Black cellular spreading and motility assay.
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Antibodies, Monoclonal↗
Biomedical subjects
A E al Moustafa
Publications and source records attributed to A E al Moustafa.
Expression of the gastrin-releasing peptide receptor confers a growth response to bombesin in immortalized human bronchial epithelial cells.
We have introduced a human gastrin-releasing peptide receptor expression vector into an immortalized human bronchial epithelial cell normally unresponsive to the ligand bombesin. Successfully transfected cells express specific binding sites at a density similar to that found at the surface of human lung cancer cells and show an elevation of intracellular calcium concentration in response to bombesin. We found that cellular strains expressing the receptor showed a growth stimulation in response to bombesin in proportion to cell surface receptor density. We conclude that expression of bombesin receptors contributes to the growth potential of human bronchial epithelial cells.
Animals↗
Avian myeloblastic cell lines transformed by two nuclear oncoproteins, P135gag-myb-ets and p61/63myc: a model of retinoic acid-induced differentiation not abrogated by v-erbA.
We previously demonstrated that the retroviral construct MHE226 transducing both the P135gag-myb-ets and p61/63myc nuclear proteins induces solid hemopoietic tumors in early chicken embryos. In the present paper, we report the characterization of two MHE226-transformed cell lines established from such hemopoietic tumors retrieved from the heart of a 13-day embryo. Cytological analysis indicated a myeloblastic phenotype. These MHE226 cell lines were positive for the MEP17 monoclonal antibody but were negative for the myeloblast-specific 51/2 monoclonal antibody. MHE226 cell lines displayed a doubling time of about 20-24 h and were maintained for at least 1 year. Contrary to E26 myeloblastic cell lines, MHE226 cell lines were independent of chicken myelomonocytic growth factor and could be maintained in serum-free medium. MHE226 cell lines could be induced to differentiate toward the monocytic lineage by retinoic acid. Retinoic acid inhibited proliferation of MHE226 cell lines as early as day 1. After 3 days, MHE226 cells displayed cytological, enzymatic (alpha-naphthyl acetate esterase and chloroacetate esterase), and functional (phagocytosis) characteristics of monocytic cells. The retinoic acid-induced differentiation of MHE226 cells could not be inhibited by v-erbA. Thus, MHE226-transformed cell lines represent a novel model of cell transformation by two nuclear oncoproteins. Furthermore, they provide a model to study molecular mechanisms implicated in the monocytic differentiation program.
Animals↗
Patterns of integration and expression of retroviral, non-replicative vectors in avian embryos: embryo developmental stage and virus subgroup envelope modulate tissue-tropism.
We previously demonstrated that Avian Leukemia Viruses (ALV) carrying the v-myc gene specifically induce two types of tumors, cardiomyocytic tumors when the virus is injected before embryonic day 3 (E3), skin tumors when the virus is injected at E3 or E5. Aiming to elucidate the mechanisms which determine this time-dependent change in target, we infected chick and quail embryos at E3 and E5 with replication-deficient, lacZ gene-carrying, ALV-based viruses produced by a packaging cell line. Three constructs driven by 3 different Long Terminal Repeats (LTRs) were tested and yielded similar results. When the constructs were inoculated at E3 and the lacZ gene product revealed 5 days later, around 70% of the embryos carried lacZ+ clones in the heart, around 50% had positive clones in the skin anywhere on the body, while a few embryos displayed clones in internal organs (liver, stomach, lungs). Immunocytological identification of the heart cell type(s) expressing the virus revealed that the only cells infected were cardiomyocytes. When the constructs were inoculated at E5, no lacZ+ clones appeared in the heart but all were located in the cephalic skin. In order to examine the relationship between viral integration and expression, DNA of different organs or tissues from lacZ stained embryos was analyzed by PCR. A tight correlation between integration and expression in the heart and in the skin was revealed in most cases. In contrast, a significant PCR signal was often detected in the liver or the stomach despite weak or absent expression as revealed by lacZ+ clones. We then investigated the influence of envelope glycoprotein subgroups on the tropism of these constructs. The lacZ vector driven by RAV-2 LTRs was packaged as subgroups A, B or E viral particles. The A subgroup, used in the part of the study described above, infects both chick and quail while the B and E subgroups are specific for chick or quail respectively. These B and E subgroups induced lacZ+ clones in the heart (after E3 injection) while no clones or only a few were detected in the skin either after E3 or E5 injection. The following conclusions can be drawn: 1) cardiomyocytes are at E3 the major target for integration and expression of ALV-derived viruses in vivo; 2) targets change rapidly with embryonic age; and 3) tissue-specific infections depend on the envelope subgroup, thus presumably on the presence of the cognate receptor.(ABSTRACT TRUNCATED AT 400 WORDS)
Alpharetrovirus↗
Immortal cell lines isolated from heart differentiate to an endothelial cell lineage in the presence of retinoic acid.
Previously, we isolated a single line of transgenic mice which develop an enlarged heart due to the expression of the immortalizing gene, polyomavirus large T antigen. Immortal cell lines were isolated from adult transgenic but not from nontransgenic hearts. All of the 24 cell lines expressed vimentin and fibronectin but not desmin or myosin heavy chain. We conclude that the cell lines are of non-muscle origin. Six cell lines were chosen for further study. All six cell lines demonstrate profound morphological and biochemical effects when incubated with 10(-4) M to 10(-7) M retinoic acid. The retinoic acid-treated cell lines showed arrested cellular proliferation and aligned to form rows and vesicle-like structures. Cycloheximide inhibited these retinoic acid-induced changes, indicating a need for continued protein synthesis. Retinoic acid-treated, but not untreated, cells lost expression of vimentin and fibronectin, gained the ability to incorporate acetylated low density lipoprotein, and expressed Factor VIII-related antigen. Retinoic acid did not induce expression of desmin or myosin heavy chain. Incubation of the cell lines with transforming growth factor beta 1, dimethyl sulfoxide, or phorbol esters had no biochemical or morphological effect. We conclude that these cell lines differentiate to an endothelial lineage in the presence of retinoic acid.
Animals↗
Tumorigenic effects mediated in the avian embryo by one or more oncogenes associated with v-myc.
We have previously shown that introduction of the v-myc oncogene in chick or quail embryos at E3 induces rapidly growing heart rhabdomyomas. We now report that a retrovirus containing one or two other oncogenes induces additional pathologies specified by the v-myc-associated oncogene. The v-mil/myc combination introduced at E3 induces, in addition to heart rhabdomyomas, tumors of proliferating cells aggregated onto the luminal aspect of vessels in both chick and quail embryos. In the quail these cells react positively with the quail-specific mAb QH1, which recognizes endothelial and most hemopoietic cells, while chick intravascular cells do not react with the chick-specific mAb VIA2 that recognizes hemopoietic cells. Thus the v-mil/myc tumors appear to be of endothelial origin. The v-myb-ets/myc combination injected at E3 induces cardiorhabdomyomas and aggressive VIA2-positive hemopoietic tumors in chick embryos, but only the v-myc-induced cardiorhabdomyomas in quail embryos. When injected into hatched animals, v-myc alone transforms hemopoietic and perhaps endothelial cells, but not cardiac cells. Thus the developmental stage at which a cell type can be transformed by v-myc and another associated oncogene depends on as yet undefined species-specific factors. More importantly, several examples of oncogene cooperation in vivo are adduced by these experiments. The type of cell transformed is specified by the viral oncogene combination.
Animals↗
Oncogenes and avian development.
In order to detect signs of oncogene activity and elucidate their possible role in avian ontogeny we implemented two different strategies. One was to detect either the protein product or messenger RNA in situ at various stages of development. The other was to try and disturb development with retroviruses carrying one or several oncogenes in their activated forms. Time- and tissue-specific expression of c-myc was apparently not related to particular phases of cell evolution, such as population amplification. Rather the presence of c-myc immunoreactive product at particular stages appeared to depend on cell types. c-myb and c-ets messenger RNAs were found expressed preferentially in the blood system, respectively in hemopoietic and differentiating endothelial cells. The developing embryo heart was found to be uniquely sensitive to the effect of retroviruses provided that two conditions were respected. The first was the injection of the virus or construct prior to E3.5. The second was the presence of the v-myc gene, whether alone or associated with one or several other v-onc. In such cases a large proportion (70%) of chick and all quail embryos developed multiple heart rhabdomyosarcomas within 10 days. In chickens the association of a second v-onc or of two others induced the formation of secondary tumors, whose type was determined by the nature of the other oncogene(s).(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Targets of v-myc tumorigenesis in the avian embryo depend on time and not on site of retroviral infection.
The present study extends our previous data, showing that the v-myc oncogene induces heart tumors and skin anomalies in young avian embryos [Saule et al., Proc. Natl. Acad. Sci. USA 84, 7982-7986 (1987)]. We now report that the target cells which become transformed are the same, whether the MC29 retrovirus is injected at E3 in various sites of the embryo (coelom, heart, brain, lateral plate mesoderm) or deposited on the embryo. Furthermore we confirm, in the quail, the time-specific pattern previously observed in the chick. In the quail, the incidence of heart tumors falls from 100% to 28% when injection is delayed from E3 to E4. By contrast, the incidence of skin anomalies rises from 30% to 64% when injection is delayed from E3 to E4. The skin defect, which consists of the presence of bell-shaped cornified feathers, could be assigned to hyperkeratinization of the epidermis. Both the dermis and the epidermis displayed hyperproliferation, whereas skin muscle hypertrophy during the embryonic period could not be confirmed. The presence of myc gene products was investigated using an antibody that recognizes both the c- and v-myc proteins. In the skin of control embryos, nuclei were well stained at E12-E13. At E14 the signal had disappeared. In abnormal skin patches from infected embryos, the antibody still marked heavily epidermal and dermal nuclei at E18. Finally we injected MC29 through the chorioallantoic vein in E10 chickens. No tumors were found during embryonic life, but 81% of the chickens developed tumors of hemopoietic or endothelial origin from the 14th posthatching day onwards. Studies of MC29 integration sites demonstrated that these tumors were derived from only a few transformed cells. Thus, contrasting with in vitro experiments, in vivo this virus has a restricted number of targets varying with the time of injection.
Animals↗
Expression of P185erbB-2, P160erbB-3, P180erbB-4, and heregulin alpha in human normal bronchial epithelial and lung cancer cell lines.
The receptors erbB-3 and erbB-4 are members of the type 1 tyrosine kinase receptor family which also comprises epidermal growth factor receptor (EGF-R) and erbB-2. ErbB-3 and erbB-4 receptors are known to bind a family of related proteins termed heregulins. In this study, we report differential expression of P185erbB-2, P160erbB-3 and P180erbB-4, and their ligand heregulin alpha, in normal bronchial epithelial, and non-small cell lung carcinoma (NSCLC) cell lines. Expression of P185erbB-2 and P160erbB-3 vary from very low to a high level in NSCLC cell lines and a low level in normal bronchial cells. In contrast, P180erbB-4 was detected only in NSCLC cell lines but not in normal bronchial cells. Heregulin alpha is expressed at intermediate levels in the normal and cancer cell lines studied. Immunoprecipitation, using antibodies to erbB-2, erbB-3 or erbB-4 receptors, coupled to phosphotyrosine Western blot analysis indicates that these three receptors are constitutively tyrosine phosphorylated in lung cancer cell lines, but only erbB-2 and erbB-3 are autophosphorylated in normal cells. These data suggest that constitutive activation of erbB-2, erbB-3 and erbB-4 receptors could be induced by heregulin alpha via an autocrine loop mechanism, and that the active forms of erbB-4 may cooperate with the other members of the EGF-receptor family in human lung carcinogenesis.
Bronchi↗