Molecular Cloning of the Rat IL-13 Alpha 2 Receptor CDNA and Its Expression in Rat Tissues

The interleukin 13 alpha 2 receptor (IL-13R2) is highly expressed in human glioma cells. As a consequence this receptor has been proposed as a potential target for immunotherapeutic approaches for treating brain tumors. In developing animal models that may utilize the IL-13R2 receptor as an immunotherapeutic target, only the murine gene sequence has thus far been elucidated. The purpose of the present study, therefore, was to determine the gene sequence and tissue distribution of IL-13R2 in the rat. A search of the NCBI expressed sequence tag (EST) database with human and mouse IL-13R2 gene sequences identified a rat EST with high homology to the human and mouse IL-13R2 conserved region. Based on the sequence information, a 1917 bp rat IL-13R2 cDNA was cloned using the 5 and 3 RACE PCR technique. The cloned rat IL-13R2 cDNA contains a full-length 1158 bp open reading frame. The deduced protein is 91.2% and 54.2% homologous to mouse and human IL-13R2, respectively, at the amino acid level. Analysis shows that the rat IL-13R2 is structurally conserved and similar to human and mouse. It has a very short cytoplasmic domain, an extracellular domain containing an N-terminal fibronectin type III domain, four putative N-glycosylation sites, and a growth factor and cytokine receptor family motif WSEWS. Using RT-PCR techniques, the mRNA of rat IL-13R2 was detected in rat brain, spleen, liver, thymus, stomach, testis, and three rat glioblastoma cell lines C6, A15A5 and 9L. The cloning of rat IL-13R2 may be helpful to establish a rat model for IL-13R2 related glioma therapies.     

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.     

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.     

Inhibition of hormone-dependent and independent breast cancer cell growthin vivo andin vitro with the antiestrogen toremifene and recombinant human interferon-α2

The antiproliferative action of the antiestrogen toremifene and recombinant human interferon-2a (IFN-2a) were examined on human breast cancer cell lines grown in culture and in the athymic mouse.Solid tumors grew from an inoculation of a 99:1 ratio of hormone dependent (MCF-7) and hormone independent (MDA-MB-231) breast cancer cells without estrogen administration. However, estradiol supplementation significantly increased the rate of tumor growth. The daily administration of 1.35 × 10⁶ U of recombinant human IFN-2a resulted in a marked reduction of tumor growth in both estradiol-treated and non-treated mice. Toremifene administration (130 µg/day from a sustained release preparation) markedly inhibited estradiol stimulation of mouse uterine weight and partially reduced estradiol-stimulated tumor growth. The combination of IFN-2a (1.35 × 10⁶ u/day) with toremifene (130 µg/day) reduced estradiol-stimulated growth much below that of toremifene alone but not below that seen with interferon alone.Toremifene (10^–10-10^–6M) did not inhibit the growth of hormone-independent MDA-MB-231 breast cancer cellsin vitro whereas it did inhibit the growth of hormone-dependent MCF-7 cells in phenol red containing media. IFN-2a (1–10,000 u) inhibited the growth of both MCF-7 and MDA-MB-231 cells in culture; however, MCF-7 cells were approximately 10-fold more sensitive to interferon inhibition. This was consistent with the MCF-7 cells showing a greater sensitivity to interferon than MDA-MB-231 cells in the induction of 25-oligoadenylate synthetase.The heterogeneous tumor model in the athymic mouse suggests that differential sensitivities of breast cancer cells to the antiproliferative actions of interferon may influence the effectiveness of combination therapies.     

Experimental down-regulation of intermediate biomarkers of carcinogenesis in mouse mammary epithelial cells

Summary The polycyclic aromatic hydrocarbon 7,12-dimethylbenz(a)anthracene (DMBA) is a metabolism-dependent procarcinogen whose tumorigenicity is modified by dietary and endocrine manipulationsin vivo. DMBA initiates molecular and cellular alterations in the mammary tissue, while dietary components and estrogens affect the post-initiational phase of tumorigenic transformation. The mechanism(s) responsible for modulation of tumorigenic transformation remain unclear. This study examines the effects of selected tumor suppressing agents and estradiol (E₂) metabolites onin vitro DMBA carcinogenesis utilizing a newly established mouse mammary epithelial cell line C57/MG. Alteration in DNA repair synthesis, metabolism of E₂ via the C2- and C16-hydroxylation pathways, and acquisition of anchorage-independent growth were utilized as molecular, endocrine, and cellular biomarkers to quantitate the cellular transformation by DMBA and its modulation by tumor suppressing agents and E₂ metabolites. A single 24 hr exposure of 0.78 µM DMBA to C57/MG cells resulted in a 193.9% increase in DNA repair synthesis and a 73.1% decrease in C2/C16 hydroxylation of E₂. The DMBA treated C57/MG cells also exhibited increased anchorage-independencein vitro prior to tumorigenesisin vivo. A simultaneous treatment of cells with DMBA and with the highest non-cytotoxic doses of the tumor suppressing agents 5 µM N-(4-hydroxyphenyl) retinamide (HPR), 50 µM indole-3-carbinol (I3C), or 1 µM tamoxifen (TAM) resulted in a 35.6% to 63.9% decrease in DNA repair synthesis, a 23.8% to 1347.6% increase in C2/C16 hydroxylation of E₂, and a 53.8% to 72.4% decrease in anchorage-independent growth. The E₂ metabolites at the highest non-cytotoxic doses of 0.76 µM estrone (E₁), 0.69 µM 2-hydroxyestrone (2-OHE₁), and 0.66 µM 2-methoxyestrone (2-MeOHE₁) suppressed DMBA-induced DNA repair synthesis by 56.0% to 68.8%. These tumor suppressing agents and E₂ metabolites also effectively suppressed post-initiational, anchorage-independent growth by 24.9% to 72.4%. These results indicate that DMBA induces cellular transformation in part by causing DNA damage, altering C2/C16 hydroxylation in favor of C16-hydroxylation, and inducing anchorage-independent growth prior to tumor development. Effective downregulation of these genotoxic, endocrine and proliferative end points by prototypic tumor suppressing agents and by E₂ metabolites generated via the C2-hydroxylation pathway suggest that these agents may influence mammary tumorigenesis by inhibiting early occurring initiational and/or post initiational events.Abbreviations DMBA 7,12-dimethylbenz(a)anthracene - HPR N-(4-hydroxyphenyl) retinamide - I3C indole-3-carbinol - TAM tamoxifen - E₂ 17-estradiol - E₁ estrone - 2-OHE₁ 2-hydroxyestrone - 2-MeOHE₁ 2-methoxyestrone - 16-OHE₁ 16-hydroxyestrone - E₃ estriol - DME/F12 Dulbecco's modified Eagle's medium - F12 Ham's medium - HU hydroxyurea - PBS phosphate buffered saline - NaOH sodium hydroxide - SDS sodium dodecyl sulfate - TCA trichloroacetic acid - [C2-³H] E₂ estradiol labeled at C2 position - [C16-³H] E₂ estradiol labeled at C16 position - ANOVA analysis of variance     

In vivo effects in mice produced by a 3′-branched homologue of α-2′-deoxythioguanosine

Summary Studies with a 3-branched chain homolog (-3-BCTGdR) of 2-deoxythioguanosine (-TGdR) showed that it did not prolong the survival of mice bearing the Mecca lymphosarcoma. Host toxicity was quite profound and resembled that seen with 6-thioguanine (6-TG). Evidence was obtained that this nucleoside derivative was not appreciably converted to 6-TG in the mouse. Mice treated with toxic doses of 6-TG or -3-BCTGdR were found to have very similar pathological changes. The granulocytes were eliminated from the peripheral blood, bone marrow was acellular, and some more limited damage was seen in the intestinal crypts. Experiments with radiosulfur-labeled drugs demonstrated that -3-BCTGdR was incorporated into the DNA of mouse bone marrow, predominantly in the chain-terminating position, with the result that shorter chains of DNA accumulated. The new homolog, unlike -TGdR, was phosphorylated in bone marrow as well as in tumor, and incorporated well into the DNA both of bone marrow and of the neoplastic cells. In devising other homologs attention must be given to the specificity of the kinases, i.e., to whether phosphorylation is superior in tumor cells or in the growing normal cells.Abbreviations used are 6-TG 6-thioguanine - ,-TGdR ,-2-deoxythioguanosine - -3-BCTGdR 6-thioguanine--2,3-dideoxy-3-(hydroxymethyl)-d-erythro-pentofuranose - ³H-TdR Tritium-labeled (methyl) thymidine     

Ligand-Induced Regulation of ERα and ERβ is Indicative of Human Breast Cancer Cell Proliferation

Two estrogen receptors (ER), ER and ER, are expressed in breast cancer but their role in treatment response is unclear. The overall objective of this study was to determine if the presence of ER protein in breast cancer cell lines is an indicator of a poor prognosis based on cell proliferation. In addition, we determined the effect of estradiol (E2) and selective estrogen receptor modulators (SERMs), such as tamoxifen and genistein, on ER and ER protein regulation, to help in the understanding of the mechanism behind their role in modulating cell proliferation. Using western blot and immunofluorescence analysis, the ER positive cell lines, MCF-7 and T47D, were found to contain both ER and ER, and thus were used as model systems. E2 and genistein, which increased cell proliferation in both cell lines, induced an up regulation of ER in both cell lines. This suggests that an estrogenic response in breast cancer cells is indicated by an increase in ER expression. Tamoxifen decreased cell proliferation in both cell lines, while up regulating ER in both cell lines, suggesting that antiestrogenic response is indicated by an increase in ER expression. Although a change in the ER/ER ratio may play a role in the effect seen in cell proliferation, this study indicates that ER is a poor prognosticator of cell proliferation in breast cancer and that ER is a positive prognosticator of responsiveness to antiestrogen treatment.     

Ultrastructural Changes of the Vascular Endothelium after Intra-arterial Administration of Tumor Necrosis Factor-alpha (TNFα) in Rat Gliomas

The ensuing ultrastructural changes in tumor vascular endothelial cells following intra-arterial administration of tumor necrosis factor- (TNF) were studied in an experimental rat glioma model. C6 glioma cells were implanted in Wistar rats and then after 14 days, 5×10³U of human natural-type TNF (1.7×10⁵U/m²) was administered through the carotid artery. The animals were sacrificed at 3 or 24h after TNF treatment. A detailed examination with transmission electron microscope revealed swelling of the tumor vascular endothelial cell nuclei and mitochondria with matrix densities at 3h. At 24h, these cells demonstrated the presence of high amplitude mitochondrial swelling or the violent blebbing characteristic of damaged mitochondria; the cytoplasm was swollen enormously and there were dissolution of cytoplasmic organelles and rupture of the plasma membrane. The observed findings were typical of cell necrosis and confirms yet another mechanism by which TNF exerts its anti-tumor effects, that is, necrotizing effects on tumor vascular endothelium. The information appears to be important in the context of clinical application of intra-arterial TNF in the treatment of malignant gliomas.     

Blood-brain barrier changes following intracerebral injection of human recombinant tumor necrosis factor-α in the rat

Summary Human recombinant tumor necrosis factor- (rTNF-) was administered to normal Fischer 344 rats by stereotaxic intracerebral (IC) injection. Animals receiving a single injection of either 6 x 10⁴ U rTNF- or an equivalent volume of excipient (vehicle) in their right parietal lobe. In order to demonstrate any effects rTNF- might have on the blood-brain barrier (BBB), two studies were conducted, one employing exogenous horseradish peroxidase (HRP, 44 kD) as a tracer of BBB permeability and the other using endogenous IgG (150 kD). Rats given rTNF- showed transitory BBB permeability to HRP by 24 hours post-injection; this BBB compromise was determined to be no longer than 60 hours. In the other study, IgG was seen to cross the BBB by 48 hours post-rTNF- injection. Alternatively, rats injected IC with excipient showed only limited BBB opening as a result of injection-induced trauma. We conclude that human rTNF-, injected IC into normal rats triggers a temporary breakdown in BBB integrity which begins sometimes between 12 and 24 hours post-injection, is large enough to permit macromolecules of at least 150 kD to pass, and resolves by 72 hours post-injection.     

Effect of exogenous epidermal-like growth factors on mammary gland development and differentiation in the estrogen receptor-alpha knockout (ERKO) mouse

The development of the mouse mammary gland requires the interaction between several different ovarian and pituitary hormones such as estrogen, progesterone and prolactin as well as several locally-derived growth factors in the mammary gland such as epidermal growth factor (EGF), transforming growth factor (TGF), amphiregulin (AR) and heregulin (HRG). The focus of this study was to investigate the degree of mammary growth and differentiation in the adult, virgin mammary gland of wild type (wt) and estrogen receptor knockout (ERKO) females that lack estrogen receptor (ER) after reciprocal transplantation into the cleared mammary fat pad of virgin wt or ERKO mice. In addition, we assessed the local response of ERKO mammary tissue to TGF or HRG1 delivered from slow release-Elvax pellets. Our initial results indicated that when we transplanted virgin wt mammary tissue into ERKO mammary fat pads, mammary morphogenesis failed to occur. However, when transplanted virgin ERKO mammary tissue was transplanted into fat pads of virgin or pregnant wt mice, the development and differentiation of lobuloalveoli was readily observed. In addition, treatment of the virgin ERKO mammary gland with TGF or HRG1 stimulated ducts to undergo localized branching and growth and both growth factors induced secretory differentiation as evidenced by the production of milk proteins, caseins and/or whey acidic protein (WAP). The results from this study imply that in ERKO mammary tissue, ERKO ductal epithelium has the capacity to proliferate and differentiate in response to non-estrogenic, morphogenic stimuli.     

Pattern of distribution of cells positive for estrogen receptor α and progesterone receptor in relation to proliferating cells in the mammary gland

Since cell proliferation is indispensable for the growth and development of the breast, and estrogens are considered to play a major role in promoting cell proliferation, while progesterone influences its differentiation, the present work was designed with the purpose of verifying the relationship between cells containing steroid hormone receptors and proliferating cells in the normal human breast. Twelve breast samples were analyzed for their content of lobules type 1 (Lob1), Lob2, Lob3, and Lob4, and the number of cells containing estrogen receptor alpha (ER-), progesterone receptor (PgR), or expressing Ki67 antibody was determined by double immunocytochemical technique with specific antibodies. The highest percentage of ER-, PgR, and Ki67 positive cells was found in Lob1, with a progressive reduction in the more differentiated Lob2 and Lob3. ER- and PgR positive cells were found exclusively in the breast epithelium and were negative for Ki67, while cells positive for Ki67 did not express receptors. These findings were compared with the distribution of ER- and PgR in the autoradiographs of mammary gland of young virgin rats inoculated with 3H-thymidine for determination of the DNA labeling index (DNA-LI). Both the DNA-LI and the percentage of ER- and PgR positive cells were maximal in the epithelium of terminal end buds, and these values were reduced in alveolar buds and lobules. ER- and PgR positive cells did not proliferate, and those cells that had incorporated 3H-thymidine were negative for both receptors. Our results led us to conclude that the content of ER- and PgR in the normal mammary tissue varies with the degree of lobular development, in parallel with cell proliferation. However, the expression of receptors occurs in cells other than the proliferating cells, indicating that they represent at least two separate cell populations. These findings open new avenues towards the understanding of the mechanisms through which estrogens and progesterone affect the proliferative activity of breast epithelial cells, and their role in the initiation of the cascade of events that leads a normal cell to cancer.     

Expression of LFA-1/ICAM-1 in CNS lymphomas: possible mechanism for lymphoma homing into the brain

We examined a possible role for the adhesion molecules LFA-1 and ICAM-1 in localizing central nervous system non-Hodgkin's lymphomas (CNS-NHLs) to the brain. Fresh frozen sections from 12 monoclonal CNS NHLs (11 primary, one secondary) were stained with monoclonal antibodies to LFA-1 chain (CD11a), chain (CD18) and, ICAM-1 (CD54). Additional staining made use of rat monoclonal antibodies to the human and mouse high endothelial venule antigens HECA 452 and MECA 79 and mouse ICAM-1. The expression of these same molecules was also studied in mice with severe combined immunodeficiency (SCID) mice, bearing intracranial human lymphoblastoid cells.Eleven of the CNS-NHL tumors expressed LFA-1 (one strongly, one intermediate, nine weakly). Nine of the tumors weakly expressed LFA-1.. Nine of twelve tumors weakly expressed ICAM-1. In six of seven tumors definite blood vessels stained for ICAM-l. Non-tumor brain from two patients and non-tumor cerebral blood vessels showed no staining with CD11a, CD18 or CD54 antibodies. Strong expression of LFA- and LFA- as well as ICAM-1 was noted in human lymphoblastoid cells (LCLs)/SCID mouse CNS lymphomas. Tumor blood vessels in these mice stained for mouse ICAM-1. Normal SCID mouse brains showed no staining with CDII, CD18, CD54 or mouse ICAM-1 antibodies. Human, human/mouse CNS lymphomas, normal human, and mouse brains showed no staining with either HECA 452 or MECA 79.Our data suggests that CNS lymphomas express LFA-1, LFA-1 and to a lesser degree ICAM-1 antigen, while tumor blood vessel endothelium expresses ICAM-1. LFA-1-LFA-1/ICAM-1 interaction may play a role in large cell lymphoma homing and persistence in the brain.     

Polyamine involvement in the secretion and action of TGF-α in hormone sensitive human breast cancer cells in culture

These experiments were designed to test polyamine (PA)* involvement in the secretion and action of transforming growth factor (TGF-) in hormone responsive MCF-7 breast cancer cells in liquid culture. At the same time, we evaluated the influence of culture conditions (with serum vs. serum depleted) and subclonality of MCF-7 cells on PA involvement in estrogen (E₂) and TGF- stimulated cell proliferation. Despite inducing a profound suppression of cellular PA levels and inhibiting basal and E₂-stimulated growth, administration of the PA synthesis inhibitor -difluoromethylornithine (DFMO) did not influence either basal or E₂-induced TGF- secretion. In the same experiments, on the other hand, addition of DFMO completely blocked the growth stimulatory effect of exogenous TGF-. However, when the culture conditions were changed to serum-free medium, TGF- and E₂-induced cell proliferation was affected modestly or not at all by DFMO administration, despite similar suppression of cellular ornithine decarboxylase (ODC) activity and PA levels. In addition, different clones of MCF-7 cells differed in their sensitivity to the antiproliferative effect of DFMO as well as in basal levels of ODC activity and PA. We conclude that PAs are not involved in basal or E₂-stimulated TGF- secretion in MCF-7 breast cancer cells. On the other hand, PAs do seem to be important mediators of TGF- and E₂-induced breast cancer cell proliferation, though the degree of such involvement appears to be influenced by serum factors and clonal variability of MCF-7 cells.     

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.     

Biological and clinical evaluation of Lanreotide (BIM 23014), a somatostatin analogue,in the treatment of advanced breast cancer

Summary Recently, compounds having pure antiestrogenic activity have become available. In this study, we examined the activity of the new steroidal antiestrogen EM-170 (N-n-butyl, N-methyl-11-(16-chloro-3,17-dihydroxy-estra-1,3,5-(10)-trien-7-yl) undecanamide) on the growth of 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma stimulated by treatment with estrone (E₁), a steroid known to play an important role as precursor of 17-estradiol (E₂), especially in postmenopausal women. Twenty-five days after ovariectomy (OVX), tumor volume in control OVX animals decreased to 51.4 ± 11% of the initial volume; treatment with E₁, administered by Silastic implants, stimulated tumor growth to 179 ± 21%. Treatment with the antiestrogen EM-170 at a dose of 200 µg (twice daily) not only completely reversed the stimulatory effect of E₁, but also inhibited tumor growth to 30.5 ± 9.6%, an effect that is 41% (P < 0.01 vs OVX control) greater than that of ovariectomy alone. At a relatively low dose of 40 µg (twice daily), 20 days of treatment with EM-170 reversed by 55% the stimulatory effect of E₁ (1.0 µg, subcutaneously, twice daily) on tumor growth in OVX animals. On the other hand, the antiestrogen also induced a significant inhibitory effect on 17-hydroxysteroid dehydrogenase (17-HSD) activity in the DMBA-induced mammary tumors, an effect that is in agreement with the marked reduction caused by the same treatment on tumor estradiol (E₂) levels in E₁-treated OVX animals. The present data show that the new steroidal antiestrogen EM-170 exerts a potent inhibitory effectin vivo on E₁-stimulated growth of DMBA-induced mammary tumors, an effect that is probably mediated by both its antiestrogenic activity at the receptor level and its inhibitory effect on 17-HSD, thus inhibiting local E₂ formation and facilitating the action of the antiestrogen at the receptor level.     

Immune adjuvants for chemotherapy or radiotherapy in the 9L rat brain tumor model

Summary The therapeutic efficacy and toxicity of three biological response modifiers,Corynebacterium parvum (Cp), Chinese blister beetle extract (CBBE), recombinant human IL-1 (rhIL-1), used alone or in combination with chemotherapy or radiotherapy, were investigated in the intracerebral (ic) rat 9L brain tumor model. Used alone, Cp (2mg/rat, ip plus 70g/rat, ic), CBBE (5l of an ethanol extract, ic), or IL-1 (lg/rat, ic or 1g/rat × 3, q 3 d, ic), had no effect on animal survival compared to the untreated or saline treated controls. When combined with chemotherapy or radiotherapy, the three immunotherapeutic agents did not show any additive effects on survival compared to that observed with systemic BCNU (12mg/kg), local ic bleomycin (0.25 unit), or local radiotherapy (16 Gy). While ic IL-1 did not produce evident toxicity, there was fatal toxicity caused by ic Cp or CBBE treatment in a few animals. The combination of Cp and bleomycin produced severe neurotoxicity, resulting in the early death of animals. This study demonstrates a lack of efficacy of the nonspecific immune adjuvants IL-1, Cp or CBBE, used either alone or combined with cytotoxic chemotherapy or radiotherapy, in this rat brain tumor model.     

Glucocorticoids and androgens up-regulate the Zn-α2-glycoprotein messenger RNA in human breast cancer cells

Summary We have studied the hormonal regulation of the gene encoding Zn-₂-glycoprotein (Zn-₂-gp), a human protein with a high degree of amino acid sequence similarity to class I histocompatibility antigens that is produced by a specific subset of breast carcinomas. Northern blot analysis revealed that dexamethasone and 5-dihydrotestosterone strongly induced the accumulation of Zn-₂-gp mRNA in T-47D human breast cancer cells. Furthermore, the effect of these two hormones was shown to be additive, since the combination of both hormones produced a stimulation of Zn-₂-gp mRNA of at least 3-fold over that produced by either hormone alone. By contrast, the addition of 5-dihydrotestosterone, 17-estradiol, or progesterone failed to induce the expression of Zn-₂-gp. The stimulatory effect of glucocorticoids and androgens on Zn-₂-gp expression was produced in a time and dose dependent manner, without significantly affecting the cell proliferation rate. A time-course study demonstrated that the induction of Zn-₂-gp mRNA by androgens and glucocorticoids reached a level of 4 or 3.2-fold over the untreated control after seven days of incubation in the presence of a 10^–7 M concentration of 5-dihydrotestosterone or dexamethasone, respectively. A dose-response study showed that as little as 10^–11 M of 5-dihydrotestosterone or dexamethasone produced an accumulation of Zn-₂-gp mRNA of 2.4 or 2.1-fold over the control, respectively. On the basis of these results, we propose that Zn-₂-gp may be useful as a biochemical marker of breast carcinomas with a specific pattern of hormone responsiveness in whose development glucocorticoids and/or androgens may play a significant role.     

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     

The use of natural interferon alpha conjugated to a monoclonal antibody anti mammary epithelial mucin (Mc5) for the treatment of human breast cancer xenografts

Summary An immunoconjugate composed of natural interferon (nIFN) bound in a noncleavable fashion to a monoclonal antibody (MoAb) recognizing a breast epithelial membrane mucin (Mc5) was used to treat xenografts of a human mammary carcinoma cell line (MCF-7) growing in nude mice. The immunoconjugate (nIFN/Mc5) was administered as 20 intralesional (i.l.) injections to 1 of 2 xenografts in each animal. It was found that nIFN/Mc5 produced a significant enhancement of the growth inhibitory actions of nIFN on the injected tumors. Further enhancement was obtained when nIFN or nIFN together with Mc5 (at a dose 10 times larger than that present in nIFN/Mc5) were added to the immunoconjugate. Biodistribution experiments showed that the uptake of¹²⁵I-nIFN/Mc5 by the tumors was greater and its elimination slower than for¹²⁵I-nIFN alone or conjugated to irrelevant mouse IgG₁. In addition, the immunoconjugate up-regulated the antigenic expression of a breast epithelial membrane mucin by the carcinoma cells, an up-regulation which was not significantly different from that produced by nIFN alone. The contralateral noninjected tumors exposed to systemic levels of the immunoconjugate showed an enhancement of antitumor effects, but to a lesser extent than the injected tumors. These findings suggest that the enhancement of the growth inhibitory action of the immunoconjugate was related to the specific binding of Mc5 which targeted the IFN to the carcinoma cells and impeded its elimination. It is likely that the targeting was favored by the IFN-mediated up-regulation of antigenic expression by the carcinoma cells, thereby producing a cascade of interrelated effects. The results of this study point out the feasibility and potential usefulness of IFN treatment by means of immunoconjugates as well as the worth of pursuing and improving this form of therapy.