ABSTRACT
Hydrostatic pressure and HeLa S3 cells were used (as a model system) to investigate the relationship of the cytoskeleton and histone gene expression. Exposure of HeLa S3 cells to hydrostatic pressure of 1000 - 10,000 psi (6.89 x 10(3) - 6.89 x 10(4) kPa) disrupts the cytoskeleton and reduces H1 and core histone mRNA and actin mRNA levels as determined by hybridization to specific DNA probes. Soluble and insoluble cell fractions were isolated from HeLa cells after lysis in Triton X-100 buffered with PIPES and being subjected to low-speed centrifugation. The insoluble fraction was designated the cytoskeletal fraction. At atmospheric pressure, 76% of H4 histone mRNA is associated with the cytoskeletal fraction and 24% of the H4 histone mRNA is in the soluble fraction. At 6000 and 10,000 psi for a duration of 10 min, H4 mRNA levels in the cytoskeletal fraction were reduced to 52 and 41%, respectively. The reduction of mRNA in the cytoskeletal fraction is accompanied by a corresponding increase of mRNA in the soluble cell fraction. The other core (H2A, H2B, and H3) and H1 histone mRNA transcripts exhibited similar sensitivity to pressure treatment. The effects of pressure on histone gene regulation may be mediated through alteration of mRNA-cytoskeleton association.
Subject(s)
Cytoskeleton/chemistry , Histones/biosynthesis , RNA, Messenger/analysis , Actin Cytoskeleton/chemistry , Cell Compartmentation , Gene Expression Regulation , HeLa Cells , Humans , Hydrostatic Pressure , Microtubules/chemistryABSTRACT
A reduction in tubulin mRNA levels up to 35-40 min following pressure (10,000 psi for 5 min) was shown by cDNA hybridization in log growth phase and deciliated Tetrahymena. The level of tubulin mRNA increased to a maximum 1 hour after pressure release. Poly(A+) mRNA derived from pressure-treated and atmospheric control cells following deciliation was translated in vitro. The profile for tubulin synthesized in vitro closely resembled the tubulin mRNA profiles. In vivo tubulin synthesis measured by 35S methionine incorporation was suppressed 60%, 40 min following pressure release. The data supports the hypothesis that both transcription and post transcriptional events are sensitive to hydrostatic pressure.
Subject(s)
Tetrahymena/physiology , Tubulin/physiology , Animals , Gene Expression Regulation , Hydrostatic Pressure , RNA, Messenger/analysis , RNA, Messenger/metabolism , Tubulin/genetics , Tubulin/metabolismABSTRACT
Exposure of HeLa S3 cells to high hydrostatic pressure (6.89 x 10(3) to 6.89 x 10(4) kPa: 1000 to 10,000 lbfin-2) reduced core and H1 histone mRNA levels as determined by hybridization to specific histone DNA probes. At 4.14 x 10(4) kPa for 10 min core histone and H1 histone mRNA levels were reduced 32-38% and 56%, respectively. At 30 min postdecompression core mRNA levels returned to atmospheric control levels while H1 histone mRNA levels continued to be suppressed. Levels of macromolecular synthesis were monitored under hydrostatic pressure with radioactive precursors of RNA, DNA and protein. Macromolecular synthesis was shown to be suppressed in a dose-dependent manner with increasing magnitude and duration of pressure. To determine the influence of pressure on histone mRNA stability, actinomycin D (10 micrograms ml-1) was used to block RNA synthesis. Relative amounts of H4 and H1 mRNA were determined at atmospheric pressure and following treatment with actinomycin D (10 micrograms ml-1), pressure (4.14 x 10(4) kPa) and a combination of pressure and actinomycin D. This study shows that a synthesis component and a stability component are involved in the pressure-induced reduction of core histone mRNA. At 4.14 x 10(4) kPa for 15 min, there was a 42% reduction in core histone mRNA of which approximately one third was due a suppression of transcription and two thirds to a loss of mRNA stability. The pressure-induced reduction in histone mRNA is attributed to the instability of endogenous histone mRNA and a reduction in transcription/processing of new histone mRNA.