Purpose: The aim of this study was to assess the activity of hedgehog signaling pathway in malignant pleural mesothelioma (MPM).Experimental Design: The expression of hedgehog signaling components was assessed by quantitative PCR and in situ hybridization in 45 clinical samples. Primary MPM cultures were developed in serum-free condition in 3% oxygen and were used to investigate the effects of smoothened (SMO) inhibitors or GLI1 silencing on cell growth and hedgehog signaling. In vivo effects of SMO antagonists were determined in an MPM xenograft growing in nude mice.Results: A significant increase in GLI1, sonic hedgehog, and human hedgehog interacting protein gene expression was observed in MPM tumors compared with nontumoral pleural tissue. SMO antagonists inhibited GLI1 expression and cell growth in sensitive primary cultures. This effect was mimicked by GLI1 silencing. Reduced survivin and YAP protein levels were also observed. Survivin protein levels were rescued by overexpression of GLI1 or constitutively active YAP1. Treatment of tumor-bearing mice with the SMO inhibitor HhAntag led to a significant inhibition of tumor growth in vivo accompanied by decreased Ki-67 and nuclear YAP immunostaining and a significant difference in selected gene expression profile in tumors.Conclusions: An aberrant hedgehog signaling is present in MPM, and inhibition of hedgehog signaling decreases tumor growth indicating potential new therapeutic approach.

BackgroundOptimizing the safety and efficacy of standard chemotherapeutic agents such as cisplatin (CDDP) is of clinical relevance. Serum starvation in vitro and short-term food starvation in vivo both stress cells by the sudden depletion of paracrine growth stimulation.MethodsThe effects of serum starvation on CDDP toxicity were investigated in normal and cancer cells by assessing proliferation, cell cycle distribution and activation of DNA-damage response and of AMPK, and were compared to effects observed in cells grown in serum-containing medium. The effects of short-term food starvation on CDDP chemotherapy were assessed in xenografts-bearing mice and were compared to effects on tumor growth and/or regression determined in mice with no diet alteration.ResultsWe observed that serum starvation in vitro sensitizes cancer cells to CDDP while protecting normal cells. In detail, in normal cells, serum starvation resulted in a complete arrest of cellular proliferation, i.e. depletion of BrdU-incorporation during S-phase and accumulation of the cells in the G0/G1-phase of the cell cycle. Further analysis revealed that proliferation arrest in normal cells is due to p53/p21 activation, which is AMPK-dependent and ATM-independent. In cancer cells, serum starvation also decreased the fraction of S-phase cells but to a minor extent. In contrast to normal cells, serum starvation-induced p53 activation in cancer cells is both AMPK- and ATM-dependent. Combination of CDDP with serum starvation in vitro increased the activation of ATM/Chk2/p53 signaling pathway compared to either treatment alone resulting in an enhanced sensitization of cancer cells to CDDP. Finally, short-term food starvation dramatically increased the sensitivity of human tumor xenografts to cisplatin as indicated not only by a significant growth delay, but also by the induction of complete remission in 60% of the animals bearing mesothelioma xenografts, and in 40% of the animals with lung carcinoma xenografts.ConclusionIn normal cells, serum starvation in vitro induces a cell cycle arrest and protects from CDDP induced toxicity. In contrast, proliferation of cancer cells is only moderately reduced by serum starvation whereas CDDP toxicity is enhanced. The combination of CDDP treatment with short term food starvation improved outcome in vivo. Therefore, starvation has the potential to enhance the therapeutic index of cisplatin-based therapy.

Drosophila melanogaster heterochromatin protein 1 (HP1a or HP1) is believed to be involved in active transcription, transcriptional gene silencing and the formation of heterochromatin. But little is known about the function of HP1 during development. Using a Gal4-induced RNA interference system, we showed that conditional depletion of HP1 in transgenic flies resulted in preferential lethality in male flies. Cytological analysis of mitotic chromosomes showed that HP1 depletion caused sex-biased chromosomal defects, including telomere fusions. The global levels of specific histone modifications, particularly the hallmarks of active chromatin, were preferentially increased in males as well. Expression analysis showed that approximately twice as many genes were specifically regulated by HP1 in males than in females. Furthermore, HP1-regulated genes showed greater enrichment for HP1 binding in males. Taken together, these results indicate that HP1 modulates chromosomal integrity, histone modifications and transcription in a sex-specific manner.

The cancer killing efficacy of standard chemotherapeutic agents such as cisplatin (CDDP) is limited by their side effects to normal tissues. Therefore, research efforts optimizing the safety and efficacy of those agents are clinically relevant. We did screen for agents that specifically protect normal human mesothelial cells against CDDP without reducing the cancer cell killing efficacy. Lovastatin was identified from the screen. Lovastatin at a pharmacologically relevant concentration strongly arrested the proliferation of normal cells, whereas cancer cells were less affected. CDDP-induced DNA damage response was not activated and normal cells showed enhanced tolerance to CDDP when normal cells were treated with the combination of CDDP and lovastatin. We demonstrate that interfering with protein geranylgeranylation is involved in the lovastatin-mediated CDDP protective effect in normal cells. In contrast to normal cells, in cancer cells lovastatin did not change the CDDP-induced response, and cancer cells were not protected by lovastatin. Furthermore, lovastatin at the pharmacological relevant concentration per se induced DNA damage, oxidative stress and autophagy in cancer cells but not in normal mesothelial cells. Therefore, our data suggest that lovastatin has a potential to improve the therapeutic index of cisplatin-based therapy.

In the developing Drosophila eye, the precursors of the neuronal photoreceptor cells R1/R6/R7 and non-neuronal cone cells share the same developmental potential and constitute the R7 equivalence group. It is not clear how cells of this group elaborate their distinct fates. Here we show that both TTK88 and D-Pax2 play decisive roles in cone cell development and act in concert to transform developing R1/R6/R7 into cone cells: while TTK88 blocks neuronal development, D-Pax2 promotes cone cell specification. In addition, ectopic TTK88 in R cells induces apoptosis, which is suppressed by ectopic D-Pax2. We further demonstrate that Phyllopod (Phyl), previously shown to promote the neuronal fate in R1/R6/R7 by targeting TTK for degradation, also inhibits D-Pax2 transcription to prevent cone cell specification. Thus, the fates of R1/R6/R7 and cone cells are determined by a dual mechanism that coordinately activates one fate while inhibiting the other.

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The two Pax6 gene homologs eyeless and twin of eyeless play decisive early roles in Drosophila eye development. Strong mutants of twin of eyeless or of eyeless are headless, which suggests that they are required for the development of all structures derived from eye-antennal discs. The activity of these genes is crucial at the very beginning of eye-antennal development in the primordia of eye-antennal discs when eyeless is first activated by the twin of eyeless gene product. This activation does not strictly depend on the Twin of eyeless protein, but is temperature-dependent in its absence. Twin of eyeless acts also in parallel to the eyeless gene and exerts functions that are partially redundant with those of Eyeless, while Eyeless is mainly required to prevent early cell death and promote eye development in eye-antennal discs.