Biomedical subjects

B C Liu

Publications and source records attributed to B C Liu.

At least 37 records · Page 2Linked to original sources

PubMed1997

Development of esophageal metaplasia and adenocarcinoma in a rat surgical model without the use of a carcinogen.

In order to establish an animal model for studying the cause and prevention of esophageal adenocarcinoma (EAC) and its frequent precursor, Barrett's esophagus (BE), factors affecting the pathogenic processes were investigated in an esophagoduodenal anastomosis model with rats. Experiments by us and others have shown that surgical treatment produced reflux esophagitis with cell hyperproliferation, but not EAC. Additional treatment with a carcinogen has been shown to be necessary for the development of EAC, squamous cell carcinomas (SCC) or EAC/SCC mixtures. We found that the surgically treated animals developed anemia due possibly to reduced iron absorption. When the operated animals were supplemented with iron, EAC occurred at a high rate (73%) after 30 weeks, and treatment with N'-nitrosonornicotine did not enhance the rate of tumorigenesis. Treatment with carcinogen, however, induced SCC in the group of rats killed after 22 weeks. The results suggest that iron overload, which is known to cause oxidative damage, is an enhancing factor for adenocarcinogenesis. The pathogenesis of EAC in the iron-supplemented, non-carcinogen treated group resembles human esophageal adenocarcinogenesis in many features. All the BE was the specialized type with goblet cells (containing sialomucin or sulfomucin) and columnar cells (containing acid or neutral mucin) as well as an incompletely developed brush border. Almost all of the BE was located at the bottom of the esophagus and was continuous with the duodenal mucosa; dysplasia became more frequent at later time points. All of the cancers were well-differentiated mucinous EAC, and most of the EAC had an adjacent area of BE with dysplasia. The results are consistent with the proposed human sequence for pathogenic events of BE progression to 'BE with dysplasia' and then to EAC. Esophagoduodenal anastomosis and iron treatment in rats produces a high rate of BE and EAC which are morphologically similar to human BE and EAC; this may be a useful animal model to study the development and prevention of EAC in humans.

Adenocarcinoma↗

PubMed1996

Role of prostatic basal cells in the regulation and suppression of human prostate cancer cells.

Cytokeratin expression in normal and malignant prostatic tissue indicates a loss of basal epithelial cells in cancer. We investigated the ability of basal-like prostatic epithelial cells to inhibit the growth of prostatic cancer cells. Human prostate LNCaP cells were grown in medium with or without 10 nM dihydrotestosterone (DHT) on plastic culture dishes or on extracellular matrix derived from basal-like epithelial cells (primary cultures derived from normal peripheral zone of the prostate) that were grown with or without 10 nM DHT. Colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays were used to assess the growth of LNCaP cells. On plastic dishes, growth of LNCaP cells was increased 5-10% by the presence of DHT in the medium. On matrix derived from basal-like cells that were grown in the absence of DHT, growth of LNCaP cells with or without DHT was similar to that on plastic. However, on matrix derived from basal-like cells that were grown with DHT, growth of LNCaP cells was suppressed when compared to all other culture conditions (P < 0.01). To determine whether basal-like cells could alter the function of LNCaP cells, we measured prostate-specific antigen (PSA) mRNA expression with the use of comparative RT-PCR. We found a significant decrease in the mature PSA transcript in cells grown on matrix derived from basal-like cells that were grown with DHT. The expression of PSA transcript was not altered in LNCaP cells that were grown on matrix derived from basal-like cells that were grown in the absence of DHT. Furthermore, using differential display of mRNA, we demonstrated that there were induction and suppression of multiple unique transcripts in the LNCaP cells when grown on the various culture conditions. To determine a possible mechanism for these observations. We used a dot blot immunoassay for several known inhibitory factors. We determined that DHT can induce the basal-like cells to secrete transforming growth factor-beta (TGF-beta 1), and that TGF-beta 1 can inhibit the proliferation of LNCaP cells in a dose dependent manner. We conclude that basal-like epithelial cells, in the presence of DHT, secrete an extracellular matrix o matrix associated factor(s), e.g. TGF-beta 1, that suppresses proliferation and function of prostate cancer cells. Our data suggest that the disappearance of the basal cell layer may be a prerequisite for the progression of prostatic neoplasia.

Base Sequence↗

PubMed1996

Specific sequences of fibronectin activate the protein kinase C signal transduction pathway in invasive bladder cancer.

The mechanism of human bladder cancer cell invasion is not clear, but it appears that extracellular matrix components, such as fibronectin, may be involved. To investigate the role of fibronectin in tumor cell invasion and progression, we used an in vitro invasion assay to define the motility stimulating fragment of fibronectin for invasive human bladder cancer T24 cells. Using a modified Boyden chamber assay and purified fragments of fibronectin, we demonstrated that both the 120 kDa chymotrypsin generated fragment of fibronectin (containing the cell attachment RGD motif and additional sequences towards the carboxyl-terminal heparin binding domain), as well as the trypsin generated 60 kDa fragment of fibronectin (containing the carboxyl-terminal heparin binding domain and additional sequences towards the cell attachment RGD motif), were able to stimulate the migration of invasive human bladder cancer T24 cells. Control fragments containing only the amino-terminal gelatin binding region of fibronectin did not stimulate the motility of the human bladder cancer T24 cells. To determine the molecular mechanism in which these fragments may stimulate the migration of the T24 cells, we assayed for intracellular signal transduction pathway protein kinase C (PKC). We demonstrated that both the 120 kDa and the 60 kDa fragments were able to stimulate the activation of protein kinase C. Non-motility stimulating fragments of fibronectin were not able to activate protein kinase C. We conclude that the PKC signal transduction pathway may be involved in matrix mediated motility, and suggest that the inhibition of such pathway(s) may alter the malignant phenotype of human bladder cancer.

Amino Acid Sequence↗

PubMed1996

Ha-ras oncogene expression abrogates a pH dependent endonuclease activity of apoptosis in normal rat kidney cells.

To investigate the role of oncogene expression in the resistance to tumor necrosis factor-alpha (TNF), we transfected the mutated T24-Ha-ras oncogene into the murine kidney cell line NRK and an alternative murine cell line C127 cells. The resulting transfectants, NRK-Ha and HC127, were assayed for TNF mediated cytotoxicity. Cellular cytotoxicity of 45% over 48 h occurred with the NRK cells. However, ras transfectant NRK-Ha cells demonstrated 0% cytotoxicity over the same period. Both C127 cells and the ras transfectant HC127 demonstrated 40% and 25% cytotoxicity, respectively, over 48 h when incubated with TNF. Furthermore, DNA isolated from NRK, C127, HC127, but not NRK-Ha cells revealed the presence of DNA fragmentation 'ladders' indicative of successful apoptosis when the cells were incubated with TNF. To determine the possible mechanism in which the ras oncogene may have protected the NRK-Ha cells from TNF mediated cytotoxicity and apoptosis, total nuclear endonucleases from the NRK cells and the ras transfectant NRK-Ha cells were isolated. We determined that the endonuclease activity in the NRK and the ras transfectant NRK-Ha cells was a pH dependent endonuclease. Significant degradation of the target DNA was observed only in pH 4-6 buffers containing the endonuclease. Furthermore, preliminary intracellular pH analysis suggested that while the NRK cells have an intracellular pH of 6.0, the ras transfectant NRK-Ha cells have an intracellular pH of 7.2 and may have abrogated its pH dependent endonuclease. Both the C127 cells and the ras transfectant HC127 cells did not express a pH dependent endonuclease but rather a Ca2+/Mg2+ dependent endonuclease. Furthermore, preliminary intracellular pH analysis suggested that both the C127 and HC127 cells have the same intracellular pH. Our results indicate that in normal rat kidney cells, ras oncogene transfection may cause a disruption in the endonuclease activation involved in apoptosis.

Animals↗

PubMed1996

Bone extracellular matrix induces homeobox proteins independent of androgens: possible mechanism for androgen-independent growth in human prostate cancer cells.

BACKGROUND: Differences in gene expression in prostate cells are believed to be secondary to epithelial-stromal interactions. We theorized that bone matrix may provide a fertile "soil" for prostate cancer by inducing androgen-dependent genes and allowing for androgen-independent growth. METHODS: Human prostate cancer cells (LNCaP) were grown under different conditions and analyzed for differential expression of mRNA. LNCaP cells were grown in the presence of 10 nM dihydrotestosterone (DHT), on extracellular matrix (ECM) derived from bone cells (without exogenous DHT), and on plastic culture dishes without exogenous DHT. A differential display of mRNA produced by LNCaP cells grown in the above conditions was then analyzed. RESULTS: Multiple unique transcripts were present in cells that were grown in the presence of DHT and on bone ECM (without exogenous DHT), but not on plastic culture dishes without exogenous DHT. Nine of these transcripts were then cloned and analyzed. Many (5/9) of these transcripts were found to contain multiple ATTA motifs in their corresponding 3'-untranslated regions. ATTA motifs have been shown to be homeobox protein-binding sites. Homeobox proteins and their target genes are thought to regulate cellular differentiation. Consistent with this, we demonstrated by reverse transcription polymerase chain reaction (PCR) that homeobox genes were differentially expressed in LNCaP cells when the cells were grown in the presence of DHT and on bone ECM (without exogenous DHT), but not on plastic culture dishes without exogenous DHT. Furthermore, we assayed LNCaP/fetal fibroblast chimeric tumors (n = 8) that were grown in male nude mice. Some of these tumors continued to grow in these mice despite treatment with surgical castration. In blinded studies, we were able to determine which tumor samples were androgen independent by their expression of homeobox genes. All samples that were androgen independent (n = 4) expressed the homeobox genes. Finally, gel retardation assay demonstrated that the homeobox proteins were able to bind to our cloned DNA sequences. Furthermore, footprinting analysis showed that the homeobox proteins bound to the ATTA motif in the 3'-region of our target DNA. CONCLUSIONS: Bone ECM, in the absence of DHT, has the ability to regulate androgen-responsive genes. Furthermore, many of these genes contain homeobox binding sites and the expression of homeobox genes may itself be regulated by bone ECM. If so, this may partially explain the clinical observation that bone provides a fertile "soil" for prostate cancer growth and metastasis.

Adenine↗

PubMed1996

Tumor necrosis factor mediates apoptosis via Ca++/Mg++ dependent endonuclease with protein kinase C as a possible mechanism for cytokine resistance in human renal carcinoma cells.

PURPOSE: Because renal cell cancers have been found to be resistant to numerous chemotherapeutic agents, other agents including tumor necrosis factor are now being considered for clinical use. In this study, we used 2 renal cancer cell lines. SK-RC-42 and SK-RC-49, and determined the cytotoxic effects of tumor necrosis factor (TNF) and the possible mechanism of TNF resistance. MATERIALS AND METHODS: Cytotoxic assays, comparative reverse transcriptase-polymerase chain reaction (RT-PCR) and nuclease digestion analyses were used. RESULTS: Cytotoxic assays with SK-RC-42 demonstrated that TNF at 50 ng./ml. for 24 hours resulted in 19% cytotoxicity of the cells. Similar assay with SK-RC-49 only demonstrated less than 4% cytotoxicity. Based on these results, we defined our TNF-sensitive cells as SK-RC-42 and SK-RC-49 as our TNF-resistant cells. To determine whether protein kinase C (PKC), which is involved in the signal transduction pathway of a cell, could regulate endogenous basal TNF mRNA levels, comparative PCR analyses for TNF expression were used. The PCR results demonstrated that the TNF-resistant cell, SK-RC-49, had a higher basal expression of TNF mRNA than the TNF-sensitive cell SK-RC-42. With PMA, a PKC activator, for various time points, both cell lines demonstrated an induction of endogenous TNF mRNA. To further confirm our findings that PKC may regulate endogenous TNF expression, a PKC inhibitor, staurosporine, was used. When the cells were treated with staurosporine prior to PMA stimulation, no increase in TNF mRNA expression was seen. To determine whether PKC is involved in providing resistance against TNF, we incubated the SK-RC-42 and SK-RC-49 cells with staurosporine at 100 nM. and TNF at 50 ng./ml. for 24 hours. After factoring out the cytotoxicity of staurosporine, the TNF-mediated cytotoxicity increased to 39.3% and 28.7% for the SK-RC-42 and SK-RC-49 cells, respectively. Furthermore, by using a nuclease digestion assay, we demonstrated that TNF activated a Ca++/Mg++ dependent endonuclease responsible for programmed cell death or apoptosis. CONCLUSIONS: Our data suggest that protein kinase C may play a role in protecting renal cancer cells from undergoing cytokine-mediated apoptosis.

Apoptosis↗

PubMed1996

[Extracellular matrix].

Extracellular matrix is composed of four families: collagens, proteoglycans, elastin and extracellular matrix structural glycoproteins. The extracellular matrix should not be viewed as merely providing strength and physical support for tissues and organisms. It is now quite clear that this matrix exerts profound influence on both the behaviour (e.g. adherence, spreading, and migration) and the pattern of gene expression of the cells. Extracellular matrix research is an active field in biology.

Animals↗

PubMed1995

LAK cell mediated apoptosis of human bladder cancer cells involves a pH-dependent endonuclease system in the cancer cell: possible mechanism of BCG therapy.

Intravesical bacillus Calmette-Guerin (BCG) is an effective treatment for superficial bladder cancer. However, its mechanism has been only partially elucidated. We studied whether LAK cell killing of human bladder cancer cells occurs via apoptosis (programmed cell death) or necrosis. Fluorescent dye labeled T24 cells were observed to undergo morphologic changes associated with apoptosis in the presence of LAK cells when analyzed under a fluorescence microscope. Furthermore, analysis of the DNA isolated from the cytotoxic assay confirmed that the LAK cell induced death of the T24 cells occurred via apoptosis. By pretreating the LAK cells with antifibronectin antibodies, we were able to significantly inhibit the LAK cell killing of the T24 cells. The percentage of cytotoxicity was reduced from 50% to 13% (p = 0.001), and the apoptotic pattern seen on agarose gel electrophoresis was significantly diminished. There was no significant change in the viability of the LAK cells following treatment with the antibodies. Endonuclease isolation from human bladder cancer T24 cells demonstrated that these cells express a pH-dependent and not a Ca++/Mg++ dependent endonuclease. Significant degradation of a target DNA was observed only in pH 4 to pH 5.6 buffers containing endonuclease from T24 cells and not in pH 6 to pH 8 buffers containing endonuclease from T24 cells. The presence or absence of Ca++/Mg++ in the various pH buffers did not alter the endonuclease activity. Finally, we demonstrated that death of T24 cells can be induced by altering the intracellular pH of the cells to 5.6 or lower with the proton ionophore nigericin. We conclude that LAK cells induce T24 cells to undergo apoptosis and that this process involves the fibronectin molecule present on the LAK cell membrane. Furthermore, the cleavage postulate that, in vivo, LAK cells activated by IL-2 produced by BCG activated CD4+ cells may induce bladder cancer cells to undergo apoptosis. This may partially explain the mechanism whereby BCG achieves its therapeutic effect.

Antibodies↗

PubMed1995

Ceruloplasmin or fibronectin synergism with quartz dust on stimulating collagen gene transcription in human 2BS fibroblast.

Human alpha 1(I), alpha 2(I) and alpha 1(III) cDNA probes and RNA dot hybridization were employed to quantitate collagen mRNA changes after adding silica dust into the media of human 2BS fibroblasts. At all dosages used (100, 200, 500 and 1000 micrograms), the alpha 1(I), alpha 2(I) and alpha 1(III) mRNA levels increased one day after dusting. At the same dosage of silica (100 micrograms), alpha 1(III) mRNA increased earlier than type I collagen mRNA did. The type I and type III collagen mRNA contents in the experimental groups were higher than those in control on days 3, 5, 7 and 9. The effect of ceruloplasmin (Cp) and fibronectin (Fn) on collagen mRNA synthesis was also studied, after adding silica dust, Cp or Fn into the media of human 2BS fibroblast. The results showed that Cp and Fn have stimulating effect on collagen mRNA production. When both Cp and silica dust were added into cell culture media, the collagen mRNA level was increased more than those of adding either Cp or silica dust alone. Similar situations were found for Fn. Cp (or Fn) synergism with silica dust on stimulating transcription of human collagen gene was suggested.

Cells, Cultured↗

PubMed1994

Bacillus Calmette-Guérin interacts with the carboxyl-terminal heparin binding domain of fibronectin: implications for BCG-mediated antitumor activity.

Intravesical bacillus Calmette-Guérin (BCG) has been shown to be an effective treatment for superficial transitional cell carcinoma of the bladder. The mechanisms by which BCG achieves this effect remain unclear. Reports have attributed an important role to fibronectin both in the initial attachment of BCG to bladder surfaces and in the limitation of tumor cell motility. In the present study, using limited protease cathepsin B degradation followed by Western blot analyses with antibodies to various domains of the fibronectin molecule, we showed that BCG appears to bind to fibronectin near the carboxyl terminal and adjacent to the heparin binding domain. Furthermore a 51-chromium release assay with human bladder cancer cell line T24 as target cells and lymphokine activated killer (LAK) cells as effector cells showed that fibronectin was needed for tumor cytotoxicity by the LAK cells. By using antibodies and peptides to various domains of the fibronectin molecule, the heparin binding domain, but not the cell binding domain, carboxyl terminal region, or the amino terminal region of the fibronectin molecule, was identified as essential to tumor cell lysis by the LAK cells. Flow cytometric analysis showed that both peripheral blood lymphocytes and the LAK cells express fibronectin receptors VLA-3, VLA-4 and VLA-5 on their surfaces. However, the numbers of receptors are not significantly different in the two cell populations. We conclude that, by binding near the carboxyl terminal region and adjacent to the heparin-binding domain of the fibronectin molecule, BCG may protect this region of the molecule from tumor proteases, and may thus allow the antitumor activity of the host immune cells to take place.

BCG Vaccine↗

PubMed1994

Collagen gene expression in radiation interstitial pneumonitis.

By using type I and type III collagen cDNA probe and cDNA-mRNA in situ hybridization, we observed the changes of rat lung alpha 1(I) and alpha 1(III) collagen gene expression in radiation interstitial pneumonitis. The results showed that the expressed cell of type I and type III collagen were scattered within the fibroblasts in the thickened interalveolar walls. The type I and type III collagen mRNA content in irradiated animals were higher than those in the controls at 0.5, 1, 2, 3, 6, and 12 months.

Animals↗

PubMed1994

[Alternations of collagen content and collagen gene expression in rat vascular structural remodeling of pulmonary artery induced by hypoxia].

The change of collagen content and procollagen alpha 1 (I), alpha 1 (III) mRNA expression in rat vascular structural remodeling of extrapulmonary artery (EPA) induced by hypoxia were investigated. 72 wistar male rats weighing 200-300g were used and divided into 2 groups: air-breathing group (N group, 39 rats) and hypoxic group (H group, 33 rats exposed to 5000m hypobaric hypoxia for 7 days). Results showed that mPAp, Rv/(Lv+s), the wet, dry weights and hypoxyproline (HP) content in EPA of H group were increased from 2.83 +/- 0.09kPa (mean +/- smean), 0.23 +/- 0.01, 5.2 +/- 0.2mg, 1.7 +/- 0.1mg and 22.3 +/- 1.0 micrograms)/100g BW (body weight) to 4.77 +/- 0.12kPa, 0.43 +/- 0.01, 10.5 +/- 0.5mg, 4.1 +/- 0.2mg and 58.4 +/- 3.4 micrograms/100g BW (body weight) to 4.77 +/- 0.12kPa, 0.43 +/- 0.02, 10.5 +/- 0.5mg, 4.1 +/- 0.2mg and 58.4 +/- 3.4 micrograms/100g (all P < 0.01). Dot Blot and Northern Blot hybridization analyses showed that in EPA of H group procollagen alpha 1(I) and alpha 1(III) mRNAs were increased 154% and 177% (all P < 0.05). Histological study (HE) showed that hypoxia could induce an increase in the wall thickness of EPA, particularly in adventitial thickness of the pulmonary arterial trunk. Electron microscopy revealed the hypertrophied medial smooth muscle cells containing a significant increase in rough endoplasmic reticulum (RER), in extracellular ground, collagen fibers and elastin were increased. In adventitia, the fibroblasts showed both hypertrophy and hyperplasia and surrounding collagen fibers were increased. These studies suggested that fibroblast might play important role in the remodeling of EPA during the early hypoxic pulmonary hypertension and the increase of collagen gene expression might be involved in the mechanism of collagen content and remodeling of EPA induced by hypoxia.

Animals↗

PubMed1994

The effects of tetrandrine (TT) and polyvinylpyridine-N-oxide (PVNO) on gene expression of type I and type III collagens during experimental silicosis.

In the screening tests of drugs for silicosis in our laboratory, we found that TT, a type of alkaloid isolated from Stephania tetrandra, could inhibit the development of experimental silicosis of rats and the synthesis of collagen in rat lung. Chest X-rays of silicotic patients treated with TT for 1-3 years showed obvious changes. The silicotic nodules became smaller and shadows became clearer. PVNO was proved to have anti-silicotic effect on animal and clinically. This presentation reports the effect of them on collagen mRNA. Dot blot results showed that alpha 1 (I) and alpha 1 (III) mRNA levels increased significantly at 60 and 120 days after the rats were exposed to silica dust. The mRNA levels went down at 1 and 3 months after treated by TT and PVNO. In situ hybridization observation revealed that the silver grains of Type I and Type III collagen were scattered within the fibroblasts in cellular nodules and in thickened interstitium of silicosis tissue. The amounts of mRNA silver grains decreased in the lung tissue treated by TT and PVNO. It was suggested that TT and PVNO may inhibit the gene expression of collagen during silicosis.

Alkaloids↗

PubMed1994

Effects of calcium and calmodulin inhibitors on the abnormal proliferation of lung fibroblast.

The effects of alveolar macrophage (Am phi) conditioned media from interstitial lung disease (ILD) patients on fibroblast (FB), and the role of calcium (Ca2+) blockers and calmodulin (CaM) inhibitors on the proliferation of lung FB were studied. We found that the AM phi conditioned media could stimulate FB cell proliferation and this effect could be abolished by Ca2+ blockers and CaM inhibitors. The results indicated that AM phi was in activated state in ILD and released some kinds of cytokines to stimulate the proliferation of FB, and Ca,2+ CaM were partially responsible for these actions.

Bronchoalveolar Lavage Fluid↗

PubMed1993

Altered extracellular matrices influence cellular processes and nuclear matrix organizations of overlying human bladder urothelial cells.

A central issue in tumor biology is the understanding of the interactions between tumor cells and their environment. Using normal and ras oncogene transfected rat fibroblast cells, we now demonstrate that the transfected cells make altered extracellular matrices (ECM) and that their resulting ECM influence the proliferation and genetic regulation of human bladder cancer EJ cells. Using Western blot analyses, we observed that the ras transfected fibroblast cells lacked the ability to produce extracellular matrix component laminin whereas the normal parental fibroblast cells were able to produce intact laminin. Both transfected and nontransfected fibroblast cells were able to synthesize other extracellular matrix molecules such as type IV collagen and fibronectin. Human bladder tumor EJ cells were grown on ECM derived from normal and transfected rat fibroblast cells, and the proliferation rate and type IV collagen mRNA expression of EJ cells were determined. We observed that EJ cells, when grown on ECM derived from the ras transfected fibroblast cells, had a higher growth rate than when grown on ECM derived from the normal fibroblast cells (P < 0.037). Furthermore, EJ cells grown on ECM derived from transfected fibroblast cells showed up-regulation of type IV collagen mRNA expression when compared with EJ cells grown on ECM derived from nontransfected fibroblast cells. Finally EJ cells grown on purified laminin but not on collagen IV coated flasks showed the same level of type IV collagen mRNA expression as when grown on ECM derived from nontransfected parental fibroblast cells. Haptotactic/motility assays with EJ cells and ECM derived from ras transfected and nontransfected fibroblast cells demonstrated that ECM of ras transfected fibroblast cells, but not the parental fibroblast cells, provided a permissive or fertile soil for EJ tumor cell invasion. Finally, two-dimensional gel electrophoresis of 35S-labeled nuclear matrix proteins of EJ cells cultured on ECM derived from ras transfected fibroblast cells revealed expression of proteins in the molecular weight range of M(r) 35,000-45,000 and isoelectric focusing pH range of 5.5 to 6.0. These proteins were not present in EJ cells cultured on ECM derived from parental nontransfected fibroblast cells. We conclude that extracellular matrices derived from transformed stroma producing cells may influence the proliferation, genetic regulation, and maintenance of the overlying urothelial tumor cells. The mechanism by which the ECM may influence cellular behavior and phenotype may be in their ability to modulate the nuclear matrix proteins of the overlying cell.

Animals↗

PubMed1992

Abrogation of the invasion of human bladder tumor cells by using protease inhibitor(s).

It was shown previously that invasive human transitional cell carcinoma cell line EJ, but not the noninvasive RT4 cells, can degrade basement membrane laminin and that the degradation of basement membrane laminin was a result of a redistribution of activated cysteine proteinase cathepsin B to the plasma membrane of the invasive EJ cells. Using a modified Boyden chamber and an artificial basement membrane, it was found first that cysteine proteinase inhibitor E-64 can abolish the ability of the EJ cells to invade through the artificial basement membrane to the underside of the filter. Second, E-64 can prevent the degradation of purified human basement membrane laminin by the plasma membrane fraction of invasive EJ cells. Third, E-64 does not affect the ability of the EJ cells to attach to the extracellular matrix nor is the inhibitory dose toxic to the cells when assayed with trypan-blue dye exclusion. However, E-64 does affect the ability of the EJ cells to respond to autocrine motility factor-induced motility. Finally, in an in vivo model, E-64 was not toxic to the animals tested and may have limited the blood-borne metastatic ability of invasive EJ cells in the treated animals. It was concluded that proteinase cathepsin B may be involved in human bladder tumor invasion, in both extracellular matrix degradation and factor-induced cellular motility, and the authors suggested that the use of inhibitor(s) to cysteine proteinases may limit the invasive potential of human bladder cancer cells.

Animals↗

PubMed1992

Bacillus Calmette-Guerin abrogates in vitro invasion and motility of human bladder tumor cells via fibronectin interaction.

Intravesical bacillus Calmette-Guerin (BCG) has been shown to be an effective treatment for superficial transitional cell carcinoma of the bladder (TCC). The mechanisms by which BCG limits tumor cell activity have thus far been unclear. We investigated the interaction between BCG and invasive human TCC cell line EJ in an in vitro invasion assay. We observed that BCG inhibited the invasion of EJ cells through an artificial basement membrane. In terms of the steps involved in tumor cell invasion, i.e. attachment, proteolysis, and motility, BCG was found to limit tumor cell motility. Attachment and proliferation of tumor cells were not affected by BCG. The effects of BCG on tumor cell migration were mediated by fibronectin (FN), a basement membrane glycoprotein component. Abrogation of BCG-FN-tumor cell interactions with anti-FN antibodies eliminated the ability of BCG to block tumor cell invasion. Fibronectin appears to link BCG and tumor cells via independent FN binding receptors to separate domains of the FN molecule. The molecular mechanism by which BCG may limit tumor cell motility may be its ability to protect against the formation of specific FN sequences as a result of protease cathepsin B digestion. A 31 kD and 27 kD FN band were absent from purified or tumor cell associated cathepsin B digestion when incubated in the presence of BCG, but present in the absence of BCG. Furthermore when purified from SDS polyacrylamide gel electrophoresis, the fragments were shown to have motility stimulating activity for the invasive EJ cells. These findings suggest that BCG functions as a potent inhibitor of tumor cell invasion. We conclude that BCG-fibronectin-tumor cell interactions may have a direct influence on the invasive mechanisms, such as motility, of tumor cells.

BCG Vaccine↗

PubMed1992

Biochemistry of bladder cancer invasion and metastasis. Clinical implications.

In bladder cancer, the finding of infiltration signals the capability of a tumor to behave in an aggressive manner and potentially create a life-threatening situation. The ability to invade is carried in the tumor cell's complement of biochemical pathways as well as in its inability to preserve or restore rather than to disrupt normal tissue architecture. What determines the activation and deployment of these biochemical activities is unclear. That they occur, however, and can distinguish aggressive cancers likely to metastasize from cancers whose behavior is benign, is not. Recognizing and identifying these distinctions is an important step in designing new therapies to prevent a tumor's potential aggressive behavior, possibly reverse or at least contain any extensions that have occurred, and ultimately convert a potentially life-threatening situation to one with a more benign prognosis.

Animals↗