Testis peritubular myoid cells increase their motility and express matrix-metalloproteinase 9 (MMP-9) after interaction with embryonal carcinoma cells.

Today cancer research studies have highlighted the role of the cancer-stroma interaction in the regulation of invasive processes. However, very little is known about cell-to-cell relationships between germinal cancer cells and the somatic ones belong to their close environment, particularly at early invasion stages. Here, we have studied the potential role of the seminiferous peritubular myoid cells (PTCs), as potential part of the reactive stroma, like tumor myofibroblast, in the progression of embryonal carcinoma (EC). To this end, we show results on the in vitro interactions between F9 murine embryonal carcinoma cells (EC cells) and primary cultures of murine PTCs, using contact-dependent and contact-independent 2D co-cultures. In these circumstances, when EC cells interact with PTCs they change their migratory behavior and matrix-metalloproteinase 9 (MMP-9) was up-regulated in PTCs. Additionally, among a variety of cytokines implicated in tumor-stroma cross-talk, we have examined in more detail the influence of tumor necrosis factor alpha (TNF-α). In this regard, it was observed that this cytokine induced a MMP-9 secretion by PTCs in a pattern dependent on its concentration, whereas does not increase the migration capacity of cancer cells. All together, our results provide evidence for a role played by peritubular myoid cells and cancer-cell secreted TNF- α for a change in the tumor microenvironment during the early stages of EC progression.

Evidence for a role of matrix metalloproteinases and their inhibitors in primordial germ cell migration.

Understanding the mechanisms that enable migrating cells to reach their targets is of vital importance, as several pathologies, including cardiac defects and some tumours, are consequences of altered cell migration. With a view to evaluating if matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) play a role in the active migration of primordial germ cells (PGCs) from their place of origin in extra-embryonic sites towards their final destination in the developing gonads, we analysed the expression of mRNAs encoding nine MMPs and four TIMPs in migrating (E10.5) and post-migrating (E12.5) PGCs by means of quantitative polymerase chain reaction and the presence of MT1-MMP in the membrane of these cells. Our results show that PGCs express MMP-2, MMP-9, MMP-11, MT1-MMP, TIMP-1, TIMP-2 and TIMP-3 at both migrating and non-migrating stages. Comparing expression levels of MMP genes between E10.5 and E12.5 PGCs revealed higher expression in migrating PGCs of MT1- MMP (10.3-fold), MMP-2 (4.8-fold), MMP-11 (3.2-fold) and MMP-9 (2.1-fold). Similarly, the levels of TIMP gene expression were always higher in E12.5 genital ridge somatic cells: TIMP-3 (3.4-fold), TIMP-1 (2.4-fold) and TIMP-2 (1.8-fold). Moreover, the analysis at protein level showed the presence of MT1-MMP in the membrane of migrating PGCs whereas the expression of these metalloproteinase is not detected once the PGCs have reach the urogenital ridges and stop migrating. These results suggest that the change from the motile to non-motile phenotype that occurs during PGC maturation to gonocytes may be mediated in part by enhanced expression of MMPs in migrating PGCs together with higher expression of TIMPs in E12.5 genital ridges.

Peritubular myoid cell-derived factors and its potential role in the progression of testicular germ cell tumours.

The tumour surrounding stroma, known as reactive stroma, is a crucial factor to understand cancer cell growth and invasion. In the normal adult testis, the stroma contains extracellular matrix components, fibroblasts, infiltrating leucocytes, lymphocytes, macrophages and capillaries, as well as other specific cell populations, like Leydig cells and a thin myoepithelium surrounding the seminiferous tubules constituted by the peritubular cells. All these cells are an important source of proliferation and survival promoting signals, proteolytic enzymes, migratory cues and pro-angiogenic factors. Ascribable to this pro-invasive activity, the tumour reactive stroma cells, especially cancer-associated myofibroblasts, have emerged as a promising target for cancer therapy. This review is focused on the potential role of the peritubular myoid cells in the development of testicular germ cell tumours as the precursors of cancer-associated myofibroblast and on an experimental model for the study of testis germinal cancer stroma and on the differences between normal and tumour-associated stromal cells, including the molecular mechanisms that mediate the important cancer stroma crosstalk. Special attention will be paid to the cancer-associated myofibroblasts as possible therapeutic targets, because they are one of the main components of the reactive stroma and are known to secrete a variety of paracrine factors that stimulate tumour progression.

Vascularization of testicular germ cell tumours: evidence from experimental teratocarcinomas.

Testicular germ cell tumours (TGCTs) represent about 2% of male malignancies, being the most common cancer among adolescents and young adults. As in most neoplasias, TGCTs show a chaotic vascular architecture, altered blood supply and over-expression of pro-angiogenic factors, aspects closely related to tumour overgrowth and metastasis. Following this trend, our laboratory has analysed the effect of the hypoxic tumour microenvironment on cancer stem cells, particularly the expression of factors related to vascularization, such as matrix metalloproteinases, adhesion molecules, vascular endothelial growth factors (VEGF) and VEGF receptors. This review also summarizes our present knowledge on vascularization in the normal and neoplastic testis, the potential role of the factors involved in TGCT neovascularization and their importance as possible therapeutic targets.

Angiogenesis and vascular network of teratocarcinoma from embryonic stem cell transplant into seminiferous tubules.

BACKGROUND: Carcinoma in situ (CIS) of the testis is considered to be a precancerous germinal cell lesion, but the precise cellular and molecular mechanisms underlying transformation of CIS into invasive pluripotent cancer cells remain to be elucidated. Moreover, a satisfactory animal model for the experimental study of germinal tumours has not been developed to date. METHODS: We have developed a tumour model that involves the microinjection of green fluorescent protein-labelled embryonic stem (ES) cells (which are functionally equivalent to CIS cells) into syngenic mouse seminiferous tubules, a unique cell microenvironment in which germinal cells mature and CIS arise. To characterise the vascularisation of teratocarcinomas, which arise after cell transplant, we used immunohistochemistry, together with a qualitative and quantitative analysis of scanning electron microscopy images of corrosion casting samples. RESULTS: Embryonic stem cells transplanted into seminiferous tubules did not differentiate into germinal cells, but rather they behaved as invasive embryonal carcinoma (EC) stem cells. The vascular pattern of the experimental teratocarcinomas showed a highly disorganised architecture, and some of the neoplastic capillaries were derived, at least in part, from the original transplanted ES cells. CONCLUSION: The transplantation of pluripotent ES cells into seminiferous tubules efficiently recapitulates the early stages of development of teratocarcinomas. Consequently, this method constitutes a novel in vivo model to study the mechanisms of invasion and progression of experimental germinal tumours.

Structural and functional preservation of specific sequences of DNA and mRNA in apoptotic bodies from ES cells.

Retinoic acid-induced apoptosis of embryonic stem (ES) cells is an experimental system which resembles the physiological programmed cell death that occurs during differentiation in embryonic development. Our aim was to analyze the involvement of epigenetic modifications such as DNA methylation and chromatin structure in the apoptotic process and to investigate the metabolic activity of apoptotic bodies. We found a relationship between DNA methylation and apoptosis, shown by a dose-dependent induction of apoptosis after treatment with the inhibitor of DNA methylation 5-aza-2'-deoxycytidine. Interestingly, we found a slight demethylation of specific sequences of the U2afl-rs1 imprinted gene in those RA treated cells which were specifically undergoing apoptosis. In addition, apoptotic bodies exhibited an unexpected open chromatin conformation accessible to the endonuclease DNase-I. Furthermore, we observed a structural and functional preservation of specific DNA sequences and mRNA. These results suggest that biological activities, such as transcription or protein synthesis, could be maintained even towards the end of the apoptotic process.

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Implication of mitochondria-derived ROS and cardiolipin peroxidation in N-(4-hydroxyphenyl)retinamide-induced apoptosis.

We have studied the effect of N-(4-hydroxyphenyl)retinamide on either malignant human leukaemia cells or normal cells and investigated its mechanism of action. We demonstrate that 4HPR induces reactive oxygen species increase on mitochondria at a target between mitochondrial respiratory chain complex I and II. Such oxidative stress causes cardiolipin peroxidation which in turn allows cytochrome c release to cytosol, caspase-3 activation and therefore apoptotic consumption. Moreover, this apoptotic pathway seems to be bcl-2/bax independent and count only on malignant cells but not normal nor activated lymphocytes.

Suppression of spermatogenesis for cell transplantation in adult mice.

Spermatogenesis occurs within the testis of adult males by a complex and very well organized process. Breakthroughs in techniques such as cryopreservation and culture of spermatogenic cells and the maturation of these cells in exogenous testes after transplantation renewed the interest in this process. Transplantation of spermatogenic cells from a donor to a recipient animal needs a preparatory step that consists in the elimination of the endogenous population of spermatogenic cells. The most common method used to empty the seminiferous tubules is the treatment with busulfan (1,4-butanediol dimethanesulfonate). Busulfan partially eliminates stem cells because of its alkylating nature, but a residual component of stem cells survives the treatment and competes in the regeneration of the testis with transplanted cells. Estradiol has also been used as an agent that causes a delay in the process of spermatogenesis by altering its hormonal stimulation, although it does not affect the spermatogonia population. Therefore, we have tested different treatments with busulfan, estradiol benzoate, and also an agonist of the chorionic gonadotrophin-releasing hormone, leuprolide acetate, for the inhibition of endogenous spermatogenesis. We have found that a combination of estradiol, busulfan, and leuprolide can destroy the population of endogenous spermatogenic cells without altering Sertoli cells and maintains the optimal environment needed to allow the development of transplanted cells.

Presence of four stem cell populations in monolayer cultures derived from teratocarcinoma embryoid bodies.

In the present work, flow cytometry techniques together with morphologic studies were used to perform multiparametric analyses in cell cultures derived from CE44 teratocarcinoma embryoid bodies. The intrinsic cell parameters studied by flow cytometry were size (FALS), cytoplasmic complexity (ISS) and autofluorescence, expressed as LIGFL/FALS (green fluorescence intensity on a logarithmic scale/FALS). Our results showed that CE44 teratocarcinoma yields monolayers whose cells show a marked morphological heterogeneity and can be grouped according to flow cytometric criteria into four populations that remain stable throughout the entire time of culture. Moreover, these populations showed a different immunolabelling with the differentiation markers SSEA-1, TROMA-1 and anti-vimentin.

Nucleoplasmin binds to nuclear pore filaments and accumulates in specific regions of the nucleolar cortex.

Nucleoplasmin is a karyophilic protein that is involved in nucleosome formation and decondensation of chromatin, although other precise functions and modes of action of this molecule are still poorly understood. In the present paper we describe a novel nucleocytoplasmic transport assay that has enabled us to study the nuclear distribution of nucleoplasmin following its transport into the nucleus. Single Xenopus laevis oocyte nuclei were isolated and incubated with Xenopus egg extract containing colloidal gold-conjugated nucleoplasmin. After a period of incubation, each individual nucleus was processed for electron microscopy. The nuclear accumulation of nucleoplasmin was dependent upon the karyophilic properties of the protein, since BSA-conjugated gold particles did not enter the nuclear interior under the same experimental conditions. Once inside the nucleus, nucleoplasmin was detected in tracks emanating from the nuclear pores and reaching the nucleolus. Additionally, we found a striking accumulation of nucleoplasmin in specific areas of the nucleolar cortex. These perinucleolar regions were surrounded by areas of electron density similar to that of the fibrillar centers. Our results indicate that nucleoplasmin may play an important role in the transcription of ribosomal precursors. Moreover, this nucleocytoplasmic transport assay will enable the determination of the precise intranuclear localization of other karyophilic proteins.

FGFs-1 and -2, and TGF beta 1 as inductive signals modulating in vitro odontoblast differentiation.

We have studied the expression of FGF1 and FGF2 during mouse odontogenesis by immunohistochemistry. FGF1 was detected in differentiated odontoblasts and at the secretory pole of ameloblasts. Localization of FGF2 was mainly observed within the basement membrane interposed between dental epithelium and dental mesenchyme. These findings indicate that FGF1 and FGF2 may participate in the control of odontoblast and ameloblast differentiation. Thereafter, we studied the ability of FGF1 and FGF2, alone or in combination with TGF beta 1, to induce polarization and/or functional differentiation of preodontoblasts. Dental papillae (DP) obtained from first lower molars of 17-day-old mouse embryo were cultured in the presence or the absence of growth factors. DP cultured with FGF1 + TGF beta 1 showed gradients of odontoblast-like cell differentiation, which displayed alkaline phosphatase reactivity. DP treated with FGF2 + TGF beta 1 exhibited pre-odontoblast cell polarization, and the cell bodies displayed long cytoplasm processes. However, following this treatment we did not observe extracellular matrix secretion, and alkaline phosphatase activity was completely inhibited. In summary, our results show that exogenous addition of FGF1 to pre-odontoblasts induces their terminal differentiation, by synergistically acting with TGF beta 1. In contrast, FGF2 may regulate the effect of TGF beta 1, permitting cell polarization but restraining pre-odontoblast functions.

Dissection of the odontoblast differentiation process in vitro by a combination of FGF1, FGF2, and TGFbeta1.

Dental papillae (DP) isolated from first lower molars of 17-day-old mouse embryos were cultured in the presence of combinations of the following growth factors: FGF1, FGF2, and TGFbeta1. After 6 days in culture, only the DP treated with FGF1+TGFbeta1 contained differentiated odontoblast-like cells at the periphery of the explants, and these cells secreted extracellular matrix similar to predentin. Surprisingly, treatments with FGF2+TGFbeta1 induced cell polarization at the surface of the explants but no matrix secretion was observed. Electron microscopy and histochemical analysis of odontoblast markers showed that differentiated cells induced by FGF1+TGFbeta1 exhibited cytological features of functional odontoblasts with matrix vesicle secretion and mineral formation, positive alkaline-phosphatase activity, and type-I collagen production. DP cultured in the presence of FGF2+TGFbeta1 showed cell polarization and long and thin cell processes containing matrix vesicles; however, type-I collagen secretion was not detected and alkaline-phosphatase activity was completely inhibited. Our results indicate that, in our culture system, exogenous combinations of FGF1, FGF2, and TGFbeta1 interact with preodontoblasts and induce cell polarization or differentiation, which can be studied separately in vitro. Thus, FGF1 and TGFbeta1 do have a synergic effect to promote morphological and functional features of differentiated odontoblasts whereas FGF2 seems to modulate TGFbeta1 action, causing morphological polarization of preodontoblasts but limiting the functional activity of these cells in terms of type-I collagen secretion and alkaline-phosphatase activity.

The role of cleavage of cell structures during apoptosis.

In this work we have studied the behavior of some cell structures, such as actin, tubulin and chromatin during apoptosis induced in F9 cells after retinoic acid treatment. In this experimental model, all defined steps of morphological changes described for apoptosis are observed. The correlation between a partial maintenance of F-actin and microtubular structures and the spatial distribution of F-actin suggests a possible relationship between this molecule and the characteristic shape changes observed in apoptosis. Additionally, the disposition of monomeric G-actin suggests a possible relationship between the fragmentation of this molecule and the cleavage of DNA. The analysis of the U2af1-rs1 specific sequence shows that the internucleosomal fragmentation observed in this gene is randomly produced during apoptosis and is not dependent of demethylation status. The results obtained confirm that specific cleavage of these cell structures is inherent to the development of the apoptotic process and do not exclude the possibility that proteolysis of key actin and/or tubulin molecules or the cleavage of specific chromatin sequences other than the ones analyzed here, could control the different phases of the apoptotic process.

Epigenetic modulation of differentiation in CE44 teratocarcinoma.

Teratocarcinoma is a mixed germ cell tumor histologically composed of embryonal carcinoma cells and embryonic and extraembryonic tissues. In the present work we have used the CE44 teratocarcinoma, which is a tumor cell line derived from the OTT6050 experimental tumor, to appreciate the influence the microenvironment has on the modulation of tumoral differentiation. For this, we have studied the development of CE44 teratocarcinoma in primary tumors (subcutaneous and intrasplenic) and in experimental metastases (hepatic and pulmonary). CE44 teratocarcinoma shows variations in its capacity for differentiation in so far as development is concerned and, in hepatic metastases, we noticed a reparative process of the intratumoral necrotic areas which in the same cases were substituted by loose connective tissue. Our results clearly suggest that the microenvironment is decisive in the biological behaviour of the teratocarcinoma cells and that epigenetic factors influence the capacity for differentiation of the undifferentiated tumoral cells.

Polyploidization and exit from cell cycle as mechanisms of cultured melanoma cell resistance to methotrexate.

Numerous malignant neoplasias are found to contain varying proportions of high-ploidy cells. Although the role they play in the tumor is poorly understood, several lines of evidence suggest that these cells could be especially resistant to various aggressions, a possibility of great interest in cancer treatment. In the present study, we tested this hypothesis through the analysis of the presence of high-ploidy cells following the administration of the chemotherapeutic agent methotrexate. We also determined the expression of two proliferation markers, PCNA and CDK1, after methotrexate-treatment. Cultured cells from the murine melanoma B16F10 were treated with high doses of methotrexate for seven days prior to determination of DNA content and proliferation markers. Our results showed an obvious increase in the mean ploidy of this population. Specifically, there was a dramatic reduction in the proportion of tetraploid cells (predominant in the original population), and an increase in the proportion of cells with higher ploidies, particularly those whose DNA content was greater than 8c, including some cells with ploidies greater than 16c. Furthermore, there was a reduction in the number of PCNA-expressing cells and the reduction was much more marked in the case of CDK1 that was almost absent in the modal-ploidy treated cells. These alterations concerning ploidy and expression of proliferation markers had completely reverted two weeks after withdrawal of the drug. Our results indicate that methotrexate at a high dosage selects a cell population heterogeneous concerning its ploidy level, composed of one subpopulation of high-ploidy cells and another of modal-ploidy cells that, considering its lack of CDK1 expression, would remain in a latent state to evade the effects of the drug.

Effects of aFGF, bFGF, TGFbeta1 and IGF-I on odontoblast differentiation in vitro.

In this work, we investigated the effects of aFGF and bFGF alone or combined with TGFbeta1 or IGF-I on odontoblast differentiation. Trypsin-isolated dental papillae from day 17 mandibular first molar were cultured in semisolid-agar medium for 6 d. Our results demonstrated that aFGF, bFGF or combinations of these promoted cell polarization at the periphery of the dental papillae. Moreover, simultaneous addition of aFGF and TGFbeta1 to dental papillae cultures induced both polarization and functional differentiation of odontoblast-like cells, as well as extracellular matrix deposition. Combination of aFGF or bFGF with IGF-I caused cell polarization at the surface of dental papillae, but matrix secretion was restricted to a few explants. In the presence of bFGF and TGFbeta1, the explants had pronounced cell elongations but no matrix deposition. These results indicate that aFGF or bFGF is not able to induce odontoblast differentiation alone. However, both aFGF and bFGF can act synergistically with TGFbeta1 and IGF-I to strengthen their inductive effects and promote gradients of cytological and functional changes in odontoblast-like cells.

The nuclear basket of the nuclear pore complex is part of a higher-order filamentous network that is related to chromatin.

We have achieved well-preserved nuclear basket structures in amphibian nuclear envelope spreads dried by the critical point method after tannic acid fixation, and we have compared these images with those obtained from conventional sections of amphibian oocyte nuclei. In cross sections, bundles of filaments from adjacent nuclear pore complexes were interconnected at regular distances, building up a higher-order network. Sometimes these bundles were observed to extend inward to amplified nucleoli located near the nuclear envelope. Furthermore, immunoelectron analysis indicated that DNA and histones were localized at these intranuclear filaments, suggesting a close relationship between chromatin and nuclear pore complexes. A model is proposed by which the intranuclear filaments associated with adjacent nuclear pore complexes create a regular higher-order network, which extends into the nucleus.