Biomedical subjects
Publications and source records attributed to M J Droller.
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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.
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.
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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.
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.
The finding of muscle-infiltrative bladder cancer is generally considered ominous, as 50% of patients who present with this condition are likely to develop distant metastases within 2 years. However, some types of infiltrative bladder cancer appear to have a less ominous prognosis. In order to assess the results of various therapies on regionally advanced bladder cancer, it is important to characterize the distinctions between the types with a poor prognosis (5-year survival rate 15% to 30%) and those with a better prognosis (5-year survival rate 50% to 85%). The former have a nodular architecture, infiltrate more deeply and in a tentacular pattern, and more often involve the bladder wall vasculature and lymphatics than does the latter type, which has papillary architecture and infiltrates on a broad front. These two types appear to respond differently to various treatments except systemic chemotherapy.
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The absence of basement membrane components correlates with tumor stage and progression in human bladder cancers. We have previously shown that invasive tumors possess the ability to degrade basement membrane. However, the presence of basement membrane may be affected not only by its degradation, but by its synthesis and deposition as well. Our results in the present study suggest that while the invasive human transitional carcinoma cell line EJ has an increased amount of type IV procollagen mRNA when compared to the non-invasive RT4 cell line, type IV collagen staining is absent in the invasive EJ cells and intensely present in the non-invasive RT4 cells. Moreover, when EJ cells were grown on an artificial basement membrane (Matrigel), type IV procollagen mRNA expression was down-regulated to the levels seen with the non-invasive RT4 cells. We also discovered that the invasive cells, when grown on Matrigel, appeared morphologically different from the same cells grown on plastic tissue cultures. We conclude that a deficient basement membrane in invasive cancer cells may be due not only to active proteolytic activity but also to an abnormal production and deposition of extracellular matrix components. In addition, we also demonstrated that basement membrane components may have a significant effect on epithelial cell morphology and gene regulation, and that any alterations of the extracellular matrix-cytoskeleton-nuclear matrix interactions can lead to altered gene regulations and cell function.
Bladder tumors comprise a spectrum of neoplastic diathesis. Some behave in a benign fashion, and others are highly aggressive and lead rapidly to metastatic disease and death. The metastasizing potential, often described as a sequence of interrelated steps, involve 1) tumor cell adhesion to basement membranes, 2) degradation of basement membranes and underlying connective tissue stroma, and 3) migration of tumor cells through the destroyed stroma into blood and lymphatic vessels. Each of these processes involves the expression of molecular and biochemical factors identified with tumor cells. With better understanding of the molecular basis of these factors, novel prognostic and potential therapeutic agents can be generated and applied to the clinical arena.
To study the effect of the protein kinase C (PKC) inhibitor staurosporine on invasion, we selected the invasive human bladder carcinoma cell line EJ. Total PKC activity was more than twofold higher in the EJ cells than in RT4 cells (superficial human bladder carcinoma cells), which do not pass through an artificial basement membrane. There was more PKC activity in the cytosol than in the membrane of EJ cells. Staurosporine, at nontoxic concentrations, inhibited the invasion of EJ cells through an artificial basement membrane in a dose-dependent manner. Staurosporine caused a dose-dependent inhibition of cell motility but did not inhibit cell attachment. Staurosporine represents a new agent for the inhibition of tumor cell invasion and may prove useful in studying the mechanisms responsible for this phenomenon.
The interrelationship between the lateral and posterior portions of the peritoneum and Gerota's fascia allows them to be separated from each other, permitting ready visualization of the major renal vessels. The posterior relationship between Gerota's fascia and the transversalis fascia overlying the more posterior psoas muscle can be used to advantage in exposing the renal vessels posteriorly. That the fascial layers defining the retroperitoneal spaces might therefore be used in dissecting the renal hilum and vessels in the performance of radical nephrectomy prompted the present investigation. Twenty patients underwent this approach for radical nephrectomy for varying stages of renal cell carcinoma involving either upper, mid, or lower portion of right or left kidney. Postoperative recovery appeared to be shortened because of the lesser interval of ileus than had previously been encountered using a transperitoneal approach. Despite the minimal manipulation of the kidney prior to ligation of the renal vessels, there was no apparent increased tumor dissemination as detected clinically, and experimentally there appeared to be no increase in tumor cells in the venous effluent. Operative time, blood loss, and postoperative complications were comparable to those reported for the transperitoneal approach. This means of performing radical nephrectomy, based on the relationship of fascial layers in the retroperitoneum thus appeared to lend itself to easier dissection as well as lesser postoperative ileus and corresponding shorter hospital stays.
To study the biochemical mechanisms of bladder tumor invasion, we analyzed specimens of invasive transitional cell carcinoma cell line EJ and non-invasive transitional cell carcinoma cell line RT4 which had been implanted into the bladders of nude mice. Using immunoprobes specific to basement membrane laminin, we observed that superficial but not invasive tumors were surrounded by intact laminin. With immunoprobes specific to cathepsin B, a cysteine proteinase which has the ability to degrade laminin, we demonstrated that cathepsin B is localized in discrete cytoplasmic granules in the non-invasive tumors, and in a more diffuse cytoplasmic pattern in the invasive tumors. Subcellular fractionation followed by immunoblot analysis and enzymatic analysis confirmed that the invasive EJ cells had active cathepsin B localized to its plasma membrane, while non-invasive RT4 cells had cathepsin B confined to lysosomes. Furthermore, immunoblot analysis revealed that invasive EJ cells had the mature form of cathepsin B with a molecular weight of 25 kD, while non-invasive RT4 cells had predominantly precursor forms with molecular weights between 30 and 35 kD. In vitro degradation assays with plasma membrane fractions isolated from invasive EJ cells and non-invasive RT4 cells demonstrated that the plasma membrane of EJ cells but not that of the RT4 cells had the ability to degrade purified laminin, and that the degradative products were similar to those obtained with purified cathepsin B. We conclude that invasive tumor cells have enhanced cathepsin B in their plasma membranes which may be used to degrade basement membrane components such as laminin and thereby facilitate tumor invasion.
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This study examines the clinical and pathologic features of 20 consecutive patients with muscle-invasive bladder cancer diagnosed between January 1, 1983, and December 31, 1984, at The Mount Sinai Hospital. On retrospective analysis, 18 patients (90%) had muscle-invasive bladder cancer at their initial presentation. The interval between onset of symptoms and histologic documentation of cancer was less than two months in 14 cases (78%), and in the remaining 22 per cent, the intervals of six, six, twelve, and twenty-four months were attributable to delay in seeking medical attention or delay in biopsy. In contrast, in the 2 patients who had presented with superficial tumors, the intervals were thirteen and one hundred eighty months. It therefore appeared that most patients with invasive bladder cancer had no therapeutically significant prior clinical or symptomatic history of superficial disease, and that invasion either was already present when symptoms had begun, or occurred rapidly after symptoms had initially appeared. These findings confirm the broad applicability of this pattern of bladder cancer in the general population, thereby complementing similar findings reported in recent years by secondary referral centers.
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