Origin and evolution of somatic cell testicular tumours in transgenic mice.

Transgenic mice bearing a construct in which the expression of the SV40 oncogene is directed by the AMH promoter (AT mice) develop testicular tumours in adult life. We aimed to study early steps of tumour development and characterize tumours at different ages by histological, morphometric, and immunohistochemical techniques. One- to 3-month-old AT mice depicted multifocal Leydig cell hyperplasia. The testicular volume occupied by interstitial tissue was significantly higher in 3-month-old AT mice in comparison with littermate controls. Between 5 1/2 and 7 months, microscopic interstitial tumours developed that progressively evolved to form large confluent areas of high mitotic index in 7- to 14-month-old AT mice. Tumour cells had the characteristics and histoarchitecture of Leydig cells, or formed solid cord-like structures reminiscent of those seen in Sertoli cell tumours. Hyperplastic areas and tumours diffusely expressed 3beta-hydroxysteroid dehydrogenase (3beta-HSD) in Leydig cell areas. AMH expression was negative in Leydig cell conglomerates and tumours and variable in cord-like tumours. The SV40 T antigen and markers of cell proliferation (PCNA) were intensely positive in hyperplastic cells and tumours. Control mice of similar ages showed neither hyperplasia nor tumours, and SV40 T expression was always negative. In conclusion, transgenic mice develop large testicular tumours that are preceded by interstitial hyperplasia and microtumours. The histological and immunohistochemical phenotype of tumours (Leydig and Sertoli cell differentiation, positive 3beta-HSD, and variable AMH) suggests a mixed differentiation of somatic cells of the specialized gonadal stroma. The finding that an oncogene directed by a promoter specifically active in fetal Sertoli cells has given rise to testicular tumours of mixed differentiation is compatible with a common origin of Leydig and Sertoli cells from the specific stroma of the gonadal ridge, as supported by double labelling experiments in fetal mice showing co-localization of the transgene with Sertoli and Leydig cell markers.

Establishment and partial characterization of an ovine synovial membrane cell line obtained by transformation with Simian Virus 40 T antigen.

The small ruminant lentiviruses, namely caprine arthritis encephalitis virus (CAEV) and Maedi Visna virus (MVV) are grown currently in secondary synovial membrane cells. Primary and secondary cell cultures are sometimes difficult to obtain and support a low number of passages and, therefore, permissive cell lines are needed. A transformed cell line was obtained by transfection of ovine synovial membrane secondary cell culture with a plasmid containing the SV40 large T antigen gene. The transformed cell culture described in this paper showed a higher growth rate and a more homogenous population of fibroblast-like cells when compared to the original ovine synovial membrane secondary cell cultures. Karyotype analysis has indicated the induction of many random chromosome changes, leading to a decrease in chromosome number. The SV40 DNA was detected in the nucleus and in the cytoplasm of transformed cells. The putative expression of large T antigen was presumed by the detection of the corresponding mRNA by PCR. Finally, the transformed ovine synovial membrane cells were shown to be permissive to small ruminant lentiviruses, and these are suggested as a cell line for in vitro isolation and propagation of these viruses.

Suppression of AP-1 constitutive activity interferes with polyomavirus MT antigen transformation ability.

Polyomavirus (Py) encodes a potent oncogene, the middle T antigen (MT), that induces cell transformation by binding to and activating several cytoplasmic proteins which take part in transduction of growth factors-induced mitogenic signal to the nucleus. We have previously reported that the AP-1 transcriptional complex is a target for MT during cell transformation although, its activation was not sufficient for establishment of the transformed phenotype. Here we show that expression of a dominant-negative cJun mutant in MT transformed cell lines inhibits its transformation ability, indicating that constitutive AP-1 activity is necessary for cell transformation mediated by MT. Evidences also suggest that proliferation of MT transformed cells in low serum concentrations and their ability to form colonies in agarose are controlled by distinct mechanisms.

Mechanisms of cell transformation induced by polyomavirus

Polyomavirus is a DNA tumor virus that induces a variety of tumors in mice. Its genome encodes three proteins, namely large T (LT), middle T (MT), and small T (ST) antigens, that have been implicated in cell transformation and tumorigenesis. LT is associated with cell immortalization, whereas MT plays an essential role in cell transformation by binding to and activating several cytoplasmic proteins that participate in growth factor-induced mitogenic signal transduction to the nucleus. The use of different MT mutants has led to the identification of MT-binding proteins as well as analysis of their importance during cell transformation. Studying the molecular mechanisms of cell transformation by MT has contributed to a better understanding of cell cycle regulation and growth control

Mapping of polyomavirus middle T domain that is responsible for AP-1 activation.

Cell transformation by Polyomavirus middle T (MT) oncoprotein involves binding and activation of several cytoplasmic proteins that participate in growth factors-induced mitogenic signal transduction to the nucleus. We have previously reported that the AP-1 transcriptional complex is a target for MT during cell transformation. To analyse the interactions between MT and cellular proteins that are required for constitutive AP-1 activation, we compared wild type and transformation-defective MT mutant cell lines. High AP-1 activity, assessed by gel mobility shift assays, displayed by MT-overexpressing cells, is dependent on MT binding to phosphatidylinositol-3 kinase (P13K). Treatment with wortmannin (a specific P13K inhibitor) leads to decreased AP-1 activity. Supershift and Western blot analysis with specific antisera, indicate that JunB and cJun, but not cFos or FosB are present in the AP-1 complex. The results confirm the AP-1 complex as a downstream MT target and indicate that AP-1 activation may not be sufficient for cell transformation, since two transformation-defective MT mutants (250phe and MT322) display high AP-1 activity.