Long-term culture and functional characterization of follicular cells from adult normal human thyroids.

We have obtained long-term cultures of differentiated proliferating follicular cells from normal adult human thyroid glands. In vitro growth of such human cells has been sustained by a modified F-12 medium, supplemented with bovine hypothalamus and pituitary extracts and no added thyrotropin. Cultures have been expanded, cloned, frozen, successfully retrieved, and characterized. Functional characterization of these cells shows constitutive thyroglobulin production and release and thyrotropin-dependent adenosine 3',5'-cyclic monophosphate production, the latter apparently not associated with significant increases in DNA synthesis or cell proliferation. Genetic characterization of these cells by chromosome counting showed the normal diploid chromosome number. The ability to cultivate differentiated human thyroid follicular cells in long-term culture opens possibilities for investigating the transduction pathways of thyrotropin stimulation in normal and pathological human tissues, developing clinically relevant in vitro assays, and considering cellular and molecular therapies.

In vitro culture of primary plasmacytomas requires stromal cell feeder layers.

Attempts to grow primary murine plasmacytomas in vitro have, to date, been largely unsuccessful. In this study, we demonstrate that long-term in vitro growth of primary plasmacytomas is accomplished by using feeder layers composed of stromal cells from the initial site of plasmacytomagenesis. The early neoplastic lines established in this manner are dependent on physical contact with the stromal layer, which is mediated in part by CD44, for growth and survival. The stromal cells provide at least two stimuli for the plasma cells, one being interleukin 6 and the second, of unknown nature, resulting from direct physical interaction that cannot be replaced by soluble factors. These plasma cell lines have been passaged for as long as 20 months yet still maintain characteristics associated with primary plasmacytomas as they will grow in vivo only in pristane-primed animals, indicating a continued dependence on the pristane-induced microenvironment characteristic of early-stage tumors. The ability to grow primary plasmacytomas in culture and maintain their "primary" properties provides a model system for detailed analysis of early events in plasma cell tumor progression involving neoplastic cells completely dependent on physical contact with a stromal feeder layer for survival and expansion.

Cell cultures of neuroblasts from rat olfactory epithelium that show odorant responses.

We have developed procedures that permit isolation and propagation of clonal cell cultures from the olfactory epithelium of the 5- to 7-day-old rat that appear to represent the neuroblasts that repopulate the sensory neurons in the olfactory epithelium throughout life. The cell lines we report here synthesize neuron-specific enolase, which is a neuron marker, 43-kDa growth-associated protein, a protein associated with neuronal growth cones, and carnosine, a possible olfactory neurotransmitter. In two of the cell lines we have found dose-dependent cAMP accumulation following exposure to submicromolar concentrations of chemical odorants in the medium. These two cell lines show different patterns of odorant specificity when tested against a panel of six chemicals commonly used as test odorants. We anticipate that these and similarly derived cell lines will prove valuable in studying aspects of neurogenesis and olfaction.

Stable integration and expression of a bacterial gene in the mosquito Anopheles gambiae.

Foreign DNA was successfully introduced into the germline of the African mosquito vector of malaria Anopheles gambiae. Stable integration of genes into the germlines of insects had been achieved previously only in Drosophila melanogaster and related species and required the use of the P element transposon. In these experiments with Anopheles gambiae, the plasmid pUChsneo was used, which contains the selectable marker neo gene flanked by P element inverted repeats. Mosquitoes injected with this plasmid were screened for resistance to the neomycin analog G-418. A single event of plasmid insertion was recovered. Integration appears to be stable and, thus far, resistance to G-418 has been expressed for eight generations. The transformation event appears to be independent of P.

Vaccination-induced variation in the 140 kD merozoite surface antigen of Plasmodium knowlesi malaria.

Immunity to 143/140 kD schizont antigens of a monkey malaria, Plasmodium knowlesi, provides partial protection to lethal malaria infection in rhesus monkeys challenged with uncloned parasites. To determine the capacity of a cloned parasite to generate variants of the 143/140 kD antigens, immunized monkeys were challenged with a clone of P. knowlesi. Parasites recovered 8 d after inoculation with a cloned parasite retained the 143/140 kD antigens. Parasites recovered 30 d after challenge had undergone changes in the 143/140 kD antigens. Antibodies that block erythrocyte invasion in vitro of the inoculum parasites did not inhibit invasion of erythrocytes by two isolates recovered from the immunized monkeys. An isolate from one monkey recovered on day 30 contained clones expressing new 76/72 kD antigens reactive with rabbit antiserum against the 143/140 kD proteins, and other clones expressing no antigens crossreactive with antisera against the 143/140 kD proteins. An isolate from another monkey obtained 59 d after challenge expressed new antigens of 160/155, 115/113, and 87/85 kD. Using monoclonal antibodies, we found that epitopes were lost from the variant proteins, but we were unable to determine whether new epitopes had appeared. We conclude that clones of P. knowlesi can rapidly vary antigenic determinants on the 143/140 kD proteins in animals immunized with these antigens.

Bovine parathyroid cells: cultures maintained for more than 140 population doublings.

Primary cultures of bovine parathyroid cells were developed using Coon's modified Ham's F-12 medium containing low (0.3 mM) concentrations of calcium and supplements of bovine hypothalamic extract, bovine pituitary extract, epidermal growth factor, insulin, transferrin, selenous acid, hydrocortisone, triiodothyronine, retinoic acid, and galactose. These cells were cultured serially on serum-coated dishes for 140 population doublings before signs of senescence were detected. The cells were epithelioid and maintained a high degree of differentiation as evidenced by calcium regulation of both growth and secretion and by prostaglandin E1 stimulation of cAMP formation and hormone release.

Splenic requirement for antigenic variation and expression of the variant antigen on the erythrocyte membrane in cloned Plasmodium knowlesi malaria.

Variant antigens appear on the surface of Plasmodium knowlesi-infected erythrocytes as the asexual parasite matures and are detected by antibody-mediated schizont-infected cell agglutination (SICA). We now show that cloned parasites can undergo antigenic variation in nonsplenectomized monkeys. In addition, we previously described a new P. knowlesi phenotype in which uncloned parasites passaged in splenectomized monkeys were no longer agglutinable by immune sera. We have designated this new phenotype SICA[-] and the one expressing the variant antigen SICA[+]. Cloned parasites can also switch from SICA[+] to SICA[-] in splenectomized monkeys. The switch from SICA[+] to SICA[-] is a gradual process that requires sequential subpassage in several monkeys. After passage in one monkey, the agglutination titer decreased 4- to 16-fold. Decreased agglutination was associated with decreased antibody binding on all infected erythrocytes as measured by fluorescein-conjugated anti-rhesus monkey immunoglobulin. The asexual malaria parasite can therefore alter its expression of variant antigen in response to the host environment (antivariant antibody or splenectomy). When cloned SICA[-] parasites were inoculated into intact monkeys, two courses of parasitemia were observed: fulminant parasitemia (greater than 20%) and parasitemia that was controlled. Fulminant infections were associated with conversion of the parasite from SICA[-] to SICA[+], i.e., from nonexpression to expression of the variant antigen on the erythrocyte surface. Parasitized erythrocytes remained SICA[-] in those infections that were controlled. It appears, therefore, that the expression of the variant antigen on the erythrocyte surface may influence parasite virulence.

Electrophysiological and pharmacological properties of cultured rat thyroid cells.

The electrophysiological properties of a hormone-dependent, differentiated thyroid epithelial cell strain were studied using intracellular microelectrodes. The average membrane potential of solitary, isolated cells was -78.4 +/- 1.3 mV. The membrane potential depolarized 55 mV per tenfold increase in extracellular potassium concentration. Weak electrical coupling was recorded between contiguous cells. Like thyroid cells in vivo, these cells did not generate action potentials. In some cells a spontaneous, slow transition in the membrane potential from -80mV to -30 mV was accompanied by an increase in input resistance. Membrane potential transitions could be induced by perfusing cells with isotonic Hanks solutions saturated with CO2 (pH = 5.5) or by perfusing cells with hypotonic Hanks solutions (190-290 mOsm/kg). Membrane potential transitions were due to a decreased potassium permeability. Noradrenaline elicited both a fast depolarization and a slow depolarization. The fast depolarization was due to an increase in conductance of Na+ channels and of Cl- channels. Intracellular injection of Ca++ elicited the fast depolarization. Intracellular injection of EGTA or cobalt abolished the fast depolarization. Replacement of extracellular Ca++ by Mg++ did not affect the fast depolarization. Thus, the fast depolarization was due to accumulation of intracellular Ca++. The fast depolarization was abolished by the alpha adrenergic blocker phentolamine (10(-6) M), and was not abolished by the beta adrenergic blocker propranolol (10(-5) M).

Culture of hormone-dependent functional epithelial cells from rat thyroids.

Primary cultures of rat thyroid cells were made in medium supplemented with 0.1--0.5% calf serum and containing six hormones or growth factors: insulin, thyrotropin, transferrin, hydrocortisone, somatostatin, and glycyl-L-histidyl-L-lysine acetate. The FRTL strain was purified by successive colonial isolations and was found to maintain highly differentiated features (secretion into the culture medium of physiological amounts of thyroglobulin and concentration of iodide by 100-fold). The FRTL strain has been observed for more than 3 years in continuous culture. It has maintained the same biochemical and morphological characteristics that typified the primary cultures of thyroid follicular cells immediately after their enzymatic release from the rat thyroid. Thyroid epithelial cells that were grown under more conventional cell culture conditions failed to retain these specialized characteristics. We show that maintenance in vitro of these specialized functions of rat thyroid follicular cells is dependent on low serum concentrations and supplementation with hormones in the primary cultures. Our observations indicate that this culture strategem may be aplicable to the general problem of maintenance of differentiated characteristics in cultures of other epithelial cells.

Recognition of antigenic differences among neurons using antiserums to clonal neural retina hybrid cells.

Antiserums prepared against hybrid cell strains formed between freshly isolated rodent neural retina cells and a human fibroblast cell line W.I. 18, VA-2 recognize cell surface antigens that are 1) restricted to the neural retina, 2) present in both embryonic and adult retina, and 3) localized to groups of cells within the retina. Normal segregants (i.e., those lines that had retained rodent chromosomes and lost human chromosomes) were used as immunogens in rabbits to produce the antiserums. Antiserums against both whole cells and purified plasma membranes were adsorbed with human parent VA-2 cells and nonneural and neural rodent tissue to remove cross-reacting specificities. All six antiserums studied continued to react with embryonic retina after brain cross-reactivity was removed.

Production of long term steroid-producing granulosa cell cultures by cell hybridization.

Primary cultures of rat ovarian granulosa cells have been used extensively to study hormonal regulation of cellular function. To date, no long term cultures of ovarian cells which retain their differentiated functions have been developed. Hypoxanthine guanine phosphoribosyl transferase-deficient simian virus 40-transformed rat ovarian granulosa cells were fused with freshly prepared rat granulosa cells using inactivated Sendai virus. Putative hybrid cell strains obtained after selection in medium containing hypoxanthine, aminopterin, and thymidine were analyzed for progesterone synthesis. Neither the original simian virus 40-transformed granulosa cell nor its hypoxanthine guanine phosphoribosyl transferase-deficient derivative produced progesterone, but three of the hybrid strains produced progesterone at basal levels and in response to dibutyryl cAMP. One of these strains produced progesterone in a dose-responsive fashion when exposed to prostaglandin E2, cholera toxin, dibutyryl cAMP, and 2-chloroadenosine. Cell strains obtained by hybridization were remarkably similar to primary cultures of granulosa cells with respect to both the magnitude and temporal aspects of progesterone production in response to dibutyryl cAMP.

Cell fusion induced by scrapie and Creutzfeldt-Jakob virus-infected brain preparations.

Cell fusion was induced by brain extracts containing the scrapie virus and the virus of Creutzfeldt-Jakob disease. The assay involved quantitation of colony-forming ability in a double selection system, strandardized against fusion induced by Sendai virus. Correlation between the logarithm of virus dilution and the hybrid colony number gave similar curves for scrapie virus and Sendai virus. Fusion induction may explain some aspects of pathogenesis in these diseases and provide a potential in vitro assay.

The genetics of the mitochondrial DNA of mammalian somatic cells, their hybrids and cybrids.

A brief review is attempted of the status of the genetics of the mitochondria of mammalian cells in vitro. Reverse segregant human-mouse hybrid strains are unusually stable and retain mitochondrial DNA (mtDNA) of both species for many generations but eventually lose the mtDNA of the species whose chromosomes are segregated. A molecular interchange resembling recombination among mtDNA molecules can be detected in these hybrids. Eisenstadt and co-workers have shown that chloramphenicol inhibits protein synthesis less in mitochondria isolated form chloramphenicol-resistant, mutant cell strains than from sensitive strains. They have also shown that resistance is effectively transmitted by cytoplast fusion. These genetic markers are, therefore, probably in the mtDNA of mammals as they are in yeast. Although chloramphenicol resistance has not been transferred from one distinct species to another by cytoplast fusion, we find that different subspecies of mouse are able to exchange resistance. We have also transferred chloramphenicol resistance to ("transformed") 10(-3) sensitive cells by treating them with purified mtDNA from resistant cells. Treatment with DNA from Escherichia coli and/or mtDNA purified from sensitive cells does not transform. Treatment with nuclear DNA, however, from either sensitive or resistant cells does transform. We suggest that exogenously applied nuclear DNA acts as a potent mutagen in mitochondria. A potential application of the mtDNA transformation phenomenon to molecular cloning of DNA in mammalian mitochondria is proposed.

Proliferation of Buffalo rat liver cells in serum-free medium does not depend upon multiplication-stimulating activity (MSA).

A line of Buffalo rat liver cells (BRL 3A) that multiplies in the absence of serum produces a family of polypeptides termed MSA that can partially satisfy the serum requirement for growth of chick embryo fibroblasts. Temin, Pierson and Dulak (1972) proposed that BRL cells multiply in serum-free medium because they produce MSA. This does not appear to be the case. We have studied three BRL cell lines: 3A2 and 3A have diverged from the same original isolate from normal liver; 61t is a spontaneous transformant of a different isolate. All three cell lines showed a 10 fold increase in cell number during 5 days in serum-free medium. However, 3A-conditioned medium stimulated 3H-thymidine incorporation into DNA in chick embryo fibroblasts and human skin fibroblasts; 3A2- and 61t-conditioned media did not. After ion-exchange chromatography or gel filtration of the conditioned media and measurement of MSA by 3H-thymidine incorporation or radioreceptor assay, MSA again was found in the 3A medium but not in the 3A2 or 61t media. The absence of MSA in the 3A2 and 61t media was not due to inactivation of MSA by these two cell lines. Addition of partially purified MSA to 3A2 cells did not increase their multiplication rate in serum-free medium. We conclude that the ability of the BRL cells to multiply in serum-free medium is independent of the level of MSA in the medium.

Effects of thyrotropin on the thyroid cell membrane: hyperpolarization induced by hormone-receptor interaction.

Cultured thyroid cells accumulate the lipophilic cation triphenylmethylphosphonium, indicating that there is an electrical potential (interior negative) across the plasma membrane. Thyrotropin stimulates the uptake of the lipophilic cation 3-fold, and the proton conductor carbonylcyanide-m-chlorophenylhydrazone causes efflux of triphenylmethylphosphonium accumulated in the presence or absence of thyrotropin. The stimulatory effect of thyrotropin on triphenylmethylphosphonium accumulation is not mimicked by human chorionic gonadotropin, a glycoprotein hormone with a similar structure whose target organ is not the thyroid, and the effect is abolished if the thyrotropin-receptor activity of the cells is destroyed by treatment with trypsin. Analogous effects are observed with thyroid plasma membrane vesicles which are essentially devoid of mitochondrial and soluble enzyme activities. Triphenylmethylphosphonium uptake and stimulation by thyrotropin occurs when NaCl, KCl, or Tris.HCl concentration gradients are artifically imposed across the vesicle membrane ([salt](out) > [salt](in)). It seems likely, therefore, that triphenylmethylphosphonium uptake is driven by a chloride diffusion potential (interior negative) and that thyrotropin either increases the permeability of the membrane to anions or decreases its permeability to cations. Thyrotropin-stimulated triphenylmethylphosphonium uptake in the vesicle preparations reaches a quasi steady-state within 3 min; in contrast, thyrotropin-stimulated adenylate cyclase activity is negligible during this period of time, becomes measurable after about 4 min, and is optimal after 12-15 min. Thus, a primary mode of action of thyrotropin on the thyroid cell may be an alteration in the electrical potential across the plasma membrane. The relevance of this observation to the mechanism of action of other glycoprotein hormones, certain bacterial toxins, and interferon is discussed.