Delivery and pathway in MCF7 cells of DNA vectorized by cationic liposomes derived from cholesterol.

We have investigated the delivery and the pathway in tumoral MCF7 cells of DNA carried by liposomes prepared from (trimethyl aminoethane carbamoyl cholesterol iodide (TMAE-Chol), a cholesterol-based cationic lipid with a quaternary ammonium on the polar head. The structure of DNA-liposome complexes depends on the length of DNA and on the lipid-DNA charge ratio X. Spherical beads constitute fine structures of the observed complexes even when they appear as aggregates. For oligonucleotide transfer, dissociation from liposomes after transfection, penetration of the oligonucleotides into nuclei, and a long resident time were observed. For plasmid transfer, a correlation between the variation in the transfection level and the ultrastructure of complexes was demonstrated. The results showed a cellular route of lipid/plasmid complexes from the beginning by endocytosis, entrapped into endosomes, released by the latter until entry in the perinuclear area, and then penetration of plasmids inside the nuclei resulting in the observed expression of the beta-galactosidase gene.

Plasmovirus: replication cycle of a novel nonviral/viral vector for gene transfer.

We recently described a novel nonviral/viral vector for gene transfer, the plasmovirus (Noguiez-Hellin P, Robert-le Meur M, Laune S, et al. C R Acad Sci Paris, Sciences de la Vie. 1996;319:45-50; Noguiez-Hellin P, Robert-le Meur M, Salzmann J-L, et al. Proc Natl Acad Sci USA. 1996;93:4175-4180). Plasmoviruses are plasmids capable of expressing all the viral genes required for generating infectious particles and packaging a defective genome containing a transgene. Transfected as plasmids, plasmoviruses transform the transduced cells into packaging cells that release infectious replication-defective retrovirus vectors (RV) containing a transgene, which are capable of infecting nearby cells. We previously showed that such a vector can efficiently "propagate" the transgene after transfection. Here we examine in greater detail the different steps of plasmovirus replication in vitro in human (143 B TK-) and murine (NIH 3T3 TK-) cells. Molecular-biological analysis revealed plasmovirus-coded protein expression starting from 24 hours post-transfection, followed by the detection of infectious RV 48 hours post-transfection. The gag proteins were correctly processed in the released particles. Electron microscopic analysis revealed typical type C particles. Nonintegrated plasmovirus DNA was not toxic for the cells and could be detected for at least 14 days post-transfection. While the transfected gag gene and the transgene could also be detected throughout this period, we observed that env-coded proteins decreased after 72 hours post-transfection. Nevertheless, the production of RV resulted in the propagation of the transgene in the culture, with stable integration of plasmovirus proviral DNA into the host genome of infected cells. We show that this propagation results in a major improvement in therapeutic efficacy using an HSV1-TK transgene and ganciclovir treatment, when compared to that of plasmovirus constructs that cannot propagate. Altogether, these results demonstrate the functionality of this gene transfer method and suggest that improvements in the vector design enhance its efficacy.

Interactions of lymphokine-activated killer cells and A549 lung carcinoma nodules maintained in three-dimensional culture.

Lymphokine-activated killer (LAK) cells were cocultured in the presence of interleukin-2 (IL-2), with pulmonary surfactant secreting A549 lung carcinoma nodules and maintained in continuous three-dimensional culture for 2-6 days in an attempt to test the response of tumor cells which produce LAK cell inhibitory substances. The A549 nodules secrete mucus which envelops them. This mucus is also secreted inside pseudoalveolar structures characteristic of these nodules. The mucus contains the pulmonary surfactant and sialomucins, both LAK cell inhibitory substances. The spontaneous infiltration into the neoplastic tissue and the membrane contacts established between the two cell types were studied by means of histological, immunohistochemical and electron-microscopic methods. Free-floating LAK cells were allowed to sediment and adhere freely to the nodule surface. The cytostatic and cytolytic effects of LAK cells were tested using thymidine incorporation into DNA and flow cytometry. Despite the presence of a mucus envelope, LAK cells adhered to the A549 nodule surface and penetrated spontaneously into them in the presence of IL-2; they settled mainly in the pseudoalveolar structures where they became apoptotic. According to electron-microscopic observations performed on the second day of coculture, the LAK cells, which remained between the cancer cells, established mostly pinpoint contacts with the carcinoma cells, forming cytoplasmic fusions. These fusions indicate the induction of pores in both the cancer cell and the LAK cell membranes. Electron-microscopic observation also displayed LAK-cell-associated apoptotic and necrotic carcinoma cells. However, at this stage of the coculture (day 2), the DNA synthesis rate of the A549 nodules still remained unchanged; it diminished by approximately 3 times on day 4 and almost stopped on day 6: nodule disintegration was then complete. In the free-floating LAK cell component of the cocultures, DNA synthesis was already strongly inhibited (26x) by the second day. Nevertheless, their cytolytic effect remained unaltered, as was tested on A549 monolayer cells. The presence of tumor necrosis factor (TNF) in the coculture supernatant has been demonstrated, and when coculturing took place in the presence of monoclonal TNF antibody, nodule proliferation was significantly enhanced (up to 145%). Our results indicate that, despite the presence of pulmonary surfactant and sialomucins containing mucus, LAK cells were capable of killing lung carcinoma cells in three-dimensional culture at an early stage of coculture (day 2) by direct cell-to-cell contact. Total nodule disintegration, however, was complete much later (on day 6), and taking into account the low amount of LAK cells in the cancer tissue, this seemed to be the result of an indirect effect implying, in particular, the presence of soluble TNF.

Effects of lymphokine-activated killer-cells on melanoma nodules maintained in 3-dimensional culture.

Lymphokine activated killer (LAK) cells were cocultivated from 2 to 6 days with WM266 metastatic melanoma cells maintained as nodules in organotypic culture. The LAK cells in suspension were allowed to deposit freely on the nodule surface from where they could infiltrate spontaneously into the nodules. Immunohistochemical studies were done to localize the LAK cells as well as electron microscopical observations for effector/target membrane contacts. Proliferation of the nodules was tested and also that of the LAK cells after coculturing using tritiated thymidine incorporation into DNA. Cell death was determined by arrest of thymidine incorporation and total nodule disintegration. Infiltration rate of LAK cells into the nodules was low: after coculturing 5% of the nodule cells were LAK cells. Although close membrane contacts and cytoplasmic fusions between effector and target cells leading to tumor cell apoptosis were observed, this direct cytolytic process seemed to be too infrequent for the induction of total nodule disintegration at day 6. Therefore, the indirect pathway to cytolysis might be predominant implying, among other cytokines, soluble TNF. On the other hand, LAK cell proliferation diminished strongly after coculturing (down to 11%) but the cytotoxicity was significantly enhanced (18% higher) suggesting an enhancement of differentiation. This might account for the peculiar efficacy of LAK cells on melanomas in vivo and it would be of interest to study this phenomenon further.

Optic atrophy, microcephaly, mental retardation and mosaic variegated aneuploidy: a human mitotic mutation.

A 12-year-old girl presents with optic atrophy, pale papilla, amblyopia and microcephaly (-3 s.d.) with mild mental retardation and facial dysmorphism. She had mitral insufficiency with mitral prolapse and moderate short stature (-2.5 d.s.). She had normal flash visual evoked potentials, normal electroretinograms and electrooculograms and normal cranial CT scan as well as other lab tests to rule out malformations, tumors or multiple sclerosis. Her lymphocyte karyotype showed a variegated mosaicism with: 2 cells with 49, XX, +mar,+mar,+mar; 21 cells with 48, XX, +mar,+mar; 57 cells, with 47, XX,+mar; 20 cells with 46,XX; while parental karyotypes were normal. This syndrome therefore associates optic atrophy, mental retardation and microcephaly and short stature with chromosomal instability in the form of variegated mosaicism.

Lymphokine-activated killer cells induce differentiation in MCF-7 breast carcinoma nodules but not in mastosis nodules maintained in three-dimensional culture.

In order to better understand the interaction between activated lymphocytes and breast carcinoma cells, we studied the degree of infiltration, the membrane contacts established and their cytostatic and cytolytic effects in MCF-7 nodules maintained in three-dimensional culture. A comparison was made with nodules of a nonmalignant, immortalized mastosis cell line. Histological, immunohistochemical and electron microscopical observations were performed as well as DNA synthesis measurements in the two components of the coculture. The lymphokine-activated killer (LAK) cells adhered more frequently to the carcinoma nodules than to the mastosis nodules. They actively penetrated into both of them. The penetration remained peripheral, and only a few cells migrated more deeply. The LAK cells established close cell-to-cell contacts with the two types of nodules, and intercellular gaps were formed: damaged cells could be seen near the activated killer cells. In MCF-7 nodules, a 5-fold inhibition of proliferation occurred, and extensive necrotic zones developed; this was accompanied by a general tendency for glandular redifferentiation. In mastosis nodules, necrosis also developed but no cell differentiation occurred and proliferation was less inhibited (2 times). Interleukin-2 alone enhanced DNA synthesis in mastosis nodules but had no effect on MCF-7 nodules, and no extending necrosis could be seen in both types of nodules. The cytolytic effects of LAK cells combined with their redifferentiating effect in MCF-7 breast carcinoma nodules may be a useful indication for further breast cancer therapy research.

[Effects of LAK cells activated by IL-2 on MCF-7 human breast cancer cell line maintained in organotypic culture]. / Effets des cellules LAK en présence d'IL-2 sur la lignée carcinomateuse mammaire humaine MCF-7 maintenue en culture organotypique.

Lymphokine Activated Killer (LAK) cells, stimulated by interleukin 2 (IL-2) have a pronounced antitumor effect in the therapy of melanoma and renal cancers. LAK cells were cultivated in presence of the nodules of the human breast adenocarcinoma cell line MCF-7 maintained in organotypic culture to study the interactions between lymphocytes and breast tumor cells. After two days of co-culture, the proliferation of MCF-7 nodules and that of LAK cells was diminished about five folds. The cytotoxic effect of the latter, appreciated by Chrome 51 release was unchanged after the coculture. In histological sections, the penetration of the LAK cells into the MCF-7 nodules was accompanied by an increase of tumor necrosis but also by a glandular differentiation of cancerous tissue. Polarized epithelial cell formations bording neoplasic lumens with intracytoplasmic vacuoles filled with mucus, appeared in the nodules. The immunohistochemistry underlines the presence of T lymphocytes marked by UCHL1 and CD3 antibodies and of Natural Killer (NK) cells marked by IOT10, located between the MCF-7 cancer cells. In electron microscopy, the membrane contacts were tight and were accompanied by the appearance of secondary lysosomes and nuclear alterations. The relatively low infiltration level of the nodules may lead to the supposition that an indirect mechanism will intervene in this dual action of a LAK cells: increase of necrosis, although partially, and development of glandular and functional differentiation.

Phenotypes of human epithelial cell lines immortalized from benign mastopathies.

We have cultured cells from normal and benign mastopathy tissues under conditions favoring epithelial cell proliferation. However, such primary cells rapidly lose their ability to grow in vitro. Immortal lines were established after transfection with SV 40 T gene and several cell clones isolated from Normal Breast Adjacent to a tumor (NBAT 32 T2 to T7) and two benign mastopathies (NPM 21 T1 to T14 and NPM 14 T4). This immortalization of epithelial cells reduced the doubling time of cultured cells and increased the densities of the cultured cell populations. Among there cell lines we have characterized five clones and found differences in a number of aspects. Southern blot analysis showed that the SV 40 T gene was stably integrated in the genome of all the established cell lines. Two of the clones (NPM 21 T2 and NPM 21 T4) were nearly diploid showing a high degree of stability. Two other clones (NPM 14 T4 and NPM 21 T1) had near-tetraploid karyotypes, although quite heterogeneous. Comparison of the ultrastructural phenotypes shows that the two near-diploid cell lines were more differentiated than the two others. Estradiol receptors measured by Scatchard analysis and by transactivation of vitellogenin promotor were absent from all the cell lines. Progesterone receptors measured by Scatchard analysis of hormone binding were present in the NPM 14 T4 and NBAT 32 T4 cell lines. The NPM 21 T1 cell line did not contain such steroid receptors. These cell lines are persistent in long-term culture, thus providing a useful in vitro model system for studying factors involved in the proliferation and the transformation of human mammary epithelial cells.

Unbalanced karyotype due to adjacent 1 segregation of t(11;22)(q23.3;q13.2).

The 11q;22q translocations, whatever the breakpoints may be, are of particular interest because of their propensity to 3:1 segregation of the chromosomes at meiosis I. Until now, no unbalanced karyotype resulting from 2:2 adjacent segregation was published among offspring of 11q;22q translocation carriers. The authors report the case of an unbalanced karyotype due to adjacent 1 segregation of a maternal translocation (11;22)(q23.3;q13.2). The proband's karyotype was 46,XX,-22,+der(22)(11;22)(q23.3;q13.2)mat. This finding demonstrates that adjacent 1 segregation is possible in t(11;22) with breakpoints at 11q23 and 22q13, and can lead to birth of viable infants.

Chromosome analysis of multipronuclear human oocytes after in vitro fertilization.

Multipronuclear human eggs are frequent after in vitro fertilization. Their chromosome analysis can provide useful information. Before cleavage it can confirm the suspected polyploidy. Among the cleaved multipronuclear eggs it provides an estimation of the incidence of the possible return to diploidy. Ninety-four multipronuclear eggs were fixed at the first, second, or third cleavage according to the air-drying method of Tarkowski with or without colchicine exposure: 60 were successfully analysed. Twelve were stopped before cleavage (six without colchicine treatment and six with colchicine treatment). They were polyploid, confirming the cytological observation. Forty-eight eggs cleaved and were stopped by colchicine treatment and karyotyped. Seventeen eggs (35 per cent) had produced diploid embryos. Mosaicism was frequent (15 cases, 31 per cent). Triploidy was not frequent (8 eggs, 17 per cent). Haploidy constituted the remaining cases (8 eggs, 17 per cent). Our data indicate that the initial count of pronuclei is a reliable test. Multipronuclear one-cell oocytes were confirmed to be polyploid. Furthermore, the developmental capacity of the multipronuclear oocytes is variable. Most of them cleaved. However, many multipronuclear oocytes led to diploid cleaving eggs.

Cytogenetic study of human oocytes uncleaved after in-vitro fertilization.

Chromosome analysis of oocytes uncleaved after IVF allows the cause of the failure of cleavage to be determined and shows the incidence of chromosome disorders among human oocytes. A total of 198 uncleaved oocytes fixed 40 h after insemination were successfully analysed according to Tarkowski's air-drying method: 78.3% were unfertilized and arrested in metaphase II. Among them, 70% were normal (23,X) and 30% aneuploid (16% were hypohaploid, 14% were hyperhaploid). The incidence of chromosome breaks was 18%. In 12.1% of the oocytes, sperm chromosome condensation appeared premature usually in the G1 phase. This was especially observed in idiopathic infertility (7% of fertilized oocytes versus 2% in tubal infertility cases). In 8.1% of the cases, chromosome analysis showed diploidy which may be interpreted by either an absence of extrusion or a reintrusion of the polar body or by first cleavage failure during mitosis. In 1% of the cases triploidy was observed. Our results show that the main reason for failure of cleavage is related to failure of fertilization (78.3%). However, premature condensation of sperm chromosomes at the G1 phase appears to be quite frequent. This may be involved in the aetiology of some cases of idiopathic infertility. Finally, the high rate of chromosomal disorders (30%) in human oocytes may explain the high rate of chromosomal abnormalities in preimplantation embryos.

Fetal myofibroblast-like cells isolated from post-radiation fibrosis in human breast cancer.

Cells were isolated from post-radiation fibrosis biopsies of patients with recurrent breast carcinoma. These cells were identified as fibroblasts and compared with fibroblasts from normal breast tissues for their proliferative activities, chromosome number and for the presence of various components of the extracellular matrix and cytoskeleton. The proliferative activity of the fibrosis-derived fibroblasts did not significantly differ from that of normal breast fibroblasts. Both cell types required serum to grow and did not form colonies in soft agar. Cells from 2 of the 3 fibroses analyzed displayed aneuploid karyotypes with multiple structural abnormalities. All of the fibroblastic cells produced types I, III and V collagen, fibronectin and vimentin. However, in contrast to normal breast fibroblasts, fibrosis-derived cells produced high amounts of oncofetal fibronectin. In addition, fibrosis of fibroblasts also expressed the alpha-actin isoform which is specific for smooth-muscle cells. These results suggest that post-radiation fibrosis in malignant breast contains atypical fibroblasts with fetal and myofibroblastic characteristics.

[Morphometric study of ultrastructural modifications in the maternal hepatocyte during normal gestation in the Wistar rat]. / Etude morphométrique des modifications ultrastructurales de l'hépatocyte maternel au cours de la gestation normale chez la rate Wistar.

Quantitative changes in hepatocyte ultrastructures during normal gestation were studied in Wistar rats with morphometric methods. On the 18th day of the gestation, variations in nuclearcytoplasmic ratio, size of R.E.R., mitochondria, lysosomes and microbodies were observed, with variations according to the localisation of hepatocyte inside the lobule. We report the most obvious effects as follows: An increase of R.E.R. in the central and perilobular zones. Mitochondria are larger and the rounded forms are more numerous inside the two lobular zones. The number of lysosomes and microbodies are only elevated in the perilobular cells. In conclusion, it is suggested that the hepatocyte organelles are significantly increased during gestation. This is probably due to the metabolic activation. These cellular modifications are only one aspect of the liver increase.

[Quantitative cytologic and histologic study of the maternal hepatocyte and lobule on the 18th day of gestation in the Wistar rat]. / Etude cytologique et histologique quantitative de quelques aspects de l'hépatocyte maternel et du lobule au 18e jour de la gestation chez le rat "Wistar".

With the object of studying the part taken by liver cells and lobules in mother's liver weight increase (+42%), planimetric cells areas were measured. The main results are: same diversity of cells size as in reference samples, perilobular cells remaining the larger. In the same way, one can note a small increase of cells size for pregnant animals: perilobular cells (+10%), nucleus (+26%), nucleolus (+19%), cytoplasms (+12%). These facts demonstrate the metabolic part played by liver during pregnancy but do not explain the liver weight increase. But planimetrical investigations of overall lobular size lead to an increase of 40%. Finally the increase of liver weight might be explained by an increase of cells number.

[Study of the weight of the pregnant rat and the maternal liver: their relation to biometric parameters of the developing embryo and placenta]. / Etude pondérale de la ratte gravide et du foie maternel: ses relations avec quelques paramètres biométriques de l'embryon et du placenta en développement.

Observations carried out on a large sample of pregnant rats, their fetuses and placentas, lead to the following findings: continuous increase of liver weight along pregnancy from 2nd to 20th days, mainly within the 2nd week (organs making), to reach limit value on the 18th day. This liver weight increases roughly along with embryos and placentas weights specially from 14th to 20th days. At the 18th day the mother's liver weight depends on embryos and placentas weights. This weight of the mother's liver increased with embryos and placentas number but does not depend on resorption number. The overall mother's weight is proportional to liver weight and embryos number. All these facts underline metabolic relations between mother and fetus during pregnancy.

Effects of gamma-irradiation on embryonic development and nucleic acids in trigeminal neurons.

Pregnant rats were exposed to absorbed doses of 0.5, 1, 2, and 4 Gy of 60Co-gamma rays either on day 8 or on day 12 p.c.. The embryos were collected on day 18 p.c.. Irradiations both on day 8 and 12 p.c. result in a dose dependent decrement of weight. The effect is larger for irradiation on day 8 p.c.. Caryometric studies of the neurons in the trigeminal ganglion show, on the other hand, that the reduction of nuclear sizes in this tissue is more substantial for an irradiation on day 12 p.c. when the ganglion is in a stage of formation. Cytophotometric determinations of the Feulgen-DNA content and determination of the RNA content by staining and chromic gallocyanin in combination with ribinuclease digestion lead to the conclusion that the irradiation induces no significant hyperploidy and that the irradiated neurons have the nuclear RNA content that is normal at this stage of gestation. This applies both to the irradiations on day 8 and on day 12 p.c.

[Detection and localization of organ-specific antigens at early stages of lens differentiation in the rat embryo (author's transl)]. / Mise en évidence d'antigènes spécifiques aux stades précoces de la différenciation du cristallin chez l'embryon de rat.

By means of liver-absorbed rabbit anti-adult rat lens sera, and by using the indirect immunoperoxidase technique, lens-specific antigens are detectable in a few isolated cells, or in small groups of cells, of the placode shortly after the onset of invagination. In the deeply invaginated lens rudiment, all the cells, exept those in the area adjacent to the ectoderm (presumptive lens epithelial cells) display a positive reaction. Once the lens vesicle has formed, all the cells contain detectable amounts of lens-specific antigens.

[Histological study of livers conserved for long periods (author's transl)]. / Etude histologique des conservations hépatiques de lognue durée.

33 pig livers were preserved using two comparative methods: extra-corporeal perfusion and immersion in hypothermia. The duration of conservation varied between 2 and 24 hours. A macroscopic and microscopic study carried out in all cases permitted us to define four cytological stages for the lesions, completed by an ultrastructural study showing the cyto-pathological course of the hepatocytes in two distinct cell series. The interest of this study is to better appreciate the degree of aggression of the lobule in relation to the metabolic gradient and that of the hepatic parenchyma. Analysis of the results is in favour of conservation of the liver by immersion.