Prevention of tumor metastasis formation by anti-variant CD44.

A splice variant of CD44 (CD44v) originally discovered on metastases of a rat pancreatic adenocarcinoma (BSp73ASML) has been shown by transfection to confer metastatic behavior to nonmetastatic tumor cells (Günthert U., M. Hofmann, W. Rudy, S. Reber, M. Zöller, I. Haussmann, S. Matzku, A. Wenzel, H. Ponta, and P. Herrlich. 1991. Cell. 65:13). A monoclonal antibody (mAb), 1.1ASML, to the metastasis-specific domain of the CD44v molecule retards growth of lymph node and lung metastases of the metastatic tumor line BSp73ASML, and can efficiently prevent formation of metastases by the transfected line. The antibody is only effective when given before lymph node colonization. Anti-CD44v does not downregulate the expression of CD44v, and prevention of metastatic growth by anti-CD44v is not due to activation of any kind of immune defense. We suggest that the mAb interferes with proliferation of metastasizing tumor cells in the draining lymph node, most probably by blocking a ligand interaction. The interference with metastatic spread will greatly facilitate the exploration of the function of CD44v and, in particular, may also open new strategies for the therapy of human metastases.

Rat mammary tumor classification: notes on comparative aspects.

Mammary tumors (170 spontaneous and 1,613 induced with 7,12-dimethylbenz[a]anthracene) in inbred SD rats were classified histologically. The neoplasms were divided into three main categories: fibroepithelial, epithelial, and connective tissue. In the fibroepithelial category, compound tumors showing a wide range of histologic structures with variation in the arrangement of both epithelial and connective tissue elements that differ among lobules occurred in 44 (25.9%) spontaneous and 1,027 (63.7%) induced neoplasms, whereas fibroadenomas occurred in 41 (24.1%) untreated and 175 (10.9%) treated rats. Uncommon fissured tumors were found in 1 (0.7%) untreated and 6 (0.4%) treated animals, whereas carcinosarcomas were found in only 10 untreated animals. In the epithelial category, tubular adenomas occurred in 48 (28.2%) untreated and 164 (10.2%) treated animals. Cystadenomas, duct papillomas, intraductal carcinomas, and anaplastic carcinomas were found less frequently. Adenoacanthomas occurred in only 12 (0.8%) of the treated animals. Among tumors with a fibrous component only, fibromas occurred in 22 (13%) untreated and 97 (6.0%) treated animals, Fibrosarcomas were less frequent, occurring in 4 untreated (2.4%) and 54 (3.4%) treated animals.

Allelic imbalance on chromosome 13q: evidence for the involvement of BRCA2 and RB1 in sporadic breast cancer.

Recently, the breast cancer susceptibility gene BRCA2 has been identified in chromosome 13q, a region that also contains the retinoblastoma gene RB1. To elucidate a possible role of BRCA2 and RB1 in sporadic breast tumorigenesis, allelic imbalance (AI) at 13q loci was examined in 78 primary sporadic breast tumors. AI was found in 52-63% of tumors. Nine tumors showed AI only in the BRCA2 region but not at RB1. Six tumors showed AI at RB1 but not in the BRCA2 region. AI in the BRCA2 region correlated significantly with aneuploidy (P = 0.032) and AI at RB1 with small tumor size (P = 0.025). Our data suggest that BRCA2 and RB1 may be both distinct target loci for AI on chromosome 13 in sporadic breast cancer.

c-fos expression induces bone tumors in transgenic mice.

The proto-oncogene c-fos has been isolated as the cellular homolog of the v-fos gene found in the osteosarcoma inducing FBR- and FBJ-murine sarcoma viruses (MSV). Expression of the c-fos gene in transgenic mice leads to the development of bone lesions of which about half progress to bone tumors mainly chondrosarcomas. The tumors display a strong preference for males and have a latency with a mean of 9.5 months. However, also mice without visible lesions develop bone tumors with the same sex preference and latency. These consequences of c-fos expression are independent of the chosen promoter but dependent on a replacement of 3' noncoding sequences of c-fos by a long terminal repeat (LTR) of the FBJ-MSV virus.

The expression of the imprinted genes H19 and IGF-2 in choriocarcinoma cell lines. Is H19 a tumor suppressor gene?

H19 is a paternally imprinted gene with unknown function. It is located in close proximity to the maternally imprinted IGF-2 gene on chromosome 11p15.5. In this study no consistent relationship between the expression of these two genes in clones derived from JEG-3 and JAr cell lines could be detected. Nor could a consistent relationship be detected between the expression levels of these two genes and between certain characteristic tumorigenic properties of these clones. We included in this study clones, expressing low H19 levels, which after transfection with an H19 expression construct highly expressed the H19 gene. In tumors, formed by the injection of cells of JAr or JEG-3 clones into nude mice, the H19 expression was high and irrelevant to the expression level in the cells before the injection. The same phenomenon was found for IGF-2 expression during tumorigenesis caused by cells of different JEG-3 clones and in some but not all JAr derived clones. Both H19 and IGF-2 are biallelicly expressed in all the JAr and JEG-3 clones. In summary, our observations point to the conclusion that H19 is not a tumor suppressor gene. However, its high expression in all the tumors formed after injection of cells of the JAr and JEG-3 clones, leaves its role, if any, in choriocarcinogenesis an open question.

Epidermal-dermal junction during experimental skin carcinogenesis and cocarcinogenesis as revealed by scanning electron microscopy.

During epidermal carcinogenesis important changes in the epidermal-dermal junction take place. Because of methodological difficulties may be these changes, especially those concerned with three-dimensional organization of the junction, remain unsatisfactorily investigated. To obtain new information, we studied with scanning electron microscopy (SEM) the development of carcinogenic changes in the epidermis of mice induced by DMBA-TPA, DMBA alone, and by the cocarcinogen TPA alone. We correlated the results with those from transmission electron microscopy and light microscopy. Although the epidermal changes morphologically showed similarities, biologically they differed. With them, distinct changes in the epidermal-dermal junction developed, that could be followed through early, hyperplastic and neoplastic phases. With the SEM the first changes were seen in the basal layer of the epidermis and concerned the cell arrangement. SEM provided information about the localization and development of incipient epidermal carcinomas induced by DMBA-TPA and DMBA treatment, as well as about the changes in the basal lamina. These can be classified by their surface, their extent and their frequency throughout large regions. Our studies indicate that these changes vary greatly, depending upon the treatment used and the time of their development. Only the progressive disintegration of the basal lamina is characteristic of carcinogenesis.

Preclinical study on gene therapy of cervical carcinoma using adeno-associated virus vectors.

Approximately 90% of cervical carcinomas are causally linked to infections with high-risk human papillomaviruses (HPVs), whose oncogenicity has been assigned to the continued expression of two early genes, E6 and E7. Reversal of the transformed phenotype by inhibiting E6/E7 gene expression therefore provides a suitable goal for future tumor therapy. Using recombinant adeno-associated virus type 2 (AAV-2) vectors, two types of therapeutic genes were expressed in cervical carcinoma cells with the aim of suppressing the E6/E7 oncogenes: (a) antisense E6/E7 and ribozyme genes and (b) the monocyte chemoattractant protein-1 (MCP-1) gene encoding MCP-1. Previous studies have shown that the MCP-1 protein is able to indirectly repress E6/E7 gene expression and is consistently absent in tumorigenic HPV-positive cervical carcinoma cell lines. Here, the effect of these therapeutic genes on tumor formation is analyzed in nude mice after ex vivo gene transfer into a HPV16- or HPV18-positive cervical carcinoma cell line (HeLa or SiHa, respectively). Whereas AAV-2 vector-mediated transfer of antisense or even ribozyme genes did not significantly influence tumor formation from implanted SiHa cells, the transfer and expression of human MCP-1 strongly inhibited the development of tumors derived from either HeLa or SiHa cells. Similar results were also obtained after in vivo delivery of these genes into SiHa-derived tumors. This suggests that transfer of therapeutic genes mediating a systemic effect via recombinant AAV-2 vectors offers a promising approach for the development of gene therapies directed against papillomavirus-induced human cancers.

Segmentation of cell nuclei in tissue section analysis.

Image segmentation is a critical step in digital picture analysis, especially for that of tissue sections. As the morphology of the cell nuclei provides important biological information, their segmentation is of particular interest. The known segmentation methods are not adequate for segmenting cell nuclei of tissue sections; the reason for this lies in the optical properties of their images. We have developed new gradient methods of segmentation of previously presegmented images by taking these properties into account and by using the approximately circular shape of the cell nuclei as a priori information. In our first technique, the segment method, the images of the nuclei are divided into eight segments, special gradient filters being defined for each segment. This has enabled us to improve the gradient image. After searching for local maxima, the contours of nuclei can be found. In the second method, the method of transformation into the polar coordinate system (PCS), the a priori information serves to define a circular direction field for gradient computation and contour finding. In contrast with the first method, which offers a rapid, general idea about the nuclear shape, the PCS method permits precise segmentation and morphological analysis of the cell nuclei.

Reproductive physiology and penile papillae morphology of rats after sexual experience.

Research which examined acute and chronic changes in the reproductive physiology and morphology of male rats after mating is reported. A group of sexually experienced (SE) and a group of sexually inexperienced (SI) animals were castrated on day 110 of age. Those animals and a group of SE and a group of SI gonadally intact males were left undisturbed for 30 days, and all animals were killed on day 140 of age. Testes, epididymides, penes and levator ani muscles were heavier in SE males, and those males had increased titres of circulating testosterone. Moreover, testicular sperm production was higher in SE than SI males immediately after the weekly copulatory sessions. Scanning electron microscopic examination of the surfaces of the glans penes revealed that sexual experience slowed the decay of penile papillae. More importantly, SE-intact males possessed uniformly large penile papillae, while the large papillae on penes from SI-intact males were interspersed with smaller papillae in various stages of development. The results suggest that mating provokes substantial changes in the endocrine integrity, fecundity and penile morphology of male rats.

Steroidal interactions in the ageing endocrine system: absence of suppression and pathology in reproductive systems of old males from a mixed-sex socially stressful rat colony.

A paradigm using chronic social stress and multiple measures of the reproductive system were used to assess changes with ageing in the dynamics of endogenous steroid interactions. The 22- to 24-month-old male rats lived for 8 weeks in one of four types of colony, in groups of the same sex or groups of mixed sex including familiar or unfamiliar old males. Measures of endocrinology (circulating steroid levels), behaviour (exploration and sociosexual responses), physiology (body and organ weights and epididymal sperm count) and histology (adrenal and ventral prostate glands) served as markers of activation of the hypothalamic-pituitary-adrenal (HPA) or hypothalamic-pituitary-testicular (HPT) axes. Old males living under stable conditions as familiar same-sex colonies served as the comparison group. Results indicated clear chronic activation of the HPA axis in the unfamiliar all-male colonies and of the HPT axis in the familiar males from mixed-sex colonies, whereas both steroidal axes were stimulated in colonies of unfamiliar males and females. Findings from aged males under chronic stress suggested that reproductive dysfunction may be limited to situations in which activation of the HPA axis occurs without concurrent stimulation of the HPT axis. Data on steroidal interactions from mixed-sex groups suggested that (1) chronic excitation of the HPA failed to suppress function in the reproductive system of the old males, (2) their stress responses were little affected by chronic HPT activation and (3) there was no evidence for stress-induced pathology, even in the vulnerable prostate gland.(ABSTRACT TRUNCATED AT 250 WORDS)

Digital picture analysis for studying the development of experimentally induced osteosarcoma.

We used the method of digital picture analysis to make a quantitative characterization, of stages of development of experimental osteosarcomas in rabbits. DMBA in gelatine pellets placed intraosteally served as a carcinogen to induce bone sarcomas. Of 25 animals treated, eight developed osteosarcomas classified as either osteoblastic, fibroblastic, or chondroblastic. We studied the process of bone neoplasia using radiographic, scintigraphic, and histological methods. To define the progress of neoplastic growth in each tumor we processed sequential radiographs of them by digital picture analysis. For this purpose, we developed a method for quantifying radiological changes using parameters independent of the rotation of the bone. In defining the histologic type of bone sarcomas, besides microscopic features we relied on quantitative criteria such as ratios of the newly formed bone and cartilage to the sarcomatous stroma. Our results, presented in this paper, indicate that digital picture analysis may be useful in characterizing experimental osteosarcomas as a model for therapeutic studies.

Carcinogenicity of small doses of inhaled N-nitrosoacetoxymethyl-N-methylamine in Syrian golden hamsters.

Male Syrian golden hamsters inhaled 0.5-1 ppm (= 2.7-5.5 mg/m3) of N-nitroso-N-acetoxy-methyl-N-methylamine 1 h/week, for 14 weeks. The total dose per animal was calculated as 150-400 micrograms or 1-3 mg/kg. Four squamous cell carcinomas and one mucoepidermoid carcinoma of the nasal mucosa were observed. No such tumors occurred in the control group.

Quantitative analysis of the early changes of hepatocyte nuclei after treating Syrian golden hamsters with di(2-ethylhexyl)phthalate.

Although the biological action of phthalates has been widely discussed there is little information on early cellular changes indicative for toxic or carcinogenic effects. To study subtle alterations in the cell morphology, we have by means of image processing evaluated the nuclei of hamster hepatocytes after treatment with di(2-ethylhexyl)phthalate given in single i.p. doses of 30, 300, and 3000 mg/kg. The results indicate that by using specially developed methods for analysis of images of cell nuclei and chromatin structure, it is possible to recognize changes eluding detection with usual light microscopy.

Subclinical human immunodeficiency virus-related changes in oral mucosa shown by image analysis of tongue smears.

There is evidence that certain lesions of the oral mucosa, such as hairy leukoplakia (HL), in patients seropositive for human immunodeficiency virus correlate with the subsequent development of acquired immune deficiency syndrome. The authors suggest that HL is a final manifestation of alterations that gradually develop after HIV infection. To recognize inapparent early subclinical changes in oral mucosa, the authors applied methods of digital image analysis to investigate tongue smears from healthy control subjects and immunosuppressed patients after chemotherapy and HIV infection. Their studies concentrated on nuclear morphologic features and chromatin structure. The results obtained with a large set of subvisual parameters indicated significant differences in nuclear and chromatin features between the smear patterns of investigated groups. One important implication of these studies is that computerized image analysis of simply prepared tongue smears enables one to recognize subvisual HIV-related changes before clinical evidence of HL appears.

Morphologic characteristics of the interaction between normal cytotrophoblasts and their malignant counterpart in the development of trophoblastic neoplasia.

OBJECTIVES: To define the biology of the tumor-host cell interaction with regard to cellular kinetics and morphologic changes during cell-cell interaction in an in vitro model of trophoblastic neoplasia. METHODS: Using a coculture in vitro system of cytotrophoblasts and choriocarcinoma cells, we investigated the cellular kinetics and the morphologic changes in these interacting cells. A fully automatic time-lapse image system was used to record phase contrast images of the cocultured cells in a tissue culture chamber. To examine cytoskeletal structure, immunofluorescent-labeled antibodies against intermediate filaments were used. Slides were examined with a confocal laser scanning microscope and subjected to computed analysis. RESULTS: The choriocarcinoma cells attract normal cytotrophoblasts using what resembles pseudopodia to engulf the latter cells and thus form slow-growing colonies. In this process, new hybrid cells are formed, which can be differentiated from their original contributors by morphologic characteristics. CONCLUSION: This phenomenon supports our previous biochemical and molecular data on the role of cell-cell interaction in the complex process of cytotrophoblast transformation and the development of gestational trophoblastic neoplasia.

Image analysis of hepatocyte nuclei in assessing di(2-ethylhexyl)phthalate effects eluding detection by conventional microscopy.

The effect of di(2-ethylhexyl)phthalate (DEHP) administered in single intraperitoneal doses of 30, 300 and 3000 mg/kg in Syrian golden hamsters was studied by means of routine pathologic investigations, electron microscopy and image analysis. The morphological evaluations did not show apparent differences between the control and treated animals. Such differences, however, were recognized by using image analysis. They concerned morphology of the hepatocyte nuclei and were defined by quantitative parameters reflecting geometrical, optical and structural properties. Of importance for differentiating dose/effect relationships were features of chromatin structure. In order to describe those features we developed special algorithms capable of identifying and characterizing regions of condensed chromatin as subimages. These were distinguished by their size, shape and optical density and showed typical distributions within the nucleus. As our results demonstrate, image analysis methods permit detection of DEHP related pathology in animals which, as far as is evident from routine morphologic evaluations, belong to the no-effect experimental group.

Pronounced damage of intestinal tract mucosa by the combination of 1-methyl-1-nitrosourea plus 1,3-bis-(2-chloroethyl)-1-nitrosourea. Effect of drug sequence and time interval of administration.

The combination of 1-methyl-1-nitrosourea (MNU) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) has an overadditive toxicity in rats. This overadditive effect is dependent on drug sequence and time interval between the administration of both compounds. Application of MNU within 2 h prior to BCNU or simultaneous application of both compounds displayed the highest toxicity. The dose-limiting toxicity appears to be a severe damage of the intestinal mucosa.

Experimental bone tumors as models of human bone tumors.

Experimental models of human bone tumors may be classified as causal, descriptive, and selective models. These models are useful for analyzing the morphology, biology, and therapy of human bone tumors. The application of these models, however, requires us to define clearly the questions to be answered and to consider the special features of experimental and human bone tumors. The suitability of experimental bone tumors as models can be determined only on the basis of a large amount of relevant experimental and clinical data.

Quantitative evaluation of heterochromatin in epithelial atypia by an image processing method.

Digital image analysis as a method enabling quantitative description of microscopical images is especially important in studying cellular atypia. However, by using that method for characterizing the complex chromatin structure and its changes during atypia considerable difficulties arise. Defining substructures of chromatin images we have developed new method for description chromatin structure based on locally adaptive thresholding. The results obtained suggest that typical for atypia are changes in the size, optical density and distribution of the high optical density regions (heterochromatin) identified within the cell nuclei.

Quantitative aspects in defining prognostic factors of breast cancer.

Although many attempts have been made to define prognostic criteria, the long-term prediction of breast cancer prognosis remains a difficult task. In search of morphologic criteria with prognostic significance, we investigated 101 ductal invasive breast carcinomas by means of computerized image analysis. Our purpose was to define prognostic measures of the basic characteristics of breast cancers, growth rate and metastatic potential. Consequently, three principal types of analysis were performed: on Feulgen stained nuclei, on histological structures by using differential stainings and on desmosome-complexes revealed by immunofluorescence microscopy. The data obtained from analysis of the cell nuclei morphology and the chromatin structure served to establish objective nuclear grading. In comparison to the visual grading made during routine histology, the objective grading correlates better with the existence of lymph node metastases and with receptor status. The description of desmosome-complexes by using quantitative immunofluorescence microscopy indicates that in order to obtain relevant results, the histological pattern of the tumors has to be considered. In order to characterize these structures we introduced special methods of cluster analysis. The studies are an example of complex quantitative analysis of tumor morphology performed with different combined methods of microscopy and image processing.