Down-regulation of transforming growth factor-β and interleukin-10 secretion from malignant glioma cells by cytokines and anticancer drugs

The effect of treatment with interleukin-1 (IL-1), interferon- (IFN-), vincristine, and etoposide was evaluated on the secretion of transforming growth factor- (TGF-) and IL-10 and the expression of major histocompatibility complex (MHC) class I, intercellular adhesion molecule-1 (ICAM-1), and CD80 molecules by malignant glioma cells. Five malignant glioma cell lines were treated with IL-1, IFN-, and/or anticancer agents (vincristine and etoposide). Combined treatment with IL-1 and IFN- caused greater inhibition of TGF- secretion compared to treatment with IFN-, and almost the same levels of inhibition as treatment with vincristine and etoposide. The greatest inhibition of TGF- secretion was achieved by treatment with all agents. Low levels of IL-10 secretion were determined in two out of five malignant glioma cell lines. This IL-10 secretion was inhibited by treatment with IL-1, IFN-, vincristine, and/or etoposide. Treatment with both cytokines and anticancer agents increased the expression of MHC class I and ICAM-1 in all tumor cell lines. The mean increase of expression of MHC class I was 50% and that of ICAM-1 was 12-fold. No tumor cell lines expressed CD80 molecules on the cell surface, and no treatment caused CD80 expression. These results suggest that TGF- and IL-10 secretion by malignant glioma cells can be suppressed by treatment with a combination of IL-1, IFN-, vincristine, and etoposide, and the treatment up-regulates MHC class I and ICAM-1 expression on tumor cells. These results have implications for immunotherapy and chemotherapy in patients with malignant tumors.     

TGFβ Signaling Pathways and Human Diseases

Recent progress in deciphering the TGF pathway has uncovered a new signaling molecule, the Smads, and with this finding now gives us insights into how TGF-like signals are transmitted from outside the cell to the nucleus. As we learn more about how TGF regulates normal development, we also are gaining insights into diseases that are caused by mis-regulation or mutation of various components of the signaling pathways.     

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.     

A novel approach of targeted ablation of mammary carcinoma cells through luteinizing hormone receptors using Hecate-CGbeta conjugate

Recent studies have shown that human and animal mammary gland carcinoma cell line express luteinizing hormone receptors (LHRs). We have examined the cytotoxic effect of Hecate-CG conjugate, that is, fusion of a lytic peptide (Hecate) and a 15-amino acid fragment of the CG-chain in vitro. To test the hypothesis that the Hecate-CG conjugate selectively abolishes cells possessing LHR, estrogen dependent and independent human breast cancer cell lines (MCF-7; MDA-MB-231) and a mouse Leydig tumor cell line (BLT-1) were treated in vitro with Hecate-CG conjugate and Hecate alone. Cytotoxic effects of the Hecate-CG conjugate and the Hecate alone was measured by lactate dehydrogenase (LDH) release immediately after treatment. We observed that the Hecate-CG conjugate selectively, in dose-dependent manner destroys cells possessing LHR in lower concentrations of preparate comparing to the Hecate alone and that the cytotoxic effect is strongly correlated with the number of LHR. Using Western blot analysis we characterized the LHR on membranes of MDA-MB-231, MCF-7 and BLT-1 tumor cell lines. In addition, we showed the evaluation of inhibition potential of the Hecate-CG conjugate to LHR. At a concentration of 33 µM the conjugate inhibited (50%; IC₅₀) the binding of CG to LHR.We suggest further development of this novel approach for the treatment of breast cancer by the Hecate-CG for in vivo trials.     

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.     

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.     

Genetic polymorphisms of platelet adhesive molecules: association with breast cancer risk and clinical presentation

The main platelet adhesive receptors integrin 21, integrin IIb3 and glycoprotein (GP) Ib are also expressed in breast carcinoma cells. They play a key role in tumor cell-induced platelet aggregation and in adhesive interactions necessary for tumoral invasion and metastasis. Several polymorphisms affecting these molecules, two in integrin 2 (C807T and G1648A), one in integrin 3 (T1565C) and one in GP Ib (VNTR), influencing their levels, structure, and possibly their function, have been previously described and associated with cardiovascular diseases. In this study, we investigated the association of these polymorphisms with breast cancer risk or clinical presentation. We studied 101 patients with invasive breast cancer. The main prognostic variables were recorded, and genomic PCR analysis of these polymorphisms was performed. A group of 101 control subjects matched on age and sex was studied and compared with patients. No association was found between VNTR (GP Ib) polymorphism and breast cancer risk or presentation. Genotype and allele frequencies of C807T and G1648A polymorphisms of integrin 2 were not statistically different in breast cancer patients and controls, although we found an association between the 1648G/G genotype and higher disease stages (III and IV) (p = 0.02). Breast cancer risk was higher in carriers of 3 integrin T/T genotype (OR = 2.08, 95% CI = 1.04–4.16, p = 0.04). Furthermore, genotype 1565T/T was also associated with axillary nodal metastasis (p = 0.017) and with tumoral diameter greater than 2 cm (p = 0.02). Although confirmatory studies are needed, our results suggest that polymorphic genetic variation of integrins expressed in platelets and epithelial breast cells could modify the risk and the biological aggressiveness of breast carcinomas.     

Expression of a Soluble Transforming Growth Factor-β (TGFβ) receptor reduces tumorigenicity by regulating natural killer (NK) cell activity against 9L gliosarcoma in vivo

Immunotherapy of gliomas has been forwarded as an attractive alternative to standard therapeutic modalities. Numerous observations indicate some therapeutic efficacy with this approach, but it is not curative in most reports. It is well established that gliomas suppress immune reactivity via a number of mechanisms, including expression CD95 ligand (CD95L), which induces apoptosis of immune effector cells, and secretion of immunosuppressive factors such as transforming growth factor-beta (TGF). It has been hypothesized that abrogation of production or function of TGF would improve immune reactivity to gliomas. To investigate this in a fashion that is translatable into clinical practice, we utilized a retroviral vector encoding a truncated, soluble form of the Type II receptor for TGF (TFGsr) and expressed it in the rat 9L gliosarcoma line (9L-TGFsr). We then determined whether expression of TGFsr affected in vitro sensitivity of 9L to lysis by immune effector cells, whether expression of TGFsr affected tumorigenesis of 9L in vivo, and whether TGFsr affected expression of immunity to 9L. In these experiments, we determined that 9L-TGFsr was more susceptible than sham transfected 9L (9L-neo) to lysis by natural killer (NK) cells. We also determined that subcutaneously implanted 9L-TGFsr was less tumorigenic than 9L-neo in syngeneic rats. Similarly, survival was extended by 40% in rats given intracranial 9L-TGFsr compared to 9L-neo. Finally, we determined that elimination of CD161⁺ cells resulted in comparable growth of 9L-neo and 9L-TGFsr in vivo, indicating that NK or NK-like cells were responsible for the anti-tumor effects in this model.     

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 role of transforming growth factor Β in glioma progression

This review examines the apparently paradoxical conversion of transforming growth factor 's (TGF) regulatory role as a growth inhibitor among normal glial cells to that of a progression factor among glioblastomas (GM). In vitro, TGF functions as an autocrine growth inhibitor of near-diploid gliomas of any grade. In contrast, hyperdiploid glioblastoma multiforme (HD-GM) cultures proliferate in response to TGF, which is mediated by induction of platelet-derived growth factor B chain (PDGF-BB). The dominant hypothesis of TGF's pathogenetic association with malignant transformation has been predicated upon acquisition of resistance to its growth inhibitory effects. However, the lack of obvious correlation with TGF receptor (TR) expression (or loss) between the HD-GM and the TGF-inhibited GM cultures suggests the existence of intrinsically opposed regulatory mechanisms influenced by TGF. The mechanism of conversion might be explained either by the loss of a putative tumor suppressor gene (TSG) which mediates TGF's inhibition of growth or by enhancement of an active oncogenic pathway among the HD-GM. The frequency of mutations within glioma-associated TSG, such as TP53 and RB, suggests that defects in TGF's inhibitory signaling pathway may have analogous effects in the progression to HD-GM, and TGF's conversion to a mitogen. Alternative sites of inactivation which might explain the loss of TGF's inhibitory effect include inactivating mutation/loss of the TR type II, alterations in post-receptor signal transmission or the cyclin/cyclin dependent kinase system which regulates the phosphorylation of pRB. Loss or inactivation of a glial TSG with a consequent failure of inhibition appears to allow TGF's other constitutive effects, such as induction of c-sis, to become functionally dominant. Mechanistically, TGF's conversion from autocrine inhibitor to mitogen promotes 'clonal dominance' by conferring a Darwinian advantage to the hyperdiploid subpopulations through qualitative and quantitative differences in its modulation of PDGF-A and c-sis, with concomitant paracrine inhibition of competing, near-diploid elements. Abbreviations: transforming growth factor (TGF) and receptor (TR); retinoblastoma gene (RB) and protein (pRB); platelet-derived growth factor (PDGF) and receptor (PDGFR); epidermal growth factor (EGF) and receptor (EGFR); fibroblast growth factor (FGF); malignant glioma (MG), astrocytoma (AST), anaplastic astrocytoma (AAST), glioblastoma (GM); hyperdiploid glioblastoma (HD-GM); glioblastoma multiforme (GM); normal rat kidney (NRK); tumor suppressor gene (TSG); loss of heterozygosity (LOH); TP53 wild type (TP53wt); TP53 mutant (TP53m)     

Tamoxifen and fenretinide in women with metastatic breast cancer

Background: Tamoxifen and fenretinide combination therapy has been shown to be an active treatment regimen in metastatic breast cancer patients. This pilot study sought to determine whether the addition of fenretinide to tamoxifen would be associated with antitumor activity in metastatic breast cancer patients who had been previously treated with tamoxifen or who had hormone receptor negative disease. The effect of this therapy on circulating plasma transforming growth factorbeta (TGF) levels and serum lipids was also examined.Patientsand Methods: Thirtyone patients were treated with tamoxifen (20mg po daily), and fenretinide (400mg po daily with a 3day drug holiday each month). Plasma TGF testing was performed using isoform specific sandwich ELISA.Results: Twenty four of the 31 patients were evaluable for an antitumor response including 14 estrogen receptor (ER) positive patients who had failed prior tamoxifen therapy, seven ERnegative patients, and three hormone therapy naive ERpositive patients. There were no objective antitumor responses; three patients had stable disease for 8, 8, and 24 months. Five patients (16%) discontinued therapy for toxicity (one for grade 3 skin rash and four for abnormal dark adaptation). There was a statistically significant decrease in total cholesterol (median change per patient of –13.5mg/dl; p=0.049, a 6.5% decrease), and an increase in HDL levels (median change per patient of +18mg/dl, p=0.0001, a 35% increase) with tamoxifen and fenretinide therapy.TGF-1 plasma levels were normal in 26 of 28 patients, and no changes in these levels post-treatment were demonstrated.Conclusions: Tamoxifen and fenretinide therapy is not an active combination in ER negative metastatic breast cancer or in patients whose disease has progressed on tamoxifen. This combination had a beneficial effect on total serum cholesterol and HDL levels with no associated rise in serum triglyceride levels. The 400mg dose of fenretinide was associated with symptomatic nyctalopia in one-third of patients making it an unsuitable dose for use in breast cancer prevention studies.     

Suppression of TGF-β1 in Human Gliomas by Retroviral Gene Transfection Enhances Susceptibility to LAK Cells

Human glioma cell line, Onda 10 produces TGF-1. TGF-1 has a biological role for the immunosuppression of the host. We have investigated whether suppression of TGF-1 on human glioma cell enhanced the susceptibility to lymphokine-activated killer (LAK) cells. In vitro, susceptibility to LAK cells on Onda 10 cell is augmented by retroviral gene transfection with antisense TGF-1. Nude mice bearing Onda 10 cells transduced with antisense TGF-1 gene has a longer life span compared to mice carrying that of sense TGF-1 gene or vector alone. The cytotoxic activity of LAK cells induced from spleen cells of mice carrying antisense TGF-1 gene transduced cells is higher against Onda 10 cell than that of LAK cells from mice carrying vector alone transduced cells. Also, antisense TGF-1 gene transduced cells are much more sensitive to LAK cells compared to Onda 10. These suggest that the augmented host systemic immunity in mice is one of the mechanisms of the reduced tumorigenicity of antisense TGF-1 gene transduced cells and that the increased systemic immunity could be ascribed to the increased immunogenicity of the tumor cells. The gene therapy for malignant glioma with antisense TGF-1 gene is expected to be promising.     

Transforming growth factor-β in breast cancer: A working hypothesis

Transforming Growth Factor- (TGF) is the most potent knowninhibitor of the progression of normal mammary epithelial cells through thecell cycle. During the early stages of breast cancer development, thetransformed epithelial cells appear to still be sensitive toTGF-mediated growth arrest, and TGF can act as an anti-tumorpromoter. In contrast, advanced breast cancers are mostly refractory toTGF-mediated growth inhibition and produce large amounts of TGF,which may enhance tumor cell invasion and metastasis by its effects onextracellular matrix. We postulate that this seemingly paradoxical switch inthe responsiveness of tumor cells to TGF during progression is theconsequence of the activation of the latent TGF that is produced anddeposited into the tumor microenvironment, thereby driving the clonalexpansion of TGF-resistant tumor cells. While tumor cells themselvesmay activate TGF, recent observations suggest that environmental tumorpromoters or carcinogens, such as ionizing radiation, can cause stromalfibroblasts to activate TGF by epigenetic mechanisms. As thebiological effects of the anti-estrogen tamoxifen may well be mediated byTGF, this model has a number of important implications for the clinicaluses of tamoxifen in the prevention and treatment of breast cancer. Inaddition, it suggests a number of novel approaches to the treatment ofadvanced breast cancer.     

Effect of irradiation on transforming growth factor-β secretion by malignant glioma cells

Glioblastoma cells secrete transforming growth factor- (TGF-), whichhas a variety of immunosuppressive properties. We investigatedthe effect of irradiation TGF- secretion by malignantglioma cells. Three malignant glioma cell lines (T98G,A172, KG-1-C) were cultured and irradiated using 10and 50 Gy Linac radiation. After further culturefor 36 hours in serum-free culture medium, thesupernatants were collected. The TGF- activity in theculture supernatants was determined using a specific bioassay.The levels of the active form and totalTGF- in the supernatants from irradiated malignant gliomacells decreased compared to those from un-irradiated cells.However, since irradiation inhibited the growth of tumorcells, the amount of TGF- secretion per cellin irradiated cells tended to increase after irradiation.These results suggest that malignant glioma cells canstill secrete TGF- and activate latent TGF- evenafter large dose irradiation, despite the inhibition oftumor growth.     

Glioma Cell Invasion: Regulation of Metalloproteinase Activity by TGF-β

Matrix metalloproteinases (MMPs) are a family of extracellular endopeptidases that selectively degrade components of the extracellular matrix. MMPs are implicated in tumor cell invasion because they mediate the breakdown of the basal membrane. In addition, they seem to be important for the creation and maintenance of a microenvironment that facilitates tumor cell survival. Among the essential characteristics of human malignant gliomas are infiltrative growth, angiogenesis and suppression of antitumor immune surveillance. Transforming growth factor-beta (TGF-) is intimately involved in the regulation of these processes. We have previously demonstrated that TGF- promotes the migration of LN-18 and LN-229 glioma cells via a process that may involve the upregulation of _V3 integrin expression. Furthermore, we have defined a novel pathway for hepatocyte growth factor (HGF)-induced glioma cell migration and invasion which requires the induction of TGF-₂ expression. Here, we demonstrate that TGF-₂ induces MMP-2 expression and suppresses tissue inhibitor of metalloproteinases (TIMP)-2 expression and that concentration-dependently promotes the invasion of U87MG and LN-229 glioma cells in a matrigel invasion assay. Similarly, ectopic expression of the anti-apoptotic BCL-x_L protein leads to enhanced matrigel invasion by LN-18 and LN-229 glioma cells. We outline the possible interrelations of TGF-, proteins of the BCL-2 family, integrins and metalloprotease activity. By virtue of its promotion of glioma invasion and its growth regulatory and immunomodulatory properties, TGF- continues to be one of the most promising targets for the experimental therapy of human malignant glioma.     

Expression of estrogen receptor-beta in normal mammary and tumor tissues: is it protective in breast carcinogenesis?

Using messenger RNA (mRNA) in situ hybridization, we investigated estrogen receptor- (ER) mRNA levels in normal mammary, benign breast tumor (BBT), breast cancer (BC), and metastatic lymph node tissues to verify the role of ER in BC development and progression. ER expression was significantly decreased in BC and metastatic lymph node tissues compared with normal mammary and BBT tissues (p < 0.01). The intensity and extent of ER mRNA signals were also significantly lower in BC and metastatic lymph node tissues than in the normal mammary and BBT tissues (p < 0.01). An inverse relationship was found between ER mRNA level and both histologic grade (p = 0.091) and progesterone receptor expression (p = 0.052) with marginal significance, but no significant association was noted between ER expression in cancer tissues and the other clinico-pathologic data. The 3-year distant relapse-free survival probability was found to be independent of ER expression. Collectively, ER mRNA decreases in the process of BC development, but seems to be associated with poor differentiation.     

Additive effect of mifepristone and tamoxifen on apoptotic pathways in MCF-7 human breast cancer cells

MCF-7 cells growing in culture were used to study the mechanism of the antiproliferative activity of the antiprogestin mifepristone, as compared with the antiestrogen 4-hydroxytamoxifen or the combination of both. These steroid antagonists induced a significant time- and dose-dependent cell growth inhibition (cytotoxicity). This inhibition of cell survival was associated with a significant increase in DNA fragmentation (apoptosis), downregulation of bcl2, and induction of TGF1 protein. Abrogation of the mifepristone- and/or 4-hydroxytamoxifen-induced cytotoxicity by TGF1 neutralizing antibody confirms the correlation between induction of active TGF1 and subsequent cell death. The effect of a combination of mifepristone and 4-hydroxytamoxifen on cell growth inhibition, on the increase in DNA fragmentation, bcl2 downregulation, and induction of TGF1 protein was additive and significantly different (P < 0.05) from the effect of monotherapy. A translocation of protein kinase C (PKC) activity from the soluble to the particulate and/or nuclear fraction appeared to be also additive in cells treated with a combination of both 4-hydroxytamoxifen and mifepristone. These results suggest that the mechanism of the additive antiproliferative activity of mifepristone and tamoxifen could be explained at least in part by an additive induction of apoptosis in both estrogen and progesterone receptor positive MCF-7 breast cancer cells. A bcl2 downregulation, the PKC transduction pathway, and TGF1 expression seem to be involved in this additive mechanism of action. Our data further suggest that a combination of an antiprogestin with tamoxifen may be more effective than tamoxifen monotherapy in the management of human breast cancer.     

Interferon effect on glycosaminoglycans in mouse gliomain vitro

Summary The effect of mouse interferon / (MuIFN /) on the production of glycosaminoglycans (GAGs) by mouse glioma G-26in vitro was evaluated. Two GAG species secreted extracellularly by the mouse glioma G-26 were isolated using cellulose acetate electrophoresis. They were identified as hyaluronic acid (HA) and chondroitin sulfate (CS) following enzymatic digestion with enzymes: hyaluronidase and chondroitinase ABC. Further characterization of CS by enzymatic digestion with specific chondroitinases for chondroitin 4-sulfate (CSA) and chondroitin 6-sulfate (CSC), revealed that the isolated CS was neither CSA nor CSC. Therefore, it may be either chondroitin sulfate B (CSB) (dermatan sulfate) or one of the chontroitin sulfate isomers (D-H).The three day incubation of glioma G-26 cells with 8×10-8×104 U/ml of MuIFN / resulted in a dose dependent inhibition of cell proliferation measured by³H-thymidine incorporation and the MTT assay. The significant decrease of the CS (p < 0.008) but not the HA level, (measured densitometrically), was observed following 72 hours (hrs) incubation of G-26 cells with 8 × 10³ U/ml of MuIFN / (IFN treated cells: 0.03 ± 0.007 integrated optical density (IOD); control cells: 0.07 ± 0.01 IOD). The decreased CS production may be the underlying cause of IFN mediated inhibition of glioma cell proliferation.     

Transforming growth factor beta and the cell surface in tumor progression

Summary Type 1 transforming growth beta (TGF-1) is a multifunctional regulator of cellular differentiation, motility and growth. It is capable of inhibiting or stimulating these processes depending on cell type, cell density, culture conditions and TGF-1 concentration. TGF-1 regulates growth, in part, by inducing the expression and secretion of various types of collagen, which participate in the control of cell adhesion and migration, as well as growth. TGF-1 also regulates cell growth by controlling the response to epidermal growth factor (EGF) and other growth factors, in ways that can either decrease or increase their growth-promoting effects. Alterations in both negative and positive growth responses to TGF-1 play important roles in tumor progression. Loss of sensitivity to growth inhibition by TGF-1 can occur as a result of decreased expression of collagen. Acquisition of sensitivity to growth stimulation, and autocrine transformation by TGF-1, are associated with aberrant EGF receptor regulation. Aberrant growth factor receptor regulation by TGF-1 may be mediated by a protein kinase C (PKC)-dependent pathway which inhibits degradation of growth factor receptor/ligand complexes. The evidence reviewed is consistent with a minimal two-step mechanism for autocrine transformation, which involves production of growth factor and enhanced cellular response as a result of aberrant membrane traffic. Defects in membrane traffic regulation may provide an explanation for common alterations in tumor cell response to both multiple growth inhibitors and growth stimulators, and may also suggest novel approaches to cancer chemotherapy.