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Query: UNIPROT:P06889 (Mol)
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Transforming growth factor beta (TGF-beta) is a member of a family of cytokines that regulate differentiation and proliferation in a wide variety of tissues including the pituitary gland. In both the normal pituitary and tumorous cell lines TGF-beta1 has anti-proliferative activity, however the intracellular mechanisms responsible have not been defined. In the pituitary derived cell line GH(3), p27(Kip1), a key regulator of G(1)/S transition is not expressed, suggesting that this protein is not an effector of the anti-proliferative response following TGF-beta1 treatment. Among other TGF-beta responsive cell cycle regulators p15(Ink4b) has been shown to have anti-proliferative effects associated with cell cycle arrest in other cell types. We therefore examined p15(Ink4b) expression in response to TGFbeta-1 to determine if this cyclin dependent kinase inhibitor was responsible for anti-proliferative activity in GH(3) cells. Treatment of GH(3) cells with TGF-beta1 (0.5-30 ng/ml) showed significant dose dependent growth inhibition (P<0.001) as assessed by viable cell counts. Maximum growth inhibition (66%) was observed following treatment with 2 ng/ml TGF-beta1. FACS analysis carried out in parallel with the growth studies showed treatment was associated with a decrease in the proportion of cells in S-phase (22-9%) and a significant increase in the G(1) fraction from 58 to 75% relative to controls (P<0.001). The absence of a sub G(1) fraction and reversibility of the G(1) arrest over three cycles showed that these changes were not due to either an apoptotic response or cytoxicity, respectively. Semi-quantitative RT-PCR and Western blot analysis showed no change in the expression level of cyclin dependent kinase 4 (CDK4), p16(Ink4a) or p21(Cip1). However, p15(Ink4b) mRNA and protein levels showed a 10 and 8 -fold induction, respectively. Increased levels of p15(Ink4b) were accompanied by a shift in the phosphorylation status of pRb toward its active hypophosphorylated form. Furthermore, studies of the kinetics of p15(Ink4b) induction showed that arrest of cells in G(1) is preceded by induction of p15(Ink4b) mRNA and protein. These investigations would suggest that p15(Ink4b) is a functional effector of TGF-beta1 mediated cell cycle arrest in GH(3) cells. However, our present studies cannot determine if it is the sole mediator. Identification of intracellular target(s) that mediate responses to anti-proliferative signals will increase our understanding of these pathways and aberrations responsible for their dysfunction in tumorigenesis.
Mol Cell Endocrinol 2001 May 15
PMID:Decreased proliferation and cell cycle arrest in neoplastic rat pituitary cells is associated with transforming growth factor-beta1-induced expression of p15/INK4B. 1136 40

Tumor suppressors function as antiproliferative signaling proteins, and defects in these genes lead to uncontrolled cell proliferation and cancer. For example, absence of the tumor suppressor p27(Kip1), a cyclin-dependent kinase inhibitor (CKI), results in increased body size, hyperplasia of several organs including the testes, and cancer in mice. Similarly, lack of inhibins, alpha/beta heterodimeric members of the transforming growth factor-beta (TGFbeta) superfamily, causes testicular and ovarian tumors of the granulosa/Sertoli cell lineage beginning at 4 weeks of age and adrenal tumors in gonadectomized mice. Neither the cell cycle alterations in the absence of inhibin nor the cause of the increased testis size in the p27 knockout mice is known. To study the molecular (cell cycle) changes that result from absence of inhibins, we analyzed the regulation of cell cycle proteins in gonadal tumors derived from inhibin alpha knockout mice (Inha(-/-)). Northern blot analyses demonstrate that cyclin-dependent kinase 4 (Cdk4) and cyclin D2 mRNA levels are elevated, and immunohistochemistry shows that p27 protein levels are decreased in both ovarian and testicular tumors from Inha(-/-) mice. These findings suggest that increased Cdk4/cyclin D2 (positive) activity and decreased p27 (negative) activity is causal for gonadal tumor formation. To test this hypothesis, we generated double mutant mice lacking both p27 and inhibin alpha to determine whether the tumor suppressors p27 and inhibin have additive suppressor activity in the gonads. Like Inha(-/-) mice, p27(-/-)Inha(-/-) mice demonstrate elevated serum activin levels, ovarian and testicular tumors, and a resultant lethal cachexia-like syndrome. However, whereas 95% of the Inha(-/-) female mice die by 18 weeks of age, 100% of the p27(-/-)Inha(-/-) female mice are dead by 8 weeks. Similarly, 95% of the Inha(-/-) single mutant males die by 13 weeks while 100% of the p27(-/-)Inha(-/-) male mice die by 10 weeks. Moreover, tumor foci in p27(-/-)Inha(-/-) mice can be observed as early as 2 weeks of age in males and as early as 4 weeks in females. These findings demonstrate that absence of both inhibin and p27 in mice causes earlier development of ovarian and testicular tumors and earlier death compared with absence of inhibin alone.
Mol Endocrinol 2001 Jun
PMID:Inhibin and p27 interact to regulate gonadal tumorigenesis. 1137 16

In MCF-7 breast cancer cells, the insulin-like growth factor 1 receptor (IGF-1R) and the oestrogen receptor (ER) are coexpressed and the two signalling systems are engaged in a crosstalk that results in synergistic growth. However, coupling between the signalling cascades is poorly understood. Oestradiol enhances IGF-1R signalling by inducing the expression of insulin receptor substrate 1 (IRS-1), a substrate of the IGF-1R. Oestradiol induced expression of IRS-1 results in enhanced tyrosine phosphorylation of IRS-1 after IGF-1 stimulation, followed by enhanced mitogen activated protein kinase, phosphoinositide 3' kinase, and Akt activation. Oestradiol can also potentiate the effect of IGF-1 on the expression of cyclin D1 and cyclin E, and on the phosphorylation of the retinoblastoma protein (RB). These effects are greatly diminished in SX13 cells, which exhibit a 50% reduction in IGF-1R expression but few functional IGF-1Rs at the surface. Oestradiol and IGF-1 regulate the expression of two cyclin dependent kinase inhibitors, p21 and p27, differently. Whereas IGF-1 increases p21 expression and reduces p27 expression, oestradiol has no effect on p21. In summary, in MCF-7 cells, oestrogen potentiates the effect of IGF-1 on IGF-1R signalling and its effects on certain cell cycle components.
Mol Pathol 2001 Jun
PMID:Insulin-like growth factor 1 and oestradiol promote cell proliferation of MCF-7 breast cancer cells: new insights into their synergistic effects. 1137 26

Bop1 is a novel nucleolar protein involved in rRNA processing and ribosome assembly. We have previously shown that expression of Bop1Delta, an amino-terminally truncated Bop1 that acts as a dominant negative mutant in mouse cells, results in inhibition of 28S and 5.8S rRNA formation and deficiency of newly synthesized 60S ribosomal subunits (Z. Strezoska, D. G. Pestov, and L. F. Lau, Mol. Cell. Biol. 20:5516-5528, 2000). Perturbation of Bop1 activities by Bop1Delta also induces a powerful yet reversible cell cycle arrest in 3T3 fibroblasts. In the present study, we show that asynchronously growing cells are arrested by Bop1Delta in a highly concerted fashion in the G(1) phase. Kinase activities of the G(1)-specific Cdk2 and Cdk4 complexes were downregulated in cells expressing Bop1Delta, whereas levels of the Cdk inhibitors p21 and p27 were concomitantly increased. The cells also displayed lack of hyperphosphorylation of retinoblastoma protein (pRb) and decreased expression of cyclin A, indicating their inability to progress through the restriction point. Inactivation of functional p53 abrogated this Bop1Delta-induced cell cycle arrest but did not restore normal rRNA processing. These findings show that deficiencies in ribosome synthesis can be uncoupled from cell cycle arrest and reveal a new role for the p53 pathway as a mediator of the signaling link between ribosome biogenesis and the cell cycle. We propose that aberrant rRNA processing and/or ribosome biogenesis may cause "nucleolar stress," leading to cell cycle arrest in a p53-dependent manner.
Mol Cell Biol 2001 Jul
PMID:Evidence of p53-dependent cross-talk between ribosome biogenesis and the cell cycle: effects of nucleolar protein Bop1 on G(1)/S transition. 1139 Jun 53

p27 is a key regulator of cell proliferation through inhibition of G(1) cyclin-dependent kinase (CDK) activity. Translation of the p27 mRNA is an important control mechanism for determining cellular levels of the inhibitor. Nearly all eukaryotic mRNAs are translated through a mechanism involving recognition of the 5' cap by eukaryotic initiation factor 4E (eIF4E). In quiescent cells eIF4E activity is repressed, leading to a global decline in translation rates. In contrast, p27 translation is highest during quiescence, suggesting that it escapes the general repression of translational initiation. We show that the 5' untranslated region (5'-UTR) of the p27 mRNA mediates cap-independent translation. This activity is unaffected by conditions in which eIF4E is inhibited. In D6P2T cells, elevated cyclic AMP levels cause a rapid withdrawal from the cell cycle that is correlated with a striking increase in p27. Under these same conditions, cap-independent translation from the p27 5'-UTR is enhanced. These results indicate that regulation of internal initiation of translation is an important determinant of p27 protein levels.
Mol Cell Biol 2001 Aug
PMID:Control of cyclin-dependent kinase inhibitor p27 expression by cap-independent translation. 1143 53

Dynactin is a multisubunit complex that regulates the activities of cytoplasmic dynein, a microtubule-associated motor. Actin-related protein 1 (Arp1) is the most abundant subunit of dynactin, and it forms a short filament to which additional subunits associate. An Arp1 filament pointed-end--binding subcomplex has been identified that consists of p62, p25, p27, and Arp11 subunits. The functional roles of these subunits have not been determined. Recently, we reported the cloning of an apparent homologue of mammalian Arp11 from the filamentous fungus Neurospora crassa. Here, we report that N. crassa ro-2 and ro-12 genes encode the respective p62 and p25 subunits of the pointed-end complex. Characterization of Delta ro-2, Delta ro-7, and Delta ro-12 mutants reveals that each has a distinct phenotype. All three mutants have reduced in vivo vesicle trafficking and have defects in vacuole distribution. We showed previously that in vivo dynactin function is required for high-level dynein ATPase activity, and we find that all three mutants have low dynein ATPase activity. Surprisingly, Delta ro-12 differs from Delta ro-2 and Delta ro-7 and other previously characterized dynein/dynactin mutants in that it has normal nuclear distribution. Each of the mutants shows a distinct dynein/dynactin localization pattern. All three mutants also show stronger dynein/dynactin-membrane interaction relative to wild type, suggesting that the Arp1 pointed-end complex may regulate interaction of dynactin with membranous cargoes.
Mol Biol Cell 2001 Jul
PMID:Null mutants of the neurospora actin-related protein 1 pointed-end complex show distinct phenotypes. 1145 13

Previous studies have demonstrated that angiotensin II (ANG II) mediates cell cycle arrest of cultured renal tubular cells by induction of p27(Kip1), an inhibitor of cyclin-dependent kinases. However, it is not known whether ANG II exerts similar effects in vivo. Infusion of ANG II into naive rats for 7 days increased formation of reactive oxygen species in tubular cells of the kidney. Furthermore, ANG II infusion stimulated protein expression of p27(Kip1) as detected by western blotting of tubular lysates and immunohistochemistry. Infusion of ANG II reduced tubular proliferation as detected by proliferating-cell nuclear antigen (PCNA) immunohistochemistry. The increase in p27(Kip1) expression was not due to an increase in mRNA. Immunoprecipitation experiments revealed that the increased p27(Kip1) protein associates with cyclin-dependent kinase 2. Coadministration of the radical scavenger dimethylthiourea abolished this ANG II mediated p27(Kip1) expression without reducing systemic blood pressure. Furthermore, dimethylthiourea infusion attenuates the ANG II mediated G(1)-phase arrest of tubular cells. However, infusion of norepinephrine did not induce reactive oxygen species or p27(Kip1) expression, despite a significant increase in blood pressure. Thus ANG II induces p27(Kip1) expression in renal tubular cells in vivo. This effect is mediated by reactive oxygen species. Since tubular hypertrophy depends on G(1)-phase arrest and may promote subsequent development of interstitial fibrosis, administering oxygen radical scavenger may be a therapeutic tool to counteract ANG II dependent remodeling of renal tubular cells.
J Mol Med (Berl) 2001 Jul
PMID:Angiotensin II induces p27(Kip1) expression in renal tubules in vivo: role of reactive oxygen species. 1146 54

Exposure of hematopoietic cells to DNA-damaging agents induces p53-independent cell cycle arrest at a G(1) checkpoint. Previously, we have shown that this growth arrest can be overridden by cytokine growth factors, such as erythropoietin or interleukin-3, through activation of a phosphatidylinositol 3-kinase (PI 3-kinase)/Akt-dependent signaling pathway. Here, we show that gamma-irradiated murine myeloid 32D cells arrest in G(1) with active cyclin D-cyclin-dependent kinase 4 (Cdk4) but with inactive cyclin E-Cdk2 kinases. The arrest was associated with elevated levels of the Cdk inhibitors p21(Cip1) and p27(Kip1), yet neither was associated with Cdk2. Instead, irradiation-induced inhibition of cyclin E-Cdk2 correlated with absence of the activating threonine-160 phosphorylation on Cdk2. Cytokine treatment of irradiated cells induced Cdk2 phosphorylation and activation, and cells entered into S phase despite sustained high-level expression of p21 and p27. Notably, the PI 3-kinase inhibitor, LY294002, completely blocked cytokine-induced Cdk2 activation and cell growth in irradiated 32D cells but not in nonirradiated cells. Together, these findings demonstrate a novel mechanism underlying the DNA damage-induced G(1) arrest of hematopoietic cells, that is, inhibition of Cdk2 phosphorylation and activation. These observations link PI 3-kinase signaling pathways with the regulation of Cdk2 activity.
Mol Cell Biol 2001 Sep
PMID:Dna damage-induced G(1) arrest in hematopoietic cells is overridden following phosphatidylinositol 3-kinase-dependent activation of cyclin-dependent kinase 2. 1150 54

Cyclins, cyclin-dependent kinases (CDKs) and the CDK inhibitor p27(kip1) are known to be involved in the regulation of G(1)/S phase transition by estrogen in the rodent endometrium. Little is known, however, of the cell-specific location and regulation of these proteins during this process, or the way they mediate the differential effect of estrogen in the epithelium and stroma of the endometrium. Here we studied the cell-specific regulation of D-type cyclin (D(1-3)), of cyclin A and E, of CDK(2) and p27(kip1) by 17beta-estradiol in the endometrium of ovariectomized rats. Time-course changes in these proteins in the endometrium of ovariectomized rats were examined by immunohistochemistry at 2, 4, 8, 12, 20, 28 and 32 h after estrogen stimulation. The expression of proliferation cell nuclear antigen (PCNA) was also studied as a marker of proliferating cells. As expected from previous studies, all the proteins investigated were up-regulated by estrogen, with peak times from 8 to 32 h. The induction of cyclin D(1) is predominant in the glandular epithelium, whereas cyclin D(3) increases mainly in the luminal epithelium. The up-regulation of p27(kip1) is restricted to stromal cells with a 'gradient-like' expression pattern, in which the sub-epithelial (functional) layer showed stronger staining than the basal layer. The differential regulation of cyclins and p27(kip1) in the epithelium and stroma of the endometrium appear indicative of distinct actions of estrogen in different cell types in the uterus, as D-type cyclins mediate the proliferative effect of estrogen in epithelial cells while p27(kip1) might help prevent the same effect in the stroma.
J Steroid Biochem Mol Biol 2001 Aug
PMID:Cell type-specific induction of cyclin D and cyclin-dependent kinase inhibitor p27(kip1) expression by estrogen in rat endometrium. 1156 44

N-nitrosomethylbenzylamine (NMBA)-induced rat esophageal tumorigenesis is an important model for squamous cell carcinoma of the human esophagus. In this model, previous studies have shown that the GGA-->GAA Ha-ras codon 12 mutation is present in the majority of papillomas. No other Ha-ras mutation has been identified. Studies using other models of chemical carcinogenesis suggest that Ha-ras activation has a critical role during tumor initiation. We have used laser-capture microdissection and polymerase chain reaction-restriction fragment length polymorphism analysis to study the role of codon 12 Ha-ras mutation at various stages of tumor development in the rat esophagus. Our results indicate that Ha-ras mutation was present infrequently (4.3%) in premalignant lesions. The incidence of Ha-ras mutation was high in papillomas (57.1%), however, and 50% of papillomas expressed mutant Ha-ras RNA message. Additionally, there was a linear trend correlating increased incidence of Ha-ras mutation with later papilloma stage. These data suggest the role of ras activation later in neoplastic development. To evaluate the potential mechanism of action by which Ha-ras contributes to promotion and progression in this model, we compared mRNA expression of cyclin D1 and p27 in Ha-ras mutant and Ha-ras normal papillomas. We found no differences in mRNA expression of either cyclin D1 or p27 between these two papilloma populations. Our data suggest an important paradigm shift for the role of ras mutations in this model of chemical carcinogenesis, indicating a functional role of Ha-ras activation in promotion/progression and not in the initiation phase of NMBA-induced papillomagenesis.
Mol Carcinog 2001 Sep
PMID:Incidence and effects of Ha-ras codon 12 G-->A transition mutations in preneoplastic lesions induced by N-nitrosomethylbenzylamine in the rat esophagus. 1156 70


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