Identification of alpha transforming growth factor as a possible local trophic agent for the mammary gland

Biologically active alpha-transforming growth factor (alpha-TGF) has been identified in medium conditioned by rat mammary myoepithelial and, to a lesser extent, by epithelial cell lines in culture and in the rat mammary gland. The alpha-TGF has been identified by its wide spectrum of activity in promoting growth of mammary-derived cells in vitro, by its chromatographic behaviour on reversed-phase high-performance liquid chromatography (HPLC), by its competition with epidermal growth factor (EGF) for the EGF receptor, and by the presence of messenger RNA for alpha-TGF in the secreting cells. In vivo the amount of alpha-TGF isolated is sixfold greater from the mammary glands of lactating than from those of virgin female rats. It is proposed that alpha-TGF is produced by the myoepithelial cells of the mammary gland, as a local trophic agent that stimulates growth of the various cell types of the gland.     

Loss of epidermal growth factor receptors and release of transforming growth factors do not correlate with sarcoma virus-transformation in clonally-related NIH/3T3-derived cell lines.

Transformation of NIH/3T3 cells by Kirsten murine sarcoma virus (MSV) caused a dramatic reduction in the number of cell-surface receptors for epidermal growth factor (EGF). However, the number of EGF receptors remained at a very low level in a non-tumourigenic revertant cell line isolated from the virus-transformed cells, indicating that an increase in EGF receptors is not a requirement for the phenotypic reversion of Kirsten MSV-transformed 3T3 cells. Serum-free conditioned medium from normal and virus-transformed cell lines contained similar amounts of cell growth-promoting activity as assayed by the ability to stimulate DNA synthesis in quiescent Swiss 3T3 cell cultures. However, the concentrated conditioned medium from these cell lines showed no evidence of beta-transforming growth factor (TGF) activity as assayed by promotion of anchorage-independent growth of untransformed normal rat kidney (NRK) fibroblasts in agarose. The cellular release of alpha-TGF activity was assayed by measuring the ability of concentrated conditioned medium to inhibit the binding of 125I-EGF to Swiss 3T3 cells. Conditioned medium protein from the virus-transformed cell line inhibited 125I-EGF binding but only to the same extent as conditioned medium protein prepared from the untransformed cell line. The alpha-TGF secretion by these cell lines was estimated to be 30-45-fold lower than the level of alpha-TGF released by a well-characterized alpha-TGF-producing cell line (3B11). These results suggest that the induction of TGF release is not a necessary event in the transformation of NIH/3T3 cells by Kirsten MSV.     

Evidence suggesting that PrP is not the infectious agent in Creutzfeldt-Jakob disease.

It has been suggested that the infectious agents of scrapie and Creutzfeldt-Jakob disease (CJD) are 'prions' constituted by a protease resistant glycopeptide, PrP. To analyze the role of PrP in CJD infectivity we re-evaluated the biochemical characteristics of infectivity. First, when the infectious agent is not aggregated, infectivity is exquisitely sensitive to proteinase K treatment, and therefore a proteinase-K-resistant molecule (e.g. PrP) is unlikely to contain information essential for agent replication. Second, removal of sugar residues from Gp34 (the major precursor of the proteolyzed PrP band) failed to reduce infectivity. Third, one-half of the PrP peptides could be separated from significant infectivity using nondenaturing conditions with practical quantitative recovery of infectivity. These studies suggest that PrP in itself is unlikely to be the replicating component of the infectious agent. We suggest that these as yet undefined agents may consist of core protein and nucleic acid that are incompletely assembled in, and protected by, cell membranes. This hypothesis would explain the absence of conventional viral particles in these diseases, account for observed membrane pathology including altered behavior of endogenous membrane proteins, and would be consistent with the replication and transforming properties of CJD that indicate there is an agent specific nucleic acid.     

Establishment and characterization of SV40 large T antigen-immortalized cell lines derived from fetal bovine brain tissues after prolonged cryopreservation

Bovine brain cell lines with specific characteristics are useful in vitro experimental systems for molecular and cellular investigation of the interactions between bovine specific neuropathogenic agents and the host. Here, we established two novel cell lines from cultures of cryopreserved fetal bovine brain tissue by the transfection of SV40 large T antigen. Both cell lines showed cobblestone morphology in DMEM/F12 medium supplemented with 10% fetal bovine serum, epidermal growth factor and basic fibroblast growth factor. They were immunostained with endothelial marker, Von Willebrand Factor. Endothelial properties, such as capillary-like tube formation on matrigel and the incorporation of DiI-AcLDL were confirmed with these cells. Removal of growth factors increased the number of cells expressing alpha-smooth muscle actin, suggesting the potential of these cell lines to differentiate into smooth muscle cells. This study suggests an efficient protocol to immortalize brain endothelial cell lines from fetal bovine brain tissue culture.     

Identification and subcellular localization of a 21-kilodalton molecule using affinity-purified antibodies against alpha-transforming growth factor

Monospecific antibodies were generated against each of six different peptide sequences derived from rat and human alpha-transforming growth factor (alpha-TGF). The affinity-purified antibody to the 17 amino acid carboxyl-terminal portion of the molecule proved most useful in detecting alpha-TGF. When used in a peptide-based radioimmunoassay, it was possible to measure nanogram quantities of native alpha-TGF in conditioned cell culture media. When used to analyze cell lysate, these antibodies specifically recognized a 21-kilodalton protein species. Indirect immunofluorescence localization procedures revealed a high concentration of alpha-TGF in a perinuclear ring with a diffuse cytoplasmic distribution. These results suggest that a precursor form of alpha-TGF has a cellular role beyond that of an autocrine growth factor.     

Local effects of EGF, alpha-TGF, and EGF-like growth factors on lobuloalveolar development of the mouse mammary gland in vivo

Five-week-old female mice supplemented with estradiol and progesterone are able to respond to epidermal growth factor (EGF) and EGF-like growth factors (alpha-transforming growth factor [alpha-TGF] and crude mammary-derived growth factor) with local lobuloalveolar development when these growth factors are directly introduced into the mammary glands via slow-release cholesterol-based pellets. Contralateral glands receiving pellets containing only cholesterol showed no growth response. The local growth effect is maximal at 4-5 days of exposure to hormones and growth factors. The glands appear to be more sensitive to alpha-TGF than EGF, since local development is seen with one-fifth the level of the former vs. the latter growth factor and can be seen even in the absence of the systemic estrogen/progesterone supplement.     

Characterization of a glial cell line persistently infected with borna disease virus (BDV): influence of neurotrophic factors on BDV protein and RNA expression.

Borna disease virus (BDV) infects cells of the nervous system in a wide range of species. Previous work suggests that there are differences in BDV replication in neuronal cells and glial cells. Many neurons are lysed by the immunopathologic response to BDV; lysis of dentate gyrus neurons in the absence of encephalitis is seen in rats inoculated with BDV as neonates. In contrast, persistently BDV-infected astrocytes increase over the course of BDV infection. Therefore, we compared BDV replication in neuronal (SK-N-SH and SK-N-SHEP) and astrocytic (C6) cell lines. While SK-N-SH cells produced more infectious virions per cell, the C6 cells contained more BDV proteins and RNA. BDV sequences in the supernatants of both cell types were identified, despite low titers of infectious virus, suggesting the release of incomplete virions into the medium. C6 cells secreted a factor or factors into the medium that enhanced the production of BDV proteins and RNA in other cell lines. In addition, nerve growth factor treatment produced the same enhancement. Thus, BDV replication in certain neural cells in vitro may be linked to the production of cell-specific factors which affect viral replication.     

Tissue Safety in View of CJD and Variant CJD

Epidemiological studies on human transmissible spongiform encephalopathies (Creutzfeldt–Jakob Disease, CJD) have shown that the agent could be transmitted by highly infectious tissues like brain, spinal cord or retina and medicinal products derived from these tissues (i.e. human growth hormone, dura mater). A few cases of transmission of CJD by neurosurgical instruments have been reported. The transmission of the agent of variant CJD, which is suspected to be transmitted by BSE-contaminated food, by blood transfusion implies that in contrast to the agent of classical CJD this agent can also be transmitted by organs and tissues other than nerve tissues. Health authorities have implemented guidelines to reduce the risk of transmission of human and animal TSE by human and veterinary medicinal products. The high resistance of TSE agents against physical or chemical treatment hamper the development of highly efficient inactivation steps in the production of medicinal products. Donor selection is considered as an efficient measure to reduce the risk of TSE transmission. However, the development of rapid, sensitive and specific diagnostic test systems is urgently required to test blood, organs and tissue of donors.     

Steroids and the prostate

Interelationships between steroid and growth factor regulation of cell proliferation has been examined in two androgen sensitive prostatic cell lines, grown in defined medium. The cell lines used were derived from normal (CAPE) and neoplastic (LNCaP) tissues. The growth of both cell lines was elevated by challenge with serum, androgens and epidermal growth factor (EGF) used as single agents. The effects of androgen in CAPE were small, but significant while the profound effects of these agents on the growth of LNCaP were confirmatory of other studies. Androgens upregulated EGF receptor expression in LNCaP measured by both ligand binding capacity and mRNA analysis. This was not observed in the CAPE cells. Addition of serum (whole or charcoal stripped) suppressed the observed androgenic stimulation of EGF receptor expression in LNCaP. This apparent anomaly is discussed in relation to the growth enhancing properties of serum in these cell lines and in the wider context of normal and neoplastic growth control in the prostate.     

Cellular factors implicated in prion replication

Prions are the unconventional infectious agents responsible for prion diseases, which are composed mainly by the misfolded prion protein (PrP^Sc) that replicates by converting the host associated cellular prion protein (PrP^C). Several lines of evidence suggest that other cellular components participate in prion conversion, however, the identity or even the chemical nature of such factors are entirely unknown. In this article we study the conversion factor activity by complementation of a PMCA procedure employing purified PrP^C and PrP^Sc. Our results show that the conversion factor is present in all major organs of diverse mammalian species, and is predominantly located in the lipid raft fraction of the cytoplasmic membrane. On the other hand, it is not present in the lower organisms tested (yeast, bacteria and flies). Surprisingly, treatments that eliminate the major classes of chemical molecules do not affect conversion activity, suggesting that various different compounds may act as conversion factor in vitro. This conclusion is further supported by experiments showing that addition of various classes of molecules have a small, but detectable effect on enhancing prion replication in vitro. More research is needed to elucidate the identity of these factors, their detailed mechanism of action and whether or not they are essential component of the infectious particle.     

Functional and structural characteristics of EGF and its receptor and their relationship to transforming proteins

Epidermal growth factor (EGF) is a peptide which effects the growth and/or differentiated functions of many cell types. Several pieces of evidence indicate that EGF and its receptor may play a role in carcinogenesis. Functional and structural characteristics of EGF and its receptor and their relationship to transforming proteins are discussed. EGF has extensive homology with alpha-transforming growth factor (alpha-TGF), which may actually be an embryonic form of EGF. Nevertheless, both EGF and alpha-TGF elicit transformation-associated phenotypes in target cells under certain conditions. EGF effects are mediated by a receptor present on the plasma membrane. The EGF receptor is a highly complex protein having several functions in addition to binding EGF in a highly specific manner. One of these functions is to phosphorylate tyrosyl residues on certain proteins. This activity is similar to that expressed by the src family of oncogene-encoded proteins. Besides sharing functional homology the EGF receptor also exhibits structural homology to several oncogene-encoded proteins. The v-erb-B-transforming protein has a striking extent of homology (95%) to the cytoplasmic portion of the EGF receptor. These data support the concept that some aspect of EGF-stimulated metabolism is involved in cellular transformation.     

Rat pleural mesothelial cells adapted to serum-free medium as a model for the study of growth factor effects

Mesothelial cells are the putative progenitors of mesotheliomas and cell lines have been used as tools to study the responses of these cells to various stimuli, including growth factors. The present study was undertaken to develop a rat mesothelial cell line capable of sustained growth under serum-free conditions with the object of avoiding the possible confounding effects of undefined serum components. Responses of mesothelial cells to epidermal growth factor were shown to differ under serum-free versus low-serum culture conditions. In contrast, a cell line, SFM1, adapted to growth in serum-free medium was characterized and found to exhibit responses to growth factors similar to the responses reported for human cell lines. This new line should prove to be a useful model for the study of these cells in vitro .     

Search for improved culture conditions for clonogenic growth of human colorectal cancer cells in vitro

In order to analyze and define potentially better growth conditions for colonic stem cell proliferation, we chose four established human colorectal cancer cell lines that differed in biologic cell properties. We studied variables of standard cloning conditions including culture medium, serum supplement, solidifying agent, addition of specific growth factors and use of capillaries as an alternative culture vessel. While modulation of serum concentration as well as use of various standard formulations of culture base media did not result in a reproducible increase of plating efficiencies (PEs), a significant increase in colony formation (when compared to the conventional assay procedure) was achieved; by use of 0.3% agarose or boiled agar as semisolid matrix and by culturing of cells in enriched 'GMF medium'. Specific growth factors, such as EGF or glucagon resulted in "occasionally better" in vitro growth. This suggests a retention of the ability of cells in culture to respond to physiologic regulators of growth. To verify and extend these initial results obtained with continuous cell lines, growth enhancing modifications of the original cloning technique were subsequently applied to in vitro growth of 15 human colorectal cancer specimens obtained directly from patients. Specimens that grew 30 or more colonies under standard plating conditions displayed a more than two-fold increase in PEs which was reproducible for the two specific variables mentioned above, but the overall success rate of the assay could not be improved. In addition to the possibility that several deficient basic requirements for achieving optimal environmental conditions for colonic stem cell growth have not been defined, we believe a major reason for failing to improve the number of drug-assayable specimens is related to an inherent interneoplastic diversity in terms of growth requirements of human colorectal malignancies.     

Phenotypic and molecular characterization of inducible human neuroblastoma cell lines

Two new neuroblastoma (NB) cell lines, NUB-6 and NUB-7, were established from recurrent and primary NB tumours respectively and identified conclusively as NB by their phenotypic characteristics, catecholamine production and N-myc amplification. The cell lines could be distinguished on the bases of distinctive growth patterns in monolayer culture and semi-solid media (collagen gel and agarose), neurite formation and their response to four classes of growth and differentiation modulators. The NUB-6 cell line consisted of two distinct cell subtypes, small typical neuroblasts and larger spheroid-forming cells, while NUB-7 was homogeneously neuroblastic. Class-I agents (dibutyrl cyclic AMP [dbcAMP], butyrate, and papaverine) inhibited growth of both cell lines, while only dbcAMP stimulated the formation of short neurites by NUB-6 neuroblast cells in monolayer culture and collagen. Of the class-II agents (vitamins), retinoic acid inhibited growth of both cell lines and stimulated formation of long neurites by NUB-6 cells and NUB-7 cells in later passages. In contrast, vitamin E inhibited growth of NUB-6 and late-passage NUB-7, but stimulated early passage NUB-7. The class III agent (nerve growth factor) resembled vitamin E. The class-IV agents (interferons; rIFN-alpha 2a and rIFN-gamma 1) inhibited growth of both cell lines in monolayer culture and agarose, but stimulated NUB-6 neuroblasts and early passage NUB-7 cells to form long neurites. Thus phenotypically distinct NB cell lines were established in vitro and shown to be differentially influenced by various growth and differentiation modulators. The potent effect of IFN suggests a role for these modulators in NB behaviour in vivo.     

Growth of preadipocyte cell lines and cell strains from rodents in serum-free hormone-supplemented medium

Ob17 is a clonal cell line isolated from the epididymal fat pad of C57 BL/6J ob/ob mouse that differentiates into adiposelike cells in serum-supplemented medium. In serum-free medium, this cell line shows increased growth under the addition of insulin, transferrin, fibroblast growth factor (FGF), and a factor present in extract of rat submaxillary gland (SMGE). This medium is referred to as 4F. Epidermal growth factor or nerve growth factor cannot replace SMGE, whereas partially purified platelet extract can substitute for FGF but only partially for SMGE. 4F Medium is able to support the proliferation of cells from other established preadipocyte clonal lines, HGFu and 3T3-F442A, and also of preadipocyte cells isolated from the stromal-vascular fraction of rat and mouse adipose tissues. In each case 4F medium is insufficient to support the differentiation of these cells into adipocytes. Ob17 cells grown and maintained in serum-free hormone-supplemented medium retain the ability to convert to adiposelike cells after serum addition. This serum requirement for differentiation cannot be substituted by the addition of growth hormone or of other putative adipogenic factors, or both. The results are discussed with respect to the requirements for growth and differentiation of the 3T3-L1 and 1246 preadipocyte cell lines previously described.     

Immunological analysis of host and agent effects on Creutzfeldt-Jakob disease and scrapie prion proteins.

Creutzfeldt-Jakob disease (CJD) and scrapie are degenerative neurological diseases caused by unusual infectious pathogens. The term prion has been introduced to underscore the apparent distinctness of these agents from viruses and viroids. The only macromolecule shown to be associated with the infectious agent, the CJD or scrapie prion protein (PrPCJD or PrPSc, respectively), is encoded by the same gene as a normal cellular protein. In several studies biochemical differences have been reported in PrPScs derived from a common host species infected with different putative strains of the scrapie agent, suggesting agent-specific characteristics independent of the host. We analyzed various agent-host combinations by Western blotting of PrPs that were separated by size or charge. The profile of immunoreactive proteins for CJD prions isolated from mice, guinea pigs, and humans appeared distinct. Importantly, PrPCJDS purified from a human brain and from the corresponding first-passage mouse brains were clearly distinguishable. PrPCJDs isolated from CJD prions propagated in NAMRU or B10.Q mice, which are homozygous for a short-incubation-time gene; from the short-incubation-time backcross progeny of (B10.Q x I/LnJ)F1 x B10.Q; or from NAMRU mice inoculated with I/LnJ prions were identical to each other but distinguishable from those of I/LnJ mice, which are homozygous for the long-incubation-time gene. The PrPs from human CJD and ovine scrapie propagated in the same mouse strain appeared the same, but they were distinct from the same isolate of scrapie passaged in hamsters. Lastly, PrPScs purified from five different strains of scrapie propagated in C57BL mice were identical, including strains, ME7 and 139A, which were previously reported to be distinct. This evidence does not support, although it does not exclude, agent-mediated characteristics independent of host-mediated ones for scrapie and CJD.