TM-1 cells from an established human malignant glioma cell line produce PDGF,TGF-α, and TGF-β which cooperatively play a stimulatory role for an autocrine growth promotion

Summary We have previously established a human malignant glioma cell line, TM-1. TM-1 cells could proliferate in the serum-free medium. In the present study, immunochemical analysis demonstrated that platelet-derived growth factor (PDGF), transforming growth factor (TGF)-, and TGF- are present in the serum-free medium conditioned by growing TM-1 cells. While the cells appeared to possess a single type of binding sites for epidermal growth factor (EGF) with properties comparable to those determined for other tumor cells, the conditioned medium did not contain EGF. PDGF, TGF-, and EGF added exogenously to serum-free media stimulated thymidine incorporation into DNA of TM-1 cells. In addition, antibodies specific for PDGF and TGF- suppressed this activity. These results indicate autocrine and stimulatory roles of PDGF and TGF- for the proliferation of TM-1 cells. As observed for other tumor cells, TGF- by itself weakly suppressed thymidine incorporation by TM-1 cells. However, TGF- employed in combination with TGF- or EGF appeared to stimulate thymidine incorporation, suggesting that a cooperative action of TGF- with different growth factors may be involved in the stimulatory growth regulation at least for TM-1 cells.     

Prognostic relevance of transforming growth factor alpha (TGF-α) and tumor necrosis factor alpha (TNF-α) detected in breast cancer tissues by immunohistochemistry

Summary The function of different growth factors in the development and progression of malignant tumors and the role of cytotoxic cytokines in the host response generated against neoplasms have been recently studied. Anti-TGF- and anti-TNF- monoclonal antibody families have been developed and characterized previously by our laboratory. Libraries of anti-TGF- and anti-TNF- monoclonal antibodies were selected for equal immunoreactivity both in native (frozen) and in formaldehyde fixed, paraffin embedded histological sections. No differences were found between native and fixed samples demonstrated in 10 cases in the present prospective study. Retrospective investigation was performed in 35 histopathological specimens of breast cancer patients detailed clinically and observed during 5 years after the surgical treatment. Correlation between TGF- and/or TNF- expression and clinical staging - TNM score, lymph node metastasis, tumor recurrence and survival time - was analyzed. According to our present study, the TGF- positive patients had worse clinical prognosis than the TNF- positive and double positive cases during long term observation.     

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.     

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.     

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.     

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.     

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.     

Molecular Targeting with Recombinant Cytotoxins of Interleukin-13 Receptor α 2-expressing Glioma

A restricted receptor for interleukin 13 (IL-13R2) is over-expressed in high-grade astrocytoma (HGA), but not in normal organs. In order to design and examine new anti-HGA therapies, which are molecularly directed against IL-13R2, we established an IL-13R2-expressing syngeneic immunocompetent murine model of HGA. The model was obtained by transfecting G-26 murine glioma cells with IL-13R2. G-26-IL-13R2(+) cells, but not mock-transfected cells, became susceptible to IL-13 mutant-based cytotoxic proteins that kill human HGA cells. G-26-IL-13R2(+) cells maintained their tumorigenicity in immunocompetent C57BL/J6 mice and preserved their expression of IL-13R2 in vivo. These characteristics of the G-26-IL-13R2(+) tumors allowed us to test molecularly defined anti-glioma passive immunotherapy. A targeted recombinant chimera cytotoxin composed of multiply mutated IL-13 (IL-13.E13Y/R66D/S69D) and a derivative of Pseudomonas exotoxin (PE), PE1E, IL-13.E13Y/R66D/S69D-PE1E, was used in anti-tumor experiments. G-26-IL-13R2(+) cells were killed by IL-13.E13Y/R66D/S69D-PE1E in an IL-4-independent fashion. To test the cytotoxin in vivo, G-26-IL-13R2(+) tumors were established in C57BL/J6 mice and when the tumors reached a size of at least 50mm³, the mice were treated with IL-13.E13Y/R66D/S69D-PE1E. In the mice treated with the targeted fusion cytotoxin, the tumors regressed and 80% of the animals were cured. This study documents the establishment of an IL-13R2-positive model of HGA in immunocompetent rodents. Furthermore, the effectiveness and safety of the targeted IL-13-based cytotoxin against IL-13R2-expressing tumors in a more clinically relevant in vivo HGA model is promising with regard to the future clinical utility of the cytotoxin.     

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.     

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.     

Apoptosis of murine melanoma B16-BL6 cells induced by quercetin targeting mitochondria, inhibiting expression of PKC-α and translocating PKC-δ

Purpose In our previous study, quercetin was found to induce apoptosis of murine melanoma B16-BL6 cells. The cellular and molecular mechanism of quercetin-induced apoptosis was investigated in the present study.Methods Nuclear morphology was determined by fluorescence microscopy. DNA fragmentation was analyzed by electrophoresis and quantified by the diphenylamine method. The transmembrane potential of mitochondria was measured by flow cytometry. Bcl-2, Bcl-X_L, PKC-, PKC-, and PKC- were detected by Western blotting. Caspase activity was determined spectrophotometrically.Results Quercetin induced the condensation of nuclei of B16-BL6 cells in a dose-dependent pattern as visualized by Hoechst 33258 and propidium iodide dying. Phorbol 12-myristate 13-acetate (PMA), a PKC activator, significantly enhanced apoptosis induced by quercetin, while doxorubicin, a PKC inhibitor, markedly decreased it. Both PMA and doxorubicin showed a consistent effect on the fragmentation of nuclear DNA caused by various dosages of quercetin. Quercetin dose-dependently led to loss of the mitochondrial membrane potential, which was also significantly reinforced or antagonized by PMA and doxorubicin, respectively. Moreover, PMA showed reinforcement, while doxorubicin showed significant antagonization, of the quercetin-mediated decrease in the expression of Bcl-2. Quercetin promoted caspase-3 activity in a dose-dependent manner, which was also regulated by PMA and doxorubicin with a pattern similar to that seen in their effect on apoptosis, mitochondrial membrane potential and Bcl-2 expression, but none of these were directly affected by PMA and doxorubicin. Free fatty acid and chlorpromazine, a PKC activator and inhibitor, respectively, did not interfere with these effects of quercetin. B16-BL6 cells expressed PKC-, PKC-, and PKC-. Quercetin dose-dependently inhibited the expression of PKC- but not that of PKC- and PKC-. Doxorubicin almost completely blocked the effect of quercetin on the expression of PKC-. Quercetin was also involved in the translocation of PKC- from the cytosol to the nucleus. PMA enhanced the effect of quercetin on the translocation of PKC-.Conclusions These results indicate that quercetin induced apoptosis of murine melanoma B16-BL6 cells by injuring their mitochondria, increasing the activity of caspase-3, inhibiting the expression of Bcl-2 and PKC-, and inducing the translocation of PKC-. Doxorubicin inhibited these effects of quercetin by blocking the decreased expression of PKC- induced by quercetin while PMA increased these effects by enhancing the translocation of PKC- induced by quercetin.     

The effects of TGF-α and 17β-estradiol on polyphosphoinositide metabolism in MCF-7 breast cancer cells

Summary The mechanism by which transforming growth factor-alpha (TGF-) stimulates breast cancer cell proliferation is largely unknown. Furthermore, its potential role as an autocrine effector of estradiol-17 (E₂)-stimulated growth of hormone-dependent mammary tumors remains controversial. Transient changes in phosphatidylinositol (PI) turnover have been demonstrated in several tissues in response to growth factors. In these experiments, we tested the effects of TGF- and E₂ on PI metabolism in three MCF-7 breast cancer cell sublines (MCF-7B, MCF-7I, and MCF-7J). Although TGF- was mitogenic in MCF-7I and MCF-7J cells, PI hydrolysis was stimulated by the growth factor only in the MCF-7I cells. In addition, the TGF- effect was relatively modest, ranging from 23% to 42%. E₂ effects on PI turnover were tested in the MCF-7B cells, which were the most sensitive to the proliferative effect of the hormone. E₂ did not stimulate PI hydrolysis, whether or not the cells were labelled in the presence of the hormone. On the other hand, E₂ did seem to stimulatede novo synthesis of phosphatidylinositol and induce activation of PI kinases. These results demonstrate that TGF--stimulated PI hydrolysis is modest and cell type dependent. At least under certain conditions, PI metabolism is not involved in the proliferative effects of TGF- (MCF-7J) or E₂ (MCF-7B). The role of increased PI synthesis in E₂-stimulated MCF-7 cell growth remains to be established.This work is supported by a grant from the National Cancer Institute, POI CA40011.     

Glioblastoma Multiforme in an Asian Population: Evidence for a Distinct Genetic Pathway

In Singapore astrocytic tumours occur in only 25% of patients with primary brain tumours compared to 40–60% in other series. Glioblastoma multiforme arises either de novo as a primary glioblastomas associated with epidermal growth factor receptor (EGFR) and mdm2 over-expression or as a secondary glioblastomas, through malignant progression from low-grade astrocytomas, associated with p53 mutations and PDGFR- over-expression. Using immunohistochemical methods and DNA sequencing, we studied our population of glioblastomas for over-expression of EGFR, mdm2, p53, and PDGFR- as well directly for mutations of the p53 gene. While levels of over-expression of EGFR and mdm2 were consistent with levels expected for primary glioblastomas, levels of p53 and PDGFR- were consistent with levels documented for secondary glioblastomas. Notably 96% of the samples over-expressed p53 as detected with monoclonal antibody pAb 240. Of the 39 samples available for DNA sequencing 18% (7/39) had p53 mutations, including three mutations previously undocumented in glioblastomas. These results provide strong evidence that glioblastomas in Asian patients do not conform to currently accepted models of glioblastoma development, and that clinically defined glioblastomas in these patients show genetic changes consistent with both primary and 'secondary glioblastomas.     

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.     

Growth factor involvement in the multihormonal regulation of MCF-7 breast cancer cell growth in soft agar

Summary The hormone dependency of the MCF-7 breast cancer cell line, while extensively tested in liquid culture, has not been previously evaluated under conditions of anchorage-independent growth in serum-free media. Using the soft agar clonogenic assay, we demonstrate that physiologically relevant concentrations of estradiol (E₂), progesterone (Pg), and prolactin (PRL) similarly stimulated MCF-7 cell colony formation in the absence of serum. Addition of an anti-insulin-like growth factor-I (IGF-I) antibody inhibited E₂- and Pg-stimulated growth, while PRL action was not affected. Similar results were obtained with an anti-IGF-I receptor antibody, except that its inhibitory effect on Pg-induced colony formation was modest and not statistically significant. Administration of either an anti-transforming growth factor- (TGF-) antibody or an anti-epidermal growth factor (EGF) receptor antibody similarly inhibited E₂-stimulated MCF-7 cell growth in soft agar, while neither antibody influenced Pg or PRL effects. Addition of TGF-₁, -₂, -₃ similarly suppressed MCF-7 cell colony formation in a dose dependent manner to a degree comparable to that observed with 4-OH-tamoxifen (4-OH-T). Furthermore, the growth inhibitory effect of 4-OH-T was completely reversed by an anti-TGF- antibody. We conclude that IGFs and TGF- are important mediators of E₂-stimulated MCF-7 cell growth in soft agar. IGFs may also be playing a role in Pg action, while neither growth factor is involved in PRL-stimulated colony formation. Finally, TGF- appears to be an important mediator of antiestrogen-induced inhibition of tumor growth.Abbreviations IGF insulin-like growth factor - TGF transforming growth factor - E₂ estradiol-17 - Pg progesterone - PRL prolactin - 4-OH-T 4-hydroxy-tamoxifen - IMEM improved minimal essential medium     

Sex hormone-induced mammary carcinogenesis in female Noble rats: Expression of TGF-β1 and its receptors, TGF-α, and EGF-R in mammary carcinogenesis

We have established a Noble rat model to explore the mechanisms of hormonal mammary carcinogenesis, in which the role of androgen in promoting mammary carcinogenesis was highlighted. We have also established that stromal–epithelial interactions may be responsible for the promotional effects of testosterone in mammary carcinogenesis. Based on these understandings, in the present study we examined the expression of transforming growth factor beta-1 (TGF-₁) and its receptors (TGF- RI, TGF- RII), transforming growth factor alpha (TGF-), and epidermal growth factor receptor (EGF-R) in 'pre-malignant' mammary glands treated with different protocols of sex hormones, as well as in mammary cancers. We observed that TGF-₁ was strongly expressed in most mammary tumors, whereas TGF- RI and TGF- RII were negative in most mammary tumor cells. The results from comparative study of 'pre-malignant' glands further showed that when the animals were treated with testosterone, either alone or in combination with 17-estradiol, the mammary gland epithelial cells expressed high levels of TGF-₁. This over-expression of TGF-₁ can be blocked by flutamide, indicating that testosterone may be responsible for the expression of TGF-₁ in mammary glands. TGF- RI and TGF- RII were also expressed strongly in testosterone-treated mammary epithelial cells and only weakly detectable in 17-estradiol treated and control mammary epithelial cells. Furthermore, TGF- RI and TGF- RII were also expressed in stromal cells, both in mammary tumors and in hormone-treated mammary glands. These observations indicate that the mechanism of testosterone in mammary carcinogenesis may be through its regulation of expression of TGF-₁ and its receptors. On the other hand, TGF- was also expressed in all 39 mammary cancers, while only 81% of the cancers were EGF-R positive. TGF- was also strongly expressed in stromal cells in all three experimental groups, but only moderately expressed in epithelial cells when treated with a combination of testosterone and 17-estradiol. By contrast, EGF-R was strongly expressed in epithelial cells in the three experimental groups but negative in stromal cells. Flutamide or tamoxifen was unable to block the expression of TGF- induced by the combined sex hormone treatment. However, they were effective in blocking the expression of TGF- when the animals were treated with testosterone or 17-estradiol alone, respectively. These results suggest that both testosterone and 17-estradiol may be required for the over-expression of TGF- in the mammary carcinogenesis induced by sex hormones. To our knowledge, this is the first experimental study to explore the regulation of TGF-₁, TGF-, and their receptors by testosterone and 17-estradiol in mammary carcinogenesis.     

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.     

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.     

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.