Rat Bladder Carcinoma Cell Line Research Articles

Blockade of β1 Integrin–Laminin-5 Interaction Affects Spreading and Insulin Secretion of Rat β-Cells Attached on Extracellular Matrix

When attached on a matrix produced by a rat bladder carcinoma cell line (804G matrix), rat pancreatic beta-cells spread in response to glucose and secrete more insulin compared with cells attached on poly-l-lysine. The aim of this study was to determine whether laminin-5 and its corresponding cell receptor beta1 integrin are implicated in these phenomena. By using specific blocking antibodies, we demonstrated that laminin-5 is the component present in 804G matrix responsible for the effect of 804G matrix on beta-cell function and spreading. When expression of two well-known laminin-5 ligands, beta1 and beta4 integrin, was assessed by Western blot and RT-PCR, only the beta1 integrin was detected in beta-cells. Anti-beta1 integrin antibody reduced the spreading of beta-cells on 804G matrix. Blockade of the interaction between beta1 integrins and laminin-5 resulted in a reduction in glucose-stimulated insulin secretion. Blocking anti-beta1 integrin antibody also inhibited focal adhesion kinase phosphorylation induced by 804G matrix. In conclusion, anti-beta1 integrin and -laminin-5 antibodies interfere with spreading of beta-cells, resulting in decreased insulin secretion in response to glucose. Our findings indicate that outside-in signaling via engagement of beta1 integrins by laminin-5 is an important component of normal beta-cell function.

Tbx3 expression is related to apoptosis and cell proliferation in rat bladder both hyperplastic epithelial cells and carcinoma cells

In the present study, Tbx3, a member of the T-box family of transcription factors, was identified as an up-regulated gene by mRNA differential display in the regression (apoptosis) stage after uracil-induction of papillomatosis in the rat urinary bladder. Immunohistochemical analysis revealed that apoptosis cells are negative and apoptosis resistant cells are positive for Tbx3 expression. That suggests that Tbx3 is an apoptosis resistant gene rather than an apoptosis induced gene. We have found the rat bladder carcinoma cell line, BC31, to feature Tbx3 expression detectable by RT-PCR. To investigate its role in such cancer cells, they were transfected with an anti-sense Tbx3 expression vector. The obtained stable transfectant clones showed reduced expression of Tbx3 and much slower cell growth, as compared with mock transfectants, and many apoptotic cells were observed under normal culture conditions. These results indicate that Tbx3 is a negative regulator of apoptosis in bladder normal epithelial cells and suppression of Tbx3 expression causes inhibition of cell proliferation and induction of apoptosis in BC31, a rat bladder carcinoma cells.

Evidence of Gap Junctions in the Stable Nonobstructed Human Bladder

Evidence of Gap Junctions in the Stable Nonobstructed Human Bladder

Successful Diagnosis Of Orthotopic Rat Superficial Bladder Tumor Model By Ultrathin Cystoscopy

No AccessJournal of UrologyINVESTIGATIVE UROLOGY1 Feb 2003Successful Diagnosis Of Orthotopic Rat Superficial Bladder Tumor Model By Ultrathin Cystoscopy HIROSHI ASANUMA, TSUNENORI ARAI, KENJI SEGUCHI, SATOKO KAWAUCHI, HIROYUKI SATOH, MAKOTO KIKUCHI, and MASARU MURAI HIROSHI ASANUMAHIROSHI ASANUMA More articles by this author , TSUNENORI ARAITSUNENORI ARAI More articles by this author , KENJI SEGUCHIKENJI SEGUCHI More articles by this author , SATOKO KAWAUCHISATOKO KAWAUCHI More articles by this author , HIROYUKI SATOHHIROYUKI SATOH More articles by this author , MAKOTO KIKUCHIMAKOTO KIKUCHI More articles by this author , and MASARU MURAIMASARU MURAI More articles by this author View All Author Informationhttps://doi.org/10.1016/S0022-5347(05)64000-1AboutFull TextPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract Purpose: In the orthotopic animal bladder tumor model noninvasive evaluation of the tumor establishment and the therapeutic effect has been difficult. To our knowledge we present the first diagnosis of orthotopic rat superficial bladder tumor model by ultrathin cystoscopy. Materials and Methods: The established AY-27 rat bladder carcinoma cell line was transplanted orthotopically into 22 female Fischer F344 rats. A cell suspension containing 4 × 106 AY-27 cells was instilled into the bladder, which had been conditioned with mild acid washing. To evaluate tumor growth serial cystoscopy was performed via the urethra with an ultrathin endoscope (diameter 0.75 mm.) 5 to 14 days after tumor cell inoculation. At intervals after cystoscopic tumor detection the rats were sacrificed for autopsy and histological examination. Results: In all 22 rats the orthotopic bladder tumor was established 7 to 10 days after tumor cell implantation and in most it was superficial. Cystoscopy permitted inspection of the urethra and whole bladder surface. We detected all tumors as broad based papillary mass (minimal lesion 1 mm. or less) and inspected its detailed appearance and accurate location. Conclusions: The orthotopic rat superficial bladder tumor model and the diagnostic procedure by ultrathin cystoscopy would be ideal system for preclinical evaluation of new potential intravesical therapies. References 1 : Magnetic resonance imaging for detecting and treatment monitoring of orthotopic murine bladder tumor implants. J Urol1991; 145: 1297. Link, Google Scholar 2 : Characterization of a novel transplantable orthotopic rat bladder transitional cell tumour model. Br J Cancer1999; 81: 638. Crossref, Medline, Google Scholar From the Department of Urology, School of Medicine and Department of Applied Physics and Physico-Informatics, Faculty of Science and Technology, Keio University, Tokyo and Departments of Urology and Medical Engineering, National Defense Medical College, Tokorozawa, Saitama, Japan© 2003 by American Urological Association, Inc.FiguresReferencesRelatedDetailsCited bySatoh H, Morimoto Y, Arai T, Asanuma H, Kawauchi S, Seguchi K, Kikuchi M and Murai M (2018) Intravesical Ultrasonography for Tumor Staging in an Orthotopically Implanted Rat Model of Bladder CancerJournal of Urology, VOL. 177, NO. 3, (1169-1173), Online publication date: 1-Mar-2007.ASANUMA H, ARAI T, MORIMOTO Y, KAWAUCHI S, SATOH H, SEGUCHI K, KIKUCHI M and MURAI M (2018) PHOTODYNAMIC THERAPY WITH PAD-S31, A NEW HYDROPHILIC CHLORIN PHOTOSENSITIZER, IN AN ORTHOTOPIC RAT BLADDER TUMOR MODELJournal of Urology, VOL. 174, NO. 5, (2016-2021), Online publication date: 1-Nov-2005. Volume 169Issue 2February 2003Page: 718-720 Advertisement Copyright & Permissions© 2003 by American Urological Association, Inc.Keywordsratsbladderbladder neoplasmscystoscopesMetricsAuthor Information HIROSHI ASANUMA More articles by this author TSUNENORI ARAI More articles by this author KENJI SEGUCHI More articles by this author SATOKO KAWAUCHI More articles by this author HIROYUKI SATOH More articles by this author MAKOTO KIKUCHI More articles by this author MASARU MURAI More articles by this author Expand All Advertisement PDF downloadLoading ...

Gap junction intercellular communication propagates cell death in cancerous cells

Gap junction intercellular communication (GJIC) or cell coupling has an important function in maintaining tissue homeostasis and is thus a critical factor in the life and death balance of cells. While the role of GJIC in cell growth regulation has been much studied, its involvement in apoptosis remains unclear. In this study we elucidated the possibility that cell death is propagated via gap junctions, employing the rat bladder carcinoma cell line BC31. BC31 cells proliferate quickly, are tumorigenic, and are well-coupled via gap junctions that contain the gap junction protein Connexin43 (Cx43). In addition, these cells are predisposed to spontaneous death by apoptosis, particularly upon achieving confluency. We found that many dying BC31 cells express Cx43 just as their non-apoptotic counterparts do. Furthermore, Cx43 in apoptotic cells could be functionally competent, supporting coupling of these cells with their non-apoptotic neighbors, and as a result, clusters of coordinately dying cells were observed. The role of Cx43 and GJIC in propagating cell death was shown by analysing clones of BC31 cells expressing a mutant of Cx43 that is a dominant negative inhibitor of GJIC, and by using beta-glycyrrhetinic acid to inhibit intrinsic cell coupling in BC31 cells: in both cases the formation of clusters of dying cells was abrogated, and the intensity of cell death was considerably decreased. These results suggest that GJIC spreads cell-killing signals initially generated by a single cell that spontaneously initiates apoptosis, into healthy surrounding cells, thus increasing the level of cell death. Treatment of BC31 cells with the sleep-inducing lipid Oleamide, which selectively restricts gap junction permeability to Ca(2+) ions, did not abrogate coordinated cell death by clusters, indicating that Ca(2+) ions are the most probable cell-killing signals spread through gap junctions.

Increased intracellular calcium is required for spreading of rat islet beta-cells on extracellular matrix.

Rat islet beta-cells spread in response to glucose when attached on the matrix produced by a rat bladder carcinoma cell line (804G). Furthermore, in a mixed population of cells, it has been observed previously that spread cells secrete more insulin acutely in response to glucose, compared with cells that remain rounded. These results suggest bi-directional signaling between the islet beta-cell and the extracellular matrix. In the present study, the role of increased intracellular free Ca2+ concentration [Ca2+]i as an intracellular step linking glucose stimulation and beta-cell spreading (inside-out signaling) was investigated. Purified rat beta-cells were attached to this matrix and incubated under various conditions known to affect [Ca2+]i. The effect of glucose on beta-cell spreading was mimicked by 25 mmol/l KCl (which induces calcium influx) and inhibited by diazoxide (which impairs depolarization and calcium entry) and by the L-type Ca2+ channel blocker SR-7037. When a 24-h incubation at 16.7 glucose was followed by 24 h at 2.8 mmol/l, beta-cells that had first spread regained a round phenotype. In the presence of thapsigargin, spreading progressed throughout the experiment, suggesting that capture of calcium by the endoplasmic reticulum is involved in the reversibility of spreading previously induced by glucose. Spreading was still observed in degranulated beta-cells and in botulinum neurotoxin E-expressing beta-cells when exocytosis was prevented. In summary, the results indicate that increased [Ca2+]i is required for the glucose-induced spreading of beta-cells on 804G matrix and that it is not a consequence of exocytotic processes that follow elevation of [Ca2+]i.

The community effect in FGF-1 mediated tumor progression of a rat bladder carcinoma does not involve a direct paracrine signaling.

A community effect was found to occur between heterogeneous tumor cell populations leading to an overall increased tumorigenicity without a clonal dominance of the more tumorigenic clone. In the rat bladder carcinoma cell line NBT-II, this effect appears mediated by the Fibroblast Growth Factor-1 (FGF-1) through either a direct or an indirect signaling pathway. Neovascularization induced by FGF-1 was found not to be responsible for the community effect. The present study shows that the community effect does not involve a direct FGF-1 signaling since tumor cells expressing a dominant-negative FGF receptor mutant were still responding to the highly tumorigenic FGF-1 expressing cells. Tumors arising from inoculates of the FGF-1 producing NBT-II cells mixed with non tumorigenic epithelial MDCK cells contain only the tumorigenic cells indicating that MDCK cells may exerce a helper effect for the growth of the tumor not dependant on their own growth. Therefore the helper function of MDCK cells must be distinguished from a community effect where the contribution of low tumorigenic cells not only provides an in vivo growth advantage to few highly tumorigenic cells but become themselves highly tumorigenic indicating that the community effect may require cell-cell specific cooperativity independent from an helper effect.

Relationship between cell migration and cell cycle during the initiation of epithelial to fibroblastoid transition

The NBT-II rat bladder carcinoma cell line, which displays epithelial to mesenchymal transition or EMT in response to FGF-1 stimulation, was used to study the interrelationships between cell cycle and cell scattering and locomotion. Time-lapse video microscopy experiments were performed with asynchronous growing cells and lovastatin-arrested cells. FGF-1 stimulation induced cell movement in cells in all phases of the cell cycle, except G2 + M phase, in which cells did not respond to stimulation. The delay between cell stimulation and cell movement depended on the age of the cell at the beginning of cell stimulation: cells less than 4 h old when stimulated by FGF-1 had a 1-h delay whereas cells more than 4 h old had a 3-h delay. Cells stimulated before they were 4 h old were temporarily arrested in their cell cycle progression. Older cells underwent mitosis on schedule. Lovastatin-treated cells were shown to be synchronized in the G1 phase and to migrate simultaneously after FGF-1 stimulation. These results indicate that the G1 phase was a critical phase for FGF-1 induced cell migation during epithelial to fibroblastoid transition. Cell Motil. Cytoskeleton 43:288–295, 1999. © 1999 Wiley-Liss, Inc.

Characterization of the signaling pathways regulating alpha2beta1 integrin-mediated events by a pharmacological approach.

In certain instances of developing and adult organism, epithelial cells can change morphology and transform into mesenchymal-like type in order to move through the extracellular matrix. However, because of the multiplicity and complexity of signaling pathways that contribute to these processes, their molecular dissection has remained difficult. By using a pharmacological approach on the rat bladder carcinoma cell line NBT-II dispersion system, we have identified distinct signaling events for adhesion and motility in response to collagen, both activities depending on alpha2beta1 integrin. Treatment of cells with PKC inhibitors markedly impaired initial attachment on collagen without affecting the capacity of cells to move, suggesting that PKC activity is required for initial adhesion strength during cell translocation. Both adhesion and motility were diminished by tyrosine kinase inhibitors herbimycin and tyrphostin whereas tyrosine phosphatase inhibitors amplified cell scattering. The collagen-induced dispersion was insensitive to genistein which we previously showed to abrogate growth factor-induced scattering, thus demonstrating inducer specificity. Finally. Ras inhibitors and expression of a dominant negative form of Ras (N17Ras) while affecting initial cell attachment, did not prevent cell migration, and instead favored the dissociated state on collagen. The specific signaling pathways identified for adhesion and motility should help to understand the sequential processes associated with cell migration.

Relationship between cell migration and cell cycle during the initiation of epithelial to fibroblastoid transition.

The NBT-II rat bladder carcinoma cell line, which displays epithelial to mesenchymal transition or EMT in response to FGF-1 stimulation, was used to study the interrelationships between cell cycle and cell scattering and locomotion. Time-lapse video microscopy experiments were performed with asynchronous growing cells and lovastatin-arrested cells. FGF-1 stimulation induced cell movement in cells in all phases of the cell cycle, except G2 + M phase, in which cells did not respond to stimulation. The delay between cell stimulation and cell movement depended on the age of the cell at the beginning of cell stimulation: cells less than 4 h old when stimulated by FGF-1 had a 1-h delay whereas cells more than 4 h old had a 3-h delay. Cells stimulated before they were 4 h old were temporarily arrested in their cell cycle progression. Older cells underwent mitosis on schedule. Lovastatin-treated cells were shown to be synchronized in the G1 phase and to migrate simultaneously after FGF-1 stimulation. These results indicate that the G1 phase was a critical phase for FGF-1 induced cell migration during epithelial to fibroblastoid transition.

Inhibition of intrinsic gap-junction intercellular communication and enhancement of tumorigenicity of the rat bladder carcinoma cell line BC31 by a dominant-negative connexin 43 mutant

The tumor-suppressive property of the connexin gap-junction proteins was postulated from the fact that their function of cell coupling is impaired in most cancer cells. However, in conflict with this notion, certain cancer cells are able to communicate through gap junctions despite their malignancy. To explain this phenomenon, we studied by using a dominant-negative strategy the effect on tumorigenicity of loss of intrinsic gap-junction intercellular communication (GJIC) in the rat bladder carcinoma cell line BC31, which shows both expression of connexin 43 (Cx43) and intercellular communication. In cells transfected with a mutant Cx43 with seven residues deleted from the internal loop at positions 130-136 (Cx43delta), transport of the resulting connexin protein to the plasma membrane occurred normally, but the GJIC of the cells was effectively abolished at the level of permeability of established gap junctions. Dominant-negative inhibition of GJIC by Cx43delta accelerated growth of BC31 cells in nude mice. In contrast, when GJIC in BC31 cells was artificially enforced by transfection of wild-type Cx43, the cells lost the capacity to grow in vivo. Decreased phosphorylation of Cx43delta suggested close interaction of the internal loop of connexin with its commonly phosphorylated domains in the C-terminal tail and involvement of this interaction in gap-junction permeability. Therefore, we conclude that the intrinsic GJIC observed in cancer cells should be considered a tumor-suppressor factor and that its level may influence malignant growth capacity.

Inhibition of intrinsic gap‐junction intercellular communication and enhancement of tumorigenicity of the rat bladder carcinoma cell line BC31 by a dominant‐negative connexin 43 mutant

The tumor-suppressive property of the connexin gap-junction proteins was postulated from the fact that their function of cell coupling is impaired in most cancer cells. However, in conflict with this notion, certain cancer cells are able to communicate through gap junctions despite their malignancy. To explain this phenomenon, we studied by using a dominant-negative strategy the effect on tumorigenicity of loss of intrinsic gap-junction intercellular communication (GJIC) in the rat bladder carcinoma cell line BC31, which shows both expression of connexin 43 (Cx43) and intercellular communication. In cells transfected with a mutant Cx43 with seven residues deleted from the internal loop at positions 130–136 (Cx43Δ), transport of the resulting connexin protein to the plasma membrane occurred normally, but the GJIC of the cells was effectively abolished at the level of permeability of established gap junctions. Dominant-negative inhibition of GJIC by Cx43Δ accelerated growth of BC31 cells in nude mice. In contrast, when GJIC in BC31 cells was artificially enforced by transfection of wild-type Cx43, the cells lost the capacity to grow in vivo. Decreased phosphorylation of Cx43Δ suggested close interaction of the internal loop of connexin with its commonly phosphorylated domains in the C-terminal tail and involvement of this interaction in gap-junction permeability. Therefore, we conclude that the intrinsic GJIC observed in cancer cells should be considered a tumor-suppressor factor and that its level may influence malignant growth capacity. Mol. Carcinog. 23:254–261, 1998. © 1998 Wiley-Liss, Inc.

Autocrine growth induced by transferrin-like substance in bladder carcinoma cells.

Ample evidence confirms that certain cancer cells have the capacity to produce multiple peptides as growth factors and that expression of their receptor may act in tumour cell paracrine and/or autocrine loop mechanisms, either by extracellular release of the growth factor or by the tumour itself. To study the possibility of an autocrine growth mechanism in bladder carcinoma, we investigated the ability of various bladder carcinoma cell lines to proliferate in serum-free medium. A rat bladder carcinoma cell line, BC47, demonstrated exponential and density-dependent growth in serum-free medium. Furthermore, conditioned medium from BC47 cells induced growth-stimulating activity for BC47 cells themselves. Purification and further characterization of this activity was performed by chromatographic methods, SDS-PAGE and N-terminal amino acid analysis. Finally, we have identified that a transferrin-like 70-kDa protein is found to be the main growth-promoting factor in this conditioned medium. In addition, specific antibodies against transferrin and the transferrin-receptor inhibit the in vitro growth of this cell line. Our data suggest that this transferrin-like factor possibly acts as an autocrine growth factor for cancer cells.

A minimal region on the integrin beta4 subunit that is critical to its localization in hemidesmosomes regulates the distribution of HD1/plectin in COS-7 cells.

The integrin alpha6 beta4 is a major component of hemidesmosomes, in which it mediates firm adhesion to laminin 5. Previous studies have shown that the incorporation of alpha6 beta4 into hemidesmosomes requires a 303 amino acid stretch of the cytoplasmic domain of beta4, comprising part of the first fibronectin type III (FNIII) repeat, the second FNIII repeat and the segment that connects the second to the third FNIII repeat (connecting segment). Now, we have further defined sequences within beta4 that are critical for its localization in hemidesmosomes and we demonstrate that these sequences also induce the redistribution of HD1/plectin into junctional complexes containing the integrin alpha6 beta4 in COS-7 cells, transfected with cDNAs encoding alpha6A and beta4. Truncation of the cytoplasmic domain of beta4 after amino acids 1,382 or 1,355 in the connecting segment, by which a potential tyrosine activation motif (TAM) is removed, does not prevent the localization of alpha6 beta4 in hemidesmosomes in the rat bladder carcinoma cell line 804G and neither did it eliminate the ability of alpha6 beta4 to change the subcellular distribution of HD1/plectin in COS-7 cells. In contrast, beta4 subunits in which the entire connecting segment had been deleted or which were truncated after amino acid 1,328, which removes almost the complete segment, had lost both of these functions. Furthermore, when beta4 subunits with either a deletion of the second FNIII repeat or a small deletion in this repeat were co-expressed with alpha6, the integrins were not localized in hemidesmosomes and did not induce the redistribution of HD1/plectin in COS-7 cells. Finally, the fourth FNIII repeat of beta4 could not replace the second in either of these activities. These findings establish that a region in beta4, which encompasses the second FNIII repeat and a stretch of 27 amino acids (1,329-1,355) of the connecting segment, is critical for the localization of alpha6beta4 in hemidesmosomes and that it regulates the distribution of HD1/plectin.

FGF-2 and FGF-1 expressed in rat bladder carcinoma cells have similar angiogenic potential but different tumorigenic properties in vivo.

The comparative biological properties of NBT-II cells, a rat bladder carcinoma cell line constitutively expressing FGF-1 and FGF-2 were analysed in nude mice. FGF-1 is not secreted by the transfected cells unless the cDNA contains a signal sequence; conversely, NBT-II cells transfected with FGF-2 coding sequence produce and secrete the factor in a biologically active form. Bovine brain capillary endothelial cells are stimulated to proliferate upon addition of medium conditioned by the FGF-2-producing cells and this activity can be abrogated by the addition of anti-FGF-2 blocking antibodies. In addition, the FGF-2-containing medium, which cannot stimulate NBT-II cells due to absence of appropriate receptors, is able to induce scattering of NBT-II cells expressing the FGFR1. It has been reported previously that FGF-1-producing cells are highly tumorigenic in nude mice and induce carcinoma with a period of latency reduced from 6 to 5 weeks when compared to parental NBT-II cells. In contrast, NBT-II cells producing FGF-2 are no more tumorigenic than parental cells, indicating that FGF-1 and FGF-2 have different oncogenic properties in carcinoma. FGF-1 and FGF-2 are potent antiogenic factors that trigger the host endothelial cells. VEGF, another potent angiogen was found to be expressed in small amounts by NBT-II cells and to be expressed in reduced amount in the FGF-producing cells. In the NBT-II system in vivo FGF-1 and FGF-2 are highly and comparatively angiogenic in the resultant carcinoma and this occurs in the absence of production of significant amounts of VEGF by the carcinoma cells. Taken together, our results indicate that activated angiogenesis is not sufficient for rapid tumor expansion. FGF-1 behaves as a tumorigenic factor in the NBT-II bladder carcinoma cell model, whereas expression and secretion of large amounts of FGF-2 are not sufficient for increasing tumor growth.

Alpha 2 beta 1 integrin is required for the collagen and FGF-1 induced cell dispersion in a rat bladder carcinoma cell line.

We have investigated the role of integrins in the epithelial to mesenchymal transition (EMT) induced by either collagen or fibroblast growth factor-1 (FGF-1) in the rat bladder carcinoma cell line NBT-II. The major collagen-binding receptor is the alpha 2 beta 1 integrin. An increase in expression of alpha 2 beta 1 integrin coincided with EMT induced by either collagen or FGF-1. When both inducers were present, a further increase in alpha 2 expression was observed which correlated with an enhancement in the speed of locomotion. Overexpression of human alpha 2 in NBT-II cells did not trigger EMT but rendered cells more sensitive to the dispersing effect of collagen and FGF-1. Anti-human alpha 2 blocking antibodies affected cell scattering and motility induced by either collagen or FGF-1. These data demonstrate that alpha 2 beta 1 integrin is the mediator of the cell scattering effect induced by collagen. They also indicate that a functional alpha 2 integrin is essential for the motile behavior of NBT-II cells during the FGF-1 induced EMT.

Over‐expression of tissue inhibitor of matrix metalloproteinases (TIMP1 and TIMP2) suppresses extravasation of pulmonary metastasis of a rat bladder carcinoma

The balance between matrix metalloproteinases and their inhibitors is a critical factor which affects tumor invasion and metastasis. We have established a rat bladder carcinoma cell line, LMC19, which is tumorigenic, invasive and metastatic to the retroperitoneal lymph nodes and to the lungs in nude mice. LMC19 cells secrete pro-gelatinases A and B as well as tissue inhibitors of matrix metalloproteinase (TIMP1 and TIMP2). We conducted the present study to determine whether or not over-expression of TIMP1 and TIMP2 can affect the metastatic potential of LMC19 cells. We transfected the cells with an expression vector containing TIMP1 or TIMP2 cDNA, isolated several clones over-expressing TIMP1 or TIMP2 and assessed their invasive and metastatic potential by inoculation at an orthotopic site (urinary bladder) in nude mice. Our results show that the transfectants over-expressing TIMP1 and TIMP2 marginally affect primary tumor growth, local invasion or metastasis to the retroperitoneal lymph nodes but significantly inhibit extravascular growth of pulmonary tumor emboli. Our results suggest that the net activity of matrix metalloproteinases of tumor cells may be a critical factor that controls extravasation at this distant metastatic site.

Marked acceleration of the metastatic phenotype of a rat bladder carcinoma cell line by the expression of human gelatinase a

Numerous studies have reported a correlation between the production of gelatinases A and B by cancer cells and invasive and metastatic potential. It has been suggested that the expression of gelatinase A (72-kDa type IV collagenase) is associated more closely with the metastatic phenotype of malignant cells in vitro and in vivo than that of gelatinase B (92-kDa type IV collagenase). We have established a rat bladder carcinoma cell line, MYU3L, which is tumorigenic and locally invasive but is not metastatic to the distal organs in nude mice. The MYU3L cell line secretes pro-gelatinase B but not any detectable level of pro-gelatinase A. We undertook the present study to determine whether over-expression of gelatinase A can affect the metastatic potential of MYU3L cells. We transfected MYU3L cells with an expression vector containing human pro-gelatinase A cDNA under the transcriptional control of the SR alpha promoter. Two stable transfectants over-expressing gelatinase A activity were isolated. We assessed the biological behavior of the transfectants by an orthotopic site (urinary bladder) inoculation and an i.v. injection in nude mice. Our results demonstrate that the induced expression of human gelatinase A enzyme markedly accelerates the metastatic phenotype of the rat bladder carcinoma cell line MYU3L. Our results suggest that gelatinase A produced by tumor cells plays a major role in the metastatic process.

Pp60c-src is a positive regulator of growth factor-induced cell scattering in a rat bladder carcinoma cell line.

The NBT-II rat carcinoma cell line exhibits two mutually exclusive responses to FGF-1 and EGF, entering mitosis at cell confluency while undergoing an epithelium-to-mesenchyme transition (EMT) when cultured at subconfluency. EMT is characterized by acquisition of cell motility, modifications of cell morphology, and cell dissociation correlating with the loss of desmosomes from cellular cortex. The pleiotropic effects of EGF and FGF-1 on NBT-II cells suggest that multiple signaling pathways may be activated. We demonstrate here that growth factor activation is linked to at least two intracellular signaling pathways. One pathway leading to EMT involves an early and sustained stimulation of pp60c-src kinase activity, which is not observed during the growth factor-induced entry into the cell cycle. Overexpression of normal c-src causes a subpopulation of cells to undergo spontaneous EMT and sensitizes the rest of the population to the scattering activity of EGF and FGF-1 without affecting their mitogenic responsiveness. Addition of cholera toxin, a cAMP-elevating agent, severely perturbs growth factor induction of EMT without altering pp60c-src activation, therefore demonstrating that cAMP blockade takes place downstream or independently of pp60c-src. On the other hand, overexpression of a mutated, constitutively activated form of pp60c-src does not block cell dispersion while strongly inhibiting growth factor-induced entry into cell division. Moreover, stable transfection of a dominant negative mutant of c-src inhibits the scattering response without affecting mitogenesis induced by the growth factors. Altogether, these results suggest a role for pp60c-src in epithelial cell scattering and indicate that pp60c-src might contribute unequally to the two separate biological activities engendered by a single signal.

A scatter factor-like factor is produced by a metastatic variant of a rat bladder carcinoma cell line.

The rat bladder carcinoma epithelial NBT-II cell line undergoes, in vitro, a morphological transition to a fibroblast-like state in the presence of different growth factors. We have selected, in vivo, a metastatic clone, designated M-NBT-II, which has a mesenchymal phenotype and secretes into the culture medium a factor able to dissociate epithelial clusters of NBT-II or MDCK cells. This factor was designated scatter factor-like (SFL) by analogy to the HGF/SF, which has the same dissociating effect in these two cell lines. Here, we show that SFL factor and HGF/SF are different factors: (i) no HGF/SF transcripts could be detected using either specific rat HGF/SF cDNA probes or PCR; (ii) blocking antibodies against rat HGF/SF do not inhibit the SFL activity; and (iii) crude culture medium or partially purified SFL factor-containing fractions do not induce MDCK tubulogenesis, a biological assay that is specific for HGF/SF activity in vitro. We report the partial purification of the SFL factor, based on ion exchange and reverse-phase chromatography. The results indicate that the M-NBT-II metastatic variant secretes a dissociating factor sharing some common biological properties with the HGF/SF, which suggests that the SFL factor is a member of the HGF/SF family and may be involved in tumor progression.