Metabolomic Analysis of Liver Tissue from the VX2 Rabbit Model of Secondary Liver Tumors

Purpose. The incidence of liver neoplasms is rising in USA. The purpose of this study was to determine metabolic profiles of liver tissue during early cancer development. Methods. We used the rabbitVX2 model of liver tumors (LT) and a control group consisting of sham animals implanted with Gelfoam into their livers (LG). After two weeks from implantation, liver tissue from lobes with and without tumor was obtained from experimental animals (LT+/LT−) as well as liver tissue from controls (LG+/LG−). Peaks obtained by Gas Chromatography-Mass Spectrometry were subjected to identification. 56 metabolites were identified and their profiles compared between groups using principal component analysis (PCA) and a mixed-effect two-way ANOVA model. Results. Animals recovered from surgery uneventfully. Analyses identified a metabolite profile that significantly differs in experimental conditions after controlling the False Discovery Rate (FDR). 16 metabolites concentrations differed significantly when comparing samples from (LT+/LT−) to samples from (LG+/LG−) livers. A significant difference was also shown in 20 metabolites when comparing samples from (LT+) liver lobes to samples from (LT−) liver lobes. Conclusion. Normal liver tissue harboring malignancy had a distinct metabolic signature. The role of metabolic profiles on liver biopsies for the detection of early liver cancer remains to be determined

A Mitosis Block Links Active Cell Cycle with Human Epidermal Differentiation and Results in Endoreplication

How human self-renewal tissues co-ordinate proliferation with differentiation is unclear. Human epidermis undergoes continuous cell growth and differentiation and is permanently exposed to mutagenic hazard. Keratinocytes are thought to arrest cell growth and cell cycle prior to terminal differentiation. However, a growing body of evidence does not satisfy this model. For instance, it does not explain how skin maintains tissue structure in hyperproliferative benign lesions. We have developed and applied novel cell cycle techniques to human skin in situ and determined the dynamics of key cell cycle regulators of DNA replication or mitosis, such as cyclins E, A and B, or members of the anaphase promoting complex pathway: cdc14A, Ndc80/Hec1 and Aurora kinase B. The results show that actively cycling keratinocytes initiate terminal differentiation, arrest in mitosis, continue DNA replication in a special G2/M state, and become polyploid by mitotic slippage. They unambiguously demonstrate that cell cycle progression coexists with terminal differentiation, thus explaining how differentiating cells increase in size. Epidermal differentiating cells arrest in mitosis and a genotoxic-induced mitosis block rapidly pushes epidermal basal cells into differentiation and polyploidy. These observations unravel a novel mitosis-differentiation link that provides new insight into skin homeostasis and cancer. It might constitute a self-defence mechanism against oncogenic alterations such as Myc deregulation

A Cell Cycle Role for the Epigenetic Factor CTCF-L/BORIS

CTCF is a ubiquitous epigenetic regulator that has been proposed as a master keeper of chromatin organisation. CTCF-like, or BORIS, is thought to antagonise CTCF and has been found in normal testis, ovary and a large variety of tumour cells. The cellular function of BORIS remains intriguing although it might be involved in developmental reprogramming of gene expression patterns. We here unravel the expression of CTCF and BORIS proteins throughout human epidermis. While CTCF is widely distributed within the nucleus, BORIS is confined to the nucleolus and other euchromatin domains. Nascent RNA experiments in primary keratinocytes revealed that endogenous BORIS is present in active transcription sites. Interestingly, BORIS also localises to interphase centrosomes suggesting a role in the cell cycle. Blocking the cell cycle at S phase or mitosis, or causing DNA damage, produced a striking accumulation of BORIS. Consistently, ectopic expression of wild type or GFP- BORIS provoked a higher rate of S phase cells as well as genomic instability by mitosis failure. Furthermore, downregulation of endogenous BORIS by specific shRNAs inhibited both RNA transcription and cell cycle progression. The results altogether suggest a role for BORIS in coordinating S phase events with mitosis

Switch from p53 to MDM2 as differentiating human keratinocytes lose their proliferative potential and increase in cellular size

p53 transcription factor is mutated in most skin cell carcinomas and in more than 50% of all human malignancies. One of its transcriptional targets is MDM2, which in turn down-regulates p53. The role of the p53/MDM2 regulatory loop upon genotoxic stress is well documented, but less is known about its role in normal tissue homeostasis. We have explored this pathway during the different transitions of the human epidermal differentiation programme and after isolating stem cells, transit amplifying cells or differentiating cells from epidermis. Maximum expression of p53 was found in proliferating keratinocytes. A striking and transient induction of MDM2 and a down-modulation of p53 characterized the transition from proliferation to differentiation in primary human keratinocytes. These changes were delayed in late differentiating carcinoma cells, and were clearly different in suspended primary fibroblasts. Interestingly, these changes correlated with an increase in cell size, at the time of irreversible commitment to differentiation. Induction of MDM2 was also associated with suppression of proliferation in normal, or hyperproliferative, psoriatic epidermis. Moreover, both proteins were induced as keratinocytes were driven to leave the stem cell compartment by c-Myc activation. Overall, our results show a critical regulation of the p53/MDM2 pathway at the epidermal transition from proliferation to differentiation

Switch from p53 to MDM2 as differentiating human keratinocytes lose their proliferative potential and increase in cellular size

p53 transcription factor is mutated in most skin cell carcinomas and in more than 50% of all human malignancies. One of its transcriptional targets is MDM2, which in turn down-regulates p53. The role of the p53/MDM2 regulatory loop upon genotoxic stress is well documented, but less is known about its role in normal tissue homeostasis. We have explored this pathway during the different transitions of the human epidermal differentiation programme and after isolating stem cells, transit amplifying cells or differentiating cells from epidermis. Maximum expression of p53 was found in proliferating keratinocytes. A striking and transient induction of MDM2 and a down-modulation of p53 characterized the transition from proliferation to differentiation in primary human keratinocytes. These changes were delayed in late differentiating carcinoma cells, and were clearly different in suspended primary fibroblasts. Interestingly, these changes correlated with an increase in cell size, at the time of irreversible commitment to differentiation. Induction of MDM2 was also associated with suppression of proliferation in normal, or hyperproliferative, psoriatic epidermis. Moreover, both proteins were induced as keratinocytes were driven to leave the stem cell compartment by c-Myc activation. Overall, our results show a critical regulation of the p53/MDM2 pathway at the epidermal transition from proliferation to differentiation

MDM2 expression in normal and pathological skin

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Skin as a potential organ for ectopic monoclonal antibody production

International audienceThe therapeutic potential of monoclonal antibodies for treating a variety of severe or life-threatening diseases is high. Although intravenous infusion appears the simplest and most obvious mode of administration, it is not applicable to many long-term treatments. It might be advantageously replaced by gene/cell therapies, however, rendering treatments cost-effective and eliminating the short- and long-term side-effects associated with injection of massive doses of antibodies. We have tested whether skin can potentially be used as an organ for production and systemic delivery of ectopic antibodies. Normal human primary keratinocytes were shown to be capable of synthesis and secretion of a model monoclonal antibody directed against human thyroglobulin upon retroviral gene transduction in vitro. Neo- epidermis reconstructed in vitro, either in cell culture inserts or on dermal substrates, from such modified keratinocytes also produced the monoclonal antibody. Interestingly, the latter could cross the epidermis basal layer and be released in culture fluids. Finally, grafting of epidermis reconstituted in vitro on dermal substrates to SCID mice permitted sustained monoclonal antibody delivery into the bloodstream to be achieved. Our data thus show that genetically engineered keratinocytes can potentially be used for genetic antibody-based immunotherapies. They also indicate that proteins as big as 150 kDa, after release by engineered keratinocytes into skin intercellular spaces, can migrate to the general circulation, which is potentially important for a number of other gene-based therapies

Characterisation of membrane oligonucleotide-binding proteins and oligonucleotide uptake in keratinocytes.

Inadequate cellular compartmentalisation of plasmid DNA and antisense oligodeoxynucleotides (ODNs) is generally considered as a major limitation in their use. In this study, an approach combining in situ visual-isation of rhodamine-labelled ODNs and affinity modification of proteins by radiolabelled-alkylating ODN derivatives has been used to investigate the uptake of ODNs into keratinocytes. We confirm here that unmodified ODNs are efficiently taken up and accumulate in cell nuclei in primary keratinocytes as well as in HaCaT and A431 keratinocyte cell lines. Uptake is fast, irreversible, saturable and not significantly altered by incubation at low temperature. Affinity modification studies in keratinocyte cell lines has revealed two high-affinity, cell-specific interactions between ODNs and proteins of 61-63 kDa and 35 kDa. Trypsin pre-treatment of A431 cells and pre-incubation with polyanions, or with unlabelled nucleic acid competitors, inhibited the accumulation of rhodamine-labelled ODNs in nuclei as well as the affinity labelling of the 61-63 kDa doublet and 35 kDa ODN-binding proteins by reactive ODN derivatives. Finally, cell fractionation studies indicated that these ODN-binding proteins were essentially localised in the plasma membrane. Our results suggest that these ODN-binding proteins might be involved in the recognition and transport of ODNs into keratinocytes

MDM-2 protein is expressed in different layers of normal human skin

MDM-2 is one of the target genes of the p53 tumor suppressor protein. Its best characterized function is found in the inhibition of p53's ability to modulate transcription. Deregulated expression of MDM-2 could thus at least partially substitute for p53 mutation in the process of tumorigenesis. We show here that MDM-2 is highly expressed in biopsies of normal human skin or in vitro reconstituted human skin. The protein is detected in the nucleus of keratinocytes throughout the different layers of the epidermis and in reconstituted skin as early as the two to three cell layer stage. The 90 kiloDalton (kD) protein is one of the major forms detected in Western blot experiments. MDM-2 is detected in skin reconstituted from keratinocytes in which p53 is inactivated by mutation or degradation by E6 protein, providing evidence that MDM-2 expression in the skin can occur in the absence of wild type p53. Moreover, we found no correlation between the p53 status and MDM-2 expression levels in a series of basal and squamous cell carcinomas or Bowen diseases. Our data provide first evidence for the expression of MDM-2 in a differentiated adult tissue