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ABH tissue isoantigens were measured by the Specific Red Cell Adherence (SRCA) test in 66 surgical specimens of urinary bladder, including 53 transitional cell carcinomas, 2 squamous cell carcinomas and 11 controls. The SRCA test was strongly positive in 10 of 11 controls. ABH isoantigens were absent or equivocally present in 68 percent of noninvasive carcinomas (stage 0) and in 65 percent of invasive carcinomas. Clinical histories revealed that all patients with invasive carcinoma who had strongly positive SRCA test results had received prior radiotherapy to the bladder region. None of the patients with invasive bladder carcinoma with negative or weakly positive SRCA tests had been radiated. Histopathology of tumors in both groups was similar. Results of this retrospective study support the hypothesis that radiation may induce differentiation in tumors, possibly through an enhancement of Golgi apparatus function. The SRCA test should not be used as a predictor of the biological behavior of future recurrences in patients with bladder carcinoma who have received therapeutic radiation since radiation may produce "false positive" SRCA test results.

This paper describes a set of simple methods for comparative light and electron microscopy studies on tissue cultured tumour cells derived from both noninvasive and invasive carcinogen-induced rat urinary bladder carcinomas. Cells are grown on Thermanox plastic coverslips and fixed in situ. Each plastic coverslip is then divided with scissors into four parts: the first is processed for light microscopy, the second for thin-section electron microscopy, the third for freeze-fracture electron microscopy, and the fourth for scanning electron microscopy. In some experiments, portions of the culture which have first been examined by light microscopy are subsequently prepared for electron microscopy. In this way, the culture conditions are kept constant and comparison of structural features (i.e. intercellular junctions) by several preparative techniques is possible. Noninvasive and invasive rat bladder tumour cells, characterized by numerous pleomorphic microvilli, have normal zonulae occludentes at the apices of lateral surfaces of tumour cells in all cultures. In some areas of invasive tumour cells, occludens junctions are focally attenuated, consisting of only one or two strands, and occasionally the strands are discontinuous. Gap junctions, type PF-1, as well as numerous demosomes are present in all cell lines. Thus, intercellular junctions in noninvasive and invasive rate bladder epithelial cell lines bear a striking resemblance to those previously described in the comparable solid primary tumours. These culture systems may be useful for studying factors which influence the formation of intercellular junctions during malignant transformation.

KLN 205 murine squamous carcinoma cells were grown in medium supplemented with the retinoid 13-cis-retinoic acid (RA) to study the relationship between RA-induced cell surface changes and alterations of the metastatic phenotype. Modulation of the cell surface glycoconjugate expression was measured by flow cytometric analysis of the RA-treated tumor cells stained with fluoresceinated lectins. RA treatment (5 X 10(-6) and 5 X 10(-7) M) altered the glycoconjugate expression of KLN 205 cells in a selective, dose-dependent fashion. Tumor cells grown in RA-supplemented medium for more than 4 days demonstrated greatly increased binding of fluoresceinated Griffonia simplicifolia I lectin, peanut lectin, wheat-germ lectin, concanavalin A, and soybean lectin (P less than .001), but the increased binding of Ulex europaeus lectin was of a much smaller magnitude (P = .02). After 15 days of growth in these noncytotoxic or cytostatic concentrations of RA, malignant KLN 205 cells had a greatly decreased proclivity to metastasize, as measured by the lung colony assay (P = .0003). The RA-induced cell surface glycoconjugate changes preceded the decrease in experimental metastatic potential. Since enzymatic (neuraminidase) alteration of the tumor cell surface to produce glycoconjugate expression similar to that seen in RA-treated cells also reduced the ability of the KLN 205 cells to form lung colonies (P = .0022), it is suggested that RA-induced alteration of the cell surface carbohydrate antigens is related to the decreased experimental metastatic potential seen in tumor cells treated with RA.

The avascularity of epithelia may be attributed to the presence of an extractable, low-molecular-weight factor. This factor contains potent inhibitors of proteolytic enzymes, as well as a growth inhibitory activity directed against endothelial cells in vitro. It is extracted from the epithelium of bovine urinary bladders by 1 M NaCl. The extract is ultrafiltered through an Amicon XM-50 membrane, then concentrated and dialyzed into a 0.9% NaCl solution, using a UM-2 membrane. This ultrafiltrate, called the UM-2 retentate (UM-2R), contains approximately 6 micrograms protein/g tissue. The UM-2R has a low content of uronic acid and is practically devoid of hydroxyproline. SDS-PAGE reveals that the UM-2R consists of six major proteins. The UM-2R contains a Trasylol-like proteinase inhibitor that expresses strong trypsin inhibitory activity. Comparisons between bladder and serum UM-2Rs and electrophoretic mobility assays indicate that this proteinase inhibitory activity is derived from the bladder epithelium and not from the serum. The UM-2R is cytotoxic to cultured endothelial cells. Cultures of other cell types (normal and neoplastic) are not affected. The bladder-derived proteinase and endothelial cell growth inhibitory activities may protect epithelia from vascular invasion.

Carcinoma in situ of the urinary bladder has been recognized as a pathologic entity for over a quarter of a century, yet its biologic and clinical significance is controversial. It has been established that the presence of areas of carcinoma in situ in bladders harboring tumors heralds a relatively poor prognosis. However, the prognostic significance of carcinoma in situ in patients without prior or current solid tumors remains open to question. From the standpoint of cancer biology, the possibility that a morphologic mimicker of carcinoma in situ lacks the metabolic machinery or crucial structural components required to complete the process of neoplastic transformation raises interesting cells. The term carcinoma paradoxicum is introduced to describe functionally benign but morphologically anaplastic intra-epithelial neoplasms with marginal malignant potential.

Neovascularization of developing, repairing or neoplastic tissues is regulated, at least partially, by a family of proteins of low molecular mass (1000 less than mol mass less than 50 000 Da) which can be extracted from avascular tissues, such as hyaline cartilage, aorta or bladder epithelium, by mild salt solutions. These extractable proteins, functionally defined as anti-invasion factor (AIF), act as local regulators for some of the major mechanistic pathways by which endothelial cells are thought to invade tissues during neovascularization, mainly by matrix-degrading enzymes and by increased rates of migration and proliferation. AIF contains a spectrum of proteinase (collagenase) inhibitory activities, as well as an endothelial cell growth inhibitor. The endothelial cell growth inhibitor is directed against actively dividing endothelial cells in culture but has no effect on endothelial cell monolayers or any other cell lines tested. In tumours, the AIF-derived endothelial cell growth inhibitor may limit tumour growth to less than 2 mm in diameter by inhibiting tumour neovascularization.

Chondrocytes isolated from bovine articular cartilage were plated at high density and grown in the presence or absence of ascorbate. Collagen and proteoglycans, the major matrix macromolecules synthesized by these cells, were isolated at times during the course of the culture period and characterized. In both control and ascorbate-treated cultures, type II collagen and cartilage proteoglycans accumulated in the cell-associated matrix. Control cells secreted proteoglycans and type II collagen into the medium, whereas with time in culture, ascorbate-treated cells secreted an increasing proportion of types I and III collagens into the medium. The ascorbate-treated cells did not incorporate type I collagen into the cell-associated matrix, but continued to accumulate type II collagen in this compartment. Upon removal of ascorbate, the cells ceased to synthesize type I collagen. Morphological examination of ascorbate-treated and control chondrocyte culture revealed that both collagen and proteoglycans were deposited into the extracellular matrix. The ascorbate-treated cells accumulated a more extensive matrix that was rich in collagen fibrils and ruthenium red-positive proteoglycans. This study demonstrated that although ascorbate facilitates the formation of an extracellular matrix in chondrocyte cultures, it can also cause a reversible alteration in the phenotypic expression of those cells in vitro.

The invasive RBTCC-8 rat bladder carcinoma cell line (passage number, greater than 100) and its derivates, the RBTCC-8 tumor isografts and the 1-RBTCC-8 daughter cell line (fourth passage), express proteolytic activities of broad substrate specificity, which allow them to efficiently degrade extracellular (collagenous) matrices. Cell-associated, collagenolytic activity is evidenced by the release of hydroxyproline from collagen substrates of types I and IV, by visualizing the low-molecular-weight collagen breakdown products on sodium dodecyl sulfate-polyacrylamide gels, and by the depth of invasion into extracellular matrices in our bone invasion assays. Fractionated by diethylaminoethyl column chromatography, the major collagenolytic activities against collagens of types I and IV coelute in a relatively narrow peak within a NaCl gradient. The pooled collagenolytic diethylaminoethyl fractions contain: (a) two chymotrypsin-like, catheptic activities; (b) activity against a synthetic elastase substrate; (c) gelatinase activity; and (d) caseinolytic activity. Despite efficient collagenolysis, a vertebrate-type collagenase cannot be detected in any of our tumor samples, even after trypsin activation of the tumor cell extracts. The mechanism of action of these nonspecific proteinases is thought to be that of collagen "crosslinkases." The neutral proteinase activities are highest in RBTCC-8 tumor isografts, intermediate in the fourth passage 1-RBTCC-8 carcinoma cell line, and lowest in the RBTCC-8 carcinoma cell line of high passage number. The levels of these nonspecific enzyme activities are well correlated with the depth of invasion into bony matrices, as shown by our invasion assays.

The coexistence of carcinoma in situ in flat epithelium adjacent to tumors is prognostically significant and is associated with an increased incidence of tumor recurrence and invasion. Recent investigations have been aimed at finding markers that will help identify flat areas of dysplasia or low-grade papillary transitional cell carcinomas that have a high potential to evolve into invasive tumors. Both DNA and karyotype analyses, as well as cell surface markers, have been examined in some detail and could have clinical value in certain settings. The tumor DNA index as measured by flow cytometry of nuclei retrieved from deparaffined sections may be particularly useful as a predictor of tumor recurrences and, possibly, tumor invasion as well. The diagnosis of superficial disease rests on urine cytology and the histopathologic assessment of tissue biopsies; however, intra- and inter-observer reproducibility of tissue and cytologic diagnoses by pathologists is a significant problem. The application of new technologies, such as flow cytometric analysis of deparaffined nuclei, may reduce the subjectivity involved in the light microscopic assessment of pathology specimens.

Asymmetric unit membrane (AUM) is a component of the luminal membrane of urinary bladder in many species. In normal human adults it is inconspicuous, but it becomes prominent following incidental exposure to therapeutic irradiation.

The parent R3230 AC rat mammary carcinoma cell line and the two variant cell lines, R3230 AC MET and R3230 AC LR, differ with respect to their abilities to invade bony matrices and to form lung colonies (experimental metastases). Both the R3230 AC and the R3230 AC MET, a cell line selected in vivo for enhanced metastatic capability, express high potentials for invasiveness and lung colony formation, while the Con A- and WGA-resistant R3230 AC LR cell line grows expansively at the periosseus implantation site and is unable to form lung colonies after intravenous inoculation. The abilities to invade bone and to metastasize to the lung are well correlated with the fibrinolytic activity and the production of urokinase-type plasminogen activators. The contribution of plasminogen activators to invasiveness and metastasis has been ascribed to its role in the fibrinolytic and collagenolytic (i.e., activation of latent collagenase) cascades.

Our previous observation that salt-extracted, devitalized cartilage could be penetrated by malignant tumor cells but was nonpermissive to fibroblastic ingrowth led us to postulate that this matrix might be used as a test connective tissue to discriminate in vitro between noninvasive and invasive tumor cell lines. In a novel in vitro system, salt-extracted, bovine articular cartilage was therefore used as a growth surface for defined noninvasive, invasive, and metastatic carcinoma cell lines, derived from chemical carcinogen-induced tumors of the rat urinary bladder. As monitored by thin-section electron microscopy, salt-extracted cartilage was readily penetrated by the invasive and metastatic rat bladder carcinoma cell lines. The metastatic cell line could be differentiated from the invasive, nonmetastatic cell line by its greater depth of invasion. In contrast, noninvasive carcinoma cells as well as normal bladder epithelial cells lacked the capacity to erode and penetrate the extracted matrix of the articular cartilage. Using these defined cell lines, salt-extracted cartilage can be used to reproducibly discriminate between carcinomas having different invasive potentials. This assay system may have diagnostic application for the in vitro staging of tumors.

Many cancers display characteristic organ colonization patterns that do not fit simple, anatomical-mechanical trapping theories of tumor cell dissemination. Organ preferences of metastatic spread appear to be mediated partly by the selective attachment of tumor cells to organ-specific, microvascular endothelium. To study these tumor cell-endothelial cell interactions in an efficient and reproducible manner, we have designed a novel in vitro assay system wherein endothelial cells isolated from large vessels (e.g., aorta) can be modulated to assume phenotypic traits of organ-specific, microvascular endothelium. Modulation is achieved by growing bovine aortic endothelial cells (BAEC) on organ-specific matrix components, termed tumor attachment modulators (TAMs). Using monolayers of modulated BAEC in a tumor attachment assay, we show here that tumor cells which metastasize to a given organ, have a significantly higher binding affinity for BAEC grown on TAMs of the preferred, metastasized organ, than they have for BAEC grown on TAMs of any other organ not colonized by these tumor cells. Lung-metastatic tumor cells (R3230AC-MET, B16-F10) adhere preferentially to BAEC monolayers grown on lung-specific TAMs, whereas liver-metastatic tumor cells (RAW117-H10, M5076) selectively adhere to BAEC grown on liver-specific TAMs. In contrast, nonmetastatic tumors cells (R3230AC-LR, RBTCC-1, 647V) show no such adhesion preferences. Preferential tumor cell adherence is increased by growing BAEC for prolonged periods on organ-specific TAMs. Metastatic preference and organ distribution are mediated, at least in part, by urea-extractable endothelial cell surface components that are regulated by the extracellular matrix.

Normal rat urinary bladder epithelial cells contain small subunit (PF-1) and large subunit (PF-2) gap junctions, whereas carcinoma cells only contain PF-1 gap junctions. The absence of PF-2 gap junctions, which are composed of larger connections with slightly larger ionic channels, may contribute to altered metabolic coupling between urinary bladder carcinoma cells.

Epithelial cells, microvascular endothelial cells, and fibroblasts have been isolated in culture from normal urinary bladders of Fischer rats. Normal epithelial cells were cultured most efficiently when transitional epithelial sheets were plated on to collagen-coated roller flasks. The epithelial sheets were obtained by two micro-dissection techniques. In the first method, the epithelium was peeled as a large coherent sheet from the submucosal connective tissue following subepithelial injection of a collagenase solution, and after incubation of the bladders in the same enzyme solution. Epithelial sheets with intact basal cell layers were essential for culture success. On collagenous matrices, epithelial differentiation was similar to that in vivo. The in vitro transitional epithelium was composed of three cell layers, namely superficial, intermediate, and basal cells. Basal cells were attached to newly synthesized basal lamina by means of hemidesmosomes. Superficial cells were sealed at their apical lateral membranes by a junctional complex, i.e. a terminal bar. Asymmetric luminal membrane plaques were not apparent. In the second method, the epithelium was separated from the underlying connective tissue after collagenase--trypsin digestion of everted urinary bladders. Although the digest consisted mainly of epithelial cells, these rarely survived the first passage when plated on conventional plastic growth surfaces. After the third culture week, epithelial cells usually died and slowly growing colonies of fibroblasts or large flattened epitheloid cells became apparent. Epitheloid cells were identified by their typical ultrastructure as endothelial cells, showing Weibel--Palade bodies and pinocytotic caveolae. These cells were reactive with antiserum against factor VIII. The free surface of monolayer cultures was non-thrombogenic when incubated in the presence of platelets. Fibroblasts were isolated from heavily contaminated epithelial cell cultures after differential trypsinization. These three cells types represent the normal control cells of an in vitro tumor model for the study of invasiveness. All three cell types are involved in the formation and functional maintenance of the epithelial--stromal junction. The study of cell--cell and cell--matrix interactions may provide important clues for the understanding of tumor invasiveness, a process that starts at the epithelial--stromal junction and proceeds with its destruction.

The resistance of cartilage to tumor invasion was studied with the use of a novel in vitro culture system. Articular cartilage obtained from fresh metacarpophalangeal joints of preadolescent bovines was used as a growth surface for human TE-85 osteosarcoma cells and foreskin fibroblasts. Cartilage disks formed the bottoms of stainless-steel cylinders, providing closed growth chambers for these cells. Both invasive osteosarcoma cells and normal fibroblasts were unable to penetrate viable, unextracted cartilage during a 2-week culture period. When cartilage was devitalized by freezing and thawing, the tissue remained resistant to invasion. Cartilage, extracted with either 1 or 3 M guanidine hydrochloride, was invaded by osteosarcoma cells, but not by control fibroblasts. Invasion by osteosarcoma cells into salt-extracted cartilage was abolished when low concentrations of a cartilage-derived, anti-invasion factor were added to the culture medium. These data provided evidence that the resistance of cartilage to tumor invasion is regulated in part by tissue-derived proteinase inhibitors.

The sequential intravenous injections of protamine and heparin into rats results in the deposition of protamine-heparin aggregates along the lamina rara externa of the glomerular basement membrane. Morphologic studies indicate that these foreign aggregates are removed from the glomerular basement membrane by virtue of phagocytic activity by the glomerular visceral epithelial cells. We have studied the disappearance rate of protamine-heparin aggregates from the glomerular basement membrane in animals given a single dose of these compounds and in animals receiving chronic administration. The disappearance rate was measured utilizing a computerized morphometric technique to determine the numerical density of protamine-heparin aggregates per 100 micrometer glomerular basement membrane in sequential biopsy specimens taken from 15 to 600 min post-injection. The disappearance rate described a linear function in both acute and chronic animals, with a half-disappearance time of approximately 120 min. Disappearance of the aggregates appeared to be due to epithelial cell phagocytosis. This model provides a method of studying this function in the intact glomerulus in both normal and disease states.

Antigens of Micropolyspora faeni were located by immunofluorescence in apparent association with alveolar macrophages within sections of lung from cases of bovine interstitial pneumonia. Pretreatment of frozen sections with pH 2.8 glycine-HC1 buffer was a necessary prerequisite for specific staining. Bovine immunoglobulin and bovine complement were identified in similar locations.

Intercellular junctions in spontaneous canine pancreatic acinic cell adenocarcinomas were compared to those in control canine pancreas. The neoplastic cells displayed proliferation and fragmentation of tight junctions and reduction in size and number of gap junctions. Marked decrease in desmosomal density was observed only in the poorly differentiated carcinoma. In the well differentiated carcinomas a few of the desmosomes were characteristic of those found in squamous cells. No quantatitive or qualitative differences in cell junctions were noted between primary and metastatic tumor.

Murine squamous carcinoma cells (KLN205) grown in a medium supplemented with the retinoid, 13-cis retinoic acid (RA), had dose-dependent, selective increases in the expression of certain lectin receptors, which correlated with a dramatic decrease in the ability to form pulmonary colonies (P = .0003) (Couch MJ, Pauli BU, Weinstein RS, Coon JS: JNCI, 78:971-977, 1987). These findings suggest a possible relationship between the RA-induced glycoconjugate alterations and the decreased experimental metastatic behavior. We further define the mechanism of RA's action. The finding that RA treatment (5 X 10(-6) M, 5 X 10(-7) M) did not perturb the cell cycle of KLN205 cells provides further proof that the decreased metastatic behavior is not attributable to any inhibition in the rate of growth or to alterations in the cell cycle. Furthermore, since stable subpopulations with variable lectin binding could not be detected, the mechanism of RA's action does not appear to be due to selection of variant tumor-cell subpopulations. Finally, in a series of experiments designed to determine the reversibility of the RA treatment, the RA-induced decrease in metastatic behavior reverted back to a more metastatic state in the same time frame (3 days) as the reversion of the RA-induced changes in cell-surface glycoconjugate expression. This reversion provides further evidence for a close relationship between the RA-induced modulation of tumor cell-surface glycoconjugate expression and the decreased metastatic behavior; it suggests that transient, reversible modulation of the tumor cell surface may play a role in determining metastatic behavior.

Small bowel anastomoses were performed without sutures by using the Nd:YAG laser to produce welded enterotomies. Optimal energy levels for contact and noncontact laser were determined. Anastomoses produced using five target energy levels between 100 and 500 J were examined. Short-term anastomotic strength of these enterotomies was measured 1 min after the welding. Bursting pressure of the laser welded enterotomies was compared to the bursting pressure of traditional two-layer, inverting, interrupted sutured bowel anastomoses. The overall mean bursting pressure of non-contact-welded enterotomies was 50.6 mmHg. Optimal laser settings determined in this initial phase were then used to produce anastomoses in rabbits which are recovered postoperatively for 1 or 2 weeks in order to examine long-term viability and integrity of the anastomoses. All chronic rabbit preparations recovered without complication. The mean bursting pressure was 200 mmHg, not significantly different from that of uncut bowel or two-layer sutured anastomosis.

The teratogenic potency of the directly acting alkylating agent methylnitrosourea (MNU) was analysed in mice. Skeletal abnormalities were evaluated after treatment on either day 11 or 12 of pregnancy. Ectrodactyly was the predominant effect after treatment on day 11. Treatment on day 12 triggered especially double-sided microdactyly (method of analysis: measuring digit lengths). Litter variabilities were analysed using a new biometrical procedure. Using probit analysis, dose-response curves were computed from the experimental data obtained and the effective doses were calculated and compared with maternal toxicity. Low dose extrapolation was performed by use of various mathematical models which yielded very similar ED1/100 and ED1/1000 values.

The volume (Vv) and surface area (surface density; Sv) of the Golgi complex were measured by electron microscopy stereologic techniques in 17 patients with urinary bladder transitional cell carcinomas. Seven patients were irradiated prior to surgical removal, 4 had recurrent carcinomas that arose from previously irradiated bladder urothelium, and the remaining 6 patients had bladder carcinoma but no prior history of therapeutic irradiation. There were significant differences in the Vv and Sv of Golgi complexes per unit of cytoplasmic volume in carcinomas from irradiated and nonirradiated patients (p < 0.01). In carcinomas obtained from irradiated urinary bladders, the Vv was 3.45 +/- 0.24% (mean +/- SE). In contrast, the Vv was 0.98 +/- 0.15% for tumors in the nonirradiated group. Sv values were 1.51 +/- 0.09 microm2/microm3 in carcinomas from irradiated bladders, and in the nonirradiated group, the mean Sv was 0.76 +/- 0.07 microm2/microm3. These results support the hypothesis that therapeutic irradiation to the urinary bladder induces hyperplasia of Golgi complexes.

Four cases of spontaneous adenocarcinoma of the canine exocrine pancreas were studied by thin section and freeze-fracture electron microscopy. The neoplastic cells in all 4 cases were of acinar origin and showed many alterations of cytoarchitecture compared with normal acinar cells. In the neoplastic cells, zymogen granules varied in number and appearance and contained an abnormal secretory product characterized by 24-nm-thick microtubules. The nuclear volume and surface area and the nuclear cytoplasmic ratio were increased; although the nuclear pore density per nuclear surface area was significantly decreased, there was no change in the nuclear pore density per nuclear volume. There was a decrease in number and size of gap junctions; focal proliferation, fragmentation, and discontinuation of the tight junctions were also noted. The basal lamina (BL) of the neoplastic cells was discontinuous. The tumor microvasculature often appeared as sinusoids and had sparse discontinuous BL. Finally, the endothelium in both tumor and normal tissue contained "tubulo-reticular inclusions" (TRI) which simulated distemper virus and were located in the rough endoplasmic reticulum (RER) and the perinuclear cisternae.