Cellular proliferation kinetics of the murine sarcoma induced by the Moloney sarcoma virus

Abstract. Cell proliferation kinetics of the sarcoma induced by Moloney virus was studied in newborn Swiss OFl mice.
After in vivo injection of tritiated thymidine, followed by autoradiography, it was shown that the majority of cells were actively proliferating (labelling index; 31%, growth fraction 78%). The mean cell cycle was 16 hr and cell loss was relatively low (cell loss factor 48%). The study of tumour specific activity with time after a single [ in vivo ] injection of [³H]dR or [¹²⁵I]UdR did not demonstrate the same degree of cell loss as that calculated by autoradiography. This result is consistent with a massive reutilization of radioactivity released by normal tissues.     

Changes in repair of potentially lethal damage with culture age in EMT6 cells.

We have investigated the changes in the amplitude of repair of potentially lethal damage (PLD) in EMT6 cells with increasing culture age and determined the delay necessary to achieve this repair. This experimental system presents all intermediaries between the exponential growth type and a plateau with a cell turnover nearly nil. The radiosensitivity was studied by the colony method. When the percentage of surviving cells was tested immediately after irradiation it was observed that their radiosensitivity increased with culture age. This percentage fell only slightly when the cells were tested for viability 6 hours after irradiation. Therefore, the amplitude of repair increases with culture age. Repair was found to terminate 1, 1.75, 3 and 6 hours after irradiation of cultures aged respectively 2, 4, 6 and 9 days. The delay and the amplitude of repair did not vary significantly for cultures of 9, 11 and 13 days.     

15-deoxy-delta12,14-prostaglandin J2 inhibits Bay 11-7085-induced sustained extracellular signal-regulated kinase phosphorylation and apoptosis in human articular chondrocytes and synovial fibroblasts

We have previously shown that nuclear factor-kappaB inhibition by adenovirus expressing mutated IkappaB-alpha or by proteasome inhibitor increases human articular chondrocytes sensibility to apoptosis. Moreover, the nuclear factor-kappaB inhibitor BAY11-7085, a potent anti-inflammatory drug in rat adjuvant arthritis, is itself a proapoptotic agent for chondrocytes. In this work, we show that BAY 11-7085 but not the proteasome inhibitor MG-132 induced a rapid and sustained phosphorylation of extracellular signal-regulated kinases (ERK1/2) in human articular chondrocytes. The level of ERK1/2 phosphorylation correlated with BAY 11-7085 concentration and chondrocyte apoptosis. 15-Deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2) and its precursor prostaglandin (PG) D2 but not PGE2 and PGF2alpha rescued chondrocytes from BAY 11-7085-induced apoptosis. 15d-PGJ2 markedly inhibited BAY 11-7085-induced phosphorylation of ERK1/2. BAY 11-7085 also induced ERK1/2 phosphorylation and apoptosis in human synovial fibroblasts, and these reactions were down-regulated by 15d-PGJ2. Further analysis in synovial fibroblasts showed that only molecules that suppressed BAY 11-7085-induced phosphorylation of ERK1/2 (i.e. 15d-PGJ2, PGD2, and to a lesser extent, MEK1/2 inhibitor UO126, but not prostaglandins E2 and F2alpha or peroxisome proliferator-activated receptor-gamma agonist ciglitazone) were able protect cells from apoptosis. These results suggested that the antiapoptotic effect of 15d-PGJ2 on chondrocytes and synovial fibroblasts might involve inhibition of ERK1/2 phosphorylation.     

Persistence of a chimerical phenotype after hepatocyte differentiation of human bone marrow mesenchymal stem cells

Abstract.  Objectives : Recent studies have suggested the potential of mesenchymal stem cells (MSCs) to differentiate into a hepatocyte-like lineage. Here, we evaluate the efficacy of hepatocyte differentiation of MSCs by studying acquisition of hepatocyte-like features together with alteration of the native mesenchymal phenotype. Material and methods : In vitro , we have investigated protein and mRNA level expression of hepatocyte and mesenchymal markers of mesenchymal-derived hepatocyte-like cells (MDHLCs) and we have evaluated their functionality using metabolic assays. In vivo , we investigated co-expression of hepatocyte (albumin, α-foetoprotein, cytokeratin 18) and mesenchymal (fibronectin, vimentin) markers after transplantation of MSCs or MDHLCs into severe combined immune deficiency mice. Results : We observed that while in vitro these cells acquired some phenotypic and functional features of mature hepatocytes, they partially preserved their mesenchymal phenotype. After intrasplenic transplantation, engrafted MSCs with isolated expression of fibronectin and α-foetoprotein were observed. When these cells were injected into the liver, they expressed all analysed markers, confirming the chimaeric co-expression observed in vitro . Conversely, liver-engrafted MDHLCs conserved their hepatocyte-lineage markers but lost their chimaeric phenotype. Conclusions : Hepatocyte differentiation of MSCs predominantly allows the acquisition of phenotypic hallmarks and provides chimaeric cells that maintain expression of initial lineage markers. However, advanced maturation to the hepatocyte-like phenotype could be obtained in vivo by conditioning MSCs prior to transplantation or by infusing cells into the liver micro-environment.     

Umbilical cord fibroblasts: Could they be considered as mesenchymal stem cells?

In cell therapy protocols, many tissues were proposed as a source of mesenchymal stem cells (MSC) isolation. So far, bone marrow (BM) has been presented as the main source of MSC despite the invasive isolation procedure related to this source. During the last years, the umbilical cord (UC) matrix was cited in different studies as a reliable source from which long term ex vivo proliferating fibroblasts were isolated but with contradictory data about their immunophenotype, gene expression profile, and differentiation potential. Hence, an interesting question emerged: Are cells isolated from cord matrix (UC-MSC) different from other MSCs? In this review, we will summarize different studies that isolated and characterized UC-MSC. Considering BM-MSC as gold standard, we will discuss if UC-MSC fulfill different criteria that define MSC, and what remain to be done in this issue.     

Peroxisome proliferator-activated receptor-gamma1 is dephosphorylated and degraded during BAY 11-7085-induced synovial fibroblast apoptosis

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) plays a central role in whole body metabolism by regulating adipocyte differentiation and energy storage. Recently, however, PPAR-gamma has also been demonstrated to affect proliferation, differentiation, and apoptosis of different cell types. As we have previously shown that BAY 11-7085-induced synovial fibroblast apoptosis is prevented by PPAR-gamma agonist 15d-PGJ2; the expression of PPAR-gamma in these cells was studied. Both PPAR-gamma1 and PPAR-gamma2 isoforms were cloned from synovial fibroblast RNA, but only PPAR-gamma1 was detected by Western blot, showing constitutive nuclear expression. Within minutes of BAY 11-7085 treatment, a PPAR-gamma1-specific band was shifted into a form of higher mobility, suggesting dephosphorylation, as confirmed by phosphatase treatment of cell extracts. Of interest, BAY 11-7085-induced PPAR-gamma1 dephosphorylation was followed by PARP and caspase-8 cleavage as well as by PPAR-gamma1 protein degradation. PPAR-gamma1 dephosphorylation was followed by the loss of PPAR-DNA binding activity ubiquitously present in synovial fibroblast nuclear extracts. Unlike the phosphorylated form, dephosphorylated PPAR-gamma1 was found in insoluble membrane cell fraction and was not ubiquitinated before degradation. PPAR-gamma1 dephosphorylation coincided with ERK1/2 phosphorylation that accompanies BAY 11-7085-induced synovial fibroblasts apoptosis. 15d-PGJ2, PGD2, and partially UO126, down-regulated ERK1/2 phosphorylation, protected cells from BAY 11-7085-induced apoptosis, and reversed both PPAR-gamma dephosphorylation and degradation. Furthermore, PPAR-gamma antagonist BADGE induced PPAR-gamma1 degradation, ERK1/2 phosphorylation, and synovial fibroblasts apoptosis. The results presented suggest an anti-apoptotic role for PPAR-gamma1 in synovial fibroblasts. Since apoptotic marker PARP is cleaved after PPAR-gamma1 dephosphorylation but before PPAR-gamma1 degradation, dephosphorylation event might be enough to mediate BAY 11-7085-induced apoptosis in synovial fibroblasts.     

Reduced repair of potentially lethal radiation damage in glutathione synthetase-deficient human fibroblasts after X-irradiation

Using a human fibroblast strain deficient in glutathione synthetase and a related proficient control strain, the role of glutathione (GSH) in repair of potentially lethal damage (PLD) has been investigated in determining survival by plating cells immediately or 24 h after irradiation. After oxic or hypoxic irradiation, both cell strains repair radiation-induced damage. However, under hypoxic conditions, the proficient cells repair PLD as well as under oxic conditions while the deficient cells repair less PLD after irradiation under hypoxic than under oxic conditions. Therefore, the oxygen enhancement ratio (o.e.r.) for proficient cells is similar whether the cells are plated immediately or 24 h later (2.0 and 2.13, respectively). In contrast, the o.e.r. for deficient cells is lower when the cells are plated 24 h after irradiation than when they are plated immediately thereafter (1.16 as compared to 1.55). The results indicate that GSH is involved in PLD repair and, in particular, in the repair of damage induced by radiation delivered under hypoxic conditions.     

Survival curves of glutathione synthetase deficient human fibroblasts: correlation between radiosensitivity in hypoxia and glutathione synthetase activity

The role of intracellular non-protein bound sulphydryl compounds (NPSH), and in particular that of glutathione (GSH), in the response of cells to ionizing radiation under different O2 concentrations has been assessed using cell strains deficient in glutathione synthetase and exhibiting different NPSH levels. The cell strains used originated from patients with 5-oxoprolinuria and from their relatives (heterozygotes and proficient homozygotes). No correlation has been found between NPSH and GSH concentrations and radiosensitivity under oxic, aerobic and hypoxic conditions. However, a highly significant correlation has been observed between radiosensitivity under hypoxic conditions (and therefore the oxygen enhancement ratio) and the glutathione synthetase activity, suggesting that synthesis of GSH is required after irradiation. In order to explain our results we postulated, beside radical processes, the existence of a GSH-dependent enzymatic repair mechanism for N2 type damage. Hypoxic radio-sensitivity measured with survival curves would result from the interaction of both competition and biochemical repair processes.     

Hypersensitivity to very-low single radiation doses: Its relationship to the adaptive response and induced radioresistance

There is now little doubt of the existence of radioprotective mechanisms, or stress responses, that are upregulated in response to exposure to small doses of ionizing radiation and other DNA-damaging agents. Phenomenologically, there are two ways in which these induced mechanisms operate. First, a small conditioning dose (generally below 30 cGy) may protect against a subsequent, separate, exposure to radiation that may be substantially larger than the initial dose. This has been termed the adaptive response. Second, the response to single doses may itself be dose-dependent so that small acute radiation exposures, or exposures at very low dose rates, are more effective per unit dose than larger exposures above the threshold where the induced radioprotection is triggered. This combination has been termed low-dose hypersensitivity (HRS) and induced radioresistance (IRR) as the dose increases. Both the adaptive response and HRS/IRR have been well documented in studies with yeast, bacteria, protozoa, algae, higher plant cells, insect cells, mammalian and human cells in vitro, and in studies on animal models in vivo. There is indirect evidence that the HRS/IRR phenomenon in response to single doses is a manifestation of the same underlying mechanism that determines the adaptive response in the two-dose case and that it can be triggered by high and low LET radiations as well as a variety of other stress-inducing agents such as hydrogen peroxide and chemotherapeutic agents although exact homology remains to be tested. Little is currently known about the precise nature of this underlying mechanism, but there is evidence that it operates by increasing the amount and rate of DNA repair, rather than by indirect mechanisms such as modulation of cell-cycle progression or apoptosis. Changed expression of some genes, only in response to low and not high doses, may occur within a few hours of irradiation and this would be rapid enough to explain the phenomenon of induced radioresistance although its specific molecular components have yet to be identified.