Loss of calpain 3 proteolytic activity leads to muscular dystrophy and to apoptosis-associated IkappaBalpha/nuclear factor kappaB pathway perturbation in mice.

Calpain 3 is known as the skeletal muscle-specific member of the calpains, a family of intracellular nonlysosomal cysteine proteases. It was previously shown that defects in the human calpain 3 gene are responsible for limb girdle muscular dystrophy type 2A (LGMD2A), an inherited disease affecting predominantly the proximal limb muscles. To better understand the function of calpain 3 and the pathophysiological mechanisms of LGMD2A and also to develop an adequate model for therapy research, we generated capn3-deficient mice by gene targeting. capn3-deficient mice are fully fertile and viable. Allele transmission in intercross progeny demonstrated a statistically significant departure from Mendel's law. capn3-deficient mice show a mild progressive muscular dystrophy that affects a specific group of muscles. The age of appearance of myopathic features varies with the genetic background, suggesting the involvement of modifier genes. Affected muscles manifest a similar apoptosis-associated perturbation of the IkappaBalpha/nuclear factor kappaB pathway as seen in LGMD2A patients. In addition, Evans blue staining of muscle fibers reveals that the pathological process due to calpain 3 deficiency is associated with membrane alterations.

Overexpression of both catalytically active and -inactive cathepsin D by cancer cells enhances apoptosis-dependent chemo-sensitivity.

The aspartic protease cathepsin D (cath-D) is a key mediator of induced-apoptosis and its proteolytic activity has been generally involved in this event. During apoptosis, cath-D is translocated to the cytosol. Because cath-D is one of the lysosomal enzymes that requires a more acidic pH to be proteolytically active relative to the cysteine lysosomal enzymes such as cath-B and -L, it is therefore open to question whether cytosolic cath-D might be able to cleave substrate(s) implicated in the apoptotic cascade. Here, we have investigated the role of wild-type cath-D and its proteolytically inactive counterpart overexpressed by 3Y1-Ad12 cancer cells during chemotherapeutic-induced cytotoxicity and apoptosis, as well as the relevance of cath-D catalytic function. We demonstrate that wild-type or mutated catalytically inactive cath-D strongly enhances chemo-sensitivity and apoptotic response to etoposide. Both wild-type and mutated inactive cath-D are translocated to the cytosol, increasing the release of cytochrome c, the activation of caspases-9 and -3 and the induction of a caspase-dependent apoptosis. In addition, pretreatment of cells with the aspartic protease inhibitor, pepstatin A, does not prevent apoptosis. Interestingly therefore, the stimulatory effect of cath-D on cell death is independent of its catalytic activity. Overall, our results imply that cytosolic cath-D stimulates apoptotic pathways by interacting with a member of the apoptotic machinery rather than by cleaving specific substrate(s).

Pathophysiology of limb girdle muscular dystrophy type 2A: hypothesis and new insights into the IkappaBalpha/NF-kappaB survival pathway in skeletal muscle.

Limb girdle muscular dystrophies (LGMDs) are a group of clinically heterogeneous genetic diseases characterized by progressive weakness and atrophy of scapular and pelvic muscles, with either a dominant or recessive autosomic mode of inheritance. The first symptoms of the disorder appear during the first 20 years of life and progresses gradually, and a walking disability develops 10-20 years later. The gene responsible for LGMD2A has been identified and encodes calpain 3, a protease expressed mainly in skeletal muscle. Apoptotic myonuclei were recently detected in muscular biopsy specimens of LGMD2A patients, and apoptosis was found to be correlated with altered subcellular distribution of inhibitory protein kappaBalpha (IkappaBalpha) and nuclear factor kappaB (NF-kappaB), resulting in sarcoplasmic sequestration of NF-kappaB. Calpain 3 dependent IkappaBalpha degradation was reconstituted in vitro, supporting a possible in vivo sequence of events leading from calpain 3 deficiency to IkappaBkappa accumulation, prevention of nuclear translocation of NF-kappaB, and ultimately apoptosis. Therefore calpain 3, present in healthy muscle as sarcoplasmic and nuclear forms, may control IkappaBalpha turnover and indirectly regulate NF-kappaB dependent expression of survival genes. Recent data reported from a new model of LGMD2A in mice and from other muscular disorders strengthen understanding of the molecular links between calpain 3 and the Ikappaalpha/NF-kappaB pathway. Finally, in light of the lack of apoptosis observed in inflammatory myopathies, a unifying model for the control of cell survival in muscle is proposed and discussed

Intracellular calcium release induced by human immunodeficiency virus type 1 (HIV-1) surface envelope glycoprotein in human intestinal epithelial cells: a putative mechanism for HIV-1 enteropathy.

Intracellular Ca2+ ([Ca2+]i) was measured in single human epithelial intestinal HT-29-D4 cells with the Ca2+ probe Fura-2 and digital imaging microscopy. Treatment of these cells with HIV-1 surface envelope glycoprotein gp120 (or a soluble form of its precursor gp160) induced an important increase of [Ca2+]i. This effect was abolished by preincubation of the viral glycoprotein with neutralizing antibodies specific for the V3 domain of gp120. These antibodies inhibited the binding of both gp120 and gp160 to galactosylceramide (GalCer), the alternative HIV-1 receptor in HT-29-D4 cells. Moreover, treatment of HT-29-D4 cells with an anti-GalCer mAb induced an increase in [Ca2+]i and rendered the cells insensitive to HIV-1 glycoprotein stimulation. The calcium response resulted from release of Ca2+ from caffeine-sensitive intracellular stores. Finally, the viral glycoprotein specifically abrogated the calcium response to the neuropeptide agonist neurotensin, a stimulator of chloride secretion via inositol trisphosphate-mediated calcium mobilization. Reciprocally, after neurotensin stimulation, the cells did not respond to gp120, showing that neurotensin and gp120 stimulate a common pathway of [Ca2+]i mobilization. These results suggest that HIV-1 may directly alter ion secretion in the intestine and thus be the causative agent of the watery diarrhea associated with HIV-1 infection.

CD4 molecules are restricted to the basolateral membrane domain of in vitro differentiated human colon cancer cells (HT29-D4).

The CD4 glycoprotein serves as a receptor for the human immunodeficiency virus HIV, the etiologic agent of acquired immunodeficiency syndrome (AIDS). We have examined the expression of CD4 molecules in a clone (HT29-D4) derived from a human colon adenocarcinoma cell line. HT29-D4 cells synthesized a 60 kDa polypeptide immunoprecipitated with two anti-CD4 monoclonal antibodies after metabolic or cell surface labeling. This 60 kDa polypeptide was also immunodetected using the same antibodies in human acute lymphoblastic leukemia cells CEM which are known to express CD4. HT29-D4 cells can be induced to differentiate into enterocyte-like cells by removing glucose from the culture medium. Under these conditions, HT29-D4 cells form a polarized epithelial monolayer in which tight junctions separate the plasma membrane in an apical and a basolateral domain. The localization of CD4 molecules in differentiated HT29-D4 cells was exclusively restricted to the basolateral membrane domain as demonstrated by radioimmunoassay and indirect immunofluorescence studies. Therefore the HT29-D4 clonal cell line represents a unique model for polarized HIV infection of colonic epithelial cells and may be useful to understand some of the gastrointestinal disorders occurring in AIDS patients.

Replication and apical budding of HIV-1 in mucous-secreting colonic epithelial cells.

The human colonic adenocarcinoma cell line HT29 can be infected with various isolates of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2). In some cases, the virus was able to perform its complete cycle of replication as demonstrated by the persistent production of mature viral particles in the cell-free culture supernatant. We have cultured HT29 cells chronically infected with the replicative strain HIV1-NDK in a chemically defined serum-free medium. Under these conditions, the cells were able to maintain a high level of viral replication, as demonstrated by reverse transcriptase activities and in situ hybridization studies. By indirect immunofluorescence labeling and electron microscopy, we observed that serum starvation was associated with the differentiation of HIV-1-infected HT29 cells into mucous-secreting cells resembling epithelial goblet cells of the colonic mucosa. These mucous-secreting cells, which accounted for 50% of the overall population, produced mature particles of HIV through their apical membrane in the vicinity of mucous granules. These data suggest that HIV-infected goblet cells in the colonic mucosa may produce the virus in the colorectal lumen; this could explain the route of transmission of HIV in the case of anal intercourse.

Intracellular localisation of suramin, an anticancer drug, in human colon adenocarcinoma cells: a study by quantitative autoradiography.

Suramin is a polysulphonated naphthylurea currently investigated for the treatment of advanced malignancy. In the present study, we have analysed the uptake and the intracellular localisation of tritiated suramin in human colon adenocarcinoma cells (HT-29-D4), using quantitative autoradiographic techniques at the optical and electron microscopy levels. Our results show that the drug is able to enter both undifferentiated and differentiated HT-29-D4 cells. The process of suramin uptake is time-dependent, and significantly inhibited by the presence of the suramin-binding protein serum albumin in the culture medium of HT-29-D4 cells. Autoradiographic analysis revealed two distinct patterns of intracellular localisation of tritiated suramin labelling, according to the presence or absence of serum albumin. Indeed, in the absence of serum albumin, the labelling of free suramin was distributed over the nucleus, the Golgi apparatus and the mitochondria, while it was restricted to the lysosomal system when suramin was complexed with albumin. These data show that a serum factor, i.e. albumin, influences the biological activity of suramin by determining its intracellular localisation. The presence of suramin in a given compartment may account for specific effects of the drug including mitochondrial hypertrophy, altered gene expression and lysosomal perturbation.

Vectorial release of carcinoembryonic antigen induced by IFN-gamma in human colon cancer cells cultured in serum-free medium.

Confluent monolayers of intestinal cell lines are useful models for studies of intestinal epithelial structure and function. Three cell lines have retained morphological and functional properties of intestinal epithelial cells compatible with such studies: Caco-2, T84 and HT29. However, the requirement of fetal bovine serum for the culture of these cells does not facilitate the design of experiments dealing with growth factors or hormonal regulation. The clonal intestinal cell line HT29-D4 can be cultured as fully differentiated epithelial monolayers in a synthetic medium containing transferrin, selenous acid, epidermal growth factor and suramin, a potent differentiation inducer. In the present study it is shown that HT29-D4 cells grown on permeable substratum in this synthetic medium developed electrically active monolayers consisting of columnar cells with morphological characteristics of normal enterocytes. After metabolic labelling with [35S]-methionine, HT29-D4 monolayers released most of their radiolabelled secretory proteins preferentially in the basal compartment of the cell culture chamber. However, the carcinoembryonic antigen, shown to be present in the apical plasma membrane, was exclusively released apically. This oriented release was stimulated by recombinant gamma-interferon (IFN-gamma) added only in the basal chamber, suggesting a basolateral restriction for IFN-gamma receptors.

Double screening of suramin derivatives on human colon cancer cells and on neural cells provides new therapeutic agents with reduced toxicity.

Suramin is a polyanionic compound currently used under evaluation for antineoplastic activity. One of the main problems encountered during clinical trials was an adverse neurotoxic effect, probably due to a direct cytotoxic effect on neural cells. Suramin is also known to trigger differentiation of human colon cancer cells, yet a chronic treatment induces a lysosomal storage disorder. The aim of this study was to evaluate suramin analogs for their effect: (i) on the lysosomal system of the human colon cancer cell clone HT29-D4; and (ii) on C6 glioma cell growth and morphology. One of the derivatives tested, NF036, induced terminal differentiation of HT29-D4 cells without any impairment of the lysosomal system. Furthermore, in contrast to suramin, NF036 did not alter C6 cell growth and morphology. We conclude that there is a relationship between the ability of a suramin derivative to induce a lysosomal storage disorder in human colon cancer cells and its neurotoxic effect. A double screening of suramin analogs on HT29-D4 and C6 cells allowed us to identify a new candidate antineoplastic drug: NF036.

Insulin like growth factor I is an autocrine regulator of human colon cancer cell differentiation and growth.

The polyanionic compound suramin triggers enterocyte-like differentiation of the human colic adenocarcinoma cell clone HT29-D4. We now demonstrate that suramin interferes with the binding of IGF-I to its receptor at the surface of HT29-D4 cells. Half-maximum inhibition of 125I-IGF-I binding was obtained in the presence of 25 micrograms/ml suramin. Moreover, the drug was able to dissociate 125I-IGF previously bound to its cell surface receptor. Affinity labeling HT29-D4 cells were cultured in the presence of 10 micrograms/ml of alpha-IR3, a monoclonal antibody directed against the binding site of IGF-I, an inhibition of cell proliferation and a stimulation of cell differentiation was observed. After 10 days of treatment with alpha-IR3, HT29-D4 cells formed a regular monolayer of enterocyte-like cells exhibiting an apical brush border and tight junctions delimiting two domains of the plasma membrane (apical and basolateral). Furthermore, we show that IGF-I significantly increased the initial rate of glucose uptake by HT29-D4 cells, while we have previously shown that suramin decreased glucose consumption. From these data we conclude that IGF-I secreted by the cells themselves, stimulates proliferation of HT29-D4 cells via an autocrine mechanism. Blockade of this stimulation by suramin or by a specific monoclonal antibody inhibits cell growth, glucose uptake and triggers the process of enterocytic differentiation.

Autoradiographic localization of tritiated suramin in polarized human colon adenocarcinoma cells.

In this report we demonstrated that [3H]suramin enters polarized human colon adenocarcinoma cells when added to the apical side of the monolayer. Using light microscopic quantitative autoradiography, we showed that suramin was accumulated in the apical cytoplasm and in the nucleus. In contrast, a weak labeling was noted in other compartments such as the basolateral cytoplasm and the intercellular space. The accumulation of suramin in the apical region of the cells is consistent with previous data showing that suramin elicited a lysosomal storage disorder in HT29-D4 cells by a mechanism of polarized endocytosis.

Co-localization of suramin and serum albumin in lysosomes of suramin-treated human colon cancer cells.

Suramin is a polysulfonated compound currently under investigation for the treatment of various types of cancer. Pharmacokinetic studies from clinical trials in humans have shown that most of the circulating drug is associated with serum albumin. The objective of the present study was to investigate the intracellular localization of suramin and serum albumin in human colon cancer cells (HT-29-D4) upon suramin treatment. For this purpose, combined gold labeling and autoradiographic methods were performed on HT-29-D4 cells grown in serum free medium containing both [3H]suramin and colloidal gold-albumin. These morphological experiments demonstrated for the first time that suramin and serum albumin were co-localized in the same cellular compartment (i.e. the lysosomal system) of the suramin-treated HT-29-D4 cells. The albumin-directed targeting of suramin in lysosomes may allow the drug to inhibit the activity of several lysosomal hydrolases, resulting in a lysosomal storage disorder.

The concentration of glucose in the culture medium determines the effect of suramin on the growth and differentiation of the human colonic adenocarcinoma cell clone HT29-D4.

Suramin, a drug currently used for advanced malignancy, induces the differentiation of the human colonic adenocarcinoma cell clone HT29-D4 and this process is correlated with a decreased glycolytic activity. We investigated the effects of suramin on HT29-D4 cells in the presence of various glucose concentrations. The main result of this study is that suramin has only an effect on HT29-D4 cell growth and differentiation when the concentration of glucose is above 10 mM. Therefore the efficiency of suramin as an anticancer drug may be greater on poorly differentiated tumoral cells with a high proliferative capacity.

Human T-lymphoblastoid cells selected for growth in serum-free medium provide new tools for study of HIV replication and cytopathogenicity.

Human T-lymphoblastoid cells H9, CEM and CEM-clone 5 were selected for growth in RPMI 1640 supplemented with transferrin 5 micrograms/ml, insulin 5 micrograms/ml and sodium selenite 5 ng/ml. After 40 days of adaptation to serum-free medium, these cells displayed growth, morphology, and expression of CD4 similar to serum-supplemented cultures. Infection of these cells with two strains of HIV-1 (LAV and NDK) and a strain of HIV-2 (ROD) was as efficient in serum-free as in serum-supplemented medium as demonstrated by reverse transcriptase activity in the culture supernatants of infected cells. Furthermore, HIV-induced cytopathogenicity was observed in serum-free cultures, demonstrating that both HIV infection and cytopathic effect did not require the presence of serum components. Electron microscopy showed that mature viral particles were produced from infected cells cultured in serum-free medium. Finally, the ability of monoclonal antibody OKT4 A to inhibit infection by HIV-1 LAV but not by HIV-1 NDK was the same with and without serum in the culture medium, demonstrating that both CD4-dependent and CD4-independent infections can occur in the total absence of serum. Human T-lymphoblastoid cells adapted for growth in serum-free medium provide therefore a complementary tool for the study of HIV infection and cytopathogenicity under defined conditions.

Production of a highly cytopathic HIV-1 isolate from a human mucosal epithelial cell line cultured on microcarrier beads in serum-free medium.

The human colonic epithelial cell line HT-29 can be productively infected with various HIV-1 and HIV-2 isolates that are highly cytopathic for T lymphocytes. In each case, a chronically infected HT-29 cell line can be established, and progeny viruses retain their original properties including high cytopathogenicity for T cells. Inasmuch as AIDS vaccines should include viral isolates capable of infecting mucosal epithelial cells, it may be useful to produce these isolates in such cells at a large scale. We describe here a microcarrier-based culture system allowing the production of infectious viruses from HT-29 cells grown in a chemically defined serum-free medium (Dulbecco's modified Eagle's medium/F12, HEPES 15 mM, pH 7.4, transferrin 5 micrograms/ml, selenium 10 ng/ml). The yield of HIV-1 from microcarrier cultures (275 ng of p24gag/ml) was greater than the yield from conventional culture flasks (122 ng of p24gag/ml). This virus, produced in serum-free medium, can be used either as a viral stock or as a source for HIV-1 proteins.

[Suramin inhibits the proliferation and stimulates the differentiation of tumoral cell lines HT29-D4 and C6]. / La suramine inhibe la prolifération et stimule la différenciation des lignées de cellules cancéreuses HT29-D4 et C6.

Suramin is an anti-helminthic drug that has been shown to antagonize the effects of a variety of growth factors including EGF, PDGF and TGF beta. When added to the culture medium, suramin inhibited the proliferation of both human colonic adenocarcinoma cells HT29-D4 and rat glioma cells C6. Suramin also induced the differentiation of both cell lines: appearance of cellular extensions for C6 cells, enterocyte-like epithelial differentiation for HT29-D4-cells. In the latter case, suramin probably acts at the level of glucose metabolism, which is likely to be modulated by autocrine growth factors. The permanent secretion of such factors probably stimulates HT29-D4 proliferation and simultaneously inhibits their differentiation. It is hypothesized that interfering with this autocrine loop, suramin allows HT29-D4 cells to differentiate.

Simulation of cellular packing in non-proliferative epithelia.

The physical laws governing the morphogenesis of biological tissues remain largely misunderstood. In particular, the role of the mechanical interactions occurring in this process needs to be better understood and studied. Inner follicular cells surrounding the oocytes of Ciona intestinalis form an epithelial monolayer resulting from an accretion process (without mitosis or apoptosis). This epithelium is elementary and useful for morphogenesis studies: the cells exhibit polygon packing with a specific but non-systematically repeatable topology (i.e. the distribution of pentagons, hexagons and heptagons changes). To understand the role of mechanical forces in tissue formation, we propose an innovative "2D spherical" model based on the physics of divided media. This approach simulates the cellular mechanical behavior and epithelium structuration by allowing cells to adopt a large variety of shapes and to self-organize in response to mechanical interactions. The numerical parameters considered in the model are derived from experimental data in order to perform pertinent and realistic simulations. The results obtained are compared to biological observations using the same counting method to characterize epithelium topology. Numerical and experimental data appear close enough to validate the model. It is then used for exploratory studies dealing with "Tissue Development Speed" variation, which is not easily attainable by experimentation. We show that the formation speed of the tissue influences its topology and hence its packing organization.

[The in vitro differentiation of cancer cells of the human colon (HT29-D4) is associated with the presence of annulate lamellae in the basal part of the cytoplasm]. / La différenciation in vitro de cellules cancéreuses du colon humain (HT29-D4) est associée à la présence de lamelles annelées dans la partie basale du cytoplasme.

The morphological differentiation of HT29-D4 cells is associated with the occurrence of annulate lamellae in the basal part of the cytoplasm. These structures could constitute a new marker for the in vitro differentiation of intestinal epithelial cells.