Toivo Maimets

Challenges with advanced therapy medicinal products and how to meet them

Advanced therapy medicinal products (ATMPs), which include gene therapy medicinal products, somatic cell therapy medicinal products and tissue-engineered products, are at the cutting edge of innovation and offer a major hope for various diseases for which there are limited or no therapeutic options. They have therefore been subject to considerable interest and debate. Following the European regulation on ATMPs, a consolidated regulatory framework for these innovative medicines has recently been established. Central to this framework is the Committee for Advanced Therapies (CAT) at the European Medicines Agency (EMA), comprising a multidisciplinary scientific expert committee, representing all EU member states and European Free Trade Association countries, as well as patient and medical associations. In this article, the CAT discusses some of the typical issues raised by developers of ATMPs, and highlights the opportunities for such companies and research groups to approach the EMA and the CAT as a regulatory advisor during development.

Activation of p53 by nutlin leads to rapid differentiation of human embryonic stem cells.

p53 is an important regulator of normal cell response to stress and frequently mutated in human tumours. Here, we studied the effects of activation of p53 and its target gene p21 in human embryonic stem cells. We show that activation of p53 with small-molecule activator nutlin leads to rapid differentiation of stem cells evidenced by changes in cell morphology and adhesion, expression of cell-specific markers for primitive endoderm and trophectoderm lineages and loss of pluripotency markers. p21 is quickly and dose-dependently activated by nutlin. It can also be activated independently from p53 by sodium butyrate, which leads to the differentiation events very similar to the ones induced by p53. During differentiation, the activating phosphorylation site of CDK2 Thr-160 becomes dephosphorylated and cyclins A and E become degraded. The target for CDK2 kinase in p53 molecule, Ser-315, also becomes dephosphorylated. We conclude that the main mechanism responsible for differentiation of human stem cells by p53 is abolition of S-phase entry and subsequent stop of cell cycle in G0/G1 phase accompanied by p21 activation.

Characterization of a novel cytotoxic cell-penetrating peptide derived from p14ARF protein.

The tumor suppressor p14ARF is widely deregulated in many types of cancers and is believed to function as a failsafe mechanism, inhibiting proliferation and inducing apoptosis as cellular response to a high oncogene load. We have found that a 22-amino-acid-long peptide derived from the N-terminal part of p14ARF, denoted ARF(1-22), which has previously been shown to mimic the function of p14ARF, has cell-penetrating properties. This peptide is internalized to the same extent as the cell-penetrating peptide (CPP) TP10 and dose-dependently decreases proliferation in MCF-7 and MDA MB 231 cells. Uptake of the ARF(1-22) peptide is associated with low membrane disturbance, measured by deoxyglucose and lactate dehydrogenase (LDH) leakage, as compared to its scrambled peptide. Also, flow cytometric analysis of annexin V/propidium iodide (PI) binding and Hoechst staining of nuclei suggest that ARF(1-22) induces apoptosis, whereas scrambled or inverted peptide sequences have no effect. The ARF(1-22) peptide mainly translocates cells through endocytosis, and is found intact inside cells for at least 3 hours. To our knowledge, this is the first time a CPP having pro-apoptopic activity has been designed from a protein.

Oligomerization of p53 is necessary to inhibit its transcriptional transactivation property at high protein concentration

We have previously shown that transactivation by tumor suppressor protein p53 can be inhibited in vivo at elevated protein concentrations. In this study we characterize the structural requirements of this function. We show that oligomerization domain of p53 is involved in loss of transactivation at high protein concentrations: mutants not able to oligomerize are neither able to suppress transactivation, although these transactivating properties can be untouched.

p53-dependent transcription can exhibit both on/off and graded response after genotoxic stress

The p53 protein is a central player in cellular response to DNA damage. Induction of p53 by DNA-damaging agents involves elevation of its steady-state level and activation of its potency as a transcription factor. In the cell population, these responses can occur either homogeneously (where every single cell responds simultaneously and similarly to its neighbor) or heterogeneously (where only some cells of a population respond and the number of these increases with increasing dose of inducer). We have studied here the p53 response to DNA-damaging agents (camptothecin, mitomycin C) in individual cells. We show that the level of p53 protein is increased in every single cell of the population homogeneously, while the p53-dependent transcription can be subject to an on/off-type response. Depending on the structure of the target promoter, p53-dependent transcription can be regulated according to the binary or graded model. The on/off-type transcriptional activation pattern of p53 defines two distinct subpopulations of cells after DNA damage.

Nocodazole Treatment Decreases Expression of Pluripotency Markers Nanog and Oct4 in Human Embryonic Stem Cells

Nocodazole is a known destabiliser of microtubule dynamics and arrests cell-cycle at the G2/M phase. In the context of the human embryonic stem cell (hESC) it is important to understand how this arrest influences the pluripotency of cells. Here we report for the first time the changes in the expression of transcription markers Nanog and Oct4 as well as SSEA-3 and SSEA-4 in human embryonic cells after their treatment with nocodazole. Multivariate permeabilised-cell flow cytometry was applied for characterising the expression of Nanog and Oct4 during different cell cycle phases. Among untreated hESC we detected Nanog-expressing cells, which also expressed Oct4, SSEA-3 and SSEA-4. We also found another population expressing SSEA-4, but without Nanog, Oct4 and SSEA-3 expression. Nocodazole treatment resulted in a decrease of cell population positive for all four markers Nanog, Oct4, SSEA-3, SSEA-4. Nocodazole-mediated cell-cycle arrest was accompanied by higher rate of apoptosis and upregulation of p53. Twenty-four hours after the release from nocodazole block, the cell cycle of hESC normalised, but no increase in the expression of transcription markers Nanog and Oct4 was detected. In addition, the presence of ROCK-2 inhibitor Y-27632 in the medium had no effect on increasing the expression of pluripotency markers Nanog and Oct4 or decreasing apoptosis or the level of p53. The expression of SSEA-3 and SSEA-4 increased in Nanog-positive cells after wash-out of nocodazole in the presence and in the absence of Y-27632. Our data show that in hESC nocodazole reversible blocks cell cycle, which is accompanied by irreversible loss of expression of pluripotency markers Nanog and Oct4.

Electroporation and carrier DNA cause p53 activation, cell cycle arrest, and apoptosis.

Methods used in transient transfection of cells may alter cellular signaling pathways that in turn may lead to misinterpretation of the results. A variety of genotoxic agents cause the accumulation of the p53 protein leading to either apoptosis or growth arrest. Here we report the effect of electroporation and carrier DNA on the stability, cellular localization, and transcriptional activity of p53. We show that electroporation leads to p53-dependent and also p53-independent cell-cycle arrest and apoptosis. At the same time a chemical agent polyethylenimine that is also used for transient transfection of cells causes neither upregulation of p53 nor cellular response.

p53 protein accumulation in addition to the transactivation activity is required for p53-dependent cell cycle arrest after treatment of cells with camptothecin.

In this study we characterize the connections between p53-dependent G1 cell cycle arrest, transcriptional activation of the protein and the increase of its intracellular steady-state concentration. Several cell lines expressing wild-type p53 protein were treated with increasing concentrations of DNA-damaging drug camptothecin. Lower doses of the drug caused transcriptional activation of p53, but no accumulation of the protein was detected. Only after a certain threshold dose of camptothecin does the amount of the protein rapidly increase and reach its plateau levels. The threshold dose was different for different cell lines, but the general non-linear profile was similar. Increase of p53 level was accompanied by additional transcriptional activation of some p53 target genes (i.e. waf1), but not the others (mdm2). We demonstrate here that transcriptional activation of p53 after the treatment of camptothecin is not sufficient to cause p53-dependent G1 cell cycle arrest. The latter is observable only after the inrease of steady-state level of p53. Low drug concentrations, although accompanied by transcriptional activation of p53, do not cause either p53 protein accumulation nor cell cycle arrest at G1. We propose a model for p53 acting as a part of cellular sensor system detecting the severity of DNA damage.

Overexpression of leukocyte marker CD43 causes activation of the tumor suppressor proteins p53 and ARF.

CD43 or leukosialin is a transmembrane sialoglycoprotein, whose extracellular domain participates in cell adhesiveness and the cytoplasmic tail regulates a variety of intracellular signal transduction pathways involved in cell proliferation. CD43 is abundantly expressed on the surface of hematopoietic cells, but CD43 expression is also frequently found in the tumor cells of nonhematopoietic origin. In the early stages of some tumors, the accumulation of tumor suppressor protein p53 has been described. Here, we show that the expression of CD43 causes the induction of functionally active p53 protein. Moreover, we found that the activation of p53 by CD43 is mediated by tumor suppressor protein ARF. The coexpression of CD43 and ARF in ARF-null mouse embryonic fibroblasts resulted in programmed cell death, but that was not the case when CD43 alone was expressed in these cells. These data provide the first evidence of the connection between p53- and CD43-dependent pathways.

SOX2 Is Regulated Differently from NANOG and OCT4 in Human Embryonic Stem Cells during Early Differentiation Initiated with Sodium Butyrate

Transcription factors NANOG, OCT4, and SOX2 regulate self-renewal and pluripotency in human embryonic stem (hES) cells; however, their expression profiles during early differentiation of hES cells are unclear. In this study, we used multiparameter flow cytometric assay to detect all three transcription factors (NANOG, OCT4, and SOX2) simultaneously at single cell level and monitored the changes in their expression during early differentiation towards endodermal lineage (induced by sodium butyrate). We observed at least four distinct populations of hES cells, characterized by specific expression patterns of NANOG, OCT4, and SOX2 and differentiation markers. Our results show that a single cell can express both differentiation and pluripotency markers at the same time, indicating a gradual mode of developmental transition in these cells. Notably, distinct regulation of SOX2 during early differentiation events was detected, highlighting the potential importance of this transcription factor for self-renewal of hES cells during differentiation.