Histological conformity of implantation tumors produced by kidney cell lines derived from dimethylnitrosamine-treated rats, with dimethylnitrosamine-induced renal mesenchymal tumors

The histology of five implantation tumors induced in rats by the deposition of cultured cell lines derived from dimethylnitrosamine (DMN)-treated rats is described and compared with the morphology of the predominant kidney neoplasm induced in vivo by a single high dose of DMN. The cell lines leading to growth upon implantation were long-established, continuously growing cultures obtained either from a DMN-induced renal mesenchymal tumor or from rats treated shortly before with a carcinogenic dose of DMN. The latter cultures had expressed morphological transformation at subcultures 5 or 6. All of the implantation tumors were of mesenchymal type, comprising variously a range of cell forms including fibroblast-like spindle cells, smooth muscle fibers, and "giant" cells, which resembled common aspects of the parent mesenchymal tumors induced in the rat kidney by DMN. Deposition of cells intrarenally illustrated the survival of remnants of preexisting nephrons as epithelial profiles scattered through the proliferating malignant tissue, a feature most characteristic of the parent tumor. The results confirmed the malignant nature of the various cell lines tested, in keeping with their altered behavior in vitro, and they were consistent also with the premise that the in vivo-in vitro system is selecting cells in culture that represent the same target population from which the renal mesenchymal tumors are derived in vivo.     

Smooth muscle associated antigen in astrocytes and astrocytomata.

Four human and 4 rat astrocytomata and mammalian adult brain and spinal cord were examined by indirect immunofluorescence with human serum containing smooth muscle antibody. Cryostat sections of astrocytomata showed staining of the tumour cell cytoplasm and processes while in normal adult brain and spinal cord the entire astrocyte stained. Impression film and tissue culture monolayers of astrocytomata showed staining of cell processes and a fine, filamentous network in the cell body. The reaction with astrocytoma tumour cells was stronger than that with the corresponding normal astrocytes. Specificity of the staining reaction was established by its prevention on neutralization absorptions of the serum with extracts or homogeneous of smooth muscle. The presence of smooth muscle-associated antigen in astrocytes and astrocytomata is indicative of contractile protein providing a mechanism of cell movement in vivo.     

The modification of the renal carcinogenicity of dimethylnitrosamine by actinomycin D and a protein deficient diet.

The effect of a single treatment with 30 mg dimethylnitrosamine (DMN) and 6 mug actinomycin D (ACT), given at different time intervals (ACT application to DMN, 2 h before, simultaneously, 5, 9 or 48 h later), was tested in female Sprague-Dawley rats in relation to renal carcinogenesis; additionally, the animals were fed either a normal or a protein deficient diet. The ACT treatment did not significantly modify either the kidney tumour incidence or the survival time in the different groups fed a normal diet. Nevertheless, there are indications that additional ACT application may shorten the latency period for DMN induced renal neoplasms or, when administered 5 h later than DMN, a slightly decreased and delayed tumour induction can be assumed. In groups fed a protein deficient diet, a significantly higher percentage of kidney tumour bearing animals as well as a shortened latency period were found when compared with the DMN group on normal diet, but these differences were independent of the additional ACT treatment 9 h later than DMN and were due to the protein deprivation. Morphologically, the tumours were of epithelial and mesenchymal type with a clear preponderance of the former type. Biochemical and morphological aspects are discussed.     

Loss of NOS1 expression in high-grade renal cell carcinoma associated with a shift of NO signalling

In normal human kidney, NOS1 and soluble guanylate cyclase (sGC) are expressed in tubular epithelial cells, suggesting a physiological autocrine NO signalling pathway. Therefore, we investigated both NOS1 and sGC expressions in benign and malignant renal tumours. In addition, we examined the pattern of protein tyrosine nitration in normal and tumour tissue. NOS1 expression and activity were found to be downregulated, correlating with the tumour grade, as shown by immunohistochemistry, quantitative RT-PCR analysis, and histochemical detection of the NADPH-diaphorase activity of nitric oxide synthases (NOS). These results show that the autocrine NO signalling pathway is maintained in benign tumours and lost in malignant tumours. In contrast, sGC expression was maintained in renal tumours whatever the tumour type, a finding showing that tumour cells remain sensitive to the bioregulatory role of exogeneous NO(*). Finally, the staining pattern of protein tyrosine nitration, assessed by immunohistochemistry, parallelled that of NOS1 expression in normal renal parenchyma and benign tumours, supporting the concept that protein nitration was accounted for by NOS1 activity. In contrast, in malignant tumours, protein tyrosine nitration was accounted for by the production of reactive nitrogen oxide species by the inflammatory infiltrate. Altogether, these findings argue for a pattern of NO signalling similar in normal kidney and benign renal tumours, whereas it is completely different in malignant renal tumours.     

Increased expression of actin-like contractile protein in preneoplastic and neoplastic lesions in rat liver.

Cryostat sections of 16 preneoplastic and 14 neoplastic hepatic lesions induced in rats by 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) were examined by indirect immunofluorescence with human serum containing smooth-muscle antibody (SMA). Preneoplastic lesions showed strong cytoplasmic staining of proliferating oval cells and of the cell outlines of hepatocytes, in areas of nodular hyperplasia. In carcinomas, poorly differentiated hepatocytes showed staining of cell outlines, while well differentiated tumour cells forming glandular structures showed only staining of the luminar surfaces. The stromal cells also showed cytoplasmic staining. Morphologically normal areas of 3'-Me-DAB-treated livers showed weak staining of cell outlines, similar to normal liver. Specificity of the staining reactions was established by failure of staining in parallel control sections treated with normal human serum, or SMA serum neutralized by absorptions with homogenates of smooth muscle or extracts of actin. The results suggest that there is an increased expression of actin-like contractile protein in preneoplastic and poorly differentiated neoplastic liver cells.     

Induction of kidney tumours by a single dose of dimethylnitrosamine: dose response and influence of diet and benzo(a)pyrene pretreatment.

Seven days on a protein-free diet increases the susceptibility of rats to the action of DMN as a renal carcinogen. The dose response for the induction of kidney tumours by a single dose of dimethylnitrosamine (DMN) in these rats is reported. The first tumour was not found until 28 weeks after the dose. At 100 weeks the incidence ranged from 22.5% at the lowest dose (20 mg/kg) to 97% at the highest dose (60 mg/kg). The incidence in probits at any time between 50 and 100 weeks was linearly related to the log dose. Epithelial and mesenchymal tumours were produced in an approximate ratio of 2:1. The protein-free diet alters the rate of metabolism of DMN in the rat, and increases the alkylation of nucleic acids by this carcinogen in the kidney. Further treatment of the rat with benzo(a)pyrene can reverse, to some extent, the change in metabolism, but does not reverse the change in alkylation. It is shown that the change in kidney-tumour incidence produced by the change in diet, and by the treatment with benzo(a)pyrene, corresponds to the changes these treatments produce in the alkylation of kidney DNA by the carcinogen.     

Ultrastructural changes in atrial myxoma

Ultrastructural changes of 20 cases of atrial myxoma were studied. Under the electron microscope, in a large amount of amorphous matrix, the myxomal cells were either scattered or aggregated. There were numerous microvilli-like cytoplasmic processes on the surface of the tumor cells. In many aspects, the ultrastructural features of the tumor cells were similar to those of the smooth muscle cells. The abundant cytoplasmic filaments were one of the most prominent ultrastructural features. Sometimes, some of them could form dense bodies. There were cytoplasmic filaments in the cytoplasmic processes of a certain tumor cells and lots of micropinocytic vesicles beneath the inner surface of the plasmic membrane. Nuclear membrane of the tumor cells often had marked indentation which was probably related to contraction of the cytoplasmic filaments. 2 of 20 lesions showed malignant characteristics. One lesion infiltrated into the atrial myocardium, and the other involved the left and right atria. It is suggested that the atrial myxoma be a true neoplasm of the atrium with potential malignant tendency. It originates from the multipotential mesenchymal tissue and differentiates mainly towards smooth muscle cells.     

Surface proteins in normal and transformed rat liver epithelial cells in culture.

The pattern of surface proteins of different types of normal and transformed rat liver cells have been studied in culture by means of lactoperoxidase-catalysed iodination procedures, followed by SDS-gel electrophoresis. The cells examined were primary cultures of epithelial liver cells, long-term cultures of epithelial liver cells, in vitro transformed epithelial liver cell lines and liver tumour-cell lines; mesenchymal cells from liver and skin were also examined. The principal surface proteins of primary cultures of epithelial cells from adult or neonatal rats had components with mol. wts of 140,000-160,000, 100,000 and 40,000-70,000. A band that had the same position as fibronectin from mesenchymal cells was also present and this band, as well as other iodinated components, were less sensitive to trypsin than fibroblastic fibronectin. A similar pattern of iodinated proteins was seen in long-term cultures of epithelial liver cells, with a great reduction in the number and intensity of the bands in the mol. wt region below 100,000. Almost all the in vitro transformed and tumour epithelial cell lines contain a protein with a mol. wt 135,000 as one of the major iodinated bands, and in contrast to the observation in transformed fibroblasts, the fibronectin was retained by most of these transformed cell lines.     

Demonstration of the tumorigenicity of transformed rat kidney cell-lines by intravenous allotransplantation in the neonate

The tumorigenic potential of allogeneic rat kidney cell-lines transformed in vivo by a carcinogenic dose of dimethylnitrosamine (DMN) was investigated by intravenous injection of single cell suspensions into neonatal outbred rats within 24 h of birth. The cell-lines tested included mesenchymal populations obtained from DMN-induced renal mesenchymal tumors (RRMT-2,-8,-9) and transformed mesenchymal (TRKM-5,-7,-8) and epithelial (TRKE-1) cell-lines derived from the kidneys of rats treated only hours previously with the carcinogen. Additionally, spontaneous nephroblastoma-derived embryonal cell-lines (REN-1,-2) were included in the study to extend the potential of the neonatal rat system over a range of differentiation lineages. The transplantation system proved to be a rapid and efficacious assay for demonstrating the malignancy of the mesenchymal and embryonal cell-lines. The major sites for tumor growth were the lungs, heart and eye, but differences in organ predilection were observed for individual cell-lines. The transformed epithelial cell-line (TRKE-1) proved refractory to the single intravenous inoculation but was transplantation-positive when a follow-up subcutaneous dose was administered several days later. The resultant growths produced by the diverse differentiation lineages were histologically characteristic for the tumor tissue affiliate of each cell-line. The results demonstrate the utility of this transplantation system for testing the malignant potential of morphologically transformed cells across the allogeneic barrier as well as proving that DMN is capable of inducing malignant transformation in both mesenchymal and epithelial cell types of the rat kidney after a very short period of exposure in vivo.     

The ultrastructure of tumours derived from spontaneously transformed tissue culture cells

The ultrastructure of 16 tumours derived from spontaneously transformed cell lines established from young and old C57 and C3H mouse organs is described. Three types of tumour were found: myxoid (fibrosarcomatous), consisting of cells with long processes and much interstitial material; leiomyomatous, consisting of a bundle of smooth muscle-like cells with less interstitial material; or epithelial-like consisting of closely packed round cells with little interstitial material. The cell types in the tumours were similar to those found in the tissue culture cell lines from which they were derived.     

Short-term primary culture of epithelial cells derived from human breast tumours.

As experimental models for breast cancer, most studies rely on established human breast cancer cell lines. However, many of these lines were established over 20 years ago, many from pleural effusions rather than the primary tumour, so the validity of using them as representative models is questionable. This paper describes our experiences, over a 3-year period, in establishing short-term epithelial-cell-enriched preparations from primary breast tumours based on differential centrifugation followed by culture in selective media. Epithelial cells were successfully cultured from 55% of samples, but culture success did not appear to be correlated with tumour histology, stage, grade or node status. Epithelial cell-enriched cultures were immunopositive for broad-spectrum cytokeratin and epithelial membrane antigen (EMA). Positivity for keratin 19 confirmed that the cultures contained tumour-derived cells, which additionally showed significantly higher activity of the reductive pathway of the steroid-converting enzyme 17beta-hydroxysteroid dehydrogenase type I. That the cultures contained tumour and not normal epithelial cells was further substantiated by the complete absence of the calmodulin-like gene NB-1 in tumour-derived cultures; this is only associated with normal breast epithelia. Eighty-five per cent of cultures established from oestrogen receptor (ER)-positive tumours expressed ER in vitro; this was functional in 66% of cultures, although ER-positive phenotype was gradually lost over time. In conclusion, epithelial cells can be isolated and maintained as short-term cultures from primary breast tumours irrespective of histopathological or clinical details, providing a model system with a greater biological and clinical relevance than breast cancer cell lines.     

Carcinoma in situ of the female breast. 10 year follow-up results of a prospective nationwide study

Mammary spindle-cell tumours and sarcomas seem to be restricted to dogs and humans. Two cell lines from spontaneous primary canine mammary spindle-cell tumours (CMT-U304 and CMT-U309) and two cell lines from spontaneous primary canine mammary osteosarcomas (CMT-U334 and CMT-U335) were established to study the mesenchymal phenotypes of mammary tumours in the female dog. The cells from the spindle-cell tumours expressed cytokeratin, vimentin and smooth muscle actin filaments. When these cells were inoculated subcutaneously into female and male nude mice they formed different types of mesenchymal tumours such as spindle-cell tumours, fibroma and rhabdomyoid tumours (n=6/8). The cells from the osteosarcomas expressed vimentin filaments and also formed different types of mesenchymal tumours such as chondroid, rhabdomyoid, smooth muscle-like and spindle-cell tumours (n=6/10). The cell lines CMT-U304, CMT-U309 and CMT-U335 had receptors for progesterone but none of the four cell lines had receptors for estrogen. All four cell lines and their corresponding primary tumours showed identical allelic patterns in microsatellite analysis. By in situ hybridization with genomic DNA we could verify that all formed tumours but one were of canine origin. Our results support the hypothesis that canine mammary tumours are derived from pluripotent stem cells.     

Stabilization of breast cancer xenograft tumour neovasculature by angiopoietin-1

Angiopoietin-1 is a promoter of physiological vasculogenesis and angiogenesis because it induces vascular branching and smooth muscle recruitment to newly formed blood vessels. However, angiopoietin-1 expression in tumours appears to be uncommon, and angiopoietin-1 overexpression in cancer cells has been reported to lead to inhibition of xenograft tumour growth. We report here that angiopoietin-1 overexpression resulted in stabilization of tumour edge-associated blood vessels, as it prevented vessel dilation and dissociation of smooth muscle cells from existing vessels. In addition, angiopoietin-1 stimulated an infiltration of mesenchymal cells into the tumours, such that the coverage of microvessels by pericytes increased markedly, and the cancer cells were separated into small masses by the host stroma. The rates of both cancer cell proliferation and apoptosis decreased significantly in the presence of angiopoietin-1. Tie2, the receptor for angiopoietin-1, was found to be present in vascular smooth muscle cells in culture in addition to endothelial cells. These findings suggest that a vascular stabilization effect of angiopoietin-1 accounts for the inhibition of tumour growth.     

Microtubules and actin-containing filaments of normal, preneoplastic, and neoplastic mouse mammary epithelial cells.

The distribution and organization of microtubules (MT's) and actin-containing microfilaments (MF's) were examined in epithelial cells of primary cultures established from normal, preneoplastic, and neoplastic mouse mammary tissues and in cells of three clonal culture lines derived from murine mammary adenocarcinomas. No consistent differences in the cytoskeletal components were found among cell populations of the primary cultures as revealed either by indirect immunofluorescence using antibodies to tubulin and actin or by electron microscopy. Overall, the majority of cells in the three types of primary cultures possessed elaborate complexes of MT's and actin filaments after fluorescent staining with the appropriate antibodies, and abundant MT's and MF's were found in the cells at the ultrastructural level. Similar patterns of MT's and MF's were observed in cells of two of the clonal mammary tumor lines. Cells of the third line, however, exhibited intricate networks of MT's but had a reduction in actin cables detectable by the immunofluorescence procedure. Moreover, MF's were difficult to locate by electron microscopy. The results suggest that the lesion(s) in growth control in the neoplastic mammary cells may not involve any gross alterations in MT's or MF's.     

Isolation of a morphologically-transformed epithelial cell-line from rat kidney following an in vivo dose of dimethylnitrosamine

In an in vivo-in vitro system, a rat kidney epithelial cell-line was isolated into continuous culture 48 h after a single administration intraperitoneally of dimethylnitrosamine (DMN). The dose used, 60 mg/kg, was known to induce approximately a 40% incidence of renal cortical epithelial tumors, in addition to a much higher incidence of renal mesenchymal tumors. The cell-line was established from several persisting islands of epithelial cells which were cultured in Leibowitz medium supplemented with various-growth enhancing agents and periodically, the antifibroblastic agent cis-hydroxyproline. Through succeeding subcultures, the cells retained an epithelial morphology and at confluence piled-up into 3-dimensional ridges and domes above the monolayer. The cells were characterized by continuous growth in culture, the ability to form colonies at very low seeding rates, loss of anchorage-dependence as determined by growth in semi-solid media and formation of multicellular aggregates in suspension culture, and agglutination in the presence of conconavalin A. These altered properties of growth and behavior indicate that the renal epithelial cells constitute a morphologically transformed line. As such, they may represent the same target cell population from which DMN-induced epithelial tumors arise in the rat kidney.     

Cytogenetics of malignant epithelial cells and lymphoblastoid cell lines from nasopharyngeal carcinoma

The malignant epithelial cells of nasopharyngeal carcinoma (NPC) and cells of lines derived form the lymphoid cells which infiltrate this tumour have been investigated cytogenetically. Chromosome spreads of lymphoblastoid cells of lines established from 7 different NPC biopsy specimens were examined after banding staining. Banding was also applied to the epithelial tumour cells of 5 further biopsy specimens freed from non-malignant infiltrating cells by passage through nude mice; epithelial cell spreads were obtained by in vivo splindle arrest. Five of the lymphoblastoid lines were found to be diploid, and 2 tetraploid; the karyotypes were essentially normal. The squamous epithelial nature of the cells in the nude-mouse-grown NPC tumours was established by light and electronmicroscopy, and 3 tumours were found to be near-triploid, and 2 near-diploid. The cells of the near-triploid tumours contained grossly abnormal chromosomes but those of the near-diploid tumours showed only relatively minor changes. Although abnormalities were observed which were specific for cells from each individual tumour, no discernible change was common to cells from all the tumours.     

A model for evaluating selective delivery of plasmid DNA to tumours via the vasculature

A comparative study of plasmid DNA delivery in a newly established rat renal solid tumour model was undertaken. Free plasmid, plasmids bound to microspheres, and plasmids complexed with liposomes were selectively delivered to tumours via arterial catheterisation. Forty-eight hours post delivery, tumour to normal kidney tissue chloramphenicol acetyltransferase expression ratios were as follows: free (1.8:1), microspherical (3.9:1), and liposomal (1.2:1). Microspheres were able to selectively deliver the plasmids to tumours, whereas cationic liposomes distributed the plasmids to both kidney parenchymal and tumour cells. This tumour model has the potential of screening delivery vehicles as well as therapeutic agents for the capacity of selective delivery to tumours via the vasculature.     

Growth pattern of tumours in mice induced by murine Moloney sarcoma-virus and sarcoma-virus-transformed cells.

Transplantation of a Moloney sarcoma-virus (MSV-M)-transformed producer cell line (Sac(+)) induced progressively or regressively growing tumours in mice. Progressive growth always occurred after transplantation of an MSV-M non-producer transformant (Sac(-)), whereas the MSV-M released from the producer cells (Sac virus) always induced tumours which regressed. In contrast to the non-producer, the producer transformant Sac(+) as well as Sac virus induced a strong immune response, detected in vitro by cell- and antibody-mediated cytotoxicity assays, and in vivo by transplantation immunity. Implantation of Sac(-) cells led to solid, under-vascularized tumours, consisting histologically of uniform densely packed tumour cells. Sac-virus-induced tumours, however, were very well vascularized and arose by proliferation of different connective-tissue cells. After transplantation of Sac(+) cells, tumours were found to consist of typical tumour cells morphologically similar to Sac(-) cells intermingled with proliferated connective-tissue cells. Cultivation of tumour fragments from Sac(+) and Sac(-) tumours was followed by outgrowth of transformed tumour cells with the properties of the originally implanted cells. Tumour explant cultures from Sac-virus-induced tumours did not lead to growth of stably transformed cells. Co-culture of mouse embryo fibroblasts (MEF) with Sac(+) cells resulted in overgrowth of the transformed cells. Infection of MEF with Sac virus led to transiently transformed cells. It is concluded that Sac(+) cell tumours will resist the strong immune defence mechanisms they induce and grow progressively, if the inoculated cells are able to build up a solid, poorly vascularized nodule in the tissue. This always happens after implantation of 10(6) cells, but only occasionally when fewer cells are inoculated. Sac-virus-induced tumours will always regress owing to the strong immune response. The regression is furthered by the fact that MSV-M infection rarely if ever leads to a stable transformation.