Expression of TGFα in Meningiomas

The objective of this study was to examine the expression of transforming growth factor (TGF), a mitogen for many cell types, and its receptor in basic subtypes of meningiomas as well as in meningiomas of varying grade. Formalin-fixed tissues from 26 meningiomas including 15 benign (5 meningothelial, 5 transitional, and 5 fibrous variants), 6 atypical, and 5 malignant examples were immunohistochemically examined for both TGF protein and EGF/TGF receptor protein. In addition, in situ hybridization (ISH) was used to detect TGF mRNA expression. Immunostaining for TGF was strongest in fibrous and atypical meningiomas, followed closely by transitional and malignant tumors. Only weak reactivity was observed in the meningothelial variant. In all but 4 tumors (2 fibrous, 2 atypical), ISH showed TGF mRNA to be present, the signal being stronger in malignant than in conventional or atypical tumors. Lastly, immunostaining for EGF/TGF receptor was positive in all tumors studied. Strong TGF protein expression in meningiomas is commonly associated with fibrous morphology. Although the frequent detection of both TGF protein and its mRNA, as well as of EGF/TGF receptor within tumors of all type and grades, suggests that TGF serves to promote tumor growth, its possible role in tumorigenesis or malignant progression is uncertain.In summary, demonstration of these substances is of no utility in the classification or grading of this common tumor because the differences in their expression among the various meningioma subtypes were not statistically significant.     

Epidermal growth factor-related peptides in the pathogenesis of human breast cancer

Summary A number of different epidermal growth factor (EGF)-related peptides such as EGF, transforming growth factor (TGF), amphiregulin (AR), heregulin (HRG), and cripto-1 (CR-1), are coexpressed to varying degrees in both normal and malignant mammary epithelial cells. However, in general the frequency and level of expression of TGF, AR, and CR-1 are higher in malignant breast epithelial cells than in normal mammary epithelium. In addition, several of these peptides such as TGF and AR can function as autocrine and/or juxtacrine growth factors in mammary epithelial cells, and their expression is stringently regulated by mammotrophic hormones such as estrogens, activated proto-oncogenes that have been implicated in the pathogenesis of breast cancer, and other growth factors. The redundancy of expression that is observed for a number of these structurally related peptides in both normal and malignant mammary epithelial cells suggests that some of these peptides may be involved in regulating other aspects of cellular behavior such as differentiation in addition to proliferation.     

Growth inhibitory effect of recombinant α and β interferon on human glioma cells

Summary Growth inhibitory activity of recombinant and interferon on two human glioma cell lines, EFC-2 and KE cells, was determined by two different growth assays. Recombinant interferon showed slight growth inhibitory effect on EFC-2 cells at day 3, and maximum inhibition was seen on day 6 with an ID50 of 50 U/ml. Recombinant interferon showed no significant growth inhibition at any concentration. KE cells were resistant to both recombinant and interferon. The growth inhibitory activity of recombinant interferon on EFC-2 cells was not blocked by recombinant interferon, although recombinant and interferons shared same receptors on EFC-2 cells. Addition of DFMO (-difluoromethylornithine) to interferon in the media showed additive effect rather than synergistic effect in growth inhibition of glioma cells. Out of 7 glioma cell lines tested, 4 showed heterogeneous sensitivity to recombinant interferon, and all were resistant to recombinant interferon. These results suggest differential sensitivity of EFC-2 cells to recombinant interferon, as well as a heterogeneous sensitivity to recombinant interferon among different glioma cell lines.     

Induction of matrix metalloproteinases MMP-1 and MMP-2 by co-culture of breast cancer cells and bone marrow fibroblasts

Two invasive breast cancer cell lines (MDA-MB-231 and BT-549) were found to be more adherent and have greater migratory capacity on bone marrow fibroblasts than three non-invasive cell lines (MCF-7, T47D and BT-483). Antibodies to the adhesion molecules CD44, E-cadherin, ICAM-1, and integrin chains 2, 3, 4, 5, 6, v, 1, 3 and 7 failed to inhibit breast cancer cell migration through bone marrow fibroblasts. Inhibitors of matrix metalloproteases, 1, 10-phenanthroline, Ro-9790, TIMP-1 and TIMP-2 were able to attenuate the migration of MDA-MB-231 cells through bone marrow fibroblast monolayers suggesting a role for these enzymes in the migration of breast cancer cells through bone marrow adherent layers. Co-culture of MDA-MB-231 cells and bone marrow fibroblasts resulted in augmentation of the levels of the matrix metalloproteases MMP-1 and MMP-2 in culture supernatants. Soluble factors produced by bone marrow fibroblasts were responsible for the increase in MMP-1 levels. However, maximal MMP-2 production was dependent on direct contract between the breast cancer cells and the bone marrow fibroblasts. Modulation of MMP production by cell–cell contact or soluble factors suggests a mechanism by which breast cancer cells can enhance their ability to invade the bone marrow microenvironment.     

Immunological detection and quantitation of alpha transforming growth factors in human breast carcinoma cells

Summary Alpha transforming growth factors (TGFs) were immunologically detected in the concentrated conditioned medium (CM) prepared from four human breast cancer cell lines and from primary cultures of human mammary epithelial cells, and in the tissue extracts prepared from normal, benign, and malignant breast biopsies. Immunoreactive TGFs were quantitated by a competitive radioimmunoassay (RIA) using affinity-purified polyclonal sheep anti-rat TGF antibodies which react with human TGF but not with human epidermal growth factor (EGF). The relative level of RIA-detectable TGFs in the CM from the breast cancer cell lines MCF-7, ZR-75-1, T47-D, and MDA-MB-231, and from the CM of primary cultures of human mammary epithelial cells, ranged from 0.02 to 0.85 ng/ml. MCF-7 or ZR-75-1 cells grown in the presence of 17-estradiol (10^–8 M) for 48 h were found to release two- to three-fold more TGFs into their CM than the same cells grown in the absence of estrogen. In detergent extracts prepared from normal breast tissue, a benign fibrocystic lesion, fibroadenomas and primary breast carcinomas, the relative TGF concentrations were found to range from 1.5 to 6 ng/mg cell protein. No significant correlations were found between the TGF levels and the pathological state of the tissues, the estrogen receptor status of the tumors, or the relative amounts of theras gene protein p21ras in the tissues as determined by Western immunoblot analysis. The question of biological relevancy of TGF for human mammary tumors will require further studies on (a) synthesis and turnover of TGF, (b) the relationship between immunoreactivity and biological activity of TGF, and (c) differences in biological responsiveness of mammary tumor cells.     

Transforming growth factor b as a potential tumor progression factor among hyperdiploid glioblastoma cultures: Evidence for the role of platelet-derived growth factor

Among early-passage, near-diploid gliomas in vitro, transforming growth factor type (TGF) has been previously shown to be an autocrine growth inhibitor. In contrast, hyperdiploid ( 57chromosomes/metaphase) glioblastoma multiforme (HD-GM) cultures were autocrinely stimulated by the TGF. The mechanism of this conversion from autocrine inhibitor to mitogen is not understood; previous studies have suggested that platelet-derived growth factor (PDGF) might be modulated by TGF. The similar expression of TGF types 1—3, PDGF-AA, — BB, as well as the PDGF receptor and subunits (a/PDGFR) between biopsies of the HD-GM and near-diploid, TGF-inhibited glioblastomas (GM) by immunohistochemistry did not explain the discrepancy in their regulatory responses. Flowcytometry demonstrated that TGF's mitogenic effect was selective for the aneuploid subpopulations of two of three selected HD-GM cultures,while the diploid cells were inhibited. Among the HD-GM, TGF1 induced the RNA of PDGF-A, c-sis and TGF1. The amount of PDGF-AA secreted following TGF treatment was sufficient to stimulate the proliferation of a HD-GM culture. Antibodies against PDGF-AA, -BB, -AB,PDGFR and/or PDGFR subunits effectively neutralized TGF's induction of DNA synthesis among the HD-GM cell lines, indicating that PDGF served as the principal mediator of TGF's growth stimulatory effect. By comparison, TGF induced only the RNA of PDGF-A and TGF1 among the near-diploid GM; c-sis was not expressed at all. However, the amount of PDGF-A which was secreted in response to TGF1 was insufficient to prevent TGF's arrest of the near-diploid cultures in G₁ phase. Thus, the emergence of hyperdiploidy was associated with qualitative and quantitative differences in TGF's modulation of PDGF-A and c-sis, which provided a mechanism by which the aneuploid glioma cellsmight achieve clonal dominance. We hypothesize that TGF may serve as an autocrine promoter of GM progression by providing a selective advantage to the hyperdiploid subpopulation through the loss of a tumor suppressor gene which mediates TGF's inhibitory effect.     

The α6β1 and α6β4 integrins in human prostate cancer progression

Summary Prostatic secretions are formed by glands composed of basal and luminal cells and surrounded by a basal lamina. The normal basal cells express several integrins (extracellular matrix receptors) including alpha 2, 3, 4, 5, 6, v, beta 1 and beta 4. These integrin units are polarized at the base of the cells adjacent to the basal lamina. The integrin alpha 6 beta 4 is associated with hemidesmosomal-like structures.The natural history of prostate cancer involves the presence of prostatic intraepithelial neoplasia (PIN) lesions (considered precursor lesions), carcinomain situ and invasive carcinoma. Hemidesmosomal proteins and the 31 and 61 integrins (laminin receptors) are retained in the early PIN lesions. Expression of the integrins 2, 4, 5, v and 4 is lost in carcinoma. The 31 and 61 integrins remain associated with invasive carcinoma, the latter being predominant. Integrin expression in carcinoma is diffuse in the plasma membrane and not restricted to the basal aspects of the cell. The 61 integrin is fully functional as judged by an ability to adhere to laminin and contains the wild type 6A cytoplasmic signaling domain. The 61 integrin is a leading candidate for conferring the invasive phenotype in prostatic carcinoma.Tumor cells with high expression of 6 integrin are more invasive when tested in a SCID mouse model system. Following intraperitoneal injection, the human tumor cells invade the mouse diaphragm and move through the muscle on the surface of the laminin coated muscle cells. Our current working hypothesis is that the production of 61 and laminin in human tumor cells contributes to the invasive phenotype. Invasion could occur on the surfaces of laminin coated structures such as the nerves, blood vessels or muscle and account for the known patterns of human prostate tumor progression. Blockage of the expression or function of 61 or laminin or preventing the loss of 4 would be essential steps in confining the carcinoma to the prostate gland where conventional treatment has already proven effective.     

Partial characterization of glioma-derived growth factor 2: A novel mitogenic activity from human cell line D-54 MG

Summary We have shown that several human malignant glioma cell lines are stimulated by bacterial lipopolysaccharide (E. coli 0111B4, 1 g/ml) to produce a high molecular weight (> 200 kD) growth activity for BALB 3T3, clone A31 cells [1, 2]. This glioma-derived growth factor (GDGF-2) acts like a competence factor. Malignant glioma cell line D-54 MG constitutively produced GDGF-2, which we have partially characterized from serumfree conditioned culture medium. GDGF-2 is resistant to heat (100° C, 5 min), acidic (pH 2, 2 hr) or reducing (0.5 M 2 ME, 30 min) conditions as well as exposure to RNases; however, it is sensitive to > 4 freeze-thaw cycles, alkaline (pH 11, 2 hr) conditions or pre-treatment with proteolytic enzymes. GDGF-2 had a pl of 6.8 determined by preparative isoelectric focusing, bound to DEAE, with elution at 35 and 185 mM NaCl and at 43% acetonitrile from a C4 reversed phase column. GDGF-2 activity was not neutralized by antibodies to TGF, TGF, PDGF, VEGF or TNF indicating that it is not immunochemically related to these growth factors. However GDGF-2 co-chromatographed on Superose 12 HPLC (250 × 9 mm; 5% isopropanol, 6 mM CHAPS in PBS) with a substance that suppressed growth of mink lung epithelial cells (Mv1Lu), but not BALB 3T3 cells, and could be neutralized by anti-TGF antibodies. GDGF-2 activity eluted from heparin columns in 0.6 M NaCl; thus, it is not a heparin binding growth factor. D-54 MG cell line produced alpha₂-macroglobulin (₂M), which is known to bind TGF; however, immunoprecipitation of ₂M did not deplete TGF or GDGF-2 activity. Further, neither GDGF-2 or TGF can be dissociated into lower molecular weight active components by chromatography in high salt (2 M NaCl) or 2-ME (0.5 M). GDGF-2 may be a novel autocrine or paracrine mitogen, stimulating mitotic division or interfering with normal cell growth regulation.     

Cytokine‐regulated urokinase‐type‐plasminogen‐activator (uPA) production by human breast fibroblasts in vitro

It has been shown that, in breast stroma, urokinasetype plasminogen activator (uPA) mRNA is predominantly expressed by myofibroblasts located at the invasive areas of the tumor. To examine which factors present in a tumor environment are candidates responsible for the induction of these uPAproducing myofibroblasts, we studied in vitro the capacity of a paired panel of normal and tumorderived human breast fibroblasts to produce uPA protein and the myofibroblast marker smoothmuscleactin (SMA) in response to various cytokines implicated in the process of tissueremodeling during malignant transformation.We found that fibroblasts produced increased amounts of uPA protein after exposure to aFGF, bFGF, EGF, PDGFBB, and IFN, were unaffected in this respect by IL6, MCSF, GMCSF and Oncostatin M, and produced decreased amounts of uPA protein after exposure to IL1, TNF, IGFI, and IGFII. None of these cytokines were able to induce a striking increase in the fraction of SMApositive fibroblasts. On the other hand, 25pM TGF₁ increased the fraction of SMApositive fibroblasts 5fold in both normal and tumortissuederived fibroblasts. Nonetheless, the normalderived fibroblasts were unaffected in their uPAproducing capacity by TGF₁, and the tumorderived fibroblasts produced decreased amounts of uPA protein after exposure to this cytokine, implying that at least in vitro the myofibroblast phenotype is not a prerequisite for the production of uPA by human breast fibroblasts. In addition, we established that the basaluPAproduction of both normal and tumorderived fibroblasts was increased by autocrinely produced bFGFlike activity, and that the basaluPAproduction of at least the normalderived fibroblasts was decreased by autocrinely produced IGFlike activity.Altogether, our data suggest an active role for fibroblasts in the process of uPAdirected breast tumor proteolysis.     

Comparison of topoisomerase-IIalpha gene status between primary breast cancer and corresponding distant metastatic sites

Background. Topoisomerase-II (topo-II) is a key enzyme in DNA replication and a molecular target for anti-cancer drugs called topoisomerase-II inhibitors, such as anthracyclines. Its value as a predictive marker of responsiveness to these cytotoxic drugs is currently being evaluated with promising results. However, even in the metastatic setting, the choice of treatment is based on the biologic characteristics of the primary tumor. Few data are available regarding the expression of biological markers between the primary tumor and the corresponding distant metastases. Methods. Topo-II gene status was evaluated in 29 breast cancer patients in which a primary tumor sample and a corresponding metastatic sample were both available. Fluorescent in situ hybridization (FISH) with the Vysis triple probe (Vysis multi-color topo-II spectrum orange, Her-2 spectrum green and CEP17 spectrum aqua probe) was used, which allowed the concomitant evaluation of HER-2 gene status. Results. As previously reported, topo-II gene aberrations are always associated with HER-2 gene amplification; indeed no topo-II gene aberrations have been observed in the HER-2 negative tumors. Conversely, 38.5% (five patients) of the HER-2 positive primary breast tumors (13 patients) were topo-II amplified, while 61.5% (eight patients) had a normal topo-II gene. No topo-II gene deletion was found in our series. Topo-II gene amplification in the primary tumor was always associated with amplification in the corresponding metastases, and no metastases with topo-II gene amplification were seen without amplification in the primary tumor. Furthermore, the amplification level of topo-II (i.e., ratio topo-II: CEP17) remained unchanged in primary and metastatic sites. Conclusion. Despite the low number of patients, our results seem to indicate that topo-II gene status evaluation in the primary breast tumor accurately reflects its status in the corresponding distant metastases.     

Acute effects of human recombinant tumor necrosis factor-α on the cerebral vasculature of the rat in both normal brain and in an experimental glioma model

Summary The effects of intravenous (IV) infusion of human recombinant tumor necrosis factor- (rTNF-, Cetus) on normal brain and malignant glioma in rats were examined. Twelve Fischer 344 rats were given either a single injection of 10⁶U rTNF- or injections of 5 × 10⁵U rTNF- for three days. One day post-rTNF- injection (s), rats were injected IV with horseradish peroxidase (HRP) to determine blood-brain barrier (BBB) breakdown and, one hour later, were perfused with an aldehyde fixative and processed for histologic examination. Treatment of normal rats with rTNF- by either dosage or schedule caused no remarkable histopathologic changes in the brain and no alteration in BBB integrity. Human glioma models were produced by intracerebal inoculation of 10⁴ syngeneic RT-2 glioma cells into the right parietal lobe of 30 rats. Animals received single IV injections of 10⁶ U human rTNF- or its excipient (TNF-E) as above on day 3, 7, or 10 post-tumor inoculation or multiple injections of 5 × 10⁵U rTNF- beginning on day 7, 10, or 12 post-tumor inoculation. With a single IV injection of either rTNF- or its excipient, 3-day models showed a similar pattern of HRP extravasation limited to the extracellular space of the tumor inoculation site. In 7-day models treated with a single IV injection of rTNF- or TNF-E, HRP extravasated throughout the tumor, but did not exceed peritumoral margins. In 10-day models treated with a single injection of TNF-E, HRP was found only in the tumor and immediate peritumoral regions while rTNF--treated rats showed more extensive areas of BBB breakdown with HRP evident throughout the entire right hemisphere and extending via the corpus callosum into the contralateral hemisphere. Pericapillary halos were also evident around the small blood vessels within the edematous areas of the corpus callosum. Within tumors, hemorrhagic necrosis and adherence of neutrophils to vessels was observed only in animals treated with rTNF- at 10 days post-tumor inoculation. Multiple IV injections of rTNF- in 7 and 10-day models triggered widespread hemorrhagic necrosis, neutrophil adherence and infiltration in the tumor. There was also extravasation and diffusion of HRP from the site of glioma into the contralateral hemisphere. Twelve-day models treated with multiple rTNF- injections, in addition, showed irregular luminal surfaces and gaps between adjacent endothelial cells of tumor vasculature. These results, which demonstrate that rTNF- can be administered IV in dosages that create widespread necrosis within the glioma but little or no damage to normal brain, suggest that treatment with rTNF- preferentially affects newly-formed vasculature of the tumor and has little effect on the integrity of normal cerebral vessels.     

Synergistic enhancement by interleukin-1 α of cisplatin-mediated antitumor activity in RIF-1 tumor-bearing C3H/HeJ mice

Administration of interleukin-1 (IL-1 ) plus certain cytotoxic drugs causes substantially greater clonogenic tumor-cell kill and tumor-regrowth delay than does treatment with either agent alone. IL-1 itself has little effect on tumor growth despite its ability to induce acute hemorrhagic necrosis, restrict tumor blood flow, and cause microvascular injury in a variety of murine model systems. To investigate further IL-1 's ability to enhance the antitumor activity of cytotoxic drugs, we initiated studies to examine the effect of IL-1 on cisplatin (cDDP)-mediated cytotoxicity using the RIF-1 tumor system. The antitumor activity of IL-1 and cDDP was quantitated through standard clonogenic tumor-cell survival assays, a tumor hemorrhagic necrosis assay and tumor-regrowth delay studies, with the interaction between IL-1 and cDDP being analyzed through median dose-effect. In vitro, IL-1 had no enhancing effect on the cDDP-mediated tumorcell kill. For examination of the in vivo efficacy of this regimen. RIF-1 tumor-bearing C3H/HeJ mice (14 days postimplantation) were treated concurrently with single i.p. injections of IL-1 and/or cDDP at various doses. The increased clonogenic tumor-cell kill obtained with IL-1 /cDDP was dose-dependent, with significant enhancement by IL-1 being observed (P<0.001), even at the lowest doses tested (2 mg/kg and 6 g/kg for cDDP and IL-1 , respectively), but it did not correlate with an increase in tumor hemorrhage. Using median dose-effect analysis, this interaction was determined to be strongly synergistic. When treated animals were monitored for long-term antitumor effects, combinations with IL-1 significantly increased the tumor-regrowth delay and decreased the fractional tumor volume (P<0.001). These results demonstrate that IL-1 synergistically enhances cDDP mediated in vivo antitumor activity and suggest that the combination of IL-1 and cDDP may have potential therapeutic application in the design of effective treatment modalities for cancer.Abbreviations IL-1 interleukin-1 - cDDP cis-diamminedichloroplatinum (cisplatin) - BRMs biological response modifiers - TNF tumor necrosis factor - IFN interferon - Fa traction affected - D_m or ED₅₀ drug concentration necessary to produce Fa=0.5 as compared with untreated controls - CI combination index; SF, surviving fraction - ANOVA oneway analysis of variance This work was supported in part by Public Health Service grant CA-48077 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services, by the Mary Hillman Jennings Foundation, and by the Ramona DeSantis Cancer Research Fund     

Selective inhibition of cell proliferation and DNA synthesis by the polysulphated carbohydrate ι-carrageenan

-Carrageenan is a polysulphated carbohydrate that antagonises some heparin-binding growth factors. We assessed the effect of -carrageenan on the proliferation of a panel of cell lines, some of which require heparin-binding growth factors for mitogenesis. The importance of growth factor antagonism for the antiproliferative activity was also determined. Cell proliferation was determined by cell counts and a tetrazolium dye (MTT) assay, and DNA synthesis was determined by thymidine incorporation. The proliferation of the basic fibroblast growth factor (bFGF)-dependent endothelial cell line FBHE was inhibited by daily administration of -carrageenan in a dose-dependent manner [concentration inhibiting cell growth by 50% (IC₅₀ value), approx. 0.5 g/ml]. However, excess bFGF did not reverse the inhibitory effect. DNA synthesis was completely inhibited by concentrations of -carrageenan that nonetheless allowed significant protein synthesis to occur. The proliferation of the androgen-dependent prostate-carcinoma cell line LNCaP was also inhibited by -carrageenan (IC₅₀ value, 5.5 g/ml) and the cells were arrested at the G1/S boundary. -Carrageenan inhibited DNA synthesis in MCF-7 cells stimulated by bFGF and transforming growth factor (TGF) but not in those stimulated by insulin-like growth factor 1 (IGF-1). Blocking IGF-1-mediated DNA synthesis with anti-IGF-1 receptor antibody IR3 enhanced the inhibitory activity of -carrageenan against MCF-7 cells grown in serum. A number of other transformed and non-transformed cell lines were either partially inhibited or not inhibited by -carrageenan. -Carrageenan had low anti-coagulant activity. -Carrageenan is a selective anti-proliferative agent and warrants further investigation for anti-angiogenic therapy (in view of its activity against endothelial cells) and for the treatment of androgen-dependent prostate cancer.     

Pharmacokinetics of an extended-release human interferon alpha-2b formulation

The in vivo half-life of human interferon alpha-2b (hIFN--2b) is relatively short, and frequent injections over prolonged periods are required for efficacy. An extended-release formulation of hIFN--2b (Depo/IFN) was created by encapsulation into a lipid-based drug-delivery system. The capture efficiency was 51%±13% and the release half-life in human plasma at 37°C was 16 days. The pharmacokinetics of Depo/IFN was compared with that of unencapsulated standard hIFN--2b (Std/IFN) in the peritoneal cavity of male BDF₁ mice. Depo/IFN exhibited a 13-fold longer intraperitoneal (i.p.) half-life as compared with Std/IFN (20 vs 1.5 h). The release of free hIFN--2b from Depo/IFN into the peritoneal cavity was slow and protracted, with a 10-fold lower peak concentration and a 13-fold longer apparent half-life being observed in comparison with Std/IFN. The areas under the curve of free hIFN--2b in the peritoneal cavity were comparable for Depo/IFN and Std/IFN. hIFN--2b was detectable in plasma only after the i.p. administration of Std/IFN. These data suggest the possibility that Depo/IFN may be useful as an extended-release formulation of hIFN--2b.     

Alpha-interferon in combination with 5-fluorouracil and leucovorin in metastatic colorectal cancer: a phase I study

Summary A high rate of response to 5-fluorouracil (5FU) and alpha-interferon (IFN) combination therapy has been reported in metastatic colorectal cancer patients. There-fore, designed a trial of high-dose continuous-infusion 5FU, oral leucovorin (LV), and IFN in this group of patients. Because this combination has not previously been tested and severe toxicity has been reported for 5FU and IFN combination therapy, we conducted a phase I trial in which 11 patients presenting with previously untreated metastatic colorectal cancer were treated with escalating doses of IFN together with fixed doses of 5FU and LV. WHO grade III toxicity consisting mainly of oral mucositis was noted in four patients. No grade IV toxicity occurred. Although IFN may enhance the toxicity of 5FU, the toxicity of this regimen remained manageable. Three partial responses were noted.     

Quantification of thrombospondin-1 secretion and expression of αvβ3 and α3β1 integrins and syndecan-1 as cell-surface receptors for thrombospondin-1 in malignant glioma cells

Malignant glioma cells secrete thrombospondin-1 (TSP-1) which participates in the motility of glioma cells, and binds to cell surface v3 and 31 integrins, and syndecan-1. This study evaluated the amount of TSP-1 secretion from malignant glioma cells, and the expression of v3 and 31 integrins, and syndecan-1. The amounts of TSP-1 in the supernatants from 10 malignant glioma cell lines and eight non-glioma malignant tumor cell lines were measured by enzyme-linked immunosorbent assay. Expression of v3 and 31 integrins, and syndecan-1 were examined by flow cytometry. The amounts of TSP-1 secreted by malignant glioma cells were 43 to 2431 ng/1 × 10⁶ cells/24 h (mean ± SD=626 ± 792). Seven of 10 glioma cell lines secreted more than 100 ng of TSP-1 and three of these cell lines secreted more than 1 g. Seven of eight non-glioma cell lines secreted less than 100 0ng of TSP-1. All glioma cell lines expressed 31 integrin and syndecan-1, and seven of 10 glioma cell lines expressed v3 integrin. Treatment of the glioma cell lines with TGF-2 did not change the expression of v3 integrin. These results suggest that malignant glioma cells secrete high levels of TSP-1, which may be important in the migration of glioma cells via interactions with v3 and 31 integrins, and syndecan-1.     

Conjugation of interferon alpha to a humanized monoclonal antibody (HuBrE-3vl) enhances the selective localization and antitumor effects of interferon in breast cancer xenografts

Human mammary carcinoma xenografts (MCF-7) growing in nude mice were treated with natural interferon (n-IFN-) alone or conjugated to a humanized monoclonal antibody (MoAb) anti-breast mucin (HuBrE-3vl) or to irrelevant human IgG₁. The IFN and the conjugates were administered as 20 intra-lesional (i.l.) injections to 1 of 2 xenografts in each mouse, or i.p. The growth inhibitory effects of HuBrE-3vl/nIFN- were significantly greater than those of nIFN- used as a single agent or conjugated to HuIgG₁. These effects occurred locally in the tumors receiving i.l. injections and systemically, although to a slightly lesser extent, in the noninjected tumors of mice treated i.l. and in the xenografts of mice treated i.p. Biodistribution studies showed that the uptake of ¹²⁵I-HuBrE-3vl/nIFN- by the tumors 24 hours after i.l. or s.c. injection was greater than that of ¹²⁵I-HuIgG₁/nIFN-,¹²⁵ I-nIFN- alone, or by normal tissues, documenting a tumor targeting effect and favorable tumor:normal tissues (T:NT) ratios. The targeting effects and the resulting tumor growth inhibition were favored by the IFN-mediated up-regulation of the HuBrE-3vl reactive antigen, which was more prominent after 3 weeks of treatment with HuBrE-3vl/nIFN-. These results were superior to those we obtained previously with nIFN- conjugated to another MoAb of the same group (Mc5). These studies point out the potential usefulness of HuBrE-3vl/nIFN- for the local and systemic treatment of breast cancer lesions by providing a means of delivering high doses of IFN to the tumors while minimizing the amount of IFN binding to normal tissues.     

Endogenous ligands for the epidermal growth factor receptor in human breast tumors do not interfere in an epidermal growth factor receptor radioligand binding assay

Summary Ligands for the epidermal growth factor receptor (EGFR) (e.g. EGF and transforming growth factor alpha, TGF) may be included in membrane preparations from human breast tumors, either complexed or not to EGFR. Assessment of EGFR in human placental membranes (HPM) after preincubation with human EGF (hEGF) or human TGF (hTGF) indicated that the presence of these ligands in a membrane preparation did not affect the apparent number of binding sites, but only resulted in an increased apparent dissociation constant (Kd).To obviate the effect of endogenously bound EGFR ligands in a radioligand binding assay, an acid treatment procedure has been used recently. In the present study we show that acid treatment of mammary tumor membranes does not result in increased EGFR levels but does result in an EGFR enriched membrane preparation by elimination of contaminating cytosol proteins. This however also occurred by washing the membranes with assay buffer at neutral pH.From these experiments we conclude that endogenous ligands, if present in membrane fractions from human breast tumors and occupying EGFR, do not interfere in a multipoint radioligand binding assay.Abbreviations EGF Epidermal Growth Factor - TGF Transforming Growth Factor alpha - EGFR Epidermal Growth Factor Receptor - HAP Hydroxylapatite     

Antitumor activity of homo-aza-steroidal esters of [p-[bis(2-chloroethyl)amino]phenyl]acetic acid and [p-[bis(2-chloroethyl)amino]phenyl]butyric acid

Summary Three new modified steroidal alkylating agents, 3-hydroxy-13-amino-13,17-seco-5-androstan-17-oic-13,17-lactam-p-bis(2-chloroethyl)aminophenylacetate, 3-hydroxy-13-amino-13,17-seco-5-androstan-17-oic-13,17-lactam-p-bis-(2-chloroethyl)aminophenylbutyrate, and 17-hydroxy-3-aza-A-homo-4-androsten-4-one-p-N,N-bis(2-chloroethyl)-aminophenylacetate are active in treatment of L1210 and P388 leukemias. A stereoisomer of the first compound, 3-hydroxy-13-amino-13,17-seco-5-androstan-17-oic-13, 17-lactam-p-bis(2-chloroethyl)aminophenylacetate, was tested in L1210 leukemia. This stereoisomer, in which the alkylating agent is linked to the modified steroid in the axial position, is active only at much higher doses in L1210 leukemia. The results of testing these compounds and previous results from similar compounds allow certain conclusions to be drawn regarding structure-activity relationships. The presence of the lactam moiety is the major structural feature that confers activity in the murine leukemias. The steric arrangement of the alkylating moiety at position 3 and the hydrogen atom at position 5 influence toxicity and antileukemic activity.     

Response of multicellular tumor spheroids to liposomes containing TNF-α

Summary This publication describes a new model to investigate the influence of tumor necrosis factor- (TNF-) on a three-dimensional glial cell aggregate under defined, standardized, reproducible conditions using the glioma cell line A 172.The cells are initially grown as normal monolayer culture until they reach a cell density of up to 1×10⁶. Subsequently they are grown as spheroids by the liquid overlay technique. Spheroids grown in this way were divided into ten groups of more than 50 cell aggregates. Three groups were coincubated with free TNF- in increasing dosages (100 ng/ml, 200 ng/ml and 1000 ng/ml); three groups were incubated with empty liposomes (0.2 mg/ml, 0.4 mg/ml and 2 mg/ml); three groups received liposomes which had been loaded with TNF-, and one group, which received no treatment, served as control.The diameter of the spheroids ranged from 80 m to 350 m. There was no significant difference in growth between the 3 groups treated with free TNF-. Comparing spheroids treated with TNF- with those which had been coincubated with empty liposomes, there was a significant difference (p<0.001) in growth, which correlated with the amount of liposomes. Similarly, free TNF- had a significantly (P<0.001) stronger growth-inhibiting effect as compared to liposomes loaded with TNF-. Comparing the groups treated with liposomes only to those treated with liposomes loaded with TNF-, the latter exhibited a more marked (although not significantly) growth-inhibiting effect.The preliminary conclusion is that the major growth-inhibiting effect seems to be mediated by the liposomes. This phenomenon is in agreement with results obtained in monolayer cultures.