Establishment of three human breast epithelial cell lines derived from carriers of the 999del5 <Emphasis Type="Italic">BRCA2</Emphasis> icelandic founder mutation

Summary Germ line mutations in BRCA1 and BRCA2 account for a large proportion of inherited breast and ovarian cancer. Both genes are involved in DNA repair by homologous recombination and are thought to play a vital role in maintaining genomic stability. A major drawback for long-term functional studies of BRCA in general and BRCA2 in particular has been a lack of representative human breast epithelial cell lines. In the present study, we have established three cell lines from two patients harboring the 999del5 germ line founder mutation in the BRCA2 gene. Primary cultures were established from cellular outgrowth of explanted tissue and subsequently transfected with a retroviral construct containing the HPV-16 E6 and E7 oncogenes. Paired cancer-derived and normal-derived cell lines were established from one patient referred to as BRCA2-999del5-2T and BRCA2-999del5-2N, respectively. In addition, one cell line was derived from cancer-associated normal tissue from another patient referred to as BRCA2-999del5-1N. All three cell lines showed characteristics of breast epithelial cells as evidenced by expression of breast epithelial specific cytokeratins. Cytogenetic analysis showed marked chromosomal instability with tetraploidy and frequent telomeric association. In conclusion, we have established three breast cpithelial cell lines from two patients carrying the BRCA2 Icelandic 999del5 founder mutation. These cell lines from the basis for further studies on carcinogenesis and malignant progression of breast cancer on a defined genetic background. Agla J. Rubner Fridriksdottir and Thorarinn Gudjonsson contributed equally to this study.     

Induction of erythropoietin responsiveness in murine hematopoietic cells by the gag-myb-ets-containing ME26 virus.

ME26 virus, which was generated by inserting the coding region of the acute avian leukemia-inducing virus E26 into a murine retrovirus vector, encodes a 135-kDa gag-myb-ets fusion protein. Amphotropic murine leukemia virus pseudotypes of ME26 virus induce a high incidence of erythroleukemia 2 to 4 months after injection into newborn NFS/N mice. Spleen cells from the majority of these mice proliferate to high levels in the presence of the erythroid hormone erythropoietin (Epo) and can easily be established as permanent Epo-dependent cell lines. The cell lines contain multiple copies of ME26 viral DNA and express viral message and protein. An Epo receptor mRNA of normal size can be detected in these cells, and binding studies reveal a single class of lower-affinity Epo receptor with an affinity for Epo that is in the range of that previously reported for erythroid cells. The ME26 virus-induced Epo-dependent cell lines, however, appear more immature than previously described erythroid cell lines and more closely resemble early hematopoietic precursor cells, suggesting that the virus may be activating the Epo receptor in hematopoietic cells that do not normally express it. Consistent with this idea, we are able to infect an interleukin-3-dependent myeloid cell line, FDC-P2, with ME26 virus and convert it to Epo dependence. The ME26 virus-infected FDC-P2 cells, even before growth on Epo, showed a large increase in the amount of Epo receptor mRNA. However, no ME26 viral integrations can be detected adjacent to the Epo receptor gene, indicating that the virus is not activating the Epo receptor gene by promoter/enhancer insertion. Our results are more consistent with the hypothesis that the gag-myb-ets-encoded viral fusion protein, which is known to bind DNA, is directly or indirectly activating the expression of the Epo receptor gene in these cells.     

Karyotypic abnormalities associated with haemopoietic lineage switching are not linked with mutations to p53

Leukemic cells can undergo lineage switching to display the phenotypic features of another haemopoietic pathway, as exemplified by B lymphoma and erythroleukemic cell lines generating variants with a monocytic appearance. Unlike the diploid parental lines, the vast majority of myeloid derivative lines examined (12 of 13 lines) were aneuploid. As p53 is involved in the maintenance of chromosomal stability, we investigated the role of p53 in the emergence of abnormal karyotypes in cells which had undergone lineage switching. Single strand conformation polymorphism and sequence analysis of cDNA, together with protein immunoprecipitations, were used to assess the p53 status of parental and variant cell lines. Unexpectedly, four or five monocytic lines with chromosomal alterations contained wild type p53. Conversely, a p53 point mutation found in one aneuploid monocytic line was also present in the diploid parental pre-B cell. These results provide strong evidence that mechanisms other than p53 mutations are responsible for karyotypic abnormalities seen in cells that have undergone lineage switching.     

Establishment of cell lines from serial samples from two patients with lung tumours before and after chemotherapy

Six cell lines were derived from pleural effusions of two lung cancer patients and established in vitro in our laboratory. Cell line AE1 was obtained from a small cell lung cancer (SCLC) before the patient had received any chemotherapy; the other lines (AE2 and AE3) were established from tumour recurrences in the same patient after therapy. Cell lines DG1 and DG2 were derived from specimens of an untreated non-small cell lung cancer (NSCLC), while cell line DG3 originated from pleural effusions recurring in the same patient after therapy. The results of the present study show that: (a) the SCLC lines AE1, AE2 and AE3 are heterogeneous in their biological characteristics and in their chemosensitivity patterns. In particular lines AE2 and AE3 are less responsive to cis-Platinum (DDP) and Adriamycin (ADM) than line AE1, so that they may reflect resistant subpopulations existing within the original tumour, selected following therapy with these drugs. In contrast, however, line AE1 proved more resistant to Vepesid (VP16) than lines AE2 and AE3. (b) The three NSCLC lines are similar in various biological features as well as in their chemosensitivity to DDP and Vinblastine (VBL).Abbreviations NSCLC Non Small Cell Lung Cancer - SCLC Small Cell Lung Cancer Recipient of a Fellowship of the Italian Association for Cancer Research     

Selection of lineage-restricted cell lines immortalized at different stages of hematopoietic differentiation from the murine cell line 32D

Erythropoietin (Epo), granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor- (G-CSF) dependent cell lines have been derived from the murine hematopoietic cell line 32D with a selection strategy involving the culture of the cells in FBS-deprived medium supplemented only with pure recombinant Epo, GM-CSF, or G-CSF. The cells retain the diploid karyotype of the original 32D clone, do not grow in the absence of exogenous growth factor, and do not induce tumors when injected into syngeneic recipients. The morphology of the Epo-dependent cell lines (32D Epo1, -2, and -3) was heterogeneous and evolved with passage. The percent of differentiated cells also was a function of the cell line investigated. Benzidine-positive cells ranged from 1-2% (32D Epo3) to 50-60% (32D Epo1). These erythroid cells expressed carbonic anhydrase I and/or globin mRNA but not carbonic anhydrase II. The GM-CSF- and G-CSF-dependent cell lines had predominantly the morphology of undifferentiated myeloblasts or metamyelocytes, respectively. The GM-CSF-dependent cell lines were sensitive to either GM-CSF or interleukin-3 (IL-3) but did not respond to G-CSF. The G-CSF-dependent cell lines grew to a limited extent in IL-3 but did not respond to GM-CSF. These results indicate that the cell line 32D, originally described as predominantly a basophil/mast cell line, has retained the capacity to give rise to cells which proliferate and differentiate in response to Epo, GM-CSF, and/or G-CSF. These cells represent the first nontransformed cell lines which can be maintained in growth factors other than IL-3 and which differentiate in the presence of physiologic signals. As such, they may represent a model to study the molecular mechanisms underlying the process of hematopoietic differentiation, as well as sensitive targets for bioassays of specific growth factors.     

Yield and activity of theHeliothis zea single nuclear polyhedrosis virus propagated in cloned and uncloned lines ofHeliothis cells

Summary Heliothis cell lines originated from different laboratories were characterized by isoenzyme analysis and then evaluated for their ability to produce the single nuclear polyhedrosis virus ofHeliothis zea (HzSNPV). A cloned cell line (designated Hzlb3), whose homogeneity was supported by both morphological and isoenzyme analysis, was derived from a parental line (Hzl). Significantly greater yields (about 10-fold) of tissue-culture-derived, non-occluded virus (TCNOV) were obtained when compared to the parental line. The Hzlb3 clone also gave significantly higher yields of TCNOV than the Hz3 and UND-K cell lines. Although lines Hzl, Hz3, and Hzlb3 produced significantly more polyhedral inclusion bodies (PIB) than line UND-K, the infectivity of PIB from UND-K equaled that of lines Hzl and Hzlb3.     

IN VITRO PROLIFERATION OF MOUSE LYMPHOBLASTOID CELL LINES: GROWTH MODULATION BY VARIOUS POPULATIONS OF ADHERENT CELLS

The in vitro growth pattern of a number of mouse lymphoblastoid tumour cell lines was modified in the presence of adherent cell layers from various sources. The AVRij-1 and ST-4b cell lines exhibited a concentration—dependent growth pattern, i.e., they would only grow well when seeded at high starting cell concentrations. Better growth of these cells from low cell concentrations was observed in the presence of adherent cell layers from syngeneic or allogeneic bone marrow. Adherent cell layers derived from mouse spleen and pleural or peritoneal cavity could also promote the growth of the above tumour cells, but in a narrower range of cell concentrations and to a lower extent. Moreover, confluent adherent layers from the pleural and peritoneal cavities completely inhibited the growth of AVRij-1 and ST-4b cells, while adherent cell layers from the bone marrow did not inhibit growth at any cell concentration tested. The in vitro growth of concentration—independent cell lines was also affected by the presence of adherent cells from the bone marrow. Under syngeneic conditions, a slight increase in the growth of the ‘null’ or pre-B lymphoma cell line ABLS-8.1 was observed. On the other hand, the growth of tumour cells expressing more differentiated properties, such as the thymus T lymphoma tumour cell line ST-1.3 and the plasma cell tumour MPC-11.45.6.2.4, was inhibited in the presence of syngeneic bone marrow derived adherent cell layers. This inhibition was more pronounced under allogeneic conditions. Growth inhibition was also observed when concentration—independent cell lines were co-cultured with adherent cells from the pleural and peritoneal cavities. Thus, adherent cell layers from non-haemopoietic sources inhibited the growth of all cell lines tested. On the other hand, adherent cells from the bone marrow had a differential effect on growth of lymphoblastoid tumour cell lines. This depended on the in vitro growth properties of each tumour cell line and on some additional specific tumour cell properties. The latter could relate to the differentiation stage characterizing each tumour cell line. The culture method described here may serve as a model system for studies on interaction of leukaemic cell and the haemopoietic microenvironment.     

Characterization of a human glycoprotein (erythropoietin) produced in cultured tobacco cells

Erythropoietin (Epo), a glycoprotein that regulates the formation of erythrocytes in mammals, was produced in cultured tobacco BY2 cells (Nicotiana tabacum L. cv. Bright Yellow 2) by introducing human Epo cDNA via Agrobacterium tumefaciens-mediated gene transfer. Epo was correctly processed and subsequently penetrated the plasma membrane of tobacco cells. However, it remained attached to the cell wall and was not released into the culture medium. Although Epo produced by tobacco cells was glycosylated with N-linked oligosaccharides, these carbohydrates were smaller than those of the recombinant Epo produced in mammalian cells. Epo produced in tobacco exhibited in vitro biological activities by inducing the differentiation and proliferation of erythroid cells. However, it had no in vivo biological activities. A lectin-binding assay indicated the lack of sialic acid residues in the N-linked oligosaccharides of Epo, suggesting that Epo was removed from the circulation before it reached erythropoietic tissues.     

Development and Characterization of a Nonmorphogenetic Cell Line of Wheat (Triticum aestivum)

This study was conducted to compare characteristics of a wheat (Triticum aestivum L.) cell line to those of the maize (Zea mays L.) black Mexican sweet (BMS) cell line and to compare protoplasts isolated from suspension cells of these cell lines. The wheat cell line was established from immature-embryo derived callus of the experimental line ‘ND7532’ and was conditioned for growth in suspension culture. For both cell lines, measurements of packed cell volume (PCV), fresh weight (FW), and dry weight (DW) were taken at 3 day intervals from suspension cultures. Measurements of FW of calluses cultured from suspension cells of both cell lines were taken at 6 day intervals. The morphogenetic potential of the wheat ND7532 cell line was tested in both callus and suspension cultures using media promoting regeneration and/or organogenesis. Growth rates of ND7532 cells in suspension culture were comparable to those of BMS cells. However, relative growth rates of calluses recovered from ND7532 suspension cells were slower than those of calluses recovered from BMS suspension cells. The ND7532 cell line has very limited morphogenetic potential and has been maintained as rapidly growing callus tissue for 11 years. Yields of protoplasts from suspension cells of the two cell lines were comparable, though ND7532 protoplasts were typically smaller. The wheat cell line has is now designated ND7532-NM (nonmorphogenetic) and is available for cellular and molecular biology research.     

Alteration of nuclear proto-oncogene expression by erythropoietin (Epo) in Epo-responsive murine cell lines

The signal transduction system of erythropoietin (Epo) and the accompanying molecular control mechanism of proliferation and differentiation of erythroid progenitors remains largely unknown. In this study, the effect of Epo on the expression of nuclear oncogenes was investigated in two murine cell lines which respond to the hormone in different ways: ELM-I-1 cells proliferate independently of Epo, but differentiate in response to the hormone, while the growth of DA-1ER cells is absolutely dependent on Epo or interleukin (IL) 3. The cell lines were stimulated with Epo or IL-3, and total RNA was extracted. Then expression of nuclear proto-oncogenes (c-myc, c-fos and c-myb) was analyzed by northern blotting. The change in c-fos expression observed during the first two h following stimulation with either stimulant were common to both cell lines; a rapid and temporary increment. Before stimulation, c-myc and c-myb were strongly expressed in both lines. No apparent change in c-myc expression was observed during the first two h of stimulation, while c-myb expression in ELM-I-1 cells was slightly reduced 1 h after stimulation with Epo but not with IL-3. Three days after stimulation with Epo, but not with IL-3, only ELM-I-1 produced hemoglobin and expressed a lower amount of c-myb mRNA. These data suggest the importance of c-fos in the early signaling system of Epo, and the involvement of c-myb in erythroid differentiation but not in proliferation.     

Comparative recombinant protein production of eight insect cell lines

Summary A recombinantAutographa californica baculovirus expressing secreted alkaline phosphatase (SEAP) gene was used to evaluate the expression of a secreted glycoprotein in eight insect cell lines derived fromSpodoptera frugiperda, Trichoplusia ni, Mamestra brassicae andEstigmene acrea. Because cell density was found to influence protein production, SEAP production was evaluated at optimal cell densities for each cell line on both a per cell and per milliliter basis. On a per cell basis, theT. ni-derived BTI-TN-5B1-4 cells produced a minimum of 20-fold more SEAP than theS. frugiperda-derived Sf9 or Sf21 cell lines and a minimum of 9-fold more than any of the other cell lines growing in serum-containing medium. On a per milliliter basis, BTI-TN-5B1-4 cells produced a minimum of fivefold more SEAP than any of the other cell lines tested. Using cell lines that were adapted to serum-free medium, SEAP yields were the same or better than their counterparts in serum-containing medium. At 3 days postinoculation, extracellular SEAP activity ranged from 59 to 85% of total SEAP activity with cell lines grown in serum-free and serum-containing media.     

Studying Drosophila embryogenesis with P-lacZ enhancer trap lines

Summary Embryos of 171 Drosophila lines carrying a P-lacZ insertion on the second or third chromosome were analyzed regarding their pattern of lacZ expression. All lines were selected from a larger screen of about 4000 lines (Bier et al. 1989). Tissue specificity and time of onset of lacZ expression was documented for each line. Thereby, a comprehensive list of markers for the various tissue and cell types of the Drosophila embryo could be assembled. With the help of several P-lacZ lines the development of a number of structures was studied which so far had been described only insufficiently or not at all. In particular, the embryonic origin and early development of the oenocytes, imaginal discs, histoblasts, fat body, dorsal vessel, and perineurial cells was analyzed. Several previously unknown cell types associated with the dorsal vessel, trachea, and epidermis were discovered. By combining data regarding the origin of the different mesodermally derived organs it was possible to generate in some detail a fate map of the mesoderm of the stage 11 Drosophila embryo. Offprint requests to: V. Hartenstein     

Selection and breeding for salinity tolerancein vitro

Summary Selection for tolerance to NaCl inCitrus sinensis andC. aurantium has been carried out in agar and suspension cultures. Callus was subjected to culture media containing up to 0.17M NaCl for ten passages. Selected cell lines were grown for three passages on media without salt before further tests on saline media. Four stable tolerant cell lines, differing in degree of tolerance, have been selected fromC. sinensis. Four lines of similar tolerance have been selected fromC. aurantium. The stability of most lines was very satisfactory. MostC. sinensis lines grew well in media containing up to 0.2M NaCl, andC. aurantium lines in media of up to 0.15M NaCl.Embryos were regenerated in most selected cell lines fromC. sinensis and, more sporadically, fromC. aurantium. Addition of 0.5–0.6% NaCl to the media often enhanced embryogenesis. Embryos from a selected line ofC. sinensis showed higher tolerance to NaCl in the medium than comparable embryos from an unselected line.Single embryos derived from both selected and unselected cell lines ofC. sinensis were successfully cloned. A limited comparison of plantlets from one tolerant line (R14) with plantlets from unselected control lines showed better adaptation of the former to salt (0.085 to 0.12M NaCl in the medium), and a lesser degree of leaf burn symptoms.Contribution No. 1045-E, 1984 series.     

Transformation of lepidopteran and coleopteran insect cell lines by Glyptapanteles indiensis polydnavirus DNA

Summary Recently investigators showed that polydnavirus DNA from the parasitic wasp Glyptapanteles indiensis could transform gypsy moth L. dispar cell lines in vitro (McKelvey et al., 1996). Here we show GiPDV DNA is capable of transforming in vitro to varying degrees lepidopteran (IPLB-TN-R², IPLB-SF-21, IAL-PID2, IPLB-HvT1) and coleopteran (IPLB-DU182E) insect cell lines derived from various somatic tissue types. An insect cell line derived from dipteran Aedes albopictus (C7/10) could not be transformed with G. indiensis polydnavirus.     

Murine myeloid cell lines derived by in vitro infection with recombinant c-myb retroviruses express myb from rearranged vector proviruses.

To date, cellular transformation in vitro by the myb oncogene has been described for avian haemopoietic cells only. In order to exploit the well-characterized murine haemopoietic system to study transformation by myb, we have infected fetal liver cells with retroviral vectors carrying cDNAs that encode either complete or carboxy-terminally truncated c-myb proteins. We describe four cell lines which, despite our ability to efficiently infect haemopoietic target cells, were generated at low frequency. This was due, as least in part, to the requirement for a rearrangement within the vector that allowed expression of myb sequences. Three of the lines express a truncated myb protein while the fourth apparently expresses a normal c-myb protein, and thus constitutes an exception to the general association of truncation with transformation by myb. All four cell lines resemble immature cells of the myelomonocytic lineage and are dependent on colony-stimulating factors (CSFs) for their growth in vitro. One representative line could be converted to CSF-independence by infection with either Abelson murine leukaemia virus or a recombinant granulocyte-macrophage-CSF-encoding retrovirus; unlike the parental line, the resultant sublines were highly tumorigenic when injected into syngeneic mice.     

The neutrophil elastase mutant affects viability and differentiation of the human monocytic THP‐1 cell

Deficiency in neutrophils (neutropenia) caused by mutations in neutrophil elastase (NE, ELA2) has been extensively investigated. Monocytes and neutrophils are derived from a common myeloid progenitor; therefore, ELA2 mutations can also influence monocyte development. These effects have not been well described. In this study, we used the human monocytic THP‐1, to carry the human wild‐type and G185R mutant ELA2 gene. Growth, death, differentiation and BiP expression were evaluated in the two stable cell lines and in the wild‐type THP‐1 cells. Exogenous wild‐type ELA2 markedly increased THP‐1 differentiation, whereas G185R ELA2 was incompetent to promote THP‐1 differentiation in response to all‐trans retinoic acid (ATRA). Indeed, during differentiation induced by ATRA, G185R cell line showed significant cell death. Also, up‐regulated BiP expression accompanied cell death in the G185R cells, suggesting that the overexpression of G185R elastase increases apoptosis through an unfolded protein response. The G185R cells treated with lithium chloride (LiCl; a Wnt signalling activator) displayed higher BiP expression but similar cell viability compared with THP1 and HNEwt/THP1 cells treated with LiCl. This suggested that Wnt signalling might increase cellular tolerance to endoplasmic reticulum stress, enabling mutant monocyte survival. Copyright © 2012 John Wiley & Sons, Ltd.