To explore a new agent for inhibiting leukemic cells, we investigated the effects of rare earth compounds (lanthanum chloride and cerium chloride) on the growth and apoptosis of HL-60 and NB4 cells. The growth of HL-60 and NB4 cells was tested by 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide (MTT) colorimetric assay. The apoptosis was measured by light microscopy, flow cytometry, and terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) method. The effect of LaCl₃ on normal bone marrow hematopoietic progenitor cells was evaluated by colony-forming unit-granulocyte-macrophage (CFU-GM) assay. Under our experimental conditions, MTT assay showed that 48-h treatment with 1, 2, and 3 mM LaCl₃ or 48- and 72-h treatments with 1 mM LaCl₃ could significantly inhibit the growth of HL-60 cells. Treatment with 2 and 4 mM CeCl₃ for 72 h could significantly inhibit the growth of NB4 cells. Apoptosis could be detected on treatment with 2 mM LaCl₃ for 24 h in HL-60 cells by light microscopic morphology examination, flow cytometric analysis, and TUNEL method. Apoptosis could be also detected on treatment with 2 mM CeCl₃ for 72 h in NB4 cells. Treatment with 1 mM LaCl₃ could arrest the transitions from G0/G1 to S phase. The granulocyte–macrophage colony formation of normal bone marrow cells was not significantly inhibited at lower concentrations of LaCl₃ (0.5 to 2 mM). Our results indicate that at certain concentrations, the rare earth compounds may inhibit the growth of leukemic cells, induce them to apoptosis, and have no significant inhibitory effects on normal bone marrow hematopoietic progenitor cells (CFU-GM). The mechanism needs to be further investigated.

This report focuses on angiotensin II AT₁ receptor autoantibodies (anti-AT₁-AABs) in preeclamptic women. An enzyme-linked immunosorbent assay was described. Biotinylated peptide was incubated with anti-AT₁-AABs. Streptavidin-coated magnetic particles bind the protein–autoantibody complex. Detection of anti-AT₁-AABs was performed using anti-human IgG3 peroxidase-coupled antibody. The color reaction of tetramethylbenzidine solution was stopped by adding 0.5 M H₂SO₄. Optical density was measured at 450 nm (620 nm reference filter). Seventy-nine percent of anti-AT₁-AAB–positive patients (measured by bioassay) showed an increase in optical density (>145%). The same biotinylated peptide was successfully used for purification of 6/6 anti-AT₁-AABs. Chronotropic effects of purified antibodies were registered on primary cultured neonatal rat cardiomyocytes with the computer imaging system IMAGOQUANT. Western blot of coimmunoprecipitation of angiotensin II AT₁ receptor shows one band (molecular weight >40.0 kDa) in potassium thiocyanate eluate.

cDNAs representing an endogenous C-type ecotropic murine leukemia virus were isolated from a cDNA library constructed to represent mRNAs present in BC3H1 myogenic cells but not in C2C12 myogenic cells. RNA blot hybridization analysis using the cDNA inserts as probes revealed that BC3H1 cells produce MuLV-related transcirpts of at least three different size classes. A polymerase chain reaction enhanced assay for reverse transcriptase activity revealed the presence of reverse transcriptase in a viral pellet from medium conditioned by BC3H1 cells. A fungizone enhanced assay for syncitium formation provided further evidence of ecotropic retroviral particle production. Exposure of 3T3 cells to medium conditioned by BC3H1 cells, using conditions that facilitate infection, resulted in infection of the 3T3 cells, as confirmed by the syncitium formation assay. We conclude that BC3H1 cells produce an infectious ecotropic murine leukemia virus. Whether or not this feature of BC3H1 cells contributes to their inability to express some muscle-specific genes or to carry out myotube formation is unknown. Investigators will want to take into account that BC3H1 cells are virus producers when planning experiments that involve coculture of BC3H1 with other cell types, BC3H1 conditioned medium, retrovirally mediated transfection into BC3H1 cells, or study of the mCAT-1 amino acid transporter (the viral receptor) in BC3H1 cells. BC3H1 cells and the virus they produce may be of interest to those studying retroviral genomes and products and their effects.

To catalog factors that may contribute to the completion of myogenesis, we have been looking for molecular differences between BC3H1 and C2C12 cells. Cells of the BC3H1 tumor line, though myogenic, are nonfusing, and withdraw from the cell cycle only reversibly, whereas cells of the C2C12 line fuse, differentiate terminally, and express several muscle-specific gene products that BC3H1 cells do not. Relative to C2C12 cells, BC3H1 cells underaccumulated cyclin-dependent kinase inhibitor p21 and underaccumulated transcripts for p21, GADD45, CDO, decorin, osteopontin, H19, fibronectin, and thrombospondin-1 (tsp-1). Levels of accumulation of H19, tsp-1, and larger isoforms of fibronectin messenger ribonucleic acid (mRNA) were found to increase in response to expression of myogenic regulatory factors as shown by their accumulation in differentiated myogenically converted 10T1/2 cells but not in 10T1/2 fibroblasts. BC3H1s accumulated a temperature-insensitive, geldanamycin-sensitive, misfolded form of p53 incapable of transactivating a p53 responsive reporter, consistent with underexpression of p21, GADD45, and tsp-1. BC3H1 and C2C12 cells were similar with respect to upregulation of p27 protein, downregulation of mitogen-activated protein kinase phosphatase-1 (MKP-1) protein, upregulation of retinoblastoma (Rb) mRNA, and nuclear localization of hypophosphorylated Rb. Cells of both lines expressed the muscle-specific 1b isoform of MEF2D. Although nonfusing in the short term, after more than 18 d in differentiation medium, some cultures of BC3H1 cells formed viable multinucleated cells in which the nuclei did not reinitiate synthesis of DNA in response to serum. Our findings suggest participation of tsp-1 and specific isoforms of fibronectin in myogenesis and suggest additional avenues of research in myogenesis and oncogenesis.

Given a role for the deregulation of p21WAF1 in the progression of bladder tumors, we examined the growth of cultured urothelial cells from wild-type and p21WAF1 null bladders. Bladders were excised, minced from euthanized p21WAF1 and wild-type mice, treated overnight with dispase, and then placed into flasks coated with collagen type I in Dulbecco modified Eagle medium with 10% fetal calf serum. After an overnight incubation, the media was replaced with a serum-free media and a portion of explants were treated with 12-O-tetrade-canoylphorbol-13-acetate (TPA) on day 7 and continued for either 4 or 9 wk. The urothelial origin of any surviving epithelial cells was determined by reverse transcription–polymerase chain reaction (RT-PCR) using uroplakin II–specific primers, and the expression of the cell cycle–related proteins, p16INK4 and p19ARF, was examined by semiquantitative RT-PCR and Western blotting. Isolated wild-type and serially passaged p21WAF1 null epithelial-like cells were then injected subcutaneously into nude mice. We found that phorbol ester treatment at two different concentrations significantly enhanced uroepithelial colony formation from isolated wild-type mouse bladder tissue. On the other hand, significantly fewer urothelial colonies were derived from p21WAF1 null bladder cells treated with phorbol ester. Although there was apparent senescence and cell death of epithelial foci and stromal cells in phorbol ester–treated and –untreated p21WAF1 null cultures, after 3 mo there was an apparent subpopulation of epitheloid cells that overgrew each flask. There was a significant decrease in the number of these serially passaged cells in the G1 phase of the cell cycle when compared with initial explant wild-type or p21WAF1 null cells. This subpopulation of epitheloid cells expressed the mouse uroplakin II gene, indicating a urothelial phenotype, but did not express either the p16INKa or p19ARF proteins, whereas p21WAF1 null bladders express both proteins. There was also a high level of expression of the p53 protein and a significant decrease in the expression of the p19ARF transcript in both p21WAF1 null bladder and p21WAF1 null cells. These p21WAF1 null cells could be easily passaged and when injected subcutaneously into nude mice, large tumors developed. Therefore, it appears that a subpopulation of urothelial cells from the p21WAF1 null bladder can develop a tumorigenic phenotype in vitro.

The role of the adenosine triphosphate–binding cassette (ABC) superfamily of membrane transporters is well documented in tumor cell multidrug resistance. More recently, growing evidence of their influence on oral bioavailability, drug excretion rates, and drug–drug interaction potential at the intestinal level has stimulated much investigation. Our laboratory is interested in evaluating the apical (AP) ABC transporter P-glycoprotein (Pgp [mdr-1]) for its role in xenobiotic efflux at the intestinal level. We propagated Caco-2 cells in the presence of vinblastine (a cytotoxic, Pgp substrate) to promote transporter expression though selection. That is, the cell population expressing Pgp, or with the capacity to up-regulate Pgp expression, survived and expanded in the presence of vinblastine. We have used this selected cell line (Caco-2 VinB) to develop a fluorescent-based assay to study the chemical modulators of Pgp activity. Using the Caco-2 VinB cells, we have successfully demonstrated the differential potency of previously characterized Pgp inhibitors. In addition, we conducted a morphological evaluation of the two cell lines using transmission, scanning, and confocal microscopy. Both cell strains differentiated into highly functional, polarized columnar epithelium, although the vinblastine-selected cell line had lost the phenotypic diversity observed in native Caco-2 populations. Increased Pgp expression was noted in Caco-2 VinB cells compared with the native cell line on Western blot analysis, which was localized to the AP surface using confocal microscopy and functionally demonstrated using transport assays. We believe that the Caco2 VinB cell line is a versatile tool for application in pharmaceutical drug development.

Adult skeletal muscle fibers can be isolated and cultured but tend to dedifferentiate and sprout with time in culture. We examined isolated adult mouse flexor digitorum brevis muscle fibers under various culture conditions by monitoring maintenance of the same fibers at 2-d intervals using survival analysis. Fibers plated on laminin and cultured in serum-free media did not show sprouting and exhibited significantly (P < 0.0001) longer survival (median survival time, T₅₀ = 10.2 d) than fibers in serum-containing media (T₅₀ = 3.3 d). Cell proliferation was markedly suppressed in serum-free cultures. Multiple or delayed Ca² transients in response to brief field stimulation were often observed in dedifferentiated fibers after several d in serum-containing media but were not observed in fibers in serum-free media. The addition of cytosine arabinoside to serum-containing cultures did not prolong fiber survival (P = 0.39) and did not eliminate sprouting but did greatly suppress proliferation of nonmuscle cells. Fibers cultured in agarose gel with serum exhibited small, bud-like extensions but no sprouts and did not survive as long (T₅₀ = 6.2 d) as fibers plated on laminin and cultured in serum-free media (T₅₀ = 10.2 d) did. These results demonstrate that both morphological and physiological properties of fibers become modified in serum-containing media but can be retained by culturing without serum.

Wolbachia infecting the small brown planthopper, Laodelphax striatellus, were successfully maintained and cultivated in two insect and one mammalian cell lines. The bacteria with the planthopper ovary were introduced into the flasks with the cultures of the cell lines. The Wolbachia proliferated in mosquito (Aedes albopictus) and lepidopteran (Heliothis zea) cell lines and in the mouse cell line, L929. Proliferation of Wolbachia was confirmed by electron microscopy and quantitative polymerase chain reaction. This simple method for the cultivation of Wolbachia was applicable to other strains of Wolbachia, such as the one found in the lepidopteran eggs, and should facilitate fundamental and applied studies of this important group of microorganisms.