than filgrastim alone for mobilizing PBPC for harvesting and transplantation after high-dose chemotherapy.

Sweet's syndrome during treatment with all-trans retinoic acid in a patient with acute promyelocytic leukemia.

A 46 year old male with acute promyelocytic leukemia treated with all-trans retinoic acid (ATRA), developed fever, bilateral erythematous nodules in his axillary area, lower abdomen and inguinal region. Histopathologic examination of the skin biopsy revealed dense neutrophil infiltration in the dermis without vasculitis. The diagnosis of Sweet's syndrome was made. High dose methylprednisolone was administered and the lesions started to improve within 24 hours.

Intravascular large cell lymphoma coexisting within hemangiomas of the skin.

Intravascular lymphomatosis is a rare and peculiar subtype of large cell lymphoma. The authors present the pathologic, clinical, and radiologic findings of a patient with intravascular large cell lymphoma coexisting within hemangiomas of the skin. Initially the lymphoma was clinically confined to the hemangiomas and the patient was closely observed for disease progression. Within 10 months the patient developed disseminated lymphoma involving both adrenals. A clinical remission was achieved, but the patient soon relapsed, and despite further chemotherapy he died with disseminated disease 23 months after the initial diagnosis. This report presents the only known case of an intravascular large cell lymphoma coexisting within a vascular lesion and highlights the potential aggressive nature of intravascular lymphomas.

Hodgkin's disease treated with neck radiation is associated with increased antibody-dependent cellular cytotoxicity against human extraocular muscle cells.

Patients with Hodgkin's disease have higher a prevalence of thyroid function abnormalities and, perhaps, orbitopathy than the general population, but the pathophysiology of this association and its relationship to Hodgkin's disease treatment remain unclear. We analyzed the frequency of thyroid function abnormalities, autoantibodies against thyroid antigens, and autoimmunity against extraocular muscle cell antigens by Western blot analyses and antibody-dependent cellular cytotoxicity (ADCC) assays in patients with Hodgkin's disease (n = 20) and controls (n = 10). Hodgkin's disease patients were subdivided into those treated with thyroidal external beam radiation therapy (XRT, n = 15) or chemotherapy (MOPP/ABVD, n = 5). The ADCC assay against extraocular muscle cells was increased in patients with Hodgkin's disease (5.5% vs. <1.0%, p = .026) when compared with controls. In addition, Hodgkin's disease patients treated with XRT (with or without chemotherapy) had significantly higher ADCC tests than controls (9.7% vs. <1.0%, p = .010), In contrast, ADCC assays were not different between Hodgkin's disease patients treated with chemotherapy alone and controls (<1.0% vs. <1.0%, p = .53). Hodgkin's patients treated with XRT had higher ADCC assays than those treated with chemotherapy alone (p = .087), although this difference did not achieve statistical significance. Serum measurements of antithyroid peroxidase (TPO) antibodies, antithyroglobulin (Tg) antibodies, thyroid binding inhibitory immunoglobulins (TBII), and thyroid stimulating immunoglobulin (TSI) were similar in all groups. Antibodies against the 64 kDa orbital antigen were detected in 1 patient and 1 control subject. Excluding patients already treated with L-thyroxine for hypothyroidism (n = 5), free T3, but not free T4, was lower in the Hodgkin's disease group than in controls (2.2 pg/mL vs. 2.7 pg/mL, p = .008). Thyrotropin (TSH) concentrations were not statistically different between these groups. In summary, these data show: (1) ADCC against human orbital muscle cells is increased in patients with Hodgkin's disease compared with controls: (2) these differences were noted among Hodgkin's disease patients treated with thyroidal XRT, with or without chemotherapy, and not among those patients treated with chemotherapy alone; and (3) no statistically significant differences in the frequency of thyroid autoantibodies were found. These data suggest that patients with Hodgkin's disease display altered antibody-dependent immune function toward extraocular muscle cells that may possibly be related to by XRT. Larger, prospective studies assessing thyroid and orbital-related immunologic abnormalities in Hodgkin's disease are warranted.

Molecular detection of bone marrow involvement in intravascular lymphomatosis.

Intravascular lymphomatosis (IVL) is an extremely rare variant of aggressive non-Hodgkin's lymphoma characterized by confinement of neoplastic lymphocytes within vascular spaces. Although IVL is potentially curable with combination chemotherapy, diagnosis is often delayed, in part owing to the negative bone marrow biopsy specimens that are typical of this disorder. We hypothesized that use of a more sensitive method of analysis might identify small clonal B-cell populations in histologically negative bone marrow biopsy specimens from patients with IVL. With use of a recently described assay for immunoglobulin heavy chain gene rearrangement based on the polymerase chain reaction, we demonstrated clonal B-cell populations in histologically negative marrow specimens from five (100%) of five patients with IVL. None of these specimens demonstrated molecular evidence of the t(14;18) associated with follicular lymphoma, providing no evidence for a common derivation of IVL and follicular lymphoma. In summary, molecular analysis of routine bone marrow biopsy sepcimens from patients in whom the diagnosis of IVL is entertained may facilitate prompt recognition of a lympho-proliferative disorder and thereby permit timely therapeutic intervention. Moreover, these findings suggest that despite histologically negative staging bone marrow biopsy specimens, IVL typically disseminates early in its course, thus arguing against the use of localized therapy in this disorder.

Mechanisms of inhibition of IL-6-mediated immunoglobulin secretion by dexamethasone and suramin in human lymphoid and myeloma cell lines.

The cytokine IL-6 has been proposed as an autocrine growth factor in multiple myeloma, and is also required for stimulation of immunoglobulin production and secretion in normal plasma cells and myeloma cells. In this study, we showed that secreted IL-6 is detectable by Western blot analysis in a panel of lymphoid and myeloma cell lines. Previous studies in our laboratory have shown that dexamethasone and suramin inhibit cell proliferation and IL-6-mediated immunoglobulin secretion in various lymphoblastoid and myeloma cell lines. In the present study, we present study, we present data to examine mechanisms by which dexamethasone and suramin inhibit IL-6-mediated immunoglobulin secretion in the lymphoid cell line SKW 6.4. Cells treated with rIL-6 or the IC10 concentration of dexamethasone respectively undergo a doubling of intracellular IgM. Moreover, rIL-6 and dexamethasone additively stimulate cells to accumulate intracellular IgM. In contrast, cells treated with the IC10 concentration of suramin undergo no significant alteration of total cellular IgM, and do not respond to IL-6 with an increase in intracellular IgM. Northern blot analysis demonstrates that cells treated with exogenous rIL-6 and/or dexamethasone respectively undergo a coordinate one to three fold increase of kappa and mu chain mRNA expression, while there is a 30-40% decrease of kappa and mu chain mRNA when cells are treated with suramin and suramin plus rIL-6. Western blot analysis shows that levels of intracellular IL-6 modestly increase when cells are treated with exogenous rIL-6, whereas treatment with dexamethasone plus rIL-6 causes a 70% decrease of immunoreactive IL-6 protein in comparison with untreated cells. An rtPCR analysis of IL-6 mRNA expression shows an abolished signal in response to dexamethasone or rIL-6 and/or dexamethasone. Using a flow cytometric assay, it is demonstrated that suramin inhibits IL-6 binding to its receptor. Taken together, these results indicate that SKW 6.4 cells treated with rIL-6 and/or dexamethasone undergo increased expression of IgM mRNA leading to increased intracellular IgM levels. Treatment with suramin or suramin plus rIL-6 does not alter the IL-6 protein level or the mRNA levels for IL-6 and IL-6 receptor. Suramin treatment causes a moderate decrease in IgM mRNA, and this is associated with a decreased intracellular level of IgM in SKW 6.4 cells. Overall these findings support the concept that IL-6 is an autocrine factor for immunoglobulin production and secretion in myeloma cells. Suramin interferes with IL-6 binding to its receptor and/or decreases IL-6 receptor expression. Dexamethasone has neither of these effects on IL-6 receptor expression or IL-6 binding to its receptor, and we postulate that it acts through a block in secretion or in degradation of intracellular immunoglobulin by decreasing IL-6 mRNA expression and IL-6 protein content. These studies suggest that the combination of suramin and dexamethasone not only synergistically growth inhibit myeloma cells but also act in concert to inhibit immunoglobulin secretion and represent a therapeutic approach worthy of further investigation.

Dexamethasone and suramin inhibit cell proliferation and interleukin-6-mediated immunoglobulin secretion in human lymphoid and multiple myeloma cell lines.

Uncontrolled growth of neoplastic cells and unregulated production of immunoglobulin are major components of the morbidity and mortality of multiple myeloma. Suramin, a polysulfonated napthylurea, has antitumor activity in a number of malignancies, but also significant dose-related toxicity. Suramin has been reported to have major antiproliferative effects in a variety of lymphoid cell lines. Glucocorticoids have long been recognized to have activity in lymphoid malignancies and multiple myeloma while IL-6 has been reported to be an autocrine growth factor for multiple myeloma. This study examines growth inhibition and inhibition of IL-6-mediated secretion of immunoglobulin in human lymphoid and myeloma cell lines by dexamethasone and suramin. Dexamethasone and suramin show synergistic inhibition of cell proliferation at their IC10 concentrations. IL-6-mediated immunoglobulin secretion is also inhibited by both dexamethasone and suramin in an additive fashion. Both dexamethasone and suramin induce apoptosis of lymphoid cell lines, and suramin inhibits the binding of IL-6 to its receptor in a multiple myeloma cell line. These findings suggest that the synergistic growth inhibitory activities of dexamethasone and suramin may be related to induction of apoptosis by both agents and inhibition of IL-6-mediated autocrine growth stimulation and immunoglobulin production. These results indicate that the combination of low-dose suramin (in concentrations not associated with significant clinical toxicity) and dexamethasone merit further study in the treatment of myeloma or lymphoid malignancies.

Hormonal carcinogenesis in breast cancer: cellular and molecular studies of malignant progression.

We have established and characterized a series of variant cell lines in which to identify the critical factors associated with E2-induced malignant progression, and the acquisition to tamoxifen resistance in human breast cancer. Sublines of the hormone-dependent MCF-7 cell line (MCF7/MIII and MCF7/LCC1) form stable, invasive, estrogen independent tumors in the mammary fat pads of ovariectomized athymic nude mice. These cells retain expression of both estrogen (ER) and progesterone receptors (PGR), but retain sensitivity to each of the major structural classes of antiestrogens. The tamoxifen-resistant MCF7/LCC2 cells retain sensitivity to the inhibitory effects of the steroidal antiestrogen ICI 182780. By comparing the parental hormone-dependent and variant hormone-independent cells, we have demonstrated an altered expression of some estrogen regulated genes (PGR, pS2, cathepsin D) in the hormone-independent variants. Other genes remain normally estrogen regulated (ER, laminin receptor, EGF-receptor). These data strongly implicate the altered regulation of a specific subset or network of estrogen regulated genes in the malignant progression of human breast cancer. Some of the primary response genes in this network may exhibit dose-response and induction kinetics similar to pS2, which is constitutively upregulated in the MCF7/MIII, MCF7/LCC1 and MCF7/LCC2 cells.

Suramin interferes with interleukin-6 receptor binding in vitro and inhibits colon-26-mediated experimental cancer cachexia in vivo.

Neoplastic diseases are frequently associated with metabolic changes collectively known as cancer cachexia. The presence of cachexia complicates therapeutic intervention and is an important cause of death in cancer patients. At present there is no effective treatment for cachexia. Recently, the involvement of interleukin-6 (IL-6) in the wasting of colon-26 adenocarcinoma-bearing mice was demonstrated. The research presented here establishes an anticachectic role for the experimental drug suramin, since it partially blocks (up to 60%) the catabolic effects associated with the growth of this tumor in vivo. Suramin prevents the binding of IL-6 to its cell surface receptor subunits, as demonstrated by radioreceptor binding assay and affinity crosslinking experiments. Furthermore, the uptake of radioactive IL-6 by the liver is significantly reduced in suramin-treated mice. On the other hand, the drug is approximately 10-fold less potent in inhibiting the binding of tumor necrosis factor-alpha to indicator cell line in vitro and fails to block liver uptake of this cytokine in vivo. Collectively, these results suggest that suramin inhibits cancer-associated wasting, in part by interfering with the binding of IL-6 to its receptor. Whether suramin inhibits the action of other factors/cytokines that may also participate in colon-26-mediated cachexia is not yet known.

Acquisition of hormone-independent growth in MCF-7 cells is accompanied by increased expression of estrogen-regulated genes but without detectable DNA amplifications.

A hormone-independent but hormone-responsive subpopulation (MCF7/MIII) of the hormone-dependent MCF-7 human breast cancer cell line (R. Clarke et al., Proc. Natl. Acad. Sci. USA 86: 3649-3653, 1989) was further passaged in ovariectomized nude mice and re-established in vitro as the continuous cell line MCF7/LCC1. The lag time to the appearance of proliferating tumors in ovariectomized animals is significantly reduced in MCF7/LCC1 when compared with MCF7/MIII cells. In gel denaturation/renaturation analysis of tumor, genomic DNA does not reveal significant differences in the pattern of detectable DNA amplifications between parent MCF-7 cells and MCF7/LCC1 cells. In the absence of estrogen, steady-state levels of phosphoinositol turnover are similar in both MCF-7 and MCF7/LCC1 cells, but turnover is increased by estrogen only in MCF-7 cells. MCF7/MIII and MCF7/LCC1, but not MCF-7 cells, express a high baseline level of the estrogen-regulated pS2 mRNA. The baseline level of expression of progesterone receptor protein, but not mRNA, is higher in MCF7/LCC1 when compared with either MCF-7 or early passage MCF7/MIII cells. However, while the estrogen receptor is also an estrogen-regulated gene, MCF7/MIII and MCF7/LCC1 cells retain estrogen receptor levels equivalent to the parental MCF-7 cells. These data indicate that progression to hormone independence can occur without major gene amplifications or a high constitutive induction of phosphoinositide metabolism. Thus, DNA amplifications may be acquired during the early initiation and/or promotional events of carcinogenesis. Significantly, acquisition of a hormone-independent but responsive phenotype in human breast cancer is associated with perturbations in the expression of specific estrogen-regulated genes.

Anti-IgM-mediated growth inhibition of a human B lymphoma cell line is independent of phosphatidylinositol turnover and protein kinase C activation and involves tyrosine phosphorylation.

The RL cell line is an EBV-negative, surface IgM, IgD-positive B lymphoma line, which is significantly growth arrested in the presence of acrylamide-linked antibodies to the surface IgM receptor. We demonstrate here that activation of protein kinase C (PKC) with PMA abrogates anti-IgM-induced phosphoinositide turnover and Ca2+ mobilization; however, growth inhibition is not affected. In addition, inhibitors of PKC are unable to reverse the anti-IgM-mediated growth inhibition. Two-dimensional gel electrophoresis reveals a different pattern of protein phosphorylation after treatment of RL with PMA or anti-IgM. These data strongly suggest that anti-IgM-induced growth inhibition does not rely on phospholipase C-mediated phosphoinositide turnover, Ca2+ mobilization, or PKC activation. On the other hand, the phosphatase inhibitor orthovanadate results in an augmentation of proteins phosphorylated on tyrosine and the growth inhibition which follows anti-IgM treatment. Furthermore, protein tyrosine kinase inhibitors, genistein and herbimycin A, are able to reverse the anti-IgM-induced inhibition of growth. These data demonstrate that multiple signaling pathways are activated by the interaction of anti-IgM with its ligand, and suggest that tyrosine kinase activation is a critical component of the inhibitory response.

Acquired immunodeficiency syndrome-associated lymphomas.

Congenital and acquired states of immunodeficiency have long been associated with an increased incidence of malignant lymphoma. An increased incidence of non-Hodgkin's lymphomas was recognized early in the epidemic immunodeficiency state associated with the human immunodeficiency virus (HIV) infection AIDS. Although the precise etiologic mechanism of these lymphomas remains speculative, the presence of Epstein-Barr viral proteins or sequences and characteristic chromosomal translocations giving rise to altered expression of the c-myc oncogene have frequently been observed. It has been suggested that HIV infection leading to disordered T-lymphocyte function (possibly in conjunction with Epstein-Barr infection) leads to the emergence of polyclonal populations of stimulated B lymphocytes. These cells, which undergo physiologic immunoglobulin gene rearrangement, may provide the background for the occurrence of characteristic chromosomal translocations that lead to the emergence of malignant lymphomas. These lymphomas tend to present clinically with high-grade histopathologic subtype, advanced stage, and a propensity for the involvement of otherwise unusual extranodal sites, including the central nervous system. The experience with therapy for HIV-associated lymphomas has indicated that highly aggressive, dose-intensive chemotherapy regimens may be associated with inferior results. More recent regimens have stressed less myelosuppressive therapy combined with prophylaxis for central nervous system disease and pneumocystis infection. The dominant prognostic factors in the HIV-associated lymphomas appear to be primarily related to the underlying HIV infection and include total CD4 lymphocyte count, performance status, and prior AIDS diagnosis.

Phosphoinositide metabolism in human prostate cancer cells in vitro.

To understand better the mechanism by which 5-alpha-dihydrotestosterone (5-alpha-DHT) influences prostate epithelial cell function, we examined the effects of 5-alpha-DHT on phosphoinositide metabolism in human prostate cancer cell lines. Androgen receptor-positive LN-CaP cells showed dose-responsive, steady-state elevations in phosphoinositide metabolism when treated with 5-alpha-DHT. The intracellular pool of 3H-myoinositol decreased and the incorporation of 3H-myoinositol into cellular lipids increased with increasing concentrations of 5-alpha-DHT. 5-alpha-DHT increased the release of 3H-inositol phosphates into the media. The inactive stereoisomer, 5-beta-DHT, did not increase phosphoinositide metabolism. In androgen receptor-negative cells, phosphoinositide metabolism was not altered by 5-alpha-DHT. The slow induction of phosphoinositide metabolism by 5-alpha-DHT suggests that the effects may be mediated through other factors that serve as intermediates in 5-alpha-DHT modulation of intracellular signalling. We conclude that this modulation involves increased turnover of phosphatidylinositol, incorporation of myoinositol into cellular lipids, and alterations in the aqueous intracellular myoinositol pool size, possibly as a result of altered transport mechanisms.

Expression of transforming growth factor alpha and its messenger ribonucleic acid in human breast cancer: its regulation by estrogen and its possible functional significance.

We have studied the estrogenic regulation and the potential autocrine role of transforming growth factor alpha (TGF alpha) in the human breast cancer cell line MCF-7. A biologically active apparent mol wt 30 k TGF alpha was identified by gel filtration chromatography in medium conditioned by MCF-7 breast cancer cells. We previously reported induction of TGF alpha levels in medium by 17 beta-estradiol. We now report correlated increases in TGF alpha mRNA, by Northern and slot blot analysis, after estrogen treatment of MCF-7 cells in vitro. In vivo experiments confirmed these data: estrogen withdrawal from MCF-7 tumor-bearing nude mice resulted in a decline in tumor size and TGF alpha mRNA levels. To explore the functional significance of TGF alpha in MCF-7 cells, anti-TGF alpha antibody was added to MCF-7 soft agar cloning assays. Inhibition of MCF-7 growth resulted, supporting an autocrine role for TGF alpha. Further experiments using an anti-EGF receptor antibody expanded this data, demonstrating inhibition of estrogen-stimulated monolayer MCF-7 cell growth. Examining the generality of TGF alpha expression, 4.8 kilobase TGF alpha mRNAs were seen in three other human breast cancer cell lines, MDA-MB-231, ZR 75B, and T47D. Expression of TGF alpha mRNA was detected in 70% of estrogen receptor positive and negative primary human breast tumors from 40 patients when examined by slot blot and Northern analysis. Thus, we have demonstrated broad expression of TGF alpha in human breast cancer, its hormonal regulation in an estrogen-responsive cell line, and its possible functional significance in MCF-7 cell growth.