Guillemette Chevalier

1,25-DIHYDROXYVITAMIN D3 INDUCES PROGRAMMED CELL DEATH IN A RAT GLIOMA CELL LINE

1,25‐Dihydroxyvitamin D3 (1,25(OH)2D3), a seco‐steroid hormone with potential antitumoral activities, has been recently reported to exert cytotoxic effects on C6 glioma cells. However, the molecular mechanisms which trigger this cell death remain unknown. We show here that this 1,25(OH)2D3‐induced cell death is dependent upon protein synthesis and is accompanied by the expression of c‐myc, p53, and gadd45 genes. Two other genes, coding for interleukin‐6 and vaso‐endothelial growth factor, are also up‐regulated after addition of 1,25(OH)2D3. This programmed cell death can be suppressed when cells are treated with forskolin, a drug which increases intracellular cAMP concentration, or with genistein, an inhibitor of tyrosine protein kinases. However, in spite of the demonstration of fragmented DNA in 1,25(OH)2D3‐treated cells, the C6.9 cells used in this study do not show the classical morphological features of apoptosis. These results provide the first evidence for the existence of a programmed cell death triggered by 1,25(OH)2D3 in glioma cells and may provide a basis for the development of new therapeutic strategies. In addition, these data also suggest that the treatment of C6.9 cells with 1,25(OH)2D3 may be a useful model to study the molecular mechanisms involved in the programmed cell death of a cell of glial origin.

Vasoactive intestinal peptide (VIP) control of glycogenolysis in the human colon carcinoma cell line HT-29 in culture

Monique ROUSSET, Marc LABURTHE+, Guillemette CHEVALIER, Claudine BOISSARD+, Gabriel ROSSELIN+ and Alain ZWEIBAUM Groupe de Recherches sur I’lmmunologie de la Diffdrenciation, INSERM UI 78, CNRS ER 231, Hepital Broussais, 96 rue Didot, 75674 Paris Cedex 14 and ?Jnitt de Recherches de DiabPtologie et d’Etudes Radio-Immunologiques des Hormones Proteiques, INSERM U5.5, CNRS ERA 494, Hbpital Saint Antoine, 184 rue du Faubourg Saint Antoine, 75571 Paris Cedex 12, France

Cytotoxic effects of 1α,25-dihydroxyvitamin D3 and synthetic vitamin D3 analogues on a glioma cell line

1 alpha,25-Dihydroxyvitamin D3 (1 alpha,25(OH)2D3) has recently been reported to exert a toxic effect on both rat and human glioma cell lines. However the potential clinical use of 1 alpha,25(OH)2D3 in the treatment of glioma is impaired by its potent hypercalcemic effects. We have therefore investigated the effects on glioma cell growth of several vitamin D3 analogues which have previously been shown to be less calcemic in vivo than 1 alpha,25(OH)2D3. The present study shows that several analogues are able to induce, in vitro, the death of rat glioma cells (C6.9). The compound KH 1060 appears to be the most effective in the induction of cell death, while MC 1288 and CB 1093 are as potent as 1 alpha,25(OH)2D3. EB 1089 was somewhat less effective than 1 alpha,25(OH)2D3 and MC 903, which is currently used in the treatment of psoriasis, has only a weak activity on C6.9 cells. The effective doses used are around 10(-9) M for 1 alpha,25(OH)2D3 and 10(-10) M for KH 1060. Interestingly, the toxic effect exerted by 1 alpha,25(OH)2D3 and its analogues is accompanied by several of the biochemical features of apoptosis, such as DNA fragmentation and induction of the c-myc protooncogene. These findings, together with the fact that the therapies currently available for glioma are only palliative, suggest that 1 alpha,25(OH)2D3 analogues such as KH 1060, EB 1089 or CB 1093, alone or in combination with other therapeutic approaches, could be of potential interest in the treatment of brain glial tumors.

Vitamin D receptor stable transfection restores the susceptibility to 1,25‐dihydroxyvitamin D3cytotoxicity in a rat glioma resistant clone

Recently, 1,25‐dihydroxyvitamin D3 (1,25‐D3) and less hypercalcemic analogs were shown to exert a delayed cytotoxic effect on rat C6 glioma cells. 1,25‐D3 induces in these cells a programmed cell death, accompanied by the induction of c‐myc, p53 and gadd 45 genes. The involvement of the intracellular vitamin D receptor (VDR) remained to be determined. In this lethal process, we have investigated its role in a subclone of C6 cells, which was isolated on the basis of its resistance to 1,25‐D3, and in which VDR expression was not detected either at the mRNA or protein levels. The stable transfection of a rat VDR cDNA into this clone restored its susceptibility to the cytotoxic effects of 1,25‐D3. This phenomenon was accompanied by a dramatic upregulation of c‐myc mRNA expression, as already described in a C6‐sensitive clone. These results provide the first evidence that VDR expression, if not sufficient, is necessary to mediate 1,25‐D3 cytotoxic effect in C6 glioma cells. Since VDR mRNA expression has been already reported in human brain tumors, our data imply that the identification of VDR expression could become a prerequisite in any strategy of glioma treatment with vitamin D analogs. J. Neurosci. Res. 52:210–219, 1998. © 1998 Wiley‐Liss, Inc.

Peptide receptors in human lung tumor cells in culture: Vasoactive intestinal peptide (VIP) and secretin interaction with the Calu-1 and SW-900 cell lines

Abstract We have investigated the interaction of VIP and secretin with two human lung carcinoma cell lines in cultures, SW-900 and Calu-1. 125 I-labeled VIP binds to and is inactivated by SW-900 and Calu-1 cells in a time- and temperature-dependent manner. The rates of binding and of inactivation were higher at 30°C than at 15°C. At equilibrium, native VIP competitively inhibited the binding of 125 I-VIP in the 10 −10 −10 −7 M range, half-maximal inhibition being observed at 1.2 nM in SW-900 cells and at 1.1 nM VIP in Calu-1 cells. Scatchard analysis indicated two classes of binding sites with similar characteristics in both cell lines. SW-900 cells have 27 600 sites with a high affinity ( K d = 0.34 nM ) and 1062 000 sites with a low affinity ( K d = 61.4 nM ). Calu-1 cells have 36 300 sites with a high affinity ( K d = 0.33 nM ) and 1148 000 sites with a low affinity ( K d = 78.6 nM ). Secretin inhibited tracer binding but with a 5000 times lower potency than native VIP in both cell lines.

Noradrenaline inhibits the programmed cell death induced by 1,25-dihydroxyvitamin D3 in glioma.

The rat glioma cell line C6.9 has been recently reported to respond to 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) by the induction of a programmed cell death. Since, in vivo, glial cells are thought to be exposed to several neurotransmitters, we investigated the possibility of a neurotransmitter-mediated inhibition of this active cell death process. Noradrenaline and the beta-adrenoceptor agonist isoproterenol showed significant inhibition of the 1,25(OH)2D3-induced programmed cell death. The beta-adrenoceptor antagonist propanolol reversed this inhibition, while the alpha-adrenoceptor antagonist yohimbin was devoid of any effect. This suggests that the efficiency of antiproliferative vitamin D-related therapies could be influenced by endogenous levels of noradrenaline.

Monensin and forskolin inhibit the transcription rate of sucrase‐isomaltase but not the stability of its mRNA in Caco‐2 cells

Treatment of Caco‐2 cells with forskolin (25 μM) or monensin (1 μM) has previously been shown to cause a marked decrease in the level of sucrase‐isomaltase (SI) mRNA, without any effect on the expression of dipeptidylpeptidase IV (DPP‐IV). In the present work, we report that there is no significant difference in the stability of SI mRNA between control and treated cells. On the other hand, we demonstrate a decrease in the transcription rate of SI mRNA which is sufficient to account for the decrease in the steady‐state level of SI mRNA both in forskolin‐ and monensin‐treated Caco‐2 cells.

Proliferation, differentiation and maturation of a mouse epidermal keratinocyte cell line.

Abstract The spontaneous development of a cell line of neonatal mouse C3H/He epidermal cells is described. The culture has been serially passaged at 29 °C over 18 months in the absence of any dermal support. The cell morphology of the 18th passage is reported. During early growth phase, the morphology of the cell layers was similar to that observed in the basal and differentiating strata of the epidermis: numerous tonofilament bundles and desmosome-filament complexes were observed. During late growth phase, maturation and vertical stratification occurred: demonstrated by the tonofilament accumulation, cell organelle degradation, nuclear pyknosis, presence of keratohyalin granules and horny cell layers with thickened membranes. Hemidesmosome-like structures were shown. No basal lamina or membrane coating granules were detectable. The 18th passage cultured cells did not induce tumors in nude mice. This keratinocyte cell line is not permanent, however: a malignant transformation occurred after 25 subcultures which resulted in an undifferentiated cell population.

Ultrastructural and biochemical characterization of a cloned mouse keratinocyte cell line

Abstract A cloned population of mouse C3H/He keratinocytes was obtained from the 14th passage of an epidermal cell line. A two-step cloning procedure using Petriperm dishes was performed. The cloned population, grown at 34 °C, was subcultured more than 30 times over a one year period. By day 14, three cell layers were formed; the ultrastructural morphology and immunofluorescence characterization of these layers showed numerous tonofilament bundles and well organized desmosome tonofilament structures. They thereby resemble the proliferative compartment of the epidermis. High resolution acrylamide gel electrophoresis of the keratins extracted from the cloned cells showed the presence of many keratin subunits. The tonofilaments extracted from the cell layers, as well as from the supernatant cells, contained a small quantity of high MW keratins (rel. MW 63 000; apparent isoelectric point 5.5–6.2). These results indicate that the cloned keratinocyte cell line had retained a certain maturation capacity in culture.

Colon Cancer Cell Differentiation as Related to Methotrexate and 5-Fluorouracil Resistance

Colon cancer is a major health problem because of its high frequency and the poor outcome of invasive forms, due to their overall resistance to chemotherapy. Although it has been assumed that drug resistance could be associated with some particular cell populations, these have not been characterized yet. It is only in recent years that progress in the field of intestinal cell biology, based on the development of cultured cell lines and availability of immunological and molecular probes, has allowed to characterize cells at the Single cell level and to study their Organization and functions. This has led to the concept of colon cancer cell differentiation. Cellular differentiation, which should not be confused with differentiation of colon cancers as defined by pathologists, is the ability of colon cancer cells to express, in vitro and in vivo, the same morphological and functional characteristics as normal epithelial intestinal cells, i.e. enterocytes or goblet cells. Whether these cells, which behave like normal cells as to their differentiation characteristics and functions, possess particular adaptation properties which allow them to escape the cytotoxic effect of a number of stress conditions, including treatment with anti-cancer drugs, is based on recent experimental data obtained with cultured human colon cancer cell lines. The purpose of this article is to (1) summarize our knowledge on colon cancer cell differentiation and show how experience in the field of cell biology can be transferred to clinical situations and (2) focus on experimental data which suggest that drug resistance is associated with cellular differentiation.