Gene therapy of rat brain glioblastoma by an episome-based transcriptional cassette expressing antisense IGF-I cDNA.
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Animals↗
Biomedical subjects
J Trojan
Publications and source records attributed to J Trojan.
Treatment and prevention of rat glioblastoma by immunogenic C6 cells expressing antisense insulin-like growth factor I RNA.
Rat C6 glioma cells express insulin-like growth factor I (IGF-I) and form rapidly growing tumors in syngeneic animals. When transfected with an episome-based vector encoding antisense IGF-I complementary DNA, these cells lost tumorigenicity. Subcutaneous injection of IGF-I antisense-transfected C6 cells into rats prevented formation of both subcutaneous tumors and brain tumors induced by nontransfected C6 cells. The antisense-transfected cells also caused regression of established brain glioblastomas when injected at a point distal to the tumor. These antitumor effects result from a glioma-specific immune response involving CD8+ lymphocytes. Antisense blocking of IGF-I expression may reverse a phenotype that allows C6 glioma cells to evade the immune system.
Animals↗
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.
B-Lymphocytes↗
Loss of tumorigenicity of rat glioblastoma directed by episome-based antisense cDNA transcription of insulin-like growth factor I.
Malignant glioma is the most common brain tumor. The molecular basis of glioma tumorigenicity has not been defined. Cultured glioma cells accumulate high levels of insulin-like growth factor I (IGF-I) transcripts. We asked whether IGF-I expression is coupled to tumorigenicity, using a combined in vivo/in vitro system employing antisense RNA for IGF-I. An antisense IGF-I expression construct in an expression vector that incorporates Epstein-Barr virus replicative signals and the ZnSO4-inducible metallothionein I transcriptional promoter was assembled. Stable glioma transfectants were derived from C6 glioma cells, which constitutively express IGF-I. B-104 neuroblastoma cells, derived originally from the same tumor but not expressing IGF-I, were also transfected as controls. In the absence of ZnSO4, the C6 transfectants expressed high levels of IGF-I mRNA and protein as detected by in situ hybridization and immunocytochemistry, respectively. Addition of ZnSO4 in the culture medium resulted in high levels of antisense transcript accumulation and dramatically decreased levels of endogenous IGF-I mRNA and IGF-I protein. Subcutaneous injection of either nontransfected C6 parental cells or C6 cells transfected with vector without IGF-I sequences into rats resulted in large tumors after 2 weeks, as did transfected and nontransfected B-104 cells. However, the rats injected with transfected C6 cells yielded no tumors after 40 weeks of observation. Two weeks after injection of the transfected C6 cells a small cyst was apparent in six rats. Histologic sections revealed a few glioma cells infiltrated by a large number of mononuclear cells. No infiltration of mononuclear cells was apparent in the glioma tumors resulting from injection of parental (nontransfected) cells, suggesting that the parental cells, but not the antisense IGF-I transfectants, escape the host immune response.
Animals↗
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.
Animals↗
Effects of actinomycin D and cycloheximide on transcript levels of IGF-I, actin, and albumin in hepatocyte primary cultures treated with growth hormone and insulin.
The stability of several RNA transcripts in cultured hepatocytes is known to increase when serum is omitted from the culture medium. In order to investigate possible mechanisms for this phenomenon, we examined the effects of actinomycin D and cycloheximide on the levels of actin, albumin, and insulin-like growth factor I transcripts in primary cultures incubated in serum-free medium. The levels of IGF-I and albumin transcripts per culture increased for the first 4 hours following addition of actinomycin D and then declined. The levels of actin transcripts and total RNA per culture declined immediately following actinomycin D addition in a manner consistent with exponential decay. IGF-I and albumin transcript levels were relatively unaffected by cycloheximide, while actin transcript levels increased 7-fold over 7 hours. The half-lives of actin transcripts and total RNA were calculated to be 4.6 to 7.7 hours and 11 to 19 hours, respectively, with no statistically significant correlation with hormone treatment. The data suggest that the stability of albumin and IGF-I transcripts, but not actin transcripts, is controlled in part by an actinomycin D-sensitive process.
Actins↗
Alpha-fetoprotein uptake by differentiating neuroretinal structures of the chick embryo.
Internalization of exogenous fluoresceinated alpha-fetoprotein (FITC-AFP) was studied at different stages of development on embryonic chick neural retinas maintained, for short periods, in organ cultures. Cellular localization of endogenous, native AFP was carried out by immunohistoperoxidase methods. Cells which specifically internalized exogenous FITC-AFP (neurons and their processes) were precisely those showing positive immunolabeling for the endogenous, native protein. Such a result supports the hypothesis of a predominantly exogenous origin of intracellular neuroretinal AFP. A precise topography and temporal sequence of AFP labeling after internalization in retinal structures is given. AFP uptake was not displayed by undifferentiated cell precursors or germinal cell layers but was apparent in cells with phenotypic characteristics of maturing neurons. Nerve fibers and synaptic layers actively internalized FITC-AFP at specific stages of development. Fully differentiated neurons and processes did not internalize AFP. The possible role of AFP, a carrier of biologically active substances such as fatty acids, in neural retina differentiation is discussed.
Animals↗
Cellular localization of an embryonic interferon, ovine trophoblastin and its mRNA in sheep embryos during early pregnancy.
The ovine embryo produces an interferon named ovine Trophoblastin (oTP) which is involved in the maternal recognition of pregnancy and ensures the maintenance of progesterone secretion by the corpus luteum. We have used indirect immunohistofluorescence and in situ hybridization on histological sections to investigate the fate of this protein and its mRNA in ovine embryos from days 3 to 25 of pregnancy. The level of expression was measured by image analysis of the autoradiographs after in situ hybridization. Both techniques clearly demonstrated that oTP and its mRNA were specifically localized in the extra-embryonic trophoblast. Neither the embryonic cells, nor the yolk sac or the amniotic tissues produced the protein or its mRNA. The protein could be detected by d 11 of pregnancy in the elongated blastocyst. Maximum of expression is observed at d 14 and the level decreased by d 16 of pregnancy. The arrest of expression occurred in the regions of trophoblast which have established cellular contacts with the uterine epithelium during the implantation process.
Animals↗
Alpha-fetoprotein (AFP) in granulomatous inflammation of the mouse.
Inflammatory granulomas constituted of various proportions of macrophages, polynuclear (PMNs) and mononuclear cells were induced in adult pathogen-free mice by injecting polyacrylamide beads into subcutaneous pouches. Using specific anti-mouse alpha-fetoprotein (AFP) antibodies, the presence of AFP was immunocytochemically demonstrated in the cytoplasm of macrophages and of immature PMNs (mature PMNs and mononuclear cells were AFP negative). AFP-labelling started as soon as 36 h after granuloma induction and reached a maximum 60-72 h later, to disappear on day 5. The examination of different organs in these mice also showed a transitory labelling for AFP of liver hepatocytes and of elements of kidney and of exocrine pancreas. Parallel to these findings, the selective concentration inside the granulomatous pouches of radiolabelled AFP injected in the course of inflammation was observed. These results suggest that (a) shortly after the inflammatory reaction the synthesis of AFP is resumed by the liver; (b) the newly synthesized AFP is secreted in the serum and preferentially taken up by the granulomas; and (c) consequently, in adult mice, AFP behaves as a positive acute phase reactant. The physiopathological implications of these facts are discussed in relation with the biological properties of AFP.
Animals↗
Localization of H blood group antigen in ectoblastic derivatives of murine teratocarcinoma.
Ectoblastic derivatives (ectodermal and neuroectodermal components) constitute more than 90% of all structures in the murine teratocarcinoma derived from the PCC4-aza-1 line. This tumor was labeled immunocytochemically with fluoresceinated antibodies to A, B and H blood groups. A and B antigens were always noted to be absent from all structures of the three germ layers. With anti-H, however, embryonic and fetal endodermal components, e.g. alimentary or respiratory duct-like structures, gave positive staining. Mesodermal components, i.e. bone and cartilage structures, fibroblasts and myocytes of mature or embryonal type, were negative. Immature and mature ectodermal components, viz. epidermoid cysts or islets, were always positive. Neuroectodermal components, neuroblastic cysts and differentiated neuronal, glial and ependymal elements were always negative. Ectoblastic and/or neuroectoblastic individual cells or cell clusters, observed in the vicinity of positive differentiating ectodermal and neuroectodermal structures, were positive. Similar cells or clusters were negative when located close to negative neuroectodermal components. The undifferentiated, embryonal carcinoma cells and structures were always negative. These observations are compared to the staining patterns of H antigen in murine embryos and adult mice and in human teratocarcinomas. It is suggested that poorly differentiated, morphologically similar ectoblastic and/or neuroectoblastic structures are positive, if they concern ectodermal components, and negative, if they belong to neuroectodermal components, with the exception of some primary sensory cells that are positive. These last cells are also positive in normal fetal and adult tissues.
ABO Blood-Group System↗
Appearance of H and B antigens in primary sensory cells of the rat olfactory apparatus and inner ear.
In the rat olfactory apparatus and inner ear the H antigen was first detected at the 15th day of gestational age. The B antigen appeared at the 16th day. In the olfactory apparatus H and B antigens were first detected in cells of the olfactory mucosa and of the olfactory bulb primordium. Towards the 16th and 17th days many small positive cells were seen between these two areas. In newborn and adult rats, olfactory bulb H and B antigens were restricted to the processes of the peripheral primary sensory cells. Both H and B antigens were detected in the epithelium of the inner ear and were later concentrated in the hair cells of the organ of Corti, the utricle and the saccule.
ABO Blood-Group System↗
The uptake of alpha-foetoprotein by C-1300 mouse neuroblastoma cells.
Recent immunocytochemical and biochemical studies have shown the intracellular uptake of alpha-foetoprotein (AFP) by most neural crest and neural tube derivatives of developing mammals and birds. The neural crest origin of neuroblastomas has been known for a long time. While many mouse neuroblastoma cell lines can express several neuronal properties, other lines lack specialized neural functions and may re-express embryonal or foetal antigens, suggesting some reversion towards an earlier stage of differentiation. We have therefore tested the C-1300 Jackson mouse neuroblastoma cell line for its ability to incorporate AFP. The results obtained confirm the significant internalization of protein by these cells, both in vitro and in vivo. External photoscans of mice bearing tumours after injection with [131I]-AFP have proven the usefulness of the protein as a radiotracer for neuroblastoma localization.
Animals↗
Inhibitory effect of mouse interferon on the growth of an embryonal carcinoma in mice.
Embryonal carcinoma cells (F9) were resistant to the inhibitory effects of interferon (IFN) on viral multiplication and cell division in vitro. Nevertheless, daily administration of IFN resulted in a marked inhibition of the growth of this carcinoma injected intraperitoneally (ip) and increased mouse survival time. An incidental finding was that daily IFN treatment increased the serum level of alpha-fetoprotein in normal 129 mice.
Animals↗
Intracellular uptake of alpha-fetoprotein: a marker of neural differentiation.
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Animals↗
[Localization of alpha fetoprotein in neuroepithelial derivatives of murine teratocarcinomas].
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Aging↗
Intracellular alpha-fetoprotein and albumin in the developing nervous system of the baboon.
The intracellular presence of alpha-fetoprotein (AFP) and of albumin in the brain and other neural derivatives of three Baboon fetuses (9,13 and 17 weeks of gestational age, respectively), was demonstrated by immunocytochemical techniques. The largest localization of both proteins was observed in the 9 week old fetus, where most neural tube and neural crest derivatives showed intracytoplasmic labelling. The staining decreased abruptly in the 17 week old fetus. In the brain, of the youngest fetus examined, the penetration of AFP and albumin into the cerebral wall is transependymal. The route seems to possess differential activity since IgG molecules never appeared internalized in spite of their presence, like AFP and albumin, in the choroid plexus and the ventricular spaces of this fetus. It is also worth noting that the labelling declines as myelination and glial cell development progress. The wide distribution of AFP and albumin labelling supports the conclusion that the intracellular uptake of these proteins is probably general in immature nervous structures. Attention is brought to the binding and transport of polyunsaturated fatty acids by AFP and albumin as a possible explanation for the transitory presence of these proteins in the developing nervous system.
Albumins↗
Immunocytochemical localisation of alpha-fetoprotein (AFP) and serum albumin (ALB) in ecto-, meso- and endodermal tissue derivatives of the developing rat.
Recent work has provided evidence of the intracellular presence of AFP and serum albumin (ALB) in the central nervous system of rats, mice and primates. We report here the immunocytochemical localization of these proteins in tissue derivatives of the three germ layers (with the exclusion of nervous system components) during rat fetal and neonatal development. Positive labelling for AFP and ALB was transitorily observed in all tissue derivatives examined. For example, labelling for AFP in the epidermis started around the 16th day of fetal life and localized in some cell aggregations of the peridermis. Between the 17th and the 19th day AFP positive cells were observed in the basal layer of epidermis, while the stratum germinativum was always negative. The maximum intensity of labelling was at birth. At this time, AFP positive cells were visible in the strata spinosum, granulosum and corneum. On day 6 to 8 after birth, the labelling become negative. The localisation of ALB showed a similar pattern with the only difference of a short (2 to 5 days) displacement in time. It can be concluded that the intracellular uptake of AFP in the structures studied is transitory and follows the stage of cell and tissue differentiation. Relative to AFP, labelling for ALB seems to correspond to a more advanced stage of morphologic development.
Animals↗
Immunocytochemical demonstration of alpha-fetoprotein uptake by primary cultures of fetal hemisphere cells from mouse brain.
Mouse alpha-fetoprotein (AFP) was added for 24 h to primary cultures of mechanically dissociated mouse brain fetal hemisphere cells grown in a serum-free medium. The intracellular presence of AFP could be demonstrated by immunocytochemical methods mainly in the cytoplasm of numerous differentiating neurons. The staining occurred only after 6 days in culture. The whole network of neurites, straight processes and filaments also appeared positively labeled. No AFP was noticeable in control cultures grown in the absence of the protein.
Animals↗