RNA, HeLA cell

Legagneux, V., Morange, M., Bensaude, O. (1990). Heat-shock and related stress enhance RNA polymerase II C-terminal-domain kinase activity in HeLa cell extracts. Eur. J. Biochemistry 193, 121-126. 

An example of miR-dependent deadenylation of mRNA measured by this method is shown in Fig. 6.3C. In this case, HeLa cells (which express let-7) were transfected with a pDNA R-luc construct encoding three let-7 binding sites in the 3 UTR (3 X bulge), or with control constructs encoding either no sites (plasmid) or three mutated let-7 sites (3 x bulge mut) (constructs described in Pillai et al, 2005). Cells were harvested 24 h after transfection, RNA was purified for the PAT assay, and luciferase activity was measured from cell lysates. As reported previously, the presence of functional let-7 target sites results in specific repression of luciferase expression with very minor effects on mRNA stability (Pillai et al., 2005). The experiment in Fig. 6.3C demonstrates that the let-7 targeted reporter mRNA is selectively deadenylated. 

De Benedetti, A., Joshi-Barve, S., Rinker-Schaeffer, C., and Rhoads, R. E. (1991). Expression of antisense RNA against initiation factor eIF-4E mRNA in HeLa cells results in lengthened cell division times, diminished translation rates, and reduced levels of both eIF-4E and the p220 component of eIF-4F. Mol. Cell Biol. 11, 5435-5445. 

By using an HeLa cell-free system together with microsomes from dog pancreas, four proteins can be made from the 26 S RNA the capsid protein, the E1 protein, a protein with an of 62,000 (the p62 protein),... 

In the laboratory the immortal cell lines are those derived from cancer. The most famous is the HeLa cancer cells that many of us use as one of the cell lines tested against various anticancer agents. The name HeLa cells is derived from Henrietta Lacks, a black woman, who died from cervical cancer. The cells are so strong that they invade other cell lines, both healthy and other cancer cell lines, giving contaminated or mutated cell lines. HeLa cells have good telomerase levels. If antisense RNA is added to HeLa cells so that the RNA contains the opposite message to the ordinary RNA in the telomerase, the effect is that the telomerase is blocked and the HeLa cells are no longer immortal and die after about 25 replications. 

Kam et al. [38-40] SW CNT-r cytochrome C,RNA, DNA CNT transferred cytochrome C to the cancer cells accumulation of SW CNT-RNA conjugates in cytoplasm and nucleus of HeLa cells... 

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... 

Figure 28-17 (A) Electron micrograph of the 45S precursor of rRNA from HeLa cells after spreading from 80% formamide and 4 M urea. The molecule is shown in reverse contrast. (B) Tracing of molecule in (A) showing several regions of secondary structure as hairpin loops. The 28S and 18S rRNA regions are indicated. (C) 32S rRNA. (D) 28S rRNA. Notice that the same secondary structure can be seen in the 28S RNA as in its 32S and 45S precursors. From Wellauer and Dawid.527...

In HeLa cells and presumably in normal mammalian nuclei, the initial cleavage at the 5 end of the in-tron leaves a 3 phospho group (Fig. 28-18B, step a), which is cyclized, probably in an ATP-dependent process, to the 2, 3 -cyclic phosphate (step b). This is ligated to the other piece of RNA by a direct displacement on the cyclic phospho group (step c).602... 

Narciclasine (215) is an antitumor agent which exerts an antimitotic effect during metaphase by immediately terminating protein synthesis in eukaryotic cells at the step of peptide bond formation (97,101,141,142), apparently by interaction with the ansiomycin area of the ribosomal peptidyl transferase center (142). The alkaloid has also been found to inhibit HeLa cell growth and to stabilize HeLa cell polysomes in vivo (97). Although DNA synthesis was retarded by narciclasine, RNA synthesis was practically unaffected (97,142). Sev-... 

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). 

As mentioned above, one consequence of stalled RNA polymerase II at a DNA adduct is activation of transcription-coupled repair [27], This effect may depend on the type of polymerase, however, since the removal of some types of DNA damage is slower from RNA-polymerase I transcribed ribosomal DNA than from a nuclear gene [160], The lower level of repair in the nucleolus could also reflect the influence of other transcription factors, such as the HMG-domain protein UBF, which bind to cisplatin-mod-ified DNA [145]. When HeLa cells were exposed to cisplatin at concentrations which did not seem to affect nuclear transcription, inhibition of rDNA gene expression was associated with the redistribution of UBF, along with other factors responsible for rRNA transcription [138], These observations indicate how cisplatin might exert a combination of effects. Transcription is stopped due to titration of essential factors by the platinum-DNA adducts, and the same proteins could shield the lesions from the repair activity. 

Huang F, Khvorova A, Marshall W, Sorkin A. Analysis of clathrin-mediated endocytosis of epidermal growth factor receptor by RNA interference. J. Biol. Chem. 2004 279 16657-16661. Hinrichsen L, Harborth J, Andrees L, Weber K, Ungewickell EJ. Effect of clathrin heavy chain- and alpha-adaptin-specific small inhibitory RNAs on endocytic accessory proteins and receptor trafficking in HeLa cells. J. Biol. Chem. 2003 278 45160-45170. Wu AM, Senter PD. Arming antibodies prospects and challenges for immunoconjugates. Nat. Biotechnol. 2005 23 1137-1146. 

Perkins KK, Furneaux HM, Hurwitz J. RNA splicing products formed with isolated fractions from HeLa cells are associated with fast-sedimenting complexes. Proc. Natl. Acad. Sci. U.S.A. 1986 83 887-891. 

Akaboshi M, Kawai K, Maki H, Akuta K, Ujeno Y, Miyahara T. The number of platinum atoms binding to DNA, RNA and protein molecules of HeLa cells treated with cisplatin at its mean lethal concentration. Japanese J. Cancer Res. 1992 83 522-526. 

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