Studies on the promoter region of the c-Ha-ras gene in FRTL5 rat thyroid cells

The functional activity of the promoter region of the rat c-Ha-ras gene was examined in FRTL5 rat thyroid cells, the cell type from which this promoter was cloned. A plasmid (p035-ras-CAT) was constructed containing the untranslated-1 exon as well as 172 base pairs (bp)5' to this exon inserted upstream of the chloramphenicol acetyl transferase (CAT) reporter gene. These 172 bp of 5'-flanking region contain two 10 bp GC box consensus sites and two CAAT boxes. Very weak promoter activity was observed in experiments involving transient transfection of FRTL5 cells with this plasmid, as well as with another plasmid (p5kb-ras-CAT) containing a much more extensive (3.5 kb) 5'-flanking region of the gene. In contrast, strong promoter activity was observed when the same plasmids were transfected into mouse 3T3 fibroblasts. When other promoters (pfos, RSV, and MMTV) were used to drive CAT activity, CAT activity in FRTL5 cells was about 10-fold less than in NIH-3T3 cells and rat embryo fibroblasts. However c-Ha-ras promoter activity was reduced out of proportion in FRTL5 thyroid cells relative to the other cell types (approximately 50-fold less). DNA gel-shift assays performed using crude extracts of FRTL5 and 3T3 nuclear proteins revealed quantitatively similar binding to the same promoter region in the c-Ha-ras 5'-flanking sequence. These data demonstrate that promoter activity of the rat c-Ha-ras gene is contained within the 172 bp 5'-flanking region of the gene. This promoter activity is expressed at a much lower level in slow-growing FRTL5 cells relative to other more rapidly growing cell types.     

An improved CAT assay for promoter analysis in either transgenic mice or tissue culture cells.

We have developed an improved method for determining CAT activity directed by stably (transgenic mice) or transiently (tissue culture cell lines) introduced CAT reporter gene constructs. The procedure is based on the use of a new buffer system which considerably increases the stability of the CAT enzyme during the preparation of the crude cell extracts. When compared to other procedures, our method enables an increase of up to 100-fold in the sensitivity of the assay, depending on the transgenic tissue tested. Furthermore, a strong increase (up to 23-fold) was also observed with various promoter/CAT constructs transiently transfected in established tissue culture cell lines. This increase in sensitivity provides a significant reduction in the time required to perform the CAT assay when strong promoters are studied (from 18 to 1 hr) and is also very useful for the analysis of CAT gene expression driven by weak promoters.     

Advantages of firefly luciferase as a reporter gene: application to the interleukin-2 gene promoter

The chloramphenicol acetyltransferase (CAT) gene is widely used in recombinant constructs employed to study promoter and enhancer control of gene expression. However, CAT-based assays require a laborious, multi-step procedure for quantitation of promoter activity. We have applied the recently described firefly luciferase (LUC) reporter gene to the study of the interleukin-2 (IL2) promoter and have further defined the properties of this reporter gene system. We find that IL2-LUC constructs have multiple advantages over IL2-CAT constructs. The LUC assay is highly sensitive and requires 1/10 the cells used in the CAT system. A final quantitative measure of promoter activity can be obtained within 25 h following transfection with IL2-LUC, compared to 108-160 h with IL2-CAT. Light emission significantly (fourfold) above background is detectable 3 h after induction in a direct assay of extracts from transfected cells. We have described the variability of the assay, the minimum number of transfected cells required to detect light, the stability of luciferase in cell extracts, the effect of Triton X-100 on the assay, and a rapid cell lysis procedure. The luciferase system is a simple, rapid, and sensitive method for the study of promoter activity in transfected cells, particularly for weakly expressed genes such as IL2 which give low activity in the CAT assay.     

Mitoxantrone resistance in HL-60 leukemia cells: reduced nuclear topoisomerase II catalytic activity and drug-induced DNA cleavage in association with reduced expression of the topoisomerase II beta isoform.

Mitoxantrone-resistant variants of the human HL-60 leukemia cell line are cross-resistant to several natural product and synthetic antineoplastic agents. The resistant cells (HL-60/MX2) retain sensitivity to the Vinca alkaloids vincristine and vinblastine, drugs that are typically associated with the classical multidrug resistance phenotype. Mitoxantrone accumulation and retention are equivalent in the sensitive and resistant cell types, suggesting that mitoxantrone resistance in HL-60/MX2 cells might be associated with an alteration in the type II DNA topoisomerases. We discovered that topoisomerase II catalytic activity in 1.0 M NaCl nuclear extracts from the HL-60/MX2 variant, as measured by the decatenation of Crithidia fasciculata kinetoplast DNA, was reduced 4- to 5-fold compared to that in the parental HL-60 cells. Total cellular topoisomerase II activity in HL-60/MX2 cells was only 50% lower than that in HL-60 cells, however, because the "cytosolic fraction" of the HL-60/MX2 nuclear preparation contained high levels of decatenating activity. Antisera to calf thymus topoisomerase II defined a distinctive immunoreactive pattern of topoisomerase II proteins in crude nuclear extracts from the HL-60/MX2 cells. Both alpha (170 kDa) and beta (180 kDa) forms of topoisomerase II were detected in the HL-60 cell extracts, but only the alpha form was detected in extracts from HL-60/MX2 cells. This finding was associated with the appearance of a new 160-kDa immunoreactive species in nuclear extracts from HL-60/MX2 but not HL-60 cells. Studies were designed to minimize the proteolytic degradation of the topoisomerase II enzymes by extraction of whole cells with hot SDS.(ABSTRACT TRUNCATED AT 250 WORDS)     

A nonchromatographic assay for expression of the chloramphenicol acetyltransferase gene in eucaryotic cells

A rapid procedure is described for assaying chloramphenicol acetyltransferase (CAT) enzyme activity following transfection of the CAT gene into eucaryotic cells. CAT enzyme activity in cell extracts catalyzes the transfer of [14C]acetyl groups from labeled acetyl coenzyme A to unlabeled chloramphenicol. Labeled reaction product is quantitated by liquid scintillation counting after extraction into ethyl acetate. The method is valid for use with transfected cell extracts only if the extracts are first heated to 65 degrees C to remove a factor which degrades acetyl coenzyme A. The revised procedure offers considerable advantages in speed and ease of performance over the chromatographic assay in current use.     

Induction by retinoic acid of NAD+-glycohydrolase activity of myelomonocytic cell lines HL-60, THP-1 and U-937, and fresh human acute promyelocytic leukemia cells in primary culture

HL-60, a human promyelocytic leukemia cell line, is induced to differentiate by retinoic acid to mature granulocytes. We have now found that after the addition of 1 μM retinoic acid to HL-60 cultures an increase in NAD⁺-glycohydrolase (NADase) activity is detected by 6 hr and after a 33-fold increase in activity reaches a plateau by 24 hr. Cycloheximide inhibits completely the retinoic acid-induced increase in NADase activity indicating that enzyme induction requires protein synthesis $\text{de}\text{,}\text{novo}$. An increase of NADase activity was found not only in HL-60 cells but also in two human monoblast cell lines (U-937 and THP-1) and fresh cells in primary culture from two patients with acute promyelocytic leukemia. An increase in synthesis $\text{de}\text{,}\text{novo}$ of NADase does not appear to be obligatory for differentiation of HL-60 because there was no increase of NADase activity in HL-60 cells induced to differentiate with either dimethylsulfoxide, hypoxanthine, butyrate, or 1, 25-dihydroxycholecalciferol and there were marked increases in NADase activity at concentrations of retinoic acid having little or no effect on differentiation.     

Identification of the differentiation-associated p93 tyrosine protein kinase of HL-60 leukemia cells as the product of the human c-fes locus and its expression in myelomonocytic cells

A differentiation-associated 93-kDa tyrosine kinase (p93) was purified previously from the human promyelocytic leukemia cell line HL-60. The present study conclusively identifies p93 as the c-fes proto-oncogene product and shows that expression of p93c-fes and its associated tyrosine kinase activity are marked in mature granulocytes, monocytes, and human myeloid leukemia cell lines. Antisera to peptides obtained by expression of c-fes cDNA fragments in Escherichia coli reacted strongly with p93 purified from HL-60 cells. Western blots using one of these antisera demonstrated high levels of p93c-fes protein in normal human granulocytes and monocytes, as well as the cell lines KG-1, THP-1, HEL, and U-937, all of which can be induced to differentiate along the myelomonocytic pathway. Conversely, in cell lines resistant to myeloid differentiation, p93c-fes expression was either very low or absent. Expression of immunoreactive p93c-fes in these cell lines showed a strong positive correlation with p93c-fes tyrosine kinase activity, which was measured in cell extracts using a nondenaturing gel assay. Finally, the expression of p93c-fes, its tyrosine kinase activity, and the binding of 125I-granulocyte-macrophage colony-stimulating factor (GM-CSF) were all coordinately increased in HL-60 cells treated with the granulocytic differentiation inducer dimethyl sulfoxide, while all three parameters were low in untreated or differentiation-resistant HL-60 cells. These results suggest that expression of p93c-fes tyrosine kinase activity may be an essential component of myeloid differentiation and responsiveness to granulocyte-macrophage colony-stimulating factor.     

New aspects in the synthesis and secretion of lysozyme by cultured human monocyte cell lines

The main purpose of this study was to investigate lysozyme synthesis and secretion in three human monocyte cell lines: U-937, HL-60, and THP-1, using sensitive fluorescence-based assay of lysozyme activity. PMA and hIFN-γ were evaluated for inducing lysozyme activity. Using well-defined cell lines from the cell culture collection, no lysozyme activity could be detected in the cultured U-937 cells either with or without addition of the inducing factors. These data suggested, contrary to previous reports, that U-937 cell line cannot synthesize or secrete active lysozyme. THP-1 and HL-60 cells were proved to produce enzymatically active lysozyme in increasing amounts with the time course. PMA and hIFN-γ had no significant inducing effect on the production or the release of active lysozyme in THP-1 and HL-60 cells. We showed inhibiting effect of PMA and hIFN-γ on the lysozyme activity, particularly in HL-60 cell line.     

The effects of promoter on transient expression in conifer cell lines

Summary Protoplasts from suspension cultures of somatic embryos of white spruce (Picea glauca Moench Voss) were electroporated with plasmids containing the chimeric genes for chloramphenicol acetyl transferase (CAT) or -glucuronidase (GUS), under control of one of three promoters. Transient CAT gene expression of approximately equal magnitude resulted when the CAT gene was fused to either the cauliflower mosaic virus (CaMV) 35S promoter or the nopaline synthase (NOS) promoter. When the CAT gene was fused to a tandem repeat CaMV 35S promoter (pPBI-363), CAT enzyme activity compared to NOS or 35S promoters increased up to eightfold (cell line WS-34), and were up to 100-fold greater than control (electroporated without plasmid). Comparatively, protoplasts of black spruce (Picea mariana Mill) and jack pine (Pinus banksiana Lamb.), electroporated with pPBI-363, produced increases in CAT activity compared to control of 90-fold and 70-fold, respectively. White spruce (WS-34) protoplasts were subsequently electroporated with the GUS gene fused to the tandem repeat CaMV 35S promoter. Comparatively, GUS enzyme activity increased up to tenfold compared to GUS fused to a CaMV 35S promoter. The results indicated that transient expression of the CAT and GUS genes was influenced by the type of promoter and cell line used, as well as by electroporation conditions.NRCC No. 30498     

Immunochemical evidence that three protein kinase C isozymes increase in abundance during HL-60 differentiation induced by dimethyl sulfoxide and retinoic acid

Activity of the Ca2+/phospholipid-dependent protein kinase C has been shown to increase during differentiation of the human promyelocytic leukemia cell line HL-60 by dimethyl sulfoxide and retinoic acid (Zylber-Katz, E., and Glazer, R. I. (1985) Cancer Res. 45, 5159-5164). Antipeptide antibodies were prepared that specifically recognize the alpha, beta, and gamma isozymes of protein kinase C in rat brain cytosol and HL-60 cell extracts. The three isozymes do not share a common tissue distribution pattern. The gamma enzyme is abundant in brain but a relatively minor component in HL-60 cells; the opposite is true for the alpha enzyme. All three isozymes increase at least 2-fold in abundance in HL-60 cells exposed to 1.2% dimethyl sulfoxide for 48 h. The increase in abundance of the alpha and beta isoforms reaches 7- and 5-fold, respectively, by 96 h without further increase in the abundance of the gamma isozyme. Similarly, all three isozymes increase at least 1.5-fold in abundance after 48 h and 3-fold after 96 h with 1 microM retinoic acid. No further increase in the abundance of any of the isozymes is seen between 96 and 144 h of incubation with retinoic acid. The increase in protein kinase C activity is not limited to the cytosolic forms of the enzyme; a parallel increase in membrane-associated protein kinase C is also observed during differentiation. Approximately 10% of total protein kinase C activity is membrane-associated in both control and differentiating cells. These studies provide the first immunochemical evidence that all three protein kinase C isozymes increase during HL-60 cell differentiation, and they suggest that the increase in the isozyme levels may be coordinately regulated.     

2’-羟基二氢黄酮诱导白血病HL-60细胞凋亡过程中抗氧化酶活性的变化

目的：观察2’-羟基二氢黄酮诱导白血病细胞HL-60凋亡过程中胞内抗氧化酶活性的变化,并探讨其抗肿瘤作用机制。方法：采用CASY-TT亚流式细胞术测定2’-羟基二氢黄酮对HL-60细胞存活率的影响;Annexin V／PI双染流式细胞仪检测分析细胞凋亡变化;化学比色法测定2’-羟基二氢黄酮作用后,HL-60细胞胞内抗氧化酶SOD、CAT、GSH—Px的活性变化。结果：2’-羟基二氢黄酮显著降低HL-60细胞存活率,其作用呈剂量和时间依赖性;凋亡分析结果显示,20μM的2’一羟基二氢黄酮作用HL-60细胞后,细胞凋亡率逐渐升高,并在12h后显著高于作用前水平;酶活性检测表明,20μM2’一羟基二氢黄酮作用后,HL-60细胞胞内抗氧化酶SOD、CAT、GSH．Px活性均显著降低,脂质过氧化产物MDA含量显著升高。结论：2’-羟基二氢黄酮显著降低白血病HL广60细胞存活率并诱导细胞凋亡,其中伴随着胞内抗氧化酶活性的显著降低。