Early G1 growth arrest of hybridoma B cells by DMSO involves cyclin D2 inhibition and p21[CIP1] induction

Ponzio, Gilles; Loubat, Agnès; Rochet, Nathalie; Turchi, Laurent; Rezzonico, Roger; Far, Dariush Farahi; Dulić, Vjekoslav; Rossi, Bernard

doi:10.1038/sj.onc.1202040

Cited by 36 publications

(27 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…For this purpose, cells were incubated for 24 h in di erent conditions of growth arrest (see Materials and methods), then harvested and their cell cycle was analysed by¯ow cytometry. As reported previously in other cell systems (Liu et al, 1994;Lucas et al, 1992;Ponzio et al, 1998;Rezzonico et al, 1995;Takase et al, 1992;Teroaka et al, 1996), 7TD1 cells were blocked in early G 1 phase by IL-6 depletion or treatment with DMSO and 8Br-cAMP, in late G 1 phase by aphidicolin and in G 2 /M phase by nocodazole treatment (Table 1). Cells treated in these conditions were then lysed and protein extracts analysed by Western blot using a polyclonal antibody raised against the amino acids 1 ± 90 of p27 (p27(N90)).…”

Section: Resultssupporting

confidence: 82%

“…The rabbit anti-p27 antibodies, p27(N90) directed against the amino acids 1 ± 90 of p27 was produced in our laboratory and immunopuri®ed as described previously (Ponzio et al, 1998). Rabbit polyclonal antibodies to CDK4 (C-22), CDK2 (M2), N-terminal (N20) or C-terminal (C19) part of p27 and the polyclonal goat anti-human PARP antibody, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).…”

Section: Antibodiesmentioning

confidence: 99%

“…Immunoblotting experiments were carried out as described previously (Ponzio et al, 1998). Puri®ed anti-p27 polyclonal antibodies (N90), (N20) and (C19) were used at the concentration of 1, 0.2 and 0.5 mg/ml respectively.…”

Section: Immunoblottingmentioning

confidence: 99%

“…Cellular extracts (0.5 ± 2 mg of protein) prepared as described previously (Ponzio et al, 1998) were incubated for 2 h at 48C with anti-CDK2 (3 mg) or anti-CDK4 (3 mg) antibodies and then with protein A-sepharose beads for 1 h. Immunocomplexes were collected and analysed by Western blotting as described above.…”

Section: Immunoprecipitationmentioning

confidence: 99%

See 3 more Smart Citations

Evidence for a p23 caspase-cleaved form of p27[KIP1] involved in G1 growth arrest

et al. 1999

Self Cite

View full text Add to dashboard Cite

p27 [KIP1] (p27) is a cyclin dependent kinase inhibitor, involved in the negative regulation of G 1 progression in response to a number of anti-proliferative signals. In this study we show, in growing mouse hybridoma (7TD1) and human myeloma (U266) cell lines, that p27 is highly expressed but slightly upregulated when cells are arrested, regardless to the phases of the cell cycle. In contrast, the speci®c blockade of these cells in early G 1 phase reveals the induction of a protein of 23 kDa (p23) speci®cally recognized by polyclonal anti-p27 antibodies raised against the NH2 terminal part of p27 but not by anti-p21 [CIP1] antibodies. Experiments using caspase inhibitors strongly suggest that p23 results from the proteolysis of p27 by a`caspase-3-like' protease. This cleavage leads to the cytosolic sequestration of p23 but does not alter its binding properties to CDK2 and CDK4 kinases. Indeed, p23 associated in vivo with high molecular weight complexes and coprecipitated with CDK2 and CDK4. We demonstrate by transfection experiments in SaOS-2 cells that p23 induces a G 1 phase growth arrest by inhibition of cyclin/CDK2 activity. In summary we describe here a caspase-cleaved form of p27, induced in absence of detectable apoptosis and likely involved in cell cycle regulation.

show abstract

Section: Resultssupporting

confidence: 82%

Section: Antibodiesmentioning

confidence: 99%

Section: Immunoblottingmentioning

confidence: 99%

Section: Immunoprecipitationmentioning

confidence: 99%

See 2 more Smart Citations

Evidence for a p23 caspase-cleaved form of p27[KIP1] involved in G1 growth arrest

et al. 1999

Self Cite

View full text Add to dashboard Cite

show abstract

“…Cells were then released into drug-free medium and harvested at 0 h (G 1 /S phase), 3 h (early S phase), 5 h (mid-S phase), or 7 h (late S phase) after removal of aphidicolin. To synchronize cells in G 0 /G 1 phase, Raji 525-7 cells were grown for 15 h in RPMI-1640 medium supplemented with 1% fetal calf serum (BRL-Life Technologies, Grand Island, NY, USA), 1% penicillin-streptomycin (BRL-Life Technologies, Grand Island, NY, USA) and 1.5% dimethylsulfoxide (DMSO; Sigma, St Louis, MO, USA) and harvested (Sawai et al, 1990;Ponzio et al, 1998). Replicate cultures of cells were trypsinized, fixed in ethanol, and stained with propidium iodide.…”

Section: Cell Culture and Synchronizationmentioning

confidence: 99%

Identification of novel pRb binding sites using CpG microarrays suggests that E2F recruits pRb to specific genomic sites during S phase

et al. 2003

View full text Add to dashboard Cite

The retinoblastoma (Rb) tumor suppressor protein is an important regulator of cell proliferation and differentiation. Many studies have shown that pRb can negatively regulate the activity of the E2F family of transcription factors during G 0 and G 1 phases of the cell cycle, perhaps by serving as a bridge between the E2Fs and transcriptional repressors such as histone deacetylases and methylases. However, pRb has also been shown to localize to discrete DNA foci during S phase, a time at which pRb is thought to be dissociated from E2F. Numerous other DNA binding proteins have been shown to interact with pRb, suggesting that pRb may control progression through S phase by binding to sites in the genome distinct from E2F target gene promoters. To test this hypothesis, we have identified novel pRb binding sites within the human genome using an unbiased approach which relies upon a combination of chromatin immunoprecipitation and CpG microarray analysis. To provide the greatest opportunity of finding distinct sets of pRb binding sites, we examined pRb binding in chromatin obtained from human Raji cells synchronized in either G 0 /G 1 phase or S phase. These experiments have allowed us to identify a large set of new genomic binding sites for the pRb protein. We found that some sites are occupied by pRb only during G 0 / G 1 phase, as would be predicted from previous models of pRb function. We also identified sites to which pRb bound only during S phase and other sites which were bound constitutively by pRb. Surprisingly, we found that E2F1 was present at most of the CpG islands bound by pRb, independent of the phase of the cell cycle. Thus, although pRb has the potential to interact with numerous transcription factors, our data suggest that the majority of DNA-bound pRb is recruited to E2F target promoters during both G 0 /G 1 and S phases.

show abstract

Cell Cycle Synchronization

Holmes

Al‐Rubeai

2000

Encyclopedia of Cell Technology

View full text Add to dashboard Cite

Early G1 growth arrest of hybridoma B cells by DMSO involves cyclin D2 inhibition and p21[CIP1] induction

Cited by 36 publications

References 41 publications

Evidence for a p23 caspase-cleaved form of p27[KIP1] involved in G1 growth arrest

Evidence for a p23 caspase-cleaved form of p27[KIP1] involved in G1 growth arrest

Identification of novel pRb binding sites using CpG microarrays suggests that E2F recruits pRb to specific genomic sites during S phase

Cell Cycle Synchronization

Contact Info

Product

Resources

About