Thenaa K. Said

Organic and inorganic selenium compounds inhibit mouse mammary cell growth in vitro by different cellular pathways.

Selenium, both organic and inorganic forms, inhibit mammary tumorigenesis in vivo and mammary cell growth in vitro. In the present study, sodium selenite was compared to methylselenocysteine (MSC) for their individual effects on cell growth, cdc2/cdk2 kinase activities and the levels of cyclins D1, E and A bound to cdk2 in a mouse mammary epithelial cell culture model. Selenite arrested the growth of cells in S-G2-M phase in contrast to MSC which arrested or delayed the cells in G1. In MSC-treated cells there was a 57% drop in the cdk2 kinase activity accompanied by a 73.5% decrease in cyclin E-cdk2 content as compared to the control cells. Selenite treatment increased the cdk2 kinase activity by 30% without any appreciable change in either of the cyclins D1, E or A bound to cdk2 when compared to the control cells. These data support the hypothesis that selenite and MSC have distinct modes of action in the inhibition of cell growth in vitro. Selenite has a strong genotoxic effect on the tumor cells; in contrast, MSC appears to inhibit cell growth via specific inhibition of cell cycle regulatory proteins.

Functional analysis of cyclin D2 and p27 Kip1 in cyclin D2 transgenic mouse mammary gland during development

Two mammary gland phenotypes were detected in pregnant MMTV-cyclin D2 transgenic mice; line D2–53 exhibited a lack of alveologenesis and failure to nurse, whereas line D2–58 featured a reduction in alveologenesis, but retained normal nursing behavior. In pregnant mammary glands, cyclin D2 protein levels were twofold (P<0.107) and 3.8-fold (P<0.0076) higher in line D2–58 and D2–53, respectively, compared to wild type. Concomitantly with the increase in cyclin D2 was a fivefold decrease in cyclin D1 hyper-phosphorylated isoform in mammary glands of pregnant cyclin D2–58 mice. Because cyclin D1 is a critical molecule in normal mammary lobuloalveolar development, these data suggest that overexpression of cyclin D2 may block mammary lobuloalveolar development through inhibition of cyclin D1 phosphorylation. During mammary gland development, p27kip1 protein level oscillated in a similar profile in wild type and cyclin D2 transgenic mice, but was consistently higher in the cyclin D2 mice suggesting that p27kip1 functions downstream of cyclin D2. The ratio of p27kip1-cdk4/p27kip1-cdk2 was 6.5-fold (P<0.0003) higher in cyclin D2 mammary glands compared to wild type in pregnant animals. This ratio reversed to 2.2-fold (P<0.005) higher in wild type compared to cyclin D2 mammary glands in involution suggesting that overexpression of cyclin D2 moderately induced apoptosis during pregnancy but accelerated involution. Collectively, the effects of cyclin D2 overexpression on mammary gland development during pregnancy and involution are attributed to two major factors, altered p27kip1 protein level and inhibition of cyclin D1 phosphorylation.

Mechanisms of suberoylanilide hydroxamic acid inhibition of mammary cell growth

The mechanism of suberoylanilide hydroxamic acid in cell growth inhibition involved induction of pRb-2/p130 interaction and nuclear translocation with E2F-4, followed by significant repression in E2F-1 and PCNA nuclear levels, which led to inhibition in DNA synthesis in mammary epithelial cell lines.SynopsisBackgroundHybrid polar compounds (HPCs) have induced cell growth arrest, terminal differentiation and/or apoptosis in various transformed cell lines. We have previously reported that the prototype HPC (hexamethylene bisacetamide [HMBA]) was able to arrest the growth of transformed mammary (TM) 2H cells (p53 null), a highly tumorigenic mouse mammary epithelial cell line, by inhibiting G1 kinase activities, concomitant with an increase in the cyclin D2 protein level and hypophosphorylated isoforms of the three pRb pocket proteins, which led to the formation of stable cyclin D2/pRb complexes and G1 cell arrest. It has been reported that the second generation of HPCs (suberoylanilide hydroxamic acid [SAHA]), structurally related to but 2000-fold more potent than HMBA, was an inhibitor of histone deacetylase activity and caused accumulation of hyperacetylated histone H4 in murine erythroleukemia.ObjectivesTo determine the mechanism of SAHA in cell growth inhibition in TM10 (p53 wt) and TM2H (p53 null) hyperplastic mouse mammary cell lines.MethodsTM10 and TM2H cells were examined in the presence or absence of 2.5 μM SAHA for cell growth rate by [3H]-thymidine uptake, DNA synthesis by flow cytometry after cells were labeled with BrdU, G1/S cyclin-dependent kinase (cdk) activities, phosphorylation levels of pRb pocket proteins, protein levels of E2F-1, PCNA and p21, pRb-2/p130 interaction, and nuclear localization with E2F-4 by western blot, immunoprecipitation and immunostaining assays.ResultsSAHA was able to arrest cell growth at G1, and inhibited DNA synthesis in both TM10 and TM2H cell lines. Cell growth arrest was accompanied by increases in histone H3 and H4 protein and acetylation levels, a profound increase in the interaction and nuclear localization of pRb-2/p130–E2F-4 complexes, significant reductions in E2F-1 and PCNA protein levels, inhibition in G1/S cdk activities and increases in the levels of hypophosphorylated isoforms of three pRb pocket proteins.ConclusionA novel mechanism of SAHA mediated growth inhibition through significant increases in the formation and nuclear localization of pRb-2/p130–E2F-4 complexes, which resulted in cell growth arrest and significant repression in the levels of two key molecules, E2F-1 and PCNA, essential for DNA synthesis in two mouse mammary epithelial cell lines. These responses to SAHA were independent of the p53 status of the cell; however, reversibility of SAHA-mediated growth correlated with the wild type p53 status.

Interaction of retinoblastoma protein and D cyclins during cell-growth inhibition by hexamethylenebisacetamide in TM2H mouse epithelial cells

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 G1; association of D‐type cyclins with cyclin‐dependent kinase (cdk) 4 and cdk6; G1 kinase activity; association of retinoblastoma protein (pRb) and phosphorylated pRb with D‐type cyclins; and association of p16INK4a, p15INK4b, and p27Kip1 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 G1 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 G1), 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 p27Kip1 and, at 9 h, a fourfold increase in cdk6‐associated p27Kip1 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 p27Kip1 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.

Cell Cycle Genes in a Mouse Mammary Hyperplasia Model

Human mammary epithelial cells emerge spontaneously from senescence, exhibiting eroding telomeric sequences, and ultimately enter crisis to generate the type of chromosomal abnormalities seen in early stages of breast cancer. In a mouse mammary tumor model, the spontaneous escape of senescence can be observed as an increase in DNA synthesis that is reflected by alterations in the cell cycle profile and increases in the expression levels and activities of cell cycle molecular components. This review provides an overview of gene alterations in the cell cycle components in mouse mammary hyperplasia.

Hormones, Growth Factors, and Gene Expression in Preneoplasias of the Mouse Mammary Gland

Breast cancer, in humans as in mice and rats, is thought to be the result of sequential changes in the epithelial cells of the mammary gland (1–3). In mice, the intermediate stages are well-defined and their biological properties extensively studied (2, 4). These intermediate stages are visualized as ductal or alveolar hyperplasias, both of which contain cells with an enhanced probability for tumor formation (2, 4, 5). These cell populations, termed preneoplasias, have a common set of biological properties. The essential properties are considered to be the following: 1) immortality, 2) morphological hyperplasia, and 3) enhanced tumorigenicity. The most frequently studied, the alveolar hyperplasias, can be induced by mouse mammary tumor virus (MMTV) (2), chemical carcinogens (2), hormones (2), or spontaneously, as a result of cell culture (6). Alveolar hyperplasias can be transplanted into the cleared fat pads of syngeneic mice, resulting in stable hyperplastic outgrowth lines (1,4). Regardless of the etiology of the alveolar hyperplastic outgrowth lines, the essential biological properties are similar for the different lines. The outgrowth lines are essentially clonal derivatives of the primary hyperplastic alveolar nodules, as was demonstrated by examination of MMTV integration sites (7). Although some of the critical molecular changes associated with MMTV-induced and chemical carcinogen-induced tumorigenesis have been defined in terms of wnt gene (8) and ras gene activation (9), respectively; these molecular events do not explain the biological properties associated with most mammary alveolar hyperplasias, particularly those induced in the absence of these oncogenic agents. Recently, we have examined a new series of transformed mouse mammary cells for which the biological properties of immortality, hyperplasia and tumorigenicity are independently assortable. The in-situ outgrowth lines and their in-vitro cell line counterparts provide a means to examine the molecular events associated with each of the essential changes acquired during the evolution to the tumor phenotype (6, 10, 11).