Modulated expression of cell surface molecules and in vivo outgrowth of modified melanoma cells.

Modulation of cell surface molecules involved in immune recognition and cellular interactions (class I major histocompatibility complex or MHC-I, B7.1 or CD80, integrin alpha4 or CD49d, tetraspanins CD9, CD81) was examined in modified B16 melanoma cells displaying either inhibited IGF-I expression or transfected OVA encoding gene. It was shown that inhibiting IGF-I expression or inserting OVA encoding gene did not lead to modification relevant to the presence of MHC-I or B7.1. However downregulation of tetraspanin CD9 was observed in modified IGF-I but not in OVA encoding gene inserted melanoma cells. Expression of tetraspanin CD81 and integrin alpha4/CD49d remained unchanged. Inoculated into syngeneic recipients, the modified melanoma cells exhibited significant delayed outgrowth with a reduction in the percentage of lethal tumors observed essentially in hosts injected with inhibited IGF-I expression cells.

Proposition of treatment of cancer cells by nuclear protein mixtures from embryonic cells.

Several kinds of cancer cells are shown to revert to normal state by action of chemical or biochemical differentiation agents. The analogy between cancer and embryonic cells, with regard to the expression of oncogenes, and the presence, in young embryos, of regulating factors, lead to the proposition of treatment of cancer cells by extracts of nuclei from embryo cells. Preliminary experiments with these extracts, on hepatoma cells in culture, have shown growth and DNA synthesis inhibitions, without cell toxicity, and a prolongation of survival of rats injected with the treated cancer cells.

Prolongation of survival of rats injected with hepatoma cells treated by nuclei extracts from mouse and rat embryo cells.

Cancer cells express particular genes, part of which are normally active during the embryonic development. On the other hand, young embryos are able to differentiate teratocarcinoma or leukemia cells, likely by producing differentiation factors. In this work, rat and mouse embryo cell nuclei extracts were tested on hepatoma carcinoma cells LFCl2A: they inhibit the cell growth in culture and increase the survival of syngeneic rats injected with hepatoma cells incubated with these extracts. This inhibition is correlated with a decrease of DNA synthesis without toxic effect: It seems to be due to the mixture of tight binding DNA proteins, possibly transcriptional factors.

Induction of apoptosis in rat hepatocarcinoma cells by expression of IGF-I antisense c-DNA.

BACKGROUND/AIMS: We have developed a gene therapy strategy based on the observation that insulin-like growth factor I (IGF-I) is necessary for the acquisition and maintenance of the transformed phenotype in hepatocarcinoma. This strategy consists in transfecting the rat hepatoma cell line with an episomal vector expressing the antisense IGF-I c-DNA under the control of the metallothionein I promoter inducible by zinc, decreasing therefore the level of IGF-I in these cells. The transfected clones lost their tumorigenic properties, and were able to induce, in vivo, the regression of an established tumor in syngeneic rats. To understand the loss of tumorigenic properties of these transfected clones, we have quantified, by different approaches, the number of apoptotic cells according to the level of IGF-I expression. METHODS: IGF-I antisense synthesis in transfected cells was stimulated using zinc. We then characterized and quantified apoptosis, in these transfected clones, by morphological and DNA fragmentation analyses, flow cytometry and comet assay. RESULTS: We have demonstrated that IGF-I inhibits the development of apoptosis in parental cells, that the transfected clones are able to restore the spontaneous apoptotic programme, and that apoptosis increases massively when overexpression of IGF-I antisense is caused by zinc stimulation of the metallothionein I promoter. CONCLUSION: The present results allow us to conclude that the level of apoptotic pathway in liver cell lines is directly related to the amount of IGF-I deficiency.

Antisense insulin-like growth factor I transferred into a rat hepatoma cell line inhibits tumorigenesis by modulating major histocompatibility complex I cell surface expression.

We have established a hepatocarcinoma cell line (LFCI2 A) that produces voluminous tumors when injected subcutaneously into syngeneic Commentry rats. These neoplastic cells express both insulin-like growth factors (IGF) I and II. When transfected with an episomal cassette-expressing IGF-I antisense RNA, the modified LFCI2 A cell lines become poorly tumorigenic and, when injected subcutaneously, are associated with inhibition of the growth of the parental tumoral cells and/or induction of regression of established tumors. By contrast, cell lines isolated after transfection with the IGF-II antisense-expressing vector were as tumorigenic as the parental cell lines. The results are discussed in terms of protective immunity induced by the tumoral cells transfected by the IGF-I antisense vector. In the transfected hepatocarcinoma cells that do not produce IGF-I, the expression of major histocompatibility complex class I antigen was increased at least 4-fold compared with parental cells. The introduction of these cells in vivo induced a tumor-specific immunity that was associated with CD8 T cells.

Use of human lymphoblastoid cells to detect the toxic effect of chloramphenicol and metabolites possibly involved in aplastic anemia in man.

Some Chloramphenicol (CAP) metabolites are suspected to be involved in the etiology of bone marrow aplasia in man. The objective of the present study was to investigate the cytotoxicity as well as the genotoxicity of CAP and six of its metabolites on human bone marrow cells (RiBM cells) and to compare these results with those obtained on human peripheral blood lymphocytes in order to estimate the relative sensitivity of the two types of cells. Three CAP metabolites NO-CAP, DH-CAP and NPAP inhibited 3H thymidine incorporation in RiBM cells at concentrations ranging from 2.10(-5) M to 2.10(-4) M. NO-CAP appeared as the most potent cytotoxic compound. CAP itself and NAPD presented some toxic effect at high concentration (1-2.10(-3) M). CAPG and HAP did not present any cytotoxic effect. By comparison, the response of human lymphocytes to CAP and its metabolites showed a similar pattern but DH-CAP was the most inhibitory compound. Concerning the genotoxic potential, NO-CAP and DH-CAP induced DNA single strand breaks in RiBM cells at concentrations of 1 and 2.10(-4) M with a dose response relationship. CAP and other metabolites were completely devoid of genotoxicity up to 4.10(-3) M. The results clearly showed that RiBM cells were much less susceptible to the genotoxic effect of CAP metabolites than human lymphocytes.

[Immunotherapy of tumors expressing IGF-I]. / Immunothérapie des tumeurs exprimant l'IGF-I.

Recently we demonstrated that rat glioma cells when transfected with a vector encoding antisense IGF-I c-DNA lost tumorigenicity and induced a tumor specific immune response involving CD8+ lymphocytes. Here we showed, using immunostaining flow cytometry analysis, that the transfected cell lines, rat C-6 glioma and rat LF hepatoma, expressed an increase level of MHC-class I, and even the amount of MHC-I was found to be higher in the transfected hepatoma, than in the transfected glioma cells. This increased expression of MHC-I could contribute to the final immune recognition of tumour immunogenicity.

Expression of serum albumin and of alphafetoprotein in murine normal and neoplastic primitive embryonic structures.

Alphafetoprotein (AFP), a major serum protein synthesized during the embryo-fetal and postnatal period (in the yolk sac, then in the liver), is also an oncoprotein. The intracellular presence of AFP and of serum albumin (SA) in normal and neoplastic neural crest and neural tube derivatives was previously demonstrated. In this work we have studied the comparative expression of AFP and SA in primitive neuroectoblastic structures of mouse embryos (6 and 7 days "post coitum") and mouse teratocarcinomas (derived from the PCC4 cell line). Using immunofluorescence technique, antibodies to SA gave a positive reaction in embryos of 7 days, while AFP was not detected during this period. By mRNA in situ hybridization, SA mRNA gave a strong signal in both 6 and 7 day embryos, whereas AFP mRNA gave a weak signal only in 7-day embryos. The distribution of SA and AFP and their mRNAs was investigated in primitive neuroectoblastic structures of the teratocarcinomas by in situ hybridization and immunostaining. Only SA protein was detectable by immunostaining. SA mRNA gave a strong signal in differentiating structures as well as in undifferentiated cell clusters. AFP mRNA was observed only in differentiating structure. Dot-blot hybridization indicated that the level of SA transcripts was at least 6-fold higher than that of AFP transcripts in the teratocarcinomas investigated. In teratocarcinoma-bearing mice injected intraperitoneally with 125I-radiolabeled SA and AFP, significant accumulations of both SA and AFP were demonstrated in the tumors, SA being about 3-fold higher than that of AFP after normalization to quantity of uptake in liver. External in vivo photoscanning confirmed this relationship of accumulated radiolabeled proteins. The last observation could be useful in vivo for diagnosis of teratocarcinoma. We conclude that the expression of SA relative to AFP and the external cellular uptake of SA relative to AFP are similar in normal embryonic developing tissues and in the corresponding morphologically neoplastic tissues of the teratocarcinomas. The same SA:AFP relationship constitutes an oncofetal marker of primitive neuroectoblastic structures.

Cellular genes possibly involved in the transformation process of the human melanoma cell line XP44 RO (Mel).

P44 Ro (Mel) is a human malignant melanoma cell line derived from a testicular metastasis in a DNA repair deficient, xeroderma pigmentosum patient. This line harbors a N-ras gene mutated in codon 61. To investigate other cellular genes possibly contributing to the expression of its transformed phenotype, four XP44 revertant cell lines were isolated by different selection procedures and the association of the level of expression of various oncogenes (including N-ras) and tumor suppressor genes with the selection for the revertant phenotype was determined. The revertants exhibited a significant but variable degree of phenotypic reversion, according to the selective pressure to which they were submitted, and a phenotypic stability dependent on their constant maintenance in selective medium. Back-revertant lines were isolated by culturing revertant lines in control medium for several weeks. The comparison between parental, revertant and back-revertant cells has revealed that, beyond the mutation in codon 61 of N-ras, two groups of genes appear to be also implicated in the transformation process of XP44 RO (Mel) cells: one group, comprising pim A, trk, Rb and p53, whose expression is independent of the cell selection conditions; the other group, comprising Ha-ras, N-ras, neu 1, fos and met H, whose expression is more or less dependent upon such conditions. The myc gene is apparently not involved in this phenomenon. These results, besides strengthening the concept that carcinogenesis is a multigenic process, suggest that diverse mechanisms can lead to the transformed phenotype, but that these mechanisms might have some pathway(s) in common.

Cytotoxicity and DNA damaging potency of chloramphenicol and six metabolites: a new evaluation in human lymphocytes and Raji cells.

Chloramphenicol (CAP) is an antibiotic which has been implicated in the etiology of aplastic anemia in man. This product is also used in veterinary medicine. The medical use of chloramphenicol has been limited to cases where the drug is indispensible but veterinary use may lead to the presence of residues in the meat of treated animals and it is essential to establish acceptable levels of intake of such residues in order to protect human health. CAP is metabolized into at least 6 metabolites: nitroso-CAP (NO-CAP), formed in the liver, 3 excretion products: the glucuronide (CAP-G), the CAP base (NAPD), and an alcoholic derivative, HAP. Dehydro-CAP (DH-CAP) and the dehydro-CAP base (NPAP) are formed by enterobacteria in the large bowel. The objective of the present study was to investigate (1) the cytotoxicity of CAP and its metabolites and (2) their ability to induce DNA damage in human cells. This work was performed with human peripheral blood lymphocytes (PBL) and with a lymphoma cell line (Raji).

Activation of an alpha-fetoprotein/receptor pathway in human normal and malignant peripheral blood mononuclear cells.

Alpha-fetoprotein (AFP) is mainly synthesized by the fetal liver and the yolk sac with minor contributions of several non-hepatic fetal tissues, variable according to the species considered. Most fetal cells, whatever their origin, possess the ability to bind and to endocytose the protein. This property, which is considered to be lost in differentiated cells of the adult, may be resumed in tumoral cells and is due to the expression of specific AFP receptors at the cell surface. Cytochemical and immunological approaches, combined with in situ hybridization, were used to investigate the specific uptake and synthesis of human AFP in several classes of peripheral blood mononuclear cells (PBMC) and in several malignant cell lines of hematopoietic origin. With the exception of quiescent T lymphocytes, all cells investigated specifically bound AFP. Both normal and malignant blood mononuclear cells expressed mRNA transcripts of AFP which were translated into the protein during a well established period of cellular growth. These results suggest that an AFP/receptor autocrine system might operate in normal and malignant blood mononuclear cells. Its physiological role is discussed in relation to recent work from our laboratory--providing experimental evidence that AFP, throughout its interaction with specific cell receptors, regulates and facilitates the entry of fatty acids into living cells undergoing growth and differentiation.

In vitro evaluation of the differential cytotoxicity of keto-C-glycosides toward normal and malignant cells.

In a previous work, we have shown that some members of the family of keto-C-glycosides (KCGs) possess interesting biological properties as they exhibited cytotoxic effects at the nanomolar level on malignant cells. In this report, we selected six KCGs in order to investigate their selective cytotoxicity on several malignant epithelial and lymphoblastoid cells, as well as on their normal counterparts. For this purpose, we compared the activities of KCGs upon hepatoma cells and hepatocytes and upon lymphoma cells, normal lymphocytes and bone marrow cells. The tested drugs showed real discriminating cytotoxic effects since the cytotoxicity was several log greater on malignant than on non malignant cells. An in vitro comparative study of KCGs and some conventional chemotherapeutic agents showed that two of them were more potent than 5-fluorouracil, cis-platinum and etoposide. It is interesting to note that KCGs showed very low cytotoxic effects on either murine splenocytes, human peripheral blood lymphocytes or human bone marrow cells, indicating a weak immunosuppressive activity. The results presented here strongly suggest the selective cytotoxic activity of KCGs toward tumoral cells.

Polyamine content and oncogene expression in hepatoma cells in culture during methionine deprivation and refeeding.

From an hepatocarcinoma cell line (LFCL.2A), unable to grow in a culture medium in which methionine was replaced by L-homocysteine, we had previously isolated revertant clones presenting a low growth rate, a loss of tumorigenicity and an inhibition of transcription of three oncogenes: c-Ki-ras, c-Ha-ras and c-myc. Here we showed that long-term deprivation of methionine led to a depletion of spermine, while putrescine and spermidine contents remained unchanged. When the revertant cells were shifted in a medium containing methionine, the oncogene transcription (except the p53 gene) started very rapidly in parallel with an increase in the putrescine content. By contrast, spermidine and spermine contents decreased during the first hours but were not significantly different from control values after numerous subcultures in methionine-containing medium.

Potential antineoplastic activity of keto-C-glycosides--a new family of cytostatic agents.

We have examined the biological activity of keto-C-glycosides (KCGs), a new family of drugs displaying antiproliferative and cytotoxic properties on tumor cells. KCG1, the most powerful drug tested on epithelial derived neoplastic cells, was 25-125 times more cytostatic on epithelial cells than on lymphoma. By contrast, KCG10 proved to be more cytostatic on lymphoma than on epithelial cells. Correlations were found between the cytostaticity of KCGs and their lipophilicity, and are discussed within the framework of the structure-activity and the structure-selectivity relationships.

Spontaneous metastatic potential of rat hepatocarcinoma cells after cell fusion or DNA transfection.

Eight LF x ICIG cell hybrid clones, isolated upon fusion of normal ICIG-7 human fibroblasts with tumorigenic, non-metastatic LF Cl.2A cells derived from a DAB-induced rat hepatocarcinoma, were studied. They were all highly tumorigenic and were capable of developing spontaneous lung metastases in syngeneic animals. All the hybrids were characterized by a rapid loss of human chromosomes. However, in long-term culture, they all revealed a persistence of human genetic information as assessed by Southern blotting. In hybrid lines in which human chromosomes were still visible, the most recurrent were numbers 7 and 9. Neither chromosome 7, previously reported to bear some of the genes controlling metastasis in human X mouse T-cell hybrids, nor chromosome 9 appeared to be correlated with the metastatic potential of LF X ICIG hybrids. The same conclusion applied (1) to a human 3.3-kb EcoRI DNA fragment which was amplified (approx. 10-fold) only in metastases induced by one out of 3 metastatic hybrids tested; (2) to the transcription level of c-Ha-ras and c-Ki-ras genes which was enhanced (approx. 4-fold) in metastatic and non-metastatic lines as well. Co-transfection of LF Cl.2A cells with pHSG 272 selectable marker DNA and genomic DNA from normal ICIG-7 human cells or from a hybrid-induced metastasis, reproducibly gave rise to geneticin-resistant transfectants capable of producing spontaneous lung metastases. Neither transfectants nor transfectant-induced metastases harbored detectable human DNA sequences but all harbored pHSG 272 DNA. These results again call for caution in gene transfer studies of the metastatic process.

Endogenous and exogenous alpha-fetoproteins as differential markers of cultured neonatal mouse Schwann cells and fibroblasts.

alpha-Fetoprotein (AFP) and AFP-gene transcripts were demonstrated in vitro in Schwann (S) cell and fibroblast (F) cell cultures of neonatal mouse origin. All S and F cells of primary cultures and of established cell lines expressed the AFP gene. AFP mRNA was detected by an in situ hybridization technique using a 35S-AFP-cDNA probe. AFP was localized by immunocytoperoxidase labelling using purified anti-AFP antibodies. The amounts of stained endogenous AFP, estimated semiquantitatively, were about 3-fold higher in S cells than in F cells. After incubating the cultures with exogenous mouse AFP, both S and F cells showed significant ability to take up the protein; the amount of internalized protein was found to be higher in F cells than in S cells. Moreover, the uptake of AFP fluorescein conjugates (FITC-AFP), estimated quantitatively by fluorometry, also gave higher values for F cells. The cytoplasm of F cells exhibited a characteristic fluorescence pattern, strongly illuminated and dispersed grains; the cytoplasm of S cells was regularly labelled. If exogenous FITC-AFP uptake could be used to distinguish labelled F cells from S cells (with application for identification and selection of F cells), the immunocytochemically stained endogenous AFP could allow S cells to be distinguished from F cells (using the dilutions of antibodies still staining the S cells but which lead to the absence of F-cell labelling). The two procedures, which can be used independently or together, may constitute differential markers for S cell and F cultures in, i.e., nerve regeneration of neurofibroma studies using the model of mixed S and F culture also containing other types of cells.

Increased endotoxin sensitivity following T-2 toxin treatment is associated with increased absorption of endotoxin.

Oral exposure to T-2 Toxin (T-2) in experimental animals results in a syndrome similar to that observed in endotoxemia. Endotoxins are lipopolysaccharide, outer-membrane components of gram-negative bacteria which induce acute, inflammatory responses. In the present study, several aspects of endotoxin pathophysiology were investigated in mice following simultaneous exposure to T-2 and endotoxin, including mortality, hypothermia, tumor necrosis factor-alpha (TNF-alpha) and corticosterone production, and thymic weight. The disposition of endotoxin was also assessed, Acute, simultaneous exposure to T-2 (4 mg/kg, po) and endotoxin (3 micrograms/mouse, ip) resulted in increased mortality, hypothermia, TNF-alpha production, and thymic atrophy compared to treatment with either T-2 of endotoxin alone. Pretreatment of mice with endotoxin, a regime that renders the animals resistant to the effects of endotoxin, reduced many endotoxin effects in animals treated simultaneously with T-2 and endotoxin. Upon further investigation, it was observed that T-2 increased the absorption rate of endotoxin: as the peak height of serum endotoxin increased, the time-to-peak decreased, and the area under the curve was unchanged in animals treated simultaneously with T-2 and endotoxin. It was concluded that increased endotoxin absorption accounted for the increases in mortality, hypothermia, and TNF-alpha associated with T-2 exposure.

Modulation of P-450 IIC7 and IIIA1,2 mRNA in pre-neoplastic liver. Effect of promotion by phenobarbital.

P-450 IIC7 and IIIA2 mRNAs are constitutively expressed in the hepatic tissue under developmental control. Both forms--as well as IIIA1, 90% homologous to IIIA2 mRNA--display positive modulation by phenobarbital a prototype inducer of the liver monooxygenases and a strong promoter of experimental chemical hepatocarcinogenesis. In the present work the variations in the concentration of these P-450 mRNA were studied in rats submitted to the hepatocarcinogenic protocol of Solt and Farber. We demonstrate that a decrease in the relative concentrations of P-450 IIC7 and IIIA1, 2 mRNA is set up along the tumor promotion stage. Animals--starting the experimental carcinogenic protocol at pubertal age--show a partial inhibition of the physiological expression of P-450 IIIA1,2 mRNA associated to male sex maturation. Administration of phenobarbital results in an acceleration of the pre-neoplastic process which is concomitant with an induction of P-450 IIC7 as well as IIIA1,2 at the earlier promotion stages. P-450 mRNA concentration markedly decreases as the preneoplastic process develops. While an impaired P-450 IIIA1,2 mRNA relative abundance is observed, an inversion of the modulation of P-450 IIC7 as well as of the male phenotype marker alpha-2u-globulin mRNA arises as the tumor promotion stage progresses, both mRNA becoming repressed in response to phenobarbital.

Transplacental transfer of T2-toxin: pathological effect.

Radioactivity was recovered in fetuses of pregnant rats following administration of radioactive T2-toxin. The transplacental effects of T2-toxin were studied by ip administration or oral feeding of pregnant rats at doses equivalent to natural contaminations and compatible with the maintenance of pregnancy. Body weights of the newborn rats from treated females were similar to the body weights of the control animals but their thymuses were atrophic. This atrophy was reversible in one week. Since the measurement of antibody production for fetuses and newborns is not feasible, the lymphoblastic response to mitogen of the splenic and thymic cells of baby rats from treated and control females was tested. At 4 and 6 days after birth, a good response to PHA for the thymic cells of the mother treated young rats was observed. Histological examination of the thymus showed that one day after birth the cortex was atrophic while the medulla was proliferative; on day six the situation was reversed. For the spleen, both B and T cells were impaired and their responsiveness to PHA and LPS decreased. On days 1 and 6, the periarteriolar sheats, as well as the follicles, appeared atrophic. These results show that T2-toxin easily passes the placental barrier and that T2-toxin injection or feedings at levels (2 ppm) similar to those in naturally contaminated foods, produced an impairment of the immune system of the newborn.