Trigeminal ganglion neurons comprise three main cell body-size types. This cell size heterogeneity provides an excellent neuronal model to study the cell size-dependent organization and dynamics of the nucleoli, Cajal (coiled) bodies (CBs), and nuclear speckles of pre-mRNA splicing factors, nuclear structures that play a key role in the normal neuronal physiology. We have analyzed the number of nucleoli and CBs and the structural and molecular organization of CBs and nuclear speckles in the three neuronal types by using immunofluorescence with antibodies that recognize nucleoli (fibrillarin), CBs (coilin), and nuclear speckles (snRNPs), confocal microscopy, and electron microscopy. Whereas the mean number of nucleoli per neuron decreases as a function of cell size, the number of CBs per cell significantly increases in large neurons in comparison with the small ones. In addition, large neurons have a higher proportion of CBs associated with the nucleolus. In all neuronal types, CBs concentrate coilin, fibrillarin, snRNPs, and the survival motor neuron protein (SMN). Immunostaining for snRNPs shows small speckle domains and extensive areas of diffuse nucleoplasmic signal in large neurons, in contrast with the large nuclear speckles found in small neurons. Furthermore, flow cytometric analysis shows that all neurons are in the range of diploid cells. These findings indicate that the fusion behavior of nucleoli, the formation of CBs and their relationships with the nucleolus, as well as the compartmentalization of the pre-mRNA splicing machinery, is related to cell body size in the trigeminal ganglion neurons. Because transcriptional activity is a basic determinant mechanism of cell size in diploid cells, we suggest that our findings reflect a distinct transcription-dependent organization of the nucleolus and splicing machinery in the three cell types of trigeminal ganglion neurons.

Melatonin is an indolic hormone produced mainly by the pineal gland. The former hypothesis of its possible role in mammary cancer development was based on the evidence that melatonin down-regulates some of the pituitary and gonadal hormones that control mammary gland development and which are also responsible for the growth of hormone-dependent mammary tumors. Furthermore, melatonin could act directly on tumoral cells, as a naturally occurring antiestrogen, thereby influencing their proliferative rate. The first reports revealed a low plasmatic melatonin concentration in women with estrogen receptor (ER)-positive breast tumors. However, later studies on the possible role of melatonin on human breast cancer have been scarce and mostly of an epidemiological type. These studies described a low incidence of breast tumors in blind women as well as an inverse relationship between breast cancer incidence and the degree of visual impairment. Since light inhibits melatonin secretion, the relative increase in the melatonin circulating levels in women with a decreased light input could be interpreted as proof of the protective role of melatonin on mammary carcinogenesis. From in vivo studies on animal models of chemically induced mammary tumorigenesis, the general conclusion is that experimental manipulations activating the pineal gland or the administration of melatonin lengthens the latency and reduces the incidence and growth rate of mammary tumors, while pinealectomy usually has the opposite effects. Melatonin also reduces the incidence of spontaneous mammary tumors in different kinds of transgenic mice (c-neu and N-ras) and mice from strains with a high tumoral incidence.In vitro experiments, carried out with the ER-positive MCF-7 human breast cancer cells, demonstrated that melatonin, at a physiological concentration (1 nM) and in the presence of serum or estradiol: (a) inhibits, in a reversible way, cell proliferation, (b) increases the expression of p53 and p21WAF1 proteins and modulates the length of the cell cycle, and (c) reduces the metastasic capacity of these cells and counteracts the stimulatory effect of estradiol on cell invasiveness; this effect is mediated, at least in part, by a melatonin-induced increase in the expression of the cell surface adhesion proteins E-cadherin and β 1 -integrin.The direct oncostatic effects of melatonin depends on its interaction with the tumor cell estrogen-responsive pathway. In this sense it has been demonstrated that melatonin down-regulates the expression of ERα and inhibits the binding of the estradiol-ER complex to the estrogen response element (ERE) in the DNA. The characteristics of melatonin's oncostatic actions, comprising different aspects of tumor biology as well as the physiological doses at which the effect is accomplished, give special value to these findings and encourage clinical studies on the possible therapeutic value of melatonin on breast cancer.

Melatonin inhibits proliferation of the estrogen-responsive MCF-7 human breast cancer cells. The objective of this work was to assess whether melatonin not only regulates MCF-7 cell proliferation but also induces apoptosis. In this experiment we used 1,25-dihydroxycholecalciferol (D3) as a positive control because it inhibits MCF-7 cell proliferation and induces apoptosis. MCF-7 cells were cultured with either I nM melatonin, 100 nM D3 or its diluent to determine their effects on cell proliferation, cell viability, cell-cycle phase distribution, population of apoptotic cells, and expression of p53, p21WAF1, bcl-2, bcl-X(L) and bax proteins. After 24 or 48 hr of incubation, both melatonin and D3-treatment significantly decreased the number of viable cells in relation to the controls, although no differences in cell viability were observed between the treatments. The incidence of apoptosis, measured as the population of cells falling in the sub-G1 region of the DNA histogram, or by the TUNEL reaction, was similar in melatonin-treated and control cells whereas, as expected, apoptosis was higher among cells treated with D3 than in controls. The expression of p53 and p21WAF1 proteins significantly increased after 24 or 48 hr of incubation with either melatonin or D3. No significant changes in bcl-2, bcl-XL and bax mRNAs were detected after treatment with melatonin whereas in D3-treated cells, a significant drop in bcl-XL was observed. These data support the hypothesis that melatonin reduces MCF-7 cell proliferation by modulating cell-cycle length through the control of the p53-p21 pathway, but without clearly inducing apoptosis.

We have used an experimental model of tellurium(Te)-induced demyelinating neuropathy in the rat to study cellular mechanisms involved in regulating Schwann cell (SC) numbers during remyelination. Starting at postnatal day 21, weaned rats were fed a diet containing 1.1% elemental Te. Following 7 days of Te treatment and at several time points of post-tellurium treatment (PTe), the animals were processed for ultrastructural analysis, SC nuclei quantification and teased fibre preparations. It is well-established that Te induces a transient demyelinating/remyelinating sequence in sciatic nerves. The loss of the myelin sheath in this neuropathy produces active proliferation and overproduction of immature SCs. By electron microscopy analysis most mitotic SCs were located along demyelinated segments. Quantitative determination of SC nuclei per transverse section of sciatic nerve revealed a dramatic increase of SCs at 2 days PTe relative to control nerves. The number of SC nuclei then decreased progressively during the long-term period of recovery studied (330 days PTe). In Te-treated rats, SCs undergoing cell death were regularly found within the nerve fibre compartment, especially on demyelinated segments. Dying cells exhibited morphological features of apoptosis and appeared enclosed by lamellar processes of adjacent healthy SCs in extracellular compartments. Both healthy immature SCs and endoneurial macrophages were involved in the phagocytosis of apoptotic SCs. Particularly during remyelination, supernumerary endoneurial SCs were observed surrounding myelinated fibres. These cells progressively became atrophic with a morphological phenotype similar so that of "onion bulb" cells. On the other hand, teased fibre measurements revealed a remarkable permanent internodal shortening in remyelinated fibres from Te-treated sciatic nerves. These results indicate that a portion of redundant immature SCs are susceptible to elimination by apoptosis. However, other distinct biological mechanisms such as the persistence of supernumerary SCs in the endoneurium and the shortening of internodal lengths are also involved in regulating SC numbers during the remyelination stage.

We have used an experimental model of tellurium (Te)-induced demyelinating neuropathy in the rat to study cellular mechanisms involved in the early response of myelinating Schwann cells (SCs) to injury, prior to demyelination. Starting at postnatal day 21, weaned rats were fed a diet containing 1.1% elemental Te. The animals were killed daily within the 1st week of Te diet and the sciatic nerves were processed for the ultrastructural and immunocytochemical studies. Immunohistochemistry revealed that Te induces an increased nuclear expression of c-Fos in SCs. By electron microscopy analysis, the early cytoplasmic alteration was a dramatic disorganization of the rough endoplasmic reticulum (ER) with cisternal dilations and redistribution and loss of membrane-bound ribosomes. This was followed by a prominent activation of the macroautophagy in SCs. This process involved the formation of autophagosomes containing well-preserved cell organelles, autolysosomes with cellular remnants in various phases of degeneration and lysosomes. Te treatment also induced the expression of free ubiquitin in the perikaryal region of the SC cytoplasm. Immunogold electron microscopy showed the subcellular distribution of ubiquitin in the cytosol, around of dilated ER cisterns and in the matrix of autolysosomes and residual bodies. At the nucleolar level, fibrillarin immunofluorescence revealed nucleolar segregation in SCs exposed to Te. The ultrastructural study confirmed the segregation of the nucleolar components with a peripheral distribution of the dense fibrillar component. These results support the hypothesis that the depletion of cholesterol induced by Te treatment triggers a stress response in myelinating SCs mediated by immediate early genes of the fos family. The cellular response includes a severe disruption of the protein synthesis machinery, namely the rough ER and nucleolus, with the subsequent activation of both ubiquitin and autophagic pathways of proteins and cell organelle degradation. This cytoplasmic remodeling may represent a cytoprotective mechanism in the response of SCs to a neurotoxic stress. Furthermore, it must be a prerequisite for the induction of phenotypic changes and cell repair mechanisms in SCs.

Melatonin exerts a direct antiproliferative effect on estrogen-responsive MCF-7 cells in culture. Recently, the importance of the anti-invasive actions of melatonin as a part of the oncostatic action of this indolamine has been reported. Gap junctional intercellular communication is known to be involved in controlling cell proliferation and differentiation, and a decrease in intercellular junctional communication has been described in highly invasive mammary cancer cells. Because melatonin at physiological doses (1 nM) shifts MCF-7 cells to a lower invasive status, we postulate that melatonin could modulate the levels of gap junctional intercellular communication in these tumor cells. To test our hypothesis, we studied gap junctional intercellular communication in MCF-7 human breast cancer cells previously (7-8 days) treated, or not, with melatonin (10 microM or 1 nM). Using the scrape-loading assay dye-transfer technique to introduce 0.05% Lucifer yellow into cells, we measured the ability of the tumor cells to transfer dye to adjacent cells. Rhodamine dextran (0.05%) was used as a control dye to verify that dye-transfer occurs through intercellular junctions. The presence of melatonin (10 microM or 1 nM) in the culture medium significantly increased (P < 0.01) the transfer of the dye to adjacent cells through gap junctions. This increase was greater at 10 microM melatonin, and averaged scan profiles of cells treated with melatonin 10 microM showed a statistically significant increase (P < 0.01) in the integrated optical density values, and a broadening of the densitometric scan. These findings suggest that melatonin could exert its antitumor action, at least in part, by increasing regulatory signals that are passed between adjacent epithelial cells through intercellular junctions.

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It is well established that forskolin-induced elevation of cAMP results in activation of DNA synthesis in Schwann cell cultures. This promitotic response is partially mediated by the Cdk2, which is required for the transition from the G1 to the S phase of the cell cycle. In the present study, we analyze the effects of cAMP elevation in cultured Schwann cells on the transcriptional activity and on the organization of two nuclear compartments involved in pre-mRNA processing: Cajal bodies (CBs) and splicing factor compartments. Our immunofluorescence and quantitative studies show that forskolin treatment induces a 5.6-fold increase in the proportion of S phase Schwann cells, detected by a short pulse (20 min) of BrdU incorporation. This increase in DNA synthesis correlates with an activation of global transcription, as is indicated by the higher nuclear incorporation of BrU in nascent RNA. Forskolin treatment significantly increases the percentage of Schwann cells containing typical CBs, which concentrate spliceosomal snRNPs and the survival motor neuron (SMN) protein. This increase in the number of CBs closely correlates with the activation of transcription. Moreover, the occurrence of CBs is significantly higher in BrdU (+) cells than in BrdU (-) cells, indicating that entry in the S phase promotes the formation of CBs. During the S phase, Schwann cell nuclei display higher Cdk2 nuclear staining and concentrate this kinase in CBs. Forskolin also induces a redistribution of the pre-mRNA splicing factors in Schwann cells. Primary cultures of Schwann cells provide an excellent physiological model to demonstrate that the assembly of CBs is a transcription- and replication-dependent cellular event. Moreover, the S phase accumulation of Cdk2 observed in Schwann cells supports a functional link between CBs and DNA replication, which is mediated by the possible participation of CBs in the regulatory control of histone gene expression.