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Biomedical subjects

H Land

Publications and source records attributed to H Land.

At least 37 records · Page 2Linked to original sources
PubMed1990

Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line.

We report the development of an advanced system for transfer and expression of exogenous genes in mammalian cells based on Moloney murine leukemia virus (Mo MuLV). Extensive deletion/mutagenesis analysis to identify cis-acting signals involved in virus transmission has led to the design of a family of novel, highly efficient retroviral vectors and a partner helper-free packaging cell line. The pBabe retroviral vector constructs transmit inserted genes at high titres and express them from the Mo MuLV Long Terminal Repeat (LTR). Each of these vectors has been constructed with one of four different dominantly acting selectable markers, allowing the growth of infected mammalian cells in the presence of G418, hygromycin B, bleomycin/phleomycin or puromycin, respectively. The high titre ecotropic helper free packaging cell line, omega E, was designed in conjunction with the pBabe vectors to reduce the risk of generation of wild type Mo MuLV via homologous recombination events. The omega E cell line was generated with separate gagpol and ecotropic env expression constructs with minimal sequence overlap and decreased sequence homology achieved by 'codon wobbling'. Homologous env coding sequences were deleted from the pBabe vectors without diminishing recombinant vector titre. Together, the pBabe vectors and omega E cell line should prove useful in experiments where highest frequencies of gene transfer, or concomitant expression of several different genes within a single cell are required with minimal risk of helper virus contamination.

Amino Acid Sequence↗
PubMed1990

Control of division and differentiation in oligodendrocyte-type-2 astrocyte progenitor cells.

Oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells give rise to oligodendrocytes and type-2 astrocytes in cultures of rat optic nerve. These progenitors are one of the few cell types in which most aspects of proliferation and differentiation can be manipulated in a defined in vitro environment. When exposed to platelet-derived growth factor (PDGF), O-2A progenitors divide a limited number of times before clonally related cells differentiate into oligodendrocytes with a timing similar to that seen in vivo. In contrast, O-2A progenitors grown in the absence of mitogen do not divide but differentiate prematurely into oligodendrocytes, and progenitors exposed to appropriate inducing factors differentiate into type-2 astrocytes. O-2A progenitors can become immortalized through at least two different mechanisms. First, when O-2A progenitors are exposed to a combination of PDGF and basic fibroblast growth factor (bFGF) these cells undergo continuous self-renewal in the absence of differentiation. In contrast, the application of bFGF alone is associated with premature oligodendrocytic differentiation of dividing O-2A lineage cells. Thus, cooperation between growth factors can modulate O-2A progenitor self-renewal in a defined chemical environment by eliciting a novel programme of division and differentiation which cannot be predicted from the effects of either factor examined in isolation. A further mechanism which allows prolonged self-renewal in the O-2A lineage is the generation of a stem cell. O-2A progenitors isolated from optic nerves of perinatal rats also have the capacity to give rise to a population of cells called O-2Aadult progenitors, which differ from their perinatal counterparts in many characteristics. Most importantly, O-2Aadult progenitors have a slow cell cycle, divide and differentiate asymmetrically and appear to have the capacity for prolonged self-renewal. Thus, immortalization in this lineage can also be achieved by the generation of a cell with stem cell-like characteristics from a rapidly dividing progenitor population.

Aging↗
PubMed1990

Negative autoregulation of c-myc transcription.

The introduction of activated c-myc and v-myc genes into a variety of non-established and established cells results in the suppression of endogenous c-myc expression. As measured in Rat-1 fibroblasts, the suppression occurs at the level of transcriptional initiation. Moreover, the extent of the down-regulation is proportional to the cellular concentration of c-myc protein, and the critical concentration range in which the endogenous c-myc RNA is effectively suppressed corresponds to that found in non-transformed cells. In addition, the autoregulatory mechanism is not only dependent on c-myc protein, but also requires additional trans-acting factors. These results support a role for c-myc in the regulation of cellular gene transcription and suggest that a negative feedback mechanism can act as a homeostatic regulator of c-myc expression in vivo.

Animals↗
PubMed1990

Cooperation between two growth factors promotes extended self-renewal and inhibits differentiation of oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells.

Bipotential oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells, which give rise to oligodendrocytes and type-2 astrocytes in cultures of rat optic nerve, are one of the few cell types in which most aspects of proliferation and differentiation can be manipulated in a defined in vitro environment. Previous studies have shown that O-2A progenitors exposed to platelet-derived growth factor (PDGF) divide as migratory bipolar cells a limited number of times, with a cell cycle time of 18 hr, before clonally related progenitors differentiate into nondividing oligodendrocytes with a timing similar to that seen in vivo. In contrast, O-2A progenitors grown in the absence of mitogen do not divide but instead differentiate prematurely into oligodendrocytes, and progenitors exposed to appropriate inducing factors differentiate into type-2 astrocytes. We now have found that O-2A progenitors can be induced to undergo continuous self-renewal in the absence of oligodendrocytic differentiation by exposure to a combination of PDGF and basic fibroblast growth factor (bFGF). With the exception of the inhibition of differentiation, the O-2A progenitors exposed to PDGF and bFGF behaved similarly to those exposed to PDGF alone. In contrast, progenitors exposed to basic bFGF alone were multipolar, had a cell-cycle length of 45 hr, showed little migratory behavior, underwent premature oligodendrocytic differentiation, and did not cease division upon expression of oligodendrocyte marker antigens. Thus, inhibition of differentiation required the presence of both mitogens. Our results demonstrate that PDGF and bFGF act on O-2A progenitors as both inducers of division and as regulators of differentiation that modulate multiple aspects of O-2A progenitor development and, additionally, reveal a previously unrecognized means of regulating self-renewal processes, wherein cooperation between growth factors promotes continuous division in the absence of differentiation.

Animals↗
PubMed1990

Domains of human c-myc protein required for autosuppression and cooperation with ras oncogenes are overlapping.

Amino acids 106 to 143 and 354 to 433 of the human c-myc protein (439 amino acids) were shown to be required for the protein to suppress c-myc gene transcription and were found to exactly overlap with those necessary for c-myc to cooperate with ras oncogenes in the transformation of rat embryo fibroblasts. The essential carboxyl-terminal region harbors structural motifs (a basic region, a helix-loop-helix motif, and a "leucine zipper"), which, in other proteins, can mediate dimerization and sequence-specific DNA binding.

Animals↗
PubMed1990

Establishment of rat endometrial cell lines by retroviral mediated transfer of immortalizing and transforming oncogenes.

To study hormone responsive genes in differentiated epithelial cells and as a model for endometrial carcinoma, lines were established from primary rat endometrial cells by infection with replication-defective retroviruses carrying oncogenes and the selectable gene neo. The initial step involved immortalization through the large T antigen of SV40 to generate a line we designate RENT4, or with the E1a gene of adenovirus to generate lines referred to as RENE1 and RENE2. Additionally, lines generated by large T antigen of SV40 were superinfected with a replication-defective retrovirus harboring the v-Ha-ras oncogene and selected by the ability to form colonies in soft agar. The latter cell lines appeared fully transformed and were designated RENTR01 and RENTR03. Five established lines were characterized for steroid hormone receptors, alkaline phosphatase activity and their complement of the intermediate filaments vimentin and cytokeratin. With the exception of the RENE1 cells all other lines have normal levels of glucocorticoid receptor, whereas only RENE1, RENE2 and RENT4 were positive for the progesterone receptor. RENTR01, RENTR03 and, to a lesser extent, RENE1 exhibited differential expression of cytokeratins dependent upon whether the cells were grown on a substrata of NIH3T3 cells. When grown on formalin-fixed NIH3T3 cells, RENTR01 and RENTR03 cells appeared to differentiate or rearrange themselves in culture. Individual islands of cells showed a heterogeneous pattern of intermediate filaments with vimentin-positive cells localized to the outer portion of the islands whereas cytokeratin-positive cells are seen on the insides of these structures.

Alkaline Phosphatase↗
PubMed1990

[Induction of primitive neuroectodermal tumors by oncogene complementation].

Primitive neuroectodermal tumors (PNET) represent a family of undifferentiated neural neoplasms which predominantly occur in children. PNETs are likely to originate from precursor cells and exhibit a marked potential for neuronal, glial and ependymal differentiation. A prominent example is the medulloblastoma of the cerebellum. In a model system using a novel transgenic CNS transplantation model, we have introduced a combination of ras and myc oncogenes into cell suspensions from fetal forebrain (E14) and postnatal cerebellum (P2) of the rat. Oncogene transfer into fetal forebrain grafts resulted in a high incidence of anaplastic neural tumors predominantly derived from glial precursors. Cell lines established from these neoplasms expressed high levels of both oncogenes. In a second experiment, the ras/myc vector was introduced into cell suspensions from neonatal cerebellum. The transformed cells were cultured for 3 weeks. Following stereotaxic transplantation, tumors of a similar morphology were observed. However, one animal developed a neoplasm with features of a cerebellar medulloblastoma. A cell line which exhibits a marked capacity for neurite extension and synaptogenesis was established from this tumor. Since these cells neither express ras nor myc, an insertion mutagenesis event appears to be responsible. Experiments to characterize this mutation are in progress. Our results indicate a potent transforming effect of ras and myc on neural precursor cells in vivo.

Animals↗
PubMed1990

C-MYC: evidence for multiple regulatory functions.

The nuclear c-myc proto-oncogene promotes cell proliferation and can inhibit terminal differentiation as well as induce immortalisation in its oncogenic form. There is increasing evidence that c-myc exerts these biological activities by modulating transcription and by directly affecting the initiation of DNA replication. The regulation of these disparate activities may involve the carboxyl end of the c-myc protein, which is essential for transformation and autosuppression of c-myc transcription. Conserved motifs in this region of the c-myc protein may mediate complex formation and sequence-specific nucleic acid binding.

Amino Acid Sequence↗
PubMed1990

Biculturality and homosexuality.

In an effort to sensitize service providers to bicultural socialization, a small but growing literature has surfaced in which theories are proposed explaining the socialization process, cultural identification, and evolution of self-concept for those functioning in two cultures. In this article, the authors examine the extent to which previously developed explanatory theories of cultural and ethnic identity and bicultural socialization apply to the experience of sexual minorities, and offer a conceptual framework for adequately assessing the experience of homosexual clientele. The multifaceted nature of the practitioner's role is discussed, and gender-sensitive practice methods are given that may assist sexual minorities in developing optimum functioning.

Acculturation↗
PubMed1989

Multistage carcinogenesis induced by ras and myc oncogenes in a reconstituted organ.

ras and myc oncogenes were able to induce distinct phenotypic alterations, resembling different types of premalignant lesions, when introduced into approximately 0.1% of the cells used to reconstitute the mouse prostate gland. While ras induced dysplasia in combination with angiogenesis, myc induced a hyperplasia of the otherwise normally developed organ. ras and myc together induced primarily carcinomas. However, tumor progression was also associated with additional genetic alterations involving gene amplification. Our data indicate that specific types of benign premalignant lesions may reflect the activation of different single oncogenes, and that the consecutive activation of multiple oncogenes could be a causal event in the step-like progression of tumorigenesis.

Animals↗
PubMed1989

The ras and myc oncogenes cooperate in tumor induction in many tissues when introduced into midgestation mouse embryos by retroviral vectors.

Midgestation embryos were infected with replication-defective retroviral vectors that either transduced the myc oncogene, the ras oncogene, or both oncogenes simultaneously. The myc virus induced tumors in diverse organs at a very low frequency and with a long latency period, while approximately 20% of the mice derived from embryos infected with the ras virus developed tumors in the skin with a latency of 4-8 weeks. In contrast, infection of embryos with the ras/myc double oncogene virus resulted in 27% of the animals developing rapidly growing and malignant tumors in a great variety of tissues after a median latency period of 2-3 weeks. All tumors were of monoclonal origin, as shown by Southern analysis using the provirus as a molecular marker. Our results are consistent with the hypothesis that the ras and myc oncogenes cooperate in transforming cells, but that additional alterations are necessary for realization of the fully malignant phenotype. Our observations also suggest that a much wider range of cell types become targets for malignant transformation when the embryos are exposed to the myc and the ras oncogenes simultaneously than when exposed to the same oncogenes separately. Infection of mouse embryos with vectors carrying different oncogenes or oncogene combinations may be an efficient and rapid method for evaluating the spectrum of cell types at risk for malignant conversion following mutation of a protooncogene to a transforming gene.

Animals↗
PubMed1989

Transforming growth factors-beta 1 and beta 2 are mitogens for rat Schwann cells.

Transforming growth factor-beta 1 (TGF-beta 1) and TGF-beta 2 were found to be potent mitogens for purified rat Schwann cells, each stimulating DNA synthesis in quiescent cells and also increasing their proliferation rate. Half-maximal stimulation of DNA synthesis occurred at approximately 0.1 ng/ml TGF-beta 1 or TGF-beta 2. Mitogenic stimulation by TGF-beta 1 and TGF-beta 2 was enhanced by forskolin, which activates adenylate cyclase, at concentrations up to 0.5 microM forskolin. However, at 5 microM forskolin, the synergistic interaction between forskolin and TGF-beta 1 was abolished. These results are in contrast to the observed synergy between forskolin and another Schwann cell mitogen, glial growth factor (GGF). Both 0.5 and 5 microM forskolin were found to enhance the stimulation of DNA synthesis by partially purified GGF (GGF-CM). As well as being functionally distinct, TGF-beta 1 and GGF-CM activities were also physically separable by chromatography on a Superose 12 gel permeation column. Thus, TGF-beta 1 and beta 2 are rat Schwann cell mitogens, and Schwann cells are one of the few normal cell populations to respond mitogenically to TGF-beta.

Animals↗
PubMed1989

An in situ transgenic enzyme marker for the midgestation mouse embryo and the visualization of inner cell mass clones during early organogenesis.

In order to study the deployment of cells during gastrulation and early organogenesis, it is necessary to have an in situ cell marker which can be used to follow cell fate. To create such a marker a transgenic mouse strain, designated Tg(Act-lac Z)-1, which carries 6 copies of the Escherichia coli lac Z gene under the control of the rat beta-actin promoter, was made by pronuclear injection of DNA. Staining early postimplantation hemizygous mouse conceptuses, during gastrulation and early organogenesis, for beta-galactosidase activity shows that lac Z expression is ubiquitous and constitutive in all epiblast derivatives of the 10th day conceptus. No activity is seen in trophectoderm and primitive endoderm derivatives. Postimplantation grafts of [3H]thymidine-labelled transgenic cells establish the cell autonomy of this transgenic marker. Preliminary observations on the distribution of inner cell mass (ICM) descendant clones, identified in situ in midgestation conceptuses, confirm the pluripotency of individual ICM cells. The implications regarding patterns of cell growth in nascent fetal primordia are discussed.

Animals↗
PubMed1988

Ras-mediated cell cycle arrest is altered by nuclear oncogenes to induce Schwann cell transformation.

The cellular responses to ras and nuclear oncogenes were investigated in purified populations of rat Schwann cells. v-Ha-ras and SV40 large T cooperate to transform Schwann cells, inducing growth in soft agar and allowing proliferation in the absence of added mitogens. Expression of large T alone reduces their growth factor requirements but is insufficient to induce full transformation. In contrast, expression of v-Ha-ras leads to proliferation arrest in Schwann cells expressing a temperature-sensitive mutant of large T at the restrictive temperature. Cells arrest in either the G1 or G2/M phases of the cell cycle, and can re-enter cell division at the permissive temperature even after prolonged periods at the restrictive conditions. Oncogenic ras proteins also inhibit DNA synthesis when microinjected into Schwann cells. Adenovirus E1a and c-myc oncogenes behave similarly to SV40 large T. They cooperate with Ha-ras oncogenes to transform Schwann cells, and prevent ras-induced growth arrest. Thus nuclear oncogenes fundamentally alter the response of Schwann cells to a ras oncogene from cell cycle arrest to transformation.

Animals↗
PubMed1986

Behavior of myc and ras oncogenes in transformation of rat embryo fibroblasts.

The requirements for transformation of rat embryo fibroblasts (REFs) by transfected ras and myc oncogenes were explored. Under conditions of dense monolayer culture, neither oncogene was able to transform REFs on its own. However, the introduction of a ras oncogene together with a selectable neomycin resistance marker into REFs allowed killing of the normal nontransfected cells and the outgrowth of colonies of ras transformants, 10% of which survived crisis and became tumorigenic. These cells expressed greater than 10-fold-higher levels of ras p21 than tumorigenic cells cotransfected with ras and myc oncogenes. The myc oncogene similarly was unable to induce tumorigenic conversion of REFs unless especially refractile colonies of oncogene-bearing cells, produced by use of a cotransfected selectable marker, were picked and subcultured. Tumorigenic conversion of REFs by single transfected oncogenes appears to require special culture conditions and high levels of gene expression.

Animals↗