Claudia Giachino

Growth stimulation of colorectal carcinoma cells via the c‐kit receptor is inhibited by TGF‐β1

Activation of the receptor tyrosine kinase c‐kit by the kit‐ligand, also known as stem cell factor (SCF), is essential to melanocyte and germ cell development and during the early stages of hematopoiesis. Deregulated expression of c‐kit has been reported in malignancies affecting these lineages, i.e., myeloid leukemias, melanomas, and germ cell tumors. In addition, c‐kit and SCF are coexpressed in some breast and colorectal cancer (CRC) cells, raising the question of whether c‐kit serves an autocrine role in normal or malignant epithelial tissues. In this study, we demonstrate that human colorectal carcinomas, but not normal colorectal mucosa cells, coexpress SCF and c‐kit in situ. Expression of c‐kit was also observed in mucosa adjacent to colorectal tumor tissue. Consistent with a growth‐regulatory role of SCF in CRC cells, exogenous SCF stimulated anchorage‐dependent and anchorage‐independent growth in four out of five CRC cell lines. Exogenous transforming growth factor (TGF)‐β1 added at nanomolar concentrations to HT‐29 CRC cells, which express the type I, II, and III TGF‐β receptors, downregulated c‐kit expression to background levels and inhibited c‐kit–dependent proliferation. Similarly, TGF‐β1 inhibited SCF‐dependent proliferation of three first‐passage CRC cell lines. In summary, expression of the potential autocrine SCF/c‐kit axis is a tumor‐associated phenomenon in colorectal cancer that can be suppressed by TGF‐β1 in TGF‐β–responsive CRC cells. J. Cell. Physiol. 172:1–11, 1997.

Multiple facets of histone variant H2AX: a DNA double-strand-break marker with several biological functions

In the last decade, many papers highlighted that the histone variant H2AX and its phosphorylation on Ser 139 (γH2AX) cannot be simply considered a specific DNA double-strand-break (DSB) marker with a role restricted to the DNA damage response, but rather as a ‘protagonist’ in different scenarios. This review will present and discuss an up-to-date view regarding the ‘non-canonical’ H2AX roles, focusing in particular on possible functional and structural parts in contexts different from the canonical DNA DSB response. We will present aspects concerning sex chromosome inactivation in male germ cells, X inactivation in female somatic cells and mitosis, but will also focus on the more recent studies regarding embryonic and neural stem cell development, asymmetric sister chromosome segregation in stem cells and cellular senescence maintenance. We will discuss whether in these new contexts there might be a relation with the canonical DNA DSB signalling function that could justify γH2AX formation. The authors will emphasize that, just as H2AX phosphorylation signals chromatin alteration and serves the canonical function of recruiting DSB repair factors, so the modification of H2AX in contexts other than the DNA damage response may contribute towards creating a specific chromatin structure frame allowing ‘non-canonical’ functions to be carried out in different cell types.

A rapid flow cytometry test based on histone H2AX phosphorylation for the sensitive and specific diagnosis of ataxia telangiectasia

Ataxia telangiectasia (A‐T) is a progressive neurodegenerative disease with onset in early childhood, caused by mutations in the ATM (ataxia‐telangiectasia mutated) gene. Diagnosis relies on laboratory tests showing high levels of serum alphafetoprotein, cell sensitivity to ionizing radiation (IR) and absence or reduced levels of ATM protein. Many tests, however, are not sufficiently sensitive or specific for A‐T, have long turnaround times, or require large blood samples. This prompted us to develop a new flow cytometry method for the diagnosis of A‐T based on the measurement of histone H2AX phosphorylation. We established normal ranges of histone H2AX phosphorylation after 2 Gy IR by testing T‐cell lines, lymphoblastoid cell lines (LCLs) and/or peripheral blood mononuclear cells (PBMCs) or both from 20 genetically proven A‐T and 46 control donors. To further evaluate the specificity and sensitivity of the test, we analyzed cells from 19 patients suspected of having A‐T, and from one Friedreich Ataxia, one Ataxia with Oculomotor Apraxia type 2, and one Nijmegen Breakage Syndrome patients. Phosphorylated histone H2AX mean fluorescence intensity of irradiated A‐T cells was significantly lower than that of healthy donors. The intrastaining, intraassay, and interassay imprecisions were ≤13.22%. Sensitivity and specificity were virtually 100% when the test was performed on PBMCs. Screening of 19 consecutive new patients with suspected A‐T classified 15 patients as non‐A‐T and four as A‐T; diagnosis of the latter four was subsequently confirmed by DNA sequencing to identify ATM mutations. The Friedreich Ataxia patient, the Ataxia with Oculomotor Apraxia type 2 patient and the Nijmegen Breakage Syndrome patient were classified as non‐A‐T. This flow cytometry test is very sensitive, specific and rapid, and requires only 2 ml of blood. It may thus be proposed for the early differential diagnosis of A‐T as an alternative to methods requiring the production of LCLs.

Senescence in Human Mesenchymal Stem Cells: Functional Changes and Implications in Stem Cell-Based Therapy

Regenerative medicine is extensively interested in developing cell therapies using mesenchymal stem cells (MSCs), with applications to several aging-associated diseases. For successful therapies, a substantial number of cells are needed, requiring extensive ex vivo cell expansion. However, MSC proliferation is limited and it is quite likely that long-term culture evokes continuous changes in MSCs. Therefore, a substantial proportion of cells may undergo senescence. In the present review, we will first present the phenotypic characterization of senescent human MSCs (hMSCs) and their possible consequent functional alterations. The accumulation of oxidative stress and dysregulation of key differentiation regulatory factors determine decreased differentiation potential of senescent hMSCs. Senescent hMSCs also show a marked impairment in their migratory and homing ability. Finally, many factors present in the secretome of senescent hMSCs are able to exacerbate the inflammatory response at a systemic level, decreasing the immune modulation activity of hMSCs and promoting either proliferation or migration of cancer cells. Considering the deleterious effects that these changes could evoke, it would appear of primary importance to monitor the occurrence of senescent phenotype in clinically expanded hMSCs and to evaluate possible ways to prevent in vitro MSC senescence. An updated critical presentation of the possible strategies for in vitro senescence monitoring and prevention constitutes the second part of this review. Understanding the mechanisms that drive toward hMSC growth arrest and evaluating how to counteract these for preserving a functional stem cell pool is of fundamental importance for the development of efficient cell-based therapeutic approaches.

Re-expression of RAG-1 and RAG-2 genes and evidence for secondary rearrangements in human germinal center B lymphocytes

V(D)J recombination occurs in immature B cells within primary lymphoid organs. However, recent evidence demonstrated that the recombination activating genes RAG‐1 and RAG‐2 can also be expressed in murine germinal centers (GC) where they can mediate secondary rearrangements. This finding raises a number of interesting questions, the most important of which is what is the physiological role, if any, of secondary immunoglobulin (Ig) gene rearrangements. In the present report, we provide evidence that human GC B cells that have lost surface immunoglobulin re‐express RAG‐1 and RAG‐2, suggesting that they may be able to undergo Ig rearrangement. Furthermore, we describe two mature B cell clones in which secondary rearrangements have possibly occurred, resulting in light chain replacement. The two clones carry both κ and λ light chains productively rearranged, but fail to express the κ chain on the cell surface due to a stop codon acquired by somatic mutation. Interestingly, the analysis of the extent of somatic mutations accumulated by the two light chains might suggest that the λ chain could have been acquired through a secondary rearrangement. Taken together, these data suggest that secondary Ig gene rearrangements leading to replacement may occur in human GC and may contribute to the peripheral B cell repertoire.

A New Paradigm in Cardiac Regeneration: The Mesenchymal Stem Cell Secretome

The potentialities to apply mesenchymal stem cells (MSCs) in regenerative medicine have been extensively studied over the last decades. In the cardiovascular disease (CVD) field, MSCs-based therapy is the subject of great expectations. Its therapeutic potential has been already shown in several preclinical models and both the safety and efficacy of MSCs-based therapy are being evaluated in humans. It is now clear that the predominant mechanism by which MSCs participate in heart tissue repair is through a paracrine activity. Via the production of a multitude of trophic factors endowed with different properties, MSCs can reduce tissue injury, protect tissue from further adverse effects, and enhance tissue repair. The present review discusses the current understanding of the MSCs secretome as a therapy for treatment of CVD. We provide insights into the possible employment of the MSCs secretome and their released extracellular vesicles as novel approaches for cardiac regeneration that would have certain advantages over injection of living cells.

High Basal γH2AX Levels Sustain Self‐Renewal of Mouse Embryonic and Induced Pluripotent Stem Cells

Phosphorylation of histone H2AX (γH2AX) is known to be the earliest indicator of DNA double‐strand breaks. Recently, it has been shown that mouse embryonic stem cells (mESCs) have very high basal levels of γH2AX, even when they have not been exposed to genotoxic agents. As the specialized role of high basal γH2AX levels in pluripotent stem cells is still debated, we investigated whether H2AX phosphorylation is important in maintaining self‐renewal of these cells. Here, we report that not only mESCs but also mouse‐induced pluripotent stem cells (miPSCs), have high basal levels of γH2AX. We show that basal γH2AX levels decrease upon ESC and iPSC differentiation and increase when the cells are treated with self‐renewal‐enhancing small molecules. We observe that self‐renewal activity is highly compromised in H2AX−/− cells and that it can be restored in these cells through reconstitution with a wild‐type, but not a phospho‐mutated, H2AX construct. Taken together, our findings suggest a novel function of H2AX that expands the knowledge of this histone variant beyond its role in DNA damage and into a new specialized biological function in mouse pluripotent stem cells. STEM CELLS2012;30:1414–1423

Diverse expansion potential and heterogeneous avidity in tumor-associated antigen-specific T lymphocytes from primary melanoma patients.

While tumor‐associated antigen (TAA)‐specific CD8+ T lymphocytes have been detected in metastatic melanoma patients, immune response in early disease phases has not yet been carefully evaluated. We looked for circulating cytotoxic T lymphocytes (CTL) directed against Melan‐A / MART1, tyrosinase, gp100 and MAGE‐3 antigens in patients with a diagnosis of primary cutaneous melanoma by using fluorescent HLA‐A2 tetramers. In five out of six cases high numbers of CD8+ / tetramer+ cells could be detected by flow cytometry, and in four patients lymphocyte populations specific for two different melanoma antigens (Melan‐A / MART1 and tyrosinase) were contemporarily present. The TAA‐specific cells could represent as much as 1 / 220 T lymphocytes in the circulating CD8+ population. When tetramers were used to monitor the in vitro expansion of TAA‐specific CTL precursors upon antigen‐specific stimulation, a diverse expansion potential was evidenced in CTL from the different donors and, more strikingly, in CTL specific for the different TAA. Melan‐A / MART1‐specific CTL clones derived from two patients exhibited a broad range of avidity. Only the highest avidity clones, representing about 50 % of the cases analyzed, were tumor specific. By correlating tetramer staining with clone avidity, we found that tetramer fluorescence intensity could represent a good indicator of TCR affinity, but not of overall clone avidity.

Cutting Edge: Recombinase-Activating Gene Expression and V(D)J Recombination in CD4+CD3low Mature T Lymphocytes

The recombinase-activating genes, RAG-1 and RAG-2, can be expressed by a subset of B cells within germinal centers, where they mediate secondary V(D)J rearrangements. This receptor revision mechanism could serve either receptor diversification or tolerance-induced functions. Alternatively, it might rescue those cells the receptors of which have been damaged by somatic mutation. Less is known about the occurrence of similar mechanisms in T cells. Here we show that mature T cells with defective TCR surface expression can express RAG genes and are capable of initiating secondary V(D)J rearrangements. The possibility that a cell rescue mechanism based on the generation of a novel Ag receptor might be active in peripheral T cells is envisaged.

Dominant TCR-α Requirements for a Self Antigen Recognition in Humans

TCR-α and -β chains are composed of somatically rearranged V, D, and J germline-encoded gene segments that confer Ag specificity. Recent crystallographic analyses revealed that TCR-α has more contacts with peptide than TCR-β, suggesting the possibility that peptide recognition predominantly relies on TCR-α. T cells specific for the self Ag Melan-A/MART-1 possess an exceptionally high precursor frequency in human histocompatibility leukocyte Ag-A2 individuals. This provided a unique situation for assessment of the structural relationship between TCR and peptide/MHC ligand at both the pre- and postimmune levels. Molecular and phenotypic analysis of many different Melan-A-specific T cell populations revealed that a structural constraint is imposed on the TCR for engagement with Melan-A peptides presented by HLA-A2, namely the highly preferential use of a particular TCRAV segment, AV2. Examination of CD8 single-positive thymocytes indicated that this preferential use in forming the Melan-A-specific TCR is mainly imposed by intrathymic positive selection. Our data demonstrate a dominant function of TCRAV2 segment in forming the TCR repertoire specific for the human self Ag Melan-A/MART-1 and support the view that Ag recognition is mediated predominantly by TCR-α.