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.     

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.     

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.     

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)     

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.     

TGFβ and Wnt pathway cross-talk

Transforming growth factor-s (TGF) and Wnts represent two distinct families of secreted molecules each of which utilizes different signaling pathways to elicit their biological effects. These factors regulate numerous developmental events and mutations in components of both pathways have been described in human cancers including colorectal carcinomas. Several studies have demonstrated that TGF and Wnt ligands can cooperate to regulate differentiation and cell fate determination by controlling gene expression patterns. In addition, their cooperation in promoting tumorigenesis in mice has been described. Here, we focus on reviewing our current understanding of the molecular mechanisms that may mediate these cooperative effects.     

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.     

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

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

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.     

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.     

Role of transforming growth factor beta in the growth inhibition of human breast cancer cells by basic fibroblast growth factor

Recent studies from our laboratory have revealed that basic fibroblast growth factor (bFGF) selectively inhibits the proliferation of human MCF-7 breast cancer cells. It has also been shown to enhance cis-platinum-induced apoptosis, decrease levels of the anti-apoptotic gene product bcl-2, and increase levels of the cyclin-dependent protein kinase inhibitor p21/WAF1/Cip1. Transforming growth factor beta-1 (TGF₁), a cell growth regulator has been found to have an inhibitory effect on breast cancer cells. The aim of the present study was to evaluate the possible role of TGF₁ in the antiproliferative effects of bFGF in MCF-7 breast cancer cells. We found that exogenous, as well as endogenous (overexpressed) bFGF increased TGF₁ mRNA expression in the cells and enhanced the secretion of TGF₁ into culture medium. However, exogenous addition of TGF₁ neither led to a decrease in bcl-2 nor induced an increase in the levels of p21/WAF1/Cip1 and neutralizing antibodies to TGF₁, did not reverse bFGF-induced G₁ arrest nor the increase in p21/WAF1/Cip1 level. In contrast, antisense oligonucleotides to TGF₁ abrogated the antiproliferative effects and inhibited the induction of p21/WAF1/Cip1 by bFGF in MCF-7 cells. These data suggest that the anti-proliferative effects of bFGF in human MCF-7 breast cancer cells are mediated by endogenous TGF₁, while exogenous TGF₁ does not mimic all the effects of bFGF on these breast cancer cells. These findings provide an important basis for further investigations into the autocrine and paracrine processes that control the growth of breast cancer cells.     

Escape from negative regulation of growth by transforming growth factor β and from the induction of apoptosis by the dietary agent sodium butyrate may be important in colorectal carcinogenesis

There are a number of lines of evidence suggesting that transforming growth factor (TGF) has an important role in the control of intestinal growth and differentiation.In vivo localization studies show that TGF expression occurs predominantly in the differentiated non proliferating cells of the intestinal epithelium. The use of an antisense expression vector for TGF resulted in an increased tumorigenicity in an antisense-transfected cancer cell line.In vitro proliferation studies showed colorectal premalignant adenoma cells to be more sensitive to the growth inhibitory effects of TGF than colorectal cancer cells. Furthermore the conversion of an adenoma to a carcinoma was accompanied by a reduced response to the inhibitory effects of TGF. The acquisition of partial or complete resistance to the inhibitory effects of TGF may be an important late event in colorectal carcinogenesis.Of further interest is the possibility that clonal selection could occur even more rapidly in colorectal tumour cells which not only had lost response to TGF inhibition but produced TGF and were growth stimulated by it. This could have the advantage of not only inhibiting the growth of surrounding less malignantly advanced cells but of also escaping from their potential growth suppressive influence. Carcinogenesis is not, however, simply losing response to negative regulators of growth; the fully malignant cell has to acquire new characteristics of invasiveness and metastatic potential. Growth factors including TGF may have a role in the complex cascade of events leading to the activation of proteolytic enzymes which are involved in progression to an invasive phenotype.Cell proliferation in the large bowel, as well as being under the control of endogenous growth factors, is also under the influence of dietary components in the lumen such as the naturally occurring fatty acid sodium butyrate. Sodium butyrate at physiological concentrations induces apoptosis (programmed cell death) in colonic tumour cell lines. Since sodium butyrate occurs naturally in the colorectum, being produced by bacterial fermentation of dietary fibre, it may be involved in the control of cell death in human colorectal epithelium. This could, in part, explain the apparent protective effects of dietary fibre. Clonal evolution and tumour progression in colorectal carcinogenesis could therefore involve loss of response to endogenous growth factors such as TGF and an escape from the induction of programmed cell death by dietary factors.     

Glycogen‐rich carcinomas of the breast display unique characteristics with respect to proliferation and the frequency of oligonucleosomal fragments

We determined the proliferation rate and apoptotic activity of glycogenrich carcinomas of the breast as opposed to nonclear cell tumors by means of MIB1 immunohistochemistry and in situ detection of oligonucleosomal fragments (TUNEL reaction). The retrospective biopsy series included six invasive clear cell carcinomas of the glycogenrich type as well as 15 randomly selected cases of invasive ductal carcinoma without evidence of glycogen storage. Three patients in the clear cell group and seven patients in the control cohort developed lymphnode metastasis. The MIB1 labeling index of glycogenrich carcinomas averaged 9.05%, while that of the controls was 30.03%. Apoptotic nuclei were present in a mean of 1.26% of glycogenrich carcinoma cells. The control tumors exhibited an average apoptotic frequency of 5.85%. Tumor size, hormone receptor status, and presence or absence of lymph node involvement were found not to correlate with either proliferation or apoptosis. We conclude that glycogenrich breast carcinomas are characterized by a peculiar low proliferationlow apoptosis cell kinetic profile. The aggressive clinical behavior of these neoplasms may possibly be accounted for by an ineffective apoptotic elimination of otherwise slowly proliferating tumor cells.     

Human Astrocytomas Co-expressing Fas and Fas Ligand also Produce TGFβ2 and Bcl-2

Human astrocytomas frequently co-express Fas (APO-1/CD95) and Fas ligand (FasL), yet do not appear to be overly susceptible to suicidal, fratricidal and immune-mediated elimination. This suggests that these gliomas have acquired mechanisms to prevent Fas-mediated apoptosis from occurring. Candidates for such a role include transforming growth factor-(₂ (TGF2) and B-cell lymphoma/leukemia-2 (Bcl-2). TGF2 effectively functions by hiding tumor cells from the immune system. This may potentially prevent the delivery of FasL from cytolytic T cells to Fas bearing astrocytomas. Bcl-2 works by rendering gliomas resistant to Fas-mediated apoptosis. Using immunohistochemistry, we analyzed seventy-six human astrocytomas (11 World Health Organization (WHO) grade I, 17 grade II, 17 grade III, and 31 grade IV) for the expression of Fas, FasL, (TGF₂) and Bcl-2 in vivo. Positive immunoreactivity was found to significantly increase with increasing tumor grade for Fas (p<0.0002), FasL (p<0.0001), TGF2 (p<0.001) and Bcl-2 (p<0.01). In addition, Fas/FasL co-expression, a counter-intuitive combination of factors in regards to glioma survival, also increased with WHO grade. Forty-five of 76 (59%) astrocytomas co-expressed Fas and FasL. Of those co-expressing Fas and FasL, 44 of 45 (98%) produced TGF2, and 26 of 45 (58%) expressed Bcl-2. We found a significant positive correlation between Fas/FasL co-expression and TGF2 (p<0.002) and Bcl-2 (p<0.005) production. We conclude that Fas and FasL are frequently co-expressed in human astrocytomas and these tumors are likely to produce other immunosuppressive and antiapoptotic factors such as TGF2 and Bcl-2.     

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.     

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.     

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.     

Phase II of doxorubicin/taxol in metastatic breast cancer

Purpose. To assess the response rate, survival, and toxicity of Taxol®(paclitaxel) as 1h infusion plus doxorubicin as firstline treatment for patients with metastatic breast cancer (MBC).Patients and methods. Seventysix patients with untreated MBC were recruited. All of them had measurable disease and were evaluable for toxicity. Fiftyfive percent of the patients had visceral involvement. The dose of doxorubicin was fixed at 50mg/m² as a short intravenous infusion, followed by 200mg/m² of Taxol as a 1h intravenous infusion. Doxorubicin was administered during the first seven cycles, continuing with Taxol only up to a maximum of ten cycles.Results. Neutropenia was the most important toxicity: 30% grade 3 and 18% grade 4. Only 2 patients showed a decrease in the left ventricular ejection fraction (LVEF) which caused discontinuing the treatment. No clinical congestive heart failure (CHF) was observed. Seventyfour patients were eligible for response evaluation: 10 (14%) achieved complete response (CR) and 46 (62%) achieved partial response (PR). The mean duration of response was 13.47± 1.35 months (95% confidence interval (CI): 10.82; 16.12) and the mean survival was 21.50± 1.42 months (95% CI: 18.72; 24.29).Conclusion. The overall response (OR) rate was 76%. No CHF was assessed and 2 patients stopped treatment due to LVEF decrease. Although doxorubicin 50mg/m² followed by Taxol 200mg/m² in 1h intravenous infusion presents a toxicity profile which demands a close followup, it represents a convenient outpatient schedule with similar activity rate compared to longer Taxol infusions.     

β-Glucuronidase levels in patients with fibrocystic breast disease

Certain enzymes in tissues and body fluids may, through reversal of the detoxification process, influence the composition and availability of steroid hormones, toxins, and carcinogens. The ubiquitous enzyme -glucuronidase, which hydrolyzes glucuronide conjugates, thereby reversing one of the main detoxification and excretion pathways, was found to vary in concentration in different cysts over a 300-fold range. The distribution was a continuum, devoid of discrete sub-populations. Evidence obtained on selected cyst fluids of high and low -glucuronidase activities indicated that the level of the enzyme significantly influenced the ratio of unconjugated: glucuronidated estradiol. The patients with fibrocystic breast disease fell into 2 distinct subpopulations on the basis of their serum -glucuronidase activity. In one group the activity was near normal, while in the second group the average serum -glucuronidase activity was 3-fold higher than in the women who did not have benign breast disease.