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Cell density related gene expression: SV40 large T antigen levels in immortalized astrocyte lines.

Frisa PS, Jacobberger JW - BMC Cell Biol. (2002)

Bottom Line: When an Rb-binding mutant T antigen transcribed from the LTR was tested, levels decreased.When transcribed from the herpes thymidine kinase promoter, Tag levels decreased.The hypothetical terms, expression at zero cell density and expression at minimum G1 phase fraction, were introduced to simplify measures of expression potential.

View Article: PubMed Central - HTML - PubMed

Affiliation: Comprehensive Cancer Center, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 4106-4944, USA. jwj@po.cwru.edu

ABSTRACT

Background: Gene expression is affected by population density. Cell density is a potent negative regulator of cell cycle time during exponential growth. Here, we asked whether SV40 large T antigen (Tag) levels, driven by two different promoters, changed in a predictable and regular manner during exponential growth in clonal astrocyte cell lines, immortalized and dependent on Tag.

Results: Expression and cell cycle phase fractions were measured and correlated using flow cytometry. T antigen levels did not change or increased during exponential growth as a function of the G1 fraction and increasing cell density when Tag was transcribed from the Moloney Murine Leukemia virus (MoMuLV) long terminal repeat (LTR). When an Rb-binding mutant T antigen transcribed from the LTR was tested, levels decreased. When transcribed from the herpes thymidine kinase promoter, Tag levels decreased. The directions of change and the rates of change in Tag expression were unrelated to the average T antigen levels (i.e., the expression potential).

Conclusions: These data show that Tag expression potential in these lines varies depending on the vector and clonal variation, but that the observed level depends on cell density and cell cycle transit time. The hypothetical terms, expression at zero cell density and expression at minimum G1 phase fraction, were introduced to simplify measures of expression potential.

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Related in: MedlinePlus

Flow cytometry measurements of Tag and DNA. This is an example of the cytometric data collected for this study. P0-3D tkT#5 cells were immunofluorescently labeled with PAB 416 (anti-Tag) or IgG2a (isotype control) and FITC-conjugated goat anti-mouse secondary antibody. DNA was stained with propidium diiodide. Mean linear immunofluorescence of the G1 sub-population for the isotype control (Fb) was subtracted from that of the Tag-stained sample (Ft) to generate the measurement of Tag expression (Fs).
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Figure 4: Flow cytometry measurements of Tag and DNA. This is an example of the cytometric data collected for this study. P0-3D tkT#5 cells were immunofluorescently labeled with PAB 416 (anti-Tag) or IgG2a (isotype control) and FITC-conjugated goat anti-mouse secondary antibody. DNA was stained with propidium diiodide. Mean linear immunofluorescence of the G1 sub-population for the isotype control (Fb) was subtracted from that of the Tag-stained sample (Ft) to generate the measurement of Tag expression (Fs).

Mentions: A priori, one might expect that most cell cycle related genes will be less expressed in growth arrested, confluent, or G0 cells relative to proliferating cultures due to a generalized decrease in metabolic activity, cell protein content, and cell size. However, based on unpublished observations and previous work (see Introduction,) [11], we expected that the Tag expression-cell density profiles would look like that in Figure 3. To test this idea, we measured Tag expression by immunofluorescence flow cytometry (see Figure 4) in 6 Tag-immortalized mouse astrocyte lines plated at a range of cell densities and cultured for 2–3 days as previously described [15]. The upper densities ensured in most cases that the cells would be in either the "transition" or "plateau" phase for some of the measurements. Since Tag accumulates throughout the cell cycle, populations with different phase fractions will have different average levels, therefore, we measured Tag from the G1 population to generate measurements independent of the cell cycle phase fraction distribution as previously described [3].


Cell density related gene expression: SV40 large T antigen levels in immortalized astrocyte lines.

Frisa PS, Jacobberger JW - BMC Cell Biol. (2002)

Flow cytometry measurements of Tag and DNA. This is an example of the cytometric data collected for this study. P0-3D tkT#5 cells were immunofluorescently labeled with PAB 416 (anti-Tag) or IgG2a (isotype control) and FITC-conjugated goat anti-mouse secondary antibody. DNA was stained with propidium diiodide. Mean linear immunofluorescence of the G1 sub-population for the isotype control (Fb) was subtracted from that of the Tag-stained sample (Ft) to generate the measurement of Tag expression (Fs).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC111187&req=5

Figure 4: Flow cytometry measurements of Tag and DNA. This is an example of the cytometric data collected for this study. P0-3D tkT#5 cells were immunofluorescently labeled with PAB 416 (anti-Tag) or IgG2a (isotype control) and FITC-conjugated goat anti-mouse secondary antibody. DNA was stained with propidium diiodide. Mean linear immunofluorescence of the G1 sub-population for the isotype control (Fb) was subtracted from that of the Tag-stained sample (Ft) to generate the measurement of Tag expression (Fs).
Mentions: A priori, one might expect that most cell cycle related genes will be less expressed in growth arrested, confluent, or G0 cells relative to proliferating cultures due to a generalized decrease in metabolic activity, cell protein content, and cell size. However, based on unpublished observations and previous work (see Introduction,) [11], we expected that the Tag expression-cell density profiles would look like that in Figure 3. To test this idea, we measured Tag expression by immunofluorescence flow cytometry (see Figure 4) in 6 Tag-immortalized mouse astrocyte lines plated at a range of cell densities and cultured for 2–3 days as previously described [15]. The upper densities ensured in most cases that the cells would be in either the "transition" or "plateau" phase for some of the measurements. Since Tag accumulates throughout the cell cycle, populations with different phase fractions will have different average levels, therefore, we measured Tag from the G1 population to generate measurements independent of the cell cycle phase fraction distribution as previously described [3].

Bottom Line: When an Rb-binding mutant T antigen transcribed from the LTR was tested, levels decreased.When transcribed from the herpes thymidine kinase promoter, Tag levels decreased.The hypothetical terms, expression at zero cell density and expression at minimum G1 phase fraction, were introduced to simplify measures of expression potential.

View Article: PubMed Central - HTML - PubMed

Affiliation: Comprehensive Cancer Center, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 4106-4944, USA. jwj@po.cwru.edu

ABSTRACT

Background: Gene expression is affected by population density. Cell density is a potent negative regulator of cell cycle time during exponential growth. Here, we asked whether SV40 large T antigen (Tag) levels, driven by two different promoters, changed in a predictable and regular manner during exponential growth in clonal astrocyte cell lines, immortalized and dependent on Tag.

Results: Expression and cell cycle phase fractions were measured and correlated using flow cytometry. T antigen levels did not change or increased during exponential growth as a function of the G1 fraction and increasing cell density when Tag was transcribed from the Moloney Murine Leukemia virus (MoMuLV) long terminal repeat (LTR). When an Rb-binding mutant T antigen transcribed from the LTR was tested, levels decreased. When transcribed from the herpes thymidine kinase promoter, Tag levels decreased. The directions of change and the rates of change in Tag expression were unrelated to the average T antigen levels (i.e., the expression potential).

Conclusions: These data show that Tag expression potential in these lines varies depending on the vector and clonal variation, but that the observed level depends on cell density and cell cycle transit time. The hypothetical terms, expression at zero cell density and expression at minimum G1 phase fraction, were introduced to simplify measures of expression potential.

Show MeSH
Related in: MedlinePlus