Expression of keratin and vimentin intermediate filaments in rabbit bladder epithelial cells at different stages of benzo[a]pyrene-induced neoplastic progression.

Rabbit bladder epithelium, grown on collagen gels and exposed to the chemical carcinogen benzo[a]pyrene, produced nontumorigenic altered foci as well as tumorigenic epithelial cell lines during 120-180 d in culture. Immunofluorescence studies revealed extensive keratin filaments in both primary epithelial cells and benzo[a]pyrene-induced altered epithelial foci but showed no detectable vimentin filaments. The absence of vimentin expression in these cells was confirmed by two-dimensional gel electrophoresis. In contrast, immunofluorescence staining of the cloned benzo[a]pyrene-transformed rabbit bladder epithelial cell line, RBC-1, revealed a reduction in filamentous keratin concomitant with the expression of vimentin filaments. The epithelial nature of this cell line was established by the observation that cells injected into nude mice formed well-differentiated adenocarcinomas. Frozen sections of such tumors showed strong staining with antikeratins antibodies, but no detectable staining with antivimentin antibodies. These results demonstrated a differential expression of intermediate filament type in cells at different stages of neoplastic progression and in cells maintained in different growth environments. It is apparent that the expression of intermediate filaments throughout neoplastic progression is best studied by use of an in vivo model system in parallel with culture studies.

Rhodamine-123 selectively reduces clonal growth of carcinoma cells in vitro.

Rhodamine-123, a cationic laser dye, markedly reduced the clonal growth of carcinoma cells but had little effect on nontumorigenic epithelial cells in vitro. This selective inhibitory effect of Rhodamine-123 on some carcinomas is unusual since known anticancer drugs, such as arabinosyl cytosine and methotrexate, have not been shown to exhibit such selectivity in vitro.

Identification of p53 gene mutations in bladder cancers and urine samples.

Although bladder cancers are very common, little is known about their molecular pathogenesis. In this study, invasive bladder cancers were evaluated for the presence of gene mutations in the p53 suppressor gene. Of 18 tumors evaluated, 11 (61 percent) were found to have genetic alterations of p53. The alterations included ten point mutations resulting in single amino acid substitutions, and one 24-base pair deletion. In all but one case, the mutations were associated with chromosome 17p allelic deletions, leaving the cells with only mutant forms of the p53 gene products. Through the use of the polymerase chain reaction and oligomer-specific hybridization, p53 mutations were identified in 1 to 7 percent of the cells within the urine sediment of each of three patients tested. The p53 mutations are the first genetic alterations demonstrated to occur in a high proportion of primary invasive bladder cancers. Detection of such mutations ex vivo has clinical implications for monitoring individuals whose tumor cells are shed extracorporeally.

Differential pp60c-src activity in well and poorly differentiated human colon carcinomas and cell lines.

The results presented in this report demonstrate increased pp60c-src kinase activity associated with moderate to well differentiated colon tumors, corroborating previous observations by other groups. Extension of this analysis to include a small number of poorly differentiated colon carcinomas revealed src kinase activity comparable to that observed in normal colonic mucosa, considerably less than that observed in moderate/well differentiated lesions. Correlations of src kinase activity with differentiation was confirmed within a panel of colon cell lines where increased activity, associated with moderate/well differentiated lines, was accompanied by increased expression of pp60c-src protein. Use of an antiphosphotyrosine antibody in immunoprecipitation revealed the presence of novel phosphotyrosyl cellular substrates in human colon cell lines displaying elevated pp60c-src kinase activity. These observations suggest a role for the src protooncogene in colonic differentiation pathways.

Effects of donor age on neoplastic transformation of adult mouse bladder epithelium in vitro.

Neoplastic transformation of C57BL/lcrf-a' mouse bladder epithelium was induced in long-term primary cultures by a single 24-hour treatment with 7,12-dimethylbenz[a]anthracene on day 2 of culture. Transformed foci appeared earlier (40--60 days) and at a higher frequency (28%) in cultures from old donors (28--30 mo) compared with 100 days and 0.9% in cultures from young adult donors (5--7 mo). After transplantation into syngeneic mice, transformed cells produced carcinomas. Spontaneous epithelial transformation occurred in dimethyl sulfoxide-treated old cultures after the same interval (40--60 days) as in the carcinogen-treated cultures but at a lower frequency (5.3%). Spontaneous epithelial transformation did not occur in cultures from young donors.

Identification of H-ras mutations in urine sediments complements cytology in the detection of bladder tumors.

BACKGROUND: Urinary cytology has long been used as a noninvasive screen for the detection of urinary tract cancer but is limited by the generation of false positive and false negative results. More recently, molecular changes associated with urothelial neoplastic progression have been identified in DNA from urine sediments, demonstrating an alternative approach for identifying neoplastic change in the bladder. PURPOSE: The purpose of this prospective study was to determine the value of detection of H-ras (also known as HRAS) mutations in urine sediment DNA as a clinical indicator of tumor presence, recurrence, and/or progression. METHODS: Urine sediments were collected from 100 patients presenting with bladder tumors, with follow-up samples collected from 19 patients. DNA extracted from urine sediments was analyzed for changes in exon 1 of the H-ras gene, using single-strand conformation polymorphism (SSCP) analysis. A representative number of aberrant H-ras/SSCP migrating bands were excised and sequenced to confirm the presence of a mutation. Human bladder specimens were obtained from patients (93 of the 100 patients initially and 18 of the 19 patients studied by follow-up) and histologically evaluated for tumor content and grade. RESULTS: Mutations in exon 1 of the H-ras gene were detected in urine sediments from 44% (44 of 100) of the patients; concordant results were obtained by cytologic analysis, where 33% (31 of 93) of the patients displayed positive cytology. Analysis of the distribution of abnormalities with tumor grade revealed greater detection of low-grade (1-2) lesions using ras analysis (47%) compared with cytology (16%). In contrast, cytology was more effective in identifying the presence of carcinoma in situ. Combined results from these two approaches substantially increased the sensitivity of tumor detection, resulting in the identification of tumors in 60% of patients. CONCLUSIONS: Identification of H-ras mutations in DNA from urine sediments facilitates the detection of low-grade bladder tumors and, in combination with cytology, increases the overall tumor detection from 33% to 60%. Preliminary results in patient follow-up suggest that detection of H-ras mutations may have some clinical utility in detecting the presence of abnormal cells in the absence of an overt lesion following cystoscopy or positive cytology.

Monoclonal antibodies raised against cell membrane components of human bladder tumor tissue recognizing subpopulations in normal urothelium.

Monoclonal antibodies to human bladder carcinoma membrane antigens were produced by fusion of MOPC-21 NS/1 mouse myeloma cells with spleen cells from BALB/c mice immunized against a crude membrane extract from a metastatic bladder carcinoma. Hybrids were screened for antibody production in a solid-phase radioimmunoassay and selected for their reactivity with subpopulations of urothelial cells on normal bladder tissue sections. Three antibody groups were defined: Group I (4-72-2) was urothelium specific and stained the basal and intermediate cells in normal urothelium; group II (3-48-2, 48-1, and 3-50-3) showed reactivity with intermediate and superficial cells; group III (8-30-3, 77-1, 2-94-2, 3-71-1, and 94-3) was restricted to antigens on the luminal membrane of superficial cells. All antibodies recognized antigenic determinants in fixed paraffin-embedded material and within groups showed a range of staining patterns in other tissues. Studies on sections representing different stages of neoplastic progression showed disruption in the antibody-staining pattern in urothelium and, in all cases, a strong distinct staining of invasive tumor areas and metastatic secondary tumors. Biochemical analysis of the antigens defined at least three antigenic systems, two of which consisted of molecules having Mr of 250,000 and 300,000 as judged by Western blot analysis. Antigenic determinants recognized by some antibodies (3-48-2, 48-1, 3-50-3, 8-30-3, 77-1, and 3-71-1) were shown to be carbohydrate by reactivity with glycolipid fraction and suggest that antibodies within groups recognize different epitopes on the same molecule.

Localization of a Mr 52,000 keratin in basal epithelial cells of the mouse bladder and expression throughout neoplastic progression.

In this study, we report the isolation of a Mr 52,000 keratin from a nontumorigenic bladder epithelial cell line and its localization, by immunofluorescence, in bladder frozen sections at different stages of neoplastic progression and in various carcinogen-transformed bladder epithelial cells and human bladder carcinoma-derived cell lines. The results showed that: (a) the Mr 52,000 keratin is present in basal epithelial cells of the normal bladder but absent from the intermediate and superficial epithelial cell layers; (b) hyperplastic bladder lesions, induced in mice after the administration of butyl-(4-hydroxybutyl)-nitrosamine, resulted primarily from the proliferation of cells in the basal compartment; (c) butyl-(4-hydroxybutyl)nitrosamine-induced bladder carcinomas displayed differential staining with the anti-Mr 52,000 keratin antiserum reflecting divergent differentiated status within a tumor and a subpopulation of cells with reduced overall keratin expression; (d) primary cultures of bladder carcinomas revealed a subpopulation of cells with limited filamentous keratin as was observed in vivo; (e) mouse bladder epithelial cell lines transformed in culture by a chemical carcinogen showed a loss or reduction in expression of the Mr 52,000 keratin; (f) two human bladder carcinoma cell lines displayed very limited expression of the Mr 52,000 keratin. Although the loss or reduction in expression of a specific keratin is unlikely to be responsible for transformation, it may contribute to the heterogeneity in the differentiated state and morphology of bladder epithelial cells during neoplastic progression both in vivo and in culture.

Selective toxicity of rhodamine 123 in carcinoma cells in vitro.

The study of mitochondria in situ has recently been facilitated through the use of rhodamine 123, a mitochondrial-specific fluorescent dye. It has been found to be nontoxic when applied for short periods to a variety of cell types and has thus become an invaluable tool for examining mitochondrial morphology and function in the intact living cell. In this report, however, we demonstrate that with continuous exposure, rhodamine 123 selectively kills carcinoma as compared to normal epithelial cells grown in vitro. At doses of rhodamine 123 which were toxic to carcinoma cells, the conversion of mitochondrial-specific to cytoplasmic-nonspecific localization of the drug was observed prior to cell death. At 10 microgram/ml, greater than 50% cell death occurred within 7 days in all nine of the carcinoma cell types and lines of different origin studied, while six of six normal epithelial cell types and lines remained unaffected. Cotreating carcinoma cells with 2-deoxyglucose and rhodamine 123 enhanced the inhibition of growth by rhodamine 123 alone in clonogenic survival assays. The observation of the selective toxicity of rhodamine 123 appears to be unique in view of the absence of selective toxicity reported in vitro for the various antitumor agents currently in clinical use. Preliminary results with rhodamine 123 in animal tumor systems indicate antitumor activity for carcinomas.

Elevated expression of pp60c-src in low grade human bladder carcinoma.

The results presented in this report demonstrate that the tyrosine-specific protein kinase activity of pp60c-src is elevated over that recorded in normal bladder mucosa in a subset of human bladder carcinomas. Increased kinase activity was observed mainly in low grade bladder lesions and was associated, at least in part, with elevated levels of pp60c-src expression. Extension of this analysis to a panel of human bladder carcinoma cell lines confirmed previous observations of low pp60c-src kinase activity in three lines established from high grade (GIII) bladder tumors and revealed increased kinase activity in three alternative bladder lines derived from GI or GII tumors. Use of an anti-phosphotyrosine antibody in Western blot analysis revealed the presence of novel phosphotyrosyl cellular substrates in human bladder cell lines and tumors displaying elevated pp60c-src kinase activity. These observations suggest an association for the src protooncogene in urothelial cell differentiation events.

c-Ha-ras-I oncogene-induced differentiation and natural killer cell resistance in a human colorectal carcinoma cell line.

Natural killer (NK) activity is primarily a peripheral blood function of a lymphocyte population capable of spontaneous lysis of many transformed and metastatic targets. However, NK-susceptible targets tend to be relatively poorly differentiated. We have previously shown that poorly differentiated human colorectal carcinoma are lysed by NK cells. Well-differentiated and chemically differentiated colorectal carcinomas are insensitive to NK lysis. The present study demonstrates that transfection of the c-Ha-ras-I oncogene into a poorly differentiated colorectal carcinoma cell line also renders it NK resistant. This resistance is accompanied by a more differentiated colorectal carcinoma phenotype. Two ras-transfected lines (Clone-A-5 and Clone-A-4) showed a 30-66% decrease in susceptibility to NK lysis as compared to the parental line in standard cytotoxicity assays. The resistance of these transfectants was strictly dependent on expression of the activated p21, the H-ras protein product. Studies to assess the integrity of the initial binding step in NK lysis showed a significant decrease in the ability of these transfectants to form conjugates with fresh NK cells. It is likely that transfection with c-Ha-ras-I has selectively modulated critical NK target recognition structures.

Oncogene-mediated transformation of fetal rat colon in vitro.

Short-term maintenance of fetal rat colonic tissue in vitro has been demonstrated using a collagen matrix organ culture system. The introduction of single (v-myc, v-rasH, v-src) oncogenes or combinations of oncogenes (v-myc/rasH, v-myc/src) into normal colon mucosal elements was established using retroviral vectors, resulting in enhanced proliferation and migration of epithelial cells from the lumen of tissue implants. Expression of a single oncogene in normal colon epithelium did not result in the establishment of cell lines. In contrast, expression of cooperating oncogenic elements resulted in cell lines in greater than 80% of experiments, revealing different morphological characteristics dependent upon the oncogene combination used. Confirmation of the expression of viral transcripts was determined using Northern blot analysis and viral oncoprotein expression using Western blot analysis (p21) and an immunoprecipitation kinase assay (src). Expression of keratin filaments was lost following passaging of cell lines but could be induced by the myc/ras transformants by growth on Rat-1 feeder layers. This induction phenomenon was not observed with myc/src lines, and although these expressed high levels of sucrase isomaltase the epithelial origin of these cells is unclear. Karyotypic analysis performed on three myc/ras-transformed cell lines revealed a normal chromosome complement associated with transformation. In this report we describe a novel in vitro transformation system for normal rat colonic epithelium mediated by the introduction of oncogene elements using different retroviral vector constructs. The potential to generate cell lines representing different stages of neoplastic progression using relevant genetic components presents significant advantages for the study of cellular and molecular interactions underlying colon neoplastic progression.

ras induced lesions in a heterotopic mouse bladder.

To determine the in vivo phenotype elicited in bladder epithelium following the expression of a ras oncogene we have introduced the HaSV ras transforming gene into transplants of normal urothelium. Stripped adult bladder mucosa incubated with HaSV, in the presence or absence of helper virus, was transplanted beneath the renal capsule of syngeneic animals and maintained for periods up to three months. Control implants, exposed to helper virus alone, formed heterotopic bladders lined by urothelium displaying focal areas of full differentiation when associated with underlying submucosal elements. Exposure of urothelium to HaSV resulted in increased proliferative potential of mucosal and submucosal elements with the appearance of a normal differentiated heterotopic bladder 10 days post-implantation. Implants left for 28 days presented a range of hyperplastic lesions (mild-severe) characterized from histological evaluation and antibody markers recognizing basal (D66) and superficial cell populations (H10) in normal bladder mucosa. Staining of mild hyperplastic lesions revealed an increased basal cell compartment and a loss of fully differentiated cells lining the lumen of the implanted bladder. In severe hyperplasia no superficial cells were observed but mucosal elements stained throughout with antibody D66. This phenotype was accompanied by an irregular laminin staining pattern associated with a disorganized basement membrane and increased blood vasculature. Similar experiments conducted with HaSV and helper virus resulted in the generation of mesenchymal lesions at 28 days with little surviving urothelium. The H-ras oncogenic protein can induce hyperplastic lesions in normal urothelium which were characteristic of preneoplastic changes identified in bladder carcinogenesis.

Preneoplastic lesions induced by myc and src oncogenes in a heterotopic rat colon.

With the use of viral vectors harboring myc and src oncogenes, we have assessed the potential contribution of these different elements to colonic neoplasia using a transplantation technique resulting in the formation of a heterotopic colon in Wistar Furth rats. While myc alone induced atypia and some dysplasia, src induced focal dysplastic lesions throughout the colon mucosa with evidence of metaplasia. In contrast, lesions induced by myc and src acting cooperatively, were highly dysplastic with evidence of tumor formation after protracted periods. These results indicated the formation of histologically distinct preneoplastic lesions elicited by the action of a single oncogene in colon implants with the production of adenocarcinomas when such oncogenic elements act cooperatively. This model provides an opportunity for studies of the action of different oncogenes, acting singly or in combination, in the multi-step progression to colon tumor formation.

Expression of the c-erbB-2 gene product (p185) at different stages of neoplastic progression in the colon.

The c-erbB-2 gene has been found amplified in a number of human adenocarcinomas leading to elevated levels of expression of the p185 protein product. Increased expression of this putative growth factor receptor has been reported to occur by molecular mechanism other than gene amplification and for this reason we have studied the expression of the p185 protein in normal colon and in lesions representing different stages of neoplastic progression. We report amplification of the c-erbB-2 gene in 3 of 44 colon carcinomas and 1 of 5 preneoplastic polyps studied. Confirmation of expression of the p185 protein product was established in Western blot analysis and by immunocytochemical staining of tissue sections. An extended study, involving adenomatous polyps and carcinomatous material in immunostaining, revealed detectable presence of the p185 protein in 20% of carcinomas, consistent with immunoprecipitation data derived using established cell lines. In contrast, a high percentage of polyps showed strong staining with both p185 antibodies used, indicating elevated levels of expression of the c-erbB-2 protein associated with preneoplastic lesions. Staining of normal human colon revealed a restricted localization of this putative receptor to cells on the luminal colonic surface, with no expression in cells of the crypt. Histologically normal mucosa, adjacent to the tumor, showed a more extensive distribution involving the crypt suggestive of a disturbance in the normal expression of c-erbB-2. These results indicate that elevated expression of the c-erbB-2 protein is associated with early stages of colonic neoplasia but do not establish it as a primary factor in these events. The occurrence of multiple copies of the c-erbB-2 in a percentage of colon lesions, however, suggests a possible role for this gene in some colon malignancies.

Increased incidence of p53 mutations is associated with hepatic metastasis in colorectal neoplastic progression.

Within a panel of 15 colon carcinoma cell lines we have characterized the p53 gene status using immunocytochemistry (ICC), SSCP and direct sequence analysis. Extension of this analysis to the use of ICC on 104 colonic lesions, representative of different stages of colonic neoplastic progression, showed an absence of detectable p53 nuclear staining in preneoplastic polyp lesions (20 cases) with staining of 52% (25/48) of primary colon carcinomas and 81% (29/36) of hepatic metastases, suggestive of an increased incidence of p53 mutations in late stage lesions of colonic cancer. To address this issue more directly, we analysed 18 primary colon carcinomas and hepatic metastases excised coincidentally from the same patients. In ICC, p53 nuclear staining was recorded in matching lesions from eight individuals where direct sequencing revealed identical mutations in each case. In four individuals no ICC staining was detected in either lesion and molecular analysis revealed wild type sequence in exons 4-9. In six individuals p53 nuclear staining was observed in the hepatic metastases of patients but not the primary lesion. Molecular analysis revealed point mutation events in hepatic metastases from these patients which were not detected in the primary tumor. The point mutations identified in colon carcinomas were predominantly transition events (83%) located in previously characterized colon hotspot regions. These results demonstrate an increased incidence of p53 mutations associated with secondary lesions of colorectal tumors suggestive of a role for p53 in the establishment of colorectal hepatic metastases.

Somatic mutation of PTEN in vulvar cancer.

PTEN, a candidate tumor suppressor gene located at chromosome 10q23.3, has been shown to be mutated in approximately 40% of endometrial cancers. Such mutations have also been identified in endometrial hyperplasia, indicating that inactivation of the PTEN tumor suppressor gene is an early event in the genesis of some endometrial cancers. In this study, we have extended the analysis of PTEN in gynecological cancer to include adenocarcinoma of the cervix and vulvar carcinomas. Microdissected tissue (including normal tissues), preneoplastic, and neoplastic lesions were analyzed from 9 patients with cervical cancer and 10 patients with vulvar cancer. Only 1 cervical adenocarcinoma displayed a PTEN mutation. In contrast, five of eight vulvar carcinomas studied harbored PTEN mutations. Alterations were identified in carcinoma in situ as well as squamous cell carcinoma of the vulva. In two patients, PTEN mutations were identified in mucosal regions with mild or focal dysplasia. These results suggest that PTEN is frequently altered in vulvar carcinomas and can be found associated with early dysplastic changes in vulvar mucosa.

Expression of individual ras proteins in normal and neoplastic colon.

In this study, we report the use of a panel of four ras antibodies in Western blot analysis which characterize the expression of individual ras members in different cell populations. The specificity of the different antibodies to H-, N- or K-ras proteins was established in a panel of NIH/3T3 transfectants and human cell lines harboring known ras oncogenic events. Localization of individual ras members in one dimensional gel electrophoresis revealed slower migration of K-ras p21 products, whilst H- and N-ras proteins co-migrated in this system. Identification of migrational differences between activated proto-oncogene products was facilitated using ras member specific probes. Application of this approach to normal gastrointestinal mucosa.revealed over-representation of K-ras p21 in both stomach and colon epithelium in the rat. This differential ras profile was maintained in normal human colon mucosa and colon tumors from the same individual. Use of such antibodies now enables dissection of ras proto-oncoprotein expression in different tissues and compartments within tissue with the ability to characterize ras mutational events in neoplastic progression.

Timing of induction of tgf-alpha in ras induced human bladder neoplastic progression.

Introduction of a H-ras oncogene into an SV-40 immortalized human urothelial cell lines (SV-HUC) results in morphologically altered cell clones which acquire tumorigenic potential following serial passaging in culture. Early and late passage cells, from individual ras transfected clones exhibiting different tumorigenic potential, display increased growth factor synthesis in mitogenic assays. Northern blot analysis revealed induction of TGF-alpha mRNA concomitant with the introduction of a H-ras oncogene with no modulation in EGF receptor expression observed throughout neoplastic progression. Consistent with completion of an autocrine loop, down modulation and activation of EGF receptors was observed in early passage cells coincident with TGF-alpha expression. In this human urothelial progression model TGF-alpha secretion follows the introduction of a H-ras oncogene prior to the acquisition of tumorigenic potential.

Expression of classic cadherins type I in urothelial neoplastic progression.

Loss or reduced expression of E-cadherin has been shown to be associated with poor survival in patients with bladder cancer. In numerous cases, loss of E-cadherin expression in bladder tumors has been accompanied by continued association of catenins with the membrane, suggestive of the expression of an alternative cadherin member. In this study we examined 75 bladder tumors using immunohistochemistry for the expression of E-, P-cadherin, and alpha-, beta-, and gamma-catenins. As reported previously, loss or reduced E-cadherin expression is a frequent event in late stage bladder cancer, accompanied by less frequent alterations associated with different catenin family members. Analysis of 51 tumors for expression of E-, P-, and N-cadherin showed P-cadherin localized to the basal cell layers of normal urothelium, with retention of expression in the majority of tumors. In low-grade tumors P-cadherin was found localized to an expanded basal cell compartment, contrasting with the more extensive staining observed in late stage tumors. Membranous P-cadherin staining was often found in the absence of E-cadherin staining. N-cadherin is not expressed in normal bladder mucosa, but detection of this cadherin member was recorded in 39% (20/51) of bladder tumors. Unlike P-cadherin, membranous N-cadherin was detected in focal regions within tumors, representing novel expression in urothelial neoplastic progression. Although focal N-cadherin staining was observed in 3 noninvasive lesions, the majority of tumors expressing N-cadherin were invasive (17/20). Coexpression of E-, P-, and N-cadherin was recorded in 5 grade 2 bladder tumors. Expression of P-cadherin is maintained throughout bladder tumorigenesis, accompanied by aberrant expression of N-cadherin. Clearly, neither P- nor N-cadherin act in an invasive-suppressor mode in bladder cancer, but whether they have a primary role to play in urothelial neoplastic progression has yet to be established.