Altered expression and transcription of the topoisomerase II gene in nitrogen mustard-resistant human cells.
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Cell Line↗
Biomedical subjects
K B Tan
Publications and source records attributed to K B Tan.
Elevated DNA topoisomerase II activity in nitrogen mustard-resistant human cells.
A human Burkitt lymphoma cell line, Raji-HN2, made 10-fold more resistant to nitrogen mustard (HN2) than the parental Raji cell line, exhibited the following characteristics when compared to the parental Raji cells: (i) decreased HN2-induced DNA interstrand crosslinking; (ii) increased (3-fold) DNA topoisomerase II [DNA topoisomerase (ATP-hydrolyzing), EC 5.99.1.3] activity; (iii) increased (4- to 11-fold) sensitivity to topoisomerase II inhibitors; (iv) increased (2-fold) glutathione content; and (v) increased (2-fold) cell doubling time. The resistant phenotype was unstable and was maintained by weekly treatment of the cells with HN2. Growing the resistant cells in the absence of HN2 resulted in a time-dependent decrease in both resistance to HN2 and topoisomerase II activity and an increase in DNA interstrand crosslinking induced by HN2. We hypothesize that HN2 resistance is due to enhanced monoadduct repair with resultant decreased DNA crosslinking and that this process is mediated by topoisomerase II.
Antineoplastic Agents↗
Elevated topoisomerase II activity and altered chromatin in nitrogen mustard-resistant human cells.
A human Burkitt's lymphoma cell line (Raji-HN2) made resistant to nitrogen mustard, a bifunctional alkylating agent, was used to study the mechanism of resistance to nitrogen mustard. A comparative study of Raji-HN2 and the parental sensitive Raji cell lines revealed the following: (1) The DNA of Raji-HN2 cells was crosslinked by nitrogen mustard to a lower extent than Raji DNA; (2) once interstrand crosslinks were formed, they were repaired at the same rate in both cell lines; (3) DNA crosslink formation in Raji-HN2, but not in Raji cells, was enhanced by novobiocin, a topoisomerase II inhibitor; (4) Raji-HN2 cells had elevated topoisomerase II activity and were hypersensitive to topoisomerase inhibitors (amsacrine, novobiocin, teniposide); (5) similar amounts of topoisomerase I were found in both cell lines; and (6) the chromatin of Raji-HN2 but not of Raji cells, was hypersensitive to DNase I digestion. The relationship between DNA repair, topoisomerase II activity, chromatin structure and drug resistance is discussed.
Amsacrine↗
Normal and neoplastic human cells have different histone H1 compositions.
The H1 histone is the least conserved of the five major groups of histone proteins. There are as many as five subtypes of H1 histone (1-3). These H1 subtypes occur in different amounts in different animal species and also show tissue specificity (1-3). Normal and neoplastic tissues from the same animal, e.g. rat or calf, contain the same H1 subtypes but in different relative amounts (4, 5). Because H1 subtypes exhibit tissue specificity, it is therefore difficult to identify the changes in their composition that are associated with neoplasia reported in studies utilizing tissues of different origins (4, 5). Tissue culture cells and their in vitro transformed neoplastic counterparts, on the other hand, offer an excellent system in which to study these changes because the cells are derived from the same origin. We have examined normal and neoplastic human cells and found a relationship between the H1 composition and the ability of the cells to form tumors in nude mice. The ratio of H1A to H1B in normal human cells is considerably lower than that in neoplastic cells and this ratio increases with the increased ability of the cells to form tumors in nude mice.
Animals↗
Chromosomal proteins of mouse teratocarcinoma cells.
We have analyzed chromosomal proteins extracted from murine teratocarcinoma-derived stem cell lines (F9 and 12-1) and from their differentiated derivatives (12-1a) because of the differential sensitivity to DNase I digestion of these two cell types. The chromosomal DNA of stem cells is more sensitive to DNase I digestion than that of differentiated cells. Stem cell core histones are more highly acetylated than their differentiated counterparts, and certain high-mobility group (HMG) proteins from stem cells (HMG 1 and HMG 2) are found in greater amounts than in the differentiated cells though others (HMG 14 and HMG 17) occur in similar amounts. We have also identified a new HMG protein (HMG 9) that is present in stem cells and is lost following differentiation.
Acetylation↗
Transcriptional control of the expression of mouse globin genes in myeloma x erythroleukemia cell hybrids.
Fusions were made between thymidine kinase deficient (TK-) Friend Cells inducible for hemoglobin production, and immunoglobulin-producing, hypoxanthine-guanine phosphoribosyltransferase-deficient (HGPRT-) myeloma cells. Hybrids were selected in hypoxanthine-aminopterin-thymidine (HAT) and identified by isozyme analysis and chromosome counts. All hybrids resembled the myeloma cell line in mode of growth and were immunoglobulin secretors. All hybrids did not express hemoglobin and were uninducible for hemoglobin production with dimethyl sulfoxide (DMSO). Hybridization of genomic globin DNA probes with hybrid-derived nuclear and cytoplasmic mRNAs blotted to nitrocellulose filter indicated that lack of expression of the globin genes in the hybrids was due to lack of transcription.
Animals↗
Nuclear tubulin of tissue culture cells.
Tubulin has been implicated as a nuclear protein because of the role it plays in mitosis. In this paper we examined the role of tubulin in the nuclei of nonmitotic cells. Tubulin was found distributed throughout the nucleus and particularly in association with the chromatin. It comprised about 6.5% of the nonhistone chromosomal proteins. Nuclear tubulin appeared to be nonmicrotubular in form. Fluorescence microscopy data on metaphase chromosomes revealed that tubulin was present on the outside of the chromosomes. These data suggest a structural role for chromatin-associated tubulin.
Animals↗
Studies on viral DNA protein complexes isolated at different times after infection of monkey kidney cells with simian virus 40.
The major protein components of the DNA complex, isolated from SV40-infected monkey cells, are the major viral structural polypeptide VP1 and cellular histones. At early times (24 h) after infection, VP1 is present in large amounts relative to histones, whereas at late times (48 h), the complex contains mostly histones. The amount of VP1 in the complex can be correlated to the amount of "free" VP1 present in the cells, i.e. VP1 not yet incorporated into virus particles. At early times about 40% of VP1 is "free" VP1; at late times, most of the VP1 is incorporated into virus particles. In contrast, viral DNA is produced in huge excess and only about 13% is incorporated into virions. In agreement with the above result, we find that only about 16% of the DNA in the DNA complex can be chased into virions. There is, apparently, no turnover of newly synthesized VP1 that is associated with the DNA complex at late times after infection.
Animals↗
Vesicular stomatitis virus deficient in protein kinase is infectious.
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Cell Line↗
Nucleoprotein complexes from simian virus 40-infected monkey cells: association of viral DNA with histones and the major viral structural protein.
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Capsid↗
The effect of arginine deprivation on DNA, thymidine kinase and RNA polymerase synthesis in simian virus 40-infected monkey kidney cells.
In arginine-deprived cells infected with simian virus 40 (SV40), both viral DNA and viral structural proteins were synthesized but infectious virus was not produced. The syntheses of cellular DNA, thymidine kinase and DNA polymerase were induced in virus-infected cells deprived of arginine but the maximum rate of synthesis of these enzymes occurred much later than that in infected cells incubated in the presence of arginine.
Arginine↗
Effect of streptovaricin on the incorporation of uridine into cellular and viral RNA.
Poxvirus replication is inhibited by streptovaricin. The most readily observed effect is the inhibition of incorporation of [3H]uridine into viral mRNA, suggesting an inhibition of RNA synthesis. Streptovaricin also inhibits the incorporation of [3H]uridine into cellular RNA but not as severely as viral RNA. On the other hand, [3H]uridine incorporation into the RNA of Semliki Forest virus (SFV), which contains a positive strand RNA genome, does not seem to be inhibited by streptovaricin. The inhibitory effect of streptovaricin is completely reversible after removal of the inhibitor. In addition to inhibiting RNA synthesis, streptovaricin also may inhibit the methylation of cellular RNA. Viral RNA is stable in the presence of streptovaricin.
Cell Line↗
Histones: metabolism in simian virus 40-infected cells and incorporation into virions.
The infection of confluent monkey cells with simian virus 40 induced the synthesis of both cellular DNA and histones. However, during the course of infection, cellular histone synthesis was uncoupled from cellular DNA replication and became coupled to viral DNA replication. The synthesis of all five host histone species was induced after virus infection and they appeared to be more highly phosphorylated than their couterparts in uninfected cells. At late times after infection, the cells contained twice as much histones as did uninfected cells. All the histone species except H1 were incorporated into virions. Compared to cellular histones, virion histones were enriched in the arginine-rich species H3 and H4. Although both old and newly synthesized cellular histones were incorporated into virions, there were about 5 times more newly synthesized than old histone polypeptides in virions.
Cell Line↗
Enhancement of simian virus 40 uptake by permissive, semi-permissive and non-permissive cells.
The uptake of simian virus 40 (SV40) by cells that are both non-permissive for virus replication and resistant to virus infection could be enhanced markedly by infecting the cells in presence of DEAE-dextran. Virus uptake by semi-permissive and permissive cells was also enhanced by DEAE-dextran. Optimum enhancement of virus uptake occurred at 100 microgram of DEAE-dextran per ml and under the conditions employed, the polycation was not toxic to cells. The increased cellular uptake of virus may result from the uptake of virus aggregates formed in the presence of DEAE-dextran.
Adsorption↗
Structural and enzymatic characterization of viper C-type virus.
The structural polypeptides of purified viper range in molecular weight from 11,000 to 97,000 daltons and consist of 3 major and about 13 minor polypeptides. The virus contains both protein kinase and reverse transcriptase activities. Several of the structural polypeptides are phosphorylated in vitro by the virus-associated protein kinase. However, most (possibly all) of the viral structural polypeptides are not phosphorylated in vivo. DeoxyATP is as efficient as ATP in donating phosphate for in vitro phosphorylation of viral proteins. In vitro protein phosphorylation always precedes transcription and the virus-associated protein kinase and reverse transcriptase activities can be partially separated by sedimentation in a sucrose gradient.
Adenosine Triphosphate↗
Comparative study of the protein kinase associated with animal viruses.
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Animals↗
Fate of parental simian virus 40 DNA in permissive monkey kidney cells.
The fate of parental SV40 DNA in monkey kidney cells was investigated by infecting the cells with purified virus labelled with [3-H]-thymidine. 5-Bromodeoxyuridine (200 mug/ml) was added to the cells at 2 h after infection to label virus progeny DNA. At 72 h post-infection the cells were harvested and virus DNA was extracted and fractionated by isopycnic sedimentation in CsCL solution. The following DNAs with characteristic densities were found: light (LL), 1.70 g/ml; hybrid (HL), 1-75 g/ml and heavy DNA (HH), 1-80 g/ml. Of the total cell-associated [3-H]-radioactivity (derived from parental virus), more than 90% was recovered in unreplicated parental DNA(LL DNA), about 2% was recovered in the HL DNA and about 1-7% was associated with the HH DNA. The unreplicated parental DNA was present as uncoated intact DNA complexed with proteins present in the infected cell. The HL DNA contains one light parental and one heavy progeny DNA strand. The nature of the radioactivity present in the HH DNA remains to be determined.
Adenine↗
Effect of hypertonic conditions on protein synthesis in cells productively infected with simian virus 40.
Hypertonic medium selectively suppressed the synthesis of most host cell polypeptides relative to the synthesis of simian virus 40 capsid polypeptides and a minority of cellular polypeptides, notably histones. Under optimal hypertonic conditions, the synthesis of the major capsid polypeptide (VP1) is enhanced about sevenfold relative to host polypeptide synthesis. Because of the small amounts of the other nonhistone capsid polypeptides (VP2) and VP3) present in cell lysates, it was difficult to quantitate the extent, if any, of their enhancement. The maintenance of the restricted pattern of protein synthesis caused by hypertonic medium was dependent on continual peptide chain initiations. The resistance of viral protein synthesis to hypertonic conditions provides a means of detecting relatively low levels of intracellular viral protein synthesis. Analysis of the specific activity of the acid-soluble [3H]lysine pool indicated that the rate of incorporation of [3H]lysine into protein was an overestimation of the actual rate of overall protein synthesis occurring in cells exposed to hypertonic as compared to isotonic conditions. Since it is likely that both cellular and viral protein synthesis draw lysine from a single pool, this change in pool specific activity does not affect the analysis of relative rates of protein synthesis at a given level of tonicity.
Cell Line↗