HeLa cells enzymes

Winters TA, Weinfeld M, Jorgensen TJ (1992) Human HeLa cell enzymes that remove phosphoglyco-late 3 -end groups from DNA. Nucleic Acids Res 20 2573-2580 Wirths A, Jung H (1972) Single-strand breaks induced in DNA by vacuum-ultraviolet radiation. Pho-tochem Photobiol 15 325-330... 

PSM has been described in porcine submaxillary gland (McGuire, 1970 Section V,A,2) the HeLa cell enzyme appears to difiFer from this salivary gland enzyme in pH optimum, cation requirements, and Km for GDP-fu-cose. The fucosyltransferase which acts on protein-bound N-acetylglucos-aminyl termini may be related to one of the blood group fucosyltrans-ferases, discussed in Section V,B,2,c, which attach fucose into either (a, l->3) or (a, l->4) linkage with N-acetylglucosamine. 

A CL ISH assay for the detection of human papillomavirus (HPV) DNA was developed, in which the hybridization reaction was performed using either digoxigenin-, biotin-, or fluorescein-labeled probes [64], The hybrids were visualized using AP as the enzyme label and a highly sensitive 1,2-dioxetane phosphate as chemiluminescent substrate. This assay was applied to biopsy specimens from different pathologies associated with HPV, which had previously proved positive for HPV DNA by polymerase chain reaction (PCR). The analytical sensitivity was assessed using samples of HeLa and CaSki cell lines, whose content in HPV DNA is known (10-50 copies of HPV 18 DNA in HeLa cells and 400-600 copies... 

During in vivo studies under biologically relevant conditions, the cis-Pt loading of the DNA is much lower than for the above-mentioned in vitro studies. It has been calculated that mortality of HeLa cells occurs at an value of 10 5 (i.e., one bound cis-Pt molecule per 105 nucleotides) (64a). This excludes atomic absorption spectroscopy for identification of the in vivo adducts. Immunochemical techniques, however, have shown to be very promising, and high sensitivity and selectivity levels have been reached. At the moment, only a few studies in which antibodies are raised against cis-Pt-treated DNA (64) or against synthetic cis-Pt adducts with mono- or dinucleotides are available (64a). With the latter method, quantitation of the different platinum-DNA adducts formed under in vivo conditions is possible. At the moment, femtomole (10-15 mol) amounts of the adducts can be detected with competitive enzyme-linked immunosorbent assay (ELISA) techniques. It has been demonstrated in this manner that the GG-Pt adduct is also the predominant adduct under in vivo conditions. 

Other nonhistone nuclear proteins. Polyacrylamide gel electrophoresis revealed more than 450 components in HeLa cell nuclei. Most are present in small amounts of <10,000 molecules per cell and are not detectable in cytoplasm.112 Among the more acidic proteins are many enzymes including RNA polymerases. There are also gene repressors, hormone-binding proteins, protein kinases, and topoi-somerases.113 Among the six most abundant nonhistone nuclear proteins in the rat are the cytoskeletal proteins myosin, actin, tubulin, and tropomyosin.114... 

Regulation of alkaline phosphatase activity was discussed by Cox and Griffin 65). Steroids such as hydrocortisone or prednisolone (1 (ig/ml of medium) can induce a 3- to 20-fold rise in certain HeLa cell cultures, while other cell lines are induced by 15 mM phenyl phosphate. Recently, it has been shown that fibroblast cell cultures are also stimulated by prednisolone (( ( ). According to one report, Pi may have a control function here a decrease in the level of rat kidney P produced by a low phosphate diet was accompanied by an increase in alkaline phosphatase. Nine other enzymes monitored were unaffected 67). 

So far the only well-characterized uncompetitive inhibitor is L-phenyl-alanine, shown to be uncompetitive for intestinal (180) and placental phosphatases (42). By contrast, D-phenylalanine has no inhibitory effect. The L isomer apparently acts by preventing the breakdown of phosphoryl phosphatase (170), possibly by blocking the acceptor site mentioned in Section III,D,4. A list of concentrations required to produce 50% inhibition of a wide variety of phosphatases showed that all the enzymes were affected though there was a spread in susceptibilities from 0.8 mM for HeLa cell culture phosphatase to 26 mM for mouse intestinal phosphatase... 

Butyrate treated Hela cells (2) and KB cells showed marked increases in the amounts of G 3 gangliosides and elevated levels of the enzyme, CMP sialic acid lactosylceramide sialosyltrans-ferase, required for its synthesis. 

Exposure of HeLa cells to butyrate had no effect on the activity of GM3-sialidase when GM3 specifically labeled in the sialic acid residue was used as substrate (Fig. 3a). We were unable to detect any "ecto"-sialidase activity in either control or butyrate-treated cells (14) although others have postulated that such an enzyme is important in regulating plasma membrane gangliosides (15,16). In contrast, the activity of the specific sialyl transferase involved in GM3 biosynthesis increased over 20-fold following butyrate treatment (Fig. 3b). The effect was specific as activities of the other glycosphingolipid transferases that could be measured in HeLa cells were not altered in butyrate-treated cells (4,8,17). 

HeLa cells were cultured for 24 hr with (open bars) and without (closed bars) 5 mM sodium butyrate, harvested, and assayed for indicated enzyme activity. A GM3-sialidase activity assayed with GM3 specifically labeled with [,iC]- N-acetylneuraminic acid as substrate ("14JJ. B Sialyltrasferase activity assayed with CMP-[, acetylneuraminic acid and lactosylceramide as substrates, and synthesis of labeled GM3 determined (17). Data... 

Pretazettine (395) has been the subject of numerous biological studies, and it has been shown to exhibit a number of interesting activities (96,97,101,178-187). For example, 395 was found to inhibit HeLa cell growth as well as protein synthesis in eukaryotic cells by interfering with the peptide bond formation step (97,101). Furthermore, pretazettine inhibited the purified RNA-dependent DNA polymerase (reverse transcriptase) from avian myeloblastosis virus, a typical C-type virus (178), in an unusual fashion since it physically combined with the polymerase enzyme itself rather than interacted with the nucleic acid template. Pretazettine also exhibited antiviral activity against the Rauscher leukemia virus in mouse embryo cell cultures by suppressing viral replication (179). 

Understandably, there are examples of oxygen-tolerant cell lines which have much higher levels of constitutive antioxidant enzymes than normal [249]. The ability of other cells to protect themselves by means of antioxidant enzymes is limited by their biosynthetic response to oxidative stress. In vitro exposure to paraquat induces an increase in SOD and catalase activities in human and hamster fibroblasts [250,251]. In cultured HeLa cells, a chronic exposure to paraquat induces a simultaneous increase in Mn- and Cu/Zn-SOD activities in surviving cells [252], Interestingly, such cells have apparently acquired resistance to paraquat, and their higher level of SOD activities persists for many months following the exposure to paraquat. In Chinese hamster fibroblasts, a chronic exposure to H2O2 results in a 5-40-fold increase in catalase activity [253], which... 

Intermolecular strand transfer has also been demonstrated for the type I enzyme from HeLa cells (Halligan et al., 1982). This enzyme can transfer a single-stranded ( donor ) DNA to a range of different accep-... 

Interesting indeed are the recent studies of Griffin and Cox (G20, G21) on the mechanism of adrenocorticoid induction of alkaline phosphatase in human cell cultures. Two temporal phases in the induction of enzyme in HeLa cells are reported a first 12-hour period exhibiting a slow rise in activity, followed by a sudden marked linear increase extending from 15 to 80 hours. The elevation of activity is marked (around 10-fold). The fact that puromycin blocked induction immediately, and actinomycin D... 

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