Histochemical analysis of the development of estradiol-induced kidney tumors in male Syrian hamsters

Chronic administration of the estrogen 17 beta-estradiol induces kidney tumors in male Syrian hamsters within 6 months of initial exposure. Although these tumors have previously been studied histologically and histochemically and have been postulated to be derived from proximal tubular and/or interstitial cells, there exists no unambiguous evidence for an epithelial or mesenchymal origin. To elucidate the histogenesis of these neoplasms, kidney sections of hamsters treated with estradiol for 4, 5, and 6 months and age-matched untreated controls were investigated histologically and histochemically. Proliferating foci were observed in kidneys exposed to estradiol for 5 and 6 months. They consisted of clusters of spindle-shaped cells forming solid blocks, cords, or branches located between tubules. These foci were judged to be precursors of larger tumors identified in the latter treatment group. The histological and histochemical profile of foci and tumors matched closely. These lesions were marked by very high activities of alkaline phosphatase, adenyl cyclase, and glucose 6-phosphate dehydrogenase. In contrast, glycogen content and activities of glucose 6-phosphatase, succinate dehydrogenase, and gamma-glutamyl transpeptidase were low or absent. Immunofluorescence of the intermediate filaments revealed that foci and tumors solely expressed vimentin and desmin but not cytokeratin. The morphology, enzyme histochemical pattern, and immunofluorescence strongly support a mesenchymal origin of the estradiol-induced hamster kidney tumors studied. The neoplasms were probably derived from vascular smooth muscle cells of a cell subtype particularly sensitive to hormonal stimulation and transformation.     

Differentiation dependent expression of P-glycoprotein in the normal and neoplastic human kidney.

Adult renal cell carcinoma (RCC) is clinically resistant to chemotherapy. However, in nephroblastoma (NBL) chemotherapy has increased survival dramatically. We studied the P-glycoprotein (P-gp) expression of 18 RCC and 9 NBL as well as 1 benign renal adenoma and fetal renal tissue using three different monoclonal antibodies (MRK-16, C-219, JSB-1). P-gp was found positive with all three antibodies in 12/18 RCC, while only 2 tumors were completely negative. Staining varied with respect to intensity and number of positive cells [5%-90%]. Intense staining was seen at the apical side of malignant tubules in well differentiated parts of RCC and in tubular structures of the benign renal adenoma. Poorly differentiated parts of the tumors showed less staining. In NBL blastemal parts were negative. In 4/8 specimens showing focal epithelial differentiation, however, the luminal side of more differentiated tubular structures did stain, strongly resembling P-gp staining in the developing fetal human kidney. These results indicate that P-gp expression in normal (fetal) human kidney as well as in benign and malignant tumors derived from this organ depends on the degree of differentiation of tubules, which may have implications for chemotherapy sensitivity in both malignant tumors.     

Immunofluorescent characterization of rat kidney tumors according to the distribution of actin as revealed by specific antiactin antibody.

A series of 15 mesenchymal and 10 cortical epithelial tumors induced in the rat kidney by dimethylnitrosamine was investigated for immunofluorescent reactivity with a human antiactin antibody. Cells of epithelial tumors showed staining restricted to peripheral sites, corresponding to the brush border region. All various neoplastic cells forms comprising renal mesenchymal tumor were characterized by cytoplasmic staining in pattersn that varied with cell type. Epithelial profiles in the form of tubules and islands of epithelium showed staining patterns, or absence of them, consistent with their identity as sequestered segments of preexisting nephrons. It is suggested that the difference in actin distribution within the cytoplasm of cells of the two types of renal neoplasm, mesenchymal and epithelial, might reflect their difference in local invasive growth.     

Histochemical and immunohistochemical characterization of surgically resected and heterotransplanted Wilms' tumor

Nine surgically resected Wilms' tumors (WIT) and nude mouse heterotransplants from one WIT were studied by histochemistry and immunohistochemistry. Histochemistry showed acid phosphatase in all cells, while alkaline phosphatase and gamma-glutamyl transpeptidase were present in only some tubules. Using immunohistochemistry, antibodies to the intermediate filaments cytokeratin and vimentin distinguished tubular epithelium and mesenchyme, respectively. WIT tubules were also identified using antibody against a structural component (epithelial membrane antigen) and a secretory product (uromucoid) associated with distal convoluted tubules of normal kidney. Basement membrane surrounding the tubules of WIT was demonstrated using antibody to type IV collagen plus laminin. Different blastema subpopulations were negative or stained positively with antibodies to cytokeratin and vimentin. Production of basement membrane by blastema was also shown. Fetal antigen expression in WIT was examined using the monoclonal PI 153/3 and J5 antibodies. The blastema and tubules of WIT were strongly stained by PI 153/3, which did not label normal adult kidney, and weakly stained by J5, which strongly labeled glomeruli and proximal convoluted tubules of normal kidney. These studies show that WIT blastema is heterogeneous in intermediate filament subtypes, while WIT tubules more closely resemble distal than proximal convoluted tubules of adult kidneys but also retain expression of fetal antigens.     

Categorization of pediatric neoplasms by immunostaining with antiprekeratin and antivimentin antisera

Forty-six tumors in children were examined using light microscopy and subsequently frozen sections were stained with antiprekeratin and antivimentin antisera, so that the tumors could be classified by tissue of origin. Except for two adrenal cortical carcinomas and four liver tumors, most epithelial neoplasms continued to produce prekeratin filaments, a characteristic of normal epithelial cells. Tumors and cells of epithelial origin did not produce vimentin filaments, whereas normal and neoplastic mesenchymal cells did. Tumors with both epithelial and mesenchymal components produced vimentin filaments in mesenchymal areas and prekeratin in epithelial areas. Tumors of lymphoid origin showed variable production of vimentin filaments, depending on the amount of cell cytoplasm, but did not contain prekeratin filaments. Of the neuroectodermal tumors, only the ganglioneuroma contained vimentin filaments and none contained prekeratin filaments. Thus, antibodies to both prekeratin and vimentin filaments are useful in diagnosing childhood neoplasms and studying their histogenesis.     

Squamous cell carcinoma of the renal pelvis with sarcomalike stroma: a light and electron microscopic study with immunohistochemical analysis

Primary neoplasms of the renal pelvis are rare. Most are malignant, and most of these are transitional cell carcinomas. We report the unusual occurrence of a squamous cell carcinoma with sarcomatoid stroma arising from the renal pelvic mucosa in a patient with renal lithiasis. Immunohistochemical stains for keratin intermediate filaments failed to demonstrate their presence in the spindle cell portion of the tumor. Transmission electron microscopic study did not reveal structures of an epithelial nature in these same cells. Our findings support the contention that the spindle cells of the stroma are not squamous in nature, but represent either a reactive or a neoplastic transformation of these underlying stromal elements.     

Smooth-muscle-associated contractile protein in renal mesenchymal tumour cells and in transformed cells from DMN-injected rats.

Cryostat sections and established in vitro cultures of dimethylnitrosamine(DMN)-induced renal mesenchymal tumours and monolayer cultures of transformed kidney cells derived from rats treated with a carcinogenic dose of DMN were examined by indirect immunofluorescence with human serum containing smooth muscle antibody. Eight mesenchymal tumours examined showed filamentous cytoplasmic staining of spindle cells infiltrating between renal tubules, whilst in normal kidneys interstitial cells were only weakly positive. In established in vitro cultures from 6 mesenchymal tumours, different patterns of staining were observed in morphologically different cell forms, ranging from fine filamentous staining in giant cells to diffuse cytoplasmic fluorescence in small bipolar cells, and cell outline staining in polygonal cells. In addition filamentous staining of microvillous projections and nucleolar staining were observed in some tumour cells. Monolayer cultures of transformed kidney cells showed strong staining of coarse, randomly-orientated cytoplasmic filaments, whilst fibroblasts cultured from normal rat kidney demonstrated an ordered array of fine, parallel filaments. Specificity of the immunofluorescent staining reaction was established by failure to obtain staining with normal serum, with smooth muscle antibody serum neutralized by homogenates of smooth muscle or extracts containing actin derived from smooth muscle. These results indicate that there is an apparent increase of actin-like contractile microfilaments in transformed cells and in renal mesenchymal tumours. The cytoplasmic contracile microfilaments in these cells may play a role in tumour cell mobility and invasion.     

Expression and prognostic value of Wilms' tumor 1 and early growth response 1 proteins in nephroblastoma.

Wilms' tumor is one of the most common solid tumors of children. The protein product of the tumor-suppressor gene, Wilms' tumor 1 (WT-1), binds to the same DNA sequences as the protein product of the early growth response 1 (EGR-1) gene. There is experimental evidence that EGR-1 is involved in controlling cell growth. The expression of both genes in Wilms' tumor was studied by others, mainly at the mRNA level. The present study evaluates the prognostic value of WT-1 and EGR-1 in 61 Wilms' tumors of chemotherapeutically treated patients at the protein level, using an immunohistochemical approach. WT-1 was expressed in normal kidney tissues and in the blastemal and epithelial component of Wilms' tumor, whereas stromal tissue was negative. EGR-1 was expressed in normal kidney tissues and in the three main cell types of Wilms' tumor. In 59 and 56% of Wilms' tumor, the blastemal cells stained for WT-1 and EGR-1, respectively. The blastemal expression of WT-1 and EGR-1 and the epithelial expression of WT-1 were statistically significantly correlated with clinical stage. WT-1 immunoreactivity correlated with EGR-1 expression. Univariate analysis showed that blastemal WT-1 and EGR-1 expression were indicative for clinical progression and tumor-specific survival, whereas epithelial staining was of no prognostic value. Multivariate analysis showed that blastemal WT-1 expression is an independent prognostic marker for clinical progression other than stage. We conclude that a relationship exists between WT-1 and EGR-1 expression in clinical nephroblastomas. Blastemal WT-1 and EGR-1 expression is related to prognosis.     

RNA expression of the WT1 gene in Wilms' tumors in relation to histology.

BACKGROUND: On the basis of accumulating data, the recently isolated WT1 gene is a Wilms' tumor gene and a putative tumor suppressor gene. These findings include expression in developing fetal kidney, intragenic deletions in tumors, and germline mutations in predisposed individuals. Wilms' tumors, which exhibit a broad range of differentiation, are composed of three cell types: blastema, epithelium, and stroma. PURPOSE: The purpose of this study was to investigate the relationship between WT1 gene expression and histologic composition in Wilms' tumors in an effort to elucidate how the WT1 gene functions in proliferation of these histologic components. METHODS: We used Northern blot hybridization to study WT1 gene expression by messenger RNA (mRNA) accumulation in 20 tumors of varying histology and in adjacent uninvolved kidney tissue. In two patients, tumors were also compared before and after therapy. RESULTS: Tumors that were predominantly blastemal expressed high amounts of WT1 mRNA, whereas predominantly stromal tumors expressed either low or undetectable amounts. Blastemal tumors that were predominantly poorly differentiated expressed WT1 mRNA at higher levels than those that were more well differentiated. Although we expected that a putative tumor suppressor gene like WT1 would generally be expressed at lower levels in tumor than in normal kidney, this was true only in predominantly stromal cells. One of the two patients studied before and after therapy had a dramatic response to therapy accompanied by a decline in WT1 gene expression and disappearance of blastemal and epithelial elements. CONCLUSIONS: A correlation was observed between WT1 gene expression and histology of the tumors. Level of expression was inversely related to the degree of differentiation in blastemal tumors and in the patient with a dramatic response to therapy. These results, in conjunction with the observation that WT1 mRNA is abundant in normal fetal kidney, suggest that WT1 gene expression is related to kidney development, especially in differentiation of blastemal components. IMPLICATIONS: Further studies to search for alterations of the WT1 gene in tumors and to identify regulatory factors in gene expression will increase understanding of the role of this gene in normal development and tumorigenesis.     

Autoradiographic analysis of proliferative activity in rat kidney epithelial and mesenchymal cell subpopulations following a carcinogenic dose of dimethylnitrosamine.

In a system that yields 100% incidence of renal mesenchymal tumors and a 30 to 40% incidence of renal cortical epithelial neoplasms, the proliferative activity of renal epithelial and mesenchymal cell subpopulations following a single dose of dimethylnitrosamine (DMN) was traced by autoradiographic analysis of [methyl-3H]thymidine uptake during the 3 weeks immediately posttreatment. The initial response to DMN was a depression in DNA synthesis and mitosis to near 0 levels in all segments of the nephron and in attendant mesenchymal cells for a period of 1 to 3 days. Following the period of inhibition, increased DNA synthetic activity was observed in certain subpopulations of both epithelium and mesenchyme and these patterns were matched by equivalent mitotic activity. A stimulation of DNA synthesis was observed in cells of the proximal and distal tubules of Zones 1 and 2 but in no other epithelial segments. The increased activity was most intense in Zone 1 epithelium reaching a peak at the 10th day after DMN injection 4 days after epithelial cell necrosis had commenced. In renal mesenchyme, the major response involved only the interstitial cells of Zones 1 and 2. At Day 3, there was a wave of increased DNA-synthetic and mitotic activity in the free interstitial cells of the cortex, followed by a 2nd, more intense peak of activity at Day 6. The cells responding at Day 3 appeared to involve the resident population of cortical fibrocytes while the major contribution to the Day 6 peak came from infiltrating mononuclear inflammatory cells, although resident fibrocytes and capillary endothelium also contributed. A significant wave of increased activity involved the intestitial cells of Zone 2, but the peak, although of equivalent intensity to the response in Zone 1, was single and occurred 3 days later at Day 9. Apart from a small, brief, and variable wave of activity in interstitial cells of Zone 3 from Days 8 to 10, no toerh mesenchymal cell populations in the kidney were stimulated by the injection of DMN.     

MIB-1 (KI-67) proliferation index and cyclin-dependent kinase inhibitor p27(Kip1) protein expression in nephroblastoma.

PURPOSE: A number of studies have indicated that the tumor proliferation marker MIB-1 and cell cycle inhibitor p27(Kip1) expression are of prognostic importance in a variety of cancers. The present study was performed to evaluate the prognostic value of these molecules in Wilms' tumors. EXPERIMENTAL DESIGN: MIB-1 and p27(Kip1) expressions were investigated by the means of immunohistochemical analysis of 62 Wilms' tumor. Patients were preoperatively treated by chemotherapeutic agents and had a mean follow-up of 5.7 years. RESULTS: MIB-1 and p27(Kip1) were expressed in normal kidney tissues and in the three main components of Wilms' tumor, i.e., the blastemal, epithelial, and stromal cells. In Wilms' tumors, the percentage of MIB-1-positive cells in the blastema ranged between 0 and 42% (mean, 9.4%) and in the epithelial component between 0 and 53% (mean, 19.9%), with a significant difference (P < 0.01). The percentage of blastemal p27(Kip1)-positive cells ranged between 3 and 85% (mean, 55.1%) and for the epithelial component between 1 and 87% (mean, 59%). There was a significant inverse relationship between blastemal MIB-1 and p27(Kip1) expression in Wilms' tumor. Univariate analysis showed that blastemal MIB-1 and p27(Kip1) expression were indicative for clinical progression and tumor-specific survival. In a multivariate analysis, blastemal MIB-1 and p27(Kip1) protein expression proved to be an independent prognostic for clinical progression besides stage. CONCLUSIONS: It was concluded that both MIB-1-based proliferative activity and p27(Kip1) protein expression in the blastema have prognostic impact in Wilms' tumor.     

Overexpression of the met/HGF receptor in renal cell carcinomas

The c-met oncogene encodes the receptor for hepatocyte growth factor/scatter factor (HGF/SF), a multifunctional cytokine able to mediate morphogenesis as well as mitogenesis, motogenesis and invasiveness of epithelial cells. HGF/SF has been implicated in branching tubulogenesis of the developing kidney and in regeneration after renal injury and nephrectomy. We have examined the expression of the met/HGF receptor in normal human kidney and tissues of the genito-urinary tract, and in 50 kidney neoplasms of different histotypes, using monoclonal antibodies (MAbs) against the met/HGF receptor and immunohistochemistry. In normal kidneys, weak staining restricted to the distal tubules was observed. Transitional cell carcinomas were consistently negative, whereas increased expression at various levels was found in 87% of renal cell carcinomas with different cytological features and histological patterns. Western blot analysis of samples showed that the met/HGF receptor found in the malignant cells exhibits features of the normal receptor. The met/HGF receptor is also overexpressed in a renal cell carcinoma cell line, whose motility is triggered by HGF/SF. Our data suggest that expression of the met/HGF receptor may be involved in the onset and progression of renal cell carcinomas. © 1996 Wiley-Liss, Inc.     

Immunocytochemical study of an endometrial diffuse clear cell stromal sarcoma and other endometrial stromal sarcomas

Intermediate filament composition was studied in the following endometrial stromal tumors: low-grade stromal sarcoma (endolymphatic stromal myosis), high-grade stromal sarcoma with an associated adenocarcinoma (collision tumor), diffuse clear cell stromal sarcoma and a mesodermal mixed tumor (carcinosarcoma). The tumor cells of the stromal tumors as well as the mesenchymal elements of the mixed mesodermal tumor were decorated exclusively with antibodies to vimentin. Desmin was not demonstrated in these tumor cells. A biochemical study of the cytoskeletal filaments present in the low-grade stromal sarcoma revealed, in addition to vimentin, beta and gamma actin as seen in normal endometrial stroma. Cytokeratins were only identified in epithelial components which were present in some of these tumors. Intermediate filament typing in these endometrial neoplasms contributes to the elucidation of histogenetic problems, may delineate mesenchymal from epithelial elements, may separate muscle from stromal lesions and in one instance helped to define a hitherto unreported diffuse clear cell stromal sarcoma.     

Expression of class I and II major histocompatibility complex antigens in Wilms' tumour and normal developing human kidney

The Wilms' tumour is a solid childhood tumour of the kidney, consisting of blastema, tubules and mesenchyme. Embryonic tumours, such as Wilms', may arise as a result of a developmental disturbance in differentiation. The expression of class I and II major histocompatibility complex (MHC) antigens was investigated on 6 Wilms' tumours and related to that in the developing human kidney in this immunohistological study, using a panel of monoclonal antibodies. The Wilms' tumour blastemal cells were class I MHC antigen negative, but differentiated structures were positive. Class II MHC antigens were not observed in Wilms' tumours. In the developing human kidney class I MHC antigen expression was observed on glomeruli from 8 weeks and on tubules from 13 weeks gestational age. Class II MHC antigen expression was observed on glomeruli from 11 weeks and on tubules from 13 weeks gestation. These results suggest that the blastemal cells within the Wilms' tumour may reflect an early stage of development with respect to the expression of MHC antigens.     

Kidney tumors induced in rats by the antischistosomal drug niridazole.

An increased incidence of kidney tumors was found in MRC rats fed the antischistosomal drug niridazole at four dose levels in the diet. Histologically, the adenomas and adenocarcinomas were solid papillary, clear cell, and tubular types, with the latter type predominating. Seven mesenchymal tumors were found among the 107 renal epithelial neoplasms. Severe nephrosclerosis occurred in both treated and control rats and has been suggested as important in renal carcinogenesis. Niridazole is considered a potent inducer of epithelial kidney tumors.     

Transplacental effect of N-diethylnitrosamine on chromosomal aberrations in fetal tracheal cells of the Syrian hamster: comparison between epithelial and mesenchymal cells

Chromosomal aberrations were investigated in tracheal cellsof fetal Syrian hamsters after transplacental administrationof N-diethylnitrosamine (DEN). On the 15th day of pregnancy,Syrian hamsters were injected s.c. with a tumorigenic dose ofDEN (50,100 and 200 mg/kg body weight). Two hours later, thefetal tracheae were isolated, the epithelial tissue was separatedfrom the mesenchymal tissue by collagenasetreatment, and theneach cell population was transferred to cell culture after Dispasetreatment. At 24, 48 and 72 h after the cell cultivation, chromosomaldamage was examined. The results clearly showed that a highincidence of chromosomal aberrations, especially chromatid-typeexchanges, was seen in the epithelial cells of DEN-treated groups.However, significant induction of chromosomal aberrations wasnot observed in the mesenchymal cells from DEN-treated groups.     

Rejection of adenovirus 2-transformed cell tumors and immune responsiveness in Syrian hamsters.

Transplantation of adenovirus type 2-transformed cell-induced newborn tumor lines to different aged hamsters revealed that the cell-mediated host defenses responsible for tumor graft rejection matured early in the second week of life. When light microscopic examinations were performed during the course of tumor development, the primary histopathological difference between progressing tumors removed from newborn or thymectomized weanling hamsters and regressing lesions from normal weanlings was the lack of an early, mononuclear cell infiltrate in neoplasms from newborn and thymectomized hosts. These results suggest that the maturation of cellular immunity determines resistance to tumor transplantation in this system. This conclusion was supported by the in vitro detection of concanavalin A-responsive lymphocytes in spleens from tumor-resistant suckling but not tumor-susceptible neonatal hamsters. Although the incomplete seeding of thymus-dependent lymphocytes to the peripheral lymphoid tissues of newborn hamsters may partially explain the deficient concanavalin A responses of neonatal spleen cells, there appears to be an additional requirement for a radioresistant, adherent accessory cell population. These findings suggest that the development of a cell-mediated immune response is necessary for the rejection of adenovirus type 2-transformed cells and transformed cell-induced tumors and that this response requires the interaction of T-cells and accessory cell populations.