A four-gene methylation marker panel as triage test in high-risk human papillomavirus positive patients.

Cervical neoplasia-specific biomarkers, e.g. DNA methylation markers, with high sensitivity and specificity are urgently needed to improve current population-based screening on (pre)malignant cervical neoplasia. We aimed to identify new cervical neoplasia-specific DNA methylation markers and to design and validate a methylation marker panel for triage of high-risk human papillomavirus (hr-HPV) positive patients. First, high-throughput quantitative methylation-specific PCRs (QMSP) on a novel OpenArray™ platform, representing 424 primers of 213 cancer specific methylated genes, were performed on frozen tissue samples from 84 cervical cancer patients and 106 normal cervices. Second, the top 20 discriminating methylation markers were validated by LightCycler® MSP on frozen tissue from 27 cervical cancer patients and 20 normal cervices and ROCs and test characteristics were assessed. Three new methylation markers were identified (JAM3, EPB41L3 and TERT), which were subsequently combined with C13ORF18 in our four-gene methylation panel. In a third step, our methylation panel detected in cervical scrapings 94% (70/74) of cervical cancers, while in a fourth step 82% (32/39) cervical intraepithelial neoplasia grade 3 or higher (CIN3+) and 65% (44/68) CIN2+ were detected, with 21% positive cases for ≤CIN1 (16/75). Finally, hypothetical scenario analysis showed that primary hr-HPV testing combined with our four-gene methylation panel as a triage test resulted in a higher identification of CIN3 and cervical cancers and a higher percentage of correct referrals compared to hr-HPV testing in combination with conventional cytology. In conclusion, our four-gene methylation panel might provide an alternative triage test after primary hr-HPV testing.

Signals that determine the fate of osteoblastic cells.

Signals that determine skeletal cell fate, function and apoptosis are critical to normal bone remodeling. Skeletal cells synthesize growth factors; some regulate cell replication, others regulate osteoblastic differentiation. Bone morphogenetic proteins (BMP) and Wnts induce the differentiation of mesenchymal cells toward mature osteoblasts. The action of BMPs is regulated by extracellular antagonists. These often act by binding to BMPs and preventing their binding to cell surface receptors. Wnt is essential for osteoblastogenesis, and mutations of Wnt co-receptors are associated with changes in bone mass. Wnt activity, like BMPs, is regulated by extracellular and intracellular antagonists. Notch, a family of transmembrane receptors, opposes Wnt signaling and inhibits osteoblastic differentiation. BMP, Wnt and Notch play a central role in osteoblastic cell fate.

NF- kappa B2/p100 induces Bcl-2 expression.

The NF-kappaB2/p100 and bcl-3 genes are involved in chromosomal translocations described in chronic lymphocytic leukemias (CLL) and non-Hodgkin's lymphomas, and nuclear factor kappaB (NF-kappaB) protects cancer cells against apoptosis. Therefore, we investigated whether this transcription factor could modulate the expression of the Bcl-2 antiapoptotic protein. Bcl-2 promoter analysis showed multiple putative NF-kappaB binding sites. Transfection assays of bcl-2 promoter constructs in HCT116 cells showed that NF-kappaB can indeed transactivate bcl-2. We identified a kappaB site located at position -180 that can only be bound and transactivated by p50 or p52 homodimers. As p50 and p52 homodimers are devoid of any transactivating domains, we showed that they can transactivate the bcl-2 promoter through association with Bcl-3. We also observed that stable overexpression of p100 and its processed product p52 can induce endogenous Bcl-2 expression in MCF7AZ breast cancer cells. Finally, we demonstrated that, in breast cancer and leukemic cells (CLL), high NF-kappaB2/p100 expression was associated with high Bcl-2 expression. Our data suggest that Bcl-2 could be an in vivo target gene for NF-kappaB2/p100.

Genetic imbalances in preleukemic thymuses.

To understand the molecular mechanisms involved in preleukemia, the suppression subtractive hybridization method was used in a murine radiation-induced thymic lymphoma model. Seventeen mRNAs overexpressed in preleukemic thymuses were identified: mouse laminin binding protein (p40/37LBP), E25 protein, Rattus norvegicus clone BB.1.4.1, profilin, poly(A) binding protein (PABP), mouse high mobility group protein 1, topoisomerase I, clusterin, proteasome RC1 subunit, rat prostatein C3 and C1 subunits; two ESTs and four unknown genes. The overexpression of PABP, clusterin, profilin, and the p40/37LBP mRNAs was confirmed in preleukemic thymuses and can be related to some cellular events observed during the preleukemic period, i.e., alterations of cell cycle and apoptosis properties. The p40/37LBP and 67-kDa laminin receptor proteins were upregulated during the preleukemic period. The data suggest that additional studies on p40/37LBP and 67-kDa laminin receptor regulation are required to evaluate their potential role in the lymphoma prevention by TNF-alpha and IFN-gamma.

Regulation of NF-kappaB activity by I kappaB-related proteins in adenocarcinoma cells.

Constitutive NF-kappaB activity varies widely among cancer cell lines. In this report, we studied the expression and the role of different I kappaB inhibitors in adenocarcinoma cell lines. High constitutive NF-kappaB activity and low I kappaB-alpha expression was found in a number of these cell lines. Moreover, some of these cells showed a high p100 expression, responsible for the cytoplasmic sequestration of most of p65 complexes. Treatment of these cells with TNF-alpha or other NF-kappaB activating agents induced only weakly nuclear NF-kappaB activity without significant p100 processing and led to a very weak transcription of NF-kappaB-dependent reporter gene. Induction of NF-kappaB activity can be restored by expression of the Tax protein or by treatment with antisense p100 oligonucleotides. In MCF7 A/Z cells stably transfected with a p100 expression vector, p65 complexes were sequestered in the cytoplasm by p100. These cells showed a reduced nuclear NF-kappaB induction and NF-kappaB-dependent gene transcription following TNF-alpha stimulation. As a consequence of a competition between I kappaB-alpha and p100, cells expressing high levels of p100 respond poorly to NF-kappaB activating stimuli as TNF-alpha.

Regulation of major histocompatibility complex class I expression by NF-kappaB-related proteins in breast cancer cells.

Downregulation of MHC Class I antigens has been observed in many cancers and usually results from a decreased gene transcription. A reporter CAT gene dependent on the MHC Class I kappaB site or on a longer promoter is transactivated by NF-kappaB complexes containing p65 or RelB. p100 as well as IkappaB-alpha are potent inhibitors of this transcription and p100 sequesters RelB and p65 complexes in the cytoplasm of breast cancer cells. However, although p100 is highly expressed in a number of breast cancer cell lines, MHC Class I antigen expression was observed on all the cell lines we analysed and could be further induced by stimulation with the cytokines IFN-gamma or TNF-alpha. Stable transfection of a unresponsive mutated IkappaB-alpha Ser 32-36 expression vector showed that TNF-alpha induced MHC Cl I expression in an NF-kappaB-dependent way while IFN-gamma did it independently of any NF-kappaB activation.