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Deregulation of DNA polymerase beta by sense and antisense RNA expression in mouse 3T3 cells alters cell growth
1Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Abstract:
DNA polymerase beta (beta-pol) and its mRNA are maintained at constitutive levels during the cell cycle and during stages of cell growth in culture. To study biological consequences of variations in the level of this DNA repair enzyme and/or its mRNA, we prepared expression vectors in which cDNA for human beta-pol is inserted under the control of a metallothionein promoter (pMT) in the sense and antisense orientation, respectively, and these vectors then were used for stable transformation of mouse 3T3 cells. Vectors also contained the mouse DHFR gene, such that culture of transformants in medium with increasing concentrations of methotrexate resulted in amplification of inserted DNA. The levels of sense and antisense transcripts are strongly increased by culture of transformants in medium with 65 microM Zn2+, although some expression is detected even without Zn2+ induction. After five days of induction, the beta-pol level was about threefold higher in sense cells and about 10-fold lower in antisense cells than in parallel cultures without induction. The antisense line has a threefold increased cell doubling time in the presence of 65 microM Zn2+ compared with the absence of Zn2+. Zn2+ (65 microM) induction for the sense line results in normal growth for the first three days and, thereafter, a complete cessation of growth. Yet, these blocked cells remain fully viable. The results indicate that sudden deregulation of beta-pol expression alters cell growth in mouse 3T3 cells.
Insights
Altering levels of DNA polymerase beta (beta-pol), a DNA repair enzyme, significantly impacts cell growth. Overexpression halts growth, while reduced levels slow it, demonstrating beta-pol
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- DNA polymerase beta (beta-pol) is a crucial DNA repair enzyme with constitutive expression levels.
- Understanding the biological impact of altered beta-pol levels is essential for cell cycle regulation research.
Purpose of the Study:
- To investigate the consequences of deregulating beta-pol expression on mouse 3T3 cell growth.
- To establish stable cell lines with modulated beta-pol mRNA and protein levels.
Main Methods:
- Human beta-pol cDNA was inserted into expression vectors (sense and antisense) under a metallothionein promoter.
- Stable mouse 3T3 cell transformants were generated, with DNA amplification facilitated by the DHFR gene and methotrexate selection.
- Beta-pol expression was induced using zinc sulfate (Zn2+).
Main Results:
- Zinc induction significantly increased sense and antisense transcripts, leading to threefold higher beta-pol in sense cells and tenfold lower in antisense cells.
- Antisense cells exhibited a threefold increase in doubling time upon zinc induction.
- Sense cells showed normal growth for three days post-induction, followed by a complete cessation of growth, though cells remained viable.
Conclusions:
- Sudden deregulation of beta-pol expression profoundly alters cell growth dynamics in mouse 3T3 cells.
- Beta-pol plays a critical role in maintaining normal cell proliferation and viability.