Potential hazards to embryo implantation: A human endometrial in vitro model to identify unwanted antigestagenic actions of chemicals.

Embryo implantation is a crucial step in human reproduction and depends on the timely development of a receptive endometrium. The human endometrium is unique among adult tissues due to its dynamic alterations during each menstrual cycle. It hosts the implantation process which is governed by progesterone, whereas 17ß-estradiol regulates the preceding proliferation of the endometrium. The receptors for both steroids are targets for drugs and endocrine disrupting chemicals. Chemicals with unwanted antigestagenic actions are potentially hazardous to embryo implantation since many pharmaceutical antiprogestins adversely affect endometrial receptivity. This risk can be addressed by human tissue-specific in vitro assays. As working basis we compiled data on chemicals interacting with the PR. In our experimental work, we developed a flexible in vitro model based on human endometrial Ishikawa cells. Effects of antiprogestin compounds on pre-selected target genes were characterized by sigmoidal concentration-response curves obtained by RT-qPCR. The estrogen sulfotransferase (SULT1E1) was identified as the most responsive target gene by microarray analysis. The agonistic effect of progesterone on SULT1E1 mRNA was concentration-dependently antagonized by RU486 (mifepristone) and ZK137316 and, with lower potency, by 4-nonylphenol, bisphenol A and apigenin. The negative control methyl acetoacetate showed no effect. The effects of progesterone and RU486 were confirmed on the protein level by Western blotting. We demonstrated proof of principle that our Ishikawa model is suitable to study quantitatively effects of antiprogestin-like chemicals on endometrial target genes in comparison to pharmaceutical reference compounds. This test is useful for hazard identification and may contribute to reduce animal studies.

CEACAM1 creates a pro-angiogenic tumor microenvironment that supports tumor vessel maturation.

We have studied the effects of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) on tumor angiogenesis in murine ductal mammary adenocarcinomas. We crossed transgenic mice with whey acidic protein promoter-driven large T-antigen expression (WAP-T mice) with oncogene-induced mammary carcinogenesis with CEACAM1null mice, and with Tie2-Ceacam1 transgenics, in which the Tie2 promoter drives endothelial overexpression of CEACAM1 (WAP-T × CEACAM1(endo+) mice), and analyzed tumor vascularization, angiogenesis and vessel maturation in these mice. Using flat-panel volume computed tomography (fpVCT) and histology, we found that WAP-T × CEACAM1(endo+) mice exhibited enhanced tumoral vascularization owing to CEACAM1(+) vessels in the tumor periphery, and increased intratumoral angiogenesis compared with controls. In contrast, vascularization of CEACAM1null/WAP-T-derived tumors was poor, and tumor vessels were dilated, leaky and showed poor pericyte coverage. Consequently, the tumoral vasculature could not be visualized in CEACAM1null/WAP-T mice by fpVCT, and we observed poor organization of the perivascular extracellular matrix (ECM), accompanied by the accumulation of collagen IV-degrading matrix metalloproteinase 9(+) (MMP9(+)) leukocytes and stromal cells. Vascular instability and alterations in ECM structure were accompanied by a significant increase in pulmonary metastases in CEACAM1null/WAP-T mice, whereas only occasional metastases were observed in CEACAM1(+) hosts. In CEACAM1(+) hosts, intratumoral vessels did not express CEACAM1, but they were intact, extensively covered with pericytes and framed by a well-organized perivascular ECM. MMP9(+) accessory cells were largely absent. Orthotopic transplantation of primary WAP-T- and CEACAM1null/WAP-T tumors into all three mouse lines confirmed that a CEACAM1(+) host environment is a prerequisite for productive angiogenic remodeling of the tumor microenvironment. Hence, CEACAM1 expression in the tumor periphery determines the vascular phenotype in a tumor, whereas systemic absence of CEACAM1 interferes with the formation of an organized tumor matrix and intratumoral vessel maturation.

In vitro-Ishikawa cell test for assessing tissue-specific chemical effects on human endometrium.

The human endometrium is a fertility-determining factor. Its receptivity during the implantation window may be altered by chemicals. Since human embryo implantation is unique chemical risk assessment cannot be based solely on animal studies. We established a tissue-specific in vitro test based on human endometrial adenocarcinoma (Ishikawa) cells. Progesterone receptor (PR) was selected as primary target gene for estrogenic effects. Changes of mRNA levels were investigated by reverse transcription quantitative real-time PCR. Sigmoidal dose-response curves for up-regulation of PR mRNA and EC(50) values were established for 17beta-estradiol, diethylstilbestrol and the weak xenoestrogen bisphenol A. Nonylphenol also had a clear PR mRNA up-regulating effect. Several other chemicals were characterized as negative compounds. Among them was methoxyacetic acid which may produce false positive results in reporter gene assays. Up-regulation of PR protein by 17beta-estradiol, diethylstilbestrol, bisphenol A and nonylphenol was confirmed by Western Blotting.

Rb2/p130 is the dominating pocket protein in the p53-p21 DNA damage response pathway leading to senescence.

The different pocket proteins are established as negative cell cycle regulators. With regard to the repressor functions of pocket proteins in cellular senescence, studies so far have mainly focused on pRb/p105. Here, we show that in a broad range of wild-type p53-expressing human tumor cells, and in human diploid fibroblasts, Rb2/p130 is the dominating pocket protein in replicative and in accelerated senescence. Senescent cells are arrested at the transition from late G1- to early S-phase, as indicated by the absence of S- and G2-phase cyclins A and B. Expression of cyclin A and entry into S-phase resumed after RNA interference-mediated knockdown of Rb2/p130. Activation of different upstream pathways by overexpression of either p21 or p16 converged on Rb2/p130 accumulation and induced senescence. In contrast, p53- or p21-negative cells treated with DNA-damaging agents failed to accumulate Rb2/p130 and to enter senescence. Our data suggest that Rb2/p130 is a member of the p53-p21 DNA damage signaling cascade, and represents the essential pocket protein family member needed for the induction of any type of senescence.

Gadd45 beta is a pro-survival factor associated with stress-resistant tumors.

Tumors that acquire resistance against death stimuli constitute a severe problem in the context of cancer therapy. To determine genetic alterations that favor the development of stress-resistant tumors in vivo, we took advantage of polyclonal tumors generated after retroviral infection of newborn Elambda-MYC mice, in which the retroviral integration acts as a mutagen to enhance tumor progression. Tumor cells were cultivated ex vivo and exposed to gamma-irradiation prior to their transplantation into syngenic recipients, thereby providing a strong selective pressure for pro-survival mutations. Secondary tumors developing from stress-resistant tumor stem cells were analysed for retroviral integration sites to reveal candidate genes whose dysregulation confer survival. In addition to the gene encoding the antiapoptotic Bcl-x(L) protein, we identified the gadd45b locus to be a novel common integration site in these tumors, leading to enhanced expression. In accord with a thus far undocumented role of Gadd45beta in tumorigenesis, we showed that NIH3T3 cells overexpressing Gadd45beta form tumors in NOD/SCID mice. Interestingly and differently to other known 'classical' antiapoptotic factors, high Gadd45beta levels did not protect against MYC-, UV- or gamma-irradiation-induced apoptosis, but conferred a strong and specific survival advantage to serum withdrawal.

Notch1 activation reduces proliferation in the multipotent hematopoietic progenitor cell line FDCP-mix through a p53-dependent pathway but Notch1 effects on myeloid and erythroid differentiation are independent of p53.

Signaling mediated by activation of the transmembrane receptor Notch influences cell-fate decisions, differentiation, proliferation, and cell survival. Activated Notch reduces proliferation by altering cell-cycle kinetics and promotes differentiation in hematopoietic progenitor cells. Here, we investigated if the G(1) arrest and differentiation induced by activated mNotch1 are dependent on tumor suppressor p53, a critical mediator of cellular growth arrest. Multipotent wild-type p53-expressing (p53(wt)) and p53-deficient (p53(null)) hematopoietic progenitor cell lines (FDCP-mix) carrying an inducible mNotch1 system were used to investigate the effects of proliferation and differentiation upon mNotch1 signaling. While activated Notch reduced proliferation of p53(wt)-cells, no change was observed in p53(null)-cells. Activated Notch upregulated the p53 target p21(cip/waf) in p53(wt)-cells, but not in p53(null)-cells. Induction of the p21(cip/waf) gene by activated Notch was mediated by increased binding of p53 to p53-binding sites in the p21(cip/waf) promoter and was independent of the canonical RBP-J binding site. Re-expression of p53(wt) in p53(null) cells restored the inhibition of proliferation by activated Notch. Thus, activated Notch inhibits proliferation of multipotent hematopoietic progenitor cells via a p53-dependent pathway. In contrast, myeloid and erythroid differentiation was similarly induced in p53(wt) and p53(null) cells. These data suggest that Notch signaling triggers two distinct pathways, a p53-dependent one leading to a block in proliferation and a p53-independent one promoting differentiation.

Interactions of mutant p53 with DNA: guilt by association.

Since the very early days of p53 research, the gain of oncogenic activities by some mutant p53 proteins had been suspected as an important factor contributing to cancer progression. Considerable progress towards understanding the biology of mutant p53 has been made during the last years, the quintessence being the realization that the impact of mutant p53 proteins on the transcriptome of a tumor cell is much more global than previously thought. The emerging role of mutant p53 proteins in coordinating oncogenic signaling and chromatin modifying activities reveals an until now unsuspected function of these proteins as important modifiers of the oncogenic transcriptional response. Notwithstanding the fact that the sequence-specific DNA binding activity of mutant p53 proteins is impaired, they are still able to associate with specific loci on DNA by utilizing different mechanisms. The ability to associate with DNA appears to be crucial for the master role of mutant p53 proteins in coordinating oncogenic transcriptional responses.

Regulation of cellular senescence by Rb2/p130.

Growth regulatory functions of Rb2/p130, which aim at a sustained arrest such as in quiescent or differentiated cells, qualify the protein also to act as a central regulator of growth arrest in cellular senescence. In this respect, Rb2/p130 functions are connected to signaling pathways induced by p53, which is a master regulator in cellular senescence. Here, we summarize the pathways, which specify pRb2/p130 to control this arrest program and distinguish its functions from those of pRb/p105.

Dissection of transcriptional and non-transcriptional p53 activities in the response to genotoxic stress.

Following genotoxic stress, p53 either rescues a damaged cell or promotes its elimination. The parameters determining a specific outcome of the p53 response are largely unknown. In mouse fibroblasts treated with different irradiation schemes, we monitored transcriptional and non-transcriptional p53 activities and identified determinants that initiate an anti- or a pro-apoptotic p53 response within the context of p53-independent stress signaling. The primary, transcription-mediated p53 response in these cells is anti-apoptotic, while induction of p53-dependent apoptosis requires an additional, transcription-independent p53 activity, provided by high intracellular levels of activated p53. High intracellular levels of p53 were selectively generated after apoptosis-inducing high-dose UV-irradiation, and correlated with a strongly delayed upregulation of Mdm2. Following high-dose UV-irradiation, p53 accumulated in the cytoplasm and led to activation of the pro-apoptotic protein Bax. As p53-dependent Bax-activation is transcription-independent, we postulated that certain transcription-deficient mutant p53 proteins might also exert this activity. Indeed we found an endogenous, transcription-inactive mutant p53 that upon genotoxic stress induced Bax-activation in vivo. Our results demonstrate the impact and in vivo relevance of non-transcriptional mechanisms for wild-type and mutant p53-mediated apoptosis.

Cooperation between p53 and p130(Rb2) in induction of cellular senescence.

To determine pathways cooperating with p53 in cellular senescence when the retinoblastoma protein (pRb)/p16INK4a pathway is defunct, we stably transfected the p16INK4a-negative C6 rat glioma cell line with a temperature-sensitive mutant p53. Activation of p53(Val-135) induces a switch in pocket protein expression from pRb and p107 to p130(Rb2) and stalls the cells in late G1, early S-phase at high levels of cyclin E. Maintenance of the arrest depends on the functions of p130(Rb2) repressing cyclin A. Inactivation of p53 in senescent cultures restores the pocket proteins to initial levels and initiates progression into S-phase, but the cells fail to resume proliferation, likely due to DNA damage becoming apparent in the arrest and activating apoptosis subsequent to the release from p53-dependent growth suppression. The data indicate that p53 can cooperate selectively with p130(Rb2) to induce cellular senescence, a pathway that may be relevant when the pRb/p16INK4a pathway is defunct.

Comparative assessment of the functional p53 status in glioma cells.

BACKGROUND: p53 is the most frequently mutated gene in human cancers and its functional integrity is an important predictor of treatment response and clinical outcome. The majority of mutations found in different types of cancer cluster within the DNA binding domain encoded by exons 5-8. In clinical specimens the functional status of p53 is, therefore, often evaluated by direct mutation analysis of exons 5-8 or indirectly by immunostaining and evaluation of the subcellular localization pattern or protein accumulation. MATERIALS AND METHODS: In a panel of glioma cell lines, the status of the P53 gene was analyzed by temperature gradient gel electrophoresis (TGGE) of exons 5-8 and direct sequencing of all p53 exons. The nuclear accumulation of p53 in unstressed cells was assessed by immunostaining. These data were correlated with stress induction of the p53 protein, nuclear translocation and a direct determination of the transcriptional activity of endogenous p53 protein and induction of p53 target genes. RESULTS: Our analysis demonstrated that a p53 gene mutation analysis limited to exons 5-8 and analysis of immunostaining patterns can not serve as reliable predictors of functional p53 in tumor cells. Conversely, in some presumably rare cases, the transcriptional activity of p53 may be retained in tumor cells in the presence of a mutation and a pathological immunostaining pattern. In our analysis, the constitutive dephosphorylation at Ser 376 correlated with the nuclear accumulation of p53, but not with the transcriptional activity of the protein. This suggests that constitutive dephosphorylation at Ser376 may be one of the factors determining stabilization of mutant and wild-type p53, which is frequently observed in glial tumors. CONCLUSION: The incidence of a dysfunctional p53 protein in gliomas may be higher than expected, based on a single parameter evaluation by mutation analysis of exons 5-8 or assessment of p53 accumulation and subcellular localization by immunostaining.

Expression patterns of CRH, CRH receptors, and CRH binding protein in human gestational tissue at term.

Recent research suggests a significant role for placental corticotropin-releasing hormone (CRH) in controlling human parturition. This paper describes the expression of CRH, CRH receptors 1 and 2, and CRH binding protein (CRH-BP) in gestational tissue in late pregnancy. Placenta, myometrium, decidua, and fetal membranes were collected after uncomplicated pregnancies at term caesarian section before the onset of labour. The localisation and mRNA expression of CRH, CRH receptors, and CRH-BP were studied by immunohistochemistry and reverse transcription (RT)-PCR. CRH receptors were detected in placenta, myometrium, decidua, and fetal membranes. We demonstrated for the first time the presence of CRH receptors on resident macrophages and on endothelial cells. CRH receptor 1 mRNA was detected in all tissues investigated by RT-PCR, whereas CRH receptor 2 mRNA was restricted to myometrium and decidua. CRH mRNA was widely expressed in all tissue under study. Novel findings are also presented on the expression of CRH-BP in the myometrium. This widespread expression of the CRH system in gestational tissue suggests a paracrine role for CRH in the birth process (e.g. effects on macrophages and endothelial cells).

Simian virus 40 large-T-antigen-specific rejection of mKSA tumor cells in BALB/c mice is critically dependent on both strictly tumor-associated, tumor-specific CD8(+) cytotoxic T lymphocytes and CD4(+) T helper cells.

Protective immunity of BALB/c mice immunized with simian virus 40 (SV40) large T antigen (TAg) against SV40-transformed, TAg-expressing mKSA tumor cells is critically dependent on both CD8(+) and CD4(+) T lymphocytes. By depleting mice of T-cell subsets at different times before and after tumor challenge, we found that at all times, CD4(+) and CD8(+) cells both were equally important in establishing and maintaining a protective immune response. CD4(+) cells do not contribute to tumor eradication by directly lysing mKSA cells. However, CD4(+) lymphocytes provide help to CD8(+) cells to proliferate and to mature into fully active cytotoxic T lymphocytes (CTL). Depletion of CD4(+) cells by a single injection of CD4-specific monoclonal antibody at any time from directly before injection of the vaccinating antigen to up to 7 days after tumor challenge inhibited the generation of cytolytic CD8(+) lymphocytes. T helper cells in this system secrete the typical Th-1 cytokines interleukin 2 (IL-2) and gamma interferon. Because in this system TAg-specific CD8(+) cells secrete only minute amounts of IL-2, it appears that T helper cells provide these cytokines for CD8(+) T cells. Moreover, this helper effect of CD4(+) T cells in mKSA tumor rejection in BALB/c mice does not simply improve the activity of TAg-specific CD8(+) CTL but actually enables them to mature into cytolytic effector cells. Beyond this activity, the presence of T helper cells is necessary even in the late phase of tumor cell rejection in order to maintain protective immunity. However, despite the support of CD4(+) T helper cells, the tumor-specific CTL response is so weak that only at the site of tumor cell inoculation and not in the spleen or in the regional lymph nodes can TAg-specific CTL be detected.

A murine tumor progression model for pancreatic cancer recapitulating the genetic alterations of the human disease.

This study describes a tumor progression model for ductal pancreatic cancer in mice overexpressing TGF-alpha. Activation of Ras and Erk causes induction of cyclin D1-Cdk4 without increase of cyclin E or PCNA in ductal lesions. Thus, TGF-alpha is able to promote progression throughout G1, but not S phase. Crossbreeding with p53 null mice accelerates tumor development in TGF-alpha transgenic mice dramatically. In tumors developing in these mice, biallelic deletion of Ink4a/Arf or LOH of the Smad4 locus is found suggesting that loci in addition to p53 are involved in antitumor activities. We conclude that these genetic events are critical for pancreatic tumor formation in mice. This model recapitulates pathomorphological features and genetic alterations of the human disease.

IC261, a specific inhibitor of the protein kinases casein kinase 1-delta and -epsilon, triggers the mitotic checkpoint and induces p53-dependent postmitotic effects.

The p53-targeted kinases casein kinase 1delta (CK1delta) and casein kinase 1epsilon (CK1epsilon) have been proposed to be involved in regulating DNA repair and chromosomal segregation. Recently, we showed that CK1delta localizes to the spindle apparatus and the centrosomes in cells with mitotic failure caused by DNA-damage prior to mitotic entry. We provide here evidence that 3-[(2,4,6-trimethoxyphenyl)methylidenyl]-indolin-2-one (IC261), a novel inhibitor of CK1delta and CK1epsilon, triggers the mitotic checkpoint control. At low micromolar concentrations IC261 inhibits cytokinesis causing a transient mitotic arrest. Cells containing active p53 arrest in the postmitotic G1 phase by blockage of entry into the S phase. Cells with non-functional p53 undergo postmitotic replication developing an 8N DNA content. The increase of DNA content is accompanied by a high amount of micronucleated and apoptotic cells. Immunfluorescence images show that at low concentrations IC261 leads to centrosome amplification causing multipolar mitosis. Our data are consistent with a role for CK1delta and CK1epsilon isoforms in regulating key aspects of cell division, possibly through the regulation of centrosome or spindle function during mitosis.

Role of heteroduplex joints in the functional interactions between human Rad51 and wild-type p53.

Our previous work (Dudenhöffer et al., 1999) unveiled a link between the capacity of p53 to regulate homologous recombination processes and to specifically bind to heteroduplex junction DNAs. Here, we show that p53 participates in ternary complex formation after preassembly of nucleoproteins, consisting of the human recombinase hRad51 and junction DNA. The cancer-related mutant p53(273H), which is defective in inhibiting recombination processes, displays a reduced capacity to associate with hRad51-DNA complexes, even under conditions which support DNA-binding. This suggests that hRad51-p53 contacts play a role in targeting p53 to heteroduplex joints and indicates an involvement in recombination immediately following hRad51-mediated strand transfer. To study the initial phase of strand exchange, when heteroduplex joints arise, we applied oligonucleotide based strand transfer assays. We observed that hRad51 stimulates exonucleolytic DNA degradation by p53, when it generates strand transfer intermediates. In agreement with this observation, artificial 3-stranded junction DNAs, designed to mimic nascent recombination intermediates, were found to represent preferred exonuclease substrates, especially when comprising a mismatch within the heteroduplex part. From our data, we propose a model according to which, p53-dependent correction of DNA exchange events is triggered by high-affinity binding to joint molecules and by stabilizing contacts with hRad51 oligomers. Oncogene (2000) 19, 4500 - 4512.