Andrea Sacchetti

Human Trop-2 is a tumor-associated calcium signal transducer.

Trop‐2/EGP‐1/GA733‐1 is a recently identified cell surface glycoprotein highly expressed by human carcinomas. The cytoplasmic tail of Trop‐2 possesses potential serine and tyrosine phosphorylation sites and a phosphatidyl‐inositol binding consensus sequence. Thus, we investigated whether Trop‐2 might be a functional signaling molecule. Using the fluorescent probe Fura‐2, we assayed the cytoplasmic calcium levels in human cancer cells stimulated with anti‐Trop‐2 or control antibodies. Three anti‐Trop‐2 MAbs, Rs7‐7G11, MOv16 and 162‐46.2 specifically induced a transient intracellular calcium level increment in up to 40% of the experiments performed. Polyclonal antisera recognizing recombinant Trop‐2 molecules possessed a much lower stimulation efficiency. The average latency of antibody‐induced Ca2+ rise for OvCa‐432 cells was 64 ± 26 sec. Internal Ca2+ concentrations reached peaks of 190 ± 24 nM vs<0R>. basal levels of 61 ± 4 nM and returned to baseline within 193 ± 37 sec. Similar values were obtained in MCF‐7 cells. For comparison, stimulation of P2‐purinergic receptors on MCF‐7 and OvCa‐432 cells induced a Ca2+ rise in most cases, leading to average internal Ca2+ concentrations of 297 ± 41 and 391 ± 39 nM, respectively. Our findings show that Trop‐2 transduces an intracellular calcium signal, are consistent with the hypothesis that it acts as a cell surface receptor and support a search for a physiological ligand. Int. J. Cancer 76:671–676, 1998.© 1998 Wiley‐Liss, Inc.

Paneth Cells in Intestinal Homeostasis and Tissue Injury

Adult stem cell niches are often co-inhabited by cycling and quiescent stem cells. In the intestine, lineage tracing has identified Lgr5 + cells as frequently cycling stem cells, whereas Bmi1 +, mTert +, Hopx + and Lrig1 + cells appear to be more quiescent. Here, we have applied a non-mutagenic and cell cycle independent approach to isolate and characterize small intestinal label-retaining cells (LRCs) persisting in the lower third of the crypt of Lieberkühn for up to 100 days. LRCs do not express markers of proliferation and of enterocyte, goblet or enteroendocrine differentiation, but are positive for Paneth cell markers. While during homeostasis, LR/Paneth cells appear to play a supportive role for Lgr5 + stem cells as previously shown, upon tissue injury they switch to a proliferating state and in the process activate Bmi1 expression while silencing Paneth-specific genes. Hence, they are likely to contribute to the regenerative process following tissue insults such as chronic inflammation.

IDH1 R132H decreases proliferation of glioma cell lines in vitro and in vivo

A high percentage of grade II and III gliomas have mutations in the gene encoding isocitrate dehydrogenase (IDH1). This mutation is always a heterozygous point mutation that affects the amino acid arginine at position 132 and results in loss of its native enzymatic activity and gain of alternative enzymatic activity (producing D‐2‐hydroxyglutarate). The objective of this study was to investigate the cellular effects of R132H mutations in IDH1.

Interplay between metabolic identities in the intestinal crypt supports stem cell function

The small intestinal epithelium self-renews every four or five days. Intestinal stem cells (Lgr5+ crypt base columnar cells (CBCs)) sustain this renewal and reside between terminally differentiated Paneth cells at the bottom of the intestinal crypt. Whereas the signalling requirements for maintaining stem cell function and crypt homeostasis have been well studied, little is known about how metabolism contributes to epithelial homeostasis. Here we show that freshly isolated Lgr5+ CBCs and Paneth cells from the mouse small intestine display different metabolic programs. Compared to Paneth cells, Lgr5+ CBCs display high mitochondrial activity. Inhibition of mitochondrial activity in Lgr5+ CBCs or inhibition of glycolysis in Paneth cells strongly affects stem cell function, as indicated by impaired organoid formation. In addition, Paneth cells support stem cell function by providing lactate to sustain the enhanced mitochondrial oxidative phosphorylation in the Lgr5+ CBCs. Mechanistically, we show that oxidative phosphorylation stimulates p38 MAPK activation by mitochondrial reactive oxygen species signalling, thereby establishing the mature crypt phenotype. Together, our results reveal a critical role for the metabolic identity of Lgr5+ CBCs and Paneth cells in supporting optimal stem cell function, and we identify mitochondria and reactive oxygen species signalling as a driving force of cellular differentiation.

Wnt Signaling Regulates the Lineage Differentiation Potential of Mouse Embryonic Stem Cells through Tcf3 Down-Regulation

Canonical Wnt signaling plays a rate-limiting role in regulating self-renewal and differentiation in mouse embryonic stem cells (ESCs). We have previously shown that mutation in the Apc (adenomatous polyposis coli) tumor suppressor gene constitutively activates Wnt signaling in ESCs and inhibits their capacity to differentiate towards ecto-, meso-, and endodermal lineages. However, the underlying molecular and cellular mechanisms through which Wnt regulates lineage differentiation in mouse ESCs remain to date largely unknown. To this aim, we have derived and studied the gene expression profiles of several Apc-mutant ESC lines encoding for different levels of Wnt signaling activation. We found that down-regulation of Tcf3, a member of the Tcf/Lef family and a key player in the control of self-renewal and pluripotency, represents a specific and primary response to Wnt activation in ESCs. Accordingly, rescuing Tcf3 expression partially restored the neural defects observed in Apc-mutant ESCs, suggesting that Tcf3 down-regulation is a necessary step towards Wnt-mediated suppression of neural differentiation. We found that Tcf3 down-regulation in the context of constitutively active Wnt signaling does not result from promoter DNA methylation but is likely to be caused by a plethora of mechanisms at both the RNA and protein level as shown by the observed decrease in activating histone marks (H3K4me3 and H3-acetylation) and the upregulation of miR-211, a novel Wnt-regulated microRNA that targets Tcf3 and attenuates early neural differentiation in mouse ESCs. Our data show for the first time that Wnt signaling down-regulates Tcf3 expression, possibly at both the transcriptional and post-transcriptional levels, and thus highlight a novel mechanism through which Wnt signaling inhibits neuro-ectodermal lineage differentiation in mouse embryonic stem cells.

Activation of c-MET Induces a Stem-Like Phenotype in Human Prostate Cancer

Prostate cancer consists of secretory cells and a population of immature cells. The function of immature cells and their mutual relation with secretory cells are still poorly understood. Immature cells either have a hierarchical relation to secretory cells (stem cell model) or represent an inducible population emerging upon appropriate stimulation of differentiated cells. Hepatocyte Growth Factor (HGF) receptor c-MET is specifically expressed in immature prostate cells. Our objective is to determine the role of immature cells in prostate cancer by analysis of the HGF/c-MET pathway. Gene-expression profiling of DU145 prostate cancer cells stimulated with HGF revealed induction of a molecular signature associated with stem cells, characterized by up-regulation of CD49b, CD49f, CD44 and SOX9, and down-regulation of CD24 (‘stem-like signature’). We confirmed the acquisition of a stem-like phenotype by quantitative PCR, FACS analysis and Western blotting. Further, HGF led to activation of the stem cell related Notch pathway by up-regulation of its ligands Jagged-1 and Delta-like 4. Small molecules SU11274 and PHA665752 targeting c-MET activity were both able to block the molecular and biologic effects of HGF. Knock-down of c-MET by shRNA infection resulted in significant reduction and delay of orthotopic tumour-formation in male NMRI mice. Immunohistochemical analysis in prostatectomies revealed significant enrichment of c-MET positive cells at the invasive front, and demonstrated co-expression of c-MET with stem-like markers CD49b and CD49f. In conclusion, activation of c-MET in prostate cancer cells induced a stem-like phenotype, indicating a dynamic relation between differentiated and stem-like cells in this malignancy. Its mediation of efficient tumour-formation in vivo and predominant receptor expression at the invasive front implicate that c-MET regulates tumour infiltration in surrounding tissues putatively by acquisition of a stem-like phenotype.

Green Fluorescent Protein variants fold differentially in prokaryotic and eukaryotic cells

Better‐folding Green Fluorescent Protein (GFP) mutants selected from bacterial screenings are commonly used in widely different cellular environments. However, it is unclear if the folding efficiency of GFPs is invariant in different cell types. In this work, we have analysed the folding properties of GFP variants in bacteria versus mammalian cells. Remarkably, S65T was found to fold at comparable levels with the wild type GFP in bacteria, but at 10‐fold lower levels in mammalian cells. On the other hand, Bex1 folded 3–4 times better than the wtGFP or S65T in E. coli, and 10–20‐fold or more than 95‐fold better, respectively, in mammalian cells. The Vex1 mutant demonstrated similar properties to Bex1. No evidence of differential GFP unfolding in vivo or of preferential degradation of unfolded GFP molecules was found. Moreover, no relationship between GFP folding efficiency and expression levels, or protein stability was detected. Trivial Aconfounding factors, like GFP unfolding caused by different pH or fluorescence quenching due to molecular crowding, were also excluded. In summary, our results demonstrate that specific GFP variants follow different folding trajectories in mammalian versus bacterial cells. The specificity of this differential folding supports a role of chaperones in guiding the folding of GFP in vivo. J. Cell. Biochem. Suppl. 36: 117–128, 2001.

Oligomerization of DsRed is required for the generation of a functional red fluorescent chromophore.

The coral red fluorescent protein (DsRed) absorbs and emits light at much higher wavelengths than the structurally homologous green fluorescent protein, raising questions about the properties of its chromophore. We have analyzed the relationship between the aggregation state and fluorescence of native, 6‐histidine‐tagged, or maltose‐binding protein‐fused DsRed. In all cases, newly synthesized DsRed molecules were largely monomeric and devoid of covalently closed chromophores. Maturation in vitro induces the expression of red fluorescent chromophores but only in oligomeric forms of the protein, whereas monomers are essentially devoid of fluorescence. NaOH‐denatured samples demonstrated a generalized breakdown of the DsRed oligomers to monomers, which refolded after neutralization into weakly green fluorescent and still monomeric species. Red fluorescent chromophores were regenerated only upon oligomerization. These findings demonstrate that ‘red’ chromophores form and are functional only as oligomers, and suggest that the smallest red fluorescent functional unit is a dimer. A comparison of alkali‐, acid‐ and guanidinium‐denatured DsRed indicates that stabilization of the DsRed chromophore by concerted steps of folding and oligomerization may play a critical role in its maturation process.

Identification of Quiescent, Stem-Like Cells in the Distal Female Reproductive Tract

In fertile women, the endometrium undergoes regular cycles of tissue build-up and regression. It is likely that uterine stem cells are involved in this remarkable turn over. The main goal of our current investigations was to identify slow-cycling (quiescent) endometrial stem cells by means of a pulse-chase approach to selectively earmark, prospectively isolate, and characterize label-retaining cells (LRCs). To this aim, transgenic mice expressing histone2B-GFP (H2B-GFP) in a Tet-inducible fashion were administered doxycycline (pulse) which was thereafter withdrawn from the drinking water (chase). Over time, dividing cells progressively loose GFP signal whereas infrequently dividing cells retain H2B-GFP expression. We evaluated H2B-GFP retaining cells at different chase time points and identified long-term (LT; >12 weeks) LRCs. The LT-LRCs are negative for estrogen receptor-α and express low levels of progesterone receptors. LRCs sorted by FACS are able to form spheroids capable of self-renewal and differentiation. Upon serum stimulation spheroid cells are induced to differentiate and form glandular structures which express markers of mature Műllerian epithelial cells. Overall, the results indicate that quiescent cells located in the distal oviduct have stem-like properties and can differentiate into distinct cell lineages specific of endometrium, proximal and distal oviduct. Future lineage-tracing studies will elucidate the role played by these cells in homeostasis, tissue injury and cancer of the female reproductive tract in the mouse and eventually in man.

Green fluorescent flowers

The Green Fluorescent Protein (GFP) is efficiently expressed in plant cells and its use as a flower fluorescent dye is appealing. However, the generation of fluorescent flowers has remained as yet elusive. In this work we demonstrate the generation of green-fluorescent flowers. Non-fluorescent GFP-transgenic flowers were analysed by Western blot and spectrofluorimetry, and were demonstrated to efficiently express GFP. This indicated that the lack of GFP fluorescence could be due to opacity to the exciting light. Thus, flowers that are largely transparent to UV, Eustoma grandiflorum (Lisianthus), or that present a transparent petal cuticle, Osteospermum ecklonis, were selected for gfp transformation. Strikingly, the transformed Eustoma flowers fluoresced brightly upon illumination with UV light. GFP-transgenic Osteospermum flowers also appeared distinctly green upon UV illumination. GFP was expressed in comparable amounts by the fluorescent Osteospermum and by the non-fluorescent Limonium petals, confirming that an efficient excitation of GFP fluorescence is a limiting factor in its detection. These results demonstrate the feasibility of using GFP as a fluorescent dye for flower petals. Chromophores excited at longer, more deeply penetrating wavelengths, e.g. DsRed, may extend the use this technology also to flowers opaque to UV light.