Cyclin E, a redundant cyclin in breast cancer

Cyclin E is an important regulator of cell cycle progression that together with cyclin-dependent kinase (cdk) 2 is crucial for the G₁/S transition during the mammalian cell cycle. Previously, we showed that severe overexpression of cyclin E protein in tumor cells and tissues results in the appearance of lower molecular weight isoforms of cyclin E, which together with cdk2 can form a kinase complex active throughout the cell cycle. In this study, we report that one of the substrates of this constitutively active cyclin E/cdk2 complex is retinoblastoma susceptibility gene product (pRb) in populations of breast cancer cells and tissues that also overexpress p16. In these tumor cells and tissues, we show that the expression of p16 and pRb is not mutually exclusive. Overexpression of p16 in these cells results in sequestering of cdk4 and cdk6, rendering cyclin D1/cdk complexes inactive. However, pRb appears to be phosphorylated throughout the cell cycle following an initial lag, revealing a time course similar to phosphorylation of glutathione S-transferase retinoblastoma by cyclin E immunoprecipitates prepared from these synchronized cells. Hence, cyclin E kinase complexes can function redundantly and replace the loss of cyclin D-dependent kinase complexes that functionally inactivate pRb. In addition, the constitutively overexpressed cyclin E is also the predominant cyclin found in p107/E2F complexes throughout the tumor, but not the normal, cell cycle. These observations suggest that overexpression of cyclin E in tumor cells, which also overexpress p16, can bypass the cyclin D/cdk4-cdk6/p16/pRb feedback loop, providing yet another mechanism by which tumors can gain a growth advantage.     

Mutations and altered expression of p16INK4 in human cancer.

Cell cycle arrest at the G1 checkpoint allows completion of critical macromolecular events prior to S phase. Regulators of the G1 checkpoint include an inhibitor of cyclin-dependent kinase, p16INK4; two tumor-suppressor proteins, p53 and RB (the product of the retinoblastoma-susceptibility gene); and cyclin D1. Neither p16INK4 nor the RB protein was detected in 28 of 29 tumor cell lines from human lung, esophagus, liver, colon, and pancreas. The presence of p16INK4 protein is inversely correlated with detectable RB or cyclin D1 proteins and is not correlated with p53 mutations. Homozygous deletions of p16INK4 were detected in several cell lines, but intragenic mutations of this gene were unusual in either cell lines or primary tumors. Transfection of the p16INK4 cDNA expression vector into carcinoma cells inhibits their colony-forming efficiency and the p16INK4 expressing cells are selected against with continued passage in vitro. These results are consistent with the hypothesis that p16INK4 is a tumor-suppressor protein and that genetic and epigenetic abnormalities in genes controlling the G1 checkpoint can lead to both escape from senescence and cancer formation.     

p27kip1 Regulates cdk2 activity in the proliferating zone of the mouse intestinal epithelium: potential role in neoplasia

BACKGROUND & AIMS: Reduced p27(kip1) expression is a marker of poor prognosis in colorectal neoplasia, and inactivation of p27 in mice (p27(Delta51/Delta51)) causes increased intestinal epithelial cell proliferation and small and large intestinal neoplasia in a diet-dependent manner. Here, we addressed the role of p27 in untransformed intestinal epithelial cells in vivo and the consequence of its targeted inactivation. METHODS: A sequential fractionation procedure was used to isolate murine intestinal epithelial cells relative to their position along the crypt-villus axis, and the levels of cyclins, cyclin-dependent kinases (cdks), and cdk inhibitors and of the complexes formed among them was determined by immunoprecipitation-immunoblotting and kinase assays. RESULTS: As cells exited the proliferative crypt compartment, expression and activity of both cdk2 and cdk4 decreased, in parallel with reduced expression of cyclin A and proliferating cell nuclear antigen (PCNA); expression of cyclin D1, D2, and cyclin E showed little change. As expected, expression of the cdk inhibitors p21, p57, and p16 was highest in differentiated villus cells. Unexpectedly, p27 protein expression was highest in cells of the proliferative crypt compartment where it bound both cdk2 and cdk4. Cdk2 activity was increased in crypt cells from p27(Delta51/Delta51) mice, although cyclin D-associated kinase activity was unchanged (indeed, cyclin D1/2-cdk4 complex levels were reduced). Importantly, cdk2 activity was unchanged in crypt cells from p21(-/-) mice, which do not develop intestinal tumors. CONCLUSIONS: We propose that p27 contributes to intestinal epithelial homeostasis by regulating cdk2 activity in proliferating cells, thus gating cell cycle progression and suppressing intestinal neoplasia.     

Functional regulation of D-type cyclins by insulin-like growth factor-I and serum in multiple myeloma cells

D-type cyclin genes are universally dysregulated in multiple myeloma (MM), but the functional consequences are unclear as D-type cyclin gene expression does not correlate with proliferation or disease progression. We examined the protein expression and regulation of D-type cyclins and other cell cycle regulators in human myeloma cell lines and primary CD138(+) plasma cells (PCs). Cyclin D1, cyclin D2, cyclin dependent kinase (CDK) 4, CDK6, p27(Kip1) p18(INK4C) and retinoblastoma protein (pRb) were absent in normal PCs, heterogeneously expressed in primary MM cells and positively correlated with disease activity/progression. Cyclins D1 and D2 complexed with both CDK4 and CDK6, suggesting that both phosphorylate pRb in MM. Furthermore, cyclin D2 expressed via either t(14;16) or t(4;14) IgH translocations was functionally upregulated by fetal calf serum or insulin-like growth factor-I, leading to pRb phosphorylation and cell cycle entry/progression, and in some cases inversely correlated with p27(Kip1). However, pRb phosphorylation and cell cycle progression mediated by cyclin D1 expressed via t(11;14) was less dependent on exogenous stimuli. These data suggest that the presence or absence of specific IgH translocations underlying aberrant D-type cyclin expression may influence their response to mitogens in the bone marrow microenvironment. We showed for the first time that D-type cyclins are functionally regulated in MM, differentially responsive to exogenous growth factors and upregulated with disease progression.     

Alterations of retinoblastoma protein and p16INK4 protein expression in extrahepatic bile duct carcinomas

BACKGROUND/AIMS: Human cancer development results from dysfunction of G1-phase regulators of the cell cycle. Retinoblastoma protein and p16INK4 are the most essential links between cell cycle control and cancer. We examined the expression of p16INK4 and pRb and their possible prognostic relevance in 34 extrahepatic bile duct carcinomas. METHODOLOGY: Expression of pRb and p16INK4 was determined using immunohistochemical techniques. Associations between expression of pRb and p16INK4 and the clinicopathological features were analyzed by using the chi 2 test and survival analysis was performed by Log-rank test. RESULTS: Two (6%) extrahepatic bile duct carcinomas were pRb negative, 26 (76%) showed pRb overexpression, and 6 (18%) demonstrated moderate expression. Twenty-two (65%) tumors were p16INK4 negative and 12 (35%) were p16INK4-positive. Cases with pRb-negative or pRb overexpression were significantly correlated with tumor progression (P = 0.004) and TNM stage (P = 0.009). Alterations in pRb and p16INK4 expression did not correlate with patient outcome. CONCLUSIONS: Alterations of pRb and p16INK4 expression are frequently involved in extrahepatic bile duct carcinomas, and that aberrant pRb expression significantly associates with tumor progression.     

Deregulation of G1/S transition is a common event in carcinoma of the ampulla of vater

BACKGROUND/AIMS: Aberrant expression of cell cycle regulators and subsequent deregulation of G1/S transition is one of the most important characteristics of human cancer. The aim of this study was to determine the overall pattern of deranged expression of the cell cycle regulators involved in the G1/S transition in ampullary carcinoma. METHODOLOGY: Using immunohistochemistry, we investigated the expression of p21WAF1/CIP1, p27Kip1, p16INK4, cyclin D1, cyclin E, pRb and p53 in 14 resected specimens of ampullary carcinoma and defined the proliferative activity of each tumor by quantifying Ki-67 antigen. RESULTS: Decreased expression of p21WAF1/CIP1, p27Kip1, and p16INK4 was detected in 6 (43%), 11 (79%), and 4 (29%) tumors, respectively. Four tumors (29%) overexpressed cyclin D1 and 8 (57%) overexpressed cyclin E. Eight tumors (57%) overexpressed pRb. Aberrant accumulation of p53 was observed in 10 (71%) of the tumors. Overall, the expression of two or more of these cell cycle regulators was altered in all of the 14 tumors. Decreased p21WAF1/CIP1 expression was related to higher TMN stage (P = 0.04) and lymphatic invasion (P = 0.04). The proliferative index was higher in tumors with decreased p27Kip expression (P = 0.005), and in tumors with cyclin E overexpression (P = 0.06). CONCLUSIONS: Our observations suggest that deregulation of G1/S transition is a very common event in ampullary carcinoma, and that altered expression of cell cycle regulators is associated with the aggressive behavior of this tumor. Correcting the G1/S transition regulatory machinery may provide a novel therapy for this malignancy.     

G1 cyclins are involved in the mechanism of cardiac myocyte hypertrophy induced by angiotensin II

The importance of the cell cycle in proliferating cells is well known, but little is known about the role of cell cycle regulatory proteins in cardiac myocytes, which are fully differentiated cells. The present study determined, in vitro, the effect of angiotensin II (Ang II) treatment of neonatal rat cardiac myocytes on protein levels of cyclins and retinoblastoma gene product (pRb) phosphorylation. The role of G1 cyclin/cdk in Ang II-induced cardiac myocyte hypertrophy by overexpressing cdk inhibitor p21Cip1/Waf1 or p16INK4a was also examined using recombinant adenoviral vectors encoding these genes. Western blot analysis revealed that Ang II stimulated cyclin D1, D2, D3 and A protein levels in cardiac myocytes. Moreover, Ang II phosphorylated pRb on serine 780, which is known to occur in mitotic cells during cell cycle progression. Cultured cardiac myocytes treated with Ang II and infected with either control or recombinant adenovirus indicated that expression of p21 and p16 inhibited Ang II-induced cardiac myocyte hypertrophy, [3H]leucine incorporation into total cellular proteins, and skeletal alpha-actin (SK-A) and atrial natriuretic peptide (ANP) mRNA accumulation. Control virus had no effects on these parameters. These results suggest that G1 cyclins play an important role in cardiac myocyte hypertrophy stimulated by Ang II.     

Mutant parathyroid hormone-related protein, devoid of the nuclear localization signal, markedly inhibits arterial smooth muscle cell cycle and neointima formation by coordinate up-regulation of p15Ink4b and p27kip1

Arterial expression of PTH-related protein is markedly induced by angioplasty. PTH-related protein contains a nuclear localization signal (NLS). PTH-related protein mutants lacking the NLS (DeltaNLS-PTH-related protein) are potent inhibitors of arterial vascular smooth muscle cell (VSMC) proliferation in vitro. This is of clinical relevance because adenoviral delivery of DeltaNLS-PTH-related protein at angioplasty completely inhibits arterial restenosis in rats. In this study we explored the cellular mechanisms through which DeltaNLS-PTH-related protein arrests the cell cycle. In vivo, adenoviral delivery of DeltaNLS-PTH-related protein at angioplasty markedly inhibited VSMC proliferation as compared with angioplastied carotids infected with control adenovirus (Ad.LacZ). In vitro, DeltaNLS-PTH-related protein overexpression was associated with a decrease in phospho-pRb, and a G(0)/G(1) arrest. This pRb underphosphorylation was associated with stable levels of cdks 2, 4, and 6, the D and E cyclins, p16, p18, p19, and p21, but was associated with a dramatic decrease in cdk-2 and cdk4 kinase activities. Cyclin A was reduced, but restoring cyclin A adenovirally to normal did not promote cell cycle progression in DeltaNLS-PTH-related protein VSMC. More importantly, p15(INK4) and p27(kip1), two critical inhibitors of the G(1/S) progression, were markedly increased. Normalization of both p15(INK4b) and p27(kip1) by small interfering RNA knockdown normalized cell cycle progression. These data indicate that the changes in p15(INK4b) and p27(kip1) fully account for the marked cell cycle slowing induced by DeltaNLS-PTH-related protein in VSMCs. Finally, DeltaNLS-PTH-related protein is able to induce p15(INK4) and p27(kip1) expression when delivered adenovirally to primary murine VSMCs. These studies provide a mechanistic understanding of DeltaNLS-PTH-related protein actions, and suggest that DeltaNLS-PTH-related protein may have particular efficacy for the prevention of arterial restenosis.     

The expression of p53, p16, cyclin D1 in esophageal squamous cell carcinoma and esophageal dysplasia]

BACKGROUNDS/AIMS: p53 is known to play a central role in sensing and signaling for the growth arrest and apoptosis in cells with DNA damage. Mutation of p53 is a frequent event in esophageal squamous cell carcinoma (ESCC). p16 protein binds to cyclin dependent kinase 4 (CDK4) inhibiting the ability of CDK4 to interact with cyclin D1, and stimulates the passage through the G1 phase of cell cycle. We observed the expression patterns and frequencies of p53, p16, and cyclin D1 in esophageal dysplasia and in esophageal squamous cell carcinomas. METHODS: In 15 patients of ESCC, 5 patients of esophageal dysplasia and 5 volunteers with normal esophagus, tissue specimens were taken from esophageal lesions during the operation or endoscopic examination. We used specific monoclonal antibodies for p53 protein, p16(INK4 ) protein and cyclin D1. Immunoreactivity was scored. RESULTS: Mean age of all groups was 66 years old (range 47-93) and men to women ratio was 19:1. p53 mutation was observed in 87% (13/15) of ESCC, in 80% (4/5) of esophageal dysplasia, in 0% (0/5) of normal mucosa (p=0.001). p16 expression was seen in 40% (2/5) of esophageal dysplasia, 27% (4/15) of ESCC and 100% (5/5) of normal mucosa (p=0.016). Cyclin D1 expression was not significantly different among 20% (1/5) of esophageal dysplasia, 53% (8/15) of ESCC and 20% (1/5) of normal mucosa. Either the expression of p53 mutation or the loss of p16 occurred in 80% (4/5) of esophageal dysplasia and in 93% (14/15) of ESCC. CONCLUSIONS: The expression of p53 mutation and the loss of p16 might play a central role in the pathogenesis of esophageal squamous cell carcinoma (ESCC), and contribute to the development of precancerous lesion such as dysplasia. In addition, there is a possibility that the mutations of p53 and p16 silencing would be the early events in ESCC development.     

Expression of thymidylate synthase in human gastric and colorectal adenocarcinomas is upregulated by p16/INK4

BACKGROUND/AIMS: We observed the relationship between the expression of thymidylate synthase protein (pTS) and cell cycle regulators in gastric and colorectal adenocarcinoma tissues. METHODOLOGY: This study included 80 gastric and 50 colorectal adenocarcinomas. Immunohistochemical staining was performed using a polyclonal antibody to recombinant human pTS, and monoclonal antibodies to p53, p21/WAF1CIP1, p16/INK4, cyclin D1 and pRB. Each staining was quantified using computerized image analysis on a CAS 200 system. We selected the mean expression values as the cutoff values to distinguish between high and low expression of these substances. RESULTS: There was no relationship between pTS expression and p21/WAF1CIP1, cyclin D1, or pRB expression in gastric and colorectal carcinomas. In both gastric and colorectal carcinomas, the pTS expression was significantly low in the high p16/INK4 expression subgroup compared with the low p16/INK4 expression subgroup (P < 0.05). Further, the pTS expression was significantly high in the high p53 expression subgroup compared with the low p53 expression subgroup in colorectal adenocarcinomas (P < 0.05). CONCLUSIONS: pTS expression regulation in human gastric and colorectal adenocarcinomas in complex, and upregulated by p16/INK4.     

Role of cdk4, p16INK4, and Rb expression in the prognosis of bronchioloalveolar carcinomas

BACKGROUND: The p16(INK4) protein has been identified as a potent inhibitor of cyclin-dependent kinase (cdk)4 by blocking cdk4-mediated phosphorylation of the tumor suppressor retinoblastoma (Rb) protein, thus allowing Rb-mediated growth suppression. OBJECTIVES: Loss of p16(INK4) has been associated with a poor cancer prognosis, but its potential significance in bronchioloalveolar carcinomas (BACs) has not been explored. METHODS: We examined immunohistochemical expression of p16(INK4), cdk4, and Rb proteins in 38 BACs and correlated their expression levels with known clinicopathological features of the disease. RESULTS: All BACs expressed cdk4, while 89 and 82% expressed p16(INK4) and Rb proteins, respectively. None of the clinicopathological factors correlated with p16(INK4), cdk4, or Rb expression separately. A low p16(INK4)/cdk4 ratio was significantly associated with a high disease stage (p = 0.04), and the ratio tended to be lower in mucinous than nonmucinous tumors. BACs with a low p16(INK4)/cdk4 ratio showed significantly higher Rb expression levels (p = 0.02). Univariable survival analyses showed a significantly lower 5-year survival probability in patients with a high stage (p = 0.002) or low p16(INK4)/cdk4 ratio (p = 0.01). CONCLUSIONS: The results suggest a role of the cdk4/p16(INK4) pathway in the prognosis of BACs. Further studies are warranted to clarify whether a low p16(INK4)/cdk4 ratio may identify tumors that are destined to behave unfavorably.     

Frequent alterations in the expression of tumor suppressor genes p16INK4A and pRb in esophageal squamous cell carcinoma in the Indian population

PURPOSE: Alterations in the cell cycle regulatory p16INK4a/Cyclin D1/pRb pathway play a pivotal role in tumorigenesis. Knowledge of alterations in the tumor suppressor protein pRb and its negative regulator, p16CDKN2/MTS1/INK4a in esophageal squamous cell carcinoma (ESCC) from the Indian subcontinent is meager. To gain insight into the mechanisms underlying tumorigenesis and to search for diagnostic molecular markers for ESCC, we analyzed the expression of p16INK4a and pRb in ESCCs in the Indian population. METHODS: Immunohistochemical analysis of pRb and p16INK4a proteins was carried out in paraffin-embedded sections from 61 surgically resected ESCCs and matched normal tissues, and the results correlated with clinicopathological parameters using chi square and Fisher's exact tests. Dual immunohistochemical analysis has been carried out to demonstrate the concomitant loss of expression of p16INK4a and pRb. RESULTS: Fifty-nine of 61 (97%) cases showed aberration(s) in either or both of these proteins confirming their critical role in esophageal tumorigenesis. Loss of pRb was observed in 51 of the 61 (84%) and loss of p16INK4a was observed in 35 of 61 (57%) cases. Loss of pRb showed significant association with dedifferentiation of the tumor (P = 0.004). p16-/pRb-, and p16+/pRb- phenotypes were significantly associated with nodal metastasis (P = 0.017 and 0.027, respectively), while p16-/pRb+ phenotype was associated with dedifferentiation of the tumor (P = 0.012). CONCLUSION: pRb/p16INK4a pathway plays a critical role in esophageal tumorigenesis in the Indian population. The dual hits (concomitant loss) of pRb and p16INK4a expression suggest that these two components are not mutually exclusive, and can both be altered in a significant proportion of primary ESCCs serving as putative diagnostic markers for esophageal cancer. However, the impact of dual hit on tumor behavior and disease prognosis remains to be determined.     

Expression of the telomerase catalytic subunit, hTERT, induces resistance to transforming growth factor beta growth inhibition in p16INK4A(-) human mammary epithelial cells

Failures to arrest growth in response to senescence or transforming growth factor beta (TGF-beta) are key derangements associated with carcinoma progression. We report that activation of telomerase activity may overcome both inhibitory pathways. Ectopic expression of the human telomerase catalytic subunit, hTERT, in cultured human mammary epithelial cells (HMEC) lacking both telomerase activity and p16(INK4A) resulted in gaining the ability to maintain indefinite growth in the absence and presence of TGF-beta. The ability to maintain growth in TGF-beta was independent of telomere length and required catalytically active telomerase capable of telomere maintenance in vivo. The capacity of ectopic hTERT to induce TGF-beta resistance may explain our previously described gain of TGF-beta resistance after reactivation of endogenous telomerase activity in rare carcinogen-treated HMEC. In those HMEC that overcame senescence, both telomerase activity and TGF-beta resistance were acquired gradually during a process we have termed conversion. This effect of hTERT may model a key change occurring during in vivo human breast carcinogenesis.     

Expression of cell-cycle regulatory proteins in endometrial carcinomas: correlations with hormone receptor status and clinicopathologic parameters

PURPOSE: The normal human endometrium is characterized by hormone-dependent variations in the levels of cell-cycle regulatory proteins during the menstrual cycle. As this tightly controlled system is disturbed in endometrial carcinomas, we analyzed which cell-cycle regulators are involved in endometrial carcinogenesis. METHODS: We performed Western blot analysis of five cell-cycle stimulating (cyclins D1, E, B1, cdk2, cdk4) and three cell-cycle inhibiting (p16(INK4a), p21(WAF1), Rb) proteins in 41 endometrial carcinoma specimens. In addition, expression of the estrogen and progesterone receptors (ER, PR), Ki67, and, in selected cases, p16, cyclin E, and cyclin B1 was studied by immunohistochemistry. RESULTS: We found upregulation of all analyzed cell-cycle regulators in most tumors compared to normal endometrial tissue samples. Overexpression of cyclin E, cyclin B1, and p21 was associated with a less differentiated phenotype. In addition, high levels of cyclin E, cdk2, and cdk4 correlated with weak/absent ER expression, and p16 and p21 overexpression was significantly associated with low PR immunoreactivity. Cyclin B1 expression correlated with cyclin E, cdk2, cdk4, p21, Rb, and Ki67, and cyclin E expression with cyclin D1 and Rb. CONCLUSIONS: We conclude that cyclin E and cyclin B1 might be the major cell-cycle regulators involved in proliferation and reduced differentiation of endometrial carcinomas. In addition, p16, p21, and Rb appear to be uncoupled from their normal cell-cycle inhibiting function in many endometrial carcinomas.