Low Cyclin Research Articles

Aberrant expression of p27(Kip1) is associated with malignant transformation of the rat urinary bladder epithelium.

Alteration in cell cycle regulators is considered to play an important role in carcinogenesis. In order to cast light on changes in reversible hyperplastic and irreversible tumorigenic lesions in the rat urinary bladder, expression of p27(Kip1), cyclin D1 and cyclin E proteins was sequentially compared. In the first study, 3% uracil was fed for 4 weeks to cause urinary calculi and consequent hyperplasia and papillomatosis, both regressing after withdrawal of the insult. Compared with normal bladder epithelium, in papillomatosis at week 4, the BrdU index and immunohistochemical positivities for cyclin D1 and cyclin E were significantly elevated, whereas values for p27(Kip1) tended to be reduced. One week after withdrawal of uracil, the BrdU index and positivities for cyclin D1 and cyclin E were decreased to below the control levels, while positivity for p27(Kip1) was dramatically increased, with a strong staining intensity. In a second study, rats were initiated with a bladder carcinogen, N-butyl-N-(4-hydroxybutyl)nitrosamine for 4 weeks, then fed 3% uracil for 8 weeks. During this latter period, expression of cyclin D1, cyclin E and p27(Kip1) in hyperplastic urothelium were comparable with those in the first study. One week after withdrawal of uracil, most urothelial lesions regressed, showing high p27(Kip1) and low cyclin D1 and cyclin E staining. Two weeks after uracil withdrawal, transitional cell carcinomas, with a low p27(Kip1) and high cyclin D1 and cyclin E staining pattern, could be easily distinguished from surrounding regressing epithelium. These data indicate that during regression of papillomatosis after cessation of a proliferative stimulus, expression of p27(Kip1)is elevated, accompanied by a lowering of cyclin D1 and cyclin E. In irreversible tumorous bladder lesions, on the other hand, persistent low expression of p27(Kip1) and elevated cyclin D1 and cyclin E are characteristic.

P27kip1 expression in breast carcinomas: An immunohistochemical study on 512 patients with long-term follow-up

p27(Kip1) (p27), a cyclin-dependent kinase inhibitor, has an important role in the progression of cells from G(1) into S phase of the cell cycle. p27 may act as a tumor suppressor, and several reports suggest that loss of its expression in breast carcinoma is related to tumor progression and poor prognosis. We evaluated p27 immunohistochemical expression in 512 consecutive cases of breast carcinoma with 9 years of median-term follow-up. p27 expression was heterogeneous and frequently less intense than in normal cells. Low p27 expression (<50% of reacting cells) was associated with grade III tumors, N0 status, estrogen receptor-negative status, and low cyclin D1 expression. In the whole series of cases, p27 expression did not predict outcome. In node-negative cases (249 patients), high p27 expression indicated poor prognosis. p27 was not prognostically relevant in the group of 223 patients with pT1 disease or in the group of 154 patients <50 years of age. We also investigated the prognostic value of the combined expression of p27 and cyclin D1, but no differences in survival were seen in this bivariate analysis.

Small hyperechoic nodules in chronic liver diseases include hepatocellular carcinomas with low cyclin D1 and Ki-67 expression.

In spite of the importance of periodic screening for hepatocellular carcinoma (HCC) by ultrasonography (US) in patients with underlying liver disease, the clinicopathological characteristics of hyperechoic nodules have not been clearly evaluated. The aim of this study was to characterize the pathological and proliferating features of small hyperechoic nodules. Tissue specimens of 55 hyperechoic and 107 hypoechoic nodules less than 20 mm in diameter in patients with chronic liver disease were obtained by echo-guided needle biopsy and examined histopathologically. Of these, 42 (76%) hyperechoic and 56 (52%) hypoechoic nodules were diagnosed as HCC, and 82% of hyperechoic HCCs contained fatty change and/or clear cell change. In addition, immunohistochemical staining using cyclin D1, p53, and Ki-67 was examined. A high-level expression of cyclin D1 was found in only 5% of hyperechoic HCCs, in contrast to 38% of hypoechoic HCCs (P <.02). The labeling index of Ki-67 in hyperechoic HCCs was lower than in hypoechoic HCCs (4.2% vs. 8.9%; P <.003). However, there was no difference on p53 staining between them. Retrospective follow-up study revealed that hyperechoic nodules showed slow growth (doubling time, median: 1,403 days) initially, and came to show rapid growth (doubling time, median: 56 days). From these results, small hyperechoic nodules in chronic liver diseases are worth notice as candidates for well-differentiated HCC with low cyclin D1 and Ki-67 expression.

Expression and Activity of Cell Cycle-Regulatory Proteins in Normal and Transformed Corneal Endothelial Cells

Corneal endothelial cells have a limited capacity for proliferation. Upon transformation with the SV40 large T antigen, however, these cells undergo division and grow rapidly. In order to gain insight into the control mechanisms that determine this proliferative switch, we investigated the expression level and activity of various known cell cycle-regulatory proteins in these cells. Primary human and rabbit corneal endothelial cells were transduced in vitro with a replication-defective adenovirus containing SV40 large T antigen, and subsequently the expression and activity of cell cycle-regulatory proteins was analyzed. Cells transduced with large T antigen exhibited strongly increased activity of cyclin-dependent kinases. This increase correlated with the elevated expression of various cyclin-dependent kinase subunits, such as cyclin A, and to a lesser extent, cyclin D, cdk2, and cdk4. Furthermore, the expression of two cyclin-dependent kinase inhibitors, p21(WAF1) and p27(KIP1), which was high in primary human cells (but not in primary rabbit cells), was strongly reduced in large T-antigen transduced cells. Thus, the remarkably low proliferative activity of normal human corneal endothelial cells appears to be regulated at two levels: the expression of certain cell cycle-regulatory proteins that are essential for cell cycle progression is extremely low (cyclin A) or somewhat low (cdk2 and cdk4); but the amount of p21 and p27, inhibitors of cell cycle progression, is very high. As a consequence, the enzymatic activity of cyclin-dependent kinase is below detectable levels. However, the growth-inhibitory status of these components is clearly reversible: upon transduction with large T antigen, the expression of cyclin A, cyclin D, cdk2, and cdk4 is induced, whereas the expression of p21 and p27 is inhibited, and the cells proliferate. Thus, our study provides insight into the molecular basis of the attenuated proliferation of corneal endothelial cells and suggests potential targets that could be manipulated for the purpose of therapeutic interventions aimed at renewed cell growth.

The Concurrent Expression of p27kip1and Cyclin D1 in Epithelial Ovarian Tumors

Mammalian cell-cycle progression is regulated by the combined action of cyclins/cyclin-dependent kinases (cdks) and cdk inhibitors. Abnormal expression as well as interaction of these proteins may result in malignant transformation of cells. To further address alterations and roles of these cell-cycle proteins in the development of epithelial ovarian carcinomas, we analyzed the expression of the p27kip1, cyclin D1, cyclin E, and cdk2. A panel of 79 epithelial ovarian tumors was selected. Immunohistochemical staining of serial paraffin sections was performed using antibodies to p27kip1, cyclin D1, cyclin E, and cdk2. The results showed that p27kip1and cyclin D1 were concurrently expressed in epithelial ovarian tumors, and the expression was down-regulated in ovarian carcinomas. There was an inverse relationship between the expression level of p27kip1and cyclin D1 and the histological tumor grades. On the other hand, the expression of cyclin E and cdk2 was enhanced in ovarian carcinomas. The results suggest that low expression of p27kip1and cyclin D1 as well as high expression of cyclin E and cdk2 promotes the development of ovarian tumors. p27kip1and cyclin D1 expression are negatively correlated with the malignant degree of epithelial ovarian tumors. Thus, the ovarian tumors with high p27kip1and cyclin D1 expression may generally have a somewhat better prognosis, while those with low p27kip1and cyclin D1 expression may have a worse prognosis.

Cyclin E dependent kinase activity in human breast cancer in relation to cyclin E, p27 and p21 expression and retinoblastoma protein phosphorylation.

The cell cycle machinery is regulated by cyclin dependent kinases and sets of activating and inhibitory proteins. The G1-S control mechanism is often deregulated in tumours supposedly leading to increased kinase activity, phosphorylation of substrates and subsequent S phase entrance. Increased kinase activity has been proposed to be essential in cell cycle aberrations, but few studies have actually shown enhanced kinase activity related to specific cell cycle defects in primary tumours. In the present study we have determined the cyclin E dependent kinase activity (cyclin E(kinase)) in 59 primary breast cancers, using an H1-kinase assay, and related the activity to the expression of cyclin E, p27 and p21. In a subgroup of 48 tumours, we further characterized the association between cyclin E(kinase), in vivo phosphorylation of the retinoblastoma protein (pRb) and proliferation. The cyclin E(kinase) correlated significantly with cyclin E content and inversely with p27 and p21 expression. P27, but not p21, was associated with low cyclin E(kinase) in specimens with normal/low levels of cyclin E. At elevated cyclin E levels, suppression of cyclin E(kinase) seemed to require high levels of both p21 and p27. The cyclin E(kinase) correlated with the phosphorylation status of pRb as well as with proliferation. Surprisingly, pRb phosphorylation did not correlate with proliferation. Our results support that pRb is a substrate for cyclin E(kinase) in primary breast cancer and that deregulation of cyclin E and p27 act through increased CDK-kinase activity, but cyclin E associated events beside pRb phosphorylation might be rate-limiting for entrance into S phase.

Hypoxia-induced pRB hypophosphorylation results from downregulation of CDK and upregulation of PP1 activities.

Exposure of CV-1P cells to hypoxic conditions results in reversible cell cycle arrest concomitant with accumulation of pRB in the hypophosphorylated, growth suppressive form. Similar to cell cycle arrest induced by serum starvation, we show here that hypoxia-induced arrest is accompanied by a decrease in pRB-directed CDK4 and CDK2 activities, lower cyclin D and E protein levels, and by an increase in p27 protein abundance. Immunoprecipitation studies reveal an increase in p27 association with cyclin E-CDK2 complexes. In contrast to cell cycle arrest induced by serum starvation, hypoxia increases PP1-mediated pRB dephosphorylation. These data reveal that synergy between decreased pRB-directed cyclin/CDK activity and increased pRB-directed phosphatase activity contribute towards inducing and maintaining pRB in its hypophosphorylated, growth suppressive state during hypoxia.

Expression of Cyclin E and the Cyclin-Dependent Kinase Inhibitor p27 in Malignant Lymphomas—Prognostic Implications

Cyclin E and the cyclin-dependent kinase inhibitor p27 are two important regulators of the G1-S transition modulating the activity of cyclin-dependent kinases. Aberrations in the cell cycle control are often observed in tumors and might even be mandatory in tumor development. To investigate the importance of cell-cycle defects in malignant lymphomas we have characterized the expression of cyclin E and p27 in 105 newly diagnosed lymphomas using immunohistochemistry. A significant, inverse correlation between p27 and cyclin E expression was observed (rs = −.24, P = .02) and both proteins correlated with the S-phase fraction (rs = −.35, P &lt; .001 andrs = .45, P &lt; .001, respectively). The inverse relationship between p27 expression and proliferation was abrogated in some lymphomas, suggesting that p27 downregulation can represent a genuine aberration. Survival analysis was performed in 105 patients with a median observation time of 86 months. Low p27 and high cyclin E expression were significantly associated with a poor prognosis (P = .0001 and .03, respectively). In a multivariate Cox analysis, p27 expression, stage, serum lactate dehydrogenase level, grade, and age were independent prognostic factors, in contrast to S-phase fraction and cyclin E expression. This is the first report showing that p27 expression in malignant lymphomas has independent prognostic significance, which necessitates future studies regarding its more precise biological role in lymphoid tumorogenesis. © 1998 by The American Society of Hematology.

Increased Cyclin D1 in Human Pancreatic Cancer Is Associated with Decreased Postoperative Survival

Cyclin D1 belongs to a family of protein kinases that have been implicated in cell cycle regulation. In the present study we characterized cyclin D1 expression in 6 cultured human pancreatic cancer cell lines and in normal and cancerous human pancreatic tissues. A 4.4-kb cyclin D1 mRNA transcript was present in all cell lines and in all pancreatic tissues. Cyclin D1 mRNA levels were 2.1-fold higher in the pancreatic cancers than in normal pancreatic tissues (p < 0.0002). Cancer patients with lower cyclin D1 levels (n = 16) had a median survival of 15.5 months whereas patients with higher levels (n = 16) had a median survival of 6.5 months (p < 0.007). These data indicate that cyclin D1 expression may serve as a predictor of postoperative survival in pancreatic cancer patients, and raise the possibility that treatment modalities blocking cyclin D1 activity may have a future role in the therapy of these patients.

Ectopic expression of cyclin D1 but not cyclin E induces anchorage-independent cell cycle progression.

Normal fibroblasts are dependent on adhesion to a substrate for cell cycle progression. Adhesion-deprived Rat1 cells arrest in the G1 phase of the cell cycle, with low cyclin E-dependent kinase activity, low levels of cyclin D1 protein, and high levels of the cyclin-dependent kinase inhibitor p27kip1. To understand the signal transduction pathway underlying adhesion-dependent growth, it is important to know whether prevention of any one of these down-regulation events under conditions of adhesion deprivation is sufficient to prevent the G1 arrest. To that end, sublines of Rat1 fibroblasts capable of expressing cyclin E, cyclin D1, or both in an inducible manner were used. Ectopic expression of cyclin D1 was sufficient to allow cells to enter S phase in an adhesion-independent manner. In contrast, cells expressing exogenous cyclin E at a level high enough to overcome the p27kip1-imposed inhibition of cyclin E-dependent kinase activity still arrested in G1 when deprived of adhesion. Moreover, expression of both cyclins D1 and E in the same cells did not confer any additional growth advantage upon adhesion deprivation compared to the expression of cyclin D1 alone. Exogenously expressed cyclin D1 was down-regulated under conditions of adhesion deprivation, despite the fact that it was expressed from a heterologous promoter. The ability of cyclin D1-induced cells to enter S phase in an adhesion-independent manner disappears as soon as cyclin D1 proteins disappear. These results suggest that adhesion-dependent cell cycle progression is mediated through cyclin D1, at least in Rat1 fibroblasts.

Phosphorylation and proteolysis: partners in the regulation of cell division in budding yeast

The budding yeast cell cycle oscillates between states of low and high cyclin B/cyclin-dependent kinase (CLB/CDK) activity. Remarkably, the two transitions that link these states are governed by ubiquitin-mediated proteolysis. The transition from low to high CLB activity is triggered by degradation of the CLB/CDK inhibitor SIC1, and the complementary excursion is propelled by the proteolytic destruction of CLBs. The extracellular environment controls this two-state circuit by regulating G 1 cyclin/CDK activity, which is directly required for SIC1 proteolysis. Thus, stable oscillations of chromosome replication and segregation in budding yeast are propagated by the interplay between protein phosphorylation and protein degradation.

Deregulation of cyclin E and D1 in breast cancer is associated with inactivation of the retinoblastoma protein.

Inactivation of the retinoblastoma protein (pRB) by mutations or abnormal phosphorylation is a mechanism by which tumour cells can subdue normal growth control. Among molecules involved in control of pRB phosphorylation, cyclin D1 and E have been found to be deregulated and overexpressed in various types of cancers. In order to study the cell cycle regulatory mechanisms in breast cancer, we have analysed the protein expression of cyclin D1 and E in 114 tumour specimens from patients with primary breast cancer using Western blotting. Twenty-five out of 34 tumours with overexpression of cyclin E showed uniform low cyclin D1 expression, and by immunohistochemical analysis of pRB we present evidence for the existence of pRB defects in approximately 40% of these tumours in contrast to no pRB defects in the other group of tumours. This result was supported by a high protein expression of the cyclin-dependent kinase inhibitor p16 in 44% of the tumours with high cyclin E and low D1 expression, and all immunohistochemical pRB defect tumours showed a high p16 protein level. Additionally, an abnormal low pRB phosphorylation in relation to a high proliferative activity and loss of heterozygosity of the retinoblastoma susceptibility gene locus were found in all but one tumour with immunohistochemical defect pRB. Interestingly, tumours with high cyclin E and low D1 expression were generally oestrogen receptor negative suggesting a role for cell cycle regulators in the mechanisms leading to oestrogen independent tumour growth. Furthermore, the prognosis differed markedly for the patients in the various groups of tumours, indicating that the heterogeneous nature of breast cancer pathogenesis and the clinical course in part could be explained by different and distinctive sets of cell cycle defects.

In VivoRegulation of the Early Embryonic Cell Cycle inXenopus

We report here the first extensivein vivostudy of cell cycle regulation in theXenopusembryo. Cyclin A1, B1, B2, and E1 levels, Cdc2 and Cdk2 kinase activity, and Cdc25C phosphorylation states were monitored during earlyXenopusembryonic cell cycles. Cyclin B1 and B2 protein levels were high in the unfertilized egg, declined upon fertilization, and reaccumulated to the same level during the first cell cycle, a pattern repeated during each of the following 11 divisions. Cyclin A1 showed a similar pattern, except that its level was lower in the egg than in the cell cycles after fertilization. Cyclin B1/Cdc2 kinase activity oscillated, peaking before each cleavage, and Cdc25C alternated between a highly phosphorylated and a less phosphorylated form that correlated with high and low cyclin B1/Cdc2 kinase activity, respectively. Unlike the mitotic cyclins, the level of cyclin E1 did not oscillate during embryogenesis, although its associated Cdk2 kinase activity cycled twice for each oscillation of cyclin B1/Cdc2 activity, consistent with a role for cyclin E1 in both S-phase and mitosis. Although the length of the first embryonic cycle is regulated by both the level of cyclin B and the phosphorylation state of Cdc2, cyclin accumulation alone was rate-limiting for later cycles, since overexpression of a mitotic cyclin after the first cycle caused cell cycle acceleration. The activity of Cdc2 closely paralleled the accumulation of cyclin B2, but cell cycle acceleration caused by cyclin B overexpression was not associated with elevation of Cdc2 activity to higher than metaphase levels. Tyrosine phosphorylation of Cdc2, absent during cycles 2–12, reappeared at the midblastula transition coincident with the disappearance of cyclin E1. Cyclin A1 disappeared later, at the beginning of gastrulation. Our results suggest that the timing of the cell cycle in theXenopusembryo evolves from regulation by accumulation of mitotic cyclins to mechanisms involving periodic G1cyclin expression and inhibitory tyrosine phosphorylation of Cdc2.

Functional analysis of the P box, a domain in cyclin B required for the activation of Cdc25

Cyclin B-cdc2 complexes are kept inactive by inhibitory phosphorylations on Thr-14 and Tyr-15 of cdc2 until they are dephosphorylated at the end of G2 by the phosphatase cdc25. Recent work has suggested that a small region of cyclin B, which we call the P box, may contribute part of a phosphatase-activating domain to cdc25. Individual conservative substitutions at three invariant residues within the P box yield mutant cyclin B proteins that bind cdc2 in vitro and then show the predicted cell cycle arrest, with cdc25 remaining in the low activity interphase form and cyclin B-cdc2 complexes remaining phosphorylated and inactive. While the low activity interphase form of cdc25 cannot act on cdc2 complexed with a mutant P box cyclin, the high activity M phase form of cdc25 can. These results demonstrate that the P box domain of cyclin B is required for cdc25 activation and support a two-step mechanism for the cdc25-dependent activation of cyclin B-cdc2.

Crimping in rat tail tendon collagen: morphology and transverse mechanical anisotropy

Examination of rat tail tendon units in scanning electron microscopy (SEM) after the removal of the endotendinium by the use of swelling agents and in comparison with controls confirms and extends our knowledge of a substantially planar crimping along the fibre axis. Polarizing optical microscopy of intact units subjected to lateral compression of controlled direction indicates a definite transverse mechanical anisotropy directly related to the morphological defined crimp plane, sensitive to shear disruption but capable of reconstitution on low strain cyclin.