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Interaction of retinoblastoma protein and D cyclins during cell‐growth inhibition by hexamethylenebisacetamide in TM2H mouse epithelial cells
Author(s) -
Said Thenaa K.,
Medina Daniel
Publication year - 1998
Publication title -
molecular carcinogenesis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.254
H-Index - 97
eISSN - 1098-2744
pISSN - 0899-1987
DOI - 10.1002/(sici)1098-2744(199806)22:2<128::aid-mc8>3.0.co;2-i
Subject(s) - biology , cyclin dependent kinase 6 , retinoblastoma protein , cyclin d3 , cyclin dependent kinase , cell cycle , cyclin d1 , cyclin , microbiology and biotechnology , cyclin e , cyclin d , kinase , growth inhibition , cyclin b , cancer research , cell growth , cell , biochemistry
To explore the regulation and function of D‐type cyclins in breast cancer cells, the mouse mammary hyperplastic epithelial cell line TM2H was treated with 5 mM hexamethylenebisacetamide (HMBA), a polar differentiation factor. The resulting growth‐inhibitory effect of HMBA was completely reversible and was analyzed in terms of percent cells in G 1 ; association of D‐type cyclins with cyclin‐dependent kinase (cdk) 4 and cdk6; G 1 kinase activity; association of retinoblastoma protein (pRb) and phosphorylated pRb with D‐type cyclins; and association of p16 INK4a , p15 INK4b , and p27 Kip1 with cdk4 and cdk6. Synchronized TM2H cells were examined at 0, 3, 5, 9, 12, and 24 h after exposure to 5 mM HMBA. Inhibition of DNA synthesis, as measured by thymidine uptake, was first observed at 5 h (40%) and peaked at 24 h (80%). Flow cytometry at 9 h showed treated cells to be in G 1 arrest. Western blot analysis showed weakly detectable cyclin D1 but readily detectable cyclin D2 and D3 proteins at 0 h; thereafter, cyclin D2 and D3 protein levels remained higher while cyclin D1 levels declined significantly in treated versus untreated cells. By 5 h (early G 1 ), HMBA had markedly inhibited cdk4 and cdk6 kinase activity (67% and 75%, respectively) in treated versus untreated cells. By 9 and 12 h, pRb levels had increased 3.4‐fold in treated versus untreated cells. At 5 h, cyclin D–associated pRb was totally hypophosphorylated in treated cells and hyperphosphorylated in untreated cells. The levels of pRb associated with cyclin D2 and D3 increased 2.89‐fold and 4.6‐fold, respectively, in treated versus untreated cells. At 5 h, treated cells showed a fivefold increase in cdk4‐associated p27 Kip1 and, at 9 h, a fourfold increase in cdk6‐associated p27 Kip1 over control levels. In confirmation of these data, HMBA was found to inhibit the growth of Rb‐positive Du/145Rb cells but not their Rb‐negative parental Du/145 cells. The data suggest that HMBA‐induced growth inhibition is due to multifactorial mechanisms involving decreases in total cyclin D1 and inhibition of cdk4 and cdk6 kinase activities through elevation of levels of cdk4‐ and cdk6‐associated p27 Kip1 and concomitant increases in hypophosphorylated pRb and stable cyclin D2/pRb and cyclin D3/pRb complexes that help maintain pRb in a functional state. Mol. Carcinog. 22:128–143, 1998. © 1998 Wiley‐Liss, Inc.