Long-term fate of terminally differentiated skeletal muscle cells following E1A-initiated cell cycle reactivation

We have previously shown that E1A reactivates the cell cycle in 'irreversibly' growth arrested, terminally differentiated (TD) cells. The molecular events following E1A-mediated reactivation of TD skeletal muscle cells have been extensively investigated. However, the long-term fate of the reactivated cells has not been directly determined. In this paper, E1A is used to reactivate TD myotubes derived from established cell lines or primary myoblasts. We show that the reactivated muscle cells continue proliferating beyond the end of the first cell cycle and progress through at least a second one. Experiments performed with an inducible E1A/estrogen receptor chimera indicate that the reactivated cell cycle is self-sustained, since E1A is exclusively necessary to reactivate TD cells, but is dispensable for both the continuation of the first cycle and the progression into the following one. Finally, we report that E1A-mediated reactivation of muscle cells results in apoptotic cell death that can be delayed by the antiapoptotic, adenoviral E1B 55 kDa oncogene.     

Stanniocalcin in terminally differentiated mammalian cells

Stanniocalcin (STC) is a glycoprotein hormone originally found in teleost fish, where it regulates the calcium/phosphate homeostasis, and protects the fish against toxic hypercalcemia. STC was considered an exclusive fish protein, until the cloning of cDNA for human (in 1995) and murine (in 1996) STC. We originally reported a high constitutive content of STC in mammalian brain neurons, and found that the expression of STC occurred concomitantly with terminal differentiation of neural cells. Since then, we have investigated the expression of STC in relation to terminal cell differentiation also in mammalian hematopoietic tissue, and fat tissues. In this review we summarize our findings on STC expression during postmitotic differentiation in three different cell systems; in neural cells, in megakaryocytes and in adipocytes. We also present findings, suggesting that STC plays a role for maintaining the integrity of terminally differentiated mammalian cells.     

Endogenous lectin from terminally differentiated epidermal cells

We isolated a concanavalin A (Con-A)-binding glycoprotein from human stratum corneum by nonionic detergent extraction, lectin affinity chromatography, and preparative gel electrophoresis. This glycoprotein migrates as a single band at 40 kilodaltons at sodium-dodecyl-sulfate gel electrophoresis with or without the presence of 2-mercaptoethanol. It was shown to have a heterogeneous distribution between pH 5.6 and 7.6 by isoelectric focusing. The glycoprotein is histidine rich (10.4%) but is distinct from other histidine-rich proteins (epidermal filaggrin and the histidine-rich glycoprotein from serum). It does not bind to lectins specific for L-fucose or alpha-D-galactose. We prepared a monospecific polyclonal antibody to the 40-kilodalton glycoprotein; at the ultrastructural level, it cytoimmunolocalizes exclusively to the membranes of the stratum corneum. A unique feature of the glycoprotein is that it is an endogenous lectin: it hemagglutinates trypsinized and gluteraldehyde-fixed rabbit erythrocytes. The inhibition of its hemagglutination was found to be greatest with amino sugars, down to a saccharide concentration of 10(-5) mM. Such a high affinity of binding at the cell surface suggests that this glycoprotein is a major carbohydrate-binding, cross-linking molecule that holds adjacent corneocytes together in the stratum corneum. We hypothesize that this lectin plays a role in the adhesion and desquamation of the stratum corneum.     

Histones of terminally differentiated cells undergo continuous turnover

In contrast to the widely accepted idea of the nearly absolute metabolic stability of histones, our experiments support the view that the histones of nonproliferating, terminally differentiated cells undergo continuous replacement. This conclusion is based on the incorporation of labeled amino acids into the histones of mouse kidney and liver cells after their intraperitoneal introduction. We have found that the intranuclear uptake of the histones made in the absence of replicative synthesis and their integration into chromatin proceed with striking delay. The metabolic rates of individual histones measured by calculating their half-lives suggest that each histone turns over at a specific rate. With regard to the basic chromatin structure, the nucleosome, such unequal turnover should mean that the histone core does not participate in this process as a single unit but rather as a protein mosaic in which each partner follows its own rate of removal. Additional experiments suggested that intact nucleosomes take part in the replacement, but the relative proportion of the nucleosomes involved should be limited. The nonnucleosomal H1A and H1 degree histones have been found to undergo faster replacement than the core histones. Moreover, in comparison to each other, these two histone subfractions are also replaced at a different rate. The results of autoradiography of isolated kidney and liver nuclei after continuous labeling with [3H]-thymidine suggest that the histone replacement is not associated with the repair of DNA.     

A robust cell cycle control mechanism limits E2F-induced proliferation of terminally differentiated cells in vivo

Terminally differentiated cells in Drosophila melanogaster wings and eyes are largely resistant to proliferation upon deregulation of either E2F or cyclin E (CycE), but exogenous expression of both factors together can bypass cell cycle exit. In this study, we show this is the result of cooperation of cell cycle control mechanisms that limit E2F-CycE positive feedback and prevent cycling after terminal differentiation. Aberrant CycE activity after differentiation leads to the degradation of E2F activator complexes, which increases the proportion of CycE-resistant E2F repressor complexes, resulting in stable E2F target gene repression. If E2F-dependent repression is lost after differentiation, high anaphase-promoting complex/cyclosome (APC/C) activity degrades key E2F targets to limit cell cycle reentry. Providing both CycE and E2F activities bypasses exit by simultaneously inhibiting the APC/C and inducing a group of E2F target genes essential for cell cycle reentry after differentiation. These mechanisms are essential for proper development, as evading them leads to tissue outgrowths composed of dividing but terminally differentiated cells.     

DNA repair kinetics in irradiated undifferentiated and terminally differentiated cells

The brains of male Fisher 344 rats bearing 80-150 mg intracerebral 9L/Ro tumors were irradiated with doses of 1,250-5,000 rads of x- or gamma-rays. At various times after irradiation, the cerebellum and tumor were excised, dissociated into single cells and the DNA from these cells sedimented through alkaline sucrose gradients in zonal rotors with slow gradient reorienting capability. Quantitation of the DNA repair kinetics demonstrated that the process in both tumor cells and neurons has a fast and slow phase. Although all other alternatives cannot be completely eliminated, we suggest that these two phases are most reasonably interpreted as representing repair of lesions in very accessible and less accessible regions of the genome rather than 1) repair of different types of lesions such as single- or double-strand breaks or 2) removal of immediate breaks and breaks induced during excision repair of latent base damage. The slow repair phase is saturable, but not inducible in both tumor cells and neurons. The data suggest that tumor cells restore their chromosomal DNA structure to the unirradiated state faster than neurons because 1) they contain more of the repair system per unit of DNA and 2) a larger proportion of their genetic material is comprised of very accessible regions. The data also suggest that the entire tumor cell genome may be accessible to the repair enzyme(s), while it is possible that a portion of the neuronal genome may be completely inaccessible.     

Mechanisms of radioresistance in terminally differentiated cells of mature retina

Retinopathy of animals is induced by many agents damaging DNA. This fact shows that DNA lesions may initiate retinal degeneration. The aim of our work was to study the effects of gamma and proton irradiation, and methylnitrosourea (MNU) on mice retina. We evaluated morphological changes, DNA damage and repair in retina, and expression of 5 proteins participating in apoptosis: p53, ATM, FasR, PARP and caspase 3 active. Dose of 14 Gy is equitoxic in terms of induction of DNA single strand breaks by both gamma and proton irradiation. But protons were 2 fold more effective than gamma-rays in induction of DNA double strand breaks. All breaks were repaired within < or =10 h. Irradiation resulted in increased expression of p53 and ATM. But no sings of cell death and retinal degeneration were observed during 7 days after irradiation. Proton irradiation in dose of 25 Gy resulted in increasing over time destructive changes localized mainly in photoreceptor layer of retina. These changes were followed by increased expression of proapoptotic proteins. A single systemic administration of MNU (70 mg/kg) increased intracellular levels of p53, PARP, FasR, caspase 3 active, which was followed by destructive changes in retina with sings of apoptosis of photoreceptors. As in the case of irradiation, the 2-fold dose reduction of MNU abrogated cytotoxic effect of MNU on retina. High level of spontaneous DNA damage such as apurine and apyrimidine sites were observed in mouse retina. The results of our study demonstrate the occurrence of genotoxic threshold in the initiation of retinal cell death in vivo. Topoisomerase 2 of retina is suggested to translate primary DNA damage to cytotoxic effect.     

Mechanisms of radioresistance in terminally differentiated cells of mature retina

Retinopathy of animals is induced by many DNA-damaging agents. This fact shows that DNA lesions may initiate retinal degeneration. The aim of our work was to study the effects of gamma and proton irradiation and single administration of methylnitrosourea (MNU) on mice retina. We assessed morphological changes, DNA damage and repair, as well as expression of proteins (p53, ATM, PARP, FasR, and caspase 3) participating in apoptosis in retina. 14 Gy was the equitoxic dose for induction of DNA single-strand breaks by both gamma- and proton irradiation. However, protons were twice as effective as γ rays in induction of DNA double-strand breaks. All breaks have been repaired for ≤10 h. Irradiation resulted in increased expression of p53 and ATM. Seven days after irradiation, no signs of cell death and retinal degeneration were observed. Proton irradiation with 25 Gy resulted in destructive changes in retina localized mainly in the photoreceptor layer. These changes were accompanied by enhanced expression of proapoptotic proteins. A single systemic administration of MNU (70 mg/kg) increased intracellular levels of p53, PARP, FasR, and Caspase 3 followed by destructive changes in retina with sings of apoptosis in photoreceptors. Similarly to irradiation, a halved MNU dose did not exhibit a cytotoxic effect on retina. A high level of spontaneous DNA damage at apurine and apyrimidine sites were observed in mouse retina. The results show that there is a genotoxic threshold in initiation of retinal cell death in vivo. It is suggested that topoisomerase 2 translates primary DNA damage into a cytotoxic effect in retina.     

Increased proteolysis in chromatin of terminally differentiated and quiescent cells

Endogenous proteolysis in chromatin of terminally differentiated, quiescent, and actively proliferating cells was studied by measuring the released acid-soluble radioactivity of [³H]tryptophan-prelabelled nuclear proteins, and by following the specific quantitative and qualitative changes in electrophoregrams of chromosomal proteins. The experiments suggest that the chromatin of differentiated mouse kidney and liver cells, as well as chromatin from Friend cells induced to commit terminal differentiation, exhibit increased proteolysis in comparison with that of chromatin isolated from actively proliferating cells. Enhanced proteolysis was found also for the slowly renewing and quiescent cells from adult mice. The control experiments designated to discriminate between the two possible alternatives explaining the difference—increased activity of the proteolytic enzymes associated with chromatin, or increased susceptibility of the chromosomal proteins to proteases—supported the latter alternative.     

Gamma-ray enhanced reactivation of gamma-irradiated adenovirus in human cells.

An enhanced reactivation of γ-irradiated human adenovirus type 2 (Ad 2) was detected following the infection of normal human fibroblasts which had been pre-irradiated with γ-rays. γ-irradiated or non-irradiated fibroblasts were infected with either non-irradiated or γ-irradiated Ad 2, and at 48 hours after infection cells were examined for the presence of viral structural antigens (Vag) using immunofluorescent staining. Pre-irradiation of the cells with 1 Krad immediately prior to infection resulted in a 5 to 15 fold increase in the survival of this viral function following different γ doses to the virus up to 3 Mrad. For a fixed γ dose of 2 Mrad to the virus this enhancement increased with pre-irradiation dose to the cells up to a maximum factor of 5 to 30 for a dose of 2 Krad. When infection was delayed until 48 hours after irradiation of the cells, this enhancement was reduced to about half the level found for immediate infection.     

Transduction of terminally differentiated neurons by avian sarcoma virus

Recent studies have demonstrated that avian sarcoma virus (ASV) can transduce cycle-arrested cells. Here, we have assessed quantitatively the transduction efficiency of an ASV vector in naturally arrested mouse hippocampal neurons. This efficiency was determined by comparing the number of transduced cells after infection of differentiated neurons versus dividing progenitor cells. The results indicate that ASV is able to transduce these differentiated neurons efficiently and that this activity is not the result of infection of residual dividing cells. The transduction efficiency of the ASV vector was found to be intermediate between the relatively high and low efficiencies obtained with human immunodeficiency virus type 1 and murine leukemia virus vectors, respectively.     

The binding of ORC2 to chromatin from terminally differentiated cells

Nuclei from terminally differentiated Xenopus erythrocytes lack essential components of the prereplication complex, including the origin recognition complex (ORC) proteins XORC1 and XORC2. In Xenopus egg extract, these proteins are able to bind erythrocyte chromatin from permeable nuclei, but not from intact nuclei, even though they are able to cross an intact nuclear envelope. In this report we use both permeable and intact erythrocyte nuclei to investigate the role of cyclin-dependent kinase activity in modulating the binding of XORC2 to chromatin. We find that elevating the level of cyclin A-dependent kinase in egg extract prevents the binding of XORC2 to chromatin from permeable nuclei and that kinase inhibition reverses this effect. We also observe a nuclear transport-dependent accumulation of H1 kinase activity within intact nuclei incubated in the extract. However, inhibiting this kinase activity does not facilitate the binding of XORC2 to chromatin, suggesting that other molecules and/or mechanisms exist to prevent association of XORC proteins with replication origins within intact nuclei from terminally differentiated cells.     

Glycoproteins modulate adhesion in terminally differentiated keratinocytes

Summary The stratum corneum can be dissociated into single squames by homogenization in ether. We have reaggregated the free corneocytes into a multilayered lamellar structure resembling an intact stratum corneum. The reconstituted stratum corneum reacts with fluorescein-conjugated lectins, unlike the intact tissue. We infer that the lack of binding in the intact tissue is due to masking of saccharide sites by lipids (which are extracted by the ether). In an extension of the procedure, the ether is removed and replaced by acetone. This system permits us to modulate corneocyte reaggregation by the addition of appropriate agents. We have used this system to corroborate our hypothesis that a 40 kD cell-surface glycoprotein (an endogenous lectin specific for amino sugars), which we have isolated from the stratum corneum, is instrumental in adhesion of corneocytes by cross-linking with amino sugar sites on adjacent cells. The reaggregation is inhibited by the antibody to the 40 kD glycoprotein. It is also inhibited by either the addition of amino sugars which bind to the endogenous lectin, or the addition of exogenous lectins specific for amino sugars which bind to the ligand.     

Purification and characterization of carboxypeptidase from terminally differentiated rat epidermal cells

A tissue carboxypeptidase-A-like enzyme was purified to apparent homogeneity from terminally differentiated epidermal cells of 2-day-old rats by potato inhibitor affinity chromatography followed by FPLC Mono Q column chromatography. The enzyme has an Mr of 35,000 as determined by SDS-polyacrylamide gel electrophoresis and HPLC gel filtration. It has a pH optimum of 8.5 for hydrolysis of benzyloxycarbonyl-Phe-Leu (Km = 0.22 mM, kcat = 57.9 s-1). The enzyme does not hydrolyze substrates with Arg, Lys and Pro at the C-terminal and Pro at the penultimate position. Angiotensin I was effectively hydrolyzed (Km = 0.06 mM, kcat = 6.48 s-1) and produced both des-Leu10-angiotensin I and angiotensin II. The enzyme activity, relatively stable at 4 degrees C and pH 8.0-10.5, was inactivated at pH values higher than 12.0 and lower than 5.0 or at 65 degrees C for 10 min. Inhibitor profiles of the epidermal enzyme also differed slightly from those of tissue carboxypeptidase A of pancreatic or mast cell origin.     

Mitotic division of differentiated neurosecretory cells in the hypothalamus of adult amphibians

By means of multiple 3H-thymidine administration to adult frogs, a direct electron microscopic radioautographic evidence is first by presented regarding the possibility of an in vivo mitotic division of differentiated hypothalamic peptidergic neurosecretory cells. The absence of any signs of cytoplasmic dedifferentiation, as well as a poor development of spindle microtubules in this cell may be suggestive of a polyploidizing, possibly acytokinetic mitotic process occurring in such kind of cells.