ABSTRACT
Human granulocytic ehrlichiosis (HGE) is an emerging vector-borne disease caused by an Ehrlichia species similar or identical to E. equi and E. phagocytophila. Previous studies have shown that the pathogen can be cultivated in vitro in permissive cells such as human promyelocytic HL-60 leukemia cells. The mechanism(s) of its infection and propagation in target cells, however, is not well understood, due in part to lack of a method capable of quantitatively determining the amount of the infectious agent. Although several assays currently exist for the HGE agent, they are mostly qualitative and have a number of limitations. In this report, size differences between prokaryotic and eukaryotic rRNAs are utilized to quantitatively assay the HGE agent in HL-60 cells. By comparing the integrated intensity of agarose gel resolved HGE-specific rRNA in host cells, with identically prepared and analyzed rRNA isolated from known quantities of E. coli (JM 109), it is possible to calculate the E. coli-equivalence of the HGE agent present in HL-60 cells according to the equation: Y (E. coli, in viable cells x 10(8)) = -2.573 + 0.11X (% infection by the HGE agent in HL-60 cells). The method described is reproducible, sensitive, and is not limited by availability of antisera. Furthermore, since the assay has no designer primer and repeated amplification requirements, it can be easily disseminated to and standardized in other laboratories.
Subject(s)
Ehrlichia/isolation & purification , Ehrlichiosis/microbiology , Granulocytes/microbiology , RNA, Bacterial/analysis , RNA, Ribosomal/analysis , Cell Count , Ehrlichia/genetics , Ehrlichia/growth & development , Ehrlichiosis/blood , Ehrlichiosis/diagnosis , Electrophoresis, Agar Gel , Escherichia coli/genetics , Escherichia coli/growth & development , Ethidium , Granulocytes/pathology , HL-60 Cells , Humans , Molecular Weight , New York , RNA, Bacterial/genetics , RNA, Ribosomal/genetics , Reproducibility of Results , Sensitivity and Specificity , Time FactorsABSTRACT
Newly synthesized proteoglycans (PG) in proliferating HL-60 cells were labeled with [35S]-sulfate and fractionated on DEAE-cellulose columns. Peak fractions were digested with chondroitinase ABC and separated by paper chromatography. Chondroitin sulfate was identified as the major PG. Treatment of cells with 16 nM TPA resulted in a 1.8-fold increase in total sulfate incorporation into PG, a shift in its location from the cellular to the extracellular compartment, and an increase in the total charge of PG (based on the profile of elution from DEAE-cellulose columns), compared to controls. Incorporation of [3H]-glucosamine into cellular PG was markedly decreased in TPA-treated cells; the sulfate/glucosamine ratio showed a 13.5-fold decrease in the newly synthesized cellular PG. The sulfate/glucosamine ratio, however, was increased by 7.6-fold in secreted PG.
Subject(s)
Cell Differentiation , Proteoglycans/biosynthesis , Cell Division , Cell Line , Cholecalciferol/pharmacology , Chromatography, DEAE-Cellulose , Chromatography, Gel , Glucosamine/metabolism , Humans , Leukemia, Promyelocytic, Acute , Proteoglycans/isolation & purification , Sulfates/metabolism , Sulfur Radioisotopes , Tetradecanoylphorbol Acetate/pharmacology , Tritium , Tumor Cells, CulturedABSTRACT
The proliferation of the human prostatic cancer JCA-1 cells showed a complex response to different concentrations of TPA. Whereas cells exposed for 24 h had growth reduction which was proportional to the concentration of TPA added to the culture media, they showed resistance to low (0.016 and 0.16 nM) but not high (> or = 1.6 nM) doses of TPA after 72 h. Growth-inhibited, treated cells also displayed a distinct cell morphology compared with the controls. Since c-myc expression has previously been shown to be correlated with cellular proliferation, we determined changes in its steady-state level in control and treated cells by Northern analysis. Following a 24h, 48h, and 72h treatment, with 16 nM TPA, c-myc mRNA was suppressed by 91%, 83%, and 78%, respectively, in good agreement with the extent of growth reduction observed. At the low dose of TPA (0.16 nM), however, the c-myc mRNA expression remained inhibited by 85% even though cell growth was only reduced by 10-14%. No difference in the total amount of c-myc protein could be detected between control and treated cells by Western analysis.
Subject(s)
Gene Expression Regulation, Neoplastic/genetics , Prostatic Neoplasms/pathology , Proto-Oncogene Proteins c-myc/genetics , Tetradecanoylphorbol Acetate/pharmacology , Androgens/pharmacology , Blotting, Northern , Blotting, Western , Cell Count/drug effects , Cell Division/drug effects , Cell Division/genetics , Cell Survival/drug effects , DNA, Complementary/genetics , Dose-Response Relationship, Drug , Gene Expression Regulation, Neoplastic/drug effects , Genes, myc , Humans , Infant, Newborn , Male , Prostatic Neoplasms/genetics , Proto-Oncogene Mas , Proto-Oncogene Proteins c-myc/drug effects , RNA, Messenger/biosynthesis , Tumor Cells, CulturedABSTRACT
The steady-state level of the PCP mRNA was investigated during HL-60 cell differentiation. When cells were induced to differentiate into granulocytes with 1.3% dimethylsulfoxide or 1 microM cis or trans retinoic acid, no significant changes in PCP mRNA levels were observed. In contrast, cells cultured in the presence of the phorbol ester TPA, which promotes the cells to be differentiated into monocytes/macrophages, were associated with a marked decrease in the steady-state concentration of PCP mRNA. When cells were simultaneously treated with a combination of TPA and staurosporine, a protein kinase C (PKC) inhibitor, the TPA-elicited decrease in PCP mRNA was partially prevented. These data suggest that PCP mRNA expression is not directly linked to HL-60 cell differentiation but appears to involve the participation of PKC.
