In the present study we investigated the expression of the cell cycle inhibitor p27 in endometrial neoplasia using immunohistochemistry with a p27-specific antibody. Expression of p27 in endometrial carcinomas was compared with expression in the normal endometrium throughout the cycle. Normal endometrial cells showed strong nuclear expression of p27. Expression was present throughout the cycle and was stronger during the secretory phase. We found strongly reduced or abolished expression of p27 in endometrial carcinoma (85.3% of cases). The 41 tumours analysed were classified according to p27 staining intensity and percentage of positive cells into the following categories of p27 expression: negative/very low (56.0%); low (29.3%); moderate (14.7%) and high (0.0%). All the p27-positive tumours were well-differentiated endometrioid carcinomas of malignancy grade G1. Comparison with the p53 status showed that all tumours with strong p53 expression had low/negative p27 staining, while those that were positive for p27 had negative/low p53 staining. Reduced or absent p27 levels were also observed by Western blot analysis both in tumour samples and in HEC-1B endometrial adenocarcinoma cells. It thus seems that p27 expression is essential for the control of normal endometrial proliferation, and reduced or absent p27 expression may be an important step in endometrial carcinogenesis.

The Fas (Apo-1/CD95) ligand (FasL) belongs to the tumor necrosis factor family and acts through its receptor (FasR/ Apo-1/CD95) to induce apoptosis in target cells. FasL is expressed in several immunologically privileged sites. Induction of apoptosis by FasL in invading lymphocytes acts as a mechanism of immune privilege and is important in preventing graft rejection. Furthermore, FasL is expressed in certain malignancies and it has been implicated as a possible key mechanism in immune privilege of these tumors. Since the invading placental trophoblast is another very important site with a privileged immune status, we investigated whether FasL is expressed in the normal and tumoral human placenta. For this purpose, mRNA was extracted from first and third trimester human placental samples as well as from JEG3 choriocarcinoma cells and reverse transcribed to obtain cDNAs. These were used as templates for PCR analysis of FasL expression, in which specific primers were employed to amplify an 853 bp fragment spanning the whole FasL coding region. A product of the appropriate length was amplified from normal placenta as well as from the choriocarcinoma cells. Expression of FasL protein was confirmed by Western Blot and was localized to trophoblast by immunohistochemistry using a FasL-specific antibody. Expression of FasL in the human placenta indicates that induction of apoptosis in lymphocytes by the invading trophoblast could be an important mechanism implicated in the immune tolerance of the fetal semi-allograft.

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Downregulation of carcinoembryonic antigen-related cell adhesion molecule (CEACAM1), a cell adhesion molecule with tumor suppressing properties has been observed in a high percentage of carcinomas of the endometrium and other malignancies. The mechanisms for the dysregulation and the role of hormones and cytokines on the expression of CEACAM1 in endometrial carcinomas is unknown. We therefore studied the effect of estradiol, medroxyprogesterone acetate (MPA), RU486, gamma-interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), 12-O-tetradecanoylphorbol-13-acetate (TPA) and calcium ionophore A23187 on the expression in the non-expressing endometrial tumor cell lines Hec1B and Skut1B, respectively. No induction of CEACAM1 expression was observed in Hec1B endometrial adenocarcinoma cells in response to hormones and cytokines whereas treatment with TPA and calcium ionophore A23187 resulted in the strong expression of endogenous CEACAM1 on the mRNA and protein levels. In contrast, no induction of CEACAM1 expression was observed in endometrial mixed mesenchymal Skut1B cells. Studies of other members of the CEACAM family revealed that the re-expression in Hec1B carcinoma cells is restricted to CEACAM1 suggesting a cell type-specific and cell type-independent mechanism of CEACAM1 activation via the protein kinase C (PKC) pathway. Induction of CEACAM1 expression was dependent on protein kinase C protein synthesis and luciferase reporter assays with CEACAM1 promoter constructs demonstrated that the re-expression of CEACAM1 is regulated at the transcriptional level. This is the first report demonstrating that activators of PKC are able to specifically induce the expression of CEACAM1 in human carcinoma cells and our findings may provide a basis for the therapeutic inhibition of tumor growth in malignancies in which CEACAM1 is downregulated.

Leukaemia inhibitory factor (LIF) is a pleiotropic cytokine which has been found to be expressed in the human endometrium and to play an important role in human reproduction. In the present study we investigated expression and regulation of the human LIF promoter in HEC-1B endometrial adenocarcinoma cells using a luciferase reporter plasmid bearing a 666 bp promoter fragment (h666LIF-Luc) in transient transfection assays. HEC-1B cells were first shown by reverse transcription-polymerase chain reaction (RT-PCR) to be able to produce endogenous LIF mRNA. The LIF promoter was efficiently transcribed in HEC-1B cells, showing much higher levels of basal activity than in the previously studied Jurkat T-lymphoma cells and SKUT-1B uterine mesodermal tumour cells. The activity of the LIF promoter was stimulated in HEC-1B cells by a combination of phorbol ester (TPA) and ionomycin, which we had previously found to strongly induce its activity in Jurkat T-lymphoma cells. We next studied the effect of progestin (medroxyprogesterone acetate; MPA) on the LIF promoter activity in HEC-1B cells. The LIF promoter was not stimulated by MPA treatment in the presence of transfected progesterone receptor B (PR-B) expression vector in HEC-1B cells, while we had previously described its induction by MPA in SKUT-1B cells. This indicates that progestin-dependent regulation of the LIF promoter in uterine tumour cells is different in cells of epithelial and mesodermal origin.