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Independent regulation of human D-type cyclin gene expression during G1 phase in primary human T lymphocytes
F Ajchenbaum1, K Ando, J A DeCaprio
1Division of Tumor Immunology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115.
Abstract:
Cyclins and cyclin-dependent kinases are critically involved in controlling cell cycle progression in virtually all cells. The recent identification of candidate G1 cyclins in mammalian cells has been a major advance in this field, but the exact functions of these cyclins are unknown. The expression of three D-type cyclins (D1, D2, and D3) was investigated in primary human T lymphocytes as these cells were induced to leave G0, traverse G1, and enter S phase by T cell-specific mitogens. G0 phase T cells expressed low levels of cyclin D2, but not cyclin D3. Treatment of these cells with phytohemagglutinin and 12-O-tetradecanoylphorbol-13-acetate in the presence of fetal calf serum resulted in rapid induction of cyclin D2 RNA in early G1 and slower induction of cyclin D3 in late G1. Cyclin D1 was not detected in T cells under any condition tested. Treatment of T cells with hydroxyurea to arrest cells at G1/S did not block induction of either D2 or D3. However, arrest of cells in mid G1 with deferoxamine blocked D3 expression without affecting D2. Cyclosporin A blocked the induction of both cyclin D2 and D3. Polyclonal antisera were prepared in rabbits against both cyclin D2 and cyclin D3 glutathione S-transferase fusion proteins and used to examine cyclin D2 and D3 proteins in [35S]methionine-labeled T cells. Protein levels were found to correlate closely with RNA levels for both cyclins. No detectable histone H1 kinase activity could be precipitated with either cyclin. However, several cellular proteins were observed to coprecipitate with the cyclins, including several proteins that were observed to associate only with D3. These results indicate that striking differences exist in the induction and regulation of two candidate G1 cyclins in human T cells and suggest that these cyclins could participate in multiple cell cycle checkpoints during G0, G1, or S phase.
Insights
This study reveals distinct regulation of cyclin D2 and D3 in human T cells during cell cycle progression. These findings highlight differences in G1 cyclin induction, impacting cell cycle checkpoints.
Area of Science:
- Cell Biology
- Immunology
- Molecular Biology
Background:
- Cyclins and cyclin-dependent kinases (CDKs) are crucial regulators of the cell cycle.
- Understanding G1 cyclins in mammalian cells is key to cell cycle control.
- D-type cyclins (D1, D2, D3) are candidate G1 cyclins with largely unknown functions.
Purpose of the Study:
- Investigate the expression and regulation of D-type cyclins (D1, D2, D3) in primary human T lymphocytes.
- Determine how T cell activation by mitogens affects cyclin D expression during cell cycle entry from G0.
- Explore the differential regulation of cyclin D2 and D3 in response to cell cycle arrest and specific inhibitors.
Main Methods:
- Primary human T lymphocytes were stimulated with mitogens (phytohemagglutinin, PMA, fetal calf serum) to induce cell cycle progression.
- Cells were arrested at different phases of G1 using hydroxyurea and deferoxamine.
- Cyclin D1, D2, and D3 RNA and protein levels were analyzed using molecular biology techniques, including Northern blotting and Western blotting (using [35S]methionine labeling).
- Cyclin-associated kinase activity and protein interactions were assessed using immunoprecipitation and kinase assays.
Main Results:
- Cyclin D2 RNA was rapidly induced in early G1, while cyclin D3 RNA was induced later in G1 following T cell activation. Cyclin D1 was not detected.
- Hydroxyurea arrest at G1/S did not affect D2 or D3 induction, but deferoxamine arrest in mid-G1 blocked D3 expression, not D2.
- Cyclosporin A inhibited the induction of both cyclin D2 and D3.
- Protein levels correlated with RNA levels; no histone H1 kinase activity was detected, but both cyclins associated with other cellular proteins, with some unique to D3.
Conclusions:
- Significant differences exist in the induction and regulation of cyclin D2 and D3 in human T cells.
- These distinct regulatory patterns suggest roles for cyclin D2 and D3 in multiple cell cycle checkpoints during G0, G1, and S phases.
- The findings provide insights into the complex control of G1 progression in T lymphocytes.