Induction of transformation and continuous cell lines from normal human mammary epithelial cells after exposure to benzo[a]pyrene.

Rapidly growing primary cultures of normal human mammary epithelial cells (HMEC) were exposed to 1 microgram of benzo[a]pyrene (B[a]P) per ml for two or three 24-hr periods. The B[a]P-treated populations consistently contained cells displaying a longer period of active growth in culture compared to the untreated control cells. Widespread heterogeneity in morphology and growth patterns was evidenced in these "extended life" (EL) cultures, with multiple sequential changes in these parameters occurring during the course of their life in culture. Two apparently immortal continuous cell lines have thus far emerged from these EL cultures. These lines have been characterized to be of human mammary epithelial origin and derived from the originally treated HMEC specimen. The continuous lines do not appear to be malignantly transformed as they do not cause tumor formation in nude mice and show little or no anchorage-independent growth. Nonetheless, they have acquired several properties characteristic of tumor-derived HMEC, which distinguish them from their normal progenitors. These cell lines, as well as the EL strains, may provide useful substrates for studies to determine what agents can induce further transforming events. Additionally, analysis of the multiple steps occurring in the El cultures, as well as in the emergence of the continuous cell lines, could potentially elucidate the processes occurring during human epithelial cell carcinogenesis and escape from senescence.     

Role of islet structure and cellular interactions in the control of insulin secretion

Close cellular proximity and correct anatomical arrangement within islets are essential for normal patterns of insulin secretion. Thus, segregation of islets into single cells is associated with a dramatic decline in stimulus secretion-coupling and glucose-induced insulin release. Generation of pseudoislets from clonal islet cell lines provides a useful model to examine islet cell interactions and insulin secretion. Such studies have highlighted the functional importance of cell adhesion molecules and connexins. Pseudoislets comprising insulin-secreting cell lines have been shown to closely mimic primary islets in both size and morphology, displaying a significantly enhanced response to glucose, nutrients and drugs over equivalent monolayer cultures. Here, we consider the influence of islet structure and cellular interactions in the control of insulin secretion. The functional characteristics of pseudoislets derived from clonal beta-cell lines or a combination of alpha-, beta- and delta-cell lines are discussed in light of normal islet function and possible therapeutic application.     

Role of islet structure and cellular interactions in the control of insulin secretion

Close cellular proximity and correct anatomical arrangement within islets are essential for normal patterns of insulin secretion. Thus, segregation of islets into single cells is associated with a dramatic decline in stimulus secretion-coupling and glucose-induced insulin release. Generation of pseudoislets from clonal islet cell lines provides a useful model to examine islet cell interactions and insulin secretion. Such studies have highlighted the functional importance of cell adhesion molecules and connexins. Pseudoislets comprising insulin-secreting cell lines have been shown to closely mimic primary islets in both size and morphology, displaying a significantly enhanced response to glucose, nutrients and drugs over equivalent monolayer cultures. Here, we consider the influence of islet structure and cellular interactions in the control of insulin secretion. The functional characteristics of pseudoislets derived from clonal beta-cell lines or a combination of alpha-, beta- and delta-cell lines are discussed in light of normal islet function and possible therapeutic application.     

Astrocyte cultures derived from human brain tissue express angiotensinogen mRNA.

We have identified human cultured cell lines that are useful for studying angiotensinogen gene expression and its regulation in the central nervous system. A model cell system of human central nervous system origin expressing angiotensinogen has not previously been available. Expression of angiotensinogen has not previously been available, Expression noninduced human astrocytes, since astrocytic cell lines derived from human glioblastomas or nonneoplastic human brain tissue invariably produced angiotensinogen mRNA. In situ hybridization histochemistry revealed that angiotensinogen mRNA production was not limited to a subpopulation of astrocytes because greater than 99% of cells in these cultures contained angiotensinogen mRNA. These cell lines will be useful in studies of the molecular mechanisms controlling angiotensin synthesis and the role of biologically active angiotensin in the human brain by allowing us to examine regulation of expression of the renin-angiotensin system in human astrocyte cultures.     

Mammalian cell cultures as models for Mycobacterium tuberculosis-human immunodeficiency virus(HIV) interaction studies:A review

Mycobacterium tuberculosis(MTB) and human immunodeficiency virus(HIV) co-infections have remained a major public health concern worldwide,particularly in Southern Africa.Yet our understanding of the molecular interactions between the pathogens has remained poor,primarily due to lack of suitable preclinical models for such studies.We reviewed the use,this far,of mammalian cell culture models in HIV-MTB interaction studies.Studies have described the use of primary human cell cultures,including monocyte-derived macrophage(MDM) fractions of peripheral blood mononuclear cell(PBMC),alveolar macrophages(AM),cell lines such as the monocyte-derived macrophage cell line(U937),T lymphocyte cell lines(CEMx174,ESAT-6-specific CD4+ T-cells) and an alveolar epithelial cell line(A549) and special models such as stem cells,three dimensional(3D) or organoid cell models [including a blood-brain barrier(BBB) cell model] in HIV-MTB interaction studies.The use of cell cultures from other mammals,including:mouse cell lines [macrophage cell lines RAW 264.7 and J774.2,fibroblast cell lines(NIH 3T3,C3 H clones),embryonic fibroblast cell lines and T-lymphoma cell lines(S1A.TB,TIMI.4 and R1.1)]; rat(T cells:Rat2,RGE,XC and HH16,and alveolar cells:NR8383) and primary guinea pigs derived AMs,in HIV-MTB studies is also described.Given the spectrum of the models available,cell cultures offer great potential for host-HIV-MTB interactions studies.     

Expression patterns of gamma-aminobutyric acid type A receptor subunit mRNAs in primary cultures of granule neurons and astrocytes from neonatal rat cerebella.

Using a competitive polymerase chain reaction assay, we have quantitated the absolute amounts of mRNA encoding 14 distinct subunits of the gamma-aminobutyric acid type A (GABAA) receptor in primary cultures of rat cerebellar granule neurons and cerebellar astrocytes. We found that the total amount of GABAA receptor subunit mRNA in astrocytes was 2 orders of magnitude lower than in neuronal cells. Furthermore, granule cell cultures expressed all 14 different GABAA subunit mRNAs, while the astroglial cultures contained detectable amounts of all the subunits expressed by granule cells except the alpha 6 and the gamma 2L subunits. Of the alpha subunit family members, the alpha 1, alpha 5, and alpha 6 mRNAs were prominent in granule cells, while the alpha 1 and alpha 2 mRNAs were abundant in astrocytes. Of the beta receptor subunit mRNAs, the beta 1 and beta 3 mRNAs were abundantly expressed in both cultures. The gamma 2S and gamma 2L mRNAs constituted the great majority of gamma subunit mRNAs in neurons, while the gamma 1 subunit mRNA was the most abundant gamma subunit mRNA in astrocytes. When various allosteric modulators of GABAA receptors were tested electrophysiologically, methyl 6,7-dimethoxy-4-ethyl-beta-carboline- 3-carboxylate (DMCM) was the only one to modulate chloride currents elicited by GABA in a significantly different manner in granule cells (negative modulation) compared with astrocytes (positive modulation). The latter effect was previously observed in transiently expressed recombinant GABAA receptors containing a gamma 1 instead of a gamma 2 subunit. Our quantitative mRNA results suggest that an important molecular determinant responsible for the DMCM-positive modulatory effect on astroglial native GABAA receptors is the presence of the gamma 1 subunit in the receptor assembly.     

Bipotential precursors of putative fibrous astrocytes and oligodendrocytes in rat cerebellar cultures express distinct surface features and "neuron-like" gamma-aminobutyric acid transport

When postnatal rat cerebellar cells were cultured in a chemically defined, serum-free medium, the only type of astrocyte (defined by the expression of the glial fibrillary acidic protein, GFAP) present was unable to accumulate gamma-[3H]aminobutyric acid (GABA), did not express surface antigens recognized by two monoclonal antibodies, A2B5 and LB1, and showed minimal proliferation. In these cultures, nonneuronal A2B5+, LB1+ stellate cells exhibiting "neuron-like" [3H]GABA uptake formed cell colonies of increasing size and were GFAP-. After about one week of culturing, the A2B5+, LB1+, GABA-uptake positive cell groups became galactocerebroside (GalCer) positive. Immunocytolysis of the A2B5+ cells at 3 and 4 days in vitro prevented the appearance of the A2B5+, LB1+, GABA-uptake positive cell colonies, and also of the GalCer+ cell groups. If 10% (vol/vol) fetal calf serum was added to 6-day cultures, the A2B5+, LB1+, GABA-uptake positive cell groups expressed GFAP and not GalCer. If the serum was added to the cultures 2 days after lysing the A2B5+ cells, only A2B5-, LB1-, GABA-uptake negative astrocytes proliferated. It is concluded that the putative fibrous astrocytes previously described in serum-containing cultures (which had a stellate shape and were A2B5+, LB1+, GABA-uptake positive) derive from bipotential precursors that differentiate into oligodendrocytes (GalCer+) in serum-free medium or into astrocytes (GFAP+) in the presence of serum, while the epithelioid A2B5-, LB1-, GABA-uptake negative astrocytes originate from a different precursor not yet identified.     

Properties of angiotensin II receptors in glial cells from the adult corpus callosum.

The existence and the properties of angiotensin II receptors in the adult bovine and human corpus callosum (CC) were investigated by using Xenopus oocytes and primary glial cell cultures. In oocytes injected with CC mRNA, angiotensin II elicited oscillatory Cl- currents due to activation of the inositol phosphate/Ca(2+)-receptor-channel coupling system. The receptors expressed in oocytes and in CC cultures were pharmacologically similar to the AT1 receptor type as assayed by binding. Northern blot analysis and in situ hybridization studies in sections from CC and in glial cultures revealed that the receptors were molecularly related to the AT1 receptor and that they were present in astrocytes. In these cells, activation of the receptors with angiotensin II increased de novo DNA synthesis, promoted the release of aldosterone, and induced c-Fos expression. These findings indicate that CC astrocytes possess functional AT1 receptors that participate in various physiological processes.     

Transfection of human lymphoblastoid cells with herpes simplex viral DNA.

The "calcium/dimethyl sulfoxide shock" method of transfection was adapted for use in human lymphoid cell cultures. One microgram of herpes simplex virus type 1 DNA regularly initiated virus replication in four lymphoblastoid cell lines. Per 10(5) cells exposed to 1 microgram of DNA, 0.5--5 cells formed an infectious center. The minimal infective dose of DNA was approximately 500 ng.     

In vitro expression of the endothelial phenotype: comparative study of primary isolated cells and cell lines,including the novel cell line HPMEC-ST1.6R

Endothelial cell lines are commonly used in in vitro studies to avoid problems associated with the use of primary endothelial cells such as the presence of contaminating cells, the difficulty in obtaining larger numbers of cells, as well as the progressive loss of cell viability and expression of endothelial markers in the course of in vitro propagation. We have analyzed the characteristics defining distinctive endothelial phenotypes in the cell lines EA.hy926, ECV304, EVLC2, HAEND, HMEC-1, ISO-HAS-1 and a cell line recently generated in our laboratory, HPMEC-ST1.6R, and have compared these phenotypes with those found in primary human endothelial cells isolated from umbilical vein (HUVEC), lung (HPMEC), and skin (HDMEC). The analysis revealed significant differences in phenotype expression between primary cells and the cell lines. Constitutive expression of von Willebrand factor, CD31, and CD34 and induced expression of cell adhesion molecules, ICAM-1, VCAM-1, and E-selectin and cytokines, IL-6, IL-8, MCP-1, and GM-CSF on stimulation with proinflammatory stimuli, as well as the uptake of DiI-Ac-LDL and the formation of cord-like structures on Matrigel, were typically observed in the primary cells. However, most cell lines exhibited only a few of these endothelial characteristics. Only HPMEC-ST1.6R exhibited the major constitutive and inducible endothelial cell characteristics and showed an angiogenic response on Matrigel similar to that of primary HPMEC. Thus, HPMEC-ST1.6R will be a valuable in vitro model system in which to study pathomechanisms and angiogenesis of the mature microvascular endothelium in vitro.     

Immortalization of bipotential and plastic glio-neuronal precursor cells.

Permanent clonal cell lines from newborn mouse striatum have been established after transfer of the simian virus 40 large tumor oncogene by means of a retroviral vector. Some of the lines obtained displayed properties of bipotential and plastic glio-neuronal precursors. Depending on the culture conditions, these cells express either the glial fibrillary acidic protein or neurofilaments. In addition, the cells can display adrenergic, D1 and D2 dopaminergic, muscarinic, and 5-hydroxytryptamine type 2 serotoninergic receptors, which are coupled either to the adenylate cyclase or to the phosphatidylinositol signaling pathways. The panel of receptors for neurotransmitters exhibited by these lines closely resembles that of primary striatal neurons. Results suggest that plastic common precursors of astrocytes and neurons persist in the striatum at a late developmental stage. As these permanent cell lines constitute an unlimited source of homogenous cell material, we suggest that they should be useful for molecular and pharmacological studies on the mechanisms and regulation of signal transduction as well as the commitment, plasticity, and differentiation of neural cells.     

Adrenocortical cell lines

The human adrenal cortex is a complex endocrine organ that secretes mineralocorticoids, glucocorticoids and adrenal androgens. These steroids arise from morphologically and biochemically distinct zones of the adrenal gland. Studying secretion of these distinct steroid hormones can make use of cells isolated from the adrenal gland but this requires animal sacrifice and the need for continued isolation for long-term studies. In addition primary cultures of adrenal cells have a limited life-span in culture and the cultured cells are often contaminated by the presence of non-steroidogenic cells. For that reason in vitro cell culture models have several benefits for research on adrenocortical function. Herein we discuss the available adrenocortical cell lines and their uses as model systems for adrenal studies. Focus is placed on the human NCI-H295 and mouse Y-1 adrenal cell lines, which have been used extensively as adrenocortical model systems. These cell lines have proven to be of considerable value in studying the molecular and biochemical mechanisms controlling adrenal steroidogenesis. The current review will discuss the attributes and limitations of the currently available adrenocortical cell lines as models for adrenal studies.     

Human T-cell leukemia virus type I infection of monocytes and microglial cells in primary human cultures.

The pathogenesis of progressive spastic paraparesis [HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP)], a serious consequence of human T-cell leukemia virus type I (HTLV-I) infection, is unclear. T and B lymphocytes can be naturally infected by HTLV-I, but the susceptibility to HTLV-I infection of other cell types that could contribute to the pathogenesis of HAM/TSP has not been determined. We found that a human monocyte cell line (THP-1), primary human peripheral blood monocytes, and isolated microglial cells but not astrocytes or oligodendroglial cells derived from adult human brain were infected by HTLV-I in vitro. Infection with HTLV-I enhanced the secretion of interleukin 6 in human microglial cell-enriched cultures but did not stimulate the release of interleukin 1 from monocytes or microglial cells. Tumor necrosis factor alpha production was stimulated by HTLV-I infection of monocytes and microglial cells and could be enhanced by suboptimal amounts of lipopolysaccharide. Since both tumor necrosis factor alpha and interleukin 6 have been implicated in inflammatory demyelination and gliosis, our findings suggest that human microglial cells and monocytes infected with and activated by HTLV-I could play a role in the pathogenesis of HAM/TSP.     

Lentiviral vectors with a defective integrase allow efficient and sustained transgene expression in vitro and in vivo

Lentivirus-derived vectors are among the most promising viral vectors for gene therapy currently available, but their use in clinical practice is limited by the associated risk of insertional mutagenesis. We have overcome this problem by developing a nonintegrative lentiviral vector derived from HIV type 1 with a class 1 integrase (IN) mutation (replacement of the 262RRK motif by AAH). We generated and characterized HIV type 1 vectors carrying this deficient enzyme and expressing the GFP or neomycin phosphotransferase transgene (NEO) under control of the immediate early promoter of human CMV. These mutant vectors efficiently transduced dividing cell lines and nondividing neural primary cultures in vitro. After transduction, transient GFP fluorescence was observed in dividing cells, whereas long-term GFP fluorescence was observed in nondividing cells, consistent with the viral genome remaining episomal. Moreover, G418 selection of cells transduced with vectors expressing the NEO gene showed that residual integration activity was lower than that of the intact IN by a factor of 500-1,250. These nonintegrative vectors were also efficient in vivo, allowing GFP expression in mouse brain cells after the stereotactic injection of IN-deficient vector particles. Thus, we have developed a generation of lentiviral vectors with a nonintegrative phenotype of great potential value for secure viral gene transfer in clinical applications.     

Glucose metabolism in human gliomas: Correspondence ofin situ andin vitro metabolic rates and altered energy metabolism

The rates of disappearance of glucose from the medium of 13 human glioma-derived cell lines and one cultured of normal human cortical astrocytes were determined by ftuorometric techniques. High-grade glioma-derived cultures showed a range of glucose consumption between 1 and 5 nmol/min/mg protein. Normal astrocyte cultures and cultures derived from grades I–III gliomas had a glucose consumption rate of 2–3 nmol/min/mg protein. Seven high-grade glioma lines were derived from surgical samples taken from patients who had been scanned by¹⁸F-2-deoxy-d-glucose positron computed tomography. The rate of glucose consumption in these high-grade glioma-derived lines was close to the maximum local cerebral metabolic rate for glucose (LCMRglc) measuredin situ in the tumors from which the cultures were derived. In cultured glioma-derived lines, approximately one-half of the glucose consumed was recovered as lactate and pyruvate, suggesting a reliance of glioma cells on aerobic glycolysis. ATP and phosphocreatine (PCr) levels were variable in the gliomaderived lines, and ATP was lower in the glioma-derived lines than in the normal astrocytes. Levels and regulation of glycogen differed significantly among the various glioma-derived cell lines. Glycogen content did not diminish as glucose was consumed, suggesting that glycogen utilization is not tightly regulated by the glucose metabolic rate. These results suggest that human glioma-derived cell cultures (1) adequately reflect the metabolic capacity of gliomasin situ and (2) are significantly altered in several aspects of their glycolytic metabolism.     

Growth requirements in vitro of oligodendrocyte cell lines and neonatal rat brain oligodendrocytes.

I have defined the basic requirements for the proliferation of cell lines expressing oligodendrocyte properties and for the survival of galactocerebroside-positive oligodendrocytes derived from neonatal rat brains. Conventional serum-containing medium can be replaced by 01 medium, a chemically defined medium supplemented with insulin, transferrin, sodium selenite, and biotin. Thyroid hormone is not required. When cells are plated directly into O1 medium, the substratum has to be modified by precoating with polylysine and adding fibronectin to the medium prior to the cells. Both cell lines and brain cells can be subcultured numerous times in O1 medium without initial culture in serum-containing medium. Brain cultures can be maintained in O1 medium for several months and contain a significantly higher percentage of mature oligodendrocytes, a lower number of astrocytes, and no fibroblasts as compared to cells maintained in serum-containing medium.     

Critical role of enhanced CD4 affinity in laboratory adaptation of human immunodeficiency virus type 1

Strains of human immunodeficiency virus type 1 (HIV-1) that use the coreceptor CXCR4 (X4 strains) become laboratory adapted (LA) when selected for ability to replicate in leukemic T cell lines such as H9. Compared with patient X4 viruses, the gp120-gp41 complexes of LA viruses have a constellation of common properties including enhanced affinities for CD4, greater sensitivities to inactivations by diverse antibodies and by soluble CD4, increased shedding of gp120, and improved abilities to infect HeLa-CD4 cell clones that contain only trace quantities of CD4. These common characteristics, which may result from a concerted structural rearrangement of the gp120-gp41 complexes, have made it difficult to identify a specific feature that is critical for laboratory adaptation. To test the hypothesis that replication of patient X4 HIV-1 is limited by the low CD4 concentration in H9 cells (7.0 x 10(3) CD4/cell), we constructed H9 derivatives that express at least 10 times more of this receptor. Interestingly, most patient X4 isolates readily grew in these derivative cells, and the resulting virus preparations retained the characteristics of primary viruses throughout multiple passages. In contrast, selection of the same viruses in the parental H9 cells resulted in outgrowth of LA derivatives. We conclude that a weak interaction of patient X4 HIV-1 isolates with CD4 is the primary factor that limits their replication in leukemic T cell lines.     

Molecular phenotyping of human chondrocyte cell lines T/C‐28a2, T/C‐28a4, and C‐28/I2

Objective

Because the immortalized chondrocyte cell lines C‐28/I2, T/C‐28a2, and T/C‐28a4 have become a common tool in cartilage research, permitting investigations in a largely unlimited and standardized manner, we investigated the molecular phenotype of these cell lines by gene expression profiling.

Methods

Complementary DNA–array analysis as well as online quantitative polymerase chain reaction were used to identify the gene expression profiles of the 3 cell lines cultured in monolayer and alginate beads, as compared with the expression profiles of cultured human adult primary chondrocytes.

Results

A similar, but not identical, gene expression profile was established for all 3 cell lines. SOX9 was expressed at a significant level in all 3 cell lines. Extracellular matrix proteins and matrix‐degrading proteases were rarely expressed. In contrast, genes involved in the cell cycle were strongly up‐regulated, as compared with the expression levels in physiologic chondrocytes.

Conclusion

The expression of SOX9, the master gene of chondrocytic cell differentiation, reflects the basically chondrocytic phenotype of these cells. However, the major issue appears to be that these cell lines mainly proliferate and show less expression of genes involved in matrix synthesis and turnover. In this respect, C‐28/I2 cells display the highest levels of matrix‐anabolic and matrix‐catabolic genes and thus are presumably preferable for use in investigating chondrocyte anabolic and catabolic activity and its regulation. None of the 3 cell lines appears to be a direct substitute for primary chondrocytes. A successful approach will have to validate the findings obtained with chondrocyte cell lines by using primary chondrocytes or cartilage‐tissue cultures. This would permit the establishment of reproducible in vitro models and subsequently allow investigators to relate the findings to the physiologic situation.