Asparagine sustains cellular proliferation and c‑Myc expression in glutamine‑starved cancer cells.

During tumorigenesis, oncogene activation and metabolism rewiring are interconnected. Activated c‑ upregulates several genes involved in glutamine metabolism, making cancer cells dependent on high levels of this amino acid to survive and proliferate. After studying the response to glutamine deprivation in cancer cells, it was found that glutamine starvation not only blocked cellular proliferation, but also altered c‑Myc protein expression, leading to a reduction in the levels of the canonical c‑Myc isoform and an increase in the expression of c‑Myc 1, a c‑Myc isoform translated from an in‑frame 5' CUG codon.

Life style factors, tumor cell plasticity and cancer stem cells.

Cancers are heterogeneous tissues and a layer of heterogeneity is determined by the presence of cells showing stemness traits, known as cancer stem cells (CSCs). Evidence indicates that CSCs are important players in tumor development, progression and relapse. Oncogenic transformation of normal stem cells can give rise to CSCs, but CSCs can also originate from de-differentiation of bulk tumor cells.

Cellular response to glutamine and/or glucose deprivation in in vitro transformed human fibroblasts.

Neoplastic transformation is characterized by metabolic rewiring to sustain the elevated biosynthetic demands of highly proliferative cancer cells. To obtain the precursors for macromolecule biosynthesis, cancer cells avidly uptake and metabolize glucose and glutamine. Thus, targeting the availability or metabolism of these nutrients is an attractive anticancer therapeutic strategy.

Cells with stemness features are generated from in vitro transformed human fibroblasts.

Cancer stem cells (CSCs) have been involved in the maintenance, progression and relapse of several tumors, but their origin is still elusive. Here, in vitro transformed human fibroblasts (cen3tel cells) and the tumorsphere assay were used to search for and possibly characterize CSCs in transformed somatic cells. Cen3tel cells formed spheres showing self-renewal capacity and Sox2 overexpression, suggesting that they contained a subset of cells with CSC-like features.

Snail levels control the migration mechanism of mesenchymal tumor cells.

Cancer cells use two major types of movement: Mesenchymal, which is typical of cells of mesenchymal origin and depends on matrix metalloproteinase (MMP) activity, and amoeboid, which is characteristic of cells with a rounded shape and relies on the activity of Rho-associated kinase (ROCK). The present authors previously demonstrated that, during neoplastic transformation, telomerase-immortalized human fibroblasts (cen3tel cells) acquired a ROCK-dependent/MMP independent mechanism of invasion, mediated by the downregulation of the ROCK cellular inhibitor Round (Rnd)3/RhoE. In the present study, cen3tel transformation was also demonstrated to be paralleled by downregulation of Snail, a major determinant of the mesenchymal movement.

A comprehensive strategy for the analysis of acoustic compressibility and optical deformability on single cells.

We realized an integrated microfluidic chip that allows measuring both optical deformability and acoustic compressibility on single cells, by optical stretching and acoustophoresis experiments respectively. Additionally, we propose a measurement protocol that allows evaluating the experimental apparatus parameters before performing the cell-characterization experiments, including a non-destructive method to characterize the optical force distribution inside the microchannel. The chip was used to study important cell-mechanics parameters in two human breast cancer cell lines, MCF7 and MDA-MB231.

Telomere and telomerase stability in human diseases and cancer.

Telomeres are the nucleoprotein structures at the end of linear eukaryotic chromosomes required for genome stability. Telomerase is the specialized enzyme deputed to their elongation. Maintenance of a proper telomere structure, an accurate regulation of telomerase biogenesis and activity, as well as a correct telomere-telomerase interaction and a faithful telomeric DNA replication are all processes that a cell has to precisely control to safeguard its functionality.

Super-telomeres in transformed human fibroblasts.

Telomere length maintenance is critical for organisms' long-term survival and cancer cell proliferation. Telomeres are kept within species-specific length ranges by the interplay between telomerase activity and telomeric chromatin organization. In this paper, we exploited telomerase immortalized human fibroblasts (cen3tel) that gradually underwent neoplastic transformation during culture propagation to study telomere composition and length regulation during the transformation process.

Telomere-independent functions of telomerase in nuclei, cytoplasm, and mitochondria.

Telomerase canonical activity at telomeres prevents telomere shortening, allowing chromosome stability and cellular proliferation. To perform this task, the catalytic subunit (telomerase reverse transcriptase, TERT) of the enzyme works as a reverse transcriptase together with the telomerase RNA component (TERC), adding telomeric repeats to DNA molecule ends. Growing evidence indicates that, besides the telomeric-DNA synthesis activity, TERT has additional functions in tumor development and is involved in many different biological processes, among which cellular proliferation, gene expression regulation, and mitochondrial functionality.

Poly(ADP-ribosylation) and neoplastic transformation: effect of PARP inhibitors.

Poly(ADP-ribose) polymerases (PARPs) and poly(ADP-ribosylation) play essential roles in several biological processes, among which neoplastic transformation and telomere maintenance. In this paper, we review the poly(ADP-ribosylation) process together with the highly appealing use of PARP inhibitors for the treatment of cancer. In addition, we report our results concerning poly(ADP-ribosylation) in a cellular model system for neoplastic transformation developed in our laboratory.

Cross-analysis of gene and miRNA genome-wide expression profiles in human fibroblasts at different stages of transformation.

We have developed a cellular system constituted of human telomerase immortalized fibroblasts that gradually underwent neoplastic transformation during propagation in culture. We exploited this cellular system to investigate gene and miRNA transcriptional programs in cells at different stages of propagation, representing five different phases along the road to transformation, from non-transformed cells up to tumorigenic and metastatic ones. Here we show that gene and miRNA expression profiles were both able to divide cells according to their transformation phase.

Relocalization of cell adhesion molecules during neoplastic transformation of human fibroblasts.

Studying neoplastic transformation of telomerase immortalized human fibroblasts (cen3tel), we found that the transition from normal to tumorigenic cells was associated with the loss of growth contact inhibition, the acquisition of an epithelial-like morphology and a change in actin organization, from stress fibers to cortical bundles. We show here that these variations were paralleled by an increase in N-cadherin expression and relocalization of different adhesion molecules, such as N-cadherin, α-catenin, p-120 and β-catenin. These proteins presented a clear membrane localization in tumorigenic cells compared to a more diffuse, cytoplasmic distribution in primary fibroblasts and non-tumorigenic immortalized cells, suggesting that tumorigenic cells could form strong cell-cell contacts and cell contacts did not induce growth inhibition.

Reduced expression of the ROCK inhibitor Rnd3 is associated with increased invasiveness and metastatic potential in mesenchymal tumor cells.

Background: Mesenchymal and amoeboid movements are two important mechanisms adopted by cancer cells to invade the surrounding environment. Mesenchymal movement depends on extracellular matrix protease activity, amoeboid movement on the RhoA-dependent kinase ROCK. Cancer cells can switch from one mechanism to the other in response to different stimuli, limiting the efficacy of antimetastatic therapies.

Drug treatment of cancer cell lines: a way to select for cancer stem cells?

Tumors are generally composed of different cell types. In recent years, it has been shown that in many types of cancers a subset of cells show peculiar characteristics, such as the ability to induce tumors when engrafted into host animals, self-renew and being immortal, and give rise to a differentiated progeny. These cells have been defined as cancer stem cells (CSCs) or tumor initiating cells.

Transcription of Satellite III non-coding RNAs is a general stress response in human cells.

In heat-shocked human cells, heat shock factor 1 activates transcription of tandem arrays of repetitive Satellite III (SatIII) DNA in pericentromeric heterochromatin. Satellite III RNAs remain associated with sites of transcription in nuclear stress bodies (nSBs). Here we use real-time RT-PCR to study the expression of these genomic regions.

Structural and functional characterization of noncoding repetitive RNAs transcribed in stressed human cells.

Thermal and chemical stresses induce the formation in human cells of novel and transient nuclear structures called nuclear stress bodies (nSBs). These contain heat shock factor 1 (HSF-1) and a specific subset of pre-mRNA processing factors. Nuclear stress bodies are assembled on specific pericentromeric heterochromatic domains containing satellite III (SatIII) DNA.

RNA recognition motif 2 directs the recruitment of SF2/ASF to nuclear stress bodies.

Heat shock induces the transcriptional activation of large heterochromatic regions of the human genome composed of arrays of satellite III DNA repeats. A number of RNA-processing factors, among them splicing factor SF2/ASF, associate with these transcription factors giving rise to nuclear stress bodies (nSBs). Here, we show that the recruitment of SF2/ASF to these structures is mediated by its second RNA recognition motif.

Transcriptional activation of a constitutive heterochromatic domain of the human genome in response to heat shock.

Heat shock triggers the assembly of nuclear stress bodies that contain heat shock factor 1 and a subset of RNA processing factors. These structures are formed on the pericentromeric heterochromatic regions of specific human chromosomes, among which chromosome 9. In this article we show that these heterochromatic domains are characterized by an epigenetic status typical of euchromatic regions.