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.

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.

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.

Interest of growth hormone-releasing hormone administration for improvement of ovarian responsiveness to gonadotropins in poor responder women.

OBJECTIVE: We have investigated the beneficial effect of a somatotroph axis stimulation on ovarian response to gonadotropin. DESIGN: Growth hormone-releasing hormone (GH-RH) was administered in a prospective study in women undergoing an in vitro fertilization protocol. PATIENTS: Twelve patients were selected for their poor ovarian response to previous stimulations using gonadotropin-releasing hormone analog (GnRH-a) and human menopausal gonadotropins (hMG). INTERVENTIONS: Five hundred micrograms of GH-RH1-29 were administered two times daily concomitantly with GnRH-a and hMG from day 2 of the cycle to the time of ovulation. MAIN OUTCOME MEASURES: Stimulation of somatotroph axis was appreciated by measuring over-night urinary growth hormone (GH) output, plasma GH, and insulin-like growth factor I (IGF-I) and follicular fluid (FF) IGF-I. The effects of GH-RH administration on ovarian function were determined by plasma estradiol levels and follicular data. RESULTS: Administration of GH-RH was associated with a significant improvement of urinary (P less than 0.025) and plasma (P less than 0.001) GH concentrations and of the hormonal response to hMG (P less than 0.01). Levels of IGF-I followed a biphasic plasma variation, and a slight increase in recruited follicles, retrieved oocytes, and FF IGF-I content was also observed. CONCLUSIONS: Activation of the somatotroph axis by GH-RH enhances the hormonal ovarian response to hMG and may be an adjunctive therapy to improve follicular maturation.

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.

De novo interstitial deletion of the long arm of chromosome 7:46,XY,del(7)(q23;q32).

A de novo interstitial deletion of the long arm of chromosome 7 is reported in a newborn boy. Our observation is compared with seven others deletions of the same bands. Clinical features showed the following: hypotonia, microcephalia, difficulty in swallowing, low-set dysplastic ears, an abnormal cry, upslanting and small palpebral fissures, and abnormalities of the hands and feet. Delayed mental and physical development is the general rule, and visceral malformations are uncommon. Our patient had genital abnormalities and a cardiac malformation.