Effects of transforming growth factor β1 expression in a rat colon carcinoma: growth inhibition, leukocyte infiltration and production of interleukin-10 and tumor necrosis factor α

The cytokine transforming growth factor β-1 (TGFβ1), was transfected into a TGFβ1-negative rat colon carcinoma. The growth of isografts of TGFβ1-expressing tumors was compared to that of vector control transfectants. The TGFβ1 transfectant grew significantly more slowly after intrahepatic isografting than did vector control and wild-type tumors. The TGFβ1-transfected tumor tissue had significantly greater infiltration of both CD4⁺ and CD8⁺ T lymphocytes than did the vector control tumor. The tumor-infiltrating leukocytes (TIL) from TGFβ1-transfected tumor secreted significantly more of the cytokines interleukin-10 (IL-10) and tumor necrosis factor α (TNFα) than did TIL from the vector control tumor. The TGFβ1 transfectant also demonstrated a significantly slower outgrowth in immunodeficient SCID mice, supporting a non-T-lymphocyte-dependent mechanism for the tumor retardation. In SCID mice, the TGFβ1-transfected tumor demonstrated significantly greater infiltration of both granulocytes and macrophages than did the vector control transfectant. We also demonstrated a direct inhibitory effect of rat TNFα on tumor proliferation in vitro. These results suggest that TGFβ1 induces a local secretion of immunomodulating cytokines and that this may influence monocytes, lymphocytes and granulocytes to retard tumor outgrowth. Received: 7 July 1999 / Accepted: 12 August 1999     

Human ex vivo carcinoma cells produce transforming growth factor β and thereby can inhibit lymphocyte functions in vitro

 We tested 20 human carcinoma samples for the production of transforming growth factor β (TGFβ) in vitro. Tumour cell suspensions without obvious contamination with non-malignant cells were kept in culture conditions for 16 h and their supernatants were added to CCL-64 cells. The proliferation of these cells is inhibited by TGFβ. According to this assay, the supernatants contained both active and latent TGFβ. In addition, the supernatants were found to suppress the spontaneous cytotoxic function and activation of T-cell-enriched lymphocyte populations. A specific monoclonal antibody (mAb) counteracted these effects and therefore we concluded that they were mediated to a large extent by TGFβ. In line with the results obtained with the supernatants, activation of lymphocytes could also be inhibited by tumour cells and their inhibitory effect was weaker in the presence of the TGFβ-specific mAb. It is important to note that, when TGFβ-specific mAb was added to autologous mixed lymphocyte/tumour cell cultures, lymphocyte activation occurred more often. These results thus substantiate the assumption that production of TGFβ may help the survival of potentially immunogenic tumour cells in immunocompetent patients. Received: 21 August 1996 / Accepted: 12 November 1996     

TGFβ and bFGF synthesis and localization in Dupuytren''s disease (nodular palmar fibromatosis) relative to cellular activity, myofibroblast phenotype and oncofetal variants of fibronectin

Summary Nodular palmar fibromatosis is a self-limited proliferation of fibro-/myofibroblasts associated with growth factor synthesis and abundant fibronectin extracellular matrix deposition. bFGF and TGFβ are potent modulators of fibro-/myofibroblast proliferation and differentiation. Moreover,in vitro investigations evidenced a TGFβ1-dependent regulation of alternative splicing of fibronectin mRNA. To investigate a possible implication of these growth factors in the tissue formation process of palmar fibromatosis, TGFβ1/2 and bFGF synthesis, as well as TGFβ1/3 and bFGF tissue distribution, is demonstrated by RNAin situ hybridization and/or immunohistochemistry in relation to myofibroblast phenotype development (α-smooth muscle actin, desmin immunohistochemistry), expression of different fibronectin isoforms (ED-A⁺, ED-B⁺ and oncofetal glycosylated fibronectin immunohistochemistry, fibronectin RNAin situ hybridization) and cellular activity (cyclin RNAin situ hybridization, Ki-67 immunolabelling). The myofibroblast phenotype (α-smooth muscle actin, desmin), the growth factor synthesis (TGFβ1 and 2, bFGF), fibronectin matrix synthesis (RNAin situ hybridization with cDNA) and ED-A⁺, ED-B⁺ and oncofetal glycosylated fibronectin immunostaining are exclusively localized in the active proliferative nodules (Ki-67 immunolabelling and cyclin mRNA demonstration). Whereas the growth factor synthesis is restricted to the proliferative areas of the fibromatosis only, TGFβ1, TGFβ3 and bFGF proteins can also be detected immunohistochemically with a lower intensity in the surrounding aponeurotic tissue. The spatial correlation of myofibroblast phenotype, TGFβ and bFGF synthesis and the occurrence of the oncofetal molecular fibronectin variants (ED-B⁺ and oncofetal glycosylated fibronectin) in the active proliferative fibromatosis nodules suggests a pathogentic role of these growth factors and matrix components in the tumorous tissue formation process. The presence of the bFGF and TGFβ1/3 proteins in fibroblasts neighbouring the proliferative nodules may point to a recruitment of quiescent aponeurotic fibroblasts in the fibromatous tissue formation process.     

Transforming growth factor β , (TGFβ) secreted by immunogenic ex vivo human carcinoma cells, counteracts the activation and inhibits the function of autologous cytotoxic lymphocytes. Pretreatment with interferon γ and tumor necrosis factor α reduces the production of active TGFβ

 We present the results obtained with an in vitro model system that resembles the in vivo tumour microenvironment, where malignant cells are in close contact with the infiltrating lymphocytes. Unmanipulated blood lymphocytes were cytotoxic against the autologous ex vivo tumour cells in 3/19 patients and this function was generated in 6-day mixed cultures in five additional cases. Production of transforming growth factor β (TGFβ) by the freshly separated tumour cells was determined in parallel. Cytotoxicity was generated by a small number of tumour cells (2–5/100 lymphocytes), while a large number (10–20/100 lymphocytes) inhibited not only the generation but also the existing lytic activity. The presence of a neutralising TGFβ-specific mAb (2G7) potentiated the activation of lymphocytes and suspended the suppression inflicted by the tumour cells. In those tumours, which expressed relatively high levels of MHC class I and ICAM-1 molecules, the quantity of secreted TGFβ interfered with the ability of tumour cells to generate cytotoxic lymphocytes. In the tumours with low expression of class I, such a correlation was not detected, indicating the primordial role of MHC class I expression in the regulation of autologous tumour recognition. Our results demonstrate the involvement of TGFβ in the impaired lymphocyte-mediated reactivity against immunogenic tumours and support a mechanism that contrasts the tolerance or anergy. Since presence of TGFβ in the microenvironment of tumours counteracts the function of cytotoxic T lymphocytes, production of this cytokine can contribute to the failure of immunotherapy. Received: 19 June 1997 / Accepted: 14 August 1997     

Transglutaminase 2 silencing reduced the beta-amyloid-effects on the activation of human THP-1 cells

The aberrant expression and activation of transglutaminase 2 (TG2), the ubiquitous enzyme which catalyzes calcium-dependent protein cross-linking reactions, has been reported in many inflammatory diseases. Chronic inflammation, mediated by prolonged activation of brain-resident immunocompetent cells, appears to be involved in the pathogenesis of several age-related diseases, such as Alzheimer’s disease. Given that increased TG2 expression has been observed in AD brains, this study was aimed to characterize the role of TG2 in THP-1 monocytes stimulated with amyloid-beta (Aβ). Aβ_1–42 treatment dose-dependently increased TG2 expression in THP-1 cells. In particular, a fivefold up-regulation of TG2, compared with control cells, was observed in the presence of 0.5 μM Aβ_1–42. At the same concentration, Aβ_1–42 was able to promote monocyte maturation as suggested by increased expression of the cell surface antigen CD14 as well as the adhesion-promoting factor fibronectin. The stimulation of THP-1 cells with Aβ_1–42 also led to a significant up-regulation of tumor necrosis factor α (TNF-α) and matrix metalloproteinase 9 (MMP-9). Interestingly, THP-1 cell transfection with small interfering RNA directed against TG2 was able to reduce Aβ_1–42 increased levels of all the examined markers of monocyte maturation (CD14, fibronectin), and activation (TNF-α, MMP-9). These results indicate that TG2 up-regulation is required for the functional THP-1 monocyte activation induced by Aβ_1–42. This work suggests that TG2 inhibition may represent a therapeutic target to ameliorate the inflammation and progression in Alzheimer’s disease.     

Transforming growth factor β3 in the fetal and neonatal rat testis: immunolocalization and effect on fetal Leydig cell function

The localization of transforming growth factor β3 (TGFβ3) in the fetal and neonatal testis (from fetal day 13.5 to postnatal day 6) was investigated by immunohistochemical staining with a specific polyclonal antibody raised against a synthetic peptide corresponding to residues 50–75 of TGFβ3. This antibody recognized 0.5 ng TGFβ3 in western blot analysis, but did not detect 25 ng TGFβ1 or TGFβ2. The immunolocalization of TGFβ3 in the fetal and neonatal testis changed throughout development. Immunostaining was present in the gonocytes by fetal day 13.5, persisted until postnatal day 3, and was heterogeneous in spermatogonia on postnatal day 6. The Sertoli cells contained no immunoreactivity at any age. The fetal-type Leydig cells were first immunostained for TGFβ3 on day 16.5 and staining became very intense from day 18.5 onward. Staining disappeared when the antibody was presaturated with the synthetic peptide, but persisted when the antibody was presaturated with a tenfold excess of the corresponding peptide from TGFβ2. Furthermore, we researched whether TGFβ3 could act as a local regulator of fetal Leydig cell function. In a dispersed fetal testicular cell system, TGFβ3 inhibited the LH-stimulated testosterone production by Leydig cells from 20.5-day-old fetuses. The inhibitory effect of TGFβ3 was equal to that observed with TGFβ1 or TGFβ2. When compared with our previous studies showing the immunolocalization of TGFβ1 and TGFβ2, the present study shows that TGFβ3 may have a specific role in the developing rat testis, but may also overlap the action of TGFβ1 and TGFβ2. Accepted: 8 June 1999     

Activity identification of ribozyme and U1 snRNA chimeric ribozyme against TGFβ1 in cell-free system and in hepatic stellate cells

Transforming growth factorβ1 (TGFβ1) is known to be intimately involved in many cellular processes. To explore the mechanism of TGFβ1 in these processes, the non-chimeric hammer-head ribozyme and U1 snRNA chimeric ribozyme against TGFβ1 were designed to down-regulate TGFβ1 expression. The activity of non-chimeric ribozyme and U1 snRNA chimeric ribozyme against TGFβ1 in vitro and in activated hepatic stellate cells (HSCs) was detected. Cleavage reactions of both ribozymes in vitro demonstrated that non-chimeric ribozyme possessed better cleavage activity in vitro than U1 snRNA chimeric ribozyme. The further study showed U1 snRNA chimeric ribozyme inhibited TGFβ1 expression more efficiently than non-chimeric ribozyme in transfected HSC cells. So it indicates that the U1 snRNA chimeric ribozyme provides an alternative approach for the research on the precise mechanism of TGFβ1 in many cellular processes and a potential therapeutic candidate for TGFβ1-related diseases.     

Fibroblast migratory factor derived from mouse colon carcinoma cells: potential roles of fibronectin in tumor stroma formation

Mouse colon carcinoma cell line colon 38 was used to investigate migratory factor for fibroblasts because marked fibrotic tissue was associated with these cells growing at transplanted sites and liver metastases in vivo. Migration-inducing activity was detected in the serum-free culture supernatants of colon 38 cells, as shown by the Boyden chamber assays using NIH3T3 cells as target cells. The active substance was partially purified by a combination of anion-exchange, hydrophobic, and gel-permeation chromatography. An approximate relative molecular mass of the active substance was estimated to be between 100,000 and 400,000, judging from the eluting position in the gel-permeation chromatography. The migratory activity in the partially purified preparation was removed by incubation with beads coated with an anti-mouse fibronectin antibody. NIH3T3 cells incubated in the presence of culture supernatants of colon 38 cells exhibited higher growth rate, organized actin filaments, and increased chondroitin sulfate and hyaluronan. Fibronectin did not elicit such effects and partially purified migratory factor showed relatively low activity in these regards. Thus, colon 38 cells seem to secrete fibronectin and other soluble substances, which induce tumor stroma formation through migration and activation of host fibroblasts.     

Activity identification of ribozyme and U1 snRNA chimeric ribozyme against TGFβ1 in cell-free system and in hepatic stellate cells

Transforming growth factorβ1 (TGFβ1) is known to be intimately involved in many cellular processes. To explore the mechanism of TGFβ1 in these processes, the non-chimeric hammerhead ribozyme and U1 snRNA chimeric ribozyme against TGFβ1 were designed to down-regulate TGFβ1 expression. The activity of non-chimeric ribozyme and U1 snRNA chimeric ribozyme against TGFβ1 in vitro and in activated hepatic stellate cells (HSCs) was detected. Cleavage reactions of both ribozymes in vitro demonstrated that non-chimeric ribozyme possessed better cleavage activity in vitro than U1 snRNA chimeric ribozyme. The further study showed U1 snRNA chimeric ribozyme inhibited TGFβ1 expression more efficiently than non-chimeric ribozyme in transfected HSC cells. So it indicates that the U1 snRNA chimeric ribozyme provides an alternative approach for the research on the precise mechanism of TGFβ1 in many cellular processes and a potential therapeutic candidate for TGFβ1-related diseases.     

Ligand-dependent and -independent interactions with the transforming growth factor type II and I receptor subunits reside in the aminoterminal portion of the ectodomain of the type III subunit

Summary The type III receptor for transforming growth factor beta (TGFβ), which exhibits no kinase activity, binds TGFβ1 and TGFβ2 and is involved in assembly and activity of the multi-subunit TGFβ signal transduction complex. Recently we showed that TGFβ receptor type III (TβRIII) can participate in a complex composed of the dimeric TGFβ ligand and a type III, II, and I receptor subunit. The interaction of the TβRIII subunit with TβRII is TGFβ-dependent, whereas interaction with TβRI is TGFβ-independent. Here we use coexpression of the three types of TGFβ receptors in baculoviral-infected insect cells to determine which parts of the unglycosylated TβRIII receptor participate in the binding of TGFβ, the TGFβ-dependent interaction with TβRII and the TGFβ-independent interaction with TβRI. The results suggest that the first 500 amino acid residues in the aminoterminal portion of TβRIII exhibit all three properties.     

The Combination of Selenium and Vitamin E Inhibits Type I Collagen Formation in Cultured Hepatic Stellate Cells

This study investigated the effects of sodium selenite (Se) and of vitamin E (d-α-tochopherol) on the deposition of type I collagen by human LX-2 stellate cells. The cultured cells were treated with or without Se or vitamin E and with or without transforming growth factor β1 (TGFβ1). The combination of Se and vitamin E, but not either alone, protected against hepatic fibrosis by decreasing TGFβ1-mediated collagen secretion and accumulation by the stellate cells. This protective effect is due to a combination of decreased formation, decreased stability and increased degradation of the collagen. Effects of Se and vitamin E in decreasing α₁(I) collagen mRNA and increasing apoptosis of stellate cells indicate decreased formation of collagen, while decreases in transglutaminase 2, which catalyze cross-linking of collagen, lead to decreased stability of the secreted collagen. Effects of Se and vitamin E on reducing tissue inhibitor metalloproteinase 1 (TIMP-1) are associated with increased degradation. The combination of Se and vitamin E decreased lipid peroxidation, while Se alone increased the activity of the antioxidant enzyme thioredoxin reductase. In conclusion, the combination of Se and vitamin E protected against TGFβ1-mediated hepatic fibrosis by decreasing TGFβ1-mediated type I collagen accumulation by stellate cells. This effect is due to a combination of decreased formation, decreased stability and increased degradation of the collagen.     

Restoration of interleukin-2 production in tumor-bearing rats through reducing tumor-derived transforming growth factor &;b by treatment with bleomycin

 We studied mechanisms of immunosuppression caused by tumor-derived transforming growth factor-β (TGFβ) and restoration of the immune response by treatment with bleomycin in rats bearing KDH-8 hepatoma. Interleukin-2 (IL-2) production from splenocytes of KDH-8-tumor-bearing rats progressively decreased as the KDH-8 tumor grew. IL-2 production from concanavalin-A-stimulated normal rat splenocytes was signficiantly inhibited by in vitro cultured KDH-8-tumor-cell-conditioned medium; this inhibition could be blocked by neutralizing the conditioned medium with anti-TGFβ antibody. TGFβ activities were found in KDH-8-tumor-tissue-conditioned medium without acid treatment and were found in tumor-cell-conditioned medium after acid treatment; TGFβ mRNA and TGFβ protein were found in cultured KDH-8 tumor cells. These results suggested that the KDH-8-tumor-derived TGFβ might be involved in the inhibition of IL-2 production from splenocytes. To determine whether bleomycin chemotherapy could reduce tumor-derived TGFβ and restore the immune responses, we treated KDH-8 tumor-bearing rats with bleomycin (5 mg/kg, one shot) at an appropriate time (before the occurrence of immunosuppression) resulting in a significiant reduction of TGFβ activity in KDH-8 tumor tissues and restoration of IL-2 production from splenocytes of tumor-bearing rats; KDH-8 tumor growth ultimately regressed. In vitro experiments also showed that TGFβ activity, mRNA expression, and protein synthesis in KDH-8 tumor cells were reduced by bleomycin treatment, and that bleomycin-treated-KDH-8-tumor-cell-conditioned medium did not inhibit IL-2 production from normal rat splenocytes. These results suggest that bleomycin treatment restored IL-2 production in tumor-bearing rats through reducing the tumor-derived TGFβ. Received: 12 June 1995 / Accepted: 3 November 1995     

In vitro wounding: effects of hypoxia and transforming growth factor β<Subscript>1</Subscript> on proliferation,migration and myofibroblastic differentiation in an endothelial cell-fibroblast co-culture model

The adequate reconstitution of human soft tissue wounds requires the coordinated interaction of endothelial cells and fibroblasts during the proliferation phase of healing. Endothelial cells assure neoangiogenesis, fibroblasts fill the defect and provide extracellular matrix proteins, and myofibroblasts are believed to support the reconstitution of microvessels. In the present study, we combined in vitro-wound size measurement and multicolour immunocytochemical staining of co-cultured human dermal microvascular endothelial cells and normal human dermal fibroblasts, recently introduced as co-culture scratch-wound migration assay. Applying antibodies for α-smooth-muscle actin, von Willebrand factor, extra domain A fibronectin and endothelin-1, we were able to monitor proliferation, migration and the differentiation process from fibroblasts to myofibroblasts as a response to hypoxia. Furthermore, we verified, whether transforming growth factor β₁ (TGFβ₁) and endothelin-1 are able to mediate this response. We show, that proliferation and migration of endothelial cells and fibroblasts decreased under hypoxia. The additional administration of TGFβ₁ did not significantly attenuate this decrease. Solely the myofibroblast population in co-culture adapted well to hypoxia, when cultures were supplemented with TGFβ₁. Considerating the data concerning TGFβ₁ and endothelin-1, we propose a model explaining the cellular interaction during early and late proliferation phase of human wound healing.     

Overexpression of transglutaminase 2 accelerates the erythroid differentiation of human chronic myelogenous leukemia K562 cell line through PI3K/Akt signaling pathway

Transglutaminase 2 (TG2) is a GTP-binding protein with transglutaminase activity. Despite advances in the characterization of TG2 functions and their impact on cellular processes, the role of TG2 in Human chronic myelogenous leukemia K562 cell line is still poorly understood. To understand the biological significance of TG2 during the differentiation of K562 cells, we established and characterized K562 cells that specifically express TG2. Non-transfected K562 cells showed the increase of membrane-bound-TG2 level after 3 days in the response to Hemin and all trans-retinoic acid (tRA), indicating that membrane recruitment of TG2 is occurred during the erythroid differentiation. However, membrane recruitment of TG2 in TG2-transfected cells revealed within earlier time period, compared with that in vector-transfected cells. The ability of membrane-bound-TG2 to be photoaffinity-labeled with [alpha-32P]GTP was also increased in TG2-transfected cells. TG2-transfected cells activated Akt phosphorylation and inactivated ERK1/2 phosphorylation, compared with vector-transfected cells. Furthermore, phosphorylation of CREB, one of the Akt substrates, was increased in TG2-transfected cells and this phenomenon was confirmed by RT-PCR analysis of several marker genes related with erythroid lineage in the absence of PI3K specific inhibitor, Wortmannin, indicating that PI3K/Akt signaling pathway also involved in the differentiation of the cell. Finally, as results of benzidine positive staining as well as hemoglobinization analysis, overexpression of TG2 revealed acceleration of the erythroid differentiation of K562 cells. Taken together, there was no increased TG2 expression level in the response of Hemin/tRA and delayed differentiation in vector transfected cells than in TG2-transfected cells, suggesting that suppression of TG2 expression may retard the erythroid differentiation of K562 cells. Therefore, our study may give a new insight for another aspect of the development of this disease.     

Expression of HSP27, HSP72 and MRP proteins in <Emphasis Type="Italic">in vitro</Emphasis> co-culture of colon tumour cell spheroids with normal cells after incubation with rhTGF-<Emphasis Type="Italic">β</Emphasis>1 and/or CPT-11

We studied the expression of inducible heat shock protein (HSP27, HSP72) and multidrug-resistance protein (MRP) in co-cultures of human colon carcinoma cell spheroids obtained from different grades of tumour with normal human colon epithelium, myofibroblast and endothelial cell monolayers. We also measured the influence of recombinant human transforming growth factor β1 (rhTGF-β1) and camptothecin (CPT-11), added as single agents or in combination, on the levels of the HSPs, MRP, interleukin (IL)-6 and nitric oxide (NO). An immunoblotting analysis with densitometry showed that rhTGF-β1 and/or CPT-11 increased HSP27, HSP72 and MRP expression in tumour cells and myofibroblasts, as well as in co-cultures compared with appropriate controls. By contrast, in colonic epithelium, inhibition of HSPs and MRP was comparable with that of the control. In endothelial cells, HSP72 was undetectable.     

Pioglitazone inhibits TGFβ induced keratocyte transformation to myofibroblast and extracellular matrix production

Phenotype transformation of corneal keratocyte to myofibroblast plays an important role in the wound healing process of cornea and TGFβ is considered to be the most important mediator to induce myofibroblast trans-differentiation. Peroxisome proliferator-activated receptors-γ (PPAR-γ) activation has been proved to exert anti-fibrotic effect in many tissues. In this study, we investigated the effect of PPAR-γ agonist, pioglitazone, on myofibroblast transformation, extracellular matrix production and cell proliferation. The results showed pioglitazone inhibited the TGFβ-driven myofibroblast differentiation, as determined by F-actin fluorescence staining, α-smooth muscle actin-specific immunocytochemistry and western blot analysis. Pioglitazone also potently attenuated TGFβ induced type I collagen and fibronectin mRNA and protein production. Moreover, pioglitazone showed inhibitory effect on TGFβ induced cell proliferation. The irreversible PPAR-γ antagonist GW9662, partially reversed the inhibition of collagen I and fibronectin expression but not myofibroblast transformation, suggesting both PPAR-γ dependent and PPAR-γ independent mechanisms were involved in the action of pioglitazone. Therefore, our study indicates pioglitazone has a potential application in therapy of corneal fibrosis and PPAR-γ might be a promising therapy target.     

Use of a DNA microfluorometric assay to measure proliferative response of mink lung cells to purified TGFβ and to TGFβ activity found in prostate cell conditioned medium

Summary The proliferative response of Mv1Lu cells to purified TGFβ₁, or TGFβ-like activity released by various cells into medium conditioned over a 24-h period was quantitated by adapting a rapid DNA fluorometric assay. Acid activation of the conditioned medium allowed the amount of biologically latent versus active TGFβ to be quantitated. A neutralizing antibody specific for TGFβ_{1, 1.2, and 2.0} completely blocked the growth inhibition observed treating Mv1Lu cells with either purified TGFβ₁ or medium containcing secreted TGFβ-like activity conditioned by DU145 prostate cells. In contrast to other assays commonly used to measure TGFβ activity, the proliferative response is related directly to DNA content rather than as a reflection of enzymatic activity or incorporation of³H-thymidine. The necessity for radioactive isotope usage has been eliminated, and the biological response can be quantitated over a period of days.     

Interferon γ and antisense transforming growth factor β transgenes synergize to enhance the immunogenicity of a murine mammary carcinoma

Transforming growth factor β (TGFβ) is an immunosuppressive cytokine that contributes to the immunological escape of tumor cells. In a previous study we demonstrated that inhibition of TGFβ production by EMT6 murine mammary tumor cells expressing an antisense TGF-β transgene reduces their tumorigenicity. On the basis of this observation we hypothesized that down-regulation of TGFβ production coupled with interferon γ (IFNγ) stimulation would induce an immune response superior to that generated by either strategy alone. In this study, EMT6 tumor cells expressing antisense TGFβ were transduced with the murine IFNγ gene. Tumor cells expressing either or both transgenes grew more slowly than mock-transduced tumors. Dual-transgene-expressing tumor cells were more immunogenic than tumor cells expressing either transgene alone. Studies in mice depleted of T cell subsets indicated that CD8⁺ T cells are the primary effectors of the antitumor activity observed. These results suggest that down-regulation of immunosuppression combined with cytokine-mediated immune augmentation is a useful strategy to improve antitumor immunity. Received: 6 October 1998 / Accepted: 15 January 1999     

Matrix Metalloproteinase-2 Cleavage of the β1 Integrin Ectodomain Facilitates Colon Cancer Cell Motility

Cancer cell invasion is a key element in metastasis that requires integrins for adhesion/de-adhesion, as well as matrix metalloproteinases (MMPs) for focalized proteolysis. Herein we show that MMP-2 is up-regulated in resected colorectal tumors and degrades β1 integrins with the release of fragments containing the β1 I-domain. The β1 cleavage pattern is similar to that produced by digestion of α5β1 and α2β1 with MMP-2. Two such fragments, at 25 and 75 kDa, were identified after immunoprecipitation, with monoclonal antibody BD610468 reacting with the NH₂-terminal I-like ectodomain followed by SDS-PAGE and microsequencing using electrospray (ISI-Q-TOF-Micromass) spectrometry. Cleavage of the β1 integrin can be abolished by inhibition of MMP-2 activity; it can be induced by up-regulation of MMP-2 expression, as exemplified by HT29 colon cancer cells transfected with pCMV6-XL5-MMP-2. Co-immunoprecipitation studies of colon cancer cells showed that the β1 integrin subunit is associated with MMP-2. The MMP-2-mediated shedding of the I-like domain from β1 integrins resulted in decreased adhesion of colon cancer cells to collagen and fibronectin, thus abolishing their receptivity. Furthermore, such cells showed enhanced motility as evaluated by a “wound healing-like” assay and time-lapse microscopy, indicating their increased invasiveness. Altogether, our data demonstrate that MMP-2 amplifies the motility of colon cancer cells, not only by digesting the extracellular matrix components in the vicinity of cancer cells but also by inactivating their major β1 integrin receptors.