RESUMO
The effect of chlorambucil on the synthesis of histone variants of a cancer cell line HEp-2 is analysed and compared to that of nontreated and hydroxyurea treated cells. Cell proteins were labelled with [14C]lysine and [14C]arginine and histone variants resolved by one- or two-dimensional electrophoresis. Chlorambucil shows no significant decrease in total protein synthesis but shows a significant decrease in histone biosynthesis. It does not selectively inhibit the synthesis of the S-phase variants, i.e., H2A.1, H2A.2, H3.2 or the G1/G2 phase (basal) histone variants, i.e., H2A.Z, H2A.X and H3.3. On the contrary, hydroxyurea treated cells, which also show no significant decrease in amino acid incorporation into total cellular protein but do exhibit a significant inhibition of histone biosynthesis, show a selective inhibition of the synthesis of S-phase variants, but have no effect on the synthesis of basal histone variants. On the basis of histone variants being synthesized in the presence of chlorambucil, it is shown that although chlorambucil shows a specificity for histone synthesis inhibition it has a general action over the whole variant complement and is not coupled to S-phase synthesis in a way typical for DNA synthesis inhibiting drugs.
Assuntos
Clorambucila/farmacologia , Histonas/biossíntese , Autorradiografia , Divisão Celular/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Eletroforese em Gel Bidimensional , Humanos , Hidroxiureia/farmacologia , Cinética , Biossíntese de Proteínas , Células Tumorais CultivadasRESUMO
Multiple microgel comet assay (MMCA) is a metho-dological adaptation of the single-cell gel electrophoresis assay in which we have introduced the use of standard agarose plug molds in an attempt to improve and expand the applications of the assay. We focused on the study of the heterogeneity of peripheral blood mononuclear cells (PBMC) at the level of the basal single-strand breakage and the DNA damage induction caused by ionizing radiation. Differences among subpopulations were also investigated at the level of chromatin organization and methylation after NotI digestion of microgel-embedded cells. In parallel experiments, the NotI-digested nucleoids were also analyzed with the use of pulsed-field gel electrophoresis (PFGE) and the DNA migration patterns were compared with the corresponding patterns from the MMCA. Significant heterogeneity in the distribution of the oxidative DNA damage, as well as intracellular variations in the NotI digestion patterns were observed in the cell population of PBMC. The combined use of both the comet assay and PFGE provides a useful model for analysis of variation in DNA damage in individual cells as well as information on size of DNA fragments.
Assuntos
Ensaio Cometa/métodos , DNA/análise , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/fisiologia , Cromatina/fisiologia , Ilhas de CpG , DNA/química , Dano ao DNA/efeitos da radiação , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Eletroforese/métodos , Eletroforese em Gel de Campo Pulsado , Humanos , MasculinoRESUMO
The pattern of histone variants from the meal moth Plodia interpunctella was compared to the mouse histone variant pattern. Plodia contains histones which comigrate on two dimensional gels with H3.2, H3.3, H4 and H2A.Z in mouse. Plodia H2A.1 and H2B.1 migrate somewhat differently from the respective mouse histones. Comparison of the iodinated tryptic peptides of H2A.1 and H2A.Z from mouse and Plodia showed that the H2A.Z proteins have two iodinated peptides that comigrate in the two species and three more that are different. The H2A.1 proteins in the two species have one iodinated peptide which comigrates and two more which migrate very close to each other. The histone variants from three developmental stages, larval, pupal and adult of Plodia interpunctella were also identified and compared. The same histone variant pattern is found through all stages of development. It is concluded that histone gene expression does not change during metamorphosis in Plodia .
Assuntos
Histonas/análise , Lepidópteros/análise , Metamorfose Biológica , Mariposas/análise , Animais , Eletroforese , Larva/análise , Leucemia L1210/metabolismo , Camundongos , Proteínas de Neoplasias/análise , Pupa/análiseRESUMO
The effect of chlorambucil, a bisalkylating agent, on the biosynthesis of the 5% PCA extractable protein fraction of the cancer cell line, HEp-2, has been analyzed. It was found that the synthesis of all the high mobility group proteins as well as that of the H1 and H1o histone proteins are inhibited by this agent. HMG 14 and the H1, H1o proteins are inhibited to the same extent as that reported for the core histones of the same cell line [7], while slightly higher levels of inhibition were found for the HMG 1, 2 and 17 proteins. The proteins, P1 and HMG I exhibited the highest level of inhibition of the entire fraction. These findings extend previous findings regarding the histone proteins and may be correlated to a dysfunction in the normal process of chromatin condensation and a potential cytotoxic effect of this agent during the G2 phase.