Ofer Shoshani

Chronic centrosome amplification without tumorigenesis

Significance Centrosomes organize the microtubule cytoskeleton in interphase and mitosis. During mitosis, the centrosomes are important for the formation and positioning of the bipolar mitotic spindle on which chromosomes are segregated. The presence of more than two centrosomes can drive mitotic chromosome segregation errors and the formation of aneuploid cells. Centrosome amplification is a common feature of aneuploid cancer cells, but a long-standing question is whether this is a cause or a consequence of tumor development. To assess this question, we generated mice in which centrosome amplification can be induced widely. Despite chronic centrosome amplification, tumorigenesis was not enhanced, demonstrating that an excess of centrosomes is not sufficient to drive tumor development. Centrosomes are microtubule-organizing centers that facilitate bipolar mitotic spindle assembly and chromosome segregation. Recognizing that centrosome amplification is a common feature of aneuploid cancer cells, we tested whether supernumerary centrosomes are sufficient to drive tumor development. To do this, we constructed and analyzed mice in which centrosome amplification can be induced by a Cre-recombinase–mediated increase in expression of Polo-like kinase 4 (Plk4). Elevated Plk4 in mouse fibroblasts produced supernumerary centrosomes and enhanced the expected mitotic errors, but proliferation continued only after inactivation of the p53 tumor suppressor. Increasing Plk4 levels in mice with functional p53 produced centrosome amplification in liver and skin, but this did not promote spontaneous tumor development in these tissues or enhance the growth of chemically induced skin tumors. In the absence of p53, Plk4 overexpression generated widespread centrosome amplification, but did not drive additional tumors or affect development of the fatal thymic lymphomas that arise in animals lacking p53. We conclude that, independent of p53 status, supernumerary centrosomes are not sufficient to drive tumor formation.

Stress as a fundamental theme in cell plasticity.

Over a decade of intensive investigation of the possible plasticity of mammalian cells has eventually substantiated that mammalian species are endowed with a remarkable capacity to change mature cell fates. We review below the evidence for the occurrence of processes such as dedifferentiation and transdifferentiation within mammalian tissues in vivo, and in cells removed from their protective microenvironment and seeded in culture under conditions poorly resembling their physiological state in situ. Overall, these studies point to one major conclusion: stressful conditions, whether due to in vivo tissue damage or otherwise to isolation of cells from their in vivo restrictive niches, lead to extreme fate changes. Some examples of dedifferentiation are discussed in detail showing that rare cells within the population tend to turn back into less mature ones due to severe cell damage. It is proposed that cell stress, mechanistically sensed by isolation from neighboring cells, leads to dedifferentiation, in an attempt to build a new stem cell reservoir for subsequent regeneration of the damaged tissue. This article is part of a Special Issue entitled: Stress as a fundamental theme in cell plasticity.

Polyploidization of Murine Mesenchymal Cells Is Associated with Suppression of the Long Noncoding RNA H19 and Reduced Tumorigenicity

Mesenchymal stromal cells (MSC) are used extensively in clinical trials; however, the possibility that MSCs have a potential for malignant transformation was raised. We examined the genomic stability versus the tumor-forming capacity of multiple mouse MSCs. Murine MSCs have been shown to be less stable and more prone to malignant transformation than their human counterparts. A large series of independently isolated MSC populations exhibited low tumorigenic potential under syngeneic conditions, which increased in immunocompromised animals. Unexpectedly, higher ploidy correlated with reduced tumor-forming capacity. Furthermore, in both cultured MSCs and primary hepatocytes, polyploidization was associated with a dramatic decrease in the expression of the long noncoding RNA H19. Direct knockdown of H19 expression in diploid cells resulted in acquisition of polyploid cell traits. Moreover, artificial tetraploidization of diploid cancer cells led to a reduction of H19 levels, as well as to an attenuation of the tumorigenic potential. Polyploidy might therefore serve as a protective mechanism aimed at reducing malignant transformation through the involvement of the H19 regulatory long noncoding RNA.

Mammalian Cell Dedifferentiation as a Possible Outcome of Stress

Differentiation cascades are arranged hierarchically; stem cells positioned at the top of the hierarchy generate committed progenitors that, in turn, proliferate and further differentiate stepwise into mature progeny. This rigid, irreversible structure ensures the phenotypic stability of adult tissues. However, such rigidity may be problematic under conditions of tissue damage when reconstitution is required. Although it may seem unlikely that the restrictions on changes in cell phenotypes would be lifted to enable tissue reconstitution, it is nevertheless possible that mammalian tissues are endowed with sufficient flexibility to enable their adaptation to extreme conditions.

Cell Isolation Induces Fate Changes of Bone Marrow Mesenchymal Cells Leading to Loss or Alternatively to Acquisition of New Differentiation Potentials

Mesenchymal stromal cell populations include a fraction, termed mesenchymal stem cells, exhibiting multipotency. Other cells within this population possess a lesser differentiation range. This was assumed to be due to a mesenchymal cellular cascade topped by a multipotent cell, which gives rise to progeny with diminishing differentiation potentials. Here, we show that mesenchymal cells, a priori exhibiting a limited differentiation potential, may gain new capacities and become multipotent following single‐cell isolation. These fate changes were accompanied by upregulation of differentiation promoting genes, many of which also became H4K20me1 methylated. Early events in the process included TGFβ and Wnt modulation, and downregulation of hypoxia signaling. Indeed, hypoxic conditions inhibited the observed cell changes. Overall, cell isolation from neighboring partners caused major molecular changes and particularly, a newly established epigenetic state, ultimately leading to the acquisition of new differentiation potentials and an altered cell fate. Stem Cells 2014;32:2008–2020

Relative genomic stability of adipose tissue derived mesenchymal stem cells: analysis of ploidy, H19 long non-coding RNA and p53 activity.

IntroductionMesenchymal stem cells (MSCs) are multipotent and have been derived from various tissues. Although MSCs share many basic features, they often display subtle tissue specific differences. We previously demonstrated that bone marrow (BM) MSCs frequently become polyploid in culture. This tendency was mediated by a reduction in the expression of H19 long non-coding RNA during the transition from a diploid to a polyploid state.MethodsMSCs were derived from both BM and adipose tissue of mice and expanded under normoxic and hypoxic culture conditions. Cells were stained by propidium iodide and their ploidy was evaluated by FACS. Gene expression of independent MSC preparations was compared by quantitative real time PCR and protein expression levels by Western blot analysis. p53 silencing in MSCs was performed by a specific small hairpin RNA (shRNA).ResultsWe set to examine whether genomic instability is common to MSCs originating from different tissues. It is demonstrated that adipose derived MSCs (ASCs) tend to remain diploid during culture while a vast majority of BM MSCs become polyploid. The diploid phenotype of ASCs is correlated with reduced H19 expression compared to BM MSCs. Under hypoxic conditions (3% oxygen) both ASCs and BM MSCs demonstrate increased RNA expression of H19 and Vascular endothelial growth factor A. Importantly, ASC gene expression is significantly less variable than BM MSCs under both oxygen conditions, indicating to their superior homogeneity. Gene expression analysis revealed that p53 target genes, often induced by DNA damage, are up-regulated in ASCs under basal conditions. However, p53 activation following treatment with DNA damaging agents was strongly elevated in BM MSCs compared to ASCs. We found that p53 is involved in maintaining the stable diploid state of ASCs as p53 shRNA induced ploidy changes in ASCs but not in BM MSCs.ConclusionsThe increased genomic stability of murine ASCs together with their lower H19 expression and relative homogeneity suggest a tissue specific higher stability of ASCs compared to BM MSCs, possibly due to higher activity of p53. The tissue specific differences between MSCs from a different tissue source may have important consequences on the use of various MSCs both in vitro and in vivo.

Transition of Endothelium to Cartilage and Bone

Mesenchymal stromal cells (MSCs) are capable of differentiating into bone-forming osteoblasts. A recent Nature Medicine study (Medici et al., 2010) shows that the mislocalized bone in the human disease fibrodisplasia ossificans progressiva (FOP) originates from vascular endothelium that gives rise to MSCs.

DNA replication-mediated error correction of ectopic CENP-A deposition maintains centromere identity

Chromatin assembled with the histone H3 variant CENP-A is the heritable epigenetic determinant of human centromere identity. Using genome-wide mapping and reference models for 23 human centromeres, CENP-A is shown in early G1 to be assembled into nucleosomes within megabase, repetitive α-satellite DNAs at each centromere and onto 11,390 transcriptionally active sites on the chromosome arms. Here we identify that DNA replication acts as an error correction mechanism to sustain centromere identity through the removal of the sites of CENP-A loading on the chromosome arms, while maintaining centromere-bound CENP-A with the same DNA sequence preferences as in its initial loading.

The Role of Mesenchymal Cells in Cancer: Contribution to Tumor Stroma and Tumorigenic Capacity

Mesenchymal stromal cells were first isolated from the bone marrow, where they serve as a component of the tissue microenvironment. These cells provide a physical support for the other cells of the tissue; i.e., the hemopoietic cell lineage, and further participate in the formation of bone structures. Most importantly, stromal cells regulate the growth and differentiation of hemopoietic stem cells. The mesenchyme is not specific to the bone marrow: such cells are found body-wide, and serve similar regulatory functions. By the same token, the mesenchymal stroma contributes to tumor formation by providing regulatory signals. In addition, the stromal cells themselves may undergo transformation, and subsequently form tumors. This chapter discusses these two major aspects of stromal cell involvement in the tumorigenic process.

Potent in vitro and in vivo effects of polyclonal anti-human-myeloma globulins

Introduction Multiple myeloma is still incurable in most cases. Polyclonal anti T lymphocyte globulins (ATG) have been reported to kill human myeloma cells in vitro and in mouse models. Methods Anti-human-myeloma globulins (AMG) were produced by immunizing rabbits with human myeloma cell lines RPMI-8226 (AMG-8226) or KMS-12-BM (AMG-12-BM). Cytotoxicity of the polyclonal antibodies was analyzed in vitro and in a xenograft NOD-SCID mouse model. Results Both AMG had stronger cytotoxicity against myeloma cells compared to ATG. In primary T cells, AMG-8226 showed greater complement-dependent cytotoxicity (CDC) than ATG, whereas complement-independent cytotoxicity did not differ. Effects on non-hematopoietic cell lines were also similar. Competitive blocking assays revealed fourfold more antibodies against CD38 in AMG-8226 compared to ATG. Low concentrations of AMG-8226 and ATG increased ADCC. At higher concentrations, ATG inhibited ADCC more potently than AMG-8226. Combinations of ATG and AMG-8226 with melphalan or bortezomib showed additive to synergistic cytotoxicity on myeloma cells. The cytotoxic effects of AMG and ATG were confirmed in the xenograft NOD-SCID mouse model. Conclusion Our data show more potent antimyeloma effects of AMG compared to ATG. These results lay the ground for the development of polyclonal antibodies for the treatment of multiple myeloma.