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Transcription factor AP-2 has been previously shown to play an important function in embryonal development and cell differentiation. We have investigated the possibility that AP-2 function in embryonic development is exerted by a multigene family of AP-2 related transcription factors. Here we describe the molecular cloning of such an AP-2 related gene, AP-2 beta, and prove that it encodes for a functional transcription factor. In situ hybridizations of murine embryo sections revealed a temporally restricted and tissue-specific expression pattern that indicates a function of AP-2 beta in the development of the midbrain in the differentiation of sensory neurons for taste, olfaction and palpation.

An overview is given on three topics of prostate carcinoma: 1. New data on genetic factors in the pathogenesis, 2. molecular activation mechanisms in metastasis and 3. endocrine dedifferentiation during the course of the disease causing androgen independent state. Recent epidemiological analyses have clearly shown, that a specific part of prostate cancer is inherited. By DNA-interphase analyses specific allelic losses have been demonstrated. Metastatic progression is both associated with oncogene ras activation and chromosomal deletions. Endocrinological dedifferentiation cannot be simply explained by an overgrowth by androgen-receptor negative tumor cells, but by a more complex deregulation with strong influences by stromal-epithelial interactions. Downregulation of the promoter region of the androgen receptor may play a crucial role in the development of "endocrine deafness".

We have applied a series of cell clones established from the human teratocarcinoma cell line PA-1 to study the effect of malignant transformation by ras-oncogenes on the regulation of cell growth and differentiation. A particular aim of this study was to identify nuclear gene-regulatory factors that are affected by ras-transformation. We show that a key nuclear target of ras is the transcription factor AP-2. AP-2 function is inhibited through the ras-controlled signal transduction cascade by at least two different mechanisms, i.e. inhibition of an AP-2 coregulatory factor and by expression of an alternatively spliced inhibitory AP-2 protein.

In order to find additional ways to classify bladder cancer, multiparameter analysis with antibodies against urothelial associated-glycoproteins (UAGAb: Uro1, -5, -9, -10; Signet) and cytokeratins (CKAb: KL1, Immunotech) were used in parallel with DNA staining. Single cell suspensions of 21 bladder cancer specimens (4pTaG1, 9pTaG2, 1pT1G2, 2 > pT2G2, 5 > pT2G3) were stained. Preliminary data showed that the proportion of UAGAb positive cells have to be related to the pan-urothelial marker Uro5, since percentage of urothelial cells was variable (30-97%). Phenotypic differences found in different stages of tumor will be described. Selection of tumor cells by UAG did result in higher precision to determine tumor S-phase fraction, and helped select tetraploid tumors. The methodology is best applicable to pTa and pT1-tumors and prospective analysis of these tumors has started.

The distribution of four antibodies against urothelial-associated glycoproteins (UAGAb: Uro1, -5, -9, -10, SIGNET) was studied by dual parameter flow-cytometry (UAGAb and DNA staining) in normal non-urothelial cancer cell lines (2), normal urothelial cell lines (2) and bladder cell lines (5). When cell lines were compared as plateau monolayers, high proportions of Uro1 and Uro10 were found in all cell lines, in contrast to Uro5 and Uro9, both showing specificity for urothelial cells. Though being described as a pan-urothelial marker, Uro5 is not found in all cell lines. In two cell lines (RT4 and J82) three different growth states (exponential and plateau monolayer and multicellular spheroids were compared, and dependence on spatial configuration could be shown for Uro9. Cell cycle relation of expression was found for Uro1 and Uro10.

Expression of two nucleolar antigens, p120 and B23, was studied in a prostatic carcinoma cell line (LNCaP) and in frozen and paraffin embedded tissue sections of 40 benign and malignant prostatic lesions. The percentage of p120 negative G0/G1 phase cells rose significantly during transition from exponential to plateau growth phase in vitro (from 9% to 32%). In contrast, B23 was equally expressed throughout different cell cycle and growth phases. Thus, nucleoli of almost all stromal and epithelial cells were stained by B23 in tissue sections. P120, however, selectively stained nucleoli of proliferating prostatic epithelium. Whereas 88% (13/15) of benign hyperplasia were p120 negative this was the case in only 24% (6/25) of carcinomas. Using microwave procedure both MoAbs reacted in paraffin sections, but the percentage of p120 negative cases doubled. A routine application to formalin fixed and paraffin embedded tissue cannot be recommended thus far.

The sensitivity of prostate epithelial cells to androgens is mediated by the androgen receptor (AR). Analyses of cell lines in vitro and prostate carcinomas in vivo provide evidence that loss of AR expression parallels the loss of sensitivity to androgens. The aim of our work is to analyze the genetic control elements of the AR gene and answer the question of why these elements fail to function in hormone-insensitive prostate cancer cells. Therefore, we have cloned and fully sequenced a 5700 base pair sequence 5'-upstream of the AR gene. This DNA fragment contains promoter and enhancer elements that are active in androgen-sensitive but inactive in androgen-insensitive prostate cancer cell lines.

Multiparameter-flow-cytometry was used to compare the levels of two proliferation-associated antigens, proliferating cell nuclear antigen (PCNA) and the Ki-67-related antigen, in squamous carcinoma cells grown as monolayers, multicellular spheroids (MCTS), and xenograft tumors. While the level of Ki-67-positive cells decreased with time of culture, the percentage of PCNA-positive cells stayed high in all experimental states investigated (up to 5 weeks of growth). The reduction of the mean fluorescence of PCNA/cell indicated a different way of regulation of this antigen in squamous carcinoma cells in comparison to other cell types.