KB Cell Nuclei Research Articles

Lipo-Oligomer Nanoformulations for Targeted Intracellular Protein Delivery.

Here, we report novel lipo-oligoaminoamide nanoformulations for targeted intracellular protein delivery. Formulations are generated by first bioreversibly conjugating a sequence-defined amphiphilic lipo-oligomer 728 to the cargo protein via disulfide bonds, followed by formulation of the formed 728-SS-protein conjugate with different helper lipids in various compositions. The triblock oligoaminoamide 728 contains cysteines for reversible covalent protein conjugation and cross-link-stabilization of formed nanoparticles, polyethylene glycol (PEG) for shielding, and providing a hydrophilic domain, eight cationizable succinoyl tetraethylene pentamine (Stp) repeats for endosomal buffering and escape into the cytosol, and a tetra-oleic acid block for hydrophobic stabilization. The added helper lipids are supposed to enhance serum stability of the nanoparticles and provide targeting by lipid-anchored folic acid (FA)-PEG. The optimized protein nanoparticles, including 728, DOPS, cholesterol, DMPE-PEG2000, and the FA-PEG conjugated lipid 1042, presented a high colloidal stability without significant size increase in 72 h. Using cytotoxic ribonuclease A (RNase A) as cargo protein, FA-728-DOPS-DMPE-RNase A nanoformulation could be identified with highest potency of targeted RNase A-mediated folate-receptor-positive KB carcinoma cell killing among all tested formulations, resulting in 85% KB cell killing at a low concentration of 2 μM. These approximately 50 nm sized nanoparticles induced superior 70% KB cell killing even in the presence of 20% serum. Efficient targeted cytosolic delivery by coformulation with helper lipids was also demonstrated by FA-728-DOPS-DMPE-nlsEGFP nanoformulation using enhanced green fluorescent protein (EGFP) as cargo. Furthermore, partial nlsEGFP was imported into the nuclei of KB cells, validating effective endosomal escape, and following nuclear transport mediated by nuclear localization signal on nlsEGFP. As demonstrated, the screening and optimization of nanoformulations with helper lipids and coformulation agents is considered to be an important and rational next step in the development of intracellular biopharmaceuticals, following initial protein conjugate synthesis.

구강암 세포주를 이종 이식한 누드마우스에서 apigenin의 경구투여에 따른 항암효능에 관한 연구

Apigenin은 과일과 야채에 들어있는 플라보노이드로 다양한 악성 세포에 항증식효과를 보여준다. 세포성장 저해효과를 확인하기 위하여 KB 구강암세포주를 96 well plate에 <TEX>$6{\times}10^3$</TEX> cells/well로 분주하고 24시간 후에 apigenin을 24시간 동안 처치하여 MTT assay를 수행하였다. Apigenin은 배양 후 용량 의존적으로 세포사를 유도하였다. Apigenin 100 <TEX>${\mu}M$</TEX>을 24시간 동안 처치하고 대조군과 세포성장을 비교하였을 때 유의적인 감소를 확인하였다. KB 구강암세포주에서의 apoptosis를 확인하기 위해 DAPI 염색을 수행하였다. Apigenin을 처치한 세포에서 핵의 응축이 존재함을 형광현미경으로 확인하였다. 우리는 누드마우스에 KB 구강암세포주를 이식하여 세포 성장 억제 효과를 알아보았다. Apigenin을 마우스에 25, 50 mg/kg을 0.2 ml의 PBS에 녹여 경구투여 하였다. 종양 사이즈는 대조군과 25, 50 mg/kg apigenin 투여군을 비교하였을 때 유의적으로 감소하였다. Apoptosis 분석을 위해 TUNEL염색을 수행하였다. 25 mg/kg apigenin 투여군과 대조군을 비교하였을 때 apoptosis의 유의적인 증가를 확인하였다. 육안적 소견을 위한 H&E 염색은 이상이 없었다. 본 연구는 apigenin이 구강암세포주 성장 억제를 apoptosis의 유도를 통하여 확인하였다. Apigenin (4', 5, 7-trihydroxyflavone), a common dietary flavonoid abundantly present in fruits and vegetables, has shown remarkable anti-proliferative effects against various malignant cell lines. To observe the anti-proliferative effects, oral cavity cancer cell lines, <TEX>$6{\times}10^3$</TEX> cells/well (96 well plate) of KB oral cavity tumor cells were plated and 24 hr later treated with apigenin for one day, after which MTT assay was performed. Apigenin induced cell death in a dose-dependent manner after incubation. Cell viability was significantly decreased in the group treated with 100 <TEX>${\mu}M$</TEX> apigenin for 24 hr (p<0.05) compared to the control group. To assess apoptosis, the nuclei of KB cells were stained with DAPI. The presence of chromatin condensation in the apigenin treated cells was detected on a fluorescent microscope (<TEX>${\times}200$</TEX>). We investigated the in vivo growth inhibitory effects of apigenin on oral cavity cancer KB tumor xenograft subcutaneously implanted in male nude mice. Apigenin was administered to mice by gavage at doses of 25 and 50 mg/kg/day in 0.2ml of PBS. Tumor volume was significantly decreased in 25 and 50 mg/kg apigenin-administration groups compared to the control group. For apoptosis analysis, TUNEL staining was performed. A significant increase in TUNEL positive cells was found in the 25 mg/kg apigenin administration group compared to the non- apigenin administration group. Histopathological changes were not observed. These results indicate that apigenin inhibits oral cavity cancer cell growth through the induction of apoptosis.

Activation of Epidermal Growth Factor Receptor Gene Transcription by Phorbol 12-Myristate 13-Acetate Is Mediated by Activator Protein 2

The response of the epidermal growth factor (EGF) receptor gene to phorbol 12-myristate 13-acetate (PMA) was analyzed using nuclei and nuclear extracts prepared from PMA-treated KB cells. Transient transfection assays and nuclear run-off experiments showed that PMA increased EGF receptor gene transcription. Cell-free transcription with promoter mutants revealed that the region of the promoter containing nucleotides -150 to -16 was sufficient for PMA inducibility. A promoter fragment containing nucleotides -167 to -105 showed increased binding of a factor present in extracts prepared from PMA-treated cells. When this factor was partially purified by column chromatography, it showed specific PMA-dependent binding to an EGF receptor promoter fragment. This binding was competed by an SV40 fragment containing binding sites for Sp1, AP1, and AP2. Purified AP2 was used in DNase I footprinting experiments to show that this factor can bind to the EGF receptor promoter. Oligonucleotides corresponding to the AP2 binding sites found in the EGF receptor promoter showed the ability to bind AP2 and compete for the binding of a factor induced by PMA treatment. The addition of AP2 to nuclear extract resulted in increased transcription from the EGF receptor promoter. These results demonstrate that AP2 can activate EGF receptor gene expression and may mediate the PMA response of this gene.

Electrophoretic characterization of low molecular weight RNAs from prokaryotic and eukaryotic cells.

Low molecular weight RNAs (small RNAs=sRNAs) from prokaryotic and eukaryotic cells were analyzed parallely by electrophoresis in an agarose-polyacrylamide composite gel and a 7M urea-10% polyacrylamide gel.Induced by chloromycetin, three major sRNAs (approx. 190, 240 and 320 nucleotides long) were found to be synthesized during germination of Bacillus subtilis spores. These RNAs were distinct from the commonly occurring 4S, 4.5S and 5S RNAs.The U-series small nuclear RNAs (U-snRNAs) from KB cell nuclei were also separated by gel electrophoresis. The number of U-snRNA molecules in the cell nucleus was calculated using a reported value for U1 RNA as the standard. When the number of U1 RNA was 1×106 molecules in a cell, those of U2, U4, U5 and U6 RNAs were estimated to be 2.7×105, 1.3×105, 5.4×104 and 1.3×105 molecules, respectively.In F9 cells, a mouse teratocarcinoma cell line, U1a and U1b RNA were observed as to U1 RNA.In each gel electrophoresis, the logarithms of the (assummed) molecular weights of sRNAs were inversely proportional to the mobility.

Structural and enzymological characterization of a deoxyribonucleic acid dependent adenosine triphosphatase from KB cell nuclei.

We have purified to near homogeneity the single DNA-dependent ATPase activity that we have identified in extracts of KB cell nuclei. The protein structure of the enzyme was defined by sodium dodecyl sulfate gel electrophoresis, which revealed a single protein band of 75000 daltons that was coincident with the profile of ATPase activity resolved by the final step of agarose-ATP chromatography or by isoelectric focusing. The enzyme has a pI of 8.5, a Stokes' radius by gel filtration of 3.8 nm, and a sedimentation coefficient in high salt of 5.3 S. At low ionic strength the enzyme activity sediments at 7.0 S, suggesting that it may dimerize under these conditions. The purified enzyme has a specific activity of 5.9 X 10(5) nmol of ATP hydrolyzed per h per mg of protein and is devoid of endonuclease, exonuclease, RNA or DNA polymerase, nicking-closing, and gyrase activities at exclusion limits of 10(-6)-10(-8) of the ATPase activity. The enzyme can hydrolyze only ATP or dATP, to generate ADP or dADP plus Pi, but the other NTPs and dNTPs are competitive inhibitors of the enzyme with respect to ATP. A divalent cation (Mg2+ greater than Mn2+ greater than Ca2+) as well as a nucleic acid cofactor is required for activity. Single-stranded DNA or deoxyhomopolymers are most effective, but blunt-ended linear and nicked circular duplex DNA molecules are also used at Vmax values approximately 20% of that obtained with single-stranded DNA. Intact duplex DNA and polyribonucleotides are unable to support ATP hydrolysis. Velocity gradient sedimentation studies corroborate the interpretations of the kinetic analyses and demonstrate enzyme binding to single-stranded DNA and nicked duplex DNA but not to intact duplex DNA. Although we have not succeeded directly in demonstrating DNA unwinding by this protein, preliminary results suggest that in the presence of ATP, the ATPase can stimulate the reactivity of homogeneous human DNA polymerases alpha and beta on nicked duplex DNA substrates.

Adenovirus type 12 VA RNA I. Synthesis in productive infection and gene mapping

VA RNA synthesized late in adenovirus type 12 (Ad12)-infected cells has been analyzed by two-dimensional polyacrylamide gel electrophoresis and by a sequence of three different gel electrophoresis separations. Each of these procedures resolves two distinct VA RNA species in Ad2-infected cells; no minor species was detected in either nuclei or cytoplasm of Ad12-infected KB cells. VA RNA synthesized in vitro in isolated nuclei of KB cells 18 hr after Ad12 infection also showed only one distinct species upon electrophoretic analysis. VA RNA synthesis in isolated nuclei was resistant to α-amanitin at concentration of 1 μg/ml but was inhibited by a high concentration of α-amanitin (100 μg/ml), which indicates that Ad12 VA RNA is synthesized by RNA polymerase III. The gene for VA RNA was mapped on Ad12 genome by blot hybridization experiments. It is located around position 0.30 of the unit map as determined by hybridization to fragments G and H of the BamHI digest of Ad12 DNA. The T 1 ribonuclease oligonucleotide fingerprint analysis of RNA selected by hybridization to fragments G and H of the BamHU digest of Ad12 DNA has so far failed to provide evidence for two species of VA RNA of distinctly different structure in this adenovirus serotype.

Transcription of adenovirus-associated virus RNA in isolated KB cell nuclei

Transcription of the genomes of the defective parvovirus, adenovirus-associated virus (AAV) type 2, and a helper virus, adenovirus (Ad) type 2, was studied in nuclei isolated from infected KB cells. As measured by the incorporation of [ 3H]UTP into acid-insoluble product, nuclei prepared 15.5 hr after coinfection with AAV and Ad (AAV-Ad nuclei) synthesized RNA for 10 min. Total RNA synthesis in AAV-Ad nuclei was greater than in uninfected nuclei but was only about 1 2 that observed in nuclei from cells infected with Ad alone (Ad nuclei). DNA-RNA hybridization with RNA samples from the AAV-Ad nuclei revealed that the in vitro synthesis of AAV-RNA (14% of total) was twice that of Ad-RNA (6% of total). When Ad or AAV-Ad nuclei were assayed at various times after infection, total RNA production was comparable until AAV transcription was well established (12 hr); thereafter, a relative depression of total RNA synthesis was observed in the AAV-Ad nuclei. However, from 12 hr on, more AAV-RNA than Ad-RNA was synthesized in AAV-Ad nuclei, and a comparison with Ad nuclei suggested a preferential inhibition of Ad-RNA transcription in the AAV-Ad nuclei. Finally, it was found that AAV, Ad, and total RNA synthesis shared the same optimum salt concentration [0.02 M (NH 4) 2SO 4], and that 95% of Ad-RNA synthesis and greater than 99% of AAV-RNA synthesis were inhibited by levels of α-amanitin which blocked cellular RNA polymerase II activity. These data suggest that the AAV genome is also transcribed by the same polymerase that transcribes the bulk of Ad-RNA and cellular heterogeneous nuclear RNA.

Association of adenovirus type 2 early proteins with a soluble complex that synthesizes adenovirus DNA [formula omitted

A soluble Ad2 DNA synthesizing complex was prepared from Ad2-infected KB cell nuclei and purified by exclusion chromatography on a BioGel A-50m column. The purified complex was able to synthesize DNA from all regions of the virus genome, as indicated by Eco RI restriction endonuclease analysis of in vitro labeled DNA. Experiments were performed to identify Ad2-induced early polypeptides present in the complex. Ad2-infected and mock-infected cells were labeled with [ 35S]methionine 7–10 h postinfection, then incubated for 8 h to allow the 35S-labeled early polypeptides to become associated with the complex. The polypeptides in the purified complex and each of the cell fractions were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. The major components of the purified complex were the 73K DNA binding phosphoprotein and 11K, two adenovirus 2-induced early polypeptides. The 11K has a preferred nuclear location. Small quantities of other Ad2-induced early proteins, 21K, 15K, and possibly 8.3K were also associated with the complex.

Visualization and mapping of late nuclear adenovirus RNA

Nuclei of KB cells harvested at late stages of productive infection with adenovirus type 2 (Ad2) harbor RNA molecules which measure up to 13 μm in length, as determined by electron microscopy of denatured RNA. While some of the molecules display features of secondary structure that are characteristic for precursor rRNA, our interest was in those showing almost no intramolecular folding. When hybridized to double-stranded viral DNA under conditions which favor RNA:DNA duplex formation, nuclear AD2 RNA displaces the homologous DNA region and generates R loop structures whose size is proportional to the length of the hybridizing RNA. Slowly sedimenting RNA forms small R loops, whereas RNA of high sedimentation velocity generates loops that span a large proportion of the DNA length. Using SV40 sequences within Ad2 +ND 4 hybrid DNA as a position marker, we oriented many of the R loops on the conventional Ad2 map. Our analysis was restricted to the most abundant sequences of late Ad2 nuclear RNA participating in R loop formation. A small but significant proportion of large RNA generates loops between map positions 0.3 and 0.9. The much more frequent RNA of intermediate size (although larger than mRNA) hybridizes with midpoints near map positions 0.55 and 0.88 — that is, near the gene locations for hexon and fiber. Our findings are compatible with the idea that the nuclear RNAs visualized in this study are intermediates in a processing pathway leading to mature forms of late Ad2 mRNA.

Endogenous protein kinase activity in nuclear RNP particles from HeLa cells.

To elucidate the mechanisms which achieve and regulate the transport from the nucleus to the cytoplasm of those sequences of heterogeneous nuclear RNA (HnRNA) destined to become cytoplasmic mRNA, we have started a systematic investigation of the proteins associated with these RNA species in the form of the so-called ‘RNP particles’. The general occurrence of such ribonucleoprotein complexes in eukaryotic cells is widely recognised although they have only recently been described in HeLa cells1,2. The occurrence of phosphorylated proteins in rat brain nuclear particles3, together with the observation that mRNA release from adenovirus-infected KB cell nuclei in vitro is an ATP-dependent process4, has prompted a search for protein kinase activity in HeLa nuclear RNP particles. The role of protein phosphorylation in the regulation of enzyme activity is now recognised5 and we have observed such an activity which does not require the addition of exogenous substrates like histones. The endogenous protein substrates present in HeLa nuclear RNP particles have also been characterised.

Crystallographic study of intranuclear adenovirus type 5 crystals.

Crystallographic analysis of adeno-virus type 5 crystalline patterns within KB cell nuclei revealed that the crystals belonged to the face centered cubic system, with four asymmetric units (viz. four

Effects of ultraviolet micro-irradiation on different parts of the cell: II. Cytological observations and unscheduled DNA synthesis after partial nuclear irradiation

Summary KB cell nuclei were partially irradiated with a 5 μm diameter microbeam of ultraviolet light (275 nm). A dose of approx. 10−3 ergs/μm2 did not produce any morphological changes that could be discerned by electron microscopy, but it did inhibit mitosis for at least 24 h. When the same cells were incubated with 3H-TdR for 3 h, the radio-autograms showed the nucleoside was incorporated in the irradiated zone. When the same dose of irradiation was given to cells normally undergoing DNA synthesis (S phase), the cells contained label throughout the nucleus and the number of grains was the same in these cells as in non-irradiated control cells in S phase. However, 40% of these cells showed a reduced incorporation of 3H-TdR within the irradiated spot, as compared with the non-irradiated part of the nucleus. The localized 3H-TdR incorporation may represent irradiation-induced DNA repair; the remainder of the nucleus did not replicate its DNA and proved the cells were not in S phase. Irradiation at higher doses (i.e., 10−1 ergs/μm2) induced focal electron microscopic lesions and DNA synthesis was impaired throughout the nucleus. These lesions remained fixed in the nucleus at the site of irradiation.

Particles of nuclear origin carrying rapidly labelled RNA.

Nuclei of KB cells have been isolated and incubated at 37° in pH 8.0 saline. The experiments described in this paper provide evidence that: a) Isolated nuclei release well defined nucleoprotein particles, which contain RNAs not detectable in untreated nuclei. b) These particles carry rapidly labelled RNA. c) In cytoplasm they cosediment with the long known smaller ribosomal particles. d) Chase experiments suggest that nuclear particles are precursors of cytoplasmic particles.