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The third isoform (MT-3) of the metallothionein gene family is unique in that it has a limited tissue distribution, is not induced by metals, has a neuronal growth inhibitory activity, and sequesters zinc more effectively under zinc-depleted conditions. The goal of the present study was to determine whether MT-3 was absent in normal breast tissue, was overexpressed in breast cancers, and if MT-3 overexpression would be associated with disease outcome. A combination of immunohistochemistry and reverse-transcription polymerase chain reaction was used to demonstrate that the normal breast had no detectable expression of MT-3 mRNA or protein. Using immunohistochemistry, it was shown that MT-3 was overexpressed in 25 of 34 cases of breast cancer. In all cases of positive staining, MT-3 was diffusely localized to the cytoplasm. The tumors from these 34 cases were divided as to outcome based on known 5-year survival, with 20 patients being disease free at 5 years (good outcome) and the other 14 having recurring disease within 5 years (bad outcome). When analyzed for MT-3 staining, it was shown that there was a trend for increased MT-3 immunoreactivity in the group having bad outcomes. However, when the tumor subgrouping was further defined on the basis of carcinoma in situ (CIS), there was a marked significant difference in MT-3 staining between patients with good and bad outcomes. Limited to DCIS, MT-3 staining was significantly increased in patients with bad outcomes compared to those with good outcomes. Thus, these studies demonstrate that MT-3 is overexpressed in selected breast cancers and that overexpression is associated with tumors having a poor prognosis.

The goal of this study was to determine which of the 10 functional metallothionein (MT) genes are expressed in four human breast cancer cell lines and whether expression varies among the cell lines. Using reverse transcription polymerase chain reaction (RT-PCR) technology, it was shown that there was no expression of mRNA for the MT-1A, MT-1B, MT-1F, MT-1G, MT-1H, MT-3, and MT-4 genes in any of the four cell lines. All four cell lines were shown to express mRNA for the MT-2A and MT-1X genes. The expression level of mRNA for the MT-2A gene demonstrated modest differences among the cell lines, whereas expression of the MT-1X gene was consistent. In contrast, mRNA for the MT-1E gene was expressed in only two of the four cell lines and expression correlated to the estrogen receptor status of the cell lines. The two estrogen-receptor-positive cell lines showed no mRNA expression for the MT-1E gene. In the two estrogen-receptor-negative cell lines, mRNA expression for the MT-1E gene was elevated with expression levels similar to the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase. The cellular content of MT protein was also shown to be elevated in the estrogen-receptor-negative cell lines that express MT-1E mRNA. These results suggest a possible relationship between estrogen receptor status and MT-1E gene expression in human breast cancer.

Clinically the anaplastic variant of Wilms' tumor differs from the classical Wilms' tumor by its poor prognosis. To begin to understand and characterize the distinctive biology of this rare form of Wilms' tumor, a study of the histology, ultrastructure, and mRNA expression was performed on the anaplastic tumor and its associated cell line. The anaplastic tumor generated mouse heterotransplants that were readily used to establish epithelial cell cultures. The epithelial cultures, in turn, produced tumors when reinjected into nude mice. Microscopic evaluation revealed that the anaplastic epithelial cells were less differentiated than their epithelial counterpart in classical Wilms' tumors. In general the molecular profile of the anaplastic tumor was more consistent with that of an epithelial-rich classic Wilms' tumor than with the classic triphasic Wilms' tumor. Unlike the classic triphasic Wilms' tumor that contains blastema, stroma, and epithelial tubules, the anaplastic tumor expressed only marginal levels of insulin-like growth factor 2 (IGF-2) mRNA and imperceptible levels of the Wilms' tumor gene (WT-1), Pax-2, and Pax-8 mRNA. In common with the classic Wilms' tumor, the anaplastic variant retained the expression of the N-myc gene while failing to express C-myc. A comparison of cultures derived from an epithelial-rich, classic Wilms' tumor and the anaplastic Wilm's tumor indicated that both lacked IGF-2 and WT-1 mRNA expression. However, the well-differentiated epithelial cell culture derived from the classic Wilms' tumor expressed C-myc, Pax-8, and Pax-2 mRNA, none of which were expressed by the anaplastic epithelial cells. Furthermore, the well-differentiated epithelial cell component failed to express N-myc, which was expressed by both the primary triphasic Wilms' tumor and the anaplastic tumor. Overall, the findings indicate that patterns of gene expression within a single component do not correlate with the aggressive clinical behavior of the anaplastic Wilms' tumor.

The ability to establish cell cultures representing the epithelial component of Wilms' tumor was determined for 18 cases of classic Wilms' tumors. From these 18 cases only two resulted in the culture of epithelial cells. Although the tumors from both cases were composed of a prominent epithelial component, other classic tumors not producing epithelial cell cultures also possessed appreciable epithelial components. Likewise, heterotransplants of these two primary tumors failed to give rise to epithelial cell cultures, although cultures of the blastemal element were produced. This suggests that Wilms' tumors may be prone to differentiate in different directions at varying times during tumor growth, possibly dependent on local tumor environment. Epithelial cells from these two classic cases were grown in culture in basal medium composed of a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium, supplemented with selenium, insulin, transferrin, hydrocortisone, tri-iodothyronine, and epidermal growth factor, on a collagen type I matrix with absorbed fetal calf serum proteins. One of the two cases also required the addition of bovine pituitary extract, ethanolamine, prostaglandin E1, and putrescine for optimum growth. Morphological analysis disclosed that the cultured cells were very similar to normal renal tubular cells in culture, except that the cells displayed little evidence for differentiated active ion transport and tended to grow in a multilayered arrangement. The culture of the epithelial cells from classic Wilms' tumors provides a model system for the study of tumor differentiation and progression.

The histology, ultrastructure, and messenger RNA expression of heterotransplants derived from the G401 cell line (American Type Culture Collection) have been characterized by comparison with Wilms' and rhabdoid tumors of the kidney. This analysis illustrates that the properties of G401 heterotransplant were consistent with a rhabdoid phenotype rather than that of a Wilms' tumor. The G401 cell line has been utilized in recent experiments to demonstrate the central role of chromosome 11 in Wilms' tumor. However, the present results suggest that these experiments may be more relevant to define the involvement of chromosome 11 in rhabdoid tumor of the kidney, a malignancy distinct from Wilms' tumor. This is clinically relevant since the rhabdoid tumor of the kidney is very aggressive and associated with an extremely poor prognosis.

Homozygous inactivation of WT1, a Wilms' tumor gene located on chromosome 11 at p13, is believed to predispose to Wilms' tumor and therefore may be a common occurrence in this cancer. The expression of this gene in primary Wilms' tumors was examined by northern and quantitative RNA slot blot analysis and compared with clinical, histologic, and molecular features of each case. The characteristic 3.2 kb RNA was readily detected in most primary tumors although there was marked variation in the level of WT1-specific transcripts. No abnormal-sized RNA products were detected and expression of WT1 was not coordinated with that of several other oncofetal genes: N-myc, insulinlike growth factor 2 (IGF-2), and c-myc. The relative abundance of WT1-specific RNA did correlate with the histologic category of Wilms' tumor such that those tumors with heterologous differentiation have, in general, lower relative levels of WT1 transcripts than those tumors without heterologous differentiation. These results further establish the heterogeneity of Wilms' tumor with respect to the expression of tumor-associated oncofetal genes and suggest a relationship between WT1 expression and cellular differentiation.

Wilms' tumor has been proposed to originate from a developmental abnormality of the metanephric blastema. This undifferentiated component of Wilms' tumors has previously eluded efforts for in vitro growth. Blastema from a "classical" Wilms' tumor was transplanted into nude mice and passaged through 12 generations of heterotransplantation. Tumors from heterotransplants were grown for 12 serial passages in a serum-free growth medium supplemented with hormones and conditioned media from human kidney proximal tubule cells. The blastema initially grew on a collagen-fetal calf serum matrix as multicellular spheroids, and the cells proliferating from the rim of the spheroids had a flattened shape. Pulse-labeling with bromodeoxyuridine (BrdU) identified the proliferating cell population as blastemal in origin. Except for a loss of extracellular matrix, ultrastructural studies demonstrated morphologic similarities in the cultured cells, compared with the primary tumor and heterotransplants. Lectin histochemical stains for the peanut lectin (PNA) and immunohistochemical stains for cytokeratin (CYTO), vimentin (VIM), and epithelial membrane antigen (EMA) were performed on the original tumor, successive heterotransplants, and cells grown in vitro. The PNA stained the surface of the blastemal cells after sialidase digestion in the original tumor, heterotransplants, and cultured cells. The blastema of the original tumors was negative for CYTO and EMA but reactive for vimentin. This lack of differentiation was maintained in heterotransplants through 12 passages. However, blastemal cells demonstrated coexpression of CYTO and VIM intermediate filaments when grown in a serum-free medium on a matrix material. These studies demonstrate that the blastemal component of Wilms' tumor can be successfully grown in culture, passaged in nude mouse heterotransplants, and shown to undergo early stages of blastemal differentiation in vitro by growth in serum-free medium. This in vitro system provides a model for testing the factors that influence the growth and differentiation of the blastemal component of Wilms' tumors.

The role of the type I insulinlike growth factor (IGF) receptor in regulating growth of Wilms' tumor (WT) was evaluated by examining the effect of antibody-mediated inhibition of this receptor on tumor growth in cell cultures and as heterotransplants in athymic mice. An antibody to the human type I IGF receptor (alpha IR-3) inhibited 125I-IGF-1 binding and prevented stimulation of thymidine incorporation by IGF-1 in vitro. Intraperitoneal administration of alpha IR-3 to nude mice bearing WT heterotransplants prevented tumor growth for 4 weeks and resulted in partial regression of established tumors. These data indicate the importance of IGF action in control of WT growth in vivo, and suggest potential therapeutic application using antigrowth factor receptor antibodies to block growth factor action.

Skeletal muscle differentiation within a Wilms' tumor is a well-documented histopathologic entity thought to occur at a relatively low incidence and influence prognosis. A serum-free hormonally defined growth medium has been developed, allowing the long-term growth of the skeletal muscle component of Wilms' tumors. Eight Wilms' tumors have been grown under these conditions. Three cases grew a homogeneous population of cells which ultrastructurally displayed all stages of myogenesis through myotubule formation. They also possessed immunoreactivity for skeletal muscle myosin and myoglobin and synthesized the M and B subunits of creatine kinase. Of interest was the finding that the ability to yield skeletal muscle cultures was limited to those cases which exhibited skeletal muscle fibers in vivo. This technique is also a very sensitive marker for identifying Wilms' tumors possessing a myoid component. A second serum-free hormonally defined medium has also been developed that supports the long-term culture of a unique cell type from Wilms' tumors which contain a myoid component. These cells are spindle-shaped and exhibit all of the characteristics of early myoblasts.

A human rhabdomyosarcoma (RMS) cell line was established from a case of childhood small cell sarcoma, which in vivo showed no evidence of differentiation, but which demonstrated myogenic differentiation in tissue culture. In a serum-free culture medium, the tumor cells demonstrated continuous growth without ultrastructural or biochemical evidence of differentiation. Heterotransplanted RMS cells gave rise to tumors in nude mice which also showed no myogenic differentiation. However, RMS cells grown in the presence of either retinoic acid (5 microns), phorbol ester (1 nM), prostaglandin E1 (10 ng/ml), or 2% fetal calf serum gave rise to myotubes with a biochemical shift in the creatine kinase isoenzyme pattern from the embryonic to the mature skeletal muscle form. The karyotype of the RMS cells revealed a translocation of Chromosomes 2 and 13, which may represent a nonrandom aberration unique to this morphologic subtype. In addition, the RMS cells gave evidence for gene amplification in the form of double minute chromosomes. This human RMS cell line provides a valuable in vitro system for study of myogenesis and factors which induce differentiation.

Insulinlike growth factors (IGFs) are peptide hormones that regulate proliferation and differentiation of several types of normal and neoplastic cells. Recent studies of Wilms' tumor, a childhood kidney neoplasm, have detected increased expression of IGF-2 mRNA and protein. The present report describes detection of Type 1 IGF receptors in specimens of Wilms' tumor and adjacent nonneoplastic kidney tissue. These receptors recognize both IGF-1 and IGF-2, and binding of either peptide activates endogenous tyrosine kinase activity of the Type 1 IGF receptor beta subunit. These data indicate that Wilms' tumors contain receptors that recognize and respond to exogenous IGF in vitro, and that autocrine IGF production might contribute to the increased proliferation and abnormal differentiation of these cells in vivo.